Metabolic engineering of Bcat1, Adh5 and Hahdb towards controlling metabolic inhibitors and improving performance in CHO cell-cultures

Abstract

CHO cells are known to secrete growth inhibitory metabolites during growth and production phases, which hampers cellular performance and negatively impacts final productivity and product quality attributes. Previous studies have identified different metabolic by-products derived from CHO metabolism and demonstrated their negative impacts on growth and titer productivity. This work presents a control strategy that incorporated genetic engineering and inhibitory metabolites pathway analysis to regulate cellular metabolism. In this study, three different metabolic genes involving the metabolism of branched-chain amino acids (Adh5, Bcat1 and Hadhb) were cloned from CHO reversely synthesized cDNA to study for gene functionality towards regulating cellular metabolism. Identified metabolic genes were individually engineered into expression vector and transfected to cells at plasmid concentration of 40 µg/µL. For all engineered conditions, the peak VCD profile of cells on Day 4 realized a 18–20% increase with cumulative VCD profile realizing 16–19% increase in terms of total viable cells collected on harvest day. Increase in IgG1 titer production and also more complex glycosylation profiles formation were also observed from all engineered subclones. This study hereby successfully demonstrated rewiring cellular metabolism through up-regulation of key metabolic enzymes can effectively control the accumulation of process inhibitors which therefore allowed improvement desirable critical process attributes.