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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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