Effect of Sodium Butyrate on glycosylation of recombinant Erythropoietin

Abstract

The effect of Sodium Butyrate (NaBu) on the N-linked oligosaccharide structure of Erythropoietin (EPO) was investigated. Recombinant human EPO was produced by CHO cells grown in an MEM alpha medium with or without 5 MM NaBu, and purified from the culture supernatants using a heparin-sepharose affinity column and immunoaffinity column. The N-linked oligosaccharides were released enzymatically and isolated by paper chromatography. The isolated oligosaccharides were then labeled with a fluorescent dye, 2-aminobenzamide, and analyzed with MonoQ anion exchange chromatography and GlycosepN amide chromatography for the assignment of a GU (glucose unit) value. A glycan analysis by HPLC showed that the most significant characteristic effect of NaBu was a reduction in the proportion of glycans with tri-and tetrasialylated oligodaccharides from 21.30% (tri-) and 14.86% (tetra-) in the control cultures (without NaBu) to 8.72% (tri-) and 1.25% (tetra-) in the NaBu-treated cultures, respectively. It was also found that the proportion of asialoglycan increased from 12.54% to 23.69% when treated with NaBu.