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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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