重组腺相关病毒中游离与错误包装宿主细胞DNA检测的两种前处理方法的比较  

Comparison of two pretreatment methods for detection of free and mispackaged host cell DNA in recombinant adeno⁃associated virus

作　　者：王光裕[1] 胡倩 史新昌[1] 闫书美 周勇[1] 刘宾 WANG Guangyu;HU Qian;SHI Xinchang;YAN Shumei;ZHOU Yong;LIU Bin(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;不详)

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]上海泰昶生物技术有限公司,上海201413 [3]上海天泽云泰生物医药有限公司,上海201203

出　　处：《中国生物制品学杂志》2024年第5期527-531,共5页Chinese Journal of Biologicals

基　　金：国家重点研发计划(2023YFC3403305);国家药品质量标准提高课题(2022S07);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资金(2023-PT350-01).

摘　　要：目的采用DNA酶(deoxyribonuclease,DNase)处理与填料亲和两种前处理方法检测重组腺相关病毒(recombinant adeno-associated virus,rAAV)游离与错误包装宿主细胞DNA(host cell DNA,HCDNA)残留量,并进行比较。方法分别采用DNase处理法和填料亲和法分离游离与错误包装HCDNA,再进行核酸提取及qPCR检测。比较两种前处理方法检测HCDNA残留量的准确度和重复性。结果采用DNase处理的方式,DNase未处理组核酸定量检测结果为HCDNA总残留量,DNase处理组为错误包装HCDNA量,二者差值为游离HCDNA残留量。DNase未处理和处理组回收率均为75%以上,重复性RSD小于30%。采用亲和提取方式,填料偶联亲和配基,结合rAAV,检测错误包装HCDNA回收率为75%以上,检测游离HCDNA回收率仅为36%。结论DNase处理法可有效检出游离与错误包装HCDNA,可为后续研究奠定基础。Objective To compare the application of two pretreatment methods,deoxyribonuclease(DNase)treatment and ligand affinity,in detecting the residual amount of free and mispackaged host cell DNA(HCDNA)in recombinant adenoassociated virus(rAAV).Methods Free and mispackaged HCDNA were isolated by DNase treatment and ligand affinity respectively,and then the nucleic acid was extracted and detected by qPCR.The accuracy and reproducibility of two pretreatment methods for detecting HCDNA residues were compared.Results Using DNase treatment,the result of nucleic acid quantitative detection in non-DNase-treated group was the total residual amount of HCDNA,that in DNase-treated group was the amount of mispackaged HCDNA,and the difference between them was the residual amount of free HCDNA.The recovery rates of both the untreated and treated groups were more than 75%,and the RSD of reproducibility was less than 30%.Using affinity extraction method,with the affinity ligand combined with rAAV,the result of recovery rate of mispackaged HCDNA was over 75%,and that of free HCDNA was only 36%.Conclusion DNase treatment method can effectively detect free and mispackaged HCDNA,laying a foundation for further research.

关 键 词：腺相关病毒 DNA酶 亲和填料 PCR 宿主细胞DNA 游离 错误包装 

分 类 号：R917[医药卫生—药物分析学]