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

Area of Science:

• Biophysical characterization
• Analytical ultracentrifugation
• Viral vector technology

Background:

• Accurate characterization of viral vector capsid species is crucial for gene therapies and vaccines.
• Sedimentation velocity analytical ultracentrifugation (SV-AUC) is the gold standard but has limitations in size range and data analysis complexity.
• Existing methods for viral vector characterization can be size-limited and require specialized software.

Purpose of the Study:

• To present optimized methods for Density Gradient Equilibrium Analytical Ultracentrifugation (DGE-AUC).
• To demonstrate the utility of DGE-AUC for high-throughput analysis of viral vector packaging.
• To establish DGE-AUC as a simplified, sensitive alternative to SV-AUC for viral capsid characterization.

Main Methods:

• Density Gradient Equilibrium Analytical Ultracentrifugation (DGE-AUC) using cesium chloride gradients.
• Optimization of DGE-AUC protocols for enhanced resolution and sensitivity.
• High-throughput analysis of adenovirus (AdV) packaging, accommodating up to 21 samples in 80 minutes.

Main Results:

• DGE-AUC provides high-resolution separation of viral capsids based on density (e.g., empty vs. full).
• The method demonstrates significantly improved sensitivity (estimated 56-fold) compared to SV-AUC.
• DGE-AUC is serotype-agnostic, amenable to multiwavelength analysis, and requires less specialized software for interpretation.
• Successful high-throughput packaging analysis of AdV was achieved.

Conclusions:

• DGE-AUC is a simplified, highly sensitive, and robust method for characterizing viral vector capsid species.
• This technique is particularly advantageous for larger viral particles like AdV and offers a more accessible alternative to SV-AUC.
• Optimized DGE-AUC enables efficient, high-throughput analysis crucial for gene therapy and vaccine development.