Adeno-Associated Viral vectors (AAVs) have emerged as a delivery vehicle in gene therapy which have become the vector of choice for the treatment of rare diseases. Viral vectors intended for clinical and commercialization use must meet increasing regulatory standards for product-and process-related impurities, therefore, an adequate characterization of the drug product is essential. For that purpose, Viralgen has developed a high resolutive and sensitive Capillary Electrophoresis (CE-SDS) based method to assess vector purity and provide a semi-quantitative estimate of the vector.

Developing the CE-SDS analytical method for AAV8 and AAV9 for GMP standards encountered challenges related to sample concentration and salt removal of the product. Applying this method to other serotypes such as AAV2, AAV3b and AAV6 faced further implications, as their Heparin binding motif presents interaction with membranes and filters, hindering the salt removal process. Thus, an alternative buffer exchange method was developed for Heparin binding serotypes, working through the barriers it entails.

As a CDMO specialized in the production of multiple grades of AAV, validation of the analytical methods is critical to demonstrate that the method is suitable for its intended purpose. Hence, we will take the opportunity to discuss the challenges faced by Viralgen when transferring the purity assessment from SDS-PAGE to CE-SDS in terms of serotype, salt content, and sample concentration, together with the validation process of the method for AAV8 and AAV9.