重组腺相关病毒(rAAV)已成功用于体内基因递送以治疗多种人类疾病。为了维持rAAV基因治疗产品的生长,迫切需要发展准确而稳健的分析方法。TCID50测定(50%组织培养感染剂量)是广泛用于确定AAV感染性的体外基于细胞的方法,并且该测定由于其高变异性而历史上被视为挑战。目前,qPCR用作检测感染后复制的病毒基因组的量的终点方法。这里,我们通过调整液滴数字PCR(ddPCR)的终点检测来优化TCID50测定。我们在18次独立运行中使用ATCCAAV-2参考标准储备材料进行了TCID50测定。然后使用qPCR和ddPCR终点分析来自TCID50测定的细胞裂解物,以允许两种方法之间的直接比较。qPCR和ddPCR之间的长期一年并排比较作为终点测量证明了当使用ddPCR方法时提高的测定间精度。特别是,在添加了用于单个孔的感染性评分的新的二级阈值后,通过qPCR和ddPCR,18次运行的平均感染滴度分别为6.45E+08,%CV为42.5和5.63E+08,%CV为34.9。在这项研究中,我们提供了感染滴度测定的改进方法:(1)通过采用ddPCR作为终点方法而无需制备标准曲线,从而提高了测定间的精密度;(2)在感染性评分中确定第二个"设定阈值",从而提高了测定精密度;(3)应用统计分析来确定感染滴度值的接受范围.一起来看,我们提供了一种优化的TCID50方法,该方法具有改进的测定间精密度,这对于在工艺开发和制造过程中进行rAAV感染滴度测试非常重要.
Recombinant adeno-associated virus (rAAV) has been utilized successfully for in vivo gene delivery for treatment of a variety of human diseases. To sustain the growth of recombinant AAV gene therapy products, there is a critical need for the development of accurate and robust analytical methods. Fifty percent tissue culture infectious dose (
TCID50) assay is an in vitro cell-based method widely used to determine AAV infectivity, and this assay is historically viewed as a challenge due to its high variability. Currently, quantitative PCR (qPCR) serves as the endpoint method to detect the amount of replicated viral genome after infection. In this study, we optimize the
TCID50 assay by adapting endpoint detection with droplet digital PCR (ddPCR). We performed
TCID50 assays using ATCC AAV-2 reference standard stock material across 18 independent runs. The cell lysate from
TCID50 assay was then analyzed using both qPCR and ddPCR endpoint to allow for direct comparison between the two methods. The long-term 1-year side-by-side comparison between qPCR and ddPCR as endpoint measurement demonstrated improved interassay precision when the ddPCR method was utilized. In particular, after the addition of a novel secondary set threshold for infectivity scoring of individual wells, the average infectious titer of 18 runs is 6.45E+08 with % coefficient of variation (CV) of 42.5 and 5.63E+08 with % CV of 34.9 by qPCR and ddPCR, respectively. In this study, we offer improvements of infectious titer assay with (1) higher interassay precision by adapting ddPCR as an endpoint method without the need of standard curve preparation; (2) identification of a second \"set threshold\" value in infectivity scoring that improves assay precision; and (3) application of statistical analysis to identify the acceptance range of infectious titer values. Taken together, we provide an optimized
TCID50 method with improved interassay precision that is important for rAAV infectious titer testing during process development and manufacturing.