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

Abstract

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 are identified and surprisingly 58% are classified as differentially expressed, including 2900 with a fold change higher than 1.5. The largest differences are found for gene-transcripts belonging to regulation of apoptosis, cell death, and protein intracellular transport GO term classifications, which are found to be significantly enriched in the high producing cell line. RNA sequencing is also performed on subclones, where down-regulation of genes encoding secreted glycoproteins is 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.

Transcriptomic analysis of IgG4 Fc‐fusion protein degradation in a panel of clonally‐derived CHO cell lines using RNASeq

“…Comparison of the high and low proteolysis groups identified 138 genes (110 upregulated and 28 downregulated) that were significantly differentially expressed (≥±1.2 fold change between the two groups along with a Benjamini Hochberg (BH) adjusted p value < 0.05; Figure 3a and Table S3). In comparison to previously published transcriptomics studies in CHO (Orellana et al, 2018), this number of differentially expressed genes is relatively small. This result, however, is not unexpected given the experimental design used in this study.…”

Bioinformatic analysis of Chinese hamster ovary host cell protein lipases

“…Pnliprp1 and Liph have also previously been identified as differentially expressed in sodium butyrate treated CHO cells when compared to non‐treated cells . In addition, a higher than 1.5‐fold change in expression of Pnliprp1 was observed between a low‐producing and a high‐producing cell line . Three of the five genes ( Pnliprp2 , Pnliprp1 , and Lipi ) had errors in their annotations for the CHO‐K1 genome, which could be seen in the multiple sequence alignment against the corresponding protein in mouse, rat, human, and CH PICR.…”

Attenuating apoptosis in Chinese hamster ovary cells for improved biopharmaceutical production

“…Regulatory and metabolic information can be extracted from omics comparisons of different cell lines (with varying q P ) and culture conditions, offering a blueprint to reverse engineer desirable phenotypes. Many CHO omics studies have already been performed and can be of great value to find new gene targets to improve cells lines for biopharmaceutical production (Baik et al, 2006;Birzele et al, 2010;Carlage et al, 2009;Han et al, 2017;Joon, Gatti, Philp, Yap, & Hu, 2008;Lee, Kim, Kim, & Lee, 2003;Meleady et al, 2008;Nissom et al, 2006;Orellana et al, 2018;Orellana, Marcellin, Munro, Gary, & Nielsen, 2015;Shen et al, 2010;Yee, Gerdtzen, & Hu, 2009). Furthermore, the latest CHO genome scale reconstruction (Hefzi et al, 2016) is a valuable tool to perform in silico analysis on some of the discussed targets.…”