Recombinant human interferon-γ. Differences in glycosylation and proteolytic processing lead to heterogeneity in batch culture

Abstract

Recombinant human interferon-gamma (Hu-IFN-gamma) produced by Chinese-hamster ovary (CHO) cells was analysed by immunoprecipitation and SDS/PAGE. Up to twelve molecular-mass variants were secreted by this cell line. Three variants were recovered after enzymic removal of all N-linked oligosaccharides or when glycosylation was inhibited by tunicamycin. The presence of three polypeptide forms rather than a single form suggested that proteolytic cleavage had occurred at two sites in both the glycosylated and non-glycosylated forms. Proteolytically cleaved IFN-gamma was more prevalent in cell lysates than in the secreted glycoprotein. In common with naturally produced IFN-gamma, both fully glycosylated IFN-gamma (asparagine residues 28 and 100 occupied) and partially glycosylated product (thought to be substituted at position Asn28) were secreted. This was deduced from the Mr of the glycosylated products and the relative amounts of sialic acid expressed by each variant. In contrast with naturally produced IFN-gamma, non-glycosylated IFN-gamma was also secreted by the transfected CHO cells. When the cells were grown in batch culture in serum-free medium under pH and dissolved-oxygen control, the proportion of non-glycosylated IFN-gamma increased from 3 to 5% after 3 h, to 30% of the total IFN-gamma present after 195 h. This change in the proportion of glycosylated protein produced was not seen when metabolically labelled IFN-gamma was incubated for 96 h with cell-free supernatant from actively growing CHO cells. This implied that an alteration in intracellular glycosylation was occurring rather than a degradation of oligosaccharide side chains after secretion. The decrease in IFN-gamma glycosylation was independent of the glucose concentration in the culture medium, but could be related to specific growth and IFN-gamma production rates, as these declined steadily after 50 h of culture, in line with the increased production of non-glycosylated IFN-gamma.

The availability of glucose to CHO cells affects the intracellular lipid-linked oligosaccharide distribution, site occupancy and the N-glycosylation profile of a monoclonal antibody

“…There is no link found between macroheterogeneity in N-glycosylation and the extracellular hydrolytic degradation (Burteau et al, 2003;Curling et al, 1990); consequently, the cause for the diminution in N-glycosylation site occupancy only relies on the intracellular effects. Our results are consistent with previous findings, as it has been recognized for some time that glucose starvation decreases glycosylation efficiency.…”

Analysis of the glycosylation sites of hepatitis C virus (HCV) glycoprotein E1 and the influence of E1 glycans on the formation of the HCV glycoprotein complex.

“…It has also been suggested that N-linked glycosylation is required for folding, transport, cell surface expression, secretion of glycoproteins (Helenius, 1994), protection from proteolytic degradation and enhancement of glycoprotein solubility (Doms et al, 1993 ;Rademacher et al, 1988). Although the sequon is essential for core glycosylation, numerous examples of glycoproteins have been observed containing sequons which are either unglycosylated or glycosylated at a low level (Curling et al, 1990 ;Pohl et al, 1984). In this study, the glycosylation sites used in HCV glycoprotein E1 have been defined and the role of individual oligosaccharide chains in the formation of HCV glycoprotein complexes has been investigated.…”

Disparate Intracellular Processing of Human IL-12 Preprotein Subunits: Atypical Processing of the P35 Signal Peptide

“…This process must also be quite rapid, as none of this form of the protein can be detected intracellularly by immunoprecipitation. Unlike p40 and many other secreted proteins (22)(23)(24), p35 appears to require glycosylation for secretion, since inhibition of N-linked glycosylation by tunicamycin completely inhibits secretion.…”