Optimizing high-throughput viral vector characterization with density gradient equilibrium analytical ultracentrifugation

Abstract

Viral vector-based gene therapies and vaccines require accurate characterization of capsid species. The current gold standard for assessing capsid loading of adeno-associated virus (AAV) is sedimentation velocity analytical ultracentrifugation (SV-AUC). However, routine SV-AUC analysis is often size-limited, especially without the use of advanced techniques (e.g., gravitational-sweep) or when acquiring the multiwavelength data needed for assessing the loading fraction of viral vectors, and requires analysis by specialized software packages. Density gradient equilibrium AUC (DGE-AUC) is a highly simplified analytical method that provides high-resolution separation of biologics of different densities (e.g., empty and full viral capsids). The analysis required is significantly simpler than SV-AUC, and larger viral particles such as adenovirus (AdV) are amenable to characterization by DGE-AUC using cesium chloride gradients. This method provides high-resolution data with significantly less sample (estimated 56-fold improvement in sensitivity compared to SV-AUC). Multiwavelength analysis can also be used without compromising data quality. Finally, DGE-AUC is serotype-agnostic and amenable to intuitive interpretation and analysis (not requiring specialized AUC software). Here, we present suggestions for optimizing DGE-AUC methods and demonstrate a high-throughput AdV packaging analysis with the AUC, running as many as 21 samples in 80 min.

Methods for the Design and Analysis of Analytical Ultracentrifugation Experiments

“…This means that the density resolution of ABDE experiments is very high for such systems and the sample requirements are very low, which is important for expensive samples that are available only in limited amounts (Henrickson et al, 2023). For example, this can be very useful for the analysis of viral vectors (Henrickson et al, 2023;Richter et al, 2023;Savelyev et al, 2023;Sternisha et al, 2023) where the degree of capsid loading needs to be quantified. Because the densities of proteins and nucleic acids are quite different, the ABDE approach provides a high degree of resolution between empty, partially, and fully loaded capsids.…”