新型冠状病毒重组蛋白疫苗中受体结合域二聚体纯度检测方法的建立及验证  

Establishment and verification of purity detection method for receptor⁃binding domain dimer in SARS⁃CoV⁃2 recombinant protein vaccine

作　　者：段艳 李文东 李珉 何欢 李潇 DUAN Yan;LI Wendong;LI Min;HE Huan;LI Xiao(Beijing Institute for Drug Control(Beijing Center for Vaccine Control),NMPA Key Laboratory for Safety Research and Evaluation of Innovative Drugs,Beijing Key Laboratory of Analysis and Evaluation on Chinese Medicine,Beijing 102206,China;不详)

机构地区：[1]北京市药品检验研究院北京市疫苗检验中心,国家药品监督管理局创新药物安全研究与评价重点实验室,中药成分分析与生物评价北京市重点实验室,北京102206 [2]北京市医疗器械检验研究院,北京市医用生物防护装备检验研究中心,北京100070

出　　处：《中国生物制品学杂志》2024年第10期1239-1244,1250,共7页Chinese Journal of Biologicals

基　　金：北京市科技新星计划项目(Z211100002121007)。

摘　　要：目的建立应用生物分析仪(Agilent 2100)检测新型冠状病毒重组蛋白疫苗中受体结合域(receptor-binding domain,RBD)二聚体纯度的方法,并对其进行验证。方法对新型冠状病毒重组蛋白疫苗进行解析附后,超滤浓缩蛋白,采用生物分析仪(Agilent 2100)检测完全及非完全变性(二硫键保留)蛋白的纯度;对建立的方法进行系统适用性、专属性、重复性、中间精密度和准确度验证后,用其检测厂家A 4批样品。结果系统适用性验证中,8个峰均可清晰区分基线;空白溶剂在目标成分出峰位置无干扰,专属性良好;在非完全和完全变性条件下,2名实验员12次重复检测结果的RSD均小于1%,重复性和中间精密度良好;3种不同蛋白浓度(50%、80%和100%)主峰(1+2)及主峰面积占比的RSD分别为0.68%和0.31%,准确度良好,其中,10%蛋白浓度样品结果不稳定;4批样品RBD二聚体纯度分别为70.5%、70.2%、73.2%和69.6%。结论建立的方法专属性、精密度、准确度良好,可用于重组蛋白疫苗中蛋白纯度的检测。Objective To establish and verify a bioanalyzer(Agilent 2100)method for the determination of the receptorbinding domain(RBD)dimer purity in SARS-CoV-2 recombinant protein vaccine.Methods The protein in the SARS-CoV-2 recombinant vaccine was analyzed and then concentrated by ultrafiltration,and the purity of fully denatured and partially denatured(disulfide bond retention)proteins was detected by bioanalyzer(Agilent 2100).The established method was verified for the applicability,specificity,reproducibility,precision and accuracy,and used to detect four batches of samples from manufacturer A.Results In the system applicability test,eight peaks were clearly distinguished from the baseline.The blank solvent had no interference at the peak position of the target component,indicating the good specificity.Under the fully denatured and partially denatured conditions,the RSDs of 12 repeated test results by two experimenters were both less than 1%,showing good repeatability and precision.The RSDs of the proportion of the main peak(1+2)and the main peak at three different protein concentrations(50%,80%and 100%)were 0.68%and 0.31%,respectively,and the accuracy was good.Among them,the results of the 10%protein concentration sample were unstable.The purity of RBD dimer in four batches was 70.5%,70.2%,73.2%and 69.6%,respectively.Conclusion The established method has good specificity,precision and accuracy,and can be used for the detection of protein purity in recombinant protein vaccines.

关 键 词：新型冠状病毒 重组蛋白疫苗 受体结合域 二聚体 蛋白纯度 

分 类 号：R917[医药卫生—药物分析学]