全柱成像毛细管等电聚焦电泳分析尤瑞克林电荷异质性方法的建立与应用  

Establishment and Application of ICIEF for the Analysis of Charge Heterogeneity of Human Urinary Kininogenase

作　　者：许蓉蓉 刘博[1] 范慧红[1] 王悦[1] XU Rongrong;LIU Bo;FAN Huihong;WANG Yue(NMPA Key Laboratory for Quality Research and Evaluation of Chemical Drugs,National Institutes for Food and Drug Control,Beijing 102629,China)

机构地区：[1]中国食品药品检定研究院,国家药品监督管理局化学药品质量研究与评价重点实验室,北京102629

出　　处：《中国药学杂志》2024年第16期1532-1539,共8页Chinese Pharmaceutical Journal

基　　金：中国食品药品检定研究院关键技术研究基金(GJJS-2022-5-2)。

摘　　要：目的 建立全柱成像毛细管等电聚焦电泳(image capillary isoelectric focusing,iCIEF)方法分析尤瑞克林(human urinary kininogenase,HUK)的电荷异质性。方法 取35μL 1%甲基纤维素、10μL 200 mmol·L^(-1)的亚氨基二乙酸、4μL两性电解质溶液、48 mg尿素、等电点(pI)为6.14和7.05的等电点标记物各0.5μL及水,配制为100μL供试品体系进行聚焦分析,聚焦条件为预聚焦电压1 500 V、持续时间1 min,聚焦电压3 000 V、持续时间6 min。结果 优化后的方法专属性良好、准确度验证中回收率在90%~110%之间;线性验证结果的r^(2)为0.995 7;重复性验证中的各异构体的pI的相对标准偏差(RSD)均小于0.2%,定量限为0.013 mg·mL^(-1);IDA及两性电解质等添加量的耐用性良好。采用该方法可以有效分离不同厂家的HUK电荷异构体。结论 建立的iCIEF方法具有良好的专属性、精密度、准确度和耐用性,能够很好地解决蛋白质类产品的电荷异质性评价的难题,对该类产品在电荷异质性角度的质量控制具有重要意义。OBJECTIVE To establish an analysis method of the charge heterogeneity of human urinary kininogenase(HUK) by using image capillary isoelectric focusing(iCIEF) and complete the methodological verification.METHODS The 35 μL 1% methyl cellulose,10 μL 200 mmol·L~(-1) IDA,4 μL pharmalyte electrolyte,48 mg urea,0.5 μL pI marker with pI 6.15 and 7.05 were added in the sample solution.The focusing condition was pre-focusing voltage 1 500V,duration 1 min,focusing voltage 3 000 V,duration 6 min.RESULTS The optimized method has stable baseline,and the target protein was significantly different from the unrelated protein,The recovery rate of accuracy verification was within 90%-110%,and the linearity verification result had r~2 of 0.995 7,The RSD of each isomer pI in the repeatability verification was less than 0.2%.The limit of quantification was 0.013 mg·mL~(-1).The concentration of urea durability,IDA durability and the electrolyte pharmalyte durability are good.Using this method,the charge isomers of HUK from different manufactures were analyzed effectively.CONCLUSION The developed iCIEF method has good specificity,precision,linearity and durability,and can solve the problem of charge heterogeneity evaluation of protein products,which is of great significance to the quality control of such products from the perspective of charge heterogeneity.

关 键 词：尤瑞克林 糖蛋白 电荷异质体 全柱成像毛细管等电聚焦电泳(iCIEF) 

分 类 号：R917[医药卫生—药物分析学]