Rapid high-resolution size distribution analysis for adeno-associated virus using high speed SV-AUC

Abstract

AbstractWhen optimized, sedimentation velocity analytical ultracentrifugation (SV-AUC) provides the most-accurate, broadest-range, and highest-resolution size distribution analysis of any method. Generating simulated data for an adeno-associated virus (AAV) sample consisting of four species differing only in their DNA content and having closely spaced sedimentation coefficients, allows manipulation of the SV-AUC experimental protocol to optimize the size distribution resolution. In developing this high speed SV-AUC (hs-SV-AUC) protocol several experimental challenges must be overcome: 1) the need for rapid data acquisition, 2) avoiding optical artifacts from steep boundaries and 3) overcoming the increased potential for convection. A protocol, hs-SV-AUC, has been developed that uses high rotor speeds, interference detection and low temperatures to overcome these challenges. By confining data analysis to a limited radial-time window and using a very short run time (< 20 min after temperature equilibration), the need to match the sample and reference solvent composition and meniscus positions is relaxed, making interference detection is as simple to employ as absorbance detection. Experimental size distributions from the same AAV sample by hs-SV-AUC at 45K rpm and 10 °C versus low-speed SV-AUC at 15K rpm, and 10 °C illustrates the improved size distribution resolution offered by the hs-SV-AUC protocol.