media optimization strategies for t cell therapy manufacturing...cryo lot release and...

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Angel Varela

BioProduction

Cell Culture & Cell Therapy R&D

Media Optimization Strategies for T cell Therapy Manufacturing

2

Pharma: Past, Present and Future

Increasing Complexity

Chemical

Penicillin

(243 Da)

Antibody

Immunoglobulin G

(150,000 Da)

Protein

Lysozyme

(14,700 Da)

Virus

Influenza

(176 x106 Da)

Cell

Human cell

(~2 ng)

Our industry has moved past chemical drugs into biologics with

an increased interest and investment in gene and cell

therapies

Cell Therapy requires manufacture of very complex therapies

where the cell is the drug

Proprietary & Confidential

3

Main Cell & Gene Therapy Production Workflows

Gene-

Modified T

cell (auto)

Gene Therapy/

Vector

Production

Cell isolation and

activation Cell engineering Cell expansion

Wash, fill, finish, and

cryo

Lot release and

characterization

Plasmid

construction

Cell line

transfection Viral production Viral purification

Wash, fill, finish and

cryo

Lot release and

characterization

MSC (allo) Cell

isolation Cell expansion Cell modification

Lot release and

characterization

Wash, fill, finish and

cryo


4

Allogeneic Vs. Autologous Therapies

Master Cell Bank Lot Tested

Patient Doses Lot Tested

Cell E

xp

an

sio

n

Cell E

xp

an

sio

n

Testing

Patient or Donor

Cell Ampoule or Dose

Submitted for Testing

Allogeneic / Universal Donor

Autologous / Patient Specific


5

CAR and TCR Gene Transfer: Most Common T cell Engineering Modalities

CAR: Reacts to Surface Proteins TCR: Reacts to Intracellular and Surface Proteins

Targets are native surface proteins

HLA-independent

CD19 CD20

Mesothelin CD38

HER2/neu

EGFRvIII

Targets are peptides derived from intracellular and

surface proteins presented in HLA molecules

Viral

antigens

MAGE

NY-ESO MART-1

WT-1


6

1. Obtain cells from patient and enrich

or isolate particular cells of interest

4. Large scale cell expansion

Reinfuse

cells

5. Remove beads and wash cells

2. Stimulate cells to

grow

3. Insert therapeutic genes

into cells

6. Quality Control What are the best T cells

for immunotherapy?

What is the best way

to expand T cells?

What is the best way

to engineer T cells?

How many cells do we really

need?

How do we

monitor adoptive

T cell therapies?

What assays will be predictive

of in vivo efficacy and safety?

Process & Challenges: Adoptive T cell Immunotherapy

How to manufacture

more efficiently?


7

Limited availability of serum as a manufacturing raw material


8

Challenges of Serum for T cell Therapy Manufacturing

• Performance concerns

• Undefined composition

• Lot-to-lot variability

• Lot qualification time and effort

• Regulatory/safety concerns

• Potential for adventitious agents

• Supply chain concerns

Treating 10,000 patients per year with CTL019

(CART19):

• 80,000 liters of cell culture media

• 4,000L of huAB serum (!)

Sera variability impacts process and product

Transduction Efficiency

T cell Expansion

control

huAB serum #1

huAB serum #2

huAB serum #3

huAB serum #4

huAB serum #5

huAB serum #6

control

huAB serum #1

huAB serum #2

huAB serum #3

huAB serum #4

huAB serum #5

huAB serum #6

20-40% variability in transduction

15-50% variability

in T cell yields


9

CTS™ Immune Cell SR

CTS™ Immune Cell SR is the first defined, T-cell qualified xeno-free replacement for human serum

that provides consistent serum-like performance without supply risk.

Parameter SPEC

Functional T Cell Assay Acceptable vs. internal assay control

pH >=7.0 to <=7.7

Osmolality > =430 to < =500

Mycoplasma testing (qPCR) Negative

USP Sterility testing Negative

Endotoxin testing (LAL) >=0.0 to <=10.0

HBsAg, HIV1&2, HCV virus testing Non-reactive (Ab)

Specifications


10

CTS™ Immune Cell SR

Security of Supply

Consistent Performance

Flexible

Primary

T cell tested

Regulatory friendly

Scalable, cGMP

(21CFR part 820)

CE-IVD

USP 1043

ISO 13485

Full Traceability: COO, COA, donor

testing, DMF

Direct replacement for human

serum

Cost competitive

No need to pre-qualify lots

Clinical & Translational Immunology (2015) 4, e31; doi:10.1038/cti.2014.31;

published online 16 January 2015

Comparable expansion performance to human serum supplementation in

commercial T cell media formulations

QA/QCrelease assay based on CTS

Dynabeads CD3/CD28 and primary

human T cells


11

CTS™ Immune Cell SR – In vivo testing

NSG mice

Leukemia

1x106

Control of

leukemia

T cell

engraftment

T cell

persistence

CART-19

10x106

Mouse

survival

Expansion

Transduction

Phenotype

We wanted to test CTSTM Immune Cell SR in an in vivo model of CAR-T immunotherapy to validate comparable

performance to human AB serum


12

CA

RT

-19

2% hu AB Serum 2% Immune Cell SR

CCR7

CD62L

CTS™ Immune Cell SR Similar Expansion, Phenotype and Transduction Efficiency

Data from James L. Riley, University of Pennsylvania


13

In vivo efficacy of CART-19 cells expanded in CTS™ Immune Cell SR

Mock transduced

OpTmizer +2% hAB

OpTmizer +2% ICSR

Data from James L. Riley, University of Pennsylvania


14

• Serum introduces challenges and risks for cell therapy manufacturing

• CTSTM Immune Cell SR can replace human AB serum in CAR-T manufacturing workflows,

maintaining

• Expansion

• Function

• Phenotype

• Efficacy

• Potency

• CTSTM Immune Cell SR is a practical and scalable solution for T cell therapy manufacturing

processes

Summary 1


15

0

1

2

3

4

5

6

7

8

0 5 10 15 20

VCD

(x10

6vi

able

cel

ls pe

r mL)

culture time (days)

0%

100%

200%

300%

400%

0 5 10 15 20

IgG

titer

(% o

f bat

ch)

culture time (days)

CD CHO+40% EFB

CD CHO+40% EFB+14% F3.4

Cell Culture Media: State of the Art

• Protein Production/Vaccines

• Cells produce the therapy

• Homogenous cell population

• Cells can be adapted to new media

• Well established/scalable processes

• Cellular Therapies

• The cells are the therapy

• Heterogeneous cell population

• Media must support cell growth directly

• Divergent/emerging culture systems

Current media and supplement technologies enable

improvements in IgG and viral titers

CHO (IgG) BHK-21 (Virus)

?

Unmet need to provide media and scalable workflows to

increase productivity

Increased Productivity


16

Better T Cell Drugs – Thermo Fisher Scientific Innovation

Leukemia

HIV

Solid

tumors

Autoimmune

diseases

other blood cancers

Dynabeads™ & Gibco®

products for

activation & expansion of

T cell subsets

Can Next Gen Technologies Improve T Cells Drug

Safety, Efficacy and Persistence?


17

Signals to Drive T cell Subset Differentiation

Th1

Th9

Th2

Th22

Th17

Treg

Naïve

T cells

Signal 1 + 2 Signal 3

Cytokines

&

Growth Factors

Signal 4

T cell subsets use different

metabolic pathways

Cytokines support

expansion of T cell

subsets


18

Better T Cell Drugs – Cell Culture Product Innovation

Signal 1 + 2 Optimized biological effect of

signal 1 + 2 + 3 + 4

Media: analyze metabolic

pathways & nutrient consumption

rate for optimal balanced

formulation

3

2 1

Beads/antibodies: balanced

antibody conjugation to a solid

surface for optimal action &

proliferation

Cytokines: defined

mix & concentration for

optimal T cell subset expansion

4 Naïve

T cells

Th1

Th9

Th2

Th22

Th17

Treg


19

Learning From T Cell Metabolic Pathways for Media Development

Buck, et al. J. Exp. Med. 2015 Vol. 212 No. 9 1345–1360


20

0%

500%

1000%

1500%

2000%

2500%

3000%

3500%

4000%

4500%

5000%

0 2 4 6 8 10 12

Pe

rce

nta

ge

Culture time, day

Alanine

MM4

MM7

MM8

MM15

0%

10%

20%

30%

40%

50%

60%

70%

80%

90%

100%

0 2 4 6 8 10 12

Pe

rce

nta

ge

Culture time, day

Asparagine

MM4

MM7

MM8

MM15

Media Development Strategy

Amino AcidConsumption Rate

(mmol/day)

L-ALANINE -0.251

L-ARGININE 0.055

L-ASPARAGINE 0.054

L-ASPARTIC ACID 0.045

L- CYSTINE 0.083

L-GLUTAMIC ACID 0.055

GLYCINE 0.068

L-HISTIDINE 0.024

L-ISOLEUCINE 0.048

L-LEUCINE 0.046

L-LYSINE 0.043

L-METHIONINE 0.036

L-PHENYLALANINE 0.061

L-PROLINE 0.057

L-SERINE 0.07

L-THREONINE 0.05

L-TRYPTOPHAN 0.039

L-TYROSINE 0.062

L-VALINE 0.064

Calculate consumption

rates

Analyze metabolic pathways/

surface marker expression/cell phenotype/effector

function

Optimized balanced formulation

Perform spent media

analysis

Sample target process Analyze growth/viability


21

D139 started 30Aug13 Recombinant Study

Time (day)

0 2 4 6 8 10 12 14

Cum

ulat

ive

Tce

lls (

tc x

10E

6)

0

100

200

300

400

500

600

XVivo + 5% huABS

Tcell Prototype SFM 1


Case Study: CAR-T cell Medium Optimization

Control + serum

Medium 1

A2184 started 6Sept13 Recombinant Study

Time (day)

0 2 4 6 8 10 12 14 16

Cum

ulat

ive

Tce

lls (

tc x

10E

6)

0

100

200

300

400

500

600

700

XVivo + 5% huABS



Serum-free

Media Prototypes Media Variant Screen + Statistical Analysis =

Results:

Prototype xeno-free serum-free media meet or exceed the performance of

serum-containing commercial medium


22

Metabolic Control by Media Optimization

0

1

2

3

4

5

6

7

8

0 2 4 6 8 10 12

0

0,5

1

1,5

2

2,5

3

Day 0 Day 3 Day 5 Day7 Day 10 Day 12

Lact

ate

g/L

P

op

ula

tio

n D

ou

blin

gs

0

20

40

60

% IF

Ng+

IFNy

IL-2

Medium 4 Medium 8 Medium 10

Media can be tailored to achieve a balance of the

desired effector function and favorable metabolic profile

Days Post Stimulation


23

Luminex™ -based 35-plex Potency Assay

Useful tool to dissect T cell function in response to media modifications

1

10

100

1000

10000

pg

/ml

AOF SF X-VIVO

Invitrogen Cytokine Human Magnetic 35-Plex Panel for Luminex™

control


24

Transcriptomic Analysis to Verify Gene Expression Profiles

Full transcriptomic analysis can be used to

understand gene expression dynamics in new

serum-free vs control media


25

Regulatory T cell Media Development

Treg are immunosuppresive T cells that express

the transcription factor FoxP3

Treg therapies are being explored as treatments

for autoimmunity and chronic inflammation

Tregs favor OxPhos over glycolysis; we exploited

this for media development

Treg Dynabead prototype was used as activation

reagent for media optimization


26

Treg Serum-free Media Development - DOE Challenges

Expansion efficiency and FoxP3 enrichment resulted in opposing responses

FoxP3

Growth


27

Po

pu

lati

on

Do

ub

lin

gs

Time (days)

Treg >70% Treg ~40%

10%huSerum

+ Treg Dynabead Treg serum-free

+ Treg Dynabead

Additional Optimization – Treg Media Development

Result: Optimal Culture System To Expand Treg cells


28

• Treg SFM and Dynabeads prototypes

expand stable and potent Tregs

• X-VIVO 15 +10% hAB -expanded Tregs require

Rapamycin during expansion to maintain

suppressive capacity

• Treg SFM -expanded Tregs maintain suppressive

function even when Rapamycin is added only on

day 0 of expansion process.

Serum-free Expansion of Stable and Potent Tregs

99%

12%

99%

44%

99%

99%

Neg Ctr

Pos Ctr

10% hAB

Rapamycin

10% hAB

Rapamycin day 0

Treg SFM

Rapamycin

Treg SFM

Rapamycin day 0

1:32 (Treg:Tconv)


29

• Defined, consistent solutions are needed to enable scalable, robust and

commercially viable manufacturing of cell therapies

• Media development in combination with appropriate characterization techniques

and in vivo models should be used where possible to validate critical to quality

parameters: phenotype, function, potency, persistence in vivo

• Thermo Fisher Scientific can help you develop technologies to manufacture cell

therapies with optimal balance of potency, efficacy and persistence

Conclusions


31

Verification at scale +

0.0E+00

1.0E+05

2.0E+05

3.0E+05

4.0E+05

5.0E+05

6.0E+05

0 2 4 6 8 10 12

cells

/mL

Time (days)

Un-optimized

Optimized

Cell

Den

sit

y (

ce

lls

/ml)

DOE

Adapted from dos Santos et al. Biotechnology and Bioengineering, 2014

Case Study: MSC Medium/Process Optimization

Results: Efficient GMP-compliant MSC expansion process

Optimization resulted in 2-fold

improvement in cell yields


media optimization strategies for t cell therapy manufacturing...cryo lot release and...

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