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
秦玺 1裴德宁 1肖乐 2史新昌 1郭莹 1陶磊 1李响 1梁成罡1
作者信息
- 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
- 折叠
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55 s,电压为1 500 V,聚焦时间为1 min;随后将电压调整至3 000 V,并继续聚焦10 min.激发波长设为280 nm,发射荧光检测曝光时间设为80 s.以rAAV 5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rAAV 5型实心颗粒(rAAV-F)样品的电荷异质性.结果 rAAV-E样品在pH 6.9～7.1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％,pI 的RSD分别为0.10％和0.11％;rAAV-E浓度在(4.08～6.12)× 1012vp/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=54 888 x-76 556,R2=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× 1012 vp/mL,Peak2定量限为0.76 × 1012 vp/mL.rAAV-F样品呈现Peak1 和 Peak2外,在酸性区域(pI＜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.
Abstract
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 1 500 V,focusing for 1 min,and then voltage adjusted to 3 000 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(Peak1 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 pI values of the two main peaks Peak1 and Peak2 of rAAV-E sample were 6.91 and 7.04 respectively with both RSDs of 0.14％,and the average propor-tions 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 pI were 0.10％and 0.11％,respectively.In the range of(4.08-6.12)× 1012 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=54 888 x-76 556,R2=0.977.The recovery rates of the the main peak content in rAAV-E samples at 80％,100％and 120％expected concentra-tions were all in the range of 80％-120％.The LOQ was 1.02 × 1012 vp/mL for Peak 1,and 0.76 × 1012 vp/mL for Peak 2.In addition to Peak 1 and Peak 2,obvious extra peaks were observed in the acidic region(pI＜5.85)of rAAV-F sample,which was the main characteristic of rAAV-F compared with rAAV-E,and also indicated that rAAV-F had complex charge heterogeneity distribution.Conclusion The established iCIEF method has good specificity,precision and accuracy,and can be used to analyze the charge heterogeneity of rAAV capsid proteins.
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Key words
Imaged capillary isoelectric focusing(iCIEF)/Recombinant adeno-associated virus(rAAV)/Capsid proteins/Charge heterogeneity引用本文复制引用
基金项目
国家重点研发计划(2021YFF0600804)
出版年
2024
NETL
NSTL
万方数据