CHO cells with their unique characteristics, represent the major expression system within the biopharmaceutical industry. However, one of the major challenges in cell line development is to identify those rare, high-producing clones in a huge population of non-expressing or low-expressing cell lines. This leads to laborious and time consuming cell line development processes. This webinar will educate the audience about challenges faced with traditional expression systems and how the CHO cell line with the glutamine synthethase knock-out via Zinc Finger Nucleases provides benefits for fast and efficient cell line development as well as stable and high titer expression. We will explore additional cell line engineering targets that can be modified to engineer a cell line that mitigates risks and removes bottlenecks throughout the biopharmaceutical process. In this webinar, you will learn: • What are the benefits of using an optimized/engineered expression system? • What can be done throughout the cell line development process to mitigate risks and remove bottlenecks? • Applications of cell line engineering for further upstream biopharmaceutical enhancements.

1. Merck KGaA
Darmstadt, Germany
Reducing risk and decreasing timelines
through optimized reagents and processes
Streamlining
Biopharmaceutical
Cell Line Development
Kate Achtien, Sr. Scientist
Webinar
30th November 2017

2. 2
The life science business of
Merck KGaA, Darmstadt, Germany
operates as MilliporeSigma
in the U.S. and Canada.
Streamlining Biopharmaceutical Cell Line Development | Webinar

3. Webinar
Goals
Streamlining Biopharmaceutical
Cell Line Development
• Finding the needle in the haystack
What are the goals and challenges of cell
line development?
• Foundation blocks
Defining the components of a robust cell
line development platform
• Striking a balance
Optimizing the cell line development
process
• The future of cell line development
processes
Advancing through genetic engineering
Streamlining Biopharmaceutical Cell Line Development | Webinar3

4. Finding the
needle in the
haystack
The Goals and Challenges
of Cell Line Development
Streamlining Biopharmaceutical Cell Line Development | Webinar4

5. Streamlining Biopharmaceutical Cell Line Development | Webinar
Poll Question
5

6. Streamlining Biopharmaceutical Cell Line Development | Webinar
What makes a robust biotherapeutic production process?
Cell culture
Harvest
Clarification
Chromatography
Virus
removal
Tangential flow
filtration
Sterile
filtration
Formulation and
Final Fill
Cell line
development
Process EconomicsSpeed to Clinic Business Continuity
6

7.  Fast development process
 Low clone screening efforts
 Screen molecules earlier in discovery process
 Maintain/enable favorable PQ attributes
 Scalable process
 Keep development costs low
 High titers and productivity
 Product homogeneity
 Culture longevity
 Clone Stability
 Exclusion of unnecessary additives
 Increased process robustness
 Concentrated feeds
 High growth rate and cell densities
 Reduce product and process impurities
 Control/reduce metabolic waste
 Clear technology IP path
 Regulatory compliance
 Reliable supply chain for paired media
 Reduce regulatory and risk considerations
Streamlining Biopharmaceutical Cell Line Development | Webinar
A robust cell line development process leads to strong benefits for
development & production
Process EconomicsSpeed to Clinic Business Continuity
7

8. Expression
Vector
Gene of
Interest
Transfection
Cell line
Cloning
The cell line development challenge
Finding the needle in the haystack
Need for robust
processes to Reduce
Time, Resources and
Cost
Streamlining Biopharmaceutical Cell Line Development | Webinar8

9. Streamlining Biopharmaceutical Cell Line Development | Webinar
Poll Question
9

10. Traditional methods
Wild Type CHO
Glutamate + Ammonia
Glutamine
Biological Function
MSX Glutamine
SynthetaseMethionine
sulfoximine
Folic acid
Dihydrofolic acid
Tetrahydrofolic acid
Dihydrofolate
Reductase
Dihydrofolate
Reductase
Purine Metabolism
X
X
X
DG44 or DUXB11
(CHO)
MTX
MTX
Wild Type CHO
Neomycin
Puromycin
Selection achieved through addition of a selection agent
Dihydrofolate Reductase
based selection
Glutmine Synthetase
based selection
Antibiotic
based selection methods
Streamlining Biopharmaceutical Cell Line Development | Webinar10

11. Weaknesses of additive-based selection methods
Technical, economic and ecological challenges
Requirement for amplification
 Significant clone screening efforts
 Increase of gene copy and risk for clone instability
 Time consuming process
Application of antibiotics
 Regulatory review less smooth - clearance to be
proven
 Disposal of hazardous drug for environmental
compliance
 Absence of antibiotic-based selection pressure in
the production process increases the risks for
reduced productivity
 Longer timelines
 Increased resources needs
 Increased development costs
 Higher risk for loss of productivity
Streamlining Biopharmaceutical Cell Line Development | Webinar11

12. Additive independent selection
Wild Type CHO
Glutamate + Ammonia
Glutamine
Biological Function
MSX Glutamine
SynthetaseMethionine
sulfoximine
Glutmine Synthetase
based selection
GS knock out cell line allows additive independent selection
CHO GS-/-
Glutamate + Ammonia
Glutamine
Biological Function
Glutamine
Synthetase
Glutamine Synthetase
KO based selection
X
Streamlining Biopharmaceutical Cell Line Development | Webinar12

13. Benefits of a glutamine synthetase auxotroph cell line
GS-/ provides extensive benefits for development & production
processes
Fewer clones need to be
evaluated
No amplification needed
Drug selection not required
Stringent selection
Streamlining Biopharmaceutical Cell Line Development | Webinar
Shortened timelines
Reduced cost and lowered regulatory hurdles
Increased clone stability
Lower resources required
✓
✓
✓
✓
13

14. Building a
Foundation
Defining the components of a
robust cell line development
platform
Streamlining Biopharmaceutical Cell Line Development | Webinar14

15.  Fast development process
 Low clone screening efforts
 Screen molecules earlier in discovery process
 Maintain/enable favorable PQ attributes
 Scalable process
 Keep development costs low
 High titers and productivity
 Product homogeneity
 Culture longevity
 Clone Stability
 Exclusion of unnecessary additives
 Increased process robustness
 Concentrated feeds
 High growth rate and cell densities
 Reduce product and process impurities
 Control/reduce metabolic waste
 Clear technology IP path
 Regulatory compliance
 Reliable supply chain for paired media
 Reduce regulatory and risk considerations
A robust cell line development process
leads to strong benefits for development & production
Process EconomicsSpeed to Clinic Business Continuity
Streamlining Biopharmaceutical Cell Line Development | Webinar15

16. Building a strong foundation for an efficient platform process
 Fast development process
 Low clone screening efforts
 Screen molecules earlier in discovery process
 Maintain/enable favorable PQ attributes
 Scalable process
 Keep development costs low
 High titers and productivity
 Product homogeneity
 Culture longevity
 Clone Stability
 Exclusion of unnecessary additives
 Increased process robustness
 Concentrated feeds
 High growth rate and cell densities
 Reduce product and process impurities
 Control/reduce metabolic waste
 Clear technology IP path
 Regulatory compliance
 Reliable supply chain for paired media
 Reduce regulatory and risk considerations
Cell line design parameters
Media design parameters
Streamlining Biopharmaceutical Cell Line Development | Webinar
Process EconomicsSpeed to Clinic Business Continuity
16

17. Streamlining Biopharmaceutical Cell Line Development | Webinar
Step 1
Develop robust host
Supension adapted
• Adapted to fast and
high suspension
growth
Strong origin
 Cells originated from
ECACC CHO K1 Ease of use
 Optimized for cell line
development, scale up
and production
processes
Regulatory friendly
• Grown in chemically
defined media
• Animal component
free
• cGMP banked
• Full viral testing,
complete traceability
2
1
43
17

18. Technical Benefits
• Introduces site specific double strand
breaks in the DNA
• High specificity
• Limited off target effects
Regulatory Benefits
• Clear IP path
• Regulatory acceptance
• Non-viral based technology
Targeted
Mutagenesis
Step 2
Genetic engineering of host through regulatory friendly means
Zinc Finger Nucleases
Genetic engineering using Zinc Finger Nucleases (ZFNs)

19. Development of a paired media for efficient production processes
Step 3
Business Continuity
• Robust and sustainable supply
chain
• Proven manufacturability and
consistency
• Multisite manufacturing
redundancy
• Scalable GMP ready products
• Animal component free
Streamlining Biopharmaceutical Cell Line Development | Webinar19

20. Comprehensive expression platform & services
To accelerate your development
CHOZN® GS-/- cell line
GS auxotroph cell line with
clear IP path
Traceability
Documentation
Comprehensive cell line history
documentation to support
regulatory filing
Expression Vector
IP free GS expression vector
suitable for mAbs/ recombinants
Process Guidance &
Protocols
Protocols for entire workflow
Media and Feeds
Fed Batch & Perfusion media
produced under GMP
Technical Support
Comprehensive product and
process technical support to
ensure your success
Streamlining Biopharmaceutical Cell Line Development | Webinar
CHOZN®
GS-/-
Expression
Platform
20

21. Striking a balance:
Titer and Speed
Developing a Robust Cell Line
Streamlining Biopharmaceutical Cell Line Development | Webinar21

22. Striking a balance
Development requires the right balance of clone performance and
resources
Speed
Resources
Equipment costs
Throughput
Clone performance
Streamlining Biopharmaceutical Cell Line Development | Webinar22

23. Time investment to develop a stable pool reduces resources needed
in single cell cloning stage
Titer
Heterogeneity
Early screens for protein
quality
Proving “clonality”
High throughput early
screening
High titers
Scalable process
Protein quality attributes
Streamlining Biopharmaceutical Cell Line Development | Webinar
Stable Pool
Development
Single Cell Clone
Isolation
Time intense Resource intense
23

24. Optimized cell line development process
Balancing Resources and Performance
Transfect Selection
via “minipools”
Screen
minipools in
96-well plates
Scale-up
top producing
minipools
Screen
7-day
TPP/shake flask
Fed-batch
assay on top
minipools
(TPP/shake)
Single Cell
Cloning
200 pools 100 pools 100 pools 20 pools
3-4 weeks 1 week 2 weeks 1 week 2 weeks 10-12 weeks
Streamlining Biopharmaceutical Cell Line Development | Webinar24

25. CHOZN® GS-/- platform performance
High expressing stable pools and clones
0
1
2
3
4
5
Stable Pools - Fed Batch Clones - Fed Batch
2 – 2.5 g/L
4 – 4.5 g/L CHOZN® Platform
Benefits
• High expressing pools can be
quickly identified for early
protein evaluation
• Clones isolated typically produce
at least twice the titer of the pool
• Clones commonly produce 4-
5g/L prior to process
development
Titer Range of Top IgG Expressing
GS-/- Pools and Clones
mAbs (IgG1 and 4, IgM), Fc Fusions, Fabs,
r-proteins, Bi-specifics
Streamlining Biopharmaceutical Cell Line Development | Webinar25

26. CHOZN® GS-/- platform performance
Performance of 4 CHOZN® GS-/- clones isolated from mini-pools
0
500
1000
1500
2000
2500
3000
3500
4000
4500
Productivity,Peak(mg/L)
Peak Productivity
0
20
40
60
80
100
120
CellSpecificProductivity
pg/Cell/Day
Cell Specific Productivity (Qp)
Streamlining Biopharmaceutical Cell Line Development | Webinar26

27. CHOZN® GS-/- platform stability
Confidence in consistent production
75%75%
High stability
Studies performed on
top ten producing
clones show above
industry standard
stability.
80-90% of CHOZN®
clones are found to
have stable
productivity.
Streamlining Biopharmaceutical Cell Line Development | Webinar27

28. Streamlining Biopharmaceutical Cell Line Development | Webinar
Process Scalability
Consistent performance between vessel sizes
0
5
10
15
20
25
- 2 4 6 8 10 12 14 16 18 20
1e6Cells/ml
Days
Viable Cell Density
0
500
1000
1500
2000
2500
- 2 4 6 8 10 12 14 16 18 20
mg/L
Days
Titer
Shake
Flask
EX-CELL® Advanced CHO Fed Batch
No process development or
optimization performed
Mobius® 50L
Bioreactor
Mobius® 3L Single Use
bioreactor
28

29. Streamlining Biopharmaceutical Cell Line Development | Webinar
Process Predictability
Glycan profiles similar across multiple scales
Shake
Flask
Mobius® 3L Single Use
bioreactor
Mobius® 50L
Bioreactor
29

30. The Future of Cell
Line Development
Processes
Advancing Cell Line Development
Through Genetic Engineering
Streamlining Biopharmaceutical Cell Line Development | Webinar30

31. Cell Line Engineering by application of gene editing technologies
Targeted Integration
Reduce CLD timelines; increase consistency and stability
Glycoengineering
Removal of immunogenic and half-life reducing glycoforms
Expression Platform
Enhanced selection process, isolate high producing stable clones
Manufacturing Security
Reduce risk of viral contamination
Robust Cell Line
Suspension adapted to chemically defined medium
CHO K1
CHOZN® GS-/-
Protein
Quality
Mitigate
Risk
TI
Streamlining Biopharmaceutical Cell Line Development | Webinar31

32. Streamlining Biopharmaceutical Cell Line Development | Webinar
Poll Question
32

33. Targeted Integration
Fast, Easy, Stable
Reduced variability/enhanced cell line performance
 Clone to clone consistency
 Decreased characterization
Decreased cell line development timelines
 More homogeneous stable pools
 More top clones in your pools
Increased pool and clonal stability
 Use of well characterized safe harbor sites
 Remove stability from the critical path
Aron M. Geurts, and Carol Moreno, Clin. Sci.
2010:119:303-311
GOI
GOI
Streamlining Biopharmaceutical Cell Line Development | Webinar33

34. Targeted Integration
Ongoing Research
32 Increased Efficiency
1 Feasibility Hot Spot Identification
 Short RFLP donors
 GFP reporters
 IgG and Fc r-proteins
 Decreasing NHEJ
 Increasing HR
 Tagged ZFNs
 Optimized donor designs
 TI landing pads
 Transcriptomic analysis of
constitutively expressed genes
 Reverse engineering of high
expressing clones
 Random integration of a
landing pad followed by
characterization
Streamlining Biopharmaceutical Cell Line Development | Webinar34

35. Cell culture
Harvest
Clarification
Chromatography
Virus
removal
Tangential flow
filtration
Sterile
filtration
Formulation and
Final Fill
Cell line
development
Process EconomicsSpeed to Clinic Business Continuity
Streamlining Biopharmaceutical Cell Line Development | Webinar
A robust cell line development process leads to strong benefits for
development & production
Finding the needle in the haystack
The goals of cell line development
Foundation blocks:
Designing a robust platform
Striking a balance
Optimizing the cell line development process
The future of cell line development processes
Advancing through genetic engineering
35

36. Questions
Streamlining Biopharmaceutical Cell Line Development | Webinar36

37. Kate Achtien
Senior R+D Scientist
2909 Laclede Ave
St. Louis, MO 63103
USA
Phone: +1 314 289 8496 ext. 1939
Email: Kate.Achtien@sial.com
CONtact:
The vibrant M, CHOZN, Mobius, SAFC, MilliporeSigma and 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.
© 2017 Merck KGaA, Darmstadt, Germany and/or its affiliates. All Rights Reserved.