|www.genzyme.com

Jade (with her mother) Fabry disease USA

UPSTREAM DEVELOPMENT OF HIGH CELL DENSITY, PERFUSION PROCESSES FOR CONTINUOUS

MANUFACTURINGTim 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 BiomanufacturingDrivers

Predictable Performance

Simplicity

Universal StandardizationFlexible

Core Drivers

Manufacturing,Process, &

Business DriversReduced Tech Transfer Risks

Efficient

time

Steady State Processes &

ProductQuality

Reduced Footprint

Variable

Steady state

Qua

lity

indi

cato

r

VariableProblem

Capture Intermediate Purification PolishClarified

HarvestBioreactorMedia Harvest Hold Clarification Unform

DS

Perfusion

Fed-Batch

Current State – Biomanufacturing Processes Limited Standardization, large and complex

CaptureClarified HarvestBioreactorMedia Harvest

Hold Clarification

Perfusion

Continuous Biomanufacturing

ActionSteady-State

High Cell DensityHigh Productivity

Key Technology

High Sp. Production RateLow Perfusion Rate

Continuous Biomanufacturing

Action

Benefit

Steady-StateHigh Cell DensityHigh Productivity

CaptureClarified HarvestBioreactorMedia Harvest

Hold Clarification

Perfusion

Reduced Bioreactor SizeSUBs now feasibleStandardized Size

Universal – mAbs/Enz

Key Technology

High Sp. Production RateLow Perfusion Rate

Continuous Biomanufacturing

Action

Benefit

Continuous flowBioreactor Capture

CaptureBioreactorMedia

Perfusion

Removes: • Hold steps• Clarification Ops.

Simplified Process

Key Technology

SimultaneousCell Separation and

Clarification

Continuous Biomanufacturing

Action

Benefit

Continuous captureCaptureBioreactorMedia

Perfusion

Reduced column size and buffer usage

Key Technology

Periodic Counter-CurrentChromatography

CaptureBioreactorMedia

Future State – Continuous BiomanufacturingStandard, Universal, Flexible

Integrated ContinuousBiomanufacturing

Unform.Drug

Substance

Predictable Performance

Universal StandardizationFlexible

Reduced Tech Transfer Risks

Efficient

time

Steady State Processes &

ProductQuality

Reduced Footprint

Variable

Steady state

Qua

lity

indi

cato

r

Variable

Future State – Continuous BiomanufacturingStandard, Nearly Universal, Flexible

PAT & Control

Process Knowledge

Robust Equipment & Design

Facilitating Aspects Predictable Performance

Universal StandardizationFlexible

Reduced Tech Transfer Risks

Efficient

time

Steady State Processes &

ProductQuality

Reduced Footprint

Variable

Steady state

Qua

lity

indi

cato

r

Variable

Steady-state cell densitySteady-state nutrient availability

Steady-state metabolism Steady-state product quality

Steady-StateUpstream 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-stateproduction andproduct quality CQA #3

Volumetric Productivity

CQA #1

CQA #2

Glycosylation Profiling

Steady-State Product QualityOver 60 days

Peak 1 Peak 4 Peak 5

Peak 7 Peak 8 Peak 11

break-even

• OPEX drivers for continuous biomanufacturing Vs. fed-batch

− High cell density

− High volumetric productivity

High Cell Density – High ProductivitymAb 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

Volu

met

ric

Prod

ucti

vity

(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

F1F2F3F4

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 DevelopmentDesign of Experiments

S.S.

Perf

usio

nFe

d-ba

tch

~11-15 weeks

15 weeks

SET 1 SET 2 SET 3 SET 4

F1F2F3F4

Measureresponse

shift

SET 1 SET 2 SET 3 SET 4

F1F2F3F4

• 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 DevelopmentDesign of Experiments

ATFExchange Rate

Design of ExperimentsResults

• Culture generally stable over the ranges tested

• Cell Specific Perfusion Rate is the most significant factor

• Little interaction effectsSP

RGr

owth

Rate

Viab

ility

Prod

uct

Qua

lity

#1

Cell SpecificPerfusion

Rate

pH DOATF

ExchangeRate

Operational Space

• Determine acceptable operational space− Fixed cell specific perfusion rate

ATF Exchange

Rate

Acceptable Space

pH Out of Spec RegionsGreen – ViabilityRed – Growth rateBlue – Product Quality #1

Dissolved Oxygen

Reactor ProductivityCapture

Yield

CombinedProductivity

Optimum pH

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

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 ResultsGrowth and Metabolism

Cell Density Oxidative Glucose Metabolism

• Growth rate and metabolism are as expected

Scale-up ResultsProductivity

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 - 35DS

Warikoo, Veena, et al. Integrated continuous production of recombinant therapeutic proteins. Biotech. & Bioeng. v109, 3018-3029; 2012Godawat, 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 ConsiderationsProductivity Possibilities

50L can meet some low demand products500L 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 DevelopmentCommercial Cell Culture DevelopmentPurification DevelopmentProcess Analytics

Early Process Development

Analytical Development

Translational Research

Many other colleagues at Genzyme

GE Healthcare

Acknowledgements