Altering the central carbon metabolism of HEK293 cells: impact on recombinant glycoprotein quality

The accumulation of metabolic by-products remains a critical challenge in the development of mammalian cells culture processes as it impacts cellular growth, productivity and product quality. Although the overexpression of the PYC2 gene was shown to significantly improve the nutrient metabolism efficiency of mammalian cells, its impact on recombinant protein quality has not been investigated yet. In this study, we assess the effect of this metabolic engineering strategy on the quality of a recombinant therapeutic glycoprotein, the human interferon \u3b12b (IFN\u3b12b). As inferred from densitometry analysis of SDS-PAGE gels, PYC2-overexpressing cells sustained a higher percentage of intact glycosylated IFN\u3b12b at the late stage of batch cultures, which was correlated with prolonged viability and reduced accumulation of waste metabolites. Contrarily to the IFN\u3b12b produced by the PYC2 cells, LC\u2013MS analysis confirmed the presence of less glycosylated IFN\u3b12b as well as the occurrence of proteolytic cleavage in the IFN\u3b12b produced in the parental cells. Taken together, these results indicate that PYC2-overexpression in mammalian cells leads to extended favorable conditions for glycosylation and offer an attractive approach to mass-produce high-quality recombinant proteins.Peer reviewed: YesNRC publication: Ye