摘要
目的建立重组腺相关病毒(recombinant adeno-associated virus,rAAV)感染细胞后目的蛋白表达水平的检测方法,并进行方法验证,以期用于rAAV9生产过程中不同阶段产物的质量监控。方法将rAAV9供试品用感染增强剂Envirus-AAV处理后,感染经羟基脲(hydroxyurea,HU)作用的人脑星形胶质母细胞瘤细胞U87-MG,以rAAV9参考品为标准,采用ELISA法检测细胞中目的蛋白戊二酰辅酶A脱氢酶(glutaryl-CoA dehydrogenase,GCDH)的表达水平,并验证方法的专属性、准确性、精密性、线性范围、定量限及耐用性。采用建立的方法检测8批rAAV9供试品。结果供试品缓冲液的A_(450)-A_(630)为0.3,略低于蛋白定量四参数标准曲线最低稀释点(1 ng/mL);150%、100%、50%理论相对效价水平样品平均回收率均在100.0%~107.3%范围内;同1名实验员重复3次及不同实验员检测3个理论相对效价水平样品的目的蛋白表达水平RSD均<25%;rAAV9供试品在50%~150%理论相对效价水平范围内,与相应的目的蛋白表达水平呈良好的线性关系,直线回归方程为y=1.077 x-0.022,R~2为0.984;方法的定量限为0.59,即6.0×10^(12)vg/mL;U87-MG细胞用HU孵育不同时间(18、21、24 h)、细胞培养上清液于不同条件(室温放置0.5 h、-60℃以下放置12 h、-60℃以下放置24 h)保存后,目的蛋白表达水平RSD均<25%。1~8批rAAV9供试品目的蛋白表达水平分别为111%、121%、72%、65%、86%、75%、102%、91%。结论建立的rAAV感染细胞后目的蛋白表达水平检测方法具有良好的专属性、准确性、精密性及耐用性,可用于rAAV9生产过程中不同阶段产物的质量监控。
Objective To establish and validate a method for the determination of the interesting protein expression level of recombinant adeno-associated virus(rAAV)infected cells,so as to monitor the product quality in different stages of rAAV9production process.Methods After incubation of serial diluted rAAV samples with infection enhancer Envirus-AAV,the human malignant glioblastoma cells(U87-MG)pretreated with hydroxyurea(HU)were infected.Using rAAV9 reference as the standard,the expression level of glutaryl-CoA dehydrogenase(GCDH)was detected by ELISA,and the specificity,accuracy,precision,linear range,limit of quantitation(LOQ)and durability of the method were verified.Eight batches of rAAV9 samples were detected by the established method.Results The A_(450)-A_(630)value of the sample buffer was 0.3,which was slightly lower than the lowest dilution point(1 ng/mL)of the four-parameter standard curve for protein quantification.The average recoveries of samples with 150%,100%and 50%theoretical relative titer levels were in the range of 100.0%-107.3%.The RSDs of the target protein expression level of the samples with three theoretical relative titer levels detected by the same experimenter three times and different experimenters were all less than 25%.There was a good linear relationship between rAAV9 samples and the target protein expression levels in the range of 50%-150%theoretical relative titer levels,and the linear regression equation was y=1.077 x-0.022,R~2=0.984.The LOQ of the method was 0.59,namely 6.0×10^(12)vg/mL.After U87-MG cells were incubated with HU for different time(18,21,24 h),and the culture supernatant was stored under different conditions(room temperature for 0.5 h,below-60℃for 12 h,below-60℃for 24 h).The RSDs of target protein expression levels were all less than 25%.The target protein expression levels of 1-8 batches of rAAV9 samples were 111%,121%,72%,65%,86%,75%,102%and 91%,respectively.Conclusion The established method for the determination of the target protein expression level after rAAV infection has good specificity,accuracy,precision and durability,and can be used for the quality control of products in different stages of rAAV9 production.
作者
裴德宁
闫书美
史新昌
胡倩
周勇
刘宾
PEI Dening;YAN Shumei;SHI Xinchang;HU Qian;ZHOU Yong;LIU Bin(不详;National Institutes for Food and Drug Control,NHC Key Laboratory of Research on Quality and Standardization of Biotech Products,NMPA Key Laboratory for Quality Research and Evaluation of Biological Products,Beijing 100050,China)
出处
《中国生物制品学杂志》
CAS
CSCD
2024年第6期646-650,655,共6页
Chinese Journal of Biologicals
基金
国家重点研发计划(2023YFC3403305)
中国医学科学院中央级公益性科研院所基本科研业务费专项(2023-PT350-01)。
