The document outlines the cell line characterization and viral safety strategies employed by Merck KGaA, focusing on the importance of assessing potential viral contaminants and the methodologies used for testing cell lines. It emphasizes the need for thorough identity and purity assays, alongside viral safety risk assessments, to ensure safe sourcing and processing of biological materials. Various assays and testing protocols are discussed to confirm the integrity, stability, and safety of cell lines for medicinal products.

1. The life science business of Merck KGaA, Darmstadt, Germany
operates as MilliporeSigma in the U.S. and Canada.
Kathryn Martin Remington, Ph.D.
21 January 2021
Does Your Cell Line
Have a Secret?
Characterizing can Avoid a
Surprise

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

3. Agenda
Assessing the Risk
Identity Assays
Purity Assays
1
2
3
Testing for MCB/WCB/CAL
4

4. 4
Viral Safety Strategy
Safe sourcing and testing
of raw materials
Verify absence
of viral contaminants
at appropriate stages
Verify capacity of manufacturing
process to remove or inactivate
potential viral contaminants
Characterization
of Cell Lines
If viral
clearance
potential is
reduced, then
importance of
testing
increases

5. 5
Start with a Virus Safety
Risk Assessment
Risk assessment should consider:
 Species of origin
 Organ, tissue, fluid of origin
 Potential contaminants in view of origin of the raw
material and history of donor(s)
 Potential contaminants from the manufacturing process
 Infectivity and pathogenicity of the potential
contaminants for the recipients
 Route of administration of product
 Amount of material used to produce a dose of medicinal
product
 Controls carried out on the donor(s), on the raw material,
during production and on the final product
 The manufacturing process and its capacity to
remove/inactivate viruses
 Patient population being treated and disease indication
K
S
I
Factors that may influence the potential level of infectious particles in
the medicinal product and factors related to the use of the medicinal
product determine the viral risk to the recipients
EP 5.1.7 Viral safety
(01/2008:50107)

6. Safety
testing
strategy
Virus safety
risk
assessment
Regulations
& guidance
documents
Regulatory
updates
Building a Robust Safety Testing Strategy

7. Verify species of cell line
Identity
• Bacteria, fungi
• Mycoplasma (Spiroplasma)
• Mycobacterium
• Viruses (Endogenous and
Adventitious)
Expression Construct
Genetic Stability
Purity
Cell Line
Characterization
in Brief

8. Verify species of cell line
Identity
Cell Line
Characterization
in Brief

9. 9
Genotypic approaches:
• DNA fingerprinting (short tandem repeats): identifies cells of same species
• Cytochrome c oxidase subunit I (CO1) PCR and barcoding: identifies species and
subspecies
Karyology
• Chromosome number and marker chromosomes identify cells of same species
• Recommended for new cell lines and for diploid cell lines, but not necessary for well
characterized cell lines such as CHO, Sp2/0, NS0
Identity testing of Cell Banks
One of the above methods is performed on MCB and EOP/CAL
Required to confirm the species origin of cell lines

10. Conserved mitochondrial coding region
 Lack of introns
 Limited exposure to recombination
 Haploid inheritance
Universal primers for CO1 gene
 Robust primers
 Enable recovery of its 5’ end from all animal phyla
Assay based on CO1 gene
 CO1 possesses a greater range of phylogenetic signal than any
other mitochondrial gene, allowing for clear species
identification
 Method of choice for taxonomic identity and for cell line identity
at cell culture collections
 Greater number of reference species, minimal subjectivity
relative to isozyme analysis
10
CO1 Barcode Assay
Compared to DNA Fingerprinting, CO1 Barcode assay has
greater robustness, reduced sample requirements and
shorter turn-around time

11. • Bacteria, fungi
• Mycoplasma (Spiroplasma)
• Mycobacterium
• Viruses (Endogenous and
Adventitious)
Purity
Cell Line
Characterization
in Brief

12. 12
 TSB media (total aerobes) – Incubated at 20-25°C
 THIO media (microaerophiles/anaerobes) – Incubated at 30-35°C
• Observed for microbial growth on day 3, 4 or 5, day 7 or 8, and
day 14.
• Assay is performed on 1% of total bank, but not less than 2 vials
Sterility Testing
Direct
Inoculation
Membrane
Filtration
Rapid Sterility
• BacT/Alert® Detection System
• US FDA Amendment to Sterility Test Requirements for Biological
Products (21 CFR 610.12, revised 2012, effective 04 Jun 2012)
• Turbidity from debris/cellular material does not interfere
• Non-destructive technology, which allows for subculture of
positive broths

13. • Bacteria, fungi
• Mycoplasma (Spiroplasma)
• Mycobacterium
• Viruses (Endogenous and
Adventitious)
Purity
Cell Line
Characterization
in Brief

14. 14
Culture-Based Mycoplasma Testing

15. 15
* Validated against Mycoplasma orale, Mycoplasma hyorhinis, Mycoplasma
synoviae, Mycoplasma fermentans, Mycoplasma arginine, Mycoplasma
pneumoniae, Acholeplasma laidlawii, Spiroplasma citri.
Parameter Pinnacle QPCR Conventional Methods
Method Real Time PCR
Agar & broth amplification;
indicator detection system
Endpoint Fluorescence Mycoplasma colony count
Duration 1 day 28 days
Sensitivity 10 cfu/mL 10-100 cfu/mL
Specificity High High
Validation Yes Yes
Range of Species 102 species* Viable
Rapid Mycoplasma Assay

16. • Bacteria, fungi
• Mycoplasma (Spiroplasma)
• Mycobacterium
• Viruses (Endogenous and
Adventitious)
Purity
Cell Line
Characterization
in Brief

17. • Infectivity assay (56 days) meets requirements of EP monograph and
EP and USP recommendations that all cells susceptible to infection
with Mycobacterium spp should be tested in an assay for this microbe
• PCR assay for mycobacterium is a rapid alternative to the infectivity
assay
• If cells are tested for mycobacterium by one of these assays, then
Guinea pigs are not required in the in vivo assay
17
Mycobacteria

18. • Bacteria, fungi
• Mycoplasma (Spiroplasma)
• Mycobacterium
• Viruses (Endogenous and
Adventitious)
Purity
Cell Line
Characterization
in Brief

19. 19
To detect unknown and a wide range of possible contaminants
must utilize a number of different assays
Broad specificity assays
• in vitro and in vivo virus assays, electron microscopy
Assays to detect contaminants associated with specific species
• Rodent, bovine, porcine, human viruses
Assays to detect retroviruses
• Infectivity assays
• Molecular biology assays (PCR)
• Biochemical assays (reverse transcriptase)
• Morphological assays (electron microscopy)
Assays to detect specific viruses
• Molecular biology assays (PCR)
Rationale for virus assays

20. 20
Common Detector Cells
• MRC-5 (Human Diploid)
• Vero (Simian)
• Same Species and Tissue - SP2/0, BHK, CHO, NS0 etc.
In Vitro Adventitious Virus Assay
Test
Article
Passaging of cells
Cell Monolayer
Day 14
CPE/HA/HAD/IFA
Passage conditioned
medium
Day 28
CPE/HA/HAD/IFA
End of Assay
9CFR assays are similar, but use CPE, HAD and IFA endpoints

21. 21
In vitro virus assays
•Use selected cell lines permissive to a wide variety of viruses
•Endpoints for viral detection may be
Cytopathic effects (CPE)
Hemagglutination or hemadsorption
IFA
Advantages:
Screens for a broad range of viruses
Low Limit of Detection (LOD), approximately 1 TCID50
Limitations:
•Only detect viruses that
- Can grow in culture
- Are detected by endpoint assay
•Cultures susceptible to toxicity as well as viral effects
•Practical limitations in the number of detector cells lines used
Broad specificity in vitro virus assays

22. 22
• Inoculate mice (adult/suckling),
guinea pigs, embryonated eggs
• Viruses detected by appearance of
disease symptoms (animals), loss of
viability (eggs) or by hemagglutination
assay (egg fluids)
• Antibody production assays in
mice/rat/hamster*
• Serum screened by ELISA for
production of virus-specific Abs
In vivo virus assays
Advantages
• Ethical concerns
• Toxic effects of test material
• Lengthy assay
Limitations
Broad specificity in vivo virus assays
*Blazar™ Rodent Panel is a molecular
alternative to the rodent antibody
production assays, using multiplexed,
degenerative PCR. Increases assay
sensitivity, robustness, detection of viral
variants and significantly reduces TAT

23. 23
Assays to Detect Retroviruses
Q-PERT assay to detect reverse
transcriptase activity that is
associated with retroviruses
Transmission Electron Microscopy
to detect viruses, virus-like
particles, and other microbes
Co-Cultivation of test article with
retrovirus-susceptible cells followed
by a focus-forming or other
endpoint assay for retroviruses

24. 24
• PCR for MMV (mouse parvovirus) for rodent cell lines
• PCR for Vesivirus 2117 for CHO cell lines
• Specific bovine or porcine viruses
• Human virus panel for human cell lines
Virus-Specific Assays (PCRs)

25. 25
• Is the finding reproduced in a repeat test?
• What is the host range, appearance of
cytopathic effect, hemadsorption/
hemagglutination pattern?
• Does the isolate remain infectious after
passage to fresh cells? Viral infections are
typically progressive resulting in the eventual
destruction of the cell population.
• Conduct specific PCRs and/or virus specific
IFAs for likely candidates
• Conduct electron microscopy of infected cells
to obtain size and morphology of virus
• Next Generation Sequencing (NGS)
Investigation of a Positive Adventitious Virus Result

26. Expression Construct
Genetic Stability
Cell Line
Characterization
in Brief

27. 27
27
Typically performed in Phase 3
using cells from MCB and EPC/CAL
Verifies that expression
system has not undergone
any changes that would
impact integrity of the
product
Molecular studies required to verify
• Correct sequence made and
incorporated into host cell
• Structure and copy # maintained to
end of production
ICH Q5B: Analysis of the
Expression Construct in Cells used
for Production of rDNA
Genetic Stability Testing

28. 28
Testing Assays MCB WCB CAL
Identity CO1 Barcode Analysis X X X
Microbial Detection
Sterility X X X
Mycoplasma X X X
Mycobacteria X X X
Virus Detection
In vitro virus assay X X X
In vivo virus assay X X
Antibody production assay/Blazar rodent panel D
TEM X X
Retrovirus infectivity assay D D
QPERT D D
PCR for Specific viruses X X
Bovine virus assay B/P B/P
PCR for Bovine Parvoviruses B/P B/P
Porcine virus assay/PCR for PCV & HepE B/P B/P
Karyotyping D D
Cell Line Characterization
D = Dependent on cell line
B/P = If bovine or porcine-derived materials have been used with cells

29. 29
• FDA or a Global submission?
• What is the species of the cell line?
• What is the source/history of the cell line?
• Was serum or trypsin used during the history of the cell line?
• Was cell line exposed to other cell species or ingredients from other species?
• Were the cells grown in medium containing antibiotics or ingredients possibly
inhibitory or toxic to adventitious agents (e.g., methotrexate)?
• Is the sample matrix biocompatible with the test system?
What are the Appropriate Tests for a Cell Bank?

30. 30
 A risk assessment directs the viral safety strategy and cell line
characterization program a cell line
 While some of the assays for characterization of cell lines are used for
all cell lines, some assays are specific to particular cells
 Use of animal-derived products at any point in the establishment of
the cell line presents a potential risk of contamination by an animal
virus. Testing the cell line should include tests for potential
contaminants
 Testing for the master and cells at the limit is more extensive
than testing for the working cell bank.
Unlock the Secrets with Cell Line Characterization

31. Technical Consultant
Field Technology Management
kathy.remington@milliporesigma.com
240 447 6280
Kathryn Martin Remington, Ph.D.
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