促红素N-糖链的超高压液相色谱分析方法验证  

Validation of ultra-high pressure liquid chromatography method for epoetin N-glycan profile

作　　者：李响[1] 秦玺[1] 裴德宁[1] 于雷[1] 史新昌[1] 周勇[1] LI Xiang;QIN Xi;PEI De-ning;YU Lei;SHI Xin-chang;ZHOU Yong(NHC Key Laboratory of Research on Quality and Standardization of Biotech Products,NMPA Key Laboratory for Quality Research and Evaluation of Biological Products,National Institutes for Food and Drug Control,Beijing 100050,China)

机构地区：[1]中国食品药品检定研究院,国家卫生健康委员会生物技术产品检定方法及其标准化重点实验室,国家药品监督管理局生物制品质量研究与评价重点实验室,北京100050

出　　处：《中国新药杂志》2024年第6期598-604,共7页Chinese Journal of New Drugs

基　　金：国家质量基础设施资助项目(2021YFFO600804)。

摘　　要：目的:应用超高压液相色谱方法对促红素N-糖链分析并进行方法学验证,为相应质控方法的建立和验证提供参考。方法:用N-糖苷酶将促红素N-糖链酶切下后,进行2-AB衍生化标记,采用磁珠法纯化后进行液相分析,以pH 4.4的100 mmol·L^(-1)甲酸铵溶液为流动相A液,70%乙腈为流动相B液,梯度洗脱70.0 min,荧光检测器检测,激发光波长260 nm、发射光波长430 nm。对该方法进行了相应的方法学验证。结果:考察了方法进样和制备重复性,峰簇1~5的峰面积百分比的RSD不高于4.08%,保留时间的RSD均不高于2.89%。方法中间精密度考察结果显示,峰簇1~5的峰面积百分比的RSD不高于11.85%,保留时间的RSD均不高于8.05%。方法准确度考察中,峰簇2,3,4相对峰面积回收率分别为98.28%~113.01%,98.68%~102.96%,98.51%~100.75%。在蛋白含量为2.5~7.5 mg·mL^(-1)(即促红素蛋白量50~150μg)时,峰簇1~5峰面积线性回归的决定系数均>0.99。设定方法定量限为蛋白量50μg,所制备的样品4℃放置48 h内稳定。结论:实验结果显示该方法的专属性、精密度、准确度和线性均良好,可为促红素N-糖质控方法的建立和验证提供参考。Objective:To analyze N-glycan of epoetin by ultra-high pressure liquid chromatography and validate the method,thus providing reference for the establishment and validation of corresponding quality control method.Methods:The epoetin sample was enzymatically digested with PNGase F,and the released N-glycan was labelled with 2-AB derivatization,followed by purification with magnetic beads.Liquid chromatography analysis with gradient elution for 70.0 min was performed,with 100 mmol·L^(-1) ammonium formate solution(pH 4.4)as mobile phase A and 70% acetonitrile as mobile phase B.Fluorescence detection was applied with excitation wavelength of 260 nm and emission wavelength of 430 nm.Comprehensive method validation was performed.Results:The injection and preparation repeatability were investigated,with RSDs of no more than 4.08% and 2.89% for relative peak area and retention time of peak groups 1~5 respectively.The intermediate precision analysis showed that the RSDs were no more than 11.85% and 8.05% for relative peak area and retention time of peak groups 1~5 respectively.The accuracy test demonstrated that the relative peak area recoveries were 98.28%~113.01%,98.68%~102.96% and 98.51%~100.75% for peak groups 2~4 respectively.When the protein concentration was 2.5~7.5 mg·mL^(-1),which corresponded to the protein content analyzed as 50~150μg,the determination coefficients of linear regression for peak groups 1~5 were all greater than 0.99.The quantitative limit of the method was set as protein content of 50μg.The prepared sample was stable after being placed at 4℃ for 48 h.Conclusion:The method has good specificity,precision,accuracy and linearity,providing a reference for the establishment and validation of the quality control method for epoetin.

关 键 词：N-糖链 促红素 方法学验证 2-AB标记 质量控制 

分 类 号：R917[医药卫生—药物分析学]