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目的 分析国产重组人促红细胞生成素 (rHuEPO)的 3个氮连接寡糖位点的微不均一性。方法 利用糖肽中肽段的辅助离子化作用 ,将唾液酸和寡糖作为一个整体分析。将国产rHuEPO用Glu C酶解 ,HPLC分离 ,ESIMS在线监测含糖位点 ,并用在线ESIMS分析了 83和 38位点氮连接寡糖的微不均一性 ,MALDI/TOFMS分析了3个糖基化位点的寡糖的微不均一性。结果 国产EPO的寡糖唾液酸乙酰化的程度高 ,大部分寡糖的唾液酸都被乙酰化了 ,我们首次发现了 83位点四天线 + 2LacNAc + 4SA、四天线 + 3LacNAc + 4SA的唾液酸乙酰化 ,这种多天线唾液酸乙酰化有助于EPO在体内的抗酶解 ,降低代谢速率 ,提高生物活性。结论 3个位点主要是含唾液酸多天线寡糖 ,二天线寡糖主要在 2 4位点上。
Objective To analyze the inhomogeneity of three nitrogen linked oligosaccharides sites in domestic recombinant human erythropoietin (rHuEPO). Methods Using the assisted ionization of peptides in glycopeptides, sialic acid and oligosaccharides were analyzed as a whole. Domestic rHuEPO was enzymatically digested with Glu C and separated by HPLC. ESIMS was used to monitor the site of sugars on-line. The on-line ESIMS was used to analyze the inhomogeneity of nitrogen-linked oligosaccharides at sites 83 and 38. MALDI / TOFMS analysis of three glycosylation Sites of oligosaccharides micro-heterogeneity. As a result, the degree of acetylation of oligosaccharides in domestic EPO was high, and most of the sialic acids in the oligosaccharides were acetylated. We found for the first time the sialic acid at the position of 83 + 4L + 2LacNAc + 4SA + 4L + 3LacNAc + 4SA Acetylation, this multi-antenna sialic acid acetylation contributes to the anti-enzymatic degradation of EPO in vivo, reduces the metabolic rate and enhances the biological activity. Conclusions The three sites mainly contain sialic acid multi-antenna oligosaccharides and the two-antenna oligosaccharides are mainly at 24 sites.