development of single-use bayshake -technology for the cultivation of mammalian cells · 2017. 1....
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Development of Single-use BaySHAKE®-Technology for the Cultivation of Mammalian Cells K.H. Schneider , B. Bödeker
Bayer Healthcare Pharmaceuticals,
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Process Development for Mammalian Expression at GB-Biotech Development, Wuppertal
Situation
- New biologics strategy being successfully implemented
- Resulting in 2 – 3 new projects (D3) per year
Full length monoclonal antibodies (short: mAbs)mAb - Derivates
Current GMP Production for PHI/II clinicals
- Scale: 200L- Mode: fed-batch
Goal for 2011
3 different proteins to produce clinical material
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New Pilot Plant for GB-Biotech Development, Wuppertal
Project
- Pilot plant for GMP-production of recombinant proteins with cell culturetechnology
- Supply of Ph IIb/III clinical studies
- Currently under construction, ready foroperation by January 2013
Equipment
- Bioreactor scale: 2500 L
- Fermentation mode: fed-batch
- Package units for flexible use of equipment
Single-use Technology
- Liquid transfer
- Cell separation
- Bioreactors for 2. fermentation line
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Why Single-use in Biologics Development and Production?
The current Environment
- Cost pressure- Need for high speed development- Conflicting process requirements for
mammalian cells ( oxygen supply vs. limited shear tolerance)
Single-use Technology Advantages
- Minimized planning and setup time, installation and change efforts
- Speed-up of product turnover and product changeover
- Reliable operation of multi-purpose plants by eliminating
cleaning & cleaning validation risk of cross contamination
- Increased production capacity
Single-use Technology: Cost Advantages
- Invest costs- Operational costs
Parameter Hard- Single-piped use
Titer g/L 3 3Capacity t/a 1 1.2Capital Cost Mio € 350 145 Capital Cost €/kg 100 35 Gas Supply % 100 12 Electr. Supply % 100 37 Water Supply % 100 8 Production Area % 100 17 Staff % 100 41
Comparison of Reusable and Single-use Upstream Technology
Case Study of Elan Corp (Roebers, IBC Life Sciences Bioprocessing Meeting, Oct 2009)
Hard Piped Facility: 2 x 15000L fermentationSingle-use Facility: 10 x 2000L fermentation
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Single-use Competitors and BaySHAKE®
Systems in Market under Development
- Rockers GE, Sartorius
- Stirred / Sparged Bioreactors Xcellerex, Sartorius, HycloneATMI, PBS, Xellexus
- Shaked BioreactorsXCellGene
Technology Requirements
- Comparability to existing fermentertechnology
Low shear operation- Process Robustness
Sterility / bag reliabilityNo foam
- Scalability > 1000L
Challenge: Biological Comparability
- Cell growth and viability- Productivity and product quality
BaySHAKE® Bioreactor Technology
Agitation Principle : Rotary Oscillation“Taxonomy” : Shaker Principle : Surface AerationBag Design : Cubical & Empty
Low-shear operation as result of narrow velocity distribution within inertial liquid phase
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Cooperation Partners
Project Start in 2007
Bayer Partners
Bayer Schering PharmaBiotech Development Wuppertal
- Biological evaluation
Bayer Technology Services - Reactor development- Hydrodynamics
Sartorius-Stedim Biotech- Bags - Control units
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TemperatureControl
Reactor Concept
Oscillating Drive - High robustness- Low noise
- Security housing concept
Container- Leakage-proof bag housing
suitable for biological liquids beyond S1
- Easy cleanability- Frontal service - Pyramidal bottom enabling
Full drainingLow starting volume (10%)Reduction of seeding steps
Production Systems50L, 200L and 1000L
Drive
GlassHousing
Container
PyramidalBottom
BeltHousing
Sensors SupplyDrain
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Bag Configuration
Bag- PE/PP laminate with high
mechanical stress resistance - Connection to environment
by silicone tubing using sterile connectors / tube welders
Media Supply / Product Harvest- Drain at low position for
maximal product harvest - Sideway ports built as single
port or multiport
Sensors- Disposable hose barbs for
Optical, fluorescence based measurement:pH, PO2PT100 reusable sensor
Hose barbs for sensors:pH, PO2, T
Multiport welds for media supplyand drainContainer
with 1000L bag
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Hydrodynamical Characterization of BaySHAKE®
Mass Transfer kLa = f( P/V)
- Supports cell density of 8 – 10 mio cells / mL with oxygen at low shear forces
- Linear scalability from 50 L to larger 1000L
Mixing Θ∗ = f(Fr)- Excellent performance by
axial liquid convection- Comparable to standard
mixer systems like RushtonTurbine
Differentiation to Single-use Sparged and Agitated Systems
- Low foam formation- No pressure build-up
Rushton(α/π=1, H/D=1)
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Biological Evaluation: Approach
Test for 3 development projects
1 Antibody-derivative
2 full length antibodies
Evaluation- cell growth, viability, productivity,
product quality of purified protein- oxygen supply, pH/T control, foam
formation
Detailed results will be shown for antibodies though results were identical for 3 different products
250 mL Flask 3x 500 mL Flask 3x 1000 mL Flask
Shake-Incubator (12- 15 d)T 37 °C
1. Thawing
2. Centrifugation3. Resuspension
1. Thawing
2. Centrifugation3. Resuspension
MCB vial
1. Cell Propagation (CHO cells)
2. Fermentation
Cell Separation
Inoculation
HarvestSeed culture Bioreactor (6 d)
100 L
100 L
100 L
100 L
Feeding solution
Feeding solution
Feeding solution
90 SP 0,2
µm
10 SP
90 SP90 SP 0,2
µm 0,2µm
10 SP10 SP
Production Bioreactor or BayShake (10 d)
Scales used and compared - Standard deep tank: 10, 20, 100, 200 L - BaySHAKE® fermenter: 50, 200, 1000 L
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- Low foam formation upon shaking operation
- No pressure build-up
- Excellent cell culture performance(cell density, viability, product yield) and product quality:
Conclusions :
Performance of BaySHAKE® fermenterswas in all aspects comparable to deep tank fermenters
Product quality between BaySHAKE® and deep tank fermenter is very comparable(assessed by analytical size exclusion chromatography)
Biological Evaluation: Results
mAb: 1000 L BaySHAKE® Fermenter Run
BaySHAKE®1000 L
Deep tank fermenter 200 L
Time [d]
Tite
r
Viability
Viable Cell
Via
ble
Cel
lCou
nts
[ 106
/mL]
Tite
r[%
], V
iabi
lity
[%]
0 2 4
15 Time [min]1529
.87
Ant
ibod
y
0
40
80
30 45
29.8
7 A
ntib
ody
45
Count
mAU
FinalBulk
10060
30
00
69,436,554,955,7Titer mg/L
6,096,7210,976,98max. viable count.
x106 cells/mL
100 L20 L1000 L200 LScale
Standard FermenterBayShake Fermenter
69,436,554,955,7Titer mg/L
6,096,7210,976,98max. viable count.
x106 cells/mL
100 L20 L1000 L200 LScale
Standard FermenterBayShake Fermenter
69,436,554,955,7Titer mg/L
6,096,7210,976,98max. viable count.
x106 cells/mL
100 L20 L1000 L200 LScale
Standard FermenterBayShake Fermenter
69,436,554,955,7Titer mg/L
6,096,7210,976,98max. viable count.
x106 cells/mL
100 L20 L1000 L200 LScale
Standard FermenterBayShake Fermenter
94 92 61 100Titer [%]
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Conclusions and Outlook
BaySHAKE® is a very simple and efficient alternative to deep tank fermenters for the fed-batch cultivation of mammalian cells up to the 1000 L scale
- Performance comparable to deep tank cultivations regarding product qualityfor mAb and mAb-derivatives
- Significant contribution to enhanced speed of development
- Cost efficacy
Next stepsBD plans to install a 1000 L unit into our small scale GMP facility, which will increase product output for preclinical and phase I/II clinical trials tremendously
Strategic FitThe BaySHAKE® project represents one of several cooperation projects between GB-BD and BTS in the field of optimizing biologics development and production
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