CGE-LIF方法分析重组腺相关病毒(rAAV)衣壳蛋白的比例含量  

Analysis of the proportion content of recombinant adeno-associated virus(rAAV)capsid protein by CGE-LIF

作　　者：李响[1] 高铁 史新昌[1] 陈泓序 唐红梅 秦玺[1] 周勇[1] LI Xiang;GAO Tie;SHI Xing-chang;CHEN Hong-xu;TANG Hong-mei;QIN xi;ZHOU Yong(National Institutes for Food and Drug Control,Key Laboratory of the Ministry of Health for Research on Quality and Standardization of Biotech Products,Beijing 100050,China;SCIEX China,Bejing 100015,China)

机构地区：[1]中国食品药品检定研究院卫生部生物技术产品检定方法及其标准化重点实验室,北京100050 [2]SCIEX中国,北京100015

出　　处：《药物分析杂志》2023年第11期1833-1839,共7页Chinese Journal of Pharmaceutical Analysis

基　　金：国家质量基础设施资助(2021YFF0600804)。

摘　　要：目的:建立毛细管凝胶电泳串联激光诱导荧光检测器(CGE-LIF)方法分析重组腺相关病毒(rAAV)衣壳蛋白VP1、VP2和VP3比例含量并进行方法学验证。方法:CGE-LIF分析rAAV衣壳蛋白方法为:rAAV样品加入4%SDS-150 mmol·L^(-1)NEM的溶液预变性(70℃,5 min);再加入P503染料标记(70℃,10 min);再加入1%SDS进行变性(70℃,5 min),然后混匀上机检测。方法验证包括准确性、精密度、线性、检测限、定量限,耐用性等。另外考察了P503、TAMRA、FQ 3种染料条件及rAAV不同血清型和部分批间一致性。结果:CGE-LIF分析不同血清型rAAV(包括2、5、8、9、10型)可将其衣壳蛋白VP1、VP2和VP3基线分离并且峰形尖锐。准确性显示衣壳蛋白VP1、VP2和VP3的实测结果与预期结果的相关系数r>0.99;重复性结果显示每个衣壳蛋白VP1、VP2和VP3的迁移时间RSD均<1.5%,校正峰面积百分比的RSD均<5%;中间精密度结果显示衣壳蛋白VP1、VP2和VP3的迁移时间RSD均<1.5%,校正峰面积百分比RSD均<5%;线性结果显示在0.6×10^(12)~3.0×10^(12)vg·mL^(-1)范围内与衣壳蛋白校正峰面积线性关系良好;VP3检测限和定量限分别为7.2×10^(10)vg·mL^(-1)和2.2×10^(11)vg·mL^(-1)。用3种不同染料(P503、FQ和TAMRA)进行CGE-LIF检测,结果良好。该方法的批间稳定结果较为理想。结论:建立了rAAV衣壳蛋白CGE-LIF分析方法,所建方法可以对rAAV产品3种衣壳蛋白进行分离和相对定量。Objective:To established a capillary gel electrophoresis tandem laser induced fluorescence detector(CGE-LIF)method to analyze the proportion content of recombinant adeno-associated virus(rAAV)capsid proteins VP1,VP2 and VP3,and to verify the methodology.Methods:The CGE-LIF analysis method for rAAV capsid protein was as follows:rAAV samples were pre denatured with a solution of 4%SDS-150 mmol·L^(-1)NEM(70℃,5 min);labeled with P503 dye(70℃,10 min);denatured with 1%SDS(70℃,5 min),then the processed rAAV samples were mixed well and tested.The method validation included accuracy,repeatability,linearity,detection limit,quantification limit,durability,etc.In addition,the conditions of P503,TAMRA,and FQ dyes,as well as the consistency between different serotypes and between some batches of rAAV were investigated.Results:CGE LIF analysis of different serotype of rAAV(including types 2,5,8,9,and 10)could separate their capsid proteins VP1,VP2,and VP3 at baseline and had sharp peaks.The accuracy showed that the correlation coefficient r was>0.99 between the measured results of capsid proteins VP1,VP2,and VP3 and the expected results.The repeatability results showed that the migration time RSD of each capsid protein VP1,VP2,and VP3 was less than 1.5%,and the peak area percentage RSD was less than 5%.The intermediate precision results showed that the migration time RSD of capsid proteins VP1,VP2,and VP3 were all less than 1.5%,and the peak area percentage RSD were all less than 5%.The linear result was displayed from 0.6×10^(12)vg·mL^(-1)to 3.0×10^(12)vg·mL^(-1),within the range,there was a good linear relationship with the peak area of capsid protein.The detection limit and quantification limit of VP3 were 7.2×10^(10)vg·mL^(-1)and 2.2×10^(11)vg·mL^(-1).Three different dyes(P503,FQ,and TAMRA)were used for CGE-LIF detection,and the results were good.The inter batch stability results of this method were relatively ideal.Conclusion:A CGE-LIF analysis method for rAAV capsid protein has been established,which can se

关 键 词：毛细管凝胶电泳 重组腺相关病毒(rAAV) 衣壳蛋白 LIF检测器 比例含量 

分 类 号：R917[医药卫生—药物分析学]