Janet Chusainow and Yuan Sheng Yang contributed equally to this study.
A study of monoclonal antibody-producing CHO cell lines: What makes a stable high producer?†
Article first published online: 8 OCT 2008
Copyright © 2008 Wiley Periodicals, Inc.
Biotechnology and Bioengineering
Volume 102, Issue 4, pages 1182–1196, 1 March 2009
How to Cite
Chusainow, J., Yang, Y. S., Yeo, J. H.M., Toh, P. C., Asvadi, P., Wong, N. S.C. and Yap, M. G.S. (2009), A study of monoclonal antibody-producing CHO cell lines: What makes a stable high producer?. Biotechnol. Bioeng., 102: 1182–1196. doi: 10.1002/bit.22158
- Issue published online: 20 JAN 2009
- Article first published online: 8 OCT 2008
- Accepted manuscript online: 8 OCT 2008 12:00AM EST
- Manuscript Accepted: 25 SEP 2008
- Manuscript Revised: 24 SEP 2008
- Manuscript Received: 25 MAR 2008
- Agency for Science, Technology and Research (A*STAR), Singapore
- Chinese hamster ovary (CHO);
- monoclonal antibody (mAb);
- dihydrofolate reductase (dhfr);
- gene amplification;
- clone stability
Generating stable, high-producing cell lines for recombinant protein production requires an understanding of the potential limitations in the cellular machinery for protein expression. In order to increase our understanding of what makes a stable high producer, we have generated a panel of 17 recombinant monoclonal antibody expressing Chinese hamster ovary subclones (CHO-mAb) with specific productivities ranging between 3 and 75 pg cell−1 day−1 using the dihydrofolate reductase (dhfr) expression system and compared the molecular features of these high- and low-producer clones. The relative heavy chain (HC) and light chain (LC) transgene copy numbers and mRNA levels were determined using real-time quantitative PCR (RT qPCR). We observed that not only higher transgene copy numbers and mRNA levels of both HC and LC were characteristic for the high-producer clones as compared to the low-producer clones but also a more favorable HC to LC transgene copy numbers ratio. By studying the long-term stability of the CHO-mAb subclones in the absence of methotrexate (MTX) selective pressure over 36 passages we observed a 35–92% decrease in volumetric productivity, primarily caused by a significant decrease in HC and LC mRNA levels with little change in the transgene copy numbers. Using Southern blot hybridization we analyzed the HC and LC transgene integration patterns in the host chromosome and their changes in course of gene amplification and long-term culturing. We observed that MTX-induced gene amplification caused chromosomal rearrangements resulting in clonal variability in regards to growth, productivity, and stability. No further obvious DNA rearrangements occurred during long-term culturing in the absence of MTX, indicating that other mechanisms were responsible for the decreased transcription efficiency. Our results implicate that the amplified transgene sequences were arranged in tandem repeats potentially triggering repeat-induced gene silencing. We hypothesize that the decline in transgene mRNA levels upon long-term culturing without MTX was mainly caused by transgene silencing consequently leading to a loss in mAb productivity. The exact molecular mechanisms causing production instability are not yet fully understood. The herein described extensive characterization studies could help understand the limitations to high-level, stable recombinant protein production and find ways to improving and accelerating the process for high-producer cell line generation and selection. Biotechnol. Bioeng. 2009;102: 1182–1196. © 2008 Wiley Periodicals, Inc.