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Parag Kolhe Biotherapeutic Pharmaceutical Sciences AAPS Annual Meeting 2017 Formulation and fill finish process development: CAR-T cell therapy case study

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Parag Kolhe

Biotherapeutic Pharmaceutical Sciences

AAPS Annual Meeting 2017

Formulation and fill finish process development: CAR-T cell therapy case study

BioTherapeutics Pharmaceutical Sciences

+ R&D SITESInnovating to Excel9 + COLLEAGUES

Each Having an Impact850 + DEVELOPMENTALMedicines under Our Wing50

The scope and responsibilities of PFIZER BioTherapeutics Pharm Sci

DiscoveryProjects

Bioprocess,Analytical,

FormulationAnd Device

Development

RegulatoryFilings

Clinical Supplies Manufacturing

Technology Transfer ApprovedMedicines

2

3

Pfizer Biologic Medicines You May Recognize

Cell Therapy Field Is Growing Rapidly

4

Natalie Mount – Cell Therapy Catapult, UK, 2013

Recent BLA approvals

Kite’s Yescarta™ (Axicabtagene Ciloleucel) Becomes First CAR T Therapy Approved by the FDA for the Treatment of Adult Patients With Relapsed or Refractory Large B-Cell Lymphoma After Two or More Lines of Systemic Therapy

Novartis receives first ever FDA approval for a CAR-T cell therapy, Kymriah(TM) (CTL019), for children and young adults with B-cell ALL that is refractory or has relapsed at least twice

T-Cells Recognition and Tumor Killing

• Contact with tumor antigen stimulates release of chemicals that induce lysis or apoptosis of tumor cell

Wickramasinghe, Disc Med 2014

Tumor cell

T cell

Cytolytic synapse

T cell

CD3

MHC/peptide

complex

T cell receptor

complex (TCR)Co-receptors

Tumor cell

5

CAR-T Cell Overview

• Chimeric antigen receptor-expressing T (CAR-T) cells

– T-cells that have been engineered to express antigens on their cell surface specific to proteins that are expressed on the surface of cancer cells

– Direct immunological activities of T-cell to cancer cells

– Advantages: recognize variety of types of antigen (protein, carbohydrate, lipid), antigen does not need to be presented by MHC

T cell

Peptide, lipid, or carbohydrate

Chimeric Antigen Receptor (CAR)

Tumor cell

7

CAR-T Cell Overview (cont.)

• Chimeric antigen receptors contain 3 sections:

– ectodomain that consists of an antigen binding region of an antibody

– hinge transmembrane domain

– intracellular cytoplasmic signaling domain of a TCR

8Source: Wikipedia

CAR-T Cell Overview (cont.)

• Addition of co-stimulatory domains to intracellular domain for 2nd and 3rd

generation CARs

Linker

Hinge/Spacer

Activation

Proliferation

Persistence

Extr

acel

lula

r D

om

ain

Intr

acel

lula

r D

om

ain

Tran

smem

bra

ne

Do

mai

n

9

Source:Clin Cancer Res; 2012; 18 (10); 2780-90World J Stem Cells. Aug 26, 2015; 7(7): 1022-1038

Autologous vs Allogeneic Approach

Adapted from Lipowska-Bhalla et al., Cancer Immunol Immunother 2012

Autologous CAR Therapy (self)

Patient cells

Irradiated or lymphodepleted to

enhance engraftment

lymphodepleted recipients

TCR knockout

Healthy

donor

• Donor T cells can create GvHD – Donor cell TCR recognizes host MHC as

foreign

– TCR knockout technology is required to avoid this

• Cells from one donor could be used to treat many patients

Allogeneic CAR Therapy (donor)

• Avoids GvHD (Graft vs Host disease)

10

Typical Allogenic CAR-T Manufacturing Process

11

Healthy Donor

Apheresis

Gene Knock-out

Gene Transduction

T- cell activation

Cell Expansion

Formulation /Fill/Finish

Cryopreservation

Delivery

Patient

11

Formulation, Process, and Delivery Challenges

12

Delivery

Process

Formulation

• Identifying optimized infusible cryopreservation media containing various formulation components

• Optimizing amount of DMSO in cryopreservation media to increase hold time and cell viability

• Optimize cryopreservation process• Design process capable of handling

extremely challenging hold times

• Suitable container closure • Supply chain • Define delivery approach

within hold time window• Define thaw processes and

methodology

12

Formulation Development – Things to Consider

Formulation/Fill/Finish Cryopreservation Delivery

Design formulation which can

• Provide adequate in-process hold after addition of cryopreservation media

• Maximize cell viability

• Stable to cryopreservation stresses

• Has long term stability in vapor phase liquid nitrogen

• Maximize cell viability

• Stable after thaw• Can provide longer hold times in

cryopreservation media• Maximize cell viability

13

Stresses To Consider For Formulation, Process, And Delivery Aspects

Intermediate formulation

Final Formulation

Controlled Rate Freezing Storage Packaging Shipping Delivery/Administration

Hold time

Impact of formulation excipients on cell

viability?

Hold time

Impact of cryopreservative on cell

viability?

Freezing rate

Impact of cryopreservation

process on cell viability?

Storage temp

Impact of storage temperature and

time?

Time out of storage temp

Define time out of storage/shipping

temperature

Time out of storage/ shipping temp

Thaw timeThaw methodHold timeInjection speed

Define and evaluate thaw methods, impact of hold time, injection

speed etc.

14

Cryopreservation Challenges

15

Source:Sampling scienceCampbell et al, Recent Advances in Cryopreservation

Proper choice of cryoprotective agent (CPA) and optimum freezing rate are critical parameters to consider during cryopreservation

Lipid peroxidationMetabolic imbalance

Cell/organelle Membrane Integrity

(CPA and cell dependent)

Transient osmotic excursions; solute

effects

Cryopreservation- induced delayed-onset cell death (DOCD) is characterized by a significant decrease in viability 12-24 hours post thaw

15

Main Types of Cell Death

Baust, Advances in Biopreservation

Cellular fragmentation

Apoptosis

Phagocytosis

Cellular shrinking

Formation of apoptotic bodies

Necrosis

Cellular and organelle swelling

Breakdown of membrane, nucleus, and organelles; leakage of contents

Inflammatory responses

Healthy cell

• Necrosis– Energy independent

– Caused by external stressors

– Occurs rapidly

– Loss of membrane integrity

– Cell lysis

• Apoptosis

– Energy dependent

– Normal physiological process

– Phospholipid inversion

– Formation of apoptotic blebs

16

Can We Use T- Cells To Predict CAR-T Cell Behavior?

• Material availability is typically challenging for preforming full blown formulation development

• Each vial is precious in terms of material

• So how can pharmaceutical scientists balance understanding of science and balance business needs?

• Can we use model T-cells to predict the behavior of CAR-T cells?

1717

Understanding Effects of DMSO

18

Pre-Freeze Hold (hr):

Post-Thaw Hold (hr):

Recovery Time (days):

• Amount of DMSO has an impact on cell recovery.• Impact is more pronounced after 1 day of recovery.

Saline (7.5 % DMSO) Saline + CS10 (5 % DMSO)

Ave

VC

D(1

0E6

/mL)

3

2

1

0

0 1 2

0 1 0 1 0 1

1 2 1 2 1 2 1 2 1 2 1 2

0 1 2

0 1 0 1 0 1

1 2 1 2 1 2 1 2 1 2 1 2

18

Impact Of Basal Medium – Saline Vs CSB

19

Pre-Freeze Hold (hr):Post-Thaw Hold (hr):

Recovery Time (days):

• Significant difference in run 2.• CSB may provide beneficial effects as basal medium.

Ave

Via

bili

ty (

%)

Run 1 Run 2

CSB – CS10 Saline – CS10 CSB – CS10 Saline – CS10

95

90

85

80

75

70

65

60

55

19

Cryopreservation Formulation Screen and Study Design

20

Formulation ID Basal medium 2X cryopreservation medium Final DMSO

concentration

F1 Saline (0.9%) PBS, HSA, 15% DMSO 7.5% DMSO

F2 Saline (0.9%) CryoStor CS10 5% DMSO

F3 CSB* CryoStor CS10 5% DMSO

F4 CSB* PBS, HSA, 10% DMSO 5% DMSO

Pre-freeze hold

3 hr CryopreservationThaw at 37 °C(water bath)

1 and 2 hr hold 2-8

°C

1 and 2 hr hold 25 °C

1 and 2 hr hold 37 °C

Cell

Recovery

* CSB- Cryostor Basal Solution

20

Cell Viability At 2-8 °C Hold

F1: Saline/PBS/HSA7.5% DMSO

F2: Saline/CS105% DMSO

F3: CSB/CS105% DMSO

F4: CSB/PBS/HSA5% DMSO

• Formulation containing HSA and saline basal medium demonstrated low recovery.

Ave

Via

bili

ty (

%)

100

95

90

85

80

75

70

Pre-Freeze Hold (hr):

Post-Thaw Hold (hr):

Recovery Time (days):

21

Cell Viability At 25 °C Hold

• Formulation containing HSA and saline basal medium demonstrated low recovery.

Pre-Freeze Hold (hr):

Post-Thaw Hold (hr):

Recovery Time (days):

Ave

Via

bili

ty (

%)

100

95

90

85

80

75

70

F1: Saline/PBS/HSA7.5% DMSO

F2: Saline/CS105% DMSO

F3: CSB/CS105% DMSO

F4: CSB/PBS/HSA5% DMSO

22

Cell Viability At 37 °C Hold

• Formulation containing HSA and saline basal medium demonstrated significantly low recovery.

Recovery Time (days):

Ave

Via

bili

ty (

%)

100

95

90

85

80

75

70

65

60

Pre-Freeze Hold (hr):

Post-Thaw Hold (hr):

F1: Saline/PBS/HSA7.5% DMSO

F2: Saline/CS105% DMSO

F3: CSB/CS105% DMSO

F4: CSB/PBS/HSA5% DMSO

23

How does cell density affect the viability?

24

Saline- 7.5% DMSOSaline- CS10 (5% DMSO)

1 h

r.

Pre

-Fre

eze

-ho

ld

2 h

r.

Pre

-Fre

eze

-ho

ld

• Cell density dependency is a function of DMSO concentration.• Formulation and DMSO exposure could have incremental impact

Ave

Via

bili

ty (

%)

95

85

75

65

55

45

95

85

75

65

55

45

Total VCD (10E6/mL):

Recovery Time (days):

24

Overall population in model-T cells post thaw

25

1 h r 2 h r 1 h r 2 h r 1 h r 2 h r 1 h r 2 h r

0 .0

0 .1

0 .2

0 .3

8 0

8 5

9 0

9 5

1 0 0

O v e r a ll p o p u la t io n s

% o

f p

aren

t p

op

ula

tio

n

L y m p h o c y t e s

C D 3 + in C D 4 5 + ( T c e lls )

C D 3 - in C D 4 5 + ( B c e lls + N K c e lls )

5 0 E 6 1 0 0 E 6 5 0 E 6 1 0 0 E 6

C S 1 0 S a lin e

• No change in overall cell population for model T-cells

1 h r 2 h r 1 h r 2 h r 1 h r 2 h r 1 h r 2 h r

0 .0

0 .1

0 .2

0 .3

8 0

8 5

9 0

9 5

1 0 0

O v e r a ll p o p u la t io n s

% o

f p

aren

t p

op

ula

tio

n

L y m p h o c y t e s

C D 3 + in C D 4 5 + ( T c e lls )

C D 3 - in C D 4 5 + ( B c e lls + N K c e lls )

5 0 E 6 1 0 0 E 6 5 0 E 6 1 0 0 E 6

C S 1 0 S a lin e

25

26

PB

S/H

SA

/7.5

%D

MS

O

CS

B/5

%D

MS

O

CS

B/2

.5%

DM

SO

CS

B/1

%D

MS

O

0

5

1 0

1 5

5 0

7 5

1 0 0

A p o p to s is /N e c ro s is P ro file

%

H e a lth y

E a r ly A p o p to s is

L a te A p o p to s is /N e c ro s is

Pre-Freeze

1 2 3 4

7 0

8 0

9 0

1 0 0

V ia b ilty

%

PB

S/H

SA

/7.5

%D

MS

O

CS

B/5

%D

MS

O

CS

B/2

.5%

DM

SO

CS

B/1

%D

MS

O

0 .0

2 .5

5 .0

V ia b le C e ll D e n s ity

ce

lls

/mL

(x

E6

)

T = 0 m in

T = 6 0 m in

7.5% 5% 2.5% 1 % 7.5% 5% 2.5% 1 %

DMSO Amount (%) DMSO Amount (%)

Reducing %DMSO on Pre-Freeze Apoptosis Profile of Cryopreserved T-Cells

26

Reducing DMSO Concentration on Post-Thaw Viability and Viable Cell Density

• Manifestation of DOCD is most apparent in cells cryopreserved in 1% DMSO

• Slowest cell growth observed in cells preserved in 1% DMSO formulation

• Quick recovery in cells preserved in 5% DMSO (followed by 2.5% DMSO)

27

Reducing %DMSO on Post-Thaw Apoptosis Profile of Cryopreserved T-Cells

Late Apoptotic/ Necrotic Cells Post Thaw

Early Apoptotic Cells Post Thaw

%

• In contrast to viability assay showing decreased viability w/ increasing DMSO, annexin V assay indicates lower health of cells in 1% & 7.5% DMSO

– More cells in necrosis/ late stages of apoptosis when cryopreserved in 1% DMSO

– More cells in early stages of apoptosis when cryopreserved in 7.5% DMSO

28

CAR-T Cells : Effect of DMSO Concentration

• Similar trends as model-T cells• High DMSO results in low recovery compared to low levels of DMSO

Recovery Time (days):

Ave

VC

D

Pre-Freeze Hold (hr):

Pre-Fill Hold (hr):

Saline- 7.5% DMSO Saline- CS 10 (5% DMSO)115

105

95

85

75

65

55

45

0 0.08 1 2

10 2

1 1 20 2

10 2

1 1 20 2

1 0 2

1 1 20 2

10 2

1 1 20 2

0 0.08 1 2

10 2

1 1 20 2

10 2

1 1 20 2

10 2

1 1 20 2

10 2

1 1 20 2

29

How do model-T cell data stacks up with CAR-T cells in final selected formulation?

• Model T cells predicted the behavior for CAR-T cells• It is important to highlight that the final formulation screen should be confirmed with CAR-T cells as

demonstrated by slight differences in behavior

Recovery Time (days):

Ave

Via

bili

ty (

%)

Pre-Freeze Hold (hr):

100

90

80

70

60

50

Run 1 Run 2 Run 3

CART(CSB:CS10) Model T cells (CSB:CS10)

1 3

0 000

0 1

1 212 21 21

2 3 0 1

1 212 21 21

2 3 0 1

1 212 21 21

2 30 2

30

Summary

• DMSO concentration in the final cryopreservation formulation affects the cell viability.

• Cryostor CSB basal medium performed better compared to saline as basal medium.

• Cryostor CS10 (5% DMSO in final cryopreservation formulation) provided the optimum cryopreservation

• Cell density impacts viability in conjunction with DMSO concentration.

• Model T cells can provide a platform to assess various variables and the learnings can be used to develop optimum cryopreservation formulation.

3131

Acknowledgements

• Michael Tennant

• Rachel Witts

• Ken Chrobak

• Tihami Qureshi

• Kim Rota

• Yajin Ni

• Christineh Hartoonian

• Mark Leonard

• Bruce Thompson

3232