Characterization and Quantification of Adeno-Associated Virus Capsid-Loading States by Multi-Wavelength Analytical Ultracentrifugation with Ultrascan
Amy Henrickson, Xiaozhe Ding, A. Seal, Zhe Qu, Lauren A. Tomlinson, John Forsey, Viviana Gradinaru, Kazuhiro Oka, Borries Demeler
Abstract
Aim: We present multi-wavelength (MW) analytical ultracentrifugation (AUC) methods offering superior accuracy for adeno-associated virus characterization and quantification. Methods: Experimental design guidelines are presented for MW sedimentation velocity and analytical buoyant density equilibrium AUC. Results: Our results were compared with dual-wavelength AUC, transmission electron microscopy and mass photometry. In contrast to dual-wavelength AUC, MW-AUC correctly quantifies adeno-associated virus capsid ratios and identifies contaminants. In contrast to transmission electron microscopy, partially filled capsids can also be detected and quantified. In contrast to mass photometry, first-principle results are obtained. Conclusion: Our study demonstrates the improved information provided by MW-AUC, highlighting the utility of several recently integrated UltraScan programs, and reinforces AUC as the gold-standard analysis for viral vectors.