Constraints on the transport and glycosylation of recombinant IFN-gamma inChinese hamster ovary and insect cells

In this study we compare intracellular transport and processing of a recomb inant glycoprotein in mammalian and insect cells. Detailed analysis of the N-glycosylation of recombinant human IFN-V by matrix-assisted laser-desorpt ion mass spectrometry showed that the protein secreted by Chinese hamster o vary and baculovirus-infected insect Sf9 cells was associated with complex sialylated or truncated tri-mannosyl core glycans, respectively. However, t he intracellular proteins were predominantly associated with high-mannose t ype oligosaccharides (Man-6 to Man-g) in both cases, indicating that endopl asmic reticulum to cis-Golgi transport is a predominant rate-limiting step in both expression systems. In CHO cells, although there was a minor intrac ellular subpopulation of sialylated IFN-gamma glycoforms identical to the s ecreted product (therefore associated with late-Golgi compartments or secre tory vesicles), no other intermediates were evident. Therefore, anterograde transport processes in the Golgi stack do not limit secretion. In Sf9 inse ct cells, there was no direct evidence of post-ER glycan-processing events other than core fucosylation and de-mannosylation, both of which were glyco sylation site-specific. To investigate the influence of nucleotide-sugar av ailability on cell-specific glycosylation, the cellular content of nucleoti de-sugar substrates in both mammalian and insect cells was quantitatively d etermined by anion-exchange HPLC. In both host cell types, UDP-hexose and U DP-N-acetylhexosamine were in greater abundance relative to other substrate s. However, unlike CHO cells, siaiyltransferase activity and CMP-NeuAc subs trate were not present in uninfected or baculovirus-infected Sf9 cells. Sim ilar data were obtained for other insect cell hosts, Sf21 and Ea4. We concl ude that although the limitations on intracellular transport and secretion of recombinant proteins in mammalian and insect cells a re similar, N-glyca n processing in Sf insect cells is limited, and that genetic modification o f N-glycan processing in these insect cell lines will be constrained by sub strate availability to terminal galactosylation. (C) 1999 John Wiley & Sons , Inc.