重组人红细胞生成素中N-连接糖的毛细管凝胶电泳-激光诱导荧光测定法

CHARACTERIZATION OF N GLYCANS FROM RECOMBINANT HUMAN ERYTHROPOIETIN BY CAPILLARY ELECTROPHORESIS WITH LASER INDUCED FLUORESCENCE

目的：表征重组人红细胞生成素中影响体内生物学活性的Ｎ连接糖谱。方法：以毛细管凝胶电泳激光诱导荧光检测法（ＣＧＥＬＩＦ）测定了重组人红细胞生成素（ｒＨｕＥＰＯ）中Ｎ连接的多个糖基化形式，并以此为基础研究结构与活性的关系。结果：同一生产线上的样品，其Ｎ连接糖的ＣＧＥＬＩＦ图谱基本一致；不同表达载体所表达的ｒＨｕＥＰＯ，其Ｎ连接糖的形式不同；活性不同的样品，各Ｎ连接糖组分的相对含量存在很大差异。结论：建立的方法可用于判断产品的来源及批间差异。

AIM: To characterize the profiles of N glycans in highly glycosylated protein recombinant human erythropoietin (rHuEPO). METHODS: Capillary gel electrophoresis (CGE) of sugars in recombinant human erythorpoietin derivatized by reductive amination with 8 aminopyrene 1,3,6 trisulfonate(APTS) and detected by laser induced fluorescence (LIF) has been performed to study the relationships between structure and bioactivities of rHuEPO. RESULTS: The results showed that the CGE LIF profiles of N glycans in rHuEPO from the same cell line were almost identical, but clear different profiles emerged when the sample was from different expression vector. Significant differences in CGE LIF mappings of rHuEPO with different in vivo bioactivities were also observed. CONCLUSION: The approach developed in this article can be used to control the first order structure of rHuEPO by combining with peptide mapping.