Monoclonal

Antibodies(MAb’s)
(Introduction &
Production)
 Contents
• Introduction

• Production of MAb’s

• Miscellaneous approaches of MAb Production

• Advantages of MAb’s

• Limitations of MAb’s
 Introduction
• Antibodies are glycoprotein molecules
present in serum ,produced in response to an
antigen.
• Antibodies are secreted by a class of blood cells
known as B-lymphocytes.
• A person can synthesize different type of
antibodies depending upon the type of
stimulant involved.
• The antibody reacts with the specific antigen by
binding non-covalently to it.
Structure of an Antibody
 Poly &Mono-clonal Antibodies

Polyclonal Antibodies Monoclonal Antibodies

• Derived from different B • Derived from a single B cell
Lymphocytes cell lines . clone.

• MAb offer reproducible,

• Batch to Batch variation predictable & potentially
affecting Ab reactivity & inexhaustible supply of Ab
titre with exquisite specificity.

• Enable the development of

• Not powerful tools for secure immunoassay
clinical diagnostic tests systems
• Fundamental Principles:
• Specificity:
• mAbs are characterized by their exceptional specificity,
binding to a single epitope on an antigen. This contrasts
with polyclonal antibodies, which recognize multiple
epitopes.
• This high specificity makes mAbs invaluable in research,
diagnostics, and therapeutics.
• Clonality:
• mAbs are derived from a single B-cell clone, ensuring
homogeneity and reproducibility. This is crucial for
standardized applications.
1975, by Georges Köhler and Cesar Milstein
- Be awarded a Nobel Prize in1984
 Production of MAb’s

Four basic steps are involved in the production of

Monoclonal antibodies :-
1.Immunization
2.Cell Fusion
3.Selection and Screening of resulting clones
4.Cloning of hybridomas
 I. Immunization

ANTIGEN ( Intact cell/ Whole

cell membrane/ micro-
organisms ) + ADJUVANT
(emulsification)

Spleen
removed Antibody reached the
serum
 II. Cell Fusion
PEG Fusion

Spleen Cells Mylenoma cells

Feeder Cell
Growth medium

Hybridoma Cells
HAT Medium
III. Selection & Screening of
Resulting Clones
Pure clones obtained-must be screened for desired
antibody specificity . Two methods are used for
assay of a particular antigen specificity .

ELISA RIA

In both assays the antigen that reacts with

antibody is bound to the bottom of 96-well plastic
culture plates and washed to remove unbound
antigen .

Supernatant is added to separate well & incubated

for desired time & then optical density is measured
at a particular λ max
 IV. Cloning of Hybridomas
A. Clone Each +ve Culture
B. Test Each Supernatant for
Antibodies

C. Expand +ve Clones

Tissue Mouse
culture Ascites
method Method
 Mouse Ascites method
• In this method hybridoma cells are injected into the
peritoneal cavity of mice.

• The mice are pretreated by i.p. injection of pristane

to irritate the peritoneal cavity and to provide
environment conditions that facilitates the growth
of ascitic tumor.

• The fluid produced can contain a high concentration

of monoclonal antibodies 3 to 15 mg/ml, and 3 to 5
ml or more can be harvested per mouse
Miscellaneous Approaches of MAb Production

 Engineered MAb’s

 MAb heteroconjugates

 Chimeric MAb

 Anti-idiotype

 T-cell hybridomas
 Engineered MAb’s:-

• Mouse MAb’s are recognized as foreign &

evoke antibody response.
• The induced human anti mouse antibodies
reduce effectiveness of immunotoxin by
clearing it from blood stream.
• Use of human MAb’s can be a solution to the
above problem.
• Researchers have been engineering MAb using
rDNA technology to avoid above difficulties.
 Chimeric MAb’s
• An antibody is engineered by cloning rDNA
containing the promoter , leader &variable region
sequences from a mouse antibody gene & the
constant region exons from human antibody gene.
• The antibody encoded by such a recombinant gene
is a mouse-human chimera or humanized antibody.

 ADVANTAGES
• They are less immunogenic than mouse MAb’s.
• Retain biological effector functions.
 MAb Heteroconjugates
• Hybrids of two different antibody molecules
known as heteroconjugates have been
designed.
• One half has specificity for tumour cell while
other half has specificity for a surface
molecule on an immune effector cell such as
NK cell, activated macrophage , or a cytotoxic
B-lymphocyte.
 Anti-idiotype
• Idiotype-The individual antigenic determinants of variable
regions. An antibody could contain multiple idiotypes.
• In some cases idiotypes & actual antigen combining sites
may be identical & referred as paratope

• Anti –idiotype antibody which are internal image of the

original antigen can serve as vaccine to induce an immune
response to a pathogenic antigen
• Anti-idiotype vaccine can induce protective immunity in mice
against hepatitis B , rabbis, Streptococcous pneumoniae etc.
 T-cell hybridomas

• Produced by fusing primed T cells with

cancerous T cells called thymoma cells.

• T cell hybridoma do not secrete antibody but

rather possess other immunologic functions.
e.g. secretion of cytokinins & expression of T
cell receptors specific for a particular antigen.
 Advantages of MAb’s
• Pure one molecular species only.
• Specificity for one antigenic determinant.
• Antibodies with high avidity can be produced.
• In vitro or in vivo production is possible with
high production rate.
• Maintenance of farm/animals is not required
for immunization and bleeding.
• High reproducibility with respect to specificity
and avidity.
 Limitations of MAb’s
• Major problem of monoclonal antibody in
human is that they are usually mouse
antibodies and recognized as foreign—to
overcome the problem human monoclonal
antibodies are to be used.
• Much costly.
• Time consuming
 References

• Vyas S.P , Dixit V.K. , Textbook of

Pharmaceutical Biotechnology, Published by:-
CBS Publishers & Distributors , New Delhi, Pg:-
486-499
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 Molecular Mechanisms of
Therapeutic Antibody

• Direct Cell Action Modification: mAbs can directly interfere

with cell signaling by binding to target molecules, either inhibiting
or activating them.
• Apoptosis Induction: In some cases, mAb binding can directly
trigger programmed cell death (apoptosis) in target cells.
• Immune Effector Targeting: A major mechanism of mAb
therapy is to recruit and activate the immune system to attack
tumor cells.
• Payload Delivery: mAbs can act as delivery vehicles, carrying
therapeutic payloads (e.g., drugs, toxins) directly to target cells.
• CAR T-cell Development: mAbs' specificity is crucial in
developing chimeric antigen receptor (CAR) T-cell therapies, a
novel approach to cancer treatment.
 Molecular Mechanisms of
Therapeutic Antibody
• Unmodified mAb Efficacy: Unmodified mAbs have
shown significant efficacy in managing various cancers.
• Antigen Binding: mAbs exert their therapeutic effects
by binding to antigens on cancer cells, other cells, or
proteins.
• Variety of Treatment Strategies: Various treatment
strategies utilizing unmodified mAbs are effective in
cancer management.
• Immune Response Initiation: mAbs can initiate and
enhance the immune response against cancer cells.
• Targeted Therapy: mAb therapy provides targeted
therapy, minimizing damage to healthy tissues.
Advantages:
•High Specificity:
• mAbs target specific antigens, minimizing off-
target effects.
• This aligns with the "Ehrlich's magic bullet"
concept.
•Enhanced Efficacy:
• mAbs often demonstrate superior therapeutic
effects compared to other treatments.
•Improved Safety Profile:
• Generally, mAbs have lower toxicity and fewer
side effects.
•Targeted Delivery:
• mAbs can be used to deliver drugs or other
therapeutic payloads directly to target cells
(ADCs).
•Immune System Modulation:
• mAbs can enhance or suppress the immune
response as needed.
•Diagnostic and Research Applications:
• mAbs are valuable tools in various scientific
and medical applications.
•Increased Patient Survival:
• mAb treatments can contribute to better
patient outcomes.
Disadvantages:
•High Production Costs:
• mAbs are complex proteins requiring expensive
manufacturing processes.
• Large-scale mammalian cell cultures and
extensive purification are needed.
•Pharmacokinetic Limitations:
• mAbs may have limited absorption, distribution,
metabolism, and excretion (ADME) profiles.
•Tissue Penetration Issues:
• Large size can hinder mAbs from reaching certain
tissues effectively.
•Immunogenicity:
• mAbs can sometimes trigger an immune
response in patients.
•Limited Immune System Interactions:
• In some cases the desired immune system
interactions are not as strong as needed.
•Implementation Challenges:
• Various obstacles hinder their widespread
clinical use.
•Need for Further Research:
• More research is needed to overcome
limitations and improve efficacy.
Humanization of mAbs
• The humanization of mouse mAbs
is considered on bulk quantities
due to the ease of obtaining, low
cost, and rapid development of
mouse mAbs.
• Typical nonhumanizedmurine
mAbs have a number of
limitations as therapy, such as
prompt HAMAs whenpatients are
medicated with them.
• When humanized mAbs are used
to reduce the immunogenicity of
Generation of humanized mAbs
• In humanized mAbs, only the complementary-
determining region (CDRs) of the light and heavy
chains are murine, which were authorized in
clinical trials in early 1988 [53].
• CDR grafting is a well-known approach for
producing humanized mAbs that was first
proposed by Gregory P. Winter in 1986 [54].
• Nonhuman CDR sequences are added tohuman
framework sequences using this method,
allowing the antibody to maintain itstarget-
specific antigen response.
• It was the development of daclizumab in 1997,
which attaches to the IL-2 receptor and inhibits
transplant rejection [55], which was the
firstCDR-grafted humanized monoclonal antibody
to be approved by the FDA.
• Various approaches have been developed to
• Human and humanized mAbs
• There is already a plethora of antibodies
that are therapeutic on the market for
thetreatment of a variety of medical
problems, including immune-mediated
chronic inflammation.
• Numerous clinical systems have been
implemented, and the insufficiency ofhead-
to-head comparative trials in distinguishing
the relative clinical efficiency and safety
profiles of one monoclonal antibody vs
another can be a difficult problem.
• Human and humanized mAbs
• The ability to determine whether a
monoclonal antibody is entirely human or
humanized is one distinguishing feature of
dermatologists when explaining clinical
trial data.
• This finding illustrates the differences and
similarities between totally human and
humanized monoclonal antibodies in terms
of nomenclature, engineering, and
therapeutic prospects.
• Human and humanized mAbs
• While there are several variances among
different types of monoclonal antibodies,
recent research indicates that this
categorization has little bearing on the
overall clinical efficacy and safety profiles of
a specific treatment.
• It is obvious from the molecular insights
provided in this perspective that any
monoclonal antibody, whether humanized or
not, will have the same effect on cells, and
must be examined separately for its medical
influence in terms of safety and efficiency.
Novel drug delivery systems for mAbs
• Though mAbs have had a lot of success for
therapeutic purposes over the last 40 years,
some constraints such as deficient
pharmacokinetics, poor pharmacodynamics,
and systemic toxicity have limited their
prospective usage.
• Advanced research is being directedtoward
numerous new techniques, including changed
Fc function antibodies, bispecific antibodies,
intrabodies, and antibody fragments, to
achieve superior pharmacokinetics, improved
selectivity, and increased efficiency [57].
Novel drug delivery systems for mAbs
• In clinical application, both patients
aswell as health professionals need
additional competent and more secured
mAbs to initiation improved concurrence.
• The following section discusses a variety
of novel drugdelivery approaches that
have been used to solve obstacles
encountered during the development of
mAb-based medicines (Fig. 4.4).
Nanoparticles
1. Nanoparticles (NPs) are a type of particulate delivery
system that come in sizes rangingfrom 10 to 1000 nm and
are widely used in pharmaceutical drug delivery systems
2. There are numerous polymers accessible for creating NPs,
one of which being poly lacticco-glycolic acid (PLGA), which
is a popular biodegradable polymer.
3. This polymer hydrolysates into glycolic acid and lactic acid,
which are easily metabolized in-vivo. Because theEMA and
FDA have approved this polymer for a variety of uses in the
development of drug delivery systems, PLGA-conjugated
NPs are frequently employed in clinical trials[59].
4. When compared to free anti-OX40 mAbs, PLGA-based NPs
loaded with mAbs areable to target tumor necrosis factor
(TNF) receptor OX40, which is predominantlyexpressed on
activated T cells, and are found to be more efficient.
• MICROSPHERES
• Microspheres are being evaluated as a viable drug delivery
carrier for IgGs or monoclonalantibodies, as they enable
consistent therapeutic release and extended stability in the
systemiccirculation.
• IgG-MPEG-PCL-MPEG microspheres (6 m) were developed
utilizing the doubleemulsion solvent evaporation technique,
using copolymer of poly(ethylene glycol)-poly(-caprolactone)
(PEG-PCL).
• At the time of irradiation, it was found that IgG-MPEG-PCL-
MPEG microspheres had lower IgG destabilization rates and
higher entrapment efficacy than IgG-PCL microspheres [62].
• The s/o/w (solid-in-oil-in-water) method was established and
optimized toprepare microspheres without compromising the
stability issues of IgG or mAb.
• The combination of the s/o/w emulsion method and spray-
drying leads to successful entrapment of IgGmicroparticles into
the PLGA microsphere core.
• The full length anti-human TNF-α mAb-entrapped microspheres of
PLGA were positively prepared by the s/o/s process with capability
to load bioactives as well as single mAbs in microspheres.
• The combination of mesenchymal stem cells and anti-BMP2 mAbs,
as well as their entrapment in alginate microspheres, significantly
boosted the osteogenesis associated with human bone marrow
mesenchymal stem cells [64].
• Ethylene-vinyl acetate copolymer(EVAc) was used to load IgG in the
form of dry powder and characterized for efficient polymer-
fabricated antigen delivery strategy [65].
• EVAc microspheres can also be used for topical administration,
sustained release injectables, and extended-release IgG.
Anotherexample of polymer-based vaginal delivery system was a
vaginal ring, which delivers anAb in the mouse model [66].
• Direct, local delivery of Abs by applying aqueous gels madeup of
carboxymethyl cellulose may be effective antiinfective technique
after surgical procedures. Abs have been delivered to the CNS for a
long time using a hyaluronic acid-bound Ab hydrogel with a covalent
link [67].
HYDROGELS
1. Using a novel drug-loaded hydrogel
system, the stability of epidermal growth
factor was successfully enhanced [68].
2. The porous architecture and higher water
content are ideal for encapsulating mAbs.
3. Furthermore, three dimensional structures
of hydrogel were capable of entrapping the
mAbs in complex networking of hydrogel
networks [69].
4. For the construction of therapeutically safe
hydrogels capable of prolonged release of
mAbs, a lot of research hasbeen done on
both natural materials (chitosan) and
synthetic polymers (PLGA-PEG-PLGA).
5. mAb delivery can be improved by applying
 Formulation challenges of
•MAB STRUCTURAL INSTABILITY:
•mAb's complex tertiary structure makes them susceptible
mAbs
to structural changes throughout development.
•Physical stress during synthesis, processing, and storage
can alter mAb structure.
•PROCESSING-INDUCED MODIFICATIONS:
•Non-physiological conditions during processing can lead to
unwanted structural variations.
•This is a significant challenge compared to small molecule
drug development.
•FORMULATION CONCERNS:
•Buffer selection, processing changes, and container
selection can impact mAb stability.
•Finished formulations with non-native proteins must be
discarded.
•DOSAGE UNIFORMITY:
•Accumulation during vial extraction can lead to non-
uniform dosing.
•ENVIRONMENTAL SENSITIVITY:
•mAbs are sensitive to pH, temperature, stress, and
•Concentration-Dependent Degradation:
•High-concentration mAb formulations are prone to aggregation and
degradation.
•Viscosity is a problem in concentrated formulations.
•Administration Challenges:
•mAb-based therapeutics face challenges in intravenous and
subcutaneous administration of lyophilized and liquid formulations.
•Stability Impact on Yield:
•mAb instability leads to product loss during downstream processing.
•Byproduct Accumulation:
•Unwanted byproducts can accumulate in HEK 293 expressed
mAbs.
•Immunogenicity Issues:
•Hybridoma-derived mAbs can be immunogenic.
•CHO-expressed mAbs can have immunogenic glycosylation
profiles.
•Therapeutic Response and Adverse Events:
•Instability can reduce therapeutic response or cause adverse
 FUTURE TRENDS OF MAbs
• Growing Therapeutic Option: Therapeutic antibodies are increasingly
recognized as a valuable treatment approach.
• Continued Growth Potential: The therapeutic antibody field anticipates
significant future expansion.
• Traditional Applications: Antibodies are historically used for cancer,
autoimmune, and infectious disease management.
• Migraine Treatment: Anti-CGRP receptor antibodies (erenumab,
galcanezumab, fremanezumab) are used for migraine management.
• Hypercholesterolemia Treatment: Anti-PCSK9 antibodies (evolocumab,
alirocumab) treat hypercholesterolemia.
• Targeted Therapy: mAbs offer potent therapy when disease mechanisms
and specific pathogenic proteins are well-defined.
• X-linked Hypophosphatemia Treatment: Anti-FGF23 antibody
(burosumab) treats X-linked hypophosphatemia.
• Rheumatoid Arthritis Treatment: Anti-IL6R antibodies (sarilumab,
tocilizumab) treat rheumatoid arthritis.
• Hemophilia A Treatment: Anti-factor IXa/Xa antibody (emicizumab) is a
promising treatment for hemophilia A.
• Thrombotic Thrombocytopenic Purpura Treatment: Anti-von Willebrand
factor antibody (caplacizumab) treats thrombotic thrombocytopenic purpura.
 FUTURE TRENDS OF MAbs
• Future Antibody Approvals: More antibodies are expected to receive approval for
new therapeutic uses.
• Naked Antibody Therapy: Therapeutic mAbs are broadly classified into naked
antibodies used directly for disease therapy.
• Cancer Treatment Mechanisms: Naked antibodies in cancer therapy employ
mechanisms like ADCC/CDC, direct apoptosis induction, tumor microenvironment
targeting, and immune checkpoint targeting.
• Immune Cell Recruitment: Antibodies can kill cancer cells by recruiting natural killer
cells and other immune cells.
• ADCC/CDC Enhancement: Novel methods are being used to improve the
therapeutic efficacy of ADCC and CDC.
• Fc Point Mutations: Antibody Fc point mutations are employed to enhance cancer
cell killing.
• Glycosylation Alteration: Altering antibody glycosylation is used to enhance cancer
cell killing.
• Molecular Mechanisms: The success of mAb therapy relies on understanding the
molecular mechanisms of diseases.
• Distinct Proteins: Identifying distinct proteins linked to pathogenesis is vital for
effective mAb therapy.
• Therapeutic Advancement: Therapeutic antibodies represent a significant
advancement in targeted medical treatments.
MICROSPHERES AS CARRIER FOR MAbs

1. Encapsulation:
• mAbs can be encapsulated within the matrix of
the microsphere. This involves incorporating
the antibodies during the microsphere
formation process.
• This method protects the mAbs from
degradation in the body and allows for
controlled release over time.
• The microsphere material (often a polymer)
gradually degrades, releasing the encapsulated
mAbs.
MICROSPHERES AS CARRIER FOR MAbs

2. Surface Attachment:
• mAbs can be attached to the surface of microspheres
through various chemical or physical methods.
• This allows the mAbs to be presented on the exterior of the
microsphere, enabling them to bind to target cells.
• Methods include:
• Adsorption: mAbs are passively adsorbed onto the
microsphere surface.
• Covalent Coupling: mAbs are chemically bonded to the
microsphere surface, providing a more stable attachment.
• Linker molecules: linker molecules are used to bridge the
microsphere surface and the monoclonal antibody.
• This is especially useful for targeted drug delivery, where the
mAbs guide the microspheres to specific cells or tissues.
Key Considerations:
•Polymer Selection: The choice of polymer for the
microsphere is crucial. It must be biocompatible,
biodegradable (if desired), and suitable for the chosen
method of mAb incorporation.
•Release Kinetics: The release rate of mAbs from
microspheres can be controlled by adjusting the polymer
composition, microsphere size, and other factors.
•Targeting: Surface modification with targeting ligands (in
addition to the mAbs) can further enhance the specificity of
microsphere delivery.
•Stability: it is very important to maintain the stability and
activity of the mAb during the microsphere preparation and
release.