In this webinar, you will learn about: - Risk assessment approaches in upstream process development - How early cell line development stage is linked to subsequent steps in the bioprocess to assure the quality of the final product - Benefits of having a completely chemically defined cell line development process Detailed description: Chinese Hamster Ovary (CHO) cells are the preferred host for producing biotherapeutics where cell line development (CLD) is the foundation of the bioprocess. CLD processes are expected to be robust while meeting a myriad of regulatory requirements. The choice of production cell line, culture conditions, and having a chemically defined (CD) CLD process by using CD cloning media can impact the subsequent measures for the CMC (Chemistry, manufacturing, and controls). In this presentation, we will discuss these choices and their impacts on subsequent bioprocess and CMC testing required by regulations and the benefits of incorporating CD cloning media into the CHOZN® expression platform.

1. The life science business of Merck KGaA,
Darmstadt, Germany operates as
MilliporeSigma in the U.S. and Canada.
Risk Mitigation in Cell Line
Development : Regulatory
Considerations and Impact on Quality
Assurance
Chemically Defined Cloning Media for Cell Line Development Process
Janice Tan, Ph.D.
7 Dec 2021

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

3. Agenda
1
2
3
Importance of a
chemically-defined process
Performance of Cellvento® 4CHO-C
cloning medium
Stability of Cellvento® 4CHO-C
cloning medium

4. Importance of Chemically
defined process

5. Risk Assessment and Mitigation in BioProcessing
Process economics
High titer
Exclusion of unnecessary additives
Concentrated feeds
High growth rate and cell densities
Culture longevity
Reduce product and process impurities
Business continuity
Regulatory and IP compliance
Reliable supply chain
Quality risk management approach
Reduce regulatory and risk
considerations
Reduced analytical workload
Product stability through harvest
Clone stability
Product homogeneity
Speed to market
Fast cell line development process
Screen molecules earlier in discovery
process
Maintain/enable favorable product quality
attributes
Scalable process

6. Clone Isolation is an Essential Part of the Risk Control Strategy
Process economics
High titer
Exclusion of unnecessary additives
Concentrated feeds
High growth rate and cell densities
Culture longevity
Reduce product and process impurities
Business continuity
Regulatory and IP compliance
Reliable supply chain
Quality risk management approach
Reduce regulatory and risk
considerations
Reduced analytical workload
Product stability through harvest
Clone stability
Product homogeneity
Speed to market
Fast cell line development process
Screen molecules earlier in discovery
process
Maintain/enable favorable product quality
attributes
Scalable process

7. Cell Line Development is the Foundation for Successful
Bioprocessing
Cell line
development
Harvest
Clarification
Chromatography
Virus
removal
Tangential flow
filtration
Sterile
filtration
Formulation and
final fill
Media and process
development
Feed
optimization
Rapid
scale-up
Robust cell line
development

8. Cloning Medium Enables Cell Survival of Selection and
Cloning Processes in Cell Line Development
Scale-up
and screen
4-5 weeks
CHOZN® GS-/-
mAb
Expression
Vector
Minipool
selection
4-5 weeks
Scale-up
and screen
4-5 weeks
Limiting Dilution
Cloning
4-5 weeks
Metabolic selection of cells
plated at a low density
(i.e., 5,000 cells per well)
Isolation of single cells
to generate a clonally-
derived population for
manufacturing
Specially formulated
media needed
Specially formulated
media needed

9. Clonality is an Essential Part of the Risk Control Strategy
 “Critically, though the non-clonally derived bank
may result in the same CQA mean value at the
time of harvest as the clonally derived bank, its
divergent starting point during cloning could
potentially result in great variability of one of
more CQA’s, and potentially higher susceptibility
to drift, shift, and unforeseen selective
pressures during manufacturing lifetime,
depicted by the manufacturing change shown
as the blue arrow”
Clonality reduces risk of protein quality variability
• Many current cloning methods involve un-defined media contain serum, hydrolysates
and other additives
• Cloning media that is not chemically defined introduces regulatory risk
Welch JT, Arden NS. Considering "clonality": A regulatory perspective on the importance of the clonal derivation of mammalian cell banks in biopharmaceutical development. Biologicals. 2019
Nov;62:16-21. doi: 10.1016/j.biologicals.2019.09.006. Epub 2019 Oct 3. PMID: 31588011.

10. A Cloning Medium is Essential for Single-Cell Cloning
In the face of these challenges, a good cloning medium is critical to ensure high clone
recovery during cell line development.
Cellvento® 4CHO-C Cloning Medium replaces hydrolysate containing, non
chemically defined Ex-Cell® CHO Cloning medium.
A specially formulated cloning media is required to support cell growth and viability during
stressful, low-density steps during cell line development
Single-cell cloning
• Single cells are isolated from stable pools
• Not all clones will survive and outgrow
• Many colonies will need to be eliminated if clonality cannot be demonstrated via imaging
Individual cells
are isolated
heterogeneous
minipools
Many cells are
unable to
survive these
conditions
More will be
eliminated if
clonality cannot
be demonstrated

11. 3 Benefits of a Fully Chemically-Defined Process
1 Fast cell line development
High rates of clone and minipool recovery
2
3
Reduced regulatory risk
Reduced risk of protein quality variability
Process robustness
Consistent performing chemically defined medium

12. Performance of Cellvento®
4CHO-C cloning medium

13. Cellvento® 4CHO-C Cloning Medium
Robust Clone Outgrowth Across Cell Lines
0
0.2
0.4
0.6
0.8
1
1.2
1.4
Minipools CHOZN® GS-/- CHO-K1
Wells
with
Outgrowth
(normalized
to
control)
EX-CELL® CHO Cloning Medium Cellvento® 4CHO-C Cloning Medium
Cells were plated at 0.5 cells per well and plates were imaged on day 7 to quantify outgrowth

14. Cellvento® 4CHO-C Cloning Medium
Robust Clone Outgrowth is Reproduceable
0
0.2
0.4
0.6
0.8
1
1.2
1.4
Clone 1 Clone 2 Clone 3 Clone 4
Wells
with
Outgrowth
(normalized
to
control)
EX-CELL® CHO Cloning Medium Cellvento® 4CHO-C Cloning Medium
Different clones were plated at 0.5 cells per well and plates were imaged on day 7 to quantify outgrowth

15. Cellvento® 4CHO-C Cloning Medium
Conditioned Medium Supports Clone Outgrowth
Inoculate from a high-
viability culture in mid-
logarithmic growth
phase at 1E6 viable
cells/mL
1 Incubate for 24 hours
and sample to ensure
high viability and
appropriate doubling
time
2 Centrifuge to pellet cells
and cell debris
3 Transfer supernatant to a
fresh tube, discarding
the cell pellet
4 Supplement the cloning
medium with conditioned
medium at 20% of the
total plating volume
Store at 4°C for up to 1
week
5
Conditioned medium is thought to contain beneficial growth factors and other components
secreted by healthy cell cultures that help cells survive low-density conditions

16. Conditioned Medium Supports Clone Outgrowth
Cellvento® 4CHO-C Cloning Medium
0
0.2
0.4
0.6
0.8
1
1.2
1.4
EX-CELL®
CHO Cloning
Medium
EX-CELL®
CHO Cloning
Medium +
Conditioned
Medium
Cellvento®
4CHO-C
Cloning
Medium
Cellvento®
4CHO-C
Cloning
Medium +
Conditioned
Medium
Wells
with
Outgrowth
(normalized
to
control)
Normalized Outgrowth
(Minipools)
0
0.2
0.4
0.6
0.8
1
1.2
1.4
EX-CELL®
CHO Cloning
Medium
EX-CELL®
CHO Cloning
Medium +
Conditioned
Medium
Cellvento®
4CHO-C
Cloning
Medium
Cellvento®
4CHO-C
Cloning
Medium +
Conditioned
Medium
Wells
with
Outgrowth
(normalized
to
control)
Normalized Outgrowth
(CHOZN® GS-/-)

17. Cellvento® 4CHO-C Cloning Medium
Robust Clone Outgrowth Relative to Industry Benchmarks
Cells were plated at 0.5 cells per well and plates were imaged on day 7 to quantify outgrowth
0.00
0.20
0.40
0.60
0.80
1.00
1.20
1.40
EX-CELL® CHO
Cloning Medium +
Conditioned Medium
Cellvento® 4CHO-C
Cloning Medium +
Conditioned Medium
Ham F12 - 100% 90% Ham F12 + 10%
FBS
Wells
with
Outgrowth
(normalized
to
control

18. Cellvento® 4CHO-C Cloning Medium
Cloning Medium Supports Minipool Generation
A specially formulated cloning media is required to support cell growth and viability during
stressful, low-density steps during cell line development
Minipool selection
• Creates heterogeneous populations and enhances the probability of finding high-producing, stable
clones
• 1-2 days after transfection, cells are plated at a low density in the presence of a selection pressure
Plated at 5,000
cells per well in
selection medium
1-2 days post
transfection
Cells without
expression of the
selection marker die
~7 days post-plating
Low cell density
Surviving cells
proliferate and can be
screened for productivity
2-3 weeks post-plating

19. Cellvento® 4CHO-C Cloning Medium
Robust Minipool Recovery
0
20
40
60
80
100
120
140
160
180
Titer
(mg/L)
Static Assay of Top 80 Minipools
EX-CELL® CHO Cloning Medium Cellvento® 4CHO-C Cloning Medium

20. Stability of Cellvento®
4CHO-C cloning medium

21. Cellvento® 4CHO-C Cloning Medium
Stable performance for up to 1 year
0.0
0.2
0.4
0.6
0.8
1.0
1.2
1.4
Wells
with
Outgrowth
(normalized
to
control)
Cellvento® 4CHO-C Cloning Medium (Newly Prepared)
Cellvento® 4CHO-C Cloning Medium (Aged 6 mo.)
Cellvento® 4CHO-C Cloning Medium (Aged 12 mo.)
EX-CELL® CHO Cloning Medium
Storage at
2-8°C

22. Cellvento® 4CHO-C Cloning Medium
Stable pH for up to 8 weeks after opening
7
7.2
7.4
7.6
7.8
8
0 7 14 21 28 35 42 49 56
pH
Days
Cellvento® 4CHO-C Cloning Medium EX-CELL® CHO Cloning Medium
pH development of products stored at 2-8°C

23. Cellvento® 4CHO-C Cloning
Medium
✓ Supports robust minipool and
clone outgrowth
✓ Comparable to EX-CELL® CHO
Cloning Medium
✓ Superior consistency between lots
✓ Ease of use by improved pH
stability
A Chemically-Defined
Cloning Medium
Allows a Fully
Defined Process

24. Amber N. Petersen, Ph.D.
R&D Manager
Biopharmaceutical Expressions
System Team
Head of Upstream MSAT
Asia Pacific excluding China
Contributor
Janice Tan, Ph.D.
The vibrant M, Cellvento, CHOZN, EX-CELL 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.
© 2021 Merck KGaA, Darmstadt, Germany and/or its affiliates. All Rights Reserved.

25. The life science business of Merck KGaA,
Darmstadt, Germany operates as MilliporeSigma
in the U.S. and Canada.
Risk Mitigation in Cell Line
Development:
Regulatory Considerations and
Impact on Quality Assurance
Chemically Defined Cloning Media for Cell Line
Development Process
Edmund Ang, PhD
Senior Technical Expert
Field Technology Management, Asia Pacific

26. Towards a chemically-defined process
Impacts on CMC testing and product quality

27. 3
3 main concerns with the use of
animal production cells:
1. Presence of adventitious viruses
2. Oncogenic host cell DNA
3. Genetic stability

28. 4
How to best assure production cell homogeneity
and transgene integrity?
Single
cell
clone
RCB MCB WCB EPCB
Use of
cloning media
Use of culture media
There is regulatory expectation to assure integrity of the transgene
and its expressed biotherapeutic product with CMC testing
throughout the bioprocess

29. 5
Testing approach to meet regulatory expectation
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 √√ √
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 √ √
Required tests based on
risk assessment of the
bioprocess, in particular
to take into consideration
the species origin and
culture history of the
production cells, including
exposure to raw/starting
materials.

30. Clonality vs product quality
6
WHO TRS 978 Annex 3
…In the process of cloning a cell culture, single cells should be
selected for expansion. The cloning procedure should be carefully
documented, including the provenance of the original culture, the
cloning protocol and reagents used. Cloning by one round of
limiting dilution will not necessarily guarantee derivation from
single cells; additional subcloning steps should be performed.
Alternatively, or in addition to limiting dilution steps, the cloning
procedure can include more recent technology such as single-cell
sorting and arraying or colony-picking from dilute seeds into semi-
solid media. In any case, the cloning procedure should be fully
documented, with details of imaging techniques and/or
appropriate statistics…
…It is important to bear in mind that, even following single-cell
cloning, epigenetic variation may result in a cloned culture
showing evidence of heterogeneity (i.e. more than one clone). This
should not preclude the use of such a culture for production unless
there are indications of instability that could affect the quality
and/or safety of the final product.

31. Cell culture consistency vs product quality
7

32. 8
Cell culture consistency vs product quality
► The process of subcloning itself does
not contribute to an improved
karyotypic homogeneity of a population
► The selection for a specific cell property
inherently can provide evolutionary
pressure that may lead to improved
chromosomal stability as well as to a
more homogenous population.
Process consistency is key to improved production cell homogeneity

33. 9
Best data to be obtained at single cell clone establishment stage
FAQ Confirmation of clonality
Confirmation of clonality?
Best data to be obtained at single cell clone establishment stage…where sequential
images can be obtained to show that a cell colony did indeed arise from a single cell.
How can we determine clonality once the MCB has been generated?
❑ No easy way!
❑ FISH can be used for comparative analysis of the chromosomal integration site(s)
❑ Interpretation of results not straightforward due to instability of CHO genome
❑ SKY-FISH could be better
❑ NGS is not yet suitable for analysis of CHO genomes (no suitable genetic stability
method at present)
❑ Generate & analysis of sub-clones with genetic stability methods

34. 10
Genetic stability study
SKY-FISH for CHO
12XCHamster (MetaSystems Probes)
https://metasystems-probes.com/en/probes/mfish/d-1526-060-di
► Cells grown to
mid-log phase
► Arrest mitotic
cells in
metaphase with
Colcemid
► Fixed
metaphase cell
nuclei
Sample
Prep
•Experimental probe
specific for transgene
•Positive control probe is
genomic DNA of the
same species
•A single copy standard
probe (GS)
FISH
12 painting
probes specific
for the 12
different CHO
chromosomes
SKY

35. 11
SKY-FISH for CHO
► Help obtain data to support the
integrity of the transgene and
clonality of the production cells
► A powerful method to help track
chromosomal translocation(s)
that might have taken place at
single cell level

36. 12
Testing approach to meet regulatory expectation
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 √√ √
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 √ √
May reduce testing for
adventitious agents due to
animal-derived material
Better results may be obtained
to support integrity of
transgene and homogeneity of
production cells
Use of CD media
CD process

37. 13
Conclusion
The use of chemically-defined process is
expected to contribute towards better
consistency of production cell culture
performance and product quality.

38. Senior Technical Expert
Field Technology Management, Asia Pacific
+65 81331794
Edmund Ang, PhD
Thank You!
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.
© 2021 Merck KGaA, Darmstadt, Germany and/or its affiliates. All Rights Reserved.