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Glycosylation is the most extensive of all the posttranslational modifications, and has important functions in the secretion, antigenicity and clearance of glycoproteins. In recent years major advances have been made in the cloning of glycosyltransferase enzymes, in understanding the varied biological functions of carbohydrates, and in the accurate analysis of glycoprotein heterogeneity. In this review we discuss the impact of these advances on the choice of a recombinant host cell line, in optimizing cell culture processes, and in choosing the appropriate level of glycosylation analysis for each stage of product development.

The use of hydrazine to release unreduced N- and O-linked oligosaccharides from glycoproteins has been investigated using several "standard" glycoproteins of previously defined glycosylation. It is shown that hydrazinolysis can be used to release intact N- and O-linked oligosaccharides in an unreduced form. The release of O-linked oligosaccharides occurs with a lower temperature dependence than the release of N-linked oligosaccharides, and the kinetic parameters governing release of oligosaccharides from these standard glycoproteins have been determined. These parameters allow a definition of reaction conditions under which anhydrous hydrazinolysis can be used to selectively release O-linked oligosaccharides (60 degrees C, 5 h) or release both N- and O-linked oligosaccharides (95 degrees C, 4 h) in high yield (> 85%) from all glycoproteins investigated (n = 11). Under these reaction conditions, the recovered N- and O-linked oligosaccharides are structurally intact (as judged by 600-MHz 1H-NMR, laser-desorption mass spectrometry, HPAEC-PAD, gel filtration, and glycosidase digestion), with the possible exception of certain N- and O-acyl substituents of sialic acid. This use of mild hydrazinolysis therefore allows both the simultaneous and sequential chemical release from glycoproteins of O- and N-linked oligosaccharides in their intact unreduced form.