Pattern And Influencing Factors Of RhEPO N-Glycosylation In CHO Cells Culture

The research in the work is based on an rCHO cell strain for industrial use and a practical production process in large-scale. The investigation into the rules of the N-glycan pattern changes of rhEPO produced by the CHO cell strain in perfusion mode and the influencing factors during the production may serve as guidance for the optimization of this process.The cell strain was firstly cultured in static mode and the supernatant was collected. It was found that, as the culture time prolonged, the quality of rhEPO protein declined. The proteins in the supernatant were run into IEF and preformed with RCA-I for lectin precipitation, and the results showed that the terminal of the N-glycans on the proteins lost their sialic acids gradually during the culture. The cells viability decreased while the sialidase activity in the supernatant increased in this process.The investigation on the samples from the perfusion culture process in industry showed that the sialic acid content dropped rapidly to low level as the culture condition was changed from serum-supplemented medium to serum-free medium. With the production time extending, the cell gradually lost their viability and the extracellular sialidase activity existed constantly.The sialidase in the extracellular environment could play a negative role on the quality of rhEPO protein produced by CHO cells, which was confirmed by the addition of sialidase inhibitor into the production medium to improve the N-glycosylation quality of the protein products. The investigation into the effect of sodium butyrate on the glycosylation pattern of rhEPO produced by CHO cells as well as the expression level and the extracellular activity of the sialidases gave the results that the addition of sodium butyrate could significantly decrease the transcriptional level of CHO sialidases and the extracellular sialidase activity. The quality of the proteins was improved although the cell viability was negatively influenced.