Assessing production variability in empty and filled adeno-associated viruses by single molecule mass analyses

Area of Science:

• Biotechnology
• Molecular Biology
• Gene Therapy

Background:

• Adeno-associated viruses (AAVs) are promising gene therapy vectors due to their stability and low immunogenicity.
• However, variations in AAV production lead to product heterogeneity, potentially impacting gene delivery efficacy.
• Precise characterization of AAV structure and genetic payload is crucial for consistent gene therapy outcomes.

Purpose of the Study:

• To evaluate the utility of mass photometry and charge-detection mass spectrometry for characterizing AAVs.
• To assess AAV heterogeneity across different serotypes and production platforms.
• To establish robust methods for quality control in AAV-based gene therapy.

Main Methods:

• Utilized mass photometry and Orbitrap-based charge-detection mass spectrometry for single-molecule analysis of AAVs.
• Analyzed AAV serotypes 8 and 2 produced by three distinct platforms.
• Characterized both empty and genome-packaged AAV particles.

Main Results:

• Successfully resolved heterogeneous AAV particle populations using both techniques.
• Demonstrated significant differences in AAV particle distributions across production methods.
• Identified common issues in packaged AAVs, including under/overpackaging and residual empty particles.

Conclusions:

• Mass photometry and charge-detection mass spectrometry are efficient and accurate tools for AAV characterization.
• These single-particle techniques can reveal critical insights into AAV production quality.
• The findings support the use of these methods for quality control of AAV vectors in gene therapy development.