Is your lot release testing strategy ready for global commercialization? In this webinar, you will learn: • CMC testing requirements with CHO production platform for global commercialization • Lot release testing of product intermediates and final product • Product-specific qualification study • Alternative rapid testing methods to advance lot release testing CHO cells continue to serve as a key cell substrate for the manufacturing of recombinant proteins that span beyond therapeutic monoclonal antibodies and including subunit vaccines. In this presentation, we will cover the CMC testing requirements with CHO production platform for global commercialization, Lot release testing of product intermediates and final product, product-specific qualification study and highlight the application of new testing methods and the benefits they bring to advance Lot Release Testing.

1. Edmund Ang, PhD
Senior Technical Specialist
Field Technology Management, Asia Pacific
Broad experience in drug discovery,
bioproduction, and assay technologies:
• Technical consultative support for biosafety testing of
biologics
• Bioproduction product management at Roche Diagnostics and
Thermo Fisher Scientific
• Drug discovery R&D at Ionis Pharmaceuticals and Eli Lilly
• Cell signaling R&D at Edinburgh University (PhD), Stanford
University and National Cancer Centre, Singapore (postdoc)

2. The life science business of Merck KGaA,
Darmstadt, Germany operates as MilliporeSigma
in the U.S. and Canada.
Setting up for
successful lot
release testing
Is your lot release testing strategy
ready for global commercialization?
Edmund Ang

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

4. Agenda
CHO Bioproduction Platform
CMC testing requirements
Lot Release Testing
Alternative rapid testing methods
Product Specific Qualification Study
Support lot release testing
1
2
3

5. CHO
Bioproduction
Platform
CMC testing
requirements

6. 6
3 main concerns with the use
of animal production cells:
1. Presence of adventitious viruses
2. Oncogenic host cell DNA
3. Genetic stability

7. 7
ICH Q5A(R1) – I. INTRODUCTION
Guidance and Regulation on Viral Safety
Three principal, complementary approaches have evolved to control the potential viral contamination
of biotechnology products:
 Selecting and testing cell lines and other raw materials, including media components, for the
absence of undesirable viruses which may be infectious and/or pathogenic for humans;
 Assessing the capacity of the production processes to clear infectious viruses;
 Testing the product at appropriate steps of production for absence of contaminating infectious
viruses.
Prevent: Raw material
control and barrier technology
Detect: Testing for presence
of viruses
Remove: Process’s ability to
reduce levels of infectious
viruses

8. 8
Viral Safety Assurance – Overview from clone/raw materials to final product
Sources of Contamination & Risk Mitigation Approaches
Routes of
contamination
Product Characterization – Identity, Purity, Potency
(Physical Characteristics, Immunoassay, Cell-Based Assays)
Lot Release Testing
(Sterility, Molecular Biology, Virology)
Virus Clearance Study
Purified Bulk
Drug Substance
Final Drug
Product
Unprocessed
Bulk Product
Chromatography
resin selection, yield
optimization, product
characterization
Formulation
development, stability
testing, fill & finish
Downstream bioprocessing
Upstream bioprocessing
Bioreactor
Research
Cell Line
Production
Cell Line
Cloning, cell selection
& expansion
Media development,
upstream process
optimization
(“scale up”)
Cell & Virus Characterization
(Sterility, Molecular Biology, Virology)
Raw Material Testing
(Sterility, Molecular Biology, Virology)
Cell & Virus Seed Stock Banking
Single-Use
Systems
Sterile bags &
connectors
Reduced risk of
contamination
Perfusion
Culture
Higher cell
density
Extended culture
duration
Cell & product
stability
Continuous
Bioprocessing
Closed system
Increased
productivity
When/What to
test?
Rapid testing
methods
Platform
Bioprocessing
Standardized
raw
materials
and workflow
Proof-in-
principle data
package?
Advanced
Therapies
ATMPs such
as genes,
cells, tissues
Rapid testing
methods
Raw
materials
MCB
WCB
CAL

9. 9
Testing approach in accordance to global guidance
Hamster cells (CHO and BHK)
1. ICH Q5A Quality of Biotechnological Products: Viral Safety Evaluation of Biotechnology Products
Derived from Cell Lines of Human or Animal Origin (1997)
2. ICH Q5B Quality of Biotechnological Products: Analysis of The Expression Construct in Cell Lines Used
for The Production of rDNA Derived Protein Products (1996)
3. ICH Q5D Derivation and Characterization of Cell Substrates Used for Production of
Biotechnological/Biological Products, (1997)
4. US FDA Points to Consider in the Characterization of Cell Lines Used to Produce Biologicals (1993)
5. US FDA Points to Consider in the Manufacture and Testing of Monoclonal Antibody Products for Human
Use (1997)
6. WHO Recommendations for the Evaluation of Animal Cell Cultures as Substrates for the Manufacture of
Biological Medicinal Products and for the Characterization of Cell Banks (2010) TRS 978, Annex 2
7. EP Monoclonal Antibodies for Human Use. 01/2012:2031
8. EMA Guideline on Virus Safety Evaluation of Biotechnological Investigational Medicinal Products. (2008)
Testing strategy is based on risk assessment and scientific rigor

10. 10
Conventional testing approach
Characterization of CHO Production Cell Banks
Test MCB WCB CAL
Identity CO1 barcode √ √ √
Purity
Sterility Q+√ √ √
Mycoplasma Q+√ √ √
Mycobacterium Q+√ √ √
Safety
Adventitious agent (in vitro) √ √ √
Adventitious agent (in vivo) √ √
Retroviruses (TEM, infectivity) √ √√
Specific rodent viruses
HAP
MAP
MMV
Other viruses (eg. bovine, porcine, ovine) √ (√)
Genetic
Stability
Sequencing of expressed gene √ √
Restriction Enzyme analysis by Southern blot √ √
Design and qualification of a QPCR assay for transgene copy
number
√ √
Transgene copy number by QPCR √ √
Sequence of 5’ and 3’ regions flanking transgene √ √
Localization of DNA sequences by FISH or SKY-FISH √ √

11. Conventional testing approach
Lot Release Testing of UBH, DS, and DP
11
Unprocessed Bulk Harvest Purified Bulk Drug Substance Final Drug Product
Assay description
Microbiology
Sterility by direct inoculation
Qualification for sterility assay
OR
Bioburden by direct inoculation
Bioburden suitability testing
Mycoplasma
Qualification of mycoplasma
PCR for mycoplasma
Adventitious virus testing
In vitro virus assay for presence of
adventitious viral contaminants
Retroviruses
Transmission electron microscopy
Specific Rodent viruses
MVM
Assay description
Microbiology
Sterility by membrane filtration
Qualification for sterility by
membrane filtration
Endotoxin (LAL)
Residual impurities
Residual CHO DNA
Qualification study for CHO
residual DNA
Host cell proteins
Protein A (if monoclonal)
Protein chemistry lot release
assays
Total protein
Capillary electrophoresis
Isoelectric focusing
Size by size exclusion
chromatography
Peptide mapping
Monosaccharide content
Biological activity
Assay description
Identity and potency
Size by size exclusion
chromatography
Biological activity
Microbiology
Sterility by membrane filtration
Qualification for sterility by
membrane filtration
Endotoxin (LAL)
Characteristics of final product
Total protein
Osmolality
pH
Moisture (Karl Fischer; freeze
dried products)
Extractable volume
Appearance
Sub-visible particles

12. Viral Safety Assurance – Detect
Limitations with Detection Assays
12
You only find what you are looking for…
▪ Screening assays are mostly designed to assess known/past pathogens
▪ How to detect unknown viruses?
You may not find what is truly there…
▪ Sensitivity of assay limits detection
▪ All assays have a LOD
▪ Sample volume limitation
▪ Cell lines may not be permissive for some known or novel viruses
▪ Interference/matrix effects with the test article
▪ Anti-virus antibodies in FCS used in in vitro assays, cytotoxicity of indicator cells, inhibition of PCR
assay enzymes
That’s why the 3-pronged approach, Detect and including Prevent
and Remove, is so important to assure viral safety of biotherapeutics
“Absence of evidence does not
equal evidence of absence.”

13. 1. Not sure what data is required to satisfy regulators
2. Cost/time in conducting comparability study to justify change in technology
3. Don’t want to be the first to present new technology data to regulators. Will wait until method has
been accepted
4. The past approach to testing has always been accepted. Why bother with a different approach?
5. New methods are not mentioned in any regulatory guidance
What factors in your company are influencing the adoption of new testing
technologies? (select all appropriate answers)
Poll Question 1
13

14. Lot Release
Testing
Alternative
rapid testing
methods

15. 15
Therapeutic mAb producers are looking to accelerate bulk
harvest lot release testing for cost and market access benefits
DSP ~7 days
CMO
• Increase facility utilization
• Faster gene to IND
Large Pharma - mAbs
• Reduce risk into DSP
• Lower inventory cost
Benefit of accelerated biosafety testing
Bulk Harvest Biosafety Testing: 35+ days Timing
mismatch

16. Cell Line Characterization
16
An issue with traditional biosafety testing methods is they
rely on biological amplification which can take several weeks
potential bottleneck for
entry into clinic
increasing pressure on
manufacturing speed
Bulk Harvest Lot Release

17. 17
Testing approach with rapid methods
Characterization of CHO production cell banks
Test MCB WCB CAL
Identity CO1 barcode √ √ √
Purity
Sterility Q+√ √ √
Mycoplasma Q+√ √ √
Mycobacterium Q+√ √ √
Safety
Adventitious agent (in vitro) √ √ √
Adventitious agent (in vivo) √ √
Retroviruses (TEM, infectivity) √ √√
Specific rodent viruses
HAP
MAP
MMV
Other viruses (eg. bovine, porcine, ovine) √ (√)
Genetic
Stability
Sequencing of expressed gene √ √
Restriction Enzyme analysis by Southern blot √ √
Design and qualification of a QPCR assay for transgene copy
number
√ √
Transgene copy number by QPCR √ √
Sequence of 5’ and 3’ regions flanking transgene √ √
Localization of DNA sequences by FISH or SKY-FISH √ √
X
Blazer™ panel
NGS
ddPCR
BacT/ALERT
QPCR
QPCR
QPCR

18. 18
Testing approach rapid methods
Lot Release Testing of Unprocessed Bulk Harves (UBH)
RVLP QPCR
BacT/ALERT
Mycoplasma QPCR
Unprocessed Bulk Harvest
Assay description
Microbiology
Sterility by direct inoculation
Qualification for sterility assay
OR
Bioburden by direct inoculation
Bioburden suitability testing
Mycoplasma
Qualification of mycoplasma
PCR for mycoplasma
Adventitious virus testing
In vitro virus assay for presence of adventitious
viral contaminants
Retroviruses
Transmission electron microscopy
Specific Rodent viruses
MVM

19. 19
Compendial Culture Method
 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.
Sterility Testing
Direct
Inoculation
Membrane
Filtration
Rapid Sterility
BacT/Alert® Detection System
 Samples are automatically monitored for CO2 production and
read every 10 minutes
 Turbidity from debris/cellular material does not interfere
 Non-destructive technology allows for subculture of positive
broths to perform identification.

20. 20
Compendial Culture-based Mycoplasma Test
(USP/EP/JP/PTC)
20

21. European Pharmacopoeia 10, 2.6.7. Mycoplasmas (2008)
“NAT may be used as an alternative to one or both of the other methods after suitable
validation”
US Pharmacopoeia 35, Chapter 63, Mycoplasma Tests (2012)
“A validated NAT may be used to detect Mycoplasma, provided such a method is shown to be
comparable to both methods”
US FDA Guidance for Industry: Characterisation and Qualification of Cell Substrates
and Other Biological Starting Materials used in Production of Viral Vaccines for the
Prevention and Treatment of Infectious Diseases (2010)
“PCR-based assays may be used to detect mycoplasma, provided that such an assay can
be shown to be comparable to the agar and broth procedure and the indicator cell culture
procedure”
November 2012
Biopharmaceutical from Roche approved. Testing used a PCR based mycoplasma assay
2017: Our validated mycoplasma qPCR submitted in product filing which received approval
Regulatory guidance
Mycoplasma Nucleic Acid Test
21

22. 22
RVLP QPCR Assay
• An alternative method to TEM for the
quantification of CHO retroviral particles in
unpurified bulk harvest
• More sensitive, accurate, robust, and rapid
quantification of CHO retroviral particles

23. 23
Biologicals 2000 Sep;28(3):137-48
Retrovirus-like Particle (RVLP) QPCR assay
ICH Q5A
“…it is important to estimate the
amount of virus in the
unprocessed bulk. This estimate
should be obtained using assays
for infectivity or other methods
such as transmission electron
microscopy (TEM).”
de Wit C, Fautz C, Xu Y. Biologicals. 2000 Sep;28(3):137-48.

24. 24
>39 citations of Biologicals 2000 Sep;28(3):137-48
Retrovirus-like Particle (RVLP) QPCR assay
“…Also presented in this session is the feasibility of the use of
retrovirus-like particle (RVLP) in the prevalidation of virus
removal and the use of quantitative polymerase chain reaction
(qPCR) as an alternative to infectivity assays in virus validation
studies as well as its use as an alternative to quantitative TEM
analysis for determining RVLP count in the bulk harvest of a
perfusion bioreactor...”
“…Feedback from the 2017 Viral Clearance Symposium indicates that qRT-PCR
would be an acceptable method of quantitating RVLPs. Furthermore, feedback
from the meeting also indicates that using permeate RVLP levels from a perfusion culture
to calculate RVLP burden could be acceptable for a clinical-phase regulatory filing,
particularly if the qPCR testing was performed as a potential IPC. The accessibility and
the high-throughput nature of the qPCR test method enable this type of strategy to be
implemented for a manufacturing process…”

25. 25
A better alternative to TEM for the quantification of RVLP
Retrovirus-like Particle (RVLP) QPCR assay
Examples of electron micrographs from testing performed at our facility
TEM RVLP QPCR
Historical standard Good correlation with TEM
Highly variable Reproducible
Requires concentration of sample No concentration required
Low sensitivity (1.4e5 VLPs/mL from 20
mL TA)
Highly sensitive (1e3 VLPs/mL from 0.13
mL TA)
Results in months Results in days
Comparison of TEM to RVLP QPCR methods

26. 6. Lot Release
Testing
1. Cell Line
Development
2. Cell Banking &
Storage
3. Cell Line
Characterization
4. Product
Characterization
5. Viral Clearance
Unpurified Bulk
Harvest
Lot
release
testing
Lot
release
testing
Lot
release
testing
26
For the assessment of virus clearance target
Quantification of retroviral particles in CHO UBH
VC
LRF
RVLP
VLP/ml
Volume of UBH per dose
VLP/dose =
X
LRF
VLP/ml
Virus Clearance Target of ≤1 VLP/106 doses (ICH Q5A, Appendix 5)

27. Regulatory Opinion (Apr 2020, Martin Wisher, PhD, Senior Regulatory Consultant)
Supporting the rapid entry into First in Human (FIH)
clinical trials of new medicinal products to treat COVID-19
27

28. 1. No current plans to switch
2. Planning to adopt rapid alternatives for sterility testing
3. Planning to adopt rapid alternatives for mycoplasma
4. Planning to adopt rapid alternatives for in vivo based testing
5. Planning to adopt rapid alternatives for in vitro viral detection
6. Planning to adopt multiple rapid alternatives (A lead)
Are you considering or have you already switched to molecular
methods as an alternative to culture or in vivo based
adventitious agent testing?
Poll Question 2
28

29. Product
Specific
Qualification
Study

30. 30
To support lot release testing
Product Specific Qualification (PSQ) Study
Unprocessed Bulk Harvest
Assay description
Microbiology
Sterility by direct inoculation
Qualification for sterility assay
OR
Bioburden by direct inoculation
Bioburden suitability testing
Mycoplasma
Qualification of mycoplasma
PCR for mycoplasma
Adventitious virus testing
In vitro virus assay for presence of adventitious
viral contaminants
Retroviruses
Transmission electron microscopy
Specific Rodent viruses
MVM

31. 31
To support lot release testing
Product Specific Qualification (PSQ) Study
What? Why? When? How?
PSQ Reports
to support
subsequent
LRT

32. 32
To support lot release testing
Product Specific Qualification (PSQ) Study
What is PSQ study?
• A study which takes representative batches of a defined production process to
establish the level of interfering effects, if any, of the test article (TA) matrix on a
generically validated assay.
• For a proper PSQ study, TA from a minimum of three batches should be tested for a
consistency assessment between batches of potential interfering effects to the assay
used for lot release testing.
• One-time qualification of the TA for subsequent lot release testing of a drug product and
its intermediates as long as the production process remains unchanged.

33. 33
To support lot release testing
Product Specific Qualification (PSQ) Study
Why perform PSQ study?
• Examine and quantify the effect of a TA matrix on the performance of an assay –
without this we can only ‘hope’ the TA has no effect on the assay which will not
be acceptable by the regulators as the test result would not be valid.
• Regulatory requirements for testing of batches of drug in GMP assays for
consistency assessment.
• PSQ study provide an opportunity to address any assay interference due to the
TA by modifying the testing conditions where possible (e.g. Dilution of TA) for a
valid lot release test result.

34. 34
To support lot release testing
Product Specific Qualification (PSQ) Study
When to perform PSQ study?
• Typically performed before or during Phase III clinical development once the
biomanufacturing process is fixed.
• PSQ certificates of analysis are required as part of the regulatory submission
process for a drug license.
• PSQ reports that summarized the results from 3 batches should be in place before
regulatory approval is sought, or a post-approval undertaking will be requested by a
regulator.

35. 35
To support lot release testing
Product Specific Qualification (PSQ) Study
How is PSQ performed?
• PSQ will be performed where TA are typically spiked at the system
suitability control level, and at one and two orders of magnitude lower, in
order to reach the detection limit.
• Release of batch may also be achieved here where batch is tested without
virus spikes, but not necessarily, and can depend on the particular assay.

36. 36
Conclusion
▪ Continued advancement of CHO bioproduction
platform has called for better CMC testing
methods to help realize its full potential and
productivity
▪ Innovation in biosafety testing and product
characterization will contribute towards
bringing safe and effective biotherapeutics to
patients sooner
▪ We are committed to working closely with
biopharma and to provide state of the art
solutions for bioprocessing

37. © 2021 Merck KGaA, Darmstadt, Germany and/or its affiliates. All Rights Reserved.
The vibrant M, and BioReliance are trademarks of Merck KGaA, Darmstadt, Germany or its affiliates. All
other trademarks are the property of their respective owners. Detailed information on trademarks is
available via publicly accessible resources.
Senior Technical Specialist
Field Technology Management, Asia Pacific
edmund.ang@emdgroup.com
+65 81331794
Edmund Ang, PhD
Thank You!