Quantitation of adeno-associated virus in a dual-vector system using sedimentation velocity analytical ultracentrifugation

Abstract

Sedimentation velocity analytical ultracentrifugation (SV-AUC) has become the "gold standard" for characterization of the empty, partial, and full capsids of gene therapy products (e.g., AAV and Adenovirus vectors). Other techniques, such as SEC-MALS, TEM, and mass photometry, are commonly used for capsid quantitation, however, the resolving power of these techniques is lacking. In this body of work, SV-AUC was implemented in the characterization of a dual-vector AAV system where the difference in packaged genomes was ∼400 nucleotides. The instrument parameters and SV-AUC analysis were optimized to accurately quantitate both AAV vectors with less than 8% error and with highly correlated linearity (R² > 0.99) as compared to ddPCR. The results of this work highlight the resolution and accuracy of dual-vector capsid quantitation by SV-AUC and demonstrate the use of the powerful Bayesian analysis implemented in the SEDFIT analysis software.

Keywords: Adeno-associated virus; Analytical ultracentrifugation; Biopharmaceutical characterization; Gene therapy; Physicochemical properties; Polymerase chain reaction; Triple-vector; Viral vector.