CHO cells in fed-batch cultures consume large amounts of nutrients and divert a significant fraction of them towards formation of metabolic byproducts, some of which are growth inhibitory in nature. A major fraction of these inhibitory metabolites are byproducts or intermediates of amino acid catabolism. The branched chain amino acid (BCAA) catabolic pathway is responsible for production of three growth inhibitory byproducts including isovalerate, 2-methylbutyrate and isobutyrate from leucine, isoleucine and valine, respectively. The branched chain aminotransferase 1 (BCAT1) gene encodes for the enzyme that catalyzes the first step in the catabolism of all three BCAAs into inhibitory byproducts. In this study, knockout (KO) of BCAT1 was performed using CRISPR CAS9 technology in an antibody producing CHO cell line. Three clones identified to have the knockout event produced negligible amounts of all the three inhibitory byproducts in fed-batch cultures. The near-complete elimination of BCAA pathway inhibitory byproducts in fed-batch cultures extended the practical culture length from 12 to 20 days due to sustained viabilities, resulted in about 20E6 cells/mL higher peak cell densities, and doubled the productivities when compared to the original producer cell line (wildtype) culture. Metabolomic analysis performed on the fed-batch cultures of a BCAT1 KO clone and wildtype cell line showed significant differences in key metabolic pathways other than BCAA catabolic pathway. The presentation will go into details of these additional understandings.

Evaluation of CHO cells metabolically engineered towards reduced biosynthesis and accumulation of growth inhibitory catabolic byproducts of branched chain amino acids in fed-batch cultures

2019 Spring National Meeting

The official archive and repository for all ACS Meetings & Expositions abstracts and select posters or presentations.