Presentation on Solentim Cell Metric by Takeda at last week's cell line development conference in San Francisco

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

21. Takeda California
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