Metabolic effects on recombinant interferon-? glycosylation in continuous culture of Chinese hamster ovary cells

Abstract

Asparagine linked (N‐linked) glycosylation is an important modification of recombinant proteins, because the attached oligosaccharide chains can significantly alter protein properties. Potential glycosylation sites are not always occupied with oligosaccharide, and site occupancy can change with the culture environment. To investigate the relationship between metabolism and glycosylation site occupancy, we studied the glycosylation of recombinant human interferon‐γ (IFN‐γ) produced in continuous culture of Chinese hamster ovary cells. Intracellular nucleotide sugar levels and IFN‐γ glycosylation were measured at different steady states which were characterized by central carbon metabolic fluxes estimated by material balances and extracellular metabolite rate measurements. Although site occupancy varied over a rather narrow range, we found that differences correlated with the intracellular pool of UDP‐N‐acetylglu‐ cosamine + UDP‐N‐acetylgalactosamine (UDP‐GNAc). Measured nucleotide levels and estimates of central carbon metabolic fluxes point to UTP depletion as the cause of decreased UDP‐GNAc during glucose limitation. Glucose limited cells preferentially utilized available 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 batch and fed‐batch culture have confirmed that UDP‐sugar concentrations are correlated with UTP levels in the absence of glutamine limitation. Glutamine limitation appears to influence glycosylation by reducing amino sugar formation and hence UDP‐GNAc concentration. The influence of nucleotide sugars on site occupancy may only be important during periods of extreme starvation, since relatively large changes in nucleotide sugar pools led to only minor changes in glycosylation. © 1999 John Wiley & Sons, Inc. Biotechnol Bioeng 62: 336–347, 1999.

Tailoring recombinant protein quality by rational media design

“…While the limitation of glucose in the culture medium allows to reduce lactate production 77 , it has been observed that critical limitation of the former leads to glycosylation heterogeneity 78 , due to a decreased UDP-acetylglucosamine (UDP-GlcNAc) availability 79 . The absence of glucose affects the synthesis of oligosaccharide precursors, as glucose-starved cells alter its synthesis process 80 .…”

“…Metabolomic analysis of fed-batch cultures revealed increasing ornithine levels coincided with higher high mannose glycan levels 285 . In continuous CHO cell culture producing interferon-γ, site glycosylation occupancy was linked with the intracellular UDP-N-acetylgalactosamine concentration 79 . Ammonium plays an active part in the regulation of the pH of acidic intracellular compartments of CHO expressing recombinant immunoadhesin tumor necrosis factor-IgG (TNFR-IgG), including trans-Golgi and it affects intracellular nucleotide sugar pools, especially UDP-N-acetylglucosamine (UDP-GlcNAc) and UDP-N-acetylgalactosamine 237 .…”

Dynamic Modeling of Apoptosis and its Interaction with Cell Growth in Mammalian Cell Culture

“…A number of studies have been reported in characterization of growth and metabolism over a wide range of dilution rates for CHO cells (Heidemann et al, 1998;Nyberg et al, 1999), hybridoma cells (Frame and Hu, 1991;Ray et al, 1989;Vriezen and van Dijken, 1998) and BHK cells (Carrondo et al, 1999;Hu et al, 2000).…”

Metabolomic study of baculovirus infected insect cells to identify metabolite based strategies for improving virus yields

“…This result may be explained by a high level of peptides consumed by infected Sf9 cells in Sf900™III at the low ICD. There is evidence that very small peptides contribute to the nutritional value of a medium as a source of AAs (Heidemann et al, 2000;Nyberg et al, 1999) and so it is possible that the use of small oligo-peptides as an AA source is preferable than the use of free AAs in some situations. Di-peptides and tri-peptides are taken up more readily in the intestine than corresponding free amino acids (Grimble et al, 1987;Silk et al, 1985).…”