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Application of Disposable Bioreactors for Biopharmaceutical Production
Andreas Castan, PhDGE Healthcare Bio-Sciences AB
2Nordic Bioprocess Improvement Seminar 2012
3/29/2012
Overview
• Introduction• Disposables and GE Healthcare
• Case studies: Production of biopharmaceuticals in disposable WAVE Bioreactors™
• High yield antibody production using process intensification
• Vaccine manufacturing platform using disposables
• Conclusions
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Accelerate process development
Market growth• 15% annual growth
• >Vaccine growth
• >150 MAbs in clinics
Single-Use Approach
Capacity bandwidth
Cost pressure• Healthcare reform
• Biogenerics
• Follow-on drugs
Smaller markets• Fewer blockbusters
• Personalized medicine
• Genetic diagnostics
New technology• Higher yields
• Potent compounds
• Drug delivery
Smaller batch sizes
Pressure on R&D budgets
Why bio-disposables?
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ReadyToProcess™ platform
Bioreactor feed and media filtration
Bioreactor Cell liquid clarification filtration
Connectivity Column protection Chromatography Final fill filtration
SPEED SIMPLICITY SAFETY
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Case study 1:High yield antibody production-Process intensification using perfusion culture in a WAVE Bioreactor™
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Challenges in recombinant protein production
Increase overall productivity
Optimize product concentration
Develop a robust process
Maintain/improve protein quality
Reduce process and changeover time
Efficient use of facility
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Process intensification with perfusion in comparison to batch and fed-batch
High cell concentrationStable culture conditions
Time for protein expression extendedHigh titers with optimized media
Use of disposables facilitatedIncreased flexibility
Continuous process
Improved productivity
Smaller bioreactors
Optimized equipment utilization
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Experimental setup
Schneider 2 (S2) insect cells producing mAb against hemagglutiningrown in a disposable WAVE Bioreactor™ system
Comparison of cultures in batch and perfusion mode
Perfusion control via loadcell, cells retained by filter integrated into Cellbag™
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Culture conditions
Protein expression induced at cell concentration of 10 to 20x106 c/mlDO control: O2 concentration in airflow adjusted automatically, manual increase of agitation
Cell Line S2 cells, producing mAb against hemagglutinin, inducible expression system
Working volume 0.85 L in Cellbag™ 2L with or without internal filter
Agitation 22 rpm/8° – 25 rpm/9° (batch), 22 rpm/8° – 27 rpm/9°(perfusion)
Aeration Headspace aeration, O2 supplementation to 50% O2
Batch culture Inoculated from shake flask, terminated at 60% viability
Perfusion culture Inoculated from shake flask, perfusion rate initially 0.3 cultivation volumes (CV), increased to max. 1.5 CV, based on keeping residual glucose conc. at 3-4 g/L
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Process time [d]
0 2 4 6 8 10 12 14 16 18 20 22
Cel
l con
c. [c
/ml]
1,0e+6
2,0e+6
4,0e+66,0e+61,0e+7
2,0e+7
4,0e+76,0e+71,0e+8
2,0e+8
Via
bilit
y [%
]
102030405060708090100 Cell conc. batch
Cell conc. perfusion Viability batch Viability perfusion
Increased viable cell concentration
Cumulated viable cell integral ~ 10 times larger than in batch
Perfusion started day 6, controlled via reactor weightCell free harvest through filterNutrient supply stabilized viability in perfusion culture
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Process time [d]
0 2 4 6 8 10 12 14 16 18 20 22
Glc
[mg/
L]
0
2000
4000
6000
8000
10000
Lac
[mg/
L]
0
200
400
600
800
1000
Perf.
rate
[vvd
]
0,0
0,5
1,0
1,5
2,0Glc Batch Glc Perfusion Lac Batch Lac PerfusionPerfusion rate
Stabilized metabolite concentrations
Perfusion to keep residual glucose around 3 g/LStable lactate concentration in perfusionNutrient limitation in batch culture
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qGlc
[pg/
c/d]
-80
-60
-40
-20
0
qLac
, qP
[pg/
c/d]
0
2
4
6
8
10
12
14q Glc batch uninduced q Glc batch induced q Glc perfusion uninduced q Glc perfusion induced q Lac batch uninduced q Lac batch induced q Lac perfusion uninducedq Lac perfusion induced Av. qP batchAv. qP perfusion
Metabolic activity maintained
Recombinant protein production increases metabolic load Perfusion stabilized cell metabolismBatch culture with higher Glc/Lac conversion
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Process time [d]0 8 10 12 14 16 18 20 22
STY
[mg/
L R
V/d
]
1
10
100
1000
10000Ig
G [m
g/L]
0200400600800
10001200 STY batch
STY perfusion Product conc. batch Product conc. perfusion
High producitvity and titer
High product conc. ensured cost effective use of mediumProtein production induced on day 10
Productivity maintained throughout process time
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Protein quality assessment
After protein G purification:Comparable product from batch and perfusion culture according to WB and Coomassie blue stain
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Summary batch and perfusion culture
Perfusion: 10 x higher cell concentration 20 x higher volumetric productivity 85% reduced upstream consumable cost
Batch Culture
Perfusion Culture
Process time [d] 16 21
Working volume [L] 0.85 0.85
Total media consumption [L] 0.85 10.80
Maximum viable cell conc. [c/ml] 1.06E+07 1.04E+08
Average specific productivity [pg/c/d] 5.5 12.9
Average volumetric productivity [mg/L/d] 46.3 969.8
Cumulated antibody production [g] 0.24 7.42
Consumable cost per 100 mg IgG [USD] 120 18
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Challenges in recombinant protein production
Increase overall productivity Optimize product concentration Develop a robust process Maintain/improve protein quality Reduce process and changeover time Efficient use of facility
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Conclusion• Viable cell conc. of 100x106 c/ml maintained for 8
days with perfusion filter integrated in Cellbag
• Perfusion culture drastically increased volumetric productivity and reduced upstream production cost
• Process stable during 3 weeks operation time
• Limited process complexity due to single use bioreactor with internal cell retention
Read more in recent publication:
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Case study 2:Vaccine manufacturing platforms using ReadyToProcess™
IntroductionCell culture in WAVE Bioreactor™ systemsScale up of microcarrier culturesHarvest using filtrationSummary
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Why use ReadyToProcess™ platform in vaccine production?
• Vaccines often manufactured in relatively small batch sizes –scale suitable for disposables
• Campaign manufacturing is common – several products can be produced in the same facility using disposables
• Efficient change over procedures between campaigns and different products with disposables
• Less risk of adventitious virus propagation in closed systems
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Cell culture in WAVE Bioreactor™ systems
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Selecting a cell line for virus production
• Cell substrate evolution from primary to diploid to continuous cell lines…
• Modern options: Vero, MDCK, EBx™, PER.C6® …
• Requirements– Suitable for GMP production – Good safety track record– Good virus propagation– Broadly and highly permissive– Scalable to high volume production
from: Pereira et al. Biotech Bioeng; 2004; 85; 5
Cytodex 1 microcarriers without (E) and with cells (F)
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Development of vaccine microcarrier applications in WAVE Bioreactor™
Protocols available for 2 L scale, under development for 10 & 100 L
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Process development: Vero cells in WAVE Bioreactor™
Preparation of microcarriers
Equilibration Inoculation
Cell attachmentto microcarriers Cell growth
Virus Infection HarvestDownstream
processing
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Process development: The effect of cell culture media
Medium 1 Medium 2 Medium 3
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Scalability: Vero cell growthCellbag™ 10 L, wv 2 L and 50L, wv 10 LCytodex™ 1 (3g/L)Continuous mixing during cell attachmentNo animal derived components and serum free medium
0
0,2
0,4
0,6
0,8
1
1,2
1,4
1,6
0 20 40 60 80 100 120
Cel
l Con
c. [c
ells
/ml]
Process Time [h]
Cellbag 50L
Cellbag 10L
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Influenza virus production workflow in WAVE Bioreactor™
Cell expansion
Cell inoculation on microcarriers in
WAVE Bioreactor at ~0.4 x 106 cells/ml
Virus infectionA/Solomon Islands/H1N1
MOI : 0,004
Harvest of supernatant
TOI ~ 48 h TOH ~ 72 h
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Scale-up of microcarrier cultures
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Vero WAVE Bioreactor™ scale-up
System: WAVE Bioreactor 20/50 WAVE Bioreactor 20/50 WAVE Bioreactor 200Cellbag™ size: 10 L 50 L 200 LWork Vol: 2 L 10 L 50 L
WAVE Bioreactor 20/50 System
WAVE Bioreactor 200 System
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Microcarrier transfer 2 L 10 L
TrypsinMicro-
carriersCells
WAVECellbag™ 10
Working volume: 2L
Medium +Micro-
carriersCells
PBS-EDTAMicro-
carriersCells
Wash (PBS-EDTA)
Trypsin is added
CellsWAVE
Cellbag50Working volume: 10 L
Cytodex 1 added
Incubation with gentle agitation
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Scale-up from 2 to 50 L - resultsWAVE
Bioreactor™2L
WAVE Bioreactor
10L
WAVEBioreactor
50L
Start cell density (cells/mL) 0.17 x 106 0.27 x 106 0.5 x 106
Final cell concentration (cells/mL) 2 x 106 3.4 x 106 3.35 x 106
Microcarrier transfer recovery (%) 67.5 70.5 NA
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Influenza virus infection – Example 10 L
Rocking parameters 5 rpm/8°
Start cell density 3.3 x 106 cells/mL
Volume 10 L
Time of infection Day 5
Trypsin conc 25 µg/mL
Temperature 37°C
Time of harvest 72 h
20 h post infection
72 h post infection
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Harvest
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Influenza Harvest ApplicationInfluenza vaccine process
Cell culture and infectionCell culture using microcarriers
Normal flow filtrationRemoval of cell debris
Cross flow filtrationConcentration of influenza and removal of DNA and Host cell
impurities
ChromatographyBind/elute or flow through mode
removal of DNA and Host cell impurities
Cross flow filtrationFormulation - concentration and buffer
exchange
Normal flow filtrationSterile filtration
Harvest
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Influenza Harvest ApplicationWaste time reduction
System preparation
Classic CFF
ReadyToProcess™ CFF
System preparation including CIP
Controlled run
Post-CIP and -SIP/disposal
Controlled runCircuit disassembly/disposal
System preparation
Classic NFF
ReadyToProcess™ NFF
System preparation including autoclaving and CIP
Controlled run
Post-CIP and -SIP/disposal
Controlled run
Circuit disassembly/disposal
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Influenza Harvest ApplicationWaste time reduction
System preparation
Classic CFF
ReadyToProcess™ CFF
System preparation including CIP
Controlled run
Post-CIP and -SIP/disposal
Controlled runCircuit disassembly/disposal
System preparation
Classic NFF
ReadyToProcess™ NFF
System preparation including autoclaving and CIP
Controlled run
Post-CIP and -SIP/disposal
Controlled run
Circuit disassembly/disposal
Using ReadyToProcess decreases the non added value time (waste) for a single harvest lab process run
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Conclusions
Disposable processing enables:• Safe and easy operations
• Sterile processing/ bioburden control
• Short process lead time
• Same process output/results compared to conventional formats
• Low capital investment
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Acknowledgements
High Yield Antibody Production• GE China Research and
Development Center
Jianjun Yang
• GE Healthcare Europe GmbH
Christian Kaisermayer
• Institut Pasteur, ShanghaiPaul ZhouLulan WangHongxing HuFeng Wang
Vaccine Manufacturing Platforms• GE Healthcare Bio-Sciences AB
Therese Lundström
Ann-Christin Magnusson
Johanna Tschöp
Mats Lundgren
• GE Healthcare Europe GmbH
Christian Kaisermayer
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Thank youCellbag, Cytodex, ReadyCircuit, ReadyToProcess, ULTA, and WAVE Bioreactor are trademarks of GE Healthcare companies. GE, imagination at work, and GE monogram are trademarks of General Electric Company.
PER.C6 logo and name are registered trade marks of Crucell Holland B.V. EBx is a registered trademark of Vivalis SA.
© 2012 General Electric Company – All rights reserved.
All goods and services are sold subject to the terms and conditions of sale of the company within GE Healthcare which supplies them. GE Healthcare reserves the right, subject to any regulatory and contractual approval, if required, to make changes in specifications and features shown herein, or discontinue the product described at any time without notice or obligation. Contact your local GE Healthcare representative for the most current information.
GE Healthcare Bio-Sciences AB, a General Electric Company.
www.gelifesciences.com/bioprocess
GE Healthcare Bio-Sciences ABBjörkgatan 30751 84 UppsalaSweden
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