Characterization and quantification of adeno-associated virus capsid loading states by multi-wavelength analytical ultracentrifugation with UltraScan


  • Current AAV (adeno-associated virus) characterization methods have low throughput, need larger sample quantities, and tend to overestimate the full capsid quantity.
  • Traditional AAV characterization techniques have limitations in accurately characterizing or quantifying product-related impurities.
  • Challenges in AAV formulation characterization relate to the accuracy, resolution, throughput, cost, and sensitivity of the analysis.
  • Traditional AAV methods overestimate filled capsids due to protein and nucleic acid spectral overlap.
  • Sedimentation Velocity (SV), a popular AAV characterization method, has limitations like being low throughput, requiring significant training and time investment, and needing larger sample amounts.


  • Introduction of multi-wavelength analytical ultracentrifugation (MW-AUC) methods for characterizing and quantifying AAV capsids. This provides high statistical certainty, unambiguous quantification of all capsid types, and the identification of contaminants.
  • The latest Beckman Optima AUC instruments and new software modules for UltraScan have been developed to facilitate MW-AUC analysis.
  • MW-AUC offers a second spectral dimension for separation, allowing for the resolution and identification of AAV loading states, distinguishing full capsids from contaminants, and detecting even small amounts of contaminants.
  • Application of multi-wavelength capabilities to both SV and analytical buoyant density equilibrium (ABDE) experiments to analyze AAV capsid loading states.
  • AUC, when combined with MW, offers improved statistics, enhanced resolution, and reduced sample requirements, making it superior to methods like TEM and MP .


  • The study validated multi-wavelength analytical ultracentrifugation (MW-AUC) for accurately analyzing adeno-associated virus (AAV) capsid loading states in various AAV serotypes.
  • MW-AUC offers superior accuracy over traditional dual-wavelength AUC and can detect partially filled capsids, which TEM cannot quantify correctly.
  • Unlike mass photometry, MW-ABDE gives independent results and distinguishes protein signals from DNA, requiring 20-40 times less sample than SV-AUC.
  • MW-ABDE identified two previously unknown high-density capsid species, potentially from AAV exposure to CsCl, necessitating further research.
  • The UltraScan software meets the 21 CFR Part 11 compliance requirements, suggesting AUC's potential as the gold standard for viral vector analysis.
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