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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

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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

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1,000

1,200

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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