Track: Manufacturing and Analytical Characterization - Biomolecular - Analytical - Modality Specific Methods - Gene Therapy
Category: Late Breaking Poster Abstract
Utilizing Two PCR Technology Platforms to Support Biosafety and Release Testing of Recombinant Adeno-Associated Virus (rAAV) Vectors for Gene Therapy
Purpose: Recombinant adeno-associated virus (rAAV) vectors have been widely used for in vivo gene therapy with Luxturna™ and Zolgensma™ already approved by FDA as commercial products. The Safety Tripod concept and practice, including screening of starting materials, testing of process intermediates, and incorporating virus clearance steps, have been applied to cGMP manufacturing of rAAV gene therapy products to mitigate viral safety concerns. Therefore, there is a strong demand for technology platform(s), which can rapidly detect viral contaminants in starting material and process-related residual DNAs in intermediates and final products. In addition, as rAAV products are designed to not replicate by themselves neither in vitro nor in vivo, genome copy number has been commonly used for clinical dosing. As a result, it is critical to be able to measure the genome titer of rAAV products with accuracy and precision. Methods: The real-time PCR (qPCR) platform is a powerful tool for rapid adventitious agents detection. The Sf9 cell line has been used as a cell substrate to manufacture rAAV products. A RT-qPCR assay can readily detect Sf-rhabdovirus, a newly discovered virus in Sf9 cell line, to safeguard the final products. Due to the limitation of DNA clearance, a significant level of residual host cell DNA as a process impurity generally would be present in rAAV products. qPCR assays can quantify not only residual host cell DNA with sizing evaluation but also any residual specific viral sequences and/or oncogene carried over from the cell substrate. In addition, a wild type AAV-specific qPCR assay can detect the presence of replication competent AAV (rcAAV) in the final product after several rounds of cell-based amplification. Droplet Digital PCR (ddPCR) technology provides high degree of accuracy and unparalleled degree of precision in terms of rAAV genome titration by absolute quantification per Poisson statistics without the need of a DNA standard as well as by higher tolerance of matrix interference per fractionation of sample into approximately 20,000 individual PCR reaction in each droplet. Results: The data will be presented in the poster for qPCR-based Sf-rhabdovirus detection assay, qPCR-based residual host cell DNA quantification assay, cell-based rcAAV assay with qPCR read-out, as well as ddPCR-based rAAV genome titration assay, respectively. The Results will show assay sensitivity as the Limit of Detection (LOD) for viral detection assays while as the Limit of Quantification (LOQ) for residual DNA quantification and rAAV genome titration assays, as well as specificity by various negative controls. More importantly, the accuracy and precision of rAAV genome titration assay will be demonstrated by reference materials from ATCC with known titers. Conclusion: XXXXXX has been successfully utilizing both qPCR and ddPCR technology platforms to develop, qualify, and validate various qualitative and quantitative analytical methods per ICH Q2 (R1) guidance in order to support biosafety and release testing of rAAV-based gene therapy products.