1. Optimization of T cell expansion in a perfusion bioreactor
Clive Glover PhD
Product Leader, Cell Bioprocessing

2. ? Perspective Scaling UP? Scaling OUT? “Home” Industry
Wikipedia.com
123RF.com ?
Industry
What does this even look like?
123RF.com

3. Chimeric Antigen Receptor T cells- CARTsTH TH TH TC TC
Antibody variable region
T cell Receptor intracellular signalling component

4. Clinical Trials Results
Approach
# patients CR PR
CAR T cells (anti CD19) 8
4 (50%)
2 (25%)
Rosenberg et.al. B-cell depletion and remissions of malignancy along with cytokine-associated toxicity in a clinical trial of anti-CD19 chimeric-antigen-receptor-transduced T cells. Blood; 119(12) March, 2012 3
2 (66%)
1 (33%)
June et.al. T cells with chimeric antigen receptors have potent antitumor effects and can establish memory in patients with advanced leukemia. Sci Transl Med.; 10(3) Aug, 2011

5. Lentiviral – expressing Chimeric Antigen Receptor
CART – Chimeric Antigen Receptor T cells +
T cells
Lentiviral – expressing Chimeric Antigen Receptor
Cell Infusion into Patient
Cell Harvest & Concentration
CAR T cells

6. Typical cell dose = 1x108/kg
20 kg patient
= 2 x 109 cells
100 kg patient
= 1 x 1010 cells

7. Factory Scale
Cell Separation
Cell Collection
Cell Selection
Cell Activation & Expansion
Cell Harvest & Concentration
Cell Infusion into Patient
Cell Separation
Cell Collection
Cell Selection
Cell Activation & Expansion
Cell Harvest & Concentration
Cell Infusion into Patient
5000 patients Process time = 10 days Number of patients in parallel = 140

8. Key Requirements of Cell Therapy Manufacturing Processes
Scalable.
Sample contained in 1 vessel
Easy to scale out to make most efficient use of manufacturing space
Automatable to minimize the chance of human error
Single Use and Traceable to eliminate cross contamination with other patient cells
Closed system to eliminate chance of contamination with adventitious agents due to handling
Robust and Compliant. To ensure consistency of product and satisfaction of regulatory requirements

9. WAVE 2/10 Closed. Automated. Single-use

10. Growth kinetics
Total Cell No.
Day of Culture

11. Optimization Studies
Objective: Maximize the expansion of viable T cells in a 10 day period
2,2
2,6
2,9
Angle
Rocking Speed
10,2
10,9
10,6
18,2
18,9
18,6
Speed (rpm) 2 10 18
Angle (º) 6 9
# of expts 3 1 5

12. Experimental Design 5 6 7 8 9 10 Daily monitoring of:
Day of culture 5 6 7 8 9 10 1 2 3 4
Culture to 1L
Perfuse 500mls
Perfuse 750mls
Daily monitoring of:
Cell proliferation/viability
Glucose/Lactate/Ammonia
Perfuse 1L

13. Experimental Design 5 10 Phenotype monitoring of: CD4/CD8 ratio
QC analysis 5 10 1 2 3 4 6 7 8 9
Phenotype monitoring of:
CD4/CD8 ratio
CD27/CD28 expression to assess differentiation state
CD57 expression to assess the presence of senescent cells
CD62L expression to assess migratory ability

14. Results
No significant effects of angle or rpm on cell health

15. Results
Significant effect of rocking speed on cell expansion

16. Optimization Optimized speed and angle: 15.02 rpm, 5.625 º
Fold expansion sum
Optimized speed and angle:
15.02 rpm, º

17. Optimization
Cell count (106/ mL)
Day

18. Summary
Autologous cellular immunotherapies have unique scalability requirements WAVE systems provide robust and reliable expansion of functional T cells 10% increase in cell yield using optimized bioreactor settings Higher cell densities and a closed and automated system make them ideal for therapeutic use