464. RCL-Pooling Assay: A Simplified Method for the Detection of Replication Competent Lentiviruses in Vector Batches Using Sequential Pooling

Non-replicative recombinant HIV-1 derived lentiviral vectors (LV) are increasingly used in gene therapy clinical trials for various genetic diseases, infectious diseases or cancer. Before they are used in man, preparations of LV must undergo extensive biochemical and biological quality control testing. In particular, the legislation stipulates that the absence of replication-competent lentiviruses (RCL) must be demonstrated with suitable methods, on representative fractions of batches. Current standard and widely used methods based on cell culture are challenging because high titers of vector batches achieved translate into high volumes of cell culture that have to be tested. Since vector batch titers and sizes are continuously-increasing due to the improvement of production and purification methods, it is necessary to modify the current cell culture method. Here, we propose a practical optimization of the p24-decrease-based culture assay developed by Escarpe et al. (2003) using a pairwise pooling strategy enabling the test of higher vector inoculum volumes. These modifications significantly decrease material handling, operator time, leading to a cost effective method, while maintaining optimal sensibility of the RCL testing. This optimized RCL-pooling assay ameliorates the feasibility of the quality control of large-scale batches of clinical-grade LV while maintaining the same sensitivity.