Perfusion

Report
UPSTREAM DEVELOPMENT OF HIGH CELL DENSITY,
PERFUSION PROCESSES FOR CONTINUOUS
MANUFACTURING
Tim Johnson, Ph.D.
October 21, 2013
Jade (with her mother) Fabry disease USA
www.genzyme.com
|
Discussion Points
• Perspectives on Continuous Manufacturing
• Upstream Development
− Steady-State Control
− Approach to Process Development
− Scale-Up
• Conclusions
Continuous Integrated Biomanufacturing
Drivers
Predictable Performance
Manufacturing,
Process, &
Business Drivers
Efficient
Flexible
Universal
Standardization
Reduced Footprint
Reduced Tech Transfer Risks
Core Drivers
Problem
Steady State
Processes &
Product
Quality
Quality indicator
Steady state
Variable
Variable
time
Simplicity
Current State – Biomanufacturing Processes
Limited Standardization, large and complex
Media
Bioreactor
Fed-Batch
Perfusion
Harvest
Hold
Clarification
Clarified
Harvest
Capture
Intermediate
Purification
Polish
Unform
DS
Continuous Biomanufacturing
Action
Steady-State
Media
Bioreactor
Harvest
Hold
Clarification
Clarified
Harvest
Capture
High Cell Density
High Productivity
Key Technology
High Sp. Production Rate
Low Perfusion Rate
Perfusion
Continuous Biomanufacturing
Action
Steady-State
Media
Bioreactor
Harvest
Hold
Clarification
Clarified
Harvest
Capture
High Cell Density
High Productivity
Key Technology
High Sp. Production Rate
Low Perfusion Rate
Perfusion
Benefit
Reduced Bioreactor Size
SUBs now feasible
Standardized Size
Universal – mAbs/Enz
Continuous Biomanufacturing
Action
Media
Bioreactor
Capture
Continuous flow
Bioreactor  Capture
Key Technology
Simultaneous
Cell Separation and
Clarification
Perfusion
Benefit
Removes:
• Hold steps
• Clarification Ops.
Simplified Process
Continuous Biomanufacturing
Action
Media
Bioreactor
Capture
Continuous capture
Key Technology
Periodic
Counter-Current
Chromatography
Perfusion
Benefit
Reduced column size
and buffer usage
Future State – Continuous Biomanufacturing
Standard, Universal, Flexible
Integrated Continuous
Biomanufacturing
Predictable Performance
Efficient
Bioreactor
Universal
Capture
Standardization
Reduced Footprint
Reduced Tech Transfer Risks
Unform.
Drug
Substance
Steady state
Steady State
Processes &
Product
Quality
Quality indicator
Media
Flexible
Variable
Variable
time
Future State – Continuous Biomanufacturing
Standard, Nearly Universal, Flexible
Facilitating Aspects
Predictable Performance
Efficient
Flexible
Universal
Process
Knowledge
Standardization
Reduced Footprint
Reduced Tech Transfer Risks
PAT &
Control
Robust
Equipment
& Design
Steady State
Processes &
Product
Quality
Quality indicator
Steady state
Variable
Variable
time
Steady-State
Upstream Control
Steady-state cell density
Steady-state nutrient availability
Cell Specific Perfusion Rate =
Perfusion Rate
Cell Density
 Steady-state metabolism
 Steady-state product quality
VCD
Viable Cell Mass Indicator
Cell Density Control Strategies
r2 = 0.88
r2 = 0.73
Viable Cell Mass Indicators




12
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 7
Peak 4
Peak 8
Peak 5
Peak 11
High Cell Density – High Productivity
mAb Demonstration
• OPEX drivers for continuous biomanufacturing Vs. fed-batch
− High volumetric productivity
− Low media cost
Productivity
VCD
Cell-Specific Perfusion Rate
− Low perfusion rate
Volumetric Productivity (g/L-d)
− High cell density
OPEX Savings
Favorable to
Perfusion
Viable cell density
Outline
• Perspectives on Continuous Manufacturing
• Upstream Development
− Steady-State Control
− Approach to Process Development
− Scale-Up
Process
Knowledge
• Conclusions
PAT &
Control
Robust
Equipment
& Design
Process Development
Design of Experiments
• Unrealistic timelines required to study full process (60 days/run)
• Leverage steady-state to condense experiments
S.S.
Perfusion
15 weeks
40 weeks
F1
F2
F3
F4
SET 1 SET
SET
1 2
SET 3
SET
4 2
SET
Fed-batch
~11-15 weeks
F1
F2
F3
F4
SET 2
SET 3
SET 4
Measure
response
shift
SET 1
SET 3
SET 4
Process Development
Design of Experiments
• Approach
− Four factors determined from screening studies
− Cell Specific Perfusion Rate
− pH
ATF
Exchange Rate
− Dissolved Oxygen
− ATF Exchange Rate
− Custom design with interaction effects  24 conditions
Design of Experiments
Results
• Little interaction effects
Viability
Rate is the most significant
factor
Product
Quality
#1
• Cell Specific Perfusion
Growth
Rate
over the ranges tested
SPR
• Culture generally stable
Cell Specific
Perfusion
Rate
pH
DO
ATF
Exchange
Rate
Operational Space
• Determine acceptable operational space
− Fixed cell specific perfusion rate
pH
ATF
Exchange
Rate
Acceptable
Space
Dissolved
Oxygen
Out of Spec Regions
Green – Viability
Red – Growth rate
Blue – Product Quality #1
Integrated Operating Spaces
Example
 Integrating upstream and downstream process knowledge
 Upstream: Productivity ↓ below critical pH value
 Downstream: Yield recovery ↓ as pH ↑
Capture
Yield
Combined
Productivity
Yield
Productivity
Reactor Productivity
Optimum pH
Solution
pH
 Optimal pH exists to maximize productivity and yield
Outline
• Perspectives on Continuous Manufacturing
• Upstream Development
− Steady-State Control
− Approach to Process Development
− Scale-Up
Process
Knowledge
• Conclusions
PAT &
Control
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
SUB
− 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
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
DS
Harvest Day 17 - 35
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

similar documents