Analytical strategies for quantification of adeno-associated virus (AAV) empty capsids to support process development.

Recombinant adeno-associated virus (rAAV)-mediated gene therapy is a fast-evolving field in the biotechnology industry. One of the major challenges in developing a purification process for AAV gene therapy is establishing an effective yet scalable method to remove empty capsids, or viral vectors lacking the therapeutic gene, from full capsids-viral product containing the therapeutic sequence. Several analytical methods that can quantify the empty-to-full capsid ratio have been reported in the literature. However, as samples can vary widely in viral titer, buffer matrix, and the relative level of empty capsids, understanding the specifications and limitations of different analytical methods is critical to providing appropriate support to facilitate process development. In this study, we developed a novel anion-exchange high-performance liquid chromatography (AEX-HPLC) assay to determine the empty-to-full capsid ratio of rAAV samples. The newly developed method demonstrated good comparability to both the transmission electron microscopy (TEM) and analytical ultracentrifugation (AUC) methods used in empty-to-full capsid ratio quantification, yet providing much higher assay throughput and reducing the minimum sample concentration requirement to 2.7E11 viral genomes (vg)/ml.