Efficient and controlled expansion of IgG1 producing CHO-DG44 cells using the ActiCHO Media System and WAVE Bioreactor™ Thomas Falkman, Eric Fäldt, Anita Vitina and Cecilia Annerén. GE Healthcare Bio-Sciences AB, Uppsala, Sweden

First presented at the Bioprocessing Summit in Boston, MA on August 20-

24, 2012

2 Efficient and controlled expansion of IgG1

producing CHO-DG44 cells using the ActiCHO Media System and WAVE

Bioreactor™

Introduction

Demands for more potent drugs, higher yields, smaller batch sizes and the cost pressure on R&D budgets push upstream bioprocessing towards single use bioreactors and cell culture media that support high density cultures and good process control.

We have developed a high cell density fed-batch process based on an IgG1 producing CHO-DG44 cell line and the ActiCHO Media System.

The process was successfully run in a conventional stainless steel stirred tank bioreactor (SS STR) up to 100L cultivation volume as well as the single use WAVE Bioreactor™ system at 10 and 100L scales. The seed-train expansion step was performed in shake flasks and the WAVE Bioreactor 20/50 system.

3 Efficient and controlled expansion of IgG1

producing CHO-DG44 cells using the ActiCHO Media System and WAVE

Bioreactor™

Workflow

4 Efficient and controlled expansion of IgG1

producing CHO-DG44 cells using the ActiCHO Media System and WAVE

Bioreactor™

ActiCHO Media System

Especially designed for easy large-scale production in fed-batch culture, using recombinant CHO-DG44 cells

All components are based on the same chemically defined raw materials.

5 Efficient and controlled expansion of IgG1

producing CHO-DG44 cells using the ActiCHO Media System and WAVE

Bioreactor™

Material CHO-DG44 cell line expressing monoclonal antibody IgG1.

WAVE Bioreactor™ 20/50 and 200 system

WAVEPOD™ II

Cellbag™ 20L and 200L with optical pH sensor. UNICORN™ DAQ 1.0 software

100 L stainless steal STR Bioreactor (Belach Bioteknik AB)

ActiCHO Media System:

ActiCHO SM CD suppl. with 6 mM L-Glut. : selective medium for adaptation of CHO cells.

ActiCHO P CD suppl. with 6 mM L-Glut. : non-selective production medium.

ActiCHO Feed-A CD & ActiCHO Feed-B CD: Supplements added in fed-batch process

6 Efficient and controlled expansion of IgG1

producing CHO-DG44 cells using the ActiCHO Media System and WAVE

Bioreactor™

Methods Seed-train expansion:

Cells were recovered in complete ActiCHO SM CD medium, a basal and nutrient limited medium suitable for cell maintenance and cell expansion. Shake flask cultures were maintained in humidified incubators at 36.8°C with 7.5% CO2 on an orbital platform at 103 rpm. Cells were sub-cultured at 0.2 x 106 cells/mL every 3 days. The last subculture up to 20 x 109 cells was performed in WAVE Bioreactor™ 20/50.

Production:

The bioreactor was filled with ActiCHO P CD medium at 60% of the maximum volume. Cells were inoculated at 0.3x106 cells/mL. Feeding of Feed A and Feed B started at day 3 post-inoculation with 43.2 g/L/day and 4.32 g/L/day respectively. Feeding of glucose was started at day 6 post-inoculation and carried out to maintain glucose level at ~6 g/L. The WAVE Bioreactor 200 culture was inoculated with 0.18 x 106 cells/mL.

Analysis:

Nutrients and physical parameters were measured off-line using the Bioprofile Flex (Nova Biomedical).

Cell density and viability were measured using CASY Cell Counter and Analyzer (Roche).

IgG titers were determined with MabSelect SuRe™ analytical chromatography and Biacore™.

7 Efficient and controlled expansion of IgG1

producing CHO-DG44 cells using the ActiCHO Media System and WAVE

Bioreactor™

Seed-train expansion in WAVE Bioreactor™ 20/50 system™ at 10L scale

The potential of the WAVE Bioreactor system to increase the volume 10-fold using the same Cellbag™ suggests that the seed-train expansion step could be initiated in the WAVE bioreactor 20/50 system using a 20L Cellbag™ going from 0,2 x 109 to 30 x 109 cells in less than a week assuming a maximum cell density of 3 x 109 cells/mL.

8 Efficient and controlled expansion of IgG1

producing CHO-DG44 cells using the ActiCHO Media System and WAVE

Bioreactor™

WAVE Bioreactor™ 20/50 system

Cell growth and viability were comparable between the two cultures in the WAVE Bioreactor 20/50 system with maximum cell densities >20 x 106 cell/mL .

The IgG production levels were also similar with final product titers consistently around 5 g/L .

9 Efficient and controlled expansion of IgG1

producing CHO-DG44 cells using the ActiCHO Media System and WAVE

Bioreactor™

WAVE Bioreactor™ 200 and 100L STR

Cell growth, viability and product titers were comparable between the two WAVE Bioreactor 20/50 cultures and the 100L STR with maximum cell densities >20 x 106 cell/mL .

The cell growth and production was a bit lower in the WAVE Bioreactor 200 culture, likely due to a lower inoculum density and a shorter production period.

The final concentration of IgG in the WAVE Bioreactor 200 system was 3.96 g/L on day 11, which was similar to the other process runs at the same time-point of the culture.

10 Efficient and controlled expansion of IgG1

producing CHO-DG44 cells using the ActiCHO Media System and WAVE

Bioreactor™

Metabolic profiles from STR culture

The ActiCHO Media System promoted low lactate

production with high viability of the cells

11 Efficient and controlled expansion of IgG1

producing CHO-DG44 cells using the ActiCHO Media System and WAVE

Bioreactor™

Metabolic profiles from WAVE Bioreactor™ cultures

The optical pH sensor in the

Cellbag™ display accurate and

precise readings with stable

regulation at a given setpoint giving

an excellent stability of parameter

control.

12 Efficient and controlled expansion of IgG1

producing CHO-DG44 cells using the ActiCHO Media System and WAVE

Bioreactor™

Conclusions

• The process consequently resulted in maximum viable cell densities around 20 x 106 cells/mL and product titers of approximately 5 g/L.

• The ActiCHO Media System promoted low lactate production with high viability of the cells

• IgG production and cell growth was independent of the type of bioreactor and scale being used showing the stability of the process and the strength of the medium platform

• Culture of IgG producing CHO-DG44 cells in the single use WAVE Bioreactor™ systems using the ActiCHO Media System is a simple and robust process option for seed-train expansion and IgG production up to at least 100L scale.

13 Efficient and controlled expansion of IgG1

producing CHO-DG44 cells using the ActiCHO Media System and WAVE

Bioreactor™

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© 2012 General Electric Company―All rights reserved.

First published 2012.

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Acknowledgments