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

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

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

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

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

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

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

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

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

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

Steady-State Upstream Control Steady-state cell density Steady-state nutrient availability Steady-state metabolism Steady-state product quality Cell Specific Perfusion Rate = Perfusion Rate Cell Density Viable Cell Mass Indicator VCD

Cell Density Control Strategies Viable Cell Mass Indicators Capacitance Oxygen sparge Oxygen uptake rate Others r2 = 0.70

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

Steady-State Product Quality Over 60 days Glycosylation Profiling Peak 1 Peak 4 Peak 5 Peak 7 Peak 8 Peak 11

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

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

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

Process Development Design 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

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

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

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

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

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

Scale-up Results Growth and Metabolism Cell Density Oxidative Glucose Metabolism Growth rate and metabolism are as expected

Scale-up Results Productivity Product Quality #1 Productivity and product quality are as expected

Scale-up Results Continuous Chromatography Integration Capture operation using three column PCC Fully automated Steady-state performance UV Chromatogram SDS PAGE for Capture Elution 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

Reactor Scale Considerations Productivity 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

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

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