(337cv) Biomanufacturing of Recombinant Adeno-Associated Virus (rAAV) for Gene Therapy: Production, Purification, and Evaluations

Adeno-Associated Virus (AAV) has been shown to be one of the safest and most effective viral vectors for gene therapy since its discovery. However, it is important to note that no gene therapy approach is completely risk-free and continued research is necessary to improve the safety and efficacy of AAV-based gene therapy. Moreover, recombinant AAV is a promising tool for clinical applications due to its low toxicity and ability to provide stable, long-lasting expression of therapeutic genes. Currently, adherent HEK293 human cell line has been widely used for AAV production despite its limited ability to scale up. Meanwhile, scalable AAV production from bioreactor, a type of suspension culture, has been explored recent years. Considering the advantages of suspension cultures, it highlights the potential of cost effective AAV production process. This study aimed to compare three different suspension cultures, from shaker flask, spinner flask, to bioreactor, and evaluate the obtained AAV’s quality. The average titer obtained was approximately 5E+10 vg/ml, and the bioreactor showed its capability to yield the highest titer among the three methods. In addition, purification methods were developed using anion exchange based Next Generation Chromatography (Bio-Rad) as the purity of AAV is a crucial deciding factor for the success of gene therapy. additionally, biochemical analysis and IVIS imaging were conducted for the virus quality and control.