Analysis of carbohydrates on IgG preparations.
Characterization of monoclonal antibodies (MAbs) produced for therapeutic or diagnostic purposes increasingly includes an assessment of their carbohydrate content. Using high-performance anion exchange chromatography with pulsed amperometric detection (HPAEC/PAD), we have analyzed the PNGase F released oligosaccharides of several IgG preparations including human polyclonal IgG, a humanized monoclonal IgG (MAb M115), and a murine monoclonal IgG (MAb MY9-6) derived respectively from serum, hybridoma cultures, and ascites fluid. The N-linked oligosaccharides released by PNGase F treatment of the above IgGs were found to consist mainly of neutral, fucosylated, biantennary species. Comparison of glycosylation of human polyclonal IgG, MAb M115, and MAb MY9-6 revealed differences in the levels of galactosylation and in the levels as well as the form of sialic acid present. HPAEC/PAD oligosaccharide profiling, combined with the use of enzymes (PNGase F, endoglycosidase F2, endoglycosidase H, neuraminidase, beta-galactosidase, and beta-N-acetylhexosaminidase), and monosaccharide analysis allowed making of tentative structural assignments. By performing monosaccharide analysis directly on PVDF electroblotted heavy and light chain bands separated by SDS-PAGE, it was verified that IgGs used in this study were glycosylated predominantly in their heavy chain.
Weitzhandler
M
M
Dionex Corp., Sunnyvale, CA 94088.
Hardy
M
M
Co
M S
MS
Avdalovic
N
N
D000581:Amidohydrolases / Q000378:metabolism; D000911:Antibodies, Monoclonal / Q000032:analysis*; D002240:Carbohydrate Sequence; D002241:Carbohydrates / Q000032:analysis*; D002626:Chemistry, Pharmaceutical; D002852:Chromatography, Ion Exchange; D004563:Electrochemistry; D006023:Glycoproteins / Q000032:analysis / Q000378:metabolism; D006026:Glycoside Hydrolases / Q000378:metabolism; D006031:Glycosylation; D007074:Immunoglobulin G / Q000032:analysis*; D008969:Molecular Sequence Data; D009844:Oligosaccharides / Q000032:analysis; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase
D000911:Antibodies, Monoclonal; D002241:Carbohydrates; D006023:Glycoproteins; D007074:Immunoglobulin G; D009844:Oligosaccharides; D006026:Glycoside Hydrolases; D000581:Amidohydrolases; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase