Removal of empty AAV capsids to undetectable levels using orthogonal purification steps – Product quality and process robustness considerations

Typical AAV Gene Therapy production systems generate cell culture harvests with a greater abundance of empty capsids (lacking DNA genomes) than full capsids (containing the desired DNA genome).  Removing the empty capsids during purification is a central production challenge since empty and full capsids possess similar physio-chemical properties. Design of a robust purification process capable of completely separating empty capsids from full capsids is presented in this work. This process employs orthogonal purification steps (Ion Exchange Chromatography and Zonal Ultracentrifugation) for removing empty capsids and controlling other process- and product-related impurities to assure the safety of AAV products. Scalability of this process and features that allow the combination of these orthogonal steps to provide robust product quality are also presented.