Development of Single-use BaySHAKE®-Technology for the Cultivation of Mammalian Cells K.H. Schneider , B. Bödeker

Bayer Healthcare Pharmaceuticals,

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

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

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

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

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

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

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

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)

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

- 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 [%]

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