Dissolved oxygen concentration in serum-free continuous culture affects N-linked glycosylation of a monoclonal antibody.
The murine B-lymphocyte hybridoma, CC9C10, was grown at steady state in serum-free continuous culture at dissolved oxygen (DO) concentrations of 10, 50, and 100% of air saturation. The secreted mAb, an IgG1, was purified and subjected to both enzymatic deglycosylation using PNGase F and chemical deglycosylation by hydrazinolysis. Both methods resulted in complete removal of N-linked oligosaccharide chains. Isolated N-glycan pools were analyzed by fluorophore-assisted carbohydrate electrophoresis (FACE) and high pH anion exchange chromatography with pulsed amperometric detection (HPAEC-PAD). The FACE profiles and corresponding HPAEC-PAD chromatograms of N-linked oligosaccharides obtained by PNGase F digestion and hydrazinolysis provided complementary and corroborating information. The predominant N-linked structures were core-fucosylated asialo biantennary chains with varying galactosylation. There were also minor amounts of monosialylated, and trace amounts of afucosyl, oligosaccharides. A definite shift towards decreased galactosylation of glycan chains was observed as DO concentration in continuous culture was reduced. The vast majority of N-linked glycosylation occurred on the heavy chain. There was no evidence for N-linked glycosylation of the light chain or for O-linked glycosylation of the mAb.
Department of Chemistry, University of Manitoba, Winnipeg, Canada.
J P
JP
Kunkel
D C
DC
Jan
J C
JC
Jamieson
M
M
Butler
D000818:Animals; D000911:Antibodies, Monoclonal / Q000737:chemistry; D001402:B-Lymphocytes / Q000378:metabolism*; D002240:Carbohydrate Sequence; D018929:Cell Culture Techniques / Q000379:methods; D004591:Electrophoresis, Polyacrylamide Gel; D006026:Glycoside Hydrolases / Q000378:metabolism; D006031:Glycosylation / Q000187:drug effects; D006825:Hybridomas / Q000378:metabolism*; D007074:Immunoglobulin G / Q000737:chemistry; D051379:Mice; D008969:Molecular Sequence Data; D009844:Oligosaccharides / Q000737:chemistry; D010100:Oxygen / Q000494:pharmacology*; D011134:Polysaccharides / Q000737:chemistry
D000911:Antibodies, Monoclonal; D007074:Immunoglobulin G; D009844:Oligosaccharides; D011134:Polysaccharides; D006026:Glycoside Hydrolases; D010100:Oxygen