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

Transcription

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

2 2 BioTherapeutics Pharmaceutical Sciences The scope and responsibilities of PFIZER BioTherapeutics Pharm Sci Discovery Projects Bioprocess, Analytical, Formulation And Device Development Technology Transfer Clinical Supplies Manufacturing Regulatory Filings Approved Medicines 9 + R&D SITES Innovating to Excel COLLEAGUES Each Having an Impact 50 + DEVELOPMENTAL Medicines under Our Wing

3 Pfizer Biologic Medicines You May Recognize 3

4 Cell Therapy Field Is Growing Rapidly 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 Recent BLA approvals Natalie Mount Cell Therapy Catapult, UK, 2013 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 4

5 T-Cells Recognition and Tumor Killing Tumor cell T cell T cell Cytolytic synapse Co-receptors CD3 T cell receptor complex (TCR) Wickramasinghe, Disc Med 2014 MHC/peptide complex Tumor cell Contact with tumor antigen stimulates release of chemicals that induce lysis or apoptosis of tumor cell 5

6 How Tumors Avoid the Immune System? Tumor antigens are only mildly immunogenic Down-regulation of MHCs expressing tumor antigens Up-regulation of inhibitory coreceptors Secretion of various immunosuppressants 6

7 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 Peptide, lipid, or carbohydrate T cell Tumor cell Chimeric Antigen Receptor (CAR) 7

8 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 Source: Wikipedia 8

9 CAR-T Cell Overview (cont.) Transmembrane Domain Extracellular Domain Intracellular Domain Addition of co-stimulatory domains to intracellular domain for 2 nd and 3 rd generation CARs Linker Hinge/Spacer Activation Proliferation Source: Clin Cancer Res; 2012; 18 (10); World J Stem Cells. Aug 26, 2015; 7(7): Persistence 9

10 Autologous vs Allogeneic Approach Autologous CAR Therapy (self) Allogeneic CAR Therapy (donor) TCR knockout Patient cells Healthy donor lymphodepleted recipients Irradiated or lymphodepleted to enhance engraftment Avoids GvHD (Graft vs Host disease) Adapted from Lipowska-Bhalla et al., Cancer Immunol Immunother 2012 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 10

11 Typical Allogenic CAR-T Manufacturing Process Healthy Donor Apheresis Delivery Patient Cryopreservation T- cell activation Formulation /Fill/Finish Gene Transduction Gene Knock-out Cell Expansion 11 11

12 Formulation, Process, and Delivery Challenges 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 Process Delivery Suitable container closure Supply chain Define delivery approach within hold time window Define thaw processes and methodology 12 12

13 Formulation Development Things to Consider Design formulation which can Formulation/Fill/Finish Cryopreservation Delivery 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

14 Stresses To Consider For Formulation, Process, And Delivery Aspects Intermediate formulation Final Formulation Controlled Rate Freezing Storage Packaging Shipping Delivery/ Administration Hold time Hold time Freezing rate Storage temp Time out of storage temp Time out of storage/ shipping temp Thaw time Thaw method Hold time Injection speed Impact of formulation excipients on cell viability? Impact of cryopreservative on cell viability? Impact of cryopreservation process on cell viability? Impact of storage temperature and time? Define time out of storage/shipping temperature Define and evaluate thaw methods, impact of hold time, injection speed etc. 14

15 Cryopreservation Challenges Transient osmotic excursions; solute effects (CPA and cell dependent) Cryopreservation- induced delayed-onset cell death (DOCD) is characterized by a significant decrease in viability hours post thaw Lipid peroxidation Metabolic imbalance Cell/organelle Membrane Integrity Proper choice of cryoprotective agent (CPA) and optimum freezing rate are critical parameters to consider during cryopreservation Source: Sampling science Campbell et al, Recent Advances in Cryopreservation 15 15

16 Main Types of Cell Death Necrosis Energy independent Caused by external stressors Occurs rapidly Loss of membrane integrity Cell lysis Apoptosis Necrosis Healthy cell Cellular and organelle swelling Apoptosis Cellular shrinking Energy dependent Normal physiological process Phospholipid inversion Formation of apoptotic blebs Breakdown of membrane, nucleus, and organelles; leakage of contents Cellular fragmentation Formation of apoptotic bodies Phagocytosis Inflammatory responses Baust, Advances in Biopreservation 16

17 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? 17 17

18 Understanding Effects of DMSO Ave VCD (10E6/mL) 3 Saline (7.5 % DMSO) Saline + CS10 (5 % DMSO) 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

19 Impact Of Basal Medium Saline Vs CSB Ave Viability (%) Run 1 Run 2 CSB CS10 Saline CS10 CSB CS10 Saline CS Pre-Freeze Hold (hr): Post-Thaw Hold (hr): Recovery Time (days): Significant difference in run 2. CSB may provide beneficial effects as basal medium

20 Cryopreservation Formulation Screen and Study Design Pre-freeze hold 3 hr Cryopreservation Thaw at 37 C (water bath) 1 and 2 hr hold 2-8 C 1 and 2 hr hold 25 C Cell Recovery 1 and 2 hr hold 37 C 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 * CSB- Cryostor Basal Solution 20 20

21 Ave Viability (%) Cell Viability At 2-8 C Hold 100 F1: Saline/PBS/HSA 7.5% DMSO F2: Saline/CS10 5% DMSO F3: CSB/CS10 5% DMSO F4: CSB/PBS/HSA 5% DMSO Pre-Freeze Hold (hr): Post-Thaw Hold (hr): Recovery Time (days): Formulation containing HSA and saline basal medium demonstrated low recovery. 21

22 Ave Viability (%) Cell Viability At 25 C Hold 100 F1: Saline/PBS/HSA 7.5% DMSO F2: Saline/CS10 5% DMSO F3: CSB/CS10 5% DMSO F4: CSB/PBS/HSA 5% DMSO Pre-Freeze Hold (hr): Post-Thaw Hold (hr): Recovery Time (days): Formulation containing HSA and saline basal medium demonstrated low recovery. 22

23 Ave Viability (%) Cell Viability At 37 C Hold F1: Saline/PBS/HSA 7.5% DMSO F2: Saline/CS10 5% DMSO F3: CSB/CS10 5% DMSO F4: CSB/PBS/HSA 5% DMSO Pre-Freeze Hold (hr): Post-Thaw Hold (hr): Recovery Time (days): Formulation containing HSA and saline basal medium demonstrated significantly low recovery. 23

24 2 hr. Pre-Freeze-hold Ave Viability (%) 1 hr. Pre-Freeze-hold How does cell density affect the viability? 95 Saline- CS10 (5% DMSO) Saline- 7.5% DMSO Total VCD (10E6/mL): Recovery Time (days): Cell density dependency is a function of DMSO concentration. Formulation and DMSO exposure could have incremental impact 24 24

25 Overall population in model-t cells post thaw % o f p a re n t p o p u la tio n O v e r a ll p o p u la tio n s O v e r a ll p o p u la tio n s L y m p h o c y t e s L y m p h o c y t e s C D 3 + in C D ( T c e lls ) C D 3 + in C D ( T c e lls ) C D 3 - in C D ( B c e lls + N K C D 3 - in C D ( B c e lls + N K c e lls ) h r 2 h r 1 h r 2 h r 1 h r 2 h r 1 h r 2 h r 1 h r 2 h r 1 h r 2 h r 1 h r 2 h r 1 h r 2 h r 5 0 E E E E E E E E 6 C S 1 0 S a lin e C S 1 0 S a lin e No change in overall cell population for model T-cells 25 25

26 c e lls /m L (x E 6 ) % % Reducing %DMSO on Pre-Freeze Apoptosis Profile of Cryopreserved T-Cells Pre-Freeze V ia b ilty A p o p to s is /N e c ro s is P ro file P B S /H S A /7.5 % D M S O 2 3 V ia b le C e ll D e n s ity 7.5% 5% 2.5% 1 % C S B /5 % D M S O C S B /2.5 % D M S O T = 0 m in T = 6 0 m in 4 C S B /1 % D M S O P B S /H S A /7.5 % D M S O 7.5% 5% 2.5% 1 % DMSO Amount (%) DMSO Amount (%) C S B /5 % D M S O C S B /2.5 % D M S O C S B /1 % D M S O H e a lth y E a rly A p o p to s is L a te A p o p to s is /N e c ro s is 26 26

27 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

28 Reducing %DMSO on Post-Thaw Apoptosis Profile of Cryopreserved T-Cells Early Apoptotic Cells Post Thaw Late Apoptotic/ Necrotic 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

29 Ave VCD CAR-T Cells : Effect of DMSO Concentration Saline- 7.5% DMSO Saline- CS 10 (5% DMSO) Pre-Freeze Hold (hr): Pre-Fill Hold (hr): Recovery Time (days): Similar trends as model-t cells High DMSO results in low recovery compared to low levels of DMSO 29

30 Ave Viability (%) How do model-t cell data stacks up with CAR-T cells in final selected formulation? CART (CSB:CS10) Model T cells (CSB:CS10) Run 1 Run 2 Run Pre-Freeze Hold (hr): Recovery Time (days): 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 30

31 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

32 Acknowledgements Michael Tennant Rachel Witts Ken Chrobak Tihami Qureshi Kim Rota Yajin Ni Christineh Hartoonian Mark Leonard Bruce Thompson 32 32