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US-12618850-B2 - Methods for analyzing AAV capsid proteins

US12618850B2US 12618850 B2US12618850 B2US 12618850B2US-12618850-B2

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.

Inventors

  • Kunal DAUD
  • JU LI
  • Uditha deAlwis

Assignees

  • SAREPTA THERAPEUTICS, INC.

Dates

Publication Date
20260505
Application Date
20201230

Claims (17)

  1. 1 . 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 using a reverse liquid chromatography column having a stationary phase and an ultraviolet (UV)-visible spectrometer, the method comprising: (a) subjecting the AAV particle to the reverse phase liquid chromatography column, wherein the subjecting comprises: (a1) introducing the AAV particle into the reverse phase liquid chromatography column, (a2) maintaining the reverse phase liquid chromatography column at a temperature approximately between 70° C. and 90° C., (a3) contacting the AAV particle with a first mobile phase having a trifluoracetic acid volume percentage approximately between 0.05% and 0.15%, and (a4) eluting VP1, VP2, and VP3 from the reverse phase liquid chromatography to produce corresponding VP1, VP2, and VP3 capsid viral protein chromatographic peaks; (b) detecting the corresponding VP1, VP2, and VP3 capsid viral protein chromatographic peaks using the ultraviolet (UV)-visible spectrometer; and (c) 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.
  2. 2 . The method of claim 1 , further comprising determining VP1 mass, VP2mass, and VP3 mass using a mass spectrometer.
  3. 3 . The method of claim 1 , wherein the determining comprises comparing a VP1 ultraviolet chromatogram with a VP2 ultraviolet chromatogram and with a VP3 ultraviolet chromatograph and comparing the VP2 ultraviolet chromatogram with the VP3 ultraviolet chromatograph.
  4. 4 . The method of claim 1 , wherein the stationary phase comprises a plurality of silica particles bonded to hydrocarbon chains having 18, 8, or 4 carbon atoms.
  5. 5 . The method of claim 4 , wherein the plurality of silica particles have a size between about 1.2 μm and about 3.5 μm.
  6. 6 . The method of claim 4 , wherein the reverse phase liquid chromatography column has a length between about 50 mm and about 300 mm long and has an internal diameter between about 1 mm and about 4.6 mm.
  7. 7 . The method of claim 1 , wherein the AAV particle is of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV 11, AAV12, AAV13, AAVrh10, AAVrh74 serotype.
  8. 8 . The method of claim 1 , wherein the contacting comprises introducing the first mobile phase into the reverse phase liquid chromatography column at a first flow rate and a second mobile phase at a second flow rate, wherein the second mobile phase comprises a mixture of trifluoroacetic acid, acetonitrile, and water.
  9. 9 . The method of claim 8 , wherein the second mobile phase comprises an acetonitrile volume percentage between about 80% and 95%.
  10. 10 . The method of claim 8 , wherein the contacting further comprises increasing the second flow rate relative to the first flow rate.
  11. 11 . The method of claim 1 , further comprising determining one or more post translational modifications in VP1, VP2, or VP3, or a combination thereof.
  12. 12 . The method of claim 11 , wherein the one or more post translational modifications comprises loss of an amino acid, glycosylation, sialylation, acetylation, phosphorylation, deamidation, oxidation, formylation, hydroxylation, methylation, or sulfation, or a combination thereof.
  13. 13 . The method of claim 11 , wherein the first mobile phase further comprises Tris-HC1.
  14. 14 . The method of claim 13 , wherein the first mobile phase further comprises acetonitrile.
  15. 15 . The method of claim 13 , wherein the first mobile phase further comprises methionine.
  16. 16 . The method of claim 13 , wherein the first mobile phase has a formulation comprising a Tris-HC1 molar concentration approximately between 5 mM and 50 mM, an acetonitrile volume percent between approximately 5% and 20%, and a methionine molar concentration between approximately 1 mM and 50 mM.
  17. 17 . The method of claim 11 , wherein the one or more post translational modifications comprise one or more deamidation sites at N263, N514, N57, N502, N254, and N94 of AAV8 or a combination thereof, or an equivalent residue of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV9, AAV10, AAV11, AAV12, AAV 13 AAV13, AAVrh10, or AAVrh74.

Description

CROSS REFERENCE TO RELATED APPLICATIONS This application claims benefit under 35 U.S.C. § 119(e) of U.S. Provisional Application No. 62/956,681 filed Jan. 3, 2020, U.S. Provisional Application No. 63/073,188 filed Sep. 1, 2020, and U.S. Provisional Application No. 63/119,909 filed Dec. 1, 2020, the contents of each of which are incorporated herein by reference in their entireties. REFERENCE TO SEQ REFERENCE TO SEQUENCE LISTING SUBMITTED ELECTRONICALLY VIA EFS-WEB The content of the electronically submitted sequence listing in ASCII text file (Name: 4140 0600003 Seqlisting ST25; Size: 1,978 bytes; and Date of Creation: Nov. 21, 2022) is herein incorporated by reference in its entirety. TECHNICAL FIELD The disclosure relates to methods for characterizing the VP1, VP2 and VP3 capsid proteins in an adeno-associated virus (AAV) particle and the purity of an AAV composition using liquid chromatography and mass spectrometry. BACKGROUND Adeno-Associated viruses (AAVs) are quickly becoming one of the most widely used vehicles for delivering gene therapy. The excellent safety profile along with the high efficiency of transduction of a broad range of target tissues has made AAV the most widely used platform for gene therapy. AAV is a small virus belonging to the Parvoviridae family. The virus is composed of a non-enveloped icosahedral capsid containing a linear single stranded DNA genome of about 4.7 kilobases. AAV is commonly expressed recombinantly in suitable host cells. However, recombinant AAV may be contaminated by proteins from the host cell lysate. The AAV capsid includes a mixture of VP1, VP2 and VP3 proteins, which are produced from a single viral Cap gene by alternative splicing and translation, and which self-assemble to form the capsid. AAV capsid proteins play a critical role in viral infectivity, tissue tropism, and potency, and the ability to fully characterize the mass and ratios of capsid proteins is becoming increasingly important for the commercial manufacturing of AAV for gene therapy. In particular, the stoichiometry of the VPs is crucial for infectivity of viral vectors. For example, high levels of VP3 capsid was negatively associated with poor transduction efficiency and reduced potency even when the VP1/VP2 ratio was not in balance. (Gene Therapy, volume 25, pages 415-424 (2018)). Since the ratio of structural proteins VP1, VP2, and VP3 from manufacture may fluctuate in a wide range, e.g., 1:1:5 to 1:1:20 (Biotechnol Adv., 26(1):73-88 (2008)), the accurate measurement of the ratio among the three capsid proteins is important in the AAV vector quality control. However, the current methods attempted to measure the masses of capsid proteins but failed to determine the stoichiometry of each of the VPs (WO 2018/035059). Thus, robust methods for a more accurate characterization of ratios and modifications of AAV capsid proteins and the purity of rAAV compositions is needed in the gene therapy industry. SUMMARY The disclosure provides methods to characterize the VP1, VP2 and VP3 capsid proteins in an adeno-associated virus (AAV) particle using liquid chromatography and mass spectrometry. The methods disclosed herein are used to determine the ratio of VP1, VP2 and VP3 capsid proteins in AAV particle, and/or the masses of one or more of the VP1, VP2 and VP3 capsid proteins. In some aspects, the present disclosure provides a method to determine the ratio of VP1, VP2 and VP3 capsid proteins in an adeno-associated virus (AAV) particle. The method includes the steps of subjecting the AAV particle to a liquid chromatography at about 70° C. to about 90° C., wherein the masses and ratio of VP1, VP2 and VP3 capsid proteins is determined by mass spectrometry and/or ultraviolet (UV)-visible spectroscopy. In some aspects, the individual masses of the capsid proteins are measured by mass spectrometry. In some aspects, the capsids on the AAV particle is denatured into the individual VP1, VP2 and VP3 proteins in the column of the liquid chromatography. In some aspects, the capsid proteins are separated by the liquid chromatography. In some aspects, the method further includes determining the masses of one or more of the VP1, VP2 and VP3 capsid proteins in the AAV particle using mass spectrometry. In some aspects, the relative amounts of the VP1, VP2 and VP3 capsid proteins are determined by analyzing the ultraviolet (UV) chromatogram of the VP1, VP2 and VP3 capsid proteins. In some aspects the liquid chromatography is reverse phase liquid chromatography. In some aspects, the AAV particle is AAVrh74. In some aspects, the chromatography uses a first mobile phase including trifluoroacetic acid in water. In some aspects, the chromatography uses a second mobile phase including trifluoroacetic acid in the mixture of acetonitrile and water. In some aspects, the percentage of the second mobile phase, in a combination of the first mobile phase and the second mobile phase, in the chromatography is increased over time. In some