893. Using QPCR and Automation to Assign Infectious Potencies to Adenovirus Based Vaccines and Vectors for Gene Therapy

Abstract

The potencies of test articles generated during bioprocess development supporting the manufacture of Ad5 based HIV vaccine have been assigned since 1999 using a QPCR-based Potency Assay (QPA). We report here the simplification of the Ad5 QPA through (1) the introduction of a facile method for the harvest of DNA for QPCR quantitation and (2) the integration of automated liquid handling systems for performing semi-automated or completely automated QPA assays. We demonstrate semi-automated QPA operation using the Beckman Coulter Multimek for the addition of reagents, preparation of dilutions, and set-up of PCR reactions in 384 well formats, which greatly reduce reagent cost and analyst time involved with QPA assays. We show preliminary results indicating that a fully automated assay is possible using a more versatile liquid handling system such as the Tecan Freedom Evo. We also present the results of a PreValidation Exercise (PreVEx) for the semi-automated QPA assay we designate Triton Lysis with the Multimek (TLM) Ad5 QPA, which exhibits a remarkable precision. The PreVEx demonstrated that the TLM Ad5 QPA has a root variability of approximately 16.8% and a format dependent variability (1|[times]|3 assay format, with 4 infection replicates per assay) of approximately 5.8%, allowing samples differing in potency by 17.4% to be discriminated with 95% confidence. This precision equals or exceeds the precision associated with the previous Ad5 QPA.