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