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Publication Number
US-12618850-B2
Publication Date
2026-05-05
Expiration Date
Abstract
Provided are methods to characterize the VP1, VP2 and VPS capsid proteins in an adeno-associated virus (AAV) particle using liquid chromatography mass spectrometry, and/or ultraviolet (UV)-visible spectroscopy. The methods generally include the steps of (a) subjecting an AAV particle to liquid chromatography to denature and then separate the VP1, VP2 and VPS capsid proteins, and (b) subjecting the separated VP1, VP2 and VPS capsid proteins produced in step (a) to UV and mass spectrometry to determine the ratio and masses of the VP1, VP2 and VPS capsid proteins in the AAV particle. In another aspect, the disclosure provides an AAV composition comprising a post-translation modification. The disclosure also provides methods for characterizing the purity of AAV compositions using liquid chromatography mass spectrometry.
Core Innovation
The disclosure provides a method of characterizing capsid viral protein 1 (VP1), capsid viral protein 2 (VP2), and capsid viral protein 3 (VP3) in an adeno-associated virus (AAV) particle by reverse phase liquid chromatography and ultraviolet (UV)-visible detection. The method introduces the AAV particle into a reverse phase liquid chromatography column, maintains the column at a temperature approximately between 70°C and 90°C, and contacts the particle with a first mobile phase having a trifluoracetic acid volume percentage approximately between 0.05% and 0.15%. VP1, VP2, and VP3 are eluted to produce corresponding VP1, VP2, and VP3 capsid viral protein chromatographic peaks.
Corresponding VP1, VP2, and VP3 peaks are detected using the ultraviolet (UV)-visible spectrometer, and corresponding relative abundances of VP1, VP2, and VP3 are determined based on the detected chromatographic peaks. The approach is described as using UV chromatographic peaks for relative abundance determination, with dependent refinements that add further characterization beyond UV detection.
The disclosure further characterizes the AAV capsid proteins by adding mass spectrometry to determine VP1, VP2, and VP3 masses, including approaches based on comparing VP1, VP2, and VP3 ultraviolet chromatograms and using mass spectrometry-based measurements. Post-translational modifications are addressed by enabling determination of selected post-translational modifications including deamidation and oxidation, with additional refinements covering defined post-translational modification types and examples of deamidated and oxidized residues.
Claims Coverage
The document contains one independent claim directed to reverse-phase liquid chromatography with UV-visible detection for determining relative abundances of VP1/VP2/VP3, plus dependent claims that add mass determination, comparative UV chromatogram assessment, and post-translational modifications.
Reverse-phase liquid chromatography at 70°C to 90°C with trifluoracetic acid mobile phase and UV-visible detection
Subjecting the AAV particle to a reverse phase liquid chromatography column maintained at a temperature approximately between 70°C and 90°C, contacting the AAV particle with a first mobile phase having a trifluoracetic acid volume percentage approximately between 0.05% and 0.15%, eluting VP1, VP2, and VP3 to produce corresponding chromatographic peaks, and detecting the corresponding peaks using a ultraviolet (UV)-visible spectrometer.
Relative abundance determination from UV-visible chromatographic peaks
Determining corresponding relative abundances of VP1, VP2, and VP3 capsid protein based on the detected corresponding VP1, VP2, and VP3 capsid viral protein chromatographic peaks.
Mass spectrometer determination of VP1, VP2, and VP3 masses
Determining the masses of VP1, VP2, and VP3 using a mass spectrometer.
Comparing VP1, VP2, and VP3 ultraviolet chromatograms
Determining by comparing VP1, VP2, and VP3 ultraviolet chromatograms with each other.
Post-translational modification determination for selected modification types including deamidation and oxidation
Determining one or more post-translational modifications selected from amino-acid loss, glycosylation, sialylation, acetylation, phosphorylation, deamidation, oxidation, formylation, hydroxylation, methylation, and sulfation, or combinations thereof.
Across the claims, the core approach is reverse-phase liquid chromatography under specified temperature and trifluoracetic acid mobile phase conditions, followed by UV-visible detection to determine VP1/VP2/VP3 relative abundances, with refinements that add mass spectrometry-based mass determination, comparative UV chromatogram evaluation, and determination of selected post-translational modifications including deamidation and oxidation.
Stated Advantages
Not explicitly described in patent.
Documented Applications
Not explicitly described in patent.
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