| jade (with her mother) fabry disease usa upstream development of high cell density, perfusion...
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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 Standardization
Flexible
Core Drivers
Manufacturing,Process, &
Business DriversReduced Tech Transfer Risks
Efficient
time
Steady State Processes &
ProductQuality
Reduced Footprint
Variable
Steady state
Qu
ali
ty i
nd
ica
tor
VariableProblem
CaptureIntermediate Purification
PolishClarified Harvest
BioreactorMediaHarvest
HoldClarification
Unform DS
Perfusion
Fed-Batch
Current State – Biomanufacturing Processes Limited Standardization, large and complex
CaptureClarified Harvest
BioreactorMediaHarvest
HoldClarification
Perfusion
Continuous Biomanufacturing
ActionSteady-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
CaptureClarified Harvest
BioreactorMediaHarvest
HoldClarification
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 CaptureCaptureBioreactorMedia
Perfusion
Removes:
• Hold steps
• Clarification Ops.
Simplified Process
Key Technology
Simultaneous
Cell Separation and
Clarification
Continuous Biomanufacturing
Action
Benefit
Continuous captureCaptureBioreactorMedia
Perfusion
Reduced column size
and buffer usage
Key Technology
Periodic
Counter-Current
Chromatography
CaptureBioreactorMedia
Future State – Continuous BiomanufacturingStandard, Universal, Flexible
Integrated ContinuousBiomanufacturing
Unform.Drug
Substance
Predictable Performance
Universal Standardization
Flexible
Reduced Tech Transfer Risks
Efficient
time
Steady State Processes &
ProductQuality
Reduced Footprint
Variable
Steady state
Qu
ali
ty i
nd
ica
tor
Variable
Future State – Continuous BiomanufacturingStandard, 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 &
ProductQuality
Reduced Footprint
Variable
Steady state
Qu
ali
ty i
nd
ica
tor
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-state
production and
product 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-S
pec
ific
Per
fusi
on
Rat
e OPEX Savings
Favorable to Perfusion
VCD
Productivity
Volu
metr
ic P
rod
ucti
vit
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
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
.P
erf
usi
on
Fe
d-b
atc
h
~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
RG
row
thRa
teVi
abili
tyPr
oduc
tQ
ualit
y#1
Cell SpecificPerfusion
Rate
pH DOATF
ExchangeRate
Operational Space
• Determine acceptable operational space− Fixed cell specific perfusion rate
ATF Exchange
Rate
Acceptable Space
pHOut of Spec Regions
Green – 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 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 DevelopmentCommercial Cell Culture Development
Purification Development
Process Analytics
Early Process Development
Analytical Development
Translational Research
Many other colleagues at Genzyme
GE Healthcare
Acknowledgements