重组5型腺相关病毒基因组滴度测定用国家标准品的研制  

Development of national standards for genomic titer determination of recombinant type 5 adeno-associated virus

作　　者：郑红梅 于雷[1] 李永红[1] 史新昌[1] 安怡方 郭莹[1] 韩春梅[1] 饶春明[1] 周勇[1] ZHENG Hongmei;YU Lei;LI Yonghong;SHI Xinchang;AN Yifang;GUO Ying;HAN Chunmei;RAO Chunming;ZHOU Yong(Key Laboratory of the Ministry of Health for Research on Quality and Standardization of Biotech Products,National Institutes for Food and Drug Control,Beijing 100050,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

出　　处：《中国生物制品学杂志》2023年第4期429-433,共5页Chinese Journal of Biologicals

基　　金：北京市科技计划(Z221100007922015)。

摘　　要：目的研制重组5型腺相关病毒(recombinant type 5 adeno-associated virus,rAAV5)基因组滴度测定用国家标准品。方法对三质粒系统制备的rAAV5-GFP原液,按《中国药典》三部(2020版)相关要求进行鉴别、外观、pH、无菌、基因组滴度、纯度、感染滴度等检定,根据结果稀释、分装,制备成候选标准品;采用热加速试验对候选标准品进行稳定性考察;组织3家实验室,采用微滴式数字PCR(droplet digital PCR,ddPCR)法对候选标准品进行协作标定。结果候选标准品原液及候选标准品的各项检测指标均符合相关要求;在25、4、-20、-40、-80℃条件下,1、3、4、6个月基因组滴度均无明显下降;经3家实验室协作标定,候选标准品赋值为2.56×10^(12)copies/mL,95%置信区间为2.48×1012~2.64×10^(12)copies/mL。结论研制的rAAV5基因组滴度测定用国家标准品具有良好的稳定性,可用于rAAV5相关产品的质量评价。Objective To develop a national standard for genomic titer determination of recombinant type 5 adeno-associated virus(rAAV5).MethodsThe rAAV5-GFP stock solution prepared by the three-plasmid system was identified and verified for the appearance,pH,sterility,genomic titer,purity and infection titer according to the relevant requirements of Chinese Pharmacopoeia(VolumeⅢ,2020 edition),which was diluted and subpackaged to prepare candidate standards according to the results;The stability of candidate standards was investigated by thermal acceleration test;Three laboratories were organized to collaboratively calibrate the candidate standards using droplet digital PCR(ddPCR).ResultsAll the detection indexes of the candidate standard and the stock solution met the relevant requirements;The genomic titer showed no significant decrease at 25,4,-20,-40,-80℃for 1,3,4,6 months;Through collaborative calibration by three laboratories,the candidate standard was assigned a value of 2.56×10^(12)copies/mL,and the 95%confidence interval was 2.48×10^(12)copies/mL~2.64×10^(12)copies/mL.ConclusionThe developed national standard for the determination of rAAV5 genomic titer had good stability and might be used for the quality evaluation of rAAV5 related products.

关 键 词：重组5型腺相关病毒 微滴式数字PCR 基因组滴度 协作标定 国家标准品 

分 类 号：R392[医药卫生—免疫学]