qPCR在重组腺相关病毒产品大片段宿主细胞残留DNA检测中的应用  

Applied research on qPCR for determination of large fragment host cell residual DNA in recombinant adeno-associated virus products

作　　者：王光裕[1] 史新昌[1] 杨靖清[1] 李响[1] 于雷[1] 周勇[1] WANG Guangyu;SHI Xinchang;YANG Jingqing;LI Xiang;YU Lei;ZHOU Yong(NHC Key Laboratory of Research on Quality and Standardization of Biotech Products,NMPA Key Laboratory for Quality Research and Evaluation of Biological 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,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

出　　处：《中国生物制品学杂志》2024年第7期775-778,787,共5页Chinese Journal of Biologicals

基　　金：国家重点研发计划(2023YFC3403305);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：目的考察qPCR法检测重组腺相关病毒(recombinant adeno-associated virus,rAAV)基因治疗产品中大片段宿主细胞残留DNA(host cell residual DNA,HCD)的可行性。方法提取4种不同血清型rAAV核酸,采用qPCR法对宿主HEK293细胞基因组长末端重复序列(long terminal repeat sequence,LTR)中244和562 bp两个片段序列进行特异性定量,计算HCD在基因组中的占比。结果可在qPCR法定量限范围内检出4种不同血清型rAAV样品大片段HCD,占比为0.3%~5.4%,且随着检测片段长度增加,占比呈降低趋势。结论qPCR技术可用于rAAV产品中大片段HCD的检测。Objective To investigate the feasibility of using quantitative PCR(qPCR)technology to detect large fragments of host cell residual DNA(HCD)in recombinant adeno-associated virus(rAAV)gene therapy products.Methods Four different serotypes of rAAV were extracted for the nucleic acids,two fragment sequences of 244 bp and 562 bp within the long terminal repeat sequence(LTR)in the genome of host cells HEK293 were specifically quantified by qPCR,and the proportion of HCD in the total nucleic acids was calculated.Results Large fragments of HCD in qPCR quantifiable range were detected in four different serotype rAAV products,with the abundance ranging from 0.3%to 5.4%.As the length of the detected fragment increased,the abundance of HCD fragments showed a decreasing trend.Conclusion qPCR technology can be used to determine the presence of large fragments of HCD in rAAV products.

关 键 词：荧光定量PCR 重组重组腺相关病毒 基因治疗产品 宿主细胞残留DNA 

分 类 号：R917[医药卫生—药物分析学]