473. Qualification of a p24 ELISA Assay for Quantitation of Total Lentiviral Vector Concentration

Abstract

Lentiviral vectors (LVV) are efficient gene transfer vehicles used in a growing list of gene therapy applications that are currently being evaluated clinically. We are conducting clinical trials in subjects with β-thalassemia major, severe sickle cell disease, and childhood cerebral adrenoleukodystrophy, which employ autologous CD34+ cells transduced with LVV encoding a corrective transgene, βA-T87Q-Globin or ABCD1. Essential to LVV process development is the availability of reliable analytical tools to characterize the purified LVV product and in-process samples. Total lentiviral vector particle titers can be estimated by quantitation of HIV-1 p24 antigen concentration using commercial ELISA kits. In support of late-stage process characterization, it is essential to qualify the assay and demonstrate its suitability to test in-process samples in different matrices. Following ICH Q2 R1 guidelines, assay accuracy, repeatability, intermediate precision, quantitation limit, linearity, and linear range were evaluated. Additionally, qualification tests for specificity and dilutional linearity were performed on in-process samples and buffers to assess potential matrix interference. The results of these tests were used to define appropriate dilutions and acceptance criteria for p24 ELISA in-process samples. The assay has shown to be linear between 400 pg/mL and 25 pg/mL, while maintaining accuracy for p24 detection with the limit of quantitation being 25 pg/mL. To demonstrate assay repeatability, various concentrations within the linear range were tested resulting in coefficients of variation below 5%. The coefficient of variation across different days and different operators was below 8% for all concentrations tested, indicating consistent assay precision. Spike recovery studies have shown consistent recovery of spiked p24 antigen (73-126%), demonstrating assay specificity and lack of matrix interference in all in-process matrices tested while maintaining dilutional linearity. To conclude, the p24 ELISA assay used for LVV quantitation has shown precision, repeatability, specificity and accuracy, within an established linear quantitation range, adequate to support late-stage process characterization activities.