High-performance liquid chromatographic mapping of the oligosaccharides released from the humanised immunoglobulin,...

A sensitive and reproducible method for the routine mapping of oligosaccharides in a humanised immunoglobulin (IgG) is described. The method involves the enzymic release of intact glycans using the endoglycosidase glycopeptidase-F, and subsequent derivatisation with 1-phenyl-3-methyl-5-pyrazolone to facilitate analysis by high-performance liquid chromatography (HPLC). The heterogeneous oligosaccharide chains are separated by a phosphate buffer-acetonitrile gradient reversed-phase HPLC method and monitored by ultraviolet detection at 245 nm, allowing the detection of picomole amounts. A number of standard oligosaccharides are similarly derivatised to enable classification of the types of structures present from a comparison of retention times.