Characterizing Adeno-Associated Virus Capsids with Both Denaturing and Intact Analysis Methods

Area of Science:

• Biochemistry
• Analytical Chemistry
• Gene Therapy

Background:

• Adeno-associated viruses (AAVs) are crucial gene delivery vectors for treating human diseases.
• Diverse AAV serotypes target specific cells and tissues, necessitating precise composition analysis.
• Current analytical methods struggle with AAV subunit selectivity, intact capsid assessment, and empty/filled capsid differentiation.

Purpose of the Study:

• To develop and apply advanced analytical workflows for comprehensive AAV capsid characterization.
• To improve quality control for gene therapy by accurately assessing capsid composition and viral protein (VP) subunits.
• To differentiate between empty and filled AAV capsids and analyze VP sequences and modifications.

Main Methods:

• Utilized selective temporal overview of resonant ion (STORI)-based charge detection-mass spectrometry (CD-MS) for intact capsid analysis.
• Employed liquid chromatography, ion mobility spectrometry, and mass spectrometry (LC-IMS-MS) for denatured capsid subunit analysis.
• Applied this combined approach to AAV serotypes 2, 6, and 8.

Main Results:

• Successfully characterized intact empty and filled capsid ratios for AAV2, AAV6, and AAV8.
• Analyzed distinct viral protein (VP) sequences and post-translational modifications within the AAV capsids.
• Demonstrated the capability of the combined analytical workflows to overcome limitations of existing methods.

Conclusions:

• The integrated STORI-CD-MS and LC-IMS-MS approach provides enhanced selectivity and accuracy for AAV characterization.
• This methodology is vital for ensuring the quality and efficacy of AAV-based gene therapies.
• Accurate analysis of AAV capsid composition is critical for precise gene delivery and therapeutic outcomes.