The document details Takeda California's assessment of clonality in production cell lines using high-resolution imaging, emphasizing the FDA's requirement for monoclonality in commercial cell substrates. The study explores the optimization of media conditions, methods for tracking single cell growth, and the impact of additives on colony growth, concluding that lower seeding densities improve colony yield from single cells. Ongoing work aims to refine the system to reduce false negatives and validate whether one round of subcloning is adequate.

1. 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

2. Takeda California
1
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

3. Takeda California
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

4. Takeda California
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

5. Takeda California
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

6. Takeda California
UNDESIRABLE CELL MIGRATION IN LIQUID MEDIA
Day 0
Day 1

7. Takeda California
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

8. Takeda California
CELLS DON’T MIGRATE, BUT GROWTH IS POOR
Day 0 Day 1
Day 2 Day 3

9. Takeda California
DOES AN ADDITIVE HELP GROWTH?
 Using a known additive did improve growth
 Further optimization is still necessary, however
Cells at D0
Number
Observed*
Colonies at
D19*
0 50 0
1 29 5
> 1 17 5
* values represent averages observed in 96 well plates

10. Takeda California
A SINGLE CELL CAN BE TRACKED TO CONFLUENCE
Day 0 Day 1 Day 2
Day 3 Day 7 Day 19

11. Takeda California
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

12. Takeda California
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

13. Takeda California
Media #1 Only Media #1 + Additive #1
Media #1 + Additive #2a Media #1 + Additive #2b

14. Takeda California
Media #2 Only Media #2 + Additive #1
Media #2 + Additive #2a Media #2 + Additive #2b

15. Takeda California
MEDIA #2 + ADDITIVES IMPROVED GROWTH
Media Condition
Colonies 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

16. Takeda California
1.0 cell/well 0.75 cells/well
0.50 cells/well 0.25 cells/well
WHAT SEEDING DENSITY IS OPTIMAL FOR 1 CPW?

17. Takeda California
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

18. Takeda California
VALIDATON: SOME WELLS ARE EASY TO ANALYZE
Day 0 Day 1 Day 2 Day 8
Day 0 Day 1 Day 5 Day 19
1cell/well>1cel/well

19. Takeda California
GROWTH CAN BE TRACKED AT EDGE OF WELL
Day 0 Day 1 Day 2
Day 8 Day 12 Day 16

20. Takeda California
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

22. Takeda California
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?

23. Takeda California
ACKNOWLEDGEMENTS
 Takeda California
 Bhavya Kadambi
 Elizabeth Stangle
 Sanjay Patel
 Solentim
 Sky Jiang
 Dave Elverd
 Ian Taylor