High resolution ES-DMA quantifies AAV capsid DNA content by electrical mobility to mass correlation

Area of Science:

• Biotechnology
• Analytical Chemistry
• Molecular Biology

Background:

• Recombinant Adeno-Associated Virus (rAAV) is a key viral vector for gene therapy.
• Accurate assessment of rAAV DNA content is crucial for manufacturing and quality control.
• Impurities like empty or over-filled capsids reduce efficacy and increase patient risk.

Purpose of the Study:

• Introduce high-resolution electrospray differential mobility analysis (ES-DMA) for rAAV characterization.
• Demonstrate ES-DMA's ability to resolve DNA encapsidation in rAAV particles.
• Benchmark ES-DMA against charge detection mass spectrometry (CD-MS).

Main Methods:

• Utilized high-resolution electrospray differential mobility analysis (ES-DMA).
• Analyzed microliter volumes of rAAV samples.
• Compared ES-DMA results with charge detection mass spectrometry (CD-MS).

Main Results:

• ES-DMA provides angstrom-scale particle sizing sufficient for resolving rAAV DNA encapsidation.
• A strong near-linear correlation was found between electrical mobility and mass of aerosolized rAAV particles.
• ES-DMA enables rapid identification and quantification of empty, full, and partial/over-filled rAAV capsids.

Conclusions:

• ES-DMA is a novel, effective benchtop analytical method for rAAV vector characterization.
• This technique can improve the quality control of gene therapy vectors.
• ES-DMA offers a viable alternative for assessing rAAV capsid content.