Size Distribution Analysis of the Adeno-Associated Virus Vector by the c(s) Analysis of Band Sedimentation Analytical Ultracentrifugation with Multiwavelength Detection

Adeno-associated virus (AAV) vector is a promising platform for in vivo gene therapy. The accurate assessment of distribution state of particles contained in AAV vector samples is one of the most important and challenging matters and is necessary because the product-related impurities with the capsid structure (empty particles, intermediate particles, and aggregates) could be a possible cause of reducing the therapeutic efficacy and enhancing the unfavorable immune response. In this study, we report an effective approach for size distribution analysis with component identification. A small amount of AAV vectors were used by the analytical zone centrifugation c(s) analysis of band sedimentation analytical ultracentrifugation (BS-AUC) with multiwavelength detection. Using PBS/(H2O)-O-18, the concentration of each component could be determined in BS-AUC with high resolution. Compared with the sedimentation velocity AUC (SV-AUC), which generally requires 2 x 10(12) vg of AAV vectors, BS-AUC could be performed with about 1/25 of the AAV vector amount at 260 nm detection and ideally with about 1/50 of the AAV vector amount at 230 nm detection (4 x 10(10) vg), depending on the extinction coefficient of the AAV sample at each wavelength. According to the limit of quantification of this BS-AUC, 6.3 x 10(11) cp mL(-1) of empty particle (EP) and 4.4 x 10(11) vg mL(-1) of full particle (FP) could be quantified for 4 x 10(10) vg in 15 mu L of AAV8-CMV-EGFP. These results demonstrated that proposed BS-AUC approach we established here can compensate for the drawback in terms of the sample amount of SV-AUC. (c) 2022 American Pharmacists Association. Published by Elsevier Inc. All rights reserved.

Automated summary

This study presents an effective approach for size distribution analysis and component identification of AAV vector samples using a small amount of material. The proposed band sedimentation analytical ultracentrifugation (BS-AUC) method offers higher resolution and allows for accurate determination of component concentration compared to traditional sedimentation velocity AUC (SV-AUC). This approach overcomes the sample amount limitation of SV-AUC.