Optimization of T cell expansion in a perfusion bioreactor Clive Glover PhD Product Leader, Cell...
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Optimization of T cell expansion in a perfusion bioreactor Clive Glover PhD Product Leader, Cell Bioprocessing
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Transcript of Optimization of T cell expansion in a perfusion bioreactor Clive Glover PhD Product Leader, Cell...
Optimization of T cell expansion in a perfusion bioreactor
Clive Glover PhDProduct Leader, Cell Bioprocessing
PerspectiveScaling UP? Scaling OUT?
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“Hom
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Indu
stry ?123RF.com
What does thiseven look like?
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 ScaleCell
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 ProcessesScalable. Sample contained in 1 vesselEasy 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 2/10Closed. Automated. Single-use
Growth kinetics
0 2 4 6 8 10 120.0E+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
W5Static
Day of Culture
Tota
l C
ell
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
2,92,62,2
10,2 10,910,6
18,2 18,6 18,9
Angle
Rocking Speed
Cardiff University
27 June 201212
0 1 2 3 4 5 6 7 8 9 10Day of culture
Experimental Design
Culture to 1L
Perfuse 500mls
Perfuse 1L
Perfuse 750mlsDaily monitoring of: • Cell proliferation/viability• Glucose/Lactate/Ammonia
0 1 2 3 4 5 6 7 8 9 10QC 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 Sum 4th expansion d14 Contrasts
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
Screening for Cell Health Contrasts
Significant effect of rocking speed on cell expansion
Optimization Fo
ld e
xpansi
on
sum
Optimized speed and angle: 15.02 rpm, 5.625 º
Optimization
5 6 7 8 9 100
2
4
6
8
10
12
14
16
10 rpm, 6° 15 rpm, 6°
Cell
count
(10
6/
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
