Capture of adeno‐associated viruses from clarified lysate by continuous flow ultracentrifugation: a comparative study of iodixanol and sucrose gradients

Abstract BACKGROUND Adeno‐Associated Virus vectors are widely used in gene therapy due to their safety and efficacy. Despite the interest in their therapeutical application, AAV purification remains a significant challenge. Density gradient ultracentrifugation using cesium chloride, sucrose, or iodixanol gradients are common methods for AAV purification and polishing. However, when conventional ultracentrifuges are used, density gradient ultracentrifugation is not scalable and time‐consuming. To overcome these limitations, this study applies density gradient ultracentrifugation using a continuous flow ultracentrifuge which allows the development of scalable and robust AAV purification methods. RESULTS Our results showed that continuous flow ultracentrifugation with both iodixanol and sucrose density gradients is a suitable method for AAV capture. Sucrose density gradient allowed for the recovery of 72% full AAV particles, however, poor resolution of full and empty AAV particles and co‐purification of host cell derived impurities were observed. Iodixanol density gradient allowed for the recovery of 68% full AAV particles as well as enrichment of full AAV particles in the higher density fractions. Moreover, separation of AAV particles from host cell derived impurities was achieved with the iodixanol density gradient. Nevertheless, chromatin fragments were still present. CONCLUSIONS The findings of this study highlight the possibility of development of scalable AAV capture methods based on density gradient ultracentrifugation using continuous flow ultracentrifuges. © 2025 The Author(s). Journal of Chemical Technology and Biotechnology published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry (SCI).