Post on 02-May-2018
Optimization of T cell expansion in a perfusion bioreactor
Clive Glover PhD Product Leader, Cell Bioprocessing
Perspective Scaling UP? Scaling OUT?
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Chimeric Antigen Receptor T cells- CARTs
TH
TC
TC
TC TH
TH
T cell Receptor intracellular signalling component
Antibody variable region
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
CAR T cells (anti CD19) 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
CART – Chimeric Antigen Receptor T cells
Cell Infusion into Patient
Cell Harvest & Concentration
T cells
CAR T cells
+
Lentiviral – expressing Chimeric Antigen
Receptor
Typical cell dose = 1x108/kg
20 kg patient = 2 x 109 cells
100 kg patient = 1 x 1010 cells
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
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
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
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
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
Cell Separation
Cell Collection
Cell Selection
Cell Activation & Expansion
Cell Harvest & Concentration
Cell Infusion
into Patient
Cell Separation
Cell Collection
Cell Selection
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
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
WAVE 2/10 Closed. Automated. Single-use
Growth kinetics
0.0E+00
1.0E+09
2.0E+09
3.0E+09
4.0E+09
5.0E+09
6.0E+09
7.0E+09
8.0E+09
9.0E+09
1.0E+10
0 2 4 6 8 10 12
W5
Static
Day of Culture
Tota
l Cel
l No.
Optimization Studies Objective: Maximize the expansion of viable T cells in a 10 day period
Speed (rpm)
2 2 10 10 10 18 18 18
Angle (º) 6 9 2 6 9 2 6 9 # of expts 3 1 1 5 1 1 1 1
2,9 2,6 2,2
10,2 10,9 10,6
18,2 18,6 18,9
Angle
Rocking Speed
Cardiff University
27 June 2012 12
0 1 2 3 4 5 6 7 8 9 10 Day of culture
Experimental Design
Culture to 1L Perfuse 500mls
Perfuse 1L Perfuse 750mls
Daily monitoring of: • Cell proliferation/viability • Glucose/Lactate/Ammonia
0 1 2 3 4 5 6 7 8 9 10 QC analysis
Experimental Design
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
Screening for Cell Health Contrasts Term Contrast Plot of t-Ratio Lenth t-
Ratio Individual p-
Value Simultaneous p-
Value angle 0.46901 0.35 0.7532 1.0000 rpm -0.16286 -0.12 0.9111 1.0000 angle*angle -1.15236 * -0.85 0.3575 0.9994 angle*rpm -0.90016 * -0.67 0.4846 1.0000 rpm*rpm 1.18950 * 0.88 0.3415 0.9985
Results
No significant effects of angle or rpm on cell health
Results
Screening for Sum 4th expansion d14 Contrasts Term Contrast Plot of t-Ratio Lenth t-
Ratio Individual p-
Value Simultaneous p-
Value rpm 0.930832 6.88 0.0011* 0.0110* angle -0.035812 -0.26 0.8085 1.0000 rpm*rpm -0.610619 * -4.51 0.0050* 0.0472* rpm*angle -0.021562 * -0.16 0.8834 1.0000 angle*angle -0.548304 * -4.05 0.0077* 0.0671
Significant effect of rocking speed on cell expansion
Optimization Fo
ld e
xpan
sion
sum
Optimized speed and angle: 15.02 rpm, 5.625 º
Optimization
0
2
4
6
8
10
12
14
16
5 6 7 8 9 10
10 rpm, 6° 15 rpm, 6°
Cel
l cou
nt (
106 /
mL)
Day
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