【摘要】 目的　建立重组人红细胞生成素 (EPO)中影响体内生物学活性的含唾液酸和去唾液酸的N 连接糖的糖谱。方法　用肽糖苷酶F释放EPO中与天冬酰胺连接的寡糖 ,再用唾液酸酶脱去糖末端的唾液酸 ,采用荧光试剂APTS标记后 ,以毛细管凝胶电泳 激光诱导荧光法分离和检测糖的各种形态。结果　EPO样品含有大致相同的N 连接糖谱 ;去唾液酸后的N 连接糖组分的保留时间延长 ;体内生物学活性不同的样品 ,其N 连接糖也有差异。　结论此法可用于判断EPO产品的来源及批间差异 ,与肽图相结合可作为EPO的指纹鉴别图谱 ,用于EPO的常规质检更多还原

【Abstract】 Aim 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. Conclusion 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.更多还原