UPSTREAM DEVELOPMENT OF HIGH CELL DENSITY, PERFUSION PROCESSES FOR CONTINUOUS MANUFACTURING

1. UPSTREAM DEVELOPMENT OF HIGH CELL DENSITY, PERFUSION PROCESSES FOR CONTINUOUS MANUFACTURING Tim Johnson, Ph.D. October 21, 2013

2. Discussion Points • Perspectives on Continuous Manufacturing • Upstream Development • Steady-State Control • Approach to Process Development • Scale-Up • Conclusions

3. Continuous Integrated BiomanufacturingDrivers Simplicity Steady state Predictable Performance Manufacturing, Process, & Business Drivers Efficient Flexible Variable Universal Standardization Reduced Footprint Reduced Tech Transfer Risks Steady State Processes & Product Quality Core Drivers Quality indicator Variable Problem time

4. Current State – Biomanufacturing Processes Limited Standardization, large and complex Bioreactor Harvest Hold Clarification Clarified Harvest Capture Intermediate Purification Polish Media Unform DS Fed-Batch Perfusion

5. Continuous Biomanufacturing Action Steady-State High Cell Density High Productivity Bioreactor Harvest Hold Clarification Clarified Harvest Capture Media Key Technology High Sp. Production Rate Low Perfusion Rate Perfusion

6. Continuous Biomanufacturing Action Steady-State High Cell Density High Productivity Bioreactor Harvest Hold Clarification Clarified Harvest Capture Media Key Technology High Sp. Production Rate Low Perfusion Rate Perfusion Benefit Reduced Bioreactor Size SUBs now feasible Standardized Size Universal – mAbs/Enz

7. Continuous Biomanufacturing Action Continuous flow Bioreactor  Capture Bioreactor Capture Media Key Technology Simultaneous Cell Separation and Clarification Perfusion Benefit • Removes: • Hold steps • Clarification Ops. • Simplified Process

8. Continuous Biomanufacturing Action Continuous capture Bioreactor Capture Media Key Technology Periodic Counter-Current Chromatography Perfusion Benefit Reduced column size and buffer usage

9. Future State – Continuous Biomanufacturing Standard, Universal, Flexible Integrated Continuous Biomanufacturing Steady state Predictable Performance Efficient Flexible Variable Universal Standardization Bioreactor Capture Media Reduced Footprint Reduced Tech Transfer Risks Unform. Drug Substance Steady State Processes & Product Quality Quality indicator Variable time

10. Future State – Continuous Biomanufacturing Standard, Nearly Universal, Flexible Steady state Predictable Performance Facilitating Aspects Efficient Flexible Variable Process Knowledge Universal Standardization Reduced Footprint Reduced Tech Transfer Risks PAT & Control Robust Equipment & Design Steady State Processes & Product Quality Quality indicator Variable time

11. Steady-StateUpstream Control Steady-state cell density Steady-state nutrient availability • Steady-state metabolism • Steady-state product quality VCD Viable Cell Mass Indicator Perfusion Rate Cell Specific Perfusion Rate = Cell Density

12. Cell Density Control Strategies r2 = 0.88 r2 = 0.73 • Viable Cell Mass Indicators • Capacitance • Oxygen sparge • Oxygen uptake rate • Others r2 = 0.70

13. Steady-State Upstream Demonstration Steady-state metabolism Steady cell density and growth Volumetric Productivity Steady-state production and product quality CQA #1 CQA #2 CQA #3

14. Steady-State Product QualityOver 60 days Glycosylation Profiling Peak 1 Peak 4 Peak 5 Peak 7 Peak 8 Peak 11

15. High Cell Density – High ProductivitymAb Demonstration • OPEX drivers for continuous biomanufacturing Vs. fed-batch • High cell density • High volumetric productivity • Low perfusion rate • Low media cost OPEX Savings Productivity break-even Cell-Specific Perfusion Rate Volumetric Productivity (g/L-d) VCD Favorable to Perfusion Viable cell density

16. Outline • Perspectives on Continuous Manufacturing • Upstream Development • Steady-State Control • Approach to Process Development • Scale-Up • Conclusions Process Knowledge Robust Equipment & Design PAT & Control

17. Process DevelopmentDesign of Experiments • Unrealistic timelines required to study full process (60 days/run) • Leverage steady-state to condense experiments 40 weeks 15 weeks F1 F2 F3 F4 S.S. Perfusion F1 F2 F3 F4 SET 1 SET 2 SET 3 SET 4 SET 1 SET 2 SET 3 SET 4 ~11-15 weeks Measure response F1 F2 F3 F4 shift Fed-batch SET 1 SET 2 SET 3 SET 4

18. Process DevelopmentDesign of Experiments • Approach • Four factors determined from screening studies • Cell Specific Perfusion Rate • pH • Dissolved Oxygen • ATF Exchange Rate • Custom design with interaction effects  24 conditions ATF Exchange Rate

19. Design of ExperimentsResults • Culture generally stable over the ranges tested • Cell Specific Perfusion Rate is the most significant factor • Little interaction effects SPR Product Quality #1 Viability Growth Rate ATF Exchange Rate Cell Specific Perfusion Rate pH DO

20. Operational Space • Determine acceptable operational space • Fixed cell specific perfusion rate pH Out of Spec Regions Green– Viability Red– Growth rate Blue– Product Quality #1 Acceptable Space ATF Exchange Rate Dissolved Oxygen

21. Integrated Operating SpacesExample • Integrating upstream and downstream process knowledge • Upstream: Productivity ↓ below critical pH value • Downstream: Yield recovery ↓ as pH ↑ Solution • Optimal pH exists to maximize productivity and yield Reactor Productivity Capture Yield Yield Combined Productivity Productivity Optimum pH pH

22. Outline • Perspectives on Continuous Manufacturing • Upstream Development • Steady-State Control • Approach to Process Development • Scale-Up • Conclusions Process Knowledge Robust Equipment & Design PAT & Control

23. Scale-up to Single Use Bioreactor • Skid • Custom HyClone 50L Turnkey System • Bioreactor customized for perfusion • Nine control loops • Scale-up approach • Match scale independent parameters • Accounted for scale dependent parameters • Agitation: match bulk P/V • Initial Run • Conservative 40 Mcells/ml set-point • 60+ day operation • 10L satellite running concurrently SUB ATF

24. Scale-up ResultsGrowth and Metabolism Oxidative Glucose Metabolism Cell Density • Growth rate and metabolism are as expected

25. Scale-up ResultsProductivity Productivity Product Quality #1 • Productivity and product quality are as expected

26. Scale-up Results Continuous Chromatography Integration • Capture operation using three column PCC • Fully automated • Steady-state performance SDS PAGE for Capture Elution UV Chromatogram Harvest Day 17 - 35 DS S.S. Harvest Feed Consistent Capture Duration and Frequency Warikoo, Veena, et al. Integrated continuous production of recombinant therapeutic proteins. Biotech. & Bioeng. v109, 3018-3029; 2012 Godawat, Rahul, et al. Periodic counter-current chromatography – design and operational considerations for integrated and continuous purification of proteins. Biotech. Journal v7, 1496-1508; 2012

27. Reactor Scale ConsiderationsProductivity Possibilities 50L can meet some low demand products 500L can meet average demand products Further optimization * 500L 50L # * Kelly, Brian. Industrialization of mAb production technology: The bioprocessing industry at a crossroads. mAbs 1:5, 443-452; 2009

28. Summary and Conclusions Simplicity • Core drivers achieved • Achieved robust and steady-state control • Developed methodology for efficient process understanding • Successfully scaled-up upstream process to 50L SUB • Platform routinely being applied to mAbs and Enzymes • Simplicity and design for manufacturability considerations are a cornerstone of our continuous & integrated platform • Additional challenges remain

29. Acknowledgements Genzyme/Sanofi Industrial Affairs Late Stage Process Development Commercial Cell Culture Development Purification Development Process Analytics Early Process Development Analytical Development Translational Research Many other colleagues at Genzyme GE Healthcare