Monosaccharide and oligosaccharide analysis of isoelectric focusing-separated and blotted granulocyte colony-stimulating factor glycoforms using high-pH anion-exchange chromatography with pulsed amperometric detection

In this study, a sensitive, straightforward technique is developed for the analysis of glycoprotein O-linked oligosaccharides. Specifically, O-linked oligosaccharides of granulocyte colony-stimulating factor (G-CSF) are analysed by separating charged glycoforms using isoelectric focusing, electroblotting to polyvinylidene difluoride, releasing monosaccharides and oligosaccharide alditols from the blotted glycoprotein bands, and producing chromatographs using high-pH anion-exchange chromatography with pulsed amperometric detection. Using this technique, the O-linked structures of G-CSF produced by recombinant Chinese hamster ovary (CHO) cells are deduced by comparison with monosaccharide and oligosaccharide standards. Lectin blotting and peptide sequencing support the identities of the presumed G-CSF glycoforms. The two major glycoforms determined using this methodology correspond to those determined previously for CHO-produced G-CSF using NMR. Additional glycoforms are also identified in this study, presumably resulting from the presence of N-glycolyl-neuraminic acid in place of N-acetylneuraminic acid. The utility of this analytical approach is then demonstrated in an analysis of the effect of the extracellular environment on the O-linked glycosylation of G-CSF by recombinant CHO cells. Increasing the level of ammonium ion in the culture medium is shown to reduce the percentage of G-CSF produced with sialic acid linked α(2,6) to N-acetylgalactosamne.