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 i n serum-free continuous culture at dissolved oxygen (DO) concentration s of 10, 50, and 100% of air saturation. The secreted mAb, an IgG1, wa s purified and subjected to both enzymatic deglycosylation using PNGas e F and chemical deglycosylation by hydrazinolysis. Both methods resul ted in complete removal of N-linked oligosaccharide chains. Isolated N -glycan pools were analyzed by fluorophore-assisted carbohydrate elect rophoresis (FACE) and high pH anion exchange chromatography with pulse d amperometric detection (HPAEC-PAD). The FACE profiles and correspond ing HPAEC-PAD chromatograms of N-linked oligosaccharides obtained by P NGase F digestion and hydrazinolysis provided complementary and corrob orating information. The predominant N-linked structures were core-fuc osylated asialo biantennary chains with varying galactosylation. There were also minor amounts of monosialylated, and trace amounts of afuco syl, oligosaccharides. A definite shift towards decreased galactosylat ion of glycan chains was observed as DO concentration in continuous cu lture was reduced. The vast majority of N-linked glycosylation occurre d on the heavy chain. There was no evidence for N-linked glycosylation of the light chain or for O-linked glycosylation of the mAb. (C) 1998 Elsevier Science B.V. All rights reserved.