[Characterization of N-glycan mapping of bioengineering recombinant erythropoietin by capillary electrophoresis with laser-induced fluorescence].
OBJECTIVE
N-Glycans in recombinant human erythropoietin (EPO) are essential to in vivo biological activity. This paper is to develop a method for mapping sialyated or asialyated N-glycans of EPO.


METHODS
At first, N-glycans linked to asparagines in glycoprotein EPO were released by peptide N-glycosidase F. To map asialyated N-glycans, sialic acid in N-glycans were removed by incubating N-glycans with sialidase. Oligosaccharides were labeled with a sensitive fluorescent dye 8-aminopyrene-1,3,6-trisulfonate (APTS), and all of the labeled oligosaccharides released from EPO were mapped by capillary gel electrophoresis with laser-induced fluorescence. The relationship between N-glycans and bioactivity of EPO was investigated on the basis of N-glycan mapping spectra.


RESULTS
N-Glycans of seven different batches of EPO were mapped. Each sample was analysed twice, with and without sialidase treatment. The results showed that N-glycans of each sample were approximately the same. But when the expression vector was different, the types of N-glycans and their relative content were quite different. In case of asialyated N-glycan mapping, the retention time of each oligosaccharide delayed greatly, and most importantly, the resulted sialic acid peak can be used as a quantitative standard to determine sialic acid content in N-glycans of EPO. In addition, the difference of N-glycan mapping was observed when the in vivo biological activity of EPO was different.


CONCLUSIONS
The approach in this article for determining N-glycan mapping can be applied to determine the source of EPO and the difference between each batch. It is also a suitable tool for routinely controlling the inner quality of EPO by coupled with peptide mapping.
Zhou
Guo-hua
GH
Nanjing Military Area Institute for Drug and Instrument Control, Nanjing 210002, China. ghzhou@public1.ptt.js.cn
Zhang
Xiao-dan
XD
Diao
Yong
Y
Zhou
Yong
Y
Luo
Guo-an
GA
Cheng
Ya-qin
YQ
D019075:Electrophoresis, Capillary / Q000379:methods; D004921:Erythropoietin / Q000737:chemistry*; D005453:Fluorescence; D007834:Lasers; D019158:N-Acetylneuraminic Acid / Q000032:analysis; D010449:Peptide Mapping; D011134:Polysaccharides / Q000032:analysis* / Q000737:chemistry; D011994:Recombinant Proteins
D011134:Polysaccharides; D011994:Recombinant Proteins; D004921:Erythropoietin; D019158:N-Acetylneuraminic Acid