1. 01 October 2016 A Cell Banking Process for the Provision of Cryo-preserved, “Assay-Ready” Cells for Drug Discovery Programmes. Jim Cooper, ECACC

2. Background ECACC has been a commercial supplier of cryo- preserved cells for many years (core business). In the last three years: increased demand for cryo- preserved cells for cell based assays (contracts). Mostly with successful outcomes – however – some issues with consistency with rGPCR expressing lines. These issues have provided a driver to develop processes, better understand the needs of users and invest in infrastructure.

3. Industrial Process Development

4. Cell Lines for Cell Based Assay - Typical

5. A Hybrid Process to Reduce Risk and Increase Efficiency? Get to know the cell line (1) Growth Curve Profiling Live Cell Imaging Get to know the cell line (2) Metabolic Analysis DMSO Sensitivity Cryopreservation and Growth Strategy based on Assay Requirements Pilot Simulating Production at scale – directly relevant Work with Client to assure material produced is fit for purpose Production Banks As per Pilot - at required scale for assay programme, manufactured to optimised and piloted process

6. Live Cell Imaging: Invaluable Analysis and QC Tool Courtesy of Essen Instruments

7. Case Study – Recombinant GPCR CHO

9. Comparison with standard CHO: Lactate

10. Growth Curve Comparison Graphs: Incucyte™ HD

11. Qualitative Assessment Movie: Native CHO Click Here Click Here to View Movie Movie: CHO GPR Click Here Click Here to View Movie Images: Incucyte™ HD

12. Qualitative Assessment Movie: GPR CHO with Media Change at Critical Point (2 days post seeding and /or when lactate~5mM) Click Here Click Here to View Movie Images: Incucyte™ HD

13. Conclusion of Growth Profiling Seed at 1.5 x 10 4 cells / cm 2 Medium change cultures when [lactate] = 5-9mM (usually 48 hour post seed) Split of harvest when cells are optimal – i.e. 24 hours after medium exchange.

14. Outcome of not adhering to optimised process: Cells rapidly detach and round up Subculture not possible Plating for assay not possible GPR CHO 4 days in culture with no medium change Images: Incucyte™ HD

15. Optimising Harvest: DMSO Sensitivity Investigation Cells harvested 90% FBS + 10% DMSO added. Incubated at RT for 15mins, 30mins, 1hr & 2hr prior to cryopreservation Plating examined post resuscitation 2 hours DMSO exposure pre-freeze lead to rounded cells

16. DMSO Effect – plating from frozen after exposure to DMSO 15 mins30 mins120 mins Time Images: Leica ‘scope and camera

17. Cryopreservation Image courtesy of Planer

18. Assess Requirements 200 vials @ 7 x 10 6 cells/vial Specification: free of contamination, minimum 90% viability, confluent monolayer within 18-24 hours when seeded at 1.25 x 10 5 cells/cm 2 in multiwell plates. Plan Growth Strategy:

19. Tools of the Trade: Images Courtesy of Corning Inc

20. Growth Strategy – Modular Approach: Seed at 1.5 x 10 4 cell/cm 2 Medium change when lactate reaches 5-9mM Harvest at peak of log phase growth – referring to reference images and growth curve data (i.e. day after medium change) Harvest rapidly to avoid DMSO exposure Establish an intermediate, high density fill WCB, from which pilots / production runs can be generated. Pre-pilot process assessment to ensure process is robust at the limits dictated Pilot Production: modular – multiples of the pilot

21. Proposed Strategy (1) - WCB Client VialPassage  T175P+1  M/C  1 layerP+2  M/C  2 x 2 Layer cell stackP+3  M/C  Minimum 40 vials WCB (5.5 x 10e6 cells/vial) P+4

22. Proposed Strategy (2) - Pilot 1 WCB Vial (5.5 x 10e6 cells/vial) P+4  2 x T175P+5  M/C  5 Layer Cell StackP+6  M/C  ~50 vials at 7 x 10e6 cells/vialP+7

23. Proposed Strategy (3) – Production: Multiples of the pilot 1 WCB Vial (5.5 x 10e6 cells/vial)P+4  2 x T175P+5  M/C  5 Layer Cell StackP+6  M/C  50 vials at 7 x 10e6 cells/vialP+7 1 WCB Vial (5.5 x 10e6 cells/vial)P+4  2 x T175P+5  M/C  5 Layer Cell StackP+6  M/C  50 vials at 7 x 10e6 cells/vialP+7 1 WCB Vial (5.5 x 10e6 cells/vial)P+4  2 x T175P+5  M/C  5 Layer Cell StackP+6  M/C  50 vials at 7 x 10e6 cells/vialP+7 1 WCB Vial (5.5 x 10e6 cells/vial)P+4  2 x T175P+5  M/C  5 Layer Cell StackP+6  M/C  50 vials at 7 x 10e6 cells/vialP+7 200 vials at 7 x 10e6 cells / vial

24. Pre-pilot (Process development) Carried out during and as part of process development stage Grow cells to P+4 – freeze ~5 vials at 5.5 x 10e6/vial (WCB equivalent) Take one vial from WCB equivalent and produce ~5 vials at 7 x 10e6 cells/vial (Production equivalent) Test plating efficiency – Incucyte™ Send material to client for evaluation.

25. Client acceptance Pre-pilot material passes plating assessment (confluent monolayer in ~18 hours) and Functional Assessment by client. Acceptance gives go ahead for Pilot Acceptance of Pilot gives go ahead for Production

26. Results:Plating – From Frozen Incucyte™

27. Plating From Frozen Standard Incucyte™ Click Here Click Here to View Movie

28. Assay Ready Cells 18 Hours After Seeding from Frozen Standard Incucyte™

29. Functional Assessment by Client (1) Z prime: 0.6 – 0.8

30. Functional Assessment by Client (2) Z prime: 0.4 – 0.5

31. Conclusions Growth of recombinant GPCR expressing cell lines cannot always be expected to be equivalent to the host cell line. By using an industrial approach and working closely with the customer there was a successful outcome. Processes appropriate for general cell banking may not always produce frozen material fit for purpose for screening. Ongoing work required to fully optimise all parameters (addressing the vial filling bottleneck with automation, looking more closely at the harvest procedure). Live cell imaging (Incucyte™) is a key QC tool for ECACC in the provision of assay ready material.

32. Acknowledgements The ECACC General Collection Cell Banking Team:  Diane Fellows   Alex Hiscott  Liz Penn Thanks also to Nigel Jones and Pete Djali, Essen Instruments And our client; for allowing us to use their data.