Analyzing the ratio of full-to empty AAV particles, also known as the full/empty ratio, is an important aspect of AAV vector characterization. The ratio can impact the efficacy and safety of gene therapy applications.

 Here are some common methods used for empty/full ratio analysis:

• Electron Microscopy (EM): Transmission electron microscopy (TEM) or cryo-electron microscopy (cryo-EM) can directly visualize and distinguish empty and full AAV particles based on their internal contents. This method provides qualitative information about the ratio.
• Gradient Centrifugation: Density gradient centrifugation using cesium chloride (CsCl) or iodixanol can separate AAV particles based on their density. Empty and full capsids have different densities, allowing for quantification of their ratio.
• Cryo-Electron Tomography (Cryo-ET): Cryo-ET can provide three-dimensional reconstructions of individual AAV particles, allowing for visualization of the genome within the capsid and determination of their full or empty status.
• Analytical Ultracentrifugation(AUC): Analytical ultracentrifugation can separate particles based on their sedimentation rate. Empty and full AAV particles might sediment differently, allowing for ratio determination.
• Mass Photometry(MP): It is a cutting-edge technique used for analyzing the mass of individual molecules in solution by measuring the light scattering when they land on a sensor surface, such as a microscope slide. When it comes to AAV particles, mass photometry can be particularly insightful. Full AAV particles, which contain the viral DNA payload, scatter more light compared to empty capsids, which lack the genetic material. This difference in light scattering leads to full particles appearing as darker spots on the sensor surface.
• Differential Scanning Calorimetry (DSC): DSC measures the heat absorbed or released during thermal denaturation of AAV particles. Differences in thermal stability between empty and full particles can help determine their ratio.
• Analytical Size Exclusion Chromatography (SEC): SEC can separate AAV particles based on their size and structure. Differences in elution profiles can provide insights into the empty/full ratio.
• Charge detection mass spectrometry (CD-MS):  CD-MS is a cutting-edge tool for analyzing the full/empty ratio of AAV particles, offering unparalleled precision and sensitivity. Its ability to perform single-particle analysis and directly measure the mass of AAV particles makes it ideal for ensuring the quality, safety, and efficacy of AAV-based therapies. While CD-MS is not yet widely available and requires specialized expertise, its advantages make it a valuable addition to the toolkit for AAV development and manufacturing.