首页|促红素N-糖链的超高压液相色谱分析方法验证

促红素N-糖链的超高压液相色谱分析方法验证

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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50~150 µg)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50 μg,所制备的样品4℃放置48h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.
Validation of ultra-high pressure liquid chromatography method for epoetin N-glycan profile
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 50~150 pg,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 50 µg.The prepared sample was stable after being placed at 4 ℃for48 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.

N-glycanepoetinmethod validation2-AB labelingquality control

李响、秦玺、裴德宁、于雷、史新昌、周勇

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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

N-糖链 促红素 方法学验证 2-AB标记 质量控制

国家质量基础设施资助项目

2021YFFO600804

2024

中国新药杂志
中国医药科技出版社 中国医药集团总公司 中国药学会

中国新药杂志

CSTPCD北大核心
影响因子:1.039
ISSN:1003-3734
年,卷(期):2024.33(6)
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