全柱成像毛细管等电聚焦电泳分析蛋白质药物电荷异质性

Analysis of Peptide and Protein Drugs Isoelectric Point by Full Column Imaging cIEF-WCID

评价了cIEF-WCID检测多肽与蛋白质药物等电点的应用效果。测定标准多肽的等电点,验证了cIEF-WCID具有高的准确度和良好的重复性(相对标准偏差<0.50%)。人血红蛋白的4种主要异构体实现了基线分离;甘赖胰岛素的重复测试均只检出单一特征峰(p I 5.95±0.01);比较重组人生长激素原液和成品,等电点特征峰比例差异明显,且成品有新特征峰出现;对比进口及国产贝伐单抗,发现厂家1、3与原研药基本一致,厂家1的主成分迁移规律与原研药高度一致,厂家2的重链C末端K缺失、N末端焦谷氨酸环化或脱酰胺修饰影响了电荷异质性;通过考察尿素浓度、电解质范围和聚焦时间,优化了检测重组人促卵泡素等电点条件:2 mol/L尿素,两性电解质pH 2.5~5.0与pH 3.0~10.0按1∶1混合,聚焦电压1 000 V(1 min)~1 800 V(4 min)~2 200 V(1 min)。cIEF-WCID可快速、准确测定具有电荷异质性的蛋白类药物等电点,分辨率和重复性好,尤其是可以跟踪聚焦过程中样本的迁移特征,特别适合蛋白类药物复杂电荷异质性的检测。

The application effect of isoelectric points（pI） of peptide and protein drugs containing different molecular weights and complex charge heterogeneity was evaluated by capillary isoelectric focusing electrophoresis-whole column imaging detection（cIEF-WCID ）.The pI of standard polypeptide was mensured repeatedly to verify that cIEF-WCID had a high accuracy and a good repeatability（RSD〈0.50%）.Four serum protein isomers had achieved baseline separation.Gan Lai insulin was detected only a single characteristic peaks（pI 5.95±0.01） when repeating the test.The proportion differences of the characteristic peakPI were significant,when comparing the recombinant human growth hormone bulk and final,and the final appeared new characteristic peaks.Comparing the imported and domestic Avastin,manufacturer 1 and 3 were found to be consistent with the innovator drug,and the migration behavior of main component in manufacturer 1 was highly consistent with the innovator drug,however,the charge heterogeneity of manufacturer 2 was influenced because of lose K,N-term pyro-Glu.The optimized pI detecting conditions of the recombinant human follicle stimulating hormone were obtained by studying co-solvent concentration,electrolyte and focusing time,namely Co-solvent：2 mol/L urea,ampholytes at pH 2.5-5.0 and pH 3.0-10.0 mixing by 1∶1,and focus voltage：1 000 V（1 min） to 1 800 V（4 min） and to 2 200 V（1 min）.cIEF-WCID was fast and accurate,and it owned excellent resolution and reproducibility,especially,it could trace the focusing process of the analytes.Therefore,it was fit for analysing complex charge heterogeneity of protein drugs .