Recombinant adeno-associated virus (rAAV) has been widely used as an effective delivery tool in gene therapy. One of the challenges facing the production of high-quality rAAV is optimization of the production and purification methodologies. Cesium chloride density gradient ultracentrifugation (CsCl-DGUC) has been traditionally utilized for rAAV purification
however, few studies have focused on CsCl-DGUC for rAAV purification despite this technique having a great potential for clinical-grade or large-scale rAAV production. In this study, we aimed to explore the unaddressed challenges associated with rAAV purification using CsCl-DGUC. We clarified that AAV capsids assembled by the different stoichiometries of three viral proteins (VP1, VP2, and VP3) showed heterogeneous population in the CsCl density gradient and encapsidated DNA increased the buoyant density differences of these capsids, resulting in the wider distribution. We implemented CsCl-DGUC using a vertical rotor which improved throughput and enhanced the separation of desired AAV particles from impurities. Furthermore, we examined the effect of CsCl exposure during purification, and the presence of residual CsCl in the purified rAAV. This study provides valuable insights into the application of CsCl-DGUC in the manufacturing of rAAV while ensuring adequate efficacy and safety for gene therapy.