1 2 3 1Rachel Witts1 Michael Tennant2 Ken Chrobak3 Parag Kolhe1Rachel Witts1, Michael Tennant2, Ken Chrobak3, Parag Kolhe1Rachel Witts , Michael Tennant , Ken Chrobak , Parag Kolhe1 & S f C f O1Pharmaceutical R&D Biotherapeutics Pharm Sci Pfizer Inc Chesterfield MOPharmaceutical R&D, Biotherapeutics Pharm. Sci., Pfizer Inc., Chesterfield, MO2Bioprocess R&D Biotherapeutics Pharm Sci Pfizer Inc Chesterfield MO2Bioprocess R&D, Biotherapeutics Pharm. Sci., Pfizer Inc., Chesterfield, MOp p3BT PS T h & I Bi th ti Ph S i Pfi I S th S F i CA3BTxPS Tech & Innov, Biotherapeutics Pharm. Sci., Pfizer Inc., South San Francisco, CABTxPS Tech & Innov, Biotherapeutics Pharm. Sci., Pfizer Inc., South San Francisco, CA

ABSTRACT F l ti R b t St d CART C ll E l tiABSTRACT Formulation Robustness Study CART Cell EvaluationABSTRACT Formulation Robustness StudyC ll f l t d fill d i t 1 8 L i l t 50E6 ll / L d h ld t

CART Cell EvaluationCAR iti T ll f l t d i th l tf f l ti i CS10F l ti d l t f T ll i h ll i d t i Cells were formulated, filled into 1.8 mL vials at 50E6 cells/mL and held at CAR positive T-cells were formulated in the platform formulation or in CS10Formulation development for T-cells is challenging due to various Cells were formulated, filled into 1.8 mL vials at 50E6 cells/mL and held at

t t f t 3 h C ll f ft 120 i tCAR positive T cells were formulated in the platform formulation or in CS10

ith li th i t d f l t d t >50E60 ll / L iFormulation development for T cells is challenging due to various id ti i l di ti i ti f th h ld ti d i filli d

room temperature for up to 3 hours. Cells were frozen after 120 minutes with saline as the suspension reagent and formulated to >50E60 cells/mL in considerations including optimization of the hold times during filling and

p pand 180 minutes and held on stability in vPLN for up to 3 months

p g2 mL vials Cells were held pre fill up to 120 minutes and pre freeze up to ang p g g

ft th i f CART ll d i i t ti O ti i ti f thand 180 minutes and held on stability in vPLN2 for up to 3 months. 2 mL vials. Cells were held pre-fill up to 120 minutes and pre-freeze up to an

after thawing for CART cell administration. Optimization of the 2additional 120 minutes Post thaw cells were allowed to recover over 2g p

formulation of a cryopreservation medium for CART cells wasadditional 120 minutes. Post thaw, cells were allowed to recover over 2

formulation of a cryopreservation medium for CART cells was daysy pdetermined through small and large scale formulation screens utilizing a Table 1. Formulation Descriptions days. determined through small and large scale formulation screens utilizing a Table 1. Formulation Descriptions

Fi 5 A Vi bilit b F l ti P t Th CART llg g g

model T cell line Pan T cells were formulated in various combinations Formulation ID Basal medium 2X cryopreservation Final DMSO Figure 5. Average Viability by Formulation Post Thaw – CART cellsmodel T-cell line. Pan T-cells were formulated in various combinations Formulation ID Basal medium 2X cryopreservation di

Final DMSOt ti

g g y y

of harvest medium and cryopreservation medium to evaluate if any medium concentrationof harvest medium and cryopreservation medium to evaluate if any offered superior cell viability and viable cell density (VCD) following thaw F1 (platform) Saline (0 9%) PBS 8%HSA 15% DMSO 7 5% DMSOoffered superior cell viability and viable cell density (VCD) following thaw F1 (platform) Saline (0.9%) PBS, 8%HSA, 15% DMSO 7.5% DMSO

when compared to the platform formulation containing HSA and highwhen compared to the platform formulation containing HSA and high F2 Saline (0 9%) CryoStor10 5% DMSODMSO Viability and VCD were measured immediately after thaw and F2 Saline (0.9%) CryoStor10 5% DMSODMSO. Viability and VCD were measured immediately after thaw and

up to 3 days post reconstitution to determine any effect of theup to 3 days post reconstitution to determine any effect of the F3 CryoStor CSB CryoStor10 5% DMSOcryopreservation media on the cell recovery Initial screens showed that F3 CryoStor CSB CryoStor10 5% DMSOcryopreservation media on the cell recovery. Initial screens showed that

a difference between cryopreservation media could be observed anda difference between cryopreservation media could be observed and l f l ti b t t di fi d thi diff Th

F4 CryoStor CSB PBS, 8%HSA, 10% DMSO 5% DMSOlarger formulation robustness studies confirmed this difference. These

y , ,larger formulation robustness studies confirmed this difference. These f l ti f th l t d i CART ll Th lt f thformulations were further evaluated in CART cells. The results of these Following 37°C waterbath thaw cells were held at 4°C 22°C and 37°Cformulations were further evaluated in CART cells. The results of these t di fi th d l t f b t ti di

Following 37 C waterbath thaw, cells were held at 4 C, 22 C, and 37 C studies confirm the development of a robust cryopreservation medium for up to 2 hours. Viability and VCD were measured post-thaw and after 3p y pf T ll f l ti

for up to 2 hours. Viability and VCD were measured post thaw and after 3 for T-cell formulation. days of recovery.days of recovery.

Initial Formulation ScreensInitial Formulation ScreensComparison of Cryopreservation Media Figure 4 Average Viability by Formulation After Three Days of Recovery Post ThawComparison of Cryopreservation Media Figure 4. Average Viability by Formulation After Three Days of Recovery Post Thaw

f (S % S % (T0 data) The platform cryopreservation medium (Saline with 8%HSA and 15% (T0 data)The platform cryopreservation medium (Saline with 8%HSA and 15%

DMSO fi l DMSO t ti 7 5%) d t C St 10 Cells were also formulated in CS10 with CSB as the suspension reagent andDMSO; final DMSO concentration 7.5%) was compared to CryoStor10 Cells were also formulated in CS10 with CSB as the suspension reagent and ; ) p y(10% DMSO; final DMSO concentration 5%) All cells were suspended in are compared with Pan T-cells in the same formualtion(10% DMSO; final DMSO concentration 5%). All cells were suspended in are compared with Pan T cells in the same formualtion.( )saline and formulated to 50E6 cells/mLsaline and formulated to 50E6 cells/mL

Figure 6 Average Viability by Formulation Post Thaw CART cells versus Pan T cellsFigure 1 Formulation and Reconstitution Process Figure 6. Average Viability by Formulation Post Thaw CART cells versus Pan T-cellsFigure 1. Formulation and Reconstitution Process

Fill i 1 8 L H ld t RT Freeze after 60 H d H ld t RT f Measure viability andFormulate Fill in 1.8 mL

or 5 mL vialsHold at RT for 2 hours

Freeze after 60 and 120 Hand

thawHold at RT for up to 1 hour

Measure viability and VCD post-thaw and or 5 mL vials for 2 hours minutes thaw up to 1 hour pafter 3 day recovery

Figure 2 Average Viable Cell Density by Formulation in 5 mL Vials After Three DaysFigure 2. Average Viable Cell Density by Formulation in 5 mL Vials After Three Days of Recovery Post Thaw – 7.5% DMSO versus CS10of Recovery Post Thaw 7.5% DMSO versus CS10

Using a formulation with CS10 gives comparable results to the platformUsing a formulation with CS10 gives comparable results to the platform g g p pformulation Additionally comparable results were obtained from cellsformulation. Additionally, comparable results were obtained from cells formulated in CSB + CS10 when observed with CART cellsformulated in CSB + CS10 when observed with CART cells.

CONCLUSIONSCells formulated in CryoStor10 (CS10) showed better recovery than cells CONCLUSIONSCells formulated in CryoStor10 (CS10) showed better recovery than cells CONCLUSIONSformulated in the plaftorm formulation The data from the inital formulation screens showed that a discernibleformulated in the plaftorm formulation. The data from the inital formulation screens showed that a discernible

difference between formulations could be observed Pan T cellsComparison of Harvest Media difference between formulations could be observed. Pan T-cells Comparison of Harvest Media formulated in CryoStor 10 showed better recovery than cells formulatedp formulated in CryoStor 10 showed better recovery than cells formulated To determine if harvesting in Cryostor Basal (CSB) medium would provide in the platform formulation after 3 days and suspending cells in CSB To determine if harvesting in Cryostor Basal (CSB) medium would provide

dd d ti ff t li C ll f l t d t 50E6in the platform formulation after 3 days and suspending cells in CSB

added cryopreservative effects over saline. Cells were formulated to 50E6 rather than saline may provide a slight improvement in recovery fromadded cryopreservative effects over saline. Cells were formulated to 50E6 ll / L

rather than saline may provide a slight improvement in recovery from cells/mL. cryopreservationcryopreservation.

Figure 3. Average Viable Cell Density by Formulation in 5 mL vials after Three DaysFigure 3. Average Viable Cell Density by Formulation in 5 mL vials after Three Days f R P t Th (2 R ) S li CSB The large scale formulation study confirmed the platform formulationof Recovery Post Thaw (2 Runs) – Saline versus CSB The large scale formulation study confirmed the platform formulation y ( )

performed worse than the other formulations tested There was littleperformed worse than the other formulations tested. There was little difference between the results of post thaw hold at 4°C and at 22°C butdifference between the results of post-thaw hold at 4 C and at 22 C, but post-thaw hold at 37°C showed a decrease in viability and VCD for thepost-thaw hold at 37 C showed a decrease in viability and VCD for the platform formulationplatform formulation.

Examination of the formulations on CART Cells showed that using CS10Examination of the formulations on CART Cells showed that using CS10 in the formulations gave equal viabilities and VCD This allows for thein the formulations gave equal viabilities and VCD. This allows for the possibility of moving to a formulation without HSA Additionally whenpossibility of moving to a formulation without HSA. Additionally when compared to Pan T-cell data there was not a significant difference whencompared to Pan T-cell data, there was not a significant difference when formulated in CS10 with CSB In pan T-cells clumping was observed informulated in CS10 with CSB. In pan T cells, clumping was observed in some Saline containing formulations suggesting the need to move to asome Saline containing formulations, suggesting the need to move to a CSB b d f l i F h l i ill b d dCSB based formulation. Further evaluation will be conducted toCSB based formulation. Further evaluation will be conducted to d t i th fi l f l ti f th CART lldetermine the final formulation for the CART cells.determine the final formulation for the CART cells.

The platform formulation showed decreased viability and recoverable TVC ACKNOWLEDGEMENTSThe platform formulation showed decreased viability and recoverable TVC ACKNOWLEDGEMENTS(data not shown) at 37°C compared to the other formulationsCryoStor Basal media may provide some added cryopreservative effects over

C O G S(data not shown) at 37 C compared to the other formulations. CryoStor Basal media may provide some added cryopreservative effects over Thanks to the Wuxi collaboration for the formulation robustness data and the entire Cell & Gene

salinea s to t e u co abo at o o t e o u at o obust ess data a d t e e t e Ce & Ge e

Therapy and PharmSci CART teamsaline. Therapy and PharmSci CART team.