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

作　　者：孟晓光[1,2] 甄里[3] 刘悦玫 侯利平[1,2] 刘丹[1,2] 李斌 欧阳伟民 魏开华[1,2,5] MENG Xiao-guang ZHEN Li LIU Yue-mei HOU Li-ping LIU Dan LI Bin OUYANG Wei-min WEI Kai-hua(Beijing C＆N International Sci - Tech Co. , Ltd. , Beijing 102206, China National Center for Protein Sciences （ Beijing）, State of Key Laboratory of Proteomics, Beijing Proteome Research Center, Beijing 102206, China School of Nursing ＆ Medical Teehnology, Jianghan University, Wuhan 430056, China Lumiere Tech Limited, Beijing 100080, China Beijing Institute of Radiation Medicine, Military Medical Science Academy of PLA, Beijing 100850, China)

机构地区：[1]北京正旦国际科技有限责任公司,北京102206 [2]北京蛋白质组研究中心蛋白质组学国家重点实验室国家蛋白质科学中心(北京),北京102206 [3]江汉大学护理与医学技术学院,湖北武汉430056 [4]北京绿绵科技有限公司,北京100080 [5]军事医学科学院放射与辐射医学研究所,北京100850

出　　处：《分析测试学报》2017年第3期343-348,共6页Journal of Instrumental Analysis

基　　金：国家高技术研究发展计划863项目(2014AA020906);国家科技部重大仪器专项(2012YQ18011710);蛋白质组学国家重点实验室课题(SKLP-O201412)

摘　　要：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 .

关 键 词：全柱成像毛细管等电聚焦电泳 电荷异质性 重组蛋白质药物 抗体 药学研究 

分 类 号：O657.8[理学—分析化学] O629.73[理学—化学]