High-performance liquid chromatographic mapping of the oligosaccharides released from the humanised immunoglobulin, CAMPATH 1H.
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.
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Wellcome Foundation, Beckenham, Kent, UK.
D000074323:Alemtuzumab; D000581:Amidohydrolases / Q000378:metabolism; D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal / Q000032:analysis*; D061067:Antibodies, Monoclonal, Humanized; D000912:Antibodies, Neoplasm / Q000032:analysis*; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D006031:Glycosylation; D006801:Humans; D007074:Immunoglobulin G / Q000032:analysis; D008969:Molecular Sequence Data; D009844:Oligosaccharides / Q000032:analysis*; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase; D011134:Polysaccharides / Q000032:analysis; D015203:Reproducibility of Results
D000911:Antibodies, Monoclonal; D061067:Antibodies, Monoclonal, Humanized; D000912:Antibodies, Neoplasm; D007074:Immunoglobulin G; D009844:Oligosaccharides; D011134:Polysaccharides; D000074323:Alemtuzumab; D000581:Amidohydrolases; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase