Comparisons of the glycosylation of a monoclonal antibody produced under nominally identical cell culture conditions in two different bioreactors

The murine B-lymphocyte hybridoma cell line, CC9C10, was grown in serum-fre e continuous culture at steady-state dissolved oxygen (DO) concentrations o f 10%, 50%, and 100% of air saturation in both LH Series 210 (LH) and New B runswick Scientific (NBS) CelliG en bioreactors. All culture parameters wer e monitored and controlled and were nominally identical at steady state in the two bioreactors. The secreted monoclonal antibody (mAb), an immunoglobu lin G(1), was purified and subjected to enzymatic deglycosylation using pep tide N-glycosidase F (PNGase F). Asparagine-linked (N-linked) oligosacchari de pools released from mAb samples cultured in each bioreactor at each of t he three DO setpoints were analyzed by high-pH anion-exchange chromatograph y with pulsed amperometric detection (HPAFC-PAD). The predominant N-linked structures were core-fucosylated asialo biantennary chains with varying gal actosylation. There were also minor amounts of monosialyl oligosaccharides and trace amounts of afucosyl oligosaccharides. The level of DO affects the glycosylation of this mAb. A definite reduction in the level of galactosyl ation of N-glycan chains was observed at lower DO in both bioreactors, as e videnced by prominent increases in the relative amounts of agalactosyl chai ns and decreases in the relative amounts of digalactosyl chains-with the re lative amounts of monogalactosyl chains being comparatively constant. Howev er, the quantitative results are not precise matches between the two biorea ctors. The effect of DO on galactosylation is less pronounced in the NBS bi oreactor than in the LH bioreactor, particularly the shift between the rela tive amounts of agalactosyl and digalactosyl chains in 10% and 50% DO. Ther e are also perceptibly higher levels of sialylation of the mAb glycans in t he NBS bioreactor than in the LH bioreactor at all three DO setpoints. The results indicate that the DO effect is not bioreactor specific and that nom inally identical steady-state conditions in different chemostat bioreactors may still lead to some incongruities in glycosylation, possibly due to the particular architectures of the bioreactors and the design of their respec tive monitoring and control systems. The observed differences in N-linked g lycosylation of the mAb secreted by the hybridoma grown in the LH and NBS b ioreactors may be explained by the differences in oxygen supply and control strategies between the two bioreactors.