Expression of GNE mutant proteins increases CHO intracellular CMP-Neu5Ac levels without impact on bioprocess performance.
Academic Article
Overview
abstract
Modulation of various nucleotide sugar levels in cells has been demonstrated as an effective way to alter the composition of N-glycans. Previous studies have demonstrated the ability to impact CMP-Neu5Ac levels by the addition of N-acetylated mannosamine (ManNAc) to culture media. In this study, the relationship between adding varying levels of ManNAc to cell cultures and the impact on both CMP-Neu5Ac levels and cell growth were examined. Increasing the concentration of ManNAc added resulted in higher levels of CMP-Neu5Ac, but negatively impacted cell growth. Through cellular genetic engineering, we sought to devise an alternative method of increasing ManNAc levels without impacting cell growth. The UDP-N-acetylglucosamine 2-epimerase/N-acetylmannosamine-kinase (GNE) gene is the rate-limiting enzyme in which congenital mutations can cause Sialuria, a rare metabolic disorder characterized by cytoplasmic accumulation and urinary excretion of free sialic acid. A mutant form of the GNE gene, harboring three mutations (D53H, R263I, R266Q), was site-specifically integrated (SSI) into one locus in CHO cells. This mutant protein dramatically increased the intracellular concentrations of CMP-Neu5Ac, reaching the maximal level as with the addition of ManNAc. These data together indicate that the GNE mutants could provide an effective way for substituting the high-cost supplementation of ManNAc without impacting cell growth. The investigation has also demonstrated the feasibility of the dual-landing-pad SSI cell line engineering approach for improving product qualities of biotherapeutics.
