Methods for analyzing AAV capsid proteins
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.
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 . The method of claim 1 , further comprising determining VP1 mass, VP2mass, and VP3 mass using a mass spectrometer.
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 . 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 . 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 . 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 . 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 . 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 . The method of claim 8 , wherein the second mobile phase comprises an acetonitrile volume percentage between about 80% and 95%.
10 . The method of claim 8 , wherein the contacting further comprises increasing the second flow rate relative to the first flow rate.
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 . 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 . The method of claim 11 , wherein the first mobile phase further comprises Tris-HC1.
14 . The method of claim 13 , wherein the first mobile phase further comprises acetonitrile.
15 . The method of claim 13 , wherein the first mobile phase further comprises methionine.
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 . 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.