formulation and fill finish process development: car-t ... · cell therapy field is growing rapidly...
TRANSCRIPT
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
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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
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How Tumors Avoid the Immune System?
• Tumor antigens are only mildly immunogenic
• Down-regulation of MHCs expressing tumor antigens
• Up-regulation of inhibitory co-receptors
• Secretion of various immunosuppressants
6
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
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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
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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)
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Typical Allogenic CAR-T Manufacturing Process
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Healthy Donor
Apheresis
Gene Knock-out
Gene Transduction
T- cell activation
Cell Expansion
Formulation /Fill/Finish
Cryopreservation
Delivery
Patient
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Formulation, Process, and Delivery Challenges
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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
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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
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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.
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Cryopreservation Challenges
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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
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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
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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
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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
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Impact Of Basal Medium – Saline Vs CSB
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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
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Cryopreservation Formulation Screen and Study Design
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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
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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
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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
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How does cell density affect the viability?
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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.
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