Characterization and Quantification of Adeno-Associated Virus Capsid-Loading States by Multi-Wavelength Analytical Ultracentrifugation With UltraScan

Authors

Amy Henrickson
Xiaozhe Ding
Austin G Seal
Zhe Qu
Lauren Tomlinson
John Forsey
Viviana Gradinaru
Kazuhiro Oka
Borries Demeler

Publication Date

9-1-2023

Journal

Nanomedicine

DOI

10.2217/nnm-2023-0156

PMID

37877696

PMCID

PMC10652292

PubMedCentral® Posted Date

10-25-2023

PubMedCentral® Full Text Version

Post-Print

Published Open-Access

yes

Keywords

Capsid, Dependovirus, Ultracentrifugation, Genetic Vectors, Microscopy, Electron, Transmission, adeno-associated virus, analytical buoyant density gradient equilibrium, gene therapy, multi-wavelength analytical ultracentrifugation, sedimentation velocity, UltraScan, viral capsid quantification, viral vector

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.