Related effects of cell adaptation to serum-free conditions on murine EPO production and glycosylation by CHO cells.

The necessity to perform serum-free cultures to produce recombinant glycoproteins generally requires an adaptation procedure of the cell line to new environmental conditions, which may therefore induce quantitative and qualitative effects on the product, particularly on its glycosylation. In previous studies, desialylation of EPO produced by CHO cells was shown to be dependent on the presence of serum in the medium. In this paper, to discriminate between the effects of the adaptation procedure to serum-free medium and the effects of the absence of serum on EPO production and glycosylation, adapted and non-adapted CHO cells were grown in serum-free and serum-containing media. The main kinetics of CHO cells were determined over batch processes as well as the glycosylation patterns of produced EPO by HPCE-LIF. A reversible decrease in EPO production was observed when cells were adapted to SFX-CHO(TM) medium, as the same cells partially recovered their production capacity when cultivated in serum-containing medium or in the enriched SFM(TM) serum-free medium. More interestingly, EPO desialylation that was not observed in both serum-free media was restored if the serum-independent cells were recultured in presence of serum. In the same way, while the serum-independent cells did not release a sialidase activity in both serum-free media, a significant activity was recovered when serum was added. In fact, the cell adaptation process to serum-free conditions did not specifically affect the sialidase release and the cellular mechanism of protein desialylation, which appeared to be mainly related to the presence of serum for both adapted and non-adapted cells.

HPCE monitoring of the N-glycosylation pattern and sialylation of murine erythropoietin produced by CHO cells in batch processes.

Using capillary electrophoresis coupled to laser-induced fluorescence (HPCE-LIF), it was possible to profile N-linked oligosaccharides from EPO, including species containing sialic acid, during the course of batch cultures performed either in serum-free or serum-containing medium. Although an unusual high heterogeneity of the N-linked oligosaccharides was observed by both SDS-PAGE and HPCE analysis, the patterns of mEPO glycans after desialylation by mild acid hydrolysis were found to be quite constant over the course of the cultures either with or without serum supplementation. In contrast, when the protein was analyzed by HPCE without acidic desialylation, fingerprints of N-linked oligosaccharides changed with time in serum-containing conditions. This phenomenon appeared to be mainly due to the desialylation of mEPO as a result of a sialidase activity released upon cell lysis. These results demonstrate that though a higher EPO titer was obtained in serum supplemented conditions, sialylation of EPO was severely affected by the presence of serum in the culture medium.