arna andrews, csl limited (vienna, april 2016)

The Use of Imaging to Demonstrate Clonality of Production CHO Cell Lines

Experiences & Learnings

Arna Andrews Director, Cell Line Development, CSL Limited

Informa Cell Line Development & Engineering Conference

April 11th 2016

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Presentation Outline

• Introduction to CSL

• Cell Line Development at CSL

• Using imaging technology to understand the limitations of the traditional limiting dilution cloning approach

• Imaging for Cell Line Development – Experiences, Tips & Tricks • Clonality Criteria & Qualification Experiment

• CloneSelect Imager v Cell Metric

• Analysis of different 96 well plates

• Conclusions

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CSL at a glance

CSL is a global specialty biotherapeutics company headquartered in Parkville, Australia that develops and delivers innovative biotherapies that save lives, and help people with life-threatening medical conditions live full lives.

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CSL R&D

OPERATIONAL BUSINESSES CORPORATE FUNCTION

Corporate structure

Good for You. Great for Life.

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Strategy

• Maintain commitment to extracting maximum value from existing assets and supporting and improving current products.

• Develop new protein-based therapies for treating serious illnesses focusing on products that align with our technical and commercial capabilities.

• First recombinant product, IDELVION® recently approved by FDA

CSL Research & Development

Immunoglobulins

Haemophilia Products

Specialty Products

Breakthrough Medicines

Plasma Fractionation

Recombinant Technologies

Protein Science

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Cell Line Development at CSL

• Major driver is quality rather than speed

• Limited automation

Process

• CHO platform

• Screen and select lead “transfectants”

• Limiting Dilution Cloning incorporating imaging

• Screen and select lead “clonal” cell lines

Imaging Technology used

• Clone Select Imager - 2009

• Cell Metric - 2015

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Traditional limiting dilution cloning approach

Assumptions

• Cells are distributed according to a Poisson distribution

• All cells have an equal chance of survival and growth regardless of the number of cells seeded per well

Reality

• Outgrowth is not 100% at low seeding densities in CD ACF media

• Cloning efficiency is impacted by many things including: • Host cell line choice / expression product / cell status / environment

Imaging technology allows us to reassess the limitations of this approach

Cells are distributed in line with a Poisson distribution

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LD at 0.5 cells/well Number of wells that contained:

0 cells 1 cell 2 cells 3 cells 4 cells 5 cells 6 cells

Image analysis 231 105 24 3 2 1# 0

Theoretical 233 116 29 5 1 0 0

4 x 96 well plates seeded at 0.5 cells / well and imaged at 3 hours post seeding

# cells deposited as a “cell clump”


Image analysis can be used to assess the outcomes from traditional limiting dilution experiments.

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* If cell survival & growth is 100%

Based on images taken at 3hrs

Product 1

Product 2

Product 3

# wells with 1 cell # wells with >1 cell # wells with 1 cell # wells with >1 cell

1 19 12 18 142 9 4 5 83 5 5 6 34 29 12 31 115 0 6 0 46 0 2 1 27 65 29 ND ND8 28 25 25 419 7 3 10 12

10 33 45 26 4411 16 15 14 2512 22 13 13 2113 15 15 10 1914 40 17 24 2715 0 2 2 916 25 16 12 2117 33 28 32 3218 24 20 16 4019 19 13 16 2620 29 31 27 4121 38 26 23 38

Expected* 116 35 71 51

Limiting Dilution Cell Line Number

Number of wells with viable growth

4 plates seeded at 0.5 cells/well 2 plates seeded at 1 cell/well

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• Large variation in cloning efficiency from experiment to experiment

• Outgrowth never matched that expected if cell survival was 100%

• Bias towards outgrowth in wells containing > 1 cell

• Lines in which the traditional Coller & Coller statistical approach would suggest a > 99% probability of monoclonality can be shown to have arisen from wells deposited with more than one cell.

Coller & Coller (Hybridoma, 1983 2:91-96)

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Imaging – Experiences, Tips & Tricks

Defining clonality criteria

• Imaging at multiple time points is essential

• Experience at CSL has led us to routinely image at:

• t minus 1hr Allows identification of plate imperfections

• t = 3hrs

• t = 24hrs Allows for tracking of individual cells

• t = 72hrs

• t = 7, 14 & 21 days

Confirm the formation of a discrete colony with “normal” growth rate

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Defining clonality criteria – Imaging Qualification Experiment

rec mAb rec protein

Limiting dilution

Combine cells in a mixed culture

3-4 days at 37°C

CSI image analysis Designation of clonality

Screening analysis Culture supernatant analysed for

protein production

Image analysis & screening analysis Statistical calculation of probability of monoclonality

Expand all growing cells to 24 well plate

http://www.clker.com/cliparts/p/n/X/L/y/J/conical-flask-md.png



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Clonality criteria

• Clean well and well focussed images

• One cell at 3 hrs

• ≤ 2 cells at 24 hrs - in close proximity to each other

• Discrete colony with growth rate in “normal” range

• Acceptance by two independent assessors

3 hours 24 hours 3 days

Non clonal example

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CloneSelect Imager Cell Metric v

Cell Metric Advantages: • Better image resolution • Automatic Focussing • High precision positioning eliminates stitching issues • Integrated plate stacker reduces manual handling time & incubator openings • Software allows easier tracking across time points

Cell Metric Disadvantage: • Requires reduced media volumes to eliminate well edge reflections

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• Comparison between different 96 well plates using Cell Metric

Plate Type ↓ing attachment Manufacturer Cell survival / Cell growth Image Quality Minimisation of

Cell Movement

CellBIND Corning CellBIND surface (Cat # 3300) / x

Corning tissue culture treated (Cat # 3595) /

Nunc cell culture treated (Cat # 167008) /

Nunc edge plate, cell culture treated (Cat #167314) / x

Nunc non-treated (Cat # 260860) /

Nunc edge plate, non-treated (Cat # 267313) / x

Low attachment Corning ultra low attachment (Cat # 3474) / x

3D Cell Culture Nunc Sphera surface plate (Cat # 174927) /

Criteria AssessedPlates

Cell Cuture treated

Non- treated

New plate of choice

Original plate used

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There is a lot less movement of the cells on this particular well surface. This makes it easier to identify any extra cells that settle to the bottom of the well after the 3 hour time period and to increase the likelihood of discrete colonies arising from independent cells The tendency to not immediately move to the well edge was also an advantage with the imaging.

3 hours

24 hours

3 days


• Images from Nunc non-treated plates (Cat # 260860)

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Conclusions

• Imaging technology has enabled deficiencies with traditional limiting dilution approaches to be highlighted

• If performed appropriately, a single round of cloning associated with imaging outperforms two rounds of LDC assessed via Coller & Coller statistics

• Imaging at multiple time points is essential

• Define fixed clonality criteria

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Acknowledgements

• Victor Turnbull • Rebecca D’Sylva

• Pia Damm

• Sally Ashe

• Michael Wilson

• Andrew Nash