Assessment of clonality for production cell lines via high resolution imaging

Takeda California

Assessment of Clonality for Production Cell Lines via

High-Resolution Imaging

Melisa Carpio, MS

May 19, 2015

IBC’s Cell Line Development & Engineering Conference

Takeda California1

THE FDA MANDATES MONOCLONALITY FOR

ALL COMMERCIAL PRODUCTION CELL LINES

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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WHY DID WE CHOOSE THE CELL METRIC?

High quality images

Automatic focusing

Direct visual reading of whole well

Unlimited number of data points

Digital record of cells

High-throughput

10 plate incubated stacker

3-4 minutes per plate

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ASSESSMENT STRATEGY

What were our goals?

Be able to clearly track growth of a single cell to confluence

Determine media conditions that fosters growth of single cells

Reliably deposit single cells into a single well of a 96WP

How did we achieve our goals?

Evaluate liquid versus semisolid media for cell tracking

Screen different media and additives to optimize colony growth

Examine seeding densities that yield 1 cell/well

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UNDESIRABLE CELL MIGRATION IN LIQUID MEDIA

Images of single cells are clearly observed

BUT cells tend to migrate during daily handling

Day 0 Day 1

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UNDESIRABLE CELL MIGRATION IN LIQUID MEDIA

Day 0

Day 1

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DOES SEMI-SOLID MEDIA SOLVE THE PROBLEM?

Semisolid #1 Semisolid #2

Semisolid #3 Semisolid #4

Significant debris

was present in

three of the four

media tested

Semisolid #4 was

chosen for further

optimization

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CELLS DON’T MIGRATE, BUT GROWTH IS POOR

Day 0 Day 1

Day 2 Day 3

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DOES AN ADDITIVE HELP GROWTH?

Using a known additive did improve growth

Further optimization is still necessary, however

Cells at D0Number

Observed*

Colonies at

D19*

0 50 0

1 29 5

> 1 17 5* values represent averages observed in 96 well plates

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A SINGLE CELL CAN BE TRACKED TO CONFLUENCE

Day 0 Day 1 Day 2

Day 3 Day 7 Day 19

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COLONIES CAN BE SCALED TO YIELD SIMILAR TITERS

96WP 24WP 6WP SF #1 SF #2 Fed-Batch

Titer from Cell Metric Clones

375 ± 174 mg/L

Titer from Legacy Process

296 ± 208 mg/L

Scale-up was done to test direct transfer from semi-solid to liquid media

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MEDIA OPTIMIZATION TO IMPROVE COLONY GROWTH

Media Condition

Media #1 Only

Media #1 + Additive #1

Media #1 + Additive #2a

Media #1 + Additive #2b

Media #2 Only

Media #2 + Additive #1

Media #2 + Additive #2a

Media #2 + Additive #2b

1 cell/well

Imaging D0, D1,

D2, and every 3-

5 days until D19

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Media #1 Only Media #1 + Additive #1

Media #1 + Additive #2a Media #1 + Additive #2b

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Media #2 Only Media #2 + Additive #1

Media #2 + Additive #2a Media #2 + Additive #2b

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MEDIA #2 + ADDITIVES IMPROVED GROWTH

Media ConditionColonies from

1 cell/well*

Colonies from

>1 cell/well*

Edge + False

Negatives*

Media #1 Only 0 1 0

Media #1 + Additive #1 5 4 1

Media #1 + Additive #2a 9 8 0

Media #1 + Additive #2b 7 9 0

Media #2 Only 5 11 2

Media #2 + Additive #1 14 15 5

Media #2 + Additive #2a 14 27 13

Media #2 + Additive #2b 12 41 4

* values represent average number of colonies observed in a 96 well plate at Day19

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1.0 cell/well 0.75 cells/well

0.50 cells/well 0.25 cells/well

WHAT SEEDING DENSITY IS OPTIMAL FOR 1 CPW?

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LOWER SEEDING DENSITIES ARE BETTER

Both 0.50 and 0.25 cells/well had >50% of the colonies coming from 1

cell/well

Seeding Density

(cells/well)

Colonies from

1 cell/well*

Colonies from

>1 cell/well*

Edge + False

Negatives*

1.0 21 23 13

0.75 17 17 10

0.50 15 9 6

0.25 11 4 2

* values represent average number of colonies observed in a 96 well plate at Day16

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VALIDATON: SOME WELLS ARE EASY TO ANALYZE

Day 0 Day 1 Day 2 Day 8

Day 0 Day 1 Day 5 Day 19

1 c

ell/

well

>1

cel/w

ell

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GROWTH CAN BE TRACKED AT EDGE OF WELL

Day 0 Day 1 Day 2

Day 8 Day 12 Day 16

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THE MYSTERIOUSLY APPEARING CELL

Day 0

Day 16

Day 1 Day 2 Day 5

Day 0 Day 1 Day 2 Day 5

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TWO CELLS START, ONLY ONE SURVIVES

Day 0 Day 1

Day 5 Day 13

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SUMMARY OF INITIAL EVALUATION & NEXT STEPS

Be able to clearly track growth of a single cell to confluence

Semi-solid media is being used

Determine media conditions that fosters growth of single cells

A Media #2 and additive combination was identified

Reliably deposit single cells into a single well of a 96WP

Lowering the cell density increases the number of colonies

coming from 1 cell/well

Work is on-going to validate the system

Reduce the occurrence of false negatives

Is one round of subcloning sufficient?

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ACKNOWLEDGEMENTS

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Bhavya Kadambi

Elizabeth Stangle

Sanjay Patel

Solentim

Sky Jiang

Dave Elverd

Ian Taylor