Current Advance in Pharmaceutical Biotechnology

Advances in Biochemical Engineering/Biotechnology 171
Series Editor: T. Scheper
Ana Catarina Silva

João Nuno Moreira
José Manuel Sousa Lobo
Hugo Almeida Editors
Current
Applications
of Pharmaceutical
Biotechnology
171
Advances in Biochemical
Engineering/Biotechnology
Series Editor
T. Scheper, Hannover, Germany
Editorial Board
S. Belkin, Jerusalem, Israel
T. Bley, Dresden, Germany
J. Bohlmann, Vancouver, Canada
M.B. Gu, Seoul, Korea (Republic of)
W.-S. Hu, Minneapolis, USA
B. Mattiasson, Lund, Sweden
H. Seitz, Potsdam, Germany
R. Ulber, Kaiserslautern, Germany
A.-P. Zeng, Hamburg, Germany
J.-J. Zhong, Shanghai, China
W. Zhou, Shanghai, China
Aims and Scope
This book series reviews current trends in modern biotechnology and biochemical
engineering. Its aim is to cover all aspects of these interdisciplinary disciplines,
where knowledge, methods and expertise are required from chemistry, biochemistry,
microbiology, molecular biology, chemical engineering and computer science.
Volumes are organized topically and provide a comprehensive discussion of

developments in the field over the past 3–5 years. The series also discusses new
discoveries and applications. Special volumes are dedicated to selected topics
which focus on new biotechnological products and new processes for their synthesis
and purification.
In general, volumes are edited by well-known guest editors. The series editor and
publisher will, however, always be pleased to receive suggestions and supplemen-
tary information. Manuscripts are accepted in English.
In references, Advances in Biochemical Engineering/Biotechnology is abbreviated

as Adv. Biochem. Engin./Biotechnol. and cited as a journal.
More information about this series at http://www.springer.com/series/10

Ana Catarina Silva • João Nuno Moreira •
José Manuel Sousa Lobo • Hugo Almeida
Editors
Current Applications
of Pharmaceutical
Biotechnology
With contributions by
T. R. Abreu H. Almeida E. T. Baran L. S. Battaglia
J. M. S. Cabral S. Chao C. P. Costa C. L. da Silva
M. de Almeida Fuzeta A. D. de Matos Branco
A. del Pozo-Rodríguez M. M. Diogo T. G. Fernandes
A. Fernandes-Platzgummer I. Gómez-Aguado
J. Goncalves J. Gonçalves M. R. Ladisch
J. M. S. Lobo A. M. Manuel H. H. Mao J. Ministro
C. C. Miranda J. N. Moreira R. Ribeiro
J. Rodríguez-Castejón A. Rodríguez-Gascón
S. R. Rudge A. C. Silva D. B. Singh M. Á. Solinís
A. Tahmasebifar M. Vicente-Pascual B. Yilmaz
Editors
Ana Catarina Silva João Nuno Moreira
UCIBIO, REQUIMTE, MEDTECH, Center for Neurosciences and Cell Biology
Laboratory of Pharmaceutical Technology, (CNC) and Faculty of Pharmacy (FFUC)
Department of Drug Sciences, Faculty of University of Coimbra
Pharmacy Coimbra, Portugal
University of Porto
Porto, Portugal Hugo Almeida
UCIBIO, REQUIMTE, MEDTECH,
FP-ENAS (UFP Energy,
Laboratory of Pharmaceutical Technology,
Environment and Health Research Unit),
Department of Drug Sciences, Faculty of
CEBIMED (Biomedical Research Centre),
Pharmacy
Faculty of Health Sciences
University of Porto
Fernando Pessoa University
Porto, Portugal
Porto, Portugal
José Manuel Sousa Lobo

UCIBIO, REQUIMTE, MEDTECH,
Laboratory of Pharmaceutical Technology,
Department of Drug Sciences, Faculty of
Pharmacy
University of Porto
Porto, Portugal
ISSN 0724-6145 ISSN 1616-8542 (electronic)

Advances in Biochemical Engineering/Biotechnology
ISBN 978-3-030-40463-5 ISBN 978-3-030-40464-2 (eBook)
https://doi.org/10.1007/978-3-030-40464-2
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Preface
Biopharmaceuticals have been showing high therapeutic potential, especially for

medical conditions associated with unmet medical needs. Examples of these condi-
tions include cancer and autoimmune, metabolic, dermatological, neurological, and
genetic diseases. Thereby, current clinical relevance of biopharmaceuticals is
unquestionable. Since the 1980s, several biological medicines have been approved
and, due to patents expiration, many biosimilars have been marketed.
Manufacture and formulation of biopharmaceuticals comprising therapeutic pro-
teins generated by DNA recombination is challenging. Depending on the protein’s
molecular weight and structure complexity, industrial manufacturing processes have
employed different host cells. Furthermore, biopharmaceuticals formulation is fun-
damental to achieve an effective medicine, being necessary to stabilize the protein
conformation, enabling its efficacy while avoiding immunogenicity and other safety
problems. Commercial biopharmaceuticals are typically for parenteral administra-
tion, although several efforts have been made to find alternative administration
routes and/or new delivery systems enabling improved pharmacokinetic properties
and better patient compliance.
Therapeutic applications of biopharmaceuticals are highly diverse and include
active substances such as monoclonal antibodies, cytokines, growth factors, hor-
mones, blood products, therapeutic enzymes, as well as vaccines, cellular therapies
and products of gene therapy. In respect of biopharmaceuticals (including
biosimilars and orphans), the European Medicines Agency (EMA) and the Food
and Drug Administration (FDA) have approved so far 89 cytokines, 78 hormones
(insulin, glucagon, growth hormone, gonadotropins, and thyroid-stimulating hor-
mone and parathyroid hormone), 49 blood products (blood factors, anticoagulants,
and thrombolytic agents), and 22 therapeutic enzymes. Current research has been
also focused on identifying new clinical applications for existing therapeutic
proteins.
A number of different advances in the development, manufacturing, and
characterization of vaccines have enabled the prevention of infections and promoted
human health worldwide. Approved recombinant vaccines include Shingrix, Ebola
v
vi Preface
(Ad5-EBOV), meningococcal subgroup B, human papilloma virus (HPV), dengue,

enterovirus 71 (EV71), and hepatitis B (HBV), while vaccines against rVSV-
ZEBOV Ebola, respiratory syncytial virus (RSV), and malaria are under clinical
evaluation.
In addition to biopharmaceuticals and vaccines, advanced therapy medicinal
products based on the use of cells, tissues, or genes offer new therapeutic possibil-
ities. For example, cell-based therapies have been used in regenerative medicine,
disease modelling, and drug screening studies. This has also constituted an oppor-
tunity to engineer new manufacturing concepts as 3D bioprinting technology, which
have succeeded in generating living tissues and organs from a combination of cells,
biomaterial bioinks, and growth factors. Examples of bioprinted tissues include
nerve, skin, heart, bone, cartilage, skeletal muscle and soft tissues such as liver,
cornea, intestine, and bladder.
In the last years, gene therapy medicines, such as DNA-based therapies, antisense
oligonucleotides, small interfering RNA, and T-cell-based therapies, have shown
promising results to such a point that they have started to change the treatment
paradigm, as in cancer. Furthermore, the use of gene-editing approaches, such as the
CRISPR (clustered regularly interspaced short palindromic repeats), has started to be
assessed at the clinical level. Besides cancer, the clinical usefulness of gene therapy
is also being assessed in monogenic infections, inflammatory, cardiovascular, and
neurodegenerative diseases.
Within the scope of the different therapeutic tools herein addressed, pharmaco-
genomics assumes a relevant role on their performance, in terms of both efficacy and
safety. In fact, the use of a medicine adapted to the genetic variants of each patient,
reducing adverse effects and improving treatment outcomes, supports pharmaco-
genomics as a relevant component of personalized medicine.
According to the wide range of pharmaceutical biotechnology applications, this
book aims at providing the most relevant up-to-date information for healthcare
professionals, students, and researchers.
We would like to thank all the authors of this book for contributing with high
quality chapters. We are also very grateful to all the reviewers, who had the kindness
of critically evaluating the chapters. In addition, we would like to thank to the Series
Editor, Professor Thomas Scheper and to the Associate Editors, Dr. Sofia Costa and
Dr. Tanja Weyandt, for giving us the opportunity of organizing this volume, and to
the publishing team (Ms. Alamelu Damodharan and Ms. S. Dhivya Geno) for their
kind help.
Porto, Portugal Ana Catarina Silva

Coimbra, Portugal João Nuno Moreira
Porto, Portugal José Manuel Sousa Lobo
Porto, Portugal Hugo Almeida
Contents
Industrial Challenges of Recombinant Proteins . . . . . . . . . . . . . . . . . . . 1

Scott R. Rudge and Michael R. Ladisch
Insights on the Formulation of Recombinant Proteins . . . . . . . . . . . . . . 23
Rita Ribeiro, Teresa Raquel Abreu, Ana Catarina Silva, João Gonçalves,
and João Nuno Moreira
Therapeutic Antibody Engineering and Selection Strategies . . . . . . . . . . 55
Joana Ministro, Ana Margarida Manuel, and Joao Goncalves
Cytokines and Growth Factors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 87
A. C. Silva and J. M. Sousa Lobo
Hormones, Blood Products, and Therapeutic Enzymes . . . . . . . . . . . . . . 115
Ana Catarina Silva, Cládia Pina Costa, Hugo Almeida, João Nuno Moreira,
and José Manuel Sousa Lobo
Advances in Vaccines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 155
Helen H. Mao and Shoubai Chao
Human Pluripotent Stem Cells: Applications and Challenges
for Regenerative Medicine and Disease Modeling . . . . . . . . . . . . . . . . . . 189
Cláudia C. Miranda, Tiago G. Fernandes, M. Margarida Diogo,
and Joaquim M. S. Cabral
Addressing the Manufacturing Challenges of Cell-Based Therapies . . . . 225
Miguel de Almeida Fuzeta, André Dargen de Matos Branco,
Ana Fernandes-Platzgummer, Cláudia Lobato da Silva,
Bioprinting Technologies in Tissue Engineering . . . . . . . . . . . . . . . . . . . 279
Bengi Yilmaz, Aydin Tahmasebifar, and Erkan Türker Baran
vii
viii Contents
Gene Therapy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 321

Ana del Pozo-Rodríguez, Alicia Rodríguez-Gascón,
Julen Rodríguez-Castejón, Mónica Vicente-Pascual, Itziar Gómez-Aguado,
Luigi S. Battaglia, and María Ángeles Solinís
The Impact of Pharmacogenomics in Personalized Medicine . . . . . . . . . 369
Dev Bukhsh Singh
Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 395
Adv Biochem Eng Biotechnol (2020) 171: 1–22
DOI: 10.1007/10_2019_120
Published online: 18 December 2019
Industrial Challenges of Recombinant

Proteins
Scott R. Rudge and Michael R. Ladisch
Contents
1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2
2 Classes of Products (Agonists and Antagonists) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3
2.1 Agonists . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3
2.2 Antagonists . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4
2.3 Humanized Antibodies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4
2.4 Biosimilars . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6
2.5 Pharmacoeconomics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6
3 Upstream Manufacturing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6
3.1 Expression Systems . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6
3.2 Promoters . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7
3.3 High Cell Density Reactors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8
4 Single Use Manufacturing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9
4.1 Single-Use Bioreactors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
5 Perfusion Reactors and Continuous Processing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
5.1 Cell Retention by Cross-Flow Filtration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11
5.2 Cell Retention by Centrifugation or Sedimentation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12
5.3 Cell Retention by Acoustic Separation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13
5.4 Perfusion Reaction Control . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14
6 Cell Separation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14
6.1 Centrifugation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15
6.2 Depth Filtration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15
6.3 Cross-Flow Filtration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16
6.4 Sedimentation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16
7 Downstream Processing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16
7.1 Chromatography and Adsorption . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17
7.2 Intensified Chromatography . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18
S. R. Rudge (*)
RMC Pharmaceutical Solutions, Inc., Longmont, CO, USA
e-mail: srudge@rmcpharma.com
M. R. Ladisch (*)
Purdue University Laboratory of Renewable Resource Engineering, West Lafayette, IN, USA
e-mail: ladisch@purdue.edu
2 S. R. Rudge and M. R. Ladisch
8 Single Use Downstream Processing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20

9 Continuous Downstream Processing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20
10 Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21
Abstract The use of recombinant DNA methods to produce large quantities of

protein for therapeutic uses has revolutionized medicine. Industrial challenges for
manufacture of biotherapeutic proteins are related to the characteristics of these
proteins and the increasing quantities required to address needs of patients, world-
wide. A brief overview of therapies in which proteins are employed helps to frame
some of the challenges facing their industrial production. The number of molecules
and their applications have significantly expanded over the last 15–20 years, together
with the quantities used to address specific indications. Challenges addressed include
achieving cell density, protein expression, separations of cells and protein, protein
purification, and segmentation of protein production into smaller quantities with the
evolution of personalized medicine and products designed for increasingly small
patient populations. This chapter highlights some of the current challenges.
Graphical Abstract
Membrane
Inoculum Cells
Chromatography
Membrane
Protein
Bioreactor Impurities
Concentrated
(Cell Culture) Product
Keywords Biologics, Bioprocessing, Cell culture, Cell separation, Chromatograhpy,

Expression systems, Fermentation, Filtration, mAbs, Recombinant proteins,
Separations
1 Introduction
Since the early commercialization of recombinant human insulin, recombinant

human growth hormone, and recombinant bovine growth hormone, the field has
expanded to include over 174 US-approved therapeutic proteins, including 98 anti-
bodies (including biosimilars) [1, 2]. While insulin was among the first recombinant
products, its lower molecular weight (~6 kD), long history of use, and molecular
Industrial Challenges of Recombinant Proteins 3
characterization positioned it for scaling up [3]. Subsequent early products such as

erythropoietin and tissue plasminogen activator, with their higher molecular weights
and complex structures, required production through mammalian cell culture and the
many attendant scaling and regulatory factors that needed to be addressed. Along the
way, the development and laboratory culture of hybridoma cells enabled the produc-
tion of monoclonal antibodies, proteins with molecular weights in the 120–150 kD
range, and for which initial applications were limited to research tools. It required
about 20 years of further molecular engineering as biopharmaceuticals to develop this
class of proteins. Further advances have been made in yeast, specifically Pichia
pastoris (reclassified as Komagataella pastoris), to enable the production of large
and small proteins including antibodies and proinsulins. Gene therapy has now
emerged with potential to not only treat diseases but perhaps provide cures. With
the emergence of gene therapy has come the need for vectors consisting of
nanoparticulate viral capsids, i.e., structured protein assemblies, for delivery of
nucleic acid payloads. Manufacturing approaches have employed similar unit oper-
ations throughout the development of biotherapeutic products since their introduction
about 40 years ago. What has changed are cells and culture methods and the
separations media utilized during the manufacturing processes.
2 Classes of Products (Agonists and Antagonists)
Therapeutic recombinant proteins generally fall within two classes, those meant for
replacement therapy (agonists) and those meant for antagonist therapy.
2.1 Agonists
Replacement therapies are those in which the intended patient has lost the ability to
produce a protein, and that lack of ability results in disease. Type I diabetes is an
excellent example of such a disease, in that the patient has lost the ability to produce
insulin due to an autoimmune destruction of pancreatic islet cells. In such patients
exogenous insulin is required for the patient’s body to properly regulate glucose in
the blood stream. These patients were treated with insulin purified from porcine
pancreas before the introduction of recombinant protein production [2]. The injec-
tion of insulin at intervals related to meal times and times of fasting (overnight, for
example) allows these patients to regain some control, albeit imperfect, of their
blood glucose levels. In this case, the ability to inject a recombinant form of human
insulin replaces the patient’s ability to produce their own insulin. In general, low
doses of proteins in this category are required, as the protein being replaced
physiologically has a specific function. These proteins are also called “agonists,”
as their specific function is to make something happen. Other examples of agonists
are erythropoietin, granulocyte-colony stimulating factor, growth hormone, and

factor VIII.
New agonist therapies are not common, as most diseases requiring a replacement
protein are well-known and fatal and are/were being treated with a purified protein from
an animal, plasma fractionation, or cadavers. Additionally, these diseases are usually in
small patient numbers, as a rare genetic defect may be involved or an autoimmunity.
The search for new agonists continues, as these treatments are almost certain to be
clinically successful with few side effects. Progress in understanding the function of the
brain and the neurological system may be the last frontier for agonist therapies.
2.2 Antagonists
The other class of recombinant proteins is called antagonists. These proteins are used
to block some physiological action. Antagonists bind to receptors or other proteins,
or other molecules in the blood stream, and prevent them from having their normal
physiological function. An excellent example of an antagonist therapy is the che-
motherapeutic antibody Avastin or antihuman vascular endothelial growth factor
(VEGF) immunoglobulin G1. VEGF is a protein that stimulates the growth of new
blood vessels, which are produced by tumors growing in the body, which require a
source of nutrition from the blood stream. Avastin blocks the action of VEGF,
thereby depriving tumors of the source of nutrition they need to grow [4].
Antagonist therapies typically require higher doses than agonists, as the antago-
nist molecule has to battle stoichiometry and biology to bind and neutralize all the
target molecules in the patient. As the target molecules are neutralized by the
antagonist, the body tends to make more as the biological feedback loop indicates
that the desired effect is not being achieved. Additionally, the complete elimination
of a physiological mechanism is usually not desirable. For example, patients taking
Avastin will have difficulty growing new healthy tissue as well as tumor mass.
2.3 Humanized Antibodies
The development of humanized antibodies to treat human disease has been a second
major milestone in the history of recombinant protein therapies. Early attempts to use
antibodies to seek out and bind targets in humans were set back by immune responses
to the nonhuman epitopes in the antibody structure. Methods for “humanizing”
antibodies, including critically in the “complement defining region” or CDR, have
made the construction of antibodies specific for distinct biological targets possible [5].
Over the last 20 years, antibody antagonists have largely displaced recombinant
agonists as the top selling protein pharmaceuticals, as shown in Table 1 (compare [6]
to [7]) with the pipeline of therapeutic mAbs now at 560 in various stages of clinical
trials [8, 37].
Table 1 Top selling biopharmaceuticals from 1997 and 2011

1997 [6] 2011 [7]
1 Erythropoietin (agonist) Anti-TNF monoclonal antibody (antagonist)
2 Granulocyte-colony stimulating factor Anti-TNF receptor monoclonal antibody
(agonist) (antagonist)
3 Insulin (agonist) Anti-VEGF monoclonal antibody (antagonist)
4 α-Interferon (agonist) Anti-CD20 monoclonal antibody (antagonist)
5 Hepatitis B vaccine (agonist) Anti-HER2 receptor monoclonal antibody
(antagonist)
6 Glucocerebrosidase (agonist) Insulin (agonist)
7 Tissue plasminogen activator (agonist) PEGylated granulocyte-colony stimulating factor
(agonist)
8 Growth hormone (agonist) Erythropoietin (agonist)
9 GPIIb/IIIa antibody (antagonist) Anti-VEGF monoclonal antibody fragment
(antagonist)
10 Interferon β-1a (agonist) Interferon β-1a
Today, the top ten selling recombinant protein pharmaceuticals are mostly mono-
clonal antibodies. Antibody products accounted for about $114.6 billion in sales in
2018, while all biopharmaceuticals were about $237 billion. Total pharmaceutical
sales were $1,200 billion for reference. Interestingly, all of these products received
their marketing approval within the last 15 years, which demonstrates the rapid
growth of the product category and may indicate the large potential still remaining.
However, as with most technologies, the targets for antibodies and other antag-
onists are becoming harder to identify, and the targets that are being pursued are in
higher concentrations, requiring higher doses of antagonists [9]. Doses as high as
one or more grams per day are becoming more common. High doses can be
problematic because (1) they require greater purity, (2) they are harder to administer
to the patient, and (3) they require lower cost of goods. The third constraint is easily
understood, the treatment of disease is an economic proposition, and the market, or
society, will only bare so much cost. The cost of treating versus not treating must be
carefully balanced, and in cases where other therapies exist, a price is already set.
The market cares little about the mass of protein required to produce the desired
effect, and the cost of goods for an active antibody can vary between $100 and
$1,000/g, excluding testing, dosage form manufacturing, and other expenses [10].
Higher doses also require higher purity. This is best illustrated with the impurity
endotoxin, which is produced in very large quantities when Escherichia coli is used
to produce the recombinant protein. Endotoxin is part of the cell wall of gram-
negative bacteria and causes an immune response and inflammation when present in
the blood stream of humans. This activity of endotoxin, called pyrogenicity, is
measured in endotoxin units (EU) by a few standardized biological assays. The
total amount of endotoxin that a patient can be exposed to per 1 h administration is
limited by medical standards to 5 EU/kg (the kg refers to the weight of the patient). A
50 kg patient may be exposed to a total of 250 EU. If the dose of the protein is 10 mg,
the protein must be purified to at most 25 EU/mg, but if the protein dose is 2 g/day,
then the most endotoxin the product can contain is 0.125 EU/mg.
2.4 Biosimilars
With a regulatory pathway now open for biosimilar products, there will be a focus on
cost of goods for biologics like never before. When patent exclusivity expires, the
cost of making and purifying the protein will become a more significant part of the
selling price. The cost for making a purified therapeutic protein ranges from about
$100 to $1,000/g, with cell culture-derived proteins requiring higher expenditures.
Therefore, reduction of cost of goods at higher purity requirements will be a major
challenge for biopharmaceutical manufacturing in the immediate future.
2.5 Pharmacoeconomics
Finally, the political climate is one in which justification of high reimbursement for
pharmaceuticals is increasingly difficult. Pharmacoeconomics may not be sufficient
to justify reimbursement in all cases. Therefore, due to high doses, biologics
intended for treating diseases where other treatments exist, pressure from
biosimilars, and price restraints, the cost of manufacturing a therapeutic protein is
under pressure. This chapter will review some current efforts to decrease the cost of
manufacturing.
3 Upstream Manufacturing
Upstream manufacturing includes fermentation, cell culture, or another means of

expressing the therapeutic protein at high titer and reasonable purity. Upstream
manufacturing typically also includes the removal of the production cells or bio-
mass. This may be done with a centrifuge, a filter, by flocculation and settling, or
another unit operation.
3.1 Expression Systems
The expression of the protein is the first and primary step in producing the product in
its desired form. Expression systems consist of a host cell and the foreign DNA that
encodes the product. The most commonly used host cells are E. coli and Chinese
hamster ovary (CHO) cells. E. coli is used when the protein requires little posttrans-
lational modifications, while CHO cells are used when posttranslational modifica-
tions are required for the structure or function of the protein. Antibodies require
fairly extensive posttranslational modifications. For example, immunoglobulin Gs
(the most common therapeutic antibody and the simplest) require posttranslational
modifications in the form of the assembly of two heavy chains with two light chains
to form a hetero-tetramer, and glycosylation of an asparagine located on the heavy
chain constant region, so typically antibodies are made in CHO. Insulin only requires
enzymatic activation, which can be done in manufacturing with the correct protease,
so typically, insulin is made in E. coli.
Protein expression is complicated, and there are several steps that have to be
optimized to get the maximum expression of the desired protein. There are several
considerations:
• The codons in the gene for the desired protein must be optimized for the host cell.
• The gene must possess flanking regions, such as a poly-adenosine tail and a
ribosome binding site.
• The gene must be under the control of a promoter that will cause its expression,
either constitutively or in response to an inducer or derepressor.
• If it is desired to export the product to another compartment of the cell, or to
secrete the product, a leader sequence must be attached to direct the protein to that
cellular compartment.
• The gene must have the proper reading frame to ensure the appropriate sequence
is transcribed and translated.
There are numerous expression systems available, both proprietary and in the
public domain. The expression of every recombinant protein is different and
unpredictable, but the expression of antibodies tends to be fairly consistent from
antibody to antibody. A titer of 3 g/L can be expected for an antibody expressed from
nonproprietary expression constructs in CHO cells [11]. Titers as high as 15–25 g/L
have been reported in vendor marketing data; however the corresponding product
quality is unknown.
3.2 Promoters
A common promoter for protein expression in CHO cells is the cytomegalovirus

(CMV) promoter [12]. This promoter is constitutive, meaning it is always activated
and does not require induction. The titer is also dependent on the cell density – the
more cells, the more product. A typical cell density for CHO cells is 20 106 cells/
mL. However, high cell density cultures with up to 100 106 cells/mL are now
being developed which can increase the titer a commensurate fivefold. These cell
densities are a relatively new development, and their impact on downstream
processing is still being determined. Cell culture medium costs about $100/L on
average, so the media alone can contribute $10–$33/g to the cost of the product,
depending on the titer. Media used to achieve high cell density is more expensive,
but not five times more expensive, so the investment in high cell density is justified.
Product expression in E. coli is typically greater than 5 g/L and can be as high as
15 g/L. At very high expression levels in E. coli, the product is usually found in
inclusion bodies, small precipitate particles that have to be solubilized and renatured
to make them usable. A common expression system for E. coli is the T7 promoter,
which is induced with iso-propyl thio glycerol (IPTG) [13]. IPTG derepresses the T7
promoter, allowing transcription of the gene and subsequent translation. An alternate
promoter system is the xyz switch where amino acid content in the media is used to
induce insulin expression [3]. In most bacterial systems, the expression of the
foreign protein is toxic to the cells, and so inducible rather than constitutive
promoters are used. Inducer is added to the fermentation after high cell density is
reached, at which point the cells do not grow significantly. The final wet weight of
solids in the fermentation is typically 12–16% (120–160 g/L) of which the expressed
protein is about 10% (12–16 g/L as previously stated). On a dry weight basis, the
proportion of expressed protein is closer to 20–25% by weight. E. coli media is much
cheaper to prepare, usually $5–$10/L. If the expression level is reasonably high, the
cost of the media will only be $1–$2/g.
Efforts to increase protein expression are ongoing and remain important. When
the protein expression is increased, the purification is easier. However, additional
impurities are typically generated with highly expressed proteins as the protein
synthesis machinery is overtaken by the higher level of expression. One common
impurity is the mis-incorporation of norleucine for methionine residues [14]. Cova-
lent protein aggregates are also prevalent in highly expressed proteins. Some atten-
tion to the likely downstream yield purity must be paid when designing high
expression systems.
3.3 High Cell Density Reactors
Another way to increase product throughput is to grow cells to very high cell density.
In mammalian cell culture, this means densities on the order of 100 106 cells/mL,
and for bacterial and yeast fermentations, this means ODs greater than 100 and
approaching 300 or 400, and wet cell weights around 50%. These higher cell
densities are made possible by new media formulations that are rich in key nutrients,
as well as advances in oxygen transfer into and heat removal from the reactor vessel.
Increased cell density gives productivity gains linearly in proportion to the
increase of numbers of cells. CHO cultures are routinely run at 20 106 cells/mL,
so the ability to achieve 100 106 cells/mL offers an opportunity to increase the
productive time in the bioreactor given that protein expression and cell growth occur
concurrently, unlike E. coli where expression follows accumulation of cells and
subsequent induction. Since mammalian cell culture expression is typically consti-
tutive, the culture is producing product throughout, again in proportion to cell
density. The area under the cell density vs. time curve gives the basis for the product
expression. As shown in Fig. 1, 43% of the productivity of a 25-day culture can be
accounted for in the 6 days the culture achieves >80 106 cells/mL.
Bacterial expression systems are typically induced, and when they are induced,
the growth of the cells slows or stops, as metabolism is taken over by the protein
synthesis machinery of the cell. The fermentation may only last some hours after
90 12000
Cell Density x 10^6 cells/mL

80
Product produced (mg/L)

10000
70
60 8000
50
6000
40
30 4000
20
2000
10
0 0
0 5 10 15 20 25 30
Days
Cell Density Product Produced
Fig. 1 Typical cell density vs. time curve and protein production, assuming a doubling time of 24 h
and specific productivity of 1 pg/cell/day
induction of the expression system; 10–20 h is typical. In this case, the specific
productivity is unchanged as the productivity gains are directly proportional to the
cell density at induction.
Both high cell density techniques are limited in the further advantage that they
can bring to protein production. At 50% wet weight, another doubling in bacterial
fermentation productivity by this method is not possible. Extending the length of
time such high cell densities can be maintained with cells in maximum productivity
will be the most practical way to increase production in the future.
High cell density cell culture poses challenges in cell separation. These will be
discussed in a subsequent section.
4 Single Use Manufacturing
Many new products are designed for increasingly small patient populations. These
products are known as orphan products, and they are highly reimbursed. Gene and
cell therapies are also beginning to emerge in this space. Efficient expression
systems have made it possible to produce a worldwide supply of these drugs in a
few batches per year. Since it is not practical to dedicate an entire clean facility to
produce a few batches of a product per year, multi-product facilities have become
more common, and contract manufacturing organizations are being tasked for
production on a more frequent basis.
4.1 Single-Use Bioreactors
Keeping different products segregated from one another is a critical requirement of

multi-product facilities, which means extensive and detailed cleaning of the equip-
ment during product change-over. Another solution is to make inexpensive, dispos-
able equipment. Disposable bioreactors have been in use at commercial scale for
about 10 years for mammalian cell culture. Disposable systems increase the flexi-
bility of multi-product manufacturing facilities by removing the requirement for
post-use cleaning of the bioreactor. These bioreactors are typically made of plastic
and come pre-sterilized by gamma irradiation. While larger bioreactors have been
made, the most common large single-use bioreactor has a 2,000 L working volume, a
version of which is available from at least three different manufacturers. These are
accompanied by other single-use unit operations, such as filtration pods, chroma-
tography systems, and vessels. A fully “disposable factory” may still be cost
prohibitive, but it could become less expensive in the future.
While flexibility is promoted as a feature of disposable systems, particularly
bioreactors, such systems are not as flexible as might be imagined [15]. The bio-
reactors require a superstructure made of steel to support them. The single-use
bioreactors are very heavy and require a crane to lift them into the support structure
and then to lift them out again. Shipping is expensive and can result in pinholes and
creases in the plastic, which lead to bioreactor rupture or sterility failure. And while
some early economic analysis made the case that single-use plastic was more
economical and environmentally friendly than cleaning fixed stainless-steel reactors,
these analyses [16] ignored shipping, gamma irradiation, the clean room required to
fabricate the single-use reactors, and the effect of the plastic load on the biosphere.
Single-use systems are not readily available for large-scale microbial fermenta-
tion. As bacteria grow many times faster than mammalian cells, their oxygen transfer
and heat transfer requirements are much larger. Plastic bioreactors are not able to
hold significant pressure which facilitates oxygen transfer, and the magnetic drive
that is used to agitate plastic bioreactors cannot deliver the torque required for
mixing and heat and oxygen transfer.
5 Perfusion Reactors and Continuous Processing
One method for increasing bioreactor productivity is to increase the time the
fermentation or bioreaction remains at high cell density and maximum productivity.
One obvious way to do this is to continuously supply fresh nutrient medium while
removing spent medium and product (if secreted) while retaining cells. There are
two challenges in this endeavor, one is to continuously provide sterile medium, and
the other is to remove spent medium while retaining cells and maintaining sterility.
The nutrients required for mammalian cell culture are complex and not stable
to autoclaving. Therefore, the introduction of fresh medium is typically via filtration.
Mammalian cultures are also susceptible to viruses, and so viral reduction devices
such as “high-temperature-short-time” and UVC irradiators are being designed to
continuously introduce sterile, viral-free media into the bioreactor [17]. This chal-
lenge appears to have been substantially met.
Sterile cell retention is a different challenge. The bioreactor contains cells, along
with dead cells, cell fragments, lipids, and other debris. The cell separation device
must operate efficiently in this environment. Several methods that are currently in
use are:
• Cross-flow filtration
• Centrifugation and sedimentation
• Acoustic separation
It is important to separate the cells without lysing them. When lysed, cells release
proteases that can degrade the product and additional impurities that must be
subsequently removed by downstream purification processes. Ideally, the retention
device would retain only cells and allow removal of debris and unproductive solids.
Otherwise, the bioreaction will be choked off by accumulation of the unproductive
solids.
5.1 Cell Retention by Cross-Flow Filtration
The alternating tangential flow filtration device may be the most widely used device
for perfusion reactors today. It works by drawing culture fluid across a micro-filter in
tangential flow fashion. The culture fluid accumulates in an expanding bulb down-
stream of the filter. Cells are retained, and spent culture fluid with secreted product
crosses into the filtrate. Then the cycle is reversed, the bulb is collapsed, and the
retained cells are returned to the bioreactor. Since bioreactors typically operate under
slight positive pressure, the driving force for filtration is vacuum on the filtrate side
of the membrane. An example is shown in Fig. 2 [18].
The alternating tangential flow filter allows high throughput and essentially total
retention of cells. Perfusion flow rates of two volumes/reactor volume/day are
readily achievable. The technology is easy to scale up as the principals of tangential
flow filtration are well-known. The stream is already filtered and so already clarified,
so no further filtration is needed. When a filter clogs, which will happen ultimately, it
must be replaced without stopping operations. This can be done with tube welding
technology, or steam in place valves, although it is inconvenient and requires the
purchase of a second device.
The alternating tangential flow filter retains all solids, including nonproductive
solids, which can accumulate and choke the cell culture if not removed. Addition-
ally, as the perfusion rate increases, the alternating flow filter must operate faster,
increasing shear on the cells and creating additional debris. This is typically
addressed by adding a cell syphon stream where cells, debris, and medium are all
removed. This is an imperfect solution, however, and eventually the cell debris will
overtake the cell culture process.
Fig. 2 An alternating tangential flow filtration device [18]
5.2 Cell Retention by Centrifugation or Sedimentation
Centrifugation is also used as a cell retention mechanism. These centrifuge systems

use a modest centrifugal force (approximately 100–300 g) to sediment cells and
other solids and to enable withdrawal of a clear fluid stream. Care must be taken not
to damage the cells and not to pack them too tightly so that they can freely flow back
to the bioreactor. The moving parts of a centrifuge also make maintaining sterility
difficult. Because of these challenges, sedimentation has also been used as a cell
retention mechanism.
Inclined settling uses an inclined plane to settle and return live cells to a
bioreactor, while allowing product-containing medium to be removed from the
overflow. This method also allows dead cells and debris to be removed with the
product stream. A recent improvement has been made to this system by allowing the
flow over stacked cones, much like in a disk-stack centrifuge but without the added
centrifugal force [19, 20]. An example is shown in Fig. 3.
The downside to inclined settlers is the slow settling, which results in fairly large
volumes required to achieve the appropriate residence time. The large volume/long
residence time results in the settling device required being nearly as large as the
bioreactor itself, meaning cells spend significant time in an environment where pH
Fig. 3 Compact settler

diagram [20]. Media
containing cells are fed
through port 142. Product
and nonproductive solids
may be withdrawn through
port 140, and live cells can
be returned to the bioreactor
through port 139
and dissolved oxygen are not being actively controlled. Attaining high flow rates has
also been difficult in inclined settling systems, meaning high rates of turnover of
bioreactor contents are not achievable. However, as a solid/liquid separating device
with no moving parts that can be made of inexpensive, light weight disposable
materials, compact settlers have some advantages that cannot be ignored.
5.3 Cell Retention by Acoustic Separation
A final method for cell retention is the application of an ultrasound field [21]. The
ultrasound is able to retain particles of a certain size against a small flow field. This
method is also difficult to scale up, since the dimensions increase to provide a small
enough linear velocity for the flow field, the device becomes large relative to the
bioreactor, and the acoustic field heats the fluid throughout, while heat can only be
removed from the periphery of the device. This method for cell retention is rarely
used outside very small-scale lab experiments.
5.4 Perfusion Reaction Control
Continuous processing is being explored as a method for faster and less expensive
manufacturing of biologics such as therapeutic proteins and antibodies [1]. The
advantage of continuous processing is a relatively high productivity at a smaller
overall volume. Continuous processing may improve overall quality by reducing
hold times between batch steps and increase flexibility by allowing for longer
production times at higher volumetric productivities [22].
Continuous processing requires that a steady state be achieved. In typical steady-
state reactions, reactants are fed to a reactor to produce product at a constant rate,
which can then be removed and purified. In order to maintain the steady state,
feedback control is used to account for small fluctuations in conditions that can
cause variability in the rate of production, such as the concentration of reactants,
temperature and pressure of feed streams, and fouling of surfaces, for example. The
state of development of feedback control for bioreactors (and other bioprocessing
steps) is in its infancy, and much more work is needed in order to achieve true
continuous processing in biopharmaceuticals. To further develop feedback control,
more information will be needed on the effects of media, temperature, metabolite
concentrations, debris and unproductive solids, oxygen transfer, pH and osmolarity
on product quality, and production over time. This information is likely to be
complex and process specific and may need to be studied on the genetic level,
where different genes in a cell are upregulated and downregulated in response
to various environmental factors. Without models for the performance of the
cells, the time of a batch can be extended perhaps by a factor of 2–3, but without
adequate feedback control, true continuous bioprocessing is still a concept rather
than a reality [23].
6 Cell Separation
Once the bioreaction phase is over, it is typical to remove cells from the spent or
conditioned medium in order to begin purification of the product. In mammalian and
yeast culture, the product is typically secreted from the cell, so the cell is considered
part of the waste stream and need not be recovered. In bacterial culture, the product
may ultimately reside in an intracellular compartment or an extracellular compart-
ment depending on how the recombinant gene is constructed. If the product is
secreted, it is desirable to have a cell separation method that does not lyse the
cells. If the cells are lysed, proteases are released that could degrade the product,
and the released DNA, RNA, lipids, and carbohydrates increase the purification
challenge downstream. Disk stack centrifugation has been the state of the art for this
step for more than 30 years but has recently become limited by the high cell densities
now achieved with mammalian cell culture and yeast cell culture. Depth filtration is
supplanting centrifugation as a gentler separation methodology.
6.1 Centrifugation
Centrifugation is limited by shear force and solids concentration. A disk stack centri-
fuge, which can intermittently or continuously discharge a concentrated solids fraction,
is only able to achieve a solids concentration of about 50% wet weight. Above this
solids concentration, the viscosity of the solids fraction increases, and the solids cannot
be discharged through the nozzles used to regulate the flow. Yeast culture can easily
achieve 50% wet weight, so these cultures must be diluted prior to use of a disk stack
centrifuge. If dilution is used, yield is still a consideration, as the upper limit of 50% wet
weight will not be overcome. For example, if a 50% wet weight harvest broth is diluted
to 16% by adding 2.125 kg of water for every kg of harvest broth, the maximum product
that can be recovered is 81%, presuming recovery of 2.125 kg of liquid phase for every
1 kg of solid phase and assuming that the 1 kg of solid phase includes 500 g of solids
and 500 g of liquid. Unless in-line dilution is used, a tank that can contain 6,250 kg of
diluted harvest broth and another one that can collect 4,250 kg of liquid phase from the
centrifuge will be needed. The need for large tanks reduces the flexibility that is
considered one of the advantages of single-use technology.
Shear force is another consideration. In a typical disk stack centrifuge, the disks
spin at 3,000–10,000 rpm [24], producing high rates of shear at the fluid inlets and
outlets. Filtration shear rates are at least an order of magnitude lower, which matters
for mammalian cells that are fragile enough to be lysed by bubbles [25]. This limits
the applicability of centrifugation to bacteria and yeast primarily, since cell lysis is a
factor for mammalian cells.
6.2 Depth Filtration
Depth filtration has been used as a method for collecting cells and other solids. Depth
filters are designed for high solids loads but do not provide an absolute barrier to
solids of a certain size. Rather, filtration is due to adsorption or entrapment of cells in
tortuous channels. It is useful to think of depth filters as resembling cotton or glass
wool, through which a solution will be poured. Solid is captured by the random
threads of the material, and the deeper the bed of random threads, the more likely the
solid is to be captured. However, solids do eventually break through this type of
filter. Some depth filters are supplemented with filter aids, such as diatomaceous
earth, which provide more solids retention. Eventually the filters clog as a solids cake
begins to form at the surface of the filter medium, and the filtration has to be
terminated, or the filters changed out for new ones. It is not uncommon for depth
filters used as the primary means of cell separation to process only 10–100 L/m2.
Once loaded with solids, these filters are discarded, as they cannot be regenerated.
Although the filters themselves are relatively inexpensive, the waste stream is
considered biohazardous and is expensive to dispose of. As the cell density in the
bioreactor increases, more area is needed for depth filtration, increasing cost.
6.3 Cross-Flow Filtration
Given the limitations of the two traditional means of providing cell separation at
harvest, other approaches are being tested. Not unlike the cell retention devices
described above, cross-flow filtration and sedimentation are both being employed,
and both suffer from limitations as described above. Cross-flow filtration has high
shear, and the membranes used are subject to fouling. Cross-flow filtration also has a
maximum solids limitation and requires dilution for recovery of cells, although the
system in Fig. 2 overcomes some of the plugging by reversing flow every 30 s to
1 min.
6.4 Sedimentation
Sedimentation is slow and requires too much volume. In order to speed up sedi-
mentation, flocculants can be added to the harvest broth, such as polyethyleneimine
or diatomaceous earth. The ideal flocculant would have high capacity for cell solids
with low affinity for the product, a density much higher than water, no extractable
compounds, and minimal toxicity in humans. Acid can also be used to aggregate
some cells but would pose a degradation risk for some products. Significant oppor-
tunity for solid/liquid separation improvements exists for therapeutic protein
production.
7 Downstream Processing
Downstream processing is often portrayed as a bottleneck in biomanufacturing

[26]. Chromatography and ultrafiltration have been the work horses in downstream
processing since they displaced differential precipitation as a means of fine purifi-
cation for biomolecules. Chromatography is slow, with low capacity, and is typically
not optimized for throughput. Chromatography has also been difficult to adapt to
continuous operations. Ultrafiltration is usually not used for purification but rather
for the change of a buffer salt and pH and is not as rapid and inexpensive as the
alternatives of dilution and pH adjustment with acid and base, and therefore it is
avoided in general. However, a founding principal in preparative biochemical
purification is that removing water is expensive and the balance between dilution
and operating at higher concentrations is not always appreciated. Challenges, prin-
ciples, and design criteria are the subject of several books and are briefly outlined
below [24, 27, 28].
7.1 Chromatography and Adsorption
Chromatography for biotherapeutic molecules bears more resemblance to a

traditional adsorption /desorption step than to analytical chromatography, although
counterflow for product recovery is rarely employed. The objective is to purify a single
component from a multicomponent mixture, rather than to separate each component
from each other. For this reason, the preparative chromatography feed stream should be
thought of as having three components: (1) the target product molecule, (2) those
molecules that bind more tightly to the resin, and (3) those molecules that bind less
tightly or not at all. This ternary mixture is the most complicated to purify since the
desired molecule has properties in between the two others [29].
There are two operating modes that are a special case of this general one: affinity
adsorption and flow-through operation. In affinity adsorption, a ligand with high
selectivity and avidity for the product is bound to the chromatography resin, and in
principal, there should be no molecules that bind more tightly. There may, however,
be “product-related impurities” such as dimers or oxidation products that bind with
the same selectivity and avidity as the product itself.
Alternatively, some adsorption separations are made in “flow-through” mode, in
which the product does not bind to the resin at all, in which case, there is no fraction of
molecules that bind less well to the resin. Using the resin to capture only contaminants
is an excellent strategy, as contaminants such as RNA and DNA are typically in very
small amounts relative to the product (parts per million to parts per billion) so a small
amount of adsorbent can be used relative to the product. This separation mode also
does not typically separate product-related impurities from product.
When the most general condition exists, that the separation is intended to purify
the product from molecules binding more and less strongly, the separation must be
carefully thought out and tested. The most efficient separation is likely to occur when
the resin binding capacity is saturated with product although there are trade-offs in
product purity when this occurs. Consequently, multiple sequential steps may be
used based on “orthogonal” binding chemistries, i.e., ion exchange, reversed phase,
or size exclusion, where protein retention is based on different principles.
Examples of binding isotherms are shown in Fig. 4. Proteins and DNA typically
have a Langmuir isotherm form, with a very high slope at low concentrations. At
high concentrations, the resin is saturated and the isotherm flattens horizontally.
Under these conditions, the less strongly binding components will largely be
displaced from the resin. To get the most binding from the resin, a length of unused
bed (LUB) approach may be used [24]. To minimize the LUB, dispersion must be
minimized in the column, and a condition where a sharp breakthrough curve would
be obtained is favored. A sharp breakthrough curve can be generated by operating
the unit operation at high protein concentration as above, away from the linear
portion of the binding isotherm. If gradients are utilized, it is possible to encounter
a number of nonideal phenomena, and these must be accounted for in the selection of
column operating conditions (i.e., gradient slope, column load, mobile phase
composition, protein concentration, media chemistry, and particle size) [24, 27, 30].
Fig. 4 Different binding

isotherms. Proteins and
biotechnology products
often have a Langmuir
shape with a very high slope
at low concentration [24]
Dilution of the product prior to loading decreases the saturation of the resin. As the
concentration of the product goes from right to left on the x-axis in Fig. 4, the
concentration bound to the resin decreases. This moves the operation down the
binding isotherm and will lead to a more diffuse breakthrough profile and a longer
LUB. Operating at high concentration will offset dispersion caused by mechanical and
mass transfer effects that are hard to overcome in protein purification [28]. Minimized
dispersion requires good packing and good mechanical column design with even flow
distribution. It also requires the smallest resin particle size practical for the application
and a flow rate that allows sufficient mass transfer [31]. Unfortunately, small particles
are difficult to handle and pack in a manufacturing environment, and the linear velocity
that matches to the rate of inter-particle diffusion for a protein is on the order of
1 10 8 cm/s, which is not very practical. Operating at high protein concentration is
the most beneficial and easiest parameter to control for efficient adsorption behavior.
7.2 Intensified Chromatography
While operating at high protein concentration could improve the efficiency of

adsorption significantly, decreasing mass transfer path lengths could also increase
the efficiency of adsorption and the throughput of downstream processing. There
have been at least three variations on this theme with varied success, macroporous
resins, membrane adsorption, and ordered media.
Macroporous resins have been synthesized that have an outer diameter on the
order of 100 μm but have flow paths within the particles on the order of 1 μm [32]. A
type of this resin is sold under the brand name POROS and is very popular for the
separation of antibodies and other therapeutic proteins. In principal, these resins
should allow some amount of convection within the pores of the particle, so mass
transfer is not limited by pore diffusion within the particle. The diffusion length
would be closer to 0.5 μm, characteristic of a 1 μm particle, rather than the existing
50 μm diffusion path length that accompanies a 100 μm particle. This inter-particle
convection is possible, but the pressure drop across one particle diameter is the
pressure required to move fluid through a bed of 100 μm particles, so not sufficient to
drive much convection through 1 μm pores. Additionally, since the particles are
macroporous, they have lower ligand densities and consequently lower binding
capacities on a volumetric basis. This might mean that more resin is required to
process the same amount of therapeutic protein compared to a more conventional
resin. Nevertheless, there is some improvement in LUB in these resins, leading to
their popularity.
Membrane chromatography was initially conceived of in the form of parallel
hollow fiber membranes with adsorbing ligands within the membrane material
[33]. The concept provided a low Reynolds number methodology for decreasing
the diameter of the flow path to increase the rate of mass transfer. The thickness of
the hollow fiber wall was ideally as small as possible, making the largest resistance
to mass transfer the transfer of a molecule through the liquid phase to the interface
with the ligand-bearing solid phase. This methodology was reduced to practice and
showed promise. However, much like macroporous adsorption, the ligand density is
low. This technology is also difficult to scale, as the hollow fibers have to be held
tightly at each end and have a monodispersed inner diameter; otherwise the widest
fibers will have a higher flow rate at constant pressure (according to Poiseuille’s law,
flow increases as diameter to the fourth power at constant pressure), and dispersion
will be introduced that way.
The concept morphed to membrane chromatography, in which existing filtration
membranes are derivatized with ligands and the feed stream is forced through the
membrane’s tortuous flow paths [34]. This idea is categorically different from the
original idea which sought to keep the flow path straight and narrow in order to get
maximum mass transfer at minimum momentum transfer. In today’s membrane
chromatography, the pressure drop is high, and the feed stream must be completely
clear of particulates or the membrane will become clogged. Furthermore, membrane
manufacturing processes are well adapted to make membranes wider, but not deeper,
so volumetric binding capacity is problematic. Because of this, membrane chroma-
tography is almost always operated in flow-through mode, where small levels of
impurity are scavenged from a concentrated product stream. This is a very efficient
method for removing DNA, endotoxin, and RNA from a protein product, for
example. This methodology allows for an inexpensive single-use format as well.
Finally, the original idea for hollow fiber membrane chromatography was
extended into a more manageable form by utilizing a separations media consisting
of wound woven cloth [35, 36]. In this idea, a derivatized fabric is tightly rolled on
an axis and packed into a column. The directions of the fibers in the cloth are both
parallel and perpendicular to the direction of flow, but not random as in a packed
bed. Fibers on the order of 1 μm can be used to minimize the mass transfer path
length, but because the cloth is an “ordered medium,” the pressure drop is a small
fraction of the pressure drop generated in a random medium. Furthermore, cloths can
be made and derivatized inexpensively and enclosed in thermoplastic, making this

configuration a reasonable candidate for a single-use application.
8 Single Use Downstream Processing
There is significant penetration of single-use technologies in downstream

processing. Plastic bags in portable superstructures are commonly used to hold
buffers and in-process intermediates. When working at high concentration, smaller
columns and buffer volumes can be used. However, a true single-use adsorption
system has not yet been developed, primarily due to the high cost of the resins. Resin
costs can be up to $10,000/L for affinity resins, so their reuse is mandatory in an
economical process. Prepacked columns are available that are advertised as single
use, but this practice is only advised for very price-insensitive molecules, which will
be very few as biosimilar molecules become more conventional. Some of the
configurations mentioned above may be more amenable to single-use modalities if
their lower cost can be proven.
9 Continuous Downstream Processing
Continuous downstream processing is a field of active research. Simulated moving

bed chromatography has been adapted to biomolecule separations and may be
feasible for some extended batch size. However, feedback control is not yet devel-
oped for this operation, so true continuous processing will not be possible in the
short term. Large gains in volumetric efficiency and cost reduction have been
reported using simulated moving bed chromatography.
10 Conclusions
There are many challenges remaining for producing therapeutic biomolecules at

large scale for reasonable prices. Just a few have been reviewed in this chapter,
including:
• Cell cultures with higher cell densities and protein production levels
• Cell retention methodologies that allow the discharge of dead cells and cell
debris, while retaining viable cells
• Solid/liquid separations with high yield and no cell lysis
• High concentration/high intensity downstream processing
• Adsorbent development with improved mass transfer properties
Acknowledgments The authors wish to acknowledge support from the College of Engineering
from “Engineering Faculty Conversation on Future Manufacturing” and Hatch Act Support 10677
and 10646, Purdue University.
References
1. FDA (2019) Quality considerations for continuous manufacturing: guidance for industry.
https://www.fda.gov/media/121314/download
2. Quianzon CC, Cheikh I (2012) History of insulin. J Community Hosp Intern Med Perspect
2:18701
3. Ladisch MR, Kohlmann K (1992) Recombinant human insulin. Biotechnol Prog 8(6):469–478
4. Ellis LM (2006) Mechanisms of action of bevacizumab as a component of therapy for
metastatic colorectal cancer. Semin Oncol 33(5 Suppl 10):S1–S7
5. Riechmann L, Clark M, Waldman H, Winter G (1988) Reshaping human antibodies for therapy.
Nature 332:323–327
6. Thayer AM (1998) Great expectations. Chem Eng News 76:19
7. Rader RA (2012) Top 50 (or so) biopharma products. Contract Pharma
8. Kaplon H, Reichert JM (2019) Antibodies to watch in 2019. MAbs 11(2):219–238
9. Doig AR, Ecker DM, Ransohoff TC (2015) Monoclonal antibody targets and indications.
Am Pharm Rev 15:177490
10. Kelly B (2009) Industrialization of mAb production technology. MAbs 1(5):443–452
11. Johansson HJ, Cardillo D, Gerwe B (2017) Are all protein a resins the same? BioProcess Int 15
(11):1–5. https://bioprocessintl.com/sponsored-content/protein-resins-performance-compari
son-eight-different-protein-resins/
12. Wilkinson GW, Akrigg A (1992) Constitutive and enhanced expression from the CMV major
IE promoter in a defective adenovirus vector. Nucleic Acids Res 20(9):2233–2239
13. Studier FW (2018) T7 expression systems for inducible production of proteins from cloned
genes in E. coli. Curr Protoc Mol Biol 124(1):e63
14. Fenton D, Lai PH, Lu H, Mann M, Tsai L (1997) Control of norleucine incorporation into
recombinant proteins. US Patent 5,599,690
15. Rudge SR (2017) Single-use systems for biotechnology products. Eur Pharm Rev 22(2):64–66
16. Sinclair A, Leveen L, Monge M, Lim J, Cox S (2008) The environmental impact of disposable
technologies. BioPharm Int Guide 11:1–11
17. Nims R, Plavsic M (2012) Circovirus inactivation: a literature review. Bioprocess J 11(1):4–10
18. Shevitz J (2003) Fluid filtration system. US Patent 6,544,424
19. Freeman CA, Samuel PSD, Kompala DS (2017) Compact cell settlers for perfusion cultures of
microbial (and mammalian) cells. Biotechnol Prog 33(4):913–922
20. Kompala DS (2017) Particle settling devices. US patent application US 2017/0333815A1
21. Shirgaonkar IZ, Lanthier S, Kamen A (2004) Acoustic cell filter: a proven cell retention
technology for perfusion of animal cell cultures. Biotechnol Adv 22(6):433–444
22. Lee S (2017) Modernizing the way drugs are made: a transition to continuous manufacturing.
https://www.fda.gov/drugs/news-events-human-drugs/modernizing-way-drugs-are-made-transi
tion-continuous-manufacturing
23. NASEM (2019) Continuous manufacturing workshop. National Academies of Sciences, Engi-
neering, and Medicine, Washington
24. Harrison RG, Todd P, Rudge SR, Petrides DP (2015) Bioseparations science and
engineering.2nd edn. Oxford University Press, New York
25. Van den Pol LA (1998) Sparging-shear sensitivity of animal cells. Thesis Landbouwuniversiteit
Wageningen
26. 9th annual report and survey of biopharmaceutical manufacturing capacity and production: a
survey of biotherapeutic developers and contract manufacturing organizations, BioPlan Asso-
ciates, Inc., Rockville, 2012. www.bioplanassociates.com
27. Ladisch MR (2001) Bioseparations engineering: principles, practice, and economics. Wiley,
New York, 735 pp
28. Wankat P (1986) Large scale adsorption and chromatography, volume. CRC Press, Boca Raton,
p1
29. Gibbs SJ, Lightfoot EN (1986) Scaling up gradient elution chromatography. Ind Eng Chem
Fundam 25(4):490–498
30. Velayudhan A, Ladisch MR (1992) Effect of modulator sorption in gradient elution chroma-
tography: gradient deformation. Chem Eng Sci 47(1):233–239
31. Peskin AP, Rudge SR (1992) Optimization of large-scale chromatography for biotechnological
applications. Appl Biochem Biotechnol 34/45:49
32. Regnier FE (1991) Perfusion chromatography. Nature 350:634–635
33. Ding H, Yang M-C, Schisla D, Cussler EL (1989) Hollow-fiber liquid chromatography. AICHE
J 35(5):814–820
34. Arunkumar A, Etzel MR (2018) Fractionation of glycomacropeptide from whey using posi-
tively charged ultrafiltration membranes. Foods 7(10):166
35. Ladisch MR, Zhang L (2016) Fiber-based monolithic columns for liquid chromatography. Anal
Bioanal Chem 408(25):6871–6883
36. Yang Y, Velayudhan A, Ladisch CM, Ladisch MR (1993) Liquid chromatography using
cellulosic continuous stationary phases. In: Tsao GT (ed) Chromatography: advances in bio-
chemical engineering/biotechnology, vol 49. Springer, Heidelberg
37. FDA. https://www.fda.gov/media/105605/download. Accessed 31 July 2019
DOI: 10.1007/10_2019_119
Insights on the Formulation of Recombinant

Proteins
Rita Ribeiro, Teresa Raquel Abreu, Ana Catarina Silva, João Gonçalves,
and João Nuno Moreira
Contents
1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25
2 Protein Structure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25
2.1 Molecular Structure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25
2.2 Chemical Degradation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 26
2.3 Physical Degradation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27
2.4 Characterization of Proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 28
3 Formulation of Proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30
3.1 Buffers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30
3.2 Osmotic Agents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31
3.3 Stabilizers/Bulking Agents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 32
3.4 Surfactants . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 32
3.5 Preservatives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33
3.6 Antioxidants . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33
3.7 Adjuvants and Immune Potentiators . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 34
3.8 Trends in Formulation Approaches . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 34
3.9 Formulation of Biosimilars and Biobetters . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 36
R. Ribeiro, T. R. Abreu, and J. N. Moreira (*)

CNC – Center for Neuroscience and Cell Biology, Faculty of Medicine (Pólo I), University of
Coimbra, Coimbra, Portugal
FFUC – Faculty of Pharmacy, University of Coimbra, Coimbra, Portugal
e-mail: jmoreira@ff.uc.pt
A. C. Silva
UCIBIO, UCIBIO, REQUIMTE, MEDTECH, Laboratory of Pharmaceutical Technology,
Department of Drug Sciences, Faculty of Pharmacy, University of Porto, Porto, Portugal
FP-ENAS (UFP Energy, Environment and Health Research Unit), CEBIMED (Biomedical
Research Centre), Faculty of Health Sciences, University Fernando Pessoa, Porto, Portugal
J. Gonçalves
iMed.ULisboa – Research Institute for Medicines, Faculty of Pharmacy, University of Lisbon,
Lisbon, Portugal
24 R. Ribeiro et al.
4 Pharmaceutical Presentation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 37
4.1 Lyophilized Therapeutic Proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 37
4.2 Containers and Administration Devices . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 38
5 Safety and Immunogenicity of Biopharmaceuticals . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 39
5.1 Quality Framework . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 39
5.2 Mechanisms of Immunogenicity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 40
5.3 Protein Aggregation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 41
5.4 Anti-drug Antibodies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 41
6 Current Trends in Formulation and Future Perspectives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 42
6.1 Novel Excipients . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 42
6.2 Alternative Routes of Administration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 43
6.3 Other Strategies to Improve Protein Delivery . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 44
6.4 High-Throughput Characterization and Formulation Screening . . . . . . . . . . . . . . . . . . . . . . . 45
6.5 Developability Assessment and Quality by Design . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 47
7 Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 48
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 48
Abstract Recombinant proteins are large and complex molecules, whose therapeu-
tic activity highly depends on their structure. Formulation of biopharmaceuticals
aims at stabilizing protein conformation, promoting its efficacy, and preventing
safety concerns, such as immunogenicity. Currently, the rational design of formula-
tions is possible due to the availability of several techniques for molecule
characterization and an array of both well-known and new excipients. Also, high-
throughput technologies and Quality by Design approaches are trending and have
been contributing to the advancement of the field. Still, there is a search for
alternatives that ensure quality of the medicines through its life cycle, particularly
for highly concentrated formulations, such as monoclonal antibodies. There is also a
demand for strategies that improve protein delivery and more comfortable adminis-
tration to the patients, especially with the arising of recombinant proteins in the
treatment of chronic diseases, such as autoimmune conditions or heart diseases. In
this chapter, current and future advancements regarding recombinant protein formu-
lation and its impact in drug development and approval will be addressed.
Insights on the Formulation of Recombinant Proteins 25
Graphical Abstract
Post-
Drug Pre-clinical Regulatory
Basic research Clinical trials marketing
discovery testing approval
surveillance
Safety and immunogenicity

• Aggregates
• ADAs
Protein characterization • Immune reactions
• Structure
• Degradation pathways
FUTURE PERSPECTIVES
Excipients choice
• Buffers Novel excipients
• Osmotic agents
• Stabilizers/Bulking agents Alternative routes of administration
• Surfactants
• Preservatives RECOMBINANT PROTEIN Protein delivery strategies
• Antioxidants FORMULATION
• Adjuvants and immune potentiators High throughput development
Quality by Design
Pharmaceutical presentation
• Lyophilized
• Solution
Keywords Formulation, Immunogenicity, Recombinant proteins, Stability
1 Introduction
Recombinant proteins represent an increasing fraction of the therapeutic arsenal

available nowadays. Relative to small molecular weight drugs, recombinant proteins
are larger and more complex active pharmaceutical ingredients, whose efficacy and
safety highly depend on the ability to recapitulate their structure in the different
stages of the protein lifetime. Additionally, challenges arise from the development of
formulations with high concentrations of protein, as in the case of monoclonal
antibodies, as well as for low-volume formulations (as demanded for self-
administration devices). The pursuit of a formulation that can stabilize the active
protein, from manufacture to administration, in an economically effective way, is
then a crucial aspect to consider in the development of biological medicines. Herein,
the formulation of recombinant proteins will be discussed, addressing general
concepts, current trends, and future perspectives.
2 Protein Structure
2.1 Molecular Structure
Proteins are large molecules composed of one or more chains of amino acid residues,
displayed in an order that is determined by a DNA sequence. The linear polypeptide
form of the protein corresponds to the protein’s primary structure. Intermolecular

interactions promote the arrangement of these chains into secondary (alpha-helices
and beta-sheets) and tertiary structures. The latter can be assembled into quaternary
structures, when two or more subunits interact. As different amino acids have
different side chains with unique chemical properties, intra- and intermolecular
interactions vary in strength, from weak forces (hydrophobic, electrostatic, hydro-
gen, van der Waals) to strong covalent bonds (disulfide bridges), adding to the
complexity of the final structure [1].
If chemical and/or physical degradation occurs, protein may unfold, and hydro-
phobic residues that would be hidden in the native state may become exposed.
Consequently, the molecule can remain unfolded or misfolded in a thermodynam-
ically non-stable state, interacting with other proteins and causing aggregation and
precipitation [2]. The presence of aggregates in biological medicines has been
associated with immunogenicity, loss of biological activity, and renal failure [3]. Fur-
ther in this chapter, mechanisms of protein degradation will be addressed, as well as
approaches to minimize aggregates formation and assure the efficacy and safety of
the final medicine.
Protein degradation can take place at different stages of the product’s life cycle,
from manufacturing and processing to storage, administration, and delivery
[4]. Because recombinant proteins are produced in cell-based systems, purification
and recovery steps are needed to isolate the product of interest from destabilizing
impurities. Long-term shelf-life (about 18–24 months for a protein-based therapeu-
tic) could promote degradation pathways (e.g., hydrolysis or oxidation), even when
the adequate storage conditions are maintained [5]. Different delivery systems are
also to consider, since the materials and processes used for their preparation and final
presentation may impact protein stability [6].
2.2 Chemical Degradation
Chemical degradation involves modifications of covalent bonds of the protein, such

as deamidation, isomerization, hydrolysis, oxidation, and disulfide bond shuffling
[7]. It affects the primary sequence of the protein, which may lead to significant
changes in the final three-dimensional structure [3].
Deamidation is one of the most common forms of chemical degradation. It
consists in the hydrolysis of asparagine (Asn) to aspartic (Asp) and iso-aspartic
acid, or that of glutamine (Gln) to glutamic acid (Glu), via a succinimide interme-
diate. Subsequently, a neutral amino acid residue is converted into a negatively
charged polar one [8]. The reaction is favored by neutral and basic pH [9] and
temperature above 25 C [10], as well as by the steric effect of adjacent residues
(mainly in the C-terminal) and their conformation. In this respect, the higher the size
and branching of the side chain, the lower the rate of deamidation, with glycine
(Xxx-Asn-Gly-Xxx) resulting in the highest extent of deamidation [11].
Hydrolysis of a peptide bond can take place either under acidic or alkaline
conditions, or it can be enzymatically mediated. As a result, the polypeptide chain
breaks, leading to protein fragmentation. Some bonds are more susceptible to
hydrolysis, like Asn-tyrosine or Asn-proline [12].
Oxidation can be a consequence of several external factors, including exposure to
light, oxygen, transition metal ions (iron and copper [13]), or formulation degrada-
tion products (e.g., hydrogen peroxide from polysorbate autoxidation [14]). Amino
acid residues such as tryptophan (Try) and histidine (His) are more susceptible to
oxidation due to their aromatic rings, while methionine (Met) and cysteine (Cys) are
vulnerable to oxidation as the result of their highly reactive sulfur atoms [15]. The
steric exposure of the reactive residue and pH of the solution also influence the
oxidation rate [16].
Cross-linking is an additional chemical modification involving the formation of
disulfide bonds, either from the formation of new Cys-Cys bonds or exchange of
pre-existing ones. This modification is favored with increasing pH values, as thiolate
ions are the major reactive species involved [17].
2.3 Physical Degradation
Physical degradation implies changes in the secondary, tertiary, or quaternary

structure of the protein, leading to aggregation, precipitation, or adsorption to
surfaces. Mechanisms of physical degradation usually involve unfolded or semi-
unfolded intermediates and are triggered by stress factors, such as mechanical stress
(stirring, pumping, filtration, and filling during manufacturing and shaking during
administration [18]), high temperatures (during shipping and storage), and freeze/
thaw cycles. High temperatures, i.e., temperatures close to the protein’s melting
point (value at which half of the protein population is unfolded and the other half is
folded [19]), lead to the formation of aggregates via non-covalent interactions
between the unfolded proteins [20]. Freeze/thaw cycles may cause partitioning of
the protein to ice-water interface, cryo-concentration, and change in pH due to
nonuniform buffer recrystallization, affecting protein stability [21]. Cold denatur-
ation is not very common, as it takes place only below the freezing point of water,
when water molecules are ordered in a layer around the protein, changing its
thermodynamics and leading to destabilization [22]. These low temperatures (usu-
ally below 20 C) are not achieved under regular manufacturing, transport, or
storage conditions. However, upon freeze-drying for lyophilization purposes, the
glass transition temperature usually goes below 20 C, which increases the chances
of cold denaturation.
Aggregation is one the most common forms of protein physical degradation. The
trigger of aggregation and the characteristics of aggregates depend on protein’s
environment: pH, temperature, ionic strength, concentration, and presence of
co-solutes and excipients [23]. This form of degradation is particularly significant
when dealing with monoclonal antibodies, as they are usually formulated at high
concentrations [24]. Moreover, large, multidomain proteins are more prone to

aggregate, because gentle conditions, such as exposure to temperatures below their
melting temperature (Tm) [25], can be sufficient to break the non-covalent interac-
tions that usually maintain the several domains properly organized, unfold the
protein, and induce aggregation. On the other hand, small, single-domain proteins
only have their intramolecular forces disrupted under more extreme conditions, such
as pH below 3 or above 10, or temperatures above 40 C [26]. Nowadays, it is
possible to predict the extent and rate of aggregation based on the physicochemical
properties of the protein, upon experimentally testing the effect of certain residues’
mutations [27, 28], complementing with in silico prediction tools [29]. Recent
developments on statistical modeling and machine-learning enable the assessment
of aggregation potential of proteins using a high-throughput screening, and thus the
selection of a lead with the lowest risk of aggregation, in an early stage of develop-
ment [30, 31].
2.4 Characterization of Proteins
The complex structure of recombinant proteins demands a thorough characterization

in early development to better control the impact on their stability and biological
activity. Several techniques of biophysical and biochemical analysis have to be
performed in order to determine, in a rational way, the best conditions for drug
stabilization through its whole life cycle. Information derived from this early char-
acterization will be further used to establish the specifications to which the active
substance, excipients, and final product must conform to be considered acceptable
by manufacturers and regulatory authorities. In this respect, the International Con-
ference on Harmonization (ICH) Q6B guideline unifies a set of international analyt-
ical procedures and respective acceptance criteria regarding biotechnological
products, based on their molecular and biological characteristics [32].
These methodologies are based in separation (chromatographic, SDS-PAGE) and
spectroscopic methods, complemented by calorimetric and light scattering tech-
niques. They assess protein size, conformation, hydrophobicity, and hydrodynamic
features [33, 34]. The detection of aggregates requires imaging techniques, such as
microscopy and light scattering that provide information to be used in computer
modeling predictive tools, giving insight in possible degradation, aggregation, and
immunogenicity. A review of computational tools used to assess protein aggregation
can be found elsewhere [29], while the most commonly performed assays and
respective measured properties are summarized in Table 1.
Finally, it is very important to do a functional characterization of the active
protein, according to its mechanism of action, in order to evaluate its performance.
Blotting, electrophoresis, chromatography, immunoassays (e.g., ELISA), and
in vivo bioassays are examples of such methodologies [33].
Table 1 Methodologies and properties assessed for protein characterization (Adapted from Angkawinitwong [35])
Property assessed
Primary Secondary Tertiary
Method Technique Measurement structure structure structure Aggregates
Chromatographic Ion exchange chromatography Charge variants X X
Size exclusion chromatography Hydrodynamic size X
High-pressure liquid chroma- Degraded product X
tography (HPLC)
Spectroscopic UV absorbance Protein concentration X X X
Raman spectroscopy Conformation X X
Fourier-transform infrared spec- β-sheet, α-helix X X
troscopy (FTIR)
Near-/far-UV circular dichroism Thermal stability X X
Fluorescence Folding/unfolding, hydrophobic X X
surface
Insights on the Formulation of Recombinant Proteins
Optical density (OD) Turbidity X

Calorimetric Differential scanning calorime- Melting temperature (Tm), thermal X X X
try (DSC) unfolding/aggregation
Light scattering Static/dynamic light scattering Diameter and monodispersity of X
monomers
Microscopic Transmission electron micros- Particles X
copy (TEM)
Fluorescence microscopy Particles X
Atomic force microscopy Particles X
(AFM)
Mass spectrometry Size, peptide mapping, degradation X
products
SDS-PAGE Molecular weight X
Analytical Molecular weight/shape X
29
ultracentrifugation
3 Formulation of Proteins
Efficacy and safety of recombinant proteins is highly dependent on their unique

structure, which is why it is critical to develop a formulation with an optimum
qualitative and quantitative composition for each therapeutic. The choice of excip-
ients will depend on several factors, aiming at stabilizing the active protein through
the life cycle of the product, up to administration to the patient. Excipients can
promote protein stabilization by increasing thermodynamic stability, either through
strengthening protein-stabilizing forces or destabilizing the unfolded state, forcing
the refolding to the native conformation [36]. This is possible because of their direct
binding to the protein (e.g., stabilizers) or their exclusion from protein surface and
replacement by water molecules, i.e., protein hydration (e.g., sugars) [37].
Aside from the intrinsic physical and chemical properties of the active protein and
excipients, some other aspects have to be considered when choosing the most
appropriate excipients. First, special attention to chemical and physical stability
issues during manufacturing is demanded. This requires a good understanding of
the critical attributes and processes that impact quality, from early development
[38]. The formulation approach intended is important, since liquid or lyophilized
formats require different processing [39]. The final concentration of protein is
another significant aspect to consider. As mentioned before, formulations with
highly concentrated protein (50–150 mg/mL) [40], as monoclonal antibodies, are
more susceptible to aggregation. It is also important to consider possible non-
specific adsorption to surfaces, as to the primary packaging or to the administration
device, especially for hydrophobic proteins [41]. The route of administration must be
regarded too, as adjustments in formulation may be necessary to keep the required
tonicity or osmolarity and pH for parenteral administration [39].
All the components to be used in a formulation must be characterized, and their
compatibility with each other and with the therapeutic protein must be evaluated.
Depending on the concentration that is being used, the same excipient may have
different roles in different formulations, making it difficult, sometimes, to categorize
them. Herein a classification of the most common excipients used in the formulation
of recombinant proteins is presented (Table 2).
3.1 Buffers
At a pH value at the protein isoelectric point, the net neutral charge of the protein
limits electrostatic interactions, thus favoring protein-protein interactions and sub-
sequent aggregation. Buffers are, therefore, an important component of protein
formulation in order to enable a pH of the protein solution below or above of the
corresponding isolectric point [42]. As therapeutic proteins are preferably adminis-
tered parenterally, on the choice of the most appropriate pH, the physiological range
(pH ¼ 7.35–7.45) [43] necessary to avoid irritation or pain during administration
Table 2 Classification, role, and examples of commonly used excipients in the formulation of
recombinant proteins
Excipient class Role in formulation Examples
Buffers pH stabilization Amino acid (histidine, methionine, glycine,
arginine), non-amino acid (phosphate, acetate,
citrate), and salts (sodium hydroxide,
hydrochloric acid, phosphoric acid)
Osmotic agents Isotonicity Sodium chloride, potassium
maintenance
Stabilizers/ Protein stabilization Amino acids (histidine, glycine, arginine, gluta-
bulking agents and formulation mate) and sugars (mannitol, sucrose, trehalose)
support
Surfactants Solubility enhance- Sodium dodecyl sulfate and polysorbate
ment and anti- 20, polysorbate 80
aggregation
Preservatives Minimization of bacte- Benzyl alcohol, phenol, metacresol
rial contamination
Antioxidants Minimization of oxida- Ascorbic acid, acetylcysteine, glutathione
tive stress degradation
Adjuvants and Modulation of immune Aluminum salts, squalene-based oil-in-water
immune response emulsions, monophosphoryl lipid A
potentiators
must be also considered. When balancing these aspects, pH ranging between 3 and
11 may be considered acceptable [44]. Substances like amino acid, as histidine,
methionine, glycine or arginine, or non-amino acid, phosphate, acetate or citrate, are
examples of buffers used in protein formulation. Acids and bases can also be used as
buffers, either as pH modifiers or to form salts in combination with other compo-
nents of the buffer (e.g., sodium hydroxide, hydrochloric acid, phosphoric acid).
Along formulation development, the presence of other excipients or the influence of
certain processes that may promote precipitation or crystallization of the buffer,
altering the pH, should be carefully assessed. This is the case of acetic acid that being
volatile, its use in lyophilized formulations is not indicated [39]. Interestingly,
formulations with high concentrations of protein may even dismiss the need of
buffer, namely, when they have buffering amino acids in their composition, such
as aspartate, glutamate, and histidine [45].
3.2 Osmotic Agents
In medicines for parenteral administration, the maintenance of the isotonicity of the

formulation in a range between 280 and 300 mOsm/L is required, to avoid damage of
the tissues (phlebitis or infiltration) or pain at the site of injection. This is particularly
difficult for formulations with a high concentration of protein. The most common
tonicifiers are sodium chloride and potassium chloride [39]. Careful choice of the
agent is required as, for example, sodium chloride and water react to form an eutectic
mixture at 21 C. In this case, water crystallizes during freeze-drying, and the
concentration of sodium chloride increases until it precipitates. For this reason,
sodium chloride is added to the diluent for reconstitution instead to the lyophilized
product [46].
3.3 Stabilizers/Bulking Agents
The solvent surrounding the protein and the solutes interacting with it are very
important in maintaining the correct folded structure. The pharmaceutical presenta-
tion of the protein-based dosage form, either in solution or lyophilized, influences
the nature of those interactions and impacts the choice of stabilizers and bulking
agents.
The most common stabilizers are amino acids (histidine, glycine, arginine,
glutamate) and sugars (mannitol, sucrose, trehalose). They stabilize the proteins by
direct interactions or solute exclusion (stabilizer amino acids are repulsed from the
protein surface, increasing the free energy of the unfolded state and favoring folding
in the native form) [47], but their specific mechanism is sometimes difficult to assess
due to the diversity and complexity of their side chains. Mixtures of different amino
acids (e.g., arginine and glutamic acid) [48] or combinations of amino acids and
sugars (e.g., histidine and sucrose or mannitol) might enable a synergistic effect in
the protein stabilization [49].
Bulking agents are incorporated in lyophilized formulations to increase product
mass (thus preventing its collapse), to aid in rehydration (facilitating dissolution) and
to improve product appearance (by providing mechanical support) [50].
Due to their amorphous or crystalline physical state, sugars may act as stabilizers
or bulking agents, respectively. For example, sucrose maintains an amorphous state
after lyophilization, acting as a stabilizer, while mannitol is capable of crystallizing
and supporting the lyophilized cake, thus acting as a bulking agent. Another case
shows that, in a formulation of a human growth hormone, when mannitol and
glycine were used separately, both crystallized during freeze-drying. However,
when combined in the same formulation, mannitol crystallized, while glycine
remained amorphous. This partially amorphous system offered the best protein
stabilization [51]. Sugar recrystallization should be avoided, since the conversion
between amorphous and crystalline states can compromise protein stability [52].
3.4 Surfactants
Surfactants are molecules whose mechanism of action will depend on their nature
(either ionic or nonionic), acting as solubility enhancers, anti-adsorption or anti-
aggregation agents. Upon competitive binding to interfaces like air-water or
container-water, surfactants prevent the non-specific adsorption of proteins through

binding to their hydrophobic residues. They also compete with other proteins for the
binding to protein surface, preventing aggregation [53]. Their activity has a signif-
icant impact in highly concentrated formulations, which are more prone to form
aggregates.
Ionic surfactants are rarely used in protein formulations, due to their tendency
to denature proteins. Nonionic surfactants, such as sodium dodecyl sulfate and
polysorbate 20 or polysorbate 80, are efficient, less toxic, and less sensitive to the
presence of electrolytes [12]. Polysorbates are the more common surfactants found
in protein formulations, but their concentration must be selected carefully, since they
can undergo autoxidation and give origin to hydrogen peroxide, which will not only
compromise protein stability but also the safety of the product [14].
3.5 Preservatives
Preservatives are present in formulations to minimize microbial contamination. They

are particularly important in products with multiple doses, due to their likelihood
to contaminate in repeated usages. Examples of preservatives include metacresol,
phenol, and benzyl alcohol. The latter was even shown to stabilize the partial
unfolded state of a protein, inhibiting its aggregation [54]. In the case of volatile
and reactive preservatives, incorporation in lyophilized products is rather performed
in the solvent for reconstitution, than in the lyophilized cake [39]. The addition of
preservatives to a formulation may, however, impact protein stability in a negative
way. In this respect, it is proposed that preservatives interact with the active proteins,
destabilizing their weakest links (so-called hot-spots), thus decreasing conforma-
tional stability and increasing propension to aggregation [55, 56]. This issue may
be minimized upon deepening the understanding of the effect of preservatives on
proteins and by applying strategies enabling protein stability. Besides, a case of
binding of preservatives to a model peptide decreased their antimicrobial effect, thus
raising possible safety concerns [57].
3.6 Antioxidants
Proteins rich in methionine, cysteine, tryptophan, tyrosine, and histidine residues are
more susceptible to degradation due to oxidative stress. In these cases, formulation
with antioxidants is a requirement. Ascorbic acid and acetylcysteine are examples of
antioxidants often used in the formulation of proteins. Glutathione also behaves as
reducing agent, upon creating disulfide bonds with cysteine residues of the protein.
Replacement of oxygen by an inert gas in the vial is a complementary measure to
reduce oxidation [58]. Other mechanism of oxidation prevention is the chelation of
metal ions. In this regard, ethylenediaminetetraacetic acid and calcium chloride have
been used as antioxidants [59].
3.7 Adjuvants and Immune Potentiators
Recombinant peptide or protein-based vaccines have the ability to induce immune

responses against a specific antigen. Their formulation often includes adjuvants that
help to modulate or even increase immune responses. Aluminum salts are the most
commonly used adjuvants. They act in the site of injection, adsorbing the antigen,
enabling its stability and uptake by antigen-presenting cells, and inducing local
pro-inflammatory reactions [60]. Other examples are MF59 and AS03, squalene-
based oil-in-water emulsions, that consist of oil nanoparticles suspended in an
aqueous phase via a surfactant. They have shown to be more effective than alumi-
num in the avian H5 pandemic flu vaccines [61].
Currently, the objective is to go beyond antibody-mediated immunity and accom-
plish long-lasting cellular responses, like those mediated by T helper and T cytotoxic
lymphocytes. This can be achieved by the activation of Toll-like receptors (TLR) or
NOD-like receptors (NLR) by immune potentiators, like pattern-recognition recep-
tors (PRR) agonists [62]. Monophosphoryl lipid A (MPL) is a natural molecule that
targets TLR4 and has been also used as an adjuvant. Synthetic analogues have been
studied, namely, in combination with aluminum or emulsions [63].
A relevant formulation aspect relies on the assessment of the mechanism of action
of the antigen and of the adjuvants, as well as the interaction of these components
with each other, as this may impact both the efficacy and the safety of the drug
[64]. For example, aluminum salts can adsorb the antigen (with a strength that
is antigen-specific), destabilizing it and promoting protein aggregation [65]. As
for emulsions, they can lead to protein denaturation or desorption, structural
rearrangements, and inter-protein interactions, with subsequent aggregation [66].
To minimize these effects, particle-based formulations for stabilization and delivery
of the adjuvant are under development, such as nanotechnology-based delivery
systems, virus-like particles, and polymeric systems [67].
3.8 Trends in Formulation Approaches
An overview of recombinant protein medicines currently approved by European

Medicines Agency (EMA) and Food and Drug Administration (FDA) gives an idea
of the formulation approaches that are preferentially being followed. In Europe,
monoclonal antibodies comprise the major class of therapeutic proteins, which, as
mentioned earlier, implies the use of excipients appropriate for highly concentrated
products. Therefore, upon briefly running through the major classes of excipients,
buffers, surfactants, preservatives and tonicifiers arise as the major components of
current protein formulations [39]. In the United States, the scenario is similar.
Antibody therapeutics are also the highest selling class of biopharmaceuticals,
delivered by parental routes of administration and stored either in solution or powder
formats. Among these, the most commonly used excipients fall into the buffers,
bulking agents, and surfactants categories [68]. For a more detailed and practical
understanding of the formulations currently used in commercialized drugs, the
European Public Assessment Reports (EPAR) [69] of the 10 top-selling biopharma-
ceutical products in 2017 [70] were analyzed, and their excipient composition and
pharmaceutical presentation were compiled in Table 3.
Table 3 10 top-selling biopharmaceuticals’ formulations

Active Pharmaceutical
Product substance Excipients presentation
Humira® Adalimumab Mannitol, polysorbate 80, water for Solution for injection
injection
Enbrel® Etanercept Mannitol, sucrose, trometamol Lyophilizate for
reconstitution and
injection
Rituxan® Rituximab Sodium citrate, polysorbate 80, sodium Concentrated solu-
MabThera® chloride, sodium hydroxide, hydrochloric tion, for dilution and
acid, water for injection infusion
Remicade® Infliximab Sucrose, polysorbate 80, monobasic Lyophilizate for
sodium phosphate, dibasic sodium reconstitution, dilu-
phosphate tion, and infusion
Herceptin® Trastuzumab L-histidine hydrochloride monohydrate, Lyophilizate for
L-histidine, polysorbate 20, α,α-trehalose reconstitution, dilu-
dihydrate tion, and infusion
Avastin® Bevacizumab Trehalose dihydrate, sodium phosphate, Concentrated solution
polysorbate 20, water for injection for dilution and
infusion
Lantus® Insulin Zinc chloride, metacresol, glycerol, Solution for injection
hydrochloric acid, sodium hydroxide,
water for injections, polysorbate
20 (when stored in vial)
Eylea® Aflibercept Polysorbate 20, sodium dihydrogen Solution for injection
phosphate monohydrate, disodium
hydrogen phosphate heptahydrate,
sodium chloride, sucrose, water for
injection
Opdivo® Nivolumab Sodium citrate dihydrate, sodium chlo- Concentrated solution
ride, mannitol, pentetic acid, polysorbate for dilution and
80, sodium hydroxide, hydrochloric acid, infusion
water for injection
Neulasta® Pegfilgrastim Sodium acetate, sorbitol, polysorbate Solution for injection
20, water for injection
3.9 Formulation of Biosimilars and Biobetters
A biosimilar is a biopharmaceutical that is highly similar to an already approved

biologic (reference medicine) in terms of quality, biological activity, safety, and
efficacy. The complexity of proteins associated with their different levels of struc-
tural organization and high molecular weight determines a demonstration of simi-
larity between a biosimilar and the reference medicine, based on the assessment of
toxicity and efficacy in a clinical setting. This implies not only a structural similarity
between the active protein in the biosimilar and reference medicine, but also the
same posology and route of administration. Some deviations from the reference
product may be accepted, such as pharmaceutical dosage form, formulation, excip-
ients, or presentation, provided that they are properly justified [71].
In fact, both EMA and FDA allow for advancements in formulation science to be
incorporated in the biosimilar presentation, admitting excipients to differ from those
of the reference. In this respect, any relevant effects of the revised formulation on the
stability, physiochemical, and functional characteristics of biosimilars must be
assessed [72]. However, as stated before, it is hard to collect information on the
safety and immunogenicity of biopharmaceuticals based only in the characterization
of the active molecule and nonclinical data. The same is applied to their biosimilars.
Therefore, clinical trials and post-marketing vigilance are required by the regulatory
authorities to ensure at least the same efficacy and safety from the innovator
medicine [73, 74].
Biobetters are the new players in biopharmaceutical development. They act
against the same target as an already marketed medicine and include changes in
the molecular structure or formulation. However, unlike biosimilars, these alter-
ations aim to improve pharmacokinetic or pharmacodynamic properties, to decrease
toxicity or immunogenicity, or to make administration more comfortable or less
frequent, i.e., biobetters are clinically superior than the respective references either in
efficacy, safety, or compliance, or both [75]. Biobetters are not regulated yet, so they
are treated as new drugs and can only enter the market after a full application
submission, which is both expensive and time-consuming. Nonetheless, they have
been used for biopharmaceutical companies that want to extend the life cycle of
drugs whose patent is expiring, as a way to manage competition from biosimilars
[76]. For example, in 2013, Roche obtained marketing authorization for a subcuta-
neous formulation of Herceptin®, since intravenous trastuzumab patent would expire
in 2014. The new formulation presented efficacy and safety profiles similar to
the intravenous one and further enabled administration of higher volumes. Along
with the advantage of shorter duration of treatment, saving time and resources for
healthcare professionals, and improving patient compliance [77], it became a con-
cern for biosimilars of intravenous trastuzumab developers. This case illustrates the
importance of formulation development beyond the drug’s marketing authorization.
4 Pharmaceutical Presentation
Even with an appropriate choice of excipients supporting the stability of the active
protein, the dosage form and drug packaging can have an impact in maintaining the
medicine’s quality throughout its life cycle. Therefore, pharmaceutical presentation
must be regarded during formulation development [78].
4.1 Lyophilized Therapeutic Proteins
Most biopharmaceutical products are stored in the form of aqueous-based solutions

or lyophilized powders [39]. As mentioned before, protein solubility depends on
several aspects, including pH, ionic strength, and associated excipients. If stability
and solubility can be achieved, a solution is preferred over a suspension, since the
latter is harder to process and handle. Suspensions have increased chemical stability,
but not necessarily higher physical stability, due to the propensity to form agglom-
erates when exposed to mechanical stress [79].
Often, the liquid formulation does not provide the required long-term stability of
the protein and quality of the product, as the presence of water may promote physical
or chemical degradation. The dried state is thus the preferred form, and the protein
must be lyophilized.
Lyophilization is a process that comprises three sequential phases: freezing,
primary drying, and secondary drying. During the freezing step, temperature is
reduced below the eutectic temperature (Te) of the mixture (i.e., a temperature that
is lower than the melting point of each component of the formulation). Then, during
primary drying, crystallized water and water that is not bound to the protein or
excipients are removed by sublimation, with temperature still below the Te, and
under reduced pressure. Secondary drying allows the removal of water that is bound
to the protein and excipients, by slowly increasing the temperature, although still
below Te, and gradually rising the pressure [80]. The result is a dried powder that
contains the protein in a glassy phase, amorphous excipients, and some residual
water. It must have a uniform and elegant appearance [81] and be rapidly dissolved
in the reconstitution solvent.
This process of water removal from frozen solutions under reduced pressure may
damage the active protein. The incorporation of sugars and polyols (e.g., sucrose,
trehalose, sorbitol) will mostly replace the hydrogen bonds between the protein and
the surrounding water molecules by hydrogen bonds created with the excipients,
stabilizing it thermodynamically and thus acting as lyoprotectants. From a kinetic
perspective, it is proposed that these sugars stabilize the active molecule by forming
an amorphous glass matrix, so they should not recrystallize during storage [82].
Due to the crystallization of different solutes in the mixture at different stages
during freeze-drying, a differential precipitation of excipients may occur. Upon
precipitation of buffer components, a shift in the pH of the mixture may take
place, with negative consequences for protein stability. This is hardly detected as,
upon reconstitution, buffers will redissolve and the pH measured will be the targeted
one. Salts are used to minimize changes in pH and tonicity, but one must be
conscious in their choice, since pure solvent freezes first than the salts, leading to
an increase of their concentration and altering the ionic strength and compromising
the protein. Sugars like lactose and mannitol can also crystallize and separate from
the solution. They should be replaced by carbohydrates (e.g., sucrose) or amino acids
(e.g., histidine or glycine) that present amorphous characteristics [39]. During
freeze-drying carbohydrates can also react with amino groups of the protein, pro-
moting the Maillard reaction and originating a yellow-brown cake. To avoid this,
nonreducing sugars like sucrose or trehalose may be used [34].
When considering lyophilized biopharmaceuticals, the reconstitution step is
of major importance. Reconstitution solvent can interfere with protein stability.
Therefore, besides using sterile water as diluent, excipients like stabilizers and
preservatives may be added to maintain the quality of the drug until administration.
Reconstitution times may have an impact in protein stability, especially for high-
concentration therapeutics that are formulated with higher concentrations of
stabilizer excipients. This increases reconstitution time and protein propensity
for degradation. The incorporation of wetting agents, like surfactants, in the recon-
stitution solvent is a possible solution [83].
4.2 Containers and Administration Devices
Packaging of biopharmaceuticals must accompany the chosen form of pharmaceu-

tical presentation in the maintenance of drugs’ stability during shelf-life and admin-
istration, while providing ease of handling and improving patient compliance.
Biologics are stored in vials, pre-filled syringes or cartridges, ampoules, or bags,
according to the physicochemical properties of the formulation, product volume,
materials of the containers, and their intended functionality [84]. There is a high
potential of interactions between containers and components of the formulation,
especially due to phenomena of leaching (migration of unwanted particles from the
container to the medicine) and sorption (diffusion of an ingredient to the container
material, through mechanisms of adsorption, absorption or permeation) [85], thus
requiring a rigorous compatibility assessment.
Pre-filled delivery devices are increasing in popularity, as they allow for the
patients to self-administer the drug in ambulatory, without the help of a healthcare
professional, improving compliance and reducing time and cost expenses with
specialized practitioners. This is relevant in a time of increasing development of
biopharmaceuticals to treat chronic diseases (diabetes, rheumatoid arthritis). They
also reduce the risk of contamination and waste-associated costs, since there is no
need to overfill the container [58]. When choosing the excipients and packaging
material for such devices, some aspects must be considered. For example, to be
administered in a syringe, liquid solutions must have low viscosity [40]. Also, during
transportation or handling, agitation may lead to the incorporation of air, which

could potentially originate new liquid-gas interfaces, and subsequently protein
unfolding.
5 Safety and Immunogenicity of Biopharmaceuticals
5.1 Quality Framework
Safety is a major concern regarding the use of biopharmaceuticals, and it can be

influenced by the quality of the medicine. In fact, unlike small drugs, recombinant
proteins are produced in living systems and are subjected to downstream steps
of processing and purification. It has been shown that the complexity of their
manufacturing may result in contaminants or impurities that affect the medicine’s
safety [86, 87]. Moreover, the physical and chemical features of therapeutic proteins
are hard to fully characterize, as well as their potential for aggregation and precip-
itation. To add to the active protein’s intrinsic features, the corresponding formula-
tion can have a significant impact in the drug’s safety profile. In fact, excipients are
perceived as pharmacologically inactive substances, but this concept is quite sim-
plified, as the safety of a biopharmaceutical also involves the quality and toxicity of
its excipients [88]. For known excipients, it is only required to prove quality to
ensure safety, since they are well-characterized and associated with market approval.
For novel excipients, however, regulatory authorities request preclinical studies to
demonstrate the desired safety of the compound on its intended formulation, which
can be very expensive and time-consuming [89]. This justifies a strict regulatory
framework on the quality control and toxicological data of the active protein and
excipients [89].
The ICH provides guidance on the quality and safety of medicines. The ICH
Q5A-Q5E and the S6(R1) guidelines provide information on contaminants
evaluation, stability testing, and preclinical safety testing within the scope of bio-
technological products. The World Health Organization (WHO) also presents rec-
ommendations for both manufacturers and regulatory agencies on the quality, safety,
and efficacy of biotherapeutic products, giving guidance on manufacture and quality
control of recombinant protein therapeutics, and on nonclinical and clinical evalu-
ation [90]. The Good Manufacturing Practice Guide for bulk Pharmaceutical
Excipients is harmonized across the different regulatory agencies and helps manu-
facturers to evaluate the quality of raw materials, in order to comply with Good
Manufacturing Practices [91]. The European and the United States pharmacopoeias
also have a natural impact in formulation development and excipients quality
control. These documents support pharmaceutical companies on the fulfillment of
requirements set by the EMA’s regulatory guidance on excipients [92] and the FDA’s
Code of Federal Regulations (Title 21, Chapter I, Subchapter C).
Despite the effort to assess all possible safety issues during development and
manufacturing, adverse events may occur when the final medicine is administered to
patients. Biologics adverse reactions have been classified based on the already
existing classification for small drugs adverse reactions (type A to E), having into
account mechanisms of action rather than clinical symptoms [93]. Thus, to distin-
guish this classification from the one existing for chemical drugs, adverse events can
be categorized from type α to type ε, as immune stimulation, immunogenicity
(against therapeutic protein), immune deviation (immunosuppression or autoimmu-
nity), cross-reactivity (with similar molecule), and non-immunological.
5.2 Mechanisms of Immunogenicity
Immunogenicity is a major issue in recombinant proteins’ safety and efficacy,

because of their high potential to elicit anti-drug responses [94]. The immunogenic
potential of biopharmaceuticals depends on a variety of factors, such as structural
features of the active molecule or excipients, presence of impurities, route of
administration, dose and duration of treatment, and patient features [95].
Reactions against biologics can be a consequence of either breakdown of immune
tolerance to autoantigens (human homologous) or activation of the host’s immune
system to neoantigens [96]. The first mechanism is not yet well understood, but it
seems that repeated administration of therapeutic proteins that are similar to endog-
enous ones, depending on their dose, may induce immune responses and clinical
adverse effects. This is exemplified by the development of thrombocytopenia in
patients who were administered with a pegylated recombinant megakaryocyte
growth and development factor. This therapeutic protein shares the first 163 amino
acids of endogenous thrombopoietin, which led to the development of antibodies
that cross-reacted and neutralized the endogenous promoter of platelet
production [97].
The second proposed mechanism of immunogenicity is related to the degree of
non-self of the active protein, glycosylation, and protein aggregates, all of which can
be examples of neoantigens (not previously recognized by the immune system). The
degree of non-self, i.e., of structures foreigner to one’s organism, is related with the
source of the protein: animal (e.g., bovine insulin is more immunogenic than porcine
insulin, which is more immunogenic than the human recombinant protein [98]),
bacterial, or plant origin (absence of posttranslation modification mechanisms
[99]). Glycosylation is one important characteristic that influences protein three-
dimensional conformation. When a glycan composition is not recognized by the
immune system, or affects protein structure, decreasing its stability and solubility,
immune reactions against the drug may be triggered. However, in some cases, a
glycosylation pattern different from the native protein’s one can be beneficial, and it
is even a modification proposed to decrease protein immunogenicity, as it can
improve solubility through protein-solvent interaction or upon hiding antigenic
sites [100].
5.3 Protein Aggregation
Protein aggregation has been considered the most important structural change related
to immunogenicity [101]. It is hypothesized that the immune system was developed
to recognize repeated patterns of proteins, sugars, or lipids present at the surface of
invading microorganisms and develop a humoral response upon generating anti-
bodies against the active site of the molecule. In the case of therapeutic proteins, the
generated antibodies are named anti-drug antibodies (ADAs). This is likely the
reason why multimerization of proteins is a key factor in the development of an
anti-drug immune response, along with protein aggregates, regardless the size of the
monomeric or aggregated form of the protein [102]. Aggregates can be classified
according to their size (submicron soluble aggregates, insoluble particles up to
100 μm, and visible particles – above 100 μm), binding type (non-covalent or
covalent) [3], or format (aggregates of proteins in the native structure; denatured
structure, aggregated in an irreversible way; and covalently bound native and
denatured proteins) [103]. There are no regulatory limits regarding the size of
aggregates in biopharmaceutical products, but the United States [104] and the
European [105, 106] pharmacopoeias determine the recommended limits for inject-
ables and methods to assess them.
5.4 Anti-drug Antibodies
When ADAs are produced in a conformation-dependent manner (instead of the

linear form of the epitope), they will alter the pharmacokinetics and pharmacody-
namics of the protein, therefore neutralizing its activity and therapeutic efficacy
[107]. Non-neutralizing antibodies did not used to be considered clinically signifi-
cant, as they do not neutralize the activity of the protein. However, they can bind to it
and form immune complexes that are rapidly eliminated, thus altering the pharma-
cokinetics of the drug and affecting its bioavailability [94]. Consequences of ADAs
can range from clinically non-relevant to severe responses, such as hypersensitivity,
anaphylactic reactions [103], or deficiency syndromes (thrombocytopenia or pure
red cell aplasia [108]). Thus, it is important to use ADA determination as a predictive
marker of immunogenicity during drug development or as an assessment tool after
drug’s approval [109]. Nonclinical studies have a low predictive value. Despite the
efforts, there is still the need to develop more accurate in silico models that can
predict protein-immune system interaction, as well as animal models that can
overcome the differences between human and animal immune systems and their
inevitable immune reactions against recombinant human proteins [110]. This sup-
ports the requirement from the regulatory authorities to generate data on ADA
detection in clinical trials, as well as the incorporation of ADA detection methods
in the drug’s risk management plan [111]. EMA and FDA generated documents that
provide guidance and harmonization on the analytical methods to be used for ADA
detection [112–114].
6 Current Trends in Formulation and Future Perspectives
The biotech industry has had a huge development, with great scientific and technical
improvements, that increased the efficacy, safety, and quality of biopharmaceutical
products. Nonetheless, challenges associated with the efforts to better understand the
structure-function relationship, rational design of suitable formulations, engineering
novel protein formats, avoidance of aggregates formation and immunogenicity, or
development of less invasive administration routes, still remain.
6.1 Novel Excipients
The research on novel excipients is a significant aspect to consider in the develop-

ment of novel biotechnology-based drugs and delivery devices. Excipients are
considered novel when they are associated for the first time with a drug or route of
administration. Therefore, both new substances and existing compounds may fit in
this definition [92].
Novel excipients can bring numerous advantages: they can be used in smaller
concentrations even in highly concentrated solutions; they can decrease viscosity
and increase protein stability; they can improve manufacturing processes or allow to
differentiate a medicine. Therefore, they can have a central role in increasing drug’s
efficacy and safety and contribute to patient’s compliance [115]. Some novel sub-
stances have been approved or are under development. Recombinant human hyal-
uronidase has been recently used in products for subcutaneous administration. It
enhances protein bioavailability by creating an interstitial matrix that promotes
dispersion of the therapeutic protein [116]. Surfactants based on trehalose fatty
acid esters are being tested as stabilizers in shake-induced stress conditions, reducing
aggregate formation and adsorption to container surfaces. These properties make
them a plausible alternative to poly(ethylene glycol)-derived surfactants that induce
protein oxidation during static storage [117]. With the increasing development of
high-concentration antibody solutions, excipients that reduce viscosity are needed.
Combined use of arginine and glutamic acid seems to reduce intermolecular attrac-
tions, thus decreasing aggregation [48]. Hydrophobic salts, which compete with
hydrophobic protein residues in protein-protein interactions, are also helpful
in avoiding aggregation [118]. Cyclodextrins are cyclic oligosaccharides that
have been used as excipients before, but not in parenteral protein formulations
[119]. Their interest in recombinant protein formulations has been rising since
they form inclusion complexes of the drug, increasing solubility and stability, and
reducing aggregation [120].
Despite the awareness on the importance of novel excipients and the efforts made
to develop them, they are not frequently found in approved products. This is because
regulatory authorities have very strict requirements about the safety of these new
substances, making the evaluation of the application process very complex, time-
consuming, and expensive [121]. In fact, the experience associated with human use
of excipients approved by regulatory authorities determines that any or only little
additional toxicological data is needed. For novel excipients, stricter requirements
are demanded [121], such as extensive preclinical evaluations, as well as data on the
pharmacokinetics of the new excipient on its intended formulation. In Europe, the
dossier that needs to be submitted for the application of a new excipient is similar to
the one of an active substance. It requires information on manufacturing, character-
ization, in process controls and safety. It must be submitted together with the
application for marketing authorization, in accordance with the EMA Guideline on
Excipients in the Dossier for Application for Marketing Authorization of a Medicinal
Product [92]. The FDA elaborated a guidance on Nonclinical Studies for the Safety
Evaluation of Pharmaceutical Excipients that includes recommendations on how to
obtain a safety profile for these new entities [122].
One last concern regarding the regulatory requirements for new excipients is that
their development generates proprietary data that demands protection. As a way of
promoting the development of novel compounds by the pharmaceutical companies,
manufacturers of the United States of America and Japan follow an Excipient Master
File, which contains both open and confidential information on quality and toxicity
of the new excipients. By using this information, new excipients can be approved
apart from a marketing drug application, a situation that is different at the present
moment in Europe [121].
6.2 Alternative Routes of Administration
Therapeutic proteins have been formulated for parental administration, to allow for
maximum availability and minimum pre-systemic degradation. Intravenous formu-
lations are administered either in a single bolus dose or in a continuous infusion.
Subsequently, maximum bioavailability is rapidly achieved. However, this pharma-
cokinetic profile may not be the desired one for all therapeutics. In fact, for proteins
with short blood half-lives, an absorption of the drug through a prolonged period
may be beneficial. One way to obtain the desired concentration-time profile is to
change the administration route from intravenous to subcutaneous or intramuscular,
which delay the absorption of the drug into the blood circulation [123].
Nowadays, other routes of administration are being studied for biopharma-
ceuticals, in search for a better convenience of administration (noninvasive) for
the patient, improving therapeutic adhesion and self-administration in ambulatory
[124]. This is especially relevant in a time where more biopharmaceuticals to treat
chronic diseases are available. The oral administration provides the previously
mentioned advantages. However, bioavailability achieved by this route is very
low, not only because of the nature of the proteins themselves (polarity and high
molecular weight) but also due to protein degradation in the gastrointestinal tract and
in first-pass hepatic metabolism. Strategies to overcome these difficulties are being
studied and include the co-administration of absorption enhancers, which can act by
temporarily disrupting the intestinal barrier or serving as a carrier (e.g., calcium
chelators, surfactants, and bile salts), or protease inhibitors, which prevent enzymatic
degradation of the drug (e.g., aprotinin and puromycin) [125].
Inhalation and intranasal administration routes can be advantageous due to the
convenience of administration, the highly vascularized large surface area for absorp-
tion and avoidance of first-pass metabolism. Exubera® was a recombinant human
insulin that, when inhaled, was absorbed more rapidly and had a shorter half-life than
subcutaneous insulin. This profile was actually similar to the one of endogenous
insulin after a meal [126]. However, the product was withdrawn from market in 2008
due to the low bioavailability and local side effects. Intranasal administration has the
benefit of a direct delivery of the drug from the nasal cavity to the brain, without
being retained by the blood-brain barrier. This is particularly interesting in thera-
peutics for central nervous system diseases, as exemplified for some neurotrophins,
neuropeptides, and cytokines in in vivo studies [127]. On the other hand, intranasal
administration is associated with more variability in absorption, due to the physio-
logical mucociliary clearance mechanism and uncertain mucus secretion in cases of
cold, allergies, or hay fever.
Transdermal administration also offers the advantage of bypassing hepatic and
gastrointestinal degradation and allows for a prolonged drug release. However,
bioavailability is limited due to proteins’ high molecular weight and hydrophilic
nature. Penetration enhancers like cell-penetrating peptides, ethanol, propylene
glycol, dimethyl sulfoxide or surfactants can be incorporated in the formulation to
change the permeability of the skin, facilitating drug absorption [128]. However,
these passive strategies of transdermal delivery are limited to peptides and small
molecular weight proteins. In this regard, permeation techniques like sonophoration
(application of low-frequency ultrasound), iontophoresis (use of low-level electric
current), and polymeric microneedles (either coated, dissolvable, degradable, or
bioresponsive, according to the type of drug-polymer system desired) [129] have
been used to allow transdermal delivery of proteins like insulin, hormone-releasing
factor, calcitonin, and parathyroid hormone [130].
Based on the advantages associated with alternative routes of administration
stated above, a new route of administration may be an interesting strategy to extend
the life cycle of a drug already marketed in an injectable formulation. In this case,
new nonclinical and clinical data would be required, to ensure comparability of
efficacy, safety, and tolerability between the two formulations [131].
6.3 Other Strategies to Improve Protein Delivery
Manipulating the choice and concentration of excipients may not be enough to

achieve the desired pharmacokinetics of certain proteins. Therefore, other strategies
to improve drug delivery have been proposed. A controlled delivery system enables
the maintenance of drug therapeutic levels for an extended period, decreasing the
need of frequent administrations and thus improving patient comfort and compli-
ance. The components of the system must be nontoxic, non-immunogenic, biode-
gradable, and/or biocompatible [132]. Options available today include mechanical
pumps (that can deliver the drug at a constant or pulsar delivery rate), subcutaneous
osmotic mini-pumps (mechanism based on a difference of osmotic pressure between
the interior of the pump and the outside), and regulated approaches (like biosensor-
pump combinations or even self-regulating systems) [133]. Other strategies include
the delivery of the therapeutic drug encapsulated in matrix-type delivery systems.
Examples of such approach are nanoparticles and microparticles that prevent
the contact of the protein with proteases and other enzymes and, consequently, its
degradation before reaching the target site [134], or immobilization in hydrogel
networks (beneficial in maintaining protein three-dimensional structure) [135].
6.4 High-Throughput Characterization and Formulation

Screening
Due to the complexity of recombinant proteins and the diversity of excipients now
available, drug characterization and formulation development are difficult and
time-consuming. The use of high-throughput screening for protein characterization
has been increasing, with the improvement of equipment such as high-density
microplates, microfluidics and more sensitive detectors [136], and automatization
of conventional technologies like spectroscopy, light scattering, or imaging [137].
When applied to formulation development, high-throughput assays give a large
amount of information about protein stability, in a short period of time, and with a
reduced amount of sample, which is critical in an early development stage [37].
Such assays should be preceded by pre-formulation studies, in order to increase
knowledge on the physical and chemical properties of the protein and optimize
the choice of experimental conditions to perform. These studies can go from the
aforementioned characterization methods to mathematical and computer modeling
tools. Mathematical models are an interesting source on the prediction of the pro-
tein’s conformational dynamics and aggregation propensity [138, 139]. To assess
protein structure, in silico characterization software is available. It is mainly based on
the knowledge provided from protein sequence and signature databases (e.g.,
UniProt, Protein Data Bank). They combine data from amino acid sequences and
three-dimensional data, to give an insight on protein structure, hydrophobicity, or
even function [140]. For subsequent steps, several tools and online services have
been developed that allow a fast and efficient analysis of the protein’s active
conformation, protein-ligand interactions, and affinity data [141].
Forced degradation characterization must be also included in pre-formulation
studies, in order to detect and quantify possible degradation products [142]. It is
Fig. 1 Flow diagram of the

high-throughput
formulation development
concept (Adapted from
Martinus et al. [145])
assumed that when exposed to stress conditions for a short period of time, such as
elevated temperatures, freeze-thawing, extreme pH, mechanical stress, or oxidizing
environments, the degradation profile of the drug will correlate with degradation
during the medicine lifetime [143]. This will enable the determination of the
best stabilizing conditions, selection of stability parameters for quality monitoring
and comparability, and prediction of degradation upon accidental exposure to
unrecommended conditions. Most common forced degradation studies and analyt-
ical methods to assess degradation products have been reviewed by Hawe et al.
[144]. After data collection from pre-formulation studies, an experiment can be
designed, and a high-throughput platform (Fig. 1) can be put in place, either for
characterization assays or for selection of appropriate formulation. There is a first
step of sample preparation, using automated handling systems that place the samples
in microplates (96–384 wells), allowing for the screening of multiple parameters
simultaneously (concentration, pH, buffers, dosage form, and storage temperature).
This is followed by sample analysis, usually with the help of specific software
[145]. Several methodologies have been performed in high-throughput platforms.
Noninvasive methods are the ones that allow for a recovery of the formulation, thus
reducing waste. They include techniques such as UV-Vis absorbance, fluorescence
spectroscopy, and light scattering. Invasive procedures should be executed after the
noninvasive ones, to make use of the same sample. Examples of such methodologies
are calorimetry, capillary electrophoresis, and mass spectrometry.
An example of a high-throughput platform for protein characterization that has
been successfully developed is that of an automated workstation for large-scale
protein identification, using reverse liquid chromatography and mass spectrometry,
followed by analysis using SEQUEST software [146]. Regarding formulation devel-
opment, Zhao et al. engineered an automated system that can test up to 500 different
samples through a variety of analytical techniques, allowing for the screening of
more formulations in a very short time, with the same reliability than classical
approaches [147].
6.5 Developability Assessment and Quality by Design
Developability assessment is the likelihood of a drug candidate to be successfully

developed into a commercial product [148]. It comprises a set of approaches that,
when implemented in early stages of development, enable a more rational choice of
lead candidates to be developed into optimal commercial products. These strategies
include pre-formulation studies, stability evaluation, interaction between discovery
and formulation design teams, and manufacturability assessment [149]. Therefore,
developability offers the chance to re-engineer the lead molecule or to adapt the
development process according to potential undesired features discovered in early
development. This decreases the risk of failure in more resource-consuming and
costly stages of drug development [150].
Developability assessment correlates with the implementation of Quality by
Design (QbD), another trend in biopharmaceutical development and manufacture
[151]. ICH guidelines Q8 and Q8(R2) define QbD as “a systematic approach to
development that begins with predefined objectives and emphasizes product and
process understanding and process control, based on sound science and quality risk
management” [152]. With a great understanding of the active protein and excipients
from pre-formulation studies and developability assessment, it is possible to define,
monitor, and adjust the parameters that contribute the most to consistently deliver
products with a defined quality. Within the QbD, the parameters that are proven to
impact protein stability, named Critical Quality Attributes (CQAs), should be
monitored throughout the manufacturing process, such as type of excipients, con-
centration, or grade. Design of Experiments (DoE) is another important element of
QbD, defined as a tool that incorporates the possibility to change the formulation
attributes within a defined design space, whose limit is justified based on solid
scientific data. This qualitative and quantitative flexibility allows for a rapid screen
and optimization of a formulation during manufacturing [153]. Any deviations in
formulation composition made within the defined design space are not considered
changes in manufacturing, which is very attractive from the regulatory point of view.
Risk analysis can help defining CQA’s upon, for example, using tools of prediction
of aggregates formation and immunogenicity propensity, which may impact the
efficacy and safety of the medicine [100]. In respect to the establishment of the
range enabling the maintenance of the protein quality (in terms of physical stability
and activity), a risk management plan must be developed to minimize the impact of
any damage that may occur, which is contemplated in the ICH Q9 Quality Risk
Management [154]. This plan must be defined by a multidisciplinary team that can
correctly assess the major technical and logistic aspects that can impact the drug
(from physical and chemical stability problems, to operation errors, or issues asso-
ciated with raw material suppliers) [155].
7 Conclusion
Protein-based therapeutics are complex products, whose stability from manufactur-

ing to patient administration is hard to achieve. From formulation optimization to
the development of delivery systems, several strategies have been studied to try to
overcome biopharmaceuticals stability limitations and safety concerns or to improve
product quality and patient compliance. Despite these efforts, many challenges
remain unsolved, like immunogenicity and safety concerns, stability issues, and
exploration of alternative routes of administration, giving space for further research
for different solutions and technical advances in this field.
Acknowledgments Rita Ribeiro is a student of the Ph.D. Program in Pharmaceutical Sciences

from the Faculty of Pharmacy, University of Coimbra, and a recipient of the fellowship SFRH/BD/
121935/2016 from the Portuguese Foundation for Science and Technology. This work was funded
by Portuguese National funds via FCT – Fundação para a Ciência e a Tecnologia, I.P. – under
projects Cancel Stem (reference POCI-01-0145-FEDER-016390), CENTRO-01-0145-FEDER-
000012 (HealthyAging2020), Euronanomed2 (FCT reference ENMed/0005/2015), and CNC.
IBILI (FCT reference UID/NEU/04539/2019).
References
1. Dill KA, Maccallum JL (2012) The protein-folding problem, 50 years on. Science
338:1042–1047
2. Dobson CM (2003) Protein folding and misfolding. Nature 426:884–890
3. Goswami S, Wang W, Arakawa T, Ohtake S (2013) Developments and challenges for
mAb-based therapeutics. Antibodies 89:452–500
4. Krause ME, Sahin E (2019) Chemical and physical instabilities in manufacturing and storage
of therapeutic proteins. Curr Opin Biotechnol 60:159–167
5. Randolph TW, Carpenter JF (2007) Engineering challenges of protein formulations. Am Inst
Chem Eng J 53:1902–1907
6. Pisal DS, Kosloski MP, Balu-iyer SV (2009) Delivery of therapeutic proteins. J Pharm Sci
99:2557–2575
7. Manning MC, Chou DK, Murphy BM, Payne RW, Katayama DS (2010) Stability of protein
pharmaceuticals: an update. Pharm Res 27:544–575
8. Soulby AJ, Heal JW, Barrow MP, Roemer RA, Connor PBO (2015) Does deamidation cause
protein unfolding? A top-down tandem mass spectrometry study. Protein Sci 24:850–860
9. Tyler-cross R, Schirchs V (1991) Effects of amino acid sequence, buffers, and ionic strength
on the rate and mechanism of deamidation of asparagine residues in small peptides. J Biol
Chem 266:22549–22556
10. Diepold K et al (2012) Simultaneous assessment of asp isomerization and asn deamidation in
recombinant antibodies by LC-MS following incubation at elevated temperatures. PLoS One
7:1–11
11. Gervais D (2016) Protein deamidation in biopharmaceutical manufacture: understanding,
control and impact. J Chem Technol Biotechnol 91:569–575
12. Parkins DA, Lashmar UT (2000) The formulation of biopharmaceutical products. Pharm Sci
Technol Today 3:129–137
13. Stadtman ER (1990) Metal ion-catalyzed oxidation of proteins: biochemical mechanism and
biological consequences. Free Radic Biol Med 9:315–325
14. Ha E, Wang WEI, Wang YJ (2002) Peroxide formation in polysorbate 80 and protein stability.
J Pharm Sci 91:2252–2264
15. Torosantucci R, Schöneich C, Jiskoot W (2014) Oxidation of therapeutic proteins and
peptides: structural and biological consequences. Pharm Res 31:541–553
16. Li S, Schoneich C, Borchardt RT (1995) Chemical instability of protein pharmaceuticals:
mechanisms of oxidation and strategies for stabilization. Biotechnol Bioeng 48:490–500
17. Wang WEI et al (2006) Antibody structure, instability, and formulation. J Pharm Sci 96:1–26
18. Kiese S, Papppenberger A, Friess W, Mahler H (2008) Shaken, not stirred: mechanical stress
testing of an IgG1 antibody. J Pharm Sci 97:4347–4366
19. Wang W (1999) Instability, stabilization, and formulation of liquid protein pharmaceuticals.
Int J Pharm 185:129–188
20. Carpenter BJF, Kendrick BS, Chang BS, Manning MC, Randolph TW (1999) Inhibition of
stressed-induced aggregation of protein therapeutics. Methods Enzymol 309:236–255
21. Kueltzo LA, Wang WEI, Randolph TW, Carpenter JF (2008) Effects of solution conditions,
processing parameters, and container materials on aggregation of a monoclonal antibody
during freeze–thawing. J Pharm Sci 97:1801–1812
22. Dias CL et al (2010) The hydrophobic effect and its role in cold denaturation. Cryobiology
60:91–99
23. Chi EY, Krishnan S, Randolph TW, Carpenter JF (2003) Physical stability of proteins in
aqueous solution: mechanism and driving forces in nonnative protein aggregation. Pharm Res
20:1325–1336
24. Joubert MK et al (2012) Highly aggregated antibody therapeutics can enhance the in vitro
innate and late-stage T-cell immune responses. J Biol Chem 287:25266–25279
25. Andersen CB, Manno M, Rischel C, Thórólfsson M, Martorana V (2010) Aggregation of a
multidomain protein: a coagulation mechanism governs aggregation of a model IgG1 antibody
under weak thermal stress. Protein Sci 19:279–290
26. Frokjaer S, Otzen DE (2005) Protein drug stability: a formulation challenge. Nat Rev Drug
Discov 4:298–306
27. Chiti F, Stefani M, Ramponi G, Dobson CM (2003) Rationalization of the effects of mutations
on peptide and protein aggregation rates. Nature 424:805–808
28. Nielsen L, Frokjaer S, Brange J, Uversky VN, Fink AL (2001) Probing the mechanism of
insulin fibril formation with insulin mutants. Biochemistry 40:8397–8409
29. Agrawal NJ et al (2011) Aggregation in protein-based biotherapeutics: computational studies
and tools to identify aggregation-prone regions. J Pharm 100:5081–5095
30. Obrezanova O et al (2015) Aggregation risk prediction for antibodies and its application to
biotherapeutic development. MAbs 7:352–363
31. Pandya A, Howard MJ, Zloh M, Dalby PA (2018) An evaluation of the potential of NMR
spectroscopy and computational modelling methods to inform biopharmaceutical formula-
tions. Pharmaceutics 10:1–24
32. ICH (1999) ICH Q6B – specifications: test procedures and acceptance criteria for
biotechnological/biological products. 1–16
33. Beck A, Wagner-rousset E, Ayoub D, van Dorsselaer A, Sanglier-cianférani S (2013)
Characterization of therapeutic antibodies and related products. Anal Chem 85:715–736
34. Crommelin D (2013) Formulation of biotech products, including biopharmaceutical
considerations. In: Pharmaceutical biotechnology. CRC Press, Boca Raton, pp 69–96
35. Angkawinitwong U, Sharma G, Khaw PT, Brocchini S (2015) Solid-state protein formula-
tions. Ther Deliv 6:59–82
36. Solá RJ, Griebenow KAI (2010) Effects of glycosylation on the stability of protein
pharmaceuticals. J Pharm Sci 98:1223–1245
37. Jorgensen L, Hostrup S, Moeller EH, Grohganz H (2009) Recent trends in stabilising peptides
and proteins in pharmaceutical formulation – considerations in the choice of excipients. Expert
Opin Drug Deliv 6:1219–1230
38. Peters B et al (2016) Effects of cooling rate in microscale and pilot scale freeze-drying –
variations in excipient polymorphs and protein secondary structure. Eur J Pharm Sci 95:72–81
39. Gervasi V et al (2018) Parenteral protein formulations: an overview of approved products
within the European Union. Eur J Pharm Biopharm 131:8–24
40. Garidel P, Kuhn AB, Schäfer LV, Karow-zwick AR, Blech M (2017) High-concentration
protein formulations: how high is high. Eur J Pharm Biopharm 119:353–360
41. Hawe A, Frieß W (2007) Formulation development for hydrophobic therapeutic proteins.
Pharm Dev Technol 12:223–237
42. Tedeschi G, Mangiagalli M, Chmielewska S, Natalello A, Brocca S (2017) Aggregation
properties of a disordered protein are tunable by pH and depend on its net charge per residue.
Biochim Biophys Acta 1861:2543–2550
43. Hopkins E, Sharma S (2019) Physiology, acid base balance. StatPearls. http://europepmc.org/
books/NBK507807. Accessed 12 Oct 2019
44. Roethlisberger D, Mahler H, Altenburger U, Pappenberger A (2016) If euhydric and isotonic
do not work, what are acceptable pH and osmolality for parenteral drug dosage forms? J Pharm
Sci 106:1–11
45. Bahrenburg S, Karow AR, Garidel P (2015) Buffer-free therapeutic antibody preparations
provide a viable alternative to conventionally buffered solutions: from protein buffer capacity
prediction to bioprocess applications. Biotechnol J 10:610–622
46. Shire SJ (2009) Formulation and manufacturability of biologics. Curr Opin Biotechnol
20:708–714
47. Arakawa T, Tsumoto K, Kita Y, Chang B, Ejima D (2007) Biotechnology applications of
amino acids in protein purification and formulations. Amino Acids 33:587–605
48. Shukla D, Trout BL (2011) Understanding the synergistic effect of arginine and glutamic acid
mixtures on protein solubility. J Phys Chem 115:11831–11839
49. Al-hussein A, Gieseler H (2013) Investigation of histidine stabilizing effects on LDH during
freeze-drying. J Pharm Sci 102:813–826
50. Wang W (2000) Lyophilization and development of solid protein pharmaceuticals. Int J Pharm
203:1–60
51. Pikal MJ, Dellerman KM, Roy ML, Riggin RM (1991) The effects of formulation variables on
the stability of freeze-dried human growth hormone. Pharm Res 8:427–436
52. Akers MJ (2002) Excipient-drug interactions in parenteral formulations. J Pharm Sci
91:2283–2300
53. Khan TA, Mahler H, Kishore RSK (2015) Key interactions of surfactants in therapeutic
protein formulations: a review. Eur J Pharm Biopharm 97:60–67
54. Goyal MK, Roy I, Amin A, Banerjee UC, Bansal AK (2010) Stabilization of lysozyme by
benzyl alcohol: surface tension and thermodynamic parameters. J Pharm Sci 99:4149–4161
55. Hutchings RL, Singh SM, Cabello-Villegas J, Mallela KMG (2013) Effect of antimicrobial
preservatives on partial protein unfolding and aggregation. J Pharm Sci 102:365–376
56. Bis RL, Singh SM, Cabello-villegas J, Mallela KMG (2014) Role of benzyl alcohol in the
unfolding and aggregation of interferon alpha-2a. J Pharm Sci 26:1–9
57. Heljo P, Ross A, Zarraga IE, Pappenberger A, Mahler H-C (2015) Interactions between
peptide and preservatives: effects on peptide self-interactions and antimicrobial efficiency in
aqueous multi-dose formulations. Pharm Res 32:3201–3212
58. Jezek J et al (2013) Biopharmaceutical formulations for pre-filled delivery devices. Expert
Opin Drug Deliv 10:811–828
59. Kocha T, Yamaguchi M, Ohtaki H, Fukuda T, Aoyagi T (1996) Hydrogen peroxide-mediated
degradation of protein: different oxidation modes of copper- and iron-dependent hydroxyl
radicals on the degradation of albumin. Biochim Biophys Acta 1337:319–326
60. Morefield GL et al (2005) Role of aluminum-containing adjuvants in antigen internalization by
dendritic cells in vitro. Vaccine 23:1588–1595
61. Mbow ML, De Gregorio E, Valiante NM, Rappuoli R (2010) New adjuvants for human
vaccines. Curr Opin Immunol 22:411–416
62. Guy B (2007) The perfect mix: recent progress in adjuvant research. Nat Rev Microbiol
5:505–517
63. Buonsanti C, Oro UD (2017) Discovery of immune potentiators as vaccine adjuvants. In:
Immunopotentiators in modern vaccines. Elsevier, Amsterdam, pp 85–104
64. EMEA/CPMP (2004) Guideline on adjuvants in vaccines. 1–18
65. Jones LS et al (2005) Effects of adsorption to aluminum salt adjuvants on the structure and
stability of model protein antigens. J Biol Chem 280:13406–13414
66. Fox CB, Kramer RM, Lucien Barnes V, Dowling QM, Vedvick TS (2013) Working together:
interactions between vaccine antigens and adjuvants. Ther Adv Vaccines Rev 1:7–20
67. Kaurav M et al (2018) Combined adjuvant-delivery system for new generation vaccine
antigens: alliance has its own advantage. Artif Cells Nanomed Biotechnol 46:S818–S831
68. Yanan C, Ping C, Binlong C, Suxin L, Hua G (2017) Monoclonal antibodies: formulations of
marketed products and recent advances in novel delivery system. Drug Dev Ind Pharm
43:519–530
69. EPAR – Product Information. European Medicines Agency. https://www.ema.europa.eu/en.
Accessed 13 Oct 2019
70. Walsh G (2018) Biopharmaceutical benchmarks 2018. Nat Biotechnol 36:1136–1145
71. EMEA/CHMP (2015) Guideline on similar biological medicinal products. pp 1–7
72. Kirchhoff CF et al (2017) Biosimilars: key regulatory considerations and similarity assessment
tools. Biotechnol Bioeng 114:2696–2705
73. FDA (2015) Scientific considerations in demonstrating biosimilarity to a reference product. pp
1–24
74. EMEA/CHMP (2014) Guideline on similar biological medicinal products containing
biotechnology-derived proteins as active substance: non-clinical and clinical issues. pp 1–13
75. Kesik-brodacka M (2018) Progress in biopharmaceutical development. Int Union Biochem
Mol Biol 65:306–322
76. Anour R (2014) Biosimilars versus ‘biobetters’ – a regulator’s perspective. Generics Ans
Biosimilars Initiat J 3:166–167
77. Ismael G et al (2012) Subcutaneous versus intravenous administration of (neo) adjuvant
trastuzumab in patients with HER2-positive, clinical stage I–III breast cancer (HannaH
study): a phase 3, open-label, multicentre, randomised trial. Lancet Oncol 13:869–878
78. Rathore N, Rajan RS (2008) Current perspectives on stability of protein drug products during
formulation, fill and finish operations. Biotechnol Prog 24:504–514
79. Shnek DR, Hostettler DL, Bell MA, Olinger JM, Frank BH (1998) Physical stress testing of
insulin suspensions and solutions. J Pharm Sci 87:1459–1465
80. Franks F (1998) Freeze-drying of bioproducts: putting principles into practice. Eur J Pharm
Biopharm 45:221–229
81. Patel SM et al (2017) Lyophilized drug product cake appearance: what is acceptable? J Pharm
Sci 106:1706–1721
82. Mensink MA, Frijlink HW, van der Voort K, Hinrichs WLJ (2017) How sugars protect
proteins in the solid state and during drying (review): mechanisms of stabilization in relation
to stress conditions. Eur J Pharm Biopharm 114:288–295
83. Cao W et al (2013) Rational design of lyophilized high concentration protein formulations-
mitigating the challenge of slow reconstitution with multidisciplinary strategies. Eur J Pharm
Biopharm 85:287–293
84. FDA (1999) Container closure systems for packaging human drugs and biologics. pp 1–41
85. Wang M et al (2018) Interactions between biological products and product packaging and
potential approaches to overcome them. AAPS PharmSciTech 19:3681–3686
86. Raghani A, Li K, Bussiere JL, Bercu JP, Qiu J (2018) Process-related impurities in
biopharmaceuticals. In: ICH quality guidelines: an implementation guide. Wiley, Hoboken,
pp 487–507
87. Dipaolo B, Pennetti A, Nugent L, Venkat K, Venkat K (1999) Monitoring impurities in
biopharmaceuticals produced by recombinant technology. Pharm Sci Technol Today 2:70–82
88. Florence AT, Attwood D (2016) Adverse events: the role of formulations and delivery
systems. In: Physicochemical principles of pharmacy. Macmillan, Basingstoke, pp 481–511
89. Elder DP, Kuentz M, Holm R (2015) Pharmaceutical excipients – quality, regulatory and
biopharmaceutical considerations. Eur J Pharm Sci 87:1–12
90. WHO (2013) Guidelines on the quality, safety, and efficacy of biotherapeutic protein products
prepared by recombinant DNA technology. pp 1–91
91. Nally J (2007) Good manufacturing practices for pharmaceuticals. CRC Press, Boca Raton
92. EMEA/CHMP (2007) Guideline on excipients in the dossier for application for marketing
authorization of a medical product. pp 1–12
93. Pichler WJ (2006) Adverse side-effects to biological agents. Allergy 61:912–920
94. Schellekens H (2002) Immunogenicity of therapeutic proteins: clinical implications and future
prospects. Clin Ther 24:1720–1740
95. Tovey MG, Lallemand C (2011) Immunogenicity and other problems associated with the use
of biopharmaceuticals. Ther Adv Drug Saf 2:113–128
96. Schellekens H, Casadevall N (2004) Immunogenicity of recombinant human proteins: causes
and consequences. J Neurol 251:4–9
97. Li J et al (2019) Thrombocytopenia caused by the development of antibodies to
thrombopoietin. Am Soc Hematol 98:3241–3249
98. Schernthaner G (1993) Immunogenicity and allergenic potential of animal and human insulins.
Diabetes Care 16:155–165
99. Dumont J, Euwart D, Mei B, Estes S, Kshirsagar R (2016) Human cell lines for biopharma-
ceutical manufacturing: history, status, and future perspectives. Crit Rev Biotechnol
36:1110–1122
100. Robinson AS (2012) Minimizing immunogenicity of biopharmaceuticals by controlling
critical quality attributes of proteins. Biotechnol Bioeng 7:1473–1484
101. Hermeling S, Crommelin DJA, Schellekens H, Jiskoot W (2004) Structure-immunogenicity
relationships of therapeutic proteins. Pharm Res 21:897–903
102. Kijanka G et al (2018) Submicron size particles of a murine monoclonal antibody are more
immunogenic than soluble oligomers or micron size particles upon subcutaneous administra-
tion in mice. J Pharm Sci 107:2847–2859
103. Rosenberg AS (2006) Effects of protein aggregates: an immunologic perspective. AAPS J
8:501–507
104. The United States Pharmacopeial Convention (2012) <788> Particulate matter in injections.
pp 1–3
105. European Pharmacopoeia (2007) 2.9.19. Particulate contamination: sub-visible particles. pp
300–302
106. European Pharmacopoeia (2007) 2.9.20. Particulate contamination: visible particles. p 302
107. Ito H, Nakashima T, So T, Hirata M, Inoue M (2003) Immunodominance of conformation-
dependent B-cell epitopes of protein antigens. Biochem Biophys Res Commun 308:770–776
108. Casadevall N, Nataf J, Viron B, Kolta A, Patrick M (2002) Pure red cell aplasia and
antierythropoietin antibodies in patients treated with recombinant erythropoietin. N Engl J
Med 346:469–475
109. Shankar G, Pendley C, Stein KE (2007) A risk-based bioanalytical strategy for the assessment
of antibody immune responses against biological drugs. Nat Biotechnol 25:555–561
110. Groot, A. S. De, Mcmurry, J. & Moise, L. Prediction of immunogenicity: in silico paradigms,
ex vivo and in vivo correlates. Curr Opin Pharmacol 8, 1–7 (2008)
111. Shankar G et al (2014) Assessment and reporting of the clinical immunogenicity of therapeutic
proteins and peptides – harmonized terminology and tactical recommendations. AAPS J
16:658–673
112. EMEA/CHMP (2007) Guideline on immunogenicity assessment of biotechnology-derived
therapeutic proteins. pp 1–18
113. EMEA/CHMP (2012) Guideline on immunogenicity assessment of monoclonal antibodies
intended for in vivo clinical use. pp 1–10
114. FDA (2016) Assay development and validation for immunogenicity testing of therapeutic
protein products. pp 1–31
115. Shah M (2018) New perspectives on protein aggregation during biopharmaceutical develop-
ment. Int J Pharm 552:1–6
116. Bookbinder LH et al (2006) A recombinant human enzyme for enhanced interstitial transport
of therapeutics. J Control Release 114:230–241
117. Schiefelbein L et al (2010) Synthesis, characterization and assessment of suitability of
trehalose fatty acid esters as alternatives for polysorbates in protein formulation. Eur J
Pharm Biopharm 76:342–350
118. Du W, Klibanov AM (2011) Hydrophobic salts markedly diminish viscosity of concentrated
protein solutions. Biotechnol Bioeng 108:632–636
119. Serno T, Geidobler R, Winter G (2011) Protein stabilization by cyclodextrins in the liquid and
dried state. Adv Drug Deliv Rev 63:1086–1106
120. Stella VJ, He Q (2008) Cyclodextrins. Toxicol Pathol 36:30–42
121. Kozarewicz P, Loftsson T (2018) Novel excipients – regulatory challenges and perspectives –
the EU insight. Int J Pharm 546:176–179
122. FDA (2005) Nonclinical studies for the safety evaluation of pharmaceutical excipients. pp 1–9
123. Zhu L, Xu H (2015) The optimal choice of medication administration route regarding
intravenous, intramuscular, and subcutaneous injection. Dove Press J 9:923–942
124. Fayad F et al (2018) Patient preferences for rheumatoid arthritis treatments: results from the
national cross-sectional LERACS study. Dove Press J 12:1619–1625
125. Mahato RI, Narang AS, Th L, Miller DD (2003) Emerging trends in oral delivery of peptide
and protein drugs. Crit Rev Ther Drug Carrier Syst 20:153–214
126. Barnett AH, Bellary S (2007) Inhaled human insulin (Exubera®): clinical profile and patient
considerations. Vasc Health Risk Manag 3:83–91
127. Hanson LR, Ii WHF (2008) Intranasal delivery bypasses the blood-brain barrier to target
therapeutic agents to the central nervous system and treat neurodegenerative disease. BMC
Neurosci 9:1–4
128. Chaulagain B, Jain A, Tiwari A, Verma A, Jain SK (2018) Passive delivery of protein drugs
through transdermal route. Artif Cells Nanomed Biotechnol 46:S472–S487
129. Ye Y, Yu J, Wen D, Kahkoska AR, Gu Z (2018) Polymeric microneedles for transdermal
protein delivery. Adv Drug Deliv Rev 127:106–118
130. Kayser O, Warzecha H (2012) Pharmaceutical biotechnology drug discovery and clinical
applications. Wiley-Blackwell, Hoboken
131. Philippart M et al (2016) Oral delivery of therapeutic proteins and peptides: an overview of
current technologies and recommendations for bridging from approved intravenous or subcu-
taneous administration to novel oral regimens. Drug Res 66:113–120
132. Vaishya R, Khurana V, Patel S, Mitra AK (2015) Long-term delivery of protein therapeutics.
Expert Opin Drug Deliv 12:415–440
133. Crommelin DJA, Hawe A, Jiskoot W (2019) Formulation of biologics including biopharma-
ceutical considerations. In: Pharmaceutical biotechnology. Springer, Berlin, pp 83–103
134. Ye C, Venkatraman S (2019) The long-term delivery of proteins and peptides using micro/
nanoparticles: overview and perspectives. Ther Deliv 10:10–13
135. Vermonden T, Censi R, Hennink WE (2012) Hydrogels for protein delivery. Chem Rev
112:2853–2888
136. Capelle MAH, Arvinte T (2008) High-throughput formulation screening of therapeutic pro-
teins. Drug Discov Today Technol 5:71–79
137. Senisterra GA, Finerty PJ (2009) High throughput methods of assessing protein stability and
aggregation. Mol Biosyst 5:217–223
138. Carbonell F, Iturria-Medina Y, Evans AC (2018) Mathematical modeling of protein
misfolding mechanisms in neurological diseases: a historical overview. Front Neurol 9:1–16
139. Andrews JM, Roberts CJ (2007) A Lumry-Eyring nucleated polymerization model of

protein aggregation kinetics: 1.aggregation with pre-equilibrated unfolding. J Phys Chem
111:7897–7913
140. Mulder NJ, Kersey P, Pruess M, Apweiler R (2008) In silico characterization of proteins:
UniProt, InterPro and Integr8. Mol Biotechnol 38:165–177
141. Kirchmair J et al (2008) The Protein Data Bank (PDB), its related services and software tools
as key components for in silico guided drug discovery. J Med Chem 51:7021–7039
142. Tamizi E, Jouyban A (2016) Forced degradation studies of biopharmaceuticals: Selection of
stress conditions. Eur J Pharm Biopharm 98:26–46
143. Nowak C et al (2017) Forced degradation of recombinant monoclonal antibodies: a practical
guide. MAbs 9:1217–1230
144. Hawe A et al (2012) Forced degradation of therapeutic proteins. J Pharm Sci 101:895–913
145. Capelle MAH, Gurny R, Arvinte T (2007) High throughput screening of protein formulation
stability: practical considerations. Eur J Pharm Biopharm 65:131–148
146. Ducret A, Oostveen IVAN, Eng JK, In JRY, Aebersold R (1998) High throughput protein
characterization by automated reverse-phase chromatography/electrospray tandem mass spec-
trometry. Protein Sci 7:706–719
147. Zhao HUI et al (2010) Formulation development of antibodies using robotic system and high-
throughput laboratory (HTL). J Pharm Sci 99:2279–2294
148. Wang W, Ohtake S (2019) Science and art of protein formulation development. Int J Pharm
568:118505
149. Perez-ramírez B, Guziewicz N, Simler R, Sreedhara A (2015) Approaches for early
developability assessment of proteins to guide quality by design of liquid formulations. In:
Quality by design for biopharmaceutical drug product development. Springer, Berlin, pp
87–114
150. Jarasch A et al (2015) Developability assessment during the selection of novel therapeutic
antibodies. J Pharm Sci 104:1885–1898
151. Zurdo J (2013) Developability assessment as an early de-risking tool for biopharmaceutical
development. Pharm Bioprocess 1:29–50
152. ICH (2009) ICH Q8(R2) – pharmaceutical development. pp 1–24
153. Grant Y, Matejtschuk P, Bird C, Wadhwa M, Dalby PA (2012) Freeze drying formulation
using microscale and design of experiment approaches: a case study using granulocyte colony-
stimulating factor. Biotechnol Lett 34:641–648
154. ICH (2005) ICH Q9 – quality risk management. pp 1–19
155. Moreton C (2009) Functionality and performance of excipients in a quality-by-design world.
Am Pharm Rev:32–35
DOI: 10.1007/10_2019_116
Published online: 28 November 2019
Therapeutic Antibody Engineering

and Selection Strategies
Joana Ministro, Ana Margarida Manuel, and Joao Goncalves
Contents
1 Antibody Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 58
1.1 Antibody Discovery . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 58
1.2 Characteristics and Structure of Antibodies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 58
1.3 Antibody Paratope . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 60
1.4 B Cell Development and Activation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 60
1.5 Monoclonal Antibodies and Their Recognition as Tools . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 65
1.6 Antibody Engineering . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 66
1.7 Market Importance of Therapeutic Antibodies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 68
2 Antibody Libraries . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 69
2.1 Generation of In Vitro Antibody Libraries . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 69
2.2 Technologies for Antibody Selection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 72
2.3 Maturation Strategies for Directed Evolution . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 75
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 79
Abstract Antibody drugs became an increasingly important element of the thera-

peutic landscape. Their accomplishment has been driven by many unique properties,
in particular by their very high specificity and selectivity, in contrast to the off-target
liabilities of small molecules (SMs). Antibodies can bring additional functionality to
the table with their ability to interact with the immune system, and this can be further
manipulated with advances in antibody engineering.
The expansion of strategies related to discovery technologies of monoclonal
antibodies (mAbs) (phage display, yeast display, ribosome display, bacterial display,
mammalian cell surface display, mRNA display, DNA display, transgenic animal,
J. Ministro
LeanBioPro, Barcelona, Spain
A. M. Manuel and J. Goncalves (*)
iMed – Research Institute for Medicines, Faculty of Pharmacy at University of Lisbon, Lisbon,
Portugal
e-mail: joao.goncalves@ff.ul.pt
56 J. Ministro et al.
and human B cell derived) opened perspectives for the screening and the selection of
therapeutic antibodies for, theoretically, any target from any kind of organism.
Moreover, antibody engineering technologies were developed and explored to
obtain chosen characteristics of selected leading candidates such as high affinity,
low immunogenicity, improved functionality, improved protein production,
improved stability, and others. This chapter contains an overview of discovery
technologies, mainly display methods and antibody humanization methods for the
selection of therapeutic humanized and human mAbs that appeared along the
development of these technologies and thereafter. The increasing applications of
these technologies will be highlighted in the antibody engineering area (affinity
maturation, guided selection to obtain human antibodies) giving promising perspec-
tives for the development of future therapeutics.
Minibody dAb (M,~15 000)
Graphical Abstract
scFv-Fc (M, 105 000)
scFv (M, 25 000)
Library
Antibody
variants
Therapeutic Antibody Engineering and Selection Strategies
Mutagenesis
Screening
Gene
Amplification
Selected
variants
57
Keywords Anti-drug antibodies, Biosimilars, European market, Immunogenicity,

Product information, Recombinant drugs, Summary of product characteristics,
Therapeutic biologics
1 Antibody Overview
1.1 Antibody Discovery
When in the 1700s the first perceptions of the immunology field were being explored
through vaccination experiments, no one could imagine the impact of antibodies in
today’s society. In fact, immunity acquisition against a previous encountered disease
has been documented for many centuries [1]. The first reference to antibodies
appeared in 1890 from Emil von Behring and Shibasaburo Kitasato. In a break-
through experiment, they treated diphtheria-infected animals with serum from
immunized animals [2]. The potential of this therapy was immediately foreseen for
human application, and Behring was later awarded with the Nobel Prize.
The term Antikörper (antibodies) was introduced around 1900 by Paul Ehrlich,
who performed several studies about toxicity and immunology. He is considered one
of the fathers of modern immunology for his insights into the antibody mechanics
and for the suggested theory that side chains of the antibodies react with antigens and
bind them and subsequently travel around the body in the blood [3]. In the 1940s,
Linus Pauling showed that the interaction between antibodies and antigens was more
dependent on their shape than on their chemical composition, confirming the lock-
and-key theory proposed by Ehrlich [4].
The modern era of antibody research started in 1975 with the development of the
hybridoma technology, the first mechanism to produce monoclonal antibodies in
large quantities [5]. Since then, antibodies have become crucial in biotech and
pharma industry, either for research purposes or for high-profit therapies. The first
monoclonal antibody for therapy was approved in 1986 [6], and by December 2017,
the FDA had already approved 79 therapeutic antibodies, with much more currently
under evaluation in various phases of clinical trials [7, 8].
1.2 Characteristics and Structure of Antibodies
Today it is well-known that the recognition of foreign agents by immunoglobulins

(Igs) is a core function of the adaptive immune response in mammals. Igs are
glycoproteins produced by all vertebrates and can be localized on the surface of B
lymphocytes, known as B cell receptors (BCR), or free in the blood or lymph, known
as antibodies (Ab). If produced by the same cell, these two molecules are identical
Therapeutic Antibody Engineering and Selection Strategies 59
Fig. 1 General structure of antibodies. (a) A typical IgG molecule is composed of two heavy
chains (blue) and two light chains (pink), linked by disulfide bonds. CH and CL are constant
domains of heavy and light chain, respectively. VH and VL are variable domains of heavy and
light chain, respectively. CDRs stand for complementary determining regions and are responsible
for antigen binding. FRs are conserved regions that confer structure to the CDRs. The two antigen-
binding fragments (Fab) are linked to the crystallizable fragment (Fc) by the hinge region. (b) An
IgG has two antigen-binding regions, containing the VH and VL domains. Each variable region has
three CDRs, flanked by the FRs regions. Adapted from O’Kennedy et al. [20]
except for a small portion that allows membrane anchoring in BCRs and secretion in
Abs [9]. Since antibodies are soluble and can be secreted in large quantities, they are
easily obtainable and studied. They are Y-shaped molecules consisting of two heavy
and two light polypeptide chains, linked by disulfide bonds, and divided into
constant and variable domains (Fig. 1a). They can be also divided in three equal-
sized portions, two antibody fragment (Fab) arms, containing the variable domains at
both ends, and the crystallizable fragment (Fc) domain. All domains are connected
by a flexible amino acid chain, called the hinge region [10, 11]. The Fab and the Fc
domains perform the two main functions of antibodies; while the Fab recognizes a
foreign element (or an antigen), through the variable domains, the Fc induces an
immune response by activating the complement system and/or the antibody-
dependent cellular cytotoxicity [12].
All antibodies are assembled in the same way. However, two classes of constant
light chains (κ and λ) and five classes of constant heavy chains (μ, δ, γ, α, and ε) can
be distinguished. Immunoglobulins (IgM, IgD, IgG, IgA, and IgE) are named by
their class of constant domains of the heavy chain. Also called isotypes, these
different constant regions determine the functional properties of an antibody. More-
over, IgA has two sub-isotypes (IgA1 and IgA2), and IgG has four sub-isotypes
(IgG1, IgG2, IgG3, and IgG4). IgM is the largest antibody and is mainly expressed
in immature B cells. IgD is expressed in naïve B cells and activates basophils and
mast cells upon exposure to an antigen. IgE is involved in allergic responses and IgA
in mucosal immunity. IgG is the most frequent isotype, accounting for 70–85% of
total immunoglobulins in serum. Its stability, long half-life and generally high
affinity make IgG the most used isotype for therapeutic use [13–15].
1.3 Antibody Paratope
For an antibody to exert its biological function, it must bind to a specific target. The
variable domains provide the contact to the antigens and determine the specificity of
each antibody. Each variable domain forms an immunoglobulin fold that consists of
a pair of β-sheets, linked by a disulfide bond, and three hypervariable loops lying at
the end of the structure, known as complementary determining regions (CDRs).
There are three CDRs in each variable domain, and their hypervariable characteristic
allows diversification and the recognition of an almost unlimited number of antigens.
Variation of the amino acid sequences inside these loops provides the major mech-
anism for the generation of the vastly diverse set of antibodies and T cell receptors
expressed by the immune system. While CDR1 and CDR2 domains vary somewhat
between different immunoglobulins, CDR 3 differs very dramatically. This happens
because contrary to CDR1 and CDR2, CDR3 suffers imprecise rearrangements
during the process of V(D)J recombination (Sect. 1.4.1) [16]. The three CDRs of
each variable domain are distributed between four structural and less variable
regions, known as framework regions (FR). The FR regions function as a scaffold
to hold the CDRs in position to contact the antigen. Even though sequence diversity
is concentrated in the CDRs, the framework regions are also diverse to some extent,
which may be advantageous in antibody function and stability to accommodate
highly diverse CDRs [17].
The variable regions of both chains interact to form a molecular site (the paratope)
that binds strongly to a part of the antigen (the epitope), and the strength of this
binding is called antigen-binding affinity (Fig. 1b). While identification of paratopes
is often done through identification of CDRs, not all the residues within the CDRs
bind the antigen. In fact, 3D analysis of the antibody structure suggested that only
20–33% of the residues within the CDRs participate in antigen binding. Nonetheless
the residues that are directly involved in the interaction with the antigen are, in
general, the most variable ones. The paratope is ultimately a function of the folding
pattern of the association of heavy and light polypeptide chains [18, 19].
1.4 B Cell Development and Activation
The antibody producing cells are called B cells or B lymphocytes. They perform a
core function in humoral immunity not only by secreting antibodies but also by
presenting antigens (antigen-presenting cells) and by secreting cytokines [21]. In
mammals, B cells start maturation in the bone marrow, from hematopoietic stem
cells, where they undergo a process called somatic recombination or V(D)J recom-
bination (see Sect. 1.4.1). This process is responsible for the generation of the first
level of antibody diversity and comprises the rearrangement of immunoglobulin
genes through random recombinations [22]. While developing, B cells undergo both
a positive and negative selection, a process that assures that the B cell receptor
(BCR) is binding to a proper ligand and not to a self-ligand. In case this would not
occur, the cell would not receive the right signals to proceed and would stop the
development. B cells then migrate to peripheral lymphoid organs, such as the spleen,
where they become mature B cells expressing both IgM and IgD; these cells are also
called naïve B cells before antigen exposure [23, 24].
Mature B cells circulate through the blood to the secondary lymphoid organs
where they contact with antigens that circulate in the lymph. The activation of a B
cell is triggered when the BCR binds to an antigen. This event induces the cells to
migrate to the interface between the follicle and T cell zone, and it is at this stage that
the B cells present the antigen to helper T cells. At this point both cells integrate
inputs to decide between death and proliferation. B cells can then start to proliferate
and form stable connections with helper T cells near the interfollicular zone, where
there is a great number of dendritic cells. A few days after antigen exposure, cells
integrate more inputs to differentiate into either plasma cells (in order to secrete large
amounts of antibodies) or memory cells (responsible for a secondary immune
response) or to go to the germinal center for affinity maturation [25]. In the germinal
centers, cells divide quickly and hypermutate the BCR V-regions in a process called
somatic hypermutation. They also undergo a process called class switch recombi-
nation, a mechanism that changes the immunoglobulin isotype (see Sect. 1.4.2).
Depending on their BCR affinity for foreign and self-antigens, the cells may be
selected again for survival or death [9]. Some antigens can activate B cells directly in
the absence of helper T cell. The ability of B cells to respond directly to these
antigens provides a rapid response to many important pathogens. However, as these
antibodies are not subject to affinity maturation, they are therefore less variable and
less functionally versatile than those induced with T cell help [26].
B cell development comprises a series of decision points where many inputs are
integrated influencing the cells’ fate. Evidence from cell dynamics and integration of
signals support the theory that more complex principles of control also exist [27].
1.4.1 V(D)J Recombination
An efficient immune response absolutely requires genetic diversity at the immuno-

globulin gene locus. The first level of diversity consists in the rearrangement of
multiple immunoglobulin genes, in a process called V(D)J recombination. It occurs
in the bone marrow, during maturation, and before antigen encounter. In mammals,
immunoglobulin gene segments contain more than 100 variable (V), 30 diversity
(D), 6 joining (J), and 9 constant (C) exons. Random choices of these genes encode
different proteins, generating in the order of a million different antibodies and
matching an enormous panoply of antigens. Rearrangements occur orderly
(Fig. 2a). At the heavy chain locus, D–J segments assemble first, followed by V
segment gathering, and, after transcription, the heavy variable regions are connected
to the constant regions by RNA splicing. At the light chain locus, D segments are
absent; thus only V–J exons are paired. CDR 1 and CDR 2 are encoded by the V
gene segments, while CDR 3 (the most variable and exposed CDR) is generated as a
a
Germ line lgH gene V D J C(m) C(x)
S(m) S(x)
VDJ recombination
Rearranged lgH gene C(m) C(x)
S(m) S(x)
Somatic Hypermutation AID Class Switch Recombination
Distribution of mutations
Frequency
CDR1 CDR2 CDR3
Position
Fig. 2 Immunoglobulin heavy chain diversification processes. (a) Random formation of diverse
V(D)J heavy combinations generates combinatorial diversity. Curved lines with arrows indicate
gene rearrangement events. The regions where somatic hypermutation accumulates are mainly the
CDRs; (b) schematic representation of protein–DNA complexes in V(D)J recombination. The
12-RSS and 23-RSS are represented as white and black triangles, respectively. Blue and yellow
rectangles represent the coding segments to be recombined, and the red rectangle represents the
newly generated nucleotide sequence at coding junction. Gray areas represent protein complexes;
AID activation-induced cytidine deaminase, C constant regions, D diversity segments, J joining
segments, S switch regions, TdT terminal deoxynucleotidyl transferase, V variable segments.
Adapted from Di Noia and Neuberger [39] and Mansilla-Soto and Cortes [31]
result of the imprecise joining of the VDJ or VJ exon. The antibody diversity is then
further increased by the assembly of different heavy and light chains [22, 24, 28].
V(D)J recombination is activated by a V(D)J recombinase that consists of
two lymphoid specific proteins, RAG1 and RAG2, that cleave DNA [29]. The
recombinase recognizes conserved DNA sequences, as well as the recombination
signal sequences (RSS) that flank each V, D, and J segments. In order to control this
mechanism, recombination only occurs between RSSs with different spacer lengths
(the “12/23 rule”), which prevents joining of two gene segments from the same
group [30]. RAG proteins introduce a pair of site-specific double-strand breaks
Fig. 2 (continued)
between the coding segments and the RSSs, creating a circular signal joint and a
linear coding joint. This cleavage generates hairpins at the extremities, which cannot
be directly ligated (Fig. 2b). The random opening of the hairpins by the endonucle-
ase protein Artemis at each extremity generates a combination of different DNA
ends that will strive for a mismatch between the two ends, enabling therefore the
formation of junctional diversity [31]. Thus, junctional diversity arises by the

imprecise fusion of V, D, and J segments and can result in deletion and/or addition
of nucleotides in the joint region. Deletions occur through the activity of an
exonuclease that is responsible for trimming the DNA ends before ligation. In fact,
80% of human Ig genes show junctional diversity caused by deletions. A less
common process is the addition of template nucleotides (P nucleotides) in the
junction site [32]. Another source of junctional diversion is the addition of
non-template N nucleotides. These random nucleotide insertions are added to the
DNA ends by a lymphoid-specific enzyme, called terminal deoxynucleotidyl trans-
ferase (TdT), in a template-independent manner, which can add up to 15 extra
residues in the joint, further increasing junctional diversity [33]. Actually, the fact
that this rearrangement process provides around 105–106 different antibody speci-
ficities in humans is in a great extend due to TdT activity. An end-joining process
strictly restricted to fully complementary ends would be unable to ligate the coding
joints. In contrast, a versatile but conservative process, such as nonhomologous end
joining (NHEJ), is able to join these DNA ends and generate a highly diverse
immune repertoire while protecting against side genomic instability [34, 35]. During
the process of V(D)J recombination, the numerous combinations of multiple gene
segments and the junctional modifications brought about by their joining result in a
large repertoire of antigen receptors, thereby assuring the hallmark diversity of the
adaptive immune system [36].
1.4.2 Somatic Hypermutation and Class Switch Recombination
Despite the great diversity achieved by V(D)J recombination, the B cell require-
ments for antigen targets exceed largely the information presented in the cell
genome. Thus, the cell needs to achieve larger repertoires by itself. After encoun-
tering an antigen and moving to the germinal centers, immunoglobulin genes suffer
both somatic hypermutation (SHM) and class switch recombination (CSR) [37].
In mice and humans, SHM causes mutations in the variable immunoglobulin both
in the light and heavy chains locus. The mutation rate is about one million-fold
higher than the spontaneous mutation rate in other genes, and it is mainly due to
single base substitutions, with occasional insertions and deletions. The mutation
activity of SHM starts around 150 nucleotides downstream of the IgV promoter, and
it is extended for about 2 kb pairs [38]. All four bases can be mutated, but the
targeting of individual bases is, however, not random. Interestingly, certain regions,
especially those responsible for antigen binding, are intrinsically more mutable than
others. The result of these mutations may alter the specificity or affinity of the
encoded antibody, potentially enhancing the response to a specific antigen
[39, 40]. In the same B cell, the heavy chain is rearranged down the chromosome
through CSR or isotype switching (Fig. 2a). CSR is the process by which CH
domains encoding one isotype are exchanged for another, thereby influencing the
effector function of the resulting antibody. In addition, CSR is accompanied by an
increase in antigen-binding affinity [41–43].
The mechanisms of SHM and CSR are not completely elucidated, but a key factor
of the two processes is well-known: the activation-induced cytidine deaminase
(AID) [44]. Upon B cell activation, AID expression is upregulated and is associated
with the cell transcription apparatus, gaining access to single-stranded DNA regions.
In most of the cases, AID recognizes specific small sequences (hotspots), where it
deaminates cytosines and converts them to uracils, creating dU:dG mismatches. This
DNA lesion is then processed by mismatch repair (MMR) and base excision repair
(BER) pathways, resulting in point mutations [45, 46]. In the CSR process, a protein
machinery is recruited after AID deamination process creating double-strand breaks
in the switch regions (S) located upstream of the constant regions. The DNA
between the S-regions is subsequently deleted from the chromosome, removing
unwanted heavy chain constant regions. The free ends of the DNA are rejoined by
NHEJ yielding B cells that express IgG, IgE, or IgA [41].
SHM and CSR are usually more evident after a second exposure to an antigen and
are responsible for the dramatically strong secondary immune response. These
events of affinity maturation work in combination to generate an antibody repertoire
estimated to be superior to 109 in humans [47].
1.5 Monoclonal Antibodies and Their Recognition as Tools
Due to their high specificity and selectivity, antibodies have the potential to be
of great use for biochemical, diagnostic, and therapeutic purposes. Antibodies are
categorized into two groups, polyclonal or monoclonal. Polyclonal antibodies
(pAbs) are a heterogeneous mixture of antibodies directed against various epitopes
on the same antigen. These antibodies are generated by different B cell clones and, as
a consequence, are immunochemically different, with different specificities and
affinities. While they are inexpensive and easy to produce, pAbs cannot be reliable
due to the high variability from batch to batch. In contrast, monoclonal antibodies
(mAbs) originate from a single antibody-producing B cell and therefore only bind to
one epitope of an antigen. Thus, while more expensive to produce, mAbs have high
specificity to a single epitope and present batch-to-batch homogeneity [48].
The development of the hybridoma technology was a big hallmark in the anti-
body field, allowing the relatively easy production of large quantities of mAbs for
diverse applications. These antibodies were generated by fusing antibody-producing
mouse cells with myeloma mouse cells, resulting in the formation of immortal cell
lines expressing a single antibody with specificity for one particular epitope of an
antigen. Once produced, hybridomas can be cultured in vitro indefinitely [5].
Many of the current laboratory procedures use mAbs as tools to answer basic
research questions. They allow researchers to identify molecules not seen by the
naked eye, enabling conclusions about the target molecule and pathway of interest.
Routine techniques such as Western blot, flow cytometry, immunohistochemistry,
and enzyme-linked immunosorbent assay (ELISA) all rely on antibody properties.
MAbs have also become an important component of many diagnostic techniques
including detection of infections, measurement of biological markers in blood, and

recognition of allergies, having increased the speed and accuracy of many tests
[49]. Also, radiolabeled antibodies can be used in the imaging diagnostic allowing,
for instance, differentiation between cancerous and non-cancerous cells [50].
With the ability to bind an almost unlimited number of targets with high speci-
ficity and stability, mAbs were sought for therapeutic applications since the first time
they were developed in vitro. Therapeutic mAbs can act through multiple mecha-
nisms, such as blocking of targeted molecule functions or disrupting a signaling
pathway [51]. Nevertheless, it soon became clear that antibodies were facing many
problems for therapeutic use, mainly due to their murine nature. Developments in
molecular biology made possible the creation of recombinant variants of mAbs that
led to optimized antibodies and ushered the age of antibody engineering [52, 53].
1.6 Antibody Engineering
1.6.1 Reduce Immunogenicity
The success of the first therapeutic antibody, OKT3 (muromonab), as a treatment for
transplant rejection, was not immediately followed by a series of approvals as
anticipated. Although very promising, patients treated with antibodies from hybrid-
oma technology often developed an immune response that attenuated the half-life
and the efficacy of the antibodies [54]. In order to reduce immunogenicity, numerous
strategies have been pursued for the humanization of animal antibodies, replacing
constant regions (chimeric antibodies) or all the non-specificity determining residues
(humanized antibodies) with corresponding human antibody sequences [55]. Since
the late 1990s, with the introduction of chimeric and humanized antibodies, thera-
peutic antibodies have become one of the fastest-growing classes of therapeutics in
the biological drug market [56]. More recently, the generation of transgenic mice
expressing a repertoire of human heavy and light chain genes, followed by the
generation of human hybridomas, was shown to be an effective way for the gener-
ation of human antibodies against many antigens [57–59].
1.6.2 Antibody Formats
Besides increasing safety, antibody engineering allowed the development of several

antibody formats for different functions. Immunoglobulins are large proteins
(between 150 and 180 kDa) and have difficulty to penetrate tissues or to target
antigens in small areas such as enzyme active sites. In drug development a heavy
chain may not be desirable or even necessary [60]. Researchers have dismembered
antibodies into their component parts, taking advantage of the smaller sizes in
order to develop new therapeutics with exciting properties (Fig. 3). These “domain
antibodies” come in numerous formats: fragment antigen binding (Fab), single-chain
Fig. 3 Examples of antibody formats. A variety of antibody fragments are represented including
Fab, scFv, and single domains VH and VL. Multimeric formats such as minibodies, bis-scFv,
diabodies, triabodies, and tetrabodies are also depicted. Adapted from Holliger and Hudson [61]
variable fragment (scFv), or VH and VL domains [61, 62]. Disadvantages of these

small domains comprise aggregation, poor solubility, and reduced half-life in circu-
lation. However, genetic engineering also allowed multimerization of antibody
fragments, and these formats can help to overcome the decrease of affinity and
stability from fragment domains. Moreover, this has also led to the generation of
multispecific antibodies, with the ability to recognize different epitopes or different
antigens [63–65]. The large panel of antibody formats that has been developed
reflects the strong interest for these molecules. While in many cases the manufactur-
ing remains challenging, several antibody formats are currently in clinical trials with
some already approved for commercialization [61, 66].
1.6.3 Target Delivery
Recently, mAbs are being exploited for modern drug delivery systems, aiming to
direct a therapeutic agent to a specific tissue or cell for enhanced pharmacology.
Antibody-based conjugates have opened up a whole new field of clinical possibil-
ities with several platforms emerging on the market, with most promising applica-
tions in cancer therapy [60].
Without the toxicity associated with chemotherapeutic agents, antibody therapy
held a tremendous promise in the elimination of tumor cells. However, only modest
success has been achieved in patients with solid tumors, with cell elimination not being
as effective as expected [67]. Antibody-drug conjugates (ADCs) combine the targeting
ability of an antibody with the cell-killing activity of a cytotoxic drug [68]. The basic
ADC technology is composed of an antibody vehicle, the cytotoxic drug, and a linker,
and each ADC is constructed in a customized manner for the specific antibody and drug
combination. By the end of 2018, there were four ADCs already approved for cancer
therapy [69, 70].
Therapeutic efficacy can also be increased by coupling an antibody with a
nanoparticle conjugate. In the context of drug delivery, nanoparticles are used to
encapsulate a drug for enhanced drug release and reduced side effects, similar to
ADCs. While fused to nanocarriers, mAbs can be used for targeting cell surface
receptors resulting in enhanced intracellular drug accumulation [71, 72].
The understanding that multiple factors might contribute for disease progression
led to the development of bispecific antibodies, in a large variety of formats.
Bispecific T cell engagers (BiTE®) have stood out from the panoply of bispecifics
due to their function in recruiting the host immune system, more specifically T cells
cytotoxic activity, to the cancer cells. They consist of two different scFvs, one
binding to T cells, usually through the CD3 receptor, and the other to a tumor cell,
through a tumor-specific molecule. One example of a BiTE antibody construct is
blinatumomab (trade name Blincyto), approved in 2017 for relapsed or refractory B
cell precursor acute lymphoblastic leukemia [73–75].
Another innovative approach for antibody use is the chimeric antigen receptors
(CARs). CARs are engineered receptors, which combine specificity (conferred by
the antibody) with immune effector function of T cells. In cancer therapy, T cells are
removed from patients, and the receptors are modified so that they become specific
to the patient’s cancer. The engineered T cells are then reintroduced into the patient,
with the new ability to recognize and kill the cancer cells [76, 77]. A CAR therapy
has been recently approved for use against acute lymphoblastic leukemia [78].
The development of creative antibody engineering technologies together with the
progress toward deciphering disease pathways has generated robust interest,
resources, and investments in antibody discovery.
1.7 Market Importance of Therapeutic Antibodies
Over the past 40 years, therapeutic antibodies have rapidly become the leading
product within the biopharmaceutical market. The global market for monoclonal
antibodies was valued at 85.4 billion dollars in 2015 and is expected to reach a value
of 138.6 billion dollars by 2024 [79]. The growing health concerns and the increas-
ing of diagnostic techniques have boosted the demand for these therapeutics in
recent years, particularly for chronic diseases. Nevertheless, the spectrum of diseases
potentially treated by antibody therapies is massive, including cardiovascular, respi-
ratory, hematology, kidney, immunology, and oncology diseases. While antibodies
are very efficient, their cost-effectiveness has always been discussed due to their
high costs, estimated to be more than one billion dollars from preclinical develop-
ment to market approval. Consequently, therapeutic antibodies are inaccessible to
many patients in both developed and developing countries. The development of
biosimilar antibodies, which show a similar quality, efficacy, and safety as the
original antibody, may help decrease the associated costs and provide alternative
treatment options [80, 81].
2 Antibody Libraries
2.1 Generation of In Vitro Antibody Libraries
As discussed before, the immune system has the incredible ability to create a vast
antibody repertoire, combining antibody gene segments recombination and somatic
hypermutation. Since the perception of the several applications for antibody mole-
cules, scientists have been trying to mimic the vast diversity achieved inside B cells
by constructing antibody libraries in vitro [82].
It was only in 1989 when the antibody repertoire from the B cells of an organism
was recombinantly cloned in large combinatorial libraries that mAb technology
really exploded [83, 84]. Another hallmark for in vitro antibody discovery occurred
roughly 1 year after, with the development of a display technology for the isolation
of mAbs from these large collections of recombinant antibody fragments [85]. Dis-
play technologies provided a way to quickly select antibodies from libraries on the
basis of the antigen-binding behavior of individual clones and allowed to overcome
the limitations from toxicity and immune tolerance. Moreover, the continuous
development of automation techniques made it possible to identify hundreds of
different antibody leads against a single therapeutic target, opening a new field for
different kinds of libraries without the need for immunization [86].
The most important parameter for in vitro antibody discovery is the quality of the
antibody libraries. Evolution has allowed the development of a wide range of
strategies for library generation that can differ in both design and means of con-
struction. They were first focused on affinity and thus on library size and diversity,
and, more recently, they are also focused on biophysical properties and reduced
immunogenicity. Based on the source of the antibody repertoire, four types of
libraries can now be identified: naïve, immune, synthetic, and semisynthetic
(Fig. 4) [87, 88].
2.1.1 Naïve Libraries
Naïve libraries are derived from B cells of nonimmunized donors. Although other
tissues can be used for B cell extraction, most naïve libraries are originated from
peripheral B lymphocytes, where they are not biased from the process of affinity
maturation that occurs in secondary lymphoid organs. Moreover, IgM repertoire is
often preferred because it closely reflects the diversity following immunoglobulin
gene rearrangement, without tolerance or antigen selection [89]. mRNA isolated
from B cells is used as template for the amplification of the variable regions of
antibody genes using degenerate oligonucleotide primer sets. The amplicon is then
Fig. 4 Different types of antibody libraries. (a) Naïve libraries are amplified from nonimmunized
donors. (b) Immune libraries are derived from immunized or immune donors. (c) Synthetic libraries
are based on computational in silico design and gene synthesis. (d) Semisynthetic libraries comprise
a combination of natural sources with in silico design. All libraries can be cloned in suitable vectors
to be used in display techniques. Adapted from Ponsel et al. [87]
cloned into a suitable vector for display and selection against a desired target. One of
the primary limitations of naïve libraries is the fact that the immune system deletes
clones with affinity to self-antigens, in order to prevent the development of autoim-
mune diseases, which may prevent the isolation of antibodies with affinity for many
important human targets. Despite the uncertainty of its content, the main value of
these kinds of libraries relies in the large repertoires (of up to 1011 antibodies) that
can be constructed and in the fact that a single library can be used for the selection of
antibodies against several types of antigens, including toxins [90, 91].
2.1.2 Immune Libraries
Immune libraries are derived from B cells of immunized donors or previously in

contact with an antigen. The library construction is performed using the same
method, but, contrary to naïve, immune libraries are smaller in size (normally 106–
108) and are not well suited for the identification of antibodies against a large panel
of antigens. Nevertheless, the pre-exposure of the host to an antigen results in the
production of very specific and high-affinity antibodies against the particular anti-
gen, mainly due to affinity maturation, which makes immunization a very suitable
model for antibody discovery. As human libraries can only be generated from B cells
obtained from disease-affected patients, several animal models are used for the
generation of this kind of libraries, including mice, rabbits, camelids, and sharks
[92, 93]. The latter two express antibodies that are only composed of heavy chains.
Thus, antigen binding is performed by only one single domain referred to as VHHs in
camelids and vNARs in sharks. Those fragments are naturally highly stable and
soluble, and it has been shown that they can generate high-affinity binders
[94, 95]. However, there are some important limitations when considering immune
libraries, including the time required for animal immunization, the unpredictability
of the immune response to the immunized antigen, and the fact that a new library
must be generated for each desired antigen [89].
2.1.3 Synthetic Libraries
While naïve and immune libraries are based entirely on naturally occurring sequence
diversity and do not require human input during the generation of sequence diver-
sity, synthetic libraries are diversified according to design and need a strategy for the
sequence diversification. In this approach the antibody diversity is designed in silico
and then synthesized in a controlled manner [96]. A key advantage of synthetic
diversification is that the composition of the CDRs and frameworks can be exactly
defined and controlled. Synthetic libraries prevent the natural biases and redundan-
cies of in vivo antibody repertoires and allow control over the sequences of variable
genes and the introduction of diversity [53]. From simple degenerate oligonucleotide
synthesis to physiochemically optimized library design, many different strategies
have been applied since the first reports of synthetic antibodies, in 1992 [97]. Most of
the constructed synthetic antibody libraries maintain a single framework sequence
and have their diversity concentrated in the CDRs, which are generated by random
combinations of mono- and trinucleotide units [98–100]. Nonetheless, there are a
few synthetic libraries with multiple variable heavy and light chain framework
regions, such as the HuCAL libraries and Ylanthia library [101, 102]. Multiple
framework sequences can accommodate more diverse CDR structures, but it
makes library design and construction more complex, and the clones are not as
uniform in their properties as in the case of single framework libraries [103].
The increasing knowledge of sequence, structure, function, and physicochemical
properties of antibodies has allowed researchers to design and construct highly
functional and sophisticated synthetic antibody libraries. However, it is still a very
complex process to assemble synthetic libraries, and intensive study regarding
structure and folding needs to be performed in order to guarantee functional
antibodies [104].
2.1.4 Semisynthetic Libraries
In semisynthetic libraries there is a combination of naturally derived and syntheti-

cally designed parts, and the ratio of both parts may vary in different strategies
[97]. They are often created to increase the natural diversity while maintaining a
good level of functional diversity. Such libraries have been created, for example, by
combining natural CDR regions or by joining naturally rearranged and highly
functional CDR 3 sequences with synthetic CDR 1 and CDR 2 diversity [105, 106].
2.2 Technologies for Antibody Selection
Antibody libraries need to be followed by a selection step in order to collect the leads
with the required affinity, specificity, and stability. In the immune system, B cells
link genotype to phenotype through presentation of the expressed antibody on the
surface, where only the positive selected cells will succeed [107]. The most common
selection methods also rely on the display at cell surface of each antibody from a
desired library. The antibody display can be on the surface of phages, on eukaryotic
cells, or even on ribosomes following in vitro transcription. All of these methods
consist of antibody incubation with a relevant antigen followed by several rounds of
washing and recovery of the best binders. Display systems offer a number of
advantages for antibody discovery and optimization as they are significantly fast
and can be carried out in a high-throughput mode [108].
2.2.1 Phage Display
Phage display was the first display technology developed for antibody discovery and
still remains widely used due to its simplicity to present large libraries. It was first
described by George P. Smith, in 1985, when he demonstrated the display of peptides
by fusing a peptide of interest to the gene III of a filamentous phage. This technology
was further developed and improved for display of antibodies mainly by the groups of
Winter and McCafferty, at the Laboratory of Molecular Biology (Cambridge, UK),
and by the groups of Lerner and Barbas, at The Scripps Research Institute (La Jolla,
USA) [85, 109, 110]. In phage display, the library DNA is usually inserted in fusion
with the gene of the phage coat protein pIII or pVIII. After infection of bacteria, a
progeny of phages is released, each phage displaying an antibody on its surface while
containing the antibody gene inside. By immobilizing a desired target to the surface
of a microtiter plate well, it is possible to incubate the phage library, in which the
phages displaying antibodies with affinity for the target will remain bound while
others are removed by washing. Those that remain can be eluted, and this resultant
pool contains a phage mixture that is enriched with relevant antibodies. This mixture
is then amplified by infecting bacteria and subjected to another incubation step. This
Fig. 5 Sequence of events of phage display technology. Biopanning of a phage display library to
select antibody binders to an immobilized target. This cycle is usually repeated 3–4 times and
includes binding of the phage library to an antigen-coated surface, washing with the desired
stringency, elution of phages containing the antibody genes, amplification, and analysis. Reprinted
from Huang et al. [111], with permission from ASM
repeated cycling is referred to as “panning” (Fig. 5), and usually after three or four
pannings, the DNA from eluted phages is collected and sequenced [111].
Phage display of antibody libraries has become a powerful method for a fast
selection of antibodies for therapy. It was used to select synthetic human libraries,
allowing fully human antibodies to be created in vitro [112]. One of the most successful
antibodies discovered by phage display was adalimumab (HUMIRA), an antibody with
affinity to human TNF-α, the world’s first fully human antibody [113]. Nevertheless,
phage display also presents some drawbacks, as it cannot accommodate all antibody
formats and often requires reformatting to produce soluble and well-expressed anti-
bodies with properties compatible with efficient manufacturing. Also, in most of the
cases, antigens are not presented in their native conformations, which decreases the
likelihood of selecting successful leads for therapeutic uses [114].
2.2.2 Ribosome and mRNA Display
In ribosome and mRNA display, a DNA library is transcribed and transduced

in vitro. Without the need to transform cells for library selection, it is possible to
achieve much higher library diversity. While in ribosome display, the translated
protein remains connected to the ribosome and to its encoding mRNA for the
selection step, in mRNA display, the mRNA is first translated and then covalently
bound to the protein it encodes, using puromycin as an adaptor molecule. Also, a
mutagenesis-based PCR can introduce random mutations after each selection round,
allowing combination of selection with affinity maturation [115, 116].
2.2.3 Yeast Cell Display
In recent years, several cell-based screening technologies have emerged. They rely
on the multi-copy display of antibodies or antibody fragments on a cell surface in a
functional form followed by high-throughput screening. Cell-based screening har-
bors the benefit of single-cell analysis and characterization of individual library
candidates [117]. The most common method used for cell-based screening is flow
cytometry, which allows the analysis of hundreds of thousands of cells per minute
according to their size, granularity, and fluorescence properties. Selection of mono-
clonal antibodies presented on the cell surface can be performed by antibody
expression level, by display strength, or by antigen affinity [118].
The yeast display technique was first described by the group of Wittrup, where a
protein of interest was displayed in fusion to a cell wall protein of Saccharomyces
cerevisiae [119]. Although not suitable for large libraries, due to limitations on yeast
transformation which limit the complexity of these libraries [120, 121], yeast display
has still the advantageous of linking an eukaryotic secretory pathway, with the
potential for the selection of antibodies with improved folding characteristics, to
the flow cytometry system, for a high-throughput screening, making it still widely
used. It is a very effective method for the isolation of high-affinity antibodies against
labeled targets and provides the possibility to discriminate between different affin-
ities of distinct clones [122].
2.2.4 Mammalian Cell Display
Despite the clear advantages, all of the above platforms share the downside of
expressing antibodies in a nonnatural environment. As large and complex proteins
that possess several functional domains, antibodies can only be efficiently expressed
and assembled into functional forms during the mammalian expression and secretory
pathway. The display of antibodies on mammalian cell surface often requires the
fusion of a transmembrane domain to the C-terminus. The ability to identify anti-
bodies directly from mammalian cells therefore enables selection of antibodies with
desirable properties from an early stage, such as high-level expression and stability
[123, 124]. Several groups have developed different mammalian display strategies,
from directly isolation of B cells specific for an antigen to more complex approaches,
as introduction of combinatory libraries through lentivirus infection [125, 126].
Most of these systems, however, are complex and time-consuming, which signifi-
cantly hamper the application of mammalian display [127].
2.2.5 In Vitro Compartmentalization
A next generation technology, called in vitro compartmentalization, intends to

substitute cells by water droplets for antibody production and display. The water
droplets are surrounded by an oil phase and contain all components required for
protein synthesis, as well as single genes that are competent for transcription and
translation. This system allows the co-compartmentalization of DNA and protein,
and this cell-like droplets (up to 1010 per ml) can therefore be used in flow
cytometry-based selections of large libraries, with a high degree of control over
selection conditions and stringencies [128, 129].
A multitude of discovery technologies are nowadays available to isolate high-
affinity antibody binders to nearly any given target. Promising strategies are also
emerging to improve the discovery process (Fig. 6). The biggest challenge remains
on how to include other important features into the screening process as, for
example, high folding and expression, low tendency to aggregate, and correct
glycosylation patterns. Various innovative approaches are expected to appear in
next years that will include as many parameters as possible and, certainly, will
increase the likelihood of antibody molecules to successfully reach the market [117].
2.3 Maturation Strategies for Directed Evolution
One of the greatest challenges in library generation when compared to in vivo

methods is the absence of somatic hypermutation (SHM), which enables nature to
create high-affinity antibodies. The in vitro occurrence of spontaneous mutations is
generally insufficient to obtain desired gene variants on a practical time scale.
Therefore, several genetic diversification techniques can be used to direct and evolve
antibody libraries toward a defined goal, thereby accelerating the identification of
desired proteins. This bottleneck process is named directed evolution and can be
applied in vitro with the purpose of mimicking the process of natural selection
[131, 132]. A single gene or a library is evolved by being subjected to mutagenesis,
screening, and amplification. Rounds of these steps are repeated, using the best
variants from one round as the templates for the next round, in order to reach the best
improvements [133].
Some methods based on introducing a certain degree of diversity into selected,
moderate affinity candidates are used and can be generally differentiated between
targeted and nontargeted diversification strategies [134]. Nontargeted approaches,
such as error-prone PCR and the use of a mutator E. coli strain, can be used for
random introduction of nucleotides into the whole antibody. Error-prone PCR is
based on the use of low-fidelity polymerases as well as on modified reaction
conditions, creating a high error rate during amplification [135]. E. coli mutator
strains are conditional mutants that produce single-base substitutions with higher
rates than normal cells [136]. Chain shuffling is another method used for nontargeted
Fig. 6 General antibody discovery process and the emerging technologies. The most common
antibody discovery process starts with animal immunization with the target antigen. The variable
genes are isolated from the antibody-producing B cells of positive responders, and polyclonal
libraries are constructed and packaged into in vitro display technologies. The displayed libraries are
then selected and screened for binding to the target antigen. Positive binders are sequenced,
recloned, and further characterized in functional assays. Improvements in the discovery process
include DNA vaccines and virus-like particles (VLPs) to enhance immunogenicity, alternative
diversification, where one of the two antibody chains is replaced by a repertoire

while the other chain is kept constant [137].
In contrast, targeted strategies introduce diversity at predicted positions, mainly at
the ones contributing for antigen binding, the CDRs. CDR walking, an approach
where diversity is introduced in short amino acid stretches, can provide up to a
420-fold increased affinity [138]. A more recent approach for targeted modifications
relies on programmable nucleases, which can be easily customized to cleave DNA at
almost any desired site. MAGE, abbreviated from multiplex automated genome
engineering, takes advantage of a template repair where different loci on the
chromosome can be targeted simultaneously, generating a multiplex genomic
mutant library. This is achieved by homologous recombination of artificial single-
stranded oligonucleotides with flanking homology to the target region. The oligo-
nucleotides hybridize to the complementary exposed strands during the process of
replication [139].
Both target and nontargeted strategies are usually followed by stringent selections
with targeted strategies being more likely to improve affinity and least likely to
create problems with protein stability [87]. Besides, the majority of mutations are
usually deleterious, and so mutated libraries often have most of the variants with
reduced activity. Therefore, a high-throughput selection and screening is important
to find the rare variants with beneficial mutations that improve the desired properties
[140]. Another important feature of directed evolution is the genotype-phenotype
link. When functional antibodies are isolated, it is necessary that their genes are also
collected. In phage display, for example, this is performed by compartmentalization
of gene (inside the phage) and protein (phage surface). The isolated gene sequences
are then amplified by transforming host bacteria or by PCR. The best pool of
sequences are then used for the next round of mutagenesis, and this repeated
cycle can generate protein variants adapted to the applied selection pressures
(Fig. 7) [141].
In practical terms it is impossible to cover the entire possibilities of mutations in a
typical protein. The entire randomization of a decapeptide would yield 1013 unique
combinations of amino acids, which exceeds the library size of all known library
creation methods. Nonetheless, directed evolution represents already a hallmark of
protein development and is being practiced for many academic and industrial
purposes. The easy manipulation of biological systems makes them very good
substrates for engineering and for redesigning new evolutionary approaches [142].
⁄
Fig. 6 (continued) animals to increase repertoire diversity, fully synthetic libraries to eliminate the
need for immunized animals, mammalian cell display for a native presentation, high-throughput
technologies to decrease discovery time, and autocrine-based selection systems to bypass the target-
binding process and directly select for antibodies with a defined functional phenotype. Reprinted
from Kennedy et al. [130], with permission from Taylor & Francis publisher
Fig. 7 Key steps in the cycle of directed evolution. A diverse library of genes is translated into a
corresponding library of gene products and screened for functional variants in a manner that
maintains the correspondence between genotype (gene) and phenotype (protein). These functional
genes are replicated and serve as starting points for subsequent rounds of diversification and
screening or selection
Over the last 40 years, monoclonal antibodies have stood out as important
scientific tools and powerful human therapeutics. Due to their high speci-
ficity to the target, these proteins are extremely important for innumerous
therapeutic applications and are recognized as a major drug modality in a
variety of diseases. Until now, the leading methodologies for therapeutic
antibody generation, immunization, and display approaches have been
effective for the generation of antibodies against various targets and have
led to a significant number of commercialization approvals.
Despite the moderate success, the common technologies for antibody discov-
ery are time-consuming and imply complex methods. Today, there is a need
to improve the antibody discovery process, creating more efficient technol-
ogies. There is a need, for example, to avoid the cost and time-consuming
practice of humanization or de-immunization, which could be surpassed
by a system capable of developing human antibodies. Also, it is a main
concern to increase the size and quality of antibody libraries. The higher the
quality and size of antibody libraries, the higher the likelihood of finding
efficient therapeutic leads. Furthermore, the most widely used display
technique, phage display, does not allow antibody and antigen molecules
(continued)
to interact in their native conformation. Having the possibility to select

different antibody formats in their native conditions would also be a big
benefit, increasing the chances of success in further assays. Finally, it
would be more efficient if one could select good candidates for high affinity
and, at the same time, optimized candidates for large-scale production.
Hybridoma technology and phage display have revolutionized the field of
antibody discovery by providing important approaches for the isolation of
monoclonal antibodies. However, the low efficiency of the hybridoma
isolation process and the limitations of the prokaryotic expression-based
technologies have been important technical drawbacks difficult to over-
come [143, 144]. Various in vitro approaches have been developed in
recent years to display full-length IgG on the surface of eukaryotic cells,
with the aim to render discovery of therapeutic monoclonal antibodies more
efficient, with favorable biophysical properties [122, 123, 126, 145].
Despite the improvements, these technologies are limited either by the
size of the antibody libraries that can be expressed or by the lack of a stable
genotype to phenotype link. With the increased number of antibodies
reaching the market, there is a need to develop new technologies that
stem from the limitations of current discovery platforms.
References
1. Riedel S (2005) Edward Jenner and the history of smallpox and vaccination. Proc (Bayl Univ
Med Cent) 18:21–25. https://doi.org/10.1080/08998280.2005.11928028
2. von Behring E, Kitasato S (1991) The mechanism of diphtheria immunity and tetanus
immunity in animals. Mol Immunol 28:1317, 1319–1320
3. Davies DR, Chacko S (1993) Antibody structure. Acc Chem Res 26:421–427. https://doi.org/
10.1021/ar00032a005
4. Pauling L (1940) A theory of the structure and process of formation of antibodies. J Am Chem
Soc 62:2643–2657. https://doi.org/10.1021/ja01867a018
5. Köhler G, Milstein C (1975) Continuous cultures of fused cells secreting antibody of
predefined specificity. Nature 256:495–497
6. Hooks MA, Wade CS, Millikan WJ (1991) Muromonab CD-3: a review of its pharmacology,
pharmacokinetics, and clinical use in transplantation. Pharmacotherapy 11:26–37
7. Singh S, Kumar NK, Dwiwedi P et al (2018) Monoclonal antibodies: a review. Curr Clin
Pharmacol 13:85–99. https://doi.org/10.2174/1574884712666170809124728
8. Strohl WR (2017) Analysis of the current antibody landscape. In: Antibody engineering and
therapeutics. San Diego
9. Goldsby RA, Kindt TJ, Osborne BA, Kuby J (2003) Immunology, 5th edn. W. H. Freeman,
New York
10. Poljak RJ, Amzel LM, Avey HP et al (1973) Three-dimensional structure of the Fab’ fragment
of a human immunoglobulin at 2.8-A resolution. Proc Natl Acad Sci 70:3305–3310. https://
doi.org/10.1073/pnas.70.12.3305
11. Inbar D, Hochman J, Givol D (1972) Localization of antibody-combining sites within the
variable portions of heavy and light chains. Proc Natl Acad Sci 69:2659–2662. https://doi.org/
10.1073/pnas.69.9.2659
12. Presta LG (2006) Engineering of therapeutic antibodies to minimize immunogenicity
and optimize function. Adv Drug Deliv Rev 58:640–656. https://doi.org/10.1016/j.addr.
2006.01.026
13. García Merino A (2011) Monoclonal antibodies. Basic features. Neurologia 26:301–306.
https://doi.org/10.1016/j.nrl.2010.10.005
14. Cruse JM, Lewis R (2010) Atlas of immunology, 3rd edn. CRC Press, Boca Raton
15. Male D, Brostoff J, Roth D, Roitt I (2006) Immunology, 7th edn. Elsevier, Philadelphia
16. Vandyk L, Meek K (1992) Assembly of IgH CDR3: mechanism, regulation, and influence on
antibody diversity. Int Rev Immunol 8:123–133. https://doi.org/10.3109/08830189209055568
17. Morea V, Lesk AM, Tramontano A (2000) Antibody modeling: implications for engineering
and design. Methods 20:267–279. https://doi.org/10.1006/meth.1999.0921
18. Padlan EA, Abergel C, Tipper JP (1995) Identification of specificity-determining residues in
antibodies. FASEB J 9:133–139. https://doi.org/10.1096/fasebj.9.1.7821752
19. Mandigan M, Martinko J (2006) Brock biology of microorganisms, 11th edn. Pearson Prentice
Hall, Upper Saddle River
20. O’Kennedy R, Fitzgerald S, Murphy C (2017) Don’t blame it all on antibodies – the need for
exhaustive characterisation, appropriate handling, and addressing the issues that affect spec-
ificity. TrAC Trends Anal Chem 89:53–59. https://doi.org/10.1016/j.trac.2017.01.009
21. Murphy K (2012) Janeway’s immunobiology, 8th edn. Garland Science, New York
22. Hozumi N, Tonegawa S (1976) Evidence for somatic rearrangement of immunoglobulin genes
coding for variable and constant regions. Proc Natl Acad Sci 73:3628–3632. https://doi.org/10.
1073/pnas.73.10.3628
23. Goodnow CC, Basten A (1989) Self-tolerance in B lymphocytes. Semin Immunol 1:125–135
24. Janeway C, Travers JP, Walport M, Shlomchik M (2001) Immunobiology: the immune system
in health and disease, 5th edn. Garland Science, New York
25. Chan TD, Gatto D, Wood K et al (2009) Antigen affinity controls rapid T-dependent antibody
production by driving the expansion rather than the differentiation or extrafollicular migration
of early plasmablasts. J Immunol 183:3139–3149. https://doi.org/10.4049/jimmunol.0901690
26. Parra D, Takizawa F, Sunyer JO (2013) Evolution of B cell immunity. Annu Rev Anim Biosci
1:65–97. https://doi.org/10.1146/annurev-animal-031412-103651
27. Goodnow CC, Vinuesa CG, Randall KL et al (2010) Control systems and decision making for
antibody production. Nat Immunol 11:681–688. https://doi.org/10.1038/ni.1900
28. Tonegawa S (1983) Somatic generation of antibody diversity. Nature 302:575–581. https://
doi.org/10.1038/302575a0
29. Oettinger M, Schatz D, Gorka C, Baltimore D (1990) RAG-1 and RAG-2, adjacent genes that
synergistically activate V(D)J recombination. Science 248:1517–1523. https://doi.org/10.
1126/science.2360047
30. Bassing CH, Alt FW, Hughes MM et al (2000) Recombination signal sequences restrict
chromosomal V(D)J recombination beyond the 12/23 rule. Nature 405:583–586. https://doi.
org/10.1038/35014635
31. Mansilla-Soto J, Cortes P (2003) VDJ recombination: artemis and its in vivo role in hairpin
opening. J Exp Med 197:543–547. https://doi.org/10.1084/jem.20022210
32. Mak TW, Saunders ME (2006) The immunoglobulin genes. In: The immune response.
Elsevier, Amsterdam, pp 179–208
33. Motea EA, Berdis AJ (2010) Terminal deoxynucleotidyl transferase: the story of a misguided
DNA polymerase. Biochim Biophys Acta – Proteins Proteomics 1804:1151–1166. https://doi.
org/10.1016/j.bbapap.2009.06.030
34. Malu S, Malshetty V, Francis D, Cortes P (2012) Role of non-homologous end joining in
V(D)J recombination. Immunol Res 54:233–246. https://doi.org/10.1007/s12026-012-8329-z
35. Helmink BA, Sleckman BP (2012) The response to and repair of RAG-mediated DNA double-
strand breaks. Annu Rev Immunol 30:175–202. https://doi.org/10.1146/annurev-immunol-
030409-101320
36. Chu H (2013) VDJ recombination. Cell 94:411–414. https://doi.org/10.1016/S0092-8674(00)
81580-9
37. Elgert KD (2009) Immunology: understanding the immune system, 2nd edn. Wiley-Blacwell,
New Jersey
38. Song H, Nie X, Basu S, Cerny J (1998) Antibody feedback and somatic mutation in B cells:
regulation of mutation by immune complexes with IgG antibody. Immunol Rev 162:211–218.
https://doi.org/10.1111/j.1600-065X.1998.tb01443.x
39. Di Noia JM, Neuberger MS (2007) Molecular mechanisms of antibody somatic
hypermutation. Annu Rev Biochem 76:1–22. https://doi.org/10.1146/annurev.biochem.76.
061705.090740
40. Rajewsky K, Forster I, Cumano A (1987) Evolutionary and somatic selection of the antibody
repertoire in the mouse. Science 238:1088–1094. https://doi.org/10.1126/science.3317826
41. Manis JP, Tian M, Alt FW (2002) Mechanism and control of class-switch recombination.
Trends Immunol 23:31–39. https://doi.org/10.1016/S1471-4906(01)02111-1
42. Stavnezer J, Amemiya CT (2004) Evolution of isotype switching. Semin Immunol
16:257–275. https://doi.org/10.1016/j.smim.2004.08.005
43. De Silva NS, Klein U (2015) Dynamics of B cells in germinal centres. Nat Rev Immunol
15:137–148. https://doi.org/10.1038/nri3804
44. Muramatsu M, Kinoshita K, Fagarasan S et al (2000) Class switch recombination and
hypermutation require activation-induced cytidine deaminase (AID), a potential RNA editing
enzyme. Cell 102:553–563. https://doi.org/10.1016/S0092-8674(00)00078-7
45. Larson ED, Maizels N (2004) Transcription-coupled mutagenesis by the DNA deaminase
AID. Genome Biol 5:211. https://doi.org/10.1186/gb-2004-5-3-211
46. Pham P, Bransteitter R, Petruska J, Goodman MF (2003) Processive AID-catalysed cytosine
deamination on single-stranded DNA simulates somatic hypermutation. Nature 424:103–107.
https://doi.org/10.1038/nature01760
47. Lin GG, Scott JG (2012) Germinal center organization and cellular dynamics. Immunity
100:130–134. https://doi.org/10.1016/j.pestbp.2011.02.012.Investigations
48. Lipman NS, Jackson LR, Trudel LJ, Weis-Garcia F (2005) Monoclonal versus polyclonal
antibodies: distinguishing characteristics, applications, and information resources. ILAR J
46:258–268. https://doi.org/10.1093/ilar.46.3.258
49. Payne WJ, Marshall DL, Shockley RK, Martin WJ (1988) Clinical laboratory applications of
monoclonal antibodies. Clin Microbiol Rev 1:313–329. https://doi.org/10.1128/cmr.1.3.313
50. Barbet J, Bardiès M, Bourgeois M et al (2012) In: Chames P (ed) Radiolabeled antibodies for
cancer imaging and therapy BT – antibody engineering: methods and protocols, 2nd edn.
Humana Press, Totowa, pp 681–697
51. Breedveld FC (2000) Therapeutic monoclonal antibodies. Lancet 355:735–740. https://doi.
org/10.1016/S0140-6736(00)01034-5
52. Chames P, Van Regenmortel M, Weiss E, Baty D (2009) Therapeutic antibodies: successes,
limitations and hopes for the future. Br J Pharmacol 157:220–233. https://doi.org/10.1111/j.
1476-5381.2009.00190.x
53. Winter G, Milstein C (1991) Man-made antibodies. Nature 349:293–299. https://doi.org/10.
1038/349293a0
54. Khazaeli MB, Conry RM, LoBuglio AF (1994) Human immune response to monoclonal
antibodies. J Immunother Emphasis Tumor Immunol 15:42–52
55. Almagro JC, Fransson J (2008) Humanization of antibodies. Front Biosci 13:1619–1633
56. Pavlou AK, Belsey MJ (2005) The therapeutic antibodies market to 2008. Eur J Pharm
Biopharm 59:389–396. https://doi.org/10.1016/j.ejpb.2004.11.007
57. Schwarz EM, Ritchlin CT (2007) Clinical development of anti-RANKL therapy. Arthritis Res
Ther 9:S7. https://doi.org/10.1186/ar2171
58. Teeling JL, French RR, Cragg MS et al (2004) Characterization of new human CD20
monoclonal antibodies with potent cytolytic activity against non-Hodgkin lymphomas char-
acterization of new human CD20 monoclonal antibodies with potent cytolytic activity against
non-Hodgkin lymphomas. Blood 104:1793–1800. https://doi.org/10.1182/blood-2004-01-
0039
59. Murphy AJ, Macdonald LE, Stevens S et al (2014) Mice with megabase humanization of their
immunoglobulin genes generate antibodies as efficiently as normal mice. Proc Natl Acad Sci
111:5153–5158. https://doi.org/10.1073/pnas.1324022111
60. Kennedy PJ, Oliveira C, Granja PL, Sarmento B (2017) Antibodies and associates: partners in
targeted drug delivery. Pharmacol Ther 177:129–145. https://doi.org/10.1016/j.pharmthera.
2017.03.004
61. Holliger P, Hudson PJ (2005) Engineered antibody fragments and the rise of single domains.
Nat Biotechnol 23:1126–1136. https://doi.org/10.1038/nbt1142
62. Weir ANC, Nesbitt A, Chapman AP et al (2002) Formatting antibody fragments to mediate
specific therapeutic functions. Biochem Soc Trans 30:512–516
63. Fischer N, Léger O (2007) Bispecific antibodies: molecules that enable novel therapeutic
strategies. Pathobiology 74:3–14. https://doi.org/10.1159/000101046
64. Cuesta AM, Sánchez-Martín D, Sanz L et al (2009) In vivo tumor targeting and imaging with
engineered trivalent antibody fragments containing collagen-derived sequences. PLoS One 4:
e5381. https://doi.org/10.1371/journal.pone.0005381
65. Oliveira SS, Aires da Silva F, Lourenco S et al (2012) Assessing combinatorial strategies to
multimerize libraries of single-domain antibodies. Biotechnol Appl Biochem 59:193–204.
https://doi.org/10.1002/bab.1011
66. Thie H, Binius S, Schirrmann T et al (2009) Multimerization domains for antibody phage
display and antibody production. N Biotechnol 26:314–321. https://doi.org/10.1016/j.nbt.
2009.07.005
67. Lambert JM (2013) Drug-conjugated antibodies for the treatment of cancer. Br J Clin
Pharmacol 76:248–262. https://doi.org/10.1111/bcp.12044
68. Casi G, Neri D (2012) Antibody–drug conjugates: basic concepts, examples and future
perspectives. J Control Release 161:422–428. https://doi.org/10.1016/J.JCONREL.2012.01.
026
69. Chudasama V, Maruani A, Caddick S (2016) Recent advances in the construction of antibody–
drug conjugates. Nat Chem 8:114–119. https://doi.org/10.1038/nchem.2415
70. Abdollahpour-Alitappeh M, Lotfinia M, Gharibi T et al (2019) Antibody–drug conjugates
(ADCs) for cancer therapy: strategies, challenges, and successes. J Cell Physiol
234:5628–5642. https://doi.org/10.1002/jcp.27419
71. Arruebo M, Valladares M, González-Fernández Á (2009) Antibody-conjugated nanoparticles
for biomedical applications. J Nanomater 2009:1–24. https://doi.org/10.1155/2009/439389
72. Bertrand N, Wu J, Xu X et al (2014) Cancer nanotechnology: the impact of passive and active
targeting in the era of modern cancer biology. Adv Drug Deliv Rev 66:2–25. https://doi.org/
10.1016/J.ADDR.2013.11.009
73. Krishnamurthy A, Jimeno A (2017) Bispecific antibodies for cancer therapy: a review.
Pharmacol Ther. https://doi.org/10.1016/j.pharmthera.2017.12.002
74. Nagorsen D, Bargou R, Rüttinger D et al (2009) Immunotherapy of lymphoma and leukemia
with T-cell engaging BiTE antibody blinatumomab. Leuk Lymphoma 50:886–891. https://doi.
org/10.1080/10428190902943077
75. Baeuerle PA, Kufer P, Bargou R (2009) BiTE: teaching antibodies to engage T-cells for cancer
therapy. Curr Opin Mol Ther 11:22–30
76. Gross G, Eshhar Z (2016) Therapeutic potential of T cell chimeric antigen receptors (CARs) in
cancer treatment: counteracting off-tumor toxicities for safe CAR T cell therapy. Annu Rev
Pharmacol Toxicol 56:59–83. https://doi.org/10.1146/annurev-pharmtox-010814-124844
77. Pule M, Finney H, Lawson A (2003) Artificial T-cell receptor. Cytotherapy 5:211–226. https://
doi.org/10.1080/14653240310001488
78. American Association for Cancer (2017) First-ever CAR T-cell therapy approved in
U.S. Cancer Discov 7. https://doi.org/10.1158/2159-8290.CD-NB2017-126
79. Grand View Research (2016) Monoclonal antibodies (mAbs) market size worth $138.6 billion
by 2024
80. Elgundi Z, Reslan M, Cruz E et al (2016) The state-of-play and future of antibody therapeutics.
Adv Drug Deliv Rev. https://doi.org/10.1016/j.addr.2016.11.004
81. Bunnak P, Allmendinger R, Ramasamy SV et al (2016) Life-cycle and cost of goods
assessment of fed-batch and perfusion-based manufacturing processes for mAbs. Biotechnol
Prog 32:1324–1335. https://doi.org/10.1002/btpr.2323
82. Nieri P, Donadio D, Rossi S et al (2009) Antibodies for therapeutic uses and the evolution of
biotechniques. Curr Med Chem 16:753–779. https://doi.org/10.2174/092986709787458380
83. Orlandi R, Gussow DH, Jones PT, Winter G (1989) Cloning immunoglobulin variable
domains for expression by the polymerase chain reaction. Proc Natl Acad Sci
86:3833–3837. https://doi.org/10.1073/pnas.86.10.3833
84. Gussow D, Ward ES, Griffiths AD et al (1989) Generating binding activities from Escherichia
coli by expression of a repertoire of immunoglobulin variable domains. Cold Spring Harb
Symp Quant Biol 54:265–272. https://doi.org/10.1101/SQB.1989.054.01.033
85. McCafferty J, Griffiths AD, Winter G, Chiswell DJ (1990) Phage antibodies: filamentous
phage displaying antibody variable domains. Nature 348:552–554. https://doi.org/10.1038/
348552a0
86. Hoogenboom HR (2005) Selecting and screening recombinant antibody libraries. Nat
Biotechnol 23:1105–1116. https://doi.org/10.1038/nbt1126
87. Ponsel D, Neugebauer J, Ladetzki-Baehs K, Tissot K (2011) High affinity, developability and
functional size: the holy grail of combinatorial antibody library generation. Molecules
16:3675–3700. https://doi.org/10.3390/molecules16053675
88. Adams JJ, Nelson B, Sidhu SS (2014) Recombinant genetic libraries and human monoclonal
antibodies. Methods Mol Biol 1060:149–170. https://doi.org/10.1007/978-1-62703-586-6_9
89. Aires da Silva F, Corte-Real S, Goncalves J (2008) Recombinant antibodies as therapeutic
agents. BioDrugs 22:301–314. https://doi.org/10.2165/00063030-200822050-00003
90. Hust M, Frenzel A, Meyer T et al (2012) Construction of human naive antibody gene libraries.
In: Gene function analysis, methods in molecular biology. Humana Press, Totowa, pp 85–107
91. Vaughan TJ, Williams AJ, Pritchard K et al (1996) Human antibodies with sub-nanomolar
affinities isolated from a large non-immunized phage display library. Nat Biotechnol
14:309–314. https://doi.org/10.1038/nbt0396-309
92. Soon Lim T, Khim Chan S (2016) Immune antibody libraries: manipulating the diverse
immune repertoire for antibody discovery. Curr Pharm Des 22:6480–6489. https://doi.org/
10.2174/1381612822666160923111924
93. Posner B, Lee I, Itoh T et al (1993) A revised strategy for cloning antibody gene fragments in
bacteria. Gene 128:111–117. https://doi.org/10.1016/0378-1119(93)90161-U
94. Nuttall SD (2012) Overview and discovery of IgNARs and generation of VNARs. Methods
Mol Biol 911:27–36. https://doi.org/10.1007/978-1-61779-968-6_3
95. Wesolowski J, Alzogaray V, Reyelt J et al (2009) Single domain antibodies: promising
experimental and therapeutic tools in infection and immunity. Med Microbiol Immunol
198:157–174. https://doi.org/10.1007/s00430-009-0116-7
96. Adams JJ, Sidhu SS (2014) Synthetic antibody technologies. Curr Opin Struct Biol 24:1–9.
https://doi.org/10.1016/j.sbi.2013.11.003
97. Hoogenboom HR, Winter G (1992) By-passing immunisation. J Mol Biol 227:381–388.
https://doi.org/10.1016/0022-2836(92)90894-P
98. Yang HY, Kang KJ, Chung JE, Shim H (2009) Construction of a large synthetic human scFv
library with six diversified CDRs and high functional diversity. Mol Cells 27:225–235. https://
doi.org/10.1007/s10059-009-0028-9
99. Silacci M, Brack S, Schirru G et al (2005) Design, construction, and characterization of a large
synthetic human antibody phage display library. Proteomics 5:2340–2350. https://doi.org/10.
1002/pmic.200401273
100. Cunha-santos C, Figueira TN, Borrego P, Oliveira SS (2016) Development of synthetic light-
chain antibodies as novel and potent HIV fusion inhibitors. AIDS 30(11):1691–1701
101. Tiller T, Schuster I, Deppe D et al (2013) A fully synthetic human Fab antibody library based
on fixed VH/VL framework pairings with favorable biophysical properties. MAbs 5:445–470.
https://doi.org/10.4161/mabs.24218
102. Prassler J, Thiel S, Pracht C et al (2011) HuCAL PLATINUM, a synthetic fab library
optimized for sequence diversity and superior performance in mammalian expression systems.
J Mol Biol 413:261–278. https://doi.org/10.1016/j.jmb.2011.08.012
103. Shim H (2015) Synthetic approach to the generation of antibody diversity. BMB Rep
48:489–494. https://doi.org/10.5483/BMBRep.2015.48.9.120
104. Miersch S, Sidhu SS (2012) Synthetic antibodies: concepts, potential and practical consider-
ations. Methods 57:486–498. https://doi.org/10.1016/j.ymeth.2012.06.012
105. Hoet RM, Cohen EH, Kent RB et al (2005) Generation of high-affinity human antibodies by
combining donor-derived and synthetic complementarity-determining-region diversity. Nat
Biotechnol 23:344–348. https://doi.org/10.1038/nbt1067
106. Soderlind E, Strandberg L, Jirholt P et al (2000) Recombining germline-derived CDR
sequences for creating diverse single-framework antibody libraries. Nat Biotechnol
18:852–856. https://doi.org/10.1038/78458
107. Hartwell L, Hood L, Goldberg M et al (2008) Genetics: from genes to genomes, 4th edn.
McGraw Hill, New York
108. Bradbury ARM, Sidhu S, Dubel S, McCafferty J (2011) Beyond natural antibodies: the power
of in vitro display technologies. Nat Biotechnol 29:245–254. https://doi.org/10.1038/nbt.1791
109. Barbas CF, Kang AS, Lerner RA, Benkovic SJ (1991) Assembly of combinatorial antibody
libraries on phage surfaces: the gene III site. Proc Natl Acad Sci 88:7978–7982. https://doi.org/
10.1073/pnas.88.18.7978
110. Smith G (1985) Filamentous fusion phage: novel expression vectors that display cloned
antigens on the virion surface. Science 228:1315–1317. https://doi.org/10.1126/science.
4001944
111. Huang JX, Bishop-Hurley SL, Cooper MA (2012) Development of anti-infectives using phage
display: biological agents against bacteria, viruses, and parasites. Antimicrob Agents
Chemother 56:4569–4582. https://doi.org/10.1128/AAC.00567-12
112. Barbas CF (1995) Synthetic human antibodies. Nat Med 1:837–839. https://doi.org/10.1038/
340091a0
113. Bain B, Brazil M (2003) Adalimumab. Nat Rev Drug Discov 2:693–694. https://doi.org/10.
1038/nrd1182
114. Vendel MC, Favis M, Snyder WB et al (2012) Secretion from bacterial versus mammalian
cells yields a recombinant scFv with variable folding properties. Arch Biochem Biophys
526:188–193. https://doi.org/10.1016/j.abb.2011.12.018
115. Lipovsek D, Plückthun A (2004) In-vitro protein evolution by ribosome display and mRNA
display. J Immunol Methods 290:51–67. https://doi.org/10.1016/j.jim.2004.04.008
116. Ullman CG, Frigotto L, Cooley RN (2011) In vitro methods for peptide display and their
applications. Brief Funct Genomics 10:125–134. https://doi.org/10.1093/bfgp/elr010
117. Doerner A, Rhiel L, Zielonka S, Kolmar H (2014) Therapeutic antibody engineering by high
efficiency cell screening. FEBS Lett 588:278–287. https://doi.org/10.1016/j.febslet.2013.11.
025
118. Black CB, Duensing TD, Trinkle LS, Dunlay RT (2011) Cell-based screening using high-
throughput flow cytometry. Assay Drug Dev Technol 9:13–20. https://doi.org/10.1089/adt.
2010.0308
119. Boder ET, Wittrup KD (1997) Yeast surface display for screening combinatorial polypeptide
libraries. Nat Biotechnol 15:553–557. https://doi.org/10.1038/nbt0697-553
120. Cherf GM, Cochran JR (2015) Applications of yeast surface display for protein engineering.
Yeast Surf Disp Methods Protoc Appl:155–175. https://doi.org/10.1007/978-1-4939-2748-7_8
121. Koide S, Koide A, Lipovšek D (2012) Target-binding proteins based on the 10th human
fibronectin type III domain (10 Fn3). Methods Enzymol 503:135–156. https://doi.org/10.
1016/B978-0-12-396962-0.00006-9
122. Boder ET, Raeeszadeh-Sarmazdeh M, Price JV (2012) Engineering antibodies by yeast
display. Arch Biochem Biophys 526:99–106. https://doi.org/10.1016/j.abb.2012.03.009
123. Zhou C, Jacobsen FW, Cai L et al (2010) Development of a novel mammalian cell surface
antibody display platform. MAbs 2:508–518. https://doi.org/10.4161/mabs.2.5.12970
124. Mitchell Ho IP (2009) Mammalian cell display for antibody. Engineering 525:1–15. https://
doi.org/10.1007/978-1-59745-554-1
125. Zhang H, Wilson IA, Lerner RA (2012) Selection of antibodies that regulate phenotype from
intracellular combinatorial antibody libraries. Proc Natl Acad Sci U S A 109:15728–15733.
https://doi.org/10.1073/pnas.1214275109
126. Beerli RR, Bauer M, Buser RB et al (2008) Isolation of human monoclonal antibodies by
mammalian cell display. Proc Natl Acad Sci U S A 105:14336–14341. https://doi.org/10.
1073/pnas.0805942105
127. Walker JM (2012) Antibody engineering methods and protocols, 2nd edn. Humana Press,
Totowa
128. Lu WC, Ellington AD (2013) In vitro selection of proteins via emulsion compartments.
Methods 60:75–80. https://doi.org/10.1016/j.ymeth.2012.03.008
129. Tawfik DS, Griffiths AD (1998) Man-made cell-like compartments for molecular evolution.
Nat Biotechnol 16:652–656. https://doi.org/10.1038/nbt0798-652
130. Kennedy PJ, Oliveira C, Granja PL, Sarmento B (2017) Monoclonal antibodies: technologies
for early discovery and engineering. Crit Rev Biotechnol 0:1–15. https://doi.org/10.1080/
07388551.2017.1357002
131. Lutz S (2011) Beyond directed evolution – semi-rational protein engineering and design. Curr
Opin Biotechnol 21:734–743. https://doi.org/10.1016/j.copbio.2010.08.011.Beyond
132. Boder ET, Midelfort KS, Wittrup KD (2000) Directed evolution of antibody fragments with
monovalent femtomolar antigen-binding affinity. Proc Natl Acad Sci 97:10701–10705. https://
doi.org/10.1073/pnas.170297297
133. Packer MS, Liu DR (2015) Methods for the directed evolution of proteins. Nat Rev Genet
16:379–394. https://doi.org/10.1038/nrg3927
134. Thie H, Voedisch B, Dübel S et al (2009) Affinity maturation by phage display. Methods Mol
Biol 525:309–322. https://doi.org/10.1007/978-1-59745-554-1_16
135. Cadwell RC, Joyce GF (1994) Mutagenic PCR. PCR Methods Appl 3:S136–S140
136. Coia G, Hudson PJ, Irving RA (2001) Protein affinity maturation in vivo using E. coli mutator
cells. J Immunol Methods 251:187–193. https://doi.org/10.1016/S0022-1759(01)00300-3
137. Marks JD, Griffiths AD, Malmqvist M et al (1992) By-passing immunization: building high
affinity human antibodies by chain shuffling. Nat Biotechnol 10:779–783. https://doi.org/10.
1038/nbt0792-779
138. Yang W-P, Green K, Pinz-Sweeney S et al (1995) CDR walking mutagenesis for the affinity
maturation of a potent human anti-HIV-1 antibody into the picomolar range. J Mol Biol
254:392–403. https://doi.org/10.1006/jmbi.1995.0626
139. Wang HH, Isaacs FJ, Carr PA et al (2009) Programming cells by multiplex genome engineer-
ing and accelerated evolution. Nature 460:894–898. https://doi.org/10.1038/nature08187
140. Hartl DL (2015) What can we learn from fitness landscapes? Curr Opin Microbiol:213–223.
https://doi.org/10.1007/978-1-62703-673-3
141. Leemhuis H, Stein V, Griffiths AD, Hollfelder F (2005) New genotype-phenotype linkages for
directed evolution of functional proteins. Curr Opin Struct Biol 15:472–478. https://doi.org/
10.1016/j.sbi.2005.07.006
142. Arnold F, Georgiou G (2003) Directed evolution library creation. Humana Press, Totowa
143. Smith SA, Crowe JE (2015) Use of human hybridoma technology to isolate human monoclo-
nal antibodies. Microbiol Spectr 3:1–12. https://doi.org/10.1128/microbiolspec.AID
144. Chan CEZ, Lim APC, MacAry PA, Hanson BJ (2014) The role of phage display in therapeutic
antibody discovery. Int Immunol 26:649–657. https://doi.org/10.1093/intimm/dxu082
145. Ho M, Pastan I (2009) Display and selection of scFv antibodies on HEK-293T cells. In:
Antibody phage display: methods and protocols, 2nd edn. Humana Press, Totowa, pp 99–113
DOI: 10.1007/10_2019_105
Published online: 6 August 2019
Cytokines and Growth Factors
A. C. Silva and J. M. Sousa Lobo
Contents
1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 88
2 Cytokines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 89
2.1 Interferons (INFs) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 90
2.2 Interleukins (ILs) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 92
2.3 Tumor Necrosis Factors (TNFs) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 95
3 Growth Factors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 96
3.1 Hematopoietic Growth Factors (HGFs) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 96
3.2 Other Growth Factors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 100
4 Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 103
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 105
Abstract Several cytokines have been used to treat autoimmune diseases, viral
infections, and cancer and to regenerate the skin. In particular, interferons (INFs)
have been used to treat cancer, hepatitis B and C, and multiple sclerosis, while
interleukins (ILs) and tumor necrosis factors (TNFs) have been used in the manage-
ment of different types of cancer. Concerning the hematopoietic growth factors
(HGFs), epoetin has been used for anemia, whereas the colony-stimulating factors
(CSFs) have been used for neutropenia. Other growth factors have been extensively
explored, although most still need to demonstrate in vivo clinical relevance before
reaching the market.
A. C. Silva (*)
UCIBIO/REQUIMTE, MEDTECH, Laboratory of Pharmaceutical Technology, Department of
Drug Sciences, Faculty of Pharmacy, University of Porto, Porto, Portugal
Research Centre), Faculty of Health Sciences, University Fernando Pessoa, Porto, Portugal
e-mail: ana.silva@ff.up.pt; acsilva@ufp.edu.pt
J. M. S. Lobo
UCIBIO/REQUIMTE, MEDTECH, Laboratory of Pharmaceutical Technology, Department of
88 A. C. Silva and J. M. S. Lobo
This chapter provides an overview on the therapeutic applications of biological

medicines containing recombinant cytokines and growth factors (HGFs and others).
From this review, we concluded that the clinical relevance of recombinant cytokines
has been increasing. Since the 1980s, the European Medicines Agency (EMA)
and/or Food and Drug Administration (FDA) have approved 89 biological medicines
containing recombinant cytokines. Among these, 18 were withdrawn, 24 are
biosimilars, and 18 are orphans.
So far, considerable progress has been made in discovering new cytokines,
additional cytokine functions, and how they interfere with human diseases. Future
prospects include the approval of more biological and biosimilar medicines for
different therapeutic applications.
Graphical Abstract
Keywords Cytokines, Growth factors, Hematopoietic growth factors, Interferons,

Interleukins, Tumor necrosis factors
1 Introduction
Biopharmaceuticals are generally recombinant therapeutic proteins obtained by

biotechnological methods, such as genetic engineering techniques (e.g., recombinant
DNA technology) that use biological systems (e.g., microorganisms, cells, plants, or
animals) to produce proteins similar to those that occur naturally in the body.
Sometimes biopharmaceuticals are called biologicals or biological products,
although this is a broader concept that includes pharmaceutical substances produced
or extracted from living sources. Examples of biological products are vaccines,
blood-derived products, allergenics, somatic cells, gene therapy, and recombinant
therapeutic proteins [1–3].
Cytokines and Growth Factors 89
The clinical use of recombinant proteins requires the manufacture of the

corresponding biological medicine, which is defined by the European Medicines
Agency (EMA) as a medicine containing an active substance produced by a living
organism [4]. In contrast, the Food and Drug Administration (FDA) includes
biological medicines in biological products that may contain sugars, proteins,
nucleic acids, or combinations of these molecules, living cells, or tissues and are
obtained from natural sources or produced by biotechnology techniques or other
innovative technologies [3].
Biological medicines have been used to treat medical conditions that do not have
other treatments or when they are the best therapeutic option for the treatment of
some diseases. Examples of these conditions include autoimmune, cardiovascular,
metabolic, dermatological, neurological diseases, cancer, and eye and respiratory
disorders [5]. Furthermore, several biological medicines have been granted with the
orphan designation, being used for the treatment of rare life-threatening or chroni-
cally debilitating conditions [6].
The advent of biological medicines brought a new era for therapeutics, being the
first biological medicine approved in the 1980s (recombinant human insulin).
To date, the expiration of some patents led to the development of biosimilar
medicines, which are medicines with the same quality, safety, and effectiveness of
an approved biological medicine, the reference medicine [7, 8].
Biopharmaceuticals can be divided into different classes, including monoclonal
antibodies, cytokines, growth factors, hormones, blood products, enzymes, vaccines,
cells and tissues, and products of gene therapy. In this chapter, an overview is given
on the therapeutic applications of biological medicines containing cytokines and
growth factors (hematopoietic growth factors and others).
2 Cytokines
Cytokines display a crucial role in the mediation of immune response, controlling

effector cells activity on the target cells. Immune regulation, disease pathogenesis,
and management of immune-mediated diseases are among the activities of cytokines
[9, 10]. These molecules are generally proteins or glycoproteins that are secreted by
cells in small quantities and bind to other cell type’s specific receptors, triggering and
modelling the immune responses (Fig. 1). Leukocytes and other cells that interact
with hematopoietic cells produce most cytokines. These mediators are essential to
regulate immune and inflammatory responses, hematopoiesis, and wound healing.
Thereby, the administration of cytokines improves immune responses against some
viral infections and cancers and manages skin regeneration [2, 10]. Most of these
molecules are hydrophilic, being the formulation of the respective medicines chal-
lenging. Notwithstanding, they have high affinity to the binding receptors, usually
are administered in small doses and show a narrow therapeutic index. So far, several
cytokines have been identified, and some are used in clinics to treat cancer, autoim-
mune diseases, and viral infections, for example, the colony-stimulating factors to
Fig. 1 Schematic representation of the general activity of cytokines
the treatment of neutropenia; the interferons to the treatment of viral infections,

neurodegenerative disorders, and cancer; and the interleukins and tumor necrosis
factor to the management of different cancers [11, 12]. In contrast, the
overexpression of cytokines can induce diseases. Accordingly, cytokine antagonists
(e.g., cytokine-binding receptors, such as monoclonal antibodies) have been used in
clinic [10], but these products are out of the scope of this chapter.
Table 1 shows examples of the different clinical applications of interferons
(INFs), interleukins (ILs), and tumor necrosis factors (TNFs) and corresponding
biological medicines.
2.1 Interferons (INFs)
Different cell types, showing diverse biological effects, including cellular antiviral
states, regulation of immunological and inflammatory responses, cell growth pro-
cesses, and apoptosis, produce INFs. Regarding these activities, INFs have been
used in clinical practice to promote immune responses against infections and to treat
autoimmune disorders and different types of cancer [2, 15].
INF-α, INF-β, and INF-γ comprise the three human classes of INFs, where INF-α
and INF-β have similar amino acid sequences and bind to the same cell receptors,
originating identical biological activities, particularly, against viral infections and
antiproliferation of tumor cells. In contrast, INF-γ has a distinct amino acid sequence
and binds to different cell receptors, having an activity related to anti-inflammatory
and immunological responses. Owing to their different biological effects, INF-α and
INF-β are also named type I interferons, and INF-γ is classified as type II interferon.
Type I has 12 subtypes or isoforms of INF-α and 1 subtype of INF-β, while type II is
only the INF-γ. Current INFs used in clinical practice are recombinant, being
typically produced in E. coli, since they do not require posttranslational modifica-
tions to display the biological activity, although some INF-β have been produced in
CHO (Chinese hamster ovary) cells [2, 15].
Table 1 Examples of interferons (INFs), interleukins (ILs), and tumor necrosis factors (TNFs),
therapeutic indications, and marketed biological medicines
Recombinant
cytokine Therapeutic indications Marketed medicines References
INF-α Various cancers; chronic hepatitis B Intron A®/Alfatronol®, [2, 11–23]
and C Virtron®/Viraferon® a,
Roferon A®, Infergen® a,
Rebetron®, Pegasys®,
PegIntron®, Sylatron®,
Alferon N®
INF-β Multiple sclerosis Betaferon®, Betaseron®, [2, 11–15,
Avonex®, Rebif®, 24–28]
Plegridy®, Extavia®
INF-γ Chronic granulomatous disease; Actimmune® [2, 11, 14,
osteopetrosis 15, 29]
IL-1 receptor Rheumatoid arthritis; Schnitzler’s Kineret® [2, 15, 30–
antagonist syndrome; Familial Mediterranean 32]
(anakinra) fever; mevalonate kinase deficiency;
TNF receptor-associated periodic
syndrome
IL-2 Renal carcinoma; melanoma Proleukin® [11, 12,
(aldesleukin) 15, 33]
IL-2 Cutaneous T-cell lymphoma Ontak® a [11, 15,
(denileukin 34]
diftitox)
IL-3 – diph- Acute myeloid leukemia Orphan medicine [35]
theria toxin
IL-7 Progressive multifocal Orphan medicine [36]
leukoencephalopathy
IL-7 – Idiopathic CD4 lymphocytopenia Orphan medicine [37]
antibody
Pegylated Pancreatic cancer Orphan medicine [38]
IL-10
IL-11 Prevention of chemotherapy- Neumega® [2, 11, 15]
(oprelvekin) induced thrombocytopenia; avoid-
ance of platelet transfusions after
chemotherapy
IL-12 Acute radiation syndrome Orphan medicine [39]
TNF-α-1a Adjunct for surgery tumor removal; Beromun® [2, 12, 40]
(tasonermin) palliative care after irresectable soft
tissue sarcoma of the limbs
NGR-human Malignant mesothelioma Orphan medicine [41, 42]
TNF (Zafiride®)a
Hepatocellular carcinoma Orphan medicine [43]
IL interleukin, INF-α interferon alpha, INF-β interferon beta, INF-γ interferon gamma, NGR-human
TNF human TNF coupled to cngrcg peptide, TNF tumor necrosis factor
a
Medicine withdrawn
Concerning therapeutic applications of INFs, INF-α subtypes are the most used.
For example, INF-α-2a (Roferon A®) has been used to treat hairy cell leukemia,
Kaposi’s sarcoma, chronic myelogenous leukemia, cutaneous T-cell lymphoma,
chronic hepatitis B and C, follicular lymphoma, renal cancer, and malignant mela-
noma [20]. INF-α-2b (Intron A®/Alfatronol®) has been indicated to the treatment of
multiple myeloma, chronic myelogenous leukemia, chronic hepatitis B and C,
carcinoid tumor, hairy cell leukemia, follicular lymphoma, malignant melanoma,
condylomata acuminate, and Kaposi’s sarcoma [16]. IFN-α-n3 (Alferon N®) has
been used to treat condylomata acuminate [23]. Regarding other INFs types,
INF-β-1b (Betaferon®, Betaseron®, and Extavia®) and INF-β-1a (Avonex®,
Rebif®, and Plegridy®) have been used to the management of multiple sclerosis
[24–28], while INF-γ-1b (Actimmune®) is indicated to the treatment of chronic
granulomatous disease and osteopetrosis [29]. Furthermore, the use of recombinant
INF-γ has been suggested to improve atopic dermatitis treatment in patients with
predisposition for skin infections [44]. Pegylated INFs (i.e., INFs chemically linked
to polyethylene glycol – PEG) have also been marketed (Pegasys®, PegIntron®, and
Sylatron®) to increase molecules half-lives, improving administration regimens
[2, 11, 15, 18, 21, 22]. Table 1 shows examples of the different clinical applications
of INFs and their corresponding marketed biological medicines.
Depending on the dose regimen, the administration of INFs usually induces
flu-like symptoms, since they promote the production of endogenous INFs that are
also produced during influenza virus infection. These symptoms are mild and can be
relieved with paracetamol. Nonetheless, there are reports of more severe adverse
effects for INF-α and INF-β [2, 15].
To our knowledge, there are no marketed biosimilar medicines with recombinant
INFs, although some are expected in the near future. Recently, Mufarrege et al. used
of a multiplexed gene expression system, based on a human monocytic cell line, to
characterize the bioactivity of recombinant INF-β. The researchers suggested the
application of this system to compare recombinant INF-β medicines (Rebif® and
Betaseron®) with the bioactivity of the respective biosimilar candidates [45].
2.2 Interleukins (ILs)
Interleukins (ILs) are a large group of cytokines comprising several subtypes that are
produced by different cells, such as macrophages, eosinophils, vascular endothelial
cells, fibroblasts, and keratinocytes. The biological activities of ILs are complex and
not totally understood and include the regulation of normal and malignant cells
growth and differentiation and the management of immunological and inflammatory
responses. Similar to INFs, ILs bind to specific receptors on the surface of neigh-
borhood cells, stimulating the production of more ILs. The medical use of ILs is
limited, due to the lack on the knowledge of all biological functions. Thereby, only
IL-1, IL-2, and IL-11 are approved for clinical use [2, 15]. Nonetheless, the thera-
peutic potential of other ILs subtypes has been studied. For example, the use of IL-4,
IL-10, and IL-11 to the treatment of psoriasis and rheumatoid arthritis has been
tested, but the results of clinical trials were not satisfactory, since patients modestly
improved upon administration of the ILs [46]. Steen-Louws et al. proposed the use
of IL-4–10 fusion protein as an alternative to the administration of isolated IL4 and
IL-10, obtaining a synergistic therapeutic effect [47]. Tang et al. performed a clinical
study using a recombinant fusion protein consisting of two IL-22 molecules linked to
an immunoglobulin constant region, which promotes tissue repair and suppresses
bacterial infection. The results showed that the tested recombinant fusion protein
was well tolerated and can be used to treat inflammatory diseases and organ failure,
such as alcoholic hepatitis [48, 49].
The efficacy of ILs as adjuvants for cancer immunotherapy has been demon-
strated in clinical trials. Naing et al. reported promising results from a phase I trial
using pegylated recombinant IL-10 to treat advanced solid tumors, suggesting its
potential for cancer immunotherapy [50, 51]. The use of IL-12 to the treatment of
melanoma and cutaneous T-cell lymphoma was tested, being observed the occur-
rence of antitumor activity and undesired toxicity effects [46]. Currently, the
National Cancer Institute (United Sates) is carrying out a phase I clinical trial to
evaluate the efficacy of a combined therapy with monoclonal antibody
(pembrolizumab) and recombinant IL-12 to treat solid tumors. Herein, the therapeu-
tic interest of using recombinant IL-12 is related with its ability to destroy tumors, by
obstruction of the blood flow to the tumor and stimulation of the white blood cells
that kill tumor cells [52]. Coyne et al. carried out a clinical trial where it was
observed that the use of immune checkpoint inhibitors combined with recombinant
IL-15 (which promote the production of CD8+T-cells, natural killer cells, and
inflammatory cytokines) improved the antitumor immune responses [53]. In other
study, Miller et al. observed the clinical efficacy of subcutaneous recombinant IL-15
to the treatment of refractory solid tumors in cancer patients [54]. Francois et al.
carried out a clinical trial using recombinant IL-7 in oncologic and lymphopenic
patients and observed an increase on the CD4+ and CD8+ lymphocytes levels. From
their findings, these researchers proposed the use of recombinant IL-7 for the
treatment of sepsis [55].
Table 1 shows examples of the different clinical applications of ILs and
corresponding biological medicines. From our research, no commercially available
biosimilars containing recombinant ILs were found.
The two subtypes of IL-1 (IL-1α and IL-1β) bind to the same receptors and induce
similar biological activities. Among these, the main function is the pro-inflammatory
activity that originates the production of inflammatory mediators. Furthermore, it has
been described that IL-1 plays a role in the activation of T-lymphocytes and
hematopoietic cells differentiation and growth. Besides, together with IL-6, IL-1
induces hepatocytes acute-phase proteins. The extension of these effects depends on
the amount of IL-1 produced, being local for low quantities and systemic for high
concentrations. Clinical investigations related with the potential of using IL-1 for
treating several cancers and chemotherapy-induced bone marrow suppression have
been carried out, but the results were not satisfactory, since no significant antitumor
activity and toxic side effects were observed. However, regarding the ability to
mediate acute and chronic inflammation, the modulation of IL-1 levels has been
showing interesting clinical results, in particular using IL-1 receptor antagonists. In
this field, there is one marketed medicine, anakinra (Kineret®), a non-glycosylated
recombinant IL-1 receptor produced in E. coli., which was first approved to treat
rheumatoid arthritis [2, 15]. Later, this medicine was approved to treat periodic
fevers and auto-inflammatory disorders at all ages, being the first-line treatment for
Schnitzler’s syndrome and second-line treatment for Familial Mediterranean fever,
mevalonate kinase deficiency, and TNF receptor-associated periodic syndrome
[30]. Moreover, anakinra is used in combination with tocilizumab to the treatment
of adult-onset Still’s disease refractory to second-line therapy [31]. Thomas et al. are
conducting a clinical trial to assess whether the use of anakinra in combination with
corticosteroids improves outcomes in patients with acute severe ulcerative colitis
[56]. Recently, Zhu et al. performed in vivo experiments where it was observed
that the use of recombinant IL-1 provides a protective role against myocardial
ischemia-reperfusion injury in rats, suggesting its potential for the treatment of this
disorder [57].
IL-2 is the most studied type of ILs, being the first identified T-growth factor and
having a crucial role in immune response activation, tolerance, and memory induc-
tion. IL-2 is produced by T-lymphocytes and stimulates natural killer cells and
antibody production by B-lymphocytes. The clinical relevance of IL-2 is related
with its immunostimulatory activity that promotes body antitumor responses
[2, 15]. Two medicines based in IL-2 have been approved, despite only one is
currently marketed. Aldesleukin (Proleukin®) is a recombinant IL-2 approved to
treat renal carcinoma and melanoma [33]; denileukin diftitox (Ontak®) is a recom-
binant engineered fusion protein composed by diphtheria toxin fragments linked to
IL-2, which targets T cells and was used to the treatment of cutaneous T-cell
lymphoma, being discontinued by FDA in 2014 [11, 15, 34].
The use of high IL-2 concentrations originates cardiovascular, hepatic, and
pulmonary adverse effects, which limits the duration of treatments. Besides, some
adverse effects can be induced indirectly, promoting the synthesis of other cytokines
[2, 58]. Some recent works referred the potential of using IL-2 to the management of
other disorders. For example, Humrich and Riemekasten showed that a low-dose of
IL-2 is well tolerated and influences positively the clinical course of patients with
active systemic lupus erythematosus. Nonetheless, phase II clinical trials are in
progress to confirm these preliminary results [58]. In other study, Zhang et al.
proposed the use of recombinant IL-2 as an adjunctive immunotherapeutic agent
to treat tuberculosis. The results of the performed experiments suggested that the use
of IL-2 increases the proliferation of CD4+ and natural killer cells and improves
sputum culture and smear conversion in patients with pulmonary tuberculosis.
Nonetheless, the real clinical efficacy of this use needs to be demonstrated [59].
When activated by IL-1, bone marrow stromal cells and fibroblasts produce
IL-11. The latter acts as a hematopoietic growth factor (Sect. 3.1), stimulating
thrombopoiesis (i.e., platelet production) and the growth and differentiation of
bone marrow cells. Recombinant IL-11, called oprelvekin (Neumega®), is produced
in E.coli and indicated to the prevention of severe thrombocytopenia and avoidance
of platelet transfusions after chemotherapy. Despite IL-11 is well tolerated, some

adverse effects have been reported, including edema, tachycardia/palpitations, dys-
pnea, and oral moniliasis [2, 11, 15]. Kuo-Ming et al. demonstrated that the use of
pegylated recombinant IL-11 did not originate additional toxicities in primates,
when compared to the recombinant IL-11 alone. Therefore, these researchers pro-
posed the use of pegylated recombinant IL-11 as a safe long-acting treatment,
originating less fluid retention, which is most common adverse effect [60].
EMA approved the market of some recombinant ILs as orphan drugs, for
example, pegylated IL-10 (in 2017) for pancreatic cancer [38]; IL-12 (in 2016) for
acute radiation syndrome [39]; IL-7 (in 2014) for progressive multifocal
leukoencephalopathy [36]; IL-7 fused with antibody (in 2017) for idiopathic CD4
lymphocytopenia [37]; and IL-3 fused with diphtheria toxin (in 2015) for acute
myeloid leukemia [35].
2.3 Tumor Necrosis Factors (TNFs)
Tumor necrosis factors (TNFs) comprise the subtypes α and β that induce similar
biological effects. Among these, the most studied is the TNF-α, also named TNF,
cachectin, macrophage cytotoxic factor, macrophage cytotoxin, or necrosin. This
cytokine is synthetized by activated macrophages, although several cell types can
produce it, for example, natural killer cells, eosinophils, Kupffer cells, glomerular
mesangial cells, fibroblasts, B- and T-lymphocytes, polymorphonuclear leukocytes,
astrocytes, Langerhans cells, and brain microglial cells. The biological effects of
TNFs include immunological activation in response to the presence of microorgan-
isms (e.g., Gram-negative bacteria), regulation of inflammation, necrosis of some
tumor cells types, and mediation of several disorders (e.g., septic shock, cachexia,
and anorexia) [2, 61].
Table 1 shows examples of the different clinical applications of TNFs and
corresponding biological medicines. From our research, no commercially available
biosimilars containing recombinant TNFs were found. The first clinical attractive-
ness of TNF-α was for cancer therapy. Nonetheless, it has been observed that some
tumors are not susceptible to TNF, due to a reduced necrosis activity and occurrence
of adverse effects upon the administration of therapeutic doses. In contrast, some
clinical studies focused in the neutralization of the negative outcomes originated by
the overexpression of TNF. Examples of these adverse effects are induced cachexia
and tumor growth stimulation in cancer patients; tissue necrosis and vascular leakage
in patients with septic shock; inflammation in patients with rheumatoid arthritis; and
diabetes by induction of insulin resistance after pancreatic cells death. The admin-
istration of anti-TNF monoclonal antibodies or anti-TNF receptor reduces the
severity of these effects. Regarding the direct use of TNF in therapy, only one
product (tasonermin – Beromun®) is approved [2]. This medicine contains recom-
binant TNF-α-1a that has been produced in E. coli and is indicated as adjunct for
surgery tumor removal, to prevent or delay amputation, or in a palliative situation,
for irresectable soft tissue sarcoma of the limbs in combination with melphalan
[2, 12, 40]. In 2008, EMA granted the orphan designation to NGR-human TNF
(human TNF coupled to cngrcg peptide) for the treatment of malignant mesotheli-
oma [42], being the respective medicine (Zafiride®) withdrawn in 2017 [41]. Latter,
in 2009, EMA approved NGR-human TNF as an orphan medicine to the treatment of
hepatocellular carcinoma [43].
Li et al. reported the results of a retrospective trial where it was observed that the
intrapleural instillation of recombinant TNF-α controlled the malignant pleural
effusion and minimized invasive intervention in a cohort group of lung cancer
patients. However, more trials are required to define the optimal therapeutic dose
and confirm clinical efficacy [62].
3 Growth Factors
3.1 Hematopoietic Growth Factors (HGFs)
Growth factors are cytokines that stimulate cell proliferation, differentiation, and/or
activation. Among these, hematopoietic growth factors (HGFs), which are glyco-
proteins that regulate the production and maturation of blood cells (i.e., hematopoi-
esis), have been showing high therapeutic potential [2, 63, 64].
HGFs with clinical relevance have been produced by DNA recombinant techniques
and include IL-11 (Sect. 2.2), recombinant erythropoietin or epoetin and darbepoetin
alfa, and the white cells factors: granulocyte colony-stimulating factor (G-CSF) and
granulocyte macrophage colony-stimulating factor (GM-CSF) [2, 63, 65].
Erythropoietin is an uncharacteristic cytokine that acts as an endocrine hormone
and is produced by the kidneys, although the liver synthetizes a small amount. Its
main function is the stimulation and regulation of the production of red blood cells
(i.e., erythropoiesis), by a mechanism that increases the number of cells able to
differentiate in erythrocytes and promotes the migration of mature red blood cells
from the bone marrow to the peripheral circulation. In addition, anemia-related tissue
hypoxia induces erythropoietin production by the kidneys. Besides, erythropoietin
improves body resistance to exercise and well-being and reduces the need of blood
transfusions in anemic patients. This hormone is present in low concentrations in the
urine of anemic patients, being initially isolated from there for clinical use. None-
theless, due to the small amount available by this method, the erythropoietin
currently used in therapy is produced by DNA recombinant technique, in mamma-
lian cells (e.g., CHO), being called epoetin [2, 63].
The white cell factors, granulocyte colony-stimulating factor (G-CSF) and gran-
ulocyte macrophage colony-stimulating factor (GM-CSF), play a major role in the
differentiation of neutrophils from hematopoietic stem cells. G-CSF is a glycopro-
tein synthetized by different cells (bone marrow stromal cells, macrophages, and
fibroblasts) and has a biological activity related to the proliferation of neutrophils
and further activation of mature cells (i.e., gain of specific functions). Moreover,
G-CSF promotes the proliferation and migration of endothelial cells and, when
associated with other HGFs, has a synergic effect on the differentiation of various
hematopoietic cells. In contrast, GM-CSF is a glycosylated polypeptide produced by
several cells (macrophages, T-lymphocytes, fibroblasts, and endothelial cells),
which induces the proliferation of neutrophils, macrophages, eosinophils, erythro-
cytes, and megakaryocytes [2]. G-CSF and GM-CSF have been used to treat
neutropenia. Furthermore, they show therapeutic effect against infections and
some cancers and in the management of bone marrow transplants. The approved
G-CSF and GM-CSF medicines are marketed under several trade names and are
usually produced in E. coli. Recombinant G-CSF include filgrastim, pegfilgrastim
(filgrastim linked to a PEG molecule), and lenograstim, while recombinant GM-CSF
comprise molgramostim and sargramostim [2, 63].
Table 2 shows examples of the different clinical applications of HGFs and
corresponding biological and biosimilar medicines.
FDA approved the first recombinant erythropoietin in 1989 (Epogen®/Procrit® –
epoetin alfa) to treat anemia in patients with chronic renal failure that are unable to
produce this hormone due to the loss of renal function. Later, the clinical use of
epoetin was extended to treat or avoid anemia caused by other disorders, for
example, to improve the production of red blood cells in patients undergoing
myelosuppressive chemotherapy and bone marrow transplantation and those under-
taking therapy for viral infections, such as human immunodeficiency virus (HIV)
and hepatitis C. However, some adverse effects have been associated with the use of
Epogen®/Procrit®, including cardiovascular events, hypertension, seizures, stroke,
thrombosis or thromboembolism, tumor recurrence or progression, and severe
anemia [2, 63, 65, 69]. Later, several subtypes of epoetin were approved for similar
therapeutic indications. For example, epoetin beta (NeoRecormon® and Mircera®)
[70–72] and epoetin theta (Eporatio® and Biopin®) [73, 74] were approved to treat
symptomatic anemia originated by chronic renal failure or post-chemotherapy
nonmyeloid malignancies. Two medicines containing darbepoetin alfa (Aranesp®,
Nespo®) were approved to treat anemia originated by renal failure or
myelosuppressive chemotherapy, although Nespo® was withdrawn in 2009 [78–80].
With the expiration of epoetin patents, some biosimilar medicines have been
developed and more are expected for the next years. Current approved epoetin
biosimilars are based in the reference medicine Epogen®/Procrit® and include alfa
epoetin (Alfa Hexal®, Abseamed®, and Binocrit®) and zeta epoetin (Retacrit® and
Silapo®). Among these, Retacrit® is the unique approved by FDA, being all
approved by EMA. Despite the cost reduction of using biosimilar epoetin, some
clinicians have been showing skepticism regarding its similar therapeutic efficacy
and still prescribe the reference medicine [8, 111].
The most common indications of epoetin biosimilar medicines include the treat-
ment of anemia caused by several disorders, such as chronic renal failure or kidney
problems, chemotherapy treatments (reducing the need of allogenic blood trans-
fusions), and defective production of blood cells. In addition, epoetin biosimilars are
used to regulate normal blood levels before surgery in patients that undergo autol-
ogous blood transfusion and to reduce the need of allogenic blood transfusions in
Table 2 Examples of hematopoietic growth factors (HGFs), therapeutic indications, and marketed
biological and biosimilar medicines
Marketed
Recombinant HGFs Therapeutic indications medicines References
Epoetin alfa Anemia caused by several Epogen®/ [2, 8, 63,
disorders Procrit®, 66–69]
Eprex®/Erypo®,
Epoetin Alfa
Hexal® a,
Abseamed® a,
Binocrit® a
Epoetin beta Anemia originated by chronic NeoRecormon®, [2, 63, 70–
renal failure or post- Mircera® 72]
Epoetin theta chemotherapy nonmyeloid Eporatio®, [73, 74]
malignancies Biopin®
Epoetin zeta Anemia caused by several Silapo® a, [8, 75–77]
disorders Retacrit® a
Darbepoetin alfa Anemia originated by renal fail- Aranesp®, [2, 63, 78–
ure or myelosuppressive Nespo® b 80]
chemotherapy
G-CSF (granulocyte Neutropenia and avoidance of Neupogen®, [2, 63, 81–
colony-stimulating factor) febrile neutropenia in patients Tevagrastim® a, 92]
or filgrastim receiving myelosuppressive che- Zarzio® a,
motherapy, or patients undergo- Ratiograstim® a,
ing myeloablative chemotherapy Grastofil® a,
followed by bone marrow trans- Nivestim® a,
plantation; reduction of severe Nivestym® a,
neutropenia effects in patients Accofil® a,
with congenital, cyclic, or idio- Filgrastim
pathic neutropenia Hexal® a,
Biograstim® a,b,
Filgrastim
Ratiopharm® a,b
G-CSF (granulocyte Amyotrophic lateral sclerosis Orphan [93]
colony-stimulating factor) Spinal cord injury medicine [94]
or filgrastim
G-CSF (granulocyte Neutropenia and avoidance of Neulasta®, [2, 63, 95–
colony-stimulating factor) febrile neutropenia in patients Lonquex®, 108]
or pegfilgrastim receiving myelosuppressive che- Granix®,
motherapy, or patients undergo- Ziextenzo® a,
ing myeloablative chemotherapy Pelgraz® a,
followed by bone marrow trans- Pelmeg® a,
plantation; reduction of severe Udenyca® a,
neutropenia effects in patients Fulphila® a,
with congenital, cyclic, or idio- Ristempa® a,b,
pathic neutropenia; increased Neupopeg® a,b,
drug residence time in the body Efgratin® a,b,
Cavoley® a,b
GM-CSF (granulocyte Acute respiratory distress Orphan [63, 109]
macrophage colony- syndrome medicine
stimulating factor) or
molgramostim
(continued)
Table 2 (continued)
Marketed
Recombinant HGFs Therapeutic indications medicines References
GM-CSF (granulocyte Hematopoietic syndrome of acute Leukine® [2, 63,
macrophage colony- radiation syndrome 110]
stimulating factor) or
sargramostim
a
Biosimilar medicine
b
Medicine withdrawn
patients with moderate anemia that undergo major surgery (e.g., hip or knee surgery)
[66, 75, 76]. Epoetin Alfa Hexal® and Silapo® have also been used in patients with
risk of developing acute myeloid leukemia that show low levels of erythropoietin
[66, 75]. Regarding Retacrit®, it has been used for the same medical indications of
the other epoetin biosimilars, and FDA also approved its use for the treatment of
anemia in HIV patients that have been treated with the antiviral zidovudine [76, 77].
Filgrastim (Neupogen®) was the first recombinant G-CSF approved by FDA, in
1991, to reduce neutropenia and avoid febrile neutropenia in patients receiving
myelosuppressive chemotherapy, except the ones with chronic myeloid leukemia
and myelodysplastic syndrome. This medicine has also been used to reduce neutro-
penia period in patients undergoing myeloablative chemotherapy followed by bone
marrow transplantation, which show high risk of prolonged severe neutropenia.
Furthermore, filgrastim reduces the incidence of severe neutropenia effects (e.g.,
fever‚ infections, and oropharyngeal ulcers) in patients with congenital, cyclic, or
idiopathic neutropenia. Later, in 2015, Neupogen® was indicated to treat patients
acutely exposed to myelosuppressive doses of radiation, designated by hematopoi-
etic syndrome of acute radiation syndrome, promoting the recovery of neutrophils
production by bone marrow cells and improving body immunity. Nonetheless, the
use of filgrastim has been associated with the occurrence of some adverse effects,
such as fever, pain, rash, headache, cough, breath difficulty, and nose bleeding. In
2015, FDA authorized Zarxio® to the same indications of Neupogen® [90, 91]. More
recently, FDA approved sargramostim (Leukine®), which is the recombinant form of
GM-CSF, to increase the survival rate of patients showing hematopoietic syndrome
of acute radiation syndrome [110].
EMA approved the use of pegfilgrastim since 2002, by means of Neulasta®
(pegfilgrastim) and Lonquex® (lipegfilgrastim), to the same therapeutic indications
of filgrastim. The PEG linkage to filgrastim molecule increases the body circulation
time of the drug, avoiding fast elimination and, therefore, reducing the number of
required administrations [106, 107].
Two orphan designations have been attributed to filgrastim: treatment of
amyotrophic lateral sclerosis, protecting nerve cells from damages [93], and reduc-
ing the effects of spinal cord injury, due to the capacity for protecting spinal cord
cells from death [94]. Molgramostim, a recombinant form of GM-CSF, is approved
to treat acute respiratory distress syndrome, due to the ability to repair cells and
eliminate microbes from the lungs, improving the oxygen flow into the blood [109].
To date, there are eight approved filgrastim biosimilars from the reference
Neupogen® [112]: Filgrastim Hexal® [88], Accofil® [87], Nivestim® [86],
Nivestym® [92], Grastofil® [85], Ratiograstim® [84], Zarzio® [83], and
Tevagrastim® [82]. Concerning pegfilgrastim, there are five approved biosimilars
to the same indications of the reference Neulasta® [106]: Fulphila® [95], Udenyca®
[97], Ziextenzo® [99], Pelgraz® [100], and Pelmed® [101]. Furthermore, some
biosimilars have been withdrawn: Biograstim® and Filgrastim Ratiopharm® for
filgrastim [81, 89] and Ristempa®, Neupopeg®, Efgratin®, and Cavoley® for
pegfilgrastim [102–105].
The clinical use of G-CSF has been explored in the management of several
disorders. For example, Affentranger et al. reviewed the clinical trials that investi-
gated the synergic therapeutic effect of using G-CSF combined with erythropoietin
in anemic patients with lower risk of myelodysplastic syndromes and concluded that
an improved efficacy can be achieved [113]. In other revision, Hamel et al. suggested
that the use of G-CSF can accelerate the white blood cell count in patients with
leukopenia related to kidney transplant, despite more data is required to confirm this
evidence [114]. The benefits of using G-CFS in infertile women, after embryo
implantation, seem to play an important role in the clinical outcome of assisted
reproductive technology [115, 116]. Herrmann et al. carried out a pilot study and
observed that the use of G-CSF improved bone regeneration in patients with large
segmental defect, fracture non-unions, or insufficient vascularization [117].
3.2 Other Growth Factors
Apart from the HGFs, other growth factors have been applied for different thera-
peutic indications. For example, the recombinant human epidermal growth factor
(EGF) is used to treat diabetic foot ulcers (Heberprot-P®, Regen-D® 150, and
Easyef®), vascular ulcers and bed sores (Regen-D® 150) [118], and healing burns
and donor site skin grafts (Regen-D® 60) [119]. The EGF mechanism of action is
related to the improvement of keratinocytes proliferation and increase on the tensile
strength of the new skin. Current EGF-based medicines are topical, for cutaneous
administration or intralesional injection [120, 121]. Nonetheless, in 2018, EMA
released a decision on granting a waiver for all EGF approved indications [122]. Sev-
eral researches showed that the topical use of EGF in individualized formulations
(i.e., magistral formulations) is effective in the management of diverse adult skin
lesions, without showing adverse effects. However, more studies are required to
establish clinical protocols for this application [123]. Furthermore, some products
containing EGF for wound healing are currently under clinical trials, being expected
to be marketed soon [124].
The platelet-derived growth factor (PDGF), in particular the isoform BB (PDGF-
BB), is used in the management of chronic wounds, regulating the healing process.
This growth factor is released by activated platelets at the site of the damage and is a
mitogenic and chemoattractant for mesenchymal stem cells, promoting the initiali-
zation of the tissue repair process. It also plays a role in angiogenesis [2, 121]. A
single medicine containing human recombinant PDGF-BB was approved for the
treatment of chronic diabetic ulcers (Regranex®) but was withdrawn by EMA
[125, 126]. FDA approved an injectable containing PDGF-BB (Augment®) for
arthrodesis and ankle hindfoot in patients that need supplemental graft material,
being an alternative to autografts [127]. In this sense, Sun et al. conducted a meta-
analysis to evaluate the efficacy of using recombinant PDGF-BB in comparison to
autologous bone grafts, in ankle and foot fusion patients. From their study, the
authors concluded that the use of this growth factor avoids the problems associated
with the autografts procedures (e.g., pain, scarring, blood loss, and extra surgery
time), although more studies are required to confirm these evidences
[128]. Human recombinant PDGF-BB is used in dental therapy to treat periodontal
defects, by means of an osteoconductive matrix enriched with this growth factor
(GEM 21S®) [129].
Similar to PDGF-BB, the fibroblast growth factor 2 (FGF-2) or basic FGF (bFGF)
shows ability to improve wound healing, stimulating the proliferation of epithelial
and mesenchymal cells and promoting neovascularization. Furthermore, the use of
bFGF in the recovery of skin burns is also effective. Fiblast® spray (trafermin) is the
first marketed product containing bFGF, which is indicated to the treatment of skin
ulcers, including leg and burn ulcers. Latter, this medicine was approved for dental
therapy to promote the regeneration of periodontal and bone tissues
[121, 130]. EMA attributed the orphan designation to the fibroblast growth factor
19 (FGF-19) for the management of primary biliary cirrhosis, primary sclerosing
cholangitis and avoidance of liver damages, since this protein decreases the produc-
tion of bile acids [131, 132]. The keratinocyte growth factor (KGF), palifermin or
FGF-7, belongs to the FGF family. This growth factor stimulates the epithelial cells
proliferation, improving tissue formation, and was approved by EMA (Kepivance®)
for the treatment of oral mucositis in patients undergoing chemotherapy and radio-
therapy, being withdrawn in 2016 [133]. Some researchers suggest the potential of
KGF for the management of cutaneous wounds, despite human clinical studies are
required to prove this evidence [121].
The vascular endothelial growth factor (VEGF) has been explored to improve
wound healing, since it plays a major role in the angiogenesis initiation, improving
the proliferation and migration of endothelial cells. Nonetheless, to our knowledge,
there are no available products in the market. In this sense, Hanft et al. carried out
randomized controlled trials to evaluate the efficacy of using human recombinant
VEGF in patients with neuropathic diabetic foot ulcers. These researchers observed
that the topical application of VEGF originated an improved wound healing, reached
in a smaller period, when compared to the placebo [121, 134]. The orphan designa-
tion was granted by EMA to the human recombinant VEFG to treat amyotrophic
lateral sclerosis, due to the ability of this growth factor to stimulate the development
of blood vessels. This medicine is for direct brain administration, promoting the
growth of blood vessels that irrigate nerve cells, increasing their survival [135]. In
contrast, some medicines containing VEGF inhibitors are available and are used to
treat age-related macular degeneration and different cancers [136].
The placental growth factor (PlGF) is an angiogenic protein that belongs to the
VEGF family, which is highly produced during pregnancy and is fundamental to the
growth of placenta blood vessels. Thereby, PlGF is fundamental to normal preg-
nancy and baby’s development and is a useful clinical indicator to predict adverse
outcomes. For example, low levels of PlGF are associated with the occurrence of
preeclampsia [137, 138]. EMA granted the orphan designation to human recombi-
nant PlGF to restore blood PlGF levels, improving preeclampsia symptoms [139].
Transforming growth factors (TGFs) are a family of mitogenic polypeptides that
includes the subtype TGF-α, which has an activity similar to EGF and induces
angiogenesis. Some studies suggested that TGF-α promotes the reepithelialization,
but more experiments are required to confirm this activity [2, 121]. TGF-β is another
subtype that has been showing an important role in the early stages of wound
healing, which has three isoforms (β1, β2, and β3). Isoforms β1 and β2 promote
fibroblast differentiation, contraction, synthesis of the extracellular matrix, and
scarring, while isoform β3 reduces scar formation [121, 140]. So et al. carried out
successful phase I and II clinical trials with human recombinant TGF-β3
(avotermin), where observed a significant scarring reduction [141]. However, this
product failed phase III trials and is not marketed [121].
Insulin-like growth factors (IGFs) family includes the subtypes I and II, which are
structurally similar to proinsulin. Thereby, the administration of IGF decreases the
levels of insulin and glucagon and increases the cellular glucose uptake. Diverse
activities have been attributed to IGFs, regarding their ability to inhibit apoptosis and
regulate cells growth and differentiation [2]. Accordingly, high circulating levels of
IGF-I have been associated with the development and progression of different
cancers [142], including breast [143], prostate [144], and thyroid [145] cancers. In
2005, FDA approved the use of IGF-I (mecasermin – Increlex®) to treat growth
failure in children with severe IGF-I deficiency or with growth hormone insensitivity
syndrome (alterations of the growth hormone gene that unable the use of this
hormone by the body). However, IGF-I should not be used instead of growth
hormone [146]. IGF-I was also approved by FDA to treat amyotrophic lateral
sclerosis [147], and EMA granted the orphan designation to IGF-I for different
therapeutic indications. For example, the recombinant human IGF-I/insulin-like
growth factor-binding protein-3 (IGF-I/IGFBP-3) was approved to treat type A
[148] and B [149] extreme resistance insulin syndrome, inherited extreme insulin
resistance (or Rabson Mendenhall syndrome) [150], primary growth hormone insen-
sitivity syndrome (or Laron syndrome) [151], and leprechaunism [152]. Nonetheless,
all these medicines are withdrawn.
Neurotrophic factors are a large group of molecules that play an important role in
the development and survival of nerve cells [2]. Among these, the nerve growth
factor (NGF) has been the most studied, since it regulates the development and
survival of specific peripheral neurons and basal forebrain cholinergic nuclei. Fur-
thermore, NGF levels are high in several anti-inflammatory and autoimmune disor-
ders (e.g., chronic arthritis, systemic lupus erythematosus, and multiple sclerosis),
and a relation with diabetic pathology was observed. Thereby, NGF constitutes a
multifactorial modulator of neuro, immune, and endocrine systems [153].
Regarding its ability to regulate the growth and survival of retina and cornea cells,
in 2013 EMA granted the orphan designation to NGF to treat retinitis pigmentosa.
This growth factor improves the survival of retina cells, slowing the progression of
the disease and preserving vision [154]. Latter, in 2015, EMA also attributed the
orphan designation to NGF to treat neurotrophic keratitis. The use of NGF improves
the eye normal healing process and repairs the damages to the cornea, which are
typical of this disorder [155]. In 2018, FDA approved the medicine Oxervate®
containing the recombinant human NGF (cenegermin) to treat neurotrophic keratitis
[156]. The use of NGF to improve the recovery of other ocular degenerative diseases
has been suggested. For example, Mesentier-Louro et al. observed that the ocular
application of NGF reduces the retinal ganglion cell degeneration that occurs after
optic nerve crush in adult rats, suggesting the potential of this growth factor to treat
optical neuropathies, such as glaucoma [157]. Other researches have investigated the
use of NGF for different clinical applications. For example, Sacchetti et al. carried
out a phase IIa prospective, open label and multiple-dose clinical trial in 40 patients
with dry eye disease, which received eye drops of recombinant NGF during 28 days.
The results showed that the use of NGF is safe and effective to treat dry eye disease,
although randomized clinical trials are required to confirm these findings [158]. Aloe
et al. reviewed the results of the researches related with the implication of NGF in the
induction and progression of carcinogenesis that remains open to debate [159]. In
contrast, Denk et al. highlighted the clinical relevance of NGF antagonists as
effective analgesic drugs for the treatment of several conditions, including osteoar-
thritis and back pain [160].
Table 3 shows examples of the clinical applications of diverse recombinant
growth factors and corresponding biological medicines.
4 Conclusion
Among the most important activities of cytokines are the triggering of immune
responses against cancer and viral infections and the ability to regenerate the skin. In
this sense, numerous recombinant cytokines have been used in clinical practice. For
example, the INFs to the treatment of several cancers, hepatitis B and C and multiple
sclerosis, and the ILs and TNFs for the management of different cancers. Concerning
the HGFs, epoetin has been used to treat anemia caused by diverse disorders, while
colony-stimulating factors have been used for neutropenia. Regarding other growth
factors, those used for wound management have been extensively explored, although
most still need to demonstrate clinical relevance in vivo before reaching the market.
From this review, we concluded that the clinical relevance of recombinant
cytokines has been increasing. Since the 1980s, EMA and/or FDA have approved
89 biological medicines containing recombinant cytokines (INFs, ILs, TNFs, HGFs,
and other growth factors). Among these, 18 were withdrawn, 24 are biosimilars, and
Table 3 Examples of recombinant growth factors for different therapeutic indications and
marketed biological medicines
Marketed
Recombinant growth factor Therapeutic indications medicines References
EGF (epidermal growth factor) Diabetic foot ulcers Heberprot-P®, [120, 121]
Regen-D®
150, Easyef®
Vascular ulcers and bed Regen-D® 150 [118, 121]
sores
Healing of burns and Regen-D® 60 [119, 121]
donor site skin grafts
bFGF/FGF-2 (basic fibroblast Skin ulcers, regeneration Fiblast® spray [121, 130]
growth factor or fibroblast growth of periodontal and bone
factor 2) or trafermin tissues
FGF-19 (fibroblast growth factor Biliary cirrhosis Orphan [131]
19) medicine
Primary sclerosing Orphan [132]
cholangitis medicine
IGF-I (insulin-like growth factor-I) Growth hormone insensi- Increlex® [2, 161]
or mecasermin tivity syndrome
Amyotrophic lateral Iplex® a [2, 162]
sclerosis
IGF-I/IGFBP-3 (insulin-like Type A and B extreme Orphan [148, 149]
growth factor-I/insulin-like growth resistance insulin medicine
factor-binding protein-3) syndrome
Inherited extreme insulin Orphan [150]
resistance medicine
Primary growth hormone Orphan [151]
insensitivity syndrome medicine
Leprechaunism Orphan [152]
medicine
PGF (placental growth factor) Preeclampsia Orphan [139]
medicine
PDGF-BB (platelet-derived growth Ankle arthrodesis and/or Augment® [127]
factor-isoform BB) hindfoot when supple-
mental graft is needed
Periodontally related GEM 21S® [2, 129]
defects
Chronic diabetic ulcers Regranex® a [2, 125,
126]
NGF (nerve growth factor) or Retinitis pigmentosa Orphan [154]
cenegermin medicine
Neurotrophic keratitis Oxervate® [155, 156]
KGF (keratinocyte growth factor) Oral mucositis Kepivance® a [133]
or palifermin
VEGF (vascular endothelial growth Amyotrophic lateral Orphan [135]
factor) sclerosis medicine
a
Medicine withdrawn
18 are orphans. The withdrawal of the medicines from the market has been requested
by the producers and is related to economic reasons, occurrence of toxicity or
non-efficacy events. Regarding biosimilars, only the HGFs epoetin and filgrastim
were approved. This can be explained by their antiquity over other classes of
recombinant cytokines, which allowed patent expiration. Thus, the approval of
more biosimilars containing recombinant cytokines is expected for the next years.
Orphan designation was granted mainly to non-HGFs, for different clinical uses.
However, HGFs, ILs, and TNFs also have orphan medicines.
So far, considerable progress has been made in discovering new cytokines,
additional cytokine functions, and how they interfere with human diseases. Future
prospects include the approval of more biological and biosimilar medicines for
different therapeutic applications.
Acknowledgments This work was supported by the Applied Molecular Biosciences Unit-
UCIBIO and FP-ENAS, which are financed by national funds from FCT/MCTES (UID/Multi/
04378/2019 and UID/Multi/04546/2019, respectively).
References
1. Chen Y-C, Yeh M-K (2018) Introductory chapter: biopharmaceuticals. In: Chen Y-C,
Yeh M-K (eds) Biopharmaceuticals. IntechOpen, London
2. Walsh G (2013) Pharmaceutical biotechnology: concepts and applications. Wiley, Hoboken
3. FDA (2019) What are “Biologics” questions and answers. https://www.fda.gov/about-fda/
about-center-biologics-evaluation-and-research-cber/what-are-biologics-questions-and-
answers
4. EMA (2019) Biological medicine. https://www.ema.europa.eu/en/glossary/biological-
medicine
5. FDA (2019) Complete list of licensed products and establishments. https://www.fda.gov/
vaccines-blood-biologics/complete-list-licensed-products-and-establishments
6. EMA (2019) Orphan designation: overview. https://www.ema.europa.eu/en/human-regula
tory/overview/orphan-designation-overview
7. EMA (2019) Biosimilar medicines: overview. https://www.ema.europa.eu/en/human-regula
tory/overview/biosimilar-medicines-overview
8. Santos SB, Lobo JMS, Silva AC (2019) Biosimilar medicines used for cancer therapy in
Europe: a review. Drug Discov Today 24(1):293–299
9. Silk AW, Margolin K (2019) Cytokine therapy. Hematol Oncol Clin 33:261–274
10. O’Shea JJ, Gadina M, Siegel RM (2019) 9 – Cytokines and cytokine receptors. In: Rich RR,
Fleisher TA, Shearer WT, Schroeder HW, Frew AJ, Weyand CM (eds) Clinical
immunology5th edn. Elsevier, London, pp 127–55.e1
11. Lipiäinen T, Peltoniemi M, Sarkhel S, Yrjönen T, Vuorela H, Urtti A et al (2015) Formulation
and stability of cytokine therapeutics. J Pharm Sci 104(2):307–326
12. Hutmacher C, Neri D (2018) Antibody-cytokine fusion proteins: biopharmaceuticals with
immunomodulatory properties for cancer therapy. Adv Drug Deliv Rev. https://doi.org/10.
1016/j.addr.2018.09.002
13. Moorkens E, Meuwissen N, Huys I, Declerck P, Vulto AG, Simoens S (2017) The market of
biopharmaceutical medicines: a snapshot of a diverse industrial landscape. Front Pharmacol
8:314
14. Crommelin DJA, Sindelar RD, Meibohm B (2013) Pharmaceutical biotechnology fundamen-
tals and applications. Taylor & Francis, Milton Park
15. Ryff J-C, Bordens RW, Pestka S (2013) Interferons and interleukins. In: Crommelin DJA,
Sindelar RD, Meibohm B (eds) Pharmaceutical biotechnology: fundamentals and applications.
Interferons and interleukins, vol 3 edn. Informa Healthcare, New York
16. EMA (2012) EPAR summary for the public: Intron A interferon alfa 2b. https://www.ema.
europa.eu/en/documents/overview/introna-epar-summary-public_en.pdf
17. EMA (2008) European Public Assessment Assessment (EPAR): viraferon. https://www.ema.
europa.eu/en/documents/overview/viraferon-epar-summary-public_en.pdf
18. EMA (2017) EPAR summary for the public: Pegasys (peginterferon alfa-2a). https://www.
ema.europa.eu/en/documents/overview/pegasys-epar-summary-public_en.pdf
19. EMA (2006) Public statement on infergen: interferon alfacon-1. https://www.ema.europa.eu/
en/documents/public-statement/public-statement-infergen-interferon-alfacon-1-withdrawal-
marketing-authorisation-european-union_en.pdf
20. FDA (2008) ROFERON®-A: Interferon alfa-2a, recombinant
21. EMA (2012) EPAR summary for the public: PegIntron (peginterferon alfa-2b). https://www.
ema.europa.eu/en/documents/overview/pegintron-epar-summary-public_en.pdf
22. Corporation MSD (2015). Sylatron™ (peginterferon alfa-2b). https://www.merck.com/prod
uct/usa/pi_circulars/s/sylatron/sylatron_pi.pdf
23. Biopharma H (2019) Alferon N injection® (Interferon alfa-n3). https://hemispherx.net/
products/
24. EMA (2012) EPAR summary for the public: Betaferon (interferon beta-1b). https://www.ema.
europa.eu/en/documents/overview/betaferon-epar-summary-public_en.pdf
25. EMA (2011) EPAR summary for the public: Avonex (interferon beta-1a). https://www.ema.
europa.eu/en/documents/overview/avonex-epar-summary-public_en.pdf
26. EMA (2014) EPAR summary for the public: rebif (interferon beta-1a). https://www.ema.
europa.eu/en/documents/overview/rebif-epar-summary-public_en.pdf
27. EMA (2014) EPAR summary for the public: Plegridy (peginterferon beta-1a). https://www.
ema.europa.eu/en/documents/overview/plegridy-epar-summary-public_en.pdf
28. EMA (2012) EPAR summary for the public: Extavia (interferon beta-1b). https://www.ema.
europa.eu/en/documents/overview/extavia-epar-summary-public_en.pdf
29. Horizon Pharma (2018) Actimmune® (Interferon gamma-1b). https://www.actimmune.com/
30. National Health Service (NHS) (2018) E. clinical commissioning policy: anakinra to treat
periodic fevers and autoinflammatory diseases (all ages)
31. National Health Service (NHS) (2018) E. clinical commissioning policy: anakinra/tocilizumab
for the treatment of adult-onset Still’s disease refractory to second-line therapy (adults)
32. EMA (2009) Kineret (anakinra): an overview of Kineret and why it is authorised in the
EU. https://www.ema.europa.eu/en/documents/overview/kineret-epar-medicine-overview_
en.pdf
33. EMA (2008) Public summary of positive opinion for orphan designation of human interleukin-
2 (glycosylated tetrasaccharide, glycosylated trisaccharide and nonglycosylated) (inhalation
use) for the treatment of renal cell carcinoma. https://www.ema.europa.eu/en/documents/
orphan-designation/eu/3/06/417-public-summary-positive-opinion-orphan-designation-
human-interleukin-2-glycosylated_en.pdf
34. FDA (2014) Current and resolved drug shortages and discontinuations reported to FDA.
https://www.accessdata.fda.gov/scripts/drugshortages/dsp_ActiveIngredientDetails.cfm?
AI¼Denileukin+Diftitox+%28Ontak%29+Injection&st¼d&tab¼tabs-2
35. EMA (2015) Public summary of opinion on orphan designation: recombinant human
interleukin-3 truncated diphtheria toxin fusion protein for the treatment of acute myeloid
leukaemia
36. Agency EM (2014) Public summary of opinion on orphan designation: recombinant human
interleukin-7 for the treatment of progressive multifocal leukoencephalopathy. https://www.
ema.europa.eu/en/documents/orphan-designation/eu/3/12/1013-public-summary-opinion-
orphan-designation-recombinant-human-interleukin-7-treatment-progressive_en.pdf
interleukin-7 fused to a hybrid crystallisable fragment region of a human antibody for
treatment of idiopathic CD4 lymphocytopenia. https://www.ema.europa.eu/en/documents/
orphan-designation/eu/3/17/1875-public-summary-opinion-orphan-designation-recombinant-
human-interleukin-7-fused-hybrid_en.pdf
38. EMA (2017) Public summary of opinion on orphan designation: pegylated recombinant
human interleukin-10 for the treatment of pancreatic cancer. https://www.ema.europa.eu/en/
documents/orphan-designation/eu/3/16/1804-public-summary-opinion-orphan-designation-
pegylated-recombinant-human-interleukin-10-treatment_en.pdf
interleukin-12 for treatment of acute radiation syndrome. https://www.ema.europa.eu/en/doc
uments/orphan-designation/eu/3/16/1727-public-summary-opinion-orphan-designation-
recombinant-human-interleukin-12-treatment-acute_en.pdf
40. EMA (2017) European public assessment report (EPAR): Beromun. https://www.ema.europa.
eu/en/documents/overview/beromun-epar-summary-public_en.pdf
41. EMA (2017). Withdrawal of the marketing authorisation application for Zafiride (NGR-human
tumour necrosis factor alpha). https://www.ema.europa.eu/en/documents/medicine-qa/ques
tions-answers-withdrawal-marketing-authorisation-application-zafiride-ngr-human-tumour-
necrosis_en.pdf
42. EMA (2008) Public summary of positive opinion for orphan designation of NGR-human
tumour necrosis factor for the treatment of malignant mesothelioma. https://www.ema.europa.
eu/en/documents/orphan-designation/eu/3/08/549-public-summary-positive-opinion-orphan-
designation-ngr-human-tumour-necrosis-factor-treatment_en.pdf
43. EMA (2009) Public summary of positive opinion for orphan designation of NGR-human
tumour necrosis factor for the treatment of hepatocellular carcinoma. https://www.ema.europa.
designation-ngr-human-tumour-necrosis-factor-treatment_en.pdf
44. Brar K, Leung DYM (2016) Recent considerations in the use of recombinant interferon
gamma for biological therapy of atopic dermatitis. Expert Opin Biol Ther 16(4):507–514
45. Mufarrege EF, Haile LA, Etcheverrigaray M, Verthelyi DI (2019) Multiplexed gene expres-
sion as a characterization of bioactivity for interferon beta (IFN-β) biosimilar candidates:
impact of innate immune response modulating impurities (IIRMIs). AAPS J 21(2):26
46. Asadullah K, Sterry W, Trefzer U (2002) Cytokines: interleukin and interferon therapy in
dermatology. Clin Exp Dermatol 27(7):578–584
47. Steen-Louws C, Hartgring SAY, Popov-Celeketic J, Lopes AP, de Smet MBM, Eijkelkamp N
et al (2019) IL4-10 fusion protein: a novel immunoregulatory drug combining activities of
interleukin 4 and interleukin 10. Clin Exp Immunol 195(1):1–9
48. Gao B, Xiang X (2018) Interleukin-22 from bench to bedside: a promising drug for epithelial
repair. Cell Mol Immunol 16:666–667
49. Tang K-Y, Lickliter J, Huang Z-H, Xian Z-S, Chen H-Y, Huang C et al (2019) Safety,
pharmacokinetics, and biomarkers of F-652, a recombinant human interleukin-22 dimer, in
healthy subjects. Cell Mol Immunol 16:473–482
50. Zhang H, Wang Y, Hwang ES, He Y-W (2016) Interleukin-10: an immune-activating cytokine
in cancer immunotherapy. J Clin Oncol 34(29):3576–3578
51. Naing A, Papadopoulos KP, Autio KA, Ott PA, Patel MR, Wong DJ et al (2016) Safety,
antitumor activity, and immune activation of pegylated recombinant human interleukin-10
(AM0010) in patients with advanced solid tumors. J Clin Oncol 34(29):3562–3569
52. Pembrolizumab and Recombinant Interleukin-12 in Treating Patients with Solid Tumors
[Internet] (2019). https://www.cancer.gov/about-cancer/treatment/clinical-trials/search/v?
id¼NCI-2017-00091&r¼1
53. Coyne GHOS, Conlon K, Takebe N, Streicher H, Quinn MF, Bruns A et al (2018) Phase I
study of recombinant interleukin-15 in combination with checkpoint inhibitors nivolumab and
ipilimumab in subjects with refractory cancers. J Clin Oncol 36(15_suppl):TPS3128–
TPS3TPS
54. Miller JS, Morishima C, McNeel DG, Patel MR, Kohrt HEK, Thompson JA et al (2018)
A first-in-human phase I study of subcutaneous outpatient recombinant human IL15 (rhIL15)
in adults with advanced solid tumors. Clin Cancer Res 24(7):1525–1535
55. Francois B, Jeannet R, Daix T, Walton AH, Shotwell MS, Unsinger J et al (2018) Interleukin-7
restores lymphocytes in septic shock: the IRIS-7 randomized clinical trial. JCI Insight 3(5):
e98960
56. Thomas MG, Bayliss C, Bond S, Dowling F, Galea J, Jairath V et al (2019) Trial summary and
protocol for a phase II randomised placebo-controlled double-blinded trial of interleukin
1 blockade in acute severe colitis: the IASO trial. BMJ Open 9(2):e023765
57. Zhu J, Huang J, Dai D, Wang X, Gao J, Han W et al (2019) Recombinant human interleukin-1
receptor antagonist treatment protects rats from myocardial ischemia–reperfusion injury.
Biomed Pharmacother 111:1–5
58. Humrich JY, Riemekasten G (2019) Low-dose interleukin-2 therapy for the treatment of
systemic lupus erythematosus. Curr Opin Rheumatol 31(2):208–212
59. Zhang R, Xi X, Wang C, Pan Y, Ge C, Zhang L et al (2018) Therapeutic effects of
recombinant human interleukin 2 as adjunctive immunotherapy against tuberculosis: a sys-
tematic review and meta-analysis. PLoS One 13(7):e0201025
60. Yu K-M, Lau JY-N, Fok M, Yeung Y-K, Fok S-P, Zhang S et al (2018) Preclinical evaluation
of the mono-PEGylated recombinant human interleukin-11 in cynomolgus monkeys. Toxicol
Appl Pharmacol 342:39–49
61. NCI (2019) NCI dictionary of cancer terms: tumor necrosis factor. https://www.cancer.gov/
publications/dictionaries/cancer-terms/def/tumor-necrosis-factor
62. Li Q, Sun W, Yuan D, Lv T, Yin J, Cao E et al (2016) Efficacy and safety of recombinant
human tumor necrosis factor application for the treatment of malignant pleural effusion caused
by lung cancer. Thorac Cancer 7(1):136–139
63. Foote M (2013) Hematopoietic growth factors. In: Crommelin DJA, Sindelar RD, Meibohm B
(eds) Pharmaceutical biotechnology: fundamentals and applications3rd edn. Informa
Healthcare, London
64. Groopman JE, Molina J-M, Scadden DT (1989) Hematopoietic growth factors. N Engl J Med
321(21):1449–1459
65. Kaushansky K (2006) Lineage-specific hematopoietic growth factors. N Engl J Med 354
(19):2034–2045
66. EMA (2018) Epoetin Alfa Hexal (epoetin alfa): an overview of Epoetin Alfa Hexal and why it
is authorised in the EU. https://www.ema.europa.eu/en/documents/overview/epoetin-alfa-
hexal-epar-summary-public_en-0.pdf
67. EMA (2018) Abseamed (epoetin alfa): an overview of Abseamed and why it is authorised in
the EU
68. EMA (2018) Binocrit (epoetin alfa): an overview of Binocrit and why it is authorised in the
EU. https://www.ema.europa.eu/en/documents/overview/binocrit-epar-medicine-overview_
en.pdf
69. FDA (2017) Information for epogen/procrit (epoetin alfa). https://www.fda.gov/Drugs/
DrugSafety/PostmarketDrugSafetyInformationforPatientsandProviders/ucm541173.htm
70. EMA (2015) EPAR summary for the public: NeoRecormon – epoetin beta. https://www.ema.
europa.eu/en/documents/overview/neorecormon-epar-summary-public_en.pdf
71. EMA (2012) EPAR summary for the public: mircera – methoxy polyethylene glycol-epoetin
beta. https://www.ema.europa.eu/en/documents/overview/mircera-epar-summary-public_en.
pdf
72. FDA (2018) FDA approves Mircera for anemia associated with chronic kidney disease in
pediatric patients on dialysis. https://www.fda.gov/Drugs/InformationOnDrugs/
ApprovedDrugs/ucm610210.htm
73. EMA (2009) EPAR summary for the public: eporatio – epoetin theta. https://www.ema.
europa.eu/en/documents/overview/eporatio-epar-summary-public_en.pdf
74. EMA (2009) EPAR summary for the public: biopoin – epoetin theta. https://www.ema.europa.
eu/en/documents/overview/biopoin-epar-summary-public_en.pdf
75. EMA (2019) Silapo (epoetin zeta): an overview of Silapo and why it is authorised in the
EU. https://www.ema.europa.eu/en/documents/overview/silapo-epar-medicine-overview_en.
pdf
76. EMA (2011) EPAR summary for the public: retacrit – epoetin zeta. https://www.ema.europa.
eu/en/documents/overview/retacrit-epar-summary-public_en.pdf
77. FDA (2018) FDA approves first epoetin alfa biosimilar for the treatment of anemia. https://
www.fda.gov/NewsEvents/Newsroom/PressAnnouncements/ucm607703.htm
78. EMA (2013) EPAR summary for the public: Aranesp – darbepoetin alfa. https://www.ema.
europa.eu/en/documents/overview/aranesp-epar-summary-public_en.pdf
79. EMA (2009) EPAR summary for the public: NESPO – darbepoetin alfa. https://www.ema.
europa.eu/en/documents/overview/nespo-epar-summary-public_en.pdf
80. FDA (2017) Information for Aranesp (darbepoetin alfa). https://www.fda.gov/Drugs/
DrugSafety/PostmarketDrugSafetyInformationforPatientsandProviders/ucm541148.htm
81. EMA (2017) EPAR summary for the public: Biograstim. https://www.ema.europa.eu/en/
documents/overview/biograstim-epar-summary-public_en.pdf
82. EMA (2008) EPAR summary for the public: Tevagrastim. https://www.ema.europa.eu/en/
documents/overview/tevagrastim-epar-summary-public_en.pdf
83. EMA (2014) EPAR summary for the public: Zarzio. https://www.ema.europa.eu/en/docu
ments/overview/zarzio-epar-summary-public_en.pdf
84. EMA (2014) EPAR summary for the public: Ratiograstim. https://www.ema.europa.eu/en/
documents/overview/ratiograstim-epar-summary-public_en.pdf
85. EMA (2018) EPAR summary for the public: Grastofil. https://www.ema.europa.eu/en/docu
ments/overview/grastofil-epar-medicine-overview_en.pdf
86. EMA (2010) EPAR summary for the public: Nivestim. https://www.ema.europa.eu/en/docu
ments/overview/nivestim-epar-summary-public_en.pdf
87. EMA (2014) EPAR summary for the public: Accofil. https://www.ema.europa.eu/en/docu
ments/overview/accofil-epar-summary-public_en.pdf
88. EMA (2014) EPAR summary for the public: Filgrastim Hexal. https://www.ema.europa.eu/en/
documents/overview/filgrastim-hexal-epar-summary-public_en.pdf
89. EMA (2011) EPAR summary for the public: Filgrastim Ratiopharm. https://www.ema.europa.
eu/en/documents/overview/filgrastim-ratiopharm-epar-summary-public_en.pdf
90. FDA (2015) FDA approves Neupogen for treatment of patients with radiation-induced
myelosuppression following a radiological/nuclear incident. https://www.fda.gov/
EmergencyPreparedness/Counterterrorism/MedicalCountermeasures/AboutMCMi/
ucm443245.htm
91. FDA (2015) Prescribing information for Zarxio. https://www.accessdata.fda.gov/drugsatfda_
docs/label/2015/125553lbl.pdf
92. FDA (2018) Prescribing information for Nivestym. https://www.accessdata.fda.gov/
drugsatfda_docs/label/2018/761080s000lbl.pdf
93. EMA (2015) Public summary of opinion on orphan designation: filgrastim for the treatment of
amyotrophic lateral sclerosis. https://www.ema.europa.eu/en/documents/orphan-designation/
eu/3/08/532-public-summary-positive-opinion-orphan-designation-filgrastim-treatment-
amyotrophic-lateral_en.pdf
94. EMA (2015) Public summary of opinion on orphan designation: Filgrastim for the treatment of
spinal cord injury. https://www.ema.europa.eu/en/documents/orphan-designation/eu/3/08/
580-public-summary-positive-opinion-orphan-designation-filgrastim-treatment-spinal-cord-
injury_en.pdf
95. EMA (2018) EPAR summary for the public: Fulphila (pegfilgrastim). https://www.ema.
europa.eu/en/documents/overview/fulphila-epar-medicine-overview_en.pdf
96. FDA (2018) Prescribing information for Fulphila. https://www.accessdata.fda.gov/
97. EMA. EPAR summary for the public: Udenyca (pegfilgrastim). https://www.ema.europa.eu/
en/documents/overview/udenyca-epar-medicine-overview_en.pdf
98. FDA (2018) Prescribing information for UDENYCA. https://www.accessdata.fda.gov/
99. EMA (2018) EPAR summary for the public: Ziextenzo (pegfilgrastim). https://www.ema.
europa.eu/en/documents/overview/ziextenzo-epar-medicine-overview_en.pdf
100. EMA (2018) EPAR summary for the public: Pelgraz (pegfilgrastim). https://www.ema.europa.
eu/en/documents/overview/pelgraz-epar-medicine-overview_en.pdf
101. EMA (2018) EPAR summary for the public: Pelmeg (pegfilgrastim). https://www.ema.europa.
eu/en/documents/overview/pelmeg-epar-medicine-overview_en.pdf
102. EMA (2015) EPAR summary for the public: Ristempa (pegfilgrastim). https://www.ema.
europa.eu/en/documents/overview/ristempa-epar-summary-public_en.pdf
103. EMA (2009) EPAR summary for the public: Neupopeg (pegfilgrastim). https://www.ema.
europa.eu/en/documents/overview/neupopeg-epar-summary-public_en.pdf
104. EMA (2018) Withdrawal of the marketing authorisation application for Efgratin
(pegfilgrastim). https://www.ema.europa.eu/en/documents/medicine-qa/questions-answers-
withdrawal-marketing-authorisation-application-efgratin-pegfilgrastim_en-0.pdf
105. EMA (2018) Withdrawal of the marketing authorisation application for Cavoley
(pegfilgrastim). https://www.ema.europa.eu/en/documents/medicine-qa/questions-answers-
withdrawal-marketing-authorisation-application-cavoley-pegfilgrastim_en-0.pdf
106. EMA (2018) EPAR summary for the public: Neulasta (pegfilgrastim). https://www.ema.
europa.eu/en/documents/overview/neulasta-epar-medicine-overview_en.pdf
107. EMA (2013) EPAR summary for the public: Lonquex (lipegfilgrastim). https://www.ema.
europa.eu/en/documents/overview/lonquex-epar-summary-public_en.pdf
108. FDA (2012) Prescribing information for Granix. https://www.accessdata.fda.gov/drugsatfda_
docs/label/2012/125294s0000lbl.pdf
109. EMA (2016) Public summary of opinion on orphan designation: Molgramostim for the
treatment of acute respiratory distress syndrome. https://www.ema.europa.eu/en/documents/
orphan-designation/eu/3/16/1685-public-summary-opinion-orphan-designation-
molgramostim-treatment-acute-respiratory-distress_en.pdf
110. FDA (2018) https://www.fda.gov/EmergencyPreparedness/Counterterrorism/
MedicalCountermeasures/MCMIssues/ucm602102.htm. https://www.fda.gov/
EmergencyPreparedness/Counterterrorism/MedicalCountermeasures/MCMIssues/
ucm602102.htm
111. Burchiel SW, Aspbury R, Munday J (2019) The search for biosimilars and biobetters. Drug
Discov Today 24:1087–1091
112. Siegel JF, Royzman I (2017) Biosimilar approvals in Europe. https://www.biologicsblog.com/
2017-biosimilar-approvals-in-europe
113. Affentranger L, Bohlius J, Hallal M, Bonadies N (2019) Efficacy of granulocyte colony
stimulating factor in combination with erythropoiesis stimulating agents for treatment of
anemia in patients with lower risk myelodysplastic syndromes: a systematic review. Crit
Rev Oncol Hematol 136:37–47
114. Hamel S, Kuo V, Sawinski D, Johnson D, Bloom RD, Bleicher M et al (2019) Single center,
real-world experience with granulocyte colony-stimulating factor for management of leuko-
penia following kidney transplantation. Clin Transpl 33:e13541
115. Kamath MS, Kirubakaran R, Sunkara SK (2018) Granulocyte-colony stimulating factor
administration for subfertile women undergoing assisted reproduction. Cochrane Database
Syst Rev 12. https://doi.org/10.1002/14651858.CD013226
116. Zhang L, Xu W-H, Fu X-H, Huang Q-X, Guo X-Y, Zhang L et al (2018) Therapeutic role of
granulocyte colony-stimulating factor (G-CSF) for infertile women under in vitro fertilization
and embryo transfer (IVF-ET) treatment: a meta-analysis. Arch Gynecol Obstet 298
(5):861–871
117. Herrmann M, Zeiter S, Eberli U, Hildebrand M, Camenisch K, Menzel U et al (2018) Five

days granulocyte colony-stimulating factor treatment increases bone formation and reduces
gap size of a rat segmental bone defect: a pilot study. Front Bioeng Biotechnol 6:5
118. Biotech B (2019) Patient product information: REGEN-D® 150. https://www.bharatbiotech.
com/images/regend150/REGEN-D150_ppi.pdf
119. Biotech B (2019) Patient product information: REGEN-D® 60. https://www.bharatbiotech.
com/images/regend60/regen-d%2060-ppi.pdf
120. Berlanga J, Férnandez J, López E, López P, del Río A, Valenzuela C et al (2013) Heberprot-P:
a novel product for treating advanced diabetic foot ulcer. MEDICC Rev 15(1):11–15
121. Yamakawa S, Hayashida K (2019) Advances in surgical applications of growth factors for
wound healing. Burns Trauma 7(1):10
122. EMA (2017) EMA decision of 30 January 2018 on the granting of a product specific waiver for
recombinant human epidermal growth factor. https://www.ema.europa.eu/en/documents/pip-
decision/p/0038/2018-ema-decision-30-january-2018-granting-product-specific-waiver-
recombinant-human-epidermal_en.pdf
123. Esquirol-Caussa J, Herrero-Vila E (2019) Human recombinant epidermal growth factor in skin
lesions: 77 cases in EPItelizando project. J Dermatol Treat 30(1):96–101
124. Öhnstedt E, Lofton Tomenius H, Vågesjö E, Phillipson M (2019) The discovery and devel-
opment of topical medicines for wound healing. Expert Opin Drug Discovery 14(5):485–497
125. EMA (2012) EPAR summary for the public: Regranex (becaplermin). https://www.ema.
europa.eu/en/documents/overview/regranex-epar-summary-public_en.pdf
126. EMA (2012) Public statement on Regranex (becaplermin): withdrawal of the marketing
authorisation in the European Union. https://www.ema.europa.eu/en/documents/public-state
ment/public-statement-regranex-withdrawal-marketing-authorisation-european-union_en.pdf
127. FDA (2018) Summary of safety and effectiveness data: Augment® Injectable. https://www.
accessdata.fda.gov/cdrh_docs/pdf10/P100006S005b.pdf
128. Sun H, Lu P-P, Zhou P-H, Sun S-W, Zhang H-T, Liu Y-J et al (2017) Recombinant human
platelet-derived growth factor-BB versus autologous bone graft in foot and ankle fusion: a
systematic review and meta-analysis. Foot Ankle Surg 23(1):32–39
129. Biologics L (2019) GEM 21S®. https://lynchbiologics.com/products/gem-21s/
130. KAKEN Pharmaceutical CO (2019) L. Fiblast® (Recombinant human basic fibroblast growth
factor, rh bFGF). http://www.kaken.co.jp/english/business/rd_pipeline.html
131. EMA (2014) Public summary of opinion on orphan designation: variant of recombinant
human fibroblast growth factor 19 for the treatment of primary biliary cirrhosis. https://
www.ema.europa.eu/en/documents/orphan-designation/eu/3/14/1329-public-summary-opin
ion-orphan-designation-variant-recombinant-human-fibroblast-growth-factor-19_en.pdf
132. Agency EM (2016) Public summary of opinion on orphan designation: Variant of recombinant
human fibroblast growth factor 19 for the treatment of primary sclerosing cholangitis. https://
www.ema.europa.eu/en/documents/orphan-designation/eu/3/15/1584-public-summary-opin
ion-orphan-designation-variant-recombinant-human-fibroblast-growth-factor-19_en.pdf
133. EMA (2016) EPAR summary for the public: Kepivance (palifermin). https://www.ema.
europa.eu/en/documents/overview/kepivance-epar-summary-public_en.pdf
134. Hanft JR, Pollak RA, Barbul A, van Gils C, Kwon PS, Gray SM et al (2008) Phase I trial on the
safety of topical rhVEGF on chronic neuropathic diabetic foot ulcers. J Wound Care 17
(1):30–37
135. EMA (2010) Public summary of opinion on orphan designation: Recombinant human vascular
endothelial growth factor for the treatment of amyotrophic lateral sclerosis. https://www.ema.
europa.eu/en/documents/orphan-designation/eu/3/09/711-public-summary-opinion-orphan-
designation-recombinant-human-vascular-endothelial-growth-factor_en.pdf
136. Yeung AWK, Abdel-Daim MM, Abushouk AI, Kadonosono K (2019) A literature analysis on
anti-vascular endothelial growth factor therapy (anti-VEGF) using a bibliometric approach.
Naunyn Schmiedeberg’s Arch Pharmacol 392(4):393–403
137. Hayes Ryan D, McCarthy FP, O'Donoghue K, Kenny LC (2018) Placental growth factor: a
review of literature and future applications. Pregnancy Hypertens 14:260–264
138. Parchem JG, Brock C, Sibai BM (2019) 442: plasma placental growth factor and the risk of
adverse perinatal outcome. Am J Obstet Gynecol 220(1, Suppl):S298
139. EMA (2018) Public summary of opinion on orphan designation: recombinant human placental
growth factor for the treatment of pre-eclampsia. https://www.ema.europa.eu/en/documents/
orphan-designation/eu/3/18/2040-public-summary-opinion-orphan-designation-recombinant-
human-placental-growth-factor-treatment_en.pdf
140. Shpichka A, Butnaru D, Bezrukov EA, Sukhanov RB, Atala A, Burdukovskii V et al (2019)
Skin tissue regeneration for burn injury. Stem Cell Res Ther 10(1):94
141. So K, McGrouther DA, Bush JA, Durani P, Taylor L, Skotny G et al (2011) Avotermin for scar
improvement following scar revision surgery: a randomized, double-blind, within-patient,
placebo-controlled, phase II clinical trial. Plast Reconstr Surg 128(1):163–172
142. Pollak M (2008) Insulin and insulin-like growth factor signalling in neoplasia. Nat Rev Cancer
8:915
143. Key T, Appleby P, Reeves G, Roddam A, Endogenous Hormones and Breast Cancer Collab-
orative Group (2010) Insulin-like growth factor 1 (IGF1), IGF binding protein 3 (IGFBP3),
and breast cancer risk: pooled individual data analysis of 17 prospective studies. Lancet Oncol
11(6):530–542
144. Travis RC, Appleby PN, Martin RM, Holly JM, Albanes D, Black A et al (2016) A meta-
analysis of individual participant data reveals an association between circulating levels of
IGF-I and prostate cancer risk. Cancer Res 76(8):2288–2300
145. Schmidt JA, Allen NE, Almquist M, Franceschi S, Rinaldi S, Tipper SJ et al (2014) Insulin-
like growth factor-i and risk of differentiated thyroid carcinoma in the European prospective
investigation into cancer and nutrition. Cancer Epidemiol Prev Biomark 23(6):976–985
146. Biopharmaceuticals I (2019) INCRELEX®: full prescribing information. https://www.ipsen.
com/websites/Ipsen_Online/wp-content/uploads/sites/9/2019/01/21153952/Increlex_Full_Pre
scribing_Information1.pdf
147. FDA (2009) Access to Iplex for patients with ALS. https://www.fda.gov/Drugs/
ResourcesForYou/HealthProfessionals/ucm118117.htm
148. EMA (2004) Public summary of opinion on orphan designation: recombinant human insulin-
like growth factor-I/recombinant human insulin-like growth factor binding protein-3 for the
treatment of Type A extreme insulin resistance syndrome. https://www.ema.europa.eu/en/
documents/orphan-designation/eu/3/04/236-public-summary-opinion-orphan-designation-
recombinant-human-insulin-growth-factor-i/recombinant-human-insulin-growth-factor-bind
ing-protein-3-treatment-type-extreme-insulin-resi_en.pdf
like growth factor-I/recombinant human insulin like growth factor binding protein-3 for the
treatment of Type B extreme insulin resistance syndrome. https://www.ema.europa.eu/en/
documents/orphan-designation/eu/3/04/235-public-summary-positive-opinion-orphan-desig
nation-recombinant-human-insulin-growth-factor-i/recombinant-human-insulin-growth-fac
tor-binding-protein-3-treatment-type-b-extreme-insu_en.pdf
treatment of Rabson Mendenhall syndrome. https://www.ema.europa.eu/en/documents/
orphan-designation/eu/3/04/237-public-summary-positive-opinion-orphan-designation-recom
binant-human-insulin-growth-factor-i/recombinant-human-insulin-growth-factor-binding-pro
tein-3-treatment-rabson-mendnhal_en.pdf
like growth factor-I/recombinant human insulin-like growth factor binding protein-3 for the
treatment of primary growth hormone insensitivity syndrome (Laron Syndrome). https://www.
ema.europa.eu/en/documents/orphan-designation/eu/3/03/159-public-summary-positive-opin
ion-orphan-designation-recombinant-human-insulin-growth-factor-i/recombinant-human-insu
lin-growth-factor-binding-protein-3-treatment-primary-growt_en.pdf
treatment of leprechaunism. https://www.ema.europa.eu/en/documents/orphan-designation/
eu/3/04/238-public-summary-positive-opinion-orphan-designation-recombinant-human-insu
lin-growth-factor-i/recombinant-human-insulin-growth-factor-binding-protein-3-treatment-
leprechaunism_en.pdf
153. Skaper SD (2017) Nerve growth factor: a neuroimmune crosstalk mediator for all seasons.
Immunology 151(1):1–15
154. EMA (2015) Public summary of opinion on orphan designation: recombinant human nerve
growth factor for the treatment of retinitis pigmentosa. https://www.ema.europa.eu/en/docu
ments/orphan-designation/eu/3/13/1135-public-summary-opinion-orphan-designation-recom
binant-human-nerve-growth-factor-treatment_en.pdf
155. EMA (2015) Public summary of opinion on orphan designation: recombinant human nerve
growth factor for the treatment of neurotrophic keratitis. https://www.ema.europa.eu/en/docu
ments/orphan-designation/eu/3/15/1586-public-summary-opinion-orphan-designation-recom
binant-human-nerve-growth-factor-treatment_en.pdf
156. FDA (2018) Prescribing information for OXERVATE. https://www.accessdata.fda.gov/
157. Mesentier-Louro LA, Rosso P, Carito V, Mendez-Otero R, Santiago MF, Rama P et al (2019)
Nerve growth factor role on retinal ganglion cell survival and axon regrowth: effects of ocular
administration in experimental model of optic nerve injury. Mol Neurobiol 56(2):1056–1069
158. Sacchetti M, Lambiase A, Schmidl D, Schmetterer L, Ferrari M, Mantelli F et al (2019) Effect
of recombinant human nerve growth factor eye drops in patients with dry eye: a phase IIa, open
label, multiple-dose study. Br J Ophthalmol. https://doi.org/10.1136/bjophthalmol-2018-
312470
159. Aloe L, Rocco ML, Balzamino BO, Micera A (2016) Nerve growth factor: role in growth,
differentiation and controlling cancer cell development. J Exp Clin Cancer Res 35(1):116
160. Denk F, Bennett DL, McMahon SB (2017) Nerve growth factor and pain mechanisms. Annu
Rev Neurosci 40(1):307–325
161. Biopharmaceuticals I (2019) Increlex® (mecasermin). https://www.increlex.com/
162. Insmed (2009) Insmed provides update on supply of IPLEX®. https://web.archive.org/web/
20170902142334/http://investor.insmed.com/releasedetail.cfm?releaseid¼399059
DOI: 10.1007/10_2019_111
Published online: 27 September 2019
Hormones, Blood Products,

and Therapeutic Enzymes
Ana Catarina Silva, Cládia Pina Costa, Hugo Almeida, João Nuno Moreira,
and José Manuel Sousa Lobo
Contents
1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 117
2 Hormones . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 117
2.1 Insulin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 117
2.2 Glucagon . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 122
2.3 Growth Hormone . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 123
2.4 Gonadotropins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 124
2.5 Other Recombinant Hormones . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 127
3 Blood Products . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 128
3.1 Blood Clotting Factors or Coagulation Factors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 128
3.2 Anticoagulants . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 132
3.3 Thrombolytic Agents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 134
4 Therapeutic Enzymes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 134
5 Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 139
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 140
A. C. Silva (*)
UCIBIO, REQUIMTE, MEDTECH, Laboratory of Pharmaceutical Technology, Department of
Research Centre), Faculty of Health Sciences, Fernando Pessoa University, Porto, Portugal
e-mail: ana.silva@ff.up.pt
C. P. Costa, H. Almeida, and J. M. Sousa Lobo
UCIBIO, REQUIMTE, MEDTECH, Laboratory of Pharmaceutical Technology, Department of
J. N. Moreira
CNC – Center for Neuroscience and Cell Biology, Faculty of Medicine (Pólo I), University of
Coimbra, Coimbra, Portugal
FFUC – Faculty of Pharmacy, Pólo das Ciências da Saúde, Coimbra, University of Coimbra,
Coimbra, Portugal
116 A. C. Silva et al.
Abstract Therapeutic uses of biological medicines are diverse and include active
substances from different classes. This chapter provides an overview on the clinical
applications of biological medicines containing hormones, blood products, and ther-
apeutic enzymes. Currently, therapeutic hormones have 78 approved medicines,
including insulin and analogs, glucagon and analogs, growth hormone, gonadotropins
(follicle-stimulating hormone, luteinizing hormone, and human chorionic gonadotro-
pin), thyroid-stimulating hormone, and parathyroid hormone. In contrast, recombinant
blood products, and particularly blood factors, anticoagulants, and thrombolytic
agents, incorporate 49 approved biological medicines. Regarding recombinant thera-
peutic enzymes, there are 22 approved medicines. Among the referred biological
medicines, there are six biosimilar hormones, and no biosimilars have been approved
for recombinant blood products and therapeutic enzymes, which is unexpected.
Current investigations on recombinant hormones, recombinant blood products,
and therapeutic enzymes seem to follow the same directions, searching for alterna-
tive non-injectable administration routes, development of new recombinant mole-
cules with improved pharmacokinetic properties and discovering new clinical
applications for approved medicines. These approaches are showing positive results
and new medicines are expected to reach clinical approval in the coming years.
Future prospects also include the approval of more biosimilar medicines.
Graphical Abstract
Keywords Anticoagulants, Blood factors, Glucagon, Gonadotropins, Growth

hormone, Insulin, Therapeutic enzymes, Thrombolytic agents
Hormones, Blood Products, and Therapeutic Enzymes 117
1 Introduction
The approval of recombinant human insulin as the first biological medicine in the
1980s marked a new era for medical therapy that brought together the biotechno-
logical and pharmaceutical industries. Biological medicines are often based on
recombinant proteins (also named as biopharmaceuticals) and produced in living
organisms, including microorganisms, cells, plants, and animals. However, due to
cost-effective manufacturing, some biological medicines contain proteins extracted
directly from natural sources. Thus far, the number of biological medicines that
gained clinical approval is impressive along with an increased number of biosimilars
approved. Current therapeutic applications of these medicines are diverse and
include active substances from different classes, such as monoclonal antibodies,
cytokines, growth factors, hormones, blood products, enzymes, vaccines, and cel-
lular therapies [1–5]. Almost all of these medicines are of parenteral administration,
as other routes pose a number of different biological barriers that are yet to be
overcome [6].
This chapter focuses on the clinical applications of biological medicines
containing hormones, blood products, and therapeutic enzymes. Therapeutic hor-
mones that were initially extracted from natural sources are currently produced
through biotechnological techniques (i.e., recombinant-DNA technology), and
include insulin and analogs, glucagon and analogs, growth hormone, gonadotropins
(follicle-stimulating hormone, luteinizing hormone, and human chorionic gonadotro-
pin), thyroid-stimulating hormone, and parathyroid hormone. In contrast, some blood
products are generated by cost-effective methodologies from the natural source.
Nonetheless, herein, only recombinant blood products, and particularly blood factors,
anticoagulants, and thrombolytic agents, will be addressed. Similarly, some thera-
peutic enzymes are natural and others are from recombinant origin and have several
clinical applications. Each section of this chapter briefly addresses current therapeutic
uses of the respective recombinant protein, and further discusses future applications.
2 Hormones
2.1 Insulin
Insulin is composed of 51-amino acids and produced by the β-cells of the islets of
Langerhans [7]. The major function of this hormone is regulating the blood glucose
levels, but it participates in other metabolic processes, promoting glycogen synthesis
in the liver and muscles, fatty acid synthesis in the adipocytes and inhibiting the
glycogenolysis and the gluconeogenesis [8–10]. Therapeutic use of insulin is clas-
sically for the management of type 1 diabetes, which is an autoimmune metabolic
disorder characterized by a deficient or absent production of insulin. Nonetheless, in
some cases, insulin can be used for type 2 diabetes, when is observed the production
of ineffective insulin [8, 11–13].
Fig. 1 Schematic representation of the human insulin. Ala alanine, Arg arginine, Asn asparagine,
Cys cysteine, Glu glutamic acid, Gln glutamine, Gly glycine, His histidine, Ile isoleucine, Leu
leucine, Lys lysine, Phe phenylalanine, Pro proline, Ser serine, Thr threonine, Tyr tyrosine, Val
valine (adapted from [17])
Insulin manufacture started with the advent of recombinant-DNA technology.

Before that therapeutic insulin was purified from bovine and porcine pancreas.
Nonetheless, the use of animal-derived products presents disadvantages, including
immunogenicity, difficulties to obtain enough supplies, and risk of contamination.
Furthermore, the insulin amino acids sequence is different among the various species
[8, 9, 14–16].
Figure 1 shows a schematic representation of human insulin, which has a dimeric
structure formed by two polypeptide chains, A- and B-, linked by disulfide bonds
between A7–B7, A20–B19, and A6–A11. A-chain consists of 21 amino acids, whereas
B-chain contains 30 amino acids [9, 13, 16, 18, 19].
First human recombinant insulin was approved in 1982 (Humulin®), being the
first drug produced by genetic engineering techniques in Escherichia coli. Since
then, several recombinant insulins have been marketed, mostly produced in
Escherichia coli, despite some are produced in Saccharomyces cerevisiae and Pichia
pastoris [8, 15, 19–21]. Moreover, the use of genetic engineering techniques allowed
the manufacture of different types of recombinant insulin (insulin analogs), changing
the amino acid sequences of the native human insulin. These products have different
pharmacokinetic and pharmacodynamic profiles and are called fast-acting or short-
acting, intermediate-acting, and slow-acting or long-acting insulins. Fast-acting
increases the blood insulin concentration more quickly, while slow-acting enters
the bloodstream more slowly, but has a longer duration, helping to maintain basal
levels of insulin in the blood. Intermediate acting insulin with specific activity was
also produced [8, 13, 14, 19, 22].
Table 1 shows examples of marketed biological and biosimilar medicines
containing recombinant human insulin and insulin analogs approved by the
European Medicines Agency (EMA) and/or the Food and Drug Administration
(FDA).
Apart from the medicines mentioned in Table 1, new insulins have been intro-
duced in some countries. For example, Afrezza® (pulmonary insulin) was recently
authorized in Brazil [72], and Glaritus® (insulin glargine) is only approved in some
Asian and African countries [73].
Concerning insulin biosimilar medicines, the firsts were approved by EMA in
2014 (Abasaglar®) [54] and by FDA in 2015 (Basaglar®) [74, 75]. Later, more
biosimilar insulins reached the market and some have been withdrawn (e.g.,
Solumarv® and Lusduna®) [33, 56].
In addition to regulating blood glucose levels, insulin also increases intestinal
cells growth. In this sense, the orphan designation was granted by EMA to recom-
binant insulin for the treatment of short bowel syndrome, a condition where the body
cannot absorb nutrients and fluids due to a missing of part of the small bowel. Thus,
insulin can regenerate the intestine of patients with short bowel syndrome, improv-
ing the disease symptoms [76].
2.1.1 New Insulin Formulations
Excluding Afrezza®, current approved insulin medicines are for subcutaneous

administration, but researchers have been looking for alternative non-invasive
routes, improving patient’s compliance [9, 77, 78]. The results seem promising
and there are several products under clinical studies. For example, MidaForm®
PharmFilm, a buccal film containing insulin, is under phase II clinical trials
[78, 79]; and IN-105 or tregopil, which is an oral insulin analog (differs from insulin
at position B29) with improved stability against enzymatic degradation, is under
phase II/III clinical trials [80].
Regarding the subcutaneous administration of insulin, advances in formulations
have been made to improve the management of diabetes. In this sense, insulin pumps
for subcutaneous implantation have been used. These devices contain a glucose
sensor connected to an insulin delivery system that promotes an optimal glycemic
control. However, limitations to control the postprandial hyperglycemia were
observed. To circumvent this, human insulin rapid-acting analogs have been
included in implantable pumps. Currently, the only pump available is the MIP
2007D that is marketed by Medtronic® in Europe. This titanium pump is surgically
implanted into the abdominal wall [81–83]. Artificial pancreas or closed-loop system
is the most promising insulin device, which consists of a pump and an infusion
system with an integrated glucose sensor and a computer that analyzes the blood
glucose level and adjusts the insulin flow rate [8, 78, 81, 84]. Recently, more
accurate closed-loop devices, which allow the real-time monitoring of interstitial
glucose levels, by means of transcutaneous glucose sensors (glucose-oxidase-based
electrochemical sensors) and external insulin pumps, have been successfully tested
for the management of type 1 diabetes in children. These systems contain an
electronic device that measures the blood glucose level and adjusts the insulin
infusion rate, maintaining glucose within the normal range [85].
Table 1 Examples of approved biological and biosimilar medicines containing recombinant

human insulin and insulin analogs, and respective duration of action and structure
Duration of Recombinant
action protein Structure Marketed medicines References
Short-acting, Human Identical to native Humulin®; [23–33]
fast-acting, or insulin human insulin Humulin®R;
rapid-acting Novolin®R;
Insuman®;
Actrapid®;
Velosulin® BR;
Exubera®; Insulin
human Winthrop®;
Afrezza®;
Solumarv®a, b
Insulin lispro Engineered insulin: Humalog®; [34–38]
inversion of B28–B29 Liprolog®; Insulin
proline–lysine Lispro Sanofi®a;
sequence Admelog®a
Insulin aspart Engineered insulin: NovoLog®/ [39–41]
B28proline is replaced NovoRapid®;
by aspartic acid Fiasp®
Insulin aspart NovoLog Mix® [42]
and insulin
aspart
protamine
Insulin Engineered insulin: B29 Apidra® [43, 44]
glulisine lysine is replaced by
glutamic acid and B3
asparagine is replaced
by lysine
Long-acting or Human Identical to native Protaphane®; [45–47]
slow-acting insulin human insulin Monotard®;
Ultratard®
Insulin Engineered insulin: A21 Lantus®; Toujeo®; [21, 48–
glargine asparagine is replaced Suliqua®/Soliqua®; 56]
by glycine and B-chain Abasaglar®a;
elongated by two Lusduna®a, b;
arginines Semglee®a
Insulin Engineered insulin: Levemir® [57, 58]
detemir 14 fatty acids are cova-
lently attached to B29
lysine and B30 threo-
nine is deleted
Insulin Engineered insulin: B30 Tresiba®; [59–62]
degludec threonine is deleted and Xultophy®
B29 is coupled to
hexadecanedionyl-ƴ-L-
glutamate
(continued)
Table 1 (continued)
Duration of Recombinant
action protein Structure Marketed medicines References
Intermediate Human Identical to native Actraphane®; [63–66]
acting, insulin human insulin Mixtard®;
dual-acting, or Insulatard®
long-acting Insulin lispro Engineered insulin: Humalog® Mix; [67, 68]
combined with and insulin inversion of B28–B29 Liprolog Mix®
fast-acting lispro proline–lysine
protamine sequence
Insulin aspart Engineered insulin: B28 NovoMix® [69]
and insulin proline is replaced by
aspart aspartic acid
protamine
Insulin Engineered insulin: B30 Ryzodeg® 70/30 [70, 71]
degludec and threonine is deleted and
insulin aspart B29 is coupled to
hexadecanedionyl-ƴ-L-
glutamate; B28 proline
is replaced by aspartic
acid
a
Biosimilar medicine
b
Medicine withdrawn
2.1.2 Insulin Analogs Under Development
Several ultra-fast-acting insulin analogs, showing higher physiological rate than

native insulin and fast-acting insulin analogs, have been developed and some are
under clinical evaluation [78, 81]. For example, VIAject®/Linjeta® acts faster than
insulin lispro, due to the addition of ethylenediaminetetraacetic acid (EDTA) that
chelates zinc and solubilizes insulin hexamers [81, 86–88]. Biodel insulin (BIOD-
531) is a concentrated formulation of recombinant human insulin with improved
post-meal glucose control, compared to Humalog®, due to the presence of EDTA,
citrate, and magnesium that promote tissue dispersion of insulin [16, 89].
BioChaperone® Lispro contains insulin lispro and oligosaccharides that promote
insulin absorption. Clinical studies with this type of insulin showed an increase of
early insulin exposure in 168%, compared to Humalog®, and improved metabolic
profile, compared to NovoLog® and Fiasp® [90, 91]. LY900014 which is an ultra-
rapid insulin lispro that recently finished phase II clinical trials is waiting for
regulatory review [92]. Ultra-fast-acting insulin aspart, containing nicotinamide to
promote absorption and arginine as a stabilizer, showed earlier onset of action and
exposure, compared to insulin aspart [89, 93, 94].
Another approach used to improve the onset of human insulin analogs involves
combining the molecule with recombinant human hyaluronidase [78]. For example,
a study in 14 healthy volunteers showed ultra-rapid profiles with a faster onset
associated with reducing postprandial hyperglycemia for insulin analogs linked to
hyaluronidase [81, 95]. Similar results were observed in another study, where was
administered human insulin combined with hyaluronidase to 40 patients with type
1 diabetes [96].
Currently, some companies are conducting clinical trials with new insulins for
subcutaneous administration in type 2 diabetes patients. For example, Eli Lilly is
evaluating LY3209590, which is an engineered insulin fused to an antibody Fc
domain that provides a long-acting basal profile, in comparison to glargine and
degludec insulins [97, 98].
2.2 Glucagon
Glucagon is a 29 amino acids polypeptide synthetized by the α-cells of the islets

of Langerhans and other related intestinal cells. Its major function is to prevent
hypoglycemia, promoting liver glycogenolysis and gluconeogenesis, increasing the
blood glucose level. Thereby, glucagon is frequently used to prevent hypoglycemia
caused by insulin administration in patients with type 1 diabetes [99, 100]. In
addition, glucagon precursors or glucagon analogs produced by intestinal endocrine
L-cells have been identified, including glucagon-like peptide 1 (GLP-1) and
glucagon-like peptide 2 (GLP-2). The first has an opposite activity to glucagon,
stimulating the β-cells proliferation with consequent insulin secretion and reduction
of the blood glucose. In contrast, GLP-2 stimulates intestinal growth while slowing
proximal bowel motility and secretion [101–103]. Similar to insulin, initial thera-
peutic glucagon was obtained directly from porcine and bovine pancreatic tissues,
being GlucaGen® the first recombinant glucagon produced in Saccharomyces
cerevisiae approved by FDA in 1998 [8, 104]. Table 2 shows examples of recom-
binant glucagon and glucagon analogs approved by EMA and/or FDA.
The pharmacokinetic and pharmacodynamic characteristics of dasiglucagon,
a glucagon analog, were evaluated in comparison to GlucaGen®, in children with
type 1 diabetes. The results showed increased blood glucose levels for the patients
treated with dasiglucagon. From these findings, the authors suggested the use of
dasiglucagon for hypoglycemia rescue therapy [122]. However, to the best of our
knowledge, there are no marketed medicines available, although the EMA approved
this indication in 2018 [123].
2.2.1 New Glucagon Formulations
A glucagon nasal powder formulation was successfully evaluated in phase III

clinical trials for use in severe hypoglycemic episodes and is currently waiting for
regulatory review. This new medicine is indicated for emergency situations, being
the first needle-free treatment for hypoglycemia [97, 124]. A liquid glucagon rescue
pen showed positive results in phase III clinical trials, where all the patients achieved
optimal blood glucose levels up to 30 min after the administration. Thereby, this new
device was suggested as an effective alternative for ready-to-use in severe hypogly-
cemia in type 1 diabetes patients, as compared to the currently marketed injectable
glucagon [125].
Table 2 Examples of recombinant glucagon and glucagon analogs, therapeutic indications, and
respective approved biological medicines
Recombinant Marketed
glucagon medicines Therapeutic indications References
Glucagon GlucaGen®; Gluca- Severe hypoglycemia in adult [104, 105]
gon for injection™ patients with diabetes type 1 treated
with insulin
Inhibition of the gastrointestinal tract
motility for radiologic examinations
Orphan medicine Noninsulinoma pancreatogenous [106]
hypoglycemia syndrome (excessive
growth of pancreas cells with insulin
overproduction and hypoglycemia
episodes)
Congenital hyperinsulinism [107, 108]
Glucagon linked to (inherited disorder where occurs a [109]
human immunoglob- not needed increase of insulin)
ulin Fc fragment
Liraglutide (GLP-1 Saxenda® Adjunct to chronic weight manage- [110, 111]
receptor agonist) ment for obese and overweight
patients undergoing diet and physi-
cal exercise
Victoza® Adjunct to diet and exercise to [112, 113]
Semaglutide (GLP-1 Ozempic® improve glycemic control (stimulate [114, 115]
receptor agonist) insulin production) in patients with
Dulaglutide (GLP-1 Trulicity® type 2 diabetes [116, 117]
receptor agonist)
Teduglutide (GLP-2 Revestive®(orphan Short bowel syndrome [118, 119]
analogue) medicine)/Gattex®
Apraglutide (GLP-2 Orphan medicine [120]
analogue)
GLP-2 linked to [121]
human immunoglob-
ulin Fc fragment
GLP-1 glucagon-like peptide 1, GLP-2 glucagon-like peptide-2
2.3 Growth Hormone
Somatotropin, growth hormone (GH) or human growth hormone plays a major role
in the regulation of body growth, cell metabolism, and circadian rhythm. This
peptide hormone has 109 amino acids and is secreted by the hypothalamus anterior
pituitary gland, in a process regulated by the GH-releasing factor or somatorelin
(stimulatory peptide) and the GH-release inhibiting hormone or somatostatin (inhib-
itory peptide) [8, 126, 127].
GH biological effects include increase of bones, cartilage, and muscles growth,
stimulation of protein synthesis, anti-insulin and lipolytic effects, and improved
renal function. GH can act directly, binding to specific cell receptors, or indirectly,
binding to liver receptors that promote different growth effects in the body. The latter
is mediated by the insulin growth factor-1 (IGF-1), which controls the secretion of
GH [8, 126, 127].
Therapeutic use of GH started in the 1950s, being this hormone obtained directly
from cadaveric human pituitaries, which presented limitations of safety and available
amounts. Only in the 1980s, by means of recombinant DNA technology, was
produced the first recombinant GH in Escherichia coli [8, 127, 128].
Clinically approved GH is usually indicated for the treatment of children with
short stature caused by different conditions, including GH deficiency, Prader–Willi
syndrome, Turner syndrome, homeobox-containing gene deficiency, or idiopathic.
In addition, GH is used for the management of growth failure in children with
chronic renal insufficiency, in children born small for gestational age, metabolism
regulation in short bowel syndrome, acquired immunodeficiency syndrome (AIDS)-
related cachexia, and for replacement therapy in adults with GH deficiency. There is
also a widespread illicit use for athlete body building [8, 127, 129]. Table 3 shows
examples of EMA and/or FDA approved medicines containing GH or somatotropin,
where it can be seen that, despite all contain recombinant GH, the therapeutic
indications of each medicine are not the same.
2.3.1 New Growth Hormone Formulations
New approaches in the development of medicines containing recombinant GH have

focused on increasing the drug circulating half-life, reducing the required number of
administrations from daily up to weekly or monthly [147]. Examples of formulations
under clinical studies include: TransCon (prodrug containing GH bounded to a
linker carrier) for weekly administration, which is in phase II (adults) and III
(children) [148]; MOD-4023 that is a carboxy-terminal peptide-modified GH for
weekly administration, which is in phase III (adults) [149]; GX-H9 (hybrid Fc-fused
to GH) for twice-monthly administration that is in phase II (adults and children)
[150]; Somapacitan (albumin-binding GH) for weekly administration that is in phase
III (adults) [151].
2.4 Gonadotropins
Gonadotropins comprise a family of hormones secreted by gonadotrope cells of the

anterior pituitary, including the follicle-stimulating hormone (FSH), luteinizing
hormone (LH), and human chorionic gonadotropin (hCG). FSH and LH play a
major role in the reproductive function regulation and sexual characteristics, while
hCG has a central role during pregnancy. These dimeric hormones are formed by
one α-polypeptide subunit of 92 amino acids and one β-polypeptide subunit with
111 FSH, 121 LH, and 145 hCG amino acids [8, 152, 153].

growth hormone (GH) or somatotropin and respective therapeutic indications
Recombinant
GH Marketed medicines Therapeutic indications References
Growth hor- Humatrope® Children with short stature [130]
mone or associated with GH deficiency,
somatotropin Turner syndrome, homeobox-
containing gene deficiency, or
idiopathic and born small for
gestational age
Norditropin®; Tev-Tropin®; Long-term treatment of GH [131–137]
Somatropin Biopartners®a; deficiency
Valtropin®a, b; Saizen® (orphan
medicine)
Genotropin®; Zomacton®; Children with short stature [138–141]
Omnitrope®b associated with GH deficiency,
Turner syndrome, Prader–Willi
syndrome, homeobox-
containing gene deficiency, or
idiopathic and born small for
gestational age
Nutropin AQ® Children with short stature [142, 143]
associated with GH deficiency,
idiopathic short stature, Turner
syndrome, and chronic kidney
disease
Zorbtive® (orphan medicine) Short bowel syndrome in [144]
patients receiving nutritional
support
Serostim® HIV patients with wasting or [145, 146]
cachexia
a
Medicine withdrawn
b
Biosimilar medicine
In men, FSH is responsible for sperm production, targeting the testis Sertoli cells
and regulating the early stages of spermatogenesis. In women, FSH stimulates the
ovarian follicle maturation, participates in the synthesis of estrogen and glycosami-
noglycans, and regulates the reproductive function. LH stimulates the women
ovulation of mature follicles, and (together with FSH) participates in the conversion
of androgens to estrogens. In men, LH is involved in the testosterone production and
sperm maturation. In contrast, the hCG is produced by pregnant women, having an
important function during the early phase of pregnancy [8, 154, 155].
Gonadotropin hormones have been used in assisted reproductive therapies and in
the treatment of several women infertility disorders. For example, to induce or
stimulate ovulation for support of natural conception or intrauterine insemination,
and to induce multifollicular growth required for in vitro fertilization procedures. In
men, FSH and hCG stimulate sperm synthesis and are used for the management of
hypogonadotropic hypogonadism. There is also an illicit use of hCG by athletes to
stimulate testosterone production. First therapeutic FSH and LH were extracted from
women menopausal urine, while hCG was obtained from the urine of pregnant
women [8, 152, 155, 156]. Owing to the increase on the number of fertility
treatments, the quantities of urinary gonadotropins available have been scarce. For
this reason, recombinant gonadotropins (or gonadotropins analogs) have been pro-
duced in mammalian cell lines, such as Chinese hamster ovary (CHO) cells, among
others. Nonetheless, urinary gonadotropins remain in clinical use [155, 156]. Table 4
shows examples of recombinant gonadotropins approved by EMA and/or FDA,
respective therapeutic indications, and biological and biosimilar medicines.
From Table 4, it can be seen that only recombinant FSH has approved biosimilar
medicines, although a recombinant hCG biosimilar was recently evaluated to induce
ovulation in patients undergoing intrauterine dissemination. The results demon-
strated clinical equivalence to Ovitrelle®, suggesting the potential of using this
biosimilar as an alternative to the reference medicine [171].
Concerning the clinical applications of gonadotropins, there is an open field for
therapeutic uses, including the treatment of polycystic ovary syndrome, management

gonadotropins (FSH follicle-stimulating hormone, LH luteinizing hormone, hCG human chorionic
gonadotropin) and respective therapeutic indications
Recombinant Marketed
gonadotropins medicines Therapeutic indications References
Follitropin alfa Gonal-f®; Women: induction of ovulation and preg- [157–160]
(FSH) Ovaleap®a; nancy in anovulatory infertile patients;
Bemfola®a development of multiple ovulatory folli-
Follitropin beta Puregon®/ cles in patients undergoing fertility treat- [161–163]
(FSH) Follistim®; ments; patients with severe deficiencies of
Fertavid® LH and FSH
Men: stimulation of spermatogenesis in
congenital or acquired hypogonadotropic
hypogonadism
Follitropin delta Rekovelle® Development of multiple ovulatory folli- [164]
(FSH) cles in women undergoing fertility treat-
Corifollitropin alfa Elonva® ments: in vitro fertilization or [165]
(long-acting FSH) intracytoplasmic sperm injection
Lutropin alfa (LH) Luveris® Induction of ovulation in women with [166, 167]
severe LH and FSH deficiencies
Follitropin alfa/ Pergoveris® Women with FSH and LH deficiencies: [168]
lutropin alfa induction of ovulation (FSH) and eggs
(FSH/LH) release (LH)
Choriogonadotropin Ovitrelle®/ Women treated with LH and FSH to [169, 170]
alfa (hCG) Ovidrel® stimulate ovaries: trigger ovulation and
development of the corpus luteum to sup-
port pregnancy
Women undergoing fertility treatments
(in vitro fertilization)
Anovulatory or oligo-ovulatory women
a
Biosimilar medicine
of ovary and prostate cancers, prevention of postmenopausal symptoms (avoidance

of bone loss and weight gain), and as contraceptives [156].
2.4.1 New Gonadotropins Formulations
Recombinant gonadotropins have been used clinically in fertility treatments, by

means of injectable formulations. Nonetheless, some limitations have been pointed
to these medicines, regarding the necessity of performing multiple administrations,
lack of stability, and adverse effects, such as ovarian hyperstimulation syndrome.
Researches have been focused in the development of long-acting recombinant
gonadotropins with improved stability, achieving appropriate pharmacokinetic pro-
files and minimizing the number of administrations [156]. Several studies showed
that corifollitropin alfa, a long-acting FSH analog, reduces the drawbacks of the
in vitro fertilization treatments (Table 4) [172, 173]. In this sense, other strategies
have been studied to develop more long-acting recombinant FSH molecules. Exam-
ples include the addition of glycosylated peptides or polyethylene glycol – PEG
(PEGylation) to the gonadotropin molecule, and fusion of the gonadotropin with an
immunoglobulin Fc domain (fusion protein) [153, 156].
2.5 Other Recombinant Hormones
In addition to insulin, glucagon, growth hormone, and gonadotropins there are

other recombinant hormones under clinical use, which are the thyroid-stimulating
hormone (TSH) and the parathyroid hormone (PTH). TSH or thyrotropin has a
molecular structure that resembles gonadotropins, containing one β-subunit and
one α-subunit. TSH is produced by the anterior pituitary and targets the thyroid
gland, stimulating its functions, including iodine uptake, production of the iodine
containing thyroid hormones (iodothyronines) triiodothyronine (T3) and thyroxine
(T4), and promotion of thyroid growth. Furthermore, TSH participates in the pre-
vention of thyroid cells apoptosis and in thyroid ontogenesis. Recombinant TSH
produced in CHO cells has been used for the diagnostic of thyroid cancer and for the
detection of thyroid remnants in post-thyroidectomy patients [8, 174]. PTH or
human PTH is an 84 amino acids polypeptide that regulates extracellular phosphate
and calcium metabolism. In bone, PTH has a catabolic effect, stimulating osteoblasts
for bone formation. In kidneys, PTH promotes the synthesis of vitamin D that
increases the intestinal calcium absorption and inhibits the renal phosphate
reabsorption. Thereby, recombinant hPTH produced in Escherichia coli was
approved for the management of osteoporosis in women post-menopausal and in
men and for chronic hypoparathyroidism [8, 175]. Table 5 shows examples of
recombinant TSH and PTH approved by EMA and/or FDA, respective therapeutic
indications, and biological and biosimilar medicines.
In addition to Table 5, other formulations containing recombinant PTH have been
investigated. For example, TransCon PTH has successfully completed phase I
clinical trials to treat hypoparathyroidism [184].
Current approved medicines containing PTH are for subcutaneous administra-
tion, which reduces patient compliance. Nasal and transdermal routes have been
suggested as alternative routes for the administration of PTH in osteoporosis treat-
ments. The results of in vitro studies are promising, although in vivo studies are
required to confirm this application [185, 186].
3 Blood Products
Blood products of therapeutic interest are proteins that can be extracted directly from
the natural source, i.e., from the blood red and white cells, platelets, and plasma.
However, due to the large amount required for clinical use and some safety concerns,
several therapeutic blood proteins have been produced by genetic engineering
techniques, including recombinant blood factors, anticoagulants, and thrombolytic
agents [187, 188]. Concerning the scope of this chapter, we focus only in recombi-
nant blood products.
3.1 Blood Clotting Factors or Coagulation Factors
Blood clotting factors (or blood factors) have been used for the treatment of
hemophilia, which is a rare bleeding inherited disorder that reduces the normal
thyroid-stimulating hormone (TSH) and parathyroid hormone (PTH) and respective therapeutic
indications
Recombinant
TSH and
PTH Marketed medicine Therapeutic indications References
Thyrotropin Thyrogen® Thyroid cancer: detection of thyroid tissue [176, 177]
alfa (TSH) left after surgery; elimination of remaining
thyroid tissue (in combination with radioac-
tive iodine) in patients who removed the
thyroid gland
Teriparatide Forsteo®/Forteo®; Osteoporosis in postmenopausal women and [178–181]
(PTH) Movymia®a/ in men with increased risk of fracture
Terrosa®a
PTH Natpara®/ Hypocalcemia control in patients with [182, 183]
Natpar®(orphan chronic hypoparathyroidism
medicine)
a
Biosimilar medicine
blood clotting process and causes severe consequences. People with this disease
bruise very easily and show difficulty in blood coagulation after trauma, increasing
the bleeding time. There are 12 different blood clotting factors and several cofactors
that play a key role in the blood coagulation process, particularly, in the blood
coagulation cascade. A genetic defect in the expression of blood factors (or anti-
hemophilic factors) originates hemophilia, which is divided into subtypes A, B, and
C. In hemophilia A (or classical hemophilia that occurs in about 90% of the cases)
the blood clotting factor VIII is deficient or abnormal, whereas in hemophilia B the
blood clotting factor IX is deficient or abnormal [188–191]. Both blood factors VIII
and IX play vital roles in the coagulation cascade, being essential for the conversion
of prothrombin into thrombin. Afterwards, thrombin converts fibrinogen to fibrin, a
primordial fibrous protein of the blood coagulation process. Hemophilia C is a rare
type that is characterized by a deficient or abnormal blood clotting factor XI [191–
193].
Hemophilia treatment is performed by supplying the impaired blood clotting
factor, which was firstly obtained directly from the blood donors. However, this
process showed disadvantages related to the risk of virus infection that originates
severe diseases, such as AIDS, hepatitis C and B, and others. Therefore, recombinant
blood clotting factors, produced by DNA recombinant techniques in CHO cells, are
used for the management of hemophilia [188, 189, 191]. In addition, some compa-
nies are conducting clinical trials with gene therapy products for the treatment of
hemophilia A and B, which may be better than the chronic treatments with weekly
injections of recombinant blood clotting factors. Examples of such companies are:
BioMarin® [194], Sangamo Therapeutics [195], and uniQure [196].
Table 6 shows examples of recombinant blood products (blood factors, antico-
agulants, and thrombolytic agents), therapeutic indications, and names of the respec-
tive approved biological and biosimilar medicines, by EMA and/or FDA.
Table 6 Examples of recombinant blood products (blood clotting factors, anticoagulants, and
thrombolytic agents), therapeutic indications, and respective approved biological and biosimilar
medicines
Recombinant blood
products Marketed medicines Therapeutic indications References
Blood clotting factors
Eptacog alfa (factor Novoseven®; Novoseven Hemophilia A and B; fac- [197, 198]
VIIa) RT®; Niastase®; Niastase tor VII deficiency;
RT® Glanzmann’s
thrombasthenia
Octocog alfa (factor Advate®; Bioclate®; Helixate Hemophilia A [199–204]
VIII) FS®; Kogenate FS®;
Kovaltry®; Recombinate®;
Iblias®
Turoctocog alfa NovoEight®; Zonovate® Hemophilia A [199, 205]
(factor VIII)
Lonoctocog alfa Afstyla® Hemophilia A [199, 206]
(factor VIII)
(continued)
Table 6 (continued)
Recombinant blood
Susoctocog alfa Obizur® Hemophilia A [207]
(factor VIII)
Rurioctocog alfa Adynovi® Hemophilia A [208]
pegol (factor VIII)
Damoctocog alfa Jivi® Hemophilia A [209]
pegol (factor VIII)
Efmoroctocog alfa Eloctate®/Elocta® Hemophilia A [199, 210]
(factor VIII linked
to Fc fusion protein)
Moroctocog alfa Refacto AF®; Xyntha® Hemophilia A [199, 211]
(factor VIII)
Simoctocog alfa Nuwiq®; Vihuma® Hemophilia A [199, 212,
(factor VIII) 213]
Pegylated factor Adynovate® Hemophilia A [199]
VIII (factor VIII
linked to PEG)
Vonicog alfa (von Veyvondi® von Willebrand disease [214]
Willebrand factor)
Eftrenonacog alfa Alprolix® (Orphan medicine) Hemophilia B [210, 215]
(factor IX linked to
Fc fusion protein)
Nonacog alfa (factor BeneFix® Hemophilia B [216]
IX)
Nonacog gamma Rixubis® Hemophilia B [217]
(factor IX)
Nonacog beta pegol Refixia® Hemophilia B [218]
(factor IX linked to
PEG)
Albutrepenonacog Idelvion® (Orphan medicine) Hemophilia B [219, 220]
alfa (factor IX
linked to albumin)
Factor IX IXINITY®, RIXUBIS® Hemophilia B [197]
Andexanet alfa (fac- ANDEXXA® Reversal of [221, 222]
tor Xa, inactivated- anticoagulation for
zhzo) patients treated with
rivaroxaban and apixaban
Catridecacog (factor TRETTEN®; NovoThirteen® Congenital factor XIIIa [223, 224]
XIIIa) subunit deficiency
Anticoagulants
Lepirudin Refludan®a Heparin-induced thrombo- [225, 226]
cytopenia type II and
thromboembolic disease
Desirudin Revasc®/Iprivask®a Prevention of thrombosis [2, 227,
in patients undergoing hip 228]
or knee replacement
(continued)
Table 6 (continued)
Recombinant blood
surgery, and for pulmonary
embolus
Antithrombin alfa ATryn® Prevention of thromboem- [229, 230]
bolic events in congenital
antithrombin deficiency
Drotrecogin alfa Xigris®a Reduce risk of blood clots [2, 231]
during sepsis by inhibition
of clotting factors Va and
VIIIa
Thrombolytic agents
Alteplase (tissue Actilyse®; Cathflo® Management of acute [232, 233]
plasminogen Activase® myocardial infarction
activator)
Reteplase Ecokinase®a; Retavase®; [234–236]
Rapilysin®
Tenecteplase Metalyse®; Tenecteplase [237–239]
Boehringer Ingelheim Pharma
GmbH Co. KG®a; TNKase®
a
Medicine withdrawn; PEG polyethylene glycol
Recombinant human coagulation factor VIIa or eptacog alfa is a vitamin

K-dependent glycoprotein that activates factor IX and factor X, which are essential
for hemostasis. Thus, eptacog alfa is involved in the clotting process initiation and
controls bleeding disorder, being used for the treatment of patients with hemophilia
A and B, congenital factor VII deficiency, and Glanzmann’s thrombasthenia (a rare
bleeding disorder) [198, 240, 241]. Recently, Biron-Andreani and Schved revised
the pharmacodynamics and pharmacokinetics data of eptacog beta, a new type of
recombinant factor VIIa produced in the milk of transgenic rabbits. From their search
the authors concluded that, when compared to eptacog alfa, eptacog beta requires a
lower dose to obtain the same in vivo effect, being a less expensive alternative for the
management of hemophilia [242].
Recombinant human coagulation factor VIII is used for the treatment of patients
with congenital factor VIII deficiency (hemophilia A). Biological and biosimilar
medicines available are shown in Table 6 and include octocog alfa, turoctocog alfa,
lonoctocog alfa, susoctocog alfa, simoctocog alfa, rurioctocog alfa pegol, and
damoctocog alfa pegol [200–209, 212, 213, 241, 243–248]. Efmoroctocog alfa is
a recombinant factor VIII-Fc fusion protein used as an antihemorrhagic agent for the
treatment and prophylaxis of acute bleeding episodes in patients with hemophilia
A. The addition of the Fc protein (B-domain) to the recombinant factor VIII
extended the drug half-life, compared to the recombinant factor VIII alone
[191, 192, 210, 241, 247]. Pegylated factor VIII contains the human recombinant
factor VIII covalently linked to a PEG molecule, which increases the circulation half
time, improving therapeutic efficacy [249, 250].
Vonicog alfa is a recombinant von Willebrand factor used to control bleeding in
patients with von Willebrand disease (an inherited bleeding disorder), when
desmopressin is not effective [214, 251]. The hemostatic efficacy of using vonicog
alfa alone or in combination with recombinant factor VIII, in patients with severe
von Willebrand disease that are undergoing surgery, was evaluated in a phase III
clinical study. Researchers observed that the use of vonicog alfa alone originated
hemostasis 6 h after the administration and the effect lasted from 72 up to 96 h. From
these findings the authors concluded that, according to each patient risk factors, the
treatment of von Willebrand disease should be performed with vonicog alfa alone or
in combination with recombinant factor VIII [252].
Eftrenonacog alfa is a recombinant fusion protein containing the human factor IX
covalently linked to the constant region (Fc) domain of the human immunoglobulin
(IgG1), which is used for the treatment and prophylaxis of bleeding episodes in
patients with hemophilia B. This molecule restores the levels of factor IX, promoting
normal blood coagulation. Adding the Fc domain to factor IX increases its half-life,
improving the bioavailability and, consequently, the therapeutic efficacy [193, 215,
241]. Albutrepenonacog alfa is a recombinant fusion protein that comprises human
factor IX linked to albumin, which is indicated for patients with hemophilia B for the
control and prevention of bleeding episodes [219, 220]. Nonacog alfa, nonacog
gamma, and nonacog beta pegol are also recombinant human factor IX used for the
treatment of hemophilia B [188, 197, 216–218].
FDA approved the recombinant coagulation factor Xa, inactivated-zhzo, or
andexanet alfa to reverse the effects of factor Xa inhibitors, apixaban and
rivaroxaban, when an anticoagulation effect is required [222]. Some studies
suggested the use of andexanet alfa for reversing the effect of edoxaban, which is
another factor Xa inhibitor, but this indication has not been approved yet [221, 253].
Patients with deficient or abnormal coagulation factor XIII can be treated with
recombinant human factor XIIIa, also known as catridecacog [223, 224, 241,
254]. Recently, Sottilotta et al. performed a clinical study where was observed that
the use of catridecacog is effective for continued prophylaxis and for conducting
major surgical procedures in patients with congenital factor XIII deficiency [255].
3.2 Anticoagulants
Thrombus formation occurs by changes in the blood coagulation process within the
vessels and causes serious health problems or even death. Anticoagulants are able to
extend the coagulation cascade length and have been used to treat and prevent
embolic events or thrombotic disorders, avoiding the changes in the blood clotting
process [188, 256].
Heparin, dicoumarol, warfarin, and hirudin are the most studied anticoagulants.
Heparin was first extracted from the liver, but current marketed medicines contain
enoxaparin sodium (low molecular weight heparin) obtained by alkaline depolymer-

ization of the heparin benzyl ester that is extracted from porcine gastric mucosa. This
heparin derivative activates the antithrombin III and, thus, inhibits the clotting
factors Xa and IIa, being used to prevent and treat vein thrombosis, acute coronary
syndromes, pulmonary embolism and for the prophylaxis of ischemic complications
(angina and myocardial infarction). EMA and FDA approved biological (Lovenox®,
Clexane®, Clexane T®, Clexane Forte®, Klexane®, Qualiop®, Enoxaparin Sanofi®,
and Enoxaparine Sanofi®) and biosimilar (Inhixa® and Thorinane®) medicines
containing enoxaparin sodium to prevent and treat conditions associated with
blood clots. These include deep vein thrombosis (usually in the legs), unstable
angina (related to impaired blood flow to the heart), and certain types of myocardial
infarction or heart attack [241, 257–261]. When compared to recombinant biological
medicines (Table 6), heparin and derivatives are less expensive. Nonetheless, dis-
advantages have been pointed, related to the risk of severe side effects, including
bleeding and thrombocytopenia [262].
Dicoumarol and warfarin are obtained by chemical synthesis and will not be
referred, because they are not biological medicines. Hirudin is a natural small
polypeptide that exists in the saliva of bloodsucking parasites (Hirudo medicinalis)
and has anticoagulants properties, binding and inhibiting thrombin [188, 256, 263,
264]. Regarding the small amount of hirudin available for removal from the natural
source, lepirudin (Table 6), a recombinant hirudin variant-1, has been produced in
Saccharomyces cerevisiae, and is indicated for preventing thrombus or clot forma-
tion. Nonetheless, this medicine is not currently available [188, 225, 226, 265,
266]. El-Mowafi et al. conducted a clinical trial where was observed that topically
administered recombinant hirudin gel is effective and safe for the management of
symptomatic hematomas [267]. Similar products have been produced by recombi-
nant techniques. For example, desirudin produced in Saccharomyces cerevisiae was
approved by EMA and FDA (Table 6) as a selective thrombin inhibitor that prevents
deep venous thrombosis (in patients undergoing elective hip or knee replacement
surgery) and decreases the risk of pulmonary embolus, but is not currently available
[188, 227, 228].
Antithrombin is a plasma native inhibitor of the coagulation process that binds to
thrombin (factor IIA), factor IXa and Xa, and interrupts the coagulation cascade.
Antithrombin alfa (Table 6) was the first biological medicine produced in transgenic
animals, being obtained from the milk of genetically modified goats [188, 268,
269]. Antithrombin alfa is used for the prevention of thromboembolic events in
patients with congenital low levels of antithrombin [230]. Drotrecogin alfa (Table 6)
is similar to the activated human protein C that inhibits the clotting factors Va and
VIIIa and reduces the blood coagulation process, and was approved to avoid
excessive blood clotting during severe sepsis, but is not currently available
[188, 231].
3.3 Thrombolytic Agents
Thrombolytic agents or fibrinolytics convert zymogen plasminogen (glycoprotein

synthesized by the kidneys) into plasmin, which is an active enzyme that
promotes the proteolytic degradation of fibrin, dissolving blood clots. There are
several recombinant thrombolytic agents used for the treatment of thromboembolic
disease, including alteplase, reteplase, tenecteplase, and urokinase (Tables 6 and 7)
[188, 191, 269, 300, 301].
Alteplase (Tables 6 and 7) is a human recombinant tissue plasminogen activator
used for the management of acute myocardial infarction or acute stroke [232, 302].
Tenecteplase (Tables 6 and 7) and reteplase (Tables 6 and 7) are also recombinant
forms of the human tissue plasminogen activator that have been indicated for the
treatment of patients suspected or after having a heart attack. This enzyme acts by
dissolving the clots formed inside the vessels, improving the blood flow into the
heart. Compared to the other thrombolytic agents, reteplase has been showing better
clinical results, due to its longer half-life [234–239, 303].
4 Therapeutic Enzymes
Concerning some of their properties, enzymes play an important role in the phar-
maceutical field. For example, in diagnostic assays, due to the high affinity and
specificity to bind targets, and for the management of several diseases and disorders.
Some therapeutic enzymes are extracted from the natural source, while others have
been produced through recombinant-DNA techniques. The latter have been origi-
nating higher yields, regarding the possibility of producing larger quantities of
pure enzymes [188, 273, 304]. Nonetheless, when therapeutic enzymes can be easily
withdrawn from the natural source, the production of similar recombinant enzymes
is not required, which reduces costs of the final product. Examples of therapeutic
enzymes obtained from natural sources include: asparaginase derived from
Escherichia coli (Elspar®), which is used for the management of acute lymphoblas-
tic leukemia [305]; collagenase Clostridium histolyticum (Xiapex®) extracted from
the bacterium Clostridium histolyticum, which is used to break up collagen in
patients suffering from Dupuytren’s contracture and Peyronie’s disease [306]. Diges-
tive enzymes including lipases, proteases, and amylases (pancrelipase) that are used
to treat pancreatic insufficiency, caused by cystic fibrosis or chronic pancreatitis
(Creon®, Pancreaze®, Zenpep®, Pertzye®, Viokace®), are extracted from pig pan-
creas [307–311]. Nonetheless, concerning the aim of this chapter, only recombinant
therapeutic enzymes are described.
Therapeutic enzymes obtained through biotechnological processes are widely
used for several clinical applications (Fig. 2), often in enzyme replacement therapy,
when the native enzyme is lacking, and as adjuvants to the management of severe
diseases and disorders. Table 7 shows examples of EMA and/or FDA approved
medicines containing recombinant enzymes, respective therapeutic indications, and
mechanism of action.
Table 7 Examples of recombinant enzymes and respective approved biological medicines, therapeutic indications, and mechanism of action
Recombinant enzymes Marketed medicines Therapeutic indications Mechanism of action References
Alfagalsidase (alpha- Replagal®; Fabry’s disease: absent/insufficient alpha- Catalyzes the hydrolysis of [270–272]
galactosidase A) Fabrazyme® galactosidase A that breaks down the globotriaosylceramide, reducing the body
globotriaosylceramide, which is a fatty accumulation
compound that accumulates in the body and
affects the nervous system, vascular endo-
thelial cells, and major organs
Alteplase (tissue plas- Actilyse®; Cathflo® Acute ischemic stroke or acute myocardial Converts plasminogen into plasmin, [232, 235,
minogen activator) Activase® infarction dissolving the blood clots responsible for the 273, 274]
blockage of the coronary arteries
Asparaginase Kidrolase®; Acute lymphoblastic leukemia Catalyzes the hydrolysis of L-asparagine [273, 275–
(L-asparaginase) Oncaspar®; into L-aspartic acid and ammonia 277]
Spectrila® Asparagine is fundamental for cancer cells
proliferation and a reduction on its blood
levels originates death of cancer cells. Nor-
mal cells are able to produce asparagine and,
Hormones, Blood Products, and Therapeutic Enzymes
thus, are not affected by the administration

of asparaginase
Pegylated Erwinia Erwinaze® (orphan Acute lymphoblastic leukemia in patients Catalyzes the hydrolysis of L-asparagine [273, 278,
chrysanthemi medicine) who have developed hypersensitivity to into L-aspartic acid and ammonia 279]
L-asparaginase native E. coli derived asparaginase Asparagine is fundamental for cancer cells
of asparaginase
PEGylation reduces the clearance of
asparaginase from the body
(continued)
135
Table 7 (continued)
136

Asparaginase Graspa®a Acute lymphoblastic leukemia Catalyzes the hydrolysis of L-asparagine [280]
(L-asparaginase) Orphan medicine Pancreatic cancer and acute myeloid into L-aspartic acid and ammonia [281, 282]
leukemia Asparagine is fundamental for cancer cells
of asparaginase
Contains asparaginase encapsulated in
erythrocytes for increasing its circulation
time and protection against blood degrada-
tion and immunological reactions
Dornase alfa (deoxyribo- Pulmozyme® Cystic fibrosis: retention of purulent and Catalyzes the cleavage of the [2, 273,
nuclease – DNase) viscous secretions that affect lung function phosphodiester linkages of the mucus DNA. 283]
and cause infections This process reduces mucus viscosity and
promotes secretions clearance
Elosulfase alfa Vimizim® (orphan Mucopolysaccharidosis type IVA: lack of Enzyme replacement therapy, breaking [284, 285]
(N-acetylgalactosamine- medicine) N-acetylgalactosamine-6-sulfatase, which down glycosaminoglycans that stop build-
6-sulfatase) breaks down glycosaminoglycans. Accu- ing up in cells
mulation of glycosaminoglycans causes
short bones, difficulty moving and difficulty
breathing, clouding vision and hearing loss
Galsulfase Aryplase®/ Mucopolysaccharidosis type VI: lack of Enzyme replacement therapy. Galsulfase is [273, 286,
(N-acetylgalactosamine Naglazyme® (orphan N-acetylgalactosamine 4-sulfatase that taken up by cells into lysosomes and cata- 287]
4-sulfatase) medicine) breaks down glycosaminoglycans. Body lyzes the cleavage of sulfate ester from ter-
accumulation of glycosaminoglycans origi- minal N-acetylgalactosamine 4-sulfate
nates macrocephaly, short body, difficult residues of glycosaminoglycan chondroitin
moving, impaired vision and hearing loss, 4-sulfate and dermatan sulfate, which
reduced pulmonary function, cardiac abnor- increases the catabolism of
malities, etc. glycosaminoglycans
A. C. Silva et al.
Idursulfase (iduronate-2- Elaprase® (orphan Hunter syndrome or mucopolysaccharidosis Enzyme replacement therapy. Cleaves the [288, 289]
sulfatase) medicine) type II: disease caused by insufficient levels terminal 2-O-sulfate moieties from glycos-
of the enzyme iduronate-2-sulfatase. aminoglycans dermatan sulfate and heparan
Patients are not able to degrade glycosami- sulfate, increasing the catabolism of
noglycans, which accumulate in cells, orig- glycosaminoglycans
inating difficulty breathing and difficulty
walking
Laronidase (α-L- Aldurazyme® Non-neurological symptoms of Enzyme replacement therapy. Catalyzes the [290, 291]
iduronidase) mucopolysaccharidosis type I: α-L- hydrolysis of the terminal α-L-iduronic acid
iduronidase enzyme deficiency that pro- residues from the glycosaminoglycans
motes the body accumulation of glycosami- dermatan sulfate and heparin sulfate, which
noglycans, causing several dysfunctions, increases the catabolism of
including enlarged liver, difficult moving, glycosaminoglycans
reduced lung, and heart and eye diseases
Imiglucerase Cerezyme® Type 1 and 3 Gaucher disease: lack of Enzyme replacement therapy. Catalyzes the [292, 293]
(glucocerebrosidase) glucocerebrosidase or acid beta-glucosidase, hydrolysis of glucosylceramide to glucose
an enzyme responsible for the degradation and ceramide, stopping it building up in the
Hormones, Blood Products, and Therapeutic Enzymes
of the fatty waste product glucosylceramide, body

which builds up in the liver, spleen, and
bone marrow. Typical disease symptoms
include enlarged spleen and liver, anemia,
thrombocytopenia, and bone disease
Velaglucerase alfa VPRIV® (orphan Type 1 Gaucher disease: glucocerebrosidase [294, 295]
(glucocerebrosidase) medicine) deficiency that affects the liver, spleen, and
bones. Originates liver malfunction, skeletal
disorders, bone lesions, neurological com-
plications, anemia, and low blood platelet
count
Rasburicase (urate Elitek®; Fasturtec® Acute hyperuricemia: high levels of uric Catalyzes enzymatic oxidation of uric acid [296, 297]
oxidase) acid in the blood. Avoid kidney failure in to allantoin, which is easily eliminated by
patients undergoing chemotherapy the kidneys
(continued)
137
Table 7 (continued)
138

Velmanase alfa Lamzede® (orphan Mild to moderate alpha-mannosidosis: con- Enzyme replacement therapy. Degradation [298, 299]
medicine) genital absence of the enzyme alpha- of glycoproteins, avoiding tissue accumula-
mannosidase that breaks down glycosides. tion of oligosaccharides
Body accumulation of oligosaccharides
originates failure of body functions, such as
breathing and movement
a
Medicine withdrawn
A. C. Silva et al.
Fig. 2 Main clinical uses of therapeutic enzymes [273, 304]
5 Conclusion
From this review, we can conclude that biological medicines are currently a well-
established therapeutic area, enabling the treatment of various incurable diseases.
Concerning the three different therapeutic groups addressed in this chapter, the
hormones are the ones with highest number of marketed products (78 approved
medicines), where 38 are insulin and analogs, 9 are glucagon and analogs, 12 contain
GH, 12 contain different gonadotropins, 1 contains TSH, and 6 incorporate PTH.
Furthermore, insulin has 4 biosimilars, 2 of which have been withdrawn and 1 has
orphan designation. In contrast, no biosimilar glucagon was approved, which is
surprising, as the first recombinant glucagon was approved in 1998. The same is
observed for GH, which has only the first biosimilar medicine approved. Two
orphan designations and 2 withdrawn medicines were noticed for GH, while gonad-
otropins have 2 approved biosimilars.
There are 49 approved biological medicines incorporating recombinant blood

products, where 35 are blood factors (2 orphan designations), 5 are anticoagulants
(3 withdrawn), and 9 are thrombolytic agents (2 withdrawn). None biosimilar has
been approved for recombinant blood products, which is unexpected since they
reached the market in the 1990s. Similarly, from the 22 approved therapeutic
enzymes, none is a biosimilar, 3 are orphan medicines, and 1 was withdrawn from
the market.
Current investigations on recombinant hormones, recombinant blood products,
and therapeutic enzymes seem to follow the same directions, searching for alterna-
tive non-injectable administration routes, development of new recombinant mole-
cules with improved pharmacokinetic properties and discovering new clinical
applications for the approved medicines. These approaches are showing positive
results and new medicines are expected to reach clinical approval in the coming
years. Future prospects also include the approval of more biosimilar medicines.
Acknowledgments This work was supported by the Applied Molecular Biosciences Unit-
UCIBIO and FP-ENAS, which are financed by national funds from FCT/MCTES (UID/Multi/
04378/2019 and UID/Multi/04546/2019, respectively).
References
1. Chen YC, Yeh MK (2018) Introductory chapter: biopharmaceuticals. In: Yeh MK, Chen YC
(eds) Biopharmaceuticals. IntechOpen, London
2. Walsh G (2013) Pharmaceutical biotechnology: concepts and applications. Wiley, Hoboken
3. FaDA (2019) What are “biologics” questions and answers. https://www.fda.gov/about-fda/
about-center-biologics-evaluation-and-research-cber/what-are-biologics-questions-and-
answers
4. EMA (2019) Biological medicine. https://www.ema.europa.eu/en/glossary/biological-
medicine
5. Kesik-Brodacka M (2018) Progress in biopharmaceutical development. Biotechnol Appl
Biochem 65(3):306–322
6. Vass P, Démuth B, Hirsch E, Nagy B, Andersen SK, Vigh T et al (2019) Drying technology
strategies for colon-targeted oral delivery of biopharmaceuticals. J Control Release
296:162–178
7. Permutt MA, Chirgwin J, Rotwein P, Giddings S (1984) Insulin gene structure and function: a
review of studies using recombinant DNA methodology. Diabetes Care 7(4):386–394
8. Walsh G (2007) Therapeutic hormones. Pharmaceutical biotechnology: concepts and appli-
cations. Wiley, Hoboken, pp 291–328
9. Shaikh IM, Jadhav KR, Ganga S, Kadam VJ, Pisal SS (2005) Advanced approaches in insulin
delivery. Curr Pharm Biotechnol 6(5):387–395
10. Rhodes CJ, White MF (2002) Molecular insights into insulin action and secretion. Eur J Clin
Invest 32(Suppl 3):3–13
11. Docherty K (1997) Gene therapy for diabetes mellitus. Clin Sci 92(4):321–330
12. Auricchio A, Gao GP, Yu QC, Raper S, Rivera VM, Clackson T et al (2002) Constitutive and
regulated expression of processed insulin following in vivo hepatic gene transfer. Gene Ther 9
(14):963–971
13. Mane K, Chaluvaraju K, Niranjan M, Zaranappa T, Manjuthej T (2012) Review of insulin and
its analogues in diabetes mellitus. J Basic Clin Pharm 3(2):283–293
14. Bristow AF (1993) Recombinant-DNA-derived insulin analogues as potentially useful thera-
peutic agents. Trends Biotechnol 11(7):301–305
15. Johnson IS (1983) Human insulin from recombinant DNA technology. Science 219
(4585):632–637
16. Herring R, Russell-Jones DDL (2018) Lessons for modern insulin development. Diabet Med
35(10):1320–1328
17. Vajo Z, Fawcett J, Duckworth WC (2001) Recombinant DNA technology in the treatment of
diabetes: insulin analogs. Endocr Rev 22(5):706–717
18. Brange J, Langkjoer L (1993) Insulin structure and stability. Pharm Biotechnol 5:315–350
19. Beals JM, Kovach P (2008) Insulin. In: Healthcare I (ed) Pharmaceutical technology: funda-
mentals and applications. Wiley, Hoboken
20. Sanchez-Garcia L, Martin L, Mangues R, Ferrer-Miralles N, Vazquez E, Villaverde A (2016)
Recombinant pharmaceuticals from microbial cells: a 2015 update. Microb Cell Factories
15:33
21. Agency EM (2018) Semglee. https://www.ema.europa.eu/en/documents/product-information/
semglee-epar-product-information_en.pdf
22. Walsh G (2005) Therapeutic insulins and their large-scale manufacture. Appl Microbiol
Biotechnol 67(2):151–159
23. Administration FaD (1982) Humulin®. https://www.accessdata.fda.gov/drugsatfda_docs/
label/2018/018780s168lbl.pdf
24. Administration FaD (2012) Novolin®R. https://www.accessdata.fda.gov/drugsatfda_docs/
25. Administration FaD (2011) Humulin®R. https://www.accessdata.fda.gov/drugsatfda_docs/
26. Agency EM (1997) Insuman®. https://www.ema.europa.eu/en/documents/product-informa
tion/insuman-epar-product-information_en.pdf
27. Agency EM (2002) Actrapid®. https://www.ema.europa.eu/en/documents/product-informa
tion/actrapid-epar-product-information_en.pdf
28. Administration FaD (1999) Velosulin® BR. https://www.accessdata.fda.gov/drugsatfda_docs/
label/1999/21028lbl.pdf
29. Administration FaD (2006) Exubera®. https://www.accessdata.fda.gov/drugsatfda_docs/label/
2006/021868lbl.pdf
30. Agency EM (2006) Exubera®. https://www.ema.europa.eu/en/documents/product-informa
tion/exubera-epar-product-information_en.pdf
31. Agency EM (2007) Insulin human Winthrop. https://www.ema.europa.eu/en/documents/prod
uct-information/insulin-human-winthrop-epar-product-information_en.pdf
32. Administration FaD (2014) Afrezza®. https://www.accessdata.fda.gov/drugsatfda_docs/label/
2014/022472lbl.pdf
33. Agency EM (2015) Solumarv. https://www.ema.europa.eu/en/medicines/human/EPAR/
solumarv-0
34. Administration FaD (1996) Humalog®. https://www.accessdata.fda.gov/drugsatfda_docs/
35. Agency EM (1996) Humalog®. https://www.ema.europa.eu/en/documents/product-informa
tion/humalog-epar-product-information_en.pdf
36. Agency EM (2001) Liprolog®. https://www.ema.europa.eu/en/documents/product-informa
tion/liprolog-epar-product-information_en.pdf
37. Agency EM (2017) Insulin Lispro Sanofi. https://www.ema.europa.eu/en/documents/product-
information/insulin-lispro-sanofi-epar-product-information_en.pdf
38. Administration FaD (2017) Admelog®. https://www.accessdata.fda.gov/drugsatfda_docs/
39. Agency EM (1999) NovoRapid®. https://www.ema.europa.eu/en/documents/product-informa

tion/novorapid-epar-product-information_en.pdf
40. Administration FaD (2017) Fiasp®. https://www.accessdata.fda.gov/drugsatfda_docs/label/
2017/208751s000lbl.pdf
41. Agency EM (2017) Fiasp®. https://www.ema.europa.eu/en/documents/product-information/
fiasp-epar-product-information_en.pdf
42. Administration FaD (2001) NovoLog Mix. https://www.accessdata.fda.gov/drugsatfda_docs/
nda/2001/21172_Novolog_prntlbl.pdf
43. Administration FaD (2004) Apidra™. https://www.accessdata.fda.gov/drugsatfda_docs/label/
2004/021629lbl.pdf
44. Agency EM (2004) Apidra™. https://www.ema.europa.eu/en/medicines/human/EPAR/apidra
45. Agency EM (2002) Protaphane®. https://www.ema.europa.eu/en/documents/product-informa
tion/protaphane-epar-product-information_en.pdf
46. Agency EM (2002) Monotard®. https://www.ema.europa.eu/en/documents/product-informa
tion/monotard-epar-product-information_en.pdf
47. Agency EM (2002) Ultratard®. https://www.ema.europa.eu/en/documents/product-informa
tion/ultratard-epar-product-information_en.pdf
48. Administration FaD (2000) Lantus®. https://www.accessdata.fda.gov/drugsatfda_docs/label/
2000/21081lbl.pdf
49. Agency EM (2000) Lantus®. https://www.ema.europa.eu/en/documents/product-information/
lantus-epar-product-information_en.pdf
50. Administration FaD (2015) Toujeo®. https://www.accessdata.fda.gov/drugsatfda_docs/label/
2015/206538s000lbl.pdf
51. Agency EM (2000) Toujeo®. https://www.ema.europa.eu/en/documents/product-information/
toujeo-epar-product-information_en.pdf
52. Administration FaD (2016) Soliqua™. https://www.accessdata.fda.gov/drugsatfda_docs/
53. Agency EM (2017) Suliqua®. https://www.ema.europa.eu/en/documents/product-informa
tion/suliqua-epar-product-information_en.pdf
54. Agency EM (2014) Abasaglar. https://www.ema.europa.eu/en/documents/product-informa
tion/abasaglar-previously-abasria-epar-product-information_en.pdf
55. Administration FaD (2017) Lusduna. https://www.accessdata.fda.gov/drugsatfda_docs/
appletter/2017/208722Orig1s000TAltr.pdf
56. Agency EM (2017) Lusduna. https://www.ema.europa.eu/en/documents/product-information/
lusduna-epar-product-information_en.pdf
57. Administration FaD (2005) Levemir®. https://www.accessdata.fda.gov/drugsatfda_docs/label/
2005/021536lbl.pdf
58. Agency EM (2004) Levemir®. https://www.ema.europa.eu/en/documents/product-informa
tion/levemir-epar-product-information_en.pdf
59. Administration FaD (2015) Tresiba®. https://www.accessdata.fda.gov/drugsatfda_docs/label/
2015/203314lbl.pdf
60. Agency EM (2013) Tresiba®. https://www.ema.europa.eu/en/documents/product-information/
tresiba-epar-product-information_en.pdf
61. Administration FaD (2016) Xultophy®. https://www.accessdata.fda.gov/drugsatfda_docs/
62. Agency EM (2014) Xultophy®. https://www.ema.europa.eu/en/documents/product-informa
tion/xultophy-epar-product-information_en.pdf
63. Agency EM (2002) Actraphane®. https://www.ema.europa.eu/en/documents/product-informa
tion/actraphane-epar-product-information_en.pdf
64. Administration FaD (1988) Mixtard® Human 70/30. https://www.accessdata.fda.gov/scripts/
cder/daf/index.cfm?event¼overview.process&ApplNo¼019585
65. Agency EM (2002) Mixtard®. https://www.ema.europa.eu/en/documents/product-informa
tion/mixtard-epar-product-information_en.pdf
66. Agency EM (2002) Insulatard®. https://www.ema.europa.eu/en/documents/product-informa

tion/insulatard-epar-product-information_en.pdf
67. Administration FaD (1999) Humalog® Mix. https://www.accessdata.fda.gov/drugsatfda_docs/
68. Agency EM (2001) Liprolog Mix®. https://www.ema.europa.eu/en/documents/product-infor
mation/liprolog-epar-product-information_en.pdf
69. Agency EM (2000) NovoMix®. https://www.ema.europa.eu/en/documents/product-informa
tion/novomix-epar-product-information_en.pdf
70. Administration FaD (2015) Ryzodeg® 70/30. https://www.accessdata.fda.gov/drugsatfda_
docs/label/2015/203313lbl.pdf
71. Agency EM (2013) Ryzodeg® 70/30. https://www.ema.europa.eu/en/documents/product-infor
mation/ryzodeg-epar-product-information_en.pdf
72. Corporation M (2019) Afrezza® (insulin human) inhalation powder approved in Brazil. https://
www.globenewswire.com/news-release/2019/06/03/1863179/0/en/Afrezza-insulin-human-Inha
lation-Powder-Approved-in-Brazil.html
73. Bhatia A, Tawade S, Mastim M, Kitabi EN, Gopalakrishnan M, Shah M et al (2018)
Comparative evaluation of pharmacokinetics and pharmacodynamics of insulin glargine
(Glaritus((R))) and Lantus((R)) in healthy subjects: a double-blind, randomized clamp study.
Acta Diabetol 55(5):461–468
74. Administration FaD (2015) Basaglar®. https://www.accessdata.fda.gov/drugsatfda_docs/
75. Davies M, Dahl D, Heise T, Kiljanski J, Mathieu C (2017) Introduction of biosimilar insulins
in Europe. Diabet Med 34(10):1340–1353
76. Agency EM (2015) Orphan designation: treatment of short bowel syndrome. https://www.
ema.europa.eu/en/medicines/human/orphan-designations/eu3151532
77. Muheem A, Shakeel F, Jahangir MA, Anwar M, Mallick N, Jain GK et al (2016) A review on
the strategies for oral delivery of proteins and peptides and their clinical perspectives. Saudi
Pharm J 24(4):413–428
78. Shahani S, Shahani L (2015) Use of insulin in diabetes: a century of treatment. Hong Kong
Med J 21(6):553–559
79. Asquetiv (2019) MonoSol Rx announces initiation of phase 2a trial for oral insulin film.
https://aquestive.com/monosol-rx-announces-initiation-of-phase-2a-trial-for-oral-insulin-film/
80. Trials C (2017) Comparison of insulin tregopil (IN-105) with insulin aspart in type 2 diabetes
mellitus patients. https://clinicaltrials.gov/ct2/show/NCT03430856
81. Cengiz E, Bode B, Van Name M, Tamborlane WV (2016) Moving toward the ideal insulin for
insulin pumps. Expert Rev Med Devices 13(1):57–69
82. Schaepelynck P (2019) The implantable insulin pump. Handbook of diabetes technology.
Springer, Berlin, pp 47–55
83. Bally L, Thabit H, Hovorka R (2017) Finding the right route for insulin delivery – an overview
of implantable pump therapy. Expert Opin Drug Deliv 14(9):1103–1111
84. Bally L, Thabit H, Kojzar H, Mader JK, Qerimi-Hyseni J, Hartnell S et al (2017) Day-and-
night glycaemic control with closed-loop insulin delivery versus conventional insulin pump
therapy in free-living adults with well controlled type 1 diabetes: an open-label, randomised,
crossover study. Lancet Diabetes Endocrinol 5(4):261–270
85. Renard E, Tubiana-Rufi N, Bonnemaison-Gilbert E, Coutant R, Dalla-Vale F, Farret A et al
(2019) Closed-loop driven by control-to-range algorithm outperforms threshold-low-glucose-
suspend insulin delivery on glucose control albeit not on nocturnal hypoglycaemia in prepu-
bertal patients with type 1 diabetes in a supervised hotel setting. Diabetes Obes Metab 21
(1):183–187
86. Heinemann L, Nosek L, Flacke F, Albus K, Krasner A, Pichotta P et al (2012) U-100,
pH-neutral formulation of VIAject((R)): faster onset of action than insulin lispro in patients
with type 1 diabetes. Diabetes Obes Metab 14(3):222–227
87. Hompesch M, McManus L, Pohl R, Simms P, Pfutzner A, Bulow E et al (2008) Intra-

individual variability of the metabolic effect of a novel rapid-acting insulin (VIAject) in
comparison to regular human insulin. J Diabetes Sci Technol 2(4):568–571
88. Steiner S, Hompesch M, Pohl R, Simms P, Flacke F, Mohr T et al (2008) A novel insulin
formulation with a more rapid onset of action. Diabetologia 51(9):1602–1606
89. Home PD (2015) Plasma insulin profiles after subcutaneous injection: how close can we get to
physiology in people with diabetes? Diabetes Obes Metab 17(11):1011–1020
90. Adocia (2019) Biochaperone® Lispro. https://www.adocia.com/products/biochaperone-ultra-
fast-analog-insulin/
91. Danne T, Heinemann L, Bolinder J (2019) New insulins, biosimilars, and insulin therapy.
Diabetes Technol Ther 21(S1):S57–S78
92. Lilly E (2019) Regulatory review: ultra-rapid lispro. https://www.lilly.com/discovery/pipeline
93. Heise T, Hovelmann U, Brondsted L, Adrian CL, Nosek L, Haahr H (2015) Faster-acting
insulin aspart: earlier onset of appearance and greater early pharmacokinetic and pharmaco-
dynamic effects than insulin aspart. Diabetes Obes Metab 17(7):682–688
94. Nordisk N (2015) Novo Nordisk completes phase 3a trials comparing faster-acting insulin
aspart with NovoRapid® in people with type 1 and type 2 diabetes. https://www.novonordisk.
com/bin/getPDF.1906174.pdf
95. Morrow L, Muchmore DB, Hompesch M, Ludington EA, Vaughn DE (2013) Comparative
pharmacokinetics and insulin action for three rapid-acting insulin analogs injected subcutane-
ously with and without hyaluronidase. Diabetes Care 36(2):273–275
96. Garg SK, Buse JB, Skyler JS, Vaughn DE, Muchmore DB (2014) Subcutaneous injection of
hyaluronidase with recombinant human insulin compared with insulin lispro in type 1 diabetes.
Diabetes Obes Metab 16(11):1065–1069
97. Lilly E (2019) Medicines in development. https://www.lilly.com/discovery/pipeline
98. Lilly E (2019) LY3209590, “basal insulin-fc”. https://clinicaltrials.gov/ct2/results?cond¼&
term¼LY3209590&cntry¼&state¼&city¼&dist¼
99. Committee NCCCMCM, Ansite J, Balamurugan AN, Barbaro B, Battle J, Brandhorst D et al
(2017) Purified human pancreatic islets, CIT culture media with lisofylline or exenatide.
CellR4 Repair Replace Regen Reprogram 5(3):e2377
100. Wu T, Rayner CK, Marathe CS, Jones KL, Horowitz M (2018) Glucagon receptor signalling –
backwards and forwards. Expert Opin Investig Drugs 27(2):135–138
101. Patent US (2018) Glucagon analogues. https://patentimages.storage.googleapis.com/d9/06/b2/
6d4c89f64b2551/US9975939.pdf
102. Hayashi Y, Seino Y (2018) Regulation of amino acid metabolism and alpha-cell proliferation
by glucagon. J Diabetes Investig 9(3):464–472
103. Burrin DG, Petersen Y, Stoll B, Sangild P (2001) Glucagon-like peptide 2: a nutrient-
responsive gut growth factor. J Nutr 131(3):709–712
104. Administration FaD (1998) GlucaGen®. https://www.accessdata.fda.gov/drugsatfda_docs/
105. Administration FaD (1998) Recombinant Glucagon. https://www.accessdata.fda.gov/
drugsatfda_docs/nda/98/20928.pdf
106. Agency EM (2019) Orphan designation: noninsulinoma pancreatogenous hypoglycaemia
syndrome. https://www.ema.europa.eu/en/medicines/human/orphan-designations/eu3182091
107. Agency EM (2019) Orphan designations: congenital hyperinsulinism. https://www.ema.
europa.eu/en/medicines/human/orphan-designations/eu3141342
108. Agency EM (2012) Orphan designations: congenital hyperinsulinism. https://www.ema.
109. Agency EM (2018) Glucagon analogue linked to a human immunoglobulin Fc fragment (also
known as HM15136). https://www.ema.europa.eu/en/medicines/human/orphan-designations/
eu3182022
110. Agency EM (2015) Saxenda®. https://www.ema.europa.eu/en/documents/product-informa
tion/saxenda-epar-product-information_en.pdf
111. Administration FaD (2014) Saxenda®. https://www.accessdata.fda.gov/drugsatfda_docs/label/

2014/206321Orig1s000lbl.pdf
112. Administration FaD (2010) Victoza®. https://www.accessdata.fda.gov/drugsatfda_docs/label/
2010/022341lbl.pdf
113. Agency EM (2009) Victoza®. https://www.ema.europa.eu/en/documents/product-informa
tion/victoza-epar-product-information_en.pdf
114. Agency EM (2018) Ozempic®. https://www.ema.europa.eu/en/documents/product-informa
tion/ozempic-epar-product-information_en.pdf
115. Administration FaD (2017) Ozempic®. https://www.accessdata.fda.gov/drugsatfda_docs/
116. Agency EM (2014) Trulicity®. https://www.ema.europa.eu/en/documents/product-informa
tion/trulicity-epar-product-information_en.pdf
117. Administration FaD (2014) Trulicity™. https://www.accessdata.fda.gov/drugsatfda_docs/
nda/2014/125469Orig1s000Lbl.pdf
118. Agency EM (2012) Revestive®. https://www.ema.europa.eu/en/documents/product-informa
tion/revestive-epar-product-information_en.pdf
119. Administration FaD (2012) Gattex®. https://www.accessdata.fda.gov/drugsatfda_docs/label/
2012/203441Orig1s000lbl.pdf
120. Agency EM (2018) Orphan designation: apraglutide. https://www.ema.europa.eu/en/documents/
orphan-designation/eu/3/18/2102-public-summary-opinion-orphan-designation-apraglutide-treat
ment-short-bowel-syndrome_en.pdf
121. Agency EM (2019) Orphan designation: human glucagon-like peptide-2 analogue linked to a
human immunoglobulin Fc fragment. https://www.ema.europa.eu/en/documents/orphan-designa
tion/eu/3/18/2126-public-summary-opinion-orphan-designation-human-glucagon-peptide-2-ana
logue-linked-human_en.pdf
122. Hovelmann U, Bysted BV, Mouritzen U, Macchi F, Lamers D, Kronshage B et al (2018)
Pharmacokinetic and pharmacodynamic characteristics of dasiglucagon, a novel soluble and
stable glucagon analog. Diabetes Care 41(3):531–537
123. Agency EM (2018) Dasiglucagon. https://www.ema.europa.eu/en/documents/pip-decision/p/
0220/2018-ema-decision-19-july-2018-agreement-paediatric-investigation-plan-granting-defer
ral_en-1.pdf
124. Company ELa (2015) Intranasal glucagon: phase III clinical trials. http://lilly.mediaroom.com/
index.php?s¼9042&item¼137474
125. Pharmaceuticals X (2018) Xeris Pharmaceuticals announces positive phase 3 clinical trial data
on its investigational ready-to-use glucagon rescue pen. https://investors.xerispharma.com/
node/6566/pdf
126. Caicedo A, Rosenfeld R (2018) Challenges and future for the delivery of growth hormone
therapy. Growth Hormon IGF Res 38:39–43
127. Marian MO, Growth Hormones J (2008) In: Healthcare I (ed) Pharmaceutical technology:
fundamentals and applications. Wiley, Hoboken, pp 281–292
128. Lal RA, Hoffman AR (2018) Long-acting growth hormone preparations in the treatment of
children. Pediatr Endocrinol Rev 16(Suppl 1):162–167
129. Administration FaD (2015) Somatropin information 2015. https://www.fda.gov/drugs/
postmarket-drug-safety-information-patients-and-providers/somatropin-information
130. Administration FaD (1987) Humatrope®. https://www.accessdata.fda.gov/drugsatfda_docs/
131. Administration FaD (2000) Norditropin®. https://www.accessdata.fda.gov/drugsatfda_docs/
132. Administration FaD (2011) Tev-Tropin®. https://www.accessdata.fda.gov/drugsatfda_docs/
133. Agency EM (2013) Somatropin Biopartners. https://www.ema.europa.eu/en/documents/prod
uct-information/somatropin-biopartners-epar-product-information_en.pdf
134. Administration FaD (1987) Saizen®. https://www.accessdata.fda.gov/drugsatfda_docs/label/

2017/019764s086lbl.pdf
135. Agency EM (2006) Valtropin®. https://www.ema.europa.eu/en/documents/product-informa
tion/valtropin-epar-product-information_en.pdf
136. Administration FaD (2007) Valtropin®. https://www.accessdata.fda.gov/drugsatfda_docs/
137. Agency EM (2013) Recombinant modified human growth hormone for the treatment of growth
hormone deficiency. https://www.ema.europa.eu/en/documents/orphan-designation/eu/3/12/
1087-public-summary-opinion-orphan-designation-recombinant-modified-human-growth-hor
mone-treatment_en.pdf
138. Administration FaD (2001) Genotropin®. https://www.accessdata.fda.gov/drugsatfda_docs/
139. Administration FaD (1995) Zomacton®. https://www.accessdata.fda.gov/drugsatfda_docs/
label/2018/180717s048s049s050s051lbl.pdf
140. Agency EM (2006) Omnitrope®. https://www.ema.europa.eu/en/documents/product-informa
tion/omnitrope-epar-product-information_en.pdf
141. Administration FaD (2006) Omnitrope™. https://www.accessdata.fda.gov/drugsatfda_docs/
142. Administration FaD (2000) Nutropin AQ®. https://www.accessdata.fda.gov/drugsatfda_docs/
143. Agency EM (2001) NutropinAq®. https://www.ema.europa.eu/en/documents/product-informa
tion/nutropinaq-epar-product-information_en.pdf
144. Administration FaD (2003) Zorbtive™. https://www.accessdata.fda.gov/drugsatfda_docs/
label/2003/20604s026_zorbtive_lbl.pdf
145. Administration FaD (1987) Serostim®. https://www.accessdata.fda.gov/drugsatfda_docs/
146. Agency EM (2000) Orphan designation: somatropin for AIDS wasting. https://www.ema.
147. Yuen KCJ, Miller BS, Biller BMK (2018) The current state of long-acting growth hormone
preparations for growth hormone therapy. Curr Opin Endocrinol Diabetes Obes 25
(4):267–273
148. Pharma A (2018) TransCon hGH. https://ascendispharma.com/wp-content/uploads/2018-09-
26-Boulder-Ascendis-Presentation.pdf
149. Strasburger CJ, Vanuga P, Payer J, Pfeifer M, Popovic V, Bajnok L et al (2017) MOD-4023, a
long-acting carboxy-terminal peptide-modified human growth hormone: results of a phase
2 study in growth hormone-deficient adults. Eur J Endocrinol 176(3):283–294
150. Ku CR, Brue T, Schilbach K, Ignatenko S, Magony S, Chung YS et al (2018) Long-acting
FC-fusion rhGH (GX-H9) shows potential for up to twice-monthly administration in
GH-deficient adults. Eur J Endocrinol 179(3):169–179
151. ClinicalTrials.gov USNLoM (2018) Trial to compare the efficacy and safety of NNC0195-
0092 (Somapacitan) with placebo and Norditropin® FlexPro® (Somatropin) in adults with
growth hormone deficiency. https://clinicaltrials.gov/ct2/show/NCT02229851
152. Banker MJ, Hinduja R, Sathe S, Arora S (2019) Infertility and assisted reproductive
technology1st edn. Jaypee Brothers Medical Publishers, New Delhi
153. Ben-Menahem D (2018) Preparation, characterization and application of long-acting FSH
analogs for assisted reproduction. Theriogenology 112:11–17
154. Bernard DJ, Li Y, Toufaily C, Schang G (2019) Regulation of gonadotropins. Oxford
University Press, Oxford
155. Sam T (2008) Follicle-stimulating hormones. In: Healthcare I (ed) Pharmaceutical technology:
fundamentals and applications. Wiley, Hoboken, pp 399–404
156. Anderson RC, Newton CL, Anderson RA, Millar RP (2018) Gonadotropins and their analogs:
current and potential clinical applications. Endocr Rev 39(6):911–937
157. Agency EM (1995) Gonal-f®. https://www.ema.europa.eu/en/medicines/human/EPAR/gonal-f
158. Administration FaD (1995) Gonal-f®. https://www.accessdata.fda.gov/drugsatfda_docs/label/

2004/20378scf015_gonal_lbl.pdf
159. Agency EM (2013) Ovaleap®. https://www.ema.europa.eu/en/documents/product-informa
tion/ovaleap-epar-product-information_en.pdf
160. Agency EM (2014) Bemfola®. https://www.ema.europa.eu/en/medicines/human/EPAR/
bemfola
161. Agency EM (1996) Puregon®. https://www.ema.europa.eu/en/medicines/human/EPAR/
puregon
162. Administration FaD (2004) Follistim® AQ. https://www.accessdata.fda.gov/drugsatfda_docs/
label/2004/21211_Follistim_lbl.pdf
163. Agency EM (2009) Fertavid®. https://www.ema.europa.eu/en/medicines/human/EPAR/
fertavid
164. Agency EM (2016) Rekovelle®. https://www.ema.europa.eu/en/medicines/human/EPAR/
rekovelle
165. Agency EM (2010) Elonva®. https://www.ema.europa.eu/en/documents/product-information/
elonva-epar-product-information_en.pdf
166. Agency EM (2000) Luveris®. https://www.ema.europa.eu/en/medicines/human/EPAR/luveris
167. Administration FaD (2004) Luveris®. https://www.accessdata.fda.gov/drugsatfda_docs/nda/
2004/021322s000_LuverisTOC.cfm
168. Agency EM (2007) Pergoveris®. https://www.ema.europa.eu/en/medicines/human/EPAR/
pergoveris
169. Agency EM (2001) Ovitrelle®. https://www.ema.europa.eu/en/medicines/human/EPAR/
ovitrelle
170. Administration FaD (2000) Ovidrel®. https://www.accessdata.fda.gov/drugsatfda_docs/label/
2000/21149lbl.pdf
171. Majumdar A, Sachan R, Nandanwar YS, Mayekar RV, Soni N, Banker MR et al (2019) A
multicenter, randomized, equivalence trial of a new recombinant human chorionic gonadotro-
pin preparation versus ovitrelle((R)) for ovulation in women undergoing intrauterine insem-
ination following ovarian stimulation. J Hum Reprod Sci 12(1):53–58
172. Zander-Fox D, Lane M, Hamilton H, Tremellen K (2018) Sequential clomiphene/
corifollitrophin alpha as a technique for mild controlled ovarian hyperstimulation in IVF: a
proof of concept study. J Assist Reprod Genet 35(6):1047–1052
173. Cozzolino M, Vitagliano A, Cecchino GN, Ambrosini G, Garcia-Velasco JA (2019)
Corifollitropin alfa for ovarian stimulation in in vitro fertilization: a systematic review and
meta-analysis of randomized controlled trials. Fertil Steril 111(4):722–733
174. Szkudlinski MW, Fremont V, Ronin C, Weintraub BD (2002) Thyroid-stimulating hormone
and thyroid-stimulating hormone receptor structure-function relationships. Physiol Rev 82
(2):473–502
175. Goltzman D (2018) Physiology of parathyroid hormone. Endocrinol Metab Clin 47
(4):743–758
176. Agency EM (2000) Thyrogen®. https://www.ema.europa.eu/en/medicines/human/EPAR/
thyrogen
177. Administration FaD (1998) Thyrogen®. https://www.accessdata.fda.gov/drugsatfda_docs/
nda/98/20898_Thyrogen_prntlbl.pdf
178. Agency EM (2003) Forsteo. https://www.ema.europa.eu/en/documents/product-information/
forsteo-epar-product-information_en.pdf
179. Administration FaD (2002) Forteo. https://www.accessdata.fda.gov/drugsatfda_docs/label/
2002/21318_forteo_lbl.pdf
180. Agency EM (2017) Terrosa®. https://www.ema.europa.eu/en/documents/product-information/
terrosa-epar-product-information_en.pdf
181. Agency EM (2017) Movymia®. https://www.ema.europa.eu/en/documents/product-informa
tion/movymia-epar-product-information_en.pdf
182. Agency EM (2017) Natpar®. https://www.ema.europa.eu/en/documents/product-information/

natpar-epar-product-information_en.pdf
183. Administration FaD (2015) Natpara®. https://www.accessdata.fda.gov/drugsatfda_docs/label/
2015/125511s000lbl.pdf
184. Pharma A (2019) TransCon PTH. https://ascendispharma.com/product-pipeline/transcon-pth/
185. Williams AJ, Jordan F, King G, Lewis AL, Illum L, Masud T et al (2018) In vitro and
preclinical assessment of an intranasal spray formulation of parathyroid hormone PTH 1-34 for
the treatment of osteoporosis. Int J Pharm 535(1–2):113–119
186. Cho M, Han S, Kim H, Kim KS, Hahn SK (2018) Hyaluronate – parathyroid hormone
peptide conjugate for transdermal treatment of osteoporosis. J Biomater Sci Polym Ed 29
(7–9):793–804
187. Avecilla ST (2019) Coagulation factor products. In: Shaz BH, Hillyer CD, Reyes Gil M (eds)
Transfusion medicine and hemostasis3rd edn. Elsevier, Amsterdam, pp 251–260
188. Walsh G (2007) Recombinant blood products and therapeutic enzymes. In: Wiley (ed)
Pharmaceutical biotechnology: concepts and applications. Wiley, Hoboken, pp 329–370
189. Bhopale GMN, Recombinant RK (2005) DNA expression products for human therapeutic use.
Curr Sci 89(4):614–622
190. Steinberg FM, Raso J (1998) Biotech pharmaceuticals and biotherapy: an overview. J Pharm
Pharm Sci 1(2):48–59
191. Modi NB (2008) Recombinant coagulation factors and thrombolytic agents. In: Crommelin
DJASR, Meibohm B (eds) Pharmaceutical biotechnology: fundamentals and applications:
informa healthcare, pp 293–308
192. Frampton JE (2016) Efmoroctocog alfa: a review in haemophilia A. Drugs 76(13):1281–1291
193. Hoy SM (2017) Eftrenonacog alfa: a review in haemophilia B. Drugs 77(11):1235–1246
194. BioMarin (2019) Current clinical trials: hemophilia A. https://www.biomarin.com/hemo
philia-a
195. Therapeutics S (2019) Hemophilia B: FIXtendz study and hemophilia A: ALTA study 2019.
https://www.sangamo.com/clinical-trials
196. uniQure (2019) GENE THERAPY: hemophilia. http://www.uniqure.com/gene-therapy/hemo
philia.php
197. FaDA (2018) Coagulation factors. https://www.fda.gov/vaccines-blood-biologics/approved-
blood-products/coagulation-factors
198. EMA (2019) NovoSeven (eptacog alfa): an overview of NovoSeven and why it is authorised in
the EU. https://www.ema.europa.eu/en/documents/overview/novoseven-epar-medicine-over
view_en.pdf
199. FaDA (2018) Antihemophilic factor (recombinant). https://www.fda.gov/vaccines-blood-bio
logics/approved-blood-products/antihemophilic-factor-recombinant
200. EMA (2018) EPAR summary for the public: Advate® (octocog alfa). https://www.ema.europa.
eu/en/documents/overview/advate-epar-summary-public_en.pdf
201. EMA (2018) EPAR summary for the public: Kogenate Bayer (octocog alfa). https://www.ema.
europa.eu/en/documents/overview/kogenate-bayer-epar-summary-public_en.pdf
202. EMA (2018) EPAR summary for the public: Helixate NexGen (octocog alfa). https://www.
ema.europa.eu/en/documents/overview/helixate-nexgen-epar-summary-public_en.pdf
203. EMA (2018) EPAR summary for the public: Kovaltry (octocog alfa). https://www.ema.
europa.eu/en/documents/overview/kovaltry-epar-summary-public_en.pdf
204. EMA (2016) EPAR summary for the public: Iblias (octocog alfa). https://www.ema.europa.eu/
en/documents/overview/iblias-epar-summary-public_en.pdf
205. EMA (2018) NovoEight (turoctocog alfa): an overview of NovoEight and why it is authorised
in the EU. https://www.ema.europa.eu/en/documents/overview/novoeight-epar-medicine-over
view_en.pdf
206. EMA (2017) EPAR summary for the public: Afstyla (lonoctocog alfa). https://www.ema.
europa.eu/en/documents/overview/afstyla-epar-summary-public_en.pdf
207. EMA (2015) EPAR summary for the public: Obizur (susoctocog alfa). https://www.ema.
europa.eu/en/documents/overview/obizur-epar-summary-public_en.pdf
208. EMA (2018) EPAR summary for the public: Adynovi (rurioctocog alfa pegol). https://www.
ema.europa.eu/en/documents/overview/adynovi-epar-summary-public_en.pdf
209. EMA (2018) Jivi (damoctocog alfa pegol): an overview of Jivi and why it is authorised in the
EU. https://www.ema.europa.eu/en/documents/overview/jivi-epar-medicine-overview_en.pdf
210. EMA (2018) Elocta (efmoroctocog alfa): an overview of Elocta and why it is authorised in
the EU. https://www.ema.europa.eu/en/documents/overview/elocta-epar-medicine-overview_
en.pdf
211. EMA (2016) EPAR summary for the public: ReFacto AF (moroctocog alfa). https://www.ema.
europa.eu/en/documents/overview/refacto-af-epar-summary-public_en.pdf
212. EMA (2018) EPAR summary for the public: Nuwiq (simoctocog alfa). https://www.ema.
europa.eu/en/documents/overview/nuwiq-epar-summary-public_en.pdf
213. EMA (2019) Vihuma (simoctocog alfa): an overview of Vihuma and why it is authorised in the
EU. https://www.ema.europa.eu/en/documents/overview/vihuma-epar-medicine-overview_
en.pdf
214. EMA (2018) Veyvondi (vonicog alfa): an overview of Veyvondi and why it is authorised in
the EU. https://www.ema.europa.eu/en/documents/overview/veyvondi-epar-medicine-over
view_en.pdf
215. EMA (2016) EPAR summary for the public: Alprolix (eftrenonacog alfa). https://www.ema.
europa.eu/en/documents/overview/alprolix-epar-summary-public_en.pdf
216. EMA (2015) EPAR summary for the public: BeneFIX (nonacog alfa). https://www.ema.
europa.eu/en/documents/overview/benefix-epar-summary-public_en.pdf
217. EMA (2015) EPAR summary for the public: Rixubis (nonacog gamma). https://www.ema.
europa.eu/en/documents/overview/rixubis-epar-summary-public_en.pdf
218. EMA (2017) EPAR summary for the public: Refixia (nonacog beta pegol). https://www.ema.
europa.eu/en/documents/overview/refixia-epar-summary-public_en.pdf
219. FaDA (2017) Prescribing information for IDELVION®. https://www.fda.gov/media/96526/
download
220. EMA (2016) EPAR summary for the public: Idelvion (albutrepenonacog alfa). https://www.
ema.europa.eu/en/documents/overview/idelvion-epar-summary-public_en.pdf
221. Baker DE (2018) Coagulation factor Xa (Recombinant), inactivated-zhzo (Andexanet Alfa).
Hosp Pharm 53(5):286–291
222. FaDA (2018) ANDEXXA (coagulation factor Xa (recombinant), inactivated-zhzo). https://
www.fda.gov/vaccines-blood-biologics/cellular-gene-therapy-products/andexxa-coagulation-
factor-xa-recombinant-inactivated-zhzo
223. FaDA (2018) TRETTEN®. https://www.fda.gov/vaccines-blood-biologics/approved-blood-
products/tretten
224. EMA (2012) EPAR summary for the public: NovoThirteen (catridecacog). https://www.ema.
europa.eu/en/documents/overview/novothirteen-epar-summary-public_en.pdf
225. EMA (2012) Refludan (lepirudin). https://www.ema.europa.eu/en/medicines/human/EPAR/
refludan
226. FaDA (2004) Prescribing information for REFLUDAN®. https://www.accessdata.fda.gov/
227. EMA (2007) Revasc (desirudin). https://www.ema.europa.eu/en/medicines/human/EPAR/
revasc
228. FaDA (2014) Prescribing information for IPRIVASK®. https://www.accessdata.fda.gov/
229. FaDA (2018) ATryn. https://www.fda.gov/vaccines-blood-biologics/approved-blood-prod
ucts/atryn
230. EMA (2016) EPAR summary for the public: ATryn (antithrombin alfa). https://www.ema.
europa.eu/en/documents/overview/atryn-epar-summary-public_en.pdf
231. EMA (2009) EPAR summary for the public: Xigris (drotrecogin alfa (activated)). https://
www.ema.europa.eu/en/documents/overview/xigris-epar-summary-public_en.pdf
232. EMA (2002) Actilyse. https://www.ema.europa.eu/en/medicines/human/referrals/actilyse
233. FaDA (2018) Cathflo® Activase® (Alteplase). https://www.accessdata.fda.gov/drugsatfda_
docs/label/2018/103172s5260lbl.pdf
234. EMA (2000) Ecokinase (reteplase). https://www.ema.europa.eu/en/medicines/human/EPAR/
ecokinase#product-information-section
235. EMA (2016) EPAR summary for the public: rapilysin (reteplase). https://www.ema.europa.eu/
en/documents/overview/rapilysin-epar-summary-public_en.pdf
236. FaDA (2010) Reteplase product approval information – licensing action. https://web.archive.org/
web/20130523120940/https://www.fda.gov/Drugs/DevelopmentApprovalProcess/HowDrugsare
DevelopedandApproved/ApprovalApplications/TherapeuticBiologicApplications/ucm093343.
htm
237. EMA (2005) Tenecteplase Boehringer Ingelheim Pharma GmbH Co. KG (tenecteplase).
https://www.ema.europa.eu/en/medicines/human/EPAR/tenecteplase-boehringer-ingelheim-
pharma-gmbh-co-kg
238. EMA (2014) EPAR summary for the public: Metalyse (tenecteplase). https://www.ema.
europa.eu/en/documents/overview/metalyse-epar-summary-public_en.pdf
239. FaDA (2018) TNKase® (Tenecteplase). https://www.accessdata.fda.gov/drugsatfda_docs/
240. Dutta TK, Verma SP (2014) Rational use of recombinant factor VIIa in clinical practice. Indian
J Hematol Blood Transfus 30(2):85–90
241. Moorkens E, Meuwissen N, Huys I, Declerck P, Vulto AG, Simoens S (2017) The market of
biopharmaceutical medicines: a snapshot of a diverse industrial landscape. Front Pharmacol
8:314
242. Biron-Andreani C, Schved J-F (2019) Eptacog beta: a novel recombinant human factor VIIa
for the treatment of hemophilia A and B with inhibitors. Expert Rev Hematol 12(1):21–28
243. Ezban M, Vad K, Kjalke M (2014) Turoctocog alfa (NovoEight(R))--from design to clinical
proof of concept. Eur J Haematol 93(5):369–376
244. Ahmadian H, Hansen EB, Faber JH, Sejergaard L, Karlsson J, Bolt G et al (2016) Molecular
design and downstream processing of turoctocog alfa (NovoEight), a B-domain truncated
factor VIII molecule. Blood Coagul Fibrinolysis 27(5):568–575
245. Takedani H, Hirose J (2015) Turoctocog alfa: an evidence-based review of its potential in the
treatment of hemophilia A. Drug Des Devel Ther 9:1767–1772
246. Lieuw K (2017) Many factor VIII products available in the treatment of hemophilia A: an
embarrassment of riches? J Blood Med 8:67–73
247. Baunsgaard D, Nielsen AD, Nielsen PF, Henriksen A, Kristensen AK, Bagger HW et al (2018)
A comparative analysis of heterogeneity in commercially available recombinant factor VIII
products. Haemophilia 24(6):880–887
248. Keating GM, Dhillon S (2012) Octocog alfa (Advate(R)): a guide to its use in hemophilia
A. BioDrugs 26(4):269–273
249. Tiede A, Brand B, Fischer R, Kavakli K, Lentz SR, Matsushita T et al (2013) Enhancing the
pharmacokinetic properties of recombinant factor VIII: first-in-human trial of glycoPEGylated
recombinant factor VIII in patients with hemophilia A. J Thromb Haemost 11(4):670–678
250. Wynn TT, Gumuscu B (2016) Potential role of a new PEGylated recombinant factor VIII for
hemophilia A. J Blood Med 7:121–128
251. Peyvandi F, Kouides P, Turecek PL, Dow E, Berntorp E (2019) Evolution of replacement
therapy for von Willebrand disease: from plasma fraction to recombinant von Willebrand
factor. Blood Rev. https://doi.org/10.1016/j.blre.2019.04.001
252. Peyvandi F, Mamaev A, Wang J-D, Stasyshyn O, Timofeeva M, Curry N et al (2019) Phase
3 study of recombinant von Willebrand factor in patients with severe von Willebrand disease
who are undergoing elective surgery. J Thromb Haemost 17(1):52–62
253. Crowther M, Levy GG, Lu G, Leeds J, Lin J, Pratikhya P et al (2014) A phase 2 randomized,
double-blind, placebo-controlled trial demonstrating reversal of edoxaban-induced
anticoagulation in healthy subjects by andexanet alfa (PRT064445), a universal antidote for
factor Xa (fXa) inhibitors. Blood 124(21):4269
254. Korte W (2014) Catridecacog: a breakthrough in the treatment of congenital factor XIII
A-subunit deficiency? J Blood Med 5:107–113
255. Sottilotta G, Luise F, Oriana V, Piromalli A, Santacroce R, Lelio AD (2019) Use of
Catridecacog in a patient with severe factor XIII deficiency undergoing surgery. Hematol
Rep 11(1):7912
256. Fenton JW, Ofosu FA, Brezniak DV, Hassouna HI (1998) Thrombin and antithrombotics.
Semin Thromb Hemost 24(02):87–91
257. EMA (2016) EPAR summary for the public: thorinane (enoxaparin sodium). https://www.
ema.europa.eu/en/documents/overview/thorinane-epar-summary-public_en.pdf
258. EMA (2016) Lovenox and associated names. https://www.ema.europa.eu/en/medicines/
human/referrals/lovenox-associated-names
259. EMA (2019) EPAR summary for the public: Inhixa (Enoxaparin sodium). https://www.ema.
europa.eu/en/documents/overview/inhixa-epar-summary-public_en.pdf
260. FaDA (2015) Lovenox (enoxaparin) information. https://www.fda.gov/drugs/postmarket-
drug-safety-information-patients-and-providers/lovenox-enoxaparin-information
261. Lee S, Gibson CM (2007) Enoxaparin in acute coronary syndromes. Expert Rev Cardiovasc
Ther 5(3):387–399
262. Greinacher A, Warkentin TE, Chong BH (2019) Heparin-induced thrombocytopenia. In:
Michelson AD (ed) Platelets4th edn. Academic Press, Cambridge, pp 741–767
263. Rydel TJ, Ravichandran KG, Tulinsky A, Bode W, Huber R, Roitsch C et al (1990) The
structure of a complex of recombinant hirudin and human alpha-thrombin. Science 249
(4966):277–280
264. Rydel TJ, Tulinsky A, Bode W, Huber R (1991) Refined structure of the hirudin-thrombin
complex. J Mol Biol 221(2):583–601
265. Lubenow N, Eichler P, Lietz T, Greinacher A (2005) Lepirudin in patients with heparin-
induced thrombocytopenia – results of the third prospective study (HAT-3) and a combined
analysis of HAT-1, HAT-2, and HAT-3. J Thromb Haemost 3(11):2428–2436
266. Tardy B, Lecompte T, Boelhen F, Tardy-Poncet B, Elalamy I, Morange P et al (2006)
Predictive factors for thrombosis and major bleeding in an observational study in 181 patients
with heparin-induced thrombocytopenia treated with lepirudin. Blood 108(5):1492–1496
267. El-Mowafi H, El Araby A, Kandil Y, Zaghloul A (2018) Randomized, double-blind, placebo-
controlled, interventional phase IV investigation to assess the efficacy and safety of r-hirudin
gel (1120I.U) in patients with hematomas. Res Pract Thromb Haemost 2(1):139–146
268. Corral J, de la Morena-Barrio ME, Vicente V (2018) The genetics of antithrombin. Thromb
Res 169:23–29
269. Echelard Y, Meade HM, Ziomek CA (2008) The first biopharmaceutical from transgenic
animals: ATryn®. Wiley, Hoboken, pp 995–1020
270. EMA (2013) EPAR summary for the public: Fabrazyme (agalsidase beta). https://www.ema.
europa.eu/en/documents/overview/fabrazyme-epar-summary-public_en.pdf
271. EMA (2015) EPAR summary for the public: Replagal (agalsidase alfa). https://www.ema.
europa.eu/en/documents/overview/replagal-epar-summary-public_en.pdf
272. FaDA (2018) Prescribing information: Fabrazyme (agalsidase beta). https://www.accessdata.
fda.gov/drugsatfda_docs/label/2018/103979s5303lbl.pdf
273. Vellard M (2003) The enzyme as drug: application of enzymes as pharmaceuticals. Curr Opin
Biotechnol 14(4):444–450
274. FaDA (2018) Prescribing information for Activase (alteplase). https://www.accessdata.fda.
gov/drugsatfda_docs/label/2018/103172s5259lbl.pdf
275. EMA (2018) Oncaspar (pegaspargase): an overview of Oncaspar and why it is authorised in
the EU. https://www.ema.europa.eu/en/documents/overview/oncaspar-epar-medicines-over
view_en.pdf
276. EMA (2015) EPAR summary for the public: Spectrila (asparaginase). https://www.ema.
europa.eu/en/documents/overview/spectrila-epar-summary-public_en.pdf
277. FaDA (2013) Prescribing information: ELSPAR® (asparaginase). https://www.accessdata.fda.
278. EMA (2012) Public summary of opinion on orphan designation: pegylated recombinant
Erwinia chrysanthemi L-asparaginase for the treatment of acute lymphoblastic leukaemia.
https://www.ema.europa.eu/en/documents/orphan-designation/eu/3/11/889-public-summary-
opinion-orphan-designation-pegylated-recombinant-erwinia-chrysanthemi-l_en.pdf
279. FaDA (2011) Prescribing information: ERWINAZE (asparaginase Erwinia chrysanthemi).
https://www.accessdata.fda.gov/drugsatfda_docs/label/2011/125359lbl.pdf
280. EMA (2018) Withdrawal of the marketing authorisation application for Graspa
(L-asparaginase). https://www.ema.europa.eu/en/documents/medicine-qa/questions-answers-
withdrawal-marketing-authorisation-application-graspa-l-asparaginase_en.pdf
281. EMA (2009) Public summary of positive opinion for orphan designation of L-asparaginase
encapsulated in erythrocytes for the treatment of pancreatic cancer. https://www.ema.europa.
designation-l-asparaginase-encapsulated-erythrocytes_en.pdf
282. EMA (2013) Public summary of opinion on orphan designation: L-asparaginase encapsulated in
erythrocytes for the treatment acute myeloid leukaemia. https://www.ema.europa.eu/en/docu
ments/orphan-designation/eu/3/13/1106-public-summary-positive-opinion-l-asparaginase-encap
sulated-erythrocytes-treatment-acute_en.pdf
283. FaDA (2014) Prescribing information: PULMOZYME® (dornase alfa). https://www.
accessdata.fda.gov/drugsatfda_docs/label/2014/103532s5175lbl.pdf
284. EMA (2014) EPAR summary for the public: Vimizim (elosulfase alfa). https://www.ema.
europa.eu/en/documents/overview/vimizim-epar-summary-public_en.pdf
285. FaDA (2014) Prescribing information: VIMIZIM (elosulfase alfa). https://www.accessdata.
286. EMA (2010) EPAR summary for the public: Naglazyme (galsulfase). https://www.ema.
europa.eu/en/documents/overview/naglazyme-epar-summary-public_en.pdf
287. FaDA (2013) Prescribing information: Naglazyme (galsulfase). https://www.accessdata.fda.
288. EMA (2016) EPAR summary for the public: Elaprase (idursulfase). https://www.ema.europa.
eu/en/documents/overview/elaprase-epar-summary-public_en.pdf
289. FaDA (2018) Prescribing information: ELAPRASE® (idursulfase). https://www.accessdata.
290. EMA (2015) EPAR summary for the public: Aldurazyme (laronidase). https://www.ema.
europa.eu/en/documents/overview/aldurazyme-epar-summary-public_en.pdf
291. FaDA (2003) Laronidase product approval information – licensing action. https://web.archive.org/
web/20170118085125/https://www.fda.gov/Drugs/DevelopmentApprovalProcess/HowDrugsare
DevelopedandApproved/ApprovalApplications/TherapeuticBiologicApplications/ucm080438.
htm
292. EMA (2010) EPAR summary for the public: Cerezyme (imiglucerase). https://www.ema.
europa.eu/en/documents/overview/cerezyme-epar-summary-public_en.pdf
293. FaDA (2003) Proposed text of the labeling of the drug: cerezyme (imiglucerase for injection).
https://www.accessdata.fda.gov/drugsatfda_docs/label/2005/20367s066lbl.pdf
294. EMA (2016) EPAR summary for the public: VPRIV (velaglucerase alfa). https://www.ema.
europa.eu/en/documents/overview/vpriv-epar-summary-public_en.pdf
295. FaDA (2010) Prescribing information for VPRIV™ (velaglucerase alfa for injection). https://
www.accessdata.fda.gov/drugsatfda_docs/label/2010/022575lbl.pdf
296. EMA (2015) EPAR summary for the public: Fasturtec (rasburicase). https://www.ema.europa.
eu/en/documents/overview/fasturtec-epar-summary-public_en.pdf
297. FaDA (2012) Rasburicase product approval information – licensing action 7/12/02. https://
web.archive.org/web/20120303004138/https://www.fda.gov/Drugs/
DevelopmentApprovalProcess/HowDrugsareDevelopedandApproved/ApprovalApplications/
TherapeuticBiologicApplications/ucm080525.htm
298. EMA (2018) Lamzede (velmanase alfa): an overview of Lamzede and why it is authorised in
the EU. https://www.ema.europa.eu/en/documents/overview/lamzede-epar-summary-public_
en.pdf
299. Harmatz P, Cattaneo F, Ardigò D, Geraci S, Hennermann JB, Guffon N et al (2018) Enzyme
replacement therapy with velmanase alfa (human recombinant alpha-mannosidase): novel
global treatment response model and outcomes in patients with alpha-mannosidosis. Mol
Genet Metab 124(2):152–160
300. Collen D, Lijnen RH (2005) Thrombolytic agents. Thromb Haemost 93(04):627–630
301. Hilleman DE, Tsikouris JP, Seals AA, Marmur JD (2007) Fibrinolytic agents for the manage-
ment of ST-segment elevation myocardial infarction. Pharmacotherapy 27(11):1558–1570
302. FaDA (2019) Drugs@FDA: FDA approved drug products – search results for “alteplase”.
https://www.accessdata.fda.gov/scripts/cder/daf/index.cfm?event¼BasicSearch.process
303. Moussaddy A, Demchuk AM, Hill MD (2018) Thrombolytic therapies for ischemic stroke:
triumphs and future challenges. Neuropharmacology 134:272–279
304. Kunamneni A, Ogaugwu C, Goli D (2018) Enzymes as therapeutic agents. In: Nunes CS,
Kumar V (eds) Enzymes in human and animal nutrition. Academic Press, Cambridge, pp
301–312
305. FaDA (2003) Prescribing information for ELSPAR ® (asparaginase). https://www.accessdata.
306. EMA (2015) EPAR summary for the public: Xiapex (collagenase clostridium histolyticum).
https://www.ema.europa.eu/en/documents/overview/xiapex-epar-summary-public_en.pdf
307. EMA (2012) Prescribing information for CREON (pancrelipase). https://www.accessdata.fda.
gov/drugsatfda_docs/label/2013/020725s016lbl.pdf#page¼16
308. FaDA (2010) Prescribing information for PANCREAZE™ (pancrelipase). https://www.
accessdata.fda.gov/drugsatfda_docs/label/2010/022523lbl.pdf
309. FaDA (2009) Prescribing information for ZENPEP (pancrelipase). https://www.accessdata.
310. FaDA (2012) Prescribing information for PERTZYE (pancrelipase). https://www.accessdata.
311. FaDA (2012) Prescribing information for VIOKACE (pancrelipase). https://www.accessdata.
DOI: 10.1007/10_2019_107
Advances in Vaccines
Helen H. Mao and Shoubai Chao
Contents
1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 157
1.1 Most Effective Tools in Controlling Infectious Diseases . . . . . . . . . . . . . . . . . . . . . . . . . . . . 157
1.2 Vaccine Development Life Cycle . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 159
1.3 Economics of Vaccine Development . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 160
1.4 Promoting Innovation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 160
2 Manufacturing of Vaccines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 161
2.1 General Considerations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 161
2.2 Vaccine Technologies Development . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 162
2.3 New Trends in Manufacturing of Vaccines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 164
2.4 Vaccine Manufacturing Challenges . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 165
3 Characterization of Vaccines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 166
3.1 Characterization of Bacterial Seeds . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 167
3.2 Characterization of Viral Seeds, Cell Banks, and Other Biological Materials Used
in Viral Vaccine Manufacturing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 167
3.3 Advances in Vaccine Characterization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 170
4 Clinical Aspects of Vaccine Development . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 171
4.1 Design of Clinical Trials for Vaccines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 171
4.2 Consistency Lots . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 172
4.3 Vaccine Immunogenicity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 172
4.4 Combination Vaccines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 173
4.5 Special Populations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 174
4.6 Accelerated Approval . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 174
5 Overview of Recent Approved Vaccines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 175
5.1 Shingrix Vaccine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 175
5.2 Ebola Vaccine: Ad5-EBOV . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 176
5.3 Meningococcal Subgroup B Vaccine (MenB) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 178
5.4 HPV Vaccine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 179
5.5 Dengue Vaccine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 180
5.6 EV71 Vaccine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 180
5.7 HBV Recombinant Vaccine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 181
6 Vaccines Under Development . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 182
H. H. Mao (*) and S. Chao

CanSino Biologics Inc., Tianjin, China
e-mail: helen.mao@cansinotech.com; shoubai.chao@cansinotech.com
156 H. H. Mao and S. Chao
6.1 rVSV-ZEBOV Ebola Vaccine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 182

6.2 RSV Vaccine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 183
6.3 Malaria Vaccine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 183
6.4 Other Vaccines Under Development . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 183
7 Challenges We Face . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 184
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 185
Abstract Vaccines represent one of the most important advances in science and
medicine, helping people around the world in preventing the spread of infectious
diseases. However, there are still gaps in vaccination programs in many countries.
Out of 11.2 million children born in EU region, more than 500,000 infants did not
receive the complete three-dose series of diphtheria, pertussis, and tetanus vaccine
before the first birthday. Data shows that there were more than 30,000 measles cases
in the European region in recent years, and measles cases are rising in the USA.
There are about 20 million children in the world still not getting adequate coverage
of basic vaccines. Emerging infectious diseases such as malaria, Ebola virus disease,
and Zika virus disease also threaten public health around the world. This chapter
provides an overview of recent advances in vaccine development and technologies,
manufacturing, characterization of various vaccines, challenges, and strategies in
vaccine clinical development. It also provides an overview of recently approved
major vaccines for human use.
Graphical Abstract
Regulatory Future
Strategies Trends
Vaccine Vaccine
Discovery Characterization
ation
Vaccine
Clinical
Trials
New Vaccine
Development Manufacturing
Beneits of Ebola
Vaccines Disease
Outbreaks
Process
Scale up
WHO/FDA
Newly
approved
Vaccines
Keywords Characterization, Dengue vaccine, Ebola vaccine, Recombinant

technology, Shingles vaccine, Vaccine clinical trials, Vaccine development,
Vaccine manufacturing, Viral vaccine
Advances in Vaccines 157
1 Introduction
Vaccines represent one of the best advances in science and medicine, helping people
around the world in eliminating and preventing the spread of infectious diseases.
Although many human vaccines have been developed and are in use, infectious
diseases are still threats to people’s health, especially during epidemic outbreaks.
During the 2003 SARS outbreaks in Asia, there were more than 8,000 cases, and
more than 800 deaths occurred, and the economic impact of SARS exceeded US$50
billion, according to the World Health Organization (WHO) report [1]. During the
largest Ebola outbreak in West Africa in 2014–2015, more than 28,000 cases were
reported, and there occurred more than 11,000 fatalities. It is estimated that Guinea,
Liberia, and Sierra Leone have endured more than US$2 billion loss in economic
growth as a result of Ebola virus disease outbreaks [2]. Based on the experiences
from West Africa Ebola epidemic outbreaks, the WHO published a prioritized list of
11 pathogens that likely to cause outbreak situations, including Ebola virus, Lassa
virus, Marburg virus, MRSA, Zika virus, etc. [3]. Some of these epidemic infectious
disease vaccines are currently under development. As pointed out by the WHO, it
will be a common goal for all countries to provide equitable access to high-quality,
safe, affordable vaccines and immunization services throughout the life course.
1.1 Most Effective Tools in Controlling Infectious Diseases
Vaccines have a long list of achievements in the past. Vaccines have been tremen-
dously beneficial in protecting individuals and communities from serious infectious
diseases and reducing healthcare costs around the world. In developed countries,
vaccines are more readily available to infants, children, and adults. In the European
region, more than 90% of children receive at least basic vaccination during infancy
[4]. In China, overall vaccination rates for basic vaccines that are recommended to
infants and children exceeded 90%, according to data published by the Chinese
Center for Diseases Control and Prevention (CCDC) [5].
In the USA, the Advisory Committee on Immunization Practices (ACIP)
recommends routine vaccination programs, and each vaccine is approved by the
regulatory agency US FDA. The immunization schedules are published by US CDC
for different age groups including infants and children, adolescents, and adults. For
example, the immunization schedule for infants from birth to 24 months includes
vaccines against 14 potentially serious illnesses. US CDC data from the 2017
National Immunization Survey-Child (NIS-Child) database show that the vaccina-
tion rates are higher than 80% in the USA nationally [6].
Huge progress in vaccination has been made in the last 30 years. Now about 85%
of children worldwide (about 116 million) receive essential, lifesaving vaccines,
protecting them from infectious diseases including measles, diphtheria, tetanus,
pertussis, hepatitis B, and polio. As vaccination rates increase from about 20% in
Fig. 1 Number of pertussis and measles cases and vaccine coverage % of measles, polio,
and DTP3
1980 to about 85% in 2017 (as shown in Fig. 1), the numbers of cases for measles
and pertussis have decreased significantly, according to the WHO report [7]. This
represents dedication and hard work by all, including public health workers,
researchers, pharmaceutical industries, policy makers, parents, and communities.
However, there are still gaps in vaccination programs in many countries. Out of
11.2 million children born in the EU region in 2012, more than 500,000 infants did
not receive the complete three-dose series of diphtheria, pertussis, and tetanus
vaccine before the first birthday. Data shows that there were more than 30,000
measles cases in the European region in recent years [4]. According to preliminary
WHO data, measles increased by around 300% globally in the first 3 months of
2019, compared with the same time last year, with sizable increases in all regions of
the world [7]. The reasons for children not getting their vaccines are diverse for
different regions in the world; the major reasons are lack of access to vaccination
services, and with Sub-Saharan Africa region that has the lowest coverage and the
greatest burden of cases. There are about 20 million children in the world that are still
not getting adequate coverage of basic vaccines.
As indicated in European Vaccine Action Plan 2015–2020 (EVAP) [4], EVAP’s
goal is to guide countries in the European region toward their joint vision of a region
free of vaccine-preventable diseases. It establishes six goals which include sustain-
ing polio-free status, eliminating measles and rubella, controlling hepatitis B infec-
tion, meeting regional vaccination coverage targets at all administrative levels
throughout the region, making evidence-based decisions on introduction of new
vaccines, and achieving financial sustainability of national immunization programs.
1.2 Vaccine Development Life Cycle
Vaccine development involves many stakeholders and multiple disciplines including

sciences, medicine, public health, regulatory agencies, manufacturers, healthcare
professionals, vaccine safety professionals, and consumers [8]. In many countries,
the government agencies play important roles in vaccine innovation, development,
and commercialization. For example, in the USA, the National Institutes of Health
(NIH) conducts and supports basic research, translational research, and clinical
evaluation to identify new vaccine targets and to advance new vaccine candidates
through product development pipelines. The regulatory agency US FDA is involved
in vaccine review and licensing, regulatory sciences, manufacturing inspection,
and post-licensure safety monitoring. The US Centers for Disease Control and
Prevention (CDC) identifies, controls, and prevents infectious diseases through
surveillance, detection and response, vaccine use recommendations, vaccine pur-
chasing and service delivery, health communications, and post-marketing vaccine
safety and effectiveness monitoring. Vaccines are highly regulated products and
require extensive safety monitoring.
In the USA, vaccines are regulated by the FDA Center for Biologics Evaluation
and Research (CBER) and Office of Vaccines Research and Review (OVRR), where
the authority resides in Section 351 of the US Public Health Service Act and the
Federal Food, Drug, and Cosmetic Act. CBER conducts thorough review of labo-
ratory, manufacturing, and clinical data to ensure the safety, efficacy, purity, and
potency of the vaccine products. Figure 2 illustrates the typical process of vaccine
development and licensure.
Clinical Investigational Plan Phase 4
Pre-clinical IND Stage BLA Stage Inspection

Post approval
commitment
Tox Studies Phase 1 Phase 2 Phase 3 Safety Monitoring
Lot Release
Safety Immunoge Safety Validation
Acute Tolerability nicity Immuno- Data to
toxicity support
Repeat Dose Immunogen Safety genicity
icity Dose Efficacy licensing
Toxicity Post approval
Dose ranging Ranging approval
Immunogeni Inspection changes
city New indications
Labels
Manufacturing
equipment
Facilities
Fig. 2 Stages of vaccine review and regulation

1.3 Economics of Vaccine Development
Vaccine developments are long, expensive processes with high financial risks. It
may take more than 10 years and more than US$1 billion to develop a new
innovative vaccine. In fact, cost data of developing new vaccines is usually scarce.
According to Dimitrios Gouglas [9], the average cost of successfully developing an
epidemic infectious disease vaccine from preclinical to Phase 2a is estimated to be
US$84–112 million (excluding the cost of facilities). Substantial investments are
needed to develop the vaccines for these new targets. Innovation is the key to the
future development of new vaccines to combat infectious diseases [10].
Although the cost of vaccine development is high, the benefits of vaccines are
also significant. The development and licensure of pneumococcal conjugate vaccine
(PCV) is a good example. The bacteria S. pneumoniae is the leading cause of
pneumonia mortality globally and accounted for more deaths than all other causes
(etiologies) combined in 2016. Most of these deaths occur in countries in Africa
and Asia. Each year Streptococcus pneumoniae causes approximately 3,300 cases of
meningitis, 100,000–135,000 cases of pneumonia requiring hospitalization, and six
million cases of otitis media annually in the USA [11]. Pneumococcal conjugate
vaccine 7-valent (PCV7) was approved in year 2000 for its use in the USA. It was
designed to cover the seven serotypes that account for about 80% of invasive
infections in children younger than 6 years of age. In 2007, the WHO published a
position paper recommending all countries to include PCV as part of the routine
infant immunization schedule. PCV7 and later PCV13 (13-valent pneumococcal
conjugate vaccine) have been widely used in the world. There are 142 countries
having put PCV13 into national immunization programs.
Following the introduction of the pneumococcal conjugate vaccines in the USA
(PCV7 in 2000 and PCV13 in 2010), there are about 90% reduction of pneumococ-
cal diseases. Invasive pneumococcal disease decreased from 100 cases per 100,000
people in 1998 to 9 cases per 100,000 in 2015, according to the data published by US
CDC [6]. Currently there are two approved PCV vaccines in the USA, including
PCV10 developed and marketed by GSK and PCV13 (Prevnar 13) developed and
marketed by Wyeth Pharmaceuticals (Pfizer).
1.4 Promoting Innovation
Vaccine industries have achieved tremendous successes in the development of

human vaccines that are currently in use. There are more than 200 vaccine clinical
trials ongoing with more than 120 vaccine candidates for more than 40 infectious
disease targets [12]. However, for the remaining targets, there are many significant
challenges in developing new vaccines for these targets. The remaining infectious
disease (ID) vaccine targets include those diseases affecting large populations such
as respiratory syndrome virus (RSV) disease, human immunodeficiency virus (HIV),
malaria, etc. and emerging infectious diseases such as Lassa, Marburg, Ebola,
MERS, and Zika listed in the WHO vaccine pipeline tracking sheet. As the devel-
opment of new vaccines against these remaining targets faces significant challenges,
the vaccine policy makers and the industries need to work together and encourage
innovations through collaboration and support [13, 14].
To continue promote innovations in vaccine research and development, policy
makers and regulatory agencies are recognizing the evolving development in
sciences and medicine and using new approaches in vaccine review and approval
(fast-track approach), as well as in the clinical trial design (demonstrated in the Ebola
clinical trials in Africa during the 2018–2019 outbreaks) [15].
2 Manufacturing of Vaccines
2.1 General Considerations
Vaccine manufacturing is a complex process. Vaccines take a long time to manu-

facture, ranging typically from 6 months to 36 months. During the manufacturing
process, more than 50% of production time is usually dedicated to the quality control
tests. During a vaccine manufacturing process, several hundred quality control tests
may be required before releasing a batch of vaccine products. The ability to
manufacture at the commercial scale is also very important.
New vaccine commercialization is a complex and costly process [16]. Licensing
of a new vaccine is based upon the demonstration of safety and effectiveness
(through clinical evaluation) and the ability to be manufactured in a consistent
manner (through process development and validation). The critical path toward
developing safe and effective vaccines includes the following:
• Speedy development of new technologies
• Well-designed and well-operated facilities
• Improved manufacturing methods and scale-up
• Improved analytical evaluation tools and reference standards
• Streamlined preclinical and clinical evaluations
• Improved international cooperation
• Effective vaccine review and release process
• Improved product safety monitoring programs
In the early phases of vaccine development and manufacturing, the following
information are critical for addressing product safety aspects:
• Source (biological seed or cell bank) characterization
• Raw materials including biologically derived materials (such as cell culture,
media, etc.)
• Initial scalable process development
• Initial product characterization
• Testing/qualification/clearance of impurities, contaminants

• Process control especially for safety of the products (e.g., sterilization, virus
clearance where applicable)
During the clinical development stages, the following chemical and manufactur-
ing control (CMC) activities are important, and gradually phased-in approaches are
usually taken:
• Process and product characterization
• Formulation development
• Raw material qualifications and supply management
• Component characterization
• Process and analytical method qualification
• Specification development
• Stability studies
• Manufacturing process scale-up and development
• Process control and validation
• Packaging development and label design
• Cold-chain establishment and vendor qualification
During the vaccine development and manufacturing process, applying current
good manufacturing practice (cGMP) is very important to ensure product safety,
efficacy, and consistency. It is highly recommended that cGMP be in effect for
manufacture of products used in clinical studies – starting from Phase 1. One should
follow the general approaches and principles that are broadly applicable, tailoring
cGMP applications to specific product, process, and facilities by assessing potential
risks and taking appropriate actions (risk-based approach).
2.2 Vaccine Technologies Development
A wide range of technologies have been used in developing successful vaccines,

including:
• Live attenuated bacteria and viruses (e.g., BCG, MMR, etc.)
• Inactivated bacteria and viruses (e.g., whole-cell pertussis, IPV, etc.)
• Proteins (e.g., diphtheria and tetanus toxoids)
• Polysaccharides (PneumoVax)
• Conjugated polysaccharides (e.g., meningococcal conjugate vaccine, PCV13)
• Viruslike particles (VLPs)
• Recombinant proteins
• Application of mRNA
• Adjuvant development and applications
Many vaccines involve pathogens; thus there are special requirements for
facility and equipment design depending on the biosafety levels. The manufacturing
methods include traditional egg-based influenza manufacturing or newly developed

cell culture-based bioreactor manufacturing. Traditional egg-based processes use
millions of eggs each year and a large number of manual handlings during
manufacturing and testing. Advances in automation of these steps have greatly
enhanced the process consistency and reduced the potential contamination from
human intervention. Fermentation processes often use stainless steel fermenters for
the manufacturing of many current bacterial vaccines on the markets. Cell culture-
based production processes are used in many viral vaccine products. The investiga-
tional new vaccine candidates often use single-use bioreactors. The advantages of
single-use bioreactors include faster and lower cost of initial installation, faster
development cycle, and quicker delivery of clinical trial materials. The disadvan-
tages include higher cost of operations (disposable bioreactors) and potential issues
with mechanical strengths in the joints and ports of the bioreactors.
A typical vaccine manufacturing and product release process include the follow-
ing steps:
1. Culture process: Selected bacterial strains or virus seeds or cells grow in
appropriate culture media through fermentation or cell culture to generate the
desired antigens.
2. Inactivation: Pathogens from the above culture are usually inactivated using
chemical agents (e.g., formalin) or heat (e.g., 65 C).
3. Harvesting: Remove cells and cell debris from the product stream. Antigens are
separated from the cells through filtration and/or centrifugation.
4. Purification: Impurities are removed from the harvest through purification
methods such as ultrafiltration, chromatography, etc. The antigens are also
concentrated during purification processes.
5. Detoxification of toxins: The pathogenicity is suppressed, and the immunoge-
nicity is maintained in the product.
6. Bulk drug substance: The desired antigen is collected and stored under con-
trolled temperatures for future use.
7. Formulation: Target antigens are assembled together with excipients to make
final formulation.
8. Filling process: The final formulation is filled with automated filling machine
under aseptic conditions into glass vials or prefilled syringes or other packaging
containers.
9. Outer packaging: The filled vials or syringes are packaged into the secondary
containers for future storage and shipping.
10. QC testing: Quality control laboratories conduct the quality control tests of the
intermediates and the final product.
11. QA release: Quality assurance confirms that the product has been manufactured
and tested according to approved specifications and procedures and can be
released.
12. Final release for clinical trials: For clinical trials conducted in EU region, a
qualified person (QP) releases the final product batch into the clinical trial usage.
13. Final release for distribution: For commercial vaccine products, many countries
require that the National Regulatory Agency (NRA) release the final product for
distribution into the market, such as in the USA, European Union, and China.
14. Product shipping: Most vaccine products currently are stored and shipped under
cold-chain management, typically under 2–8 C, with very few products shipped
under 60 C.
15. Product monitoring: After the product is released into the markets, product
safety monitoring for serious adverse events (SAEs) is tracked and reported
back to the manufacturers and to the regulatory agencies accordingly.
2.3 New Trends in Manufacturing of Vaccines
In recent years, there are new trends in the manufacturing of vaccine products, and
these include:
1. Use of recombinant technology
It is more often that vaccine constructions are derived from recombinant technol-
ogy, such as Shingrix vaccine, adenovirus-based Ebola vaccine (Ad5-EBOV), and
rVSV-ZEBOV Ebola vaccine [17, 18]. Recombinant technology is used in a new
recombinant pertussis vaccine development which provides higher product yield and
less impurities.
2. Single-use technology
In recent years, single-use technology has been used more and more often in
many new vaccine development and manufacturing processes. The advantages of
using single-use technologies in vaccine development and manufacturing include:
• Minimizing potential contamination
• No need for equipment cleaning between batches
• No need or less requirement for cleaning validation
• Less initial capital costs
• Fast and easy installation
The disadvantages of single-use technologies include the following:
• Need mechanical strength to avoid component breakup.
• Installation of testing probes.
• Mixing may not be as good as in stainless steel tanks.
• Potential leachable and extractable materials from the bags.
• Scalability depending on the bioreactor design.
• More suitable for viral products than bacterial products.
3. Continuous manufacturing
Recent advances in disposable manufacturing technologies and process
analytical technologies (PAT) have made the continuous manufacturing possible.
The application of continuous process in antibody manufacturing has been reported
in a number of conferences by companies such as WuXi Biologics and Amgen. The
development of purification technologies has enabled integration of continuous
processes from upstream through to downstream to final bulk drug substances
manufacturing. Application of continuous manufacturing in vaccines has gained
attention from nonprofit organizations such as Bill and Melinda Gates Foundation. It
is reported (private conversation) that application of continuous manufacturing
could result in a much lower cost of goods. The goal is to make vaccines affordable
to everyone in the world. This will greatly improve the accessibility and affordability
of vaccines in less developed nations, especially for GAVI (Global Alliance for
Vaccines and Immunisation) countries.
4. Use of animal-free components
The raw materials and media used for vaccine fermentation and purification
processes are mostly animal component-free to avoid the potential BSE/TSE risks,
especially for final products.
2.4 Vaccine Manufacturing Challenges
One of the big challenges in the manufacturing of vaccine products is that materials
used in Phase 3 clinical trials are typically manufactured in a commercial-scale
facility. Many different technology platforms are used for various vaccines. It is
difficult to standardize facilities and equipment. Unique facility and equipment are
usually necessary for each vaccine or for each family of vaccines. There is a long
lead time for building up a new commercial-scale manufacturing facility (typically
3–5 years), and large capital investment is often required.
The successful scale-up requires deep understanding of the processes and
conducting well-designed experiments to complete process scale-up. As demon-
strated in the assessment of safety and immunogenicity of two different lots of
diphtheria, tetanus, pertussis, hepatitis B, and haemophilus influenzae type b vaccine
manufactured using small- and large-scale manufacturing process, successful scale-
up was completed and proved through clinical trial results [19].
During the manufacturing of biological products including vaccines, it is often
considered that process is product, and the process/product is tightly linked with
clinical experiences and outcomes. Major changes in manufacturing facilities or
manufacturing processes may require regulatory approval or even conducting addi-
tional clinical trials. Thus for vaccine manufacturing, once a process is confirmed, it
is usually not changed unless there is a strong reason to make a major change. This
situation makes process improvements for existing vaccines challenging.
Another manufacturing challenge lies in the large-scale process validation. It is an

expensive exercise to produce full-scale process validation lots to meet regulatory
filing (e.g., line-specific real-time stability requirements for fill and finish in the
USA). In addition, vaccines are biological products that are typically with low fill
volume but very high throughput (with million to tens of million doses annually);
thus it is challenging to run product filling lines under aseptic conditions for a long
period of time. Employee training and/or utilizing isolation technology is important.
Moreover, live viral vaccine (such as FluMist, measles vaccine) or live bacterial
vaccine (such as BCG vaccine) requires dedicated fill-and-finish facility to avoid
potential contaminations.
In addition, as current vaccines are typically temperature sensitive, thus they need
cold-chain storage under 2–8 C or even lower temperatures (20 C or 60 C).
This generates significant challenges for handling in-process holding and final
product storage as well as supply chain management. Large cold rooms are required
in the manufacturing areas to meet the cold-chain storage requirements. Consistent
electrical power supplies to these cold storage areas are essential to maintain the cold
temperature control all the time. Most vaccine producers maintain emergency
backup power supply in the event of power outages; the power supply can be
switched to the backup power supply quickly.
Another significant challenge is the unpredictability of demand for seasonal
vaccines (e.g., flu vaccines) and vaccines for emergency use (such as Ebola vaccine).
The vaccine manufacturers will need advanced procurement and significant lead
time to prepare raw materials, facilities and equipment, quality control, and human
resources for the production of these vaccines.
3 Characterization of Vaccines
Vaccines, unlike other pharmaceutical products, are often perceived as being not
well characterized due to their complex structures and properties. The implications
are that Phase 3 clinical trials need to be conducted using clinical trial materials made
in large-scale manufacturing facilities to ensure the consistency of Phase 3 clinical
materials with the commercial products. The other approach is to conduct clinical
bridging studies to demonstrate equivalence of Phase 3 clinical trial materials with
commercial materials. The potential impact of this requirement is twofold: (1) delay
in final facility readiness and (2) requirement of large capital investment in facilities
much ahead of time. Thus it is very important to perform characterization of vaccine
products as early as possible, to avoid or minimize costly changes later in the Phase
3 clinical trial stages.
In general, vaccines are very heterogeneous in structure. As greater characteriza-
tion of vaccines becomes more prevalent, it may be possible to connect structural
changes in the vaccine components with changes in potency and toxicity. This, in
turn, may provide a better understanding of how certain vaccines function and
interact with the immune system. Information gained in this area will undoubtedly
improve the effectiveness and safety of future vaccines.
Vaccines can be divided into three major categories: live vaccines, killed or
attenuated vaccines, and component (subunit) vaccines. The component vaccines
are generally the more easily characterized. They usually consist of a relatively small
number of immunogenic components. The live or killed/attenuated vaccines include
complex biological components such as attenuated or killed viruses and intact
bacteria or multiple bacterial components. Advances in proteomics make the char-
acterization of even these difficult vaccines more manageable.
3.1 Characterization of Bacterial Seeds
Characterization of vaccine generally involves analysis of bacterial strains, virus

seeds, cell banks, intermediates, bulk drug substance, formulated final bulk, and
finished product. Various physical, chemical, and biological tests are used for the
analyses of these different biological materials. The purpose of these characteriza-
tion tests is to ensure product quality and consistency.
For genetically modified bacterial strains used for vaccine manufacturing, the
following tests need to be performed in addition to generally accepted characteriza-
tion tests of bacterial strains:
• Genotype verification by PCR
• Verification of linear plasmid DNA
• Gene sequence verification
• Verification of flanking sequence
Table 1 is an example of quality tests on a pre-master seed for a genetically
modified bacterial seed (recombinant pertussis vaccine under development) for
GMP material manufacturing.
3.2 Characterization of Viral Seeds, Cell Banks, and Other

Biological Materials Used in Viral Vaccine
Manufacturing
Cell-based processes are used in viral vaccine development and manufacturing. The
characterization of both viral seeds and cell lines is required. In addition, biological
raw materials used in manufacturing processes should also be characterized with
clear source of origin with potential risks of introduction of adventitious agents or
viruses into the process and product stream. For viral product, US FDA guidance
“Characterization and Qualification of Cell Substrates and Other Biological
Materials Used in the Production of Viral Vaccines for Infectious Disease
Table 1 Quality control tests of a genetically modified seed lot

Items Methods Results
Culture LB medium culture No growth
characteristics Bordet-Gengou medium Small, round, smooth, convex, silver-gray,
culture opaque colonies and without abnormal
colonies
Biochemical Glucose biochemical No utilization of glucose
tests medium
Nitrate peptone water No reduction of nitrate
medium
Urea medium No urease reaction
Citrate medium No use of citrate as a carbon source and
nitrogen source
Semisolid nutrient agar No flagellar power
medium
Morphology Gram stain Gram negative
Serological tests Serum agglutination test Serum agglutination: positive
Phenotype tests ELISA Positive for target antigen
Genotype tests PCR and restriction (1) The results of the recovered PCR products
digestion showed that the restriction enzyme map of the
original strain is consistent with the size of the
theoretical sequences. (2) The sequence is
identical to the sequence of target strain
OD600 Culture in flask 3.1
Target protein ELISA >5
expression
(μg/mL)
Skin necrosis Intradermal injection of Positive
test bacterial suspension (rabbit
model)
Indications” has clearly outlined the requirements for characterization of cell sub-
strates, cell banking, viral seeds, vaccine intermediates, and biological raw materials
at different stages of vaccine development [20].
For viral subunit vaccine product manufacturing, the most important factor is to
prevent contaminations from adventitious materials and other contaminants in all
stages of processes. It is important to demonstrate viral clearance through process
validation.
However, live attenuated viruses, whole inactivated virus, or viruslike particles
often cannot be purified as rigorously as viral subunit vaccines. Unlike the produc-
tion of protein products, it is not possible to introduce validated viral inactivation or
removal steps to mitigate these risks. Addressing these issues requires application of
GMP principles and approaches in development programs at a very early stage,
focusing on the history, purity, and stability of the viral seeds, cell banks, and
biological raw materials.
For viral vector-based vaccine development, it is now common for a viral vector
to be rescued from synthesized plasmids, allowing for traceability of the viral vector
and full sequencing to be performed of the plasmids and resulting viral vector.
Studies can also be performed to demonstrate genetic stability of vectors at an
early stage of development. For manufacturing cell lines, it is essential that the
origins are known with clear history and that they are free from adventitious agents
and that generation of cell banks for both process development and production meet
GMP requirements. In addition, it is very important to maintain segregation in the
processes throughout development programs to prevent potential contamination of
viral stocks and cell banks.
Although the production of viral vectors poses a number of technical challenges
in cell culture, recovery, characterization, and analytical perspective, but with
recombinant viral vector systems, where the virus is essentially used as a delivery
vehicle and the manufacturing approach is independent of the genes it carries, a
platform process can be developed. Therefore, the application of a platform can be
developed for the production of those vaccines based on viral vectors, such as
adenovirus, adeno-associated virus (AAV), and lentivirus.
For cell substrates (cell lines) intended for vaccine manufacturing, the following
characterization tests or documentation needs to be provided:
• Cell substrate properties such as plasmid sequence, phenotype, and expression of
antigens
• Source of the cell line including species of origin and the tissue type
• Donor’s medical history and the results of tests performed on the donor for the
detection of adventitious agents
• Culture history of the cell line, including methods used for the isolation of the
tissues from which the line was derived
• Passage history, medium used, and history of passage in animals
• Documentation of the history of human-derived and animal-derived materials
used during passage of the cells
• Documentation of any genetic material introduced into the cell substrate
• Identity test, cytogenetic characteristics
• Results of all available adventitious agent testing
• Growth characteristics
• Expression characteristics
• Susceptibility to adventitious agents
• Generation of cell substrate
• Long-term storage conditions
• Stability of cell lines
Based on the above thorough understanding of the cell substrates, the cell
banking systems with primary cell bank (PCB), master cell bank (MCB), and
working cell bank (WCB) can be established.
The passage history and derivation history of viral seeds intended for vaccine
manufacturing should also be well documented, including:
• Sourcing of each biological starting material (e.g., plasmids, parental viruses)

• Donor screening, testing, and donor medical history
• Any manipulation of the viral phenotype, such as cold adaptation and develop-
ment of temperature sensitivity
• Any attenuation of virulence and genetic manipulations such as reassortment or
recombination
• Nucleic acid sequences
• Growth characteristics on production cell substrate
• Genetic markers
• Long-term storage conditions
• Viability during storage
• Genetic stability through production
• Absence of adventitious agents
Other biological raw materials such as serum and other biologically sourced raw
materials should also be controlled to prevent adventitious material contaminations.
3.3 Advances in Vaccine Characterization
The continuous development of safe, effective, and innovative vaccines around the
world calls for new technologies, not only in the vaccine discovery areas but also the
new technologies for characterization of vaccines.
The traditional methods of vaccine characterization rely on the study of physical-
chemical properties using methods such as differential scanning colorimetry (DSC)
and thermogravimetric analysis (TGA), pH, various stress conditions (agitation,
freeze-thaw, etc.) based on particulate formation, and methods of quantitating
protein content as well as elemental composition. While these methods are capable
of determining whether or not the end product is consistent with previous batches,
they are unable to detect small changes that can result in a vaccine with reduced or
even lost immunogenicity. Not all changes to the structure of the vaccine compo-
nents have physical consequences, but many of them result in reduced vaccine
effectiveness. Most of these techniques lack sensitivity when it comes to detecting
small changes in the structure of the vaccine components that can cause them to fail
during use. Some changes can cause severe side reactions even in small quantities.
TGA and DSC are used to analyze the denaturing point of the vaccine protein or
nucleotide. These tests generally give indirect indications of changes in the vaccine
with time and stress. Changes in protein sequence or modifications can substantially
affect denaturing kinetics, but these techniques provide no way to correlate these
changes with actual changes in the structure of the molecule.
Appearance and pH are used to monitor major changes in the composition of the
vaccines and are relatively insensitive to these changes. Other physical characteris-
tics that affect vaccine function include particle size and particle size distribution.
Clumping of the vaccine antigen can degrade the function of the vaccine and can
cause unwanted side effects. Specific tests for quantitating proteins or oligonucleo-
tides, such as elemental analysis and total protein content (bicinchoninic acid or
BCA) tests, can provide vital quality control data for troubleshooting manufacturing
problems, but they are of limited value in analyzing degradation of the vaccines
since elemental composition changes from degrades represent only a small percent-
age of the overall elemental composition. In addition, most protein degrades will still
be identified as proteins in a total protein analysis. The conditions used for these
assays also break up clumped proteins or oligonucleotides and are insensitive to
most changes caused by minor structural modifications of these molecules.
Besides the traditional physical and chemical tests for vaccines, advanced tech-
nologies such as high-resolution mass spectrometry (MS) and nuclear magnetic
resonance (NMR) spectroscopy, chromatography, and polymerase chain reaction
(PCR) are used in the new vaccine development. ICP-MS, GC-MS, HPLC-MS, etc.
are the examples of MS used for vaccine characterization. ICP-MS can be used for
quantitatively measurement of metals in the vaccine intermediates and finished
product. GC-MS and HPLC-MS can be used to determine the molecular weight
of vaccines and detect if there are changes in the molecular structure. The mass
spectrometry method can detect the structure change of functional proteins and lipids
in vaccine with a high degree of accuracy, which is often linked with vaccine
immunogenicity. Other biophysical tools such as florescence spectroscopy, light-
scattering spectroscopy, etc. are also used for macromolecule characterization.
4 Clinical Aspects of Vaccine Development
Like other pharmaceutical products, the development of new vaccines will include
clinical testing phases. Through well-controlled clinical trials, data will be collected
to demonstrate that the vaccine product has an effect on clinical endpoint or a
surrogate endpoint that is reasonably likely, based on clinical, serologic, epidemio-
logic, therapeutic, pathophysiologic, or other evidence, to provide clinical benefits.
4.1 Design of Clinical Trials for Vaccines
Generally, clinical trials for vaccines include clinical trials from Phase 1 to Phase
3 and Phase 4 post-approval. Phase 1 clinical trial is to test the initial safety and
tolerability of the candidate vaccine; Phase 2 clinical trials test the safety, immuno-
genicity, and dose ranges; and Phase 3 clinical trials are for additional safety data,
immunogenicity, and efficacy.
Prior to initiating Phase 3 clinical trials, it is recommended that the vaccine
developer has continuous dialogue with regulatory agencies to discuss study details
such as disease prevention or treatment; study sites; subject selection; choice of
control group; trial design including endpoints, case definitions, diagnostic tests,
dose selection, dosing schedule, study duration, concomitant vaccinations, and

medications; and safety assessments to ensure that they meet the intended goals
for clinical trial and product licensure. Vaccines are approved for licensure from
regulatory agencies if clinical trial data show that the product is safe, pure, and potent
and that the manufacturing facility meets the standards designed to assure that the
vaccine product continues to be safe, pure, effective, and non-inferior when admin-
istered with concomitant vaccines.
From regulatory point of view, the proof of effectiveness of a vaccine would
consist of controlled clinical investigations that are adequate and well-controlled
studies, unless waived as not applicable to the vaccine product or when an alternative
method is adequate to substantiate effectiveness, such as using serological response
data where a previously accepted correlation with clinical effectiveness exists [21].
The effectiveness may be proven by a well-designed, single clinical study. In the
case of preventive vaccines, one adequate and well-controlled clinical trial may be
supported by compelling animal challenge/protection models, human serological
data, passive antibody data, or pathogenesis information.
4.2 Consistency Lots
Phase 3 vaccine clinical trials usually use the same scale of manufacturing and make
clinical trial materials in the intended commercial manufacturing facilities. This
requirement has made it challenging for the vaccine developers, as the facilities
need to be ready long before the vaccine can be approved for commercial launch.
Unlike other therapeutic biological products, the efficacy of Phase 3 clinical trials
for vaccines usually needs to demonstrate the protection of healthy people from a
particular disease in the target population. Thus depending on the disease burden of
a particular candidate vaccine, the Phase 3 efficacy clinical trial can be very large,
enrolling several thousand or more healthy volunteers. For example, the recently
approved Shingrix vaccine had Phase 3 clinical trials with more than 17,000 people
enrolled. Thus Phase 3 clinical trials for preventive vaccine are usually very lengthy
and costly. It is estimated that the cost of Phase 3 clinical trials of preventive
vaccines may exceed US$100 million [9].
4.3 Vaccine Immunogenicity
Vaccine immunogenicity is often tested in the clinical trials. For example, during the
clinical trials conducted for Ad5-EBOV Ebola virus disease vaccine, the Ebola-
specific antibody responses against the vaccine-matched 2014 Zaire-Makona glyco-
protein (GP) were assessed with enzyme-linked immunosorbent assay (ELISA)
method, and anti-adenovirus type-5 neutralizing antibody titers were detected with
a serum neutralization assay before and after vaccination. GP-specific IgG titers are
the important data for the immune response of the vaccine. Anti-Ad5 neutralizing
antibody detection was used to analyze the vector immunity and the preexisting
immunity influence on the vaccine’s immunogenicity. Specific T-cell response was
quantified by enzyme-linked immunospot (ELISpot) assay (IL-2, IFN-γ, and
TNF-α), and IFN-γ, tumor necrosis factor-α (TNF-α), and interleukin-2 (IL-2) in
peripheral blood mononuclear cells when phase I and phase Ib clinial trials were
conducted in China. Another phase 2 clinical trial was conducted in Sierra Leone in
2015 [22–24]. The study results demonstrated that one shot of intramuscular injec-
tion with Ad5-EBOV vaccine could elicit strong humoral and cellular immune
response in three clinical trials. Ad5-EBOV vaccine produced a considerable GP
antibody response and GP-specific T-cell response in healthy African participants in
China after 14 days [22]. GP antibody response peaked at day 28 and lasted more
than 6 months. With a booster at the sixth month, the GP antibody response can last
more than 12 months. The pre-exsiting immunity to Ad5 can be overcome with
selected vaccine dosage.
Another example is Shingrix vaccine. Shingrix is a recombinant, subunit vaccine
designed to restore VZV immunity in individuals who are at increased risk of
developing Shingles due to age or immunodeficiency [25]. VZV gE is the most
abundant envelope glycoprotein, predominantly expressed on the surface of virus-
infected cells. The VZV gE protein plays a critical role in virus infectivity since it is
involved in virus entry and cell-to-cell spread, harboring sites for N- and O-linked
glycosylation. The subunit vaccine elicits stronger virus-specific CD4+ T-cell
response as well as antibody B-cell response to gE, compared to the currently used
live attenuated vaccine (Zostavax®). Evidence indicates that the gE protein induces
both neutralizing antibodies and T-cell responses. The gE antigen component of
Shingrix is derived from a VZV strain that was isolated from a patient with severe
varicella disease. This antigen is a recombinant truncated form of VZV gE. The
recombinant protein is then produced in Chinese hamster ovary (CHO) cells that
were genetically modified to express the VZV gE gene [26].
There are many vaccine clinical trials ongoing for various vaccine candidates, and
these are published in the website either at clinicaltrial.gov or at clinicaltrialsregister.
eu.
4.4 Combination Vaccines
A combination vaccine consists of two or more live organisms, inactivated organ-

isms, or purified antigens either combined by the manufacturer as a single product or
mixed immediately before administration. It is intended to prevent multiple diseases
or to prevent one disease caused by different strains or serotypes of the same
organism. Meningococcal conjugated vaccine MCV (A, C, Y, W135), DTcP, and
Prevnar 13 are good examples of combination vaccines.
The clinical studies are designed to demonstrate the safety, immunogenicity, and
efficacy of combination vaccines through randomized, controlled studies. The
efficacy of each component should be demonstrated in clinical studies. Ideally,

clinical trials will be prospective, randomized, and controlled. Endpoints used to
evaluate efficacy in these trials can range from disease incidence to a well-
established correlation of protection [27].
4.5 Special Populations
Vaccine development and clinical trials generally take stepwise approaches starting
from adults to children. For routine vaccines aiming for pediatric applications,
clinical trials in children’s population groups are normally required, such as menin-
gococcal vaccine, PCV vaccine, DTcP vaccine, etc. However, for some global
infectious diseases such as malaria or Ebola virus disease, children suffer the same
or even greater risks as their immune systems are not as strong as those of adults. It
was reported that during the Ebola outbreaks in the Democratic Republic of the
Congo, about one third of the reported cases were from children or adolescents under
18 years of age [28]. Thus it is important to include children and adolescents in the
vaccine development programs for global infectious diseases unless a waiver is
granted by the regulatory agencies. In some cases, if the course of the disease and
the effects of the drug are sufficiently similar in adults and pediatric patients, the
regulators may conclude that pediatric effectiveness can be extrapolated from
adequate and well-controlled studies in adults, supplemented with other information
obtained in pediatric subjects, such as immune response studies, as pointed out in the
US FDA guidance [29].
For vaccine clinical trials including pregnant women in the population groups,
reproductive toxicity studies need to be completed prior to including them.
4.6 Accelerated Approval
For certain global infectious diseases such as tuberculosis, malaria, and human
immunodeficiency virus/acquired immunodeficiency syndrome (AIDS) that are
serious and/or life-threatening, accelerated approval may be granted using a surro-
gate endpoint or a clinical endpoint other than survival or irreversible morbidity for a
vaccine that provides meaningful clinical benefits for preventing the spread of
infectious diseases or therapeutic benefits to patients over existing treatments [29].
For some special vaccine products, approval may be granted based on evidence of
effectiveness from studies in animals when human efficacy studies are not ethical or
feasible [30]. In such cases, after approval, a sponsor must conduct post-marketing
studies, such as field studies, to verify and describe the biological product’s clinical
benefit and to assess its safety when used as indicated in circumstances where
such studies are feasible and ethical. One example of applying “animal rule” is the
approval of anthrax vaccine by the US FDA [31].
5 Overview of Recent Approved Vaccines
In recent years, several new vaccines are approved in different countries around the
world. Brief descriptions are given in the following sections.
5.1 Shingrix Vaccine
Shingles causes painful rash of fluid-filled blisters and sometimes causes chronic
pain. Shingles is a virus that results from reactivation of the varicella zoster virus
(type 3 herpes zoster), the virus that causes chicken pox. Chicken pox is the initial
infection, and shingles is the reactivation of the virus years later. Shingles may be
developed at any age but most common for people aged over 50 years. Shingles
causes substantial pain that can interfere with activities of daily living and reduce the
quality of life. The estimated average overall incidence of shingles is about 3.4–4.8
per 1,000 person years which increases to more than 11 per 1,000 person years in
those aged over 80 years in Europe, according to Robert W. Johnson’s study
[32]. Shingles is the third most common cause of chronic neuropathic pain in the
USA, with an estimate of approximately 500,000 cases yearly.
Vaccination is an effective way to reduce the incidence of shingles. Two vaccines
against shingles have been approved by the US FDA. Merck developed zoster
vaccine live (ZVL, Zostavax), and it has been in use since 2006 for people
60 years and older.
The second vaccine for shingles has been developed by GSK Company. Shingrix
is a recombinant zoster vaccine with adjuvant and is indicated for prevention of
herpes zoster (shingles) in adults aged 50 years and older. It has two doses; the
second dose is administered 2–6 months after the first dose [33].
Since its approval in 2017, Shingrix has obtained tremendous success in market-
place in the USA and internationally, as the sales of Shingrix exceeded US$1 billion
in 2018. It is recommended by ACIP as the preferred shingles vaccine for people
aged 50 years and older.
Shingrix is a suspension for injection supplied as a single-dose vial of lyophilized
varicella zoster virus glycoprotein E (gE) antigen component reconstituted with the
accompanying vial of AS01B adjuvant suspension component. After reconstitution,
a single dose of Shingrix is 0.5 mL. The gE antigen is obtained by culturing
genetically engineered Chinese hamster ovary (CHO) cells, which carry a truncated
gE gene, in media containing amino acids, with no albumin, antibiotics, or animal-
derived proteins. The gE protein is purified by several chromatographic steps,
formulated with excipients, filled into vials, and lyophilized. The adjuvant suspen-
sion component is AS01B, which is composed of 3-O-desacyl-40 -monophosphoryl
lipid A (MPL) from Salmonella minnesota and QS-21, a saponin purified from plant
extract Quillaja saponaria Molina, combined in a liposomal formulation. The
liposomes are composed of dioleoyl phosphatidylcholine (DOPC) and cholesterol
in phosphate-buffered saline solution containing disodium phosphate anhydrous,

potassium dihydrogen phosphate, sodium chloride, and water for injection. After
reconstitution, each 0.5 mL dose is formulated to contain 50 μg of the recombinant
gE antigen, 50 μg of MPL, and 50 μg of QS-21. Each dose also contains 20 mg of
sucrose (as stabilizer), 4.385 mg of sodium chloride, 1 mg of DOPC, 0.54 mg of
potassium dihydrogen phosphate, 0.25 mg of cholesterol, 0.160 mg of sodium
dihydrogen phosphate dihydrate, 0.15 mg of disodium phosphate anhydrous,
0.116 mg of dipotassium phosphate, and 0.08 mg of polysorbate 80. After reconsti-
tution, Shingrix is a sterile, opalescent, and colorless to pale brownish liquid.
Shingrix does not contain preservatives. Each dose may also contain residual
amounts of host cell proteins (3.0%) and DNA (2.1 picogram) from the
manufacturing process [33].
Shingrix has gone through multiple clinical trials. Overall, 17,041 adults aged
50 years and older received at least 1 dose of Shingrix in 17 clinical studies. The
safety of Shingrix was evaluated by pooling data from 2 placebo-controlled clinical
studies (Studies 1 and 2) involving 29,305 subjects aged 50 years and older who
received at least 1 dose of Shingrix (n ¼ 14,645) or saline placebo (n ¼ 14,660)
administered according to a 0- and 2-month schedule. Both studies were conducted
in North America, Latin America, Europe, Asia, and Australia. In the overall
4 population, the majority of subjects were white (74.3%), followed by Asian
(18.3%), black (1.4%), and other racial/ethnic groups (6.0%), and 58% were female.
The safety profile was acceptable. Phase 3 clinical trials demonstrated that Shingrix
has been shown to be highly efficacious with VE against shingles of 97.2% in
subjects over 50 years of age and 91.3% in subjects over 70 years of age [34].
Shingrix is currently under significant shortage in the USA and around the world.
This supply issue is a common problem with many successful vaccines. When the
products do well because of their effectiveness, vaccine developers are struggling to
supply. It is good to address these supply issues and how to mitigate it ahead of time.
GSK announced in April 2019 that it plans to expand its manufacturing facility in
Montana with a capital investment of US$100 million.
5.2 Ebola Vaccine: Ad5-EBOV
Ebola viruses (EBOVs) are enveloped, non-segmented, negative-stranded RNA

viruses belonging to the family Filoviridae. They are known to cause lethal hemor-
rhagic fever in humans and nonhuman primates with a mortality rate of 40–90%
[35, 36]. Ebola virus disease (EVD), formerly known as Ebola hemorrhagic fever,
is a severe, often fatal illness in humans. EBOVs are transmitted among humans
through close contact with infected blood, bodily fluids, or tissues; moreover, the
intentional release of EBOVs would probably result in mucosal infection by small-
particle aerosol dispersion.
From 1976 when the Ebola virus was first discovered to June 2019, there have
been more than 30 epidemic outbreaks in Africa causing tens of thousands of deaths.
The species of Ebola viruses were mainly Zaire and Sultan types, and the outbreak
sites were concentrated in Central Africa, in countries such as Congo, Sultan,
Uganda, and South Africa. The 2014 outbreak in West Africa has been one of the
worst Ebola epidemics. On August 8, 2014, the WHO director general declared this
outbreak a Public Health Emergency of International Concern [37].
Many methods have been used to develop EBOV vaccines. Some of the vaccine
candidates have been tested in nonhuman primates (NHP) and showed good pro-
tection. These vaccine candidates include inactivated vaccine, subunit vaccine,
non-replicated virus vector vaccine, and replicated virus vector vaccine. Most of
them were developed to protect against Zaire ebolavirus. In the past a few years, the
protective mechanism of vaccine has also been studied, and the results showed that
the antibody protection is essential.
A recombinant Ebola virus disease vaccine (Ad5-EBOV) has been successfully
developed jointly by Beijing Institute of Biotechnology and CanSino Biologics Inc.
(CanSinoBIO). The preclinical research of the recombinant Ebola virus disease
vaccine (adenovirus type-5 vector) (Ad5-EBOV) was initiated in 2006. The key
technology in vaccine preparation and evaluation was based on the Ebola GP
antigen. A significant gene sequence variation of the GP antigen in the Zaire
Ebola virus strain has been identified in the 2014 Ebola epidemic outbreaks in
West Africa. To ensure the effectiveness of the vaccine, Ad5-EBOV vaccine was
designed according to the 2014 Ebola virus genotype. The vaccine candidate was
tested in animal models to confirm the immunogenicity, safety, and efficacy.
Challenge studies in guinea pigs and NHPs (cynomolgus monkeys) conducted in
biosafety level 4 (BL-4) lab in Public Health Agency of Canada also confirmed that
the vaccine candidate is 100% effective in protecting guinea pigs and NHPs from
Ebola virus infection.
CanSinoBIO started the development of recombinant Ebola virus disease vaccine
at the end of 2014 as the Ebola virus disease outbreaks occurred in West Africa. The
HEK293 cell line used for Ad5-EBOV production was licensed from National
Research Council (NRC), Canada. The production process has been scaled up
successfully. All key materials, intermediates, and final product (including virus
seeds, cell banks, purified bulk, and final product) are QC tested and released
internally. The entire production process for EBOV vaccine is animal component-
free. The process of large-scale production of adenoviruses has been well character-
ized and optimized. Several vaccines and biological products have been developed
using the Ad5 vector-based technology [38]. The platform technology applied to
Ad5 vector-based production has been demonstrated by Ad5-EBOV to be robust and
scalable and with high productivity.
In 2015, CanSino obtained a clinical trial permit from the Chinese Food
Drug Administration (NMPA, formerly CFDA) and from the Government of Sierra
Leone. The Phase 1 and Phase 2 clinical trials conducted in China and Sierra Leone
involved a total of 681 subjects, and the clinical trial results indicated that the
Ad5-EBOV vaccine is safe and immunogenic. The immune response level in
human is comparable with that of rVZV-ZEBOV vaccine developed by Merck.
Clinical trial results demonstrated that Ad5-EBOV is well tolerated with good safety
profile in tested subjects between ages 18 and 60. The GMTs of anti-GP antibody
peaked around 28 days after vaccination regardless of the dose levels. Satisfactory
immune response can be reached at dosage of 8 1010 VP per dose. Pre-existing
immunity to Ad5 vector can be overcome by proper dose selection (8 1010
VP/dose), and the conversion rate is 100%. Ad5-EBOV response is fast and long-
lasting, and these features could offer help in Ebola virus disease outbreak situation.
In October 2017, the new drug registration application (NDA) for recombinant
Ebola virus vaccine (adenovirus type-5 vector) was approved by the Chinese Food
and Drug Administration (CFDA). The manufacturing facilities, including QC
testing center, are fully validated and in operation at CanSinoBIO.
5.3 Meningococcal Subgroup B Vaccine (MenB)
Currently approved meningococcal ACWY conjugate vaccine (MenACYW135) is

given to preteens and teens at age 11 or 12 years with a booster dose at 16 years to
protect against serotypes A, C, Y, and W135. In 2014–2015, another meningococcal
subgroup B vaccine (Bexsero and Trumenba) [39, 40] were approved for adolescent
and adult population aged 10–25 years who are at increased risk of meningococcal B
diseases. Bexsero is an FDA-approved vaccine to prevent invasive disease caused by
Neisseria meningitidis serogroup. It is not recommended for routine vaccination at
this point.
In fact, meningitis subgroup B infection is very rare in the USA. Among all
11–23-year-old adolescents and young adults in the USA, between 50 and 60 cases
are reported annually, with 5–10 deaths. According to the CDC, there were 7 out-
breaks on college campuses from 2009 to 2013, with 41 cases and 3 deaths [41].
Bexsero was developed by Novartis and is now marketed by the GSK Company.
It is a sterile suspension of three recombinant proteins and meningococcal outer
membrane vesicles (OMV). The recombinant proteins neisserial adhesin A (NadA),
neisserial heparin binding antigen (NHBA), and factor H binding protein (fHbp) are
individually produced in Escherichia coli and purified. The OMV component is
produced from N. meningitidis strain NZ98/254 (expressing outer membrane protein
P or A serosubtype P1.4). The antigens are adsorbed onto aluminum hydroxide.
Each 0.5 mL dose of Bexsero is formulated to contain 50 μg each of recombinant
proteins NadA, NHBA, and fHbp, 25 μg of OMV, 1.5 mg aluminum hydroxide,
3.125 mg sodium chloride, 0.776 mg histidine, and 10 mg sucrose at pH 6.4–6.7
according to the product insert [39]. Bexsero is a suspension for intramuscular
injection in 0.5 mL single-dose prefilled syringes. Two doses are given at least
1 month apart from the first dose.
Another approved meningococcal group B vaccine is Trumenba® developed by
Pfizer, initially approved in 2014. Trumenba is a bivalent meningococcal group B
vaccine that contains two factor H binding proteins (fHBP) from Neisseria
meningitidis (N. meningitidis) serogroup B. fHBP is a conserved, outer membrane
lipoprotein and a virulence factor that contributes to the ability of the bacteria to
avoid host defenses.
Trumenba is a sterile suspension of two recombinant lipidated factor H binding
protein (fHBP) variants, one from each of the two antigenically distinct fHBP
subfamilies subfamily A and subfamily B (A05 and B01, respectively). These
proteins are also known as rLP2086 proteins. The proteins are individually produced
in Escherichia coli and subsequently purified. Each 0.5 mL dose of Trumenba is
formulated to contain 60 μg of each fHBP variant subtype (120 μg total protein),
0.018 mg of polysorbate 80, and 0.25 mg of Al3+ as AlPO4 in 10 mM histidine-
buffered saline at pH 6.0, according to the product insert [40]. Trumenba is approved
for use in individuals from 10 through 25 years of age.
Later in 2017, Trumenba vaccine was approved for three doses schedule (a dose
administered at 0, 1–2, and 6 months) by US FDA. Additional clinical trial results
also demonstrated that it is safe to co-administer Trumenba vaccine with meningo-
coccal (groups A, C, Y, and W135) polysaccharide diphtheria toxoid conjugate
vaccine and tetanus toxoid, reduced diphtheria toxoid, and acellular pertussis vac-
cine, adsorbed in persons 10 years to less than 13 years of age.
5.4 HPV Vaccine
Human papillomavirus (HPV) is a sexually transmitted virus. It is passed through

genital contact or by skin-to-skin contact. HPV infection causes benign and malig-
nant dysplastic anogenital disease in men and women. Nearly 100% of cervical
cancers and 90% of anal cancers are caused by oncogenic HPV types.
GARDASIL 9, human papillomavirus 9-valent recombinant vaccine, was devel-
oped by Merck and initially approved by US FDA in 2014. Prior to the licensure
of GARDASIL 9, Merck’s 4-valent HPV vaccine, GARDASIL, was licensed in
2006. GARDASIL protects against disease caused by HPV types 6, 11, 16, and 18.
GARDASIL 9 includes the original four HPV types in GARDASIL, plus an
additional five types, HPV 31, 33, 45, 52, and 58. GARDASIL 9 is indicated in
girls and women aged 9–26 years for the prevention of cervical, vulvar, vaginal, and
anal cancer caused by human papillomavirus (HPV) types 16, 18, 31, 33, 45, 52, and
58 and genital warts (condyloma acuminata) caused by HPV types 6 and 11.
GARDASIL 9 is also indicated in boys and men aged 9–26 years for the prevention
of anal cancer caused by HPV types 16, 18, 31, 33, 45, 52, and 58 and genital warts
(condyloma acuminata) caused by HPV types 6 and 11.
A 0.5 mL dose of GARDASIL 9 contains recombinant viruslike particles (VLPs)
of the major capsid (L1) protein of HPV types 6, 11, 16, 18, 31, 33, 45, 52, and
58 adsorbed on preformed aluminum-containing adjuvant (amorphous aluminum
hydroxyphosphate sulfate or AAHS). The amounts of HPV type L1 protein in each
dose are as follows: 30 μg/40 μg/60 μg/40 μg/20 μg/20 μg/20 μg/20 μg/20 μg,
respectively. It is available as a suspension in 0.5 mL single-dose vials or prefilled
syringes, for intramuscular administration in two doses at months 0 and 2–6 month
or three doses at months 0, 2, and 6 according to product insert [42].
5.5 Dengue Vaccine
Dengue infection is caused by dengue virus which includes four known serotypes
(dengue virus 1, 2, 3, and 4), all transmitted primarily by Aedes aegypti mosquitos,
as well as other members of the Aedes mosquito family. Annually, an estimated
390 million dengue infections occur worldwide, of which approximately 100 million
are associated with clinical manifestations, 500,000 with hospitalization, and 20,000
with death [43]. Dengue disease is a major public health concern in more than
128 countries. It is endemic in Asia, the Pacific area, Africa, and Latin America with
the four dengue virus serotypes found in tropical and subtropical regions, including
some European territories. Dengue is endemic in the US territories of American
Samoa, Guam, Puerto Rico, and the US Virgin Islands [44–46].
A dengue tetravalent vaccine is developed by Sanofi Pasteur Inc. and is approved
by US FDA in May 2019. DENGVAXIA is a live, attenuated, tetravalent, chimeric
virus vaccine, containing the replication genes and the capsid gene from the atten-
uated yellow fever [17D] virus and the Pre-M and ENV genes from each of the four
serotypes (CYD). Each CYD virus is purified from Vero cells.
The indication of DENGVAXIA vaccine is for the prevention of dengue disease
caused by dengue virus serotypes 1, 2, 3, and 4 in individuals 9 through 16 years of
age with laboratory-confirmed previous dengue infection and living in endemic
areas. Previous dengue infection can be assessed through a medical record of a
previous laboratory-confirmed dengue infection or through current serotesting. The
safety and effectiveness of the vaccine were determined in three randomized,
placebo-controlled studies involving approximately 35,000 individuals in dengue-
endemic areas, including Puerto Rico, Latin America, and the Asia-Pacific region.
The vaccine was approximately 76% effective in preventing symptomatic,
laboratory-confirmed dengue disease in population 9 through 16 years of age who
previously had laboratory-confirmed dengue disease. DENGVAXIA has already
been approved in 19 countries and has been approved by the European Union.
DENGVAXIA is supplied as a vial of lyophilized powder containing each of the
four virus components that is reconstituted at the time of use with the supplied
sodium chloride diluent (0.4% NaCl). After reconstitution, each 0.5 mL dose of
DENGVAXIA is formulated to contain 4.5–6.0 log10 CCID50 of each of the CYD
virus components. The reconstituted vaccine is administered subcutaneously in three
doses at 6-month intervals (at day 0, month 6, and month 12) according to product
insert [47].
5.6 EV71 Vaccine
Enterovirus 71 (EV71) is one of the major causative agents of outbreaks of hand,

foot, and mouth disease or herpangina in Asia. An EV71 (Enterovirus 71) vaccine
has been developed by Sinovac Biotech (China) using Vero cell and EV71 C4
subgenotype. The Phase 2 study of inactivated vaccine (Vero cell) against EV71
virus has been completed in December 2011 in China. The purpose of the Phase
2 clinical study was to demonstrate the safety and immunogenicity of EV71 vaccine
in preventing hand, foot, and mouth disease caused by EV71 in a total of 10,000
healthy infant volunteers aged from 6 to 35 months old. The data from Phase 1 and
2 clinical studies suggested that the inactivated EV71 vaccine had clinically accept-
able safety and good immunogenicity for healthy Chinese infants. A Phase 3 clinical
trial was conducted in China in 2014 with 10,007 healthy infants and young children
(6–35 months of age). The vaccine efficacy against EV71-associated hand, foot,
and mouth disease or herpangina was 94.8%. Vaccine efficacies against EV71-
associated hospitalization and hand, foot, and mouth disease with neurologic com-
plications were both 100%. In the immunogenicity subgroup (1,291 children), an
anti-EV71 immune response was elicited by the two-dose vaccine series in 98.8% of
participants at day 56. An anti-EV71 neutralizing antibody titer of 1:16 was associ-
ated with protection against EV71-associated hand, foot, and mouth disease or
herpangina [48–55]. EV71 vaccine was approved by the Chinese Drug Administra-
tion (formerly CFDA) in 2017.
5.7 HBV Recombinant Vaccine
Hepatitis B virus infection is a serious public health issue. More than 250 million
persons are infected with hepatitis B virus (HBV) worldwide. Approximately
887,000 deaths worldwide were reported in 2015, mostly due to chronic hepatitis
B and resultant end-stage liver disease and/or hepatocellular carcinoma [56, 57].
A new HBV vaccine, recombinant, with adjuvant (Heplisav-B) has been devel-
oped by Dynavax Technologies Corporation for preventing hepatitis B virus infec-
tions. The product contains 20 μg recombinant hepatitis B surface antigen with
3,000 μg adjuvant 1018, which is a novel cytosine phosphoguanine (CpG)-enriched
oligodeoxynucleotide (ODN) phosphorothioate adjuvant. The indication and usage
are for immunization against infection caused by all known subtypes of hepatitis B
virus in adults 18 years of age and older. The product Heplisav-B (rHBsAg-1018
ISS), recombinant hepatitis B surface antigen (rHBsAg), subtype adw, is produced
in yeast cells. The dosage contains two 0.5 mL doses administered 4 weeks apart.
Heplisav-B is supplied as single-use vials of 0.5 mL volume. Each 0.5 mL dose
contains 20 μg HBsAg, 3,000 μg CpG 1018 adjuvant, 8 mM sodium phosphate,
154 mM sodium chloride, 0.01% w/w polysorbate 80, and pH 7.0 buffer. The
vaccine does not contain preservatives. The shelf life of the final container product
is 36 months at 5 3 C from the date of manufacture according to the product insert
[58–60].
The Heplisav-B vaccine was approved by the US FDA in 2017. This was the first
hepatitis B vaccine approved in the USA for the last 25 years.
6 Vaccines Under Development
Many vaccines are under development around the world; below are a few examples
of those vaccines under development.
6.1 rVSV-ZEBOV Ebola Vaccine
Although rVSV-EBOV has not been officially approved for licensing yet, it is in the
process of applying licensure both with US FDA and with EMA in Europe. It has
obtained fast-track review and breakthrough status. It has completed Phase 3 clinical
trials and demonstrated its safety and efficacy [61].
In July 2015, clinical results of the Ebola ça Suffit ring vaccination Phase
3 cluster-randomized trial of the rVSV-ZEBOV vaccine in Guinea were obtained
and published in Lancet. On the basis of interim analysis, the trial showed 100%
vaccine efficacy, with 75.1% vaccine effectiveness at the cluster level, including
herd immunity of unvaccinated members of clusters.
In March 2016, an rVSV-ZEBOV expanded access and compassionate use trial
was conducted, named as “Ring vaccination with rVSV-ZEBOV under expanded
access in response to an outbreak of Ebola virus disease in Guinea,” and the interim
results were published in Lancet. The ring vaccination strategy was used between
Ebola and the contacts. A total of 1,510 individuals were vaccinated in four rings in
Guinea, including 303 individuals aged between 6 years and 17 years and 307 front-
line workers. The results show that a ring vaccination strategy can be rapidly and
safely implemented at scale in response to Ebola virus disease outbreaks in rural
settings.
Although there are a lot of progress made in recent combat to Ebola outbreaks in
Congo, and the data published by Gsell and Camacho [62] regarding the ring
vaccination results demonstrated 100% efficacy of rVSV vaccine, it is worrisome
that a survivor of Ebola virus disease previously in Guinea in the 2014 outbreak
had Ebola virus after 531 days and caused another round of Ebola infections among
the people he was in contact with. We need much better tools to monitor the
situation [63].
In addition, despite significant progress in the characterization of the response to
vaccination, correlation of protection (CoP) against Ebola virus infection has not
been established in humans [64]. This holds true even for rVSV-ZEBOV, the most
advanced Ebola vaccine candidate and the only one with demonstrated efficacy in
humans so far when this chapter is published. This is especially challenging in
emergency outbreaks settings, and major efforts would be required to ensure that
samples and data are collected from the clinical trial participants. During emergency
outbreaks, it is very difficult to track those people and to collect samples for further
testing. Integration of data from preclinical and clinical vaccine studies together with
data from disease survivors will thus be essential to identify Ebola vaccine correlates
of protection. The information generated for rVSV-ZEBOV may help identify the
CoP of the other Ebola vaccine candidates, although these may also be different.
6.2 RSV Vaccine
Respiratory syncytial virus (RSV) is an important cause of viral lower respiratory

tract illness in infants and children globally, but no vaccine is currently approved
to protect these vulnerable populations. RSV is transmitted by direct and indirect
contact with nasal or oral secretions and causes repeat infections throughout life and
significant disease in pediatric and elderly populations [65–70]. The pathogen is an
enveloped, non-segmented, single-stranded, negative-sense RNA pneumovirus
belonging to the family Paramyxoviridae.
Many development programs for RSV vaccine are ongoing, some of them are in
the preclinical stage, and 16 RSV candidate vaccines are in clinical development
[71, 72]. RSV candidate vaccine developed by Novavax is currently in Phase
3 clinical trial stage. RSV vaccine development had a setback recently as Novavax
Phase 3 efficacy trial for RSV F vaccine in maternal immunization did not reach its
Phase 3 primary efficacy endpoint (www.novavax.com. Accessed 12 Apr 2019).
4,636 third-trimester pregnant women were enrolled in this large clinical trial. The
clinical trial results demonstrated effectiveness in severe RSV cases. So far there is
no approved RSV vaccine on the market yet. The development work is continuing,
and more work remains to be done.
6.3 Malaria Vaccine
In 2013, there were an estimated 584,000 deaths and 198 million clinical illnesses
due to malaria, the majority of which is in sub-Saharan Africa. Development of
malaria vaccine has been ongoing for more than 40 years. The fact that malaria is
caused by parasites that makes the development of effective vaccine against malaria
more difficult than the development of vaccines against bacteria and viruses [73–78].
There is no vaccine approved on the market for prevention of malaria at present
time. Clinical trials for malaria vaccines are ongoing.
6.4 Other Vaccines Under Development
Other vaccines currently under development include 15-valent and 20-valent

pneumococcal conjugated vaccines, HIV vaccine, TB booster vaccine, Zika vaccine,
Lassa vaccine, Middle East respiratory syndrome coronavirus (MERS-CoV)
vaccine, chikungunya vaccine, Nipah virus vaccine, and so on [78]. Development of

mRNA vaccines has achieved significant progresses in recent years [79].
7 Challenges We Face
There are many challenges in providing adequate vaccination around the world.
Firstly, vaccine hesitancy is still a problem in many countries including the USA, the
UK, and also China [80]. The vaccine hesitancy does not include situations where
vaccine uptake is low because of poor availability, e.g., lack of vaccine, lack of offer
or access to vaccines, unacceptable travel/distances to reach immunization clinics,
poor vaccine program communication, etc. Vaccine hesitancy reflects concerns
about the decision to vaccinate oneself or one’s children. Vaccine hesitancy and
refusal, a growing trend in recent years, hinders the elimination and eradication of
diseases. As of March 2019, there are multiple measles outbreaks in the USA, and
parts of New York City declared emergency due to measles outbreak.
Although concerns about vaccine safety can be linked to vaccine hesitancy, it is
only one factor that may be related to hesitancy; many other factors also contribute to
the issue. Vaccine hesitancy and refusal factors may include:
• Compulsory nature of vaccines
• Coincidental temporal relationships to adverse health outcomes
• Unfamiliarity with vaccine-preventable diseases
• Lack of trust in corporations and public health agencies
In some areas, people are allowed to not participate in vaccination programs for
religious reasons. In some other areas, people have limited access to education, and
they do not know much about the benefits of vaccines. As a result, they refuse
vaccination.
Overall, vaccine hesitancy is a complex and rapidly changing global problem that
requires ongoing monitoring. Each country and each region need to come up with
specific strategies to address the issue.
Another challenge is the resurgence of pertussis diseases. Despite the vaccination
of DTP to infants and children over three decades, the occurrence of pertussis in
adolescent and adult population is under recognized. Although the adolescents and
adults do not have significant clinical symptoms when pertussis bacteria infect them,
they can transmit the disease to infants and young children around them. Studies in
European region (2019 ECDC Report on Pertussis) found that young adolescents
older than 15 years old had the highest infection rate and young infants less than
6 months old had the most severe infections. The study results demonstrated that the
majority of cases above the age of 30 years were unvaccinated. These data stress
the need to refine vaccination strategies with the final aim of protecting infants. In
the USA and many European countries, immunization of Tdap for adolescents
and adults is recommended in the national immunization programs. Thus it is
recommended that Tdap immunization of adolescents and adults should be
implemented in China and other countries that do not have Tdap immunization for
adults and adolescents.
In summary, vaccines have made tremendous contributions to the society so far.
However, we have more work to do to continue developing safe and effective
vaccines to control the infectious diseases and help people around the world to
live a healthy and long life [81–83].
References
1. GBD 2016 Lower Respiratory Infections Collaborators (2018) Estimates of the global, regional,
and national morbidity, mortality, and aetiologies of lower respiratory infections in 195 coun-
tries, 1990–2016: a systematic analysis for the Global Burden of Disease Study 2016. Lancet
Infect Dis 18:1191–1210
2. Doherty M et al (2016) Vaccine impact: benefits for human health. Vaccine 34:6707–6714
3. WHO. https://www.who.int/immunization/research/vaccine_pipeline_tracker_spreadsheet.
Accessed 25 Apr 2019
4. WHO (2014) European vaccine action plan 2015–2020. http://www.euro.who.int/en/health-
topics/disease-prevention/vaccines-and-immunization/publications/2014/european-vaccine-act
ion-plan-20152020-2014
5. China CDC. www.china.cdc.cn. Accessed 12 Apr 2019
6. USCDC. www.CDC.gov. Accessed 25 Apr 2019
7. WHO report on Measles. www.who.int. Accessed 12 Apr 2019
8. EMA (2019) European Centre for Disease Prevention and Control, annual epidemiological
report for 2017. ECDC, Stockholm
9. Gouglas D, Le TT et al (2018) Estimating the cost of vaccine development against epidemic
infectious diseases: a cost minimisation study. Lancet Glob Health 6:e1386–e1396
10. Mahmoud A et al (2017) Achieving a “Grand Convergence” in global health by 2035. Vaccine
35:A2–A5
11. USCDC. www.uscdc.gov. Accessed 27 Apr 2019
12. USFDA (2017) Encouraging vaccine innovation: promoting the development of vaccines that
minimize the burden of infectious diseases in the 21st century report to congress. www.fda.gov
13. Hardt K et al (2016) Vaccine strategies: optimising outcomes. Vaccine 34:6691–6699
14. Lal H et al (2018) Immunogenicity, reactogenicity and safety of 2 doses of an adjuvanted herpes
zoster subunit vaccine administered 2, 6 or 12 months apart in older adults: results of a phase III,
randomized, open-label, multicenter. Vaccine 34:148–154
15. WHO Website. www.who.int. Accessed 2 Apr 2019
16. Plotkin S et al (2017) The complexity and cost of vaccine manufacturing – an overview.
Vaccine 35:4064–4071
17. Jeyanathan M, Shao Z, Yu X, Harkness R et al (2015) AdHu5Ag85A respiratory mucosal boost
immunization enhances protection against pulmonary tuberculosis in BCG-primed non-human
primates. PLoS One 10(8):e0135009
18. Regules JA et al (2014) A recombinant vesicular stomatitis virus Ebola vaccine. N Engl J Med
376:330–341
19. Sharmaa HJ et al (2012) Assessment of safety and immunogenicity of two different lots
of diphtheria, tetanus, pertussis, hepatitis B and Haemophilus influenzae type b vaccine
manufactured using small and large scale manufacturing process. Vaccine 30:510–516
20. USFDA, FDA Guidance (2010) Characterization and qualification of cell substrates and other
biological materials used in the production of viral vaccines for infectious disease indications.
www.fda.gov/BiologicsBloodVaccine/guidence
21. USFDA. FDA guidance for industry providing clinical evidence of effectiveness for human
drug and biological products. Accessed 18 Apr 2019
22. Zhu FC, Hou LH, Li JX et al (2015) Safety and immunogenicity of a novel recombinant
adenovirus type-5 vector-based Ebola vaccine in healthy adults in China: preliminary report of a
randomised, double-blind, placebo-controlled, phase 1 trial. Lancet 385(9984):2272–2279
23. Wu L, Zhang Z, Gao H et al (2017) Open-label phase I clinical trial of Ad5-EBOV in Africans
in China. Hum Vaccin Immunother 13(9):2078–2085
24. Zhu FC, Wurie AH, Hou LH et al (2017) Safety and immunogenicity of a recombinant
adenovirus type-5 vector-based Ebola vaccine in healthy adults in Sierra Leone: a single-
centre, randomised, double-blind, placebo-controlled, phase 2 trial. Lancet 389
(10069):621–628
25. Kovac M et al (2018) Complications of herpes zoster in immunocompetent older adults:
incidence in vaccine and placebo groups in two large phase 3 trials. Vaccine 36:1537–1541
26. Chlibek R et al (2013) Safety and immunogenicity of an AS01-adjuvanted varicella-zoster virus
subunit candidate vaccine against herpes zoster in adults 50 years of age. J Infect Dis
208:1953
27. USFDA (1997) FDA guidance for industry, for the evaluation of combination vaccines for
preventable diseases: production, testing and clinical studies. Accessed 10 Apr 2019
28. WHO. WHO website. www.who.int. Accessed 23 Apr 2019
29. USFDA (2011) FDA guidance for industry general principles for the development of vaccines
to protect against global infectious diseases
30. 21 CFR Part 601, Subpart H. https://www.law.cornell.edu/cfr/text/21/part-314/subpart-H.
31. USFDA. Biothrax vaccine. https://www.fda.gov/vaccines-blood-biologics/vaccines/biothrax.
Accessed 3 Apr 2019
32. Johnson RW et al (2015) Herpes zoster epidemiology, management, and disease and economic
burden in Europe: a multidisciplinary perspective. Ther Adv Vaccines 3(4):109–120
33. Shingrix Product Insert. www.fda.gov. Accessed 26 Mar 2019
34. Diez-Domingo J et al (2015) Comparison of intramuscular and subcutaneous administration of
a herpes zoster live-attenuated vaccine in adults aged 50 years: a randomised non-inferiority
clinical trial. Vaccine 33:789–795
35. Baize S, Pannetier D, Oestereich L et al (2014) Emergence of Zaire Ebola virus disease in
Guinea. N Engl J Med 371(15):1418–1425
36. Kucharski AJ, Edmunds WJ (2014) Case fatality rate for Ebola virus disease in West Africa.
Lancet 384(9950):1260
37. WHO (2014) Ebola virus disease. http://www.who.int/mediacentre/factsheets/fs103/en/.
Accessed 23 Mar 2019
38. Sheets RL, Stein J, Bailer RT et al (2008) Biodistribution and toxicological safety of adenovirus
type 5 and type 35 vectored vaccines against human immunodeficiency virus-1 (HIV-1), Ebola,
Marburg are similar despite differing adenovirus serotype vector, manufacturer’s construct,
gene inserts. J Immunotoxicol 5(3):315–335
39. USFDA. Bexsero product insert. www.fda.gov. Accessed 27 Mar 2019
40. USFDA. Trmenba product insert. www.fda.gov. Accessed 27 Mar 2019
41. USCDC. www.uscdc.gov. Accessed 27 Mar 2019
42. USFDA (2016) Gardasil 9 product insert. www.fda.gov
43. Bhatt S, Gething PW, Brady OJ et al (2013) The global distribution and burden of dengue.
Nature 496:504–507
44. Morens DM, Fauci AS et al (2008) Dengue and hemorrhagic fever: a potential threat to public
health in the United States. JAMA 299:214–216
45. WHO. Dengue and dengue haemorrhagic fever, Fact sheet No.117. http://www.who.int/
mediacentre/factsheets/fs117/en/. Revised Apr 2016
46. WHO (2014) Dengue: guidelines for diagnosis, treatment, prevention and control: new edition.
Geneva 2009. WHO, Geneva
47. USFDA (2019) Dengue vaccine, VRBPAC briefing document. www.fda.gov

48. McMinn PC et al (2002) An overview of the evolution of enterovirus 71 and its clinical and
public health significance. FEMS Microbiol Rev 26:91–107
49. Xu J, Qian Y, Wang S, Serrano JMG, Li W, Huang Z et al (2010) EV71: an emerging infectious
disease vaccine target in the far east? Vaccine 28:3516–3521
50. Li YP, Liang ZL, Gao Q, Huang LR, Mao QY, Wen SQ et al (2012) Safety and immunogenicity
of a novel human enterovirus 71 (EV71) vaccine: a randomized, placebo-controlled, double-
blind, phase I clinical trial. Vaccine 30:3295–3303
51. Zhu F et al (2014) Efficacy, safety, and immunogenicity of an enterovirus 71 vaccine in China.
N Engl J Med 370(9):818–828
52. Chong P et al (2012) Production of EV71 vaccine candidates. Hum Vaccin Immunother
8(12):1775–1783
53. Solomon T, Lewthwaite P, Perera D, Cardosa MJ, McMinn P, Ooi MH (2010) Virology,
epidemiology, pathogenesis, and control of enterovirus 71. Lancet Infect Dis 10:778–790
54. Chone P et al (2015) Review of enterovirus 71 vaccines. Clin Infect Dis 60(5):797–780
55. Wu CY et al (2019) The mature EV71 virion induced a broadly cross-neutralizing VP1 antibody
against subtypes of the EV71 virus. PLoS One 14(1):e0210553
56. Vaccines and Related Biological Products Advisory Committee Meeting July 28, 2017. FDA
briefing document heplisav-B (Hepatitis B vaccine recombinant and 1018 ISS adjuvant).
www.fda.gov
57. Heplisav-B Product Insert. www.USFDA.gov. Accessed 12 Apr 2019
58. Kuan RK et al (2013) Cost-effectiveness of hepatitis B vaccination using HEPLISAV™ in
selected adult populations compared to Engerix-B® vaccine. Vaccine 31(37):4024–4032
59. Gilbert CL et al (2011) Safety and immunogenicity of a modified process hepatitis B vaccine in
healthy adults 50 years. Hum Vaccin 7(12):1336–1342
60. Splawn LM et al (2018) Heplisav-B vaccination for the prevention of hepatitis B virus infection
in adults in the United States. Drugs Today (Barc) 54(7):399–405
61. Strezova A et al (2017) A randomized lot-to-lot immunogenicity consistency study of the
candidate zoster vaccine HZ/su. Vaccine 35:6700–6706
62. Gsell PS, Camacho A et al (2017) Ring vaccination with rVSV-ZEBOV under expanded access
in response to an outbreak of Ebola virus disease in Guinea, 2016: an operational and vaccine
safety report. Lancet Infect Dis 7:1276–1128
63. Daouda Sissoko BD et al Resurgence of Ebola virus disease in guinea linked to a survivor with
virus persistence in seminal fluid for more than 500 days. Clin Infect Dis 63(10):1353–1356
64. Medaglini D et al Correlates of vaccine-induced protective immunity against Ebola virus
disease. Seminars in immunology. Academic Press, Cambridge
65. Lozano R, Naghavi M, Foreman K, Lim S, Shibuya K, Aboyans V et al (2012) Global and
regional mortality from 235 causes of death for 20 age groups in 1990 and 2010: a systematic
analysis for the global burden of disease study 2010. Lancet 380:2095–2128
66. Falsey AR, Hennessey PA, Formica MA, Cox C, Walsh EE (2005) Respiratory syncytial virus
infection in elderly and high-risk adults. N Engl J Med 352:1749–1759
67. Hall CB et al (2012) The burgeoning burden of respiratory syncytial virus among children.
Infect Disord Drug Targets 12:92–97
68. Dudas RA, Karron RA (1998) Respiratory syncytial virus vaccines. Clin Microbiol Rev
11:430–439
69. Nair H, Nokes DJ, Gessner BD, Dherani M, Madhi SA, Singleton RJ et al (2010) Global burden
of acute lower respiratory infections due to respiratory syncytial virus in young children: a
systematic review and meta-analysis. Lancet 375:1545–1555
70. WHO (2012) Trends in maternal mortality: 1990–2010 WHO, UNICEF, UNFPA, and The
World Bank estimates. WHO, Geneva. http://apps.who.int/iris/bitstream/10665/44874/1/
9789241503631eng.pdf
71. Higgins D et al (2016) Advances in RSV vaccine research and development - a global agenda.
Vaccine 34(26):2870–2875
72. Villafana T et al (2017) Passive and active immunization against respiratory syncytial virus for
the young and old. Expert Rev Vaccines 16(7):1–13
73. Hoffman SL et al (2015) The march toward malaria vaccines. Vaccine 33(Suppl 4):D13–D23
74. Lyke KE (2017) Steady progress toward a malaria vaccine. Curr Opin Infect Dis 30(5):463–470
75. Keitany JG et al (2014) Live attenuated pre-erythrocytic malaria vaccine. Hum Vaccine
Immunother 10(10):2903–2909
76. Richie TL et al (2015) Progress with Plasmodium falciparum sporozoite (PfSPZ)-based malaria
vaccines. Vaccine 33(52):7452–7461
77. Olotu A, Urbano V, Hamad A, Eka A et al (2018) Advancing global health through develop-
ment and clinical trials partnerships: a randomized, placebo-controlled, double-blind assess-
ment of safety, tolerability, and immunogenicity of PfSPZ vaccine for malaria in healthy
equatoguinean men. Am J Trop Med Hyg 98(1):308–318
78. CEPI. Targeting diseases with epidemic potential. CEPI. www.CEPI.net. Accessed 26 Mar
2019
79. Zhang C, Maruggi G et al (2019) Advances in mRNA vaccines for infectious diseases.
Front Immunol 10:594. https://doi.org/10.3389/fimmu.2019.00594
80. WHO (2014) Report “WHO SAGE Vaccine Hesitancy Working Group report”. https://
www.who.int/immunization/sage/meetings/2014/october/SAGE_working_group_revised_rep
ort_vaccine_hesitancy.pdf
81. Pinti M et al (2016) Aging of the immune system: focus on inflammation and vaccination. Eur J
Immunol 46:2286–2301
82. Kaufmann SH et al (2014) Challenges and responses in human vaccine development. Curr Opin
Immunol 28:18–26
83. Cunningham AL et al (2016) Vaccine development: from concept to early clinical testing.
Vaccine 34:6655–6664
DOI: 10.1007/10_2019_117
Published online: 19 November 2019
Human Pluripotent Stem Cells:

Applications and Challenges
for Regenerative Medicine and Disease
Modeling
Cláudia C. Miranda, Tiago G. Fernandes, M. Margarida Diogo,

Contents
1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 191
2 Human Pluripotent Stem Cells: The Tool . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 192
2.1 Methods for Isolation and Derivation of hES and hiPS Cells . . . . . . . . . . . . . . . . . . . . . . . . 192
2.2 Safety and Efficiency of hiPS Cell Reprogramming . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 193
2.3 Epigenetic Memory in hiPS Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 194
3 Human Pluripotent Stem Cells and Regenerative Medicine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 195
3.1 Clinical Challenges and Legislation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 195
3.2 Cell Sources . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 196
3.3 Current Clinical and Preclinical Trials Involving hPS Cell-Derived Products . . . . . . 196
4 Bioprocesses for hPS Cell Expansion, Differentiation, and Purification . . . . . . . . . . . . . . . . . . . 200
4.1 hPS Cell Expansion as 3D Aggregates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 202
4.2 hPS Cell Differentiation as 3D Aggregates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 204
4.3 Purification of hPS Cell-Derived Products . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 206
4.4 Bioengineering Strategies for the Improvement of hPS Cell-Derived Organoids . . . 207
5 Human Pluripotent Stem Cells and Disease Modeling . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 208
5.1 Modeled Diseases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 209
5.2 Organoids and Disease Modeling . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 210
5.3 Disease Modeling Using CRISPR/Cas9 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 212
5.4 Drug Discovery . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 213
6 Conclusions and Future Perspectives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 214
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 215
C. C. Miranda, T. G. Fernandes, M. M. Diogo (*), and J. M. S. Cabral

iBB - Institute for Bioengineering and Biosciences and Department of Bioengineering,
Instituto Superior Técnico, Universidade de Lisboa, Lisbon, Portugal
e-mail: margarida.diogo@tecnico.ulisboa.pt
190 C. C. Miranda et al.
Abstract In recent years, human pluripotent stem (hPS) cells have started to emerge
as a potential tool with application in fields such as regenerative medicine, disease
modeling, and drug screening. In particular, the ability to differentiate human-
induced pluripotent stem (hiPS) cells into different cell types and to mimic structures
and functions of a specific target organ, resourcing to organoid technology, has
introduced novel model systems for disease recapitulation while offering a powerful
tool to provide a faster and reproducible approach in the process of drug discovery.
All these technologies are expected to improve the overall quality of life of the
humankind. Here, we highlight the main applications of hiPS cells and the main
challenges associated with the translation of hPS cell derivatives into clinical settings
and other biomedical applications, such as the costs of the process and the ability
to mimic the complexity of the in vivo systems. Moreover, we focus on the
bioprocessing approaches that can be applied towards the production of high
numbers of cells as well as their efficient differentiation into the final product and
further purification.
Graphical Abstract
Keywords Bioprocessing, Disease modeling, Human pluripotent stem cells,

Organoids, Regenerative medicine
Human Pluripotent Stem Cells: Applications and Challenges for. . . 191
1 Introduction
The possibility of producing virtually any type of cell of the human body from
human pluripotent stem (hPS) cells has introduced a myriad of possibilities regard-
ing their applications in innovative fields such as regenerative medicine, disease
modeling, and drug discovery.
Besides the self-renewal capacity and differentiation ability associated with stem
cells in general, hPS cells provide the opportunity of generating every cell type
derived from the three embryonic germ layers – endoderm, ectoderm, and mesoderm
[1]. Importantly, the breakthrough discovery that somatic cells can be reprogrammed
back to a pluripotent stem cell state, awarded with the Nobel Prize of Physiology or
Medicine in 2012, has introduced the opportunity of using these cells in personalized
medicine approaches, both including regenerative medicine and drug discovery [2].
The opportunity of generating organoids from hPS cells amplifies the relevance
of this stem cell source within the fields of disease modeling and drug discovery
[3]. Besides allowing the recapitulation of the inherent differentiation of embryo-like
cells into complex and organized structures that are found within a normal embry-
onic development process, disease-specific hPS cell-derived organoids also provide
a unique opportunity of understanding the underlying mechanisms of a particular
disease and how they will affect the final phenotypic characteristics [3]. On the other
hand, organoid technology opens the door for new methods of drug screening that
may reduce the need to resort to animal models that do not fully represent a human
response and that are associated with ethical issues [4].
Stem cell bioprocessing includes the main strategies for scaling up or scaling out
of stem cell expansion, differentiation, and purification for the upcoming use of stem
cells or their derivatives in the fields of regenerative medicine, disease modeling, and
drug screening. To fulfill the previously described applications, scalable processes
that involve the use of laboratory-scale (e.g., spinner flasks) and fully controlled
bioreactors for the production of clinically relevant numbers of cells are currently
under development and optimization. So far, several robust and efficient
bioprocesses have been developed including the xeno-free scalable expansion of
hPS cells while retaining their pluripotent potential [5] as well as the directed
differentiation of human induced pluripotent stem (hiPS) cells into cardiomyocytes
[6]. Moreover, scale-out approaches that envisage novel methodologies for drug
screening and testing processes have been described and hopefully will be integrated
in the drug discovery pipeline in a near future [7].
In this chapter, we address the main challenges and opportunities regarding the
application of hPS cell derivatives towards regenerative medicine, disease modeling,
and drug screening, and we revise the main bioprocessing tools that are necessary to
take full advantage of the potential of hPS cells.
2 Human Pluripotent Stem Cells: The Tool
2.1 Methods for Isolation and Derivation of hES and hiPS

Cells
The properties of human pluripotent stem cells confer them a remarkable potential
towards biomedical and pharmaceutical applications. Embryonic stem (ES) cells
were firstly isolated from mouse embryos, and their pluripotent potential was
demonstrated by showing that these cells gave rise to teratocarcinomas when grafted
into adult mice [8]. Later, in 1998, it was also possible to isolate ES cells from human
embryos [1]. Soon – 4 to 5 days – after the fertilization of an egg in human embryos,
there is the formation of the blastocyst, a hollow sphere composed of an outer layer
of cells, the trophectoderm, and an inner cell mass, from where embryonic stem cells
are isolated [9]. These cells are known for their pluripotency, as in vitro they can give
rise to any type of cell originated from the three embryonic germ layers (ectoderm,
mesoderm, and endoderm). Nevertheless, they are unable to form a whole new
individual, since the placenta is derived from the trophectoderm and therefore cannot
be formed [1]. The pluripotency of human ES (hES) cells can be assessed by
embryoid body formation, directed differentiation into cells of the three embryonic
germ layers, and teratoma formation assays [1].
Despite their enormous potential in cell therapy, drug discovery, and disease
modeling, the requirement of destroying a human embryo for obtaining hES cells
raises ethical concerns, namely, the ending of a potential human life. The ethical
implications and reduced availability of hES cells led to search for alternative paths
to obtain human pluripotent stem cells. Several methods were explored, the first ones
being nuclear transfer [10] and cell fusion [11] which in spite of creating pluripotent
stem cell lines retained the ethical concerns and the impossibility of clinical use,
respectively. Soon after that, it was discovered another technology for pluripotency
induction, capable of bypassing these two problems, along with the possibility of
reducing the risk of immune rejection, hiPS cells, obtained by direct reprogramming
of somatic cells by defined transcription factors [12]. Furthermore, hiPS cell tech-
nology offers the possibility of developing personalized medicine approaches, since
hiPS cells can be induced from somatic cells of a specific patient.
The technique of nuclear transfer requires a somatic cell and an unfertilized
oocyte from which the nucleus was removed. It consists in the transfer of the
DNA content of the somatic cell into the oocyte, where the union will occur, leading
to the creation of an “embryonic-like” cell [13]. However, this process still maintains
the ethical concerns associated with hES cells, due to the origin of a cell that could
theoretically form a human being.
Cell fusion, as the name indicates, involves the fusion between two cells,
generally an embryonic stem cell and a somatic cell. The fusion comprises that the
reprogramming factors that exist in the cytoplasm of embryonic stem cells will
induce the somatic nucleus towards a pluripotent stem cell state, expressing Oct4,
Sox2, and Nanog. However, their tetraploid nature prevents their use in clinical
applications [13–15].
In the past few years, scientific research was directed towards bypassing the
ethical concerns associated to human embryonic stem cells. Takahashi and
Yamanaka [12] discovered in 2006 that terminally differentiated cells could be
reprogrammed back to a state of pluripotency, therefore achieving an embryonic-
like state [16]. This process was firstly performed using mouse embryonic fibro-
blasts, but 1 year later the process of cell reprogramming was also validated for
human cells, by using adult human fibroblasts to generate human iPS cells for the
first time [2]. In this study, four transcription factors were shown to be essential for
the generation of iPS cells, those being Oct3/4, Sox2, c-Myc, and Klf4. These four
factors were introduced into the somatic cells by viral vectors. One of the most
surprising results of this work was that Nanog, a transcription factor present in ES
cells, was not found to be essential in the reprogramming process. It is important to
notice that the optimal duration of transgene expression will depend on the type of
somatic cell that will be reprogrammed, but still, once the cell achieves the
pluripotency hallmark, the endogenous expression of the factors is sufficient to
maintain the pluripotent state [17].
Human iPS cells are identical to hES cells in terms of colony and cell morphol-
ogy, gene expression, and in vitro differentiation ability. At the same time, these cell
types share the expression of surface pluripotency markers SSEA-3, SSEA-4,
TRA-1-60, and TRA-1-81, as well as the transcription factors Oct3/4, Sox2, and
Nanog [2, 18]. Despite the great similarity observed between hES and hiPS cells
regarding morphologic characteristics and signaling pathways, there are some dif-
ferences that distinguish these two types of cells. Firstly, there are some indications
that differential promoter binding of the reprogramming factors leaves a signature in
the gene expression of hiPS cells [19]. Also, hiPS cells exhibit a different methyl-
ation pattern when compared to hES cells. Moreover, it has also been argued that
hiPS cells may retain somatic mutations acquired before reprogramming, which
would make the reprogrammed cells genetically different from its embryonic equiv-
alents. In addition, it was previously shown that hiPS cells have the ability of
forming teratomas with an efficiency of 100% when transplanted subcutaneously
or intratesticularly into immunocompromised mice, whereas this percentage
decreases for 81 and 91% with hES cells, with subcutaneous and intratesticular
implantation, respectively [20].
2.2 Safety and Efficiency of hiPS Cell Reprogramming
The production of hiPS cells without genomic integration of reprogramming factors

is one key feature that facilitates their approval mainly for regenerative medicine
applications. In fact, methods based on genomic integration can cause insertional
mutagenesis and potentially transform the cells towards a malignant pathway [21].
Although the first report of derivation of hiPS cells was based on retroviral
vectors that encoded key reprogramming factors with reprogramming efficiencies
averaging 0.02% [2], several different approaches have also been reported that avoid
integration of the genetic material in the genome. Amid integration-free methods,

episomal transfection has demonstrated the ability to generate iPS cells in a techni-
cally simple and reproducible manner [22, 23]. Despite that the reprogramming
efficiencies of non-integrating methods tend to be lower when compared to viral
vectors, averaging 0.01%, a recent study demonstrated that there are no significant
differences in terms of DNA methylation pattern, marker expression levels, or
developmental potential in hiPS cells derived using different non-integrating tech-
niques [24]. Another way to evade introducing genetic material into the genome
consists in the direct delivery of synthetic RNAs, modified to prevent antiviral
response mechanisms from the cells, to reprogram somatic cells back to a pluripotent
state with an efficiency of more than 2%, which translates to a reprogramming
efficiency of two orders of magnitude higher comparing to viral methods [25].
Although hiPS cells represent an alternative for overcoming the ethical issues
often associated with hES cells, they still retain the risk of forming teratomas if not
efficiently differentiated. A safer approach may reside in using direct reprogramming
to derive a somatic cell from another somatic cell type or from an induced tissue-
specific stem (iTS) cell. These strategies allow bypassing of the pluripotent state,
while at the same time reduce the duration of the periods of hiPS cell derivation and
differentiation from several months to a few weeks [26]. As a good example of direct
reprogramming from a somatic cell type to another, the reprogramming of mouse
fibroblasts into cardiomyocytes was performed by overexpression of cardiac-specific
factors Gata4, Mef2c, and Tbx5 [27]. The derivation of functional neurons from
fibroblasts using lentiviral vectors to induce the expression of Ascl1, Brn2, and
Myt1l was also reported [28]. Still, a major drawback of direct reprogramming into
somatic cells relies on the reduced proliferative capacity of the derived cells, as well
as a lack of population diversity, which in the end may compromise the regenerative
medicine potential of the cells [29]. Another alternative relies on the use of iTS cells,
which are derived by transfection of somatic cells with a plasmid that harbors the
genes for the four pluripotency induction factors in combination with tissue-specific
selection genes [30]. Using this process, mouse pancreatic and liver iTS cells were
derived by using transient overexpression of reprogramming factors and combina-
tion with Pdx1+ or HNF4α+ cell selection, respectively. These cells expressed
genetic markers for endoderm and pancreatic or hepatic progenitors and were able
to further differentiate into insulin-producing cells and hepatocytes.
2.3 Epigenetic Memory in hiPS Cells
Induction of pluripotency of somatic cells also involves epigenetic changes in the

DNA. Therefore, there is the need for specific DNA methylation and methylation/
acetylation of histones that will allow the required genes for pluripotency to be
expressed [17]. Although DNA methylation patterns are dynamic throughout devel-
opment and cell differentiation, some of these patterns can be retained as a form of
epigenetic memory.
The DNA methylation of Oct3/4 and the modification of histones observed by

Takahashi and Yamanaka [12] were the first suggestion that iPS cells were caught in
an intermediate epigenetic state between the initial somatic cell and an ES cell state.
Later, Bar-Nur et al. observed that hiPS cells derived from pancreatic islet β cells had
increased potential to differentiate into insulin-producing cells [31]. After analyzing
the open chromatin structure, it was possible to determine that this hiPS cell line
retained a methylation pattern and an open chromatin structure at key β-cell genes,
introducing the hypothesis that methylation patterns in hiPS cell lines are influenced
by their tissues of origin. Therefore, the epigenetic memory associated with the
reprogramming process was also shown to improve the efficiency of differentiation
into mature cells of the same lineage of the donor cell.
Nevertheless, the method of iPS cell generation can also influence the epigenetic
memory of the cells. Kim et al. compared iPS cells derived by transcription factor-
based treatment and by nuclear transfer [32]. While transcription factor-based
reprogramming demonstrated some residual DNA methylation patterns characteris-
tic of the somatic donor cells, reprogramming by nuclear transfer yielded DNA
methylation signatures that closely resemble the ones of ES cells [32]. Still, in
transcription factor-based approaches, the predisposal of iPS cells to favor differen-
tiation towards the same lineage of the donor cell could be bypassed by sequential
differentiation and reprogramming of iPS cells, as well as with treatment with
chromatin-modifying drugs [19].
3 Human Pluripotent Stem Cells and Regenerative

Medicine
3.1 Clinical Challenges and Legislation
Given that regenerative medicine products provide a high-risk-high-reward

approach, some countries, including the United States and Japan, have begun to
review the laws of delivery of new regenerative medicine-based products or even
create new laws to fulfill the gaps in legislation.
Since late 2014, there are two laws in Japan that provide a fast track for product
approval for the commercialization of cell therapy products within the country.
Therefore, companies are allowed to receive a conditional marketing approval and
commercialize cell-based products while clinical trials proceed to later stages. The
first law, Act on the Safety of Regenerative Medicine (Law No. 85/2013), states the
guidelines that allow the acceleration of clinical application and commercialization
of innovative regenerative medicine products, covering clinical research and medical
practice. The second law, Pharmaceuticals and Medical Device (PMD) Act (Law
No. 84/2013), introduces a specific regulatory framework for regenerative medicine
products, providing a provisory marketing approval of 7 years after exploratory
clinical trials have demonstrated safety and basic efficiency data. During this period,
companies are required to continue to submit new clinical trials data to Japan’s
Pharmaceuticals and Medical Devices Agency and, in the end, apply for final
marketing approval or withdrawal of the product. In this Act, regenerative medicine
products include cell and gene therapy products, as well as tissue-engineered
products.
In the United States, the Food and Drug Administration (FDA) approved a
directive – the 21st Century Cures Acts – in order to accelerate the process of giving
access to innovative products to patients in need and to reduce the regulatory hurdles
for FDA approval. In this Act, there is also a pathway to accelerate FDA approval
and market entry of regenerative medicine therapies, including cell therapies and
other human tissue products, known as the regenerative medicine advanced therapies
(RMAT).
Among the main challenges of hPS cell-based product applications in cell
therapies, the high cost of the cell manufacturing process remains as one of the
main obstacles to tackle [33]. For example, the costs of the preclinical studies of the
first clinical trial using hES cell-derived oligodendrocyte progenitors to treat spinal
cord injury were estimated to be 200 million dollars [34].
3.2 Cell Sources
Although hiPS cells offer the possibility of patient-specific cell therapies, the
reprogramming, differentiation, and purification steps may require months of
processing before reaching clinically relevant cell numbers with high quality and
functionality. Thus, allogeneic therapy using universal hiPS cells from healthy
donors can be used instead of customized hiPS cells. In fact, hiPS cells from donors
with homozygous human leukocyte antigen (HLA) who match 20% of the
Japanese population at major HLA loci have been generated and banked and
potentially provide an excellent alternative to autologous stem cell-based therapy
[23]. It was already demonstrated that upon hiPS cell-derived dopamine neurons
engraftment in nonhuman primates, major histocompatibility matching reduces the
immunological response, through suppression of immune cells in the site of the graft
and increases the survival of the grafted neurons [35].
3.3 Current Clinical and Preclinical Trials Involving hPS

Cell-Derived Products
Most of the clinical trials that have employed hPS cell-based products are directed
towards treatment of ophthalmologic, neurological, and neurodegenerative diseases.
Furthermore, there is a clear observation that so far there has been more resourcing to
hES cells than to hiPS cells for differentiation into the final cell product (Tables 1
and 2). One of the major debates concerning the clinical applications of hPS
Table 1 hES cell-based products in clinical trials

Company/
Disease sponsor ID Status Country
AMD Southwest NCT02749734 Phase I China
Hospital, China
AMD Chinese Acad- NCT02755428 Phase I China
emy of Sciences
AMD Chinese Acad- NCT03046407 Early phase I China
emy of Sciences
Dry-form AMD Astellas Institute NCT02463344 Follow-up of USA
for Regenerative NCT01344993
Medicine
Dry-form AMD Cell Cure NCT02286089 Phase I/II Israel/USA
Neurosciences
Dry-form AMD CHA Biotech NCT01674829 Phase I/II Korea
Dry-form AMD Astellas Institute NCT01344993 Phase I/II USA
for Regenerative
Medicine
Dry-form AMD Regenerative NCT02590692 Phase I/II USA
Patch
Technologies
Wet-form AMD Pfizer NCT01691261 Phase I UK
Macular degenerative Astellas Institute NCT03167203 Phase I/II USA
disease for Regenerative
Medicine
Outer retinal Federal Univer- NCT02903576 Phase I/II Brazil
degenerations sity of São Paulo
Parkinson’s disease Chinese Acad- NCT03119636 Phase I/II China
emy of Sciences
Severe heart failure Assistance NCT02057900 Phase I France
Publique –
Hôpitaux de
Paris
Spinal cord injury Asterias NCT02302157 Phase I/II USA
Biotherapeutics
SMD CHA Biotech NCT01625559 Phase I Korea
SMD Astellas Institute NCT02941991 Follow-up of UK
for Regenerative phase I/II
Medicine
SMD Astellas Institute NCT01469832 Phase I/II USA/UK
for Regenerative
Medicine
SMD Astellas Institute NCT01345006 Phase I/II USA
for Regenerative
Medicine
SMD Astellas Institute NCT02445612 Long-term USA
for Regenerative follow-up of
Medicine NCT01345006
(continued)
Table 1 (continued)
Company/
Disease sponsor ID Status Country
Type 1 diabetes ViaCyte NCT02239354 Phase I/II USA/Canada
mellitus
Type 1 diabetes ViaCyte NCT02939118 Observational, USA
mellitus follow-up of
NCT02239354
Type 1 diabetes ViaCyte NCT03162926 Phase I USA/Canada
mellitus
Type 1 diabetes ViaCyte NCT03163511 Phase I/II USA
mellitus and hypogly-
cemia unawareness
AMD age-related macular degeneration, SMD Stargardt’s macular dystrophy
Table 2 hiPS cell-based products in clinical trials

Company/ Type of
Disease sponsor ID Status Country therapy
AMD Moorfields Eye NCT02464956 Observational UK Autologous
Hospital NHS
Foundation
Trust
Wet-AMD RIKEN UMIN000011929 Interventional Japan Autologous
Graft-vs- Cynata NCT02923375 Phase I Australia/ Allogenic
host disease Therapeutics UK
Neovascular Kobe City UMIN000026003 Interventional Japan Allogenic
AMD Medical Center
General
Hospital
Thalassemia The Third Affil- NCT03222453 Not China Autologous
iated Hospital of applicable
Guangzhou
Medical
University
AMD age-related macular degeneration, SMD Stargardt’s macular dystrophy, Not applicable trials
without FDA-defined phases, including trials of devices or behavioral interventions
cell-based products resides in whether to transplant mature cells with full function-
ality or progenitor cells that still have some plasticity and may integrate better within
the host tissues. Several examples of both scenarios have been reported, including
the transplantation of fully differentiated cells, such as dopaminergic neurons [35] or
cardiomyocytes [36], or the transplantation of dopaminergic [37], oligodendrocyte
[38] or pancreatic [39] precursor cells.
The first clinical trial involving a hPS cell-based product dates back to 2014 and
was started by Geron Corporation, in the United States. This study was directed
towards spinal cord injury and relied on a preclinical trial that used hES cell-derived
oligodendrocyte precursor (OP) cells to remyelinate rat spinal cords [40]. In fact, it
was demonstrated that OP cells were able to not only survive but also migrate and
further differentiate into oligodendrocytes, restoring motor function. Currently, this
trial is in phase I/II by Asterias Biotherapeutics, with the main objective of optimiz-
ing the injected cell dose.
Neurodegenerative diseases are potentially one of the main targets for iPS cell
therapies. Parkinson’s disease (PD), which is characterized by a neurological dys-
function based on the loss of dopaminergic neurons, was the first condition to be
tackled using iPS cell-based therapy. In 2011, the first evidence that dopaminergic
progenitor cells derived from hES cells were able to engraft and mature upon
injection was attained using a rat model [41]. In addition, the mature dopaminergic
neurons survived for long periods, retaining the ability to form synaptic connections
and restoring motor functions that were lost due to PD [42].
A preclinical trial in monkeys was initially run to confirm the safety and efficacy
of a treatment for PD resorting to hiPS cells [37]. In this study, dopaminergic
precursor cells were derived from hiPS cells and injected into the putamen of
macaque monkeys. For that, hiPS cells were firstly directed towards midbrain
dopaminergic progenitor cells, and then cells expressing CORIN – a floor plate
marker – were sorted in order to obtain a more purified population of cells expressing
FOXA2 and Tuj1, markers for floor plate and immature neurons, respectively. In the
end, this approach yielded more than 85% of FOXA2+Tuj1+ cells, while more than
15% of the cells expressed a marker of midbrain dopaminergic neurons (NURR1).
Furthermore, no pluripotent Oct4+ cells were detected, as well as neural rosette-
forming cells (Sox1+Pax6+), that might also contribute to tumor formation. Upon
transplantation, implanted cells were able to survive for at least 2 years without
harmful effects in the body and were able to even integrate with monkey’s brain
cells. In October 2018, the first clinical trial comprising the implantation of hiPS cell-
derived dopaminergic precursor cells has been initiated in Japan. In the first
approach of the procedure performed in a single individual, 2.4 million cells were
implanted into 12 different sites known to contain dopamine activity, and, if no
complications arise in the first 6 months, the procedure will be repeated. Until the
end of 2020, there is the hope to confirm the safety and efficacy of the technique by
treating six more patients with PD, with the final goal to make the therapy available
by 2023 [43].
A rat model was used for validation of a preclinical trial to address radiation
injury to the brain, a clinically important need for cancer survivors. The goal was to
use hES cell-derived oligodendrocyte precursor (OP) cells that upon engraftment
into the forebrain had the ability to remyelinate the brain and salvage cognitive
deficits [38]. Also, upon injection of 250,000 O4+ cells in the cerebellum, there was
an improvement in motor tasks of the rats, while there was no indication of teratoma
formation. Further efficacy and safety data in preclinical animal studies has already
demonstrated that OP cell distribution is limited to the spinal cord and that there were
no adverse clinical observations [44], supporting the initiation of a Phase I/IIa
clinical trial in the United States.
It was recently announced that in 2019 the first clinical trial to tackle spinal cord
injury using hiPS cell-derived neural precursors will be held in Japan [45]. The
preclinical trial has taken place in 2012, during which it was demonstrated that the
procedure, involving the injection of neural precursors 2–4 weeks after the injury,
promoted regeneration of the spinal cords and increased the mobility in monkeys
[46]. In the clinical trial that will take place, two million neural precursors will be
injected within the same timeframe of injury, starting with a group of four patients.
Tissue engineering approaches combining the use of cells with scaffolds, bio-
molecules, or devices promise an evolution in the regenerative medicine field.
Generation of pancreatic progenitor (PP) cells from hPS cells has promised to
establish an almost unlimited source of islet cells for transplantation in diabetic
patients [39]. Furthermore, these cells have demonstrated the ability of treating
diabetes upon transplantation into mice [47]. ViaCyte, a biotechnology company,
has been conducting a series of clinical trials in the past years that are currently on
Phase I/II (Table 1). Their products comprise an islet-like cell population differen-
tiated from hES cell-derived PPs, which is microencapsulated within a permeable
device [48]. Upon transplantation into the patient, the islet-like cells can further
mature in vivo and generate glucose responsive β-like cells.
Although heart disease represents the main cause of death worldwide, it was not
until 2018 that the first clinical trial results started to emerge. The main aim of these
therapies involves the replacement of cells lost due to acute heart failure and
employs strategies such as cell sheets containing hPS cell-derived cardiomyocytes
[49] or fibrin patches comprising hPS cell-derived endothelial and smooth muscle
cells [50]. Recently, a clinical trial for the treatment of severe ischemic left ventric-
ular dysfunction has demonstrated the safety of hES cell-derived cardiovascular
progenitor (CVP) cells. In this safety study, a combination of hES cell-derived CVP
cells embedded in a fibrin patch was delivered to patients during a coronary artery
bypass, and it was demonstrated that this product improved the patient’s symptoms
while avoiding the formation of teratomas or the presence of arrhythmias, a common
complication of cardiac cell therapy [51]. Importantly, a preclinical trial that tested
the efficiency and safety in primates was already performed [36]. Here, it was
observed that hiPS cell-derived cardiomyocytes were able to survive up to
12 weeks post-implantation and, importantly, were able to connect electrically
with host cardiomyocytes and thus improve cardiac contractile function. This
study will be one of the bases for the initiation of a small clinical trial led by the
cardiac surgeon Yoshiki Sawa, which is supposed to start in early 2019. In this trial,
allogeneic hiPS cell-derived cardiomyocyte sheets comprising 100 million cells each
will be implanted onto the hearts of 3 patients suffering from advanced heart failure.
4 Bioprocesses for hPS Cell Expansion, Differentiation,

and Purification
A bioprocess for production of hiPS cell derivatives, starting with the collection of
donor somatic cells and ending with the delivery of a purified hiPS cell-derived
product, is a long pathway that can take up to a few months. Most of these
bioprocesses still rely on culturing cells under 2D conditions, in the absence of

control over culture parameters, such as pH and oxygen, and without automation,
which can lead to operator variability and, consequently, low reproducibility and
higher risks of contamination [52]. Therefore, to fulfill the potential of hPS cells in
regenerative medicine, there is the need to develop robust and scalable platforms that
can produce large numbers of high-quality hPS cells and/or hPS cell-derived
products.
Most of the cells within the human body, upon in vitro culture, depend on the
adhesion to a substrate to survive and proliferate. Under static conditions, this
dependence can be addressed by coating tissue culture plates with the appropriate
ECM molecules. However, in scaling up to dynamic culture systems, there is the
need to increase the surface-to-volume ratio of the adhesion substrate and, at the
same time, provide a system that can be used under dynamic conditions to afford a
homogeneous environment to the cells. This can be attained using microcarriers.
Although the use of solid microcarriers has been extensively studied for hPS cell
expansion and differentiation either in spinner flasks [5, 53–55] or vertical wheel
bioreactors [56], their use towards regenerative medicine applications requires an
additional separation step. This limitation can be potentially overcome by the use of
dissolvable microcarriers, which allow the integrated cell expansion and harvesting
inside the bioreactor while avoiding the use of filtration procedures [54]. Neverthe-
less, although 2D culture can sustain the growth of almost all cell types, it fails to
recapitulate in vivo conditions, as cells in vivo are usually surrounded either by other
cells or ECM. Thus, 3D aggregate-based culture provides a better recapitulation of
the in vivo microenvironment while avoiding the need for matrices that are typically
required for cell adhesion, either in adherent or microcarrier cultures, hence reducing
the number of components within the culture. Thus, in this chapter we will focus on
this culture format for scalable expansion and differentiation of hiPS cells.
The first described method for aggregate generation was the hanging drop, which
relies on the spontaneous aggregation of cells contained in droplets hanging from a
surface [57]. By controlling the cell number and volume in each droplet, the size of
the outcoming aggregate can be adjusted. Another technique relies on the seeding of
V-shaped well plates, allowing generation of one aggregate per well [58]. This
method can be adapted, in order to be reduced to a microscale level, where the
droplets are formed in microwells, allowing the formation of smaller aggregates
[59]. Moreover, the simplest technique involves the plating of a single-cell suspen-
sion into static or dynamic non-adherent surfaces, leading to spontaneous aggrega-
tion of the cells [60]. Cell density is an important factor, since the more cells are in
the suspension, higher the probability of starting to form aggregates [61]. In addition,
manually dissociated clumps can be passed through a mesh filter and fragmented
into smaller aggregates [62]. Microfabrication methods can be used to produce
microwell arrays made from a PDMS stamp in agarose that comprises an inverted
pyramid shape where a cell suspension is seeded and, by centrifuging the plate
containing the cells, will be entrapped in the microwell and generate size-controlled
aggregates [63].
Scaling up of hPS cell culture exposes the cells to fluctuations in physicochemical

parameters and to the hydrodynamic shear stress inside the bioreactors [64]. Impor-
tantly, homogeneity of size and morphology of hPS cell aggregates can be improved
using forced aggregation in microwells [63], and the negative effect of shear stress
under dynamic conditions can potentially be controlled by cell encapsulation
[65]. Nevertheless, the use of dynamic suspension cultures also leads to increased
spheroid homogeneity, as well as increased cell yields [66, 67]. However, it has to be
considered that increased agitation speeds leads to an augmentation of shear forces,
thus compromising the viability of the cells [61]. Polysulfated compounds have
confirmed their capacity of reducing cell aggregation and promoting cell survival by
reducing apoptosis [68]. Dextran sulfate has recently been demonstrated to improve
aggregate size homogeneity on hPS cells without compromising their pluripotency,
which will improve their efficiency in terms of expansion [69].
An important highlight on the scale-up of cell culture resides in the reduction of
the overall cost of the process. For example, it has been demonstrated that scaling up
of the production of β cells from hPS cells to provide insulin for type 1 diabetes
would be able to reduce the cost per patient from $430,000 to $160,000 [70].
4.1 hPS Cell Expansion as 3D Aggregates
An initial approach towards expansion of hPS cells as suspension aggregates

included the use of mouse embryonic fibroblast (MEF)-conditioned medium
(MEF-CM), which was supplemented with bFGF and ROCKi [71], but this strategy
carried the risk of contamination from xenogeneic products. A number of systems
using animal-free conditions have emerged since, including human foreskin
fibroblast-CM, which was used to scale up hPS cell expansion with a yield of
eightfold in 7–10 days [72]. Amit et al. developed one of the first protocols for
hPS cell expansion as suspension aggregates using serum-free media supplemented
with interleukins, bFGF, and ROCKi [73]. This system was able to attain a 25-fold
increase in 10 days, while maintaining the pluripotency and differentiation potential
of the cells. At the same time, Steiner et al. developed a system that employed
ECM-rich culture medium in combination with bFGF, activin A, and a serum
replacement [74]. Since then, several defined culture systems have emerged
and established major improvements in terms of robustness and reproducibility
(Table 3), as well as compliance with good manufacturing practices (GMP) for
eligibility for future clinical trials.
Culture of hPS cells as suspension aggregates in chemically defined conditions
using commercially available culture media, such as mTeSR [61, 76, 78–80, 82, 85–
87], StemPro [77, 88], and E8 [75, 80], has been extensively reported, leading to a
maximum 25-fold increase in cell number after 6 days [78]. As another improvement
to this culture system, single-cell inoculation and consequent aggregation of hPS
cells have been used instead of passaging cells as aggregates, resulting in a higher
level of expression of pluripotency markers [73, 76, 87]. 3D aggregate culture
Table 3 Scalable approaches for hPS cell expansion as suspension aggregates

Vessel Seeding Expansion
Volume Feeding density
Type (mL) strategy (cells/mL) Media Yield Ref
Spinner flask 45 Repeated 4–5 105 E8 4.5-fold in [75]
batch 4 days
50 Repeated 3 104 to DMEM/ 25-fold in [60]
batch 1 106 F12 + KOSR+ 10 days
bFGF+IL6RIL6
50 Repeated 0.33–1 106 mTeSR1 Sixfold in [76]
batch 4–7 days
50 Repeated 0.3 105 DMEM/ Eightfold [72]
batch F12 + Glutamax + in 7–
bFGF 10 days
50 Repeated 1 106 mTeSR1 Twofold [61]
batch in 7 days
60 Repeated 2.5 105 StemPro SFM Fourfold [77]
batch in 4 days
100 Repeated 1.8 104 mTeSR1 25-fold in [78]
batch 6 days
100 Batch 2 104 mTeSR1 11–12- [79]
fold in
6 days
BioLevitator 30 Fed batch 0.3 105 mTeSR1 20-fold in [80]
4 days
30 Fed batch 0.3 105 E8 14-fold in
4 days
3D hollow 17 Perfusion 2.9 106 mTeSR1 100-fold [81]
fiber in 15 days
bioreactors
Controlled 100 Repeated 4–5 105 mTeSR1 Fourfold [82]
bioreactor batch in 7 days
125 Perfusion 5 105 mTeSR1 Sixfold in [83]
7 days
150 Perfusion 5 105 E8 6.7-fold in [84]
7 days
systems have been performed using several scalable culture platforms, including
shaking flasks [60, 86], spinner flasks with a wide range of working volumes [61, 72,
73, 75–79, 82], and bioreactors with no impellers, using a gentle tube rotation [80],
or using 3D hollow fibers [81]. In this type of systems, factorial design was used to
maximize cell productivity, by allowing simultaneous optimization of the different
culture parameters, including the agitation speed, and inoculation density. This
mathematical tool allowed an increase in cell number of 12-fold after only 6 days
of culture [79]. The feeding regimen of the reactor also plays an important role, with
the use of perfusion yielding a final cell density of 2.85 106 cells/mL, which
represents an increase of 47% in cell yield when compared with batch cultures
[83]. Furthermore, a recent approach by Manstein et al. describes the use of four
parallel bioreactors that allows the generation of two million hPS cells using
chemically defined medium under a perfusion feeding regimen during 7 days [84].
Importantly, the quality of hPS cells cultured in bioreactors and other scalable
systems is closely monitored in terms of maintenance of the expression of
pluripotency markers, assessment of pluripotent potential by multilineage differen-
tiation or ability to generate teratomas in immunocompromised mice, and karyotypic
analysis [75, 79].
4.2 hPS Cell Differentiation as 3D Aggregates
Although hPS cell-derived products represent an amazing opportunity for regener-

ative medicine, there is still the need to produce clinically relevant numbers of cells,
which can attain up to 109 cells per patient [89]. The development of strategies for
production of lineage-differentiated derivatives from hPS cells on a large scale has
been one of the foci in the field of stem cell bioprocessing in the last years (Table 4).
The use of small molecules and chemically defined and xeno-free culture media has
increased the efficiency and robustness of differentiation protocols.
In the case of neural induction, it was determined that an average aggregate
diameter of approximately 140 μm was optimal to efficiently generate neural
progenitor (NP) cells from hiPS cells [90]. Furthermore, after only 6 days of
induction using a dual SMAD inhibition protocol using small molecules – 10 μM
SB431542 and 100 nM LDN193189 – nearly 14 million Pax6+ NP cells were
successfully derived from hiPS cells, in spinner flasks, under chemically defined
conditions with efficiencies averaging 62% [91].
Table 4 Scalable approaches for hPS cell directed differentiation as suspension aggregates
Culture type Differentiation
Type Directed lineage Yield (%) Ref
Spinner flask Neural progenitors ~80 [90]
Neural progenitors ~62 [91]
Cardiomyocytes 27 [77]
Hematopoietic progenitors 25–80 [75]
Hematopoietic progenitors 5–6 [67]
Cardiomyocytes 90 [6]
Cardiomyocytes 80–99 [88]
Islet cells ~90 [95]
Rotary orbital shaker Cardiomyocytes >60 [86]
Pancreatic progenitors 7–32 [94]
Controlled bioreactor Cardiomyocytes 85 [86]
Cardiomyocytes ~80 [119]
As previously mentioned, the use of 3D conditions has numerous advantages

comparing to adherent monolayer conditions. In the case of cardiomyocyte deriva-
tion from hPS cells, 3D conditions have demonstrated to promote an earlier
mesendoderm lineage differentiation, as well as a faster maturation of the
cardiomyocytes, either structurally or functionally [92].
Several groups have reported different methodologies that were able to generate
large numbers of cardiomyocytes [6, 77, 85, 88] by culturing hiPS cells under 3D
conditions in stirred bioreactors. The majority of these protocols are based in a Wnt
signaling pathway modulation, as established by Lian et al. [93], which consists in
an early Wnt signaling activation at early stages of differentiation by using a GSK3
inhibitor such as CHIR99021, followed by an inhibition of this signaling pathway by
using IWP to specify cardiac differentiation of the mesendoderm-directed cells.
Using this protocol for Wnt signaling modulation, hPS cell cardiac differentiation
using a fully controlled stirred bioreactor has led to the production of up to 40 million
cardiomyocytes with up to 95% of efficiency [85, 86].
Chen et al. highlighted the importance of size control of hPS cell aggregates
before cardiac induction [88]. Here, an average hPS cell aggregate diameter of
200 20 μm was shown to be less susceptible to changes in CHIR99021 concen-
trations, which could affect the efficiency of the differentiation protocol. Therefore,
the optimized protocol was translated to a 1 L scale and was able to produce up to
96% of cardiac troponin T (cTnT) positive cells at a density of 1.4 0.4 106 cells
per mL.
Furthermore, Fonoudi et al. developed a modified protocol that also used the
initial inhibition of the Wnt signaling pathway but that, in the inhibition phase, also
inhibited the TGF-β pathway and activated the sonic hedgehog (SHH) pathway
[6]. Here, an optimal hPS cell initial aggregate diameter of 175 25 μm promoted
spheroid beating efficiency averaging almost 100% and was able to derive up to 90%
of cardiomyocytes in only 10 days at a 100 mL bioreactor scale.
A four-stage protocol to differentiate pancreatic progenitors (PPs) from hPS cells
was implemented under suspension conditions using ultra-low attachment plates and
rotary agitation [94]. The first step involved the specification into definitive endo-
derm, by using a medium supplemented with activin A and Wnt3A for 1 day,
following another day without Wnt3A. Then, KGF and TGF-β RI kinase inhibitor
IV were used to promote primitive gut tube, and TTNPB, KAAD-cyclopamine, and
Noggin supplementation favored posterior foregut formation. Finally, pancreatic and
endocrine progenitors were generated by further supplementation with Noggin,
KGF, and EGF. In the end, it was possible to obtain functional glucose-responsive,
insulin-secreting cells. Nevertheless, this protocol still employs the use of fetal
bovine serum in the first stages of differentiation, which will need to be replaced
before considering this approach towards clinical applications. A similar protocol
was translated to a 30 mL spinner flask, promoting the generation of islet-like cells
with an efficiency of more than 90% of PDX1+ cells that, upon engraftment in mice,
were able to revert hyperglycemia [95].
4.3 Purification of hPS Cell-Derived Products
The major risk of the application of hPS cell-derived products resides in the
possibility of teratoma formation due to an uncontrolled proliferation of a small
number of pluripotent cells that remain in the final cell therapy product. In order to
prevent this, clinical-scale purification of hPS cell derivatives for cell-based thera-
pies needs to be addressed [96, 97].
Fluorescent-activated cell sorting (FACS) is probably the most widely used and
more accurate method for purification of cells. This technique involves labeling the
target cells with an antibody linked to a fluorescent molecule and has demonstrated
efficiencies of separation above 95% [98]. For example, Kikuchi et al. included a
sorting step at day 12 of neural induction of hPS cells that allowed the selection of
more than 90% CORIN-positive cells, which would further differentiate into mid-
brain dopaminergic neurons [37]. In this study, on day 26 of differentiation, there
were less than 1% of cells expressing pluripotency markers. FACS sorting of
O4-positive oligodendrocyte progenitors led to an enriched cell population
containing more than 93% of these cells that upon animal engraftment did not
originate teratomas [38].
A widely used example of a target antigen for cell purification is SSEA-1, because
this surface marker is absent from hPS cells [99]. Since the efficiency of the
differentiation protocol of hES cells into CVPs only led to approximately 64% of
efficiency, Menasche et al. integrated within the production pipeline a purification
step taking advantage of this marker [51, 100]. The purification strategy consists in
the depletion of hPS cells in culture by magnetically labeling cells with SSEA-1 and
then using magnetic activated cell sorting (MACS) to select SSEA-1-positive cells,
which had lost their pluripotency. Furthermore, the final product, which was
required to contain less than 0.1% of undifferentiated cells, was also tested for the
expression of Nanog.
For the treatment of Stargardt’s macular dystrophy (SMD) and dry-age-related
macular degeneration (AMD), hES cell-derived retinal pigment epithelium (RPE)
cells were purified by exposure to type IV collagenase followed by manual isolation
of RPE using a glass pipette [101]. Still, long-term safety studies in preclinical
models have not detected any uncontrolled cell proliferation, indicating that no
teratomas were generated upon implantation of hES cell-derived RPE [102]. An
approach to address spinal cord injury by using hES cell-derived OP cells has
demonstrated that a final product containing up to 5% of undifferentiated hES
cells did not lead to teratoma formation [103]. However, the OP cell differentiation
protocol has an efficiency that leads to less than 1% of hES cells in the final product.
Metabolic purification of hPS cell-derived products has also been focused in the
last few years, mainly due to its reduced cost and high technical feasibility, which
relies on the supplementation of the culture medium with components that are
already present in most culture media formulation. Furthermore, these methods
can be easily integrated within bioreactor culture systems without the need for cell
singularization, contrary to methods like FACS or MACS. One good example of the
application of these methods is for hPSC-derived cardiomyocyte purification. In fact,

the two main substrates for energy production by cardiomyocytes are glucose and
fatty acids, but cardiomyocytes can also use lactate as an energy source in anaerobic
metabolism [104]. In the first report of hPS cell-derived cardiomyocyte purification
using a metabolic approach, a glucose-depleted and lactate-supplemented culture
medium was capable of purifying the cardiomyocyte population up to 99%, both in
mouse and human cells [105]. Still, glucose remains the main substrate for primary
ATP production in immature cardiomyocytes, whereas fatty acids play an important
role on the energy demand of mature cardiomyocytes. It has been demonstrated that
glucose depletion associated with fatty acid supplementation is capable of reducing
the number of hPS cells in the final product while increasing the maturation of hPS
cell-derived cardiomyocytes [106].
Contrary to hPS cells, hPS-differentiated cells have mechanisms that prevent
excessive uptake of L-alanine – an amino acid present in the human body – which
would lead to cell swelling and, consequently, death. A novel approach towards
selective elimination of hPS cells relies on the use of high concentrations of
L-alanine to deplete the culture from pluripotent stem cells, and that can be a
cost-effective alternative for purification of hPS cell-derived products to use in cell
therapies [107].
4.4 Bioengineering Strategies for the Improvement of hPS

Cell-Derived Organoids
Organoids are complex structures that can be generated from hPS cells or from
organ-specific progenitor cells and that are capable of differentiating into multiple
cell types while self-organizing themselves in a structure similar to the represented
organ and, at the same time, recapitulating some of the organ functions, such as
neural activity or contraction [108].
The application of bioprocessing strategies is crucial towards overcoming the
limitations often associated with organoids. One of the main limitations resides in
the heterogeneity among different organoids often associated with poor efficiency of
differentiation of hPS cells into a specific lineage. It was already demonstrated that
the initial hPS cell aggregate diameter is critical for the optimal differentiation into a
specific cell lineage [90, 109]. For that, strategies that employ the use of microwells
to control hPS cell aggregate size have proven effective to direct hPS cells into
cardiomyocytes [63]. Arora et al. demonstrated that the passage from a spheroid
containing hindgut progenitor cells into a pre-organoid is favored by the size and
morphology of the spheroid [110]. Here, the sorting of aggregates with diameters
greater than 75 μm favored the further development into intestinal organoids in 3.8-
fold when compared with non-sorted populations.
As the size of the organoids increases, mass transport to the inner areas of the
organoid becomes critical, since diffusional limitations often arise in spheroids with
diameters greater than 300 μm [111]. The use of agitated systems, such as spinner
flasks, that enhance the mixing of nutrients and promote gas transfer has led to a
rapid development of brain organoids [3, 112]. Nevertheless, in the absence of
medium agitation, a vascularized liver organoid has already been developed, by
combining hPS cell-derived hepatic endoderm with mesenchymal stem cells and
human umbilical vein endothelial cells [113].
Another limitation of organoid technology relies on organoid encapsulation,
since many of the processes developed so far depend on the embedding of organoids
in a supporting extracellular matrix that helps maintaining the organoid in a favor-
able microenvironment for growth and maintenance [3]. Still, mechanical properties
of the substrate in which the organoids are embed also play an important role.
Although intestinal organoids present self-organization, embedding the organoids
within a collagen gel allowed the inner cystic structures to align, generating macro-
scopic hollow tubes composed of multiple cells, a process that was not observed
without this matrix [114]. A bioprocessing approach that employs the use of a
two-fluidic electrostatic co-spraying technique has tackled the issue of large-scale
production of encapsulated organoids [115]. Here, the organoids are encapsulated in
a Matrigel core-shell that promotes cell growth and structural support and an outer
alginate shell that protects the capsule from shear stress in suspension culture inside
bioreactors.
Lastly, the main challenge associated with organoids and other tissues derived
from hPS cells remains in the display of fetal-like characteristics [116], which may
not adequately model adult diseases. Human intestinal organoids derived from hPS
cells have demonstrated to resemble human fetal intestine instead of adult tissue
[117], but upon in vivo transplantation are able to further mature and become more
adult-like intestinal tissue. For cardiac derivatives generated from hPS cells, physical
conditioning using electromechanical stimuli over a period of 4 weeks in culture was
sufficient to accelerate cardiomyocyte maturation into adult-like human cardiac
tissue [118].
5 Human Pluripotent Stem Cells and Disease Modeling
The ability to generate disease-specific hiPS cells has brought the opportunity of
unveiling disease mechanisms that until recently were unknown. In the past few
years, and mainly by using a combination between hiPS cells and organoid tech-
nologies, a myriad of disease models have been developed, from neurological
[3, 120] to cardiac [121] or gut [122] diseases.
5.1 Modeled Diseases
Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disorder where loss of

motor neurons in the spinal cord and motor cortex induces progressive paralysis and,
ultimately, death [123]. One of the first reports using hiPS cell technology consisted
in the generation of ALS-specific hiPS cell lines that efficiently differentiated into
motor neurons [124]. Still, only recently a model that incorporates 3D skeletal
muscle bundles with light-sensitive channelrhodopsin-2-induced motor neuron
spheroids derived from ALS patients was developed [125]. Using this platform, it
is possible to analyze muscular contraction through light activation of the motor
neurons. When ALS-specific motor neurons were used instead of healthy cells, there
was less muscular contraction, together with more motor neuron degradation and
decrease in their viability.
Second to Alzheimer’s disease, Parkinson’s disease (PD) is the most common
chronic progressive neurodegenerative disorder, characterized by loss of dopami-
nergic neurons. Although patient-specific PD hiPS cells were already derived [126],
since the onset of PD occurs on average at 50 years of a human life, additional cues –
such as exposure to oxidative stress or neurotoxins – are needed to replicate the
pathological mechanisms of PD in vitro.
Spinal muscular atrophy (SMA) is a genetic disorder based on the mutation of the
SMN1 gene and is characterized by selective degeneration of lower α-motor neurons
[127]. Derivation of hiPS cells from a child with SMA followed by neural differen-
tiation into motor neurons demonstrated deficits in either cell number or size when
compared with a control from a parent [128].
Rett syndrome, a neurological disease commonly caused by mutations in the
MECP2 gene of the X chromosome, is related with impairment of motor function
and autistic-like behavior [120]. Using patient cells and hiPS cell technology, it was
possible to recapitulate an altered process of neurogenesis in cells of these patients,
as well as the presence of an inferior number of neurons and less complex
neurites [129].
Familial dysautonomia, or Riley-Day syndrome, is a fatal autosomal recessive
disease caused by a mutation in the IKBKAP gene and characterized by the degen-
eration of sensory and autonomic neurons [130]. Neural induction of patient-specific
hiPS cells has demonstrated that in vitro these cultures present neural crest pre-
cursors with lower Tuj1+ neuron differentiation potential, in addition to defects in
migration behavior [131].
Schizophrenia affects around 1% of the worldwide population and possesses a
hereditary genetic component in almost 85% of the cases [132]. It was shown that
neural differentiation of hiPS cells from schizophrenic patients led to decreased
neural connectivity and decreased number of neurites and synaptic protein levels
while presenting a normal electrophysiological behavior and spontaneous calcium
transient activity [133].
Heart diseases have also been addressed as a target for disease modeling using
hiPS cells. Recapitulation of familial long QT syndrome was achieved by generating
hiPS cell-derived cardiomyocytes from patients, recreating electrophysiological

features of the disorder, such as prolonged action potentials [121].
Dilated cardiomyopathy occurs in 1 of 250 adults and is characterized by
progressive left ventricular dilation and, eventually, heart failure [134]. This disease
may arise from mutations that truncate the sarcomere protein titin, which was
verified by derivation of disease-specific hiPS cells that, upon cardiac differentiation,
resulted in the generation of cardiomyocytes with sarcomere deficiency, combined
with decreased responses to stress, as well as to activation from growth factors [135].
Another example of a heart disease modeled using hiPS cell technology consists
in Duchenne muscular dystrophy (DMD), which is characterized by the progressive
weakness of the skeletal and cardiac muscle [136]. Cardiomyocytes derived from
patients with DMD displayed dystrophin deficiency when compared with controls
from healthy donors. Furthermore, disease-specific cardiomyocytes demonstrated
higher levels of apoptotic markers, including increased levels of cytosolic Ca2+ or
caspase-3, since these cells are more sensitive to stress-induced damages [137].
5.2 Organoids and Disease Modeling
Neural differentiation of hPS cells has been thoroughly investigated, since

neuroectoderm represents the default differentiation pathway upon withdrawal of
pluripotency maintenance culture conditions [138]. Brain organoids derived from
hPS cells were the first complex 3D models that were able to recapitulate certain
aspects of in vivo brain development such as progenitor zone organization and the
organization of outer radial glial stem cells [3]. Using Matrigel as an extracellular
matrix to support the process of self-organization, it was possible to obtain brain
organoids that exhibited cortical self-organization, as well as specification of hind-
brain and forebrain regions. Since then, other brain-specific organoids have been
recreated using specific morphogen gradients, including the cerebellum [139] and
the optic cup [140], as well as organoids from other tissues, such as the lung [116] or
even liver buds [113]. Furthermore, it was recently demonstrated that brain
organoids from different regions could be fused to mimic interactions between
brain regions. The coculture of brain organoids with dorsal and ventral forebrain
identities led to the generation of a model of a dorsal-ventral axis that demonstrated
the migration of CXCR4-dependent GABAergic interneurons from ventral to dorsal
forebrain [141].
The combination between hiPS cell differentiation and organoid technology has
opened the possibility of using patient-specific cells to recreate disease mechanisms
in vitro using more relevant models of in vivo tissues, as certain structural charac-
teristics of a disease can also be replicated in a 3D structure. Several disease models
have been developed using organoids, ranging from neurological diseases [3, 142–
144], using brain organoids, to heart conditions, using cardiac organoids [121, 135],
or cystic fibrosis [145], using lung organoids, as listed in Table 5.
Table 5 Disease modeling using hiPS cells and respective observed in vitro characteristics
Modeled disease Model characteristics Ref
Alagille syndrome • Impaired chloride transport [144]
Autism • Normal early neuronal differentiation [143]
• Imbalance in inhibitory GABAergic neurons over
glutamatergic
Cystic fibrosis • Impaired forskolin-induced swelling [145]
Dilated • Sarcomere insufficiency [135]
cardiomyopathy • Impaired responses to mechanical and β-adrenergic stress
Familial adenomatous • Increased cell proliferation [122]
polyposis • Increased nuclear localization of β-catenin
Familial dysautonomia • Defects in neural differentiation [131]
Familial long QT • Prolonged action potential in cardiomyocytes [121]
syndrome
Hirschsprung’s disease • Impaired organization of enteric nervous system [146]
Microcephaly • Premature neuroepithelial differentiation [3]
• Aberrant glial orientation
• Smaller areas of differentiated tissue
Miller-Dieker • Poor neurite growth [142]
syndrome • Apoptosis in the ventricular zone
• Impaired neuronal migration
Rett syndrome • Impaired neurogenesis [120]
• Reduced neuronal migration
Schizophrenia • Decreased neural connectivity, neurite formation, and [133]
synaptic protein expression
Spinal muscular • Motor neuron differentiation [128]
atrophy
In the field of neurological diseases, the study of microcephaly was the first that
was addressed using hiPS cell-derived brain organoids [3]. Using fibroblasts from a
patient with microcephaly, caused by truncating mutations in CDK5RAP2, it was
possible to generate a patient-specific hiPS cell line that, under pluripotency main-
tenance conditions, behaved similar to a hiPS cell control line. Still, upon induction
to neuroectoderm as a 3D structure, a smaller neuroepithelial tissue with fewer
progenitor zones and an increased premature neuronal outgrowth was observed.
Autism spectrum disorders affect 1 in every 60–70 children and have as main
symptoms behavioral deficits in social interaction, communication, and interests,
along with repetitive behaviors [147]. Using patient-specific hiPS cell-derived neural
organoids, normal neuronal differentiation was observed, while there was an imbal-
ance in the number of inhibitory GABAergic neurons over glutamatergic neurons,
which suggests an underlying mechanism for this neurological disease [143].
Miller-Dieker syndrome is a genetic neurological disorder caused by large het-
erozygous deletions of human band 17p13.3, and it is characterized by a major
absence of cortical folding of the brain, leading to microcephaly, mental retardation,
and intractable epilepsy [148]. Using brain organoids derived from patient-specific
hiPS cells, Bershteyn et al. demonstrated that there were apoptotic areas in the
subventricular zone, poor neurite growth, and impaired neural migration [142].
The enteric nervous system (ENS) of the gastrointestinal tract is crucial for the
functions of this organ including motility, secretion, and blood flow [149]. The
development of a normal intestinal enteric nervous system has been recapitulated by
merging hiPS cell-derived neural crest cells and human intestinal organoids, which
were able to mediate contractile waves [146]. Using this model, it was possible to
recapitulate Hirschsprung’s disease, characterized by congenital lack of enteric
ganglia [150], and to verify that in vitro there was an impaired neural crest and
ENS development [146].
A model of vascular disease based on blood vessel damage induced by diabetes
was recently developed [151]. Here, endothelial cells were derived from hiPS cells,
and, using a basement membrane, blood vessel organoids were created. Further-
more, upon transplantation to mouse models and exposure to a diabetic environment,
there was thickening of vascular basement membrane, characteristic of diabetic
vasculopathy.
Cystic fibrosis (CF) is a genetic disease that affects several organs, such as the
liver and intestine, but mostly the lungs. Contrary to other lineage specifications
from hPS cells, differentiation protocols towards cells of the respiratory track tend to
be complex. Still, directed differentiation of hPS cells into NKX2.1+ airway pro-
genitor cells followed by low Wnt signaling activation led to the generation of lung
organoids that contained cells from the goblet, basal, and secretory lineages
[145]. Furthermore, using hiPS cells derived from CF patients, it was possible to
recapitulate key features of the disease, including impaired forskolin-induced
swelling.
5.3 Disease Modeling Using CRISPR/Cas9
Despite the several examples of genetic diseases modeled using hiPS cell technology
(Table 5), a new challenge that emerges is how to clearly discriminate if the
differences observed in the phenotype are due to the mutation or to the individual’s
genetic background. A state-of-the-art approach relies on the use of genetic manip-
ulation tools that correct the mutation of a given clone, creating an isogenic hiPS cell
line. The most used tool is the clustered regularly interspaced short palindromic
repeats (CRISPR) modified with two components – Cas9, which is the enzyme
responsible for DNA cleavage, and guide RNA, which binds to Cas9 and pairs with
the desired DNA site [152]. The CRISPR/Cas9 technology allows a footprint-free
gene modification and at the same time has the advantage of being a simpler
manipulation procedure comparing to other gene editing techniques such as zinc
finger nucleases [153]. In addition to this strategy, CRISPR/Cas9 can also be used
for the insertion of a mutation to recreate a mutated phenotype.
Huntington disease (HD) is caused by a CAG repeat in HTT and results in
impaired neural rosette formation and deficits in mitochondrial respiration. Using a
HD-specific hiPS cell line and a corrected isogenic hiPS cell line, it was possible not
only to differentiate the cells into forebrain neurons but also to rescue phenotypic
abnormalities in the isogenic hiPS cell line [154]. Several other genetic diseases, like
amyotrophic lateral sclerosis [155], Alzheimer’s disease [156], and β-thalassemia
[157], among others, have been studied using this approach. On the other hand,
CRISPR/Cas9 has also been used to introduce the CCR5del32 mutation in hiPS
cells, which originated monocytes resistant to HIV infection [158].
Despite all the apparent advantages, CRISP/Cas9 technology has been thor-
oughly debated, since recent reports have demonstrated that this methodology may
not be as specific as initially presented, since significant mutations near the target site
have been detected [159].
5.4 Drug Discovery
Both organoids and hiPS cell-derived cells are currently part of a paradigm change in
the industry of drug screening and development. Apart from the use of such
technologies for studying the response of in vitro human tissues to specific drugs,
the possibility of attaining personalized treatments is also possible due to hiPS cell
technology.
We have previously reported the use of hiPS cell-derived NPs to predict the effect
of commonly used compounds, such as valproic acid (VPA), during the early stages
of the process of neurodevelopment [7]. Exposure of cells to VPA during the initial
stage of brain tissue formation, replicated in vitro using hiPS cell spheroids, has led
to disorganized neural rosette structures, decreased cell viability, and delayed neu-
ronal differentiation.
As previously mentioned in this chapter, using a combination of 3D skeletal
muscle bundles and light sensitive spheroids of motor neurons derived from hiPS
cells of an ALS patient, it was possible to analyze muscular contraction based on
light activation of the motor neurons [125]. This platform was used to test the
efficacy of drug candidates to treat ALS, such as rapamycin and bosutinib. In the
presence of these drugs, the levels of caspase-3/7 decreased, indicating fewer cell
death, which may suggest a neuroprotection mechanism of such drugs.
Another application of hiPS cell-based products relies on the validation of drugs
to inhibit viral infection of neural tissues. Derivation of NP cells from hiPS cells has
also been addressed to screen for compounds that block Zika virus infection and, at
the same time, clear the virus from already infected NP cells [160].
Acute myeloid leukemia (AML) is an oncologic disease that is caused by a
translocation in the MLL gene and that compromises normal hematopoiesis, by
uncontrolled proliferation of myeloid progenitor cells. iPS cells derived from
AML patients were able to differentiate into hematopoietic progenitors, while still
retaining their malignant characteristics [161]. Importantly, this study has demon-
strated that clonal differences in different hiPS cell lines derived from patients with
AML can represent a powerful tool to understand drug sensibility of individual hiPS
cell-derived subclones.
Although the efficiency of a drug is important, other aspects comprised within the
drug metabolism process cannot be neglected. Intestinal drug absorption is important
for the oral drug delivery system, and, for that, using hiPS cell-derived epithelial
cells, a model of prediction of drug absorption was developed [162]. The derived
epithelial cells displayed similar characteristics to their in vivo counterparts, such as
the presence of thigh junctions, metabolic enzymes, and drug transporters, that
closely mimic the mechanisms of oral drug absorption, which makes this platform
a powerful tool towards the prediction of drug absorption in vivo.
Organ-on-a chip platforms provide invaluable tools for drug screening, since they
combine the opportunity of recapitulating the multicellular architectures of an
organoid with the possibility of testing different drugs on a high-throughput scale.
The derivation of multi-organ platforms from hPS cells and their application for drug
screening have already been reviewed in Miranda et al. [4]. Still, we highlight the
development of a multi-organ platform that incorporates three different modules
containing organoids from the liver, heart, and lung [163]. This device has the great
advantage of allowing to analyze an individual response to a drug for each organoid
or to combine the response between the organoids from different tissues, which gives
a more complete preview of the in vivo behavior. Here, the effect of bleomycin – a
chemotherapeutic drug used to treat lung cancer – was studied in separated organ
modules and in an interconnected manner. Using an isolated module setting, as
predictable, there was an increase in inflammatory markers in the lung module and
no cardiotoxic effect in the separate heart module. Still, in a three-organ setting, it
was possible to observe a decrease in heart rate due to inflammatory factor-driven
cardiotoxicity, which highlights the importance of a combined response between
different organs in order to efficiently predict cell behavior upon drug exposure.
Overall, although organ-on-a-chip platforms provide an opportunity towards drug
discovery applications, most of them still uses primary cell sources and immortalized
cell lines. Despite the fact that primary cells retain similar characteristics, such as
metabolic profiles and functional properties to the original tissues, they tend to lose
these properties when cultured in vitro for prolonged periods of time [164]. On the
other hand, immortalized cell lines frequently fail to recapitulate the original tissue
and are known for the accumulation of karyotypic abnormalities that may compro-
mise their response to stimuli [165]. Therefore, hPS cell derivatives can address
some of these issues, adding the possibility of generating disease- and patient-
specific cells that promote a more realistic approach to drug screening applications.
6 Conclusions and Future Perspectives
The advancement in hiPS cell technology has been impressive since the first
derivation of these cells, providing great opportunities in the fields of regenerative
medicine, disease modeling, and drug screening. Here, we summarized recent pro-
gresses in the biomedical applications of hiPS cells as well as the bioprocessing
challenges that have to be surpassed to fulfill these applications, namely, to allow
their translation either into clinical settings or for study of disease mechanisms or
towards drug discovery.
Some limitations still need to be addressed prior to a complete shift towards these
systems. First, there is the need to keep developing robust protocols for hiPS cell
lineage specification, since even protocols that employ the use of defined culture
medium and small molecules are not always reproducible. Moreover, there is the
need to develop robust, safe, and scalable protocols for the large-scale production of
these derivatives with high quality and functionality. Second, even though patient-
specific hiPS cell lines can be derived, the individual hiPS cell clones may display
some variability. Furthermore, although isogenic hiPS cell lines are considered state
of the art, it must be considered that the single-cell cloning procedures may result in
clones that possess different properties from the original cells.
Currently, most of the hPS cell-derived organoids represent only some aspects of
the target organ, displaying a limited number of cell types [166], which can hinder a
correct structural organization and thus function of the tissue. In the future, it will be
necessary to introduce protocols that will differentiate hPS cells into a representative
number of cell types, in order to accurately represent the physical interactions and
organization between cells. Future approaches also include further maturation of
organoids, since current organoids obtained from hPS cells represent an early
developmental stage, often compared with fetal tissues [167]. It is expected that
hiPS cell-based disease models using organoids will improve preclinical drug
screening and accelerate candidate therapies into clinical trials. Incorporation of
organoid technology combined with organ-on-a-chip technology will accelerate the
readouts of each experiment, while maintaining a humanized response in compari-
son with the currently available animal models.
Acknowledgments C.C.M. acknowledges support from FCT – Fundação para a Ciência e a

Tecnologia (CEECINST_IJ3/IST-ID). This work was further financially supported by FCT
Portugal through iBB, Institute for Bioengineering and Biosciences (UID/BIO/04565/2013), and
from Programa Operacional Regional de Lisboa 2020 (Project No. 007317), PTDC/EMD-TLM/
29728/2017 and PTDC/EQU-EQU/29653/2017 projects, and project PRECISE – Accelerating
progress toward the new era of precision medicine (PAC-PRECISE-LISBOA-01-0145-FEDER-
016394, SAICTPAC/0021/2015).
References
1. Thomson JA, Itskovitz-Eldor J, Shapiro SS, Waknitz MA, Swiergiel JJ, Marshall VS et al
(1998) Embryonic stem cell lines derived from human blastocysts. Science 282
(5391):1145–1147
2. Takahashi K, Tanabe K, Ohnuki M, Narita M, Ichisaka T, Tomoda K et al (2007) Induction of
pluripotent stem cells from adult human fibroblasts by defined factors. Cell 131(5):861–872
3. Lancaster MA, Renner M, Martin CA, Wenzel D, Bicknell LS, Hurles ME et al (2013)
Cerebral organoids model human brain development and microcephaly. Nature 501
(7467):373–379
4. Miranda CC, Fernandes TG, Diogo MM, Cabral JMS (2018) Towards multi-organoid systems
for drug screening applications. Bioengineering 5(3):E49
5. Badenes SM, Fernandes TG, Cordeiro CS, Boucher S, Kuninger D, Vemuri MC et al (2016)
Defined essential 8 medium and vitronectin efficiently support scalable xeno-free expansion of
human induced pluripotent stem cells in stirred microcarrier culture systems. PLoS One 11(3):
e0151264
6. Fonoudi H, Ansari H, Abbasalizadeh S, Larijani MR, Kiani S, Hashemizadeh S et al (2015) A
universal and robust integrated platform for the scalable production of human cardiomyocytes
from pluripotent stem cells. Stem Cells Transl Med 4(12):1482–1494
7. Miranda CC, Fernandes TG, Pinto SN, Prieto M, Diogo MM, Cabral JMS (2018) A scale
out approach towards neural induction of human induced pluripotent stem cells for
neurodevelopmental toxicity studies. Toxicol Lett 294:51–60
8. Evans MJ, Kaufman MH (1981) Establishment in culture of pluripotential cells from mouse
embryos. Nature 292(5819):154–156
9. Bongso A, Lee EH (2005) Stem cells: their definition, classification and sources. In:
Bongso A, Lee EH (eds) Stem cells: from bench to bedside. World Scientific, Singapore, pp
1–13
10. Campbell KH, McWhir J, Ritchie WA, Wilmut I (1996) Sheep cloned by nuclear transfer from
a cultured cell line. Nature 380(6569):64–66
11. Cowan CA, Atienza J, Melton DA, Eggan K (2005) Nuclear reprogramming of somatic cells
after fusion with human embryonic stem cells. Science 309(5739):1369–1373
12. Takahashi K, Yamanaka S (2006) Induction of pluripotent stem cells from mouse embryonic
and adult fibroblast cultures by defined factors. Cell 126(4):663–676
13. Gurdon JB, Melton DA (2008) Nuclear reprogramming in cells. Science 322
(5909):1811–1815
14. Patel M, Yang S (2010) Advances in reprogramming somatic cells to induced pluripotent stem
cells. Stem Cell Rev 6(3):367–380
15. Pralong D, Trounson AO, Verma PJ (2006) Cell fusion for reprogramming pluripotency:
toward elimination of the pluripotent genome. Stem Cell Rev 2(4):331–340
16. Zeuschner D, Mildner K, Zaehres H, Scholer HR (2010) Induced pluripotent stem cells at
nanoscale. Stem Cells Dev 19(5):615–620
17. Okita K, Yamanaka S (2010) Induction of pluripotency by defined factors. Exp Cell Res 316
(16):2565–2570
18. Draper JS, Pigott C, Thomson JA, Andrews PW (2002) Surface antigens of human embryonic
stem cells: changes upon differentiation in culture. J Anat 200(Pt 3):249–258
19. Chin MH, Mason MJ, Xie W, Volinia S, Singer M, Peterson C et al (2009) Induced pluripotent
stem cells and embryonic stem cells are distinguished by gene expression signatures. Cell
Stem Cell 5(1):111–123
20. Gutierrez-Aranda I, Ramos-Mejia V, Bueno C, Munoz-Lopez M, Real PJ, Macia A et al
(2010) Human induced pluripotent stem cells develop teratoma more efficiently and faster than
human embryonic stem cells regardless the site of injection. Stem Cells 28(9):1568–1570
21. Okita K, Ichisaka T, Yamanaka S (2007) Generation of germline-competent induced plurip-
otent stem cells. Nature 448(7151):313–317
22. Yu J, Hu K, Smuga-Otto K, Tian S, Stewart R, Slukvin II et al (2009) Human induced
pluripotent stem cells free of vector and transgene sequences. Science 324(5928):797–801
23. Okita K, Matsumura Y, Sato Y, Okada A, Morizane A, Okamoto S et al (2011) A more
efficient method to generate integration-free human iPS cells. Nat Methods 8(5):409–412
24. Schlaeger TM, Daheron L, Brickler TR, Entwisle S, Chan K, Cianci A et al (2015)
A comparison of non-integrating reprogramming methods. Nat Biotechnol 33(1):58–63
25. Warren L, Manos PD, Ahfeldt T, Loh YH, Li H, Lau F et al (2010) Highly efficient
reprogramming to pluripotency and directed differentiation of human cells with synthetic
modified mRNA. Cell Stem Cell 7(5):618–630
26. Kelaini S, Cochrane A, Margariti A (2014) Direct reprogramming of adult cells: avoiding the
pluripotent state. Stem Cells Cloning 7:19–29
27. Ieda M, Fu JD, Delgado-Olguin P, Vedantham V, Hayashi Y, Bruneau BG et al (2010) Direct
reprogramming of fibroblasts into functional cardiomyocytes by defined factors. Cell 142
(3):375–386
28. Vierbuchen T, Ostermeier A, Pang ZP, Kokubu Y, Sudhof TC, Wernig M (2010)
Direct conversion of fibroblasts to functional neurons by defined factors. Nature 463
(7284):1035–1041
29. Margariti A, Winkler B, Karamariti E, Zampetaki A, Tsai TN, Baban D et al (2012)
Direct reprogramming of fibroblasts into endothelial cells capable of angiogenesis and
reendothelialization in tissue-engineered vessels. Proc Natl Acad Sci U S A 109
(34):13793–13798
30. Noguchi H, Saitoh I, Tsugata T, Kataoka H, Watanabe M, Noguchi Y (2015) Induction of
tissue-specific stem cells by reprogramming factors, and tissue-specific selection. Cell Death
Differ 22(1):145–155
31. Bar-Nur O, Russ HA, Efrat S, Benvenisty N (2011) Epigenetic memory and preferential
lineage-specific differentiation in induced pluripotent stem cells derived from human pancre-
atic islet beta cells. Cell Stem Cell 9(1):17–23
32. Kim K, Doi A, Wen B, Ng K, Zhao R, Cahan P et al (2010) Epigenetic memory in induced
pluripotent stem cells. Nature 467(7313):285–290
33. Trounson A, McDonald C (2015) Stem cell therapies in clinical trials: progress and challenges.
Cell Stem Cell 17(1):11–22
34. Keirstead H (2008) Challenges to the clinical viability of stem cell technology. In: Spinal cord
injury – what are the barriers to cure? Bedford Center Workshop 29
35. Morizane A, Kikuchi T, Hayashi T, Mizuma H, Takara S, Doi H et al (2017) MHC matching
improves engraftment of iPSC-derived neurons in non-human primates. Nat Commun 8
(1):385
36. Shiba Y, Gomibuchi T, Seto T, Wada Y, Ichimura H, Tanaka Y et al (2016) Allogeneic
transplantation of iPS cell-derived cardiomyocytes regenerates primate hearts. Nature 538
(7625):388–391
37. Kikuchi T, Morizane A, Doi D, Magotani H, Onoe H, Hayashi T et al (2017) Human iPS cell-
derived dopaminergic neurons function in a primate Parkinson’s disease model. Nature 548
(7669):592–596
38. Piao J, Major T, Auyeung G, Policarpio E, Menon J, Droms L et al (2015) Human embryonic
stem cell-derived oligodendrocyte progenitors remyelinate the brain and rescue behavioral
deficits following radiation. Cell Stem Cell 16(2):198–210
39. Kroon E, Martinson LA, Kadoya K, Bang AG, Kelly OG, Eliazer S et al (2008) Pancreatic
endoderm derived from human embryonic stem cells generates glucose-responsive insulin-
secreting cells in vivo. Nat Biotechnol 26(4):443–452
40. Keirstead HS, Nistor G, Bernal G, Totoiu M, Cloutier F, Sharp K et al (2005) Human
embryonic stem cell-derived oligodendrocyte progenitor cell transplants remyelinate and
restore locomotion after spinal cord injury. J Neurosci 25(19):4694–4705
41. Kriks S, Shim JW, Piao J, Ganat YM, Wakeman DR, Xie Z et al (2011) Dopamine neurons
derived from human ES cells efficiently engraft in animal models of Parkinson’s disease.
Nature 480(7378):547–551
42. Grealish S, Diguet E, Kirkeby A, Mattsson B, Heuer A, Bramoulle Y et al (2014) Human
ESC-derived dopamine neurons show similar preclinical efficacy and potency to fetal neurons
when grafted in a rat model of Parkinson’s disease. Cell Stem Cell 15(5):653–665
43. Cyranoski D (2019) ‘Reprogrammed’ stem cells implanted into patient with Parkinson’s
disease. https://www.nature.com/articles/d41586-018-07407-9
44. Manley NC, Priest CA, Denham J, Wirth 3rd ED, Lebkowski JS (2017) Human embryonic
stem cell-derived oligodendrocyte progenitor cells: preclinical efficacy and safety in cervical
spinal cord injury. Stem Cells Transl Med 6(10):1917–1929
45. Cyranoski D (2019) ‘Reprogrammed’ stem cells to treat spinal-cord injuries for the first time.
https://www.nature.com/articles/d41586-019-00656-2
46. Kobayashi Y, Okada Y, Itakura G, Iwai H, Nishimura S, Yasuda A et al (2012) Pre-evaluated
safe human iPSC-derived neural stem cells promote functional recovery after spinal cord
injury in common marmoset without tumorigenicity. PLoS One 7(12):e52787
47. Rezania A, Bruin JE, Riedel MJ, Mojibian M, Asadi A, Xu J et al (2012) Maturation of human
embryonic stem cell-derived pancreatic progenitors into functional islets capable of treating
pre-existing diabetes in mice. Diabetes 61(8):2016–2029
48. Agulnick AD, Ambruzs DM, Moorman MA, Bhoumik A, Cesario RM, Payne JK et al (2015)
Insulin-producing endocrine cells differentiated in vitro from human embryonic stem cells
function in macroencapsulation devices in vivo. Stem Cells Transl Med 4(10):1214–1222
49. Kawamura M, Miyagawa S, Miki K, Saito A, Fukushima S, Higuchi T et al (2012) Feasibility,
safety, and therapeutic efficacy of human induced pluripotent stem cell-derived cardiomyocyte
sheets in a porcine ischemic cardiomyopathy model. Circulation 126(11 Suppl 1):S29–S37
50. Xiong Q, Ye L, Zhang P, Lepley M, Tian J, Li J et al (2013) Functional consequences of
human induced pluripotent stem cell therapy: myocardial ATP turnover rate in the in vivo
swine heart with postinfarction remodeling. Circulation 127(9):997–1008
51. Menasche P, Vanneaux V, Hagege A, Bel A, Cholley B, Parouchev A et al (2018) Transplan-
tation of human embryonic stem cell-derived cardiovascular progenitors for severe ischemic
left ventricular dysfunction. J Am Coll Cardiol 71(4):429–438
52. Trainor N, Pietak A, Smith T (2014) Rethinking clinical delivery of adult stem cell therapies.
Nat Biotechnol 32(8):729–735
53. Badenes SM, Fernandes TG, Miranda CC, Pusch-Klein A, Haupt S, Rodrigues CAV et al
(2017) Long-term expansion of human induced pluripotent stem cells in a microcarrier-based
dynamic system. J Chem Technol Biotechnol 92(3):492–503
54. Rodrigues AL, Rodrigues CAV, Gomes AR, Vieira SF, Badenes SM, Diogo MM et al (2019)
Dissolvable microcarriers allow scalable expansion and harvesting of human induced plurip-
otent stem cells under xeno-free conditions. Biotechnol J 14(4):e1800461
55. Badenes SM, Fernandes TG, Rodrigues CA, Diogo MM, Cabral JM (2015) Scalable expan-
sion of human-induced pluripotent stem cells in xeno-free microcarriers. Methods Mol Biol
1283:23–29
56. Rodrigues CA, Silva TP, Nogueira DE, Fernandes TG, Hashimura Y, Wesselschmidt R et al
(2018) Scalable culture of human induced pluripotent cells on microcarriers under xeno-free
conditions using single-use vertical-wheel™ bioreactors. J Chem Technol Biotechnol 93
(12):3597–3606
57. Kelm JM, Timmins NE, Brown CJ, Fussenegger M, Nielsen LK (2003) Method for generation
of homogeneous multicellular tumor spheroids applicable to a wide variety of cell types.
Biotechnol Bioeng 83(2):173–180
58. Burridge PW, Anderson D, Priddle H, Barbadillo Munoz MD, Chamberlain S, Allegrucci C
et al (2007) Improved human embryonic stem cell embryoid body homogeneity and
cardiomyocyte differentiation from a novel V-96 plate aggregation system highlights interline
variability. Stem Cells 25(4):929–938
59. Tung YC, Hsiao AY, Allen SG, Torisawa YS, Ho M, Takayama S (2011) High-throughput 3D
spheroid culture and drug testing using a 384 hanging drop array. Analyst 136(3):473–478
60. Amit M, Laevsky I, Miropolsky Y, Shariki K, Peri M, Itskovitz-Eldor J (2011) Dynamic
suspension culture for scalable expansion of undifferentiated human pluripotent stem cells.
Nat Protoc 6(5):572–579
61. Singh H, Mok P, Balakrishnan T, Rahmat SN, Zweigerdt R (2010) Up-scaling single cell-
inoculated suspension culture of human embryonic stem cells. Stem Cell Res 4(3):165–179
62. Otsuji TG, Bin J, Yoshimura A, Tomura M, Tateyama D, Minami I et al (2014) A 3D sphere
culture system containing functional polymers for large-scale human pluripotent stem cell
production. Stem Cell Reports 2(5):734–745
63. Ungrin MD, Joshi C, Nica A, Bauwens C, Zandstra PW (2008) Reproducible, ultra high-
throughput formation of multicellular organization from single cell suspension-derived human
embryonic stem cell aggregates. PLoS One 3(2):e1565
64. Niebruegge S, Bauwens CL, Peerani R, Thavandiran N, Masse S, Sevaptisidis E et al (2009)
Generation of human embryonic stem cell-derived mesoderm and cardiac cells using size-
specified aggregates in an oxygen-controlled bioreactor. Biotechnol Bioeng 102(2):493–507
65. Jing D, Parikh A, Tzanakakis ES (2010) Cardiac cell generation from encapsulated embryonic
stem cells in static and scalable culture systems. Cell Transplant 19(11):1397–1412
66. Carpenedo RL, Sargent CY, McDevitt TC (2007) Rotary suspension culture enhances
the efficiency, yield, and homogeneity of embryoid body differentiation. Stem Cells 25
(9):2224–2234
67. Cameron CM, Hu WS, Kaufman DS (2006) Improved development of human embryonic stem
cell-derived embryoid bodies by stirred vessel cultivation. Biotechnol Bioeng 94(5):938–948
68. Zanghi JA, Renner WA, Bailey JE, Fussenegger M (2000) The growth factor inhibitor suramin
reduces apoptosis and cell aggregation in protein-free CHO cell batch cultures. Biotechnol
Prog 16(3):319–325
69. Lipsitz YY, Tonge PD, Zandstra PW (2018) Chemically controlled aggregation of pluripotent
stem cells. Biotechnol Bioeng 115(8):2061–2066
70. Bandeiras C, Cabral JMS, Gabbay RA, Finkelstein SN, Ferreira FC (2019) Bringing stem cell-
based therapies for type 1 diabetes to the clinic: early insights from bioprocess economics and
cost-effectiveness analysis. Biotechnol J 14(8):e1800563
71. Larijani MR, Seifinejad A, Pournasr B, Hajihoseini V, Hassani SN, Totonchi M et al (2011)
Long-term maintenance of undifferentiated human embryonic and induced pluripotent stem
cells in suspension. Stem Cells Dev 20(11):1911–1923
72. Abbasalizadeh S, Larijani MR, Samadian A, Baharvand H (2012) Bioprocess development for
mass production of size-controlled human pluripotent stem cell aggregates in stirred suspen-
sion bioreactor. Tissue Eng Part C Methods 18(11):831–851
73. Amit M, Chebath J, Margulets V, Laevsky I, Miropolsky Y, Shariki K et al (2010) Suspension
culture of undifferentiated human embryonic and induced pluripotent stem cells. Stem Cell
Rev 6(2):248–259
74. Steiner D, Khaner H, Cohen M, Even-Ram S, Gil Y, Itsykson P et al (2010) Derivation,
propagation and controlled differentiation of human embryonic stem cells in suspension.
Nat Biotechnol 28(4):361–364
75. Wang Y, Chou BK, Dowey S, He C, Gerecht S, Cheng L (2013) Scalable expansion of human
induced pluripotent stem cells in the defined xeno-free E8 medium under adherent and
suspension culture conditions. Stem Cell Res 11(3):1103–1116
76. Zweigerdt R, Olmer R, Singh H, Haverich A, Martin U (2011) Scalable expansion of human
pluripotent stem cells in suspension culture. Nat Protoc 6(5):689–700
77. Chen VC, Couture SM, Ye J, Lin Z, Hua G, Huang HI et al (2012) Scalable GMP compliant
suspension culture system for human ES cells. Stem Cell Res 8(3):388–402
78. Krawetz R, Taiani JT, Liu S, Meng G, Li X, Kallos MS et al (2010) Large-scale expansion of
pluripotent human embryonic stem cells in stirred-suspension bioreactors. Tissue Eng Part C
Methods 16(4):573–582
79. Hunt MM, Meng G, Rancourt DE, Gates ID, Kallos MS (2014) Factorial experimental design
for the culture of human embryonic stem cells as aggregates in stirred suspension bioreactors
reveals the potential for interaction effects between bioprocess parameters. Tissue Eng Part C
Methods 20(1):76–89
80. Elanzew A, Sommer A, Pusch-Klein A, Brustle O, Haupt S (2015) A reproducible and
versatile system for the dynamic expansion of human pluripotent stem cells in suspension.
Biotechnol J 10(10):1589–1599
81. Greuel S, Hanci G, Bohme M, Miki T, Schubert F, Sittinger M et al (2019) Effect of inoculum
density on human-induced pluripotent stem cell expansion in 3D bioreactors. Cell Prolif 52(4):
e12604
82. Olmer R, Lange A, Selzer S, Kasper C, Haverich A, Martin U et al (2012) Suspension culture
of human pluripotent stem cells in controlled, stirred bioreactors. Tissue Eng Part C Methods
18(10):772–784
83. Kropp C, Kempf H, Halloin C, Robles-Diaz D, Franke A, Scheper T et al (2016) Impact of
feeding strategies on the scalable expansion of human pluripotent stem cells in single-use
stirred tank bioreactors. Stem Cells Transl Med 5(10):1289–1301
84. Manstein F, Halloin C, Zweigerdt R (2019) Human pluripotent stem cell expansion in stirred
tank bioreactors. Methods Mol Biol 1994:79–91
85. Kempf H, Kropp C, Olmer R, Martin U, Zweigerdt R (2015) Cardiac differentiation of human
pluripotent stem cells in scalable suspension culture. Nat Protoc 10(9):1345–1361
86. Kempf H, Olmer R, Kropp C, Ruckert M, Jara-Avaca M, Robles-Diaz D et al (2014)
Controlling expansion and cardiomyogenic differentiation of human pluripotent stem cells
in scalable suspension culture. Stem Cell Reports 3(6):1132–1146
87. Olmer R, Haase A, Merkert S, Cui W, Palecek J, Ran C et al (2010) Long term expansion of
undifferentiated human iPS and ES cells in suspension culture using a defined medium. Stem
Cell Res 5(1):51–64
88. Chen VC, Ye J, Shukla P, Hua G, Chen D, Lin Z et al (2015) Development of a scalable
suspension culture for cardiac differentiation from human pluripotent stem cells. Stem Cell
Res 15(2):365–375
89. Zweigerdt R (2009) Large scale production of stem cells and their derivatives. Adv Biochem
Eng Biotechnol 114:201–235
90. Miranda CC, Fernandes TG, Pascoal JF, Haupt S, Brustle O, Cabral JM et al (2015) Spatial
and temporal control of cell aggregation efficiently directs human pluripotent stem cells
towards neural commitment. Biotechnol J 10(10):1612–1624
91. Miranda CC, Fernandes TG, Diogo MM, Cabral JM (2016) Scaling up a chemically-defined
aggregate-based suspension culture system for neural commitment of human pluripotent stem
cells. Biotechnol J 11(12):1628–1638
92. Branco MA, Cotovio JP, Rodrigues CAV, Vaz SH, Fernandes TG, Moreira LM et al (2019)
Transcriptomic analysis of 3D cardiac differentiation of human induced pluripotent stem cells
reveals faster cardiomyocyte maturation compared to 2D culture. Sci Rep 9(1):9229
93. Lian X, Hsiao C, Wilson G, Zhu K, Hazeltine LB, Azarin SM et al (2012) Robust
cardiomyocyte differentiation from human pluripotent stem cells via temporal modulation of
canonical Wnt signaling. Proc Natl Acad Sci U S A 109(27):E1848–E1857
94. Schulz TC, Young HY, Agulnick AD, Babin MJ, Baetge EE, Bang AG et al (2012) A scalable
system for production of functional pancreatic progenitors from human embryonic stem cells.
PLoS One 7(5):e37004
95. Yabe SG, Fukuda S, Nishida J, Takeda F, Nashiro K, Okochi H (2019) Induction of functional
islet-like cells from human iPS cells by suspension culture. Regen Ther 10:69–76
96. Diogo MM, da Silva CL, Cabral JM (2012) Separation technologies for stem cell
bioprocessing. Biotechnol Bioeng 109(11):2699–2709
97. Rodrigues GM, Rodrigues CA, Fernandes TG, Diogo MM, Cabral JM (2015) Clinical-scale
purification of pluripotent stem cell derivatives for cell-based therapies. Biotechnol J 10
(8):1103–1114
98. Fukuda H, Takahashi J, Watanabe K, Hayashi H, Morizane A, Koyanagi M et al (2006)
Fluorescence-activated cell sorting-based purification of embryonic stem cell-derived neural
precursors averts tumor formation after transplantation. Stem Cells 24(3):763–771
99. Blin G, Nury D, Stefanovic S, Neri T, Guillevic O, Brinon B et al (2010) A purified population
of multipotent cardiovascular progenitors derived from primate pluripotent stem cells engrafts
in postmyocardial infarcted nonhuman primates. J Clin Invest 120(4):1125–1139
100. Menasche P, Vanneaux V, Fabreguettes JR, Bel A, Tosca L, Garcia S et al (2015) Towards a
clinical use of human embryonic stem cell-derived cardiac progenitors: a translational expe-
rience. Eur Heart J 36(12):743–750
101. Schwartz SD, Hubschman JP, Heilwell G, Franco-Cardenas V, Pan CK, Ostrick RM et al
(2012) Embryonic stem cell trials for macular degeneration: a preliminary report. Lancet 379
(9817):713–720
102. Lu B, Malcuit C, Wang S, Girman S, Francis P, Lemieux L et al (2009) Long-term safety and
function of RPE from human embryonic stem cells in preclinical models of macular degen-
eration. Stem Cells 27(9):2126–2135
103. Priest CA, Manley NC, Denham J, Wirth 3rd ED, Lebkowski JS (2015) Preclinical safety of
human embryonic stem cell-derived oligodendrocyte progenitors supporting clinical trials in
spinal cord injury. Regen Med 10(8):939–958
104. Hattori F, Chen H, Yamashita H, Tohyama S, Satoh YS, Yuasa S et al (2010) Nongenetic
method for purifying stem cell-derived cardiomyocytes. Nat Methods 7(1):61–66
105. Tohyama S, Hattori F, Sano M, Hishiki T, Nagahata Y, Matsuura T et al (2013) Distinct
metabolic flow enables large-scale purification of mouse and human pluripotent stem cell-
derived cardiomyocytes. Cell Stem Cell 12(1):127–137
106. Lin B, Lin X, Stachel M, Wang E, Luo Y, Lader J et al (2017) Culture in glucose-depleted
medium supplemented with fatty acid and 3,30 ,5-triiodo-l-thyronine facilitates purification and
maturation of human pluripotent stem cell-derived cardiomyocytes. Front Endocrinol 8:253
107. Nagashima T, Shimizu K, Matsumoto R, Honda H (2018) Selective elimination of human
induced pluripotent stem cells using medium with high concentration of L-alanine. Sci Rep 8
(1):12427
108. Lancaster MA, Knoblich JA (2014) Organogenesis in a dish: modeling development and
disease using organoid technologies. Science 345(6194):1247125
109. Peerani R, Rao BM, Bauwens C, Yin T, Wood GA, Nagy A et al (2007) Niche-mediated
control of human embryonic stem cell self-renewal and differentiation. EMBO J 26
(22):4744–4755
110. Arora N, Imran Alsous J, Guggenheim JW, Mak M, Munera J, Wells JM et al (2017) A process
engineering approach to increase organoid yield. Development 144(6):1128–1136
111. Kinney MA, Sargent CY, McDevitt TC (2011) The multiparametric effects of hydrodynamic
environments on stem cell culture. Tissue Eng Part B Rev 17(4):249–262
112. Garcez PP, Loiola EC, Madeiro da Costa R, Higa LM, Trindade P, Delvecchio R et al (2016)
Zika virus impairs growth in human neurospheres and brain organoids. Science 352
(6287):816–818
113. Takebe T, Sekine K, Enomura M, Koike H, Kimura M, Ogaeri T et al (2013) Vascularized
and functional human liver from an iPSC-derived organ bud transplant. Nature 499
(7459):481–484
114. Sachs N, Tsukamoto Y, Kujala P, Peters PJ, Clevers H (2017) Intestinal epithelial organoids
fuse to form self-organizing tubes in floating collagen gels. Development 144(6):1107–1112
115. Lu YC, Fu DJ, An D, Chiu A, Schwartz R, Nikitin AY et al (2017) Scalable production and
cryostorage of organoids using core-shell decoupled hydrogel capsules. Adv Biosyst 1
(12):1700165
116. Dye BR, Hill DR, Ferguson MA, Tsai YH, Nagy MS, Dyal R et al (2015) In vitro generation of
human pluripotent stem cell derived lung organoids. eLife 4:05098
117. Finkbeiner SR, Hill DR, Altheim CH, Dedhia PH, Taylor MJ, Tsai YH et al (2015)
Transcriptome-wide analysis reveals hallmarks of human intestine development and matura-
tion in vitro and in vivo. Stem Cell Reports 4(6):1140–1155
118. Ronaldson-Bouchard K, Ma SP, Yeager K, Chen T, Song L, Sirabella D et al (2018) Advanced
maturation of human cardiac tissue grown from pluripotent stem cells. Nature 556
(7700):239–243
119. Halloin C, Coffee M, Manstein F, Zweigerdt R (1994) Production of cardiomyocytes from
human pluripotent stem cells by bioreactor technologies. Methods Mol Biol 2019:55–70
120. Marchetto MC, Carromeu C, Acab A, Yu D, Yeo GW, Mu Y et al (2010) A model for neural
development and treatment of Rett syndrome using human induced pluripotent stem cells. Cell
143(4):527–539
121. Moretti A, Bellin M, Welling A, Jung CB, Lam JT, Bott-Flugel L et al (2010) Patient-specific
induced pluripotent stem-cell models for long-QT syndrome. N Engl J Med 363
(15):1397–1409
122. Crespo M, Vilar E, Tsai SY, Chang K, Amin S, Srinivasan T et al (2017) Colonic organoids
derived from human induced pluripotent stem cells for modeling colorectal cancer and drug
testing. Nat Med 23(7):878–884
123. Pasinelli P, Brown RH (2006) Molecular biology of amyotrophic lateral sclerosis: insights
from genetics. Nat Rev Neurosci 7(9):710–723
124. Dimos JT, Rodolfa KT, Niakan KK, Weisenthal LM, Mitsumoto H, Chung W et al (2008)
Induced pluripotent stem cells generated from patients with ALS can be differentiated into
motor neurons. Science 321(5893):1218–1221
125. Osaki T, Uzel SGM, Kamm RD (2018) Microphysiological 3D model of amyotrophic lateral
sclerosis (ALS) from human iPS-derived muscle cells and optogenetic motor neurons. Sci Adv
4(10):eaat5847
126. Soldner F, Hockemeyer D, Beard C, Gao Q, Bell GW, Cook EG et al (2009) Parkinson’s
disease patient-derived induced pluripotent stem cells free of viral reprogramming factors. Cell
136(5):964–977
127. Crawford TO, Pardo CA (1996) The neurobiology of childhood spinal muscular atrophy.
Neurobiol Dis 3(2):97–110
128. Ebert AD, Yu J, Rose Jr FF, Mattis VB, Lorson CL, Thomson JA et al (2009) Induced
pluripotent stem cells from a spinal muscular atrophy patient. Nature 457(7227):277–280
129. Fernandes TG, Duarte ST, Ghazvini M, Gaspar C, Santos DC, Porteira AR et al (2015) Neural
commitment of human pluripotent stem cells under defined conditions recapitulates neural
development and generates patient-specific neural cells. Biotechnol J 10(10):1578–1588
130. Slaugenhaupt SA, Blumenfeld A, Gill SP, Leyne M, Mull J, Cuajungco MP et al (2001)
Tissue-specific expression of a splicing mutation in the IKBKAP gene causes familial
dysautonomia. Am J Hum Genet 68(3):598–605
131. Lee G, Papapetrou EP, Kim H, Chambers SM, Tomishima MJ, Fasano CA et al (2009)
Modelling pathogenesis and treatment of familial dysautonomia using patient-specific
iPSCs. Nature 461(7262):402–406
132. Sullivan PF, Kendler KS, Neale MC (2003) Schizophrenia as a complex trait: evidence from a
meta-analysis of twin studies. Arch Gen Psychiatry 60(12):1187–1192
133. Brennand KJ, Simone A, Jou J, Gelboin-Burkhart C, Tran N, Sangar S et al (2011) Modelling
schizophrenia using human induced pluripotent stem cells. Nature 473(7346):221–225
134. Hershberger RE, Hedges DJ, Morales A (2013) Dilated cardiomyopathy: the complexity of a
diverse genetic architecture. Nat Rev Cardiol 10(9):531–547
135. Hinson JT, Chopra A, Nafissi N, Polacheck WJ, Benson CC, Swist S et al (2015) HEART
DISEASE. Titin mutations in iPS cells define sarcomere insufficiency as a cause of dilated
cardiomyopathy. Science 349(6251):982–986
136. Mendell JR, Shilling C, Leslie ND, Flanigan KM, al-Dahhak R, Gastier-Foster J et al (2012)
Evidence-based path to newborn screening for Duchenne muscular dystrophy. Ann Neurol 71
(3):304–313
137. Lin B, Li Y, Han L, Kaplan AD, Ao Y, Kalra S et al (2015) Modeling and study of the
mechanism of dilated cardiomyopathy using induced pluripotent stem cells derived from
individuals with Duchenne muscular dystrophy. Dis Model Mech 8(5):457–466
138. Zhang SC, Wernig M, Duncan ID, Brustle O, Thomson JA (2001) In vitro differentiation of
transplantable neural precursors from human embryonic stem cells. Nat Biotechnol 19
(12):1129–1133
139. Muguruma K, Nishiyama A, Kawakami H, Hashimoto K, Sasai Y (2015) Self-organization of
polarized cerebellar tissue in 3D culture of human pluripotent stem cells. Cell Rep 10
(4):537–550
140. Eiraku M, Takata N, Ishibashi H, Kawada M, Sakakura E, Okuda S et al (2011) Self-
organizing optic-cup morphogenesis in three-dimensional culture. Nature 472(7341):51–56
141. Bagley JA, Reumann D, Bian S, Levi-Strauss J, Knoblich JA (2017) Fused cerebral organoids
model interactions between brain regions. Nat Methods 14(7):743–751
142. Bershteyn M, Nowakowski TJ, Pollen AA, Di Lullo E, Nene A, Wynshaw-Boris A et al
(2017) Human iPSC-derived cerebral organoids model cellular features of lissencephaly and
reveal prolonged mitosis of outer radial glia. Cell Stem Cell 20(4):435–49 e4
143. Mariani J, Coppola G, Zhang P, Abyzov A, Provini L, Tomasini L et al (2015) FOXG1-
dependent dysregulation of GABA/glutamate neuron differentiation in autism spectrum
disorders. Cell 162(2):375–390
144. Sampaziotis F, de Brito MC, Madrigal P, Bertero A, Saeb-Parsy K, Soares FAC et al (2015)
Cholangiocytes derived from human induced pluripotent stem cells for disease modeling and
drug validation. Nat Biotechnol 33(8):845–852
145. McCauley KB, Hawkins F, Serra M, Thomas DC, Jacob A, Kotton DN (2017) Efficient
derivation of functional human airway epithelium from pluripotent stem cells via temporal
regulation of wnt signaling. Cell Stem Cell 20(6):844–857.e6
146. Workman MJ, Mahe MM, Trisno S, Poling HM, Watson CL, Sundaram N et al (2017)
Engineered human pluripotent-stem-cell-derived intestinal tissues with a functional enteric
nervous system. Nat Med 23(1):49–59
147. Baio J, Wiggins L, Christensen DL, Maenner MJ, Daniels J, Warren Z et al (2018) Prevalence
of autism spectrum disorder among children aged 8 years – autism and developmental
disabilities monitoring network, 11 sites, United States, 2014. Morb Mortal Wkly Rep Surveill
Summ 67(6):1–23
148. Nagamani SC, Zhang F, Shchelochkov OA, Bi W, Ou Z, Scaglia F et al (2009) Microdeletions
including YWHAE in the Miller-Dieker syndrome region on chromosome 17p13.3 result
in facial dysmorphisms, growth restriction, and cognitive impairment. J Med Genet 46
(12):825–833
149. Furness JB (2012) The enteric nervous system and neurogastroenterology. Nat Rev
Gastroenterol Hepatol 9(5):286–294
150. McKeown SJ, Stamp L, Hao MM, Young HM (2013) Hirschsprung disease: a developmental
disorder of the enteric nervous system. Wiley Interdiscip Rev Dev Biol 2(1):113–129
151. Wimmer RA, Leopoldi A, Aichinger M, Wick N, Hantusch B, Novatchkova M et al (2019)
Human blood vessel organoids as a model of diabetic vasculopathy. Nature 565
(7740):505–510
152. Sander JD, Joung JK (2014) CRISPR-Cas systems for editing, regulating and targeting
genomes. Nat Biotechnol 32(4):347–355
153. Mali P, Yang L, Esvelt KM, Aach J, Guell M, DiCarlo JE et al (2013) RNA-guided human
genome engineering via Cas9. Science 339(6121):823–826
154. Xu X, Tay Y, Sim B, Yoon SI, Huang Y, Ooi J et al (2017) Reversal of phenotypic
abnormalities by CRISPR/Cas9-mediated gene correction in Huntington disease patient-
derived induced pluripotent stem cells. Stem Cell Reports 8(3):619–633
155. Wang L, Yi F, Fu L, Yang J, Wang S, Wang Z et al (2017) CRISPR/Cas9-mediated targeted
gene correction in amyotrophic lateral sclerosis patient iPSCs. Protein Cell 8(5):365–378
156. Poon A, Schmid B, Pires C, Nielsen TT, Hjermind LE, Nielsen JE et al (2016) Generation of a
gene-corrected isogenic control hiPSC line derived from a familial Alzheimer’s disease patient
carrying a L150P mutation in presenilin 1. Stem Cell Res 17(3):466–469
157. Yang Y, Zhang X, Yi L, Hou Z, Chen J, Kou X et al (2016) Naive induced pluripotent stem
cells generated from beta-thalassemia fibroblasts allow efficient gene correction with CRISPR/
Cas9. Stem Cells Transl Med 5(1):8–19
158. Ye L, Wang J, Beyer AI, Teque F, Cradick TJ, Qi Z et al (2014) Seamless modification of
wild-type induced pluripotent stem cells to the natural CCR5Delta32 mutation confers resis-
tance to HIV infection. Proc Natl Acad Sci U S A 111(26):9591–9596
159. Kosicki M, Tomberg K, Bradley A (2018) Repair of double-strand breaks induced by
CRISPR-Cas9 leads to large deletions and complex rearrangements. Nat Biotechnol 36
(8):765–771
160. Zhou T, Tan L, Cederquist GY, Fan Y, Hartley BJ, Mukherjee S et al (2017) High-content
screening in hPSC-neural progenitors identifies drug candidates that inhibit Zika virus infec-
tion in fetal-like organoids and adult brain. Cell Stem Cell 21(2):274–283.e5
161. Chao MP, Gentles AJ, Chatterjee S, Lan F, Reinisch A, Corces MR et al (2017) Human
AML-iPSCs reacquire leukemic properties after differentiation and model clonal variation of
disease. Cell Stem Cell 20(3):329–44 e7
162. Akazawa T, Yoshida S, Ohnishi S, Kanazu T, Kawai M, Takahashi K (2018) Application of
intestinal epithelial cells differentiated from human induced pluripotent stem cells for studies
of prodrug hydrolysis and drug absorption in the small intestine. Drug Metab Dispos 46
(11):1497–1506
163. Skardal A, Murphy SV, Devarasetty M, Mead I, Kang HW, Seol YJ et al (2017) Multi-tissue
interactions in an integrated three-tissue organ-on-a-chip platform. Sci Rep 7(1):8837
164. Gomez-Lechon MJ, Donato MT, Castell JV, Jover R (2003) Human hepatocytes as a tool for
studying toxicity and drug metabolism. Curr Drug Metab 4(4):292–312
165. Vcelar S, Jadhav V, Melcher M, Auer N, Hrdina A, Sagmeister R et al (2018) Karyotype
variation of CHO host cell lines over time in culture characterized by chromosome counting
and chromosome painting. Biotechnol Bioeng 115(1):165–173
166. Yin X, Mead BE, Safaee H, Langer R, Karp JM, Levy O (2016) Engineering stem cell
organoids. Cell Stem Cell 18(1):25–38
167. Takasato M, Er PX, Chiu HS, Maier B, Baillie GJ, Ferguson C et al (2015) Kidney organoids
from human iPS cells contain multiple lineages and model human nephrogenesis. Nature 526
(7574):564–568
DOI: 10.1007/10_2019_118
Addressing the Manufacturing Challenges

of Cell-Based Therapies
Miguel de Almeida Fuzeta, André Dargen de Matos Branco,

Ana Fernandes-Platzgummer, Cláudia Lobato da Silva,
Contents
1 The Landscape of Cell-Based Therapies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 226
1.1 Hematopoietic Cell Transplantation: A Landmark Cell-Based Therapy . . . . . . . . . . . . . 226
1.2 Mesenchymal Stem/Stromal Cells as a New Paradigm for Paracrine Cell Therapy . . 229
1.3 Clinical Application and Challenges of Cell-Based Therapies . . . . . . . . . . . . . . . . . . . . . . . 231
2 Source and Isolation of Cells for Therapeutic Use . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 234
2.1 Sources and Tissue Collection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 234
2.2 Isolation of Target Cell Populations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 235
3 Cell Production . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 239
3.1 Culture Medium Formulation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 239
3.2 Physicochemical Parameters . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 242
3.3 Scalable Culture Vessels . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 243
3.4 Agitation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 248
3.5 Culture Monitoring . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 249
4 Downstream Processing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 254
5 Formulation and Storage . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 256
6 Cost of Goods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 257
7 Quality by Design . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 260
8 The New Cell-Free Therapeutic Paradigm . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 263
9 Concluding Remarks and Future Perspectives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 265
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 266
Authors “Miguel de Almeida Fuzeta and André Dargen de Matos Branco” contributed equally.
M. de Almeida Fuzeta, A. D. de Matos Branco, A. Fernandes-Platzgummer, C. L. da Silva (*),

and J. M. S. Cabral
Department of Bioengineering and iBB-Institute for Bioengineering and Biosciences, Instituto
Superior Técnico, Universidade de Lisboa, Lisboa, Portugal
e-mail: claudia_lobato@tecnico.ulisboa.pt
226 M. de Almeida Fuzeta et al.
Abstract Exciting developments in the cell therapy field over the last decades have
led to an increasing number of clinical trials and the first cell products receiving
marketing authorization. In spite of substantial progress in the field, manufacturing
of cell-based therapies presents multiple challenges that need to be addressed in
order to assure the development of safe, efficacious, and cost-effective cell therapies.
The manufacturing process of cell-based therapies generally requires tissue
collection, cell isolation, culture and expansion (upstream processing), cell harvest,
separation and purification (downstream processing), and, finally, product formula-
tion and storage. Each one of these stages presents significant challenges that have
been the focus of study over the years, leading to innovative and groundbreaking
technological advances, as discussed throughout this chapter.
Delivery of cell-based therapies relies on defining product targets while control-
ling process variable impact on cellular features. Moreover, commercial viability is a
critical issue that has had damaging consequences for some therapies. Implementa-
tion of cost-effectiveness measures facilitates healthy process development, poten-
tially being able to influence end product pricing.
Although cell-based therapies represent a new level in bioprocessing complexity
in every manufacturing stage, they also show unprecedented levels of therapeutic
potential, already radically changing the landscape of medical care.
Graphical Abstract
Keywords Bioreactor, Cell therapy, Hematopoietic stem/progenitor cells (HSPC),

Manufacturing, Mesenchymal stem/stromal cells (MSC), Process engineering
1 The Landscape of Cell-Based Therapies
1.1 Hematopoietic Cell Transplantation: A Landmark

Cell-Based Therapy
The development of cellular therapies began with the establishment of hematopoietic

cell transplantation (HCT). Early work in murine models led to the observation that
Addressing the Manufacturing Challenges of Cell-Based Therapies 227
supralethal radiation could be survived if affected mice were infused with a bone
marrow (BM) graft [1]. BM aspirates containing hematopoietic stem/progenitor
cells (HSPC) were able to migrate to the affected BM after radiation-derived
myeloablative treatment and reconstitute the entire hematopoietic system.
After proving to treat radiation injury, BM transplantation was considered as a
possible treatment for leukemia. In 1956, Barnes and colleagues were able to infuse
normal BM grafts on leukemic mice as a proof of concept [2]. Knocking out a
murine hematopoietic system also meant eliminating its blood-related malignancies.
Transplants of healthy grafts would then repopulate the BM and form a new
hematopoietic system. By transposing this knowledge to humans, Thomas and
colleagues were the first to successfully perform HCT in human acute leukemia
patients, paving the way toward showing the feasibility of cell therapies [3].
HCT development demonstrated that a main and contemporary threat in cell
therapies, known as rejection, could be overcome. Indeed, early on, Medawar and
colleagues were crucial in identifying rejection as an immunologic response by
observing the progressive degradation of skin grafts [4]. In 1953, Billingham and
colleagues described a breakthrough that brought down the inevitability of rejection.
The concept of acquired tolerance was described when lack of rejection was seen in
mice that were previously infused with donor cells during fetal development,
originating chimeras [5]. HCT was able to surpass rejection through acquired
tolerance with elimination of the existing immunological response by radiation
pre-conditioning.
Rapidly after the discovery of acquired tolerance, another major concern related
with cell therapies arose. Billingham and Brent observed that even with spleen or
BM cells infused to induce tolerance, skin grafts continued to fail as the age of the
recipients increased [6]. The reason behind this adverse reaction was another
immunological response, brought upon by the actual graft. Active immune cells
from the donor that accompanied the grafts were not tolerant toward the recipient.
Consequently, these would launch a hostile immune response mirroring rejection
mechanisms. However, this reaction is limited to HCT, not occurring in solid organ
transplantation, where the only mechanism of rejection is the attack triggered by the
recipient’s immune system against the transplanted organ. Initially termed runt
disease, since affected mice would not develop into their adult life, this immune-
based effect is presently known as graft versus host disease (GVHD) [7].
Rejection and GVHD possess similar modes of action, being based on an
immunologic response. Circumventing these complications in a transplant scenario
meant finding the basis of immunological identity, namely, genes encoding for the
human leukocyte antigens (HLA) [8]. With the development of HLA typing, trans-
plants could be performed between HLA-matched patients, with the hope of
avoiding rejection and GVHD. This led to the first successful HLA-matched HCT
between two siblings performed by Gatti and colleagues in 1968 to treat severe
combined immunodeficiency (SCID) [9], thus demonstrating the potential of HCT to
treat illnesses other than cancer.
HCT was the first cell therapy to be adopted in a clinical setting, ultimately
gaining widespread acceptance for treatment of genetic or oncological disease with
Fig. 1 HCT as a platform for the development of cell-based therapies. Pioneering experience in
using cells as therapeutic agents laid the foundation for the diversification of cell therapies. HCT has
been challenged with most of the crucial obstacles of cell-based therapies, from technical hurdles
due to handling, isolation, or infusion of cells to unprecedented biological concepts (e.g., rejection)
transversal to any therapy involving cells as therapeutic agents. Success in solid organ transplants
was reliant on understanding compatibility between patients. Identifying the main agents of the
immunological system was imperative to the development of immunotherapies. Also, induced
pluripotent stem cell technology gained substantial impact since it showed that lack of compatibility
could be overcome by creating autologous sources for any type of cell. HCT hematopoietic cell
transplantation
therapeutic success. The implementation of most current cell therapies can be traced
back to knowledge gained by the establishment of HCT (Fig. 1). For instances, the
field of solid organ transplantation, which is very limited by tissue tolerance, has also
benefited from HCT experience [10]. Joint transplantation of organs with their
respective BM graft was able to cause acquired tolerance toward the solid organ
and overcome HLA mismatch [11]. Gained knowledge of the potential and limits of
allogenic and autologous approaches has facilitated therapeutic objective delineation
in skin grafts. Using an allogenic source for the production of dermal and composite
substitutes (e.g., Dermagraft® and Apligraf®) has limited grafts to serve only as a
temporary barrier that promotes wound healing, without any permanent engraftment
[12, 13]. On the other hand, autologous skin grafts (e.g., EpiCel® and PermaDerm®)
integrate the skin of the patient while continuously promoting wound closure,
reducing scar tissue formation, and mitigating an inflammatory microenvironment
[14, 15].
Besides GVHD, HCT also demonstrates a graft vs. leukemia (GVL) effect against
residual malignant cells that defied conditioning treatments. This behavior has been
associated with lymphoid constituents of the transplant, which has heightened
interest in adoptive T-cell therapy. Donor lymphocyte infusions (DLI) of specific

T-cell subsets or, more recently, enhancement of their antitumoral capabilities
through chimeric antigen receptor T (CAR-T) cell technology are approaches that
have shown great success in both treating cancer and mitigating GVHD in patients
[16, 17].
Instead of tackling donor compatibility, rejection and GVHD could be avoided
altogether. Induced pluripotent stem cell (iPSC) technology has opened up the
possibility of using patients as cell sources for their own therapies. Although very
promising and prompting considerable investment, this technology is yet at its
infancy, with very few clinical trials to date and still requires significant maturation
before moving to clinical practice [18]. Nonetheless, its potential for cell therapies
and disruptive nature are founded on compatibility issues learned from HCT.
HSPC are the most studied cells in clinical trials for multiple innovative
applications [19], with over 3,500 clinical trials taking place worldwide, the vast
majority in the USA followed by Europe (Fig. 2a) (clinicaltrials.gov, accessed on
29 May 2019, using the search term “hematopoietic stem cell OR hematopoietic
progenitor cell”). Neoplasms by histologic type, immune system diseases, leukemia,
lymphoproliferative disorders, and immunoproliferative disorders are the top five
conditions with the highest numbers of undergoing clinical trials worldwide.
1.2 Mesenchymal Stem/Stromal Cells as a New Paradigm

for Paracrine Cell Therapy
Following initial developments in HCT, the BM was once more the source for the
discovery of yet another promising stem cell population, named mesenchymal stem
cells (MSC) by Caplan in 1991 [20]. The foundations for the discovery of these stem
cells can be traced back to the nineteenth century, when studies on the BM trans-
plantation to heterotopic anatomical sites resulted in de novo generation of ectopic
bone and marrow [21, 22]. However, it was only later that the work of Tavassoli and
Crosby clearly provided evidence of an inherent osteogenic potential associated with
the BM [23]. In the 1960s–1970s, Friedenstein and colleagues isolated and charac-
terized a subpopulation of adherent spindle-shaped cells from mouse BM that were
responsible for the previously described osteogenic potential [24, 25]. Moreover,
they also demonstrated that BM cell suspensions could generate colony-forming
unit-fibroblasts (CFU-F). These cells were then later designated as “mesenchymal
stem cells” and shown to have multilineage differentiation potential, including the
osteogenic, adipogenic, and chondrogenic lineages [20, 26].
Over the following decades, questions were raised over the usage of the term
“mesenchymal stem cells,” and alternative nomenclatures have been proposed by
different authors. This is due to unfractionated plastic-adherent marrow cells being
quite heterogeneous and current data being insufficient to characterize them as stem
cells. In order to address the inconsistency in the nomenclature and account for the
46
225
769
2180 360
119 37
23 14
43 20 35
35
121
18
11
210
201 303
77 7
1 24
10 15
20
28
12
Fig. 2 Worldwide distribution of clinical trials obtained from “clinicaltrials.gov” on 29 May 2019,
using the terms “hematopoietic stem cell OR hematopoietic progenitor cell” (a) and “mesenchymal
stem cell OR mesenchymal stromal cell” (b)
biological properties of these cells, the International Society for Cellular Therapy
(ISCT) proposed that plastic-adherent cells described as mesenchymal stem cells
should be termed multipotent mesenchymal stromal cells, maintaining the acronym
MSC [27].
The controversy in the appropriate nomenclature is accompanied by the incon-
sistency between investigators on the set of characteristics that define MSC. Labo-
ratories have developed different methods of isolation and expansion, as well as
different approaches to characterize these cells. Thus, an appropriate comparison
between studies may be difficult to achieve. In order to address this issue, the ISCT
has proposed minimal criteria to define human MSC: (1) adherence to plastic;
(2) expression of CD73, CD90, and CD105 and lack of expression of CD14 or
CD11b, CD79α or CD19, CD34, CD45, and HLA-DR; and (3) osteogenic,
adipogenic, and chondrogenic differentiation potential under standard culture con-
ditions [28]. Similarly, minimal criteria for the definition of adipose tissue (AT)-
derived stromal/stem cells have also been recently established by a combined panel
from ISCT and the International Federation for Adipose Therapeutics and Science
(IFATS) [29].
MSC present additional characteristics that make them attractive for therapeutic
purposes, other than their ability to give rise to different mesenchymal phenotypes.
The secretion of a broad range of bioactive molecules, such as growth factors,
cytokines, and chemokines, renders them with immunomodulatory and trophic
activities, acting both in a paracrine and autocrine manner [30, 31]. MSC trophic
activity relies on bioactive factors that assist in repair and regeneration processes.
MSC are able to inhibit scarring (fibrosis) and apoptosis, promote angiogenesis, and
support growth and differentiation of progenitor cells into functional regenerative
units [30, 31].
The panoply of beneficial effects ascribed to MSC has made them the second
most studied cells in clinical trials, immediately after HSPC [19], with over 900 clin-
ical trials taking place worldwide, receiving a special focus in China, Europe, and the
USA (Fig. 2b) (clinicaltrials.gov, accessed on 29 May 2019, using the search term
“mesenchymal stem cell OR mesenchymal stromal cell”). MSC are promising
candidates for the treatment of a wide range of diseases, which is clearly observed
from the great diversity of conditions targeted in clinical trials. Musculoskeletal
diseases, immune system diseases, wounds and injuries, central nervous system
diseases, and vascular diseases are the top five conditions with the highest numbers
of undergoing clinical trials worldwide.
Many other cell types are being studied in clinical trials including lymphocytes,
dendritic cells, hepatocytes, and endothelial cells [19]. Nonetheless, a special focus
will be given to HSPC and MSC throughout this chapter.
1.3 Clinical Application and Challenges of Cell-Based

Therapies
Since 2009, 12 cell-based therapies have been approved and received marketing
authorization in the European Union (EU) and the USA combined (Table 1) [32–
34]. The first successfully approved product was ChondroCelect, from TiGenix,
despite being withdrawn in 2016 due to commercial reasons. This product consisted
in autologous cartilage cells expanded ex vivo to treat knee cartilage defects.
Holoclar (Chiesi Farmaceutici) was the first approved stem cell product (2015),
consisting in ex vivo expanded autologous human corneal epithelial cells containing
stem cells to treat severe limbal stem cell deficiency. Other approved products
Table 1 Cell-based therapies that received MA in the USA and EU by September 2018 [32–34]
Product Date
(MA holder) Product description Therapeutic indication approved
Alofisel Expanded allogeneic mesen- Perianal fistulas in patients 2018 (EU)
(Takeda Pharma chymal adult stem cells with Crohn’s disease
A/S) extracted from adipose tissue
Yescarta (Kite Autologous T cells genetically Large B-cell lymphoma 2018 (EU)
Pharma) modified by retroviral trans- 2017 (USA)
duction to encode an anti-CD19
chimeric antigen receptor
(CAR)
Kymriah Autologous T cells genetically Acute lymphoblastic leuke- 2018 (EU)
(Novartis) modified using a lentiviral vec- mia; large B-cell lymphoma 2017 (USA)
tor to encode an anti-CD19
CAR
Spherox (co.don Spheroids of human autologous Knee cartilage defects 2017 (EU)
AG) matrix-associated chondrocytes
Strimvelis Autologous CD34+ cells trans- Severe combined 2016 (EU)
(Orchard duced with an engineered ret- immunodeficiency
Therapeutics) roviral vector encoding the
human adenosine deaminase
sequence
Zalmoxis Allogeneic T cells genetically Control mechanism for 2016 (EU)
(MolMed) modified to express a truncated graft-versus-host disease
form of the human low affinity after hematopoietic cell
nerve growth factor receptor transplantation
and the herpes simplex I virus
thymidine kinase
Holoclar (Chiesi Ex vivo expanded autologous Severe limbal stem cell 2015 (EU)
Farmaceutici) human corneal epithelial cells deficiency
containing stem cells
Provenge Autologous peripheral-blood Metastatic prostate cancer 2013 (EU)
(Dendreon) mononuclear cells activated (withdrawn
with prostatic acid phosphatase from EU in
granulocyte-macrophage col- 2015)
ony-stimulating factor 2010 (USA)
Maci (Vericel) Autologous cultured Knee cartilage defects 2016 (USA)
chondrocytes 2013 (EU)
(suspended
in EU in
2014)
GINTUIT Allogeneic cultured Mucogingival conditions 2012 (USA)
(Organogenesis) keratinocytes and fibroblasts in
bovine collagen
Laviv (Fibrocell Autologous fibroblasts Severe nasolabial fold 2011 (USA)
Technologies) wrinkles
ChondroCelect Autologous cartilage cells Knee cartilage defects 2009 (EU)
(TiGenix) expanded ex vivo expressing (withdrawn
specific marker proteins in 2016)
MA marketing authorization, USA United States of America, EU European Union
include the first CAR-T cell therapies for liquid cancers, Kymriah (Novartis) and
Yescarta (Kite Pharma), approved in 2017 in the USA and in 2018 in the EU, and
more recently, Alofisel (Takeda Pharma) that consists in expanded allogeneic
AT-derived MSC to treat perianal fistulas in patients with Crohn’s disease.
Due to their uniqueness, cell therapies have earned their own category in regu-
latory agencies with special directives concerning approval candidature. Cell-based
therapies are considered advanced therapy medicinal products (ATMP), defined by
the European Medicines Agency (EMA) as medicines for human use that are based
on genes, tissues, or cells, offering groundbreaking new opportunities for the
treatment of disease and injury [33].
In spite of the establishment of guidelines and regulations applying to cell
therapies, a number of unresolved issues remain, making the regulatory path toward
clinical approval a challenge [35]. Certain important requirements often lack in
clarity, and regulation is not specific enough. This results in products where the
appropriate classification is not entirely certain [36, 37]. Furthermore, discrepancies
between regulatory agencies from different countries hinder companies trying to
reach the market at an international level [37]. The challenging regulatory environ-
ment contributes to the need to endure over long time periods before reaching the
market. Often, cell products only gain market access 15–20 years after the company
was founded [37]. Nevertheless, the regulatory environment is gradually improving.
In order to continue this path and make wise development choices, it will be crucial
to promote a cross talk between scientists, companies developing cell therapies, and
regulators [37].
Although this millennium has been marked with considerate advances, with
regulatory victories for several ATMP, cell therapy development has a long and
considerable track record. Recent success is due to much effort in the past
uncovering and understanding all the obstacles that stood between the establish-
ments of therapeutic options based on cells. HCT was decisive as a vehicle of
problem-solving and thus has deserved its recognition as a foundation for cell
therapy development [10].
With multiple cell-based therapies already reaching the market, one of the most
pressing issues will be addressing the challenges in manufacturing these products.
Most cell-based therapies are costly and target widespread medical conditions. The
robust and scalable cell manufacturing for the cost-effective delivery of safe and
potent cell-derived ATMP (either with autologous origin (i.e., cells from the patient)
or allogeneic) relies on process engineering tools to understand the impact of cellular
features (biological, biochemical, etc.) on cell product function and performance and
how process variables influence the critical quality attributes of the cell product. In
general, the manufacturing process of cell-based therapies consists of several stages:
tissue collection, cell isolation, culture and expansion (upstream processing), cell
harvest, separation and purification (downstream processing), and finally product
formulation and storage (Fig. 3). The main advances made in the field and future
challenges will be addressed in this text, for each of the manufacturing stages.
Fig. 3 Manufacturing process for cell-based therapeutic products
2 Source and Isolation of Cells for Therapeutic Use
From a manufacturing perspective, cell-based therapies have transformed cells

and tissues themselves into a bioprocess raw material. Consequently, securing
their supply is an unprecedented initial challenge in a production pipeline, differing
from previously established engineered cell factories. For instance, cell retrieval
from human tissues can be problematic. Although the appropriation of biological
waste can minimize this issue, some cell sources may be very difficult to reach, while
potentially posing health risks for a donor or patient. Thus, management of this
supply chain has a level of complexity that is very case specific, depending on the
cellular component of the therapy [38].
2.1 Sources and Tissue Collection
Cell-based therapies depend primarily on obtaining the appropriate material

from which cells with possible therapeutic application can be isolated. So far,
multiple human tissues have been used as sources to obtain cells with therapeutic
potential [39].
Home to the hematopoiesis process, the BM harbors multiple cell types
that closely interact together, forming the so-called BM hematopoietic niche,
encompassing bone, osteoblasts, osteoclasts, HSPC, MSC, macrophages, blood
vessels, and extracellular matrix (ECM) [40]. However, the harvesting of BM
requires an invasive procedure, allowing a relatively small cell yield, which declines
with donor age [41, 42]. For example, HSPC (CD34+) frequency from BM aspirates
after leukapheresis is only 1.5% [43], and MSC frequency in a BM aspirate is only
0.001–0.01% [26].
Mobilized peripheral blood (PB) is an alternative source of HSPC. Donors are
treated with granulocyte-colony stimulating factor (G-CSF) that temporarily shifts
HSPC from extravascular BM sites into the circulating blood. This allows a painless
and less invasive harvesting procedure compared to BM aspiration. Hematopoietic
recovery from mobilized PB transplantation is similar to BM [44, 45]. However, the
risk of GVHD is lower with BM transplantation compared with PB [44, 45], and the
frequency of CD34+ cells in the PB is only 0.5%, which is lower than in BM [43].
AT obtained from subcutaneous tissue represents an abundant source for isolating
MSC reliably using simple techniques. Liposuction, the technique generally used for
harvesting AT, has the advantage of being less invasive than BM aspiration and is
associated with high MSC isolation yields [46]. Specifically, liposuction allowed a
yield of stromal vascular cells of 0.5 106 to 0.7 106 cell/g AT, and between 0.4
and 1.9% of the cells were able to adhere and proliferate in culture [46]. Moreover,
liposuction material is considered medical waste, thus being an attractive alternative
source. The expansion potential, differentiation capacity, and immunophenotype of
MSC derived from AT are nearly identical to those isolated from BM [42].
Neonatal tissues, such as the umbilical cord and placenta, are promising alterna-
tive sources to adult ones. The umbilical cord is a rich source of HSPC [47, 48] and
has been shown to be a rich source of MSC [49]. For example, about 0.6 million
MSC were obtained per gram of umbilical cord [50]. Harvesting the umbilical cord
requires a painless and noninvasive procedure. The umbilical cord is considered
medical waste and is usually discarded after birth, thus being an attractive alternative
source. Within the umbilical cord, HSPC are collected from the umbilical cord
blood (UCB). A large-scale study of 126,341 red blood cell-depleted UCB units in
the USA inventory revealed that the median frequency of CD34+ cells was 0.34%
[51]. MSC on the other hand can be isolated from the UCB as well as from the
Wharton’s jelly, the connective tissue surrounding umbilical vessels [52]. Most
studies are performed with MSC derived from Wharton’s jelly, which is commonly
referred as the umbilical cord matrix (UCM). Umbilical cord-derived MSC expand
at a higher rate when compared to BM- and AT-derived MSC [42, 53]. Furthermore,
UCB enables a better repopulation efficiency upon HCT, when compared to BM and
mobilized PB, as assessed through quantitative in vivo severe combined immuno-
deficiency (SCID)-repopulating cell assay [54], despite the limited cell number per
unit, which has set UCB particularly suited for pediatric patients.
Possibly due to their broad definition, MSC have been successfully isolated
from a number of tissues other than the previously mentioned, including synovial
membrane [55], placenta [56], dental pulp [57], brain, liver, kidney, lung, muscle,
thymus, and pancreas [58].
Notably, cells show different therapeutic capacity depending on the source they
were isolated from. For example, MSC isolated from BM, AT, and UCM revealed
different ability to suppress PB natural killer and B and T cells, when co-cultured
with phytohemagglutinin-stimulated PB mononuclear cells [59].
2.2 Isolation of Target Cell Populations
Depending on the nature of a specific cell therapy, assuring source availability

and succeeding in tissue collection may be enough to proceed to the following
bioprocessing stage. For minimally manipulated cell products, such as HCT,
heterogeneous populations are isolated and directly infused into the patient. How-
ever, newer and more advanced cell therapies are becoming ever more population
specific. Thus, bulk populations that normally result from harvesting procedures
need funneling techniques that isolate a desired cell type [60].
Still, the most commonly used method to isolate MSC is very simplistic, relying
solely on the ability that MSC have to adhere to plastic surfaces [28]. After tissue
collection, cells are plated on polystyrene-based tissue culture flasks. MSC will
adhere to the plastic surface, while contaminating cells, such as the ones from
hematopoietic lineages, are washed away after medium change and passaging
[61, 62]. Typically, when MSC are obtained from tissues such as UCM, AT, or
synovial membrane, these can be either enzymatically digested using collagenase
solutions [46, 62, 63] or simply plated directly onto plastic surfaces as explants
[64–66].
More sophisticated techniques can be used to isolate specific cell populations
following tissue collection, typically relying on affinity-based and centrifugation-
based separations. Although affinity-based separation has gained significant momen-
tum in cell therapy manufacturing, classical centrifugation techniques are still part of
typical bioproduction processes. When blood or marrow samples are used (e.g., BM,
PB, and UCB), a first isolation step with density gradient centrifugation, using a
polymeric solution (e.g., ficoll or percoll), separates the mononuclear cell (MNC)
fraction from other constituents such as plasma and erythrocytes [61]. A consider-
able part of cell therapies are centered on hematopoietic subpopulations (e.g.,
CAR-T cells, regulatory T cells (Treg), monocytes, and HSPC), whose mentioned
isolation consists of centrifugation approaches for separation and extraction
of MNC.
Several Sepax (originally developed by BIOSAFE, now GE Healthcare) cell
processing systems have brought a fully closed and automated centrifugation unit
to cell therapy production pipelines [67]. More advanced centrifugation platforms
combine different physical forces to achieve higher isolation recovery and purity.
Terumo BCT has established a continuous centrifugation system (Elutra®) that joins
centrifugal forces with counterflow [68]. Using blood, initial centrifugation separates
the MNC layer from plasma and erythrocytes. Fluid moving in counterflow separates
the buffy coat into different fractions. By achieving cell population separation based
on size and density, these platforms are able to reach much higher resolution in
separation [69]. Monocyte isolation from peripheral blood mononuclear cells using
this technology managed to concentrate monocytes in a single elutriation fraction
with a recovery of 78% and a purity of 62%. Nevertheless, in combination with a
post-elutriation density gradient, monocyte purity rose to 91% without affecting the
initial recovery [69].
Cell isolation through affinity is an ever-growing alternative due to its separation
criteria being based on biological instead of physical characteristics. Cell population
immunophenotype is commonly used to isolate specific cells from their original
sources, such as HSPC (CD34+ selection) [70, 71] and MSC (Stro-1+ selection)
[72, 73]. Typically mediated by antibody-antigen interactions, fluorescence-
activated cell sorting (FACS) and magnetic-activated cell sorting (MACS) occupy
leading roles in affinity-based separation. Through fluorescent-labeled antibodies,

FACS is able to separate cell populations based on their surface marker expression.
This technology allows for multiple marker selection with high selectivity due to
single-cell analysis [74]. In combination with cell separation, multiparametric stud-
ies can be performed simultaneously, allowing for identity and quality control.
MACS shares the same separation criteria as FACS (i.e., immunophenotype) but
achieves cell sorting with antibodies coupled to magnetic particles. While these
techniques are not novel, direct and thorough comparison has only recently been
established [75]. Whereas FACS dominates selectivity and subpopulation purity, the
respective cell sorter is not inherently prepared for a clinical setting. An expensive
hardware system combined with lack of parallelization, sterility issues, and
time-consuming protocols are some constraints that contribute against its translation.
Due to its column-based system, MACS is able to separate cells at a much faster
rate with possibility for parallel operation and is compatible with current good
manufacturing practice (cGMP) guidelines. Closed versions of MACS (e.g.,
CliniMACS Plus® by Miltenyi Biotec and CTS™ DynaMag™ by Thermo Fisher)
have been developed for clinical-scale cell isolation [76, 77]. The trade-off between
purity and recovery has burdened many isolation techniques and protocols. Natural
killer cells purified by MACS were able to achieve a 95% purity while only reaching
a median recovery of 37% [76]. Nevertheless, lack of bead detachment from cells
after isolation is a significant drawback for MACS as a cell therapy bioprocessing
unit. Besides particle contamination during the production process, epitope restrain
by antibody-magnetic particle complexes can affect cell performance and undermine
therapeutic value.
Interestingly, both techniques are complementary and therefore selection of cell
sorting technique is application dependent. Still, for cell therapy process develop-
ment, there is a clear tendency toward MACS due to previously mentioned argu-
ments. Recently, efforts have been made by both sides to overcome their limitations.
New platforms for FACS that include disposable microfluidic cartridges provide
optimism for its adaptation into production pipelines. WOLF (NanoCellect Biomed-
ical) and On-chip Sort (On-chip Biotechnologies) are two cell sorters that use this
concept to overcome typical FACS limitations. The microfluidic cartridges allow
for a closed circuit that includes the optical path and sterile sorting containers,
minimizing contamination risks. Still, these systems have intrinsically low sorting
rates that combined with demanding clinical cell dose targets make their use
unrealistic [74].
Development of the MACSQuant Tyto (Miltenyi Biotec) system has been the
latest contribution toward FACS adoption into the cell therapy bioproduction pro-
cess. With sorting speeds reaching 30,000 cells/s, MACSQuant Tyto outmaneuvers
its microchip competitors by a factor of 100 [74].
Although not possessing an established clinical platform, vortex actuated cell
sorting (VACS) has come forward as a potential challenger for MACSQuant Tyto.
This technology uses the same principles for cell sorting as FACS but possesses a
different sorting mechanism. The valves or deflection plates used to separate dis-
tinctive cell populations are substituted by a microfluidic thermal vapor bubble
actuator that deflects cells due to the formation of an inertial vortex in the flow.
Designed by Cellular Highways, the prototype sorter named Highway 1 is currently
being developed, with sorting rates reaching 43,000 cells/s. It combines full auto-
mation with closed circuits, possibility for multiplexing and a respectable sorting
speed [78].
On the other hand, strategies for MACS improvement regarding disruption
of cell-magnetic particle complexes have also been explored, focusing on the
interaction between antibody molecules and magnetic particles. Thermo Fisher’s
Dynabeads® FlowComp™ combine streptavidin-coated beads with biotin-
conjugated antibodies, enabling a post-isolation bead removal mechanism [79].
STEMCELL Technologies established their own product, termed Releasable
RapidSpheres™, that possess a tetrameric antibody complex which assures cell
and particle interaction [80]. Following cell isolation, a mild dissociation agent
cleaves the tetrameric complex, releasing the magnetic particles. Successful particle
removal will also help alleviate regulatory issues over end product safety. Conse-
quences of nano- and microparticle contaminations are still debatable, with magnetic
bead internalization being a validated concern.
Instead of improving existing techniques, novel approaches for affinity-based cell
isolation have also been investigated. Since cell therapies possess more stringent
safety criteria, delivering cells without any by-products due to bioprocessing
is crucial. Therefore, antibody removal after affinity separation is of considerable
interest.
Traceless affinity cell selection (Fab-TACS®) is an innovative technology that
explores a reversible antibody-antigen interaction. Antigen-binding fragments (Fab)
are combined with a short peptide tag (Strep-tag®II) with significant affinity toward a
derivative of streptavidin (Strep-Tactin®). By having a Strep-Tactin®-coated agarose
matrix, desired cells are retained in a Fab-TACS® column [81]. Using biotin analogs,
cells are eluted from the column due to interaction competition. Since the antibody
fragments have low affinity toward the selected antigen, Fab release occurs, leaving
the isolated cells without any separation by-product or trace. This technology
has been translated into an automated commercial device (FABian® by IBA
Lifesciences) [81].
A combinatorial approach for cell isolation has been explored by Akadeum Life
Sciences. Instead of improving or developing new techniques, an innovative method
has been devised that brings centrifugation and affinity-based separation together.
Buoyancy-activated cell sorting (BACS™) brings several above-mentioned con-
cepts together in a novel manner [82]. Biotinylated antibodies are introduced in a cell
suspension to target undesired cells (negative selection). Glass-shelled microbubbles
coated with streptavidin are mixed to capture antibody-tagged cells. These micro-
bubbles are separated from the remaining cell populations through centrifugation by
flotation [82]. Purity and recovery values higher than 80% have been reported for
this novel technique [83].
Isolation of a target cell population can have different impact depending on
a specific cell therapy, with products ranging from bulk and heterogeneous
populations to very selective subpopulations with a defined phenotype. Adequate
selection of a separation method is also dependent on the prioritization of opposing

purification concepts, such as purity and recovery [38].
3 Cell Production
The relatively low frequency of cells with therapeutic potential within the native
tissues, followed by harvesting procedures and eventually successive isolation steps,
yield a substantially low number of cells in the end. Therefore, in order to use these
cells in a clinical context, it is usually required additional steps of manipulation and
propagation ex vivo, which depend on choosing the appropriate culture medium
conditions, physicochemical parameters, and culture platforms [84].
3.1 Culture Medium Formulation
The maintenance and propagation of animal cells in vitro require a cell culture
medium, supplying nutrients and inorganic salts, as well as providing the appropriate
physicochemical conditions. Generally, medium components include glucose
(carbon source), amino acids (nitrogen source), vitamins (cofactors), inorganic
salts (maintains electrolyte balance), sodium bicarbonate (buffer, to maintain pH at
7.4), sodium chloride (adjusts osmotic pressure), antibiotics (prevent microorganism
contamination), phenol red (visual pH indicator), and growth factors and hormones
(growth stimulation) [85, 86]. These components are provided in commercially
available basal medium formulations, such as Eagle’s medium and derivatives
(e.g., Dulbecco’s Modified Eagle’s medium (DMEM), Minimum Essential Medium
Eagle alpha (αMEM)), medium from Roswell Park Memorial Institute (RPMI), and
several other well-established media, which have been subject of improvement over
the years [86].
Generally, the addition of certain molecules to basal medium formulations is
required. For cell therapies based on hematopoietic lineages, cytokines play an
important part as a culture medium component. These soluble factors have a great
role in influencing cellular signaling pathways. In the context of HSPC expansion,
factors present in the medium prevent cell differentiation while promoting the self-
renewal capability of cells. Stem cell factor, thrombopoietin, granulocyte-colony
stimulating factor, fms-like tyrosine kinase 3 ligand, and interleukin-6 are some
cytokines currently used in expansion protocols for the manufacturing of UCB
expanded grafts under study in several clinical trials [87–89].
Culture medium formulations usually require the addition of a protein-rich
supplement, containing growth and adhesion factors. The most commonly used
culture supplement is animal serum, especially fetal bovine serum (FBS). Serum is
a source of amino acids, proteins, vitamins, carbohydrates, lipids, hormones, growth
factors, and inorganic salts [86]. Moreover, it enhances cell adhesion, improves
the pH-buffering capacity of the medium, and helps reduce shear stress during
cell manipulation. However, it presents significant disadvantages such as being
ill-defined, wide batch-to-batch variability, risk of contamination with virus and
prions, and ability to transmit xenogeneic antigens, leading to increased immunoge-
nicity of cultured cells, thus limiting FBS application in the clinical setting
[90, 91]. Besides the cell biological perspective, ethical concerns and animal welfare
issues arise from the use of animal serum, as serum collection causes animal
suffering [90]. Furthermore, the global supply of FBS is declining over the years,
and this tendency is expected to continue [90]. This will eventually result in a FBS
supply that will not be able to meet the increasing demand. Therefore, given its
disadvantages, using FBS for cell culture of clinically applied cell products is
discouraged and should be avoided. By complying with current international guide-
lines and regulatory frameworks [92–94], there is need for developing alternative
culture supplements.
In the last decade, the development of serum-/xeno(geneic)-free (S/XF) culture
formulations (i.e., without serum or animal origin components) has been a priority
for the field of cell therapies. Although these media represent a valuable alternative
to FBS, as they are more consistent and standardized, they still contain a cocktail of
growth factors, proteins, and hormones derived from human serum or even plant
hydrolysates, classified as chemically undefined [95]. Chemically defined, animal
component-free media, on the other hand, consist exclusively of well-defined and
characterized components and entirely free of animal (including human) derived
products. These include purified recombinant proteins and synthetic bioactive
molecules [95].
One of the well-established supplements used in S/XF media, proposed as
an alternative to FBS, is human platelet lysate (hPL). As early as the 1980s,
hPL-supplemented medium was found to support proliferation of established cell
lines and primary fibroblasts [96, 97]. hPL is usually prepared from fresh blood or
platelet concentrates, containing bioactive molecules such as growth factors, adhe-
sion molecules, and chemokines, which originate primarily in the α-granules of
platelets [98]. Preparation of hPL from platelet concentrates can be achieved either
by repeated freeze/thaw cycles, sonication, induced platelet activation by addition of
thrombin or CaCl2, or solvent/detergent treatment [98].
Blood banks routinely prepare pooled allogeneic platelets from human blood
donations. When these are not used for transfusion, they are used for further
manufacturing into hPL, thus allowing a steady supply for manufacturing an allo-
geneic “off-the-shelf” hPL product for cell culture [98, 99].
Multiple studies have demonstrated hPL-supplemented media to be efficient for
the isolation and expansion of MSC from various origins [100–102], cultured both in
static and dynamic systems [103, 104], already with several ongoing and completed
clinical trials (clinicaltrials.gov). Moreover, it has been shown that both allogeneic
and autologous hPL-supplemented media allow improved cell proliferation when
compared to FBS-containing media [100, 102, 105, 106]. The main differences in
hPL protein content compared to FBS are the higher content of immunoglobulins
and the possible presence of fibrinogen and other coagulation factors, when hPL is
produced without thrombin activation [98].
In addition to its application for MSC expansion, hPL has been evidenced as an
efficient growth medium supplement for ex vivo expansion of other cell types such
as human gingival fibroblasts [107], chondrocytes [108], osteocytes, myocytes and
tenocytes [109], as well as endothelial cells [110], indicating its potential applica-
bility in multiple areas of cell therapies and regenerative medicine. Although hPL is
considered safer than FBS by the scientific community and regulatory agencies, it
still poses some constraints, such as the risk of transmission of human diseases by
known or unknown viruses, ill-definition, and the possibility of triggering immune
responses [111]. Nonetheless, hPL products derived from pooled units and produced
in large scale are already commercially available [99] and seem to be the most
promising alternative to FBS supplementation in cell culture medium in the near
future. Moreover, novel gamma-irradiated hPL products have been developed
toward pathogen reduction. Results showed that gamma radiation allowed 4 log10
reduction of viral titer with low impacts on the potency for cell expansion [112].
There are other commercially available S/XF media that have been successfully
applied for cell culture. StemPro® MSC SFM (Life Technologies) and
MesenCult™-XF (STEMCELL™ Technologies) are two chemically undefined
S/XF media that have been used to successfully expand MSC from different sources
[113–116].
Considering the expansion of human HSPC, most current protocols are
targeting the use of serum-free medium formulations supplemented with cytokines.
StemSpan™ H3000 (STEMCELL™ Technologies) is a S/XF medium with human-
derived components and has been used for expansion of HSPC [117]. Although
being chemically undefined, containing human-derived components, these media
formulations represent an improvement for cell culture, due to better definition and
lower batch-to-batch variability when compared to FBS- and hPL-supplemented
media.
The ideal candidate for production of clinical-grade cell-based therapies would be
a chemically defined, animal component-free media (including human), composed
exclusively of well-defined factors that could replace serum and serum-derived
products. These include synthetic bioactive molecules and purified recombinant
proteins [95]. There are multiple factors that can be combined in order to replace
serum, such as growth factors (e.g., EGF, FGF, TGF), hormones (e.g., growth
hormone, insulin), carrier proteins (e.g., albumin, transferrin), lipids (e.g., choles-
terol, fatty acids), transition metals (e.g., Se, Fe, Cu, Zn), vitamins, adhesion factors
(e.g., fibronectin, laminin), polyamines, and reductants (e.g., 2-mercaptoethanol)
[86]. The number of possibilities that result from the combination of these compo-
nents is enormous, making the selection of the most appropriate ones and their
respective concentration in a medium formulation an extremely difficult task. For
that purpose, design of experiments is possibly the best strategy to find the optimal
concentration of each component in a culture medium, especially considering likely
interactions between the components [86]. Consequently, S/XF media, especially
chemically defined culture media, are often cell type specific.
The chemically defined medium TheraPEAK™ MSCGM-CD™ (Lonza) has
been used for the expansion of MSC [118]. Successful expansion of T cells was
also achieved using chemically defined S/XF media, relying, for instance, on the
CTS™ Immune Cell Serum Replacement supplement [119, 120].
In order to disseminate the development and application of serum-free media for
cell culture, “FCS-free Database,” a freely accessible serum-free media database, is
available online (https://fcs-free.org/), providing an overview of FBS-free media for
cell culture.
3.2 Physicochemical Parameters
Besides biochemical factors such as nutrient/metabolite concentration and growth

factors, physicochemical parameters such as pH, temperature, osmolality, and oxy-
gen tension are equally important for the maintenance of animal cell cultures. The
optimal values for each of these physicochemical parameters will differ depending
on the cell product, which poses an additional challenge in cell manufacturing.
Most cell lines grow successfully at pH 7.2–7.4. However, the optimum culture
pH depends on the intended application. For example, differentiation of human MSC
into osteoblasts can be improved by changing the pH of culture medium from normal
to alkaline medium [121]. The differentiation of erythroid progenitors can also
progressively increase as pH is increased from 6.95 to 7.4 and 7.6 [122]. Usually
the pH is controlled in cell culture by using the CO2/HCO3 buffer system. Cells are
typically cultured in humidified incubators with gas phase CO2 at 5% and sodium
bicarbonate as a medium additive. The CO2 dissolved in the aqueous phase stays in
equilibrium with HCO3 , adjusting the pH [86].
The optimal temperature to cultivate human and warm-blooded animal cells is
37 C. However, cell culture at different temperatures may be advantageous for
certain purposes. For example, culturing MSC at 32 C decreased the accumulation
of oxidative damage and improved their osteogenic differentiation ability, when
compared to 37 C [123].
Although different cells have different optimal osmolality values, most cells grow
well in the range between 290 and 310 mOsm [124, 125]. As previously mentioned
(Sect. 3.1), the osmolality is mainly defined by the sodium chloride content in the
medium.
Most animal cell cultures are performed at an atmospheric oxygen level (21%
O2). However, the oxygen concentration in most tissues is lower than the atmo-
spheric one, due to gas transfer phenomena. Therefore, mimicking the in vivo
oxygen concentration might have a positive impact in cell culture as well as on the
therapeutic potential of the cultured cells. One canonical example would be the BM,
which is characterized by a hypoxic environment, with oxygen concentration rang-
ing in the interval between 1 and 6% [126, 127].
In light of this observation, several studies were performed by exposing MSC to
hypoxic conditions. Hypoxic conditions were found to have an advantage for MSC
expansion as well as in terms of differentiation [128–130]. A study performed by
Oliveira and colleagues revealed that both BM MSC and AT MSC cultured in
hypoxic conditions experienced an immediate and concerted downregulation of
genes involved in DNA repair and damage response pathways [131]. Moreover, it
revealed that AT MSC reacted to hypoxic environment more slowly than BM MSC,
as different characteristics of each cell niche (e.g., degree of vascularization, oxygen
tension, cell-cell interactions) determine distinct sensitivities to hypoxia ex vivo
[131]. Likewise, hypoxia (5% O2) enhanced the proliferation of UCB-derived
HSPC, when compared to normoxia (21% O2), as well as allowing a better preser-
vation of bone marrow repopulation in SCID mice [132]. In another study, the
ex vivo expansion of UCB-derived HSPC in co-culture with BM MSC was maxi-
mized in a 10% O2 atmosphere, when compared to other hypoxia conditions and
normoxia levels [133].
Besides the need to establish the most appropriate physicochemical conditions for
a certain cell-therapy manufacturing process, maintaining these parameters at the
correct values throughout culture is equally important. In traditional culture systems,
these parameters are often observed, but rarely controlled, thus decreasing the
robustness of the manufacturing process. This issue will be addressed in more detail
in Sect. 3.5.
3.3 Scalable Culture Vessels
Whether isolated cell populations need to undergo differentiation or expansion,

appropriate cell culture vessels and systems are necessary.
In terms of complexity, at the rear end of cell culture technology are simple
plasticware containers. Different geometries make up a broad collection of vessels in
order to cover any cell type and their projected application. Petri dishes, T-flasks,
roller bottles, and multiwell plates all incorporate cell culture plasticware and are
typically made of polystyrene that is previously treated either chemically or physi-
cally in order to gain hydrophilic functional groups (e.g., ketones, aldehydes,
hydroxyl, and carboxyl groups) [134]. Indeed, surface treatment has a dramatic
impact on adherent cell culture, with proper cell adhesion being a main concern.
Unfortunately, when using S/XF culture media, cell adhesion can be compromised
due to deficiency in serum-derived adhesion factors [135]. Commercially enhanced
plasma treatment plasticware (e.g., CellBIND® by Corning Life Sciences) and
xeno-free surface coatings (e.g., CELLstart™ by Thermo Fisher Scientific and
Synthemax® by Corning Life Sciences) have been developed to address this issue
[136, 137].
Besides allowing gas exchange through the cap region and having excellent
optical clarity, commonly used vessels are seriously limited regarding any type of
monitoring and control. Conventional plasticware as culture flasks also lack an
agitation mechanism, not being able to assure fully homogenized cell cultures.
Since their design was directed mainly toward research purposes, manufacturers
quickly identified scalability issues for large-scale production. Advanced and
scalable culture systems based on plasticware were created to avoid laborious and
unsustainable scale-out.
Although very simplistic, plastic malleable bags have a consolidated place in cell
culture. Being integrated in basic plasticware, they offer a simple closed system
solution which is critical for manufacturing under cGMP. However, limited culture
control and poor agitation severely limit their application in optimized processes.
Nevertheless, therapies based on hematopoietic cells (e.g., tumor-infiltrating lym-
phocytes, CAR-T, and HSPC) have relied on these platforms for cell culture,
reaching human use in clinical trials [87, 138, 139].
Multilayered flasks (e.g., Nunc™ Cell Factory™ System by Thermo Fisher
Scientific) were designed to increase culture area while reducing volumetric foot-
print of using multiple individual flasks. Additionally, closed versions with perfu-
sion mechanisms of these flasks were also developed to overcome the open nature of
conventional flasks. Large-scale expansion of MSC in serum-free conditions was
achieved using HYPERStack system (Corning Life Sciences), yielding an average
cell density of 2 104 cell/cm2, corresponding to a fourfold increase in total cell
number after 4 days [140]. Proprietary gas permeable films improve gas diffusion,
which do not compromise cell viability in high-density adherent cultures of tightly
packed multilayered flasks. Flask potential has been pushed further with the com-
mercialization of the CellCube® by Corning Life Sciences, a closed system com-
prising of densely packed thin individual surfaces with continuous medium supply in
laminar flow, reaching 85,000 cm2 (39 cm 25 cm) for adherent cell culture
[141]. The Xpansion® multiplate system designed by Pall Corporation takes advan-
tage of the same concept, aside from assuming a cylindrical geometry with capacity
for up to 122,400 cm2 of culture surface. Xpansion®-50 was used for large-scale
expansion of human periosteum-derived stem cells for the treatment of bone defects,
achieving a final cell density of 1.75 104 cell/cm2, corresponding to a 3.9-fold
change in total cell number after 7 days, and presenting a final recovery efficiency of
45% [142].
Roller bottles have also been optimized for large-scale manufacturing of cells.
Cord blood-derived MNC were isolated and expanded in multiple 500 mL roller
bottles with rotation assured by a bottle roller [143]. Further improvement led to the
design of RollerCell™ by Cellon, a system capable of simultaneously holding
40 roller bottles with automated robotic processors for cell handling. RollerCell™
comparison with CellCube® for cell line production yielded similar results [144].
Although planar systems have evolved to closed and scalable systems with
possibility for dynamic regimen through continuous fluid flow (e.g., CellCube®
and Xpansion®), bioreactors have been the ultimate objective for cell therapy
manufacturing, seeing that they incorporate monitoring and control, reduce process
footprint, and minimize cell handling.
Incorporating highlighted challenges of a cell-centered process requires platforms
capable of dealing with parameter complexity to deliver a safe and reproducible cell-
based product. Table 2 enumerates current cell-based therapies in clinical trials that
involve bioreactors in their production process. Innovative bioreactor designs have
come forward to challenge more classical versions.
Table 2 List of clinical trials using bioreactors for cell based-therapies

Type of
Study name bioreactor Cells Condition Phase
Extracorporeal Immune EISS- Human Severe sepsis and Phase 1
Support System (EISS) for the immune-cell donor septic shock and
Treatment of Septic Patients bioreactor granulocytes phase 2
(EISS-1)a device
Safety of Intramuscular Injec- PluriXTM 3D Placental Critical limb Phase 1
tion of Allogeneic PLX-PAD bioreactor adherent ischemia
Cells for the Treatment of system stromal cells
Critical Limb Ischemiaa
Expansion of Invariant NKT Bioreactor NKT cells Allogeneic hema- Not
Cells for a Cell Immunothera- topoietic stem cell available
peutic Approach Allowing the (HSC)
Control of GvHD and transplantation
Preserving the Graft Versus
Leukemia Effect After Allo-
geneic HSC Transplantationb
Laryngo-tracheal Tissue- Stem-cell Autologous Tracheal diseases Not
Engineered Clinical seeded stem cells applicable
Transplantationc bioartificial
tracheal
scaffold
Clinical trials were obtained from “clinicaltrials.gov,” on 21 March 2019, using the term “bioreac-
tor” and selected for cell therapy applications
Clinical trial status: acompleted; bnot yet recruiting; cunknown
Stirred tank bioreactors (Fig. 4a) maintain widespread use, with their simpler and
more standardized geometry. With extensive experience in what concerns the
production of traditional biopharmaceuticals, much knowledge regarding these bio-
reactors has been transposed to cell-based therapies. These systems have mechanical
impellers that are responsible for appropriate mixing and assuring dynamic flow.
High compatibility with monitoring probes and respective modules has made culture
control an intrinsic part of this bioreactor. Internal sparging mechanisms allow for
efficient gas transfer, although shear stress associated with bubbling can be an
issue to sensitive cells [145]. Exhaustive knowledge on fluid profiles based on
computational fluid dynamics (CFD) models have given significant predictive con-
trol on culture estimates.
While being naturally prone for suspension cultures [146], adherent cell culture
has been adapted through microcarrier development. These spherical particles pro-
vide the surface area for cell adhesion to occur. A broad variety of materials, porosity
levels, and surface coatings have been developed to fulfill specific cell needs. The
high variety of microcarriers has been extensively reviewed [147, 148].
Of notice, we have pioneered in the development of clinical-grade expansion of
MSC of different human sources (i.e., BM and AT) in scalable microcarrier-based
bioreactors using S/XF culture components, achieving the production of 1.1 108
and 4.5 107 cells for BM MSC and AT MSC, respectively, after 7 days of culture
A B
C
E
D
Fig. 4 Schematic representations of bioreactor configurations that can be potentially used in the
manufacturing of cell-based therapies: (a) stirred tank bioreactor, (b) packed bed bioreactor, (c)
hollow fiber bioreactor, (d) wave bioreactor, and (e) Vertical-Wheel™ bioreactor
[149]. Building on this platform, we have concentrated efforts in maximizing cell

productivity by changing different culture parameters. We have previously opti-
mized feeding and agitation regimes and performed microcarrier screening [114].
Furthermore, we have successfully incorporated an alternative MSC tissue source
(i.e., UCM) [150] and have implemented a different bioreactor configuration with a
vertical agitation design (Vertical-Wheel™) (Sect. 3.4) [104].
The scalability potential of stirred tank bioreactors for cell-based therapies has
been embodied by development of MSC expansion processes. While initial studies
restricted their culture scale to spinner flasks, Rafiq and colleagues managed to scale
up MSC expansion to a 5 L stirred tank bioreactor, achieving a cell concentration of
1.7 105 cell/mL, corresponding to over a sixfold expansion in total number of cells
[151]. Subsequently, Lawson and colleagues pushed the scalability of stirred tank
MSC culture forward by successfully expanding human MSC in a 50 L bioreactor,
being able to produce 177 clinical doses (70 million cells/patient assuming a 70 kg
patient) in a single run [152]. In contrast to the above-mentioned scale-up, with
contributions of multiple groups to ever-increasing culture dimensions, Schirmaier
and colleagues were able to perform an entire stepwise scale-up of AT MSC
expansion from spinner flasks to 35 L cultures, yielding 1 1010 cells at the end
(35 L scale) [153].
Consequently, both adherent and suspension cultures are firmly established

for cell culture in stirred tank bioreactors. Commercial versions of stirred tank
bioreactors include the Celligene® series by Eppendorf and the Finesse series by
Thermo Fisher Scientific.
Mammalian cells are known to be more shear sensitive which stimulated efforts
to develop non-abrasive environments during cell culture. Packed bed bioreactors
(Fig. 4b) provide a fixed chamber where microcarriers or scaffolds are located
[154]. Adhered cells that populate the chamber have translational movements
restricted, thus being able to better mimic solid tissue presence. Their constrained
movement also promotes structured organization and cell-cell interaction, leading to
high-density cultures. Low-velocity fluid flow guarantees dynamic culture without
causing shear damage to cells. Culture medium has access to the chamber providing
necessary nutrients and removing metabolites. Diffusion limitations or nutrient
deficiency can occur due to 3D culture organization. Furthermore, significant cellu-
lar organization can result in beneficial biological outcomes but will normally
complicate cell extraction and subsequent downstream processes. Expansion of
MSC in a 2.5 L CelliGen® bioreactor (New Brunswick Scientific) with Fibra-Cel®
(Eppendorf) disks demonstrated large-scale manufacturing potential for packed bed
bioreactors, achieving 9.2 107 cells after 9 days of culture, corresponding to a
9.2-fold increase in total cell number [155].
Increasing available area for cell culture while protecting cells from harsh con-
ditions has inspired innovative bioreactor designs. Hollow fiber bioreactors (Fig. 4c)
fulfill those requirements by joining thousands of hollow fibers. These fibers are
made of thin and porous material that provide a selective passage of nutrients.
Culture medium recirculates through the fibers producing interesting tangential
flow, mimicking vasculature to some extent [154]. However, significant quantity
of fibers originates successive diffusion barriers that cause concentration gradients
for nutrients, signaling factors, or gases. Similar to packed bed bioreactors, cell
extraction processes are challenging to perform due to high cell interaction and
difficulty in reaching cells uniformly inside the bioreactor.
With unprecedented tight regulatory measures, the field of bioreactors has moved
toward disposable and single-use versions. In order to avoid clean-in-place (CIP)
and steam-in-place (SIP) procedures and assure contamination-free product quality,
conventional stainless steel or other reusable bioreactors are being substituted by
plastic single-use bioreactors (SUB). They reduce cross-contamination and can be
combined with limited monitoring probes. Disposable technology has been able to
successfully adapt existing geometries, such as the Mobius series by EMD Millipore
for stirred tank bioreactors and the Quantum® bioreactor by Terumo BCT for hollow
fiber bioreactors. The latter bioreactor has been validated with adherent AT MSC,
BM MSC, periosteum-derived MSC, and neural stem cells [156–160]. However,
novel designs, such as the wave bioreactor (Fig. 4d) and the Vertical-Wheel™
bioreactor (Fig. 4e), have also shown that there is space for bioreactor innovation
that integrate single-use technology. Recently, an overview of SUB and their
applicability toward cell therapy have been investigated [161]. It was observed
that SUB designs have evolved, currently integrating well-known principles of
mass transfer and mixing. Their versatility and single-use nature align with cost
reduction and demanding regulatory guidelines associated with cell therapies. How-
ever, culture monitoring remains a challenge, and long-term bag stability must be
assured.
Numerous bioreactor designs exist for performing cell culture; nevertheless
selecting the correct culture vessel with an appropriate scalability strategy is the
actual challenge for the manufacture of cell therapies. Achieving parallelization of
individual units (scale-out) tends to be more associated with autologous therapies,
while increasing bioreactor size and maintaining culture conditions (scale-up) is
more adequate for an allogeneic production. A compromise between scalability and
optimal culture conditions is deemed necessary.
3.4 Agitation
One of the crucial factors for successful cell expansion is culture medium homog-
enization. Bioreactors require sustained agitation of the culture system, in order to
allow an appropriate mass transfer of nutrients and oxygen to the cells, as well as a
removal of waste products derived from cell metabolism. For that purpose, cells
must be maintained in suspension homogeneously, independently of whether
the cells are cultured freely in suspension, as cellular aggregates or adherent to
microcarriers/scaffolds.
However, agitation may have an impact on cellular physiology, due to increased
shear stress. In this context, shear stress can be defined as the force component acting
tangentially to a material, due to fluid motion [162]. Therefore, in bioreactor
processing, cells are exposed to shear stress originating from fluid agitation. Shear
stress has been described to have a significant impact on cell phenotype, which can
be either negative or beneficial depending on the final application. In fact, it has been
long established that animal cells in general are sensitive to shear stress, which
compromises their viability above certain levels [163–165]. Additionally, agitation
affects HSPC surface marker expression, including cytokine receptors [166] and
CD34 [167], which impacts cell expansion by enriching specific HSPC populations
in culture. On the other hand, shear stress has been demonstrated to induce osteo-
genic differentiation of BM MSC through increased expression of osteogenic
factors such as bone morphogenetic protein-2 (BMP-2), bone sialoprotein (BSP),
and osteopontin (OP) [168] and also resulted in increased intracellular Ca2+ levels
[169]. Shear stress also improved the angiogenic potential of human AT MSC
through stimulation of vascular endothelial growth factor (VEGF) secretion [170].
The importance of agitation and the impact it has on culture outcome have led to
the development of new technologies and bioreactor configurations that specifically
target this issue. Wave bioreactors (Fig. 4d) are suitable for the manufacturing of
shear-sensitive cells. Their agitation through rocking motion prevents the use of an
impeller exerting high shear forces directly in the cells. Very low level of shear stress
was found in wave bioreactors compared to classical stirred tank reactors
[171]. Wave bioreactor implementation for culture of suspension cells, with empha-
sis to hematopoietic lineages, is well-known [172, 173].
In the same line, Vertical-Wheel™ bioreactors (Fig. 4e), developed by PBS
Biotech, incorporate a vertically rotating wheel, allowing a more efficient mixing
than the traditional horizontal stirring solutions. By allowing lower agitation rates,
they are able to minimize shear stress effects. The vertical mixing allows a higher
mass transfer rate and more homogenous and gentle particle suspension, favorable
for anchorage-dependent cells on microcarriers [174]. Moreover, this technology is
fully scalable, being available at working volumes that range from 60 mL up to
500 L. Vertical-Wheel™ bioreactors have been successfully applied in microcarrier-
based cell culture systems for the expansion of MSC from multiple sources
[104, 175], as well as for human iPSC [176].
In summary, agitation can modulate culture conditions and have a significant
impact on the characteristics of expanded cells. Different agitation rates and config-
urations can be used to influence the cell culture outcome. An appropriate balance
needs to be found at an agitation rate that allows adequate mass transfer for cell
growth, without compromising cell integrity or stem cell fate due to excessive shear
stress. Different bioreactor technologies and configurations are available to fine-tune
cell culture agitation for each specific application.
3.5 Culture Monitoring
Monitoring of culture conditions is essential in any cell manufacturing process. An

ideal continuous gathering of information from every bioprocess stage would allow
for real-time informed decision-making, complete control and oversight, extensive
knowledge of the whole manufacture process, and model estimation with response
simulation. Naturally, any cell therapy manufacturing process would hugely benefit
from such observational power, especially due to inherent complexity of being based
on living organisms. The dynamic nature of cells is a significant source of instability
for process control [177].
Monitoring has been exposed to ever-increasing difficulties associated with more
advanced processes and products. Cell therapies have turned the spotlight toward
cells, and monitoring has not been able to fully respond to newfound needs.
However, regulatory agencies have implemented guidelines in order to stimulate
the improvement of monitoring tools, establishing the process analytical technology
(PAT) framework [178, 179]. It highly recommends design, incorporation, and
control of innovative analytical tools for continuous improvement of cell therapy
manufacturing. In order to ensure product quality and facilitate monitoring variable
selection strategies and prioritization, critical process parameters (CPP) must be
identified and closely followed.
As previously mentioned, cell therapies possess conventional physicochemical
process parameters (e.g., pH, temperature, and agitation speed). Nevertheless, intro-
duction of cells has brought a significant amount of unprecedented process
parameters, whose nature revolves around cellular-based concepts. For a cellular

product, assurance of cell viability and cellular fitness involves controlling a micro-
environment based on complex nutrient formulations and dissolved gas concentra-
tions. Besides parameters related with cellular well-being, complexity in cell therapy
production monitoring is associated with information on cell state, including
phenotype and functionality. Identity of a cell population during production
must meet desired standards and quality control, thus surface marker expression,
transcriptomics, and metabolic profile are also important monitoring targets.
Concisely, having cells as therapeutic products has considerably extended the list
of CPP in both length and complexity, forcing PAT to advance and to try to develop
novel tools at an unprecedented and unmanageable rate [177].
With the identification of process parameters present in cell therapy production
that can be difficult to measure, versatility in monitoring might facilitate early
development of new PAT tools. Measurement of process parameters should prefer-
ably be performed in situ, avoiding lag phases and delays in information gathering
that can endanger the whole bioprocess [180]. By measuring directly inside the
manufacturing unit, these sensors provide real-time data and do not compromise the
sterility barrier. However, another possibility includes online measurement, which
requires sample displacement and return to the unit through a bypass mechanism but
also maintains vessel sterility [181].
For techniques that have not been developed for in situ integration, off-line and
at-line monitoring are possible alternatives. These require destructive sampling
accoupled with external sample preparation and analysis. The difference between
both is related with the distance of the assay equipment, with at-line having the
advantage of close proximity to the respective manufacturing unit. Consequently,
any process control with these monitoring strategies will be performed in retrospec-
tive with delayed information [181].
Currently, cell therapy manufacturing possesses process parameters with differ-
ent kinds of monitoring methods [181]. Nevertheless, advancement of PAT aspires
for universal in situ monitoring (Fig. 5).
Conventional CPP have monitoring techniques that have existed for several
decades. The lack of modernization associated with industry resistance in
applying PAT advancements has crippled much needed innovation in cell therapy
manufacturing. Instead of having monitoring development accompany efforts in
making standard bioprocessing units, cell-centered manufacturers have paradoxi-
cally opposed it [182]. This is clearly evident for traditional univariable parameters,
whose monitoring is based on outdated techniques.
Historically, pH and dissolved oxygen (DO) tracking is performed using electro-
chemical sensors [183, 184]. Due to the requirement of a reference electrode, pH
glass electrodes tend to be bulky, and their glass envelope displays some fragility.
Likewise, DO electrodes are also limited, since consumption of local oxygen
requires constant flow around the sensor and low membrane stability restricts
shelf-life [182]. Furthermore, mostly fixed geometry configurations have impaired
adaptation of traditional electrochemical electrodes into novel bioreactor systems.
Addressing the Manufacturing Challenges of Cell-Based Therapies
Fig. 5 Comparison of different monitoring paradigms. Conventional and established systems are outdated and based mostly on off-line methods. Innovative
approaches have envisioned a new perspective that aspires to reach universal online monitoring toward a quality-assured cell-based product
251
Nevertheless, manufacturers have extensive knowledge regarding these sensors, and

their considerable reliability has made their adoption widespread.
Fortunately, advancements in optical fibers have made it possible to follow pH
and DO without the need of electrochemical probes. Optical sensors are able to
quantify these parameters through the presence of indicator or fluorescent dyes
[181]. Continuous optical monitoring of pH in a perfusion bioreactor for a baby
hamster kidney (BHK-21) cell culture was achieved using phenol red as an indicator
[185]. Additionally, instead of having bulk media constituents as indicators, dye
adsorption on a solid matrix combined with patch technology has originated viable
products and systems for cell culture during manufacture (e.g., Optical pH and DO
sensors by PreSens Precision Sensing GmbH or Ocean Optics). Optical patch
sensors were used to monitor pH and DO in a high-throughput system for simulta-
neous operation of 12 bioreactors [186]. Versatility, easy implementation, and
miniaturization potential of patch sensors have also been exploited in disposable
culture technology. Single-use bioreactors, such as BIOSTAT STR® produced by
Sartorius, have integrated patch sensors for both DO and pH. This system has been
used for scalability studies on AT MSC expansion using microcarriers in a stirred
bioreactor [153]. Although optical sensors exhibit great adaptability and allow for
continuous external monitoring, their dyes are vulnerable to photobleaching. Nev-
ertheless, a comparison between electrochemical and optical pH and DO sensors
highlighted considerable correlation between parameter measurements, easing con-
cerns regarding lesser robustness of optical sensors [187].
Cell-related CPP demand pioneering approaches and instruments that are capable
of following complex and multivariate data. For certain parameters it would be
impossible to individually follow each specific component in a parallel manner.
Culture medium formulation and cell transcriptomics are some variables that require
a widespread perspective. Spectroscopy is an attractive technique to address
multiparametric needs since a broad range of wavelengths can be covered [181].
Additionally, electromagnetic radiation interacts with any type of matter, which
makes spectroscopy also applicable to biological parameters [180]. Partition of
this wide-reaching field originates multiple techniques, with several being adapted
as PAT tools for monitoring.
Ultraviolet (UV)/visible spectroscopy focuses on consequences of sample or
analyte excitation through a UV or visible light source. Information for cell-based
bioprocessing monitoring can be retrieved by observing two different radiation
phenomena, namely, scattering and absorption. Measurement of light absorption
through a specific path length is the basis for optical density (OD) and absorbance
techniques [180]. Although restricted to suspension cultures, medium turbidity can
be correlated with cell concentration. Furthermore, significant absorption targets for
this range of the spectrum include aromatic molecules, such as fluorophores, chro-
mophores, and aromatic amino acids. The latter can be extremely useful for protein
quantification. Quality of cytokines used in medium supplementation can be certified
using this technique.
Instead of absorption, light scattering is another event that can provide parameter
information. Biomass from aggregate-based or suspension cell cultures can be
followed by the amount of scattering of an incoming light source. Growth of Chinese

hamster ovary (CHO) cells was successfully followed with a backward light
scattering platform [188]. While cell proliferation can be monitored, potential of
UV/visible spectroscopy for cell therapy manufacturing is limited, since high aro-
matic compound selectivity cannot be fully exploited in a cell-centered bioprocess.
Moving to higher wavelengths in the spectral window leads to infrared (IR)
spectroscopy. Lower-energy radiation associated with IR spectroscopy affects the
vibrational states of molecules. Fortunately, each molecule after excitation emits
its own unique radiation fingerprint [180]. Spectral monitoring of culture media
allows for multiple quantification of culture medium components and metabolic
by-products in a noninvasive manner. Unfortunately, water molecules present can
also interfere with data acquisition. Deconstruction of these measured spectra is
necessary to distinguish between individual analytes [189]. Thus, there is a signif-
icant dependence on chemometric algorithms to unravel incoming data. An in situ
near-infrared spectroscopy probe was validated in determining analyte concentra-
tions in a CHO-K1 cell culture [190]. Fourier transform infrared (FTIR) spectrom-
eters are a third generation of instruments to reach the field of IR spectroscopy. With
a higher signal-to-noise ratio, this technique has also been successfully used to
follow MSC osteogenic differentiation and the metabolic profile of MSC expanded
on microcarriers in a XF culture system [191, 192].
Molecular vibrational interactions are also responsible for originating Raman
spectroscopy. This technique is based on sporadic inelastic scattering of incident
light. In contrast to IR spectroscopy, it is less affected by water interference, and
therefore substantial focus is being given to Raman spectroscopy [180]. This method
combines possibility for versatile in situ probes, continuous and noninvasive real-
time monitoring, sensitivity for most culture components, and a high signal-to-noise
ratio. Cellular events aside from proliferation are also potential targets for Raman
spectroscopy, with differentiation of adipose-derived MSC in multiple lineages
being followed with an in situ probe [193].
Radiation-based monitoring strategies have an extensive application potential and
have unquestionably expanded monitoring capabilities. Further spectroscopy tech-
niques that have been explored as PAT tools include fluorescence spectroscopy and
dielectric spectroscopy [194]. In situ probes based on the latter (e.g., iBiomass by
Fogale Biotech and Incyte by Hamilton) have been developed for cell density
measurements and are compatible with microcarrier-based cultures [195, 196].
With an ever-growing listing of CPP, the demand for techniques or instruments
that incorporate multiple culture parameters in an efficient manner is growing. YSI
2950D by YSI is a metabolite analyzer that has been used to simultaneously measure
up to six different culture parameters using proprietary immobilized enzyme elec-
trodes [142, 197]. The leading instrument BioProfile Flex2 by Nova Biomedical has
challenged the boundaries of parameter parallelization with monitoring capacity for
16 different parameters, ranging from glucose concentration to cell viability
[198]. However, these devices do not fulfill in-line monitoring ambitions. In trying
to solve this issue, a prototype capsule (PATsule) currently in development has
heightened hopes for a real-time multiparametric in-line device [182].
The development of these techniques hopes to give PAT significant observational

power, helping assure cell therapy manufacturing needs. The need for PAT advance-
ment emphasized beforehand has culminated with a momentum in solving this issue.
Recently, Raman spectroscopy was employed as a PAT tool in an autologous
immunotherapy model for cell therapy bioprocessing [199].
4 Downstream Processing
For traditional biopharmaceuticals, cellular contribution ends after upstream

processing, where cells are taken advantage of as miniature factories to produce a
desired product. Downstream units from cell-centered bioprocesses have an unprec-
edented challenge in trying to design purification methods that give special care to
cell sensitivity without changing cell identity and potency [84]. Fortunately, cell
separation is a common practice in research, thus manufacturing units can try to scale
existing technology or develop entirely novel techniques.
After upstream manipulation, cells must be harvested from their respective
vessels to proceed to downstream processing. While cells grown in single-cell
suspensions can be easily recovered, adherent cells require surface detaching tech-
niques. Enzymatic methods disrupt cell-surface interactions by causing proteolytic
cleavage of integrins, which are proteins responsible for cell adhesion and contribute
to cell signaling by transducing ECM stimuli [200]. While damaging integrins can
affect cell phenotype or function, enzymatic detachment of cells is common in cell-
related processes [201–204], with possibilities varying between Trypsin-EDTA,
TrypLE™, and Accutase™. In order to avoid disrupting cell phenotype, approaches
focused on reversible adherence to microcarriers have been pursued.
Considering that many cell therapies will be administered intravenously, the
presence of particulates or intact microcarriers in the final cell product represents a
major safety risk [205]. In this context, different technologies have emerged that may
address this concern. For instance, Advanced Corning® Dissolvable Microcarriers
(Corning Life Sciences) have been developed to facilitate adherent cell harvesting.
These carriers are comprised of polygalacturonic acid chains cross-linked by cal-
cium ions, which can be dissolved with exposure to ethylenediaminetetraacetic acid
(EDTA) and pectinase. Human iPSC expanded in a Vertical-Wheel™ bioreactor
were successfully extracted from dissolvable microcarriers [206]. The use of dis-
solvable microcarriers for expansion of iPSC led to a detachment efficiency of
92 4% compared with 45 3% when using plastic microcarriers with common
detachment techniques.
Microcarrier functionalization is an alternative strategy to achieve reversible
microcarrier adherence. Poly(N-isopropylacrylamide) is a thermal-responsive poly-
mer which dramatically alters its conformation based on temperature fluctuations
[207]. Different microcarrier functionalization methods using this polymer with cell
culture validation of various cell types have been reviewed [207]. Recently, the same
concept was explored using carriers with larger pores (i.e., macrocarriers) for human
dermal fibroblast and MSC culture. Considering the difficulties in harvesting adher-
ent cells from macroporous carriers, this technology may give these carriers new
potential for implementation in cell therapy processes [208].
Considering their different size and density, cell-carrier separation has been
explored by filtration or centrifugation techniques. These selected approaches must
be compatible with manufacturing processes of considerate scale, while respecting
cell sensitivity. Dead-end filtration represents the scalable version for small-scale
mesh filters, with commercial versions (Opticap® series by Merck Millipore) being
applied for efficient separation of MSC from respective microcarriers after cell
expansion and detachment [209]. A screening of several filter mesh sizes between
30 and 100 μm was performed after detachment of cells from the microcarriers.
Interestingly, 30 μm pore sizes originated a significantly reduced cell recovery
(approximately 67%) when compared with the performance of meshes with 80 and
100 μm pore sizes (>90%) [209]. In order to avoid fouling and membrane clogging
associated with normal flow filtration, tangential flow filtration (TFF) might be an
improved alternative for such separation. Hollow fibers (developed by Repligen or
GE Healthcare) allow for separation with less pressure due to feed flow occurring
tangential to the membrane.
Being reliant on pressure for separation, filtration methods can cause cellular
stress. Therefore, centrifugation techniques are a suitable alternative for such sepa-
rations. Aforementioned counterflow centrifugation or fluidized bed centrifugation
minimize shear stress during cell separation and can also be implemented for
downstream purposes [210]. However, the more demanding volume scale might
limit application of previously described instruments. kSep systems developed by
Sartorius explore the same centrifugation principles, having been designed to be able
to process larger volumes and also allow for continuous cell isolation [211].
With both cell types in suspension, cell purification stages may be required after
cell manipulation. These techniques were extensively covered in Sect. 2.2, and their
application is also ever-present during downstream phases of the manufacturing
process. Depending on the production method, cell-based therapies may demand
several cell purification units throughout the pipeline.
At this point, both adherent and suspension cells converge in their respective
downstream pipeline, requiring concentration and washing units before entering the
formulation and filling stage. Fortunately, reducing volume possesses coincident
methods with cell-carrier separation. Previously mentioned TFF and counterflow
centrifugation are both viable options for concentration. TFF implementation for
MSC concentration after microcarrier detachment and clarification was studied with
a systematic breakdown of hollow fiber characteristics [209] (i.e., material and pore
size) and filtration operation modes [212]. TFF under continuous and discontinuous
operation resulted in cell recovery rates higher than 80% with a cell viability of 95%.
Although downstream stages appear to be dominated by filtration and centrifu-
gation processes, disruptive separation mechanisms are being explored for cell
therapy manufacturing applications. FloDesign Sonics has harnessed acoustic
waves to influence cell movement [213]. Using acoustophoresis, cells are restrained
in produced acoustic waves, which allows for washing and volume reduction
without negatively affecting cells. Ekko represents a continuous, closed, and scal-
able platform that has been commercially developed by FloDesign Sonics for cell
therapy manufacture.
Downstream flowcharts for cell therapy manufacturing vary between adherent
and suspension cultures. However, established filtration and centrifugation tech-
niques are mostly transversal throughout different stages. Still, the limited amount
of approaches for downstream processing is a critical issue for cell therapy
manufacturing.
5 Formulation and Storage
When purified cells are compliant with quality control objectives, their formulation
and filling is required for commercialization. Transport of cell-based products is
much more demanding than conventional biopharmaceuticals. For minimally
manipulated therapies, such as transplants, cells are delivered fresh in cold storage.
Autologous products can follow similar formulation and filling principles, since their
shelf-life is typically low. However, allogeneic cell therapies that serve a one-fit-all
business model depend heavily on cryopreservation methods. The storage of cells at
extremely low temperatures (e.g., ranges between 135 and 190 C, using liquid/
vapor phase nitrogen tanks) drastically minimizes metabolic activity, allowing the
preservation of cell viability over prolonged storage. However, cryopreserved prod-
ucts are a logistic burden as specialized infrastructure and equipment are necessary
for handling and transport [84].
Moreover, cryopreservation processes are prone to inflict cell damage through
multiple mechanisms [214]. These include abrupt temperature changes and
unwanted thermal fluctuations. Therefore, appropriate cooling rates and temperature
maintenance are important parameters for a successful cryopreservation. Cell injury
can be associated with extracellular and intracellular ice formation. In order to
prevent ice formation, cryoprotectants are usually added to cryopreserved suspen-
sions. However, cryoprotectants can also be toxic to cells. Finally, cells are also
subject to thawing injury, as a result of intracellular recrystallization.
The median viability of PB-derived HSPC cells can decrease from 98–99% (at the
time of harvest) to 71–76% after thawing these cells from a liquid/vapor phase
nitrogen freezer [215, 216]. UCM MSC, isolated through enzymatic digestion,
presented a mean viability after thawing ranging from 75 to 81%, depending on
the cryopreservation period, while viability prior to freezing was 83% [217]. When
isolated through explant culture, UCM MSC presented a mean viability after
thawing ranging from 71 to 83%, while viability of fresh cells was 93%.
In order to minimize cryopreservation-induced cell damage and enhance product
reproducibility, automated and quantitative tools can be used for cryopreservation
and thawing [218]. Programmable controlled rate freezers (CRFs) employing liquid
nitrogen as a refrigerant offer greater control and customizability of cooling rates.
New freezing systems that do not require liquid nitrogen have recently emerged such
as the Asymptote VIA Quad, Duo, and Research freezers [218]. These are suitable
for GMP cleanroom facilities where the use of liquid nitrogen tanks leads to risks in
terms of contamination and air quality.
Automated dry-thawing devices can eliminate the risks associated with manual
thawing in a water bath, such as water-borne contaminants and operator variability
[218]. Examples of this technology include the Asymptote VIA Thaw SC2 and
CB1000, Biocision ThawSTAR, Medcision ThawCB, and Sarstedt Sahara.
To some extent, it might be desirable to avoid cryopreservation at all, either to
prevent cryo-induced cell damage or to circumvent laborious and expensive freeze
and thawing procedures. Storage at 2–8 C can be sufficient, especially in situations
that only require short-term storage. Specialized hypothermic storage media such
as HypoThermosol (BioLife Solutions) allow an increased product stability at
hypothermic temperatures (e.g., 2–8 C), avoiding the need for cryopreservation
procedures [219]. In 2012, TiGenix completed the first phase I clinical trial
with AT MSC (i.e., Alofisel) stored and administered in HypoThermosol
(NCT01743222) [220].
An innovative technique has been developed that could have major implications
on tissue and cell preservation. Osiris Therapeutics has designed a lyophilization
technology (Prestige Lyotechnology) to preserve living cells and tissue. A proof-of-
concept was performed with placental tissue, which can be used as biological
dressings for treatment of burns and deep wounds [221]. Lyophilized tissue was
compared directly with cryopreserved samples concerning cell viability, ECM
components, and tissue organization, showing comparable results. Lyopreservation
of cells has major consequences, as lyophilized samples can be stored at room
temperature without the need for expensive cryopreservation equipment. For cell
therapy manufacturing, this breakthrough might imply considerable cost reductions,
with deep structural changes to formulation and filling.
6 Cost of Goods
Feasibility is a key concept concerning cell therapy development. Although

possessing great therapeutic potential, cell therapies have inherently complex
manufacturing processes that can impact their commercial viability. The introduc-
tion of cells as a therapeutic product has caused a paradigm shift in bioproduction.
Every manufacture stage has had difficulties in translating typical manufacturing
units to cell-based products. More sophisticated microenvironments during upstream
production are necessary when producing cell-based therapies, which can include
feeder layers and biomaterial-based scaffolds. During the downstream phase, sensi-
tivity of living cells to typical separation and purification processes demands inno-
vative approaches, which usually are costly. Also, end-stage product transportation
cannot yet fully rely on more conventional lyophilization options, since cells still
depend either on fresh or cryopreserved storage for transport [84].
In turn, production has a high risk of becoming exceptionally expensive, leading

to an unsustainable commercial product. Even after achieving regulatory approval,
recent products have suffered with suspicions against long-term commercial sus-
tainability. CAR-T therapies, such as Yescarta and Kymriah, have battled with
health insurers to achieve coverage deals in several countries [222]. In England, its
health cost-effectiveness watchdog (NICE) had initially ruled against acceptance of
Yescarta into the national healthcare system, claiming £300,000 per patient was an
excessive strain on its healthcare budget [223]. However, progress was made when
Yescarta-producing Gilead offered a confidential discount on the listed price, lead-
ing to the approval of Yescarta for treatment of adult patients with diffuse large
B-cell lymphoma [224]. Being potential cures for blood malignancies, their pricing
must recognize a less favorable commercial and manufacturing scenario associated
with one-time treatments. Additionally, their therapeutic indication has been for
cancer patients who have shown resistance to chemotherapy and have ruled out bone
marrow transplants. Thus, expected demand for such cell therapies is relatively low.
Every one of these constraints is a threat against reliable commercialization of these
potentially life-saving cell therapies.
CAR-T cell therapies are not isolated cases, since approved Alofisel has also
suffered from similar concerns. In early 2019, NICE released its final appraisal on
this cell-based product, advising against its adoption for treatment of perianal fistulas
in adults with non-active or mildly active luminal Crohn’s disease [225]. Although
possessing very promising clinical trial results with 50% of patients treated with
Alofisel during its Phase 3 trial showing fistula remission [226], lack of long-term
remission studies has raised suspicions on the durability of its treatment benefit.
Consequently, the proposed therapeutic value of Alofisel reflected in its pricing (list
price – £13,500/vial with one dose consisting in four vials) cannot be assured,
leading to rejection of coverage by NICE.
In order to assure the sustainability of approved therapies, with regulatory consent
and adoption by healthcare providers, detection of cost reductions at any stage of a
cell therapy bioprocess is crucial [35].
Incorporation of cost of goods (COG) analysis allows for a systemic search for
cost drivers and should be included during any decision related with the manufacture
process [39]. Assessing COG at a preliminary level facilitates process modifications
that would become laborious and critically expensive at a later stage. In the case of
cell therapies, there are some inherent characteristics that differentiate their produc-
tion backbone and are responsible for immediate distinctions in the COG analysis
approach.
Interestingly, cell origin has a profound impact on manufacture, commercializa-
tion, and business model choice. Production of allogeneic therapies is an economy of
scale, which becomes increasingly cost-effective as the demand increases. Aligned
with common concerns regarding the need of high cell doses for most cell therapies,
donor to patient treatments have a desired production profile. Scale-up allows for
reductions of consumable costs and operating labor. However, allogeneic therapies
have yet to fully harness the potential for COG reduction of an economy of scale
[39]. The above-mentioned lack of automation and closed production pipelines for
“off-the-shelf” allogeneic products have been holding these therapies back. Never-
theless, these concerns have been identified, and efforts are being made to overcome
them. Decisional tools for the manufacture process based on risk management
analysis that incorporate COG breakdown have been developed for allogeneic
cell-based therapies [227]. Recently, an open-source bioprocess economics tool
revealed that the Vertical-Wheel™ bioreactor system would allow cost savings in
the manufacturing of MSC for cell therapy purposes [104].
Autologous therapies have their own distinct scenario concerning COG.
Being patient specific, each produced lot is restricted to only one patient. Instead
of implementing a scale-up strategy, these therapies require parallelization as
their manufacturing dogma. This has clear consequences in COG analysis, with
single-use modular options becoming more attractive than traditional larger-scale
production vessels [39]. Furthermore, autologous cell therapies do not follow con-
ventional demand-supply relationships. In this case, demand is simultaneously
supply, since the patient possesses the cellular component for its own cell therapy.
This significantly reduces any implementation of cost-effectiveness measures due to
demand predictability. Although demand can be tracked, increasing cell therapy
stock is not possible for autologous therapies, as they are not an “off-the-shelf”
product. In terms of manufacturing facility policy, autologous therapies require
prioritized point-of-care. Since cells from the patient are extracted, altered/expanded,
and reinfused, proximity to the patient is critical. Accordingly, the concept of
decentralized facilities for COG reduction in autologous approaches has been
explored [228].
Tailored COG analysis models are necessary to accurately reduce costs in
autologous and allogeneic therapies. However, there have been attempts to increase
cell therapy versatility by changing its cell source requirement. By removing the
endogenous T-cell receptor (TCR) from allogeneic T-cells, advances were made
toward the creation of a universal CAR-T product [229]. Consequently, an alloge-
neic COG model was able to be developed for such a cell therapy [230]. Altering the
nature of tissue procurement may be a COG solution when trying to convert an
unsustainable cell therapy into a commercially available product. Although cell
source is able to deeply condition cost drivers due to different production models,
other manufacturing parameters also have impact on commercial sustainability.
Having recognized the importance of COG analysis in cell therapy manufactur-
ing, the ISCT conducted a survey for its members to quantify and discriminate costs
in their production pipelines [39]. Acquisition of material and consumables had the
highest mean response, being responsible for 31% of the total costs. Labor-related
costs followed, occupying 20% of the cost burden. Processing and facility costs
shared a similar slice, having been attributed 16% and 17%, respectively. Quality
control and distribution were at the rear, representing 11% and 5% of total
manufacturing costs. Although this breakdown cannot be applied as a universal
template for cell therapies, it helps in comprehending their COG scenario.
Production of an MSC-based therapy was used as a case study in order to explore
the full potential of cost optimization through COG analysis. As mentioned before-
hand, allogeneic therapies benefit from an economy of scale and an “off-the-shelf”
business model. By predicting increases in annual demand and batch sizes, COG
layout recommended adoption of specific expansion strategies [227]. While
multiplate bioreactors and multilayered flasks competed at annual demands between
1010–1011 cells and at a batch size of 109 cells, single-use bioreactors in combination
with microcarriers dominated higher targets in annual demand and batch size.
Although planar technologies are normally chosen as the initial expansion platform,
this simulation demonstrated that single-use bioreactors have comparable costs even
at the lower values of demand and batch size. This observation was an improvement
of a previous study that only considered adoption of bioreactors when confronted
with infeasible scenarios of planar technology [231]. Additionally, considerable
differences were pointed out concerning the labor associated with the explored
platforms. For the first 50 days of an annual production of 100 batches consisting
of 5 1010 cells each, multilayered flasks required 14 full-time equivalents with
a labor load surpassing 80 h in a single day [227]. In the same manufacturing
conditions, single-use bioreactors needed only two full-time equivalents with daily
labor loads constantly under 10 h. Both selection of expansion platform and labor
requirements were shown to be highly influenced by a COG breakdown as cost
drivers.
This methodology enhances decision-making, by simulating possible scenarios
and COG reaction to manufacture alterations. COG optimization should be pursued
throughout the whole bioprocess, even as development advances.
7 Quality by Design
Having cells taking the central role in a therapy has broaden therapeutic angles to
tackle innumerous diseases. However, ensuring high-quality products with consis-
tency is more challenging for such cell-based therapies. The complexity of the
cellular component associated with lack of complete comprehension of its machin-
eries demands even more stringent quality measures during manufacture.
After initial proof-of-concept research, bioprocess development must include
product quality guidelines to guarantee cellular therapeutic attributes, avoid
manufacturing failure and alleviate regulatory concerns regarding safety. Quality
assurance in the biopharmaceutical field was introduced by the US Food and
Drug Administration (FDA) to tackle alarming waste rates, nonexistence of predict-
able models, and insufficient production control [232]. cGMP was delineated to
renew the pharmaceutical sector to address this issue. Quality management of
biopharmaceuticals changed with the creation of the Quality by Design (QbD)
model [233]. Due to its increasing impact, the FDA and EMA launched a joint
pilot program with parallel application assessments in order to harmonize and
integrate QbD guidelines [234]. A holistic view of the bioprocess allied with
extensive scientific knowledge of each production component and a systematic
and iterative method of improvement serve as the basis of QbD.
Cell therapies have followed traditional biopharmaceutical development mistakes
regarding manufacturing development. Inability to apply QbD and COG analysis
Fig. 6 Global description of the QbD process. An initial QTPP requires defining end product
objectives that satisfy therapeutic needs. Translation of these objectives to their cellular features
uncovers process CQA. In turn, process variables that are responsible for influencing CQA are
identified as CPP. Controlled variation of these parameters originates a normal operating space,
which limits process operability. Continuous process monitoring and control facilitates improve-
ment implementation, creating a cycle of process optimization. QbD quality by design, QTPP
quality target product profile, CQA critical quality attribute, CPP critical process parameters
has increased manufacturing failures and unsustainable bioprocesses. Translation of

prominent clinical trial results to a scalable and cost-effective bioprocess that
has led to several letdowns with development suspension and product withdrawal
[235, 236].
A logical sequence of steps is delineated by QbD in order to advise manufacturers
regarding intelligent production pipeline development for cell therapies (Fig. 6).
Initial guidelines concern the end product, with the identification of therapeutic
objectives. These should describe with great detail cutoff goals for concepts such
as identity, potency, and purity [237]. High degree of clarity and detail in defining
end product properties will facilitate identification of critical production processes
and problem localization and solving. By creating a quality target product profile
(QTPP), the framework for QbD is established.
With the target profile set, the entire process needs to be broken down to identify
critical variables that have a direct effect on the QTPP. Raw material attributes
(RMA) need to be controlled for quality with selected checkpoints so that variability
and lack of quality cannot compromise the bioprocess from the start [233]. After
controlling process inputs, continuous monitoring with PAT throughout the entire
process should exist to assure correct transformation or manipulation of raw mate-
rials into the final product.
Considering that following every possible variable is unrealistic, critical quality
attributes (CQA) should be selected. For cell-based therapies, these attributes corre-
spond to cellular features. Since living cells are multivariate systems, isolating inputs
(e.g., signaling factors) and controlling outputs (e.g., cell expansion) are not trivial.
Knowledge on cell networks is increasing, but a complete map of cellular machinery
is still far from reach. Thus, identification of CQA, which are crucial for QbD, can be
difficult.
Throughout the bioprocess, each unit has an impact on cells and is accountable
for altering their characteristics to some extent. Controlling CQA will depend on
identifying which CPP are responsible for changing those same features. Control
strategies should build on CPP discovery, which serve as directives for selection of
monitoring techniques [233]. The holistic nature of QbD demands whole process
overview, with parallel interventions as the production pipeline moves forward.
After definition of individual CQA and respective CPP, studying their interac-
tions should follow. By uncovering and exploring networks, process knowledge
increases greatly. In turn, reaction to process variability can be achieved quickly and
pragmatically by tweaking CPP. Rapid process correction is particularly relevant for
cell therapy manufacturing. Even with RMA tightly controlled, cells have inherent
complexity that leads to aberrant and unpredictable behavior. Therefore, discerning
connections between CQA and CPP will increase process mapping and improve
decision-making [238].
Interactive effects can be revealed by analyzing a defined design space. The range
of variability of CQA caused by fine-tuning of CPP or RMA will define boundaries
for a normal operating range in the design space. Working inside the normal
operating range admits changes in CPP without compromising end product quality.
CQA-CPP connection can be very laborious and difficult to obtain, with a substantial
need for varied experimental data. In order to circumvent excess labor and costly
optimizations, design of experiments and systems biology are effective tools that
originate the same results using up only a fraction of expected time and resources
[237, 239].
Design of experiments is dedicated to evidencing relationships between input and
response variables in an optimized manner. Extensive multivariable exploration
inside a selected design window is performed using the least amount of experimental
conditions. Factorial design is responsible for generating necessary experimental
combinations. Obtained experimental data allows delineation of a response surface
for every CPP. These surfaces are described by behavior functions, which model the
above-mentioned CQA-CPP relationships [240]. Besides enhancing process knowl-
edge, behavior functions enable CPP optimization resulting in CQA improvement.
Implementation of design of experiments has caused process improvements from
pluripotent stem cell cultivation to ex vivo HSPC expansion [241–243].
Systems biology tools can also contribute to discerning CQA-CPP interactions.
Omics-based techniques, such as gene expression and protein production, can
contribute to QTTP review, updating end product identity, and potency. Also,
metabolic pathways can derive similar information without directly compromising
cells. In this context, metabolic by-products were used to construct a fed-batch
platform for HSPC expansion in order to avoid inhibitory feedback signaling
[244]. Intrinsically, large amounts of generated data can also yield reduced dimen-
sion mechanistic models, similar to behavior functions. Overall, the impact of any
CPP modifications on CQA can be more easily detected.
As QbD requires the combination of extensive fundamental knowledge with
engineering principles, applying it to cell therapies is a daunting task. Biological
variability is inherent, making standardization and quality control a struggle for any
process that includes cells. However, QbD guidelines serve as a backbone for correct
cell therapy manufacturing development. Its implementation is critical to any future
bioprocess, assuring quality and robustness throughout each unit. Additionally,
continuous improvement associated with QbD brings process evolution to the
production pipeline, making it dynamically resistant to unsustainability [237].
8 The New Cell-Free Therapeutic Paradigm
Recent advances in cell biology have led to a better understanding of cell commu-
nication processes. Cells are able to interact with their surroundings, influencing
other cells and tissues through paracrine and endocrine signaling. Multiple studies
have been developed using cellular secretome for therapeutic purposes. For instance,
MSC-conditioned medium improved hepatocellular regeneration in a rat model of
acute liver injury [245]. On the other hand, conditioned medium from genetically
modified MSC was able to limit infarct size and improve ventricular function in mice
infarcted hearts [246]. In addition, the secretome obtained from bioreactor cultures
of human BM MSC was able to induce neurogenesis and increase neuronal cell
differentiation in vivo [247].
The secretome consists of every macromolecule a cell transports to the extracel-
lular space at a given point in time. The secretome contains mainly proteins such as
growth factors, hormones, cytokines, chemokines, and interferons, as well as extra-
cellular vesicles [248, 249]. In particular, extracellular vesicles (EVs) have been
given great attention recently by the scientific community, due to their potential both
as diagnostic and therapeutic tools. EVs are lipid membrane enclosed structures
actively secreted by most cell types. These vesicles have emerged as relevant
mediators of intercellular communication, through the transfer of a cargo of proteins
and RNA (i.e., microRNA and mRNA), which prompt alterations on recipient cells
[250–252].
Depending on their biogenesis, EVs are broadly categorized as exosomes,
microvesicles, or apoptotic bodies. Of notice, exosomes (50–150 nm) are generated
via the endosomal pathway [253, 254], microvesicles (50–1,000 nm) by outward
budding of the plasma membrane [253, 254], and apoptotic bodies (up to 5 μm)
released as blebs of cells undergoing apoptosis [255]. EVs are associated with
multiple physiological processes, such as immune surveillance [250] and tissue
repair [256, 257] but also in the pathology underlying several diseases, such as
cancer [258–260] and neurodegenerative diseases [261].
Given the importance of EVs in cell communication, the scientific community
soon realized their potential to target diseased cells. EV membranes resemble the cell
membrane, allowing high biocompatibility to target cells for therapeutic purposes.
Moreover, EVs show specific targeting activity [262] and small size, ideal to cross
biological barriers, such as the blood-brain barrier [263].
The therapeutic use of EVs is twofold. On the one hand, EVs were found to
mediate some of the therapeutic effects from their original cells [264, 265]. Therefore,
these could be potentially used in substitution of their cell of origin, as a cell-free
therapy triggering equivalent therapeutic effect. On the other hand, EVs can be used
as drug delivery vehicles, through loading of EVs with therapeutic cargo [266].
Engineered MSC-derived EVs were able to successfully target and regenerate
ischemic myocardium in mice [267]. Dendritic cell-derived EVs were able to deliver
siRNA to the brain in mice, demonstrating their potential use as targeted therapy for
neurodegenerative diseases [268]. Multiple studies have successfully developed EVs
as drug delivery vehicles for cancer therapy [269–272]. As an example, intrave-
nously injected exosomes from immature dendritic cells delivered doxorubicin
specifically to tumor tissues in mice, leading to inhibition of tumor growth without
overt toxicity [269].
Despite the promising potential of EVs for therapeutic applications, robust
manufacturing processes that would increase the consistency and scalability of EV
production are still lacking, both in EV production (upstream processing) and further
isolation (downstream processing) [273, 274]. Furthermore, higher efficiency drug
loading approaches and strategies to increase EV cell-specific targeting need to be
developed [274].
In conclusion, cell-derived products such as EVs are emerging as novel thera-
peutic opportunities. These cell-free therapies present significant advantages,
avoiding the complexity and safety issues in utilizing cells themselves as therapeutic
systems in a clinical context [266, 275]. Nonetheless, this field is still in a very early
development stage and requires substantial research before being successfully trans-
lated into clinics.
9 Concluding Remarks and Future Perspectives
Exciting developments in the cell therapy field over the last decades has led to an
increasing number of clinical trials testing multiple cell products for therapeutic
purposes. This has been followed by an increasing (although still small) number of
products receiving marketing authorization by regulatory agencies. Even though
there was a substantial progress in the field, manufacturing of cell-based therapies
still presents multiple challenges that need to be addressed in order to assure the
development of safe, efficacious, and cost-effective cell therapies.
Multiple human tissues have been used to derive cells for therapy, showing
different therapeutic capacity depending on the source that cells were isolated
from. Identifying the most appropriate cell source for each application would
allow increased therapeutic efficacy. Moreover, most cells isolated from human
sources are extremely heterogeneous. Identifying and expanding specific functional
cell subpopulations could allow the development of more efficacious and reproduc-
ible products for a specific application.
FBS and other animal-derived products comprise the majority of cell culture
media supplements used for cell manufacturing. These supplements are ill-defined,
increase the risk of contamination with virus and prions, and lead to poor reproduc-
ibility, limiting their application in the clinical setting. The development and appli-
cation of S/XF culture media formulations aiming at a chemically defined medium
are essential for the clinical translation of cell-based products.
Bioreactor technology has allowed the establishment of scalable manufacturing
processes for cell expansion. However, these dynamic systems also present chal-
lenges for cell culture, namely, shear stress, which may impact product features.
New agitation designs such as the Vertical-Wheel™ and wave bioreactors have been
developed to provide a more homogenous and gentler particle suspension at lower
agitation rates. These bioreactors also present the opportunity to implement single-
use technologies, more suitable for the establishment of GMP-compliant processes.
Furthermore, bioreactors could be designed for a specific application and further
optimized relying on simulation techniques.
Computer-monitored culture systems, capable of controlling feeding regimes and
maintaining concentrations of physicochemical parameters (e.g., O2 and pH) and
certain molecules (e.g., growth factors and cytokines) within an optimal range,
would offer a great improvement in the robustness of the manufacturing process.
Currently, isolation and expansion protocols are performed in open systems,
involving a two-stage protocol, which lead to increased risk of contamination, thus
compromising the safety and standardization of the final cell product. Ideally, cell
isolation and expansion could be performed in a single-stage, closed system.
This can be achieved using the Quantum® bioreactor and other strategies such as
the one developed by Papadimitropoulos and colleagues to culture freshly isolated
BM nucleated cells within 3D porous scaffolds in a perfusion-based bioreactor
system [276].
The implementation of automated and closed systems would provide a major
contribution to achieve a GMP-compliant, clinical-grade cell manufacturing.
CliniMACS Prodigy, developed by Miltenyi Biotec, has successfully shown how

manufacturing automation and closed systems can be applied to cell therapies. This
platform has integrated an entire manufacturing process in an automated manner.
Upstream stages (cell fractionation, isolation, and cultivation) were combined with
downstream units (cell purification, formulation, and filling) to enable a fully closed
clinical scale cell therapy platform [277].
Advancements in cell therapy manufacturing and their individual process units
are fruitless if the overall production pipeline is not cost-effective. Commercial
sustainability will ultimately be responsible for dictating the future of a certain cell
therapy. Continuous COG analysis represents the most pertinent tool for identifying
critical cost drivers, which should be flagged and made priorities for improvement.
Early process development by design can avoid considerable and costly time
constraints with manufacture innovation being focused for ensuring product quality.
Continuous monitoring improves process knowledge, and respective control assures
maximized pipeline stability, promoting reproducibility of cell therapy production.
Furthermore, QbD cooperation with COG analysis can assist in parameter targeting
for optimization, contributing toward cyclical process improvements.
Although cell-based therapies represent a new level in bioprocessing complexity
in every manufacturing stage, these also show unprecedented levels of therapeutic
potential, already radically changing the landscape of medical care.
References
1. Main JM, Prehn RT (1955) Successful skin homografts after the administration of high dosage
x radiation and homologous bone marrow. J Natl Cancer Inst 15:1023–1029
2. Barnes DWH, Corp MJ, Loutit JF, Neal FE (1956) Treatment of murine leukaemia with X rays
and homologous bone marrow. Br Med J 2:626–627
3. Thomas E-D, Lochte HL, Lu WC, Ferreebee JW (1957) Intravenous infusion of bone marrow
in patients receiving radiation and chemotherapy. N Engl J Med 257:491–496
4. Gibson T, Medawar PB (1943) The fate of skin homografts in man. J Anat 77:299–310
5. Billingham RE, Brent L, Medawar PB (1953) ‘Activity acquired tolerance’ of foreign cells.
Nature 172:603–606
6. Billingham RE, Brent L (1959) Quantitative studies on tissue transplantation immunity.
IV. Induction of tolerance in newborn mice and studies on the phenomenon of runt disease.
Philos Trans R Soc Lond B Biol Sci 242:439–477
7. Ferrara JL, Levine JE, Reddy P, Holler E (2009) Graft-versus-host disease. Lancet
373:1550–1561
8. Dausset J (1958) Iso-Leuco-Anticorps. Acta Haematol 20:156–166
9. Gatti RA, Meuwissen HJ, Allen HD, Hong R, Good RA (1968) Immunological reconstitution
of sex-linked Lymphopenic immunological deficiency. Lancet 2:1366–1369
10. Chabannon C et al (2018) Hematopoietic stem cell transplantation in its 60s – a platform for
cellular therapies. Sci Transl Med 10:1–10
11. Kawai T et al (2008) HLA-mismatched renal transplantation without maintenance immuno-
suppression. N Engl J Med 358:353–361
12. Gentzkow GD et al (1996) Use of Dermagraft, a cultured human dermis, to treat diabetic foot
ulcers. Diabetes Care 19:350–354
13. Falanga V et al (1998) Rapid healing of venous ulcers and lack of clinical rejection with an
allogeneic cultured human skin equivalent. Arch Dermatol 134:293–300
14. Cuono C, Langdon R, Mcguire J (1986) Use of cultured epidermal autografts and dermal
allografts as skin replacement after burn injury. Lancet 327:1123–1124
15. Boyce ST et al (2017) Randomized, paired-site comparison of autologous engineered skin
substitutes and split-thickness skin graft for closure of extensive, full-thickness burns. J Burn
Care Res 38:61–70
16. Locatelli F et al (2019) Outcome of children with acute leukemia given HLA-haploidentical
HSCT after αβ T-cell and B-cell depletion. Blood 130:677–686
17. Brentjens RJ et al (2003) Eradication of systemic B-cell tumors by genetically targeted human
T lymphocytes co-stimulated by CD80 and Interleukin-15. Nat Med 9:279–286
18. Kimbrel EA, Lanza R (2015) Current status of pluripotent stem cells: moving the first therapies
to the clinic. Nat Rev Drug Discov 14:681–692
19. Heathman TR et al (2015) The translation of cell-based therapies: clinical landscape and
manufacturing challenges. Regen Med 10:49–64
20. Caplan AI (1991) Mesenchymal stem cells. J Orthop Res 9:641–650
21. Goujon E (1869) Recherches expérimentales sur les propriétés de la moelle des os. J
l’anatomie la Physiol Norm Pathol l’homme des animaux 6:399–412
22. Bianco P, Robey PG, Simmons PJ (2008) Mesenchymal stem cells: revisiting history,
concepts, and assays. Cell Stem Cell 2:313–319
23. Tavassoli M, Crosby WH (1968) Transplantation of marrow to extramedullary sites. Science
161:54–56
24. Friedenstein AJ, Chailakhjan RK, Lalykin KS (1970) The development of fibroblast colonies
in marrow and spleen cells. Cell Tissue Kinet 3:393–403
25. Friedenstein AJ (1990) Osteogenic stem cells in the bone marrow. In: Bone and mineral
research. Elsevier, Amsterdam. https://doi.org/10.1016/b978-0-444-81371-8.50012-1
26. Pittenger MF et al (1999) Multilineage potential of adult human mesenchymal stem cells.
Science 284:143–147
27. Horwitz EM et al (2005) Clarification of the nomenclature for MSC: the International Society
for Cellular Therapy position statement. Cytotherapy 7:393–395
28. Dominici M et al (2006) Minimal criteria for defining multipotent mesenchymal stromal cells.
The International Society for Cellular Therapy position statement. Cytotherapy 8:315–317
29. Bourin P et al (2013) Stromal cells from the adipose tissue-derived stromal vascular fraction
and culture expanded adipose tissue-derived stromal/stem cells: a joint statement of the
International Federation for Adipose Therapeutics and Science (IFATS) and the International
Society for Cellular Therapy (ISCT). Cytotherapy 15:641–648
30. Caplan AI, Dennis JE (2006) Mesenchymal stem cells as trophic mediators. J Cell Biochem
98:1076–1084
31. da Silva Meirelles L, Fontes AM, Covas DT, Caplan AI (2009) Mechanisms involved in the
therapeutic properties of mesenchymal stem cells. Cytokine Growth Factor Rev 20:419–427
32. Cuende N, Koh MBC, Dominici M, Rasko JEJ, Ikonomou L (2018) Cell, tissue and gene
products with marketing authorization in 2018 worldwide. Cytotherapy 20:1401–1413
33. European Medicines Agency. https://www.ema.europa.eu/en. Accessed 20 Mar 2019
34. United States Food and Drug Administration. Approved cellular and gene therapy products.
https://www.fda.gov/BiologicsBloodVaccines/CellularGeneTherapyProducts/ApprovedProducts/
default.htm. Accessed 20 Mar 2019
35. Kirouac DC, Zandstra PW (2008) The systematic production of cells for cell therapies. Cell
Stem Cell 3:369–381
36. Blasimme A, Rial-Sebbag E (2013) Regulation of cell-based therapies in Europe: current
challenges and emerging issues. Stem Cells Dev 22:14–19
37. Dodson BP, Levine AD (2015) Challenges in the translation and commercialization of cell
therapies. BMC Biotechnol 15:1–15
38. Ährlund-Richter L et al (2009) Isolation and production of cells suitable for human therapy:
challenges ahead. Cell Stem Cell 4:20–26
39. Lipsitz YY et al (2017) A roadmap for cost-of-goods planning to guide economic production
of cell therapy products. Cytotherapy 19:1383–1391
40. Morrison SJ, Scadden DT (2014) The bone marrow niche for haematopoietic stem cells.
Nature 505:327–334
41. Mueller SM, Glowacki J (2001) Age-related decline in the osteogenic potential of human bone
marrow cells cultured in three-dimensional collagen sponges. J Cell Biochem 82:583–590
42. Kern S, Eichler H, Stoeve J, Klüter H, Bieback K (2006) Comparative analysis of mesenchy-
mal stem cells from bone marrow, umbilical cord blood, or adipose tissue. Stem Cells
24:1294–1301
43. Krause D, Fackler M, Civin C, May W (1996) CD34: structure, biology, and clinical utility.
Blood 87:1–13
44. Gallardo D et al (2009) Is mobilized peripheral blood comparable with bone marrow as a
source of hematopoietic stem cells for allogeneic transplantation from HLA-identical sibling
donors? A case-control study. Haematologica 94:1282–1288
45. Bashey A et al (2017) Mobilized peripheral blood stem cells versus unstimulated bone marrow
as a graft source for T-cell – replete haploidentical donor transplantation using post-transplant
cyclophosphamide. J Clin Oncol 35:3002–3009
46. Oedayrajsingh-Varma MJ et al (2006) Adipose tissue-derived mesenchymal stem cell yield
and growth characteristics are affected by the tissue-harvesting procedure. Cytotherapy
8:166–177
47. Rubinstein P et al (1995) Processing and cryopreservation of placental/umbilical cord blood
for unrelated bone marrow reconstitution. Proc Natl Acad Sci U S A 92:10119–10122
48. Wyrsch A et al (1999) Umbilical cord blood from preterm human fetuses is rich in committed
and primitive hematopoietic progenitors with high proliferative and self-renewal capacity. Exp
Hematol 27:1338–1345
49. Prindull G et al (1987) CFU-F circulating in cord blood. Blut 54:351–359
50. Mennan C et al (2016) Mesenchymal stromal cells derived from whole human umbilical cord
exhibit similar properties to those derived from Wharton’s jelly and bone marrow. FEBS Open
Bio 6:1054–1066
51. Barker JN et al (2019) CD34+ cell content of 126 341 cord blood units in the US inventory:
implications for transplantation and banking. Blood Adv 3:1267–1271
52. Troyer DL, Weiss ML (2008) Concise review: Wharton’s jelly-derived cells are a primitive
stromal cell population. Stem Cells 26:591–599
53. Goodwin HS et al (2001) Multilineage differentiation activity by cells isolated from umbilical
cord blood: expression of bone, fat, and neural markers. Biol Blood Marrow Transplant
7:581–588
54. Wang JC, Doedens M, Dick JE (1997) Primitive human hematopoietic cells are enriched in
cord blood compared with adult bone marrow or mobilized peripheral blood as measured by
the quantitative in vivo SCID-repopulating cell assay. Blood 89:3919–3924
55. De Bari C, Dell’Accio F, Tylzanowski P, Luyten FP (2001) Multipotent mesenchymal stem
cells from adult human synovial membrane. Arthritis Rheum 44:1928–1942
56. Fukuchi Y et al (2004) Human placenta-derived cells have mesenchymal stem/progenitor cell
potential. Stem Cells 22:649–658
57. Karaöz E et al (2010) Isolation and in vitro characterisation of dental pulp stem cells from natal
teeth. Histochem Cell Biol 133:95–112
58. da Silva Meirelles L, Chagastelles PC, Nardi NB (2006) Mesenchymal stem cells reside in
virtually all post-natal organs and tissues. J Cell Sci 119:2204–2213
59. Ribeiro A et al (2013) Mesenchymal stem cells from umbilical cord matrix, adipose tissue and
bone marrow exhibit different capability to suppress peripheral blood B, natural killer and
T cells. Stem Cell Res Ther 4:125
60. Vormittag P, Gunn R, Ghorashian S, Veraitch FS (2018) A guide to manufacturing CAR T cell
therapies. Curr Opin Biotechnol 53:164–181
61. Lennon DP, Caplan AI (2006) Isolation of rat marrow-derived mesenchymal stem cells. Exp
Hematol 34:1606–1607
62. Lu LL et al (2006) Isolation and characterization of human umbilical cord mesenchymal
stem cells with hematopoiesis-supportive function and other potentials. Haematologica
91:1017–1026
63. Nimura A et al (2008) Increased proliferation of human synovial mesenchymal stem cells with
autologous human serum: comparisons with bone marrow mesenchymal stem cells and with
fetal bovine serum. Arthritis Rheum 58:501–510
64. De Bruyn C et al (2010) A rapid, simple, and reproducible method for the isolation of
mesenchymal stromal cells from Wharton’s jelly without enzymatic treatment. Stem Cells
Dev 20:547–557
65. Ghorbani A, Jalali SA, Varedi M (2014) Isolation of adipose tissue mesenchymal stem cells
without tissue destruction: a non-enzymatic method. Tissue Cell 46:54–58
66. Zhang S, Muneta T, Morito T, Mochizuki T, Sekiya I (2008) Autologous synovial fluid
enhances migration of mesenchymal stem cells from synovium of osteoarthritis patients in
tissue culture system. J Orthop Res 26:1413–1418
67. Aktas M, Radke TF, Strauer BE, Wernet P, Kogler G (2008) Separation of adult bone
marrow mononuclear cells using the automated closed separation system Sepax. Cytotherapy
10:203–211
68. Eyrich M et al (2014) Development and validation of a fully GMP-compliant production
process of autologous, tumor-lysate-pulsed dendritic cells. Cytotherapy 16:946–964
69. Stroncek DF et al (2014) Counter-flow elutriation of clinical peripheral blood mononuclear
cell concentrates for the production of dendritic and T cell therapies. J Transl Med 12:241
70. Kato K, Radbruch A (1993) Isolation and characterization of CD34+ hematopoietic stem cells
from human peripheral blood by high-gradient magnetic cell sorting. Cytometry 14:384–392
71. De Wynter EA et al (1995) Comparison of purity and enrichment of CD34+ cells from bone
marrow, umbilical cord and peripheral blood (primed for apheresis) using five separation
systems. Stem Cells 13:524–532
72. Simmons PJ, Torok-Storb B (1991) Identification of stromal cell precursors in human bone
marrow by a novel monoclonal antibody, STRO-1. Blood 78:55–62
73. Gonçalves R, da Silva CL, Cabral JMS, Zanjani ED, Almeida-Porada G (2006) A Stro-1+
human universal stromal feeder layer to expand/maintain human bone marrow hematopoietic
stem/progenitor cells in a serum-free culture system. Exp Hematol 34:1353–1359
74. Leong W, Nankervis B, Beltzer J (2019) Automation: what will the cell therapy laboratory of
the future look like? Cell Gene Ther Insights 4:679–694
75. Sutermaster BA, Darling EM (2019) Considerations for high-yield, high-throughput cell
enrichment: fluorescence versus magnetic sorting. Sci Rep 9:1–9
76. Koehl U et al (2004) IL-2 activated NK cell immunotherapy of three children after
haploidentical stem cell transplantation. Blood Cells Mol Dis 33:261–266
77. Neurauter AA et al (2007) Cell isolation and expansion using dynabeads. Adv Biochem Eng
Biotechnol 106:41–73
78. Rogers S, Pritchard R, Zhukov A (2018) A faster GMP therapeutic cell sorter enabled by a new
microfluidic technology: the inertial vortex sorter. Cytotherapy 20:S70
79. Feng X et al (2010) Foxp1 is an essential transcriptional regulator for the generation of
quiescent naive T cells during thymocyte development. Blood 115:510–518
80. Kokaji AI (2018) Method for the in situ formation of bifunctional immunological complexes.
US 2018/0188245A1
81. Dai X, Mei Y, Nie J, Bai Z (2019) Scaling up the manufacturing process of adoptive T cell
immunotherapy. Biotechnol J 14:1800239
82. McNaughton BH, Younger JG, Ostruszka LJ (2019) Method and system for buoyant separa-
tion. US 10,195,547 B2
83. Liou Y-R, Wang Y-H, Lee C-Y, Li P-C (2015) Buoyancy-activated cell sorting using targeted
biotinylated albumin microbubbles. PLoS One 10:e0125036
84. Aijaz A et al (2018) Biomanufacturing for clinically advanced cell therapies. Nat Biomed Eng
2:362–376
85. Burgener A, Butler M (2005) Medium development. In: Ozturck SS, Hu WS (eds) Cell culture
technology for pharmaceutical and cell-based therapies. CRC Press, Boca Raton, pp 41–64
86. Yao T, Asayama Y (2017) Animal-cell culture media: history, characteristics, and current
issues. Reprod Med Biol 16:99–117
87. de Lima M et al (2012) Cord-blood engraftment with ex vivo mesenchymal-cell coculture.
N Engl J Med 367:2305–2315
88. Horwitz ME et al (2019) Phase I/II study of stem-cell transplantation using a single cord blood
unit expanded ex vivo with nicotinamide. J Clin Oncol 37:367–374
89. Wagner JE et al (2016) Phase I/II trial of StemRegenin-1 expanded umbilical cord blood
hematopoietic stem cells supports testing as a stand-alone graft. Cell Stem Cell 18:144–155
90. Brunner D, Appl H, Pfaller W, Gstraunthaler G (2010) Serum-free cell culture: the serum-free
media interactive online database. ALTEX 27:53–62
91. Spees JL et al (2004) Internalized antigens must be removed to prepare hypoimmunogenic
mesenchymal stem cells for cell and gene therapy. Mol Ther 9:747–756
92. European Medicines Agency (2007) Guideline on human cell-based medicinal products
(EMEA/CHMP/410869/2006). Off J Eur Union
93. European Medicines Agency (2011) Note for guidance on minimising the risk of transmitting
animal spongiform encephalopathy agents via human and veterinary medicinal products
(EMA/410/01 rev.3). Off J Eur Union
94. US Food and Drug Administration (2019) Medical devices containing materials derived from
animal sources (except for in vitro diagnostic devices)
95. Karnieli O et al (2017) A consensus introduction to serum replacements and serum-free media
for cellular therapies. Cytotherapy 19:155–169
96. Hara Y, Steiner M, Baldini MG (1980) Platelets as a source of growth-Dromotina factor(s) for
tumor cells. Cancer Res 40:1212–1216
97. Umeno Y, Okuda A, Kimura G (1989) Proliferative behaviour of fibroblasts in plasma-rich
culture medium. J Cell Sci 94:567–575
98. Burnouf T, Strunk D, Koh MBC, Schallmoser K (2016) Human platelet lysate: replacing fetal
bovine serum as a gold standard for human cell propagation? Biomaterials 76:371–387
99. van der Valk J et al (2018) Fetal bovine serum (FBS): past–present–future. ALTEX 35:99–118
100. Doucet C et al (2005) Platelet lysates promote mesenchymal stem cell expansion: a safety
substitute for animal serum in cell-based therapy applications. J Cell Physiol 205:228–236
101. Reinisch A et al (2015) Epigenetic and in vivo comparison of diverse MSC sources reveals an
endochondral signature for human hematopoietic niche formation. Blood 125:249–260
102. Kinzebach S, Dietz L, Klüter H, Thierse HJ, Bieback K (2013) Functional and differential
proteomic analyses to identify platelet derived factors affecting ex vivo expansion of mesen-
chymal stromal cells. BMC Cell Biol 14:48
103. de Soure AM et al (2017) Integrated culture platform based on a human platelet lysate
supplement for the isolation and scalable manufacturing of umbilical cord matrix-derived
mesenchymal stem/stromal cells. J Tissue Eng Regen Med 11:1630–1640
104. Sousa Pinto D et al (2019) Scalable manufacturing of human mesenchymal stromal cells in the
Vertical-Wheel™ bioreactor system: an experimental and economic approach. Biotechnol
J:1800716. https://doi.org/10.1002/biot.201800716
105. Schallmoser K et al (2007) Human platelet lysate can replace fetal bovine serum for clinical-
scale expansion of functional mesenchymal stromal cells. Transfusion 47:1436–1446
106. Atashi F, Jaconi MEE, Pittet-Cuénod B, Modarressi A (2014) Autologous platelet-rich
plasma: a biological supplement to enhance adipose-derived mesenchymal stem cell expan-
sion. Tissue Eng Part C Methods 21:253–262
107. Naveau A et al (2010) Phenotypic study of human gingival fibroblasts in a medium enriched
with platelet lysate. J Periodontol 82:632–641
108. Hildner F et al (2015) Human platelet lysate successfully promotes proliferation and subse-
quent chondrogenic differentiation of adipose-derived stem cells: a comparison with articular
chondrocytes. J Tissue Eng Regen Med 9:808–818
109. Mazzocca AD et al (2012) The positive effects of different platelet-rich plasma methods on
human muscle, bone, and tendon cells. Am J Sports Med 40:1742–1749
110. Hofbauer P et al (2014) Human platelet lysate is a feasible candidate to replace fetal calf serum
as medium supplement for blood vascular and lymphatic endothelial cells. Cytotherapy
16:1238–1244
111. Hemeda H, Giebel B, Wagner W (2014) Evaluation of human platelet lysate versus fetal
bovine serum for culture of mesenchymal stromal cells. Cytotherapy 16:170–180
112. Huang C et al (2019) Gamma irradiation of human platelet lysate: validation of efficacy for
pathogen reduction and assessment of impacts on HPL performance. Cytotherapy 21:S82–S83
113. Simões IN et al (2013) Human mesenchymal stem cells from the umbilical cord matrix:
successful isolation and ex vivo expansion using serum /xeno-free culture media. Biotechnol
J 8:448–458
114. Carmelo JG, Fernandes-Platzgummer A, Diogo MM, da Silva CL, Cabral JMS (2015) A
xeno-free microcarrier-based stirred culture system for the scalable expansion of human
mesenchymal stem/stromal cells isolated from bone marrow and adipose tissue. Biotechnol
J 10:1235–1247
115. Al-Saqi SH et al (2014) Defined serum-free media for in vitro expansion of adipose-derived
mesenchymal stem cells. Cytotherapy 16:915–926
116. Chen G et al (2014) Human umbilical cord-derived mesenchymal stem cells do not undergo
malignant transformation during long-term culturing in serum-free medium. PLoS One 9:1–8
117. Spanholtz J et al (2010) High log-scale expansion of functional human natural killer cells from
umbilical cord blood CD34-positive cells for adoptive cancer immunotherapy. PLoS One 5:
e9221
118. Wang Y et al (2014) Human mesenchymal stem cells possess different biological character-
istics but do not change their therapeutic potential when cultured in serum free medium. Stem
Cell Res Ther 5:1–14
119. Smith C et al (2015) Ex vivo expansion of human T cells for adoptive immunotherapy using
the novel xeno-free CTS immune cell serum replacement. Clin Transl Immunol 4:e31
120. Lu TL et al (2016) A rapid cell expansion process for production of engineered autologous
CAR-T cell therapies. Hum Gene Ther Methods 27:209–218
121. Fliefel R et al (2016) Mesenchymal stem cell proliferation and mineralization but not osteo-
genic differentiation are strongly affected by extracellular pH. J Cranio-Maxillofac Surg
44:715–724
122. Mcadams TA, Miller WM, Papoutsakis ET (1997) Variations in culture pH affect the cloning
efficiency and differentiation of progenitor cells in ex vivo haemopoiesis. Br J Haematol
97:889–895
123. Stolzing A, Scutt A (2006) Effect of reduced culture temperature on antioxidant defences of
mesenchymal stem cells. Free Radic Biol Med 41:326–338
124. Waymouth C (1970) Osmolality of mammalian blood and of media for. In Vitro 6:109–110
125. Mather JP, Roberts PE (1998) Introduction to cell and tissue culture: theory and technique.
Plenum Press, New York
126. Eliasson P, Jönsson JI (2010) The hematopoietic stem cell niche: low in oxygen but a nice
place to be. J Cell Physiol 222:17–22
127. Spencer JA et al (2014) Direct measurement of local oxygen concentration in the bone marrow
of live animals. Nature 508:269–273
128. Valorani MG et al (2012) Pre-culturing human adipose tissue mesenchymal stem cells under
hypoxia increases their adipogenic and osteogenic differentiation potentials. Cell Prolif
45:225–238
129. Dos Santos F et al (2010) Ex vivo expansion of human mesenchymal stem cells: a more
effective cell proliferation kinetics and metabolism under hypoxia. J Cell Physiol 223:27–35
130. Lee HH et al (2013) Hypoxia enhances chondrogenesis and prevents terminal differentiation
through PI3K/Akt/FoxO dependent anti-apoptotic effect. Sci Rep 3:1–12
131. Oliveira PH et al (2012) Impact of hypoxia and long-term cultivation on the genomic stability
and mitochondrial performance of ex vivo expanded human stem/stromal cells. Stem Cell Res
9:225–236
132. Roy S, Tripathy M, Mathur N, Jain A, Mukhopadhyay A (2012) Hypoxia improves expansion
potential of human cord blood-derived hematopoietic stem cells and marrow repopulation
efficiency. Eur J Haematol 88:396–405
133. Andrade PZ et al (2015) Ex vivo expansion of cord blood haematopoietic stem/progenitor cells
under physiological oxygen tensions: clear-cut effects on cell proliferation, differentiation and
metabolism. J Tissue Eng Regen Med 9:1172–1181
134. Guruvenket S, Rao GM, Komath M, Raichur AM (2004) Plasma surface modification of
polystyrene and polyethylene. Appl Surf Sci 236:278–284
135. Jung S, Sen A, Rosenberg L, Behie LA (2010) Identification of growth and attachment factors
for the serum-free isolation and expansion of human mesenchymal stromal cells. Cytotherapy
12:637–657
136. Bryhan MD, Gagnon PE, LaChance OV, Shen Z-H, Wang H (2003) Method for creating a cell
growth surface on a polymeric substrate. US 6,617,152 B2
137. Swistowski A et al (2009) Xeno-free defined conditions for culture of human embryonic stem
cells, neural stem cells and dopaminergic neurons derived from them. PLoS One 4:e6233
138. Lee DW et al (2015) T cells expressing CD19 chimeric antigen receptors for acute lympho-
blastic leukaemia in children and young adults: a phase 1 dose-escalation trial. Lancet
385:517–528
139. Saint-Jean M et al (2018) Adoptive cell therapy with tumor-infiltrating lymphocytes in
advanced melanoma patients. J Immunol Res 2018:3530148
140. Pardo AMP, Rothenberg ME (2013) Large scale expansion of human mesenchymal stem cells
using Corning® stemgro® hMSC Medium and Corning CellBIND ® Surface HYPER Stack®
cell culture vessels. Corning, Tewksbury
141. Blasey HD, Isch C, Bernard AR (1995) Cellcube: a new system for large scale growth of
adherent cells. Biotechnol Tech 9:725–728
142. Lambrechts T et al (2016) Evaluation of a monitored multiplate bioreactor for large-scale
expansion of human periosteum derived stem cells for bone tissue engineering applications.
Biochem Eng J 108:58–68
143. Andrade-Zaldivar H, Kalixto-Sánchez MA, Barba de la Rosa AP, León-Rodriguez A (2011)
Expansion of human hematopoietic cells from umbilical cord blood using roller bottles in CO2
and CO2-free atmosphere. Stem Cells Dev 20:593–598
144. Wikström K, Blomberg P, Islam KB (2004) Clinical grade vector production: analysis of yield,
stability, and storage of GMP-produced retroviral vectors for gene therapy. Biotechnol Prog
20:1198–1203
145. Wang H, Kehoe D, Murrell J, Jing D (2017) Structured methodology for process development
in scalable stirred tank bioreactors platforms. In: Connon CJ (ed) Bioprocessing for cell-based
therapies. Wiley, Hoboken, pp 35–64
146. Bayley R et al (2018) The productivity limit of manufacturing blood cell therapy in scalable
stirred bioreactors. J Tissue Eng Regen Med 12:e368–e378
147. Martin Y, Eldardiri M, Lawrence-Watt DJ, Sharpe JR (2010) Microcarriers and their potential
in tissue regeneration. Tissue Eng Part B Rev 17:71–80
148. Chen AKL, Reuveny S, Oh SKW (2013) Application of human mesenchymal and pluripotent
stem cell microcarrier cultures in cellular therapy: achievements and future direction.
Biotechnol Adv 31:1032–1046
149. dos Santos F et al (2014) A xenogeneic-free bioreactor system for the clinical-scale expansion
of human mesenchymal stem/stromal cells. Biotechnol Bioeng 116:1116–1127
150. Mizukami A et al (2016) Stirred tank bioreactor culture combined with serum-/xenogeneic-
free culture medium enables an efficient expansion of umbilical cord-derived mesenchymal
stem/stromal cells. Biotechnol J 11:1048–1059
151. Rafiq QA, Brosnan KM, Coopman K, Nienow AW, Hewitt CJ (2013) Culture of human
mesenchymal stem cells on microcarriers in a 5 l stirred-tank bioreactor. Biotechnol Lett
35:1233–1245
152. Lawson T et al (2017) Process development for expansion of human mesenchymal stromal
cells in a 50L single-use stirred tank bioreactor. Biochem Eng J 120:49–62
153. Schirmaier C et al (2014) Scale-up of adipose tissue-derived mesenchymal stem cell produc-
tion in stirred single-use bioreactors under low-serum conditions. Eng Life Sci 14:292–303
154. Robb KP, Fitzgerald JC, Barry F, Viswanathan S (2019) Mesenchymal stromal cell therapy:
progress in manufacturing and assessments of potency. Cytotherapy 21:289–306
155. Tsai AC, Liu Y, Ma T (2016) Expansion of human mesenchymal stem cells in fibrous bed
bioreactor. Biochem Eng J 108:51–57
156. Haack-Sørensen M et al (2016) Culture expansion of adipose derived stromal cells. A closed
automated Quantum Cell Expansion System compared with manual flask-based culture.
J Transl Med 14:319
157. Mizukami A et al (2018) A fully-closed and automated hollow fiber bioreactor for clinical-
grade manufacturing of human mesenchymal stem/stromal cells. Stem Cell Rev Rep
14:141–143
158. Tirughana R et al (2018) GMP production and scale-up of adherent neural stem cells with a
quantum cell expansion system. Mol Ther Methods Clin Dev 10:48–56
159. Lechanteur C et al (2014) Large-scale clinical expansion of mesenchymal stem cells in the
GMP-compliant, closed automated Quantum® Cell Expansion System: comparison with
expansion in traditional T-flasks. J Stem Cell Res Ther 4:1000222
160. Lambrechts T et al (2016) Large-scale progenitor cell expansion for multiple donors in a
monitored hollow fibre bioreactor. Cytotherapy 18:1219–1233
161. Junne S, Neubauer P (2018) How scalable and suitable are single-use bioreactors? Curr Opin
Biotechnol 53:240–247
162. Doran PM (2013) Bioprocess engineering principles. Academic, London
163. Schürch U, Kramer H, Einsele A, Widmer F, Eppenberger HM (1988) Experimental evalua-
tion of laminar shear stress on the behaviour of hybridoma mass cell cultures, producing
monoclonal antibodies against mitochondrial creatine kinase. J Biotechnol 7:179–184
164. McQueen A, Bailey JE (1989) Influence of serum level, cell line, flow type and viscosity on
flow-induced lysis of suspended mammalian cells. Biotechnol Lett 11:531–536
165. Chisti Y (2001) Hydrodynamic damage to animal cells. Crit Rev Biotechnol 21:67–110
166. McDowell CL, Papoutsakis ET (1998) Increased agitation intensity increases CD13 receptor
surface content and mRNA levels, and alters the metabolism of HL60 cells cultured in stirred
tank bioreactors. Biotechnol Bioeng 60:239–250
167. Jing Q, Cai H, Du Z, Ye Z, Tan WS (2013) Effects of agitation speed on the ex vivo expansion
of cord blood hematopoietic stem/progenitor cells in stirred suspension culture. Artif Cells
Nanomed Biotechnol 41:98–102
168. Yourek G, McCormick SM, Mao JJ, Reilly GC (2010) Shear stress induces osteogenic
differentiation of human mesenchymal stem cells. Regen Med 5:713–724
169. Hu K, Sun H, Gui B, Sui C (2017) TRPV4 functions in flow shear stress induced early
osteogenic differentiation of human bone marrow mesenchymal stem cells. Biomed
Pharmacother 91:841–848
170. Bassaneze V et al (2009) Shear stress induces nitric oxide–mediated vascular endothelial
growth factor production in human adipose tissue mesenchymal stem cells. Stem Cells Dev
19:371–378
171. Öncul AA, Kalmbach A, Genzel Y, Reichl U, Thévenin D (2010) Characterization of flow
conditions in 2 L and 20 L wave bioreactors® using computational fluid dynamics. Biotechnol
Prog 26:101–110
172. Timmins NE et al (2009) Clinical scale ex vivo manufacture of neutrophils from hematopoietic
progenitor cells. Biotechnol Bioeng 104:832–840
173. Sutlu T et al (2010) Clinical-grade, large-scale, feeder-free expansion of highly active human
natural killer cells for adoptive immunotherapy using an automated bioreactor. Cytotherapy
12:1044–1055
174. Croughan MS, Giroux D, Fang D, Lee B (2016) Novel single-use bioreactors for scale-up of
anchorage-dependent cell manufacturing for cell therapies. In: Cabral JMS, da Silva CL,
Chase LG, Diogo MM (eds) Stem cell manufacturing. Elsevier, Amsterdam, pp 105–139.
https://doi.org/10.1016/B978-0-444-63265-4.00005-4
175. Sousa MFQ et al (2015) Production of oncolytic adenovirus and human mesenchymal stem
cells in a single-use, Vertical-Wheel bioreactor system: impact of bioreactor design on
performance of microcarrier-based cell culture processes. Biotechnol Prog 31:1600–1612
176. Rodrigues CAV et al (2018) Scalable culture of human induced pluripotent cells on
microcarriers under xeno-free conditions using single-use Vertical-Wheel™ bioreactors.
J Chem Technol Biotechnol 93:3597–3606
177. Campbell A et al (2015) Concise review: process development considerations for cell therapy.
Stem Cells Transl Med 4:1155–1163
178. U.S. Department of Health and Human Services, Food and Drug Administration, Center for
Drug Evaluation and Research (2004) Guidance for Industry: PAT, a framework for innova-
tive pharmaceutical development, manufacturing, and quality assurance. Washington
179. EMEA (2006) Mandate for Process Analytical Technology Team (EMEA/48327/2006
Mandate). European Medicines Agency
180. Claßen J, Aupert F, Reardon KF, Solle D, Scheper T (2017) Spectroscopic sensors for in-line
bioprocess monitoring in research and pharmaceutical industrial application. Anal Bioanal
Chem 409:651–666
181. Biechele P, Busse C, Solle D, Scheper T, Reardon K (2015) Sensor systems for bioprocess
monitoring. Eng Life Sci 15:469–488
182. O’Mara P, Farrell A, Bones J, Twomey K (2018) Staying alive! Sensors used for monitoring
cell health in bioreactors. Talanta 176:130–139
183. Haber F, Hlemensiewicz Z (1909) Über elektrische Phasengrenzkräfte. Z Phys Chem
67U:385–431
184. Clark LC, Wolf R, Granger D, Taylor Z (1953) Continuous recording of blood oxygen
tensions by polarography. J Appl Physiol 6:189–193
185. Jeevarajan AS, Vani S, Taylor TD, Anderson MM (2002) Continuous pH monitoring in a
perfused bioreactor system using an optical pH sensor. Biotechnol Bioeng 78:467–472
186. Ge X et al (2006) Validation of an optical sensor-based high-throughput bioreactor system for
mammalian cell culture. J Biotechnol 122:293–306
187. Hanson MA et al (2007) Comparisons of optical pH and dissolved oxygen sensors with
traditional electrochemical probes during mammalian cell culture. Biotechnol Bioeng
97:833–841
188. Ude C et al (2014) Application of an online-biomass sensor in an optical multisensory platform
prototype for growth monitoring of biotechnical relevant microorganism and cell lines in
single-use shake flasks. Sensors (Basel) 14:17390–17405
189. Lavine BK (2000) Chemometrics. Anal Chem 72:91–98
190. Arnold SA, Crowley J, Woods N, Harvey LM, McNeil B (2003) In-situ near infrared
spectroscopy to monitor key analytes in mammalian cell cultivation. Biotechnol Bioeng
84:13–19
191. Salasznyk RM, Klees RF, Williams WA, Boskey A, Plopper GE (2007) Focal adhesion kinase
signaling pathways regulate the osteogenic differentiation of human mesenchymal stem cells.
Exp Cell Res 313:22–37
192. Rosa F et al (2016) Monitoring the ex-vivo expansion of human mesenchymal stem/stromal
cells in xeno-free microcarrier-based reactor systems by MIR spectroscopy. Biotechnol Prog
32:447–455
193. Suhito IR, Han Y, Min J, Son H, Kim TH (2018) In situ label-free monitoring of human
adipose-derived mesenchymal stem cell differentiation into multiple lineages. Biomaterials
154:223–233
194. Gomes J, Chopda V, Rathore AS (2018) Monitoring and control of bioreactor: basic concepts
and recent advances. In: Bioprocessing technology for production of biopharmaceuticals and
bioproducts. Wiley, Hoboken, pp 201–237. https://doi.org/10.1002/9781119378341.ch6
195. Mercier SM et al (2016) Process analytical technology tools for perfusion cell culture. Eng
Life Sci 16:25–35
196. Courtès F, Ebel B, Guédon E, Marc A (2016) A dual near-infrared and dielectric spectros-
copies strategy to monitor populations of Chinese hamster ovary cells in bioreactor.
Biotechnol Lett 38:745–750
197. Horiguchi I, Sakai Y (2016) Serum replacement with albumin-associated lipids prevents
excess aggregation and enhances growth of induced pluripotent stem cells in suspension
culture. Biotechnol Prog 32:1009–1016
198. Dekker L, Polizzi KM (2017) Sense and sensitivity in bioprocessing – detecting cellular
metabolites with biosensors. Curr Opin Chem Biol 40:31–36
199. Baradez M-O, Biziato D, Hassan E, Marshall D (2018) Application of Raman spectroscopy
and univariate modelling as a process analytical technology for cell therapy bioprocessing.
Front Med 5:47
200. Hynes RO (2002) Integrins: bidirectional, allosteric signaling machines. Cell 110:673–687
201. Lei Y, Schaffer DV (2013) A fully defined and scalable 3D culture system for human
pluripotent stem cell expansion and differentiation. Proc Natl Acad Sci U S A 110:E5039–
E5048
202. dos Santos F et al (2014) A xenogeneic-free bioreactor system for the clinical-scale expansion
of human mesenchymal stem/stromal cells. Biotechnol Bioeng 111:1116–1127
203. Mariappan I et al (2010) In vitro culture and expansion of human limbal epithelial cells.
Nat Protoc 5:1470–1479
204. Joen VT, Declercq H, Cornelissen M (2011) Expansion of human embryonic stem cells: a
comparative study. Cell Prolif 44:462–476
205. Rowley J, Abraham E, Campbell A, Brandwein H, Oh S (2012) Meeting lot-size challenges of
manufacturing adherent cells for therapy. Bioprocess Int 10:16–22
206. Rodrigues AL et al (2019) Dissolvable microcarriers allow scalable expansion and harvesting
of human induced pluripotent stem cells under xeno-free conditions. Biotechnol J 14:1800461
207. Zhang J et al (2015) Thermo-responsive microcarriers based on poly (N-isopropylacrylamide).
Eur Polym J 67:346–364
208. Nguyen LTB, Odeleye AOO, Chui CY, Baudequin T (2019) Development of thermo-
responsive polycaprolactone macrocarriers conjugated with poly (N-isopropyl acrylamide)
for cell culture. Sci Rep 9:1–11
209. Cunha B et al (2015) Filtration methodologies for the clarification and concentration of human
mesenchymal stem cells. J Membr Sci 478:117–129
210. Schnitzler AC et al (2016) Bioprocessing of human mesenchymal stem/stromal cells for
therapeutic use: current technologies and challenges. Biochem Eng J 108:3–13
211. Mehta S, Herman T, Ross H, Iqbal K, McMahon J (2016) Methods and systems for manip-
ulating particles using a fluidized bed. US 9,279,133 B2
212. Cunha B et al (2015) Exploring continuous and integrated strategies for the up- and down-
stream processing of human mesenchymal stem cells. J Biotechnol 213:97–108
213. Wang Z, Feke D, Belovich J (2014) Acoustic device and methods thereof for separation and
concentration. US 8,889,388 B2
214. Woods EJ, Thirumala S, Badhe-buchanan SS, Clarke D, Mathew ABYJ (2016) Off the shelf
cellular therapeutics: factors to consider during cryopreservation and storage of human cells
for clinical use. Cytotherapy 18:697–711
215. Lee S et al (2008) Post-thaw viable CD34+ cell count is a valuable predictor of haematopoietic
stem cell engraftment in autologous peripheral blood stem cell transplantation. Vox Sang
94:146–152
216. Allan DS et al (2002) Number of viable CD34+ cells reinfused predicts engraftment in
autologous hematopoietic stem cell transplantation. Bone Marrow Transplant 29:967–972
217. Chatzistamatiou TK et al (2014) Optimizing isolation culture and freezing methods to preserve
Wharton’s jelly’s mesenchymal stem cell (MSC) properties: an MSC banking protocol
validation for the Hellenic Cord Blood Bank. Transfusion 54:3108–3120
218. Iyer RK, Bowles PA, Kim H, Dulgar-tulloch A (2018) Industrializing autologous adoptive
immunotherapies: manufacturing advances and challenges. Front Med 5:150
219. Peltzer J et al (2018) Mesenchymal stromal cells based therapy in systemic sclerosis: rational
and challenges. Front Immunol 9:2013
220. Coopman K, Medcalf N (2014) From production to patient: challenges and approaches for
delivering cell therapies. In: StemBook. https://doi.org/10.3824/STEMBOOK.1.97.1
221. Dhall S et al (2018) Properties of viable lyopreserved amnion are equivalent to viable
cryopreserved amnion with the convenience of ambient storage. PLoS One 13:1–19
222. Paton J (2018) Novartis’ Kymriah cancer drug priced at 320,000 euros in Germany.
Bloomberg
223. Hirschler B (2018) UK rejects Gilead’s CAR-T cancer cell therapy as too expensive. Reuters
224. Liu A (2018) Beat you to it, Kymriah: Gilead strikes discount Yescarta deal with NHS in
adults. Fierce Pharma
225. National Institute for Health and Care Excellence (2019) Darvadstrocel for treating complex
perianal fistulas in Crohn’s disease. London
226. Panés J et al (2016) Expanded allogeneic adipose-derived mesenchymal stem cells (Cx601) for
complex perianal fistulas in Crohn’s disease: a phase 3 randomised, double-blind controlled
trial. Lancet 388:1281–1290
227. Chilima TDP, Moncaubeig F, Farid SS (2018) Impact of allogeneic stem cell manufacturing
decisions on cost of goods, process robustness and reimbursement. Biochem Eng J
137:132–151
228. Trainor N, Pietak A, Smith T (2014) Rethinking clinical delivery of adult stem cell therapies.
Nat Biotechnol 32:729–735
229. Torikai H et al (2012) A foundation for universal T-cell based immunotherapy: T cells
engineered to express a CD19-specific chimeric-antigen-receptor and eliminate expression
of endogenous TCR. Blood 119:5697–5705
230. Jenkins MJ, Farid SS (2018) Cost-effective bioprocess design for the manufacture of alloge-
neic CAR-T cell therapies using a decisional tool with multi-attribute decision-making anal-
ysis. Biochem Eng J 137:192–204
231. Simaria AS et al (2014) Allogeneic cell therapy bioprocess economics and optimization:
single-use cell expansion technologies. Biotechnol Bioeng 111:69–83
232. US Drug and Food Administration (2004) Pharmaceutical CGMPs for the 21st century – a
risk-based approach. FDA
233. Rathore AS, Winkle H (2009) Quality by design for biopharmaceuticals. Nat Biotechnol
27:26–34
234. EMA & FDA (2017) Report from the EMA-FDA QbD pilot program (EMA/213746/2017)
235. Senior M (2017) After Glybera’s withdrawal, what’s next for gene therapy? Nat Biotechnol
35:491–492
236. Grover N (2014) Dendreon files for bankruptcy as cancer vaccine disappoints. Reuters
237. Lipsitz YY, Timmins NE, Zandstra PW (2016) Quality cell therapy manufacturing by design.
Nat Biotechnol 34:393–400
238. Martin-Moe S et al (2011) A new roadmap for biopharmaceutical drug product development:
integrating development, validation, and quality by design. J Pharm Sci 100:3031–3043
239. Rathore AS (2009) Roadmap for implementation of quality by design (QbD) for biotechnol-
ogy products. Trends Biotechnol 27:546–553
240. Mandenius C-F et al (2009) Quality-by-design for biotechnology-related pharmaceuticals.

Biotechnol J 4:600–609
241. Hunt MM, Meng G, Rancourt DE, Gates ID, Kallos MS (2013) Factorial experimental design
for the culture of human embryonic stem cells as aggregates in stirred suspension bioreactors
reveals the potential for interaction effects between bioprocess parameters. Tissue Eng Part C
Methods 20:76–89
242. Ratcliffe E et al (2013) Application of response surface methodology to maximize the
productivity of scalable automated human embryonic stem cell manufacture. Regen Med
8:39–48
243. Andrade PZ, Dos Santos F, Almeida-Porada G, Lobato Da Silva C, Joaquim JM (2010)
Systematic delineation of optimal cytokine concentrations to expand hematopoietic stem/
progenitor cells in co-culture with mesenchymal stem cells. Mol BioSyst 6:1207–1215
244. Csaszar E et al (2012) Rapid expansion of human hematopoietic stem cells by automated
control of inhibitory feedback signaling. Cell Stem Cell 10:218–229
245. van Poll D et al (2008) Mesenchymal stem cell-derived molecules directly modulate hepato-
cellular death and regeneration in vitro and in vivo. Hepatology 47:1634–1643
246. Gnecchi M et al (2006) Evidence supporting paracrine hypothesis for Akt-modified
mesenchymal stem cell-mediated cardiac protection and functional improvement. FASEB J
20:661–669
247. Teixeira FG et al (2016) Modulation of the mesenchymal stem cell secretome using computer-
controlled bioreactors: impact on neuronal cell proliferation, survival and differentiation. Sci
Rep 6:1–14
248. Mukherjee P, Mani S (2013) Methodologies to decipher the cell secretome. Biochim Biophys
Acta Proteins Proteomics 1834:2226–2232
249. Paltridge JL, Belle L, Khew-Goodall Y (2013) The secretome in cancer progression. Biochim
Biophys Acta Proteins Proteomics 1834:2233–2241
250. Raposo G, Nijman HW, Stoorvogel W, Liejendekker R, Harding CV, Melief CJ, Geuze HJ
(1996) B lymphocytes secrete antigen-presenting vesicles. J Exp Med 183:1161–1172
251. Ratajczak J et al (2006) Embryonic stem cell-derived microvesicles reprogram hematopoietic
progenitors: evidence for horizontal transfer of mRNA and protein delivery. Leukemia
20:847–856
252. Valadi H et al (2007) Exosome-mediated transfer of mRNAs and microRNAs is a novel
mechanism of genetic exchange between cells. Nat Cell Biol 9:654–659
253. Mathivanan S, Ji H, Simpson RJ (2010) Exosomes: extracellular organelles important in
intercellular communication. J Proteome 73:1907–1920
254. Van Niel G, D’Angelo G, Raposo G (2018) Shedding light on the cell biology of extracellular
vesicles. Nat Rev Mol Cell Biol 19:213–228
255. György B et al (2011) Membrane vesicles, current state-of-the-art: emerging role of extracel-
lular vesicles. Cell Mol Life Sci 68:2667–2688
256. Lopez-Verrilli MA, Picou F, Court FA (2013) Schwann cell-derived exosomes enhance
axonal regeneration in the peripheral nervous system. Glia 61:1795–1806
257. Barile L et al (2014) Extracellular vesicles from human cardiac progenitor cells inhibit
cardiomyocyte apoptosis and improve cardiac function after myocardial infarction. Cardiovasc
Res 103:530–541
258. Peinado H et al (2012) Melanoma exosomes educate bone marrow progenitor cells toward a
pro-metastatic phenotype through MET. Nat Med 18:883–891
259. Costa-Silva B et al (2015) Pancreatic cancer exosomes initiate pre-metastatic niche formation
in the liver. Nat Cell Biol 17:816–826
260. Yan W et al (2018) Cancer-cell-secreted exosomal miR-105 promotes tumour growth through
the MYC-dependent metabolic reprogramming of stromal cells. Nat Cell Biol. https://doi.org/
10.1038/s41556-018-0083-6
261. Sharples RA et al (2008) Inhibition of γ-secretase causes increased secretion of amyloid
precursor protein C-terminal fragments in association with exosomes. FASEB J 22:1469–1478
262. Wiklander OPB et al (2015) Extracellular vesicle in vivo biodistribution is determined by cell
source, route of administration and targeting. J Extracell Vesicles 4:1–13
263. Yang T et al (2015) Exosome delivered anticancer drugs across the blood-brain barrier for
brain cancer therapy in Danio rerio. Pharm Res 32:2003–2014
264. Lai RC et al (2010) Exosome secreted by MSC reduces myocardial ischemia/reperfusion
injury. Stem Cell Res 4:214–222
265. Bruno S et al (2012) Microvesicles derived from mesenchymal stem cells enhance survival in a
lethal model of acute kidney injury. PLoS One 7:e33115
266. Batrakova EV, Kim MS (2015) Using exosomes, naturally-equipped nanocarriers, for drug
delivery. J Control Release 219:396–405
267. Wang X et al (2018) Engineered exosomes with ischemic myocardium-targeting peptide for
targeted therapy in myocardial infarction. J Am Heart Assoc 7:1–16
268. Alvarez-Erviti L et al (2011) Delivery of siRNA to the mouse brain by systemic injection of
targeted exosomes. Nat Biotechnol 29:341–345
269. Tian Y et al (2014) A doxorubicin delivery platform using engineered natural membrane
vesicle exosomes for targeted tumor therapy. Biomaterials 35:2383–2390
270. Kim MS et al (2016) Development of exosome-encapsulated paclitaxel to overcome MDR in
cancer cells. Nanomedicine 12:655–664
271. Li Y et al (2018) A33 antibody-functionalized exosomes for targeted delivery of doxorubicin
against colorectal cancer. Nanomedicine 14:1973–1985
272. Kim MS et al (2018) Engineering macrophage-derived exosomes for targeted paclitaxel
delivery to pulmonary metastases: in vitro and in vivo evaluations. Nanomedicine 14:195–204
273. Colao IL, Corteling R, Bracewell D, Wall I (2018) Manufacturing exosomes: a promising
therapeutic platform. Trends Mol Med 24:242–256
274. Vader P, Mol EA, Pasterkamp G, Schiffelers RM (2016) Extracellular vesicles for drug
delivery. Adv Drug Deliv Rev 106:148–156
275. Conlan RS, Pisano S, Oliveira MI, Ferrari M, Mendes Pinto I (2017) Exosomes as
reconfigurable therapeutic systems. Trends Mol Med 23:636–650
276. Papadimitropoulos A et al (2014) Expansion of human mesenchymal stromal cells from fresh
bone marrow in a 3D scaffold-based system under direct perfusion. PLoS One 9:e102359
277. Hümmer C et al (2016) Automation of cellular therapy product manufacturing: results of a
split validation comparing CD34 selection of peripheral blood stem cell apheresis product with
a semi-manual vs. an automatic procedure. J Transl Med 14:1–7
DOI: 10.1007/10_2019_108
Bioprinting Technologies in Tissue

Engineering
Bengi Yilmaz, Aydin Tahmasebifar, and Erkan Türker Baran
Contents
1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 281
2 3D Bioprinting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 284
2.1 Inkjet Bioprinting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 285
2.2 Extrusion Bioprinting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 286
2.3 Laser-Assisted Bioprinting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 287
2.4 Stereolithography (SLA) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 288
2.5 Microvalve-Based Bioprinting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 289
3 4D Bioprinting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 289
4 Tissue Engineering Applications of Bioprinting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 290
4.1 Bone Tissue . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 290
4.2 Osteochondral and Cartilage . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 295
4.3 Nerve . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 296
4.4 Skin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 302
4.5 Cardiac and Skeletal Muscle . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 305
4.6 Other Soft Tissues . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 309
5 Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 312
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 312
Abstract Bioprinting technology is a strong tool in producing living functional

tissues and organs from cells, biomaterial-based bioinks, and growth factors in
computer-controlled platform. The aim of this chapter is to present recent progresses
in bioprinting of nerve, skin, cardiac, bone, cartilage, skeletal muscle, and other soft
tissues and highlight the challenges in these applications. Various composite bioinks
B. Yilmaz, A. Tahmasebifar, and ErkanT. Baran (*)

Institute of Health Sciences, Department of Tissue Engineering, University of Health Sciences,
Istanbul, Turkey
Institute of Health Sciences, Department of Biomaterials, University of Health Sciences,
Istanbul, Turkey
e-mail: erkanturker.boran@sbu.edu.tr
280 B. Yilmaz et al.
with bioactive ceramic-based scaffolds having patient-specific design and controlled

micro-architectures were used at clinical and preclinical applications successfully
for regeneration of bone. In nerve tissue engineering, bioprinting of alginate- and
gelatin-based gel bioinks by extrusion presented a controllable 3D microstructures
and showed satisfactory cytocompatibility and axonal regeneration. Bioprinting of
cardiac progenitors in biopolymers resulted in limited success, while the use of
bioinks from extracellular matrix induced satisfactory results in cardiac regeneration.
Osteochondral scaffold bioprinting is challenging due to the complex hierarchical
structure and limited chondral regeneration. Therefore, current approaches focused
on osteochondral scaffold with vascular network and mimicking hierarchical struc-
tures. The applications of bioprinting in other types of tissues were also studied, and
results showed significant potentials in regeneration of tissues such as cornea, liver,
and urinary bladder.
Graphical Abstract
Keywords Bioprinting, Bone, Cardiac, Cartilage, Nerve, Skin, Tissue engineering

Bioprinting Technologies in Tissue Engineering 281
1 Introduction
Bioprinting is a variant of 3D printing and refers to the three-dimensional

(3D) manufacturing of biological constructs through the layer-by-layer deposition
of inks with living cells. It can also be defined as the use of material science and
production techniques to build tissues and organs with viable cells and biomolecules
in specific organization to perform a particular biological function [1]. The first
international conference on bioprinting was held at the University of Manchester,
UK, in September 2004, and the definition of bioprinting was first proposed in this
conference as: “the use of material transfer processes for patterning and assembling
biologically relevant materials – molecules, cells, tissues, and biodegradable bio-
materials – with a prescribed organization to accomplish one or more biological
functions” [2]. Since the ultimate goal of bioprinting is to produce living functional
tissues and organs to be transplanted, Mironov et al. [3] proposed another term
“organ printing” which is defined as: “computer aided 3D tissue engineering of
living organs based on the simultaneous deposition of cells and hydrogels with the
principles of self-assembly.”
Bioprinting, or 3D bioprinting, was defined in “Workshop on Bioprinting,
Biopatterning and Bioassembly” at the University of Manchester, UK, in 2004, as:
“The use of material transfer processes for patterning and assembling biologically
relevant materials—molecules, cells, tissues, and biodegradable biomaterials—with
a prescribed organization to accomplish one or more biological functions” [4]. 3D
bioprinting for the production of biological structures typically involves layer-by-
layer deposition of bioink to form 3D tissue or organ structures with an input from a
computer-aided design (CAD) program [5]. 3D bioprinting has been gathering
enormous attention from the scientific community of regenerative medicine due
to its ability to manufacture organs from native cells. Figure 1 demonstrates the
increasing scientific interest to bioprinting and shows the related scientific categories
according to Web of Science database. Although the bio-additive production of a
transplantable entire organ has not been achieved yet, this technology is advancing,
and it is thought that it will soon resolve the crisis of organ shortages [6].
According to the Organ Procurement and Transplantation Network of US
Department of Health and Human Services, 114,417 people are actively waiting
for a lifesaving organ transplant in the USA, and only 30,415 organ transplants were
performed in 2018; see Fig. 2 [7]. Regenerative medicine gives hope to fill the gap
between the number of organ transplants and organ need through the engineering of
functional tissues or organs to improve or modify abnormal and necrotic tissues and
organs [8]. Furthermore, 3D printed human tissues can be a better model for
preclinical testing of potential drugs than in vivo testing on animal models in order
to predict the clinical outcome in humans [9].
There are three basic variables that should be considered in order to be able to
produce living and functional tissue structures successfully by using the bioprinting
method. The first is the cellular component which is the living part of the structure
and containing at least one or multiple cell types. The second one is the scaffolding
a Number of Journal Arcles

Keyword: 'bioprinng'
330
350 307
300 244
250
Documents
200 145
150
100 72
38 40
24 29
50 1 1 3 3 6 12
0
2004 2006 2008 2010 2012 2014 2016 2018
Years
Web of Science Categories
b
ENGINEERING BIOMEDICAL
4%
6%
MATERIALS SCIENCE BIOMATERIALS
7% 24%
BIOTECHNOLOGY APPLIED MICROBIOLOGY
9% CELL TISSUE ENGINEERING
CELL BIOLOGY
9% MATERIALS SCIENCE MULTIDISCIPLINARY

19% NANOSCIENCE NANOTECHNOLOGY
10% CHEMISTRY MULTIDISCIPLINARY

12%
MULTIDISCIPLINARY SCIENCES
Fig. 1 (a) Number of records for “bioprinting” in academic publications by year; (b) scientific
categories related to bioprinting. Source: Web of Science, December 2018
or supportive biomaterials comprising mostly proteins and polymers that can pro-
vide support and protection to cells during and after the manufacturing process.
These biomaterials can mimic the physical environment and the biochemical signal
cells as in the body and serve as a bioink bridge between cells and hardware. Third is
the actual bioprinting device in which the manufacturing process is performed. Most
importantly, the biomaterial used is a necessary bridge between the bioink, cells, and
hardware [9].
Fig. 2 According to Organ (a) Waing list candidates in US

Procurement and
Transplantation Network: 3% 1% 1%
0%
(a) number of waiting list
12% Kidney
candidates by organ type as
of December 2018 in the Kidney/Pancreas
1%
USA; (b) source of Liver
transplants performed in
January to October 2018 in Intestine
the USA [7] Heart
Lung
Pancreas
82%
(b) Transplants performed in 2018 by source
Living Donor
19%
Deceased
Donor
81%
In a historical point of view, Wyn Kelly Swainson owned a patent for a method
and an apparatus for the first time in 1971 to produce 3D structures in a medium
which is selectively sensitive to different parameters of electromagnetic radiation
[10]. Later, in 1984, Charles W. Hull patented an apparatus for the production of
three-dimensional objects by stereolithography [11], which was considered as the
world’s first 3D printer [12].
The use of 3D printing in healthcare applications was started with customized and
patient-specific surgical guides and implants in orthopedics [13]. In 2002,
Envisiontec GmbH began to sell a bioprinter named Bioplotter which was able to
produce scaffold structures from various biomaterials for tissue engineering [14]. In
2010, a research center Chemical Institute of Sarria (IQS – Instituto Químico de
Sarrià) announced two hydroxyapatite (HA) formulations for use in 3D printers
[14]. In 2011, Objet introduced a biocompatible colorless photopolymer material,
MED610, for medical and dental markets [15]. In 2012, Organovo Holdings and
Autodesk announced a collaboration to create the first commercial 3D bioprinting
software [14]. Organovo’s NovoGen MMX Bioprinter was the first commercial
bioprinter tested for 3D-printed tissues [16, 17].
2 3D Bioprinting
Custom-made and patient-specific scaffolds/tissues/organs can be printed according

to the 3D models constructed from the data acquired by medical imaging techniques,
such as 3D computed tomography (CT) and magnetic resonance imaging (MRI), or
generated digitally by using many CAD software. Figure 3 shows the flow diagram
for the manufacturing of bioprinted tissues that can be used either directly in animal
models or patients or as in vitro constructs that can be used for drug testing and
disease modeling.
3D bioprinting is a comprehensive process that requires adjustment of various
design parameters, such as imaging, modeling, printer choice, selection of bioink,
culture conditions, and development of 3D construct [8]. The manufacturing process
can be categorized in three different steps: pre-bioprinting, bioprinting, and post-
bioprinting [18]. The pre-bioprinting (modeling) step involves acquisition of 3D
images by using techniques like 3D scanner, CT, and MRI, designing a digital 3D
model suitable for printing from these imaging modalities or by using CAD-CAM
and mathematical modeling techniques, and finally the selection of bioink, bioma-
terial, and cells. In the bioprinting step, 3D-rendered model is sliced into customiz-
able horizontal portions that are imported into the bioprinter system. One or more
bioinks, which are composed of cells, bioactive molecules, and biomaterials, are
Material Cell
selection selection
Input from
imaging Slicing and setting
techniques parameters
(MRI, CT etc.)
Data converted
3D Bioprinting
to STL file
CAD design
Maturation Yes
Bioreactor
needed?
No
Implantation /
In vitro drug or
chemical testing
Fig. 3 A typical production flowchart for bioprinting

Table 1 Comparison of bioprinter types [19]

Inkjet Extrusion Laser assisted
Material 3.5–12 mPa/s 30 mPa/s to >6 107 mPa/s 1–300 mPa/s
viscosities
Gelation Chemical, photo- Chemical, photo-cross-linking, Chemical, photo-
methods cross-linking sheer thinning, temperature cross-linking
Preparation Low Low to medium Medium to high
time
Print speed Fast (1–10,000 drop- Slow (10–50 μm/s) Medium-fast
lets per s) (200–1,600 mm/s)
Resolution or <1 pL to >300 pL 5 μm to millimeters wide Microscale
droplet size droplets, 50 μm wide resolution
Cell viability >85% 40–80% >95%
Cell densities Low, <106 cells/mL High, cell spheroids Medium,
108 cells/mL
Printer cost Low Medium High
placed in the printer cartridges and printed layer by layer in aseptic conditions. The
post-printing, which is biomimetic remodeling of tissue with the help of mechanical
and chemical signals, is also an important step for obtaining mechanical integrity and
biological functionality.
The main technologies used for 3D bioprinting with biological materials are
inkjet, micro-extrusion, and laser-assisted printing [19]. Table 1 summarizes the
different features of these technologies.
2.1 Inkjet Bioprinting
2D inkjet printing is a noncontact technique that accepts a digital signal representing

an image and deposits tiny ink drops to form the image onto a substrate, which is
mostly paper. It is possible to fabricate 3D tissues by using a technology very similar
to that of a common inkjet printer. In fact, the first attempts of bioprinting started
with working on the hardware and software necessary to convert commercial
tabletop inkjet printers into cell printers [20], and the first patent for printing viable
cells with inkjet printer came in 2006 by Thomas Boland and coworkers at Clemson
University [21]. One of the early researchers Anthony Atala (the director of the
Wake Forest Institute for Regenerative Medicine in Winston-Salem, NC, USA) also
began on a desktop inkjet printer that was modified to print 3D structures [22].
Droplet-based inkjet bioprinting works in a similar manner to the inkjet technol-
ogy as described above. The most common types of inkjet printing devices that are
adapted to bioprinting have thermal and piezoelectric mechanisms to generate the
droplets in drop-on-demand principle (see Fig. 4). The devices with a thermal
printhead have an electric heating unit that vaporizes the binder material to form a
Extrusion Inkjet Laser
Piston
Screw
Pneumatic
Piezo
actuator Heater
Laser
Donor
side
Fused
particles
Collector
side
Fig. 4 Inkjet, extrusion, and laser-assisted bioprinters
vapor bubble. This vapor bubble expands due to pressure and exits as a droplet from
the printhead. Although the temperature rises to 200–300 C while forming a bubble,
the printhead temperature increases only about 10 C, because the process only takes
a few microseconds (about 2 μs) [8]. In the devices using a printhead with a
piezoelectric actuator, the voltage pulse in the printhead causes a rapid shape change
in the piezo-material, and this generates a pressure pulse in the binding fluid,
resulting in a droplet. These printers ensure fast and precise deposition of the binder
liquid.
2.2 Extrusion Bioprinting
Fused deposition modeling (FDM) or fused filament fabrication (FFF) was devel-
oped in the early 1990s, and this extrusion-based 3D printing strategy is the most
common and inexpensive type of additive manufacturing technology [8, 23]. FDM
technique is based on extrusion of molten thermoplastic polymer filaments or small
granules from a small nozzle, which then hardens to form a solid construct (see
Fig. 5) [24]. FDM offers many advantages, such as easy material switching, low
maintenance costs, compact size, and flexibility in working temperatures; however,
the narrow range of printable materials limits its applications [25].
Fig. 5 A schematic
diagram showing fused
deposition modeling
Extrusion-based (dispensing or direct writing) bioprinting originates from

FDM printing technology. These devices use pneumatic-, mechanical-, or
electromagnetic-driven needle-syringe-type systems to deposit cells and biomate-
rials [8]. The cell-laden bioinks, which are mostly composed of hydrogels and some
bioactive components, are dispensed by the printhead to form the desired 3D
structures. In order to produce 3D structures with high accuracy, bioinks must be
designed with shear-thinning or fast-solidifying properties. It is possible to obtain
multiwalled and core-shell fibers from different bioinks by using multi-compartment
type of nozzles and to adapt microfluidic strategy to extrusion bioprinting [26].
2.3 Laser-Assisted Bioprinting
In 1999, Odde and Renn [27] reported the possible tissue engineering applications of
a laser-guided direct-writing system which has the ability to organize cells spatially
into well-defined 3D arrays. Laser-assisted bioprinting is also a droplet-based system
which is also known as laser-induced forward transfer (LIFT). This system consists
of a pulsed laser source; a focusing system; a ribbon, which contains a layer of
biological material to be printed and a glass covered with a laser-energy-absorbing
layer (such as gold or titanium); and a substrate that collects the printed material
[19, 28]; see Fig. 4. The metal film is vaporized by a laser pulse, and this produces
a jet of liquid solution of organic materials (cells and molecules) which is then
deposited onto the facing substrate [29]. Although laser-assisted bioprinting is less
common than other bioprinting techniques, it has many advantages, such as having
high spatial resolution and capability of printing a variety of biological materials and
high cell viability.
2.4 Stereolithography (SLA)
Stereolithography was developed as a solid free-form manufacturing technique in

the early 1980s [30]. Today, most 3D printers and recent bioprinters accept input
files in the STL format. The acronym “STL” stands for STereoLithography, since it
was the first 3D printing process, and the STL file format emerged as a native file
format from a CAD software [31]. This STL file is sliced to generate a G-code,
which provides instructions for the stereolithography apparatus (SLA).
The main components of the system are a reservoir filled with a photocurable
polymer solution or resin, a x-y axis controlled laser, and a fabrication stage with
z-axis control [9]. A SLA is depicted in Fig. 6. Stereolithography is a laser-assisted
additive manufacturing technology that utilizes an ultraviolet (UV) laser to
photopolymerize the surface of a bath of photosensitive polymer. The stage is
gradually lowered, allowing layers to be polymerized on top of each other, thereby
forming 3D structures in a bottom-up manner. The unscanned areas stay in the liquid
phase.
The use of SLA in bioprinting was reported by the study of Dhariwala and
coworkers in 2004 for the first time [32]. They filled a vat with cells and a
photocurable hydrogel, which consists of poly(ethylene oxide) and poly(ethylene
glycol dimethacrylate) in a ratio of 3:2 and a photoinitiator (Irgacure). The vat was
placed on a table where the movement could be controlled in the vertical axis. UV
light cured designated places in the vat according to the CAD file. For each layer, the
process was repeated to produce 3D structures. High cell viability was achieved with
a laser of UV light at 365 nm. Today, there are various applications of SLA from
bioprinting to other rapid prototyping purposes.
Fig. 6 A schematic
diagram showing
stereolithography apparatus
2.5 Microvalve-Based Bioprinting
Microvalve-based bioprinting is a kind of drop-on-demand systems which is mostly

preferred for hydrogel dispensation [33]. This technology was developed by Demirci
and Montesano in 2007. The main focus of their custom-made microvalve-based
bioprinting is cell printing [34]. A conventional microvalve bioprinting system
is composed of robotic platform and electromechanical printheads which are
connected to a gas regulator [34, 35]. The gas regulator provides positive pressure
on the system as it is in conventional systems. The solenoid coil and the plunger
movement controls the microvalve opening time as low as 0.1 ms [34, 35]. The valve
can be controlled by mechanical, electrical, and magnetic forces. Thus, the positive
pressure and valve opening time controls allocating of bioink during printing [35].
The pressure, which is required for dispensing of material from the nozzle, leads
to significant shear stress on cells. In addition, the parameters such as nozzle
diameter and the viscosity of bioink have considerable effect on shear stress
[36]. Although the moderate shear stress plays a crucial role in cell signaling and
protein expression and improves stem cell differentiation, the disproportionate shear
stress may rapture cell membrane [36]. The narrow range of printable viscosity is the
main limitation of microvalve-based bioprinting systems (1–70 Mpa) [37].
3 4D Bioprinting
4D printing technology was invented by the Self-Assembly Laboratory at the

Massachusetts Institute of Technology (MIT) in collaboration with Stratasys Ltd.
[38]. The main difference from 3D printing is the production of multi-material prints
with shape-changing ability over time or the use of customized materials which can
be changed from one shape to another after being taken from the print bed. 3D
bioprinting technology relies on the assumption that the printed cells can rapidly
form tissues and start to synthesize the extracellular matrices, which can provide
geometrical shape and mechanical support. 4D bioprinting, where time is the fourth
dimension, the printed materials or living cellular constructs continue to evolve over
time after being printed [39] (see Fig. 7). The stimuli for these constructs to evolve
can be one or more of specific triggers, such as temperature, pH, humidity, electric-
ity, magnetic field, light, and acoustics [40]. Integrating new smart materials, which
can respond to these stimuli and transform with time, into 3D printing is the basis of
4D printing concept. One such smart feature is the shear-thinning property of bioinks
that allows reversible changes in the shear viscosity of printable inks to assist 4D
printing [41].
Since bioprinting requires the use of viable cells, 4D printable smart materials
should be biocompatible, and rheological properties should be adequate in order to
maintain high cell viability at optimum printability [42]. 4D printed cell-laden
structures face many challenges as it is in 3D printing technique: (1) cell damage
Fig. 7 Schematic depiction of printing of 3D and 4D objects
after printing, (2) deterioration of cell proliferation, and (3) deposition of low or
excess cell population [43].
The number of sensitive materials, which can be printed by using the 4D printing
technique, is very limited, and most are capable of responding to only one type of
stimulus. Furthermore, the deformation ability of the 4D bioprinted structures
formed with these materials is also limited to simple deformations such as folding/
unfolding or self-organization. The precise control of the spatiotemporal evolution
of the printed structure is restricted because these deformations can occur only in
macroscale [39]. The 4D printing technique also needs to overcome the major
difficulties in mimicking the complex dynamic deformation of natural tissues such
as the blood pumping of the heart and the peristaltic movement of the esophagus or
intestine [44].
4 Tissue Engineering Applications of Bioprinting
4.1 Bone Tissue
Bone tissue provides mechanical strength, creates a structural framework, and allows
the body to move. It also serves as a mineral storage and plays a vital role in
homeostasis and regulation of blood pH [45]. Bone is a connective tissue with a
complex hierarchical structure. It is composed mainly of a mineral phase, which
constitutes between 60 and 70 wt% of its total mass, and water between 5 and
10 wt%, while the remaining part is an organic matrix of collagen and other proteins
[46]. Collagen and agglomerated HA mineral crystals intertwine to form several
hundred nanometers of mineralized fibrils [47].
Bone is the second most widely transplanted tissue around the world, and more
than four million operations are performed each year by using natural or synthetic
bone grafts to treat bone defects [48]. The main solutions to facilitate bone repair are
still the autograft and allograft techniques, even though autografting is expensive,
invasive, and subject to infections and hematoma, frequently affecting both donor
sites and surgical sites, and allografts may cause an immune reaction followed by a
rejection [49]. The role of biomaterials and tissue-engineered constructs has become
more important in the last several decades. Among the most challenging and one
of the most studied tissues to reconstruct is bone tissue. The design of bone
tissue engineering scaffold should comprise a three-dimensional structure with
interconnected pore network for cell growth and transport of nutrients/waste. The
scaffold should certainly be biocompatible, and its surface chemistry should be
suitable for cell attachment, proliferation, and differentiation. Finally, the absorbable
scaffold should degrade at a rate that matches the tissue regeneration while keeping
its mechanical properties very close to that of the host tissue [50].
Several techniques previously used in bone tissue engineering to obtain an
ideal cell-laden scaffold did not yield significant successes due to many limiting
factors, majorly vascularity [51]. Another substantial challenge is the critical size
of the construct while meeting the clinical requirements of structural support,
osteoinductive property, and controllable biodegradability [52].
The main focus of bioprinting for bone tissue engineering is the advancement in
printing methods and the development of compatible ink materials. Various mate-
rials have been developed for use in 3D printing to create new alternatives to
conventional bone grafts. The material groups, such as polymers, ceramics, and
hydrogels, cannot fully mimic the properties of bone when used alone. For example,
in order to increase the bioactivity, hydrogels are generally combined with
osteoconductive elements such as calcium phosphates, tricalcium phosphate
(TCP), biphasic calcium phosphate (BCP), hydroxyapatite (HAp), silicate, or silica
and bioactive glass nanoparticles. Also soluble molecules such as bone morphoge-
netic protein-2 (BMP-2) and vascular endothelial growth factor (VEGF) are used to
induce angiogenesis and osteogenesis [51]. The simultaneous integration of these
temperature-sensitive biological agents, living cells, and the bioink materials to
create a 3D artificial bone implant or a complex bone tissue construct with controlled
cell distribution, geometry, and biodegradability, good mechanical properties, and
high osteoinductivity and osteoconductivity is today’s most important challenge in
3D bioprinting. Table 2 reviews the studies investigating these possible bioink
formulations for specific fabrication methods and their osteogenic, osteochondral,
or vasculogenic/angiogenic potential.
Compared to 3D printing of constructs, which are added in the post-printing, cell-
laden 3D bioprinting, as mentioned above, involves more complex factors, such as
narrow set of printing materials, the strategy of gelling, cell viability, and some
technical issues [62]. On the other hand, cell-free inks, or, in more accurate words,
3D printed biomaterials, offer a wide range of possible scaffolding materials and
more unconstrained operating parameters, such as high temperature. However,
controlling the cell distribution and seeding density on prefabricated 3D scaffolds
is a very hard constraint, and the results are generally poor formations of extracel-
lular matrix (ECM) microenvironment.
The advantage of using various 3D printable materials to construct cell-free
scaffolds also makes this approach attractive to bone tissue engineering applications.
Table 2 Bioprinted cells and bioinks for bone tissue engineering
292
Fabrication Main outcomes about osteogenic

Bioinksa, b Cells method 3D printer potentialb
Alginate hydrogel (3% w/v in PBS); Mouse calvaria Syringe Fab@Home Model • Chitosan was superior to alginate
alginate-HAp (20 mg/mL) + CaCl2 (2% pre-osteoblast cell line extrusion 3, Seraph Robotics, • The deposition of 48 mg Ca ion per 1 g
v/v as cross-linker); chitosan hydrogel (MC3T3-E1) USA of chitosan – HAp hydrogel was detected
(2% w/v in acetic acid); and chitosan- on 21st day of culture
HAp (20 mg/mL) [53]
VEGF-conjugated (5% w/v) GelMA with Human umbilical vein Pneumatic- NovoGen MMX • The encapsulated hMSCs formed a
low methacryloyl substitution (in core) endothelial cells based Bioprinter, Organovo, mature bone niche after 21 days of cul-
and VEGF-conjugated (10% w/v) GelMA (HUVECs), human mesen- syringe USA ture under the medium perfused
with high methacryloyl substitution and chymal stem cells (hMSCs) extrusion condition
loaded with silicate nanoplatelets • Medium perfusion resulted in signifi-
(around) [54] cant increases in gene expression levels
of osteogenic differentiation markers
with respect to nonperfused construct
Alginate (50 mg/mL)-gelatin (50 mg/mL) Human primary osteogenic Pneumatic 3D-Bioplotter, • Ca2+-polyP complex caused the cells to
hydrogel + CaCl2 (0.4% w/v as cross- sarcoma (SaOS-2) cells syringe EnvisionTEC, proliferate faster with an average gener-
linking solution) cell-laden bioink and extrusion Germany ation time of approximately 47–55 h
agarose hydrogel (0.3% in growth during the 6 days incubation period
medium) with Ca2+-polyP complex • After 7 days of culturing, the cells
(100 μM) as overlay layer [55] exposed to the osteogenic medium and
Ca2+-polyP complex exhibited signifi-
cantly higher mineralization with respect
to the cells that were not exposed to
Ca2+-polyP complex but treated with the
osteogenic medium
GMs of two different methacrylation Primary human adipose- Volumetric TR300 tabletop robot, • The changes in rheological character-
degree (7 wt% and 5 wt%)-HAM derived stem cells (hASCs) dispensing Unitechnologies SA, istics are attributed to extensive matrix
(1 wt%)-HAp (5 wt%) in PBS + LAP (extrusion) Switzerland production in the hydrogels by the
(0.135 wt% of polymer mass as encapsulated cells
photoinitiator) [56] • The expression of bone matrix
B. Yilmaz et al.
components, such as collagen I, fibro-
nectin ALP, and osteopontin, was shown
to increase with the presence of HAp but
even more with the osteogenic media
supplements after 28 days of culture
PEGDA (20% w/v)-Laponite XLG Primary rat osteoblasts Two- BioScaffolder 3.1, • 7% nanoclay was selected as the opti-
nanoclay (3, 5, 7, 10% w/v) + Irgacure (ROBs) channel GeSiM, Germany mal concentration to add to the 20%
1173 (3 wt% relative to the mass of pneumatic PEGDA aqueous solutions
PEGDA cross-linker as photoinitiator); extrusion • The viability of the printed cells was
HA (20% w/v) in PBS [57] higher than 95%
• The release of Mg2+ and Si4+ bioactive
ions from the PEG-clay scaffolds stimu-
lated the osteogenic differentiation of
ROBs
• ROB-laden PEG-clay constructs
Bioprinting Technologies in Tissue Engineering
exhibited excellent osteogenic capability

both in vitro and in vivo
Alginate (3 w/v % in PBS)-MC powder Immortalized human mes- Pneumatic BioScaffolder 3.1, • Calcium phosphate cement and bioink
(9 w/v %) [58] enchymal stem cell line extrusion GeSiM, Germany biphasic constructs were printed
expressing human telome- • Initial and local decrease in cell viabil-
rase reverse transcriptase ity was detected at the interface of
(hTERT-MSC) calcium phosphate cements, and cells
started to migrate from the alginate-MC
bioink to the calcium phosphate cement
strands between day 7 and day 21
Type B gelatin (30%), alginate (5%), and Wharton’s jelly mesenchy- Pneumatic BioScaffolder 3.1, • The HUVEC-laden bioink and molten
Ca-free DMEM [59] mal stem cells (WJMSCs), syringe and GeSiM, Germany PDACS/PCL were dispensed alternately
human umbilical vein melt in each layer of structure, and WJMSCs
endothelial cells (HUVECs) extrusion were ejected by a piezoelectric nozzle on
PDACS/PCL layers
• Expressed ion levels of both angiogenic
markers (vWF and Ang-1) of HUVECs
293
increased at day 7
(continued)
Table 2 (continued)
294
Fabrication Main outcomes about osteogenic

Bioinksa, b Cells method 3D printer potentialb
• ALP, BSP, and OC protein expression
levels of WJMSCs were highest on the
PDASC/PCL with WJMSC and alginate-
gelatin hydrogels with HUVEC group
for 14 days
Matrigel (9 mg/mL in PBS) [60] Human adipose stem cells Pneumatic Custom-modified • ASCs suspended in Matrigel bioink
(ASCs) syringe Prusa I3 A Pro with were printed with PCL/bioactive borate
extrusion two additional syrin- glass composite in chloroform
ges, Geeetech, China • The live/dead assay showed 58 11%
viable ASCs on the scaffold after 1 week
of incubation
Alginate-PVA-HAp (2.5% w/v)-collagen Mouse calvaria Ball screw- System 30 3D printer • The constructs were unable to promote
(5.0–6.5% w/v) + CaCl2 (0.5 and 1.0% as pre-osteoblast cell line driven with a modified cell proliferation over 10 days
cross-linker) [61] (MC3T3-E1) extrusion EMO-25 extruder, • Further in vitro differentiation and
HyRel 3D, USA in vivo studies are needed
a
Different cell-laden bioink formulations are separated with semicolons (;)
b
PBS phosphate-buffered saline, HAp hydroxyapatite, VEGF vascular endothelial growth factor, GelMA/GM gelatin methacryloyl/gelatin methacrylate, polyP
poly(phosphate), PVA poly(vinyl alcohol), HAM methacrylated hyaluronic acid, LAP lithium phenyl-2,4,6-trimethylbenzoylphosphinate, ALP alkaline
phosphatase, PEGDA poly(ethylene glycol diacrylate), Laponite XLG ([Mg5.34Li0.66Si8O20(OH)4]Na0.66, HA hyaluronic acid sodium salt, MC methylcellulose,
DMEM Dulbecco’s modified eagle medium, PDACS polydopamine-modified calcium silicate, BSP bone sialoprotein, OC osteocalcin, β-TCP β-tricalcium
phosphate
B. Yilmaz et al.
There are various polymers that were 3D printed with CaPs or other biomaterials to
form bone tissue engineering scaffolds. For example, poly(ε-caprolactone) (PCL)
and (1) Ca2+-poly(phosphate) microparticles (at a ratio of 2:1 w/w) [63] and
(2) silanated silica particles (5, 10, 20 wt%) [64] were printed by pneumatic melt
extrusion. Poly(lactic acid) (10 wt%) was previously printed with β-TCP (5 wt%)
with four different hydrogels by syringe extrusion method [65]. Natural polymers,
such as chitosan, are also of interest of bone tissue engineering area, for example,
nano-sized HAp-chitosan-silica (15/50/35 molar ratio, respectively) and chitosan-
silica (50/35 molar ratio, respectively) were previously printed by using pneumatic
syringe extrusion [66]. Several forms of coating were also applied on 3D printed
bone scaffolds. HAp/β-TCP (at a mass ratio of 60/40) (67% w/w) and 20 wt%
Pluronic F-127 solution (33% w/w) was previously used as 3D printed biomaterial,
and RGD (Arg-Gly-Asp)-phage nanofibers (1014 pfu/mL) and chitosan (1 mg/mL)
were used as coating material [67].
4.2 Osteochondral and Cartilage
Osteochondral tissue is composed of hyaline cartilage and subchondral bone and is

found at synovial fluid joints [68]. The main responsibility of hyaline cartilage is to
decrease surface friction and act as a shock absorber on the joint during daily
activity, while the subchondral bone tolerates load and supports hyaline cartilage
[68]. Osteochondral tissue can be degenerated through tumor, aging, or disease that
can result in poor life quality. The vascular structure of articular cartilage has limited
self-reparability, so the presence of defect in articular cartilage can progress to the
underlying subchondral bone creating osteochondral defects.
Osteochondral tissue has a hierarchical and organizational structure which is a big
challenge in designing of scaffold for osteochondral defects [69]. Articular cartilage
is composed of four different zones which, respectively, are the calcified, deep,
middle, and superficial zones [70]. These zones have different extracellular matrix
composition, collagen orientation, and chondrocyte phenotype [70]. The orientation
of collagen fibers is changed in these zones as follows: (a) parallel alignment to the
articular surface (superficial zone), (b) random alignment in the middle zone, and
(c) perpendicular form to the articular surface (deep zone); the calcified zone has
collagen X [70]. The orientation of collagen fibers has a key role in mechanical
properties of articular cartilage.
The surgical treatments are usually required in osteochondral defects [68].
There are clinical treatments such as debridement, microfracture, autologous chon-
drocyte implantation, matrix-induced autologous chondrocyte implantation, and
osteochondral autografting and allografting, but these are effective in early stages
and only postpone tissue degeneration. All of these are complicated and costly
treatments and did not result in proper biomechanical restoration [71].
Table 3 The list of commercial tissue-engineered cartilage scaffolds

Product name Company Biomaterial Cell
BioCart™II Histogenics, Waltham, Fibrinogen/ Chondrocytes
MA hyaluronic acid
BioSeed®-C BioTissue Technologies Polyglactin Fibrin and expanded
GmbH, Freiburg, 910/poly-p- autologous
Germany dioxanone fleece chondrocytes
Cartipatch® Tissue Bank of France, Agarose-alginate Chondrocytes
Lyon, France hydrogel
Chondrosphere® Co.don AG, Teltow, Spheroids of Chondrocytes
Germany neocartilage
Hyalograft® C Anika therapeutics, Hyaluronic acid Chondrocytes
Bedford, MA
Matrix-induced autolo- MACI, Vericel, Collagen II/III Chondrocytes
gous chondrocyte Cambridge, MA
implantation
NeoCart® Histogenics, Waltham, Bovine collagen I Chondrocytes
MA
NOVOCART® 3D TETEC, Melsungen, Biphasic Chondrocytes
Germany collagen I
There are different tissue-engineered cartilage products which are under clinical
trials to overcome limitations of current treatments. Table 3 reviews the list of
cartilage products [71].
Recently, there are significant research in tissue engineering approach for
osteochondral defect treatment and cartilage scaffold design [68]. The architecture
of scaffold plays an important role in vascularization, mechanical properties, cell
proliferation, and infiltration. The 3D printing technology is widely used for printing
of osteochondral and cartilage scaffolds due to its ability to create complex structures
and control pore size (Table 4).
4.3 Nerve
In the treatment of peripheral nerve and spine injuries, neural conduits have been
proposed as an effective neural regeneration matrix that support and guide
neural cells using tissue engineering approaches. Neural tissue harbors neurons
and glial cells (microglia, astrocytes, oligodendrocytes), while the vascular compo-
nent consists of endothelial cells, pericytes, and vascular smooth muscle cells
[83]. Bioprinting offers a relatively recent approach for engineering of controllable
3D scaffolds for neural tissues with diverse cell types and complex microscale
features [28]. The major points for the design of nerve guide conduits are (1) mechan-
ical support while aligning the proximal and distal nerve ends and prevent nerve
compression, (2) permeability to nutrients and waste products through the conduit
Table 4 Bioprinted biomaterials and cells for osteochondral and cartilage
Fabrication
Biomaterial Cells method 3D printer Main outcome
15% methacrylated gelatin (GelMA) Bone marrow mesen- Pneumatic Customized, pneu- The in vivo results showed that GelMa/
hydrogel for cartilage on top layer, a chymal stem cells dispensing matic dispensing nHA-based tri-layered scaffold is useful
combination of 20% GelMA and 3% system in repairing of hyaline cartilage and
nanohydroxyapatite (nHA) (20/3% subchondral bone
GelMA/nHA) hydrogel for interfacial
layer, a 30/3% GelMA/nHA hydrogel for
subchondral bone at bottom layer [68]
L2C4S4 apatite (1.8 g) + sodium alginate Rabbit chondrocytes, Three-axis posi- 3D scaffold printer The L2C4S4 extracts promote osteo-
(0.1 g) + Pluronic F-127 (1.8 g) [72] rabbit bone marrow tioning system genic differentiation and bioactivity
stem cells
HA + polycaprolactone (in different – Extrusion printing 3D-Bioplotter; The porosity is more effective than
ratios) [73] EnvisionTEC, Glad- addition of HA in terms of mechanical
beck, Germany strain

PLGA + 1% ß-TCP 3D printed as a Mesenchymal stem cell Pneumatic 3D biological printer The advantages of this system include
subchondral part and articular cartilage from the femoral mar- printing high cell utility efficiency, easy pro-
part was prepared from fresh bovine limb row cavity duction, and precise tissue repair, which
by freeze-drying [74] could provide a promising alternative to
current approaches to repair joints
defects
PLLA filament used for 3D print- Murine fibroblasts L929 Fused deposition – Gelatin concentration has significant
ing + 6% gelatin membrane prepared by (ATCC, USA) modeling effect on mechanical strength and also
electrospinning + Osteogenon drug [75] 1.5% of Osteogenon promote mechani-
cal properties of scaffold
PCL + 20% GelMa + 1:1 GelMa/cell Mesenchymal stem Microextrusion 3D discovery, The reinforced polymeric framework by
suspension [76] cell + chondrocyte and inkjet printing RegenHU, printing of spheroids and the collagen
Switzerland natural structure was mimicked
(continued)
297
Table 4 (continued)
298
Fabrication
Biomaterial Cells method 3D printer Main outcome
LCS (Li2Ca2Si2O7) powders, sodium Rabbit chondrocytes Pneumatic – The in vitro studies proved that release
alginate + LCS powder (5:100) added to printing of Li and Si augmented osteogenic dif-
20% F-127 solution [77] ferentiation of rabbit chondrocytes
HAMA-GelMa + photoinitiator as a shell Allogeneic adipose- Core-shell extru- BioPen (custom- The pilot study proved that real-time 3D
bioink derived mesenchymal sion printing made) printing promotes cartilage regeneration
HAMA-GelMa + cells as a core stem cells The shell thickness should be enough to
bioink [78] protect cells from UV light
Nanocellulose + alginate [79] IPSC Microextrusion 3D discovery, The nanocellulose+ alginate bioink is
printing RegenHU, suitable for cartilage tissue; also cell
Switzerland density in bioink plays important role in
cartilage regeneration
Gelatin methacrylate (GelMa) [80] – Pneumatic 3D-Bioplotter; The architecture of scaffold has an
printing EnvisionTEC, important role in mechanical properties
Germany
Nanocellulose/alginate bioink + cells Human nasal – – The new cell/bioink mixing system
(10:1) [81] chondrocytes (passive mixing) was used and proved
that this system increase cell viability in
comparison with other conventional
mixing
10% GelMA + different concentrations Human bone marrow Stereolithography- Custom-made The stereolithography-based 3D print-
of polyethylene glycol mesenchymal stem cell based 3D printing ing system promotes cell viability and
diacrylate + photoinitiator + TGFβ1 [82] protects growth factors after printing
B. Yilmaz et al.
wall, (3) low immunogenicity, and (4) biodegradability to eliminate the need for
secondary surgery [84].
Among the printing techniques used in neural conduit construction, extrusion
printing has been used frequently as gels can be deposited conveniently with high
speed. However, laser-based bioprinting, such as stereolithography and inkjet print-
ing, allows better print resolution, which is a significant advantage in mimicking
anisotropic architecture of nerve tissue. The selection of bioink has a tremendous
effect on neural regeneration as neural cells are relatively more sensitive to their
extracellular milieu; hence neural tissue applications should mimic the natural ECM
of the target tissue [85]. Table 5 summarizes recent studies that reported application
of bioinks in neural conduit bioprinting.
Alginate, a seaweed polysaccharide, can form stable hydrogels upon ionic
cross-link with bivalent ions like Ca+2 and Mg+2. In a recent study, Naghieh et al.
constructed alginate scaffolds at low concentration to enable effective neural
network formation by using indirect bioprinting technique [86]. After a sacrificial
gelatin framework was printed by extrusion-based device, a low-concentration
alginate incorporating primary rat Schwann cells (PRSCs) was casted into the
framework. Later, the gelatin framework was removed, and low-concentration
alginate scaffold was exposed. However, although low alginate provided favorable
conditions for cells, it was not mechanically stable, and cell viability decreased
with time.
Recent studies showed that the cell compatibility of alginate bioink would be
increased by using cell adhesion factors. In one of such studies, Sarker et al. tested
the potential of RGD or YIGSR peptide in increasing cytocompatibility of alginate
in bioprinting application [87]. An extrusion-based system was used to bioprint
RGD, YIGSR, or both peptide-conjugated sodium alginate with Schwann cells into
3D scaffolds. After 9 days of culture, the printed hydrogel constructs contained more
cells and supported long-term cell proliferation compared to 2D culture conditions
(petri dish) with respect to control alginate construct. Furthermore, the conjugation
of both RGD and YIGSR peptides in the same alginate molecules increased the
proliferation of neural cells in bioprinted constructs significantly. To increase algi-
nate biocompatibility, fibrin, hyaluronic acid, and RGD peptide were mixed [88]. A
3D plotter was used to construct a grid-type scaffold by extruding the blended
biopolymer solution mixed with Schwann cells, which was later stabilized with a
cross-linking solution containing CaCl2 and thrombin that cross-link alginate and
fibroin, respectively. As indicated by in vitro techniques, cells were able to sustain
viability and proliferation in the scaffolds effectively. Similarly, the composite
technique was used by Li et al. to increase the cell compatibility of alginate by
using gelatin [89]. With the use of a bioplotter, Schwann cells were co-extruded in
alginate-gelatin into a grid-type scaffold structure. In vitro culture of the printed
hydrogel scaffolds showed that more cells were present in the scaffolds and the
composite gel support long-term cell proliferation compared to 2D culture (petri
dish) conditions.
Hyaluronate, a highly hydrophilic polysaccharide, is also known to not support
cells like alginate. England et al. suggested the use of fibrin-factor XIII in
Table 5 The bioinks and bioprinting methods used in neural conduit construction
Bioink Fabrication method Neural regeneration outcome
Alginate [86] An indirect bioprinting pro- Decreased PRSCs viability
cess of alginate with primary with culture time. The scaf-
rat Schwann cells (PRSCs) fold with low alginate con-
centration showed mechanical
unstability during culture
Alginate conjugated with RGD 3D gel extrusion of alginate Cell viability was over 90%
or YIGSR peptide [87] peptides with Schwann cells after 1 week for RGD and
YIGSR-gel composites, while
lower viability was observed
without peptides
Composite hydrogels of algi- Extrusion of gel with Schwann Cells were able to sustain
nate, fibrin, hyaluronic acid, cell using 3D-Bioplotter viability and proliferate in the
and/or RGD peptide [88] scaffolds
Composite alginate-gelatin Rat Schwann cell in composite The printed hydrogel con-
hydrogel [89] gel was printed with an structs contained more cells
extrusion-based bioprinter and support long-term cell
proliferation (after day 9)
Fibrin-factor XIII-hyaluronate 3D gel extrusion with a Schwann cells encapsulated
hydrogel [90] bioplotter within these scaffolds during
fabrication were viable and
proliferated in culture. Extru-
sion induced longitudinal
alignment of fine fibrin fibers
which in turn enabled the
alignment of encapsulated
Schwann cells and dorsal root
ganglion neurites along the
3D printed strands
(a) Gelatin methacrylate Extrusion-based multi- (a) The printed cells did not
(GelMA) and gelatin mixed material 3D bioprinting with proliferate, and axon propa-
with fibrin (GEL/FIB) iPSC-derived neural progeni- gation was not observed in the
hydrogels tor cells for bioengineering of matrix
(b) Commercial extracellular spinal cord (b) When printed in 50%
matrix (Matrigel) [91] Matrigel concentration as the
cell-laden bioink, the overall
cell viability over 4 days in
culture was >75% for both
iPSC-derived sNPCs and oli-
godendrocyte progenitor cells
(OPCs)
Water-based biodegradable 3D dispersion extrusion with Murine neural stem cells
polyurethane dispersions fused deposition manufactur- (NSC) which was extruded in
which may later form gel at ing equipment hydrogels would proliferate
mild conditions [92] and differentiate. NSC-laden
hydrogels could reestablish
the function of impaired ner-
vous system in the zebrafish
embryo
(continued)
Table 5 (continued)
Bioink Fabrication method Neural regeneration outcome
GelMA, PEGDA [93] Engineered nerve guidance Complete sciatic nerve tran-
conduit using digital light sections of mouse models
processing (DLP)-based rapid demonstrated directional
continuous 3D-printing guidance of regenerating sci-
platform atic nerves via branching into
the microchannels and
extended toward the distal end
of the injury site
hyaluronate gel precursor for the extrusion of Schwann cells [90]. A bioplotter
extruded hyaluronan-fibrin with Schwann cells into a 3D structure by stacking of
longitudinal strands. Schwann cells encapsulated within these scaffolds during
fabrication were seen viable and proliferated in culture. Interestingly, extrusion-
induced longitudinal alignment of fine fibrin fibers in gel enabled the alignment of
encapsulated Schwann cells and dorsal root ganglion neurites along the 3D printed
strands.
GelMA gels, on the other hand, were proven to be not effective in keeping the
viability and proliferation of induced pluripotent stem cell-derived neural progenitor
cells, which are known to depend on extracellular matrix [91]. Either GelMA or
gelatin blended with fibrin gels was bioprinted via extrusion-based plotter for
construction of scaffold, intended for spinal cord regeneration. In vitro tests showed
that the printed cells did not proliferate, and axon propagation was not observed in
the matrix. Therefore, in the same study, Matrigel, which is composed of growth
factors and proteins that mimic the basement membrane and extracellular matrix
from mouse sarcoma cells, was used in bioprinting of neural progenitors [91]. The
results showed that after extrusion plotting of cell-laden bioink, which was prepared
at 50% Matrigel concentration, the overall cell viability over 4 days culture was
>75% for both iPSC-derived sNPCs and oligodendrocyte progenitor cells (OPCs).
Water-based biodegradable polyurethane dispersions were used in bioprinting
of murine neural stem cells (NSCs) which may later form gel at mild conditions
[92]. NSCs were embedded into the polyurethane nanoparticle dispersions before
gelation and extruded by using fused deposition manufacturing equipment. In vitro
results reported that NSCs could proliferate and differentiate and hence showed the
mild effect of bioprinting of cells with PU bioink. In addition, NSC-laden hydrogels
that were injected into the zebrafish embryo neural injury model could reestablish the
function of impaired nervous system.
Hydrogels based on collagen and gelatin have been proposed as an efficient
bioink for neural regeneration. Due to insolubility of collagen at neutral pH,
gelatin, which is a shorter-chain, denatured collagen product, is preferred candidate
with high water solubility at physiologic pH. Gelatin itself has a random coil
structure in solution and forms a helix below 35 C where helix-chain aggregation
causes gelation [94]. Especially, the acrylated gelatin, gelatin methacryloyl
(GelMA), has found particular interest as viable cells could be encapsulated via
photopolymerization during bioprinting process. Tao et al. attempted bioprinting

of GelMA with poly(ethylene glycol)-poly(3-caprolactone) (MPEG-PCH) nano-
particles which encapsulated Hippo pathway inhibitor that can block MST1/2 kinase
activity [95]. The bioprinted nerve conduit-drug construct induced higher rate of
recovery of sciatic injuries with respect to morphology, histopathology, and func-
tions in in vivo rat sciatic nerve model. In a similar approach, dopamine (DA) was
conjugated to GelMA molecules and used in bioprinting of a scaffold for culturing of
neural stem cells (NSCs) [96]. A custom-made stereolithography was used in
bioprinting of GelMA-DA, and UV laser beam was effective in gelation at 25 μJ
intensity. The observation of a significant neural network on the 3D printed GelMA-
DA scaffolds after 12 days of culture indicated a stimulatory effect of conjugated
dopamine on neural progenitor cell when compared to unconjugated GelMA
scaffolds. In another study, GelMA was blended with poly(ethylene glycol
diacrylate) (PEGDA) for the engineering of nerve conduit [93]. A digital light
processing (DLP) apparatus, which could generate the desired patterns, was
adapted to the laser beam processing in a rapid continuous 3D-printing platform
for engineering of nerve guidance conduit. The implantation of the conduits with
microchannels and sleeve design into mouse models of complete sciatic nerve
transections demonstrated directional guidance of regenerating sciatic nerves via
branching into the microchannels and extension toward the distal end of the
injury site.
4.4 Skin
The skin being the outermost organ is susceptible to injuries, infections, and
environmental effects. Skin tissue is composed of the upper epidermis and inner
dermal layers with their respective cells of keratinocytes and fibroblasts. Tissue
engineering of skin grafts is a lifesaving approach for patients having major
injuries, burns, and skin diseases. The membrane preparation techniques, such as
electrospinning, freeze-drying, and solvent casting, have long been used for skin
graft constructions conveniently. Bioprinting of the skin attracted ever-growing
interest due to its sophisticated and controlled production properties which would
be rather difficult with conventional skin graft production methods.
Vijayavenkataraman et al. pointed out commercial initiatives and collaborations
among cosmetic companies (L’Oréal USA, NovoGen, Procter & Gamble) and
bioprinter producers (Organovo, Rokit) mainly aimed at fulfilling the huge demand
in production of skin products to test cosmetic products [97]. The potentials of 3D
bioprinting technique for skin tissue engineering have been investigated recently by
several researchers (see Table 6). Bioprinting facilitates the specific deposition of
multiple types of skin cells simultaneously [105]. In addition, 3D bioprinting enables
the precise localization of multiple cell types and appendages within a construct
[106]. However, there are challenges that limit skin bioprinting, such as the technical
difficulties associated with nozzle blockage and shearing stresses on the cells
Table 6 Bioprinted skin scaffolds with various bioinks and cell sources
Bioink Fabrication method Skin regeneration outcome
Sodium alginate [98] Extrusion by 3D plotting Bioprinted patches were pro-
duced according to patient’s
wound size and contour
Type I collagen [99] Pneumatic extrusion by a 3D The bioprinted skin structure
plotter showed the dermal and epi-
dermal layers as well as the
terminal differentiation of the
KC that formed the stratum
corneum. MC-containing epi-
dermal layer showed freckle-
like pigmentations at the
dermal-epidermal junction
Collagen [100] The 3D bioprinted constructs Skin constructs had a higher
were fabricated using a degree of resemblance to
bioplotter with multiple native skin tissue in terms of
microvalve-based printheads the presence of well-
in two separate steps: developed stratified epidermal
1. Bioprinting of collagen layers and the presence of a
fibroblast matrices continuous layer of basement
2. Keratinocytes and melano- membrane proteins as com-
cytes were directly printed pared to the manually cast
onto the bioprinted collagen samples
fibroblast matrices
Bovine fibrinogen- 3D cell spraying with a Bioprinted layered human
thrombin [101] bioplotter dermal fibroblasts and epider-
mal keratinocytes in a hydro-
gel showed rapid wound
closure, reduced contraction,
and accelerated
reepithelialization in nude
mice model
Adipose-derived ECM mixed The simultaneous inkjet and Perfusable channels could
with bovine fibrinogen mixture extrusion 3D printing supply nutrients throughout
for hypodermal compartment. the dECM-based construct.
Skin-derived ECM (dECM) Printed HUVEC could cover
and fibrinogen mixture for the surface of channel,
dermal compartment. Gelatin forming endothelium
for vascular channels [102]
Cells embedded in collagen gel Cell lines of fibroblasts and Printed fibroblasts and
or a mixture of blood plasma keratinocytes embedded in keratinocytes proliferated and
and alginate [103] collagen were printed in 3D as were vital. Extensive forma-
a simple example for skin tion of intercellular adherens
tissue. On the principle of junctions between
laser-induced forward transfer keratinocytes and their minor
formation between fibro-
blasts, which proves the tissue
formation, were observed
(continued)
Table 6 (continued)
Bioink Fabrication method Skin regeneration outcome
Amniotic fluid-derived stem Pneumatic-driven and Bioprinted constructs of col-
(AFS) cells and mesenchymal extrusion-based 3D hydrogel lagen/fibrin gel with AFS or
stem cells (MSCs) were sepa- solutions, with or without cells MSC cells had faster wound
rately suspended in the fibrin- closure rate than the gels
ogen/collagen solution [104] without cells in vivo. High
vessel formation per unit area
was detected for both kinds of
cells as compared to con-
structs without cells in vivo
[107]. Patient-specific construction of scaffolds is one of the most important advan-

tages of 3D printing technology. There is one recent study that investigated
segmenting chronic wounds and transmitting the coordinates to a bioprinter robot
to facilitate the treatment of chronic wounds [98]. As a result, semiautomatic
segmentation of wound images and image processing methods improved the control
of bioprinting process through more accurate coordinates.
For increased vascularization and efficient epidermal growth, a perfusable,
vascularized full-thickness skin equivalent composed of epidermis, dermis, and
hypodermis was investigated by Kim et al. [102]. It was observed that the printed
HUVECs were covered on the surface of vascular channel, forming endothelium
resembling vascular structures. The results showed that keratin 10 and filaggrin were
expressed at the early and late stages of differentiation of the epidermis. Within the
dermal compartment, laminin and dermal ECMs were expressed at the boundary
between the dermis and epidermis similar to natural process.
In a different approach, Koch et al. deposited 20 layers of fibroblasts (mouse
NIH-3T3) and 20 layers of keratinocytes (human HaCaT) embedded in collagen
gel onto a sheet of MatriDerm® (decellularized dermal matrix) by laser-assisted
bioprinting for constructing dermis and epidermis layers, respectively [103]. The
printed constructs could show the presence of intercellular junctions, such as
cadherins and connexin 43 (Cx43), in the epidermis after 10 days of cultivation in
culture.
In addition, bioprinting of pigmented skin was tested to obtain a better resem-
blance to native skin [100]. Primary human keratinocytes, melanocytes, and fibro-
blasts were used in bioprinting process, while a two-step drop-on-demand
bioprinting strategy was used to deposit the cell droplets to form the melanin units
of epidermal layer. As a result, 3D bioprinted skin constructs had a better resem-
blance to native skin as the printed tissue had well-developed stratified epidermal
layers and a continuous layer of basement membrane proteins as compared to the
manually cast samples.
Skin regeneration potential of amniotic fluid-derived stem (AFS) cells and bone
marrow-derived mesenchymal stem cells (MSCs) was compared by suspending cells
in fibrin-collagen gel and printing over the wound site [104]. A bioprinter with
pneumatic-driven nozzles was used to deposit layers of fibrin-collagen and thrombin
gel solutions containing AFS cells or MSCs deposited over full-thickness wounds,
which were created in nude mice models. In vivo tests showed that the constructs of
collagen/fibrin gel with AFS cells accelerated closure of full-thickness wounds faster
than the construct without cells, and they were as effective as the gels with MSC. In
addition, the vessel formation per unit area, which is a significant indication of
regeneration, was significantly higher with both kinds of cells than only-gel printed
grafts as the histology staining revealed.
As a conclusion, the bioprinting technology has enabled controlled fabrication
platform for construction of artificial skin, which potential has yet to be realized in
skin tissue engineering [108]. However, the resolution, vascularity, optimal cell and
scaffold combinations, and cost of bioprinted skin are some issues that should be
overcome to harness the new technology [109]. Histological complexity of the skin,
such as neural and immuno-components and hair follicles, should also be considered
in bioprinting of the skin to obtain fully functional native skin [108, 110].
4.5 Cardiac and Skeletal Muscle
Irreversible loss of cardiomyocytes due to ischemic heart attack leads to lethal heart
diseases and high mortality rates. Therefore, the regeneration of damaged cardiac
muscle is the only way to restore heart functions. Cellular cardiomyoplasty is the
implantation of in vitro cultured cardiomyocytes into the damaged myocardium, to
facilitate regeneration [111]. However, when injected to the heart directly, low cell
retention and viability of cardiomyocytes are encountered. Tissue engineering of
heart muscle by using bioprinting is carrying significant potential as the scaffold
matrix architecture would be customized while cardiac cells are placed in situ. In this
part of the chapter, the skeletal tissue engineering by bioprinting approach is also
discussed. Table 7 summarizes recent applications of bioprinting in cardiac and
skeletal tissue engineering.
Adams et al. studied bioprinting of silicone rubber and poly(caprolactone) (PCL),
which are known as printable and biocompatible materials, into scaffolds for cardiac
patch construction [111]. A grid-type scaffold was 3D printed with pneumatic
syringe extrusion from silicone rubber or PCL bioink. In vitro tests showed that
PCL scaffolds had more efficacy as there was a higher percentage of adult primary
human cardiomyocyte (pHCM) attachment and more cell migration, compared to the
silicone rubber. In addition, an increase in cell density of cardiac cells was observed
on the PCL scaffold after electrical stimulation.
Laser-induced forward transfer (LIFT) cell printing technique performs con-
trolled transfer of inorganic and biological materials in conjunction with proteins,
peptides, DNA, RNA, and cells in three-dimensional (3D) patterns with survival
rates of printed cells at nearly 100% [112]. Poly(ester urethane urea) (PEUU) cardiac
patch immersed in Matrigel was used to collect patterned cells from laser beam
focused on the donor slide. Human mesenchymal stem cells (hMSCs) and endothe-
lial cells (EC) were patterned in such a way that the rectangular islands of hMSC
Table 7 Bioprinted muscle scaffolds with various bioinks and cell sources
Bioink Fabrication method Muscle regeneration outcome
Silicone rubber and poly 3D bioprinter with pneumatic PCL scaffolds showed more
(caprolactone) (PCL) [111] syringe extrusion was used in efficacy as there was a higher
the deposition of silicone rub- percentage of adult primary
ber or PCL in a grid structure human cardiomyocyte
(pHCM)) attachment and
more cell migration, com-
pared to the silicone rubber.
Electrical stimulation
increased cell density on the
PCL scaffold
Poly(ester urethane urea) Laser-induced forward transfer Increased vessel formation
(PEUU) cardiac patch (LIFT) cell (human mesen- and found significant func-
immersed in Matrigel was chymal stem cells) printing tional improvement of
used to collect patterned technique infarcted hearts following
cells [112] transplantation of a LIFT
tissue-engineered cardiac
patch in immune-deficient rat
model
Carbon nanotubes (CNT)- A UV-integrated pneumatic Human coronary artery endo-
incorporated alginate and 3D-Bioplotter system was thelial cells in MeCol gel
methacrylated collagen employed to create hybrid cell- presented significant cellular
(MeCol) [113] laden MeCol. Alginate was proliferation, migration, and
printed as the implant frame- differentiation (lumen-like
work to contain cell-laden formation) over 10 days of
MeCol within its spaces in incubation in vitro
each printed layer
Heart tissue-derived Human cardiac progenitor MixC/M (with VEFGs and
decellularized extracellular cells (hCPCs) or endothelial MSCs) and patternC/M (gen-
matrix (hdECM) bioink [114] cells were extruded in a disk- erated patches by patterning
shape structures (8 mm in of CPCs and MSCs with
diameter and 0.5 mm in thick- VEGFs) greatly promoted
ness) by a bioplotter vascularization compared
with the CPC patch after
4 weeks implantation; the
patterned patch exhibited
enhanced cardiac functions,
reduced cardiac hypertrophy
and fibrosis, increased migra-
tion from patch to the infarct
area, and neo-muscle and
capillary formation in rat
myocardial infarction model
Gelatin methacrylate Human embryonic stem cell- A contractable cardiac tissue
(GelMA) [115] derived cardiomyocytes was obtained. hESC-CMs
(hESC-CMs) were mixed with printed in isotropic slabs beat
GelMA printed into a scaffold synchronously; however they
by using micro-continuous contract without directional
optical printing (μCOP) preference, whereas hESC-
CMs encapsulated in the par-
allel line pattern contract in
the direction of patterning
(continued)
Table 7 (continued)
Bioink Fabrication method Muscle regeneration outcome
No bioink was used [116] Human-induced pluripotent Patches exhibited ventricular-
stem cell-derived like action potential wave-
cardiomyocytes (hiPSC-CMs), forms and uniform electrical
fibroblasts (FB), and endothe- conduction throughout the
lial cells (EC) were aggregated patch. In vivo implantations
to create mixed cell spheroids. of assembled cardiac patch
The 3D bioprinter picks up showed vascularization and
individual cardiospheres using engraftment of 3D bioprinted
vacuum suction and loads cardiac patches
them onto a needle array
Fibrin-based (containing Primary cardiomyocytes Dense, uniformly aligned,
gelatin and hyaluronic acid) suspended in a fibrin-based and electromechanically
bioink [117] bioink and cell-laden hydrogel coupled cardiac cells were
were sequentially printed with observed after 3 weeks of
a sacrificial hydrogel and a culture
supporting polymeric frame by
using 3D pneumatic printing
The decellularized skeletal Human skeletal muscle cells Improved cell viability,
muscle extracellular matrix (hSKMs)-encapsulated bioink myotube formation, and de
(dECM) and vascular dECM solution was 3D extruded into novo myofiber regeneration
(vdECM) bioinks [118] 3D scaffold by a plotter. in rat models of volumetric
Prevascularized muscle con- muscle loss (VML).
structs were fabricated through Improved de novo muscle
coaxial nozzle printing with fiber formation and vasculari-
dECM and vdECM bioinks zation were observed in rats
PEG-fibrinogen and Microfluidic printing head An enhanced myogenic
alginate [119] with coaxial needle was used differentiation with the for-
to extrude muscle precursor mation of parallel aligned,
cells (C2C12) into 3D con- long-range, tightly packed,
structs composed of aligned and completely striated
hydrogel fibers myotubes
were surrounded by the EC lanes, mimicking vascular tissue model on a PEUU

cardiac patch that was immersed in Matrigel. The therapeutic potential of the patch
in cardiac regeneration was tested in immune-deficient Rowett nude rat left anterior
descending ligation model with respect to cell survival, infarct wall thickness,
angiogenesis, cardiac remodeling, and functional improvement. Increased vessel
formation and significant functional improvement of infarcted hearts were observed
in in vivo results.
Carbon nanotubes (CNT) were incorporated into alginate and methacrylated
collagen (MeCol) bioink for building cardiac patch with electrical, mechanical
attributes [113]. A UV-integrated pneumatic 3D-Bioplotter system was used to
construct human coronary artery endothelial cells (HCAECs) encapsulated in
MeCol. An indirect bioprinting technique was used for MeCol bioink. Alginate
was printed as the implant framework in order to hold cell-laden MeCol within its
spaces in each printed layer. As a result, HCAECs in MeCol gel presented significant
cellular proliferation, migration, and differentiation (lumen-like formation) over

10 days of incubation in in vitro cell culture.
Jang et al. tested heart tissue-derived decellularized extracellular matrix (hdECM)
bioink to encapsulate and print human cardiac progenitor cells (hCPCs) [114].
Human cardiac progenitor cells (hCPCs) or endothelial cells were extruded in a
disk-shape structures (8 mm in diameter and 0.5 mm in thickness) by using a
bioplotter. Three patch types were constructed as: (1) only CPCs (CPC), (2) ran-
domly mixed of both CPCs and human turbinate tissue-derived mesenchymal stem
cells (MSCs) with vascular endothelial growth factors (VEGFs) (mixC/M), or
(3) generated patches by patterning of CPCs and MSCs with VEGFs alternatively
(patternC/M). Neovascularization and tissue formation potential of cell-laden con-
structs were studied in Balb/c nude mice model, while hdECM patch without cells
was used at the epicardium of the rat myocardial infarct (MI) model to study the
functional benefits of hdECM. The tests of vascularization and tissue formation of
stem cell patch in vivo showed that mixC/M and patternC/M greatly promoted
vascularization compared with the CPC patch after 4 weeks implantation. The
mixC/M and patternC/M patches both seemed to induce a potent angiogenic
response in the host tissues: blood vessels developed in the implanted patches and
appeared to be functional vessels because red blood cells could be observed in the
lumens. The cardiac functions in rat myocardial infarction model revealed that the
patterned patch could exhibit enhanced cardiac functions, reduced cardiac hypertro-
phy and fibrosis, increased migration from patch to the infarct area, and neo-muscle
and capillary formation.
In another study, gelatin methacrylate (GelMA) bioink was used in bioprinting of
human embryonic stem cell-derived cardiomyocytes (hESC-CMs) [115]. With the
aim of cardiac patch construction, hESC-CMs were mixed GelMA and bioprinted by
using Micro-continuous optical printing (μCOP) platform. μCOP system consists of
programmable digital masks to control millions of individual mirrors to pattern light
onto a liquid volume. The digital patterns are casted on photopolymerizable polymer
on a high-precision stage, which is computer controlled, to create 3D scaffolds.
hESC-CMs were mixed with GelMA and polymerized into micropatterned (parallel
lines) cardiac patch. After 3–7 days of cell culture, a contractable cardiac tissue was
obtained. hESC-CMs printed in isotropic slabs beat synchronously; however they
contract without directional preference, whereas hESC-CMs encapsulated in the
parallel lines pattern contract in the direction of patterning.
In a different approach, a vacuum-assisted bioprinter device, which picks up
individual cardiospheres using vacuum suction and loads them onto a needle
array, could be used to construct a cardiac patch without using any bioink material
[116]. Cardiospheres that were produced by cell aggregation of human-induced
pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs), fibroblast (FB), and
endothelial cells (EC) were assembled into a cardiac patch by pickup function of
dispenser needles of the bioprinter. Electrophysiological results indicated that
patches exhibited ventricular-like action potential waveforms and uniform electrical
conduction throughout the patch.
Wang et al. tested fibrin-based (containing gelatin and hyaluronic acid) bioink for
primary cardiomyocytes (infant rat hearts) by co-printing with sacrificial gel and
supportive frames [117]. Three dispensing modules for cell-laden hydrogel, sacrifi-
cial hydrogel, and PCL were operated separately. Primary cardiomyocytes
suspended in a fibrin-based bioink and cell-laden hydrogel were bioprinted along
with supporting frame (PCL) and a sacrificial hydrogel by using 3D pneumatic
printing. Bioprinted cardiac tissue constructs had a spontaneous synchronous con-
traction in culture medium. Progressive cardiac tissue development was confirmed
by immunostaining for actinin and a calcium influx spreading across the entire tissue
after 1 week of culture. After 3 weeks, cardiac tissues were formed with uniformly
aligned, dense, and electromechanically coupled cardiac cells.
In skeletal tissue engineering, Choi et al. used decellularized skeletal muscle
extracellular matrix (dECM) and vascular dECM (vdECM) as bioinks for printing
vascularized human skeletal muscle cells [118]. hSKMs were mixed in dECM while
endothels (HUVECs) were mixed in vdECM bioinks for coaxial bioprinting of them
into thick constructs by using extrusion-based 3D plotting system. The 3D muscle
constructs showed enhanced cell viability, myotube formation, and de novo
myofiber regeneration for rat models of volumetric muscle loss (VML). In vivo
results showed that coaxial nozzle printing mimicked the hierarchical architecture of
vascularized muscles, and allogenic human cells in the constructs improved de novo
muscle fiber formation, vascularization, and innervation, as well as 85% of func-
tional recovery could be observed in VML injury. In another muscle bioprinting
trial, Constantini et al. used PEG-fibrinogen- and alginate-based bioinks by
extrusion-based construction of aligned hydrogel fibers encapsulating muscle pre-
cursor cells (C2C12) [119]. With a unique approach, the printing head had a
microfluidic device with a coaxial needle that can be used to extrude C2C12 cells
into 3D constructs while producing aligned hydrogel fibers. By this method an
enhanced myogenic differentiation with the formation of parallel aligned, long-
range, tightly packed, and completely striated myotube structures could be achieved.
4.6 Other Soft Tissues
The 3D printing technique gives the chance to control the size and features at
micro-/nanoscale for various types of soft tissues. The printing of complex tissues,
such as liver and cornea, is possible, but many issues exist until today. In printing of
multicellular tissues, cells and matrix materials should be printed together to mimic
tissue micro-architecture [120, 121]. Therefore, many tissue scaffolds have been
designed to mimic native tissue with complex 3D morphology. Table 8 shows some
important researches that investigated those challenging soft tissues such as liver,
cornea, and urinary bladder.
Table 8 Biomaterials and cells used for bioprinting of selected soft tissues
310
Fabrication
Tissue Biomaterials Cells method 3D printer Main outcome
Liver [122] GelMa + PEG-bis- Human hepatic stellate Cellbricks – It was proved liver organoid
chloroacetate + photoinitiator cells, HepaRG cells printing- can be printed by using
+ cells stereolithography stereolithography technique
and hydrogel as a bioink
Liver [123] 2% w/v alginate, 3% w/v gel- Human bipotent hepatic Microextrusion Inkredible, Cellink, The desirable bioprinting was
atin, 0; 0.25; 0.5; 1, or 2 mg/ progenitor cells, human printer Gothenburg, Sweden achieved in alginate-gelatin
mL w/v hECM, 0.03 M epithelial lung carci- with 0.5 and 1 mg/mL hECM
CaSO4, and 7 106 mature noma cells bioink
HepaRG cells/mL
Urinary blad- Bone marrow-derived cell Bone marrow-derived – Regenova, Cyfuse Bio- The urinary bladder can be
der [124] spheroids cells medical K.K., Tokyo, printed by using bone mar-
Japan row stem cells, and vascular-
ization is seen after
transplantation to rabbit
Cornea [125] Sodium algi- Human corneal stromal Microextrusion Inkredible, Cellink, The bioinks with low viscos-
nate + methacrylated collagen cells printer Gothenburg, Sweden ity are suitable for 3D
printing of cornea
Hepatic struc- 3% alginate + cells Mouse embryonic fibro- Pneumatic 3D bioprinting system, The collagen is not printable
tures [126] blasts (MEFs) printing Korea Institute of alone, and alginate was
Machinery and Materials, added to modify its pH and
South Korea printing temperature
Cornea [127] Gelatin + lyophilized bovine Human corneal endothe- Extrusion INVIVO, Rokit, Seoul, 3D bioprinted HCEC-laden
AM lial cells (HCEC) printing Korea AM grafts being well
engrafted and demonstrated
significantly improved cor-
neal thickness and edema
compared to the control
conditions
B. Yilmaz et al.
Human intes- Human intestinal Human primary intesti- Extrusion Organovo, 3D NovoGen Fully human 3D intestinal
tinal tissues myofibroblast (IMF) nal epithelial cells, printing Bioprinter, San Diego, tissue model composed of
[128] interstitium, human intestinal myofibroblast USA primary cells with increased
epithelial cells (HIEC) complexity and function
compared with standard
in vitro models can be
designed
Photoreceptor- Human retinal pigmented epi- Human retinal Microvalve- RegenHU bioprinter, The microvalve-based
retinal tissue thelial cell line, human retino- pigmented epithelial cell based bioprinting Switzerland bioprinting is efficient and
[129] blastoma cell line line, human retinoblas- accurate to build the in vitro
toma cell line tissue models with the
potential to mimic natural
tissue
Cornea [130] Collagen I, Limbal epithelial stem Laser-assisted – The laser-assisted bioprinting
ethylenediaminetetraacetic cells, human adipose- bioprinting systems can be used in cell
acid, human female AB blood derived stem cells printing by optimizing laser
plasma, human recombinant source power
laminin-521, hyaluronic acid
sodium salt
311
5 Conclusion
The significant factors which should be taken into account in designing of biological
substitutes are pore size, mechanical integrity, architecture, and mass transportation.
Thus, the success of biological substitute requires a deep knowledge about contri-
bution of mechanical properties, biological factors, and material composition during
regeneration period. The 3D print technology gives opportunity to accurately control
the architecture of printing substitute which gives the ability of mimicking natural
tissues. In addition, the bioink composition has a tremendous effect on success
of bioprinting. The bioinks that mimic extracellular matrix of target tissue are a
promising approach in bioprinting. Therefore, formulations of tissue-specific bioink
for each type of tissue will become an effective strategy. 3D printing of complex
and large-scale constructs with high spatial resolution is a major issue that needs
to be investigated. Further inventions on these issues will unblock challenges in
bioprinting of functional and complex tissues with high mechanical integrity.
References
1. Sundaramurthi D, Rauf S, Hauser CAE (2016) 3D bioprinting technology for regenerative

medicine applications. Int J Bioprinting 2:9–26. https://doi.org/10.18063/IJB.2016.02.010
2. Mironov V, Reis N, Derby B (2006) Bioprinting: a beginning. Tissue Eng 12:631–634
3. Mironov V, Visconti RP, Kasyanov V, Forgacs G, Drake CJ, Markwald RR (2009) Organ
printing: tissue spheroids as building blocks. Biomaterials 30:2164–2174. https://doi.org/10.
1016/j.biomaterials.2008.12.084
4. Groll J, Boland T, Blunk T, Burdick JA, Cho D-W, Dalton PD, Derby B, Forgacs G, Li Q,
Mironov VA, Moroni L, Nakamura M, Shu W, Takeuchi S, Vozzi G, Woodfield TBF, Xu T,
Yoo JJ, Malda J (2016) Biofabrication: reappraising the definition of an evolving field.
Biofabrication 8:013001. https://doi.org/10.1088/1758-5090/8/1/013001
5. Varkey M, Atala A (2018) Current challenges and future perspectives of bioprinting. In:
Khademhosseini A, Camci-Unal G (eds) 3D bioprinting in regenerative engineering principles
and applications. CRC Press, Boca Raton, pp 363–373
6. Vyas D, Udyawar D (2019) A review on current state of art of bioprinting. In: Kumar J,
Pandey PM, Ian D (eds) 3D printing and additive manufacturing technologies. Springer,
Singapore, pp 195–202
7. U.S. Department of Health and Human Services (2018) Organ Procurement and Transplanta-
tion Network. https://optn.transplant.hrsa.gov/. Accessed 2 Dec 2018
8. Cui H, Nowicki M, Fisher JP, Zhang LG (2017) 3D bioprinting for organ regeneration. Adv
Healthc Mater 6. https://doi.org/10.1002/adhm.201601118
9. Skardal A (2018) Principles and applications of bioprinting. In: Khademhosseini A, Camci-
Unal G (eds) 3D bioprinting in regenerative engineering principles and applications. CRC
Press, Boca Raton, pp 1–19
10. Swainson WK (1977) Method, medium and apparatus for producing three-dimensional figure
product
11. Hull CW (1984) Apparatus for production of three-dimensional objects by stereolithography
12. Whitaker M (2014) The history of 3D printing in healthcare. Bull R Coll Surg Engl
96:228–229. https://doi.org/10.1308/147363514X13990346756481
13. Su A, Al’Aref SJ (2018) Chapter 1 – History of 3D printing. Elsevier, Amsterdam
14. Wohlers T, Gornet T (2016) History of additive manufacturing. Wohlers Rep 24:118
15. Butt J, Shirvani H (2018) Additive, subtractive, and hybrid manufacturing processes. In:
Bar-Cohen Y (ed) Advances in manufacturing and processing of materials and structures.
CRC Press, Boca Raton, pp 187–218
16. Jose RR, Rodriquez MJ, Dixon TA, Omenetto FG, Kaplan DL (2016) Evolution of bioinks
and additive manufacturing technologies for 3D bioprinting. ACS Biomater Sci Eng
2:1662–1678. https://doi.org/10.1021/acsbiomaterials.6b00088
17. Murphy K, Dorfman S, Smith N, Bauwens L, Sohn I, Mcdonald T, Leigh-Lancaster C, Law RJ
(2012) Devices, systems, and methods for the fabrication of tissue, p 1
18. Shafiee A, Atala A (2016) Printing technologies for medical applications. Trends Mol Med
22:254–265. https://doi.org/10.1016/j.molmed.2016.01.003
19. Murphy SV, Atala A (2014) 3D bioprinting of tissues and organs. Nat Biotechnol 32:773.
https://doi.org/10.1038/nbt.2958
20. Wilson WC, Boland T (2003) Cell and organ printing 1: protein and cell printers. Anat Rec A
Discov Mol Cell Evol Biol 271:491–496. https://doi.org/10.1002/ar.a.10057
21. Boland T, Wilson WC, Xu T (2006) Inkjet printing of viable cells. Biomaterials 26:93–99
22. Doyle K (2014) Bioprinting: from patches to parts. Genet Eng Biotechnol News 34:34–35.
https://doi.org/10.1089/gen.34.10.02
23. Tappa K, Jammalamadaka U (2018) Novel biomaterials used in medical 3D printing tech-
niques. J Funct Biomater 9:17–33. https://doi.org/10.3390/jfb9010017
24. Zein I, Hutmacher DW, Cheng K, Hin S (2002) Fused deposition modeling of novel scaffold
architectures for tissue engineering applications. Biomaterials 23:1169–1185
25. Wu W, Geng P, Li G, Zhao D, Zhang H, Zhao J (2015) Influence of layer thickness and raster
angle on the mechanical properties of 3D-printed PEEK and a comparative mechanical study
between PEEK and ABS. Materials 8:5834–5846. https://doi.org/10.3390/ma8095271
26. Liu W, Zhong Z, Hu N, Zhou Y, Maggio L, Miri A, Fragasso A, Jin X, Khademhosseini A,
Zhang Y (2017) Coaxial extrusion bioprinting of 3D microfibrous constructs with cell-
favorable gelatin methacryloyl microenvironments. Biofabrication 10:024102. https://doi.
org/10.1088/1758-5090/aa9d44
27. Odde DJ, Renn MJ (1999) Laser-guided direct writing for applications in biotechnology.
Trends Biotechnol 17:385–389
28. Zhang B, Luo Y, Ma L, Gao L, Li Y, Xue Q, Yang H, Cui Z (2018) 3D bioprinting: an
emerging technology full of opportunities and challenges. Bio-Des Manuf 1:2–13. https://doi.
org/10.1007/s42242-018-0004-3
29. Guillotin B, Souquet A, Catros S, Duocastella M, Pippenger B, Bellance S, Bareille R,
Rémy M, Bordenave L, Amédée J, Guillemot F (2010) Laser assisted bioprinting of
engineered tissue with high cell density and microscale organization. Biomaterials
31:7250–7256. https://doi.org/10.1016/j.biomaterials.2010.05.055
30. Huang Y, Zhang X, Gao G, Yonezawa T, Cui X (2017) 3D bioprinting and the current
applications in tissue engineering. Biotechnol J 12:1600734. https://doi.org/10.1002/biot.
201600734
31. Ozbolat IT (2017) 3D bioprinting fundamentals, principles and applications. Elsevier, London
32. Dhariwala B, Hunt E, Boland T (2004) Rapid prototyping of tissue-engineering constructs,
using photopolymerizable hydrogels and stereolithography. Tissue Eng 10:1316–1322.
https://doi.org/10.1089/ten.2004.10.1316
33. Ng WL, Goh MH, Yeong WY, Naing MW (2018) Applying macromolecular crowding to
3D bioprinting: fabrication of 3D hierarchical porous collagen-based hydrogel constructs.
Biomater Sci 6:562–574. https://doi.org/10.1039/C7BM01015J
34. Agarwala S (2016) A perspective on 3D bioprinting technology: present and future. Am J Eng
Appl Sci 9:985–990. https://doi.org/10.3844/ajeassp.2016.985.990
35. Ng WL, Lee JM, Yeong WY, Win Naing M (2017) Microvalve-based bioprinting – process,
bio-inks and applications. Biomater Sci 5:632–647. https://doi.org/10.1039/C6BM00861E
36. Blaeser A, Duarte Campos DF, Puster U, Richtering W, Stevens MM, Fischer H (2016)
Controlling shear stress in 3D bioprinting is a key factor to balance printing resolution and
stem cell integrity. Adv Healthc Mater 5:326–333. https://doi.org/10.1002/adhm.201500677
37. Derby B (2010) Inkjet printing of functional and structural materials: fluid property require-
ments, feature stability, and resolution. Annu Rev Mater Res 40:395–414. https://doi.org/10.
1146/annurev-matsci-070909-104502
38. Tibbits S (2014) 4D printing: multi-material shape change. Archit Des 84:116–121
39. Gao B, Yang Q, Zhao X, Jin G, Ma Y, Xu F (2016) 4D bioprinting for biomedical applica-
tions. Trends Biotechnol 34:746–756. https://doi.org/10.1016/j.tibtech.2016.03.004
40. Ashammakhi N, Ahadian S, Zengjie F, Suthiwanich K, Lorestani F, Orive G, Ostrovidov S,
Khademhosseini A (2018) Advances and future perspectives in 4D bioprinting. Biotechnol J
13:1–12. https://doi.org/10.1002/biot.201800148
41. Zhu W, Webster TJ, Zhang LG (2019) 4D printing smart biosystems for nanomedicine.
Nanomedicine 14:1643–1645. https://doi.org/10.2217/nnm-2019-0134
42. Yang GH, Yeo M, Koo YW, Kim GH (2019) 4D bioprinting: technological advances in
biofabrication. Macromol Biosci 19:1800441. https://doi.org/10.1002/mabi.201800441
43. Cidonio G, Glinka M, Dawson JI, Oreffo ROC (2019) The cell in the ink: improving
biofabrication by printing stem cells for skeletal regenerative medicine. Biomaterials
44. Li Y, Zhang YS, Akpek A, Shin SR, Khademhosseini A (2017) 4D bioprinting: the
next-generation technology for biofabrication enabled by stimuli-responsive materials.
Biofabrication 9:012001. https://doi.org/10.1088/1758-5090/9/1/012001
45. Sowjanya JA, Singh J, Mohita T, Sarvanan S, Moorthi A, Srinivasan N, Selvamurugan N
(2013) Biocomposite scaffolds containing chitosan/alginate/nano-silica for bone tissue engi-
neering. Colloids Surf B Biointerfaces 109:294–300. https://doi.org/10.1016/j.colsurfb.2013.
04.006
46. Velasco MA, Narváez-Tovar CA, Garzón-Alvarado DA (2015) Design, materials, and
mechanobiology of biodegradable scaffolds for bone tissue engineering. Biomed Res Int
2015:1–21. https://doi.org/10.1155/2015/729076
47. Venkatesan J, Bhatnagar I, Manivasagan P, Kang K-H, Kim S-K (2015) Alginate composites
for bone tissue engineering: a review. Int J Biol Macromol 72:269–281. https://doi.org/10.
1016/j.ijbiomac.2014.07.008
48. Turnbull G, Clarke J, Picard F, Riches P, Jia L, Han F, Li B, Shu W (2018) 3D bioactive
composite scaffolds for bone tissue engineering. Bioact Mater 3:278–314. https://doi.org/10.
1016/j.bioactmat.2017.10.001
49. Pacifici A, Laino L, Gargari M, Guzzo F, Velandia Luz A, Polimeni A, Pacifici L (2018)
Decellularized hydrogels in bone tissue engineering: a topical review. Int J Med Sci
15:492–497. https://doi.org/10.7150/ijms.22789
50. Gilmore J, Burg T, Groff RE, Burg KJL (2017) Design and optimization of a novel bio-loom
to weave melt-spun absorbable polymers for bone tissue engineering: design and optimization
of a novel bio-loom. J Biomed Mater Res B Appl Biomater 105:1342–1351. https://doi.org/10.
1002/jbm.b.33700
51. Ashammakhi N, Kaarela O (2017) Three-dimensional bioprinting can help bone. J Craniofac
Surg 00:1. https://doi.org/10.1097/SCS.0000000000004143
52. Zhang L, Yang G, Johnson BN, Jia X (2019) Three-dimensional (3D) printed scaffold and
material selection for bone repair. Acta Biomater 84:16–33. https://doi.org/10.1016/j.actbio.
2018.11.039
53. Demirtaş TT, Irmak G, Gümüşderelioğlu M (2017) Bioprintable form of chitosan hydrogel for
bone tissue engineering. Biofabrication 9:035003. https://doi.org/10.1088/1758-5090/aa7b1d
54. Byambaa B, Annabi N, Yue K, Santiago GT, Alvarez MM, Jia W, Kazemzadeh-narbat M,
Shin SR, Tamayol A, Khademhosseini A (2017) Bioprinted osteogenic and vasculogenic
patterns for engineering 3D bone tissue. Adv Healthc Mater 6:1700015. https://doi.org/10.
1002/adhm.201700015
55. Neufurth M, Wang X, Schröder HC, Feng Q, Diehl-Seifert B, Ziebart T, Steffen R,

Wang S, Müller WEG (2014) Engineering a morphogenetically active hydrogel for
bioprinting of bioartificial tissue derived from human osteoblast-like SaOS-2 cells. Biomate-
rials 35:8810–8819. https://doi.org/10.1016/j.biomaterials.2014.07.002
56. Wenz A, Borchers K, Tovar GEM, Kluger PJ (2017) Bone matrix production in
hydroxyapatite-modified hydrogels suitable for bone bioprinting. Biofabrication 9:044103.
https://doi.org/10.1088/1758-5090/aa91ec
57. Zhai X, Ruan C, Ma Y, Cheng D, Wu M, Liu W, Zhao X, Pan H, Lu WW (2018)
3D-bioprinted osteoblast-laden nanocomposite hydrogel constructs with induced microenvi-
ronments promote cell viability, differentiation, and osteogenesis both in vitro and in vivo.
Adv Sci 5:1700550. https://doi.org/10.1002/advs.201700550
58. Ahlfeld T, Doberenz F, Kilian D, Vater C, Korn P, Lauer G, Lode A, Gelinsky M (2018)
Bioprinting of mineralized constructs utilizing multichannel plotting of a self-setting calcium
phosphate cement and a cell-laden bioink. Biofabrication 10:045002. https://doi.org/10.1088/
1758-5090/aad36d
59. Chen Y, Shen Y, Ho C, Yu J, Wu YA, Wang K, Shih C-T, Shie M-Y (2018) Osteogenic and
angiogenic potentials of the cell-laden hydrogel/mussel-inspired calcium silicate complex
hierarchical porous scaffold fabricated by 3D bioprinting. Mater Sci Eng C 91:679–687.
https://doi.org/10.1016/j.msec.2018.06.005
60. Murphy C, Kolan K, Li W, Semon J, Day D, Leu M (2017) 3D bioprinting of stem cells and
polymer/bioactive glass composite scaffolds for tissue engineering. Int J Bioprinting 3:53–63.
https://doi.org/10.18063/ijb.2017.01.005
61. Bendtsen ST, Wei M (2017) In vitro evaluation of 3D bioprinted tri-polymer network scaffolds
for bone tissue regeneration. J Biomed Mater Res A 105:3262–3272. https://doi.org/10.1002/
jbm.a.36184
62. Huang J, Fu H, Li C, Dai J, Zhang Z (2017) Recent advances in cell-laden 3D bioprinting:
materials, technologies and applications. J 3D Print Med 1:245–268. https://doi.org/10.2217/
3dp-2017-0010
63. Neufurth M, Wang X, Wang S, Steffen R, Ackermann M, Haep ND, Schröder HC,
Müller WEG (2017) 3D printing of hybrid biomaterials for bone tissue engineering:
calcium-polyphosphate microparticles encapsulated by polycaprolactone. Acta Biomater
64:377–388. https://doi.org/10.1016/j.actbio.2017.09.031
64. Jeon HJ, Lee M, Yun S, Kang D, Park KH, Choi S, Choi E, Jin S, Shim JH, Yun WS, Yoon BJ,
Park J (2019) Fabrication and characterization of 3D-printed biocomposite scaffolds based on
PCL and silanated silica particles for bone tissue regeneration. Chem Eng J 360:519–530.
https://doi.org/10.1016/j.cej.2018.11.176
65. Aydogdu MO, Oner ET, Ekren N, Erdemir G, Kuruca SE, Yuca E, Bostan MS, Eroglu MS,
Ikram F, Uzun M, Gunduz O (2019) Comparative characterization of the hydrogel added
PLA/β-TCP scaffolds produced by 3D bioprinting. Bioprinting 13:e00046. https://doi.org/10.
1016/j.bprint.2019.e00046
66. Dong Y, Liang J, Cui Y, Xu S, Zhao N (2018) Fabrication of novel bioactive hydroxyapatite-
chitosan-silica hybrid scaffolds: combined the sol-gel method with 3D plotting technique.
Carbohydr Polym 197:183–193. https://doi.org/10.1016/j.carbpol.2018.05.086
67. Wang J, Yang M, Zhu Y, Wang L, Tomsia AP, Mao C (2014) Phage nanofibers induce
vascularized osteogenesis in 3D printed bone scaffolds. Adv Mater 26:4961–4966. https://doi.
org/10.1002/adma.201400154
68. Liu J, Li L, Suo H, Yan M, Yin J, Fu J (2019) 3D printing of biomimetic multi-layered
GelMA/nHA scaffold for osteochondral defect repair. Mater Des 171:107708. https://doi.org/
10.1016/j.matdes.2019.107708
69. Du Y, Liu H, Yang Q, Wang S, Wang J, Ma J, Noh I, Mikos AG, Zhang S (2017)
Selective laser sintering scaffold with hierarchical architecture and gradient composition
for osteochondral repair in rabbits. Biomaterials 137:37–48. https://doi.org/10.1016/j.biomate
rials.2017.05.021
70. Correia CR, Reis RL, Mano JF (2015) Multiphasic, multistructured and hierarchical strategies
for cartilage regeneration. In: Bertassoni LE, Coelho PG (eds) Engineering mineralized and
load bearing tissues. Springer, Cham, pp 143–160
71. Huang BJ, Hu JC, Athanasiou KA (2016) Cell-based tissue engineering strategies used in the
clinical repair of articular cartilage. Biomaterials 98:1–22. https://doi.org/10.1016/j.biomate
rials.2016.04.018
72. Chen L, Deng C, Li J, Yao Q, Chang J, Wang L, Wu C (2019) 3D printing of a lithium-
calcium-silicate crystal bioscaffold with dual bioactivities for osteochondral interface recon-
struction. Biomaterials 196:138–150. https://doi.org/10.1016/j.biomaterials.2018.04.005
73. Bittner SM, Smith BT, Diaz-Gomez L, Hudgins CD, Melchiorri AJ, Scott DW, Fisher JP,
Mikos AG (2019) Fabrication and mechanical characterization of 3D printed vertical uniform
and gradient scaffolds for bone and osteochondral tissue engineering. Acta Biomater
90:37–48. https://doi.org/10.1016/j.actbio.2019.03.041
74. Li Z, Jia S, Xiong Z, Long Q, Yan S, Hao F, Liu J, Yuan Z (2018) 3D-printed scaffolds with
calcified layer for osteochondral tissue engineering. J Biosci Bioeng 126:389–396. https://doi.
org/10.1016/j.jbiosc.2018.03.014
75. Rajzer I, Kurowska A, Jabłoński A, Jatteau S, Śliwka M, Ziąbka M, Menaszek E (2018)
Layered gelatin/PLLA scaffolds fabricated by electrospinning and 3D printing- for nasal
cartilages and subchondral bone reconstruction. Mater Des 155:297–306. https://doi.org/10.
1016/j.matdes.2018.06.012
76. Daly AC, Kelly DJ (2019) Biofabrication of spatially organised tissues by directing the growth
of cellular spheroids within 3D printed polymeric microchambers. Biomaterials 197:194–206.
https://doi.org/10.1016/j.biomaterials.2018.12.028
77. Deng C, Yang Q, Sun X, Chen L, Feng C, Chang J, Wu C (2018) Bioactive scaffolds with Li
and Si ions-synergistic effects for osteochondral defects regeneration. Appl Mater Today
10:203–216. https://doi.org/10.1016/j.apmt.2017.12.010
78. Di Bella C, Duchi S, O’Connell CD, Blanchard R, Augustine C, Yue Z, Thompson F,
Richards C, Beirne S, Onofrillo C, Bauquier SH, Ryan SD, Pivonka P, Wallace GG, Choong
PF (2018) In situ handheld three-dimensional bioprinting for cartilage regeneration. J Tissue
Eng Regen Med 12:611–621. https://doi.org/10.1002/term.2476
79. Nguyen D, Hägg DA, Forsman A, Ekholm J, Nimkingratana P, Brantsing C,
Kalogeropoulos T, Zaunz S, Concaro S, Brittberg M, Lindahl A, Gatenholm P, Enejder A,
Simonsson S (2017) Cartilage tissue engineering by the 3D bioprinting of iPS cells in a
nanocellulose/alginate bioink. Sci Rep 7:658. https://doi.org/10.1038/s41598-017-00690-y
80. Kuo C-Y, Wilson E, Fuson A, Gandhi N, Monfaredi R, Jenkins A, Romero M, Santoro M,
Fisher JP, Cleary K, Reilly B (2018) Repair of tympanic membrane perforations with
customized bioprinted ear grafts using chinchilla models. Tissue Eng Part A 24:527–535.
https://doi.org/10.1089/ten.tea.2017.0246
81. Thayer PS, Orrhult LS, Martínez H (2018) Bioprinting of cartilage and skin tissue analogs
utilizing a novel passive mixing unit technique for bioink precellularization. J Vis Exp
131:56372. https://doi.org/10.3791/56372
82. Zhu W, Cui H, Boualam B, Masood F, Flynn E, Rao RD, Zhang Z-Y, Zhang LG (2018) 3D
bioprinting mesenchymal stem cell-laden construct with core–shell nanospheres for cartilage
tissue engineering. Nanotechnology 29:185101. https://doi.org/10.1088/1361-6528/aaafa1
83. Potjewyd G, Moxon S, Wang T, Domingos M, Hooper NM (2018) Tissue engineering
3D neurovascular units: a biomaterials and bioprinting perspective. Trends Biotechnol
36:457–472. https://doi.org/10.1016/j.tibtech.2018.01.003
84. Dixon AR, Jariwala SH, Bilis Z, Loverde JR, Pasquina PF, Alvarez LM (2018) Bridging
the gap in peripheral nerve repair with 3D printed and bioprinted conduits. Biomaterials
85. Knowlton S, Anand S, Shah T, Tasoglu S (2018) Bioprinting for neural tissue engineering.
Trends Neurosci 41:31–46. https://doi.org/10.1016/j.tins.2017.11.001
86. Naghieh S, Sarker MD, Abelseth E, Chen X (2019) Indirect 3D bioprinting and characteriza-
tion of alginate scaffolds for potential nerve tissue engineering applications. J Mech Behav
Biomed Mater 93:183–193. https://doi.org/10.1016/j.jmbbm.2019.02.014
87. Sarker MD, Naghieh S, McInnes AD, Ning L, Schreyer DJ, Chen X (2019) Bio-fabrication of
peptide-modified alginate scaffolds: printability, mechanical stability and neurite outgrowth
assessments. Bioprinting 14:e00045. https://doi.org/10.1016/j.bprint.2019.e00045
88. Ning L, Sun H, Lelong T, Guilloteau R, Zhu N, Schreyer DJ, Chen X (2018) 3D bioprinting of
scaffolds with living Schwann cells for potential nerve tissue engineering applications.
Biofabrication 10:035014. https://doi.org/10.1088/1758-5090/aacd30
89. Li X, Wang X, Wang X, Chen H, Zhang X, Zhou L, Xu T (2018) 3D bioprinted rat Schwann
cell-laden structures with shape flexibility and enhanced nerve growth factor expression.
3 Biotech 8:342. https://doi.org/10.1007/s13205-018-1341-9
90. England S, Rajaram A, Schreyer DJ, Chen X (2017) Bioprinted fibrin-factor XIII-hyaluronate
hydrogel scaffolds with encapsulated Schwann cells and their in vitro characterization for use
in nerve regeneration. Bioprinting 5:1–9. https://doi.org/10.1016/j.bprint.2016.12.001
91. Joung D, Truong V, Neitzke CC, Guo S-Z, Walsh PJ, Monat JR, Meng F, Park SH, Dutton JR,
Parr AM, McAlpine MC (2018) 3D printed stem-cell derived neural progenitors generate
spinal cord scaffolds. Adv Funct Mater 28:1801850. https://doi.org/10.1002/adfm.201801850
92. Hsieh F-Y, Lin H-H, Hsu S (2015) 3D bioprinting of neural stem cell-laden thermoresponsive
biodegradable polyurethane hydrogel and potential in central nervous system repair.
Biomaterials 71:48–57. https://doi.org/10.1016/j.biomaterials.2015.08.028
93. Zhu W, Tringale KR, Woller SA, You S, Johnson S, Shen H, Schimelman J, Whitney M,
Steinauer J, Xu W, Yaksh TL, Nguyen QT, Chen S (2018) Rapid continuous 3D printing of
customizable peripheral nerve guidance conduits. Mater Today 21:951–959. https://doi.org/
10.1016/j.mattod.2018.04.001
94. Donderwinkel I, van Hest JCM, Cameron NR (2017) Bio-inks for 3D bioprinting: recent
advances and future prospects. Polym Chem 8:4451–4471. https://doi.org/10.1039/
C7PY00826K
95. Tao J, Zhang J, Du T, Xu X, Deng X, Chen S, Liu J, Chen Y, Liu X, Xiong M, Luo Y,
Cheng H, Mao J, Cardon L, Gou M, Wei Y (2019) Rapid 3D printing of functional
nanoparticle-enhanced conduits for effective nerve repair. Acta Biomater 90:49–59. https://
doi.org/10.1016/j.actbio.2019.03.047
96. Zhou X, Cui H, Nowicki M, Miao S, Lee S-J, Masood F, Harris BT, Zhang LG (2018) Three-
dimensional-bioprinted dopamine-based matrix for promoting neural regeneration. ACS Appl
Mater Interfaces 10:8993–9001. https://doi.org/10.1021/acsami.7b18197
97. Vijayavenkataraman S, Lu WF, Fuh JYH (2016) 3D bioprinting of skin: a state-of-the-art
review on modelling, materials, and processes. Biofabrication 8:032001. https://doi.org/10.
1088/1758-5090/8/3/032001
98. Gholami P, Ahmadi-pajouh MA, Abolftahi N, Hamarneh G, Kayvanrad M (2018) Segmen-
tation and measurement of chronic wounds for bioprinting. IEEE J Biomed Health Inform
22:1269–1277. https://doi.org/10.1109/JBHI.2017.2743526
99. Min D, Lee W, Bae I-H, Lee TR, Croce P, Yoo S-S (2018) Bioprinting of biomimetic skin
containing melanocytes. Exp Dermatol 27:453–459. https://doi.org/10.1111/exd.13376
100. Ng WL, Qi JTZ, Yeong WY, Naing MW (2018) Proof-of-concept: 3D bioprinting of
pigmented human skin constructs. Biofabrication 10:025005. https://doi.org/10.1088/1758-
5090/aa9e1e
101. Albanna M, Binder KW, Murphy SV, Kim J, Qasem SA, Zhao W, Tan J, El-Amin IB, Dice
DD, Marco J, Green J, Xu T, Skardal A, Holmes JH, Jackson JD, Atala A, Yoo JJ (2019) In
situ bioprinting of autologous skin cells accelerates wound healing of extensive excisional full-
thickness wounds. Sci Rep 9:1856. https://doi.org/10.1038/s41598-018-38366-w
102. Kim BS, Gao G, Kim JY, Cho D (2019) 3D cell printing of perfusable vascularized human
skin equivalent composed of epidermis, dermis, and hypodermis for better structural
recapitulation of native skin. Adv Healthc Mater 8:1801019. https://doi.org/10.1002/adhm.
201801019
103. Koch L, Deiwick A, Schlie S, Michael S, Gruene M, Coger V, Zychlinski D, Schambach A,

Reimers K, Vogt PM, Chichkov B (2012) Skin tissue generation by laser cell printing.
Biotechnol Bioeng 109:1855–1863. https://doi.org/10.1002/bit.24455
104. Skardal A, Mack D, Kapetanovic E, Atala A, Jackson JD, Yoo J, Soker S (2012) Bioprinted
amniotic fluid-derived stem cells accelerate healing of large skin wounds. Stem Cells Trans
Med 1:792–802. https://doi.org/10.5966/sctm.2012-0088
105. Augustine R (2018) Skin bioprinting: a novel approach for creating artificial skin from
synthetic and natural building blocks. Prog Biomater 7:77–92. https://doi.org/10.1007/
s40204-018-0087-0
106. Yan W-C, Davoodi P, Vijayavenkataraman S, Tian Y, Ng WC, Fuh JYH, Robinson KS,
Wang C-H (2018) 3D bioprinting of skin tissue: from pre-processing to final product evalu-
ation. Adv Drug Deliv Rev 132:270–295. https://doi.org/10.1016/j.addr.2018.07.016
107. El-Serafi AT, El-Serafi IT, Elmasry M, Steinvall I, Sjöberg F (2017) Skin regeneration in three
dimensions, current status, challenges and opportunities. Differentiation 96:26–29. https://doi.
org/10.1016/j.diff.2017.06.002
108. Ng WL, Wang S, Yeong WY, Naing MW (2016) Skin bioprinting: impending reality or
fantasy? Trends Biotechnol 34:689–699. https://doi.org/10.1016/j.tibtech.2016.04.006
109. Tarassoli SP, Jessop ZM, Al-Sabah A, Gao N, Whitaker S, Doak S, Whitaker IS (2018) Skin
tissue engineering using 3D bioprinting: an evolving research field. J Plast Reconstr Aesthet
Surg 71:615–623. https://doi.org/10.1016/j.bjps.2017.12.006
110. Vidal Yucha SE, Tamamoto KA, Nguyen H, Cairns DM, Kaplan DL (2019) Human skin
equivalents demonstrate need for neuro-immuno-cutaneous system. Adv Biosyst 3:1800283.
https://doi.org/10.1002/adbi.201800283
111. Adams SD, Ashok A, Kanwar RK, Kanwar JR, Kouzani AZ (2017) Integrated 3D printed
scaffolds and electrical stimulation for enhancing primary human cardiomyocyte cultures.
Bioprinting 6:18–24. https://doi.org/10.1016/j.bprint.2017.04.003
112. Gaebel R, Ma N, Liu J, Guan J, Koch L, Klopsch C, Gruene M, Toelk A, Wang W, Mark P,
Wang F, Chichkov B, Li W, Steinhoff G (2011) Patterning human stem cells and endothelial
cells with laser printing for cardiac regeneration. Biomaterials 32:9218–9230. https://doi.org/
10.1016/j.biomaterials.2011.08.071
113. Izadifar M, Chapman D, Babyn P, Chen X, Kelly ME (2018) UV-assisted 3D bioprinting of
nanoreinforced hybrid cardiac patch for myocardial tissue engineering. Tissue Eng Part C
Methods 24:74–88. https://doi.org/10.1089/ten.tec.2017.0346
114. Jang J, Park H-J, Kim S-W, Kim H, Park JY, Na SJ, Kim HJ, Park MN, Choi SH, Park SH,
Kim SW, Kwon S-M, Kim P-J, Cho D-W (2017) 3D printed complex tissue construct using
stem cell-laden decellularized extracellular matrix bioinks for cardiac repair. Biomaterials
115. Liu J, He J, Liu J, Ma X, Chen Q, Lawrence N, Zhu W, Xu Y, Chen S (2019) Rapid 3D
bioprinting of in vitro cardiac tissue models using human embryonic stem cell-derived
cardiomyocytes. Bioprinting 13:e00040. https://doi.org/10.1016/j.bprint.2019.e00040
116. Ong CS, Fukunishi T, Zhang H, Huang CY, Nashed A, Blazeski A, DiSilvestre D, Vricella L,
Conte J, Tung L, Tomaselli GF, Hibino N (2017) Biomaterial-free three-dimensional
bioprinting of cardiac tissue using human induced pluripotent stem cell derived
cardiomyocytes. Sci Rep 7:4566. https://doi.org/10.1038/s41598-017-05018-4
117. Wang Z, Lee SJ, Cheng H-J, Yoo JJ, Atala A (2018) 3D bioprinted functional and
contractile cardiac tissue constructs. Acta Biomater 70:48–56. https://doi.org/10.1016/j.
actbio.2018.02.007
118. Choi Y-J, Jun Y-J, Kim DY, Yi H-G, Chae S-H, Kang J, Lee J, Gao G, Kong J-S, Jang J,
Chung WK, Rhie J-W, Cho D-W (2019) A 3D cell printed muscle construct with tissue-
derived bioink for the treatment of volumetric muscle loss. Biomaterials 206:160–169. https://
doi.org/10.1016/j.biomaterials.2019.03.036
119. Costantini M, Testa S, Mozetic P, Barbetta A, Fuoco C, Fornetti E, Tamiro F, Bernardini S,
Jaroszewicz J, Święszkowski W, Trombetta M, Castagnoli L, Seliktar D, Garstecki P,
Cesareni G, Cannata S, Rainer A, Gargioli C (2017) Microfluidic-enhanced 3D bioprinting of

aligned myoblast-laden hydrogels leads to functionally organized myofibers in vitro and
in vivo. Biomaterials 131:98–110. https://doi.org/10.1016/j.biomaterials.2017.03.026
120. Mondschein RJ, Kanitkar A, Williams CB, Verbridge SS, Long TE (2017) Polymer structure-
property requirements for stereolithographic 3D printing of soft tissue engineering scaffolds.
Biomaterials 140:170–188. https://doi.org/10.1016/j.biomaterials.2017.06.005
121. Pati F, Ha D-H, Jang J, Han HH, Rhie J-W, Cho D-W (2015) Biomimetic 3D tissue printing
for soft tissue regeneration. Biomaterials 62:164–175. https://doi.org/10.1016/j.biomaterials.
2015.05.043
122. Grix T, Ruppelt A, Thomas A, Amler A-K, Noichl B, Lauster R, Kloke L (2018) Bioprinting
perfusion-enabled liver equivalents for advanced organ-on-a-chip applications. Genes 9:176.
https://doi.org/10.3390/genes9040176
123. Hiller T, Berg J, Elomaa L, Röhrs V, Ullah I, Schaar K, Dietrich A-C, Al-Zeer M, Kurtz A,
Hocke A, Hippenstiel S, Fechner H, Weinhart M, Kurreck J (2018) Generation of a 3D liver
model comprising human extracellular matrix in an alginate/gelatin-based bioink by extrusion
bioprinting for infection and transduction studies. Int J Mol Sci 19:3129. https://doi.org/10.
3390/ijms19103129
124. Imamura T, Shimamura M, Ogawa T, Minagawa T, Nagai T, Silwal Gautam S, Ishizuka O
(2018) Biofabricated structures reconstruct functional urinary bladders in radiation-injured rat
bladders. Tissue Eng Part A 24:1574–1587. https://doi.org/10.1089/ten.tea.2017.0533
125. Isaacson A, Swioklo S, Connon CJ (2018) 3D bioprinting of a corneal stroma equivalent. Exp
Eye Res 173:188–193. https://doi.org/10.1016/j.exer.2018.05.010
126. Kang K, Kim Y, Jeon H, Lee SB, Kim JS, Park SA, Kim WD, Yang HM, Kim SJ, Jeong J,
Choi D (2018) Three-dimensional bioprinting of hepatic structures with directly converted
hepatocyte-like cells. Tissue Eng Part A 24:576–583. https://doi.org/10.1089/ten.tea.
2017.0161
127. Kim KW, Lee SJ, Park SH, Kim JC (2018) Ex vivo functionality of 3D bioprinted corneal
endothelium engineered with ribonuclease 5-overexpressing human corneal endothelial cells.
Adv Healthc Mater 7:1800398. https://doi.org/10.1002/adhm.201800398
128. Madden LR, Nguyen TV, Garcia-Mojica S, Shah V, Le AV, Peier A, Visconti R, Parker EM,
Presnell SC, Nguyen DG, Retting KN (2018) Bioprinted 3D primary human intestinal tissues
model aspects of native physiology and ADME/Tox functions. iScience 2:156–167. https://
doi.org/10.1016/j.isci.2018.03.015
129. Shi P, Tan YSE, Yeong WY, Li HY, Laude A (2018) A bilayer photoreceptor-retinal tissue
model with gradient cell density design: a study of microvalve-based bioprinting. J Tissue Eng
Regen Med 12:1297–1306. https://doi.org/10.1002/term.2661
130. Sorkio A, Koch L, Koivusalo L, Deiwick A, Miettinen S, Chichkov B, Skottman H (2018)
Human stem cell based corneal tissue mimicking structures using laser-assisted 3D bioprinting
and functional bioinks. Biomaterials 171:57–71. https://doi.org/10.1016/j.biomaterials.2018.
04.034
DOI: 10.1007/10_2019_109
Gene Therapy
Ana del Pozo-Rodríguez, Alicia Rodríguez-Gascón,

Julen Rodríguez-Castejón, Mónica Vicente-Pascual, Itziar Gómez-Aguado,
Luigi S. Battaglia, and María Ángeles Solinís
Contents
1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 322
2 Concept of Gene Therapy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 323
3 Nucleic Acids for Gene Therapy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 324
3.1 Gene Augmentation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 325
3.2 Gene Silencing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 326
3.3 Gene Editing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 331
4 Delivery Systems Used in Gene Therapy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 333
4.1 Viral Vectors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 334
4.2 Non-viral Vectors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 341
5 Applications of Gene Therapy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 347
5.1 Gene Therapy Medicinal Products for Cancer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 351
5.2 Gene Therapy Medicinal Products for Other Applications . . . . . . . . . . . . . . . . . . . . . . . . . . 353
6 Challenges of Gene Therapy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 356
6.1 Efficacy Issues . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 356
6.2 Safety Issues . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 357
6.3 Drug Development and Manufacture Issues . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 357
6.4 Ethical Issues . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 358
6.5 Affordability . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 358
6.6 Intellectual Property Complexity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 358
7 Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 359
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 360
A. del Pozo-Rodríguez, A. Rodríguez-Gascón, J. Rodríguez-Castejón, M. Vicente-Pascual,

I. Gómez-Aguado, and M. Á. Solinís (*)
Pharmacokinetic, Nanotechnology and Gene Therapy Group (PharmaNanoGene), Faculty of
Pharmacy, Centro de investigación Lascaray ikergunea, University of the Basque Country
UPV/EHU, Vitoria-Gasteiz, Spain
e-mail: marian.solinis@ehu.eus
L. S. Battaglia
Dipartimento di Scienza e Tecnologia del Farmaco, Università degli Studi di Torino, Turin, Italy
322 A. del Pozo-Rodríguez et al.
Abstract Gene therapy medicinal products (GTMPs) are one of the most promising
biopharmaceuticals, which are beginning to show encouraging results. The broad
clinical research activity has been addressed mainly to cancer, primarily to those
cancers that do not respond well to conventional treatment. GTMPs to treat rare
disorders caused by single-gene mutations have also made important advancements
toward market availability, with eye and hematopoietic system diseases as the main
applications.
Nucleic acid-marketed products are based on both in vivo and ex vivo strategies.
Apart from DNA-based therapies, antisense oligonucleotides, small interfering
RNA, and, recently, T-cell-based therapies have been also marketed. Moreover,
the gene-editing tool CRISPR is boosting the development of new gene therapy-
based medicines, and it is expected to have a substantial impact on the gene therapy
biopharmaceutical market in the near future.
However, despite the important advancements of gene therapy, many challenges
have still to be overcome, which are discussed in this book chapter. Issues such
as efficacy and safety of the gene delivery systems and manufacturing capacity of
biotechnological companies to produce viral vectors are usually considered, but
problems related to cost and patient affordability must be also faced to ensure the
success of this emerging therapy.
Graphical Abstract
Keywords Delivery vectors, Ex vivo, Gene therapy medicinal product, In vivo,

Manufacturing, Quality control
1 Introduction
Biopharmaceuticals, pharmaceuticals produced in biotechnological processes

by molecular biology methods, have become one of the most effective clinical
treatments for a wide variety of diseases. The biopharmaceutical market includes
Gene Therapy 323
gene therapy products, based on the use of nucleic acids as active pharmaceutical
ingredients for the modulation of the gene expression. Gene therapy based on the
administration of DNA and messenger RNA (mRNA) acts by means of therapeutic
protein expression, whereas the use of small interfering RNA (siRNA), microRNA,
oligonucleotides, or aptamers provides posttranslational gene silencing. An emerg-
ing area in this field is genome editing, which corrects the disease by replacing
a sequence of a defective gene by a healthy copy in order to restore the “wild-type”
DNA. Most of those nucleic acids are produced as biopharmaceuticals, although
some of them, such as antisense oligonucleotides, are made by chemistry means.
Despite that gene therapy entered clinical trials in the early 1990s, the first nucleic
acid-based product registered in the European Union was Glybera in 2012, for
lipoprotein lipase deficiency. Currently, only 15 gene therapy medicinal products
have received approval worldwide; nevertheless, since 1989 almost 2,700 gene
therapy-based clinical trials have been completed, are ongoing, or have been
approved for a broad range of applications. Therefore, it is expected that nucleic
acid-based products will have a substantial impact on the biopharmaceutical market
in the near future.
2 Concept of Gene Therapy
Gene therapy is a novel approach to treat, cure, or ultimately prevent disease

by changing the expression of a person’s gene [1]. According to the European
Medicines Agency (EMA), a gene therapy medicinal product generally consists of
a vector or delivery formulation/system containing a genetic construct engineered
to express a specific transgene (“therapeutic sequence”) for the regulation, repair,
replacement, addition, or deletion of a genetic sequence [2]. By far the most common
vector systems used for gene therapy to date have been viral vectors and plasmid
DNA vectors. Gene therapy works by repairing, deactivating, or replacing dysfunc-
tional genes that cause disease with the aim of (re)establishing normal function.
Gene therapy has long been regarded a promising treatment for many diseases,
including those inherited through a genetic disorder (such as hemophilia, human
severe combined immunodeficiency, spinal muscular atrophy, or cystic fibrosis) or
acquired (such as cancers of different kinds).
Depending on the cell type to be modified, gene therapy can be classified into
two categories: somatic and germline gene therapy [3]. Somatic gene therapy targets
to body somatic cells such as bone marrow or blood cells. This type of gene therapy
cannot be passed on to descendants. In germline gene therapy, egg and sperm
cells (germ cells) are the objective of therapy, and the inserted gene passes on to
future generations. The idea of germline gene therapy is controversial due to ethical
concerns, and it is not permitted in many countries. In spite of that, at the end of
2018, a research team in China announced the birth of two babies whose genomes
had been edited, and there may be a third one [4]. These practices have been widely
condemned as irresponsible and as failing to conform with international norms [5].
Fig. 1 Ex vivo and in vivo approaches to gene therapy
Two fundamental strategies have evolved to restore or modify target cell func-
tion: ex vivo or in vivo gene delivery [6]. In ex vivo therapy, cells from the patient or
a donor are harvested, and the therapeutic gene is then transduced in a cell therapy
manufacturing setting. The modified cells are later reinfused into the patient. In vivo
gene therapy consists on functional modification of targets by direct transgene
injection into the patient. Figure 1 features a scheme with the ex vivo and in vivo
approaches to gene therapy.
Thanks to the advances of genetics and bioengineering, gene therapy has become
possible, although at present, this is predominantly an experimental area. However,
in the last 5 years, enormous advances have occurred, with the approval of a few
drug products by the Food and Drug Administration (FDA) and the EMA and others
that are expected to be in the near future.
3 Nucleic Acids for Gene Therapy
Historically many gene therapy approaches have been based on expression of

a transgene encoding a functional protein (i.e., the transgene product). However,
newer tools including directly acting nucleic acid sequences such as microRNA,
interference RNA (RNAi) via short hairpin RNAs (shRNA) or short interference
RNA (siRNA), molecular scissor and gene-editing approaches such as zinc finger
nucleases (ZFNs), transcription activator-like effector nucleases (TALEN), and clus-
tered regularly interspaced short palindromic repeats (CRISP)-associated nuclease
Cas9 (CRISPR/Cas9) are being extensively applied in research and for developing
new medicinal products. The development of this emerging field is still maturing,
although there are many nucleic acid-based drugs in clinical trials, and some of them
Gene Therapy 325
have been already approved. There are also other promising candidates to be used in a
variety of genetic and nongenetic disorders such a monogenic, infectious, cardiovas-
cular, inflammatory, neurodegenerative, and a wide variety of cancers.
Nucleic acids are negatively charged and high molecular weight molecules, with
physical-chemical properties very different to that of conventional drugs. They
present limited stability in the biological medium and must access to an intracellular
compartment (the cytoplasm or the nucleus); these two characteristics contribute
to the difficulties to develop a medicinal product.
Depending on the application, the objective of the therapy can be gene augmen-
tation, gene silencing, or gene editing [7].
3.1 Gene Augmentation
The objective of gene augmentation is to restore normal cellular function by

delivering a functional copy of a gene (DNA) or messenger RNA (mRNA).
3.1.1 DNA
Typically, the gene of interest is inserted into a plasmid or expression cassette, which
is a high molecular weight, double-stranded DNA construct containing transgenes,
which encode specific proteins [8]. Plasmids also contain a promoter and a termi-
nator signal to drive and end gene transcription, respectively [9]. Transfection with
DNA leads to much higher protein and persistent expression than those obtained
with mRNA. However, with mRNA, once it reaches the cytoplasm, translation starts
instantly, without the need to enter the nucleus to be functional [10]; on the contrary,
DNA has to reach the nucleus of the target cell, being this process one of the most
limiting steps for transfection.
Apart from plasmid DNA, minicircle DNA (mcDNA) are emerging due to their
safety and persistent transgene expression in both quiescent and actively dividing cells
[11]. mcDNA are episomal, covalently closed circular gene expression systems, gen-
erally biosynthesized in recombinant bacteria, that consist in minimalistic backbones
with potential to meet the clinical requirements for safe and long-lasting expression
[12]. An alternative approach to sustain prolonged gene expression is the inclusion of
scaffold matrix attachment regions (S/MAR) moieties in mcDNA constructs. Among
others, mcDNA has been proposed as a potential therapeutic strategy for cancer [13].
3.1.2 Messenger RNA
Messenger RNA (mRNA) is the template for the synthesis of proteins. The use of
synthetic mRNA to produce a desired protein in cells is a very promising technology to
apply in clinic. Contrary to plasmid DNA, mRNA-based therapeutics are still in their
infancy, in spite of important advantages. mRNA must only be delivered to the

cytoplasm where cellular translation machinery is located. From a therapeutic perspec-
tive, protein expression arising from mRNA is more transient than that from DNA.
From a safety point of view, mRNA does not integrate into the genome and so poses no
risk of insertional mutagenesis [14]. Moreover, mRNA gene therapy circumvents the
need for selecting a specific promoter, and thus the transfection process is relatively
efficient and simple [15, 16]. Another advantage of mRNA refers to the production
process, raw material synthesis, and the quality product, which are more easily stan-
dardized than that for DNA, that leads to higher reproducibility [17].
Foreign RNA possesses inherent immune-activating adjuvant properties, and this
effect has been studied for the intracellular delivery of mRNAs coding for specific
antigens, with potential application in cancer immunotherapy [18], prophylactic
vaccines [19], and allergy tolerization [20].
Since the protein expression profile of mRNA and DNA is completely different,
the codelivery of these two nucleic acids has been proposed to take advantage of
both [6].
3.2 Gene Silencing

3.2.1 Antisense Oligonucleotides
Single-stranded antisense oligonucleotides (ASO) are short sequences of modified

DNA or RNA that can be used as therapeutic tools through (1) activation of RNase H to
achieve specific knockdown of the target transcript or (2) modulation of pre-mRNA
splicing to enable the restoration of a (partially) functional protein or alternatively a
protein with reduced toxicity [21]. The ASO’s unprecedented specificity for transcripts
makes them unique as a therapeutic entity as it allows very specific targeting and
provides the opportunity to, for example, correct genetic defects for rare genetic
diseases with a current unmet medical need, modulate splice defects in autoimmune
or neurodegenerative diseases, or target transcripts expressed by tumors or viruses.
The utilization of synthetic ASOs is increasing in areas ranging from clinical
diagnostics to novel pharmaceutical therapeutics, and the efficacy and safety of
ASOs are being investigated for the treatment of various genetic disorders where
no treatment is currently available. Recently, several first-in-class ASO drugs have
been approved by the FDA or EMA, including mipomersen for the treatment of
familial hypercholesterolemia, eteplirsen for the treatment of Duchenne muscular
dystrophy, and nusinersen for the treatment of spinal muscular atrophy.
3.2.2 Aptamers
Aptamers are short single-stranded RNA or DNA oligonucleotides, normally 15–80

nucleotides, with the capacity to fold in stable three-dimensional structures. These
molecules present very high affinity with nucleic acids through structural recognition
Gene Therapy 327
and bind to them through electrostatic interactions, hydrogen bonding, van der
Waals forces, base stacking, or a combination of them [22].
Aptamers recognize and bind targets of interest just like antibodies and have
important advantages over conventional antibodies: (1) easy to synthesize by auto-
mated methods; (2) easy to modify to improve the stability, binding strength, and
specificity to the target nucleic acid; (3) structure very flexible; and (4) display low
to no immunogenicity when administered in preclinical doses 1,000-fold greater
than doses used in animal and human therapeutic applications [23].
Due to the molecular recognition of their targets, aptamers have a variety of
diagnostic and therapeutic applications, such as biosensors and target inhibitors. Due
to simple preparation, easy modification, and stability, aptamers have been used
in diverse areas within molecular biology, biotechnology, and biomedicine [24].
However, up to now, the introduction of aptamers into the market has not been very
successful, and only one aptamer-based product have been approved for clinical use,
Macugen® (pegaptanib), for the treatment of age-related macular degeneration.
3.2.3 RNA Interference
RNA interference (RNAi) is a posttranscriptional mechanism of gene silencing

through chromatin remodeling, inhibition of protein translation, or direct mRNA
degradation. RNAi is a naturally occurring process of gene regulation present in
plants and mammalian cells, and it can be used to downregulate disease-causing
genes. Typically, there are three different types of commonly used RNAi molecules
[25]:
– MicroRNA (miRNA)
– Short interfering RNA (siRNA)
– Short hairpin RNA (shRNA), also called expressed RNAi activators
miRNA
miRNA and their role in regulating normal physiological processes were discovered
in the last decade, as well as their involvement in pathological disorders such as
cancer [26]. They are noncoding RNA molecules of 18–25 nucleotide in length
that regulate at posttranscriptional level the expression of genes by binding to the
30 -UTR of target genes [27]. A miRNA can regulate different mRNAs, because
they are not specific to a single mRNA [28]. miRNA is transcribed from DNA as
primary miRNA (pri-miRNA), which is later processed into a precursor miRNA
(pre-miRNA) by two proteins: Pasha and Drosha. The pre-miRNA is transported to
the cytoplasm, where it is processed by Dicer to obtain the miRNA, which is
incorporated into the RNA-induced silencing complex (RISC), where a helicase
unwinds the miRNA. The resulting antisense stand guides the RISC to its comple-
mentary mRNA, which is cleaved (Fig. 2).
Fig. 2 Schematic representation of the mechanism of action of miRNA. Pri-miRNA is transcribed

from DNA and is later processed into pre-miRNA. In the cytoplasm, the pre-miRNA is processed
to give the miRNA, which incorporates into the RNA-induced silencing complex (RISC), where a
helicase unwinds the miRNA. Finally, the resulting antisense strand binds to its complementary
mRNA and cleaves it
miRNAs have emerged as key players in a wide array of biological processes,

and changes in their expression and/or function have been associated with plethora
of human diseases, such as myocardial infarction and stroke [29], Parkinson’s
disease [30], or cancer. The application of miRNA in cancer therapy is based on
the finding that miRNA expression is deregulated in cancer tissues and also due
to the ability of miRNA to target multiple genes and alter cancer phenotypes [31].
In fact, in neoplastic diseases, miRNA can be downregulated when they function
as tumor suppressors or overexpressed when they function as oncogenes [32].
siRNA
siRNAs are short double-stranded RNA segments with 21–23 nucleotides and are
complementary to the mRNA sequence of the protein whose transcription is to be
blocked. siRNA molecules are incorporated into the RISC complex, which bind to
the mRNA of interest and stimulate degradation of mRNA or the suppression of the
translation process [22]. Figure 3 shows the mechanism of siRNA.
The main advantage of synthesized siRNA is that these molecules do not need to
reach the nucleus to induce the therapeutic effect. As a drawback, stability must be
improved in order to optimize the efficacy [22].
Gene Therapy 329
Fig. 3 Schematic representation of the mechanism of action of siRNA. The molecules of siRNA
are incorporated into the RNA-induced silencing complex (RISC), where a helicase unwinds
it. Finally, the resulting antisense strand binds to its complementary mRNA and cleaves it
Because of their small size and low potential to elicit adaptive immune responses,
several antihuman immunodeficiency virus (HIV) RNAs have advanced to clinical
trials. A potential advantage of anti-HIV-1 siRNAs over current therapies is that their
sequences could be tailored to target a patient’s particular viral strains and provide
a personalized approach to therapy [33]. A major challenge for the development
of anti-HIV-1 siRNAs is that lymphocytes, which represent the major cell type for
HIV-1 replication, are widely distributed in the body and extremely difficult to
penetrate with existing siRNA delivery technologies [34]. Other viral infections
with limited treatment options and more easily accessible target that could be treated
with siRNA include hepatitis B virus, Ebola, and respiratory syncytial virus
[29]. Other indications of siRNA under clinical investigation are hepatocellular
carcinoma, hepatic fibrosis, dry eye syndrome, melanoma, and pancreatic ductal
adenocarcinoma.
At present there is a siRNA-based therapy (patisiran) recently approved by
the FDA and the EMA for the treatment of hereditary transthyretin-mediated amy-
loidosis, a rapidly progressive, heterogeneous disease caused by the accumulation of
misfolded transthyretin protein as amyloid fibrils at multiple sites and characterized
by peripheral sensorimotor neuropathy, autonomic neuropathy, and/or cardiomyop-
athy [35]. Another product, inclisiran, is an experimental therapeutic agent for the
treatment of hypercholesterolemia, which is being tested in late-stage clinical trials.
Fig. 4 Schematic representation of the mechanism of action of shRNA. The molecules of shRNA
are transcribed in the nucleus and are exported to the cytoplasm, where the complex Dicer processes
them to form a double-stranded siRNA. The siRNA is later incorporated into the RNA-induced
silencing complex (RISC), where a helicase unwinds it. Finally, the resulting antisense strand binds
to its complementary mRNA and cleaves it
Short Hairpin RNA
Short hairpin RNA (shRNA), also called expressed RNAi activator, is a plasmid-
coded RNA that needs to be transcribed in the nucleus to downregulate the expres-
sion of a desired gene. It can be transcribed through either RNA polymerase II or III.
The first transcript generates a hairpin-like stem-loop structure and is then processed
in the nucleus by a complex containing the RNase II enzyme Drosha. The individual
pre-shRNAs generated are finally transported to the cytoplasm by exportin 5. Once
in the cytoplasm, the complex Dicer processes the loop of the hairpin to form a
double-stranded siRNA [36]. Figure 4 features a scheme with the mechanism of
action of shRNA. Since shRNA is constantly synthesized in the target cells, more
durable gene silencing is achieved in comparison to other forms of RNAi [37].
shRNAs represent an important tool in the assessment of gene function in mammals
and are largely used as a research tool. Although shRNA has been assayed to
develop new therapies for retinal diseases [38], viral infections [22, 33], or cancer
[39], no therapeutic product based on shRNA has been approved.
Gene Therapy 331
3.3 Gene Editing
Gene-editing technology has recently emerged as a new treatment modality for

a variety of diseases, including hereditary, infectious, and neoplastic diseases. This
technology is based on programmable nucleases, which consist of a nuclease that
can be reprogrammed to cleave a precise target sequence [40, 41]. Consequently,
they induce a double-strand break (DSB) at a specific and desired location.
There are four types of gene-editing nucleases: meganucleases, ZFN, TALEN,
and CRISPR/Cas9 [36]. Meganucleases, also called homing endonucleases,
stimulate the cellular recombination and repair of DNA to fix the break by simply
copying the gene encoding them (the homing endonuclease gene) and flanking DNA
into the broken chromosome [42]. ZFN contains zinc finger proteins, the most
common class of DNA-binding proteins across all of biology, and FokI nuclease
as the DNA-binding and cleavage domains. ZFNs have now been widely used for
genome editing in many species and cell types for basic science, biotechnology, and
medical applications, such as targeted disruption of the CCR5 gene for HIV-1
therapy [43]. One important disadvantage of ZFN is that it is an expensive and
time-consuming technology. TALEN use the same FokI-derived nuclease domain as
ZFN, but differ in that they employ distinctive DNA-binding arrays: TALE effector
repeat arrays [44]. TALEN technology for the recognition of a wider range or target
gene sequences requires complicated engineering that is a matter of concern. The
CRISPR/Cas9 system is based on a prokaryotic antiviral mechanism in which
the bacteria insert a partial gene sequence from an infection source, such as bacte-
riophage, into their own genomes to defend against repeat infection [45]. This
system includes a RNA guide to bind to a complementary sequence in a target
gene, which is recognized and cut by the Cas9 [46]. Once the target gene is cleaved
by the programmed nuclease, the reparation of the DSB can be done by two different
mechanisms: nonhomologous end joining (NHEJ) and homology-dependent repair
(HDR) [43]. In the NHEJ, the target region is eliminated by joining the DSB, and it
can be used to silence or correct a pathogenic gene. On the contrary, with the HDR
modality, a homologous sequence can be introduced into the DSB, enabling donor
DNA to be inserted to either correct an existing gene or add a new one. Figure 5
shows the different modalities of DSB reparation.
Contrary to gene augmentation and suppression, therapeutics based on gene
editing can lead to a permanent effect at the genome, and therefore, this recent
technology represents a key development of gene therapy [37].
In the last years, there has been tremendous progress in gene editing, mainly
with CRISPR/Cas9, thanks to the simplicity of the manufacturing procedures in com-
parison to the earlier tools, meganucleases, ZFN, and TALEN. However, it is important
to remark that in comparison to standard gene transfer approaches, genome editing,
particularly that based on CRISPR/Cas9, is in its translational and clinical infancy.
In spite of that, several clinical trials with gene-editing technologies have been completed
or are undergoing [47]. For instance, engineering ZFN have been tested in clinical trials
to disrupt CCR5 (C-C motif chemokine receptor type 5) expressed in human T cells and
Cell with defective gene
Non-homologous
[ ± ]
Homology-directed repair
(HDR) end joining
(NHEJ)
Gene transfer of nuclease ± new DNA
X
Correction Addition Knock-down
Functional allele following

Diseased cell Corrected cell Non-functional allele Functional allele
targeted gene insertion
Fig. 5 Schematic representation of the different modalities of gene repair with nucleases
hematopoietic stem cells to provide to them resistance to human immunodeficiency virus

(HIV) infection [48]. Other clinical trials with ZFN have been approved for delivering
the factor IX gene for hemophilia, the α-iduronidase gene for mucopolysaccharidosis I,
and the iuronidate-2-sulfatase gene for mucopolysaccharidosis II. The first-in-human use
of TALEN gene-edited T cells in two infants with refractory relapsed B-cell acute
lymphoblastic leukemia led to a successful induction of molecular remission ahead of
allogeneic stem cell transplantation [49].
The rapid technological advances in genome editing have allowed manipulating
germ cells, gametes, zygotes, or embryos. In a recent study [50], the CRISPR/Cas9
technology repaired in an embryo DSBs induced at the mutant paternal allele,
by predominantly using the homologous wild-type maternal allele instead of a synthetic
DNA template. The authors were able to avoid mosaicism in cleaving embryos and
achieve a high yield of homozygous embryos carrying the wild-type MYBPC3 gene
(whose mutation causes hypertrophic cardiomyopathy), without evidence of off-target
mutation. In spite of the potential use for the correction of heritable mutations in human
embryos, much remains to be considered before clinical applications, including the
reproducibility of the technique with other heterozygous mutations.
Human application of these technologies still presents a substantial challenge.
Collaboration between regulatory bodies and the scientists developing these life-
changing treatments is very important for gene editing to progress to the clinic, with
special emphasis to the critical assessment of risk vs benefit [41]. Among all the
potential applications of gene editing, it is impossible to predict which approaches
will ultimately be successful, both in the clinic and on the market, but it is expected
that the application of gene-editing technologies is poised to change the practice of
Gene Therapy 333
medicine dramatically in the years to come [51]. The long-term follow-up of patients
who will participate in genome-editing clinical trials will likely provide invaluable
insight into the in vivo activity and specificity of programmable nucleases.
4 Delivery Systems Used in Gene Therapy
One of the main challenges of gene therapy is the development of safe and effective
administration systems that are able to overcome the main limitations of nucleic
acids when they are administered in the body [52]. Therefore, a fundamental aspect
for the success of gene therapy is the availability of delivery systems capable of
protecting the genetic material from degradation, facilitating its internalization in
target cells, and releasing them intracellularly. The ideal delivery system depends
on the target cells, the kind of nucleic acid to be delivered, and the duration of
expression [53]. The delivery systems are classified into two large groups: viral
and non-viral vectors.
Viral vectors are prepared from genetically modified viruses so that they are not
able to replicate in the target cells, but they express the therapeutic gene they
transport. Viral vectors allow high transfection efficiencies; however, they present
important safety limitations due to the oncogenic and immunogenic potential (due
to viral proteins). Another problem associated with viral vectors is the inability to
transport large nucleic acids.
Non-viral vectors are safer, simpler, cheaper, and more reproducible systems.
In addition, they do not present limitations regarding the size of the genetic material
they can incorporate. Nevertheless, a disadvantage of non-viral systems is that their
transfection efficiency is lower compared to viral vectors, although in recent years
new non-viral systems have been developed with materials that exhibit higher
transfection efficiencies. In fact, the number of clinical trials with products based
on non-viral vectors (Fig. 6) has increased in the last decade, and those based on lipid
nanocarriers (lipofection) are used in 4.1% of all trials [54].
Fig. 6 Vectors used in gene therapy clinical trials. Data consulted in gene therapy clinical trials
worldwide [54]
Table 1 Features of the most studied viral vectors in gene therapy

Retroviruses Lentiviruses Adenoviruses AAV
Viral genome RNA RNA DNA DNA
Target cells Dividing Dividing and Dividing and Dividing and
cells nondividing nondividing nondividing
cells cells cells
Integration in the host Yes Yes No Yes
genome
Response Long-term Long-term Short-term Long-term
Size of the genetic 8 kb 8 kb 7.5–30 kb 4.5 kb
material to be carried
AAV adeno-associated viruses
Fig. 7 General structure of retroviruses and retroviral genome. LTR long terminal repeats, pbs
first binding site, ppt poly-purine sequence, ψ packaging signal
4.1 Viral Vectors
Viruses used as delivery systems of genetic material include adenoviruses, adeno-

associated viruses (AAV), retroviruses, and lentiviruses among the most evaluated
in clinical trials. Other viruses, such as those derived from herpesvirus or poxvirus,
have also been studied as possible viral vectors.
The selection of the most suitable viral system in each case depends on different
factors: the organ or the target cell, the ability to integrate the genetic material carried
by the vector in the genome of the host cell, the duration of expression gene
over time (short-term or long-term response), or the size of therapeutic nucleic
acid. Table 1 shows the main characteristics of the most frequently studied viral
vectors, to be taken into account for their application in gene therapy.
4.1.1 Retroviral Vectors
Retroviruses are RNA viruses that contain two strands of RNA enveloped by
an icosahedral capsid of peptide nature (Fig. 7). The capsid is surrounded by a
phospholipid envelope, in which different types of glycoproteins act as ligands for
specific receptors on cell surfaces and therefore determine the tropism of the virus.
Gene Therapy 335
The genome of retroviruses (Fig. 7) contains three types of genes: gag genes
that encode capsid proteins, pol genes that encode the enzymes necessary for the
replicative cycle of the virus (protease, integrase, reverse transcriptase), and env
genes that encode the envelope glycoproteins. This genome also contains a packag-
ing signal, ψ, thanks to which the RNA molecules bind to the capsid proteins and are
effectively packaged, and the long terminal repeats (LTR) at each end of the viral
genome. The left LTR contains a region for the start of transcription (U3 promoter)
and a first binding site (pbs) for the start of reverse transcription. The right LTR
contains a poly-purine sequence (ppt) for replication of the second strand. For
application in gene therapy, the retroviral vectors are generated by the substitution
of the gag, pol, and env sequences of the viral genome by the therapeutic gene. As
a consequence, the therapeutic sequence in this case cannot be greater than 7–8 kb.
The replicative cycle of a retrovirus begins with entry into the cell, which is
mediated by receptors [55]. Once inside the cell, the viral reverse transcriptase
enzyme produces a DNA molecule from the viral RNA. Subsequently, the DNA
is integrated into the genome of the host cell, and the transcription yields different
RNAs, which are exported to the cellular cytoplasm, where they are translated into
structural proteins of the virus, and directs synthesis of new virion nucleocapsids.
The nucleocapsids leave the cell and keep enveloped by a plasma membrane-derived
outer coat.
It is important to point out that the DNA obtained by reverse transcription
from the RNA is not able to cross the nuclear membrane of the cell to be treated.
Therefore, retroviral vectors only transfect efficiently dividing cells, because the
DNA takes advantage of the disruption of the nuclear envelope during the mitosis,
to reach the interior of the nucleus [56].
On the other hand, the integrase enzyme allows the integration of the genetic
material in the genome of the host cell. Thanks to this, it is possible to obtain a long-
lasting expression of the therapeutic sequence; however, the insertion into the
genome of the target cell is also one of the major problems of viral gene therapy,
since if it takes place in an unwanted region of the genome of the transfected cells,
there is a risk of mutagenesis and oncogenesis. In fact, in clinical trials with these
vectors, several patients developed leukemia and dysplasia of the bone marrow due
to insertional mutagenesis [57, 58]. These adverse effects were partially reduced by
the design of so-called self-inactivating vectors in which the genome sequences of
the virus identified as responsible for mutagenesis, 30 LTR, are deleted. This idea for
self-inactivating vectors had been already described in the literature by Yu et al. [59].
Another limitation of retroviral vectors is that they are recognized and inactivated
by the complement system, which means that they are mainly used in ex vivo gene
therapy.
Despite the mentioned limitations, and due to its high transfection efficiency,
since 1989, the year in which the first clinical trial with gene therapy was launched,
514 clinical trials with retroviral vectors have been started (17.5% of the total of
clinical trials with gene therapy) [54].
One of the most commonly used retroviruses in gene therapy is the murine
leukemia virus (MLV), which has shown efficacy in different types of
immunodeficiency. In fact, recently the European Medicines Agency (EMA) has

approved a drug based on ex vivo gene therapy with this retroviral vector for the
treatment of patients with severe combined immunodeficiency due to adenosine
deaminase (ADA-SCID) deficiency that cannot be treated with bone marrow trans-
plant (Strimvelis; Orchard Therapeutics) [60].
4.1.2 Lentiviral Vectors
Lentiviruses also belong to the Retroviridae family. However, they have certain
differential genes with respect to the rest of retroviruses that facilitate the entry
of genetic material into the cell nucleus, so lentiviral vectors can also transfect
nondividing cells. One of the most known and studied viruses of this subfamily
is the human immunodeficiency virus (HIV).
The general structure of lentiviruses is the same as that described for retroviruses:
two RNA strands included in a protein capsid, surrounded by a phospholipid
envelope that includes different types of glycoproteins.
The genome of the lentiviruses (Fig. 8) shares with the retroviruses the gag, pol,
and env genes, as well as the LTR, pbs, and ppt sequences. However, it presents
other specific genes: tat genes (transcription regulators), rev genes (regulators of the
expression of viral proteins), vif genes (necessary for the infection of different
cell types), vpr genes (participate in the entrance to the nucleus), vpu genes (involved
in the release of viral particles from infected cells), and nef genes (increase the
infectivity of the virus).
The cycle of life is similar to that described for retroviral vectors, with the
difference that lentiviruses present genes encoding nuclear localization signals that
favor the entry of DNA (synthesized by the reverse transcription process) into the
nucleus.
In order to use lentiviruses as gene delivery systems, the tat gene is removed, and
the gag and pol genes are encoded on a different plasmid from that of the rev or env
genes. The final vector results from three separate plasmids containing the necessary
viral sequences for packaging [61]. In addition, the 30 LTR sequence of the viral
genome may be deleted to generate self-inactivating (SIN) lentiviral vectors [62],
as previously mentioned in the case of retroviral vectors.
Fig. 8 General structure of lentiviral genome. LTR long terminal repeats, pbs first binding site,
ppt poly-purine sequence, ψ packaging signal
Gene Therapy 337
Due to the tropism of lentiviruses and their ability to transfect cells that are not in
division, the main application of lentiviral vectors is directed to introduce genetic
material in vivo in cells of the central nervous system [63], for example, for the
treatment of Parkinson’s disease [64] or in cells of the retina [65] (suitable for
the treatment of retinitis pigmentosa). Furthermore, lentiviral vectors efficiently
transfect ex vivo cells of the hematopoietic system, which are difficult to transfect
with other vectors. In fact, ex vivo gene therapy with lentiviral vectors to genetically
modify CD34+ cells has been evaluated in more than 100 clinical trials in recent
years for the treatment of monogenic diseases (i.e., β-thalassemia [66], X-linked
adrenoleukodystrophy [67], metachromatic leukodystrophy [68], or Wiskott-
Aldrich syndrome [69]), of different types of cancer [70], and of infectious diseases
such as HIV infection [71].
4.1.3 Adenoviral Vectors
Adenoviruses encompass more than 50 different virus serotypes and are responsible
for 5–10% of respiratory infections in children and adults. In gene therapy, serotypes
2 and 5 are the most used. Adenoviruses are non-enveloped viruses that consist of
a double strand of DNA within an icosahedral capsid (Fig. 9).
The genome of an adenovirus (Fig. 9) has a size of approximately 35 kb. At the
ends are the inverted terminal repeats (ITR), and close to the left ITR, the packing
signal, ψ, is arranged. The numerous genes it contains differ in the early (E) and
late (L) regions. The latter, responsible for the coding of structural proteins, are
transcribed after replication of the viral genome, and the E regions are transcribed
before synthesizing the viral DNA, since they give rise to regulatory proteins. These
proteins alter the expression of host cell proteins that are necessary for DNA
synthesis, intervene in viral replication, and also prevent the death of infected cells
by blocking the apoptosis or avoiding recognition by the immune system.
Adenoviruses are endocytosized into the cell after binding to the CAR receptor
(coxsackievirus and adenovirus receptor). The nucleocapsids are released into the
cytoplasm, and with the help of cellular microtubules, they reach the nuclear
membrane, and the genetic material is introduced to the nucleus of the cell through
Fig. 9 General structure of adenoviruses and adenoviral genome. ITR inverted terminal repeats,
ψ packaging signal
the nuclear pores. Replication then begins, and once all the components of the virus
have been synthesized, they are assembled and released from the cell by cell
lysis induced by the virus itself. During this process the adenoviral genome does
not integrate into the genome of the host cell. This is one of the advantages of
adenoviruses which makes them safer compared to other viral systems, although this
also means that the viral life cycle is not adapted for long-term transgene expression.
To be used as vectors for gene therapy, E regions are deleted from the genome,
and various levels of attenuation can be achieved by removal of different numbers
of genes: only one E1B gene (first-generation vectors), the majority of early genes
(second-generation vectors), and even full deletion of all genetic information of an
adenovirus (so-called gutless vectors) [72]. The large size of the genome and the
possibility to delete a major part of it provide high coding capacity for these vectors:
1–2 kb can be inserted in early generation vectors and up to 30 kb in gutless vectors.
However, they need another helper virus that replicates normally and expresses all
the proteins needed to assemble the adenoviral vector [73]. Despite its advantages,
the total elimination of impurities from helper viruses is complicated and limits its
clinical application. In fact, at high doses adenoviruses are toxic [74], and one of
their main limitations is that they are very immunogenic [75], which decreases their
effectiveness.
In spite of their limitations, the advantages of adenoviral vectors have meant that
they have been evaluated in more than 500 clinical trials [76], most of them aimed at
the treatment of cancer.
4.1.4 Adeno-Associated Viral (AAV) Vectors
Adeno-associated viruses (AAV) are small non-enveloped viruses with single-

stranded DNA (Fig. 10). The genome has a size of about 4.7 kb and is composed
of three regions, called rep, cap, and aap, flanked by the corresponding ITR. The
rep region encodes nonstructural proteins involved in viral replication, packaging,
and integration into the host genome, genes in the cap region encode the structural
proteins of the capsid, and aap gene encodes the assembly-activating protein [77].
AAV vectors for clinical application are generated by replacing the rep, cap, and aap
genes with the gene of interest.
The entry of AAV into the cell takes place by endocytosis after binding to the
receptor-mediated cellular surface. Once the nucleocapsid escapes from the
Fig. 10 General structure

of AAV and AAV genome.
ITR inverted terminal
repeats
Gene Therapy 339
Table 2 AAV serotypes suitable for specific target tissues [79]

Serotype Target tissue
AAV1 Nervous system, skeletal muscle
AAV2 Nervous system, kidney, photoreceptors, retinal pigment epithelium
AAV4 Nervous system, retinal pigment epithelium
AAV5 Nervous system, lungs, photoreceptors, retinal pigment epithelium
AAV6 Skeletal muscle, lungs
AAV7 Skeletal muscle
AAV8 Nervous system, photoreceptors, retinal pigment epithelial, liver, skeletal muscle,
heart, pancreas
AAV9 Nervous system, heart, liver, skeletal muscle, lungs
AAV adeno-associated viruses
endosome and is transported to the nucleus, the viral genome is able to cross the
nuclear membrane. In the nucleus a second strand of DNA, necessary for the
replication of the virus, will be synthesized. In the presence of a helper virus
(coinfection by an adenovirus or herpesvirus), the double-helix DNA generated
can be integrated into the genome of the host cell, and replication of the virus will
take place. In the absence of a helper virus, the AAV genome usually remains latent
in the form of an episome. Replication of the viral genome and subsequent packag-
ing results in the generation of viral particles that will escape from the host cell by
lysis thereof [78].
These vectors are quite safe, can transfect cells with or without capacity of
division, and provide long-term gene expression, up to 6 years. Another advantage
of AAV is the possibility of selecting the most suitable serotype depending on
the target tissue [79]. Table 2 shows the most suitable AAV serotypes for different
tissues.
The main disadvantage of AAV is the limited size of the genetic material they can
transport, which must not exceed 4.5 kb. However, viral vectors based on AAV have
been evaluated in more than 180 clinical trials, most of them aimed at the treatment
of monogenic diseases. In fact, a medicinal product called Luxturna and based on
AAV2 have reached the market. Luxturna (voretigene neparvovec) is a gene transfer
vector that employs an AAV2 as a delivery vehicle for the human retinal pigment
epithelium 65 kDa protein (hRPE65) cDNA to the retina [80]. This medicine is
indicated for the treatment of adult and pediatric patients with vision loss due to
inherited retinal dystrophy caused by confirmed biallelic RPE65 mutations and who
have sufficient viable retinal cells to express the protein and respond to the treatment.
4.1.5 Manufacturing of Viral Vectors
Viral vector production for clinical application requires viral propagation in suitable
animal cell lines, viral recovery, concentration purification, and formulation [81].
To meet commercial and regulatory requirements, each process must be scalable
and reproducible and must yield high virus titers. For large-scale manufacturing,
suspension-adapted cell lines cultured in bioreactors are more appropriate than

adherent cells systems, conventionally used at laboratory scale [82]. Depending
on the viral vector and the cell clone used, the most suitable bioreactor must be
chosen [83].
The production of viral vectors may be carried out by transiently transfecting
the producing cells with vector and helper or packaging plasmids or by generating
stable producer cell lines. In the first case, culture cells are co-transfected
with multiple plasmids, one containing the expression cassette for the transgene
and other plasmids encoding regulatory and structural viral proteins. The main
limitations of transient transfected cells are the amount of transfected cells achieved
and the variability in transfection [84]. In addition, transient transfection results
in contaminations of the final product due to excess plasmids [85] and residual
transfection reagent. In the second case, cells are genetically modified, so that they
contain, inserted in the cellular genome, the genes that encode the structural proteins
necessary for the formation of viral particles containing the transgene of interest
[86]. Processes using stable producer cell lines are easier to scale-up and result in less
contaminated vectors, although some drawbacks have to be also considered: the
gene products necessary to produce vectors are toxic to cells, each vector-produced
cell line requires specific certifications as master cell bank, and, upon cell expansion,
high-titer vectors are not always ensured [84].
One of the limitations of viral vector manufacturing is to purify a sufficient
amount of viral particles even to start a clinical trial. The downstream processing
or purification of viral vectors aims to eliminate contaminants, whether process
or product-related. Process-related impurities derive from starting materials (residual
DNA and residual host cell protein from each cell bank) or raw materials (culture
reagents, purification reagents and equipment materials, helper viruses, and helper
virus nucleic acid used in production), whereas product-related impurities include
vectors with deleted, rearranged, hybrid, or mutated sequences. The ultimate goal
of the downstream processing is to obtain a product with high purity, potency,
and quality that can meet the guidelines of the FDA [87] and EMA [2] regulatory
agencies. With this aim, different concentration methods have been developed:
centrifugation, tangential flow filtration, ultrafiltration, polyethylene glycol precipi-
tation, two-phase extraction, membrane filtration, liquid chromatography, or adsorp-
tion chromatography [88].
4.1.6 Quality Control of Viral Vectors
Another aspect to take into account are the quality controls that these vectors must
overcome to ensure that each batch manufactured meets the specifications of purity,
potency, safety, and identity and there are consistency and comparability between
batches. This is a challenge given the high complexity of the viral systems due to the
large number of protein subunits that make up the viral capsid and the composition
of the lipid membrane present in enveloped viruses. In addition, it is necessary
to develop specific analytical methods for each type of virus and even for each
serotype. These methods can be divided into those that are similar to others already
Gene Therapy 341
validated for recombinant proteins and vaccines and those that are specific to each
vector. The former include, for example, the analysis of impurities related to the
production process, such as packaging cell proteins. Specific assays of viral vectors
include the analysis of the activity of the resulting product of the therapeutic genetic
material, as a measure of potency. It is also necessary to determine the impurities due
to the presence of residual genetic material encapsulated in the vector. In any case,
it must be considered that many of these methods developed to analyze the specific
quality controls of viral vectors are not yet validated according to the standards
established to license and market these products.
4.2 Non-viral Vectors
Non-viral systems can be defined as those physical or chemical methods that

help in the process of transfer of exogenous genetic material to the cell, facilitating
the entry and intracellular bioavailability thereof. Non-viral systems try to imitate the
capacities of viruses as gene transfer vehicles, providing greater security from
the point of view of biological risk and pathogenicity. However, reproducing with
a non-viral system what a virus performs naturally is a great challenge that has led
to the development of different strategies. The selection and design of the most
appropriate non-viral system are conditioned by its efficacy and safety, by the tissue
or target cell, and by the type of therapeutic genetic material [53].
4.2.1 Physical Methods
Physical methods are based on the application of physical forces to temporarily alter
the permeability of the cell membrane, allowing the genetic material to cross the
cytoplasmic membrane and reach the interior of the cell. It is important that there is a
balance between the efficiency of cellular internalization and the damage exerted on
the cell. These methods have been frequently evaluated as systems of administration
of naked genetic material, without the need to formulate it or include it in a viral or
non-viral vector, which gives them great simplicity. Nevertheless, physical methods
are mainly evaluated and used in preclinical studies. Table 3 summarizes the main
characteristics of physical delivery methods used in gene therapy [89].
4.2.2 Chemical Carriers
Chemical vectors are based on the use of different types of compounds capable of
encapsulating or binding, electrostatically or covalently, the genetic material.
In order to develop a suitable non-viral delivery system, the selected vector must
have the capacity to enter the cell and to overcome different barriers maintaining the
stability of the nucleic acid throughout the entire transfection process. Once within
the cell, it must guarantee the proper intracellular distribution of genetic material.
Table 3 Types of physical delivery systems in gene therapy and main features
Method Advantages Limitations
Needle injection Simple Low efficiency
Direct needle injection on a specific tissue Safe Local inflammation
Hydrofection or hydrodynamic injection High efficacy in the Hemodynamic
Intravascular injection of high volumes of a liver changes
solution containing the nucleic acids
Microinjection High efficiency Cell by cell admin-
Direct injection into host cell by microneedles istration
Time-consuming
Need of specialist
Biolistic injection or gene gun Simple and fast Low efficiency
Administration of metal microparticles at high Reproducibility Low tissue penetra-
velocity tion
Cell damage
High cost
Electroporation Noninvasive Risk of tissue dam-
Application of electric pulses that open pores Simple age
on cell membrane High efficiency Surgery necessary to
Low cost target internal
Widely employed organs
Sonoporation Noninvasive Low reproducibility
Application of ultrasounds (combined with Safe Tissue damage
microbubbles or nanocarriers) to permeabilize Targeting to specific
temporally cell membrane tissues
Magnetofection Noninvasive Effective only on
Application of external magnetic fields com- Effective in primary surface areas
bined with magnetic particles cells (difficult to Mainly applied
transfect) in vitro
Optofection Nucleic acids release Tissue damage
Application of laser pulses combined with from endosomes Inflammation
nucleic acid complexes or nanoparticles Restricted to single
cells or small areas
Therefore, the main steps that these delivery systems have to overcome to reach
the cytoplasm (in the case of RNAs) or the nucleus (in the case of DNAs) are the
following: interaction with cell membrane, entry into the cytoplasm, intracellular
distribution, and entry into the nucleus (Fig. 11). To date, different strategies for
overcoming these limitations have been proposed, and the evolution of non-viral
vector transfection has been significantly improved in recent years.
The first step in the genetic transfer process is the interaction between vectors and
cell membranes. Cationic vectors interact electrostatically with the negative charge
of the cell membrane surface, and the internalization process is started. Additionally,
in order to enhance the interaction with specific cells, different ligands can be added
to the vectors to improve binding to surface receptors [90, 91].
Once the vector has bound to the cell surface, it must penetrate into the cyto-
plasm. The internalization or entry into the cell can take place through two mech-
anisms: fusion with cell membrane or endocytosis. These two entry routes are not
Gene Therapy 343
Fig. 11 Main stages during transfection process: (1) interaction with cell membrane, (2) entry into
the cytoplasm, (3) intracellular distribution, and (4) entry into the nucleus. NPC nuclear pore
complex
exclusive, and depending on the type of cell and vector, one or the other may
predominate. However, in most cases vectors penetrate into the cells mainly through
endocytic pathways. Endocytosis begins with the formation of a vesicle from the
invagination of the plasma membrane, called an endosome. These endosomes
fuse with lysosomes by creating endolysosomes, and their hydrolytic enzymes can
degrade genetic material. Therefore, to achieve efficient gene transfer, it is necessary
for the nucleic acid material to be protected from degradation by lysosomes to ensure
release of nucleic acids into the cytoplasm. In fact, the endosomal escape represents
an important barrier to achieve efficient transfection in the case of non-viral gene
therapy [92].
In the case of DNA, once it is cytoplasm, it must be able to enter to the nucleus.
However, the nuclear membrane is a selective barrier for macromolecules, such as
DNA. The transport through this membrane is a highly regulated process, facilitated
by a series of water channels of about 10 nm, called nuclear pore complexes (NPCs).
The genetic material transported by non-viral vectors penetrates the nucleus through
two main routes: NPCs or during cellular mitosis, when the nuclear membrane is
temporally disrupted. The passage through the NPCs is carried out by means of an
energy-dependent process that generally involves the recognition of specific nuclear
localization signals (NLS) [93]. The NLS consist of one or more short sequences
of amino acids with positive charges containing arginines and lysines [94]. The
formulation of DNA with compounds containing NLS is a strategy commonly used
in non-viral gene therapy.
Chemical delivery systems or non-viral vectors are broadly categorized into
inorganic, polymeric, lipidic, or peptidic particles. In many cases, the combination
of some of different kinds of chemical compounds is used in order to improve
their profile of efficiency, cellular specificity, and safety, giving rise to hybrid
systems [53].
Inorganic Particles
Inorganic particles are nanostructured systems with different sizes, shapes, and
porosity, designed to protect the genetic material from degradation and to escape
from the reticuloendothelial system after its systemic administration. They can be
composed of different elements, being used in gene therapy calcium phosphate [95],
silica [96], gold [97], or magnetic compounds such as iron oxide [98].
These inorganic particles are of interest since they are easy to produce and
ligands can be added to their surface that facilitate the union to the genetic material
through electrostatic interactions. Cationic components are usually incorporated to
the surface of the particle. An example of this type of system consists of combining
iron oxide particles with PEI, which favors the condensation of nucleic acids, with
polyethylene glycol (PEG), which favors the colloidal stability of the particles,
and with cell penetration peptides, which favor cellular internalization [99]. In the
case of gold particles, nucleic acids are previously thiolated to covalently bound to
the delivery system [100].
Other types of inorganic materials used to develop inorganic particles that are
showing encouraging results in vitro and in vivo in animal models are graphene
[101] or fullerene [102]. However, it is still necessary to study in greater depth the
long-term safety and the influence of functionalization, size, and shape in transfec-
tion efficiency to facilitate the clinical application of these newer compounds.
Polymeric Particles
The main component of these vectors is a cationic polymer that binds and condenses
the genetic material, giving rise to the so-called polyplexes [103]. Cationic polymers
bind by electrostatic interactions the negatively charged genetic material, so that
the nucleic acid is adsorbed to the surface of the nanoparticulate system or is
encapsulated in its interior. In addition, these systems allow the incorporation
of different ligands that improve the transfection efficiency in the target tissue.
In general, polymeric vectors are more stable than lipid vectors, and even in some
cases, the progressive degradation of the polymer allows controlling the rate of
release of the genetic material once it is inside the cell.
The polymers used in the preparation of non-viral vectors can be subdivided into
synthetic and natural polymers (also called biopolymers).
Gene Therapy 345
The synthetic polymers most used in gene therapy are polyethyleneimine (PEI),
the dendrimers [104], the polyesters (i.e., poly(lactic-co-glycolic) or PLGA) [105],
or polymethacrylates [106]. Among them, PEI has been evaluated in various clinical
trials for the treatment by local gene therapy of different types of cancer [107], but
the high toxicity of this polymer has limited its application.
In the group of the biopolymers applied in gene therapy are polysaccharides,
such as chitosan, cyclodextrins, alginate, pullulan, or dextran. Some of these poly-
saccharides are used by themselves as delivery systems, but most of them are
generally used in combination with other non-viral vectors to improve their efficacy,
safety, or biodistribution [90, 108, 109].
Lipidic Particles
Lipid-based systems are the most studied non-viral vectors at the clinical level.
Up to 119 clinical trials have been documented, most of them in phases I and II, in
which lipid vectors have been used as delivery systems for the genetic material. In
most cases, vectors have been designed for the treatment of different types of cancer
but also for the treatment of cardiovascular diseases, hepatitis C virus infection, or
monogenic diseases such as cystic fibrosis [54]. Recently, a lipid-based siRNA
delivery system called patisiran (Alnylam® Pharmaceuticals) has reached the
market, as treatment of hereditary transthyretin-induced amyloidosis [35].
The main components of the lipid-based vectors are cationic lipids, formed
by hydrophobic alkyl chains, linked through an intermediate binding structure
to a polar group. The most used cationic lipids are 1,2-di-O-octadecenyl-3-
trimethylammonium propane (DOTMA), 1,2-dioleoyloxy-3-trimethylammonium
propane (DOTAP), 1,2-dimyristyloxypropyl-3-dimethyldhydroxyethylammonium
bromide (DMRIE), or 3ß-[N-(N0 , N0 -dimethylaminoethane)-carbamoyl]-cholesterol
(DC-cholesterol), although derivatives of these lipids are also being studied in order
to improve their efficacy and safety [110]. Thanks to their cationic nature, these
lipids are able to condense and protect the genetic material, as well as to bind to
the negative charges of the cell membranes. The main limitations of non-viral
vectors based on cationic lipids are the low efficacy in vivo due to the fact that
they are not stable and that they undergo rapid clearance, as well as the possibility
of generating inflammatory or anti-inflammatory responses.
Cationic lipids can be used by themselves to form complexes, known as
lipoplexes, by mixing them directly with the negatively charged genetic material,
but they are normally used to prepare colloidal systems that are then bound to
the genetic material to obtain the lipoplexes. The preparation of these colloidal
systems can involve other lipid components, which may improve the transfection
efficiency of cationic lipids, such as the phospholipid 1,2-dioleoyl-sn-glycero-3-
phosphoethanolamine (DOPE), which has fusogenic function and facilitates
endosomal escape, or polyethylene glycol (PEG), which forms a steric coating
that makes vectors more stable in vivo. The colloidal lipid systems used in gene
therapy are liposomes, nanoemulsions, and solid lipid nanoparticles (SLNs) [92].
Nanoemulsions consist of a dispersion of an oil phase stabilized in an aqueous
phase by means of a third component that acts as a surfactant, so that droplets of
about 200 nm are formed. From the technological point of view, nanoemulsions
are simple to manufacture, and they are very stable during storage. In spite of this,
the application of cationic nanoemulsions in gene therapy is still quite limited [111].
SLNs are spherical particles in the range of nanometers, formed by a core
composed of a solid lipid at room temperature surrounded by a layer of surfactants.
In the case of SLNs designed to be applied in gene therapy, cationic lipids exert part
of the surfactant effect and, in turn, confer positive charge to the surface of the
particles. SLNs have shown efficacy as systems for administering different types of
genetic material at preclinical level in vitro and in vivo, after their systemic or
local administration, showing promising results especially in ocular pathologies
[112–115], as well as in infectious diseases [37], lysosomal storage disorders
[116], and various types of cancer [92].
Liposomes are spherical vesicles composed of one or more lipid bilayers sur-
rounding an aqueous core, which show a size ranging from 20 nm to a few microns.
Cationic liposomes are effective transfection systems in very varied types of cells
in vitro and also in vivo after their local or systemic administration. In fact, in most
clinical trials using lipid vectors, these are cationic liposomes.
Peptidic Particles
Some peptides are capable of condensing nucleic acids by themselves resulting in

the formation of nanoparticulate systems. These include cationic peptides composed
of short sequences of positively charged amino acids such as histidine, arginine, or
lysine; in fact, poly-L-lysine is one of the peptide vectors with the highest transfec-
tion efficiency [117]. Proteins of natural origin, such as collagen or albumin [118],
are also used as peptide vectors. In addition, it is very common to use peptides as
ligands to functionalize some of the previously described polyplexes and lipoplexes.
In this sense, peptides may be used to target non-viral vectors to a specific tissue
(i.e., transferrin to target tumor tissue or hepatocytes [119] or RGD (arginine-
glycine-aspartic acid) sequences to target specific tissues [120]). Another application
of peptide as ligands is to improve their effectiveness by helping the genetic material
to overcome some of the barriers of the transfection process: cell penetrating
peptides [121] to improve cell entry, fusogenic peptides [122] to increase endosomal
escape, or NLS [94] to entry into the nucleus.
Manufacturing and Quality Control of Non-viral Vectors
The production of nanoparticles for clinical gene therapy presents important hurdles,
which are still hampering the translation from laboratory to patients. Non-viral
Gene Therapy 347
vectors are complex formulations that must be customized depending on the nucleic
acid to be delivered, the variety of target diseases, and the administration route
[107]. Due to their complexity, nanoparticulate systems show unique Chemistry,
Manufacturing and Controls (CMC) challenges [123]. In fact, suitable methods for
large-scale production of simple nanosystems, such as liposomes, have been devel-
oped [124]. However, when formulation becomes more complex, for example, with
the addition of surface modification or ligands, the number of steps in the production
process and the cost of the final product increase, and quality control is also
more difficult [125].
Regarding quality attributes, parameters that must be especially considered
because of their impact on biological yield are size, shape, surface charge, presence
of ligands to provide effective targeting, surface modification with PEG, impurities
associated to starting materials and the production process, and stability during
manufacturing and long-term storage and upon administration [126]. Batch-to-
batch variability of non-viral vectors can potentially led to changes in all these
parameters. Therefore, small changes in manufacturing process variables (such as
temperature, pH, time, agitation speed, quality of starting materials, etc.) can signif-
icantly affect the quality, efficacy, and safety of the final vector [127]. It is important
to stablish procedures to assess nanotherapeutics not only at final steps but also at
intermediate ones. Moreover, the application of concepts of quality by design (QbD)
based on quality guidelines introduced by the International Conference on
Harmonisation (ICH Q8, Q9, and Q10 [128]) has been proposed to address questions
related to manufacturing processes and CMC complexities [123, 127]. The aim of
QbD fundamentally aims at building quality and safety from the first design steps
of the product [129]. This methodology intends for establishing a multidimensional
design that defines process input requirements and operational ranges necessary
to ensure that the product meets critical quality attributes. Designers of new
nanotherapeutics will gain an understanding of these concepts and the role their
preliminary data plays in preparing and positioning a potential nanoparticulate
system for a gene therapy product development.
5 Applications of Gene Therapy
The first major clinical advance in the state of the field of gene therapy was in 1990,
when the adenosine deaminase (ADA) gene was administered to a 4-year-old girl
to treat the severe combined immunodeficiency (SCID) she suffered [130]. This
clinical trial fostered the launching of additional studies, one of them in the year
2000 for patients with the X-linked form of SCID [131], which was an important
landmark for gene therapy. On the one hand, it provided for the first time a
demonstration of therapeutic effect of gene transfer for the treatment of a genetic
disease. On the other hand, two of the patients developed T-cell leukemia as a result
of insertional oncogenesis related to the retroviral vector used [57], which dampened
the perspectives on gene therapy. Nonetheless, other potential hazards derived
Fig. 12 Indications addressed by gene therapy clinical trials [54]
from the use of viral vectors to administer the genetic material were already known.
In 1999, a systemic inflammatory response to the dosage of adenoviral vector
administered into the right hepatic artery for the treatment of ornithine
transcarbamylase (OTC) deficiency caused the death of Jesse Gelsinger, an
18-year-old male with partial OTC deficiency who participated in a pilot (safety)
study of gene therapy [74].
Gene therapy has survived its previous failures, and it has emerged, thanks to the
improvements of viral and non-viral vectors, the management of immune reactions,
and the use of new mechanisms of action. Actually, a large number of clinical trials,
almost 2,700 undertaken in 38 different countries, have been approved globally
since 1989, and as can be seen in Fig. 12, most of them addressed cancer [54].
The extensive research activity in this field has not led to a significant number
of gene therapy-based approvals. Since 2003, when Gencidine®, indicated for the
treatment of head and neck squamous cell carcinoma, received approval in China
as the first gene therapy medicinal product (GTMP) marketed worldwide [132],
only 15 new products have been approved. However, GTMPs are becoming an
emerging and expanding class of innovative medicinal products that can offer a more
specific and causal/targeted treatment of many rare diseases, including rare cancers
[133]. Gene therapy may be initially approved for patients who are lacking other
therapeutic options, including conditions that in absence of treatment can cause
disability or early death and conditions that require intensive and onerous mainte-
nance therapy in form of enzyme or protein replacement. For these patients, gene
therapy could offer long-term stabilization or improvement of their health, with the
ultimate objective of obtaining a cure [134]. Table 4 shows nucleic acid-based
products, including antisense oligonucleotides (ASOs) and gene-engineered cells,
commercialized until present [135–137].
Apart from the five gene therapy products approved (Gencidine®, Oncorine®,
Glybera®, Imlygic®, and Luxturna®), products based on ASOs, small interfering
RNAs (siRNA), or aptamers have been also authorized, which have yet to exert a
profound influence on the biopharma product landscape.
Kymriah®, Yescarta®, Zalmoxis®, and Strimvelis™ may be categorized as both
cell and gene therapies. In all these cases, genetic modification is undertaken ex vivo
Gene Therapy 349
Table 4 Nucleic acid-based products approved

Year/
agency
(first Indication/
Product approval) Company administration route Strategy/vector
Vitravene® 1998/ Isis CMV retinitis in In vivo – ASO
(fomivirsen) FDA Pharmaceuticals AIDS patients/
intravitreal injection
Gencidine® 2003/ SiBiono Head and neck In vivo – gene
SDFA GeneTech carcinoma/ augmentation/
intratumoral AD5
Macugen® 2004/ EyeTech Wet form of In vivo – aptamer
(pegaptanib) FDA AMD/intravitreal
injection
Oncorine® 2005/ Sunway Nasopharyngeal In vivo –
SDFA Biotech cancer/local oncolytic virus/
AD5
Glybera® 2012/ UniQure Lipoprotein lipase In vivo – gene
(alipogene EMA deficiency/ augmentation/
tiparvovec) intramuscular AAV1
Kynamro® 2013/ Kastle Familial hypercholes- In vivo – ASO
(mipomersen) FDA Therapeutics terolemia/subcutane-
ous injection
Imlygic® 2015/ BioVex Melanoma/local In vivo – oncolitic
(Talimogene FDA virus/HSV-1
laherparepvec)
Spinraza® 2016/ Biogen Spinal muscular atro- In vivo – ASO
(nusinersen) FDA phy/intrathecal
Exondys 51® 2016/ Sarepta Duchenne muscular In vivo – ASO
(eteplirsen) FDA Therapeutics dystrophy/intrave-
nous infusion
Zalmoxis® 2016/ MolMed Haploidentical HSC Ex vivo – alloge-
(nalotimagene EMA transplant/intravenous neic T cells genet-
carmaleucel) infusion ically modified
Strimvelis™ 2016/ Orchard ADA-SCID/intrave- Ex vivo – autolo-
EMA Therapeutics nous infusion gous
CD34+ geneti-
cally modified
Luxturna® 2017/ Spark Retinal dystrophy In vivo – gene
(voretigene FDA Therapeutics (RPE65)/subretinal augmentation/
neparvovec) AAV2
Kymriah® 2017/ Novartis ALL and DLBCL/ Ex vivo – autolo-
(tisagenlecleucel) FDA intravenous infusion gous
CAR T cell
Yescarta® 2018/ Kite Pharma DLBCL and PMBCL/ Ex vivo – autolo-
(axicabtagene FDA intravenous infusion gous
ciloleucel) CAR T cell
Tegsedi® 2018/ Akcea hATTR/subcutaneous In vivo – ASO
(inotersen) EMA Therapeutics injection
(continued)
Table 4 (continued)
Year/
agency
(first Indication/
Product approval) Company administration route Strategy/vector
Onpattro® 2018/ Alnylam hATTR/intravenous In vivo – siRNA/
(patisiran) FDA Pharmaceuticals infusion lipid nanoparticles
Zolgensma® 2019/ AveXis, Inc. Spinal muscular atro- In vivo – gene
(onasemnogene FDA phy/intravenous augmentation/
abeparvovec-xioi) infusion AAV9
FDA Food and Drug Administration, CMV cytomegalovirus, AIDS acquired immunodeficiency
syndrome, ASO antisense oligonucleotides, SFDA China State Food and Drug Administration,
AD5 adenovirus serotype 5, AMD age-related macular degeneration, EMA European Medicines
Agency, AAV1 adeno-associated virus serotype 1, HSV-1 herpes simplex virus type 1, HSCT
hematopoietic stem cell transplant, ADA-SCID severe combined immunodeficiency due to adeno-
sine deaminase deficiency, AAV2 adeno-associated virus serotype 1, ALL B-cell acute lymphoblas-
tic leukemia, DLBCL diffuse large B-cell lymphoma, PMBCL primary mediastinal large B-cell
lymphoma, CAR chimeric antigen receptor, hATTR hereditary transthyretin amyloidosis, siRNA
small interfering ribonucleic acid
using a viral vector to achieve transduction, followed by infusion of the genetically

modified cells into the patient. Kymriah®, Yescarta®, and Strimvelis™ use autolo-
gous cells, whereas Zalmoxis® uses allogeneic cells as a starting point. Strimvelis™
is a hematopoietic stem cell therapy, and the other three are T-cell therapies, being
Kymriah® and Yescarta® the first chimeric antigen receptor (CAR) T-cell-based
products. All four products have orphan status or target niche conditions and either
are under additional monitoring or require further post-authorization safety studies.
Out of the total of GTMPs approvals, Vitravene® and Glybera® were withdrawn
from market in 2002 and 2017, respectively. Moreover, the authorizations for use in
the EU of Kynamro® and Exondys 51® were refused in 2012 and 2018, respectively.
A major challenge that faces most of the GTMPs is high development and
production cost, which has led to pricing and reimbursement issues. A representative
example is Glybera®, an adeno-associated virus serotype 1-based gene therapy
for intramuscular administration in adult patients with familial lipoprotein lipase
deficiency, a rare autosomal recessive disorder. Glybera® was commercialized in
2012 with a price of $1m per treatment, and it was pulled from market in 2017
despite being therapeutically successful [138].
As challenging as the generally high price is the developmental timeline of
GTMPs that typically span two or even three decades from concept introduction to
commercialization [138]. For instance, Kymriah®, the first CAR T-cell therapy, was
approved by the FDA in 2017, after almost 30 years since the concept of redirecting
T cells’ potential to kill cancerous cells was introduced [139]. In the same way, in
the case of Strimvelis™, it took nearly 15 years since the onset of the preclinical
in vivo study [140] before its developer Fondazione Telethon (Italy) received orphan
designation status from the European Commission in 2005. In this sense, there is
already a similar precedent with the monoclonal antibodies; it took more than three
Gene Therapy 351
decades to get the commercialization of these products to become the primary

drivers of the pharmaceutical market.
Besides the economic factors, other aspects have contributed to the low level
of commercialization of GTMPs, such as the complexity of the technologies,
difficulties in manufacturing processes, and regulatory barriers [138]. GTMPs face
significant additional regulatory challenges when pursuing market approval due to
the risks and concerns gene therapies which must be accounted during the regulatory
process [141]. Moreover, the mismatch between the capacity of manufacturing
vectors and the requirement of these emerging therapies is an important hurdle
that is slowing down gene therapy progress [142].
Nevertheless, considering not only the intensive investigation performed but also
the recent advances in the gene-editing field and T-cell-based therapies, GTMPs will
undoubtedly have a substantial contribution on the biopharmaceutical market over
the years to come. Furthermore, with several product candidates now undergoing
regulatory review, it appears likely that clinicians will have increasing opportunities
to generate their own assessments of gene therapy as a treatment modality [7].
5.1 Gene Therapy Medicinal Products for Cancer
Cancer diseases that have been targeted by gene therapy are primarily those that
do not respond well to conventional treatment such as metastatic melanoma or
glioblastoma. As mentioned above, the first gene therapy marketed was Gencidine®,
approved in 2003 for the treatment of head and neck squamous cell carcinoma by
the China State Food and Drug Administration (SFDA), although it is not available
in the United States or Europe. Gencidine is a type 5 recombinant adenovirus, which
has the E1 region replaced by a Rous sarcoma virus promoter linked with the human
wild-type p53 gene and a poly (A) tail [143]. The tumor suppressor p53 and its target
genes are essential regulators of cell cycle control and induction of apoptosis. The
p53 signaling cascade modulates cell cycle and DNA repair to maintain the genetic
integrity of cells. If irreparable DNA damages occur, p53 activates cellular apoptotic
pathways to eliminate genetically damaged cells [144]. Gencidine®, administered
by intratumoral injection, induces the expression of the tumor suppressor protein
p53 causing growth arrest and apoptosis in tumor cells. However, the antitumor
effects depend on the expression level of transduced p53 and on the integrity in
p53-mediated cascades in the target tumors [145].
Another approach to address the treatment of cancer is the use of oncolytic
viruses (OV) that selectively replicate in tumor cells without harming normal cells.
Recombinant virus technology has allowed the development of conditionally repli-
cating viruses, being Oncorine® (H101) the first OV marketed, which received
approval in China in 2005 for treatment of nasopharyngeal cancer after intratumoral
administration [146]. Oncorine® is a type 5 adenovirus with E1B-55KD and partial
E3 deletion that cannot replicate in normal cells where p53 is active; therefore, it
can selectively infect and kill tumor cells via the targeting of pro-apoptosis. The first
OV to gain approval by the FDA and EMA as an anticancer therapy was talimogene
laherparepvec (Imlygic®), approved in 2015 for melanoma treatment. It is a modified
type 1 herpes simplex virus (HSV-1) engineered to express human granulocyte-
macrophage colony-stimulating factor (GM-CSF). The insertion of GM-CSF in
place of both loci of the ICP34.5 gene, as well as by the deletion of the ICP47
gene, increased the selective replication within tumor cells, enhancing the tumor-
specific immune response [147, 148]. The treatment is administered as a series of
subcutaneous or intranodal injections over at least 6 months, and it has an estimated
average cost of US$65,000 [134].
Targeting a sufficient number of cells, even when the vector could be injected
into the tumors directly and repeatedly, represented a serious obstacle to achieving
full efficacy. Furthermore, considering that metastasis is the source of mortality
for most cancers, systemic gene therapy is of considerable interest, and nowadays it
is available with the ex vivo infusion of genetically modified hematopoietic T cells.
In this sense, genetically modified immune T cells represent a new class of thera-
peutics that has shown encouraging success for the treatment of some types of
cancer. However, specialized manufacturing facilities and personal trained to
conduct customized procedures for such therapies are vital to ensure accessibility
and quality of care [134].
Zalmoxis® (nalotimagene carmaleucel) is an ex vivo GTMP approved by
EMA in 2016 as adjunctive treatment in haploidentical hematopoietic stem
cell transplantation (Haplo-HSCT) of adult patients with high-risk hematological
malignancies. Haplo-HSCT can be associated with prolonged immunodeficiency
posttransplantation, and Zalmoxis® aids immune reconstitution and reduces the risk
of graft-versus-host disease [149]. This GTMP is based on allogenic somatic T cells
genetically modified with a retroviral vector to express the herpes simplex thymidine
kinase (HSV-TK) suicide gene and a truncated form of the human low-affinity nerve
growth factor receptor (ΔLNGFR) genes (for identification of transduced cells).
The expression of the HSV-TK gene allows the selective killing of T cells that have
this suicide gene, upon administration of ganciclovir or valganciclovir, preventing
further development if the patient develops graft-versus-host disease [150].
The most recent therapies approved by FDA and EMA against cancer are
Kymriah® (tisagenlecleucel) and Yescarta® (axicabtagene ciloleucel), the first
chimeric antigen receptor (CAR) T cell-based products, being both CD19-directed
genetically modified autologous CAR T-cell immunotherapies. CARs consist of an
antigen-binding domain, from either an immunoglobulin molecule or a T-cell
receptor, fused to an intracellular signaling domain, from receptors such as CD28,
OX40, and CD137, that mediates activation and costimulation to enhance T-cell
function and persistence [47]. CARs recognize antigens independently of the major
histocompatibility complex (MHC), which endows the CAR T cell with a funda-
mental antitumor advantage, because a major mechanism of immunoevasion by
cancer is loss of MHC-associated antigen presentation by tumor cells. Another
advantage is that CARs target nonprotein surface molecules, like carbohydrates
and glycolipids. One limitation of current CAR T-cell strategies is that they require
extracellular surface targets on the tumor cells [151].
Gene Therapy 353
CD19 is at present the most common CAR target; CD19 displays frequent
and high-level expression in B-cell malignancies, it is required for normal B-cell
development in humans, and it is not expressed outside of the B-cell lineage, which
makes CD19 a nearly ideal target. For CAR T-cell therapy manufacture, T cells are
isolated from the blood of the patient, activated, and then genetically engineered to
express the CAR construct. T cells are modified by using a lentiviral or a retroviral
vector for Kymriah® and Yescarta®, respectively. After ex vivo expansion of the
CAR T cells, they are formulated into the final product for direct infusion [152].
However, CAR T-cell administration has been associated with serious systemic
toxicities that often require intensive care and in some instances have caused patient
deaths. To date, the most prevalent adverse effects following infusion of CAR T
cells result from on-target T-cell activation, including cytokine release syndrome,
macrophage activation syndrome, and tumor lysis syndrome [7].
Kymriah® and Yescarta® have a US list price of $475,000 and of $373,000,
respectively [134]. Both in the United States and in EU, one of the indications
of Kymriah® is the treatment of pediatric and young adult patients up to 25 years of
age with B-cell acute lymphoblastic leukemia (ALL) that is refractory, in relapse
posttransplant or in second or later relapse. The other indication is the treatment of
adult patients with relapsed or refractory diffuse large B-cell lymphoma (DLBCL),
after two or more lines of systemic therapy. Yescarta® is indicated for the treatment
of adult patients with relapsed or refractory diffuse large B-cell lymphoma (DLBCL)
and primary mediastinal large B-cell lymphoma (PMBCL), after two or more lines
of systemic therapy (EMA).
5.2 Gene Therapy Medicinal Products for Other Applications
The resurgence of gene therapy in recent years for the treatment of genetic diseases is
largely due to successes in trials that utilized both ex vivo strategies (for X-linked
SCID and adrenoleukodystrophy) and in vivo approaches (Leber congenital amau-
rosis type 2 and hemophilia B) [7]. Gene therapies to treat rare disorders caused by
single-gene mutations have made the most progress toward market availability. It
has to be considered that in many of these diseases, there are few treatment options
apart from supportive and symptomatic care. Development of gene therapies has
also been influenced by ease of administration in target tissues, i.e., diseases of
the eye and hematopoietic system.
Among the organs targeted by gene therapy, the eye has been at the forefront of
translational gene therapy largely due to appropriate disease targets and its suitable
anatomic features [153]. In fact, fomivirsen (Vitravene®) indicated for the treatment
of cytomegalovirus retinitis (CMV) in patients with AIDS was the first therapeutic
ASO approved by FDA in 1998. It was administered intraocularly, and its target was
the mRNA that encoded the CMV immediate early (IE) 2 protein, which is required
for viral replication. EMA also approved this product in 1999; however, Novartis
stopped marketing the drug in 2002 in Europe and in 2006 in the United States [154].
Likewise, pegaptanib (Macugen®), indicated for the treatment of age-related

macular degeneration, was the first therapeutic aptamer approved by FDA in 2004.
Macugen® is an RNA aptamer for intravitreal administration that consists of
28 nucleotides that binds to 165 isoform of VEGF (vascular endothelial growth
factor). Its anti-angiogenic effect not only stops the excessive growth of blood
vessels but also prevents the formation of defective blood vessels [155].
Moreover, FDA and EMA approved Luxturna® in 2017 and 2018, respectively,
for the treatment of adult and pediatric patients with vision loss due to inherited
retinal dystrophy caused by confirmed biallelic RPE65 mutations and who have
sufficient viable retinal cells. Biallelic mutations in the RPE65 gene, which encodes
the all-trans-retinyl ester isomerase, in this gene, can be described as Leber congen-
ital amaurosis type 2, retinitis pigmentosa type 20, early-onset retinal dystrophy,
and other clinical labels for severe rod-mediated inherited retinal dystrophies, which
all eventually progress to complete blindness [156]. Luxturna® was designated an
orphan medicine by EMA for two forms of the disease, retinitis pigmentosa in 2015
and Leber’s congenital amaurosis in 2012. Voretigene neparvovec (Luxturna®)
consists of a recombinant adeno-associated virus serotype 2 vector carrying a
functional RPE65 gene. This gene augmentation therapy is given by bilateral
subretinal injection and has a list price of US$425,000 (per eye treatment).
Ex vivo gene therapy approaches have mainly targeted hematopoietic system,
being Strimvelis™ the first ex vivo GTMP approved for the treatment of an inherited
disorder, adenosine deaminase deficiency-severe combined immunodeficiency
(ADA-SCID). This recessive immune disorder is caused by mutations in the ADA
gene and characterized by the absence of cellular and humoral immune function
and a fatal outcome very early in life. Strimvelis™, with orphan designation and
approved in 2016 by EMA under additional monitoring, saw the first clinical
application on a single patient in March 2017. This hematopoietic stem cell therapy
is indicated for the treatment of patients with ADA-SCID, for whom no suitable
human leukocyte antigen (HLA)-matched related stem cell donor is available [134].
Patients receive CD34+ cells transduced with retroviral vector that encodes for the
human ADA cDNA sequence. The genetically modified autologous CD34+ cells act
by repopulating the hematopoietic system with cells that express active levels of the
ADA enzyme. Strimvelis™, with a list price of €594,000, should be administered by
intravenous infusion in a specialized transplant center. These ex vivo therapies
require complex procedures and trained personnel to harvest, transduce, and reinfuse
the hematopoietic target cells, and in the case of Strimvelis™, it is currently
available only at a single center in Milan to where the patient and family are required
to go for treatment [7].
Apart from gene therapy medicines, different nucleic acid-based products have
also been approved for the treatment of diverse genetic diseases, including spinal
muscular atrophy (SMA), Duchenne muscular dystrophy (DMD), familial hyper-
cholesterolemia, and hereditary transthyretin amyloidosis (hATTR).
SMA is an autosomal recessive neurodegenerative rare disease that, in most
cases, involves homozygous deletion of the survival of motor neuron 1 (SMN1)
gene on chromosome 5q. As a consequence, patients suffer the deficiency of the
survival of motor neuron (SNM) protein which plays a critical role in motor neuron
Gene Therapy 355
development. SMN1 is one of two nearly identical genes that encode SMN; the other
is survival of motor neuron 2 (SMN2). Infants with a more severe form of the disease
(type 1 SMA) die before 2 years of age; later onset of the disease in infants is referred
to as SMA2. FDA has approved in 2019 Zolgensma® (onasemnogene abeparvovec-
xioi), a recombinant AAV9-based gene therapy designed to deliver a copy of the
gene encoding the human SMN protein. Zolgensma® is administered as infusion,
and it is indicated for the treatment of pediatric patients less than 2 years of age
with SMA with biallelic mutations in the SMN1 gene. This product, with a price of
$2.1 million, is the most expensive drug in the world.
Nusinersen (Spinraza®) is an ASO approved in 2016 by FDA and in 2017 by
EMA (orphan medicine in 2012) for treating patients with SMA. Biogen has priced
Spinraza® at $750,000 for the first year’s treatment ($125,000 per injection) and
$350,000 per year subsequently [154].
SMN protein is mainly produced from SMN1, whereas SMN2 produces a small
amount of full-length SMN protein. Typically, a higher number of copies of SMN2
are associated with a less severe phenotype of the pathology. However, since the
amount of protein formed is low, even multiple copies of SMN2 do not fully stop the
disease. The intron 7 in SMN2 contains an intronic splicing silencer (termed ISS-N1)
with binding sites for negative splicing factors (NSFs). Binding of these NSFs to
intron 7 pre-mRNA precludes the recognition of exon 7 during the splicing process.
The ASO nusinersen blocks the ISS-N1 site preventing the binding of the NSFs. As
a result, Spinraza® administered via intrathecal injections modulates the splicing of
the SMN2 mRNA transcript to include exon 7, thereby increasing the production
of full-length SMN functional protein [157].
DMD is a rare X-linked disease characterized by loss-of-function mutations in the
DMD gene coding for dystrophin, which disrupt the reading frame of the dystrophin
mRNA and cause the introduction of premature stop codons, leading to mRNA
degradation and the loss of protein synthesis in striated muscle. It is a fatal disorder
characterized by progressive muscle weakening and wasting, with boys losing
ambulation by 12 years of age or earlier; death often occurs in the 20s, usually due
to respiratory or cardiac complications [154, 158]. Eteplirsen (Exondys 51®) is a
30-nucleotide phosphorodiamidate morpholino oligomer and was approved as an
ASO drug in 2016 by FDA, although authorization for use in the EU was refused
by EMA in 2018. Eteplirsen promotes dystrophin production by restoring the
translational reading frame of DMD through specific skipping of exon 51 in defec-
tive gene variants. The therapeutic strategy of antisense-mediated “exon skipping”
is developed to force exon exclusion from mature mRNA of DMD with the purpose
of restoring reading frame. Eteplirsen is suitable for 14% of DMD patients with
DMD mutations, it is administered by intravenous injection, and it has a price of
$300,000/patient/year [159].
Familial hypercholesterolemia is an autosomal dominant genetic condition
resulting from mutations of the low-density lipoprotein cholesterol (LDL-C) recep-
tor, apolipoprotein B (ApoB), or pro-protein convertase subtilisin/kexin 9 (PCSK9)
[160]. Mipomersen (Kynamro®) is an orphan medicine approved in 2013 by FDA
but with refused authorization for use in the EU by EMA in 2012. Mipomersen is a
single-stranded synthetic DNA ASO targeting ApoB-100, resulting in suppression

of the hepatic production of the ApoB, total cholesterol, LDL-C, and non-high-
density lipoprotein (HDL) cholesterol lipoproteins in rare genetic disorder patients
with homozygous familial hypercholesterolemia. Kynamro® is available as a solu-
tion for injection under the skin. Due to the serious risk of liver toxicity, mipomersen
is labeled a black box warning hepatotoxicity by FDA [159].
hATTR is a rare, autosomal dominantly inherited, progressively debilitating,
and life-threatening disease. Misfolded transthyretin (TTR) proteins accumulate as
amyloid deposits at multiple sites culminating in intractable peripheral sensorimotor
neuropathy and, in many cases, autonomic neuropathy and/or cardiomyopathy.
Recently, two different nucleic acid-based products have been approved for the
treatment of hATTR, by using two different strategies [35]. Inotersen (Tegsedi®) is
an ASO designed to suppress the expression of both wild-type and mutant forms of
TTR. It has recently gained marketing authorization approval by EMA in 2018
(orphan designation in 2014) under additional monitoring for the treatment of stage
1 or stage 2 polyneuropathy in adult patients with hATTR and regulatory approval
from the FDA for the treatment of the polyneuropathy of hATTR. It is available as a
solution for injection under the skin in pre-filled syringes, and the recommended
dose is one injection once a week [161]. Onpattro® (Patisiran) is a siRNA designed
to target TTR to reduce the levels of both wild-type and mutant TTR. Patisiran is
formulated in lipid nanoparticles that direct the siRNA to the liver, the primary site of
TTR production. Patisiran, with the same indications that of inotersen, is available
as a solution for infusion and received regulatory approval in 2018 from the FDA
and EMA (orphan designation in 2011) [35].
6 Challenges of Gene Therapy
As has been commented in this chapter, gene therapy has still many challenges
to be overcome: the science is complex, treatment is technically difficult, and the
regulatory approval process is necessarily different to that for conventional thera-
pies. Actually, it has been considered as the most complex “drugs” ever developed
[47]. Efficacy, safety, and manufacturing issues are important challenges that must
be faced. There are also difficult questions about cost, accessibility, and social justice
that will need answers once the methods are shown to be effective and safe.
6.1 Efficacy Issues
The low efficacy, which may lead to treatment failure, is one of the most important
challenges of gene therapy [162]. The development of new delivery systems
with higher transduction rates and higher affinity to a specific cell or tissue is
necessary to increase the number of products that reach clinical evaluation. Another
Gene Therapy 357
reason that may explain the low efficacy of a gene therapy product is the presence
of endogenous natural antibodies against viral vectors or the transgene product.
This is of particular importance because it prevents transduction and limits gene
therapy product administration more than once.
6.2 Safety Issues
Potential immunogenicity and oncogenicity are the main challenges concerning

safety. When a gene vector integrates into the host genome, there is potential
for gene disruption and insertional mutagenesis giving with an increase in risk of
tumor arising from the genetically modified cells. This occurs as a consequence
of activation/upregulation of oncogenes or inactivation or downregulation of tumor/
suppressing genes [162]. To overcome the potential oncogenicity of viral vectors,
it is crucial to design new vectors that prevent the activation of oncogenic genes at
integrations sites. The use of non-integrating vectors or highly targeted genomic
integration at the desired chromosomal loci is also helpful.
Immunogenicity reactions may be due to both the viral vector and the transgene
product due to the unpredictability of innate and antigen-dependent immune
responses in humans [162]. One additional problem is that these responses are
difficult to detect in animal models, as such effects arising in animals, who may
have been given human product, are not indicative of whether such would occur in
humans. Some alternatives to prevent immunogenicity include the administration
of immunosuppressive agents prior or after the administration of the gene therapy
product, the modification of the capsid proteins of the vector, and the elimination
of viral genes.
6.3 Drug Development and Manufacture Issues
Because of its unique set of characteristics, the nonclinical development package of a

gene therapy medicinal product is rather more complex than conventional medicinal
products. One important difference with respect to a conventional drug product is
that the approval of a gene therapy product must be based not only on data of the
therapeutic transgene but also of data on the vector/delivery system.
Manufacturing of gene therapy products is an additional complexity factor.
In the near future, there is a challenge for the development of manufacturing
capacities, which must be sufficient to meet the coming demand, specifically the
AAV production. In this sense, several companies of viral vector production are
amplifying their manufacturing facilities to face the future increase demand for viral
production [142].
6.4 Ethical Issues
Currently, at least in the Western countries, clinical use of gene therapy is limited to
somatic cells for the treatment of a specific disease. As it has been explained above,
germline gene therapy leads to hereditary modifications that pass on to subsequent
generations, and therefore, it is the object of a heated discussion [163]. The recent
announcement by a Chinese research of the birth of twins whose genomes were
edited by CRISPR/Cas9 during in vitro fertilization has engendered broad condem-
nation for the premature clinical deployment of a still experimental biomedical area
[164]. The organizing committee of “the Second International Summit on Human
Genome Editing,” held in Hong Kong in November 2018 under the auspices of
the US National Academies of Science and Medicine, the UK Royal Society, and the
Hong Kong Academy of Sciences, reiterated that “the scientific understanding and
technical requirements for clinical practice remain too uncertain and the risks too
great to permit clinical trials of germline editing at this time” [163].
The potential use of gene therapy for purposes other than diseases treatment is
another important topic to be addressed. For instance, the application to eugenics,
that is, the attempt to change or improve complex human traits related to a broader
number of genes; such as, personality, intelligence, or character [162].
6.5 Affordability
Gene therapy-based medicines have a high cost of development, production, product

storage, and transportation, which lead to very high prices. For instance, the price of
Glybera was around 1,000,000 €; Strimvelis, 600,000 €; and Yescarta and Kymriah
300,000 and 400,000 €, respectively. Gene therapy medicinal products are expen-
sive to develop and to manufacture, and sometimes, they are one-time treatments.
These reasons, among others, may justify the high cost [164]. Affordability of
novel innovative and high budget impact therapies has become an important topic
in Europe, and so far, each country has come up with individual approaches
to improve affordability [165]. For example, in the case of Zolgensma®, with an
annualized cost of $425,000 per year for 5 years, the company proposes to payers
to create 5-year outcomes-based agreements and novel pay-over-time options [166].
6.6 Intellectual Property Complexity
The complexity of the intellectual property “territories” that can surround a given
gene therapy development also explains the slow progress of gene therapy [142].
In fact, a new gene therapy product under development may be conditioned by
patents involving not only the therapeutic transgene itself but also its mechanism of
Gene Therapy 359
action, the non-viral or viral vector used as the delivery system, and the method for
delivery of the nucleic acid therapy to the patient (e.g., the delivery devices, surgical
techniques, and treatment protocols to be used).
Despite the numerous challenges, in the last years, important unified efforts by
research and clinical scientists in academic, translational, and industry settings have
resulted in tangible outcomes, with several marketing authorizations and approved
commercial products. Initiatives for willingness to participate in clinical trials and
equable access of patient population to somatic gene editing as a treatment option in
clinical care are necessary to increase the opportunities to successful of gene therapy.
7 Conclusion
Gene therapy, regarded as one of the most promising and most active fields of
medicine, is beginning to show encouraging results. Current therapies are primarily
experimental with only a few gene therapy medicinal products on the market,
although several product candidates are undergoing regulatory review.
The efforts and advances made in this area have led to the development of new
therapeutic strategies to treat several disorders, many of them without currently
available treatments. The remarkable basic, translational, and clinical research
activity in gene therapy has been addressed mainly to cancer, but a significant
number of clinical trials have also targeted a broad variety of diseases including
monogenic, infectious, and cardiovascular diseases.
Nucleic acid-marketed products are based mainly on in vivo strategies. Initially
gene augmentation was the main option, although ex vivo therapies and new ASOs
seem to be major strategies at present. Moreover, the first product containing
a siRNA has been already marketed. Recent strategies also include T-cell-based
therapies, with two products marketed in 2018 for the treatment of hematological
malignancies by immunotherapy, and the gene-editing tool CRISPR, whose rapid
progress is boosting the development of new gene therapy-based medicines.
Despite the positive forecast that the nucleic acid-based products have on the
biopharmaceutical market, different hurdles are slowing down their progress.
The success of gene therapy may be compromised by two main challenges,
the cost and reimbursement of the treatments, as well as the technological issues to
ensure accessibility and quality of the treatments. The availability of specialized
manufacturing personnel and facilities to conduct customized procedures and the
progress in the manufacturing capacity of efficient and safe vectors to meet the
upcoming demand of gene therapies are essential for the advance of this emerging
therapy in the future.
Acknowledgments This work was supported by the University of the Basque Country UPV/EHU
(GIU17/32) and by the Spanish Ministry of Economy and Competitiveness (SAF2014-53092-R)
and FEDER funds. I Gómez-Aguado thanks UPV/EHU for her research grant.
References
1. American Medical Association (2015) Gene therapy. https://www.immortalitymedicine.tv/

gene-medicine/gene-therapy-american-medical-association.php. Accessed 24 Apr 2019
2. EMA (European Medicine Agency) (2018) Guideline on the quality, non-clinical and
clinical aspects of gene therapy medicinal product. https://www.ema.europa.eu/en/docu
ments/scientific-guideline/guideline-quality-non-clinical-clinical-aspects-gene-therapy-medic
inal-products_en.pdf. Accessed 23 Apr 2019
3. Genetics Home Reference (2017) Gene therapy. Lister Hill National Center for Biomedical
Communications U.S. National Library of Medicine. National Institutes of Health. Department
of Health and Human Services. https://ghr.nlm.nih.gov/primer/. Accessed 24 Apr 2019
4. Regalado A (2019) A third CRISPR baby may have already been born in China. MIT
Technological Review. https://www.technologyreview.com/s/613890/a-third-crispr-baby-
may-have-already-been-born-in-china/ Accessed 24 Jul 2019
5. Rosemann A, Balen A, Nerlich B, Hauskeller C, Sleeboom-Faulkner M, Hartley S et al (2019)
Heritable genome editing in a global context: national and international policy challenges.
Hast Cent Rep 49:30–42. https://doi.org/10.1002/hast.1006
6. Thorne B, Takeya R, Vitelli F, Swanson X (2018) Gene therapy. Adv Biochem Eng
Biotechnol 165:351–399. https://doi.org/10.1007/10_2016_53
7. Anguela XM, High KA (2019) Entering the modern era of gene therapy. Annu Rev Med
70:273–288. https://doi.org/10.1146/annurev-med-012017-043332
8. Pushpendra S, Arvind P, Anil B (2012) Nucleic acids as therapeutics. In: Erdmann VA,
Barciszewski J (eds) From nucleic acids sequences to molecular medicine. RNA
Technologies, Springer, Berlin
9. Papadakis ED, Nicklin SA, Baker AH, White SJ (2004) Promoters and control elements:
designing expression cassettes for gene therapy. Curr Gene Ther 4:89–113. https://doi.org/10.
2174/1566523044578077
10. Guan S, Rosenecker J (2017) Nanotechnologies in delivery of mRNA therapeutics using
nonviral vector-based delivery systems. Gene Ther 24:133–143. https://doi.org/10.1038/gt.
2017.5
11. Gaspar V, de Melo-Diogo D, Costa E, Moreira A, Queiroz J, Pichon C et al (2015) Minicircle
DNA vectors for gene therapy: advances and applications. Expert Opin Biol Ther 15:353–379.
https://doi.org/10.1517/14712598.2015.996544
12. Maniar LEG, Maniar JM, Chen Z-Y, Lu J, Fire AZ, Kay MA (2013) Minicircle DNA vectors
achieve sustained expression reflected by active chromatin and transcriptional level. Mol Ther
21:131–138. https://doi.org/10.1038/mt.2012.244
13. Zuo Y, Wu J, Xu Z, Yang S, Yan H, Tan L et al (2014) Minicircle-oriP-IFNg: a novel targeted
gene therapeutic system for EBV positive human nasopharyngeal carcinoma. Oncol Rep
32:2564–2570. https://doi.org/10.1371/journal.pone.0019407
14. Kauffman KJ, Webber MJ, Anderson DG (2016) Materials for non-viral intracellular delivery
of messenger RNA therapeutics. J Control Release 240:227–234. https://doi.org/10.1016/j.
jconrel.2015.12.032
15. Meng Z, O’Keeffe-Ahern J, Lyu J, Pierucci L, Zhou D, Wang W (2017) A new developing
class of gene delivery: messenger RNA-based therapeutics. Biomater Sci 5:2381–2392.
https://doi.org/10.1039/c7bm00712d
16. Tavernier G, Andries O, Demeester J, Sanders NN, De Smedt SC, Rejman J (2011) mRNA as
gene therapeutic: how to control protein expression. J Control Release 150:238–247. https://
doi.org/10.1016/j.jconrel.2010.10.020
17. Sahin U, Karikó K, Türeci Ö (2014) mRNA-based therapeutics--developing a new class
of drugs. Nat Rev Drug Discov 13:759–780. https://doi.org/10.1038/nrd4278
18. Kreiter S, Diken M, Sahin U (2014) In: Britten CM (ed) Cancer immunotherapy meets
oncology. Springer, Cham, pp 21–27
Gene Therapy 361
19. Pollard C, De Koker S, Saelens X, Vanham G, Grooten J (2013) Challenges and advances
towards the rational design of mRNA vaccines. Trends Mol Med 19:705–713. https://doi.org/
10.1016/j.molmed.2013.09.002
20. Weiss R, Scheiblhofer S, Roesler E, Weinberger E, Thalhamer J (2012) mRNA vaccination as
a safe approach for specific protection from type I allergy. Expert Rev Vaccines 11:55–67.
https://doi.org/10.1586/erv.11.168
21. Sridharan K, Gogtay JN (2016) Therapeutic nucleic acids: current clinical status. Br J Clin
Pharmacol 82:659–672. https://doi.org/10.1111/bcp.12987
22. Rozenblum GT, Lopez VG, Vitullo AD, Radrizzani M (2016) Aptamers: current challenges
and future prospects. Expert Opin Drug Discov 11:127–135. https://doi.org/10.1517/
17460441.2016.1126244
23. Eyetech Study Group (2002) Preclinical and phase 1A clinical evaluation of an anti-VEGF
pegylated aptamer (EYE001) for the treatment of exudative age-related macular degeneration.
Retina 22:143–152
24. Zhang Y, Lai BS, Juhas M (2019) Recent advances in aptamer discovery and applications.
Molecules 24:E941. https://doi.org/10.3390/molecules24050941
25. Torrecilla J, Rodríguez-Gascón A, Solinís MÁ, del Pozo-Rodríguez A (2014)
Lipid nanoparticles as carriers for RNAi against viral infections: current status and future
perspectives. Biomed Res Int 2014:161794. https://doi.org/10.1155/2014/161794
26. Iorio MV, Croce CM (2009) MicroRNAs in cancer: small molecules with a huge impact.
J Clin Oncol 27:5848–5856. https://doi.org/10.1200/JCO.2009.24.0317
27. Ha M, Kim VN (2014) Regulation of microRNA biogenesis. Nat Rev Mol Cell Biol
15:509–524. https://doi.org/10.1038/nrm3838
28. Croce CM (2009) Causes and consequences of microRNA dysregulation in cancer. Nat Rev
Genet 10:704–714. https://doi.org/10.1038/nrg2634
29. Laffont B, Rayner KJ (2017) MicroRNAs in the pathobiology and therapy of atherosclerosis.
Can J Cardiol 33:313–324. https://doi.org/10.1016/j.cjca.2017.01.001
30. Nakamori M, Junn E, Mochizuki H, Mouradian MM (2019) Nucleic acid-based therapeutics
for Parkinson’s disease. Neurotherapeutics 16:287–298. https://doi.org/10.1007/s13311-019-
00714-7
31. Lam JK, Chow MY, Zhang Y, Leung SW (2015) siRNA versus miRNA as therapeutics for
gene silencing. Mol Ther Nucleic Acids 4:e252. https://doi.org/10.1038/mtna.2015.23
32. Fernandez-Piñeiro I, Badiola I, Sanchez A (2017) Nanocarriers for microRNA delivery
in cancer medicine. Biotechnol Adv 35:350–360. https://doi.org/10.1016/j.biotechadv.2017.
03.002
33. Scarborough RJ, Gatignol A (2017) RNA interference therapies for an HIV-1 functional cure.
Viruses 10:E8. https://doi.org/10.3390/v10010008
34. Mizrahy S, Hazan-Halevy I, Dammes N, Landesman-Milo D, Peer D (2017) Current progress
in non-viral RNAi-based delivery strategies to lymphocytes. Mol Ther 25:1491–1500. https://
doi.org/10.1016/j.ymthe.2017.03.001
35. Kristen AV, Ajroud-Driss S, Conceição I, Gorevic P, Kyriakides T, Obici L (2019) Patisiran,
an RNAi therapeutic for the treatment of hereditary transthyretin-mediated amyloidosis.
Neurodegener Dis Manag 9:5–23. https://doi.org/10.2217/nmt-2018-0033
36. Moore CB, Guthrie EH, Huang MT, Taxman DJ (2010) Short hairpin RNA (shRNA): design,
delivery, and assessment of gene knockdown. Methods Mol Biol 629:141–158. https://doi.
org/10.1007/978-1-60761-657-3_10
37. Torrecilla J, del Pozo-Rodríguez A, Solinís MÁ, Apaolaza PS, Berzal-Herranz B,
Romero-López C et al (2016) Silencing of hepatitis C virus replication by a non-viral vector
based on solid lipid nanoparticles containing a shRNA targeted to the internal ribosome entry
site (IRES). Colloids Surf B Biointerfaces 146:808–817. https://doi.org/10.1016/j.colsurfb.
2016.07.026
38. Hardcastle AJ, Sieving PA, Sahel JA, Jacobson SG, Cideciyan AV, Flannery JG et al (2018)
Translational retinal research and therapies. Transl Vis Sci Technol 7:8. https://doi.org/10.
1167/tvst.7.5.8
39. Mues M, Karra L, Romero-Moya D, Wandler A, Hangauer MJ, Ksionda O et al (2019)

High-complexity shRNA libraries and PI3 kinase inhibition in cancer: high-fidelity synthetic
lethality predictions. Cell Rep 27:631–47.e5. https://doi.org/10.1016/j.celrep.2019.03.045
40. Blighe K, DeDionisio L, Christie KA, Chawes B, Shareef S, Kakouli-Duarte T et al (2018)
Gene editing in the context of an increasingly complex genome. BMC Genomics 19:595.
https://doi.org/10.1186/s12864-018-4963-8
41. Moore CBT, Christie KA, Marshall J, Nesbit MA (2018) Personalised genome editing – the
future for corneal dystrophies. Prog Retin Eye Res 65:147–165. https://doi.org/10.1016/j.
preteyeres.2018.01.004
42. Belfort M, Bonocora RP (2014) Homing endonucleases: from genetic anomalies to program-
mable genomic clippers. Methods Mol Biol 1123:1–26. https://doi.org/10.1007/978-1-62703-
968-0_1
43. Gersbach CA, Gaj T, Barbas 3rd. CF (2014) Synthetic zinc finger proteins: the
advent of targeted gene regulation and genome modification technologies. Acc Chem Res
47:2309–2318. https://doi.org/10.1021/ar500039w
44. Kim Y, Kweon J, Kim A, Chon JK, Yoo JY, Kim HJ et al (2013) A library of TAL effector
nucleases spanning the human genome. Nat Biotechnol 31:251–258. https://doi.org/10.1038/
nbt.2517
45. Makarova KS, Wolf YI, Alkhnbashi OS, Costa F, Shah SA, Saunders SJ et al (2015) An
updated evolutionary classification of CRISPR-Cas systems. Nat Rev Microbiol 13:722–736.
https://doi.org/10.1038/nrmicro3569
46. Shim G, Kim D, Park GT, Jin H, Suh SK, Oh YK (2017) Therapeutic gene editing: delivery
and regulatory perspectives. Acta Pharmacol Sin 38:738–753. https://doi.org/10.1038/aps.
2017.2
47. Dunbar CE, High KA, Joung JK, Kohn DB, Ozawa K, Sadelain M (2018) Gene therapy comes
of age. Science 359:6372. https://doi.org/10.1126/science.aan4672
48. Tebas P, Stein D, Tang WW, Frank I, Wang SQ, Lee G et al (2014) Gene editing of CCR5 in
autologous CD4 T cells of persons infected with HIV. N Engl J Med 370:901–910. https://doi.
org/10.1056/NEJMoa1300662
49. Qasim W, Zhan H, Samarasinghe S, Adams S, Amrolia P, Stafford S et al (2017) Molecular
remission of infant B-ALL after infusion of universal TALEN gene-edited CAR T cells.
Sci Transl Med 9:eaaj2013. https://doi.org/10.1126/scitranslmed.aaj2013
50. Ma H, Marti-Gutierrez N, Park SW, Wu J, Lee Y, Suzuki K et al (2017) Correction of a
pathogenic gene mutation in human embryos. Nature 548:413–419. https://doi.org/10.1038/
nature23305
51. Bailey SR, Maus MV (2019) Gene editing for immune cell therapies. Nat Biotechnol. https://
doi.org/10.1038/s41587-019-0137-8
52. Rodríguez-Gascón A, del Pozo-Rodríguez A, Solinís MA (2013) Non-viral delivery systems
in gene therapy. In: Martín Molina F (ed) Gene therapy – tools and potential application.
IntechOpen, London, pp 3–33
53. Ramamoorth M, Narvekar A (2015) Nonviral vectors in gene therapy – an overview. J Clin
Diagn Res 9:GE01–GE06. https://doi.org/10.7860/JCDR/2015/10443.5394
54. Gene Therapy Clinical Trials Worldwide (2018) Provided by the Journal of Gene Medicine.
Wiley, Hoboken. http://abedia.com/wiley/index.html. Accessed 5 Apr 2019
55. Baum C, Schambach A, Bohne J, Galla M (2006) Retrovirus vectors: toward the plentivirus?
Mol Ther 13:1050–1063. https://doi.org/10.1016/j.ymthe.2006.03.007
56. Matuskova M, Durinikova E (2016) Retroviral vectors in gene therapy. In: Saxena SK
(ed) Advances in molecular retrovirology. IntechOpen, London, pp 143–166
57. Hacein-Bey-Abina S, Garrigue A, Wang GP, Soulier J, Lim A, Morillon E et al (2008)
Insertional oncogenesis in 4 patients after retrovirus-mediated gene therapy of SCID-X1.
J Clin Invest 118:3132–3142. https://doi.org/10.1172/JCI35700
58. Howe SJ, Mansour MR, Schwarzwaelder K, Bartholomae C, Hubank M, Kempski H et al
(2008) Insertional mutagenesis combined with acquired somatic mutations causes
Gene Therapy 363
leukemogenesis following gene therapy of SCID-X1 patients. J Clin Invest 118:3143–3150.

https://doi.org/10.1172/JCI35798
59. Yu SF, von Rüden T, Kantoff PW, Garber C, Seiberg M, Rüther U et al (1986) Self-
inactivating retroviral vectors designed for transfer of whole genes into mammalian cells.
Proc Natl Acad Sci U S A 83:3194–3198
60. Stirnadel-Farrant H, Kudari M, Garman N, Imrie J, Chopra B, Giannelli S et al (2018)
Gene therapy in rare diseases: the benefits and challenges of developing a patient-centric
registry for Strimvelis in ADA-SCID. Orphanet J Rare Dis 13:49. https://doi.org/10.1186/
s13023-018-0791-9
61. Milone MC, O’Doherty U (2018) Clinical use of lentiviral vectors. Leukemia 32:1529–1541.
https://doi.org/10.1038/s41375-018-0106-0
62. Dull T, Zufferey R, Kelly M, Mandel RJ, Nguyen M, Trono D et al (1998) A third-generation
lentivirus vector with a conditional packaging system. J Virol 72:8463–8471
63. Hutson TH, Foster E, Moon LD, Yáñez-Muñoz RJ (2014) Lentiviral vector-mediated RNA
silencing in the central nervous system. Hum Gene Ther Methods 25:14–32. https://doi.org/10.
1089/hgtb.2013.016
64. Palfi S, Gurruchaga JM, Lepetit H, Howard K, Ralph GS, Mason S et al (2018) Long-term
follow-up of a phase I/II study of ProSavin, a lentiviral vector gene therapy for Parkinson’s
disease. Hum Gene Ther Clin Dev 29:148–155. https://doi.org/10.1089/humc.2018.081
65. Matet A, Kostic C, Bemelmans AP, Moulin A, Rosolen SG, Martin S et al (2017) Evaluation
of tolerance to lentiviral LV-RPE65 gene therapy vector after subretinal delivery in
non-human primates. Transl Res 188:40–57. https://doi.org/10.1016/j.trsl.2017.06.012
66. Cavazzana-Calvo M, Payen E, Negre O, Wang G, Hehir K, Fusil F et al (2010) Transfusion
independence and HMGA2 activation after gene therapy of human [bgr]-thalassaemia. Nature
467:318–322. https://doi.org/10.1038/nature09328
67. Cartier N, Hacein-Bey-Abina S, Bartholomae CC, Veres G, Schmidt M, Kutschera I et al
(2009) Hematopoietic stem cell gene therapy with a lentiviral vector in X-linked adrenoleu-
kodystrophy. Science 326:818–823. https://doi.org/10.1126/science.1171242
68. Sessa M, Lorioli L, Fumagalli F, Acquati S, Redaelli D, Baldoli C et al (2016) Lentiviral
haemopoietic stem-cell gene therapy in early-onset metachromatic leukodystrophy: an ad-hoc
analysis of anon-randomised, open-label, phase 1/2 trial. Lancet 388:476–487. https://doi.org/
10.1016/S0140-6736(16)30374-9
69. Aiuti A, Biasco L, Scaramuzza S, Ferrua F, Cicalese MP, Bar-icordi C et al (2013) Lentiviral
hematopoietic stem cell gene therapy inpatients with Wiskott-Aldrich syndrome. Science
341:1233151. https://doi.org/10.1126/science.1233151
70. Kalos M, Levine BL, Porter DL, Katz S, Grupp SA, Bagg A et al (2011) T cells with chimeric
antigen receptors have potent antitumor effects and can establish memory in patients with
advanced leukemia. Sci Transl Med 3:95ra73. https://doi.org/10.1126/scitranslmed.3002842
71. Symonds G, Bartlett JS, Kiem HP, Tsie M, Breton L (2016) Cell-delivered entry inhibitors for
HIV-1: CCR5 downregulation and blocking virus/membrane fusion in defending the host cell
population. AIDS Patient Care STDs 30:545–550
72. Giacca M (2010) Gene therapy. Springer, Mailand
73. Brunetti-Pierri N (2011) Helper-dependent adenoviral vectors for liver-directed gene therapy.
Hum Mol Genet 20:7–13. https://doi.org/10.1093/hmg/ddr143
74. Raper SE, Chirmule N, Lee FS, Wivel NA, Bagg A, Gao GP et al (2003) Fatal systemic
inflammatory response syndrome in a ornithine transcarbamylase deficient patient following
adenoviral gene transfer. Mol Genet Metab 80:148–158
75. Wold WS, Toth K (2013) Adenovirus vectors for gene therapy, vaccination and cancer gene
therapy. Curr Gene Ther 13:421–433
76. Lee CS, Bishop ES, Zhang R, Yu X, Farina EM, Yan S et al (2017) Adenovirus-mediated gene
delivery: potential applications for gene and cell-based therapies in the new era of personalized
medicine. Genes Dis 4:43–63. https://doi.org/10.1016/j.gendis.2017.04.001
77. Naumer M, Sonntag F, Schmidt K, Nieto K, Panke C, Davey NE et al (2012) Properties of the
adeno-associated virus assembly-activating protein. J Virol 86:13038–13048. https://doi.org/
10.1128/JVI.01675-12
78. Hastie E, Samulski RJ (2015) Adeno-associated virus at 50: a golden anniversary of discovery,
research, and gene therapy success--a personal perspective. Hum Gene Ther 26:257–265.
https://doi.org/10.1089/hum.2015.025
79. Naso MF, Tomkowicz B, Perry 3rd WL, Strohl WR (2017) Adeno-associated virus (AAV) as
a vector for gene therapy. BioDrugs 31:317–334. https://doi.org/10.1007/s40259-017-0234-5
80. EMA (European Medicine Agency) (2018) Luxturna: summary of product characteristics.
https://www.ema.europa.eu/en/documents/product-information/luxturna-epar-product-inform
ation_en.pdf. Accessed 5 Apr 2019
81. Walsh G (2007) Nucleic acid and cell-based therapeutics. In: Walsh G (ed) Pharmaceutical
biotechnology: concepts and applications. Wiley, Chichester, pp 419–464
82. Brindley DA, Fuerstenau-Sharp M, Smith JA, Bure K, Pettitt D, Mitrophanous K et al (2016)
Emerging platform bioprocesses for viral vectors and gene therapies. BioProcess Int 14:8–14
83. Grein TA, Weidner T, Czermak P (2017) Concepts for the production of viruses and viral
vectors in cell cultures. In: Gowder SJT (ed) New insights into cell culture technology.
IntechOpen, London, pp 173–192
84. van der Loo JC, Wright JF (2016) Progress and challenges in viral vector manufacturing.
Hum Mol Genet 25:R42–R52. https://doi.org/10.1093/hmg/ddv451
85. Sanber KS, Knight SB, Stephen SL, Bailey R, Escors D, Minshull J et al (2015) Construction
of stable packaging cell lines for clinical lentiviral vector production. Sci Rep 5:9021. https://
doi.org/10.1038/srep09021
86. Rodrigues GA, Shalaev E, Karami TK, Cunningham J, Slater NKH, Rivers HM (2018)
Pharmaceutical development of AAV-based gene therapy products for the eye. Pharm Res
36:29. https://doi.org/10.1007/s11095-018-2554-7
87. FDA, U.S. Food and Drug Administration Center for Biologics Evaluation and
Research (2018) Chemistry, manufacturing, and control (CMC) information for human
gene therapy investigational new drug applications (INDs): draft guidance for industry.
https://www.fda.gov/downloads/BiologicsBloodVaccines/GuidanceComplianceRegulatoryIn
formation/Guidances/CellularandGeneTherapy/UCM610795.pdf. Accessed 5 Apr 2019
88. Nestola P, Peixoto C, Silva RR, Alves PM, Mota JP, Carrondo MJ (2015) Improved virus
purification processes for vaccines and gene therapy. Biotechnol Bioeng 112:843–857. https://
doi.org/10.1002/bit.25545
89. Herrero MJ, Sendra L, Miguel A, Aliño SF (2017) Physical methods of gene delivery.
In: Brunetti-Pierri N (ed) Safety and efficacy of gene-based therapeutics for inherited disor-
ders. Springer, Cham, pp 113–135
90. Apaolaza PS, del Pozo-Rodríguez A, Solinís MA, Rodríguez JM, Friedrich U, Torrecilla J et al
(2016) Structural recovery of the retina in a retinoschisin-deficient mouse after gene replace-
ment therapy by solid lipid nanoparticles. Biomaterials 90:40–49. https://doi.org/10.1016/j.
biomaterials.2016.03.004
91. Gan L, Wang J, Zhao Y, Chen D, Zhu C, Liu J et al (2013) Hyaluronan-modified core-shell
liponanoparticles targeting CD44-positive retinal pigment epithelium cells via intravitreal
injection. Biomaterials 34:5978–5987. https://doi.org/10.1016/j.biomaterials.2013.04.035
92. del Pozo-Rodríguez A, Solinís MÁ, Rodríguez-Gascón A (2016) Applications of lipid
nanoparticles in gene therapy. Eur J Pharm Biopharm 109:184–193. https://doi.org/10.1016/
j.ejpb.2016.10.016
93. Kim YH, Han ME, Oh SO (2017) The molecular mechanism for nuclear transport and its
application. Anat Cell Biol 50:77–85. https://doi.org/10.5115/acb.2017.50.2.77
94. Delgado D, del Pozo-Rodríguez A, Solinís MÁ, Rodríguez-Gascón A (2011) Understanding
the mechanism of protamine in solid lipid nanoparticle-based lipofection: the importance of
the entry pathway. Eur J Pharm Biopharm 79:495–502. https://doi.org/10.1016/j.ejpb.2011.
06.005
Gene Therapy 365
95. Mostaghaci B, Loretz B, Lehr CM (2016) Calcium phosphate system for gene delivery:
historical background and emerging opportunities. Curr Pharm Des 22:1529–1533
96. Kesse S, Boakye-Yiadom KO, Ochete BO, Opoku-Damoah Y, Akhtar F, Filli MS et al (2019)
Mesoporous silica nanomaterials: versatile nanocarriers for cancer theranostics and drug
and gene delivery. Pharmaceutics 11:E77. https://doi.org/10.3390/pharmaceutics11020077
97. Bishop CJ, Tzeng SY, Green JJ (2015) Degradable polymer-coated gold nanoparticles for
co-delivery of DNA and siRNA. Acta Biomater 11:393–403. https://doi.org/10.1016/j.actbio.
2014.09.020
98. Eslaminejad T, Nematollahi-Mahani SN, Ansari M (2017) Glioblastoma targeted gene therapy
based on pEGFP/p53-loaded superparamagnetic iron oxide nanoparticles. Curr Gene Ther
17:59–69. https://doi.org/10.2174/1566523217666170605115829
99. Stephen ZR, Dayringer CJ, Lim JJ, Revia RA, Halbert MV, Jeon M et al (2016) Approach to
rapid synthesis and functionalization of iron oxide nanoparticles for high gene transfection.
ACS Appl Mater Interfaces 8:6320–6328. https://doi.org/10.1021/acsami.5b10883
100. Hu Y, Wen C, Song L, Zhao N, Xu FJ (2017) Multifunctional hetero-nanostructures
of hydroxyl-rich polycation wrapped cellulose-gold hybrids for combined cancer therapy.
J Control Release 255:154–163. https://doi.org/10.1016/j.jconrel.2017.04.001
101. Vincent M, de Lázaro I, Kostarelos K (2017) Graphene materials as 2D non-viral gene transfer
vector platforms. Gene Ther 24:123–132. https://doi.org/10.1038/gt.2016.79
102. Clancy KFA, Hardy JG (2017) Gene delivery with organic electronic biomaterials. Curr
Pharm Des 23:3614–3625. https://doi.org/10.2174/1381612823666170710124137
103. Sung YK, Kim SW (2019) Recent advances in the development of gene delivery systems.
Biomater Res 23:8. https://doi.org/10.1186/s40824-019-0156-z
104. Palmerston Mendes L, Pan J, Torchilin VP (2017) Dendrimers as nanocarriers for nucleic
acid and drug delivery in cancer therapy. Molecules 22:E1401. https://doi.org/10.3390/
molecules22091401
105. Ramezani M, Ebrahimian M, Hashemi M (2017) Current strategies in the modification
of PLGA-based gene delivery system. Curr Med Chem 24:728–739. https://doi.org/10.2174/
0929867324666161205130416
106. Xie Y, Yu F, Tang W, Alade B, Peng Z-H, Wang Y et al (2018) Chloroquine-containing
DMAEMA copolymers as efficient anti-miRNA delivery vectors with improved endosomal
escape and anti-migratory activity in cancer cells. Macromol Biosci 18:1. https://doi.org/10.
1002/mabi.201700194
107. Chen J, Guo Z, Tian H, Chen X (2016) Production and clinical development of nanoparticles
for gene delivery. Mol Ther Methods Clin Dev 3:16023. https://doi.org/10.1038/mtm.2016.23
108. Kim YM, Park SC, Jang MK (2017) Targeted gene delivery of polyethyleneimine-grafted
chitosan with RGD dendrimer peptide in αvβ3 integrin-overexpressing tumor cells. Carbohydr
Polym 174:1059–1068. https://doi.org/10.1016/j.carbpol.2017.07.035
109. Tabasum S, Noreen A, Maqsood MF, Umar H, Akram N, Nazli ZI et al (2018) A review
on versatile applications of blends and composites of pullulan with natural and synthetic
polymers. Int J Biol Macromol 120(Pt A):603–632. https://doi.org/10.1016/j.ijbiomac.
2018.07.154
110. Kulkarni JA, Cullis PR, van der Meel R (2018) Lipid nanoparticles enabling gene therapies:
from concepts to clinical utility. Nucleic Acid Ther 28:146–157. https://doi.org/10.1089/nat.
2018.0721
111. Teixeira HF, Bruxel F, Fraga M, Schuh RS, Zorzi GK, Matte U et al (2017) Cationic
nanoemulsions as nucleic acids delivery systems. Int J Pharm 534:356–367. https://doi.org/
10.1016/j.ijpharm.2017.10.030
112. Apaolaza PS, del Pozo-Rodríguez A, Torrecilla J, Rodríguez-Gascón A, Rodríguez JM,
Friedrich U et al (2015) Solid lipid nanoparticle-based vectors intended for the treatment of
X-linked juvenile retinoschisis by gene therapy: in vivo approaches in Rs1h-deficient mouse
model. J Control Release 217:273–283. https://doi.org/10.1016/j.jconrel.2015.09.033
113. Torrecilla J, del Pozo-Rodríguez A, Vicente-Pascual M, Solinís MÁ, Rodríguez-Gascón A
(2018) Targeting corneal inflammation by gene therapy: emerging strategies for keratitis. Exp
Eye Res 176:130–140. https://doi.org/10.1016/j.exer.2018.07.006
114. Torrecilla J, Gómez-Aguado I, Vicente-Pascual M, del Pozo-Rodríguez A, Solinís MÁ,

Rodríguez-Gascón A (2019) MMP-9 downregulation with lipid nanoparticles for inhibiting
corneal neovascularization by gene silencing. Nanomaterials 9:E631. https://doi.org/10.3390/
nano9040631
115. Vicente-Pascual M, Albano A, Solinís MÁ, Serpe L, Rodríguez-Gascón A, Foglietta F et al
(2018) Gene delivery in the cornea: in vitro & ex vivo evaluation of solid lipid nanoparticle-
based vectors. Nanomedicine 13:1847–1854. https://doi.org/10.2217/nnm-2018-0112
116. Ruiz de Garibay AP, Solinís MA, del Pozo-Rodríguez A, Apaolaza PS, Shen JS,
Rodríguez-Gascón A (2015) Solid lipid nanoparticles as non-viral vectors for gene transfec-
tion in a cell model of Fabry disease. J Biomed Nanotechnol 11:500–511
117. Mandal H, Katiyar SS, Swami R, Kushwah V, Katare PB, Kumar Meka A et al (2018) ε-Poly-
l-Lysine/plasmid DNA nanoplexes for efficient gene delivery in vivo. Int J Pharm
542:142–152. https://doi.org/10.1016/j.ijpharm.2018.03.021
118. Look J, Wilhelm N, von Briesen H, Noske N, Günther C, Langer K et al (2015)
Ligand-modified human serum albumin nanoparticles for enhanced gene delivery. Mol
Pharm 12:3202–3213. https://doi.org/10.1021/acs.molpharmaceut.5b00153
119. Tros de Ilarduya C, Düzgüneş N (2013) Delivery of therapeutic nucleic acids via transferrin
and transferrin receptors: lipoplexes and other carriers. Expert Opin Drug Deliv
10:1583–1591. https://doi.org/10.1517/17425247.2013.837447
120. Mohammed-Saeid W, Chitanda J, Al-Dulaymi M, Verrall R, Badea I (2017) Design and
evaluation of RGD-modified gemini surfactant-based lipoplexes for targeted gene therapy in
melanoma model. Pharm Res 34:1886–1896. https://doi.org/10.1007/s11095-017-2197-0
121. Layek B, Lipp L, Singh J (2015) Cell penetrating peptide conjugated chitosan for
enhanced delivery of nucleic acid. Int J Mol Sci 16:28912–28930. https://doi.org/10.3390/
ijms161226142
122. Alipour M, Hosseinkhani S, Sheikhnejad R, Cheraghi R (2017) Nano-biomimetic carriers
are implicated in mechanistic evaluation of intracellular gene delivery. Sci Rep 7:41507.
https://doi.org/10.1038/srep41507
123. Havel H, Finch G, Strode P, Wolfgang M, Zale S, Bobe I et al (2016) Nanomedicines:
from bench to bedside and beyond. AAPS J 18:1373–1378
124. Kraft JC, Freeling JP, Wang Z, Ho RJ (2014) Emerging research and clinical development
trends of liposome and lipid nanoparticle drug delivery systems. J Pharm Sci 103:29–52.
https://doi.org/10.1002/jps.23773
125. Tinkle S, McNeil SE, Mühlebach S, Bawa R, Borchard G, Barenholz YC et al (2014)
Nanomedicines: addressing the scientific and regulatory gap. Ann N Y Acad Sci
1313:35–56. https://doi.org/10.1111/nyas.12403
126. Tyner KM, Zou P, Yang X, Zhang H, Cruz CN, Lee SL (2015) Product quality for
nanomaterials: current U.S. experience and perspective. Wiley Interdiscip Rev Nanomed
Nanobiotechnol 7:640–654. https://doi.org/10.1002/wnan.1338
127. Tyagi P, Santos JL (2018) Macromolecule nanotherapeutics: approaches and challenges.
Drug Discov Today 23:1053–1061. https://doi.org/10.1016/j.drudis.2018.01.017
128. ICH (International Conference on Harmonisation) Quality guidelines. https://www.ich.org/
products/guidelines/quality/article/quality-guidelines.html. Accessed 10 Apr 2019
129. Bastogne T (2017) Quality-by-design of nanopharmaceuticals – a state of the art.
Nanomedicine 13:2151–2157. https://doi.org/10.1016/j.nano.2017.05.014
130. Anderson WF, Blaese RM, Culver K (1990) The ADA human gene therapy clinical protocol:
points to consider response with clinical protocol. Hum Gene Ther 1:331–362
131. Cavazzana-Calvo M, Hacein-Bey S, de Saint Basile G, Gross F, Yvon E, Nusbaum P et al
(2000) Gene therapy of human severe combined immunodeficiency (SCID)-X1 disease.
Science 288:669–672. https://doi.org/10.1126/science.288.5466.669
132. Pearson S, Jia H, Kandachi K (2004) China approves first gene therapy. Nat Biotechnol
22:3–4. https://doi.org/10.1038/nbt0104-3
133. Farkas AM, Mariz S, Stoyanova-Beninska V, Celis P, Vamvakas S, Larsson K et al (2017)
Advanced therapy medicinal products for rare diseases: state of play of incentives supporting
development in Europe. Front Med 4:53. https://doi.org/10.3389/fmed.2017.00053
Gene Therapy 367
134. Sinclair A, Islam S, Jones S (2018) Gene therapy: an overview of approved and pipeline
technologies. CADTH, Ottawa. (CADTH issues in emerging health technologies; issue 171)
135. EMA (European Medicine Agency) (2019) Human medicines. https://www.ema.europa.eu/en/
medicines/human. Accessed 16 Apr 2019
136. FDA, U.S. Food and Drug Administration (2019) Approved cellular and gene therapy products.
https://www.fda.gov/BiologicsBloodVaccines/CellularGeneTherapyProducts/ApprovedProducts/
default.htm Accessed 16 Apr 2019
137. Walsh G (2018) Biopharmaceutical benchmarks 2018. Nat Biotechnol 36:1136–1145. https://
doi.org/10.1038/nbt.4305
138. Yu TTL, Gupta P, Ronfard V, Vertès AA, Bayon Y (2018) Recent progress in European
advanced therapy medicinal products and beyond. Front Bioeng Biotechnol 6:130. https://doi.
org/10.3389/fbioe.2018.00130
139. Gross G, Waks T, Eshhar Z (1989) Expression of immunoglobulin-T-cell receptor chimeric
molecules as functional receptors with antibody-type specificity. Proc Natl Acad Sci U S A
86:10024–10028. https://doi.org/10.1073/pnas.86.24.10024
140. Ferrari G, Rossini S, Giavazzi R, Maggioni D, Nobili N, Soldati M et al (1991) An in vivo
model of somatic cell gene therapy for human severe combined immunodeficiency. Science
251:1363–1366. https://doi.org/10.1126/science.1848369
141. Halioua-Haubold CL, Peyer JG, Smith JA, Arshad Z, Scholz M, Brindley DA et al (2017)
Regulatory considerations for gene therapy products in the US, EU, and Japan. Yale J Biol
Med 90:683–693
142. Kaemmerer WF (2018) How will the field of gene therapy survive its success? Bioeng Transl
Med 3:166–177. https://doi.org/10.1002/btm2.10090
143. Ma G, Shimada H, Hiroshima K, Tada Y, Suzuki N, Tagawa M (2009) Gene medicine for
cancer treatment: commercially available medicine and accumulated clinical data in China.
Drug Des Devel Ther 2:115–122
144. Pflaum J, Schlosser S, Müller M (2014) p53 family and cellular stress responses in cancer.
Front Oncol 4:285. https://doi.org/10.3389/fonc.2014.00285
145. Cheng P-H, Wechman SL, McMasters KM, Zhou HS (2015) Oncolytic replication of
E1b-deleted adenoviruses. Viruses 7:5767–5779. https://doi.org/10.3390/v7112905
146. Castellanos MR, Pan Q (2016) Novel p53 therapies for head and neck cancer. World J
Otorhinolaryngol Head Neck Surg 2:68–75. https://doi.org/10.1016/j.wjorl.2016.05.005
147. Eissa IR, Bustos-Villalobos I, Ichinose T, Matsumura S, Naoe Y, Miyajima N et al (2018)
The current status and future prospects of oncolytic viruses in clinical trials against
melanoma, glioma, pancreatic, and breast cancers. Cancers 10:E356. https://doi.org/10.3390/
cancers10100356
148. Rehman H, Silk AW, Kane MP, Kaufman HL (2016) Into the clinic: talimogene laherparepvec
(T-VEC), a first-in-class intratumoral oncolytic viral therapy. J Immunother Cancer 4:53.
https://doi.org/10.1186/s40425-016-0158-5
149. EMA (European Medicine Agency) (2016) Zalmoxis: summary of product characteristics.
https://www.ema.europa.eu/en/documents/product-information/zalmoxis-epar-product-informa
tion_en.pdf. Accessed 16 Apr 2019
150. Mohty M, Labopin M, Velardi A, van Lint MT, Bunjes D, Bruno B et al (2016) Allogeneic
genetically modified T cells (HSV-TK) as adjunctive treatment in haploidentical hematopoi-
etic stem-cell transplantation (haplo-HSCT) of adult patients with high-risk hematological
malignancies: a pair-matched analysis from the acute leukemia working party of EBMT.
Blood 128:672
151. June CH, O’Connor RS, Kawalekar OU, Ghassemi S, Milone MC (2018) CAR T cell
immunotherapy for human cancer. Science 359:1361–1365. https://doi.org/10.1126/science.
aar6711
152. Hartmann J, Schüßler-Lenz M, Bondanza A, Buchholz CJ (2017) Clinical development of
CAR T cells – challenges and opportunities in translating innovative treatment concepts.
EMBO Mol Med 9:1183–1197. https://doi.org/10.15252/emmm.201607485
153. Solinís MA, del Pozo-Rodríguez A, Apaolaza PS, Rodríguez-Gascón A (2015) Treatment of
ocular disorders by gene therapy. Eur J Pharm Biopharm 95(Pt B):331–342. https://doi.org/10.
1016/j.ejpb.2014.12.022
154. Stein CA, Castanotto D (2017) FDA-approved oligonucleotide therapies in 2017. Mol Ther
25:1069–1075. https://doi.org/10.1016/j.ymthe.2017.03.023
155. Parashar A (2016) Aptamers in therapeutics. J Clin Diagn Res 10:BE01–BE06. https://doi.org/
10.7860/JCDR/2016/18712.7922
156. Russell S, Bennett J, Wellman JA, Chung DC, Yu ZF, Tillman A et al (2017) Efficacy and
safety of voretigene neparvovec (AAV2-hRPE65v2) in patients with RPE65-mediated
inherited retinal dystrophy: a randomised, controlled, open-label, phase 3 trial. Lancet
390:849–860. https://doi.org/10.1016/S0140-6736(17)31868-8
157. Talbot K, Tizzano EF (2017) The clinical landscape for SMA in a new therapeutic era.
Gene Ther 24:529–533. https://doi.org/10.1038/gt.2017.52
158. Lim KRQ, Maruyama R, Yokota T (2017) Eteplirsen in the treatment of Duchenne muscular
dystrophy. Drug Des Devel Ther 11:533–545. https://doi.org/10.2147/DDDT.S97635
159. Chen C, Yang Z, Tang X (2018) Chemical modifications of nucleic acid drugs and their
delivery systems for gene-based therapy. Med Res Rev 38:829–869. https://doi.org/10.1002/
med.21479|
160. Wong E, Goldberg T (2014) Mipomersen (kynamro): a novel antisense oligonucleotide
inhibitor for the management of homozygous familial hypercholesterolemia. P T 39:119–122
161. EMA (European Medicine Agency) (2018) Onpattro: summary of product char-
acteristics. https://www.ema.europa.eu/en/documents/product-information/onpattro-epar-prod
uct-information_en.pdf. Accessed 16 Apr 2019
162. Carvalho M, Martins AP, Sepodes B (2019) Hurdles in gene therapy regulatory approval: a
retrospective analysis of European Marketing Authorization Applications. Drug Discov Today
24:823–828. https://doi.org/10.1016/j.drudis.2018.12.007
163. Gonçalves GAR, Paiva RMA (2017) Gene therapy: advances, challenges and perspectives.
Einstein 15:369–375. https://doi.org/10.1590/S1679-45082017RB4024
164. Daley GQ, Lovell-Badge R, Steffann J (2019) After the storm – a responsible path for genome
editing. N Engl J Med 380:897–899. https://doi.org/10.1056/NEJMp1900504
165. Flume M, Bardou M, Capri S, Sola-Morales O, Cunningham D, Levin LA et al (2018)
Approaches to manage ‘affordability’ of high budget impact medicines in key EU countries.
J Mark Access Health Policy 6:1478539. https://doi.org/10.1080/20016689.2018.1478539
166. Novartis (2019) AveXis announces innovative Zolgensma® gene therapy access programs for US
payers and families. https://www.novartis.com/news/media-releases/avexis-announces-innova
tive-zolgensma-gene-therapy-access-programs-us-payers-and-families. Accessed 31 Jul 2019
DOI: 10.1007/10_2019_110
The Impact of Pharmacogenomics

in Personalized Medicine
Dev Bukhsh Singh
Contents
1 Pharmacogenomics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 370
2 Factors Affecting Response of Drug . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 372
2.1 Genetic Polymorphism . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 372
2.2 Epigenetic and Other Factors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 373
3 Pharmacogenomics Biomarker . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 373
4 Pharmacogenomics Guidelines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 374
5 Personalized Medicine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 375
5.1 Clinical Guidance for Personalization of Therapy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 376
5.2 Impact of Pharmacogenomics on Personalized Medicine . . . . . . . . . . . . . . . . . . . . . . . . . . . . 379
5.3 Ethical Issues Related to Personalized Medicine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 387
6 Challenges and Future Opportunities . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 387
7 Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 389
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 389
Abstract Recent advances in Pharmacogenomics have made it possible to under-

stand the reasons behind the different response of a drug. Discovery of genetic
variants and its association with the varying response of drug provide the basis for
recommending a drug and its dose to an individual patient. Genetic makeup-based
prescription, design, and implementation of therapy not only improve the outcome
of treatments but also reduce the risk of toxicity and other adverse effects. A better
understanding of individual variations and their effect on drug response, metabolism
excretion, and toxicity will replace the trial-and-error approach of treatment. Evi-
dence of the clinical utility of pharmacogenetics testing is only available for a few
medications, and FDA labels only require pharmacogenetics testing for a small
number of drugs. Although there is a great promise, there are not many examples
D. B. Singh (*)
Department of Biotechnology, Institute of Biosciences and Biotechnology, Chhatrapati Shahu Ji
Maharaj University, Kanpur, Uttar Pradesh, India
370 D. B. Singh
where Pharmacogenomics impacts clinical utility. Some genetic variants related to

different diseases have been reported, and many have not been studied yet. The
information related to the outcome of treatment with a particular drug and a genetic
variant can be used to release a warning/label for the use of that drug. There are many
limitations in the way of implementing the goal of personalized medicine. Future
advances in the field of genomics, diagnosis approaches, data analysis, clinical
decision-making, and sustainable business model for personalization of therapy
can speed up the individualization of therapy based on genetic makeup.
Graphical Abstract
Higher dose of Lower dose of

drug 'X' drug 'X'
recommended recommended
Pharmacogenomics
Tests
GV01 GV02
Patient Genetic
Therapeutic
Population Variants
Biomarker
(GV)
GV04 GV03
An alternative Optimal dose

drug 'Y' of drug 'X'
recommended recommended
Keywords Clinical decision, Genetic makeup, Personalized medicine,

Pharmacogenomics, Therapeutic response
1 Pharmacogenomics
Pharmacogenomics deals with the study of the genetic basis for varying response of
drugs among individuals. It is an emerging and challenging field of therapy with a
limited clinical utility and applicability. There are several factors such as environ-
mental factors, age, weight, gender, and metabolism which affect the efficacy of a
drug. The genetic makeup of the patient also decides the response drug of a drug
The Impact of Pharmacogenomics in Personalized Medicine 371
Fig. 1 Medical questions and use of omics and other approaches for better therapeutic decisions
and individualization of therapy
[1]. There are many genetic variants associated with a disease. All genetic variants
are not associated with the drug responses on treatment. Some genetic variants may
be associated with the risk of developing a disease. The expression level of genes in
different patients may be different. Patient with the higher expression level of a
protein will produce a different response as compared to a patient with lower or no
expression level of the same protein [2]. Many Pharmacogenomics tests have been
developed for the treatment of some disease, but the clinical applicability of these
tests is very limited.
Ethical issues related to the study and application of Pharmacogenomics should
be addressed. There is a need to protect the confidentiality of patients, and all
patients should have equal opportunity to get benefitted by personalized therapy.
Pharmacogenomics can bring a significant improvement in the issues related to the
safety and efficacy of the drug. Recent omics advances have made it possible to
understand the significance of genetic variations on individual patient’s response to a
drug. Omics technologies have enabled us to answer the many medical questions and
also promoted the personalization of therapies based on genetic makeup (Fig. 1). The
long-term goal of Pharmacogenomics is to help medical practitioners in the diagno-
sis and prescription of a drug and its dosage, based on the patient’s genetic makeup.
The major objective of Pharmacogenomics is to study and catalog all the genetic and
epigenetic variants that cause variation in drug response. Pharmacogenomics-related
data, testing, and drug label are available for only some drugs. Pharmacogenomics-
related study has been conducted for drugs used for the treatment of cancer, diabetes,
depression, immunotherapy, anticonvulsant, anti-infective, cardiovascular, and psy-
chotropic drugs, as well as for some other therapies.
Several deaths occur due to the adverse effect of the drug. Pharmacogenomics
tests have reduced the cases of adverse drug reactions and also ignored the trial-and-
372 D. B. Singh
error approach of therapy. Thus, Pharmacogenomics approaches may lower down

the cost of therapy for patients and physicians significantly. Currently, most of drug
and dose prescriptions are based on age, weight, sex, lifestyle, and level of liver and
kidney enzymes. Scientists can identify the genetic variation in a small subset of
population which is associated with varying response to a drug [3]. The Food and
Drug Administration (FDA) includes Pharmacogenomics information such as dos-
age guidelines, side effects, and efficacy of 150 drugs for patients with certain
genetic makeup.
2 Factors Affecting Response of Drug

2.1 Genetic Polymorphism
Eighty-five percent of human diversity at Short Tandem Repeat (STR) and Restric-
tion Fragment Length Polymorphism (RFLP) autosomal loci is the reason behind
differences between individuals of the same population, whereas differences
between individuals of the same continent account for 5–10% [4]. Variation in
drug response is not the only result of a mutation in a single gene but also happens
by the altered function of related genes. Variations in genes associated with phar-
macokinetics and pharmacodynamics of drug may result in toxic, altered, or no
response. These ADME-related variations decide the effective concentration of drug
that reaches to drug target and its metabolism [5].
The information of genetic variants associated with cancer or other disease is very
useful in early diagnosis of disease and also provides the basis for personalized
therapy. Next-generation sequencing (NGS) and genome-wide association studies
(GWAS) have made it possible to understand the genetic mechanisms involved in a
disease and also promote the individualization of therapy based on genetic makeup.
The GWAS have discovered many genetic variants related to different lethal dis-
eases. These variants can be used for developing a biomarker for diagnosis and
therapeutic categorization of a particular disease. The systematic cataloguing of
genetic variants and related outcome on therapy provide the information of pathways
and related enzymes and also explain the reason behind the varied response of
treatment by different patients [6]. Genetic variants can provide better and accurate
clinical decisions along with the family history of the disease. Rare genetic variants
cannot be easily identified by GWAS, and they may have a significant effect on the
risk of disease [7]. The 1000 genome projects have identified many genetic variants
at lower frequencies [8]. The Human Genome Project has covered the sequencing of
the entire human genome, including the 99.9% of the genome where all humans are
identical in genetic makeup/composition [9]. The HapMap project has characterized
the patterns of a DNA sequence within the 0.1% genome where a genetic variation
exists among individuals. The HapMap has facilitated the development of some
diagnostic tools and also guides the selection of drug target for the treatment of some
disease. Information’s on polymorphism and related risk of occurrence of a disease

can be used as an alert message for a patient.
2.2 Epigenetic and Other Factors
Epigenetic factors such as age, sex, liver and kidney function, lifestyle, previous
disease, and adverse reaction are also decides the therapeutic response of a drug
[10]. Old age patients have a high risk of adverse reaction due to the poor rate of
physiology and metabolism. Gender-specific physiological differences such as preg-
nancy and breastfeeding also affect the outcome of treatment by a drug [11]. A
significant difference between the level of different hormones between a male and
female has been observed, which may cause a different response to a drug in male
and female. Environmental chemicals, drugs, and natural compounds can alter the
efficacy of the drug by drug-drug interaction or drug-herb interactions [12]. These
factors can induce or inhibit drug-metabolizing enzymes and drug transporters.
The use of Ginkgo biloba (ginkgo) along with warfarin or aspirin results in
bleeding and raises blood pressure, when used in combination with a thiazide
diuretic [13]. The use of Ginkgo biloba (ginkgo) along with trazodone may cause
coma in patients. Herb labels should also be available to avoid the use of a drug in
combination with the herb. A drug-drug interaction (DDIs) involves pharmacoki-
netic or pharmacodynamic mechanisms which may affect the bioavailability, effi-
cacy, and response of a drug. The pharmacokinetics and pharmacodynamics of many
co-administered drugs are known, but the roles of co-administered herbs are not well
explored due to a complex mixture of herbal extracts [14]. The pharmacokinetics and
pharmacodynamics of drug-drug and herb-drug interactions cannot be ignored while
prescribing a drug for therapy.
3 Pharmacogenomics Biomarker
Biomarkers include genetic or somatic gene variants, changes in expression level,

functional irregularities, and chromosomal abnormality. Biomarkers may be used for
diagnosis of diseases or assessment of therapeutic efficacy. Pharmacogenomics
biomarker is used for prescribing the drug or its dose based on the level of biomarker
and presence or absence of its variants. Some biomarker-related treatments of
different diseases are available and can describe clinical response variability, risk
of adverse drug reaction, a genotype-specific dose of a drug, mechanism of the
drug, and polymorphic drug target. FDA-approved drugs for some disease and
their biomarkers are listed in Table 1. It represents the drugs that can only be
recommended to patients who carry a particular variant related to the biomarker.
This can be very useful for prescribing safe and effective therapy based on the
biomarker. Discovery of novel and potential biomarkers related to a disease can play
a very important role in diagnosis, prescription, and personalization of therapy.
374 D. B. Singh
Table 1 List of Pharmacogenomics biomarkers used for the therapy of diseases [15]
Referenced
Drug Therapeutic area Biomarker subgroup Outcome/efficacy
Abacavir Infectious dis- HLA-B HLA-B5701 High risk of immune-
eases (HIV) allele carriers mediated hypersensitivity
reaction and should not
receive abacavir [16]
Afatinib Oncology EGFR EGFR exon These mutants incur sen-
(tyrosine 19 deletion or sitivity to afatinib treat-
kinase exon 21 substitu- ment [17]
inhibitor) tion (L858R)
positive
Aripiprazole Psychiatry CYP2D6 CYP2D6 poor Half of the usual dose
metabolizers should be administered
[18]
Carvedilol Cardiology CYP2D6 CYP2D6 poor High plasma concentra-
metabolizers tions of carvedilol, dose
monitoring required [18]
Clobazam Neurology CYP2C19 CYP2C19 poor Avoid clobazam or start
metabolizers with a low dose (2.5 mg/
day) [19]
Celecoxib Rheumatology CYP2C9 CYP2C9 poor Half/lower dose
metabolizers recommended [20]
Cisplatin Oncology TPMT TPMT intermedi- Consider alternate drug or
ate or poor use lower dose to avoid
metabolizers ototoxicity [21]
Diazepam Psychiatry CYP2C19 CYP2C19 poor Consider lower dose to
metabolizers avoid prolonged sedation
and unconsciousness [22]
Omeprazole Gastroenterology CYP2C19 CYP2C19 poor Lower the dose to avoid
metabolizers drug-drug interaction [23]
4 Pharmacogenomics Guidelines
Genotype-based dosing guideline has been published by clinical pharmacogenetics

implementation consortium (CPIC), Dutch Pharmacogenetics Working Group
(DPWG), or other organizations [24]. CPIC provides the supports for implementa-
tion of pharmacogenetics tests into clinical practice [25]. The DPWG is a
multidisciplinary organization and includes the knowledge of experts such as
pharmacists, pharmacologists, chemists, epidemiologists, and toxicologists [26].
The DPWG is also working for therapeutic (dose) recommendations based on
pharmacogenetics data and guides the health practitioners and doctors in drug and
dose prescription [27]. PGxOne™ provides the clinical Pharmacogenomics test and
generates a relevant medical and clinical report which can guide the treatment of
patients. PGxOne™ provides the dose-related guidelines for some drugs. [28] stated
that “Justice demands that benefits of personalized medicine must be available
to individuals of all racial and socioeconomic status” [28]. Peterson-Iyer [29]
has also suggested some recommendation for consideration in the policy of

Pharmacogenomics [29].
5 Personalized Medicine
Personalized medicine is a therapeutic approach which utilizes the genetic and

epigenetic information of an individual. The current trend of clinical therapy is to
provide the medication to a particular patient based on its characteristics. Such
personalization is achieved by the use of omics approaches which enable us to
find the inter-individual variability at genomic, transcriptomic, proteomic, and
metabolomic levels. The food and drug administration has approved some genomic
companies for the screening of genetic health risks related to Alzheimer’s disease
and rare blood disorder. The current clinical treatment paradigm is the right drug, for
the right patient, at the right time. Personalization of therapy requires data collection
through diagnostic from patients, individualization of therapy based on analysis, and
an appropriate and sustainable business model. Digital and mobile medical applica-
tions have played an important role in the characteristics of disease at the individual
level. Many definitions of personalized medicine are available. Some definitions
refer to medicine at an individual patient and while some consider its scope to a
subpopulation. But, most examples of personalized medicine are not personalized.
For example, a subgroup of women with early breast cancer and HER2 positive is
suitable for treatment with trastuzumab. Personalized medicine is a subset of per-
sonalized health care. Personalized health care not only includes genomics but also
considers the other information’s which predicts the risk of disease and patients
response to treatment.
Genetic variants associated with pharmacokinetics and pharmacodynamics of a
drug decide the outcome or efficacy of treatment. For the application of Pharmaco-
genomics in cancer, the selection of a drug and target of drug both are important in
deciding the response of treatment. It is important to decide whether a drug is based
on the genetics of individuals or genetics of cancer cell or both. If a drug target to
cancer cell, then the outcome of treatment depends on the genetics of individual as
well also on the genetics of particular tumor cell.
A better understanding of heterogeneity that exists among individuals and dis-
eases can help in implementing the goal of personalized medicine. The outcomes of
the Human Genome Project have generated a lot of interest in the personalization of
therapy by identifying the individual-level differences of diseases based on genetic
makeup. Therapeutic decisions based on an individual’s genetic profile and medical
tests can be more accurate and efficacious in terms of response. The risk of disease
and possible outcome of treatment can be estimated using information such as age,
sex, genetic profile, expressions related to disease, medical tests, and history of the
disease [30]. Disease screening tests and low-cost diagnostic tools can help in
detection of fatal disease at an early stage, and these results can be utilized in making
therapeutic decisions to improve the health status of a patient. Genomic information
376 D. B. Singh
greatly affects the dose and response of treatment. Dose efficacy and safety moni-
toring tests play a significant role in improving the status and cost-effectiveness of
patient care.
As the result of omics effort, more than 10 million SNPs have been identified, and
extensive studies on SNPs and related diseases have also been performed to find its
role in clinical applications for Pharmacogenomics and personalized medicine
[31]. There is a need to validate the biomarkers for patient stratification and dose
selection. Clinical relevance, molecular mechanism, clinical evidence, and regula-
tory and clinical guidelines related to relevant SNPs can trigger the application of
personalized medicine. Genomic informations are highly valuable in making a
medical decision related to drug and dose. But, the overall therapeutic response is
not only based on genomics test alone. Moreover, a better therapeutic response can
be achieved by combining genomic test results with knowledge of age, sex, lifestyle,
size, stage, nature, and origin of the disease. The nongenetic factors, such as
environmental and clinical covariates, may provide important phenotypic informa-
tion which can be used for better therapeutic decision [32]. In addition to CYP2C9
and VKORC1, the dose of warfarin therapy also depends on age, sex, body mass
index, diet, and concomitant drug therapy.
5.1 Clinical Guidance for Personalization of Therapy
In previous decades, all patients with the disease were receiving the treatment with
the same drug. But, now therapies have become more personalized. For example,
breast cancer patients that produce estrogen receptor may be treated with the drug
that targets the estrogen receptor. A significant fraction of patients (subtype)
possessing a particular characteristic of the disease can be identified for treatment
with a drug. This can be one of the ways to achieve the goal of personalized
medicine. Different biomarkers related to a disease can be detected and quantified.
Biomarkers can be used to divide the patients into different subtypes based on the
susceptibility to disease and the outcome of treatment. Recent advances in technol-
ogy have made it possible to sequence an entire genome of an individual and
quantify all the proteins, metabolites, and microbiome in a tissue. Advance data
analysis tools and algorithms can be used to mine the clinically significant bio-
markers related to a disease. Regulatory guidelines related to the use of biomarkers
as a diagnostic tool are needed, and it will help in the discovery and use of bio-
markers in medical practice. Biomarker-based tests and related clinical guidance for
the personalized treatment of some diseases are shown in Table 2. Advanced
diagnostic approaches are allowing doctors to prescribe the drug and dose to patients
based on their molecular profile [44]. There are several advanced diagnostic tests for
different types of cancer which identifies the expression or mutation in genes related
to the disease. These diagnostic tests are a key parameter for prescribing a drug to a
patient. Personalized guidance regarding prevention of a disease can be given to a
healthy person by analyzing his genome, proteome, metabolome, and microbiome.
Table 2 Some examples of personalized medicine and related clinical guidance

Therapy Test and its description Clinical guidance Reference
Breast cancer BRCA1: deleterious Surveillance and chemo- Redekop and
BRCA1 or BRCA2 prevention therapy Mladsi [30]
mutants have a high risk
of breast and ovarian
cancer
Breast cancer HER2: tumors with Use of Trastuzumab Redekop and
HER2 overexpression recommended Mladsi [30]
Epilepsy HLA-B1502: Use of carbamazepine Redekop and
HLA-B1502-positive may be dangerous, Mladsi [30]
patients show skin reac- choose an alternative
tions on treatment with drug
carbamazepine
Atrial fibrillation CYP2C9, VKORC1: Dose of Warfarin Redekop and
dose of warfarin therapy Mladsi [30]
partly dependent on
CYP2C9 and VKORC1
genotypes
Hepatitis C HCV RNA: measures the Required duration of Redekop and
level of viral RNA after treatment Mladsi [30]
treatment with interferon
alfa and ribavirin
Antiplatelet CYP2C19: clopidogrel is Use alternative Mousa et al.
medications a prodrug that requires antiplatelet agent other [33]
metabolic activation by than clopidogrel for
CYP2C19 patients with decreased
CYP2C19
Immunosuppressive Thiopurine Patients with decreased Relling et al.
therapy methyltransferase TPMT function have a [34]
(TPMT): azathioprine is a higher risk for toxicity
prodrug converted to with azathioprine
mercaptopurine that
undergoes methylation to
inactive metabolites by
TPMT
Immunosuppressive CYP3A5: tacrolimus Carriers of CYP3A5 Renders et al.
therapy undergoes oxidative alleles may require [35]
metabolism to inactive higher doses
metabolites by CYP3A4/
3A5 enzymes
Kidney transplants CYP2C19: metabolism of Use an alternative agent Guinea et al.
and invasive fungal voriconazole occurs pre- in case of CYP2C19 [36]
infection dominantly through rapid or poor metabolizer
CYP2C19
(continued)
378 D. B. Singh
Table 2 (continued)
Therapy Test and its description Clinical guidance Reference
Hyperlipidemia SLCO1B1 Use an alternative agent Armitage et al.
SLCO1B1 is responsible or lower dose in case of [37]
for uptake of simvastatin decreased SLCO1B1
from the blood to hepato- activity
cytes (metabolized). Sim-
vastatin blood
concentrations increases
at reduced SLCO1B1
level
Chronic myeloid BCR and ABL gene are Imatinib is a BCR-ABL Druker et al.
leukemia (CML) present on chromosome tyrosine kinase inhibitor [38]
number 22 and 9, respec- and can be used for
tively. The BCR-ABL treatment of BCR-ABL-
mutation occurs when based CML
pieces of BCR and ABL
break off and switch
places. BCR-ABL con-
firms the diagnosis of
CML
Lung cancer EML4-ALK fusion gene Crizotinib works only in Kwak et al. [39]
used for diagnosis of cancer with overactive
non-small cell lung can- ALK
cer (NSCLC). Such types
of cancer can be cured by
targeting anaplastic lym-
phoma kinase (ALK)
inhibitor
Coronary artery CYP2C19 converts Use of clopidogrel may Simon et al. [40]
disease clopidogrel into an active be toxic or less effica-
metabolite. Individuals cious in poor
who carry two metabolizer. Use another
nonfunctional copies of alternative agent/drug
the CYP2C19 gene are
poor metabolizers of
clopidogrel
Autoimmune CXCL13, aberrant Antibodies targeting Leypoldt et al.
encephalitis expression of CXCL13, is CXCL13-mediated sig- [41]
linked to the development naling pathway
of autoimmune disorders
Cystic fibrosis G551D, G551D mutation Ivacaftor recommended Ramsey et al.
in ATP-binding pockets for the patients with the [42]
abolishes ATP-dependent G551D mutation
gating. It is one of the
common mutation asso-
ciated with cystic fibrosis
Thrombosis Factor V Leiden is a spe- Avoid prothrombotic Vandenbroucke
cific gene mutation that drugs for factor V et al. [43]
results in thrombophilia Leiden-positive patients
Pharmacogenomics tests are responsible for drug metabolism, transport, and

drug target. Genetic variations can increase the risk of toxicity or poor efficacy.
Pharmacogenomics helps us to select the most suitable drug and dose to achieve a
better therapeutic response. Several clinical recommendations for commonly used
anticoagulants, antiplatelets, transplant medications, and other therapies are avail-
able [45]. Clinical decision-making depends on many factors such as variants in
the patient or population and the quality of evidence, availability of testing and
Pharmacogenomics data, pharmacokinetics and pharmacodynamics of drug, drug
history, and drug-drug interactions. Service providers should also have a good
knowledge of Pharmacogenomics resources to implement an accurate clinical deci-
sion. Pharmacogenomics mainly helps in the implementation of personalized med-
icine by predicting the patients who should receive a lower dose/higher dose or an
alternative drug. The implementation of Pharmacogenomics is limited by challenges
in clinical testing, data analysis, lack of education, and ethical, legal, and social
implications. Despite the barriers to clinical Pharmacogenomics, several academic,
medical, and community centers have initiated Pharmacogenomics implementation
programs. Future advances in precision medicine and data sharing between health
service providers and patients will help in achieving the goal of personalized
medicine.
Imaging techniques can perform many diagnoses with good confidence and also
avoids the patients to go for invasive testing and unnecessary surgery. Now com-
puted tomography and ultrasonography provide better results with improved sensi-
tivity and specificity in the diagnosis of many diseases [46]. Positron-emission
tomography can detect metabolically active cancer that is not easily detectable
with traditional imaging. A rich database of electronic health records can be a
guide for clinical decision. In the future, software can be developed to identify
patients with disease risk factors or to follow screening guidelines and also for
drug selection and administration.
5.2 Impact of Pharmacogenomics on Personalized Medicine
5.2.1 Cancer
The concept of personalizing treatments provides the best possible direction for
the treatment of many cancers. Different therapeutic strategies for the treatment of
cancer are surgery, radiotherapy, chemotherapy, immunotherapy, hormone therapy,
gene therapy, and dietary therapy. We can prefer therapies from a wide range
of options based on the type, location, and stage of cancer. Current knowledge of
cancer classification, predictive markers, and combination therapies holds the
best possible avenues for cancer treatment in the future. The strategy is the most
important issue in cancer therapy. As a result of pharmacogenomic advances,
different therapeutic strategies for the treatment of various cancers have been
adopted. Gene therapy approach has been used for the treatment of prostate cancer
and as result necrosis observed at metastatic sites [47]. Personalized medicine is used
380 D. B. Singh
in cancer therapy for screening formulation in the epidermal growth factor receptor
(EGFER) gene in lung adenocarcinoma.
Variants Associated with Risk of Cancer
Studies have shown an association between genetic variants of a gene and the risk
of developing a disease. BRCA1, BRCA2, TP53, PTEN, STK11, CDH1, CHEK2,
ATM, BRIP1, PALB2, RAD51C, RAD50, and NBN genes have been identified
for breast cancer. Association of these genes with risk of disease has been further
investigated. Three variants rs80358978 (Gly2508Ser), rs80359065 (Lys2729Asn),
and rs11571653 (Met784Val) have been reported in the BRCA2 gene, and these
variants have shown significant associations with risk of developing breast cancer
[48]. Variants rs8176085, rs799923, rs8176173, and rs8176258 in the BRCA1 gene,
one common variant in the CHEK2 gene (rs9620817), and one common variant in
the PALB2 gene (rs13330119) are also associated with risk of this disease. Simi-
larly, four genetic variants, Phe139Ser in SORD, Ala350Arg in KRT6A, Gly387Cys
in SVEP1, and Gly144Arg in MRPL38, have shown significant association for risk
of liver cancer [49]. These variants can be used for early detection of liver cancer and
designing of relevant therapeutics for treatment. These variants only predict the risk
of developing a particular type of cancer and may not be used for deciding the
therapeutic outcome of treatment with a drug.
Nearly 100 genetic variants that signal an increased risk of colon cancer are
available. Several combined genetic variants may be clinically relevant and can
impact personalized screening. For breast cancer, 182 breast cancer susceptibility
SNPs are available which can be used to understand the heritability of breast cancer
[50]. Study of risk loci for breast cancer can be a guide for prevention, prognosis, and
treatment of breast cancer. The polygenic risk score can be calculated to estimate
individual risks for developing breast cancer. GWAS have identified more than
30 epithelial ovarian cancer risk loci which may involve genome editing to establish
the cell-specific carcinogenic effects [51]. Studies have also identified susceptibility
regions potentially shared between breast, ovarian, and prostate cancer. For example,
in oral cancer, an association between TNF-α variant, rs361525, and risk of an oral
precancerous lesion has been observed. An allelic variant of rs361525 SNP, located
at a transcriptional factor-binding site of the promoter region of TNF-α, disturbs the
expression of TNF-α [52]. Studies have shown that genetic alterations in immune
system genes and genes with metastatic potential are associated with oral precancer
and oral squamous cell carcinoma.
Variants Associated with Outcome of Treatment
Afatinib is a TKI inhibitor used for the treatment of patients with non-small-cell
lung cancer (NSCLC) whose tumors are activated by EGFR [53]. It is also effective
for NSCLC patients with EGFR-T790M mutation who are resistance to the TKIs
erlotinib and gefitinib. Afatinib has shown very good response among patients with
tumors having HER2 or HER4 mutations [54]. HER2 mutations are rare; afatinib has
shown high activity for NSCLC patients with HER2 mutation. Imatinib is a TKI
inhibitor used for the treatment of CML. It has also been used for other BCR-ABL-,
c-KIT-, and PDGFR-driven cancers. Imatinib is metabolized by CYP2C8 and
CYP3A4 in vitro. CYP2C8 genotype affects the metabolism of imatinib and its
systemic exposure [55]. But, no evidence regarding the metabolism of imatinib in
CYP3A4 or CYP3A5 genotype is available. Many studies have been performed to
find the association between genetic variants and the risk of developing particular
cancer. Similarly, the varied response of the same drug has been noticed in different
genotypes which may be affected by genes involved in pharmacokinetics and
pharmacodynamics. FDA label regarding the clinical use of different therapeutics
is available which may be taken into consideration while prescribing the drug and
dose for the treatment.
Recent advances have made it possible to understand the mechanisms of the
development and progression of cancer, diagnosis, and monitoring of cancer, clinical
outcome, and risk of adverse reactions. The Cancer Genomics Analysis (TCGA) and
the International Cancer Genomics Consortium (ICGC) projects have identified
several genetic mutations responsible for various cancers. Liquid biopsy is carried
out to obtain genomic information from cell-free DNA for screening, monitoring,
and relapse/recurrence of cancer [56]. Recent advances have made it possible to
detect and quantify biomarkers with high specificity and sensitivity. KRAS and
BRAS are the most frequent mutation in human cancer, and analysis of these
mutations might be an effective approach to screen cancers [57]. There are many
mutations for which effective drugs are not available. Personalized biomarker
selection can improve the response rate of targeted therapy for many cancers. A
small subset of patients with a particular type of mutation can be targeted for the
treatment with a drug. Germline variants related to ADMET are also important for
personalization of therapy.
HLA genotypes may be used to predict the risk of drug-induced severe skin
hypersensitivity and drug-induced liver injury. Different variants of HLA-B are used
as a therapeutic biomarker for some diseases including cancer. Individuals with
HLA-B15:02 or HLA-A31:01, HLAB57:01, and HLA-B58:01 variants have
shown skin hypersensitivity for carbamazepine and abacavir drugs [58]. These side
effects can be avoided by HLA genotyping of patients. Immuno-genomic analysis of
cancer cells, such as quantification of infiltrated CD8+ T cells and their T cell
receptor, can be useful for assessment of the immune response on therapy [59]. Chi-
meric antigen receptor T cell therapy and TCR-engineered T cell therapy have
shown clinical effectiveness in hematological cancers.
5.2.2 Neurodegenerative Diseases
Use of novel CSF, blood-based, and neuroimaging biomarkers specific to particular

diseases has played a very important role in the treatment of many neurodegenerative
382 D. B. Singh
diseases. The use of novel biomarkers for neurodegenerative diseases has improved
the accuracy of diagnosis, prognosis, and the efficacy of therapy. Personalized
therapy is suggested for Alzheimer’s and Parkinson’s diseases because these dis-
eases are clinically heterogeneous and have strong genetic connections. Genetic risk
for Alzheimer’s disease was found related to the APOE4 gene. Homozygous
individuals for APOEe4 allele have a 50% risk of developing Alzheimer’s disease,
while heterozygous individuals for the APOEe3/e4 genotype have a risk of 20–30%
[60]. Mutation in other genes such as APP, PSEN1, and PSEN2 are also associated
with early onset of Alzheimer’s disease. High levels of neurogranin CSF is associ-
ated with progression to Alzheimer’s disease and results in a synaptic loss. Blood-
based biomarkers are very useful in the area of Alzheimer’s disease progression.
Neuroimaging allows for preclinical identification of individuals genetically suscep-
tible to Alzheimer’s disease.
Parkinson’s disease is characterized by loss of dopaminergic neurons and loss of
striatal dopamine signaling. GWAS have identified many genes and loci related to
Parkinson’s disease. Mutations in the genes SNCA, LRRK2, PINK1, DJ-1, and
Parkin are associated with Parkinson’s disease [61]. The activity of caspase enzymes
is also associated with Parkinson’s disease. Increased activity of Cas9 is associated
with high cellular toxicity in Parkinson’s disease and has been recommended as a
therapeutic target for the treatment of Parkinson’s disease [62]. CRISPR-Cas9 may
also be used for gene therapy in Parkinson’s disease. Immunoglobulin G exerts
pro-inflammatory responses in the body and may be used as a unique biomarker for
Parkinson’s disease. Targeting ganglioside GM1 levels in patients of Parkinson’s
disease may also be a personalized therapeutic approach. Discovery of new bio-
markers of Parkinson’s disease might be more useful for the personalized therapy of
Parkinson’s disease.
Multiple sclerosis is an autoimmune disease which affects the function of the
central nervous system. The allele HLA-DRB1 is associated with an increased risk
of multiple sclerosis [63]. GWAS has identified other mutations which increase the
risk of multiple sclerosis such as SNPs in interleukin-7 receptor an (IL7R) gene. The
chemokine C-X-C motif ligand 13 (CXCL13) is used as a dual biomarker for
prognosis and therapy monitoring. A high correlation was found between increased
CXCL13 levels and multiple sclerosis [64]. Increased soluble CD163 levels in blood
and CSF have been identified in multiple sclerosis patients, and soluble CD163 may
also be used as a biomarker for multiple sclerosis [65].
Technological advances in Pharmacogenomics will ensure the clinical applica-
tion of personalized medicine. Genomics, transcriptomics proteomics, meta-
bolomics, and neuroimaging have to speed up the identification of novel
biomarkers and genetic connection of diseases. GWAS, NGS, microarray data
analysis, and identification of mRNA, miRNAs, metabolites, proteins, SNPs,
copy-number variations, and other genetic alternations can be useful to assess the
individualized risk levels for a patient and can also suggest a most effective
therapeutic approach.
5.2.3 Thrombosis
Novel approaches are required to understand the genetic basis of thrombosis which
can provide better and personalized therapy for thrombosis. Thrombosis is a com-
plex disease caused by a combination of numerous factors such as environmental
and genetic factors. Both plasma-based and genetic assays are important for
assessing the risk of disease, but genetic factors are more informative in assessing
platelet risk factors for thrombosis. Five genetic risk factors for venous thromboem-
bolism are VTE, deficiencies of antithrombin, protein C, protein S factor V Leiden,
and the G20210A prothrombin gene variant [66]. There is a lack of clinical data set
related to thrombosis which limits the goal of personalized therapy. Clopidogrel is
effective in platelet aggregation, but a subset of patients has shown no response to
clopidogrel therapy. Varying response to warfarin therapy was noticed due to the
effect of the gene variants of CYP2C9 and VCORC1 [67]. Treatment of thrombosis
can be personalized by identifying genetic factors responsible for the development
and recurrence of the disease. VCORC1 and CYP2C9 genotype-based strategies are
very useful for initiating anticoagulant therapies. For CYP2C9 and VKORC1
genotypes, edoxaban produces better and safe response than warfarin [68].
CRISPR/Cas9 gene therapy and anti-microRNA approaches have also been used
to treat thrombosis. More clinical trials on antithrombotic agents are required to
collect and analyze the impact of genetic, clinical environmental, and dietary factors
on response to therapy. Risk of thrombosis can be assessed by studying the effect of
different genetic variants related to thrombosis.
5.2.4 Cardiovascular Diseases
Cardiovascular diseases are one the leading cause of death in developed countries.
Patients with systemic lupus erythematosus and rheumatoid arthritis have increased
risk of cardiovascular disease. Two new putative risk loci associated with increased
risk for cardiovascular disease are identified [69]. An IL19 risk allele, rs17581834
(T), is associated with stroke/myocardial infarction in systemic lupus erythematosus
and rheumatoid arthritis, while another SRP54-AS1 risk allele, rs799454(G), is
associated with stroke/transient ischemic attack in systemic lupus erythematosus
but not with rheumatoid arthritis. Several SNPs related to coronary artery disease
have been identified, but their function is not clear yet. The risk allele of a common
coronary artery disease-associated marker at the TOMM40/APOE locus was found
to be associated with a lower level of high-sensitivity C-reactive protein [70].
GWAS have shown the association between coronary artery disease-associated
risk variants and common inflammatory markers. Calprotectin, an inflammatory
marker for of plaque instability, is used as a new biomarker of coronary artery
disease.
Total 53 loci with significant effects in both coronary artery diseases were
identified and at least 1 of low-density lipoprotein, high-density lipoprotein,
384 D. B. Singh
triglycerides, type 2 diabetes mellitus, C-reactive protein, systolic blood pressure,

and type 1 diabetes mellitus [71]. These genetic loci implicate novel genetic mech-
anisms involved in coronary artery disease. These genetic loci may be used for
earlier diagnosis, prevention, and individualization of coronary artery disease
therapy.
5.2.5 Anesthesia
Succinylcholine, mivacurium, and other anesthetics are substrates for enzyme pseu-
docholinesterase. There are 70 different variants of pseudocholinesterase, and most
common polymorphism is known as atypical which impairs the function of pseudo-
cholinesterase [72]. An individual homozygous for atypical allele remains paralyzed
for 2 h, while individuals who are heterozygous paralyzed for up to an hour.
Similarly, the varying response of pseudocholinesterase inhibition has been reported
for dibucaine. An individual with rare S-variant of pseudocholinesterase remains
paralyzed for up to 8 h [73]. An individual with no or lower level of pseudocholin-
esterase may have an increased risk of toxicity. Codeine is a prodrug that is
metabolized by CYP2D6 into the active metabolite, morphine. The dose of codeine
may be recommended based on poor, intermediate, extensive, or rapid metabolizer
which will depend on the expression of CYP2D6 gene [74]. Genes for the 5HT3B
receptor, dopamine D2 receptor, the ABCB1 transporter, and CYP2D6 are associ-
ated with postoperative nausea and vomiting. Some polymorphisms related to these
genes are associated with a high incidence of postoperative nausea and vomiting.
ADRB1encodes for the β-1 adrenergic receptor, and certain polymorphism related to
this gene has also been reported.
5.2.6 Type 2 Diabetes
Type 2 diabetes is one of the major causes of premature mortality. GWAS has
identified hundreds of variants associated with type 2 diabetes which can be assessed
for their clinical utility [75]. Some genetic variants related to type 2 diabetes are
known that may have clinical significance in the prevention, personalized treatment
of type 2 diabetes. TBC1D4 and nonsense variant (Arg684ter) have different
prevalence in different population and may decide the risk of type 2 diabetes
[76]. In Latinos population, a low-frequency missense variant (E508K) at HNF1A
conveys a type 2 diabetes. These variants may be useful in the assessment of clinical
utility and personalized therapy of type 2 diabetes [77]. Metformin is used for the
treatment of diabetes treatment, and some cases of metformin intolerance have been
found. Genetic variants at ATM and glucose transporter gene (SLC2A2) are asso-
ciated with the glycemic response of metformin treatment [78].
5.2.7 Depression
Many genetic variants of cytochrome p450 (CYP450) system, relevant to the

metabolism of many antidepressants, have been identified. However, all these
variants are not providing the clinically significant information which can be utilized
in decision-making [79]. Many efforts have been made to understand the impact of
common genetic variation of CYP450 on treatment in depression. CYP450 2D6
(CYP2D6) is responsible for the oxidative metabolism of most of the antidepressants
[80]. For CYP2D6, 60–85% of white individuals were found fast metabolizers,
while some individuals with two disrupted copies of CYP2D6 were poor
metabolizers. An effect of CYP450 on treatment with venlafaxine has been studied.
Individuals who are less efficient in metabolizing venlafaxine will have lower blood
concentrations of the active metabolite desvenlafaxine [81]. For many diseases,
more clinical studies are required to take a better therapeutic decision regarding
the recommendation of a drug and its dose. Other than CYP450 systems, the drug
transport protein P-glycoprotein (ATP-binding cassette, subfamily B (MDR/TAP),
member 1 (ABCB1)) was also studied due to its role in the efflux of drugs across the
BBB [82].
5.2.8 Psychiatry
CYP has more than 90 known genetic variations and more than 60 alleles. Study of
these CYP2D6 alleles may provide better clinical information related to treatment
for psychiatry. CYP2D6 metabolizes many antipsychotics and antidepressants. The
FDA has recommended the HLA-B1502 genotyping in Asians before prescribing
carbamazepine to avoid toxic outcome [83]. A better understanding of the pharma-
cokinetics and pharmacodynamics of psychiatric drugs has allowed personalized
prescription in clinical practice. Dosing recommendation of risperidone in psychi-
atric patients is based on the presence of CYP3A inducers and/or CYP inhibitors and
CYP2D6 [84]. CYP2D6 has a higher affinity for risperidone and hydroxylating it to
9-hydroxyrisperidone. Poor metabolizer can be identified by CYP2D6 genotyping or
by measuring the level of risperidone. Knowledge pharmacokinetic, pharmacody-
namic, efficacy, safety, and adverse drug reaction are required to understand per-
sonalized prescription and its applications in psychiatry. The goal of personalized
medicine can be achieved by applying the précised medical information or knowl-
edge in therapeutic practices. AmpliChip CYP450 test for analysis of the CYP2D6
and CYP2C19 genes are available to detect poor and fast metabolizer [85]. Around
5% of the Caucasian population is CYP2D6 poor metabolizers for psychiatric drugs
which may have some side effects. Metabolism of one drug depends on other drug
taken which can inhibit or stimulate the metabolism of other. CYP450 isoenzymes
(CYP1A2, CYP2C19, CYP2D6, CYP3A4) are involved in the metabolism of
psychotropic drugs and may cause adverse drug reactions [86]. A concentration-
to-dose ratio for prescribing clozapine and risperidone is known, and CYP
386 D. B. Singh
genotyping of patients can guide therapeutic dose monitoring. CYP1A2, CYP2B6,

and CYP3A4 genotyping has a small utility from a clinical point of view, while
CYP2C9 genotyping has no utility in psychiatry [87].
5.2.9 Hypertension
Variation in individual response on treatment with the same drug motivates us to

look for genetic factors associated with this variation. Studies have shown the
association between the response of blood pressure and specific gene polymorphism.
Clinical data related to genetic polymorphism and therapeutic response can guide the
way of personalized medicine for hypertension. Inhibitors of angiotensin-converting
enzyme, β-blockers, angiotensin 2 blockers, and calcium channel blocker are well-
studied inhibitors for the treatment of hypertension [88]. Effect of β1AR Arg389Gly
polymorphism on responses of blood pressure to β-blocker therapy has been studied.
On treatment with metoprolol, patients homozygous for Arg389 had shown a high
reduction in diastolic blood pressure [89]. The β1-adrenergic receptor gene (ADRB1)
encodes a 51.3 kDa protein with 477 amino acid residues. The β1-adrenergic
receptor is present in the heart, controlling heart rate. Six SNPs located near the
ADBR1 gene region are available in dbSNP. Out of six SNPs, two polymorphisms
Ser49Gly (rs1801252) and Arg389Gly (rs1801253) have been studied well
[90]. Studies have shown that siblings with Gly389 allele have a low diastolic
blood pressure than those homozygous for Arg389.
Such types of knowledge related to polymorphism and outcome of treatment can
be utilized while recommending the drug and dose to any hypertensive patient. More
investigations should be performed to know the effect of other polymorphism on the
outcome of treatment with a hypertensive drug. Studies have found that there is a
genotype group that shows a less favorable response to β-blocker therapy. For
adrenergic receptor polymorphism, the effect of drug bucindolol on different geno-
types can be assessed [91]. This may help in finding the genotypes which show
better or poor response on treatment with bucindolol. Knowledge of genetic factors
that decides the response of a drug can enable us to choose the most suitable drug
and dose for each patient based on genetic profile. Advances in “omics” technologies
have made it possible to identify genetic markers that can be used to know the
response of a particular drug and guide the way for individualized therapy [92]. A
relationship between nephrosis (NPNS1) gene variants and response to angiotensin-
receptor antagonist losartan has been found. Also, two other genes (ALDH1A3 and
CLIC5) have shown their influence on blood pressure response to hydrochlorothi-
azide treatment. Many genetic polymorphisms that affect the response of treatment
with hypertensive drugs such as enalapril, perindopril, lisinopril, metoprolol, aten-
olol, bisoprolol, losartan, and hydrochlorothiazide have been identified [93].
Some effective approaches for the personalized treatment of hypertension are aldo-
sterone measurement, renin profiling, aldosterone-to-renin ratio, SNP and haplotype
approach, and hemodynamic assessments.
5.3 Ethical Issues Related to Personalized Medicine
In hospitals and universities, numerous tests and kits are available to detect genetic
diseases. But the accuracy of some kits and validity of some biomarkers related to
disease have a low level of confidence. The ethics of personalized medicine became
an issue when a healthy person has been reported to have a high risk of breast or
ovarian cancer. Ethical issues are also associated with the use and storage of genetic
information of an individual. Ethics related to personalized medicines are also
affected by the health policies of a country [94]. The National Institutes of Health
(NIH) is trying to develop the best way of treatment and also delivering clinical
information to the health sector and patients. Many research organizations have
confined their study and tests related to a specific genetic disorder in certain groups
of people or region. Informed consent of a patient is required for the use of their body
material to protect the privacy of an individual. There is a need to assure the
protection of privacy of genetic data of an individual from the government or
insurance companies.
6 Challenges and Future Opportunities
One of the important challenges in the way of personalized medicine is to manage

the complexity associated classification of disease. A large number of new genetic
alterations are being reported by next-generation sequencing. Association of some of
these genetic alternations with a disease is well established, but the role of many
mutations/alternation was not evaluated yet. Other alternations might have alone or
combined effect on disease prognosis. Systematic storage, retrieval, and analysis
platform are required to store the clinical data and also for decision-making. Better
biomarkers of the disease can assist with detection and also can guide treatment. The
financial incentives for designing of new diagnostics are required because a better
diagnosis of the disease can improve the rate of cure. Advances in DNA sequencing
have enabled studies of the microbiome which are present in our system.
Microbiome studies have been considered for disease and may offer opportunities
for personalized therapy.
Associations between a genetic variation and related outcome should be quanti-
fied. Implementation of Pharmacogenomics is based on these associations, and
no more emphasis has been given on clinical validity and utility of the test.
Pharmacogenetics tests should be adopted based on their clinical utility and cost-
effectiveness. Pharmacogenomics-related data and tests are available only for some
drugs, but still, this knowledge has not been potentially translated into clinical
practice at a bigger scale. However, the clinical utility of Pharmacogenomics test
is limited due to a gap between clinical validity and its application to patients
through health-care providers. Pharmacogenomics-based CPIC dosing guidelines
are available for some drugs. Primary care providers have no adequate guidelines on
388 D. B. Singh
how and when to Pharmacogenomics markers [95]. There exist a gap between
evidence of association and the clinical utility of Pharmacogenomics tests, and
there is need to overcome this challenge by exploring evidence from a health-care
provider, patient, and policy perspective [96]. Well-defined steps of health care
from clinical validity to clinical utility are required for successful implementation
of Pharmacogenomics tests to patients. Many publications related to Pharmaco-
genomics reports only about association, but other pieces of evidence such as
guideline development and coverage decisions are necessary for better implemen-
tation of Pharmacogenomics. Pharmacokinetics and pharmacodynamics of many
drugs and its mechanism of interactions have been reported well, but its clinical
utility is still very limited. The lack of clinical utility of a drug delays the application
of Pharmacogenomics tests into health care [97].
Pharmacogenomics-based therapies will reduce the trial-and-error prescription
and will also improve drug safety by avoiding adverse drug reaction. Pharmaco-
genomics will reduce the time and cost of a clinical trial. Genetic profiling will also
help in recommending the use of a failed drug for a particular group of another
population [98]. Availability of large scale of data related to the association of
genetic variations with the disease across many countries will enable us to achieve
the goal of personalized medicine. Study of genetic variations also speeds up the
process of the clinical trial process by targeting the individuals for testing belonging
to a specific population. The government and other funding agencies should give
more incentive for adopting the practices related to personalized medicine. Faster,
reliable and cost-effective approaches for sequencing, screening, and diagnosis of
diseases should be developed which will bring the use of personalized medicine in
real practice. In recent years, a large amount of knowledge-related genome, prote-
ome, and metabolic pathways have been generated which enable us to use, monitor,
and assess the effect of a drug on a patient’s genotype. The government should take
an initiative to frame policies related to the use of genetic material and individual-
ization of therapy. Health-care centers, educational institutes, and hospitals may play
important in promoting research and clinical practices of personalized medicine. It is
expected that the cases of serious adverse drug reactions will reduce in the future
with the implementation of Pharmacogenomics approaches for personalized therapy.
The cost of genetic tests and its availability to rural people are also a big challenge
for many poor and developing countries. Governments will also have to think over
the issues raised as a result of discriminations based on genotype. The pharmaceu-
tical companies may take more interest in developing the drug for a genotype
belonging to the rich group of the population rather than a disease or patient confined
to a poor subgroup of the population. Pharmacogenomics has brought a big shift in
the trend of therapies for many diseases; still, many patients are not in a position to
take the benefit of pharmacogenomic knowledge in personalizing the therapy.
Benefits of personalized medicine should be utilized without waiting for more
discoveries related to Pharmacogenomics and biomarker. The personalized prescrip-
tion can be given based on genetic, environmental, or personal factors that can affect
the outcome of the treatment. The FDA has set many recommendations and guide-
lines to promote the use of Pharmacogenomics in the personalization of therapy. To
ensure the success of personalized medicine, some strategic recommendations have

been suggested [99]. These recommendations are related to the discovery of more
potential biomarkers, framing regulations for genetic privacy and confidentiality,
regulatory incentives to pharmaceutical industries and equity of access to personal-
ized medicine, standardization of genetic testing and documentation, regulation for
private genetic testing firms, and better awareness about personalized medicine
within the medical imaging community.
7 Conclusion
Pharmacogenomics has played a very important role in adopting the goal of person-
alized medicine for many diseases. It has greatly impacted the prescription of a
drug and its dose for many diseases, but a small population of the world can enjoy
the benefits of Pharmacogenomics. Personalized medicine offers the opportunity to
identify patients for whom a drug can be both effective and safe. Efficient decision-
making on clinical data and proper utilization of health-care resources can enable us
to achieve the goal of personalized therapy. Pharmacogenomics also improves the
process of drug development by focusing the companies to test the safety and
efficacy of a drug in an individual or subgroup of a population. Pharmaceutical
companies have to reduce the cost of personalized treatment so that poor patients can
also utilize the benefit of pharmacogenomics. Medical imaging techniques have
played a very important role in the diagnosis, prediction, and treatment of many
diseases and can be very helpful in achieving the goal of personalized therapy.
References
1. Hess GP, Fonseca E, Scott R, Fagerness J (2015) Pharmacogenomic and pharmacogenetic-

guided therapy as a tool in precision medicine: current state and factors impacting acceptance by
stakeholders. Genet Res (Camb) 97:e13. https://doi.org/10.1017/S0016672315000099
2. NIH (2019) NIGMS: pharmacogenomics fact sheet. http://www.nigms.nih.gov/education/
pages/factsheet-pharmacogenomics.aspx. Accessed 20 Apr 2019
3. Kitzmiller JP, Groen DK, Phelps MA, Sadee W (2011) Pharmacogenomic testing: relevance in
medical practice: why drugs work in some patients but not in others. Cleve Clin J Med
78:243–257
4. Romualdi C, Balding D, Nasidze IS, Risch G, Robichaux M, Sherry ST, Stoneking M, Batzer
MA, Barbujani G (2002) Patterns of human diversity, within and among continents, inferred
from biallelic DNA polymorphisms. Genome Res 12:602–612
5. Nadine C, Theresa F (2008) Challenges, opportunities, and evolving landscapes in pharmaco-
genomics and personalized medicine. In: Cohen N (ed) Pharmacogenomics and personalized
medicine. Humana Press, Totowa, pp 1–26
6. Cho WC (2010) Recent progress in genetic variants associated with cancer and their implica-
tions in diagnostics development. Expert Rev Mol Diagn 10(6):699–703
7. Cirulli ET, Goldstein DB (2010) Uncovering the roles of rare variants in common disease
through whole-genome sequencing. Nat Rev Genet 11(6):415–425
390 D. B. Singh
8. IGSR (2018) The International Genome Sample Resource. http://www.1000genomes.org/

about. Accessed 02 Apr 2018
9. International HapMap Consortium (2003) The international HapMap project. Nature
426:789–796
10. Alomar MJ (2014) Factors affecting the development of adverse drug reactions (Review
article). Saudi Pharm J 22:83–94
11. Soldin OP, Chung SH, Mattison DR (2011) Sex differences in drug disposition. J Biomed
Biotechnol. https://doi.org/10.1155/2011/187103
12. Ma Q, Lu AY (2011) Pharmacogenetics, pharmacogenomics, and individualized medicine.
Pharmacol Rev 63:437–459
13. Hu Z, Yang X, Ho PC, Chan SY, Heng PW, Chan E, Duan W, Koh HL, Zhou S (2005) Herb-
drug interactions: a literature review. Drugs 65:1239–1282
14. Zuo Z, Huang M, Kanfer I, Chow MS, Cho WC (2015) Herb-drug interactions: systematic
review, mechanisms, and therapies. Evid Based Complement Alternat Med 2015:239150.
https://doi.org/10.1155/2015/239150
15. Singh DB (2017) Pharmacogenomics: clinical perspective, strategies, and challenges. In: Wei
DQ, Ma Y, Cho W, Xu Q, Zhou F (eds) Translational bioinformatics and its application.
Translational medicine research. Springer, Dordrecht, pp 299–333
16. Martin MA, Kroetz DL (2013) Abacavir pharmacogenetics--from initial reports to standard of
care. Pharmacotherapy 33:765–775
17. Delaney C, Frank S, Huang RS (2015) Pharmacogenomics of EGFR-targeted therapies in
non-small cell lung cancer: EGFR and beyond. Chin J Cancer 34:149–160
18. Dean L (2016) Aripiprazole therapy and CYP2D6 genotype. In: Pratt V, McLeod H,
Rubinstein W, Dean L, Kattman B, Malheiro A (eds) Medical genetics summaries [Internet].
National Center for Biotechnology Information (US), Bethesda
19. Hashi S, Yano I, Shibata M, Masuda S, Kinoshita M, Matsumoto R, Ikeda A, Takahashi R,
Matsubara K (2015) Effect of CYP2C19 polymorphisms on the clinical outcome of low-dose
clobazam therapy in Japanese patients with epilepsy. Eur J Clin Pharmacol 71:51–58
20. Kim SH, Kim DH, Byeon JY, Kim YH, Kim DH, Lim HJ, Lee CM, Whang SS, Choi CI,
Bae JW, Lee YJ, Jang CG, Lee SY (2017) Effects of CYP2C9 genetic polymorphisms on the
pharmacokinetics of celecoxib and its carboxylic acid metabolite. Arch Pharm Res 40:382–390
21. Hagleitner MM, Coenen MJ, Patino-Garcia A, de Bont ES, Gonzalez-Neira A, Vos HI,
van Leeuwen FN, Gelderblom H, Hoogerbrugge PM, Guchelaar HJ, Te Loo MW (2014)
Influence of genetic variants in TPMT and COMT associated with cisplatin induced hearing
loss in patients with cancer: two new cohorts and a meta-analysis reveal significant heteroge-
neity between cohorts. PLoS One 9:e115869
22. Lee SJ (2013) Clinical application of CYP2C19 pharmacogenetics toward more personalized
medicine. Front Genet 3:318
23. Shirasaka Y, Sager JE, Lutz JD, Davis C, Isoherranen N (2013) Inhibition of CYP2C19 and
CYP3A4 by omeprazole metabolites and their contribution to drug-drug interactions. Drug
Metab Dispos 41:1414–1424
24. PharmGKB (2019) CPIC: Clinical Pharmacogenetics Implementation Consortium. https://
www.pharmgkb.org/page/cpic. Accessed 03 Apr 2019
25. CPIC (2019) Clinical Pharmacogenetics Implementation Consortium. https://cpicpgx.org.
26. PharmGKB (2019) DPWG: Dutch Pharmacogenetics Working Group. https://www.pharmgkb.
org/page/dpwg. Accessed 03 Apr 2019
27. Swen JJ, Nijenhuis M, de Boer A, Grandia L, Maitland-van der Zee AH, Mulder H, Rongen
GA, van Schaik RH, Schalekamp T, Touw DJ, van der Weide J, Wilffert B, Deneer VH,
Guchelaar HJ (2011) Pharmacogenetics: from bench to byte--an update of guidelines. Clin
Pharmacol Ther 89:662–673
28. Ossorio P, Duster T (2005) Race and genetics: controversies in biomedical, behavioral, and
forensic sciences. American Psychologist 60(1):115–128
29. Peterson-Iyer K (2008) Pharmacogenomics, ethics, and public policy. Kennedy Inst Ethics J
18(1):35–56
30. Redekop WK, Mladsi D (2013) The faces of personalized medicine: a framework for under-
standing its meaning and scope. Value Health 16(6 Suppl):S4–S9
31. Laing RE, Hess P, Shen Y, Wang J, Hu SX (2011) The role and impact of SNPs in pharmaco-
genomics and personalized medicine. Curr Drug Metab 12(5):460–486
32. Schwab M, Schaeffeler E (2012) Pharmacogenomics: a key component of personalized therapy.
Genome Med 4(11):93
33. Mousa AY, Broce M, Campbell J, Nanjundappa A, Stone PA, Abu-Halimah S, Srivastava M,
Bates MC, Aburahma AF (2012) Clopidogrel use before renal artery angioplasty with/without
stent placement resulted in tertiary procedure risk reduction. J Vasc Surg 56(2):416–423
34. Relling MV, Gardner EE, Sandborn WJ, Schmiegelow K, Pui CH, Yee SW, Stein CM,
Carrillo M, Evans WE, Klein TE, Clinical Pharmacogenetics Implementation Consortium
(2011) Clinical Pharmacogenetics Implementation Consortium guidelines for thiopurine
methyltransferase genotype and thiopurine dosing. Clin Pharmacol Ther 89(3):387–391
35. Renders L, Frisman M, Ufer M, Mosyagin I, Haenisch S, Ott U, Caliebe A, Dechant M,
Braun F, Kunzendorf U, Cascorbi I (2007) CYP3A5 genotype markedly influences the
pharmacokinetics of tacrolimus and sirolimus in kidney transplant recipients. Clin Pharmacol
Ther 81(2):228–234
36. Guinea J, Escribano P, Marcos-Zambrano LJ, Pela’ez T, Kestler M, Muñoz P, Vena A,
López-Fabal F, Bouza E (2016) Therapeutic drug monitoring of voriconazole helps to decrease
the percentage of patients with off-target trough serum levels. Med Mycol 54(4):353–360
37. Armitage J, Bowman L, Wallendszus K, Bulbulia R, Rahimi K, Haynes R, Parish S, Peto R,
Collins R, Study of the Effectiveness of Additional Reductions in Cholesterol and Homocys-
teine (SEARCH) Collaborative Group (2010) Intensive lowering of LDL cholesterol with
80 mg versus 20 mg simvastatin daily in 12,064 survivors of myocardial infarction: a double-
blind randomised trial. Lancet 376(9753):1658–1669
38. Druker BJ, Guilhot F, O’Brien SG et al (2006) Five-year follow-up of patients receiving
imatinib for chronic myeloid leukemia. N Engl J Med 355(23):2408–2417
39. Kwak EL, Bang YJ, Camidge DR et al (2010) Anaplastic lymphoma kinase inhibition in non–
small-cell lung cancer. N Engl J Med 363(18):1693–1703
40. Simon T, Verstuyft C, Mary-Krause M et al (2009) Genetic determinants of response to
clopidogrel and cardiovascular events. N Engl J Med 360(4):363–375
41. Leypoldt F, Höftberger R, Titulaer MJ et al (2015) Investigations on CXCL13 in anti-N-methyl-
D-aspartate receptor encephalitis: a potential biomarker of treatment response. JAMA Neurol
72(2):180–186
42. Ramsey BW, Davies J, McElvaney NG et al (2011) A CFTR potentiator in patients with cystic
fibrosis and the G551D mutation. N Engl J Med 365:1663–1672
43. Vandenbroucke JP, Koster T, Briët E, Reitsma PH, Bertina RM, Rosendaal FR (1994)
Increased risk of venous thrombosis in oralcontraceptive users who are carriers of factor V
Leiden mutation. Lancet 344(8935):1453–1457
44. O’Day E, Alsafar H (2018) Advanced diagnostics for personalized medicine. https://www.
scientificamerican.com/article/advanced-diagnostics-for-personalized-medicine. Accessed
06 Apr 2019
45. Dams SM, Crisamore KR, Empey PE (2018) Clinical pharmacogenomics: applications in
nephrology. Clin J Am Soc Nephrol 13(10):1561–1571
46. Jameson JL, Longo DL (2015) Precision medicine--personalized, problematic, and promising.
N Engl J Med 372(23):2229–2234
47. Akinobu G (2014) Personalized medicine-based strategy for prostate cancer. Pers Med Univ
3:1–3
48. Han MR, Zheng W, Cai Q, Gao YT, Zheng Y, Bolla MK, Michailidou K, Dennis J, Wang Q,
Dunning AM, Brennan P, Chen ST, Choi JY, Hartman M, Ito H, Lophatananon A, Matsuo K,
Miao H, Muir K, Sangrajrang S, Shen CY, Teo SH, Tseng CC, Wu AH, Yip CH, Kang D,
392 D. B. Singh
Xiang YB, Easton DF, Shu XO, Long J (2017) Evaluating genetic variants associated with
breast cancer risk in high and moderate-penetrance genes in Asians. Carcinogenesis 38
(5):511–518
49. Sultana N, Rahman M, Myti S, Islam J, Mustafa MG, Nag K (2019) A novel knowledge-
derived data potentizing method revealed unique liver cancer-associated genetic variants. Hum
Genomics 13(1)
50. Lilyquist J, Ruddy KJ, Vachon CM, Couch FJ (2018) Common genetic variation and breast
cancer risk-past, present, and future. Cancer Epidemiol Biomarkers Prev 27:380–394
51. Kar SP, Berchuck A, Gayther SA, Goode EL, Moysich KB, Pearce CL, Ramus SJ, Schildkraut
JM, Sellers TA, Pharoah PDP (2018) Common genetic variation and susceptibility to ovarian
cancer: current insights and future directions. Cancer Epidemiol Biomarkers Prev 27:395–404
52. Erdei E, Luo L, Sheng H, Maestas E, White KA, Mackey A, Dong Y, Berwick M, Morse DE
(2013) Cytokines and tumor metastasis gene variants in oral cancer and precancer in Puerto
Rico. PLoS One 8:e79187
53. Engle JA, Kolesar JM (2014) Afatinib: a first-line treatment for selected patients with metastatic
non-small-cell lung cancer. Am J Health Syst Pharm 71:1933–1938
54. Goss GD, Felip E, Cobo M, Lu S, Syrigos K, Lee KH, Göker E, Georgoulias V, Li W, Guclu S,
Isla D, Min YJ, Morabito A, Ardizzoni A, Gadgeel SM, Fülöp A, Bühnemann C, Gibson N,
Krämer N, Solca F, Cseh A, Ehrnrooth E, Soria JC (2018) Association of ERBB mutations with
clinical outcomes of afatinib- or erlotinib-treated patients with lung squamous cell carcinoma:
secondary analysis of the LUX-Lung 8 randomized clinical trial. JAMA Oncol 4:1189–1197
55. Barratt DT, Somogyi AA (2017) Role of pharmacogenetics in personalised imatinib dosing.
Transl Cancer Res 6:S1541–S1557
56. Togneri FS, Ward DG, Foster JM, Devall AJ, Wojtowicz P, Alyas S, Vasques FR, Oumie A,
James ND, Cheng KK, Zeegers MP, Deshmukh N, O’Sullivan B, Taniere P, Spink KG,
McMullan DJ, Griffiths M, Bryan RT (2016) Genomic complexity of urothelial bladder cancer
revealed in urinary cfDNA. Eur J Hum Genet 24(8):1167–1174
57. Taly V, Pekin D, Benhaim L, Kotsopoulos SK, Le Corre D, Li X, Atochin I, Link DR, Griffiths
AD, Pallier K, Blons H, Bouché O, Landi B, Hutchison JB, Laurent-Puig P (2013) Multiplex
picodroplet digital PCR to detect KRAS mutations in circulating DNA from the plasma of
colorectal cancer patients. Clin Chem 59(12):1722–1731
58. Nakamura Y (2015) Challenges and future directions of immunopharmacogenomics.
In: Nakamura Y (ed) Immunopharmacogenomics. Springer, Tokyo, pp 159–162
59. Deng X, Nakamura Y (2017) Cancer precision medicine: from cancer screening to drug
selection and personalized immunotherapy. Trends Pharmacol Sci 38(1):15–24
60. Scheltens P, Blennow K, Breteler MM, de Strooper B, Frisoni GB, Salloway S, van der Flier
WM (2016) Alzheimer’s disease. Lancet 388(10043):505–517
61. Lill CM (2016) Genetics of Parkinson’s disease. Mol Cell Probes 30:386–396
62. Cong L, Ran FA, Cox D, Lin S, Barretto R, Habib N, Hsu PD, Wu X, Jiang W, Marraffini LA,
Zhang F (2013) Multiplex genome engineering using CRISPR/Cas systems. Science
339(6121):819–823
63. Raffel J, Wakerley B, Nicholas R (2016) Multiple sclerosis. Medicine 44(9):537–541
64. Chatawa J (2017) Treating clinically isolated syndrome: the long game. J Neurol Neurosurg
Psychiatry 88(4):284
65. Stilund M, Reuschlein AK, Christensen T, Møller HJ, Rasmussen PV, Petersen T (2014)
Soluble CD163 as a marker of macrophage activity in newly diagnosed patients with multiple
sclerosis. PLoS One 9(6):e98588
66. Nagalla S, Bray PF (2016) Personalized medicine in thrombosis: back to the future. Blood 127
(22):2665–2671
67. Johnson JA (2012) Warfarin pharmacogenetics: a rising tide for its clinical value. Circulation
125(16):1964–1966
68. Mega JL, Walker JR, Ruff CT et al (2015) Genetics and the clinical response to warfarin and
edoxaban: findings from the randomised, double-blind ENGAGE AF-TIMI 48 trial. Lancet 385
(9984):2280–2287
69. Leonard D, Svenungsson E, Dahlqvist J, Alexsson A, Ärlestig L, Taylor KE, Sandling JK,
Bengtsson C, Frodlund M, Jönsen A, Eketjäll S, Jensen-Urstad K, Gunnarsson I, Sjöwall C,
Bengtsson AA, Eloranta ML, Syvänen AC, Rantapää-Dahlqvist S, Criswell LA, Rönnblom L
(2018) Novel gene variants associated with cardiovascular disease in systemic lupus
erythematosus and rheumatoid arthritis. Ann Rheum Dis. 77(7):1063–1069
70. Christiansen MK, Larsen SB, Nyegaard M, Neergaard-Petersen S, Ajjan R, Würtz M,
Kristensen SD (2017) Coronary artery disease-associated genetic variants and biomarkers of
inflammation. PloS one 12(7):e0180365
71. LeBlanc M, Zuber V, Andreassen BK, Witoelar A, Zeng L, Bettella F, Wang Y, McEvoy LK,
Thompson WK, Schork AJ, Reppe S, Barrett-Connor E, Ligthart S, Dehghan A, Gautvik KM,
Nelson CP, Schunkert H, Samani NJ, CARDIoGRAM Consortium, Ridker PM, Chasman DI,
Aukrust P, Djurovic S, Frigessi A, Desikan RS, Dale AM, Andreassen OA (2016) Identifying
novel gene variants in coronary artery disease and shared genes with several cardiovascular risk
factors. Circ Res 118(1):83–94
72. Soliday FK, Conley YP, Henker R (2010) Pseudocholinesterase deficiency: a comprehensive
review of genetic, acquired, and drug influences. AANA J 78(4):313–320
73. Kaye AD, Mahakian T, Kaye AJ, Pham AA, Hart BM, Gennuso S, Cornett EM, Gabriel R,
Urman RD (2018) Pharmacogenomics, precision medicine, and implications on anesthesia care.
Best Pract Res Clin Anaesthesiol 32:61–81. https://doi.org/10.1016/j.bpa.2018.07.001
74. Palmer SN, Giesecke NM, Body SC, Shernan SK, Fox AA, Collard CD (2005)
Pharmacogenetics of anesthetic and analgesic agents. Anesthesiology 102(3):663–671
75. Fitipaldi H, McCarthy MI, Florez JC, Franks PW (2018) A global overview of precision
medicine in type 2 diabetes. Diabetes 67(10):1911–1922
76. Moltke I, Grarup N, Jørgensen ME et al (2014) A common Greenlandic TBC1D4 variant
confers muscle insulin resistance and type 2 diabetes. Nature 512(7513):190–193
77. Estrada K, Aukrust I, Bjørkhaug L, SIGMA Type 2 Diabetes Consortium et al (2014) Associ-
ation of a low-frequency variant in HNF1A with type 2 diabetes in a Latino population. JAMA
311:2305–2314
78. Zhou K, Yee SW, Seiser EL, MetGen Investigators; DPP Investigators; ACCORD Investigators
et al (2016) Variation in the glucose transporter gene SLC2A2 is associated with glycemic
response to metformin. Nat Genet 48(9):1055–1059
79. Perlis RH (2014) Pharmacogenomic testing and personalized treatment of depression. Clin
Chem. 60(1):53–59. https://doi.org/10.1373/clinchem.2013.204446
80. Samer CF, Lorenzini KI, Rollason V, Daali Y, Desmeules JA (2013) Applications of CYP450
testing in the clinical setting. Mol Diagn Ther 17(3):165–184
81. Lobello KW, Preskorn SH, Guico-Pabia CJ, Jiang Q, Paul J, Nichols AI, Patroneva A,
Ninan PT (2010) Cytochrome P450 2D6 phenotype predicts antidepressant efficacy of
venlafaxine: a secondary analysis of 4 studies in major depressive disorder. J Clin Psychiatry
71(11):1482–1487
82. Finch RA, Sartorelli AC, Piwnica-Worms D (1999) Choroid plexus epithelial expression of
MDR1 P glycoprotein and multidrug resistance-associated protein contribute to the blood-
cerebrospinal fluid drug-permeability barrier. Proc Natl Acad Sci U S A 96(7):3900–3905
83. de Leon J (2009) The future (or lack of future) of personalized prescription in psychiatry.
Pharmacol Res 59(2):81–89
84. de Leon J (2006) The AmpliChip CYP450 test: personalized medicine has arrived in psychiatry.
Expert Rev Mol Diagn 6(3):277–286
85. Filaković P, Petek A (2009) Personalized pharmacotherapy in psychiatry. Psychiatr Danub
21(3):341–346
86. Skrętkowicz J, Barańska M, Rychlik-Sych M (2013) Clinical significance of pharmacogenetics
in psychiatry. Wiad Lek 66(2 Pt 2):185–191
394 D. B. Singh
87. Spina E, de Leon J (2015) Clinical applications of CYP genotyping in psychiatry. J Neural
Transm (Vienna) 122(1):5–28
88. Arneet DK, Class SA, Glasser SP (2006) Pharmacogenetics of antihypertensive treatment. Vasc
Pharmacol 44(2):107–118
89. Johnson JA, Zineh I, Puckett BJ, McGorray SP, Yarandi HN, Pauly DF (2003) Beta
1-adrenergic receptor polymorphisms and antihypertensive response to metoprolol. Clin
Pharmacol Ther 74(1):44–52
90. Bengtsson K, Melander O, Orho-Melander M (2001) Polymorphism in the beta(1)-adrenergic
receptor gene and hypertension. Circulation 104(2):187–190
91. Shin J, Lobmeyer MT, Gong Y (2007) Relation of beta(2)-adrenoceptor haplotype to risk of
death and heart transplantation in patients with heart failure. Am J Cardiol 99(2):250–255
92. Cooper-DeHoff RM, Johnson JA (2016) Hypertension pharmacogenomics: in search of
personalized treatment approaches. Nat Rev Nephrol 12(2):110–122
93. Byrd JB (2016) Personalized medicine and treatment approaches in hypertension: current
perspectives. Integr Blood Pressure Control 9:59–67
94. Kushner J (2014) The ethics of personalized medicine. Pers Med Univ 3:42–45
95. Relling M (2015) Clinical implementation of pharmacogenetics: CPIC guidelines. Clin Chem
Lab Med 53:S75
96. Jansen ME, Rigter T, Rodenburg W, Fleur TMC, Houwink EJF, Weda M, Cornel MC (2017)
Review of the reported measures of clinical validity and clinical utility as arguments for the
implementation of pharmacogenetic testing: a case study of statin-induced muscle toxicity.
Front Pharmacol 8:555
97. Tonk EC, Gurwitz D, Maitland-van der Zee AH, Janssens AC (2016) Assessment of
pharmacogenetic tests: presenting measures of clinical validity and potential population impact
in association studies. Pharmacogenomics J 17:386–392
98. Vogenberg FR, Isaacson Barash C, Pursel M (2010) Personalized medicine: part 1: evolution
and development into theranostics. P T 35(10):560–576
99. Atutornu J, Hayre CM (2018) Personalized medicine and medical imaging: opportunities and
challenges for contemporary health care. J Med Imaging Radiat Sci 49(4):352–359
Index
A anti-drug (ADAs), 41
Acoustic separation, ultrasound, 13 engineering, 66
Acquired immunodeficiency syndrome high-affinity, 75
(AIDS), 124, 129, 174, 349, 353 humanized, 4
Activation-induced cytidine deaminase libraries, 69
(AID), 65 therapeutic, 55
Acute myeloid leukemia (AML), 91, 99, Antibody-drug conjugates (ADCs), 67
136, 213 Anticoagulants, 115, 128, 132, 140, 379, 383
Acute respiratory distress syndrome, 100 Antidepressants, 385
Adalimumab (HUMIRA), 73 Anti-drug antibodies, 55
Adeno-associated viruses (AAV), 169, 334, Antioxidants, 33, 34
338, 357 Antipsychotics, 385
Adenosine deaminase (ADA), 347 Antisense oligonucleotides (ASO), 326
deficiency-severe combined Antithrombin, 133
immunodeficiency Aptamers, 323, 326, 348, 354
(ADA-SCID), 354 Asparaginase, 134–136
Adjuvants, 31, 34, 93, 134 Autism spectrum disorders, 211
Administration, therapeutic proteins, 43 Autoantigens, 40
Adrenoleukodystrophy, 353 Avastin, 4
Advanced therapy medicinal products Avotermin, 102
(ATMP), 233 Axicabtagene ciloleucel, 352
Agitation, 248
Agonists, 3
Alginate, 208, 280, 292, 299, 300, 345 B
Alteplase, 134 B cell receptors (BCR), 58
Alzheimer’s disease, 209, 213, 375, 382 Bexsero, 178
Amniotic fluid-derived stem (AFS) cells, 304 Bioavailability, 41, 43, 44, 132, 341, 373
Amyotrophic lateral sclerosis (ALS), 98, 99, Biobetters, formulation, 36
101–104, 209, 213 Biodel insulin (BIOD-531), 121
Anesthesia, 384 Biologics, 1
Antagonists, 4 Biomarkers, 373
Anthrax vaccine, 174 Biopharmaceuticals, packaging, 38
Anti-adsorption agents, 32 safety, 39
Anti-aggregation agents, 32 Bioprinting, 279
Antibodies, 1 Bioprocessing, 1, 189
395
396 Index
Bioreactors, 225 Cost of goods (COG), 257

single-use, 10 CRISPR/Cas9, 212, 324, 331, 358, 382, 383
Biosimilars, 6, 55 Critical quality attributes (CQAs), 47
formulation, 36 Cross-flow filtration, 11, 16
Blood clotting factors (blood factors), 115, 116, Culture medium homogenization, 248
128, 140 Culture monitoring, 249
Blood glucose, 3 Cyclodextrins, 42
Blood products, 89, 128 CYP2D6, 385
Bone marrow (BM), 60, 93, 99, 243, 310, Cystic fibrosis (CF), 134, 136, 210–212,
323, 335 323, 345
graft rejection, 227 Cytokines, 44, 87, 89, 231, 239, 241, 252, 263
transplant, 97, 336 release syndrome, 353
Bone sialoprotein (BSP), 248 Cytomegalovirus (CMV), 7, 350
Bone tissue, 279 retinitis, 353
Buffers, 30
Bulking agents, 32
Buoyancy-activated cell sorting, 238 D
Deamidation, 26
Degludec, 122
C Delivery devices, 38, 42, 359
Calcitonin, 44 Delivery vectors, 321
Calcium channel blockers, 386 Dengue, vaccine, 155, 180
Cancer, breast, 375–377, 380 DENGVAXIA, 180
gene therapy, 351 Depression, 385
pancreatic, 91, 95, 136, 329 Depth filtration, 15
prostate, 127, 232, 379, 380 Design of experiments (DoE), 47
thyroid, 127 Design space, 47
Carbon nanotubes (CNT), 307 Developability assessment, 47
Cardiac tissue, 279 Diabetes, 38, 95, 119, 200, 212, 371
Cardiomyocytes, 191, 205, 305, 308 type 1, 3, 117, 121, 198, 202, 384
CAR T-cell therapy, 353 type 2, 123, 384
Cartilage, 231, 279, 295 Dicoumarol, 132
printing, 295 Digital light processing (DLP), 302
CD19, 353 Diphtheria toxoid, 179
Cell retention, cross-flow filtration, 11 Disease modeling, 189
Cell separation, 1–16, 237, 255 Disposable systems, 10, 163, 165, 247, 252
Cell therapy, 192, 225–266, 324 Donor lymphocyte infusions (DLI), 229
Centrifugation, cell retention, 12 Downstream processing, 16–20, 254, 264, 340
cell separation, 15 Drotrecogin alfa, 133
Chikungunya, 184 Drug-drug interaction (DDIs), 372
Chimeric antigen receptors (CARs), 68, 229 DTcP vaccine, 174
Chromatograhpy, 1 Duchenne muscular dystrophy (DMD), 210,
Class switch recombination (CSR), 64 354, 355
Clinical decision, 369 Dupuytren’s contracture, 134
Clotting factors, 133
Coagulation factors, 128
Collagenase, 134, 206, 236 E
Colony-forming unit-fibroblasts (CFU-F), 229 Ebola, 157, 329
Colony-stimulating factors (CSFs), 87 vaccine
Combination vaccines, 173 Ad5-EBOV, 155, 172, 176
Complementary determining regions rVSV-ZEBOV, 182
(CDRs), 60 Embryonic stem (ES) cells, 192
Computer-aided design (CAD), 281 Endotoxin, 5, 19
Consistency lots, 172 Enoxaparin sodium, 133
Continuous processing, 10 Enterovirus 71 (EV71), 180
Index 397
Enzymes, therapeutic, 115, 134 Granulocyte colony-stimulating factor

Epidermal growth factor (EGF), 100, 104 (G-CSF), 4, 96
receptor (EGFER), 380 Granulocyte macrophage colony-stimulating
Epigenetic factors, 373 factor (GM-CSF), 96
Epigenetic memory, hiPS cells, 194 Growth factors, 87, 117, 210, 231, 239,
Epitopes, 4, 41, 60, 67, 237 263, 279
Epoetin, 87 Growth hormone (GH), 2, 4, 32, 102, 115, 123,
Erythropoietin, 3, 4, 97, 100 125, 127
Eteplirsen, 355 GX-H9, 124
Expression systems, 1, 6, 92, 325
Extracellular vesicles (EVs), 263
H
F Heart disease, 209
Factor VIII, 4 Heart tissue-derived decellularized extracellular
Familial dysautonomia matrix (hdECM), 308
(Riley-Day syndrome), 209 Hematopoietic cell transplantation (HCT), 226
Familial hypercholesterolemia, 355 Hematopoietic growth factors (HGFs), 87, 96
Fermentation, 1, 6, 8, 163 Hematopoietic stem/progenitor cells
Fibrinolytics, 134 (HSPC), 225
Fibroblast growth factors, 101 Hemophilia, 128, 132, 323, 332, 353
Filgrastim, 97, 99 Heparin, 132
Filtration, cross-flow, 11, 16 Hepatitis, 87
Follicle-stimulating hormone (FSH), 124 alcoholic, 93
Follitropin, 126 B, 5, 91, 103, 129, 157, 165, 181
Fomivirsen, 353 recombinant vaccine, 181
Fused deposition modeling (FDM), 286 C, 91, 97, 103, 129, 345, 377
Fused filament fabrication (FFF), 286 Heplisav-B, 181
Hereditary transthyretin amyloidosis
(hATTR), 354
G Herpes zoster, 175
GARDASIL 9, 179 High cell density reactors, 8
Gelatin methacrylate (GelMA) bioink, 308 Hirschsprung’s disease, 212
GelMA gels, 301 Hirudin, 132
Gene augmentation, 325 Homology-dependent repair (HDR), 331
Gene delivery, in vivo, 324–359 Human cardiac progenitor cells (hCPCs), 308
Gene editing, 331 Human chorionic gonadotropin (hCG), 124
Gene repair, nucleases, 332 Human coronary artery endothelial cells
Gene silencing, 323, 326, 330 (HCAECs), 307
Gene therapy, 3, 321 Human immunodeficiency virus (HIV), 97, 99,
Genetic makeup, 369 160, 213, 329–337, 374
Genetic polymorphism, 372 RNAs, 329
Ginkgo biloba (ginkgo), 372 vaccine, 183
Glargine, 122 Human leukocyte antigens (HLA), 227
Glucagon, 115, 122, 127, 139 Human papillomavirus (HPV) vaccine, 179
Glucagon-like peptides, 122 Human platelet lysate (hPL), 240
Gluconeogenesis, 117 Human pluripotent stem (hPS) cells, 189
Glycosylation, 7, 40, 75, 173 Huntington disease (HD), 212
Gonadotropins, 115, 124, 127, 139 Hyaluronate/hyaluronic acid, 296, 299, 307,
Good manufacturing practice (GMP), 39, 162, 309, 311
237, 257, 265 Hyaluronidase, 42, 121
Graft vs. host disease (GVHD), 227 Hydrogels, 301
Graft vs. leukemia (GVL) effect, 228 Hypertension, 97, 386
398 Index
I Mipomersen, 326
Immune potentiators, 34 MOD-4023, 124
Immunogenicity, 24, 55, 118, 165, 170–181, Molgramostim, 97, 99
299, 327, 357 Monoclonal antibodies (mAbs), 1, 65
mechanisms, 40 Monophosphoryl lipid A (MPL), 34
Immunoglobulins, 4, 58, 132, 240, 352, 382 MRSA, 157
IN-105 (tregopil), 119 Multiple sclerosis, 87, 91, 92, 102, 382
Induced tissue-specific stem (iTS) cells, 194 Multiplex automated genome engineering
Insulin, 2, 35, 44, 89, 102, 115–124, 127, 139, (MAGE), 77
202, 241 Mutagenesis, 75, 193, 335, 357
Insulin-like growth factors (IGFs), 102, 124 Myocardial infarction, 131, 135, 306, 308,
Interferons (INFs), 5, 87–91, 263 328, 383
Interleukins (ILs), 87–92, 173, 202, 239
receptor, 382
In vitro compartmentalization, 75 N
Isotonicity, 31 Nalotimagene carmaleucel, 352
Neisseria meningitidis, 178
Neoantigens, 40
L Nerve growth factor (NGF), 102
Laron syndrome, 102 Nerve tissue, 279
Laser-induced forward transfer (LIFT) cell printing, 296
printing, 287, 305 Neurodegenerative diseases, 90, 196, 199, 209,
Lassa virus, 157, 161 264, 326, 354, 381
vaccine, 183 Neutropenia, 87, 97–99, 103
Leber congenital amaurosis type 2, 353 Nipah virus, 184
Lenograstim, 97 NOD-like receptors (NLR), 34
Leprechaunism, 102 Nonhomologous end joining (NHEJ), 331
Luteinizing hormone (LH), 116, 124, 126 Novel excipients, 42
LY3209590, 122 Nucleic acids, gene therapy, 324
LY900014, 121 Nusinersen, 355
Lyophilization, 27, 32, 37, 257
Lyoprotectants, 37
O
OKT3 (muromonab), 66
M Oncolytic viruses (OV), 351
Malaria, 161 Organoids, 189, 210
vaccine, 183 Ornithine transcarbamylase (OTC)
Mammalian cell display, 74 deficiency, 348
Manufacturing, 225, 321 Orphan products, 9
single-use, 9 Osteopontin (OP), 248
upstream, 6
Marburg virus, 157, 161
Measles, 156–158, 184 P
vaccine, 166 Palifermin, 101
Mecasermin, 102 Pancreatic progenitors (PPs), 194, 200,
Meningococcal ACWY conjugate vaccine 204, 205
(MenACYW135), 178 Pancreatitis, 134, 194
Meningococcal subgroup B vaccine Panning, 73
(MenB), 178 Paracrine cell therapy, 229
Mesenchymal stem/stromal cells Parathyroid hormone (PTH), 44, 116, 127
(MSC), 225, 229 Paratope, 60
Metoprolol, 386 Parkinson’s disease (PD), 197, 199, 209, 328,
Middle East respiratory syndrome (MERS), 337, 382
161, 183 Pattern-recognition receptors
Miller-Dieker syndrome, 211 (PRR) agonists, 34
Index 399
PCV vaccine, 174 Rett syndrome, 209

PDGF-BB, 100 Ribosome/mRNA display, 73
Pediatric applications, 174 RNA-induced silencing complex (RISC), 327
Pegaptanib, 354 RNA interference (RNAi), 327
Pegfilgrastim, 97, 99 RNA, mRNA vaccines, 184
Perfusion reactors, 10 microRNA (miRNA), 327
Personalized medicine, 2, 192, 369, 375
Pertussis, 158
Peyronie’s disease, 134 S
Phage display technology, 72 Saccharomyces cerevisiae, 74, 118, 122, 133
Pharmacoeconomics, 6 Sargramostim, 97
Pichia pastoris (Komagataella pastoris), 3, 118 Scalable culture vessels, 243
Placental growth factor (PlGF), 102 Schizophrenia, 209
Plastic bioreactors, 10 Sedimentation, cell separation, 16
Platelet-derived growth factor (PDGF), 100 Separation, cells, 1
Pneumococcus conjugated vaccines, 160, 183 Severe combined immunodeficiency (SCID),
Polyclonal antibodies (pAbs), 65 227, 235, 347, 354
Preservatives, 33 Shingles, 175
Process engineering, 225 vaccine, 155
Product information, 55 Shingrix vaccine, 164, 173, 175
Promoters, 7 Short hairpin RNA (shRNA, expressed RNAi
Protein-protein interactions, 30 activators), 327, 330
Proteins, aggregation, 27 Short interfering RNA (siRNA), 264, 327,
characterization, 28 328, 359
chemical degradation, 26 Skin, 279
expression, 7 printing, 302
formulation, 30 regeneration, 89, 92, 103, 303
lyophilized, 37 Small molecules (SMs), 55
physical degradation, 27 Solubility enhancers, 32
recombinant, 1, 24 Somatic hypermutation (SHM), 64, 75
structure, 25 Somatotropin, 123, 125
Prothrombin, 129 Spinal muscular atrophy (SMA), 209, 354
Stability, 24
Stabilizers, 30, 32, 38, 42
Q Stem cells, human pluripotent, 189
QT syndrome, 209 Stereolithography (SLA), 283, 288
Quality by design (QbD), 47, 260 Streptococcus pneumoniae, 160
Quality control, 39, 161, 237, 263, 321, Surfactants, 32, 44, 346
340, 346 Survival of motor neuron (SNM) protein, 354
Quality risk management, 47
T
R Tangential flow filtration, alternating, 12
Recombinant drugs, 55 Tenecteplase, 134
Recombinant enzymes, 134–138 Teriparatide, 128
Recombinant proteins, 1, 24, 178, 240 Tetanus toxoid, 179
formulation, 24 Therapeutic biologics, 55
Recombinant technology, 155, 164, 351 Therapeutic response, 369, 373, 386
Regenerative medicine, 189 Thrombin, 129
Replacement therapy, 3, 124, 134 Thrombolytic agents, 115, 134
Respiratory syncytial virus (RSV) vaccine, 183 Thrombopoietin, 40
Respiratory syndrome virus (RSV) disease, 160 Thrombosis, 97, 133, 378, 383
Reteplase, 134 Thrombus formation, 132
400 Index
Thyroid-stimulating hormone (TSH), 127 clinical trials, 155, 171

Tissue engineering, 279 development, 155, 159
Toll-like receptors (TLR), 34 immunogenicity, 172
Tonicifiers, 31 manufacturing, 155, 161
Traceless affinity cell selection, 238 Valproic acid (VPA), 213
Trafermin, 101 Varicella zoster virus, 175
Transcription activator-like effector nucleases Vascular endothelial growth factor (VEGF), 4,
(TALEN), 324, 331 101, 248, 291, 308, 354
Transdermal delivery, 44 V(D)J recombination, 61
Transfection, 194, 325, 343, 346 Viral vaccine, 155, 163, 167
Transforming growth factors (TGFs), 102, Viral vectors, manufacturing, 339
205, 241 Virus-like particles (VLPs), 34, 76, 162, 168,
Transplant rejection, 66 179
Transthyretin (TTR), 356
Trastuzumab, 36
Trehalose fatty acid esters, 42 W
Trumenba vaccine, 179 Warfarin, 132, 133, 373, 383
Tuberculosis (TB), booster vaccine, 183
Tumor necrosis factors (TNFs), 73, 87, 95,
173, 380 Y
Yeast display technique, 74
U
Umbilical cord blood (UCB), 235 Z
Zika virus, 157, 161, 183, 213
vaccine, 183
V Zinc finger nucleases (ZFNs), 324, 331
Vaccines, accelerated approval, 174 Zoster vaccine, 175
characterization, 166 Zymogen plasminogen, 134

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Advances in Biochemical Engineering/Biotechnology 171

Series Editor: T. Scheper

Ana Catarina Silva

Volumes are organized topically and provide a comprehensive discussion of

In references, Advances in Biochemical Engineering/Biotechnology is abbreviated

More information about this series at http://www.springer.com/series/10

José Manuel Sousa Lobo

ISSN 0724-6145 ISSN 1616-8542 (electronic)

© Springer Nature Switzerland AG 2020

Biopharmaceuticals have been showing high therapeutic potential, especially for

(Ad5-EBOV), meningococcal subgroup B, human papilloma virus (HPV), dengue,

Porto, Portugal Ana Catarina Silva

Industrial Challenges of Recombinant Proteins . . . . . . . . . . . . . . . . . . . 1

Gene Therapy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 321

Industrial Challenges of Recombinant

Scott R. Rudge and Michael R. Ladisch

8 Single Use Downstream Processing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20

Abstract The use of recombinant DNA methods to produce large quantities of

Keywords Biologics, Bioprocessing, Cell culture, Cell separation, Chromatograhpy,

Since the early commercialization of recombinant human insulin, recombinant

characterization positioned it for scaling up [3]. Subsequent early products such as

2 Classes of Products (Agonists and Antagonists)

are erythropoietin, granulocyte-colony stimulating factor, growth hormone, and

2.3 Humanized Antibodies

Table 1 Top selling biopharmaceuticals from 1997 and 2011

Upstream manufacturing includes fermentation, cell culture, or another means of

3.1 Expression Systems

A common promoter for protein expression in CHO cells is the cytomegalovirus

3.3 High Cell Density Reactors

Cell Density x 10^6 cells/mL

Product produced (mg/L)

Cell Density Product Produced

4 Single Use Manufacturing

4.1 Single-Use Bioreactors

Keeping different products segregated from one another is a critical requirement of

5 Perfusion Reactors and Continuous Processing

5.1 Cell Retention by Cross-Flow Filtration

Fig. 2 An alternating tangential ﬂow ﬁltration device [18]

5.2 Cell Retention by Centrifugation or Sedimentation

Centrifugation is also used as a cell retention mechanism. These centrifuge systems

Fig. 3 Compact settler

5.3 Cell Retention by Acoustic Separation

5.4 Perfusion Reaction Control

6.2 Depth Filtration

6.3 Cross-Flow Filtration

Downstream processing is often portrayed as a bottleneck in biomanufacturing

7.1 Chromatography and Adsorption

Chromatography for biotherapeutic molecules bears more resemblance to a

Fig. 4 Different binding

7.2 Intensiﬁed Chromatography

While operating at high protein concentration could improve the efﬁciency of

be made and derivatized inexpensively and enclosed in thermoplastic, making this

8 Single Use Downstream Processing

There is signiﬁcant penetration of single-use technologies in downstream

9 Continuous Downstream Processing

Continuous downstream processing is a ﬁeld of active research. Simulated moving

There are many challenges remaining for producing therapeutic biomolecules at

Insights on the Formulation of Recombinant