| www.genzyme.com

Jade (with her mother) Fabry disease USA

UPSTREAM DEVELOPMENT OF HIGH CELL DENSITY, PERFUSION PROCESSES FOR CONTINUOUS

MANUFACTURING Tim Johnson, Ph.D. October 21, 2013

• Perspectives on Continuous Manufacturing

• Upstream Development

− Steady-State Control

− Approach to Process Development

− Scale-Up

• Conclusions

Discussion Points

Continuous Integrated Biomanufacturing Drivers

Predictable Performance

Simplicity

Universal Standardization Flexible

Core Drivers

Manufacturing, Process, &

Business Drivers Reduced Tech Transfer Risks

Efficient

time

Steady State Processes &

Product Quality

Reduced Footprint

Variable

Steady state

Qua

lity

indi

cato

r

Variable Problem

Capture Intermediate Purification Polish Clarified

Harvest Bioreactor Media Harvest Hold Clarification Unform

DS

Perfusion

Fed-Batch

Current State – Biomanufacturing Processes Limited Standardization, large and complex

Capture Clarified Harvest Bioreactor Media Harvest

Hold Clarification

Perfusion

Continuous Biomanufacturing

Action Steady-State

High Cell Density High Productivity

Key Technology

High Sp. Production Rate Low Perfusion Rate

Continuous Biomanufacturing

Action

Benefit

Steady-State High Cell Density High Productivity

Capture Clarified Harvest Bioreactor Media Harvest

Hold Clarification

Perfusion

Reduced Bioreactor Size SUBs now feasible Standardized Size

Universal – mAbs/Enz

Key Technology

High Sp. Production Rate Low Perfusion Rate

Continuous Biomanufacturing

Action

Benefit

Continuous flow Bioreactor Capture

Capture Bioreactor Media

Perfusion

Removes: • Hold steps • Clarification Ops.

Simplified Process

Key Technology Simultaneous

Cell Separation and Clarification

Continuous Biomanufacturing

Action

Benefit

Continuous capture Capture Bioreactor Media

Perfusion

Reduced column size and buffer usage

Key Technology Periodic

Counter-Current Chromatography

Capture Bioreactor Media

Future State – Continuous Biomanufacturing Standard, Universal, Flexible

Integrated Continuous Biomanufacturing

Unform. Drug

Substance

Predictable Performance

Universal Standardization Flexible

Reduced Tech Transfer Risks

Efficient

time

Steady State Processes &

Product Quality

Reduced Footprint

Variable

Steady state

Qua

lity

indi

cato

r

Variable

Future State – Continuous Biomanufacturing Standard, Nearly Universal, Flexible

PAT & Control

Process Knowledge

Robust Equipment & Design

Facilitating Aspects Predictable Performance

Universal Standardization Flexible

Reduced Tech Transfer Risks

Efficient

time

Steady State Processes &

Product Quality

Reduced Footprint

Variable

Steady state

Qua

lity

indi

cato

r

Variable

Steady-state cell density Steady-state nutrient availability Steady-state metabolism Steady-state product quality

Steady-State Upstream Control

VCD

Cell Specific Perfusion Rate = Perfusion Rate

Cell Density

Viable Cell Mass Indicator

Cell Density Control Strategies

12

r2 = 0.88 r2 = 0.73

r2 = 0.70

Viable Cell Mass Indicators Capacitance Oxygen sparge Oxygen uptake rate Others

Steady cell density and growth

Steady-State Upstream Demonstration

Steady-state metabolism

Steady-state production and product quality CQA #3

Volumetric Productivity

CQA #1

CQA #2

Glycosylation Profiling

Steady-State Product Quality Over 60 days

Peak 1 Peak 4 Peak 5

Peak 7 Peak 8 Peak 11

• OPEX drivers for continuous biomanufacturing Vs. fed-batch − High cell density

− High volumetric productivity

High Cell Density – High Productivity mAb Demonstration

− Low perfusion rate

− Low media cost

Viable cell density

Cel

l-Spe

cific

Per

fusi

on R

ate OPEX Savings

Favorable to Perfusion

VCD

Productivity

Vol

umet

ric

Pro

duct

ivit

y (g

/L-d

)

• Perspectives on Continuous Manufacturing

• Upstream Development

− Steady-State Control

− Approach to Process Development

− Scale-Up

• Conclusions

Outline

PAT & Control

Process Knowledge

Robust Equipment & Design

F1 F2 F3 F4

SET 1 SET 2 SET 3 SET 4

40 weeks

• Unrealistic timelines required to study full process (60 days/run)

• Leverage steady-state to condense experiments

Process Development Design of Experiments

S.S.

Pe

rfus

ion

Fed-

batc

h

~11-15 weeks

15 weeks

SET 1 SET 2 SET 3 SET 4

F1 F2 F3 F4

Measure response

shift

SET 1 SET 2 SET 3 SET 4

F1 F2 F3 F4

• Approach − Four factors determined from screening studies

− Cell Specific Perfusion Rate

− pH

− Dissolved Oxygen

− ATF Exchange Rate

− Custom design with interaction effects 24 conditions

Process Development Design of Experiments

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

SP

R G

row

th

Rate

Vi

abili

ty

Prod

uct

Qua

lity

#1

Cell Specific Perfusion

Rate

pH DO ATF

Exchange Rate

Operational Space

• Determine acceptable operational space − Fixed cell specific perfusion rate

ATF Exchange

Rate

Acceptable Space

pH Out of Spec Regions Green – Viability Red – Growth rate Blue – Product Quality #1

Dissolved Oxygen

Reactor Productivity Capture

Yield

Combined Productivity

Optimum pH

Integrated Operating Spaces Example

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

Prod

uctiv

ity

Yield

pH

• Perspectives on Continuous Manufacturing

• Upstream Development

− Steady-State Control

− Approach to Process Development

− Scale-Up

• Conclusions

Outline

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

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

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

S.S. Harvest Feed

Consistent Capture Duration and Frequency

Reactor Scale Considerations Productivity Possibilities

50L can meet some low demand products 500L can meet average demand products

* Kelly, Brian. Industrialization of mAb production technology: The bioprocessing industry at a crossroads. mAbs 1:5, 443-452; 2009

*

50L

500L

Further optimization

#

Summary and Conclusions

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

Simplicity

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

Acknowledgements