DO Effects

The effect of the DO concentration on cell metabolism and growth has been investigated for numerous mammalian cell types (74,98-104). A few resear-chers have extended their investigations to include the influence of DO on N-glycosylation and the sialic acid content of glycoproteins (74,102-104). Interestingly, these authors observed that protein production increased with increased DO (102) and N-glycosylation was less sensitive to DO changes than to growth rate (74,101). Jan et al. (103) observed that even though cell viability, cell growth rate, and MAb productivity were not significantly influenced by DO changes (10-100% air saturation), the fluxes through metabolic pathways were significantly affected for a hybridoma cell line. Ozturk and Palsson (100) investigated cell growth, amino acid, carbohydrate, energy metabolism, and antibody production for a hybridoma cell line under oxygen stress. These authors observed that amino acid utilization was significantly increased in an oxygen-stressed environment. Although the glycosylation of the MAbs was not addressed, the authors' data indicate that glycosylation may have been altered, since the cell switched metabolism to adapt to the low DO.

Lin et al. (101) investigated the influence of DO on glycosylation of tPA in perfusion reactors. Although they observed significant chances in the growth rate, metabolism, and protein productivity, they did not observe significant changes in glycosylation. The methods they used to evaluate glycosylation were in vitro activity and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), methods known to detect gross changes only. Additionally, the cells were cultivated in perfusion reactors that never reached steady state, in terms of cell density or media composition. The DO of the perfusion reactor was changed while the nutrient composition of the media was changing. Therefore, it is likely that any changes in glyco-sylation due to DO were masked by other nutrient influences on glycosylation.

One of the most complete studies on MAb glycosylation examined the influence of DO on glycosylation (104). Kunkel et al. (104) varied the DO from 10 to 100% air saturation in serum-free chemostats. Glycosylation was evaluated by fluor-ophore-assisted carbohydrate electrophoresis (FACE) and monosaccharide and oligosaccharide profiles were determined by high-pH anion-exchange chromatography with pulsed amperometric detection (HPAEC-PAD). The authors observed that galactosylation decreased as the DO decreased. This decrease in the galactose content of the oligosaccharide could be the result of disturbances in the ER. The authors also noted that cells at the lower DO concentrations had increased glycolytic flux and a decreased tricarboxylic acid cycle flux (104). The changes in the glycosylation of the MAb were directly related to changes in central metabolic pathway enzyme activity.

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