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confidentialconfidential
Reducing the Timeline to Produce a MonoclonalCell Line Expressing a Clinical Candidate Bi-specificAntibody
Camilla Wang Scientist, SystImmune Inc.
Cell Line Development & Engineering SummitSan Francisco, June 2016
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Outline
• SystImmune introduction
• Overview of our cell line development process
• What are the challenges in cell line development
• Cell Metric™ CLD system for confirming monoclonalility
• How to combine image processing and tracking system for clone picking
• Summary
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SystImmune introduction
• Founded in 2014
• Located near Seattle, WA, USA
• Subsidiary of Biokin Pharma headquartered in Chengdu (China)
• 20+ scientific staff
• Discover antibodies through own antibody discovery platform
• Develop multi-specific antibodies with focus on immuno-oncology
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Cell line development at SystImmune
• Objective: To generate a single cell clone with maximal expression level of candidate bi-specific antibody
• Limited human resource and automation
• Imaging tool: Cell Metric – Solentim 2015
Process:
• CHOZN ZFN modified GS-/- expression platform
• Screen and select top ‘minipool’
• Limiting dilution cloning incorporating imaging
• Screen and select lead single cell clones
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Overview of cell line development processLimiting dilutions(0.3 to 1 cells/well)Set up 40 plates
Transfected minipool
Shake evaluation:Cell growth/titer assessmentProtein quality
Identify clonal lines w/titerApprox. 200-300 wells
Create RCBsMycoplasma testFreeze cell pellets for gDNA analysis
Shake adaption/expansion30ml Fed-batch production run
Pick up best clones (approx. 50) to 24 W plate – analyze titer/growth, cell staining
Expand cell into 24W plate then T25 and T75
Static expansion
Upstream and Downstream Processing in bioreactor of top 8 clones:
Lead Clone selection
Expansion, screening
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What are the challenges in cell line development?
• Complex multistep process, each step from cloning to final clone which leads to the generation of research cell bank (RCB) is critical;
• Efficient processes are essential to reduce timelines and costs;
• Need to define criteria of success for every step;
• Need document to support regulatory filings from Day 1
• Demonstrate monoclonality – use of an appropriate imaging system to document and prove clonality is critical
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Regulatory Requirements for Clonality
Expectation of a Clonal Cell Line:
“For Recombinant products, the cell substrate is the transfected cell containing the desired sequences, which has been cloned from a single cell progenitor.”
-ICH Q5D
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How to achieve monoclonality for commercial production cell lines• Historically, two rounds of limiting dilution at a sufficient dilution
factor was expected, lengthy procedure and observing one final colony doesn’t guarantee single cell clone;
• Poisson distribution is used to calculate probability of clonality. But in reality, cloning efficiency is impacted by many facts such as host cell choice, cell behavior and status, environment, etc.;
• An imager which can image the entire well and provides a sharp image of a single cell on day 0 is critical and possibly allows for single round of cloning;
• Clearly defined IND submission criteria from FDA would be useful
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Cell Metric CLD by Solentim• A dedicated high-resolution bench-top
imaging system
• Enables fast, unmistakable identification
• Tracks single cell derived clones
• High resolution and high contrast imaging to observe a single cell on the day of seeding
• Colony formation can be monitored and recorded by imaging the same wells at regular intervals
• Built-in incubated stacker for batches of 10 plates
• Clonality report generated by software
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Example of a single colony generated from a single cell:
Day 0 Day 1 Day 2 Day 7
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Example of single colony image generated from two cells
Day 0 Day 1 Day 2 Day 7
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Documentation of monoclonality
• Cell Metric imaging system records original plate and well ID of the single cell progenitor and images associated with this well from day 0 so that we can easily trace all the wells throughout the cloning process;
• The software also highlights wells of interest and stores photo-evidence of monoclonality
• It is designed specifically for cell line development with emphasis on the clone screening
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Seeding day image (d0) 24h post seeding day image (d1)
Clonality report generated by Cell Metric CLD:
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48h post seeding day image (d2) 6 days post seeding day image
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7x SCC productivity assessment
0
2
4
6
8
10
12
14
16
18
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16
VC
D x
10
^6
/mL
Days in culture
Viable Cell Density
R011 R012 R013 R014
R015 R016 R017 R018
0
200
400
600
800
1,000
1,200
1,400
7 8 9 10 11 12 13 14 15 16
mg/
Lite
r
Days in culture
IgG productivity
R011 R012 R013 R014
R015 R016 R017 R018
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Summary• Generation of a manufacturing cell line is very time consuming and
typically the most rate limiting step of filing an IND
• Demonstration of monoclonality is a critical part of the overall regulatory package
• Historically, 2 rounds of Limited Dilution Cloning is required for monoclonality demonstration
• Solentim Cell Metric system which has the appropriate resolution to allow for documentation of the initial doubling steps: from 1 cell to 2 cell to 4 cells may gain permit for one round of Limited Dilution
• Elimination of second round of Limited Dilution cloning will reduce timelines significantly while meeting Regulatory requirements
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Acknowledgements
• Zeren Gao
• Jonathan Klepinger
• Zachary Caldwell
• Amanda Mak
Solentim team
Sigma CHOZN platform team