The relative infectious titre for each sample was determined using the parallel-line GANT61 analysis as described in the European Pharmacopoeia 8.0 . The analysis by extrapolation is not an appropriate approach as several parameters including the similar conditions between the in-house reference control and test samples are not considered during analysis. In this study, the correlation between test samples and the in-house reference control was assessed using PLA software version 2.0. Before PLA analysis, all C T values for the in-house reference control and test samples
were subjected to standard outlier analysis, with the limit that no more than one data point (one replicate out of the four replicates) per HSV529 dilution could be removed. Afterwards, each assay was analyzed by PLA software. The assay was considered valid if the regression, linearity, and parallelism were significant. To investigate if RT-qPCR infectivity assay is a suitable method to evaluate the mTOR inhibitor drugs stability of HSV529 test samples, a concordance study was conducted between the RT-qPCR infectivity assay and a conventional infectivity plaque assay using identical test samples. While the results illustrated a suitable correlation
(R2 ~0.91) between the qRT-PCR infectivity assay and the plaque assay, higher cost and complexity of RT-qPCR infectivity assay were AZD5153 clinical trial two drawbacks of this method compare to a traditional method. To evaluate the closeness of the analytically determined HSV529 infectious titre values, the accuracy of the method was evaluated in six independent assays by two analysts (-)-p-Bromotetramisole Oxalate on different days. The accuracy was determined as the percentage of the infectious titre values obtained by RT-qPCR versus infectious titre values by a plaque assay. The accuracy was evaluated in the range of 92.91% to 120.57%, indicating a suitable accuracy for the assay. The intermediate precision
of the assay was also evaluated to measure the variation of the obtained data. To evaluate this parameter, the assay was performed six times by two different operators over a time period of 2 months. The mean value of this run control was 16.53 log pfu/ml with a standard deviation of 0.091, resulting in a coefficient of variation of 9.19. Conclusions In this study, a RT-qPCR based approach was utilized to specifically detect and quantitate the HSV529 RNA after productive infection in AV529-19 cells. The results show that the developed RT-qPCR infectivity assay is a reproducible approach that can quantitate the HSV529 infectious titre before the plaque assay formation is visible on day 3. The described RT-qPCR infectivity approach might also be a suitable approach for determination of potency of test samples, however; further evaluation of sub-potent lots and/or assessing clinical data is required. Methods Plaque assay The infectious titre of an HSV529 (lot#10954) was determined through a plaque assay on AV529 cells by performing 30 independent plaque assays.