Lecture 7:

Introduction to Downstream
Processing
Learning Objectives
1. Outline the methods available for downstream processing of biopharmaceuticals

2. Discuss the use of Single-Use Technologies in downstream processing

3. Discuss the importance of protein stability during purification

Downstream Processing (DSP)

Topics

Harvest

Unit Operations

Single-Use

Protein Stability
Harvest
Downstream Challenge

Higher cell densities = higher titres and higher impurities

Faster harvesting times = more product to be purified in less time

Bottleneck is now in downstream processing

The Downstream Challenge Cell Culture Medium
Amino Acids
Inorganic Salts
Cell Culture Vitamins
Medium D-glucose
Other Organics
Medium
Supplements Medium
Supplements
Cells Bovine Serum
Proteins

Cells
Intact Cells
Cell Debris
HCP’s
Viruses
Nucleic Acids
Lipids

Endotoxins
The Downstream Challenge

Enormous Number of Cells

Different Types of Molecules

Different Structures of Molecules

Different Chemistry of Molecules

Similarities in Sizes and Chemistry of Impurities with Product Molecule

Overview of Bioprocessing
1. Upstream 2. Harvest 3. Downstream

4. Fill Finish
Heuristics or “Common Sense ”
Rules of DSP
1 Remove most plentiful impurities first

2 Remove the easiest to remove impurities first

3 Keep the most difficult and expensive separations last

Select processes that make use of the greatest differences

4
in the properties of the product and impurities

If it works at a laboratory scale, it does not mean it will work at

5
manufacturing scale… scale up studies are performed

Prazers, D.M., Plasmid Biopharmaceuticals: basics applications and manufacturing, John Wiley, 2011.
Goal of Harvest
Separation of the protein of interest from the cell mass whilst
maintaining structural integrity of the protein

Intracellular Extracellular
Protein Protein

Cell Cell

Extracellular Extracellular
Space Space

Typical for microbial Typical for mammalian

products products
 Harvest/Primary Recovery

Cell GEA Pathfinder GMP. GEA engineering for a better world. (n.d.). Retrieved May 31,
2022, from https://www.gea.com/en/products/centrifuges-separation/centrifugal-
separator/clarifier/separator-pathfinder-pharma-Biotech.jsp
clarifier: https://www.gea.com/en/products/centrifuges-separation/centrifugal- Stax Max Clarification Platform. Depth Filtration System | Pall Shop. (n.d.). Retrieved May 31,
separator/clarifier/separator-pathfinder-pharma-Biotech.jsp 2022, from https://shop.pall.com/us/en/biotech/depth-filtration/zidht5ftm7f
Harvest/Primary Centrifugation Depth Filtration Bioburden
Filtration
Recovery
Options
Depth Filtration Depth Filtration
(Coarse) (Fine) Bioburden
Filtration

Microfiltration Bioburden
Filtration
Single-Use in Harvest
Disk Stack Centrifuge Depth Filtration
 Disk Stack Centrifuge
Commercial scale bioprocessing uses continuous flow Disk Stack Centrifuges

✓ Low running costs.

✓ Continuous processing possible

✓ Quick processing time – runs at 12,000 rpm

Principle of Filtration
Retain Molecules > MWCO

Filter Out Permeate

Ultrafiltration & Diafiltration (UF/DF): Unchained Labs. unchainedlabs. (2022, April 5). Retrieved May 31,
2022, from https://www.unchainedlabs.com/ultrafiltration-diafiltration-uf-df/
Tangential Flow Filtration (TFF) :
A Key DSP operation
Microfiltration
✓ Highly efficient method of
separating cells from liquid
✓ Pore size of filters average at
0.45µm
✓ Uses tangential flow filtration /
cross flow filtration

× Expensive
× Long processing time
Depth Filtration
Thick mat of randomly arranged fibres – unlike membranes with specific pores

Creates a torturous path for material to travel

Traps particles throughout the medium – good for liquids with high particle densities

Depth filters also remove particles by adsorption, attracting the contaminants using either
electrokinetic or surface affinity

Nixon, B. Troubleshooting depth filtration. 2012. Pharmaceutical Manufacturing.

Poll

What is the process step that is frequently placed

immediately following centrifugation?

a. Viral filtration
b. Formulation
c. Depth filtration
d. Nano-centrifugation
Unit Operations
Overview of Bioprocessing
1. Upstream 2. Harvest 3. Downstream

4. Fill Finish
Purification in Downstream
3. Downstream

Chromatography and filtration are

the main techniques used in
Downstream Processing
Protein A/G
Enrichment

Ultrafiltration Viral Anion Cation Viral

Filtration Exchange Exchange Inactivation
Purpose of Purification
1 Remove impurities from the process

2 Inactivate and remove potential viruses and other contaminants from the process

3 Concentrate the protein to a safe, therapeutic dose

Maintain structural integrity of the protein

4
i.e. biologically active shape

Industrial-scale
chromatography
Chromatography
3. Downstream
At this point in the manufacturing process, the product
contains many product and process related impurities

Chromatography is a separation method used to purify

our protein of interest
Protein A/G
Enrichment

Ultrafiltration Viral Anion Cation Viral

Filtration Exchange Exchange Inactivation
What is Chromatography?
The purpose of chromatography is to separate the components of a mixture for more
advanced use – and thus is a form of purification

Unpurified Product Purified Product

from Upstream for Patient-use

It is based on the affinity of the components in the mixture for the

stationary or mobile phases
Poll

What principles are commonly used in chromatography

to purify proteins?

A. Affinity (specific binding to a given protein sequence)

B. Net charge
C. Size (size exclusion chromatography)
D. Hydrophilic or hydrophobic interactions
E. All of the above
Chromatography – Components

Column Stationary Phase Mobile phase

• Cylindrical vessel that • Solid matrix retained • Liquid, mostly aqueous
holds stationary phase in within column buffer
place
• Consists of small, bead- • Pumped through the
• Allows for migration of like particles column and stationary
the mobile phase phase
• Should be always kept in
buffer solution • Can be changed as
needed
Viral Clearance/Control Strategy
Risk Assessment:
Where and what is likely to occur

Validation:
Assessing the capacity of the
production processes to remove or
inactivate viruses

Testing/Detection:
Selecting and testing source material
for the absence of detectable viruses
Testing the product at appropriate
stages of production for freedom
from detectable viruses
Potential Sources Of Viral Derivation of
cell lines
Contamination from
infected
animals

Viral contamination of
biotechnology products Use of a Personnel:
may arise from: contaminated Contamination
excipient during Possible during cell
formulation sources of viral
Cell Lines (endogenous) handling
contamination
During Production Process
(adventitious)
Use of
contaminated Use of virus
biological to establish
reagents, such production
as animal serum cell lines
components
Virus Clearance Methods
Chromatography
Affinity
Pasteurisation
Ion-exchange
Lyophilisation
Viral Viral Hydrophobic interaction
Inactivation
Low pH treatment Removal Size exclusion
Solvent/detergent
Precipitation
UV irradiation
Centrifugation
Membrane filtration
Question

Why do we have viral clearance studies?

(Post your answers in the chat – get those participation marks!)

What is Ultrafiltration/Diafiltration?

Filtrate
Refill

Filter
Exchange
Sample

Ultrafiltration & Diafiltration (UF/DF): Unchained Labs.

unchainedlabs. (2022, April 5). Retrieved May 31, 2022, from
https://www.unchainedlabs.com/ultrafiltration-diafiltration-uf-df/
 Ultrafiltration/Diafiltration
Ultrafiltration/Diafiltration (UF/DF),
also called
Tangential Flow filtration (TFF)

Pellicon® 3 cassettes - pellicon 3 cassettes - EMD millipore. (n.d.). Retrieved May

31, 2022, from https://www.emdmillipore.com/CA/en/product/Pellicon-3- Spectrum® hollow fiber filter modules. Repligen. (n.d.). Retrieved May 31, 2022, from
Cassettes,MM_NF-C9947 https://www.repligen.com/technologies/spectrum-hollow-fibers/hollow-fiber-filters
What is Ultrafiltration/Diafiltration?
Ultrafiltration operates in a similar fashion to microfiltration (harvest).
However, this time the protein is retained and waste is removed.
Unwanted buffer/waste is filtered out
Vessel Vessel while the protein of interest is retained
and returned to the holding vessel
Pump
Retentate
(protein)
PRetenate
PFeed

Cassette
Filtrate / Permeate
(Unwanted buffer/waste)

Diafiltration involves the replacement of the old buffer with a new buffer.
This new buffer aids in future purification processes.
Poll

Downstream processing refers to the recovery and

purification of a therapeutic protein from cell culture.

True
or
False
Single-Use
 Single-Use

Single-use TFF
Text: Pauline Nicholson. (2012, June 8). Single-use TFF
Single-use DSP Systems. https://www.industr.com. Retrieved May 31, 2022,
from https://www.industr.com/de/single-use-tff-systems-
ÄKTA ready single-use system. Cytiva. (n.d.). 219940
Retrieved May 31, 2022, from
https://www.cytivalifesciences.com/en/us/shop/chr
omatography/chromatography-systems/akta-ready-
Single-use Bioreactor single-use-system-p-05843
Allegro STR single-use stirred tank bioreactors. Pall Shop. (n.d.). Retrieved May 31,
2022, from https://shop.pall.com/us/en/biotech/cell-culture/bioreactors/zidhslqw8fu
Single-Use Chromatography
Reduces physical size and footprint of facility whilst achieving required production scale capacity
Eliminate large-scale chromatography columns

No column packing
No resin storage
No storing of packed columns
Reduced labour

Increase plant flexibility/modularity accommodating multi-product facilities (smaller formats)

Reduce buffer usage (water, chemicals, disposal)

Resin Costs
50–80% of the production cost of
biopharmaceuticals are due to
downstream processing

A large proportion of that cost is due to

column resins

To recoup the cost of the resin it has to

be reused many times

It is not feasible to throw out resin after

each run
Membrane Adsorbers/ Chromatography
Advantages
Efficient
Higher throughput for trace impurity removal (g/h)
Economical
Save capital
No packing, regeneration
No re-use validation
95% less buffer consumption
Ease of Use
Disposable, simple set-up
Handle like a filter capsule

Disadvantages
No large-scale Protein A
Relatively new technology
Involves making change to existing bioprocess. Sartorius. (n.d.). Biopharma, Laboratory, Applied & Life Sciences. Sartorius. Retrieved May 31, 2022,
from https://www.sartorius.com/en
Single Use UF/DF
Membranes are reused but flow paths are single-use

Flow paths are still very expensive

Eliminate tank cleaning

Lessen chance of contamination

Clark, D. F. (n.d.). Implementing single-use technology in tangential flow filtration systems in clinical manufacturing.
BioPharm International. Retrieved May 31, 2022, from http://www.biopharminternational.com/implementing-single-
use-technology-tangential-flow-filtration-systems-clinical-manufacturing?id=&sk=&date=&%0A%09%09%09&pageID=2
Protein Stability
Molecular Cut-Offs of Filtration
Protein Biologics Are Very Sensitive
Activity of Biologics can be negatively affected by:
Temperature
Prolonged Storage
Denaturants
Organic Solvents
Free Clip art library. Free Clip art - Clip Art Collection - Dow nload Clipart on Oxygen
Clipart Library. (n.d.). Retrieved May 31, 2022, from http://clipart- library.com/
Changes in pH

Biologics require high-end controls in:

Manufacturing
Transport
Storage
Rare proteins collapse earlier. Rare proteins collapse earlier | ETH Zurich. (n.d.). Retrieved May
31, 2022, from https://ethz.ch/en/news-and-events/eth-news/news/2017/02/rare-proteins-
collapse-earlier.html
What Makes A Human Protein?
Polypeptide
Proteins are composed of molecular building Chain

blocks called ‘amino acids’ Amino Acids

Arranged in linear chains called ‘polypeptides’

21 different amino acids

Every protein contains a unique combination of

some, or all, of these amino acids
Genes Dictate Amino Acid Sequence

Biologically
Active Protein

Gene

Alberts et al. Molecular Biology of the Cell; Sixth Edition

Figure from: http://pdb101.rcsb.org/motm/14

The amino acids are arranged in a specific sequence which is dictated by the gene for that
protein.

The sequence is extremely important for the protein to take its biologically active shape.
Protein Instability
Protein destabilization can lead to:
1. Clipping

2. Denaturation

3. Aggregation

4. Precipitation

5. Chemical alterations

Protein aggregates
Protein aggregation and particle analysis. ProteinSimple. (n.d.). Retrieved May 31, 2022,
from https://www.proteinsimple.com/app_protein_aggregation.html
1. Clipping
Breaking apart of subunits of a protein

Breaking peptide bonds between amino acids (hydrolysis) and

degrading polypeptide chains

Also called ‘fragmentation’ or ‘truncation’

Clipping can be the result of many factors:

The presence of contaminating proteases

Photo-oxidation: reaction with light energy

Residual caustic cleaning agents (sodium hydroxide) in equipment

or low pH
2. Denaturation
The native state of a protein is its operative or functional form

Denaturation = a major change from the original native state

without alteration of the molecule's primary structure

When a protein is denatured, it loses its function

e.g. does not perform therapeutic action in the patient

It can be reversible or irreversible

3. Aggregation
Clusters of multiple proteins (can be same protein or several different proteins)
Monomer – a single protein
Dimers – 2 proteins aggregated
Trimers – 3 proteins aggregated
Multimers – multiple proteins aggregated

It can occur by several mechanisms, e.g.

Denaturation of protein leads to self-association
Contaminating non-protein particles provide attractive surface for protein
accumulation
4. Precipitation
Protein becomes insoluble as a result of:
Denaturation (heat, pH effects)
Excessively high protein concentration
Excessively high salt concentration in the buffer
Exposure to organic solvents

Normal Protein Solution + 2ml Hydrochloric Acid

Protein is fully dissolved. Protein is denatured by pH change

Solution has clear appearance. and precipitates.
Solution has cloudy appearance.
5. Oxidation (Chemical Alteration)

Oxidation:
Covalent modification of certain amino
acids with oxidising agents (oxygen,
metal ions, peroxides, reactive oxygen
species, UV light) and pH extremes

Can lead to disulfide scrambling,

conformational and functional changes

Davies, Michael J. "Protein oxidation and

peroxidation." Biochemical Journal 473.7 (2016): 805-825.
Factors Affecting Protein Stability
Disruptive influences may be of:

1. Biological origin

2. Chemical origin

3. Physical origin
Biological:
Proteolytic Enzymes (Bioreactor)

= inactive
protein!
Chemical Influences

Certain environmental factors during bioprocessing can

change the chemical structure of the protein

pH: Proteins are very sensitive to pH changes, which can

affect how amino acids interact and thus folding

Ionic Strength: Salt disrupts protein-protein interactions

Physical Influences
Protein denaturation/destruction/aggregation can be caused by many physical stressors

1. Mechanical Shear
Vigorous shaking (like whipping egg whites)

2. Temperature
All proteins function optimally at specific temperature
Freeze/thaw cycles

3. Protein Concentration
Too low: adsorption of product to container surface
Too high: precipitation

4. Light/ Ionizing Radiation

Exposure to light can trigger chemical chain reaction that can continue after
light source removed
Poll

Denaturation of a protein causes it to?

A. Lose therapeutic function

B. Gain therapeutic function
Poll

Proteolytic enzymes influence proteins by causing:

A. Aggregation
B. Clipping
C. Precipitation
D. Denturation
Topics Review

Harvest

Unit Operations

Single-Use

Protein Stability
Questions?
References
Alberts et al. Molecular Biology of the Cell; Sixth Edition Figure from: http://pdb101.rcsb.org/motm/14
Allegro STR single-use stirred tank bioreactors. Pall Shop. (n.d.). Retrieved May 31, 2022, from https://shop.pall.com/us/en/biotech/cell-culture/bioreactors/zidhslqw8fu
Clark, D. F. (n.d.). Implementing single-use technology in tangential flow filtration systems in clinical manufacturing. BioPharm International. Retrieved May 31, 2022, from
http://www.biopharminternational.com/implementing-single-use-technology -tangential-flow-filtration-systems-clinical-manufacturing?id=&sk=&date=&%0A%09%09%09&pageID=2
Davies, Michael J. "Protein oxidation and peroxidation." Biochemical Journal 473.7 (2016): 805-825.
Free Clip art library. Free Clip art - Clip Art Collection - Download Clipart on Clipart Library. (n.d.). Retrieved May 31, 2022, from http://clipart -library.com/
GEA Pathfinder GMP. GEA engineering for a better world. (n.d.). Retrieved May 31, 2022, from https://www.gea.com/en/products/centrifuges-separation/centrifugal-
separator/clarifier/separator-pathfinder-pharma-Biotech.jsp
Hydrophobic-interaction membrane chromatography for large-scale ... (n.d.). Retrieved May 31, 2022, from https://bioprocessintl.com/downstream-processing/chromatography/hydrophobic-
interaction-membrane-chromatography-for-large-scale-purification-of-biopharmaceuticals-184195/
Konstantinov K.B. and Cooney C.L. (2014). White Paper on Continuous Bioprocessing. International Symposium on Continuous Manufacturing of Pharmaceuticals
Nixon, B. Troubleshooting depth filtration. 2012. Pharmaceutical Manufacturing.
Pellicon® 3 cassettes - pellicon 3 cassettes - EMD millipore. (n.d.). Retrieved May 31, 2022, from https://www.emdmillipore.com/CA/en/product/Pellicon-3-Cassettes,MM_NF-C9947
Prazers, D.M., Plasmid Biopharmaceuticals: basics applications and manufacturing, John Wiley, 2011.
Protein aggregation and particle analysis. ProteinSimple. (n.d.). Retrieved May 31, 2022, from https://www.proteinsimple.com/app_protein_aggregation.html
Rare proteins collapse earlier. Rare proteins collapse earlier | ETH Zurich. (n.d.). Retrieved May 31, 2022, from https://ethz.ch/en/news-and-events/eth-news/news/2017/02/rare-proteins-
collapse-earlier.html
Sartorius. (n.d.). Biopharma, Laboratory, Applied & Life Sciences. Sartorius. Retrieved May 31, 2022, from https://www.sartorius.com/en
Spectrum® hollow fiber filter modules. Repligen. (n.d.). Retrieved May 31, 2022, from https://www.repligen.com/technologies/spectrum-hollow-fibers/hollow-fiber-filters
Stax Max Clarification Platform. Depth Filtration System | Pall Shop. (n.d.). Retrieved May 31, 2022, from https://shop.pall.com/us/en/biotech/depth-filtration/zidht5ftm7f
Text: Pauline Nicholson. (2012, June 8). Single-use TFF Systems. https://www.industr.com. Retrieved May 31, 2022, from https://www.industr.com/de/single-use-tff-systems-219940
Ultrafiltration & Diafiltration (UF/DF): Unchained Labs. unchainedlabs. (2022, April 5). Retrieved May 31, 2022, from https://www.unchainedlabs.com/ultrafiltration-diafiltration-uf-df/
ÄKTA ready single-use system. Cytiva. (n.d.). Retrieved May 31, 2022, from https://www.cytivalifesciences.com/en/us/shop/chromatography/chromatography-systems/akta-ready-single-use-
system-p-05843