2.9. Sedimentation Velocity Analytical Ultracentrifugation—SV-AUC

Sedimentation velocity analytical ultracentrifugation (SV-AUC) was performed on an Optima AUC instrument (Beckman Coulter, Brea, CA, USA). rAAV samples were loaded into AUC cells with standard 2-sector centerpieces (Beckman Coulter, Brea, CA, USA) into both sectors. Sedimentation of particles was monitored at 16,336× g at the wavelengths of 230 nm, 260 nm, and 280 nm in an AN Ti-50 rotor (Beckman Coulter, Brea, CA, USA). Data analysis was then performed in UltraScan III (AUC Solutions, Katy, TX, USA) using the intensity signal of each sample cell. Data were processed to contain the relevant scan range and optimal solutions for the time-invariant and radial-invariant noises, which were determined together with the optimal meniscus position. Final fitting was performed with the PCSA-SL-MC model and the statistical evaluation based on 100 Monte-Carlo simulations. Peak integration was performed to derive the relative contents of the empty (~65 S) and filled capsids (~105 S) and of the formulation components, such as small proteins, DNA fragments, and smaller unidentified species. For determination of the full/empty ratio, the same factors for 260 nm and 280 nm were used as in the analytical AEX experiments, and for 230 nm, a correction factor of 1.6 was used.