Metabolic effects on recombinant interferon-gamma glycosylation in continuous culture of Chinese hamster ovary cells (Reprinted from Biotechnology and Bioengineering, vol 62, pg 336-347, 1999)

Asparagine linked (N-linked) glycosylation is an important modification of recombinant proteins, because the attached oligosaccharide chains can signi ficantly alter protein properties. Potential glycosylation sites are not al ways occupied with oligosaccharide, and site occupancy can change with the cu Itu re environ ment. To investigate the relationship between metabolism and glycosylation site occupancy, we studied the glycosylation of recombina nt human interferon-gamma (IFN-gamma) produced in continuous culture of Chi nese hamster ovary cells. Intracellular nucleotide sugar levels and IFN-gam ma glycosylation were measured at different steady states which were charac terized by central carbon metabolic fluxes estimated by material balances a nd extracellular metabolite rate measurements. Although site occupancy vari ed over a rather narrow range, we found that differences correlated with th e intracellular pool of UDP-N-acetylglucosamine + UDP-N-acetylgalactosamine (UDP-GNAc). Measured nucleotide levels and estimates of central carbon met abolic fluxes point to UTP depletion as the cause of decreased UDP-GNAc dur ing glucose limitation. Glucose limited cells preferentially utilized avail able carbon for energy production, causing reduced nucleotide biosynthesis. Lower nucleoside triphosphate pools in turn led to lower nucleotide sugar pools and reduced glycosylation site occupancy. Subsequent experiments in b atch and fed-batch culture have confirmed that UDP-sugar concentrations are correlated with UTP levels in the absence of glutamine limitation. Glutami ne limitation appears to influence glycosylation by reducing amino sugar fo rmation and hence UDP-GNAc concentration. The influence of nucleotide sugar s on site occupancy may only be important during periods of extreme starvat ion, since relatively large changes in nucleotide sugar pools led to only m inor changes in glycosylation. (C) 1999 John Wiley & Sons, Inc. Biotechnol Bioeng 62: 336-347, 1999.