Strategies to improve CHO cell culture performance: Targeted deletion of amino acid catabolism and apoptosis genes paired with growth inhibitor supplementation

Abstract

AbstractChinese hamster ovary (CHO) cells are the predominant host of choice for recombinant monoclonal antibody (mAb) expression. Recent advancements in gene editing technology have enabled engineering new CHO hosts with higher growth, viability, or productivity. One approach involved knock out (KO) of BCAT1 gene, which codes for the first enzyme in the branched chain amino acid (BCAA) catabolism pathway; BCAT1 KO reduced accumulation of growth inhibitory short chain fatty acid (SCFA) byproducts and improved culture growth and titer when used in conjunction with high‐end pH‐controlled delivery of glucose (HiPDOG) technology and SCFA supplementation during production. Accumulation of SCFAs in the culture media is critical for metabolic shift toward higher specific productivity and hence titer. Here we describe knocking out BCKDHa/b genes (2XKO), which act downstream of the BCAT1, in a BAX/BAK KO CHO host cell line background to reduce accumulation of growth‐inhibitory molecules in culture. Evaluation of the new 4XKO CHO cell lines in fed‐batch production cultures (without HiPDOG) revealed that partial KO of BCKDHa/b genes in an apoptosis‐resistant (BAX/BAK KO) background can achieve higher viabilities and mAb titers. This was evident when SCFAs were added to boost productivity as such additives negatively impacted culture viability in the WT but not BAX/BAK KO cells during batch production. Altogether, our findings suggest that SCFA addbacks can significantly increase productivity and mAb titers in the context of apoptosis‐attenuated CHO cells with partial KO of BCAA genes. Such engineered CHO hosts can offer productivity advantages for expressing biotherapeutics in an industrial setting.