腺相关病毒基因治疗产品基因组滴度数字PCR检测方法的建立及验证
Establishment and verification of a digital PCR detection method for genome titer of adeno-associated virus gene therapy products
安怡方 1毕华 1刘家池 2魏云欢 2赵琴 2于雷 1史新昌 1裴德宁 1李响 1周勇 1秦玺 1梁成罡1
作者信息
- 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
- 2. 武汉纽福斯生物科技有限公司,湖北武汉 430014
- 折叠
摘要
目的 建立一种广泛用于腺相关病毒(adeno-associated virus,AAV)基因治疗产品基因组滴度检测的数字PCR(digital polymerase chain reaction,dPCR)法,并进行初步验证.方法 通过对同一批次AAV基因治疗产品进行dPCR检测,优化筛选最佳引物、探针、样品处理方式以及引物、探针浓度;对优化后方法进行专属性、线性、相对准确度、重复性验证,并确定方法的定量限及检测范围.结果 选择NFS-05 vector titer 5.0为引物、探针,引物终浓度为800 nmol/L,探针终浓度为400 nmol/L,样品前处理方式为经Proteinase K消化.建立的方法专属性良好;在3.3× 105～1.3 × 108vg/mL检测范围内,标准曲线相关系数(R2)＞0.999;相对准确度为70％～130％;重复性CV均≤15％;定量限为3.3× 105vg/mL.结论 建立的dPCR法专属性、线性、准确度及重复性良好,可用于NFS-05重组腺相关病毒(recombinant adeno-associated virus,rAAV)原液产品基因组滴度的检测.
Abstract
Objective To establish and preliminarily verify a digital polymerase chain reaction(dPCR)method for the detection of genome titer of adeno-associated virus(AAV)gene therapy products.Methods Through dPCR detection of the same batch of AAV gene therapy products,the primers,probes,sample treatment methods and concentrations of primer and probe were optimized.The specificity,linearity,relative accuracy and repeatability of the optimized method were veri-fied,and the limit of quantitation(LOQ)and detection range were determined.Results NFS-05 vector titer 5.0 crop primer probe was selected,the final primer concentration was 800 nmol/L,the final probe concentration was 400 nmol/L,and the sample pretreatment method was digestion by Proteinase K.The established method had good specificity.The correlation coefficient(R2)of the standard curve was greater than 0.999 in the range of 3.3 x 105-1.3 x 108 vg/mL.The relative accuracy was between 70％and 130％,the CV of repeatability was not more than 15％,and the LOQ was 3.3 x 105 vg/mL.Conclusion The established dPCR method has good specificity,linearity,accuracy and repeatability,and can be applied to the determination of the genome titer of NFS-05 recombinant adeno-associated virus(rAAV)stock solution products.
关键词
数字PCR/腺相关病毒/基因组滴度/基因治疗Key words
Digital polymerase chain reaction(dPCR)/Adeno-associated virus(AAV)/Genome titer/Gene therapy引用本文复制引用
基金项目
北京市科技计划课题资助(Z221100007922015)
出版年
2024
NETL
NSTL
万方数据