LakePharma’s CHO-GSN Platform for Stable Cell Line Generation

1. LakePharma’s CHO-GSN Platform for
Stable Cell Line Generation
June 14, 2016
Hua Tu, Ph.D.
hua.tu@lakepharma.com

2. Development Goal
Objectives
• CHO line with documentation and FTO to support
commercial manufacturing
• High yield  3 gram per liter for antibody
• Fast timeline
• Consistency in performance
Approach
• GS selection on a CHO GS knockout line
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3. History of CHO-GSN Host Cells
Parental CHO K1 stock cells
obtained by LP partner
Knock-out of GS allele 1 by LP
partner (CHO GS -/+)
Knock-out of GS allele 2 by LP
partner (CHO GS -/-)
Suspension growth and adaptation
Characterization of CHO GS-/- and
banking of CHO GS -/-
• Cells were analyzed using Infectious Microbe PCR AmplifiCation
Test (IMPACT) test panels I and III that aim to detect a wide range
of human or other pathogens
• Results were negative for all infectious agents
• Via rAAV technology, both alleles of the catalytic domain of the
Glutamine Synthetase (GS) enzyme were deleted
• Removal of the GS gene was confirmed both genetically and
phenotypically
• Validation by reversion test when a functional gene copy of GS
was restored by transfection
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4. Stable Cell Line Generation Process
Target
DNA
GS Stable
Vector
Transfect
CHO
(MaxCyte)
GS Selection
(MSX)
CHO Stable
Pool
Subclones
CHO Stable
Clones
RCB
1 ~ 2 weeks 5 weeks
10 weeks
Production
Run
(+3 weeks)
10 weeks
Production
Run
(+2 weeks)
Production
Run
(+2 weeks)
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5. Case Study 1: CHO-GSN Pool and Clones of An Antibody
• HEK293 transient titer is 0.24 g/L
• CHO-GSN pool titer is 1.3 g/L
• Best clone titer is 4.5 g/L
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mAb Titer Profile

6. Case Study 2: CHO-GSN vs. CHO-K1 of Fc-fusion
• GSN is the LP adapted suspension and
serum free CHO GS -/-
• CHO-K1 is the parental cell line (no
knock-out, suspension)
• There is significant increase in titer
when using the knockout line and even
more increase when single cell cloning
is performed
Day
CHO-K1
pool
titer
0 1.35
3 2.49
5 4.5
7 7
10 9.8
12 13.8
14 15.7
17 20.4
5
Fc-fusion Titer

7. Case Study 3: CHO-GSN Producing A Bispecific Antibody
• A bispecific antibody does not contain Fc
• Titer quantification by Octet sensors
• Product related variants presented
challenges in single cell cloning
• Through method development and custom
screening approach, LakePharma was
successful in generating a cell line expressing
the bispecific antibody
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Bispecific A Titer
4C2 3A6

8. Stable CHO-GSN Pools Have Higher Titer Than Transient
0
200
400
600
800
1000
1200
1400
1600
0 50 100 150 200 250 300
CHO-GSNPoolYield(mg/L)
Transient Yield (mg/L)
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9. Case Study 4: Stability of Cell Line Producing an Antibody
PDL Antibody Yield (mg/L)
10 892
28 960
60 920
90 1007
@ PDL 28
@ PDL 90
1
min0 2.5 5 7.5 10 12.5 15 17.5 20 22.5
mAU
0
20
40
60
80
2
min8 10 12 14 16 18
2mAU
0
100
200
300
400
1
CE-SDS SE-HPLC
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10. Case Study 5: Antibody Productivity Correlates with
Gene Copy Numbers of Both Heavy and Light Chains
• Copy Number is Determined by qPCR Method
Standard Curve with Reference Gene
Slope: -3.325 R2: 0.999
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11. Highlights of LakePharma’s CHO-GSN Platform
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• Complete cell line lineage and documentation; freedom to operate
• In-house proprietary cell lines and vectors
• MaxCyte electroporation
• High yield can be obtained at stable pool stage
• Reduce the need to screen large number of clones for high yield
• Start to finish RCB in 6 months
• Proven success with difficult proteins

12. Additional Services Related to CHO-GSN Platform
Release
Testing
Sterility,
mycoplasma, copy
number, and viral
testing
Stability
Studies
Stability over 80
generations
Scale-up
Productions
Large scale
bioreactor capacity
Bioreactor
Processing
Development
Process
optimization, scale
down and scale up
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