Selective Enrichment of Full Capsids of Adeno-Associated Virus

Gene therapies using recombinant adeno-associated virus (rAAV) have been developed to treat monogenic and acquired diseases, but are currently the most expensive drugs, limiting patient access to these treatments. The drug pricing is due, in part, to high manufacturing costs. In addition to the discontinuous production process, the cells producing rAAV generate substantial quantities of empty (50-90%) and partially filled (indeterminant) capsids. These impurities do not contribute to the potency and increase the immunogenicity; therefore, empty particles must be removed prior to formulating the bulk drug substance. The conventional separation processes inefficiently separate the empty and partially filled capsids from the biologically active full capsids. These separation processes result in substantial product losses, scale poorly, and are expensive, time-consuming, and require additional processing steps. This Article describes a one-step separation method using selective crystallization─a process which, to our knowledge, has not been used previously for rAAV purification. The process does not physically/chemically modify the target component (full capsid), is highly efficient, highly scalable, and economical, and improves product quality. Hanging-drop vapor diffusion experiments are used to scout crystallization conditions in which full and empty capsids crystallize and then to define conditions in which crystals of full, empty, or both full and empty capsids nucleate and grow. The experimental results for rAAV serotypes 5, 8, and 9 as exemplary vectors and scale-up results show that full capsids can be selectively crystallized and separated from a mixture of full, partially filled, and empty capsids as well as other protein and salt impurities with full capsid enrichment of >80%, approximately 20% higher than the existing methods, and a yield of >90%, while maintaining biological activity. The selective crystallization process rapidly (<4 h) achieves greater enrichment of full capsids in a single step than previously reported protocols. Importantly, incorporating the selective crystallization process into cGMP vector production would accelerate production timelines and substantially reduce costs by eliminating the need of specialized immunoaffinity chromatography media.