Effect of ammonia on the glycosylation of human recombinant erythropoietinin culture

Recombinant human erythropoietin (EPO) was produced by a stable transfected CHO-K1 cell done (EPO-81) grown in serum-free medium. Our previous work sh owed that there was a significant increase in the heterogeneity of the glyc oforms of EPO and a reduction of the sialylation at 20 mM NH4Cl. In the wor k presented here, the effects of ammonia on EPO N-linked oligosaccharides w ere analyzed. EPO was purified from culture supernatants by immunoaffinity chromatography. The N-linked oligosaccharides were released enzymatically a nd analyzed by fluorophore-assisted carbohydrate electrophoressis (FACE) an d HPLC. The FACE N-linked oligosaccharide profile showed that the sialylate d glycans contain one prominent band at a position corresponding to eight g lucose units. The density of the major band was greatly diminished and the width was significantly increased in cultures containing added ammonia. The proportion of tetraantennary structures was reduced by 60%, while the tri- and biantennary structures were increased proportionally in the presence o f ammonia. Glycan analysis by HPLC using a weak anion exchange column showe d that the most significant characteristic effect of ammonia was a reductio n of the proportion of glycans with four sialic acids from 46% in control c ultures to 29% in ammonia-treated cultures. Analysis of the desialylated gl ycans by normal phase chromatography indicated a distribution of tetra-, tr i-, and bianteennary structures similar to that shown by FACE. The N-linked glycan sequence was determined by sequential exoglycosidase digestion foll owed by FACE. The results indicated a typical N-linked complex oligosacchar ide structure. Glycans from ammonia-containing cultures showed the same seq uence pattern. In conclusion, we showed that ammonia in the culture medium affected EPO glycosylation, which was observed as a reduction of the tetraa ntennary and tetra-sialylated oligosaccharide structures. However, the pres ence of ammonia in the cultures did not change the oligosaccharide sequence .