Empty / Full capsid ratio is a critical parameter for AAV therapeutics production as well as patient treatment because “empty” capsids are nonfunctional as they contain no ssDNA to implement gene therapy. This parameter for two AAV lots was measured by analytical ultracentrifugation – sedimentation velocity (AUC-SV), Mass Photometry, Stunner, and immunoassay – ddPCR combination and results compared. The industry standard for this parameter evaluation has been AUC-SV method. In this method, generally either 280 or 260 nm is used as the scan monitoring wavelength, taking advantage of absorbance by capsid proteins or ssDNA presence in full capsid respectively. However, due to protein and nucleic acid spectra overlap, the empty / full capsid ratio value varies with monitoring wavelength. Due to this situation, same AAV preparation would show different ratio values when monitored either at 280 or 260 nm. In this work it is illustrated that monitoring by refractive index or at 230 nm would remove or minimize this dependency of empty / full capsid ratio on monitoring wavelength. Among the methods studied here, based on low sample volume, less sample preparation / experimental time requirements, rapid and ease of getting result, Mass Photometry seems to be the best for AAV Empty / Full capsid composition determination.
Learning Objectives:
Upon completion, participants will be able to understand AAV modality
Upon completion, participants will be able to understand the need for AAV Empty / Full capsid quantitation
Upon completion, participants will know the available tchnologies / methods for AAV Empty / Full quantitation
Upon completion, participants will know comparative advantages / disadvantages among the available tchnologies / methods for AAV Empty / Full quantitation
Upon completion, participants will be able to assesss future tchnologies / methods for AAV Empty / Full quantitation
