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

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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