毛细管电泳结合激光诱导荧光检测分析单抗N糖谱的方法学联合验证

Inter-laboratory method validation of CE-LIF for N-glycan profile analysis of monoclonal antibodies

目的:对毛细管电泳结合激光诱导荧光(CE-LIF)检测单抗N糖谱方法进行多家实验室的联合验证。方法:依据国际人用药品注册技术协调会(ICH)Q2;1指导原则和2015年版《中华人民共和国药典》通则9101,开展方法学验证。评价指标包括专属性、线性、准确度、精密度、定量限、范围和耐用性。结果:验证数据表明,该方法具有良好的特异性、准确性、精密性和耐用性。蛋白量在100~400μg范围内具有良好的线性,R;大于0.97,且回收率在81%~109%范围内。精密性评价结果显示,G0F、Man5、G1F(1,6)、G1F(1,3)和G2F的校正峰面积百分比和迁移时间的RSD值均小于10%。该方法的定量下限为0.016%,可对在0.016%~77.233%范围内的单个色谱峰进行准确定量分析。同时多个条件下的耐用性评价结果表明该方法具有耐用性,峰面积百分比的RSD小于9%,迁移时间的RSD小于0.1%。结论:组织开展了基于CE-LIF的检测单抗N糖谱的方法学联合验证,结果表明方法可行,可用于单抗N糖谱的检测。

Objective:To validate the CE-LIF method for N-glycan profile analysis of antibodies inter laboratory.Methods:The method validation was performed according to ICH;2;1 guideline and general chapter 9101 of Chinese Pharmacopoeia(2015 edition).The validation characteristics include specificity,linearity,accuracy,precision,quantitation limit,range and robustness.Results:The method showed good specificity,accuracy,precision and robustness,and showed a good linearity at a protein content ranging from 100μg to 400μg.The R;of linear regression equation were above 0.97,and the recoveries were between 81%and 109%.For precision test,the RSDs of peak area percentage and retention time of G0 F,Man5,G1 F(1,6),G1 F(1,3)and G2 F were below 10%.The quantitation limit of the method was 0.016%,and the range was from 0.016%to 77.233%,whichindicated that single peak at this range could be quantified accurately.Furthermore,robustness evaluation under a series of condition showed that this method was robust,where the RSD of peak area percentage was below 9%and RSD of retention time was below 0.1%.Conclusion:Inter-laboratory method validation of CE-LIF has been organized,and the results verifies that the method is feasible for analysis of antibodies.