Cambridge Healthtech Institute recently interviewed Dr. Noriko Yamano, Senior Scientist, Manufacturing Technology Association of Biologics and Guest Academic Staff, Graduate School of Engineering, Osaka University.
In the leadup to her presentation on “The Stability of CHO Genome: Essential for Cell Line Characterization or Not?” at the Fourth Annual CHO Cell Lines conference (January 10-11, 2018 during the 17th Annual PepTalk event in San Diego, CA), Dr. Yamano discussed with us:
CHO Cells, Chromosomal Instability, and Protein Production
To start, she considered how chromosomal instability of CHO cells affects cell line characterization, and ultimately protein expression and production. “Based on the distribution of chromosome number in the CHO-DG44 line,” she said, “we have shown that most cells contain approximately 21 chromosomes, whereas 4% harbor more than 30 chromosomes. We have shown that high chromosome number CHO cells have a greater potential to become robust producers of recombinant proteins.”
Why?
“One explanation is the increase in transgene integration sites. However, high chromosome number cell clones produce more IgG3 following the culture of single-cell clones, even under conditions that yield the same number of integrations as cells with normal chromosome numbers. These results suggest that the number of integration sites is not the only reason for the higher antibody productivity in high chromosome number cells.”
Other explanations exist. As Dr. Yamano stated, “One of the other possible reasons may be due to the chromosomal instability. Our group has previously shown that the efficiency of gene amplification by Dhfr and methotrexate increases by weakened DNA damage checkpoints. At the same time, cell lines with weakened DNA damage checkpoints had higher mRNA expression and protein production of recombinant IgG compared to the normal cell lines that had similar or more gene copies.”
“Now, we are trying to characterize cells with high chromosome number cells, which have more chromosomal instability, by methods such as transcriptome analyses.”
What about comparing the transcriptome in cells with different chromosome numbers? Are there methods that are more effective than others?
Dr. Yamano responded, “When gene expression is adjusted for RNA amount, it is difficult to determine the characteristics of specific cells, in the case that RNA amount differs between cells. As the expression of housekeeping genes can differ between cells, gene expression profiles between cells with disparate chromosome numbers were compared using RNA standards which corresponds to the cell number. RNA standards are a set of aqueous solutions of RNAs of five different nucleotide sequences based on management systems conforming to ISO GUIDE 34: 2009 and ISO/IEC 17025: 2005 (NMIJ CRM 6204-a; National Institute of Advanced Industrial Science and Technology). Diluted solutions of RNA standards were added to cell lysates, and the total RNA was extracted.”
Finally, when it comes to using aneuploid cells for biopharmaceutical production, Dr. Yamano shared this perspective: “Our unpublished data demonstrate many chromosomal rearrangements in CHO cells regardless of chromosome number. Inserted exogenous genes might be deleted by chromosomal rearrangements. Our results demonstrate that the stability of each chromosome in a CHO cell differs.”
“To exploit CHO cells with chromosome aberrations, it is speculated to be important to introduce genes into relatively stable chromosomes. Using aneuploid cells will become a powerful tool, if long-term stable expression is guaranteed.”
SPEAKER BIOGRAPHY
Noriko Yamano, Ph.D., Senior Scientist, Manufacturing Technology Association of Biologics; Guest Academic Staff, Graduate School of Engineering, Osaka University
Dr. Yamano’s credentials include: Doctor of Science, Graduate School of Frontier Bioscieneces, Osaka University, 2010; 2010-2014 Researcher, Institute of Health Biosciences, Tokushima University; 2014-2017 Project-Based Assistant Professor, Institute of Technology and Science (in 2016, the institution name was changed to “Graduate School of Bioscience and Bioindustry”), Tokushima University; 2017-present Senior Scientist, Manufacturing Technology Association of Biologics; 2017-present Guest Academic Staff, Graduate School of Engineering, Osaka University.
TO LEARN MORE ABOUT HER PRESENTATION AND PEPTALK, VISIT:
CHI-PepTalk.com/CHO-Cells/
(Attendees of the Optimizing Expression Platforms and Bioprocess Analytics conferences can join this same talk during a shared session on Thursday, January 11.)