RNA‐Seq Highlights High Clonal Variation in Monoclonal Antibody Producing CHO Cells

Camila A. Orellana, Esteban Marcellin, Robin W. Palfreyman, Trent P. Munro, Peter P. Gray, Lars K. Nielsen

The development of next-generation sequencing technologies has opened new opportunities to better characterize complex eukaryotic cells. Chinese hamster ovary (CHO) cells play a primary role in therapeutic protein production, with currently five of the top ten blockbuster drugs produced in CHO. However, engineering superior CHO cells with improved production features has had limited success to date and cell lines are still developed through the generation and screening of large strain pools. Here, we applied RNA sequencing to contrast a high and a low monoclonal antibody producing cell line. Rigorous experimental design achieved high reproducibility between biological replicates, remarkably reducing variation to less than 10%. More than 14,000 gene-transcripts were identified and surprisingly 58% were classified as differentially expressed, including 2,900 with a fold change higher than 1.5. The largest differences were found for gene-transcripts belonging to regulation of apoptosis, cell death and protein intracellular transport GO term classifications, which were found to be significantly enriched in the high producing cell line. RNA sequencing was also performed on subclones, where down-regulation of genes encoding secreted glycoproteins was found to be the most significant change. The large number of significant differences even between subclones challenges the notion of identifying and manipulating a few key genes to generate high production CHO cell lines.