Effect of Ammonia on the Glycosylation of Human Recombinant Erythropoietin in Culture

Abstract

Recombinant human erythropoietin (EPO) was produced by a stable transfected CHO-K1 cell clone (EPO-81) grown in serum-free medium. Our previous work showed that there was a significant increase in the heterogeneity of the glycoforms of EPO and a reduction of the sialylation at 20 mM NH(4)Cl. In the work presented here, the effects of ammonia on EPO N-linked oligosaccharides were analyzed. EPO was purified from culture supernatants by immunoaffinity chromatography. The N-linked oligosaccharides were released enzymatically and analyzed by fluorophore-assisted carbohydrate electrophoresis (FACE) and HPLC. The FACE N-linked oligosaccharide profile showed that the sialylated glycans contain one prominent band at a position corresponding to eight glucose 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 of ammonia. Glycan analysis by HPLC using a weak anion exchange column showed that the most significant characteristic effect of ammonia was a reduction of the proportion of glycans with four sialic acids from 46% in control cultures to 29% in ammonia-treated cultures. Analysis of the desialylated glycans by normal phase chromatography indicated a distribution of tetra-, tri-, and biantennary structures similar to that shown by FACE. The N-linked glycan sequence was determined by sequential exoglycosidase digestion followed by FACE. The results indicated a typical N-linked complex oligosaccharide structure. Glycans from ammonia-containing cultures showed the same sequence pattern. In conclusion, we showed that ammonia in the culture medium affected EPO glycosylation, which was observed as a reduction of the tetraantennary and tetrasialylated oligosaccharide structures. However, the presence of ammonia in the cultures did not change the oligosaccharide sequence.

Erythropoietin production from CHO cells grown by continuous culture in a fluidized‐bed bioreactor

“…Six discrete bands were identi®ed with a molecular size range of 33± 39 kD and pI values from 4.1 to 4.4. These are the expected ranges of glycosylated EPO with variable terminal sialylation of the N-glycan structures (Yang and Butler, 2000b). Identical patterns were obtained with EPO samples taken from T-¯asks and the stirred-tank bioreactor.…”

Towards dynamic metabolic flux analysis in CHO cell cultures

“…For example, Ozturk and Palsson [81] showed that the actual uptake of glutamine in hybridoma cell culture was three-fold lower than the apparent uptake.Ammonium that is released via glutaminolysis and glutamine decomposition is a toxic by-product in mammalian cell cultures [84]. Ammonium can accumulate to levels up to 2-10 mM in batch cultures [11], and can negatively affect cell growth [85][86][87][88], protein production [89] and protein glycosylation [9,[90][91][92][93]. Schneider et al [8] suggested that alanine aminotransferase and aspartate aminotransferase reactions act as a detoxification process for ammonium removal.…”

Glycosylation: an intrinsic sign of "Danger"

“…3 Other factors such as the level of glucose, glucoseamine, ammonia, dissolved oxygen, temperature and the time of culture have all affected the glycosyaltion of either specific protein analyzed or the overall protein glycosylation. [4][5][6][7][8][9][10] Significantly, these changes were attributed to alternations in intracellular glycosyaltion rather than degradation of carbohydrate side chains after secretion of the protein to the media. [4][5][6]9 Mechanistically, these changes can be translated to changes in intracellular compartments that are responsible for glycosylation.…”