Recombinant human erythropoietin (rhuEPO) has been extensively used as a pharmaceutical product for treating anemia in the clinic. Glycosylation of rhuEPO was crucial for affecting biological activity, immunogenicity, and pharmacokinetics. Because of the heterogeneity of glycan, the structure of rhuEPO was complex with several isoforms. Characterization of isoforms was important for quality control of rhuEPO. Here, an improved cIEF method has been established and validated. A polarity‐reversed focusing step was used by reversing both the polarity of the voltage and the catholyte and anolyte vials. A weak base (100 mM ammonium hydroxide solution) was used as a chemical mobilizer to make the acidic bands mobilize stably to the detection window. Compared with CZE method in European Pharmacopoeia, the numbers of isoforms and their peak area percentage were highly consistent. Better reproducibility and higher resolution have been obtained by the improved cIEF method. Moreover, in improved cIEF method, the isoelectric points (pI) of each isoform can be calculated and used for identification. It was also the first time that the cIEF method was fully validated for rhuEPO analysis according to the International Conference on Harmonization (ICH) guidelines.

中文翻译：

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极性反转毛细管等电聚焦分析重组人促红细胞生成素的亚型。

重组人促红细胞生成素（rhuEPO）已在临床上广泛用作治疗贫血的药物。rhuEPO的糖基化对于影响生物学活性，免疫原性和药代动力学至关重要。由于聚糖的异质性，rhuEPO的结构很复杂，具有多种同工型。同工型的表征对于rhuEPO的质量控制很重要。在这里，已经建立并验证了一种改进的cIEF方法。通过反转电压和阴极电解液和阳极电解液样品瓶的极性，使用了极性反转的聚焦步骤。使用弱碱（100 mM氢氧化铵溶液）作为化学迁移剂，以使酸性带稳定地迁移到检测窗口。与欧洲药典中的CZE方法相比，异构体的数量及其峰面积百分比高度一致。通过改进的cIEF方法获得了更好的重现性和更高的分辨率。此外，在改进的cIEF方法中，可以计算每个同工型的等电点（pI）并用于识别。这也是首次根据国际协调会议（ICH）指南对cIEF方法进行rhuEPO分析的完全验证。