重组腺相关病毒衣壳蛋白电荷异质性成像毛细管等电聚焦电泳检测方法的建立及验证  

Establishment and verification of an imaged capillary isoelectric focusing method for detection of charge heterogeneity of recombinant adeno-associated virus capsid proteins

作　　者：秦玺[1] 裴德宁[1] 肖乐 史新昌[1] 郭莹[1] 陶磊[1] 李响[1] 梁成罡[1] QIN Xi;PEI Dening;XIAO Le;SHI Xinchang;GUO Ying;TAO Lei;LI Xiang;LIANG Chengang(National Institutes for Food and Drug Control,State Key Laboratory of Drug Regulatory Science,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)

机构地区：[1]中国食品药品检定研究院药品监管科学全国重点实验室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 [2]ProteinSimple(中国),上海200051

出　　处：《中国生物制品学杂志》2024年第8期904-910,共7页Chinese Journal of Biologicals

基　　金：国家重点研发计划国家质量基础设施体系(2021YFF0600804)。

摘　　要：目的建立重组腺相关病毒(recombinant adeno-associated virus,rAAV)衣壳蛋白电荷异质性的成像毛细管等电聚焦电泳(imaged capillary isoelectric focusing,iCIEF)检测方法,并进行方法验证,以期为rAAV产品的表征研究、质量控制及生产工艺稳定性的监控提供可靠的检测方法。方法将待测样品经变性处理后,采用紫外荧光检测模式进行iCIEF分析,进样时间设为55s,电压为1500V,聚焦时间为1min;随后将电压调整至3000V,并继续聚焦10min。激发波长设为280nm,发射荧光检测曝光时间设为80s。以rAAV5型空心颗粒(rAAV-E)为待测样品,验证方法的专属性、精密性、线性范围、准确性及定量限。采用建立的方法检测rAAV5型实心颗粒(rAAV-F)样品的电荷异质性。结果rAAV-E样品在pH6.9~7.1之间呈现2个明显主峰(Peak1和Peak2),空白对照未检测到病毒衣壳蛋白峰;6次重复检测rAAV-E样品2个主峰Peak1和Peak2的pI均值分别为6.91和7.04,RSD均为0.14%,峰面积百分比均值分别为35.6%和55.8%,RSD分别为1.1%和0.4%;3个时间点检测rAAV-E样品2个主峰Peak1和Peak2峰面积百分比的RSD分别为3.45%和3.38%,pl的RSD分别为0.10%和0.11%;rAAV-E浓度在(4.08~6.12)×10^(12)vp/mL范围内,与Peak1和Peak2的总峰面积均值呈良好的线性关系,线性回归方程为y=54888x-76556,R^(2)=0.976;80%、100%和120%预期浓度rAAV-E样品2个主峰总峰面积的回收率均在80%~120%范围内;Peak1的定量限为1.02×10^(12)vp/mL,Peak2定量限为0.76×10^(12)vp/mL。rAAV-F样品呈现Peak1和Peak2外,在酸性区域(pl<5.85)还观察到明显的额外峰,这是rAAV-F区别于rAAV-E的主要特征,也表明其具有复杂的电荷异质性分布。结论建立的iCIEF法具有良好的专属性、精密性及准确性,可用于rAAV衣壳蛋白电荷异质性的分析。Objective To establish and verify an imaged capillary isoelectric focusing(iCIEF)method for analyzing the charge heterogeneity of recombinant adeno-associated virus(rAAV)capsid proteins,in order to provide a reliable detection method for characterization research,quality control and process stability monitoring of rAAV products.Methods After denaturation,the sample was analyzed by iCIEF using UV fluorescence detection imaging mode,with injection time of 55 s,voltage of 1500 V,focusing for 1 min,and then voltage adjusted to 3000 V for another 10 min.The excitation wavelength was set at 280 nm,and the exposure time of emission fluorescence detection was 80 s.Taking rAAV 5 empty particles(rAAV-E)as the research object,the specificity,precision,linear range,accuracy and limit of quantitation(LOQ)of the method were verified.The established method was used to detect the charge heterogeneity of rAAV 5 full particles(rAAV-F).Results The rAAV-E sample showed two main peaks(Peakl and Peak2)between pH 6.9 and pH 7.1,and no virus capsid protein peak was detected in the blank control.In the six repeated detections,the average pl values of the two main peaks Peakl and Peak2 of rAAV-E sample were 6.91 and 7.04 respectively with both RSDs of 0.14%,and the average proportions of peak area were 35.6%and 55.8%,with RSDs of 1.1%and 0.4%,respectively.The RSDs of peak area proportions of peak 1 and peak 2 of rAAV-E sample at three time points was 3.45%and 3.38%,and the RSDs of pl were 0.10%and 0.11%,respectively.In the range of(4.08-6.12)×10^(12)vp/mL,the rAAV-E concentration showed a good linear relationship with the average peak area of the main peaks,and the linear regression equation was y=54888 x-76556,R^(2)=0.977.The recovery rates of the the main peak content in rAAV-E samples at 80%,100%and 120%expected concentrations were all in the range of 80%-120%.The L0Q was 1.02×10^(12)vp/mL for Peak 1,and 0.76×10^(12)vp/mL for Peak 2.In addition to Peak 1 and Peak 2,obvious extra peaks were observed in the acidic region(pl<5.85)of rA

关 键 词：成像毛细管等电聚焦电泳 重组腺相关病毒 衣壳蛋白 电荷异质性 

分 类 号：Q71[生物学—分子生物学]