目的 对亲水相互作用超高效液相色谱法检测单抗N糖谱进行多家实验室的联合验证。方法 依据ICH_ Q2_R1指导原则和2015年版《中国药典》通则9101,开展方法学验证。评价指标包括:特异性、线性、准确性、精密性、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。在100~400 μg蛋白范围内具有良好的线性,r2大于0.98,且回收率在86%~117%内。精密性评价结果显示,该方法整体的RSD值小于10%。该方法的定量限为0.040%,可对在0.040%~78.751%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5%,保留时间的RSD小于1%。结论 组织开展的基于亲水相互作用超高效液相色谱法(HILIC-UPLC)的检测单抗N糖谱的方法学联合验证,为《中国药典》标准提高提供方法学验证依据。
Abstract
OBJECTIVE To validate the HILIC-UPLC method for N-glycan profile analysis of therapeutic antibodies. METHODS The interlaboratory method validation was performed according to ICH_Q2_R1 guideline and general principles of China Pharmacopoeia (2015 edition) 9101. The validation items included 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 range from 100 to 400 μg. The r2 of linear regression equation was above 0.98, and the recoveries were between 86% and 117%. Both the RSDs of peak area percentage and retention time were below 10%, which indicated good precision. The lower quantitation limit of the method was 0.040%, and the range was from 0.040% to 78.751%, which means that single peak at this range could be quantified accurately. Furthermore, robustness evaluation under a series of conditions showed that this method was robust, where the RSD of peak area percentage was below 5% and RSD of retention time was below 1%. CONCLUSION Interlaboratory validation of HILIC-UPLC provides a methodological verification basis for the improvement of Chinese Pharmacopoeia standards.
关键词
单克隆抗体 /
N糖基化修饰 /
方法学验证 /
中国药典 /
亲水相互作用色谱
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Key words
monoclonal antibody /
N-linked glycosylation /
method validation /
Chinese Pharmacopoeia /
hydrophilic interaction liquid chromatography
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中图分类号：
R917
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参考文献
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脚注
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基金
《中国药典》药品标准提高课题项目资助(445)
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