Optimizing LOD/LOQ and sample consumption during analytical ultracentrifugation to characterize AAV gene therapy vectors.

Production and development of adeno-associated viruses (AAV) for gene therapy applications requires sophisticated analytical methods to assess critical quality attributes of the product. A popular method providing relevant information on the loading status of the AAV capsid is sedimentation velocity analytical ultracentrifugation (SV-AUC). While multiwavelength SV-AUC, used along with extinction coefficients for different capsid species, delivers the relative percentages of empty, partially and filled AAV capsids, it does so at the cost of high sample consumption (typically 400 µl at ∼9*1012 viral particles/ml) and therefore high expense. We investigated the SV-AUC method for AAV characterization at three wavelengths and determined a LOQ of 7.7% empty capsids with standard SEDFIT analysis, which can be improved by using the GUSSI integration package or automated SEDFIT workflows with predefined integration ranges to 2.3%. Beyond that we find that SV-AUC allows adaptation of the method to low sample consumption based on reduced filling volume and reduced sample concentration. For this, we tested result consistency at up to 50-fold reduction of AAV particle concentration compared to standard conditions, and a 3-fold reduction of sample volume. In these measurements, it remained possible to obtain several critical sample parameters, such as reliable values for empty and filled capsid contents and values for AAV concentrations despite the reduced sample amounts. Based on these data, sample consumption can be reduced by a factor of 20 with few setbacks, which mitigates one of the main disadvantages in the usage of SV-AUC for the characterization of AAV samples.