OLIGOSACCHARIDE STRUCTURES OF GLYCOPROTEINS FROM RECOMBINANT MAMMALIAN CELL LINES

Abstract

Our group has elucidated the carbohydrate structures of a number of pharmaceutically relevant recombinant glycoproteins (IFN-8, IL-2, t-PA, AT II, EPO as well as mutant proteins thereof) expressed in several mammalian host cell lines (CHO, BHK, C127, Ltk) . After enzymatic liberation of the N-linked oligosaccharide chains by action of polypeptide:N-glycanase from the intact glycoprotein or tryptic fragments thereof, the individual oligosaccharides were separated by a combination of ion exchange chromatography and HPLC on NH,-phase. Oligosaccharide structures were elucidated using several analytical techniques: GC/MS (SIM-mode) for compositional and methylation analyses, FAB-MS for the determination of their molecular weight and the terminal substitution pattern as well as 600 MHz 7H-NMR spectrometry for the determination of anomeric configuration and linkage pattern of the monosaccharide building blocks. The recently introduced high-pH-anion-exchange-chromatography with pulsed amperometric detection (HPAE-PAD) was applied for comparison of differently charged oligosaccharide fractions after enzymatic desialylation, determination of oligosaccharide structures at individual glycosylation sites and for control of batch-to-batch consistency of biotechnologically produced glycoproteins.