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

Area of Science:

• Biophysical characterization
• Analytical ultracentrifugation
• Adeno-associated virus (AAV) analysis

Background:

• Size distribution analysis of adeno-associated virus (AAV) is crucial for characterizing viral vectors.
• Traditional SV-AUC methods can be time-consuming and may lack sufficient resolution for closely spaced components.
• Optimizing SV-AUC protocols is essential for efficient and accurate biophysical characterization.

Purpose of the Study:

• To develop a high-speed SV-AUC (hs-SV-AUC) protocol for enhanced resolution of AAV size distribution.
• To address experimental challenges associated with high-speed data acquisition and potential artifacts.
• To demonstrate the advantages of hs-SV-AUC over standard low-speed methods.

Main Methods:

• Development of a high-speed (45K rpm) SV-AUC protocol using simulated and experimental AAV data.
• Implementation of interference detection at low temperatures and a limited radial-time window.
• Comparison of hs-SV-AUC with standard low-speed (15K rpm) SV-AUC.

Main Results:

• The hs-SV-AUC protocol significantly improved size distribution resolution for AAV samples with closely spaced components.
• Run times were reduced to under 20 minutes after temperature equilibration.
• The protocol relaxed requirements for matching sample and reference solvent composition and meniscus positions.
• Experimental data validated the improved resolution and rapid data acquisition capabilities.

Conclusions:

• The developed hs-SV-AUC protocol offers a powerful tool for high-resolution size distribution analysis of AAV.
• This method provides a faster and more experimentally accessible alternative to standard SV-AUC.
• The protocol's ability to overcome challenges like optical artifacts and convection enhances its utility in biophysical studies.