Effect of Sodium Butyrate on Glycosylation of Recombinant Erythropoietin

Abstract

EPO is a glycoprotein hormone which controls the production of erythrocytes in mammalian cells and has a complex oligosaccharide structure that plays an important role in biological activity in vivo. Many research groups have studied to enhance the productivity of EPO by adding the chmicals including Sodium butyrate in the culture medium. Sodium butyrate (NaBu) is known to enhance the rate of biosynthesis of recombinant proteins in Chinese Hamster Ovary cells (CHO). However, this chemical may influence the glycoform of recombinant EPO. We studied the effect of NaBu on N-linked oligosaccharide structure of EPO. Recombinant human EPO was produced by CHO cells grown in the MEMa medium with or without 5 mM NaBu and purified from culture supernatants by heparin-sepharose affinity column and immunoaffinity column. N-linked oligosaccharides were released enzymatically and isolated by paper chromatography. The isolated oligosaccharides were labeled with a fluorescent dye, 2-aminobenzamide and analyzed with MonoQ anion exchange chromatography and GlycosepN amide chromatography for the assignment of GU (glucose unit) value. Glycan analysis by HPLC showed that the most significant characteristic effect of NaBu was a reduction of the proportion of glycans with tri- and tetrasialylated oligodaccharides from 21.30% (tri-), 14.86% (tetra-) in control cultures (without NaBu) to 8.72% (tri-), 1.25% (tetra-) in NaBu-treated cultures respectively. It was also found that portion of asialo-glycan was increased from 12.54% to 23.69% when treated with NaBu.