Details

Autor(en) / Beteiligte

• Sternisha, Shawn M.
• Wilson, Abraham D.
• Bouda, Emilie
• Bhattacharya, Akash
• VerHeul, Ross

Titel

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

Ist Teil von

• European biophysics journal, 2023-07, Vol.52 (4-5), p.387-392

Ort / Verlag

Cham: Springer International Publishing

Erscheinungsjahr

2023

Quelle

Alma/SFX Local Collection

Beschreibungen/Notizen

• 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.