Process Optimization using High Throughput Automated Micro-Bioreactors in Chinese Hamster Ovary Cell Cultivation

Summary

Here, we present a detailed procedure to run a Design of Experiment in an automated micro-bioreactor followed by cell harvest and protein quantification using a Protein A column.

Abstract

Optimization of bioprocesses to increase the yield of desired products is of importance in the biopharmaceutical industry. This can be achieved by strain selection and by developing bioprocess parameters. Shake flasks have been used for this purpose. They, however, lack the capability to control the process parameters such as pH and dissolved oxygen (DO). This limitation can be overcome with the help of an automated micro-bioreactor. These bioreactors mimic cultivation at a larger scale. One of the major advantages of this system is the integration of the Design of Experiment (DOE) in the software. This integration enables establishing a design where multiple process parameters can be varied simultaneously. The critical process parameters and optimum bioprocess conditions can be analyzed within the software. The focus of the work presented here is to introduce the user to the steps involved in process design in the software and incorporation of the DOE within the cultivation run.

Introduction

The global biopharmaceutical market was worth more than US $250 billion in 2018 and has been continuously expanding¹. Pharmaceutical companies are moving away from producing small molecular drugs to biotechnologically produced therapeutics such as recombinant proteins. These alone are responsible for a revenue of more than $150 billion¹. Mammalian cells are now extensively used for the production of these pharmaceutical recombinant proteins. In the current period, among the 68 approved products produced by mammalian cells, 57 are produced by Chinese Hamster ovary cells (CHO)². CHO cells are specif....

Protocol

1. Preculture Procedure

NOTE: Recombinant CHO DG44 cells with a viable cell concentration of 1 x 10⁷ cells/mL are used for this protocol.

1. Thaw the vial containing 1.2 mL of cells to room temperature and immediately transfer the cell suspension to a 15 mL conical centrifuge tube containing 10 mL of cold seed medium.
2. Centrifuge the conical centrifuge tube for 5 minutes at 190 x g and room temperature and discard the supernatant.
3. Pre-heat 150 mL of the seed medium in a 500 mL shake flask to 36.8 °C.
4. Gently resuspend the cell pellet in 10 mL of pre-warmed seed medium....

Results

An overview of the cultivation performed in this study is presented in Figure 2.

Figure 2: Schematic representation of the experimental conditions to test pH and stirrer speed profiles in the cultur.......

Discussion

Optimization of the process to increase the yield is of crucial importance in the biopharmaceutical industry. Shake flasks could potentially be used for the screening of the strain; however, the monitoring of the process parameters such as pH and DO are unavailable in the flasks. The micro-bioreactors have an advantage as they allow continuous monitoring and control of the process. These control loops in the micro-bioreactor also provide a condition similar to those at larger scale and thus, deliver results that are comp.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
1 mL disposable pipette tips, sterilized	Sartorius Stedim Biotech GmbH	A-0040
200 mM L-glutamine	Corning, Merck	25-005-CV
24 Well deep well plates	Sartorius Stedim Biotech GmbH	A-0038
5 mL disposable pipette tips, sterilized	Sartorius Stedim Biotech GmbH	A-0039
ambr 15 automated microbioreactor system	Sartorius Stedim Biotech GmbH	001-2804
ambr 15 Cell Culture 24 Disposable Bioreactors - Sparged	Sartorius Stedim Biotech GmbH	001-1B86
Antifoam C Emulsion	Sigma-Aldrich, Merck	A8011
Bottle Top Sterile filter	Corning, Merck	CLS431474	0.1 μm pore size
CEDEX Detergent (3% Mucosol)	Roche Innovatis AG	05-650-658-001
Cell counter	Roche Innovatis AG	05-650-216-001	CEDEX HiRes
CHO DG44 cell line	Cellca, Sartorius Stedim Biotech GmbH
CHOKO Feed Media A (FMA)	Sigma-Aldrich, Merck	CR80025
CHOKO Feed Media B (FMB)	Sigma-Aldrich, Merck	CR80026
CHOKO Production Medium	Sigma-Aldrich, Merck	CR80027
CHOKO Stock Culture Meium	Sigma-Aldrich, Merck	CR80028
Chromaster high pressure liquid chromatography system	VWR International
Conical Centrifuge tube	Corning, Merck	SIAL0790
Ethanol	Merck	1070179026
Glycine	Carl Roth	56-40-6
HPLC Vials	VWR International	SUPLSU860181
PBS	Sigma-Aldrich,Merck	P4417
Protein A Column	Thermo Fisher Scientific	1502226	POROS™ A 1.7 mL
Sodium chloride	Sigma-Aldrich,Merck	7647-14-5
Sodium phosphate dibasic anhydrous	Sigma-Aldrich,Merck	7558-79-4
Trypan Blue	VWR International	VWRVK940
YSI	YSI Inc	2900D	YSI 2900 Select