title: Characterization of monoclonal antibody glycosylation: comparison of expression systems and identification of terminal alpha-linked galactose.
abstract: CAMPATH-1H is a recombinant humanized murine monoclonal immunoglobulin (IgG1) which recognizes the CDw52 antigen of human lymphocytes, and has been the subject of clinical trials for the treatment of non-Hodgkin's lymphoma and rheumatoid arthritis. Peptide mapping by liquid chromatography-mass spectrometry was used to confirm the predicted amino acid sequences and profile glycosylation for two CAMPATH isotypes expressed in a murine myeloma cell line (NS0) and a single isotype expressed in both Chinese hamster ovary (CHO) and NS0 lines. The three major glycoforms identified in CAMPATH are fucosylated biantennary structures, containing zero, one, or two galactose residues. Glycosylation of the IgG1 form of CAMPATH expressed in CHO cells is consistent with normal human IgG. However, IgG1 and IgG4 expressed in NS0 cells include two potentially immunogenic glycoforms which contain either one or two nonreducing terminal alpha-linked galactose residues. Oligosaccharide structures were characterized by a combination of tandem mass spectrometry, methylation analysis, and exoglycosidase digestion. The strategy used here is designed to be widely applicable to recombinant glycoproteins.
authors.affiliation: Glaxo Wellcome Inc., Research Triangle Park, North Carolina 27709, USA.
authors.forename: D M
authors.initials: DM
authors.lastname: Sheeley
authors.forename: B M
authors.initials: BM
authors.lastname: Merrill
authors.forename: L C
authors.initials: LC
authors.lastname: Taylor
mesh_terms: D000074323:Alemtuzumab; D000818:Animals; D000911:Antibodies, Monoclonal / Q000737:chemistry* / Q000235:genetics*; D061067:Antibodies, Monoclonal, Humanized; D000912:Antibodies, Neoplasm / Q000737:chemistry* / Q000235:genetics*; D016466:CHO Cells; D002240:Carbohydrate Sequence; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D006224:Cricetinae; D005690:Galactose / Q000737:chemistry; D015870:Gene Expression; D006026:Glycoside Hydrolases; D006031:Glycosylation; D006801:Humans; D007074:Immunoglobulin G / Q000737:chemistry / Q000235:genetics; D051379:Mice; D008969:Molecular Sequence Data; D015394:Molecular Structure; D009844:Oligosaccharides / Q000737:chemistry; D010449:Peptide Mapping / Q000379:methods; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods
chemical_list: D000911:Antibodies, Monoclonal; D061067:Antibodies, Monoclonal, Humanized; D000912:Antibodies, Neoplasm; D007074:Immunoglobulin G; D009844:Oligosaccharides; D000074323:Alemtuzumab; D006026:Glycoside Hydrolases; D005690:Galactose