This document discusses strategies for preventing and detecting viral contamination in biologic manufacturing processes. It outlines sources of viral contamination including raw materials, facilities, and personnel. A multi-tiered approach is recommended involving screening raw materials and cell banks, in-process testing, and confirming downstream processes can clear viruses. Detection methods like in vitro and in vivo assays have limitations and next generation sequencing is presented as a powerful new tool to detect unknown viruses. Upstream prevention focuses on raw material control through pretreatment or virus-resistant cell lines while downstream processes aim to clear any contamination through viral inactivation or filtration steps. A holistic biosafety strategy applying prevention, detection, and removal approaches at all stages is emphasized.

1. Merck KGaA
Darmstadt, Germany
Alison A Armstrong, PhD
Global Head of Field Development Services
Key Considerations in the Prevention
and Detection of Viral Contamination
Viral Risk Mitigation
Strategies

2. 2
The life science business of
Merck KGaA, Darmstadt, Germany
operates as MilliporeSigma
in the U.S. and Canada.

3. Viral Risk Strategies
Content
2
1
Sources & Risks of Viral Contamination
Complexity of biomanufacturing & regulatory requirements
Developing a Biosafety Strategy
Industry expectations and detection methods
3
Viral Risk Mitigation
Prevent, Detect, and Remove
3
3

4. Sources & Risks of
Viral Contamination

5. Sources & Risks of Viral Contamination
Adventitious Agents
Medicinal products during their development, and prior to being marketed, must
meet strict criteria of Quality, Safety and Efficacy.
Safety considerations are associated with contaminants in
biological products including the following agents:
 Bacteria
 Fungi
 Mycoplasma
 Viruses
 Transmissible spongiform encephalopathies (TSE)
5

6. Growing Diversity/Complexity of Biologics
Sources & Risks of Viral Contamination
A biologic is a therapeutic substance that is produced through a biological process (often
involving biotechnology methods), rather than chemical synthesis (as for traditional
pharmaceuticals). Types of biologics include: recombinant protein, antibody, antibody-drug
conjugate, cell component or derivatives, vaccines, gene therapies, and cell therapies.
► The manufacturing of biologics is complex.
Raw materials
Assembly at
different sites
Safety testing
Timothy Moore, Senior Vice President, Global Head, Pharmaceutical Technical Operations Biologics Unit, Genentech
https://www.gene.com/stories/how-hard-can-it-be
6
6

7. Case Studies of Microbial Contamination in Biologic Product Manufacturing
Suvarna, K., Lolas, A., Hughes, P., Friedman, R. Biotechnology Manufacturing Team, Division of Manufacturing and
Product Quality, Office of Compliance, Center for Drug Evaluation and Research, Food and Drug Administration
Facility
Equipment
Process
Materials
Utilities
Personnel
Each source
is a potential
entry point for
microbial
contamination
Acholeplasma laidlawii (<0.2um)
Leptospira species (>5 um)
Sources of Adventitious Agent Contamination
Biosafety: A Challenge and Business Risk
Minute virus of mice (MVM) ~18-24nm
7

8. Routes of Contamination
Secondary
Clarification
Chromatography
Protein A
Column
Protection
Column
Protection
Final FillingFinal Sterile
Filtration
Concentration
and
Formulation
Bulk Storage
and Transport
Viral
Inactivation
Column
Protection
Chromatography
Purification
Chromatography
Polishing
Virus Filtration
Clearance
TFF
Bioreactor
Column
Protection
Chromatography
Purification
Primary
Clarification
MCB WCB Seed Train
Raw Materials
Filtration
8
8

9. Regulatory Requirements and Guidance
Sources & Risks of Viral Contamination
► To ensure the quality, safety and efficacy of biotherapeutic products intended for
use in humans
► Guidelines are based on experience gained over three decades in this technically
demanding field
9
9
• FDA: U.S. Food and Drug Administration
• FDA: Center for Biologics Evaluation and
Research
• European Pharmacopoeia
• CFDA: China Food and Drug Administration
• World Health Organization
• USP
• NIST: National Institute of Standards and
Technology
• FDA: Center for Drug Evaluation and
Research
• ICH
• European Medicines Agency
• Pmda: Pharmaceuticals and Medical Devices
Agency
• ChP
• JP: Japanese Pharmacopoeia
• Ministry of Food and Drug Safety

10. ICH Q5A: Viral safety evaluation of biotechnology products derived from cell lines of human or animal
origin (Apr 1997 to Feb 2000)
ICH Q5B: Analysis of the expression construct in cells used for production of rDNA derived protein
products (Dec 1995 to Jan 1998)
ICH Q5C: Stability testing of biotechnological / biological products (Dec 1995 to Jan 1998)
ICH Q5D: Derivation and characterisation of cell substrates used for production of
biotechnological/biological products (Sept 1997 to July 2000)
ICH Q5E: Comparability of biotechnological / biological products subject to changes in their
manufacturing process (Dec 2004 to June 2005)
Regulatory Requirements and Guidance
Sources & Risks of Viral Contamination
10

11. FDA Guidance for Industry: Characterization and Qualification of Cell Substrates and
other Biological Starting Materials Used in the Production of Viral Vaccines for the
Prevention and Treatment of Infectious Diseases (Draft 2006, Final 2010)
EMA Guideline on Virus Safety Evaluation of Biotechnological Investigational
Medicinal Products (EMEA/CHMP/BWP/398498/2005) July 2008
World Health Organization recommendations for the Evaluation of Animal Cell
Cultures as Substrates for the Manufacture of Biological Medicinal Products and for
the Characterization of Cell Banks. 2011 TRS 978 Annex 1
Regulatory Requirements and Guidance
Sources & Risks of Viral Contamination
11

12. Developing a biosafety
strategy

13. Cell Type Viral Contaminant Source of Virus
Various BVDV (non-cytopathic) Bovine serum
BHK/CHO Reovirus Bovine serum
CHO EHDV Bovine serum
(not screened)
CHO Cache Valley Virus Bovine serum
(not screened)
CHO Minute Virus of Mice Components of media
CHO Calicivirus 2117 Bovine serum?
Vero Porcine circovirus Porcine trypsin
Vero Bluetongue virus ?
Rhesus monkey kidney SV40 Primary cell line
Various SMRV Other cell lines
Insect Tn5 (High Five) Nodavirus Latent infection of cell
Insect Sf9, Sf21 Rhabdovirus Latent infection of cell
Contamination of Production Processes
13

14. Minute Virus of Mice (MVM)
A Threat Even in Animal Component Free Processes
Parvovirus (family Parvoviridae)
Small (< 20 nm) non enveloped virus with 5-kb linear, ssDNA genome
Extremely virulent
One viral particle per liter of culture sufficient to generate a contamination event
Difficult to remove from processes
Present in animal component free processes
Resistant to inactivation via chemical or physical means
14
14

15. Three Complementary Approaches
Viral Risk Mitigation
✓ Selecting and testing of starting/raw materials
 Cell line
 Culture media, sera, supplements & reagents
 Drug substance/component
✓ Testing the product intermediates at appropriate steps of
production
 MCB, WCB, EOPC
 Bulk harvests
✓ Assessment of the capacity of USP and DSP to clear viruses
 Low pH inactivation
 Chromatography
 Nanofiltration
15
15

16. You may not find what is truly there…
 Sensitivity of assay limits detection
 All assays have a LOD
 Sample volume limitations
 Cell lines may not be permissive for some known or novel viruses
 Interference/matrix effects
 Anti-virus antibodies in FCS used in in vitro assays
 Cytotoxicity of indicator cells
 Inhibition of PCR assay enzymes
You only find what you are looking for….
 Screening assays are designed to assess known/past pathogens
 Adapted from K Brorson, US FDA
Detection
Issues with Detection Assays
16

17. • Cell banks should be prepared, stored and tested under GMP conditions
• Reduces risk of contamination
 “During the establishment of the cell bank, no other living or infectious material (e.g. virus,
cell lines or cell strains) should be handled simultaneously in the same area or by the same
persons.”
 “Following the establishment of cell banks, quarantine and release procedures should be
followed. This should include adequate characterization and testing for contaminants.”
 “Cell banks should be stored and used in such a way as to minimise the risks of
contamination (e.g. stored in the vapour phase of liquid nitrogen in sealed containers)”
• EU Guidelines for GMP for Medicinal Products for Human and Veterinary Use, Annex 2,
Manufacture of Biological Active Substances and Medicinal Products for Human Use.
Production of Cell Banks under GMP Conditions
17

18. Virus Contamination Prevention Options: Detection
Secondary
Clarification
Chromatography
Protein A
Column
Protection
Column
Protection
Final FillingFinal Sterile
Filtration
Concentration
and
Formulation
Bulk Storage
and Transport
Viral
Inactivation
Column
Protection
Chromatography
Purification
Chromatography
Polishing
Virus Filtration
Clearance
TFF
Bioreactor
Column
Protection
Chromatography
Purification
Primary
Clarification
MCB
Pretreated
Raw
Materials
FiltrationHTST
γ Radiation
UV-CRaw
Materials
Virus
Resistant
Cell Line Seed Train
WCB
= Routes of contamination
= Upstream Virus Prevention
= Downstream Virus Removal
= Points for testing
18
18

19. Rigorous Screening of Cell Banks
Viral Risk Mitigation
• Starting material for the whole of the production process
• Full characterisation for microbial and viral contaminants
• One time testing
MCB
• Small number of passages beyond MCB
• Reduced package of testing on cells directly from 1st WCB
• Full characterisation for subsequent WCBs
WCB
• “Worst case” for amplification of contaminants
• Full characterisation, one time testing at production scale
• For “well characterized cell lines” may not be required at
early clinical development stages
EPC/CAL
19
19

20. Broad Range Virus Detection Approach
Viral Risk Mitigation
viruses detected by cytopathic effects (CPE), haemadsorption, haemagglutination, IFAIn vitro virus assay
•Not all viruses produce CPE
•Can only use a limited number of detector cells
•Not all viruses grow well in tissue culture
injection into suckling mice, adult mice, embryonated eggs (allantoic cavity and yolk sac)
and (Guinea pigs)In vivo virus assay
•Viruses detected by morbidity and mortality
•Classical virus isolation methods that may detect viruses that do not grow well in tissue culture
•Ongoing discussion about the sensitivity and usefulness of this assay
detects intracellular virus particlesElectron microscopy
•No amplification potential
•Required specialized resources
capable of detecting a broad range of microorganisms including the unknownsNext generation sequencing
•Use is not yet specifically defined for most applications, but may be inferred under use of ‘state of the art’ technologies
•US FDA have suggested using NGS for the analysis of gene therapy viral vectors
•Required specialized resources
20

21. A Powerful Tool for Detection of Adventitious Agents
Next Generation Sequencing
✓ Tool to detect contaminants
✓ No selection of nucleic acid prior to sequencing
✓ Depth of sequencing may allow construction of whole virus genome
Sample Processing Sequencing Bioinformatics
Next Generation Sequencing (NGS)
21
21

22. • Adventitious Agent Testing
• Raw Material Qualification
• In-process Testing
• Lot Release Testing
• MVSS Identity Genetic Purity
• Cell Line Identity/Purity
• Engineered Culture Purity
• Genetic Stability
A powerful tool for the detection of unknown adventitious agents
Applications in Use Today
Next Generation Sequencing
22
22

23. Regulatory Acceptance
Next Generation Sequencing
✓ Recent WHO and European Pharmacopoeia documents have indicated the potential
usefulness of NGS for viral vector identification/stability, recombinant cell line genetic
stability and adventitious agent detection
✓ Draft EP 5.2.14. Substitution of in vivo method(s) by in vitro method(s) for the quality
control of vaccines
‒ Novel, sensitive molecular techniques with broad detection capabilities are available, including
deep sequencing or high throughput sequencing methods.
‒ The use of these new broad molecular methods has highlighted the gaps with the existing testing
strategy by identifying previously undetected viral contaminants in final product, the cell banks
from which it was produced and intermediate manufacturing stages.
‒ The implementation of such new broad molecular methods as substitutes for existing methods
requires a comparison of the specificity (breadth of detection) and the sensitivity of the new and
existing methods.
✓ Companies have used NGS for adventitious agent detection and submitted the data to
regulatory agencies in support of the conventional testing also performed
23
23

24. Viral risk mitigation

25. A Multi-tiered Approach
Viral Risk Mitigation
Detect
Virus Risk
Mitigation
Raw material control
or barrier technology
Process’s ability to
reduce potential risk
Ensure that the approach to risk works
Plavsic, M. BioPharm International, May 2016. p.40
25
25

26. Sources of Contamination Exist at Multiple Stages
Viral Risk Mitigation
Facility registration (ISO,
cGMP, FDA), component
grade, component
concentration rodent
attractant
Origin and
Source
Raw Materials
Manufacturing
Process and Facility
Repacking of
Material
Shipping and
Warehousing
Geographical origin,
synthetic or mined,
fermented, plant or
animal derived
Added step to raw
materials
manufacturing
Warehouse control, lack of
transport vehicle control
26
26

27. Genetic Engineering of MVM Virus Resistance in CHO Cells
Targets to Reduce MVM Uptake and Propagation
Create a CHO parental cell line (CHOZN® cell
line background), that is resistant to (or cannot
propagate) an MVM infection while maintaining
robust biomanufacturing performance.
 Infection initiation and entry
- Capsid-mediated binding to one or
more surface receptor(s)
- Receptor-mediated endocytosis
 Transfer into/through the cytoplasm
- Affected by a capsid-borne
phospholipase
 Entry into the host nucleus
- Replication
27
27

28. Raw Material Risk Mitigation Approach
Viral Risk Mitigation
Eliminating or reducing the load with a layered approach
“Point-of-origin”
• Low risk vendors
• Pretreated products (HTST pre-treated
glucose or gamma irradiated serum)
• Material origin (serum sourcing for
BSE mitigation)
“Point-of-use”
• Point of origin prevention will not
mitigate all components
• Virus barrier technologies
• Mitigate contamination from shipping
28
28

29. Contamination Profile & Risk Profile Vary throughout the Process
Focus Upstream
Secondary
Clarification
Chromatography
Protein A
Column
Protection
Column
Protection
Final FillingFinal Sterile
Filtration
Concentration
& Formulation
Bulk Storage
& Transport
Viral
Inactivation
Column
Protection
Chromatography
Purification
Chromatography
Polishing
Virus Filtration
Clearance
TFF
Bioreactor
Column
Protection
Chromatography
Purification
Primary
Clarification
MCB WCB Seed Train
Filtration
Raw Materials
29
29

30. Preventing Contamination
Adventitious Agent Safety of Upstream Raw Materials
Consider virus resistant cell lines
•Centinel® technology, first commercially
available gene editing tool to confer Minute
Virus of Mice (MVM) resistance to CHO cell
lines
Replace animal derived
components in media
• Recombinant proteins (r-Insulin,
r-Trypsin, etc.)
• Serum alternatives
Source lower risk animal derived
components
• Source from lower risk geographies
• Irradiation of raw material
• Viral testing (CFR9) of raw material by
manufacturer
Adopt of chemically defined animal
derived component-free media
• Most conservative approach
1
2
3
4
30
30

31. Upstream Prevention Technologies
Prevention Strategies
102°C for 10-15
seconds
25 – 40 kGy exposure 254 nm wavelength
Size exclusion
removal
HTST
Pasteurization
Gamma
Radiation
UV-C
Radiation
Microbiological or
Virus Barrier
FiltrationPretreatment option Pretreatment option Pretreatment option
31
31

32. Preventing Contamination
Raw Material Pre-Treatment
Technology Robust Clearance Media Compatibility Point of Use Scalability Cost Effective
HTST
(~102C ~10 sec)
Yes
Component
dependent
Yes Large Scale
Yes at Large
Scale
UV-C
(254 nm)
Organism
dependent
Component
dependent
Yes
Challenging at
large scale
Yes but
challenges at
large scale
Gamma –
Radiation
Yes
Component
dependent
No Small batches Yes
Upstream Virus
Barrier Filtration
Yes by size
exclusion.
Consistent
LRV
Yes Yes Yes
Yes
upstream
virus filters
32
32

33. Prevention Strategies
Virus Barrier Filters (Media Nanofiltration)
Specifically designed for media filtration
Broadly effective against adventitious agents
Low/no capital
Easy to scale (robust scale-down model)
Easy to validate
Small system footprint
No impact to cell culture media
Low development & validation costs
High operating expenses
Media dependent throughput
33
Benefits Limitations
33

34. Contamination Profile & Risk Profile Vary Throughout the Process
Focus: Downstream
Secondary
Clarification
Chromatography
Protein A
Column
Protection
Column
Protection
Final FillingFinal Sterile
Filtration
Concentration
and
Formulation
Bulk Storage
and Transport
Column
Protection
Chromatography
Purification
Chromatography
Polishing
Virus Filtration
Clearance
TFF
Bioreactor
Column
Protection
Chromatography
Purification
Primary
Clarification
MCB WCB Seed Train
Raw Materials
Viral
Inactivation
Filtration
34
34

35. Preventing Contamination
Adventitious Agents Downstream
Employ single-use systems
• Closed systems and sterile connectors
minimize the chance for microbial ingress
from the environment and personnel
• Eliminates need for CIP SIP, sanitization
and storage
Utilize sound process design for
multi-use systems
• Minimization of dead legs
• Proper pipe sloping
• Understand impact of temperature
differentials
• Verify torque settings
Understand the risk profile of
process chemicals
• Chemicals for buffers, cleaning,
sanitization, inactivation, and storage
should be of suitable quality for
pharmaceutical GMP production
1
2
3
35
35

36. Downstream Virus Clearance Technologies
Removal Strategies
Parvovirus LRVa 1-3
Retrovirus 1-4
Parvovirus LRVb > 4
Retrovirus > 5.8
Parvovirus LRVb > 4
Retrovirus > 6
Virus Flow-Through
Protein A
Chromatography
Virus Inactivation
Low pH
Solvent/Detergent
Virus Binding
Chromatography
Anion* IEX
Size Exclusion
Virus
Nano-Filtration*
Parvovirus LRV a 3.25
Retrovirus LRV 4.22
a) Remington et. al. Viral Clearance by Protein A, Anion Exchange and Cation Exchange Chromatography
Steps, 2015 American Pharmaceutical Review.
b) Merck KGaA, Darmstadt, Germany data
* Sterilizing-grade microfiltration used
for bioburden reduction at various
points downstream36
36

37. Process Step
Clears
Enveloped Virus
Clears Non-
Enveloped
Virus
Robustness of
Step
Ease of
Downscale
Typical
Clearance
Low pH
Inactivation
Yes No
Yes; at or
below pH
3.5
Easy ≥ 4 logs
Detergent
Inactivation
Yes No Yes Easy ≥ 4 logs
Anion
Exchange/Anion
Mixed Mode
Chromatography
Potential
Exists; Virus
Dependent
Potential
Exists;
Virus
Dependent
No More Difficult
1 to ≥ 4
logs
Other
Chromatography
Potential
Exists; Virus
Dependent
Potential
Exists;
Virus
Dependent
No More Difficult
1 to 3
logs
Virus Reduction
Filtration
Yes Yes
Yes; for
viruses
above filter
pore size
Easy (with
vendor small-
scale device)
≥ 4 logs
Removal Strategies
Downstream Virus Clearance
37

38. Build a Robust Safety Strategy
• Source low risk raw materials
• Consider virus resistant cell lines
• Choose appropriate chemicals for
• GMP Production
• Implement Single Use Systems
Detect
Virus Risk
Mitigation
• Consider implementation
of upstream virus barrier
filtration
• Understand benefits and
limitations of upstream
pre-treatments
• Develop a robust virus
clearance process
• Understand limitations of methods
• Use PCR and other rapid methods
for process monitoring38
38

39. Summary
Virus Risk Mitigation
4
3
2
1
Sourcing low risk raw materials
• Moving towards serum free, chemically defined media
• Evaluate production culture for adventitious agents early in production
cycle to mitigate product loss and spread to facility
Developing a robust testing strategy
• Tailor sampling plans based on achievable viral clearance
• New genomic detection methods can improve the breadth of virus detection
Implementation of viral clearance technologies
• Incorporate robust viral inactivation steps and virus removing filter in
product purification
Overall Strategy
• Must be a strategic application of the three steps (highly inter-related)
• Optimization possible to drive risk down to the lowest theoretical level possible
39
39

40. Alison Armstrong, PhD
Global Head of Field Development Services
Life Science business of Merck KGaA, Darmstadt, Germany
+44 141 576 2440
alison.armstrong@sial.com
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