Methods for analyzing AAV capsid proteins

Abstract

To provide methods for characterizing 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.SOLUTION: 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 the 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.SELECTED DRAWING: None

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority under 35 U.S.C. §119(e) to U.S. Provisional Patent Application No. 62 / 956,681, filed January 3, 2020, U.S. Provisional Patent Application No. 63 / 073,188, filed September 1, 2020, and U.S. Provisional Patent Application No. 63 / 119,909, filed December 1, 2020, the contents of each of which are incorporated by reference in their entirety herein.

[0002] The present disclosure relates to methods for characterizing VP1, VP2, and VP3 capsid proteins in adeno-associated virus (AAV) particles, as well as the purity of AAV compositions, using liquid chromatography and mass spectrometry. [Background technology]

[0003] Adeno-associated virus (AAV) is rapidly becoming one of the most widely used vehicles for gene therapy delivery. The excellent safety profile along with high efficiency of transduction of a wide range of target tissues has made AAV the most widely used platform for gene therapy. AAV is a small virus that belongs to the Parvoviridae family. The virus consists of a non-enveloped icosahedral capsid containing a linear, single-stranded DNA genome of approximately 4.7 kilobases. AAV is generally expressed recombinantly in suitable host cells. However, recombinant AAV can be contaminated by proteins from host cell lysates.

[0004] The AAV capsid contains a mixture of VP1, VP2, and VP3 proteins, which are produced from a single viral Cap gene by alternative splicing and translation and self-assemble to form the capsid. AAV capsid proteins play important roles in viral infectivity, tissue tropism, and potency, and the ability to fully characterize the masses and ratios of capsid proteins is becoming increasingly important for commercial production of AAV for gene therapy.

[0005] Specifically, VP stoichiometry is crucial for the infectivity of viral vectors. For example, high levels of VP3 capsids were negatively associated with poor transduction efficiency and reduced potency, even when the VP1 / VP2 ratio was unbalanced. (Gene Therapy, volume 25, pages 415-424(2018)). Because the ratio of structural proteins VP1, VP2, and VP3 obtained in production can vary over a wide range, e.g., from 1:1:5 to 1:1:20 (Biotechnol Adv.,26(1):73-88(2008)), accurate measurement of the ratio between the three capsid proteins is important in the quality control of AAV vectors. However, current methods have attempted to measure the mass of capsid proteins but have been unable to determine the stoichiometry of each VP (WO2018 / 035059). Therefore, there is a need in the gene therapy industry for robust methods to more accurately characterize the ratios and modifications of AAV capsid proteins and the purity of rAAV compositions. Summary of the Invention [Means for solving the problem]

[0006] The present disclosure provides a method for characterizing 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 an AAV particle and / or the mass of one or more of the VP1, VP2, and VP3 capsid proteins.

[0007] In some aspects, the present disclosure provides a method for determining the ratio of VP1, VP2, and VP3 capsid proteins in an adeno-associated virus (AAV) particle. The method includes subjecting the AAV particle to liquid chromatography at about 70°C to about 90°C, where the mass and ratio of VP1, VP2, and VP3 capsid proteins are 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 are denatured into each of the VP1, VP2, and VP3 proteins in a liquid chromatography column. In some aspects, the capsid proteins are separated by liquid chromatography.

[0008] In some embodiments, the method further includes determining the mass of one or more of the VP1, VP2, and VP3 capsid proteins in the AAV particle using mass spectrometry.

[0009] In some embodiments, the relative amounts of VP1, VP2, and VP3 capsid proteins are determined by analyzing ultraviolet (UV) chromatograms of VP1, VP2, and VP3 capsid proteins. In some embodiments, the liquid chromatography is reversed-phase liquid chromatography. In some embodiments, the AAV particles are AAVrh74.

[0010] In some embodiments, the chromatography uses a first mobile phase comprising trifluoroacetic acid in water. In some embodiments, the chromatography uses a second mobile phase comprising trifluoroacetic acid in a mixture of acetonitrile and water. In some embodiments, in the chromatography, the percentage of the second mobile phase in the combination of the first and second mobile phases increases over time.

[0011] In some embodiments, the mass spectrometry comprises a fragmentor voltage of about 125-350V.

[0012] Deamidation is one of the common post-translational modifications (PTMs) observed in proteins that is known to have a significant impact on protein activity and stability. Deamidation is usually caused by hydrolysis of the amide side chain of asparagine, forming a mixture of aspartic acid and isoaspartic acid. In some aspects, deamidation is a hydrolysis reaction of cytosine to uracil, releasing ammonia in the process. This can occur in vitro through the use of bisulfite, which deaminates cytosine but not 5-methylcytosine. In some aspects, deamination of 5-methylcytosine results in thymine and ammonia. In some aspects, glutamine residues also undergo deamidation to form a mixture of glutamic acid and isoglutamic acid, but glutamine residues are significantly less susceptible to deamidation compared to asparagine. In some aspects, deamination of guanine results in the formation of xanthine. In some aspects, deamidation of adenine results in the formation of hypoxanthine. Deamidation of capsid proteins can affect the stability and activity of AAV formulations.

[0013] In some embodiments of the present disclosure, mass spectrometry is used to study post-translational modifications such as deamidation. In some embodiments, proteins can be denatured using reagents such as guanidine and urea. The denatured proteins are reduced using 1,4-dithiothreitol (DTT) or Tris(2-carboxyethyl)phosphine (TECP) to break disulfide bonds. The reduced disulfide bonds are then alkylated using iodoacetamide. The denaturation and alkylation steps are performed to ensure that the protein is unfolded and therefore fully accessible to proteases. The denatured and reduced proteins are then digested using one of several proteases, such as trypsin. The digested peptides are separated on HPLC / UPLC using RP-HPLC. The separated peptides are then detected using their m / z ratio on a mass spectrometer, typically a Q-ToF or Orbitrap. The peptides are identified using appropriate software and databases. Deamidation is identified as an increase of approximately 1 Da compared to the theoretical value of the peptide.

[0014] In some embodiments, the method further comprises determining a post-translational modification of at least one of the VP1, VP2, and VP3 capsid proteins, hi some embodiments, the method further comprises determining a post-translational phosphorylation or acetylation of at least one of the VP1, VP2, and VP3 capsid proteins.

[0015] The disclosure also provides a method for characterizing host cell proteins in an AAV composition, the method comprising immunoprecipitating viral capsid proteins from the composition, digesting residual host cell proteins, and analyzing the digested proteins by liquid chromatography quadrupole time-of-flight mass spectrometry (LC-QTOF-MS) to identify the host cell proteins.

[0016] In some embodiments, the immunoprecipitation comprises incubating the AAV composition with an anti-AAV VP1 antibody, an anti-AAV VP2 antibody, an anti-AAV VP3 antibody, or a combination thereof.

[0017] In some embodiments, the method further comprises analyzing the digested host cell proteins by iterative MS / MS.

[0018] In some embodiments, the digestion is carried out in solution. In some embodiments, the digestion is carried out at a temperature of about 60° C. to about 80° C. In some embodiments, the digestion is carried out at about 70° C.

[0019] In some embodiments, the method further comprises spiking the AAV composition with a known amount of at least one known protein preparation. In some embodiments, the at least one known protein preparation is a human or bovine protein preparation. In some embodiments, the method further comprises quantifying the amount of digested host cell protein compared to the at least one protein preparation.

[0020] In some embodiments, the liquid chromatography is reversed-phase liquid chromatography. In some embodiments, the reversed-phase liquid chromatography is performed using a C18 column, a C8 column, or a C4 column. In some embodiments, the liquid chromatography is performed using a C8 column. In some embodiments, the column comprises particles of about 1.2 to 3.5 μm. In some embodiments, the column comprises particles of about 1.7 μm or about 1.8 μm. In some embodiments, the column is about 50 mm to about 300 mm long and has an inner diameter of about 1 mm to about 4.6 mm. In some embodiments, the column is about 150 mm long and has an inner diameter of about 2.1 mm.

[0021] In some embodiments, the liquid chromatography is carried out at about 40° C. to about 50° C. In some embodiments, the liquid chromatography is carried out at about 45° C.

[0022] In some embodiments, the liquid chromatography comprises a first mobile phase comprising formic acid. In some embodiments, the first mobile phase comprises about 0.05% to about 0.15% formic acid by volume. In some embodiments, the first mobile phase comprises about 0.1% formic acid by volume.

[0023] In some embodiments, the liquid chromatography includes a second mobile phase comprising formic acid in a mixture of acetonitrile and water. In some embodiments, the second mobile phase comprises about 0.05% to about 0.15% formic acid by volume. In some embodiments, the second mobile phase comprises about 0.1% formic acid by volume. In some embodiments, the second mobile phase comprises about 80-95% acetonitrile by volume. In some embodiments, the second mobile phase comprises about 90% acetonitrile by volume and about 10% water by volume.

[0024] In some embodiments, in liquid chromatography, the percentage of the second mobile phase is increased over time, compared to the combination of the first mobile phase and the second mobile phase. In some embodiments, the percentage of the second mobile phase is increased from about 2% to about 50%. In some embodiments, the percentage of the second mobile phase is increased from about 2% to about 50% by volume over about 120 minutes. In some embodiments, the percentage of the second mobile phase is then increased to 100% by volume over about 25 minutes. In some embodiments, the percentage of the second mobile phase is then maintained at 100% by volume for about 1 minute. In some embodiments, the second mobile phase is then reduced to about 2% by volume over about 4 minutes. In some embodiments, the percentage of the second mobile phase is then increased to 100% by volume over about 5 minutes. In some embodiments, the percentage of the second mobile phase is then maintained at 100% by volume for about 3 minutes. In some embodiments, the second mobile phase is then reduced to about 2% by volume over about 2 minutes.

[0025] In some embodiments, mass analysis is performed using a fragmentor voltage of about 125-350 V. In some embodiments, mass analysis is performed using a fragmentor voltage of about 135 V. In some embodiments, mass analysis is performed using a capillary voltage of about 3-6 kV. In some embodiments, mass analysis is performed using a capillary voltage of about 4 kV.

[0026] Some aspects of the present disclosure are directed to recombinant AAV (rAAV) that comprise a heterogeneous population of capsid proteins that contain subpopulations with amino acid modifications. In some aspects, the modifications are deamidation or oxidation.

[0027] In some aspects, the heterogeneous population comprises a deamidated asparagine (N) at one or more of N57, N255, N256, and N263 of AAV.rh74, or the equivalent residues of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, or AAVrhlO, as determined by mass spectrometry and / or ultraviolet (UV) visible spectroscopy. In another embodiment, the heterogeneous population comprises a deamidated asparagine (N) within the peptide sequence of any one of SEQ ID NOs: 1-5, or the equivalent peptide sequence of another AAV serotype.

[0028] In some embodiments, the heterogeneous population comprises less than 70%, 60%, 50%, 40%, 30%, 20%, 15%, 10%, or 5% of capsid proteins are deamidated at N57 of the AAV.rh74 capsid. In some embodiments, the heterogeneous population comprises less than 15% of capsid proteins are deamidated at N57 of the AAV.rh74 capsid. In some embodiments, the heterogeneous population comprises less than 70%, 60%, 50%, 40%, 30%, 20%, 15%, 10%, or 5% of capsid proteins are deamidated at N254 and / or N255 of the AAV.rh74 capsid. In some embodiments, the heterogeneous population contains less than 70%, 60%, 50%, 40%, 30%, 20%, 10%, or 5% capsid protein deamidated at N263.

[0029] In some embodiments, the heterogeneous population comprises oxidized methionines at one or more of M437, M473, M526, M544, M560, and M637 in AAV.Rh74, or the equivalent residues in AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, CAAV11, AAV12, AAV13, or AAVrhlO, as determined by mass spectrometry and / or ultraviolet (UV) visible spectroscopy. In some embodiments, the heterogeneous population comprises less than 30%, 20%, 10%, 5%, or 1% of capsid proteins oxidized at M437. In some embodiments, the heterogeneous population comprises less than 30%, 20%, 10%, 5%, or 1% of capsid proteins oxidized at M473. In some embodiments, the heterogeneous population comprises less than 30%, 20%, 10%, 5%, or 3% of capsid proteins oxidized with M526. In some embodiments, the heterogeneous population comprises less than 30%, 20%, 10%, 5%, or 2% of capsid proteins oxidized with M544. In some embodiments, the heterogeneous population comprises less than 30%, 20%, 10%, 5%, or 2% of capsid proteins oxidized with M560. In some embodiments, the heterogeneous population comprises less than 30%, 20%, 10%, 5%, or 1% of capsid proteins oxidized with M637.

[0030] Those skilled in the art will recognize that the invention described herein is subject to variations and modifications other than those specifically described. It will be understood that the invention described herein includes all such variations and modifications. The invention also includes all such steps, features, compositions, and compounds referred to or labeled herein, individually or collectively, and any and all combinations of any two or more of these steps or features. (Item 1) 1. A method for characterizing VP1, VP2, and VP3 capsid proteins in an adeno-associated virus (AAV) particle, comprising: subjecting the AAV particles to liquid chromatography at about 70° C. to about 90° C.; The ratio of VP1, VP2, and VP3 capsid proteins is determined by mass spectrometry and / or ultraviolet (UV) visible spectroscopy. (Item 2) 2. The method of claim 1, wherein the individual masses of VP1, VP2, and VP3 capsid proteins are determined by mass spectrometry. (Item 3) 3. The method of claim 1 or 2, wherein the ratio of VP1, VP2, and VP3 capsid proteins is determined by comparing ultraviolet chromatograms (UV) of the VP1, VP1, VP2, and VP3 capsid proteins. (Item 4) The method according to any one of items 1 to 7, wherein the liquid chromatography is reverse phase liquid chromatography. (Item 5) 5. The method according to claim 4, wherein the reverse phase liquid chromatography is carried out using a C18 column, a C8 column, or a C4 column. (Item 6) 6. The method of claim 5, wherein the liquid chromatography is carried out using a C8 column. (Item 7) 7. The method according to item 5 or 6, wherein the column contains particles of about 1.2 to 3.5 μm. (Item 8) 8. The method according to any one of items 5 to 7, wherein the column contains particles of about 1.7 μm or about 1.8 μm. (Item 9) 9. The method according to any one of items 5 to 8, wherein the column is about 50 mm to about 300 mm in length and has an inner diameter of about 1 mm to about 4.6 mm. (Item 10) 10. The method of claim 9, wherein the chromatography column is about 100 mm in length and has an inner diameter of about 2.1 mm. (Item 11) 11. The method of any one of items 1 to 10, wherein the AAV particles are of the serotype AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAVrhlO, AAVrh74, or any natural, recombinant, or synthetic AAV particle. (Item 12) 12. The method according to any one of items 1 to 11, wherein the AAV is of the serotype AAVrh74. (Item 13) 13. The method according to any one of items 1 to 12, wherein the liquid chromatography is carried out at about 75°C to about 85°C. (Item 14) 14. The method according to any one of items 1 to 13, wherein the liquid chromatography is carried out at about 80°C. (Item 15) 15. The method according to any one of items 1 to 14, wherein the liquid chromatography comprises a first mobile phase comprising trifluoroacetic acid. (Item 16) 16. The method of claim 15, wherein the first mobile phase comprises about 0.05 to about 0.15% trifluoroacetic acid by volume. (Item 17) 17. The method of claim 15 or 16, wherein the first mobile phase comprises about 0.1% trifluoroacetic acid by volume. (Item 18) 18. The method according to any one of items 1 to 17, wherein the liquid chromatography comprises a second mobile phase comprising trifluoroacetic acid in the mixture of acetonitrile and water. (Item 19) 20. The method of claim 18, wherein the second mobile phase comprises about 0.05 to about 0.15% trifluoroacetic acid by volume. (Item 20) 20. The method of claim 18 or 19, wherein the second mobile phase comprises about 0.1% trifluoroacetic acid by volume. (Item 21) 21. The method according to any one of items 18 to 20, wherein the second mobile phase comprises about 80 to 95% acetonitrile by volume. (Item 22) 22. The method according to any one of items 18 to 21, wherein the second mobile phase comprises about 90% acetonitrile and 10% water by volume. (Item 23) 23. The method according to any one of Items 18 to 22, wherein in the liquid chromatography, a percentage of the second mobile phase is increased over time as compared to a combination of the first mobile phase and the second mobile phase. (Item 24) 24. The method of claim 23, wherein the percentage of the second mobile phase is increased by volume from about 10% to about 40%, and then from about 40% to about 45%. (Item 25) 25. The method of claim 24, wherein the percentage of the second mobile phase is increased from about 10% to about 40% by volume over about 3 minutes, and then from about 40% to about 45% over about 30 minutes. (Item 26) 26. The method of claim 25, wherein the percentage of the second mobile phase in the combination of the first and second mobile phases is then increased to 100% by volume over a period of about 1 minute. (Item 27) 27. The method of claim 26, wherein the percentage of the second mobile phase in the combination of the first and second mobile phases is then reduced to 10% by volume over about 1 minute. (Item 28) 28. The method according to any one of items 1 to 27, wherein the mass spectrometry is carried out using a fragmentor voltage of about 125 to 350 V. (Item 29) 29. The method of any one of items 1 to 28, wherein the mass spectrometry is carried out using a fragmentor voltage of about 175V. (Item 30) Items 1 to 2, wherein the mass spectrometry is carried out using a capillary voltage of about 3 to 6 kV. 10. The method of any one of claims 9 to 9. (Item 31) 31. The method according to any one of items 1 to 30, wherein the mass spectrometry is carried out using a capillary voltage of about 5 kV. (Item 32) 2. The method of claim 1, wherein the characterization further comprises determining a post-translational modification of at least one of VP1, VP2, and VP3 capsid proteins. (Item 33) 33. The method of claim 32, wherein the post-translational modification comprises one or more of amino acid deletion, glycosylation, sialylation, acetylation, phosphorylation, deamidation, oxidation, formylation, hydroxylation, methylation, and sulfation. (Item 34) 33. The method of claim 32, wherein the post-translational modification comprises one or more of N-terminal methionine deletion, deamidation, threonine deletion, phosphorylation, and acetylation. (Item 35) The method according to any one of items 1 to 33, which is carried out using the gradient program listed in Table 2. (Item 36) 33. The method according to claim 32, wherein the characterization uses a buffer containing Tris-HCl. (Item 37) 37. The method of claim 36, wherein the buffer comprises acetonitrile. (Item 38) 37. The method of claim 36, wherein the buffer contains methionine. (Item 39) 39. The method according to any one of items 36 to 38, wherein the buffer comprises 5 mM to 50 mM Tris-HCl, 5% to 20% acetonitrile, and 1 mM to 50 mM methionine. (Item 40) Item 39. The method of item 39, wherein the buffer comprises 20 mM Tris-HCl, 5% to 10% acetonitrile, and 10 mM methionine. (Item 41) 37. The method of claim 36, wherein the Tris-HCl is at pH 7.5. (Item 42) 33. The method of claim 32, wherein the post-translational modification comprises deamidation at one or more of N263, N514, N57, N502, N254, and N94 of AAV8, or the equivalent residues in AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV9, AAV10, AAV11, AAV12, AAV13, AAVr10, or AAVr14. (Item 43) 33. The method of claim 32, wherein the post-translational modification comprises deamidation at one or more of N57, N255, N256, and N263 of AAV.Rh74. (Item 44) 33. The method of claim 32, wherein the post-translational modification comprises oxidation at one or more of M437, M473, M526, M544, M560, and M637 of AAV.Rh74. (Item 45) An AAV composition comprising an AAV capsid comprising deamidation at one or more of N57, N255, N256, and N263 of AAV.Rh74, or the equivalent residues of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, or AAVrhlO. (Item 46) An AAV composition comprising an AAV capsid comprising oxidation at one or more of M437, M473, M526, M544, M560, and M637 of AAV.Rh74, or the equivalent residues in AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, CAAV11, AAV12, AAV13, or AAVrhlO. (Item 47) 1. A method for characterizing a host cell protein in an AAV composition, comprising: immunoprecipitating viral capsid proteins from said composition; digesting residual host cell proteins; and analyzing the digested proteins using liquid chromatography quadrupole time-of-flight mass spectrometry (LC-QTOF-MS) to identify host cell proteins. (Item 48) 48. The method of claim 47, wherein the immunoprecipitation comprises incubating the AAV composition with an anti-AAV VP1 antibody, an anti-AAV VP2 antibody, an anti-AAV VP3 antibody, or a combination thereof. (Item 49) 49. The method of claim 47 or 48, further comprising analyzing the digested host cell proteins by iterative MS / MS. (Item 50) 50. The method according to any one of items 47 to 49, wherein the digestion is carried out in solution. (Item 51) 51. The method of claim 50, wherein the digestion comprises rapid digestion carried out at a temperature of about 60° C. to about 80° C. (Item 52) 52. The method of claim 51, wherein the temperature is about 70°C. (Item 53) 53. The method of any one of items 47 to 52, further comprising spiking the AAV composition with a known amount of at least one known protein standard. (Item 54) 54. The method of claim 53, wherein the at least one known protein preparation is a human or bovine protein preparation. (Item 55) 55. The method of claim 53 or 54, further comprising quantifying the amount of digested host cell protein by comparison to the at least one protein standard. (Item 56) 56. The method according to any one of items 47 to 55, wherein the liquid chromatography is reverse phase liquid chromatography. (Item 57) 57. The method of claim 56, wherein the reverse phase liquid chromatography is carried out using a C18 column, a C8 column, or a C4 column. (Item 58) 58. The method of claim 57, wherein the liquid chromatography is carried out using a C8 column. (Item 59) 59. The method of claim 57 or 58, wherein the column contains particles of about 1.2 to 3.5 μm. (Item 60) 59. Any of items 57 to 59, wherein the column contains particles of about 1.7 μm or about 1.8 μm. The method according to any one of claims 1 to 5. (Item 61) 61. The method according to any one of items 57 to 60, wherein the column is about 50 mm to about 300 mm in length and has an inner diameter of about 1 mm to about 4.6 mm. (Item 62) Item 62. The method of item 61, wherein the column is about 150 mm in length and has an inner diameter of about 2.1 mm. (Item 63) 63. The method according to any one of items 47 to 62, wherein the liquid chromatography is carried out at about 40°C to about 50°C. (Item 64) 64. The method according to any one of items 47 to 63, wherein the liquid chromatography is carried out at about 45°C. (Item 65) 65. The method according to any one of items 47 to 64, wherein the liquid chromatography comprises a first mobile phase comprising formic acid. (Item 66) Item 66. The method of item 65, wherein the first mobile phase comprises about 0.05% to about 0.15% formic acid by volume. (Item 67) 67. The method of claim 65 or 66, wherein the first mobile phase comprises about 0.1% formic acid by volume. (Item 68) 68. The method according to any one of items 47 to 67, wherein the liquid chromatography comprises a second mobile phase comprising formic acid in a mixture of acetonitrile and water. (Item 69) 69. The method of claim 68, wherein the second mobile phase comprises about 0.05% to about 0.15% formic acid by volume. (Item 70) 70. The method of claim 68 or 69, wherein the second mobile phase comprises about 0.1% formic acid by volume. (Item 71) 71. The method according to any one of items 68 to 70, wherein the second mobile phase comprises about 80 to 95% acetonitrile by volume. (Item 72) 72. The method of claim 71, wherein the second mobile phase comprises about 90% acetonitrile by volume and 10% water by volume. (Item 73) 73. The method according to any one of Items 68 to 72, wherein in the liquid chromatography, a percentage of the second mobile phase is increased over time as compared to a combination of the first mobile phase and the second mobile phase. (Item 74) 74. The method of claim 73, wherein the percentage of the second mobile phase is increased from about 2% to about 50%. (Item 75) 75. The method of claim 74, wherein the percentage of the second mobile phase is increased from about 2% to about 50% by volume over a period of about 120 minutes. (Item 76) 76. The method of claim 75, wherein the percentage of the second mobile phase is then increased to 100% by volume for about 5 minutes. (Item 77) 77. The method of claim 76, wherein the percentage of the second mobile phase is then maintained at 100% by volume for about 3 minutes. (Item 78) 78. The method of claim 77, wherein the percentage of the second mobile phase is then reduced to about 2% by volume for about 2 minutes. (Item 79) 79. The method according to any one of items 47 to 78, wherein the mass spectrometry is carried out using a fragmentor voltage of about 125 to 350 V. (Item 80) 80. The method of any one of items 47 to 79, wherein the mass spectrometry is carried out using a fragmentor voltage of about 135V. (Item 81) 81. The method according to any one of items 47 to 80, wherein the mass spectrometry is carried out using a capillary voltage of about 3 to 6 kV. (Item 82) 82. The method according to any one of items 47 to 81, wherein the mass spectrometry is carried out using a capillary voltage of about 4 kV. (Item 83) A recombinant AAV (rAAV) comprising a heterogeneous population of capsid proteins containing subpopulations having amino acid modifications, wherein the amino acid modifications are post-translational modifications. (Item 84) 84. The rAAV of item 83, wherein the modification is deamidation or oxidation. (Item 85) 85. The rAAV of paragraph 83 or 84, wherein the heterogeneous population comprises a deamidated asparagine (N) at one or more of N57, N255, N256, and N263 of AAV.rh74, or the equivalent residues of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, or AAVrhlO, as measured by mass spectrometry and / or ultraviolet (UV) visible spectroscopy. (Item 86) 86. The rAAV of item 85, wherein the heterogeneous population comprises less than 70%, 60%, 50%, 40%, 30%, 20%, or 15% of capsid protein deamidated at N57 of the AAV.rh74 capsid. (Item 87) 86. The rAAV of item 85, wherein the heterogeneous population contains less than 15% capsid protein deamidated at N57 of the AAV.rh74 capsid. (Item 88) 86. The rAAV of item 85, wherein the heterogeneous group contains less than 70%, 60%, 50%, 40%, 30%, 20%, 15%, 10%, or 5% of capsid protein deamidated at N254 and / or N255 of AAV.rh74. (Item 89) 86. The rAAV of item 85, wherein the heterogeneous population comprises less than 70%, 60%, 50%, 40%, 30%, or 20% of capsid protein deamidated at N263. (Item 90) the heterogeneous population comprises an oxidized methionine at one or more of M437, M473, M526, M544, M560, and M637 of AAV.Rh74, or the equivalent residues of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, CAAV11, AAV12, AAV13, or AAVrhlO, as determined by mass spectrometry and / or ultraviolet (UV) visible spectroscopy. 85. The rAAV according to item 83 or 84. (Item 91) 91. The rAAV of item 90, wherein the heterogeneous population contains less than 30%, 20%, 10%, 5%, or 1% M437-oxidized capsid protein. (Item 92) 91. The rAAV of item 90, wherein the heterogeneous population contains less than 30%, 20%, 10%, 5%, or 1% M473 oxidized capsid protein. (Item 93) 91. The rAAV of item 90, wherein the heterogeneous population contains less than 30%, 20%, 10%, 5%, or 3% M526 oxidized capsid protein. (Item 94) 91. The rAAV of item 90, wherein the heterogeneous population contains less than 30%, 20%, 10%, 5%, or 2% of capsid protein that is M544 oxidized. (Item 95) 91. The rAAV of item 90, wherein the heterogeneous population contains less than 30%, 20%, 10%, 5%, or 2% of capsid protein that is M560 oxidized. (Item 96) 91. The rAAV of item 90, wherein the heterogeneous population contains less than 30%, 20%, 10%, 5%, or 1% of capsid protein that is M637 oxidized. (Item 97) 86. The rAAV of any one of paragraphs 83 to 85, wherein the heterogeneous population comprises a deamidated asparagine (N) within the peptide sequence of any one of SEQ ID NOs: 1 to 5, or the equivalent peptide sequence of another AAV serotype. [Brief description of the drawings]

[0031] The following figures form part of the present specification and are included to further demonstrate aspects of the present invention.

[0032] [Figure 1] FIG. 1 illustrates a UV chromatogram of AAVrh74 capsid protein, including integration to determine capsid protein ratios.

[0033] [Diagram 2] FIG. 2 depicts the total ion chromatogram of the AAVrh74 capsid protein.

[0034] [Figure 3-1] FIG. 3 shows the deconvoluted MS spectra of VP1 (FIG. 3A), VP2 (FIG. 3B), and VP3 (FIG. 3C) capsid proteins, respectively, confirming the intact masses of all three capsid proteins and the detection of post-translational modifications of the capsid proteins. [Figure 3-2] Same as above. [Figure 3-3] Ibid. Figure 3D shows the deconvoluted MS spectra of VP1 in multiple samples. [Diagram 3-4] Figure 3E shows the deconvoluted MS spectra of VP2 in multiple samples, and Figure 3F shows the deconvoluted MS spectra of VP3 in multiple samples.

[0035] [Figure 4] FIG. 4 shows the process of deamidation analysis using Tris-HCl as a buffer.

[0036] [Diagram 5]FIG. 5 shows the results of deamidation of AAV.rh74 using Tris-HCl buffer.

[0037] [Figure 6] FIG. 6 shows the results of oxidation of AAV.rh74 with Tris-HCl buffer. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0038] Provided is a method for characterizing VP1, VP2, and VP3 capsid proteins in adeno-associated virus (AAV) particles using liquid chromatography, mass spectrometry, or ultraviolet (UV) visible spectroscopy. In some embodiments, provided is a method for determining the ratio of VP1, VP2, and VP3 capsid proteins in AAV particles, and / or the mass of one or more of VP1, VP2, and VP3 capsid proteins. The present disclosure also provides a method for characterizing the purity of rAAV compositions using liquid chromatography and mass spectrometry. definition

[0039] For convenience, before further describing the present invention, certain terms used in the specification, examples, and appended claims are collected here. These definitions should be read and understood in light of the remainder of the disclosure by one of ordinary skill in the art. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. Terms used throughout this specification are defined as follows, unless otherwise limited in specific instances.

[0040] The articles "a", "an" and "the" are used to refer to one or to more than one (i.e. to at least one) of the grammatical object of the article.

[0041] As used herein, "about" is used to indicate that a value includes the error variation inherent in the device, the method employed to determine the value, or the variation that exists between test subjects. In some embodiments, "about" indicates that a deviation of more than 5% to 10% (e.g., more than a maximum of 5% to 10%) and less than 5% to 10% (e.g., less than a minimum of 5% to 10%) of a given value or range remains within the intended meaning of the recited value or range.

[0042] The term "AAV" or "adeno-associated virus" refers to a dependoparvovirus within the Parvovirus genus of viruses. As used herein, AAV can refer to wild-type virus or AAV derived from a naturally occurring wild-type virus, such as AAV derived from a rAAV genome packaged in a capsid derived from a capsid protein encoded by a natural cap gene, and / or AAV derived from a rAAV genome packaged in a capsid derived from a capsid protein encoded by a non-natural capsid cap gene, such as AAVrh.74.

[0043] The AAV can be any serotype, such as AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV-10, AAV-11, AAV-12, AAV-13, AAV rh.10, AAV rh.74, or variants and derivatives thereof. In some embodiments, the rAAV is of serotype AAVrh.74. The production of pseudotyped rAAV is disclosed, for example, in WO01 / 83692, which is incorporated by reference in its entirety. Other types of rAAV variants are also contemplated, such as rAAV with capsid mutations. See, for example, Marsic et al., Molecular Please refer to Therapy, 22(11):1900-1909(2014).

[0044] As used herein, the term "AAV particle", "AAV vector", "AAV virion", "AAV virus particle" or "AAV vector particle" is used to refer to a viral particle that is composed of an AAV capsid and an encapsidated AAV genome. In some embodiments, an AAV particle contains a heterologous polynucleotide (i.e., a polynucleotide other than a wild-type AAV genome, such as a transgene delivered to a mammalian cell). In some embodiments, the production of an AAV vector particle includes the production of an AAV vector, when such a vector is contained within the AAV vector particle.

[0045] For example, wild-type (wt) AAV virus particles that contain a linear single-stranded AAV nucleic acid genome associated with an AAV capsid protein coat. AAV virions can be either single-stranded (ss) AAV or self-complementary (SC) AAV. In some embodiments, single-stranded AAV nucleic acid molecules of either complementary sense strand, e.g., "sense" strand or "antisense" strand, can be packaged within the AAV virion, and both strands are equally infectious.

[0046] The term "recombinant AAV" or "rAAV" is defined herein as an infectious, replication-defective virus that is composed of an AAV protein shell that encapsulates a heterologous nucleotide sequence of interest flanked by AAV ITRs. In some embodiments, rAAV is produced in a suitable host cell into which an AAV vector, AAV helper functions, and accessory functions have been introduced. In this way, the host cell can encode the AAV polypeptides necessary to package the AAV vector (containing the recombinant nucleotide sequence of interest) into an infectious recombinant virion particle for subsequent gene delivery.

[0047] As used herein, the term "capsid protein" refers to a protein that forms the coat or shell of a virus. The term "AAV capsid protein" refers to a protein that forms the coat of an adeno-associated virus (AAV), which is composed of a total of 60 subunits, each subunit being an amino acid sequence, for example, viral protein 1 (VP1), VP2, or VP3.

[0048] As used herein, the term "liquid chromatography (LC)" refers to a technique used to separate, identify, and quantify components in a mixture. In column liquid chromatography, a liquid mobile phase is passed through a column, and the components of the mobile phase interact with a solid stationary phase. The composition of the mobile phase can be altered during a separation run to change the strength of interaction of the compounds of interest. As the mobile phase flows through the column, the eluent is typically collected in fractions while the concentration of compounds eluted from the column over time is monitored to generate an elution curve or chromatogram.

[0049] As used herein, the term "stationary phase" refers to the material that remains fixed in the column. The most commonly used stationary phase columns are carbon-bonded silica, phenyl-bonded silica, and cyano-bonded silica. In some embodiments, the stationary phase may include a hydrophobic alkyl chain of a specific length, such as C4, C8, or C18. In some embodiments, the reversed phase chromatography is C8 reversed phase chromatography (e.g., reversed phase chromatography utilizing a C8 stationary phase).

[0050] As used herein, the term "mobile phase" refers to water, a solvent, or a mixture of water and a solvent used to elute a compound from a column. The most common mobile phase solvents include, but are not limited to, acetonitrile, methanol, tetrahydrofuran, ethanol, or isopropyl alcohol. In some embodiments, two mobile phases are used. For example, a first mobile phase and a second mobile phase can be mixed in situ to obtain the solvent used to elute the substance from the column. In some embodiments, the volume ratio of the second mobile phase to the first mobile phase is in an increasing gradient during the elution step.

[0051] As used herein, the term "mass spectrometry" or "MS" refers to an analytical technique that measures the mass-to-charge (m / z) ratio of ions to identify and quantify molecules in simple and complex mixtures. MS techniques generally include (1) ionizing compounds to form charged compounds, and (2) detecting the mass-to-charge ratio of the charged compounds to calculate molecular weight. Compounds may be ionized and detected by any suitable means. A "mass spectrometer" generally includes an ionizer, a mass analyzer, and an ion detector. Generally, one or more molecules of interest are ionized, and then the ions are introduced into a mass analyzer, where a combination of magnetic and electric fields causes the ions to follow a path in space that depends on their mass ("m") and charge ("z"). In some methods of mass spectrometry, ions may be separated from one another using, for example, a time-of-flight (TOF), orbitrap, Fourier transform ion cyclotron resonance spectrometer, quadrupole, or ion trap, and then detected using an ion detector.

[0052] As used herein, the terms "ultraviolet-visible spectroscopy," "ultraviolet-visible spectrophotometry," "UV-Vis," or "UV / Vis" refer to absorption or reflectance spectroscopy used to determine the optical properties (transmittance, reflectance, and absorbance) of liquids and solids. In some embodiments, ultraviolet-visible spectroscopy is used to characterize the capsid protein of AAV particles.

[0053] As used herein, the term "total ion chromatogram (TIC)" refers to a type of chromatogram generated by summing the intensities of all mass spectral peaks belonging to the same scan.

[0054] As used herein, the term "AAVrh74" refers to an AAV particle having AAVrh74 VP1, VP2, and VP3 capsid proteins or variants thereof. An exemplary AAVrh74 VP1 capsid protein sequence is set forth in SEQ ID NO: 4 of U.S. Patent No. 9,909,142, the entirety of which is incorporated herein by reference. Examples of variants of AAVrh74 VP1 capsid protein are also set forth in U.S. Patent No. 9,909,142.

[0055] As used herein, the term "subpopulation" of VP proteins refers to a group of VP proteins that have at least one defined characteristic in common and include at least one group member to less than all members of a reference group, unless otherwise specified. For example, a "subpopulation" of VP1 proteins can include at least one VP1 protein to less than all VP1 proteins in an assembled AAV capsid, unless otherwise specified. A "subpopulation" of VP3 proteins can include one VP3 protein to less than all VP3 proteins in an assembled AAV capsid, unless otherwise specified. For example, VP1 proteins can be a subpopulation of VP proteins in an assembled AAV capsid, VP2 proteins can be another subpopulation of VP proteins, and VP3 is yet another subpopulation of VP proteins. In another example, VP1, VP2, and VP3 proteins may contain subpopulations with different modifications, e.g., at least one, two, three, or four highly deamidated asparagines, e.g., at asparagine-glycine pairs. Characterization of AAV VP1, VP2, and VP3 capsid proteins

[0056] In some aspects, the present disclosure provides a method for characterizing VP1, VP2, and VP3 capsid proteins in an adeno-associated virus (AAV) particle, the method comprising subjecting the AAV particle to liquid chromatography at about 70°C to about 90°C, where the mass and ratio of VP1, VP2, and VP3 capsid proteins are determined by mass spectrometry and / or ultraviolet (UV) visible spectroscopy. In some aspects, the mass and ratio of VP1, VP2, and VP3 capsid proteins are determined by mass spectrometry and ultraviolet (UV) visible spectroscopy. In some aspects, the capsid on the AAV particle is denatured into each of VP1, VP2, and VP3 proteins in a liquid chromatography column. In some aspects, the capsid proteins are separated by liquid chromatography. In some embodiments, the method includes (a) subjecting the AAV particles to liquid chromatography to separate VP1, VP2, and VP3 capsid proteins, and (b) subjecting the separated VP1, VP2, and VP3 capsid proteins resulting from step (a) to mass spectrometry and / or UV-visible spectroscopy to determine the relative amounts of VP1, VP2, and VP3 capsid proteins, thereby determining the ratio of VP1, VP2, and VP3 capsid proteins in the AAV particles. In some embodiments, the liquid chromatography is performed at about 70° C. to about 90° C. In some embodiments, the liquid chromatography is performed at about 70° C., 74° C., 76° C., 78° C., 80° C., 82° C., 84° C., 86° C., 88° C., or 90° C. In some embodiments, the liquid chromatography is performed at about 80° C.

[0057] In some embodiments, the method further includes determining the mass of one or more of the VP1, VP2, and VP3 capsid proteins of the AAV particle.

[0058] In some embodiments, the relative amounts of VP1, VP2, and VP3 capsid proteins are determined by comparing the total ion chromatograms (TIC) of VP1, VP2, and VP3 capsid proteins.

[0059] In some aspects, the liquid chromatography is reversed-phase liquid chromatography, size-exclusion chromatography, hydrophilic interaction liquid chromatography, or cation exchange chromatography. In some aspects, the liquid chromatography is reversed-phase liquid chromatography.

[0060] In some embodiments, the reverse phase chromatography is performed using a C18, C8, or C4 column. In some embodiments, the liquid chromatography is performed using a C8 column.

[0061] In some embodiments, the stationary phase of the reversed phase liquid chromatography is contained within a chromatography column having a length of about 50-300 mm and an internal diameter of about 1-4.6 mm. In some embodiments, the column is a BEH column. In some embodiments, the column has an internal diameter of 1 mm, 2.1 mm, 3 mm, or 4.6 mm. In some embodiments, the column has a length of 50 mm, 75 mm, 100 mm, 150 mm, or 300 mm. In some embodiments, the column size is 1 mm x 50 mm, 2.1 mm x 50 mm, 3 mm x 50 mm, 4.6 mm x 50 mm, 1 mm x 75 mm, 2.1 mm x 75 mm, 3 mm x 75 mm, 4.6 mm x 75 mm, 1 mm x 100 mm, 2.1 mm x 100 mm, 3 mm x 100 mm, 4.6 mm x 100 mm, 1 mm x 150 mm, 2.1 mm x 150 mm, 3 mm x 150 mm, 4.6 mm x 150 mm, 1 mm x 300 mm, 2.1 mm x 300 mm, 3 mm x 300 mm, or 4.6 mm x 300 mm. In some embodiments, the column sizes are 1.6×50 mm, 1.6×60 mm, 1.6×70 mm, 1.6×80 mm, 1.6×90 mm, 1.6×100 mm, 1.6×110 mm, 1.6×120 mm, 1.6×130 mm, 1.6×140 mm, 1.6×150 mm, 1.7×50 mm, 1.7×60, 1.7×70 mm, 1.7×80mm, 1.7×90mm, 1.7×100mm, 1.7×110mm, 1.7×120mm, 1.7×130mm, 1.7×140mm, 1 .7×150mm, 1.8×50mm, 1.8×60, 1.8×70mm, 1.8×80mm, 1.8×90mm, 1.8×100mm, 1.8×110mm, 1.8×120mm, 1.8×130mm, 1.8×140mm, 1.8×150mm, 1.9×50mm, 1.9×60mm, 1.9×70mm, 1.9×80mm, 1.9×90mm, 1.9×100mm, 1.9×110mm, 1.9×120mm, 1.9×130mm, 1.9×140mm, 1.9×150mm , 2.0×50mm, 2.0×60mm, 2.0×70mm, 2.0×80mm, 2.0×90mm, 2.0×100mm, 2.0×110mm, 2.0×120mm, 2.0×130mm, 2.0×140mm, 2.0×150mm, 2.1×50mm, 2.1×60mm, 2.1×70mm, 2.1×80mm, 2.1x90mm, 2.1x100mm, 2.1x110mm, 2.1x120mm, 2.1x130mm, 2.1x140mm, 2.1x150mm, 2.2x50mm, 2.2x60mm, 2.2x70mm, 2.2x80mm, 2.2x90mm, 2.2x100mm, 2.2x110mm, 2.2x120mm, 2.2x130 mm, 2.2×140mm, 2.2×150mm, 2.3×50mm, 2.3×60mm, 2.3×70mm, 2.3×80mm, 2.3×90mm, 2.3×10 0mm, 2.3×110mm, 2.3×120mm, 2.3×130mm, 2.3×140mm, 2.3×150mm, 2.4×50mm, 2.4×60mm, 2.4 x70mm, 2.4x80mm, 2.4x90mm, 2.4x100mm, 2.4x110mm, 2.4x120mm, 2.4x130mm, 2.4x140mm, 2.4x150mm, 2.5x50mm, 2.5x60, 2.5x70mm, 2.5x80mm, 2.5x90mm, 2.5x100mm, 2.5x110mm, 2. 5×120 mm, 2.5×130 mm, 2.5×140 mm, 2.5×150 mm, 2.6×50 mm, 2.6×60 mm, 2.6×70 mm, 2.6×80 mm, 2.6×90 mm, 2.6×100 mm, 2.6×110 mm, 2.6×120 mm, 2.6×130 mm, 2.6×140 mm, or 2.6×150 mm. In some embodiments, the reverse phase liquid chromatography stationary phase is contained within a chromatography column that is about 100 mm in length and has an internal diameter of about 2.1 mm.

[0062] In some embodiments, the stationary phase of the reversed phase liquid chromatography comprises particles between about 1.2 μm and 2.5 μm in size. In other embodiments, the stationary phase of the reversed phase liquid chromatography comprises particles of about 1.7 μm, 1.8 μm, or 2.1 μm in size. In some embodiments, the particle size is about 1.2 μm, 1.3 μm, 1.4 μm, 1.5 μm, 1.6 μm, 1.7 μm, 1.8 μm, 1.9 μm, 2.0 μm, 2.1 μm, 2.2 μm, 2.3 μm, 2.4 μm, or 2.5 μm. In some embodiments, the stationary phase of the reversed phase liquid chromatography is composed of particles of about 1.7 μm.

[0063] In some embodiments, the chromatography uses a first mobile phase comprising fluorine-substituted acetic acid in water. Fluorine-substituted acetic acid includes monofluoroacetic acid, difluoroacetic acid, and trifluoroacetic acid. In some embodiments, the chromatography uses a first mobile phase comprising trifluoroacetic acid in water.

[0064] In some embodiments, the first mobile phase comprises about 0.05 to about 0.15% by volume of fluorinated acetic acid. In some embodiments, the first mobile phase comprises about 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, 0.1%, 0.11%, 0.12%, 0.13%, 0.14%, 0.15%, 0.16%, 0.17%, 0.18%, 0.19%, or 0.2% by volume of fluorinated acetic acid. In some embodiments, the first mobile phase comprises about 0.05% or 0.1% by volume of fluorinated acetic acid. In some embodiments, the first mobile phase comprises about 0.1% by volume of fluorinated acetic acid. In some embodiments, the fluorinated acetic acid is trifluoroacetic acid. In some embodiments, the first mobile phase comprises about 0.1% by volume of trifluoroacetic acid.

[0065] In some embodiments, the chromatography uses a second mobile phase comprising fluorine-substituted acetic acid in acetonitrile. In some embodiments, the chromatography uses a second mobile phase comprising trifluoroacetic acid in acetonitrile. In some embodiments, the chromatography uses a second mobile phase comprising fluorine-substituted acetic acid in a mixture of acetonitrile and water. In some embodiments, the chromatography uses a second mobile phase comprising trifluoroacetic acid in a mixture of acetonitrile and water.

[0066] In some embodiments, the second mobile phase comprises about 0.05-0.2% fluorine-substituted acetic acid by volume. In some embodiments, the second mobile phase comprises about 0.05-0.15% fluorine-substituted acetic acid by volume. In some embodiments, the second mobile phase comprises about 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, 0.1%, 0.11%, 0.12%, 0.13%, 0.14%, 0.15%, 0.16%, 0.17%, 0.18%, 0.19%, or 0.2% fluorine-substituted acetic acid by volume. In some embodiments, the second mobile phase comprises about 0.05% or 0.1% fluorine-substituted acetic acid by volume. In some embodiments, the second mobile phase comprises about 0.1% fluorine-substituted acetic acid. In some embodiments, the fluorine-substituted acetic acid is trifluoroacetic acid. In some embodiments, the second mobile phase comprises about 0.1% trifluoroacetic acid.

[0067] In some embodiments, the second mobile phase comprises about 75-95% acetonitrile by volume. In some embodiments, the second mobile phase comprises about 75%, 80%, 85%, 90%, or 95% acetonitrile by volume. In some embodiments, the second mobile phase comprises about 90% acetonitrile and 10% water by volume.

[0068] In some embodiments, in chromatography, the percentage of the second mobile phase in the combination of the first mobile phase and the second mobile phase is increased over time. In some embodiments, the percentage of the second mobile phase is increased from about 10% to about 40% by volume. In some embodiments, the percentage of the second mobile phase is increased from about 10% to about 45% by volume. In some embodiments, the percentage of the second mobile phase is increased from about 10% to about 100% by volume. In some embodiments, the percentage of the second mobile phase is increased from about 10% to about 45% in about 30-40 minutes. In some embodiments, the percentage of the second mobile phase is increased from about 10% to about 45% in about 35 minutes. In some embodiments, the percentage of the second mobile phase is increased from about 10% to about 100% by volume in about 30-50 minutes. In some embodiments, the percentage of the second mobile phase is increased from about 10% to about 100% by volume in about 36 minutes.

[0069] In some embodiments, the percentage of the second mobile phase is increased from about 10% to about 40% by volume in about 5-10 minutes and from about 40% to about 45% in about 25-35 minutes. In some embodiments, the percentage of the second mobile phase is increased from about 45% to about 100% in about 0.5-2 minutes. In some embodiments, the percentage of the second mobile phase is reduced from about 100% to about 10% in about 0.5-2 minutes.

[0070] In some embodiments, the percentage of the second mobile phase is increased from about 10% to about 40% by volume in about 6 minutes and from about 40% to about 45% in about 29 minutes. In some embodiments, the percentage of the second mobile phase is increased from about 45% to about 100% in about 1 minute. In some embodiments, the percentage of the second mobile phase is reduced from about 100% to about 10% in about 1 minute.

[0071] In some embodiments, the liquid chromatography is high pressure liquid chromatography (HPLC). In some embodiments, the liquid chromatography is ultra high pressure liquid chromatography (UHPLC).

[0072] In some embodiments, mass spectrometry may use any ionization mode, particularly one suitable for the analysis of biomolecules, including, but not limited to, direct infusion mass spectrometry, electrospray ionization (ESI)-MS, desorption electrospray ionization (DESI)-MS, direct analysis in real time (DART)-MS, atmospheric pressure chemical ionization (APCI)-MS, electron impact (El) or chemical ionization (CI), matrix-assisted laser desorption / ionization (MALDI)-MS, and atmospheric pressure ionization-electrospray (API-ES). In some embodiments, mass spectrometry uses API-ES ionization mode.

[0073] In some embodiments, the mass spectrometry scans signals over a range of m / z from 400 to 16000. In some embodiments, the mass spectrometry scans signals over a range of m / z from 700 to 13700.

[0074] In some embodiments, the scan type of the mass spectrometry is positive polarity. In some embodiments, the data acquisition time of the mass spectrometry is about 10 to 35 minutes. In some embodiments, the data acquisition time of the mass spectrometry is about 17 to 28 minutes.

[0075] In some embodiments, the nozzle voltage of the mass spectrometry is about 400 to 600V. In some embodiments, the nozzle voltage of the mass spectrometry is about 500V. In some embodiments, the skimmer voltage of the mass spectrometry is about 60 to 70V. In some embodiments, the skimmer voltage of the mass spectrometry is about 65V. In some embodiments, the difference between the nozzle voltage and the skimmer voltage is about 400 to 450V. In some embodiments, the difference between the nozzle voltage and the skimmer voltage is about 435V.

[0076] In some embodiments, the drying gas temperature for mass spectrometry is about 200-350° C. In some embodiments, the drying gas temperature for mass spectrometry is about 300° C. In some embodiments, the drying gas flow rate for mass spectrometry is about 5-13 L / min. In some embodiments, the drying gas flow rate for mass spectrometry is about 13 L / min.

[0077] In some embodiments, the mass spectrometry uses a capillary voltage of about 3-6 kV. In some embodiments, the mass spectrometry uses a capillary voltage of about 3, 4, 5, or 6 kV. In some embodiments, the mass spectrometry uses a capillary voltage of about 5 kV.

[0078] In some embodiments, the mass spectrometry uses a fragmentor voltage of about 125-350V. In some embodiments, the mass spectrometry uses a fragmentor voltage of about 125V, 130V, 135V, 145V, 155V, 160V, 165V, 175V, 185V, 190V, 195V, 200V, 205V, 210V, 215V, 220V, 225V, 230V, 235V, 240V, 245V, 250V, 255V, 260V, 265V, 270V, 275V, 280V, 285V, 290V, 295V, 300V, 305V, 310V, 315V, 320V, 325V, 330V, 335V, 340V, 345V, or 350V. In some embodiments, the mass spectrometry uses a fragmentor voltage of about 175V.

[0079] In some embodiments, the AAV particle is AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAVrhlO, AAVrh74, or any natural, recombinant, or synthetic AAV particle.In some embodiments, the AAV particle is a recombinant AAV (rAAV) particle.In some embodiments, the AAV particle is AAVrhl74.

[0080] In some of the above-mentioned embodiments, the disclosure further includes determining the post-translational modification of at least one of the VP1, VP2, and VP3 capsid proteins. In some embodiments, the disclosure further includes determining the post-translational glycosylation, sialylation, acetylation, amino acid deletion, amidation, phosphorylation, formylation, hydroxylation, methylation, and / or sulfation of at least one of the VP1, VP2, and VP3 capsid proteins. The post-translational modification includes one or more of N-terminal methionine deletion, threonine deletion, phosphorylation, and acetylation.

[0081] In some aspects, the present disclosure includes determining the removal of the N-terminal methionine in VP1, VP2, or VP3 capsid protein. In some aspects, the present disclosure includes determining the removal of the N-terminal methionine in VP1 or VP3 capsid protein. In some aspects, the present disclosure includes determining the N-terminal acetylation after the removal of the N-terminal methionine in VP1, VP2, or VP3 capsid protein. In some aspects, the present disclosure includes determining the N-terminal acetylation after the removal of the N-terminal methionine in VP1 or VP3 capsid protein.

[0082] In some aspects, the disclosure provides methods for characterizing capsid proteins of an AAV particle based at least in part on the ratio of VP1, VP2, and VP3 capsid proteins and / or the mass of one or more of the VP1, VP2, and VP3 capsid proteins in the AAV particle.

[0083] In some aspects, the disclosure provides methods for determining the serotype of an AAV particle based at least in part on the ratio of VP1, VP2, and VP3 capsid proteins in the AAV particle, and / or the mass of one or more of VP1, VP2, and VP3 capsid proteins, where the ratio of VP1, VP2, and VP3 capsid proteins, and the mass of one or more of VP1, VP2, and VP3 capsid proteins are determined by the methods disclosed herein.

[0084] Mass spectrometry is an analytical technique for protein characterization. In some embodiments, a method is provided for characterizing the ratio of AAVrh74 capsid proteins, along with the intact masses of all three capsid proteins, by liquid chromatography and mass spectrometry. In some embodiments, AAVrh74 capsids are denatured on-column into individual capsid proteins VP1, VP2, and VP3. Denaturation is achieved by heating the column compartment to 80° C. (3, 4). Capsid proteins are then baseline separated on a Waters BEH C8 column utilizing trifluoroacetic acid as an ion-pairing agent in the mobile phase (5). The denatured proteins are first analyzed by UV to obtain capsid ratios, and then by mass spectrometry to obtain the intact masses of the individual proteins.

[0085] Deamidation is a common post-translational modification that results in the conversion of asparagine residues to a mixture of isoaspartic and aspartic acids. Deamidation of glutamine residues also occurs, but at a much slower rate. Oxidation is also a common post-translational modification that is the result of the reaction of proteins with various free radicals and reactive oxygen species. Methionine oxidation is the most common, but oxidation of several other amino acid residues such as cysteine ​​and tryptophan has also been observed. Deamidation / oxidation is also a common degradation pathway of proteins that occurs during production and storage. Deamidation can affect protein activity and stability. Oxidation can cause conformational changes in proteins and therefore affect protein activity and stability. Oxidation can also affect protein immunogenicity. Therefore, critical quality attributes (CQAs) of proteins need to be carefully monitored for post-translational modifications.

[0086] Current methods using ammonium bicarbonate either generate spurious signals or overestimate deamidation in AAV capsid proteins (Table 7). Thus, the present disclosure provides a method to more accurately measure post-translational modifications on capsid proteins using Tris-HCl. In some embodiments, the LC MS method uses a buffer containing Tris-HCl. In some embodiments, the buffer contains acetonitrile. In some embodiments, the buffer contains methionine. In some embodiments, the buffer contains 5 mM to 50 mM Tris-HCl, 5% to 20% acetonitrile, and 1 mM to 50 mM methionine. In some embodiments, the buffer contains 20 mM Tris-HCl, 5% to 10% acetonitrile, and 10 mM methionine.

[0087] In some embodiments, the post-translational modification comprises deamidation at one or more of N263, N514, N57, N502, N254, and N94 of AAV8, or the equivalent residues in AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV9, AAV10, AAV11, AAV12, AAV13, AAVrhlO, or AAVrh74. In some embodiments, the post-translational modification comprises deamidation at one or more of N57, N255, N256, and N263 of AAV.Rh74. In some embodiments, the post-translational modification comprises oxidation at one or more of M437, M473, M526, M544, M560, and M637 of AAV.Rh74. AAV composition

[0088] Some aspects of the present disclosure are directed to recombinant AAV (rAAV) that comprises a heterogeneous capsid protein population that contains subpopulations with amino acid modifications.In some aspects, the modifications can be deamidation, acetylation, isomerization, phosphorylation, or oxidation.In some aspects, the modifications are deamidation or oxidation.

[0089] In some embodiments, the rAAV capsid may contain subpopulations of VP1, VP2, and VP3 having at least one, at least two, at least three, at least four, at least five to at least about 25 deamidated amino acid residues, of which at least about 1% to about 10%, at least about 10% to about 25%, at least about 25% to about 50%, at least about 50% to about 70%, at least about 70% to about 100%, at least about 75% to about 100%, at least about 80% to about 100%, or at least about 90% to about 100% are deamidated compared to the encoded amino acid sequence of the VP protein. In some embodiments, the majority of these may be N residues. In some embodiments, Q residues may be deamidated.

[0090] In some aspects, the disclosure provides AAV compositions comprising an AAV capsid that comprises deamidation at one or more of N57, N255, N256, and N263 of AAV.Rh74, or the equivalent residues of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, or AAVrhlO, as measured by mass spectrometry and / or ultraviolet (UV) visible spectroscopy. In some aspects, deamidation is measured by any of the methods disclosed herein.

[0091] In some embodiments, the heterogeneous population comprises less than about 80%, less than about 78%, less than about 76%, less than about 74%, less than about 72%, less than about 70%, less than about 68%, less than about 66%, less than about 64%, less than about 62%, less than about 60%, less than about 58%, less than about 56%, less than about 54%, less than about 52%, less than about 50%, less than about Including less than 48%, less than about 46%, less than about 44%, less than about 42%, less than about 40%, less than about 38%, less than about 36%, less than about 34%, less than about 32%, less than about 30%, less than about 28%, less than about 26%, less than about 24%, less than about 22%, less than about 20%, less than about 18%, less than about 16%, less than about 15%, less than about 14%, less than about 13%, less than about 12%, less than about 11%, or less than about 10%.

[0092] In some aspects, the heterogeneous population comprises less than about 25%, less than about 24%, less than about 23%, less than about 22%, less than about 21%, less than about 20%, less than about 19%, less than about 18%, less than about 17%, less than about 16%, less than about 15%, less than about 14%, less than about 13%, less than about 12%, less than about 11%, or less than about 10% of capsid protein deamidated at N57 of the AAV.rh74 capsid.

[0093] In some embodiments, the heterogeneous population comprises less than about 80%, less than about 78%, less than about 76%, less than about 74%, less than about 72%, less than about 70%, less than about 68%, less than about 66%, less than about 64%, less than about 62%, less than about 60%, less than about 58%, less than about 56%, less than about 54%, less than about 52%, less than about 50%, less than about 48%, less than about 46%, less than about 44% or less of the capsid protein deamidated at N254 and / or N255 of the AAV.rh74 capsid. including less than about 42%, less than about 40%, less than about 38%, less than about 36%, less than about 34%, less than about 32%, less than about 30%, less than about 28%, less than about 26%, less than about 24%, less than about 22%, less than about 20%, less than about 18%, less than about 16%, less than about 15%, less than about 14%, less than about 13%, less than about 12%, less than about 11%, or less than about 10%, less than about 8%, less than about 7%, less than about 6%, less than about 5%, less than about 4%, less than about 3%, less than about 2%, and less than about 1%.

[0094] In some embodiments, the heterogeneous population has less than about 80%, less than about 78%, less than about 76%, less than about 74%, less than about 72%, less than about 70%, less than about 68%, less than about 66%, less than about 64%, less than about 62%, less than about 60%, less than about 58%, less than about 56%, less than about 54%, less than about 52%, less than about 50%, less than about 48%, less than about 50 ... Including less than about 46%, less than about 44%, less than about 42%, less than about 40%, less than about 38%, less than about 36%, less than about 34%, less than about 32%, less than about 30%, less than about 28%, less than about 26%, less than about 24%, less than about 22%, less than about 20%, less than about 18%, less than about 16%, less than about 15%, less than about 14%, less than about 13%, less than about 12%, less than about 11%, or less than about 10%.

[0095] In some embodiments, the AAV composition comprises an AAV capsid that comprises oxidation of one or more of M437, M473, M526, M544, M560, and M637 of AAV.Rh74, or the equivalent residues of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, or AAVrhlO, as measured by mass spectrometry and / or ultraviolet (UV) visible spectroscopy. In some embodiments, deamidation is measured by any of the methods disclosed herein.

[0096] In some embodiments, the heterogeneous population comprises less than about 40%, less than about 48%, less than about 46%, less than about 44%, less than about 42%, less than about 40%, less than about 38%, less than about 36%, less than about 34%, less than about 32%, less than about 30%, less than about 28%, less than about 26%, less than about 24%, less than about 22%, less than about 20%, less than about 18%, less than about 16%, less than about 14%, less than about 12%, less than about 10%, less than about 9%, less than about 8%, less than about 7%, less than about 6%, less than about 5%, less than about 4%, less than about 3%, less than about 2%, less than about 1%, less than about 0.9%, less than about 0.8%, less than about 0.7%, less than about 0.6%, or less than 0.5% capsid protein oxidized with M437.

[0097] In some embodiments, the heterogeneous population comprises less than about 40%, less than about 48%, less than about 46%, less than about 44%, less than about 42%, less than about 40%, less than about 38%, less than about 36%, less than about 34%, less than about 32%, less than about 30%, less than about 28%, less than about 26%, less than about 24%, less than about 22%, less than about 20%, less than about 18%, less than about 16%, less than about 14%, less than about 12%, less than about 10%, less than about 9%, less than about 8%, less than about 7%, less than about 6%, less than about 5%, less than about 4%, less than about 3%, less than about 2%, less than about 1%, less than about 0.9%, less than about 0.8%, less than about 0.7%, less than about 0.6%, or less than 0.5% capsid protein oxidized with M473.

[0098] In some embodiments, the heterogeneous population comprises less than about 40%, less than about 48%, less than about 46%, less than about 44%, less than about 42%, less than about 40%, less than about 38%, less than about 36%, less than about 34%, less than about 32%, less than about 30%, less than about 28%, less than about 26%, less than about 24%, less than about 22%, less than about 20%, less than about 18%, less than about 16%, less than about 14%, less than about 12%, less than about 10%, less than about 9%, less than about 8%, less than about 7%, less than about 6%, less than about 5%, less than about 4%, less than about 3%, less than about 2%, less than about 1%, less than about 0.9%, less than about 0.8%, less than about 0.7%, less than about 0.6%, or less than 0.5% of the capsid protein is oxidized with M526.

[0099] In some embodiments, the heterogeneous population comprises less than about 40%, less than about 48%, less than about 46%, less than about 44%, less than about 42%, less than about 40%, less than about 38%, less than about 36%, less than about 34%, less than about 32%, less than about 30%, less than about 28%, less than about 26%, less than about 24%, less than about 22%, less than about 20%, less than about 18%, less than about 16%, less than about 14%, less than about 12%, less than about 10%, less than about 9%, less than about 8%, less than about 7%, less than about 6%, less than about 5%, less than about 4%, less than about 3%, less than about 2%, less than about 1%, less than about 0.9%, less than about 0.8%, less than about 0.7%, less than about 0.6%, or less than 0.5% of the capsid protein is oxidized with M544.

[0100] In some embodiments, the heterogeneous population comprises less than about 40%, less than about 48%, less than about 46%, less than about 44%, less than about 42%, less than about 40%, less than about 38%, less than about 36%, less than about 34%, less than about 32%, less than about 30%, less than about 28%, less than about 26%, less than about 24%, less than about 22%, less than about 20%, less than about 18%, less than about 16%, less than about 14%, less than about 12%, less than about 10%, less than about 9%, less than about 8%, less than about 7%, less than about 6%, less than about 5%, less than about 4%, less than about 3%, less than about 2%, less than about 1%, less than about 0.9%, less than about 0.8%, less than about 0.7%, less than about 0.6%, or less than 0.5% of the capsid protein is oxidized with M560.

[0101] In some embodiments, the heterogeneous population comprises less than about 40%, less than about 48%, less than about 46%, less than about 44%, less than about 42%, less than about 40%, less than about 38%, less than about 36%, less than about 34%, less than about 32%, less than about 30%, less than about 28%, less than about 26%, less than about 24%, less than about 22%, less than about 20%, less than about 18%, less than about 16%, less than about 14%, less than about 12%, less than about 10%, less than about 9%, less than about 8%, less than about 7%, less than about 6%, less than about 5%, less than about 4%, less than about 3%, less than about 2%, less than about 1%, less than about 0.9%, less than about 0.8%, less than about 0.7%, less than about 0.6%, or less than 0.5% capsid protein oxidized with M637. Characterization of host cell proteins in AAV compositions

[0102] In some aspects, the present disclosure provides a method for characterizing host cell proteins in an AAV composition, such as an AAV-based gene therapy pharmaceutical. In some aspects, the method for characterizing host cell proteins in an AAV composition includes immunoprecipitating viral capsid proteins from the composition, digesting residual host cell proteins, and analyzing the digested proteins by liquid chromatography quadrupole time-of-flight mass spectrometry (LC-QTOF-MS) to identify the host cell proteins. As used herein, the term "residual host cell proteins" or "residual proteins" refers to proteins that remain in solution after immunoprecipitation. In some aspects, the method further includes analyzing the digested host cell proteins by repeated MS / MS.

[0103] In some embodiments, the immunoprecipitation comprises incubating the AAV composition with a VP antibody. In some embodiments, the VP antibody comprises an anti-AAV VP1 antibody, an anti-AAV VP2 antibody, an anti-AAV VP3 antibody, or a combination thereof. In some embodiments, the antibody can be an anti-adeno-associated virus (AAV) VP1 / VP2 / VP3 antibody from American Research Products, Inc. (Cat. No.: 03-61058).

[0104] In some embodiments, the residual host cell proteins are digested in solution. In some embodiments, the digestion is rapid digestion. In some embodiments, the rapid digestion is performed at about 60°C to about 80°C. In some embodiments, the rapid digestion is performed at about 60°C, about 61°C, about 62°C, about 63°C, about 64°C, about 65°C, about 66°C, about 67°C, about 68°C, about 69°C, about 70°C, about 71°C, about 72°C, about 73°C, about 74°C, about 75°C, about 76°C, about 77°C, about 78°C, about 79°C, or about 80°C. In some embodiments, the rapid digestion is performed at about 70°C.

[0105] In some embodiments, the AAV composition is spiked with a known amount of at least one known protein standard. In some embodiments, the at least one known protein standard is a human or bovine protein standard. In some embodiments, the method further comprises quantifying the amount of residual host cell protein relative to the at least one known protein standard.

[0106] In some aspects, the liquid chromatography is reversed-phase liquid chromatography, size-exclusion chromatography, hydrophilic interaction liquid chromatography, or cation exchange chromatography. In some aspects, the liquid chromatography is reversed-phase liquid chromatography.

[0107] In some embodiments, liquid chromatography is performed at about 35° C. to about 55° C. In some embodiments, liquid chromatography is performed at about 35° C., about 36° C., about 37° C., about 38° C., about 39° C., about 40° C., about 41° C., about 42° C., about 43° C., about 44° C., about 45° C., about 46° C., about 47° C., about 48° C., about 49° C., about 50° C., about 51° C., about 52° C., about 53° C., about 54° C., or about 55° C. In some embodiments, liquid chromatography is performed at about 45° C.

[0108] In some embodiments, the reverse phase chromatography is performed using a C18, C8, or C4 column. In some embodiments, the liquid chromatography is performed using a C8 column.

[0109] In some embodiments, the stationary phase of the reversed phase liquid chromatography is contained within a chromatography column having a length of about 50-300 mm and an internal diameter of about 1-4.6 mm. In some embodiments, the column is a BEH column. In some embodiments, the column has an internal diameter of 1 mm, 2.1 mm, 3 mm, or 4.6 mm. In some embodiments, the column has a length of 50 mm, 75 mm, 100 mm, 150 mm, or 300 mm. In some embodiments, the column size is 1 mm x 50 mm, 2.1 mm x 50 mm, 3 mm x 50 mm, 4.6 mm x 50 mm, 1 mm x 75 mm, 2.1 mm x 75 mm, 3 mm x 75 mm, 4.6 mm x 75 mm, 1 mm x 100 mm, 2.1 mm x 100 mm, 3 mm x 100 mm, 4.6 mm x 100 mm, 1 mm x 150 mm, 2.1 mm x 150 mm, 3 mm x 150 mm, 4.6 mm x 150 mm, 1 mm x 300 mm, 2.1 mm x 300 mm, 3 mm x 300 mm, or 4.6 mm x 300 mm. In some embodiments, the column sizes are 1.6×50 mm, 1.6×60 mm, 1.6×70 mm, 1.6×80 mm, 1.6×90 mm, 1.6×100 mm, 1.6×110 mm, 1.6×120 mm, 1.6×130 mm, 1.6×140 mm, 1.6×150 mm, 1.7×50 mm, 1.7×60, 1.7×70 mm, 1.7×80mm, 1.7×90mm, 1.7×100mm, 1.7×110mm, 1.7×120mm, 1.7×130mm, 1.7×140mm, 1 .7×150mm, 1.8×50mm, 1.8×60, 1.8×70mm, 1.8×80mm, 1.8×90mm, 1.8×100mm, 1.8×110mm, 1.8×120mm, 1.8×130mm, 1.8×140mm, 1.8×150mm, 1.9×50mm, 1.9×60mm, 1.9×70mm, 1.9×80mm, 1.9×90mm, 1.9×100mm, 1.9×110mm, 1.9×120mm, 1.9×130mm, 1.9×140mm, 1.9×150mm , 2.0×50mm, 2.0×60mm, 2.0×70mm, 2.0×80mm, 2.0×90mm, 2.0×100mm, 2.0×110mm, 2.0×120mm, 2.0×130mm, 2.0×140mm, 2.0×150mm, 2.1×50mm, 2.1×60mm, 2.1×70mm, 2.1×80mm, 2.1x90mm, 2.1x100mm, 2.1x110mm, 2.1x120mm, 2.1x130mm, 2.1x140mm, 2.1x150mm, 2.2x50mm, 2.2x60mm, 2.2x70mm, 2.2x80mm, 2.2x90mm, 2.2x100mm, 2.2x110mm, 2.2x120mm, 2.2x130 mm, 2.2×140mm, 2.2×150mm, 2.3×50mm, 2.3×60mm, 2.3×70mm, 2.3×80mm, 2.3×90mm, 2.3×10 0mm, 2.3×110mm, 2.3×120mm, 2.3×130mm, 2.3×140mm, 2.3×150mm, 2.4×50mm, 2.4×60mm, 2.4 x70mm, 2.4x80mm, 2.4x90mm, 2.4x100mm, 2.4x110mm, 2.4x120mm, 2.4x130mm, 2.4x140mm, 2.4x150mm, 2.5x50mm, 2.5x60, 2.5x70mm, 2.5x80mm, 2.5x90mm, 2.5x100mm, 2.5x110mm, 2. 5×120 mm, 2.5×130 mm, 2.5×140 mm, 2.5×150 mm, 2.6×50 mm, 2.6×60 mm, 2.6×70 mm, 2.6×80 mm, 2.6×90 mm, 2.6×100 mm, 2.6×110 mm, 2.6×120 mm, 2.6×130 mm, 2.6×140 mm, or 2.6×150 mm. In some embodiments, the reverse phase liquid chromatography stationary phase is contained within a chromatography column that is about 150 mm in length and has an internal diameter of about 2.1 mm.

[0110] In some embodiments, the stationary phase of the reversed phase liquid chromatography comprises particles with a size between about 1.2 μm and 2.5 μm. In some embodiments, the stationary phase of the reversed phase liquid chromatography comprises particles with a size of about 1.7 μm, 1.8 μm, or 2.1 μm. In some embodiments, the particle size is about 1.2 μm, 1.3 μm, 1.4 μm, 1.5 μm, 1.6 μm, 1.7 μm, 1.8 μm, 1.9 μm, 2.0 μm, 2.1 μm, 2.2 μm, 2.3 μm, 2.4 μm, or 2.5 μm. In some embodiments, the stationary phase of the reversed phase liquid chromatography is composed of particles of about 1.7 μm.

[0111] In some embodiments, the chromatography uses a first mobile phase comprising fluorine-substituted acetic acid in water. Fluorine-substituted acetic acid includes monofluoroacetic acid, difluoroacetic acid, and trifluoroacetic acid. In some embodiments, the chromatography uses a first mobile phase comprising trifluoroacetic acid in water.

[0112] In some aspects, the chromatography uses a first mobile phase that includes formic acid.

[0113] In some embodiments, the first mobile phase comprises about 0.05 to about 0.15% formic acid by volume. In some embodiments, the first mobile phase comprises about 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, 0.1%, 0.11%, 0.12%, 0.13%, 0.14%, 0.15%, 0.16%, 0.17%, 0.18%, 0.19%, or 0.2% formic acid by volume. In some embodiments, the first mobile phase comprises about 0.05% or 0.1% formic acid by volume. In some embodiments, the first mobile phase comprises about 0.1% formic acid by volume.

[0114] In some embodiments, the chromatography uses a second mobile phase comprising fluorine-substituted acetic acid in acetonitrile. In some embodiments, the chromatography uses a second mobile phase comprising trifluoroacetic acid in acetonitrile. In some embodiments, the chromatography uses a second mobile phase comprising fluorine-substituted acetic acid in a mixture of acetonitrile and water. In some embodiments, the chromatography uses a second mobile phase comprising trifluoroacetic acid in a mixture of acetonitrile and water.

[0115] In some aspects, the chromatography uses a second mobile phase that comprises formic acid in acetonitrile. In some aspects, the chromatography uses a second mobile phase that comprises formic acid in a mixture of acetonitrile and water.

[0116] In some embodiments, the second mobile phase comprises about 0.05-0.2% formic acid by volume. In some embodiments, the second mobile phase comprises about 0.05-0.15% formic acid by volume. In some embodiments, the second mobile phase comprises about 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, 0.1%, 0.11%, 0.12%, 0.13%, 0.14%, 0.15%, 0.16%, 0.17%, 0.18%, 0.19%, or 0.2% formic acid by volume. In some embodiments, the second mobile phase comprises about 0.05% or 0.1% formic acid by volume. In some embodiments, the second mobile phase comprises about 0.1% formic acid by volume.

[0117] In some embodiments, the second mobile phase comprises about 75-95% acetonitrile by volume. In some embodiments, the second mobile phase comprises about 75%, 80%, 85%, 90%, or 95% acetonitrile by volume. In some embodiments, the second mobile phase comprises about 90% acetonitrile and about 10% water by volume.

[0118] In some embodiments, in chromatography, the percentage of the second mobile phase in the combination of the first mobile phase and the second mobile phase is increased over time. In some embodiments, the percentage of the second mobile phase is increased from about 2% to about 50% by volume. In some embodiments, the percentage of the second mobile phase is increased from about 50% to about 100% by volume. In some embodiments, the percentage of the second mobile phase is increased from about 2% to about 50% in about 110-130 minutes. In some embodiments, the percentage of the second mobile phase is increased from about 2% to about 50% in about 120 minutes. In some embodiments, the percentage of the second mobile phase is increased from about 50% to about 100% by volume in about 20-30 minutes. In some embodiments, the percentage of the second mobile phase is increased from about 50% to about 100% by volume in about 25 minutes. In some embodiments, the percentage of the second mobile phase is then increased to 100% by volume for about 5 minutes. In some embodiments, the percentage of the second mobile phase is then maintained at 100% by volume for about 3 minutes. In some embodiments, the second mobile phase is then reduced to about 2% by volume for about 2 minutes.

[0119] In some embodiments, the percentage of the second mobile phase is maintained at about 100% by volume for about 0.5 to 1.5 minutes. In some embodiments, the percentage of the second mobile phase is maintained at about 100% by volume for about 1 minute.

[0120] In some embodiments, the percentage of the second mobile phase is reduced from about 100% to about 2% in about 1-10 minutes, hi some embodiments, the percentage of the second mobile phase is reduced from about 100% to about 2% in about 4 minutes.

[0121] In some embodiments, the liquid chromatography is high pressure liquid chromatography (HPLC). In some embodiments, the liquid chromatography is ultra high pressure liquid chromatography (UHPLC).

[0122] In some embodiments, mass spectrometry may use any ionization mode, particularly one suitable for the analysis of biomolecules, including, but not limited to, direct infusion mass spectrometry, electrospray ionization (ESI)-MS, desorption electrospray ionization (DESI)-MS, direct analysis in real time (DART)-MS, atmospheric pressure chemical ionization (APCI)-MS, electron impact (El) or chemical ionization (CI), matrix-assisted laser desorption / ionization (MALDI)-MS, and atmospheric pressure ionization-electrospray (API-ES). In some embodiments, mass spectrometry uses API-ES ionization mode.

[0123] In some embodiments, the mass spectrometry scans the signals over a range of m / z from 40 to 5000. In some embodiments, the mass spectrometry scans the signals over a range of m / z from 50 to 3000. In some embodiments, the mass spectrometry scans the signals over a range of m / z from 300 to 3000.

[0124] In some embodiments, the scan type of the mass spectrometry is positive polarity. In some embodiments, the data acquisition time of the mass spectrometry is about 1 to 130 minutes. In some embodiments, the data acquisition time of the mass spectrometry is about 2 to 120 minutes.

[0125] In some embodiments, the nozzle voltage of the mass spectrometry is about 400 to 600V. In some embodiments, the nozzle voltage of the mass spectrometry is about 500V. In some embodiments, the skimmer voltage of the mass spectrometry is about 60 to 70V. In some embodiments, the skimmer voltage of the mass spectrometry is about 65V. In some embodiments, the difference between the nozzle voltage and the skimmer voltage is about 400 to 450V. In some embodiments, the difference between the nozzle voltage and the skimmer voltage is about 435V.

[0126] In some embodiments, the drying gas temperature for mass spectrometry is about 200-375° C. In some embodiments, the drying gas temperature for mass spectrometry is about 325° C. In some embodiments, the drying gas flow rate for mass spectrometry is about 5-13 L / min. In some embodiments, the drying gas flow rate for mass spectrometry is about 12 L / min.

[0127] In some embodiments, the mass spectrometry uses a capillary voltage of about 3-6 kV. In some embodiments, the mass spectrometry uses a capillary voltage of about 3, 4, 5, or 6 kV. In some embodiments, the mass spectrometry uses a capillary voltage of about 5 kV.

[0128] In some embodiments, the mass spectrometry uses a fragmentor voltage of about 125-350V. In some embodiments, the mass spectrometry uses a fragmentor voltage of about 125V, 130V, 135V, 145V, 155V, 160V, 165V, 175V, 185V, 190V, 195V, 200V, 205V, 210V, 215V, 220V, 225V, 230V, 235V, 240V, 245V, 250V, 255V, 260V, 265V, 270V, 275V, 280V, 285V, 290V, 295V, 300V, 305V, 310V, 315V, 320V, 325V, 330V, 335V, 340V, 345V, or 350V. In some embodiments, the mass spectrometry uses a fragmentor voltage of about 135V.

[0129] Although the subject matter has been described in some detail with reference to specific embodiments thereof, other embodiments are possible, therefore, the spirit and scope of the appended claims should not be limited to the description of the specific embodiments contained therein. EXAMPLES

[0130] The present disclosure will now be described with examples, which are intended to illustrate the work of the present disclosure and are not intended to limit any limitations on the scope of the present disclosure.Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which the present disclosure belongs.Although methods and materials similar or equivalent to those described herein can be used in the practice of the methods and compositions of the present disclosure, exemplary methods, devices, and materials are described herein.

[0131] Example 1: General Methods and Equipment

[0132] 1.1 Reagents

[0133] LCMS water, acetonitrile, and trifluoroacetic acid were obtained from Fisher Scientific and ammonium bicarbonate was obtained from Sigma-Aldrich. Table 1 shows the reagents used for liquid chromatography and solution preparation.

[0134] [Table 1-1] [Table 1-2]

[0135] 1.2 Equipment

[0136] The following equipment was used to carry out the examples disclosed herein. a) Waters ACQUITY UPLC BEH C8 Column, 2.1 x 100 mm, 1.7 μm; part number 186002878 b) Pierce Detergent Removal Spin Column, 0.5 mL; Catalog No. 87777 c) A suitable analytical balance d) Automatic pipette e) Class A volumetric glassware f) HPLC vials and caps g) Spatula and weighing boat h) Agilent 1290 Infinity II UHPLC system i) Agilent 6545XT AdvanceBio quadrupole time-of-flight mass spectrometer (Q-ToF)

[0137] Example 2: Preparation of solutions

[0138] 2.1 Preparation of 100 mM ammonium bicarbonate

[0139] 0.395±0.01 grams of ammonium bicarbonate was weighed into a 50 mL Falcon® tube. Using a graduated cylinder, 50 mL of LCMS water was transferred to the tube and the ammonium bicarbonate was completely dissolved using a vortex mixer to obtain a 100 mM ammonium bicarbonate solution. The solution is stable for one month at 2-8°C.

[0140] 2.2 Preparation of the first mobile phase (0.1% trifluoroacetic acid in water)

[0141] Using a graduated cylinder, 1 L of LCMS water was transferred to a 1 L bottle. Using a pipette, 1000 μL of trifluoroacetic acid was transferred to the bottle. The trifluoroacetic acid and water were mixed well for 5 minutes to obtain the first mobile phase. The first mobile phase is stable at ambient temperature for up to 1 month.

[0142] 2.3 Preparation of the second mobile phase (0.1% trifluoroacetic acid in 90% acetonitrile and 10% water)

[0143] 900 mL of acetonitrile was added to a 1 L graduated cylinder. LCMS water was added to the 1 L graduated cylinder to make 1 L of solution. The solution was transferred to a 1 L bottle. Using a pipette, 1000 μL of trifluoroacetic acid was transferred to the bottle. The trifluoroacetic acid and solution were mixed well for 5 minutes to obtain the second mobile phase. The second mobile phase is stable at ambient temperature for up to 1 month.

[0144] Example 3: Sample preparation

[0145] The seal at the bottom of the spin column was removed and the cap of the spin column was loosened. The column was placed into a 2 mL collection tube and centrifuged at 1500 x g for 1 minute. When using a fixed angle rotor, the compressed resin was tilted upwards to leave a mark on the side of the column. The column was then placed in the centrifuge with this mark facing outwards for all subsequent steps.

[0146] 400 μL of 100 mM ammonium bicarbonate solution was added to the column and the column was centrifuged at 1500×g for 1 min. This step was repeated two more times, discarding the flow-through after each step. The column was placed in a new 2 mL collection tube. 5 μg of sample was slowly applied to the top of the compressed resin bed and incubated at room temperature for 2 min. The column was centrifuged at 1500×g for 2 min to collect the polymer-free sample. The sample volume was then adjusted up to 100 μL with 100 mM ammonium bicarbonate before being transferred to an HPLC vial.

[0147] Example 4: Characterization of VP1, VP2, and VP3 capsid proteins in AAV particles

[0148] This example describes a method to determine the ratio of VP1, VP2, and VP3 capsid proteins in AAV particles, as well as the mass of VP1, VP2, and VP3 capsid proteins. Here, AAV particles were denatured and separated into VP1, VP2, and VP3 capsid proteins by liquid chromatography. The separated VP1, VP2, and VP3 capsid proteins were first subjected to UV to determine the ratio of VP1, VP2, and VP3 capsid proteins in AAV particles, and then subjected to mass spectrometry to obtain the respective masses of VP1, VP2, and VP3 capsid proteins.

[0149] 4.1 LC operating conditions

[0150] Separation of AAV VP1, VP2, and VP3 capsid proteins was performed using ACQUITY The experiments were carried out on an ACQUITY UPLC® system using a UPLC® BEH 1.7 μm, 2.1×100 mm, C8 analytical column (part number 186002878). The mobile phase used was: First mobile phase (A): 0.1% trifluoroacetic acid in water; and Second mobile phase (B): 0.1% trifluoroacetic acid in 90% acetonitrile and 10% water.

[0151] The column temperature was maintained at approximately 80° C. and separation was achieved using increasing amounts of mobile phase B from 10% to 40% and from 40% to 45% at a flow rate of 0.4 mL / min, followed by flushing with 100% mobile phase B for 1 min and re-equilibration with the starting mobile phase composition (10% mobile phase B) for an additional 5 min.

[0152] The operating conditions of the LC are listed in Table 2. [Table 2-1] [Table 2-2]

[0153] 4.2 Mass spectrometer (MS) operating conditions

[0154] Mass spectrometry was performed using API-ES ionization in a survey scan ranging from m / z values ​​700 to 13700 m / z on an Agilent 6545XT AdvanceBio quadrupole time-of-flight mass spectrometer (Q-ToF). The capillary voltage, nozzle voltage, fragmentor voltage, and skimmer voltage were set at 5 kV, 500 V, 175 V, and 65 V, respectively. The drying gas temperature and drying gas flow rate were set at 300 °C and 13 L / min, respectively.

[0155] The operating conditions of the mass spectrometer are listed in Table 3. [Table 3]

[0156] 4.3 Analysis and Results

[0157] The capsid proteins were first denatured by heating the column compartment to 80° C. The three capsid proteins VP1, VP2, and VP3 were then baseline separated by using a Waters UPLC BEH C8 column (part number 186002878) and eluted with a combination of a first mobile phase containing 0.1% trifluoroacetic acid in water and a second mobile phase containing 0.1% trifluoroacetic acid in a mixture of acetonitrile and water, with the percentage of the second mobile phase increasing over time. The use of 0.1% trifluoroacetic acid as an ion-pairing agent in the mobile phase aids in baseline resolution.

[0158] The VP1, VP2, and VP3 capsid proteins separated by liquid chromatography were first subjected to UV to determine the relative amounts and then subjected to mass spectrometry to determine the masses of the VP1, VP2, and VP3 capsid proteins. As shown in Figure 1 and Table 4, a stoichiometry of VP1 / VP2 / VP3 near 1:1:10 was obtained by baseline integration of the UV chromatograms. [Table 4]

[0159] The three capsid protein peaks were then deconvoluted using the parameters listed in Table 5. The total ion chromatogram and the deconvoluted spectra of all three peaks are shown in Figure 2 and Figures 3A-3C. The three major masses detected under the VP1 peak (theoretical mass 81587 Da) were 81496 Da, 81578 Da, and 81658 Da. The 81496 Da peak represents the VP1 protein with a missing N-terminal methionine and a single acetylation modification. The other two peaks with a mass shift of +80 Da represent phosphorylation. Deconvolution of the VP2 peak (theoretical mass 66381 Da) showed two major masses: 66282 Da and 66360 Da. The 66282 Da peak represents the VP2 protein lacking a threonine, and the 66360 Da peak is consistent with a single phosphorylation with a mass shift of +80. The VP3 peak (theoretical mass 59750 Da) shows a single major mass of 59662 Da, which is consistent with the mass of the VP3 protein with an N-terminal methionine loss and a single acetylation. [Table 5]

[0160] Figure 3 shows detection of post-translational modifications of VP1, VP2, and VP3. Table 6 shows the mass spectrometry of intact AAV.rh74 capsid proteins. [Table 6-1] [Table 6-2]

[0161] Example 5: Characterization of AAVrh74 deamidation using LCMS

[0162] Extensive deamidation in capsid proteins can be determined by mass spectrometry, which can also determine the sites of deamidation in capsid proteins and the level of deamidation at these sites. To measure deamidation of AAV capsids, capsid proteins were denatured and reduced in the presence of 2M guanidine hydrochloride and 10 mM DTT at 90°C for 10 minutes. After the samples were cooled to room temperature, 30 mM iodoacetamide was added for alkylation and incubated in the dark at room temperature for 30 minutes. The alkylation was then quenched by the addition of 1 mL DTT. 20 mM ammonium bicarbonate was added to the samples to dilute the guanidine hydrochloride to 200 mM. The samples were then digested with trypsin at an enzyme:protein ratio of 1:20 and incubated overnight at 37°C. After overnight incubation, digestion was quenched by adding trifluoroacetic acid to a final concentration of 0.5% and samples were analyzed on a Thermo UltiMate 3000 RSLC system coupled to a Q Exactive HF using a NanoFlex source.

[0163] Table 7 shows the major deamidation sites identified and the level of deamidation at these sites (i.e., percentage of deamidation). The AAV8 data in Table 7 is disclosed in a previous publication (Molecular Therapy, Volume 26 No 12, Pages 2848-2962 (2018)). [Table 7-1] [Table 7-2]

[0164] The deamidation state of AAV.rh74 was measured using two buffers separately (ammonium bicarbonate and Tris-HCl).

[0165] For ammonium bicarbonate, samples were denatured by buffer exchange into 100 mM ammonium bicarbonate. The denatured samples were reduced by the addition of 10 mM DTT and incubated at 37° C. for 45 minutes. Alkylation was then performed by adding iodoacetamide to a final concentration of 30 mM in the samples. The denatured, reduced, and alkylated samples were then exchanged back into 100 mM ammonium bicarbonate using a 10 kDa Amicon ultrafilter. The samples were then digested with trypsin and incubated overnight at 37° C. Using this sample preparation digested in ammonium bicarbonate, similar levels of deamidation were obtained for AAVrh74 as seen in Table 7.

[0166] For Tris-HCl, 60 μg sample aliquots were buffer exchanged into 4 M guanidine and 200 mM Tris pH 7.5 using Amicon 10K centrifugal filters to remove sample matrix and concentrate proteins. Guanidine concentration was adjusted to 6 M and DTT (10 mM) was added to the 60 μg aliquots. The reaction mixture was incubated at 56° C. for 45 min and then cooled to room temperature. Iodoacetamide (30 mM) was added and incubated at room temperature for 60 min in the dark. Tris buffer (100 mM, pH=7.5) was then added to dilute the guanidine HCl concentration to 0.6 M. Trypsin / Lys-C (60 μg) was added to 60 μg of reduced and alkylated sample (enzyme:protein ratio approximately 1:1 (w:w)). Methionine was added to 10 mM during digestion to minimize artifactual oxidation. Digestion was carried out overnight (17 h) at 37° C. TFA (1%) was then added prior to LC-MS / MS analysis.

[0167] As shown in Table 7, when Tris-HCl was used as the buffer, the deamidation state was significantly lower than that when ammonium bicarbonate was used.

[0168] To further optimize the Tris-HCl method, eight separate AAV capsid samples were measured according to the flow chart in Figure 4. Samples were first denatured by buffer exchange into 6 M guanidine hydrochloride, 20 mM Tris-HCl, pH 7.5. Samples were then reduced by adding DTT to a final concentration of 10 mM and incubated at 37°C for 45 min. Alkylation was performed by adding iodoacetamide to a final concentration of 30 mM and incubated at room temperature for 1 h in the dark. Samples were buffer exchanged back into 20 mM Tris-HCl, pH 7.5 using a 10 kDa Amicon ultrafilter. Acetonitrile was then added to the samples to a final concentration of 10% and methionine was also added to a final concentration of 10 mM. Samples were digested with trypsin overnight at 37°C. Peptides are then separated on an Agilent 1290 U-HPLC using RP-HPLC. The separated peptides are then detected using an Agilent 6545XT QToF and deamidation analysis is performed using MassHunter and Bioconfirm software. The status of deamidation is shown in Figure 5 and the status of oxidation in Figure 6. Of the 52 asparagine residues present, no deamidation was observed for 48 residues (total asparagine residues in VP1: 56). Of the 39 glutamine residues present, no deamidation was observed for all 39 residues (total glutamine residues in VP1: 48). Using O18-labeled water, the small number of deamidations at N57 was shown to be an artifact related to sample preparation. No oxidation was detected for the remaining five methionine residues (total methionine residues in VP1: 11). Of the 14 tryptophan residues detected, no oxidation was observed for all 14 residues (total tryptophan residues in VP1: 15).

[0169] Ammonium bicarbonate digestion can significantly increase deamidation artifacts due to the increase in pH over time. Therefore, the higher level of deamidation observed could have been a deamidation artifact generated during sample preparation. To confirm the deamidation level, a Tris HCl-based digestion was set up in Sarepta. 20 mM Tris HCl, pH 7.5 was used as the buffer. 10% acetonitrile was added to the digestion solution since it is known to reduce deamidation artifacts. Also, 10 mM methionine was added to the digestion solution to reduce oxidation artifacts.

[0170] Therefore, the methods of the present disclosure are more accurate in measuring deamidation, oxidation, or other post-translational modifications with Tris-HCl buffer.

[0171] Example 6: Characterization of host cell proteins using LC-QTOF-MS

[0172] The purity of the rAAV-based gene therapy pharmaceutical was analyzed by characterizing the host cell proteins remaining in the AAV composition by LC-QTOF-MS.

[0173] 6.1 Sample preparation

[0174] AAVrh74 samples were spiked with known amounts of human thioredoxin 1 (HTI) protein standard from Invitrogen (catalog no. LF-P0001) and bovine carbonic anhydrase II (BCAII) protein standard from Sigma (catalog no. C7749). Human thioredoxin 1 (HTI) and bovine carbonic anhydrase II (BCAII) were selected as spiking protein standards to quantify the found human and bovine HCPs, respectively. For the immunodepletion process, 100 μL of 0.05 mg / mL anti-adeno-associated virus (AAV) VP1 / VP2 / VP3 antibody and 100 μL of sample solution are pipetted into 270 μL of IP-MS cell lysis buffer of a Pierce MS compatible magnetic IP kit along with 20 μL of 0.05 mg / mL BCAII and 10 μL of 0.1 mg / mL HTI. AAV capsid proteins were then immunoprecipitated from the samples using anti-adeno-associated virus (AAV) VP1 / VP2 / VP3 (catalog number 03-6105) and the Pierce MS-compatible Magnetic IP Kit (catalog number 90409) from American Research Products, Inc. Samples were then passed through a Pierce Detergent Removal Spin Column (catalog number 87777).

[0175] The samples were then buffer exchanged into Promega Rapid Digest Buffer (catalog no. VA1060). Samples were reduced, alkylated, and digested with Rapid Digest Trypsin at 70°C for 60-180 minutes.

[0176] 6.2 LC Operating Conditions

[0177] Separation of the digested and residual host cell proteins was performed on an Agilent 1290 HPLC system using a Waters Acquity Peptide BEH C18, 1.7 μm, 2.1×150 mm column. The mobile phase used was as follows:

[0178] First mobile phase (A): 0.1% formic acid in water; and

[0179] Second mobile phase (B): 0.1% formic acid in 90% acetonitrile and 10% water.

[0180] The column temperature was maintained at approximately 45° C. and separation was achieved using mobile phase B increasing from 2% to 50% and from 50% to 100% at a flow rate of 0.3 mL / min, followed by flushing with 100% mobile phase B for 3 min and re-equilibration with the starting mobile phase composition (2% mobile phase B) for an additional 5 min.

[0181] The operating conditions of the LC are listed in Table 8. [Table 8] 6.3 Mass spectrometer (MS) operating conditions

[0182] Mass spectrometry was performed using API-ES ionization in a survey scan ranging from m / z values ​​50 to 3000 m / z on an Agilent 6545XT AdvanceBio quadrupole time-of-flight mass spectrometer (Q-ToF). The capillary voltage, nozzle voltage, fragmentor voltage, and skimmer voltage were set at 4 kV, 500 V, 135 V, and 65 V, respectively. The drying gas temperature and drying gas flow rate were set at 325 °C and 12 L / min, respectively.

[0183] The operating conditions of the mass spectrometer are listed in Table 9. [Table 9-1] [Table 9-2]

[0184] 6.4 Analysis and Results

[0185] The data generated in Example 6.3 was processed by Byos software from Protein Metrics and searched against a Uniprot protein database. The identity and relative amount of each residual protein was calculated relative to the amount of spiked protein preparation. HCP analysis showed that only a few host cell proteins were identified by MS (two bovine proteins but no human proteins) for three lots of AAV virions (Table 10). Protein concentrations are on the order of ng / mL or ppm levels based on spiked protein preparations. [Table 10]

[0186] The following references are incorporated herein in their entirety: 1. Buller RM, Rose JA. Characterization of adenovirus-associated virus-induced polypeptides in KB cells. J Virol 25:1978, Pages 331-338. 2. Johnson FB, Ozer HL, Hoggan MD. Structural proteins of adenovirus-associated viruses. J Virol 8:1971, Pages 776-770. 3.DWBauer.et.al.Exploring the Balance between DNA Pressure and Capsid Stability in Herpesviruses and Phages.J Virol 2015,9288-98. 4.Vamseedhar Rayaprolu.et.al.Comparative Analysis of Adeno-Associated Virus Capsid Stability and Dynamics.J Virol 2013,13150-60. 5.Xiaoying Jin et.al.Direct Liquid Chromatography / Mass Spectrometry Analysis for Complete Characterization of Recombinant Adeno-Associated Virus Capsid Proteins.Human Gene Therapy Methods,Volume 38 Number 5 2017,255-267.

Claims

1. An AAV composition comprising an AAV capsid comprising deamidation at one or more of N57, N255, N256, and N263 of AAV.Rh74, or the equivalent residues of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, or AAVrhlO.

2. An AAV composition comprising an AAV capsid that includes oxidation of one or more of M437, M473, M526, M544, M560, and M637 of AAV.Rh74, or the equivalent residues of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, CAAV11, AAV12, AAV13, or AAVrhlO.

3. 1. A method for characterizing a host cell protein in an AAV composition, comprising: immunoprecipitating viral capsid proteins from said composition; digesting residual host cell proteins; analyzing the digested proteins using liquid chromatography quadrupole time-of-flight mass spectrometry (LC-QTOF-MS) to identify host cell proteins.

4. The method of claim 3, wherein the immunoprecipitation comprises incubating the AAV composition with an anti-AAV VP1 antibody, an anti-AAV VP2 antibody, an anti-AAV VP3 antibody, or a combination thereof.

5. 5. The method of claim 3 or 4, further comprising analyzing the digested host cell proteins by iterative MS / MS.

6. The method according to any one of claims 3 to 5, wherein the digestion is carried out in solution.

7. 7. The method of claim 6, wherein the digestion comprises rapid digestion carried out at a temperature of about 60°C to about 80°C.

8. 8. The method of claim 7, wherein the temperature is about 70°C.

9. The method of any one of claims 3 to 8, further comprising spiking the AAV composition with a known amount of at least one known protein standard.

10. 10. The method of claim 9, wherein the at least one known protein preparation is a human or bovine protein preparation.

11. 11. The method of claim 9 or 10, further comprising quantifying the amount of digested host cell protein by comparison to the at least one protein standard.

12. The method according to any one of claims 3 to 11, wherein the liquid chromatography is reversed-phase liquid chromatography.

13. 13. The method of claim 12, wherein the reverse phase liquid chromatography is performed using a C18 column, a C8 column, or a C4 column.

14. 14. The method of claim 13, wherein the liquid chromatography is carried out using a C8 column.

15. The method of claim 13 or 14, wherein the column contains particles of about 1.2 to 3.5 μm.

16. The method of any one of claims 13 to 15, wherein the column contains particles of about 1.7 μm or about 1.8 μm.

17. 17. The method of any one of claims 13 to 16, wherein the column is about 50 mm to about 300 mm in length and has an inner diameter of about 1 mm to about 4.6 mm.

18. 18. The method of claim 17, wherein the column is about 150 mm long and has an inner diameter of about 2.1 mm.

19. The method of any one of claims 3 to 18, wherein the liquid chromatography is carried out at about 40°C to about 50°C.

20. The method of any one of claims 3 to 19, wherein the liquid chromatography is carried out at about 45°C.

21. The method of any one of claims 3 to 20, wherein the liquid chromatography comprises a first mobile phase comprising formic acid.

22. 22. The method of claim 21 , wherein the first mobile phase comprises from about 0.05% to about 0.15% formic acid by volume.

23. 23. The method of claim 21 or 22, wherein the first mobile phase comprises about 0.1% formic acid by volume.

24. 24. The method of any one of claims 3 to 23, wherein the liquid chromatography comprises a second mobile phase comprising formic acid in a mixture of acetonitrile and water.

25. 25. The method of claim 24, wherein the second mobile phase comprises from about 0.05% to about 0.15% formic acid by volume.

26. 26. The method of claim 24 or 25, wherein the second mobile phase comprises about 0.1% formic acid by volume.

27. 27. The method of any one of claims 24 to 26, wherein the second mobile phase comprises about 80-95% acetonitrile by volume.

28. 28. The method of claim 27, wherein the second mobile phase comprises about 90% acetonitrile by volume and 10% water by volume.

29. 29. The method of any one of claims 24 to 28, wherein in the liquid chromatography, a percentage of the second mobile phase is increased over time compared to a combination of the first mobile phase and the second mobile phase.

30. 30. The method of claim 29, wherein the percentage of the second mobile phase is increased from about 2% to about 50%.

31. 31. The method of claim 30, wherein the percentage of the second mobile phase is increased from about 2% to about 50% by volume over a period of about 120 minutes.

32. 32. The method of claim 31 , wherein the percentage of the second mobile phase is then increased to 100% by volume for about 5 minutes.

33. 33. The method of claim 32, wherein the percentage of the second mobile phase is then maintained at 100% by volume for about 3 minutes.

34. 34. The method of claim 33, wherein the percentage of the second mobile phase is then reduced to about 2% by volume for about 2 minutes.

35. The method of any one of claims 3 to 34, wherein the mass spectrometry is carried out with a fragmentor voltage of about 125 to 350V.

36. The method of any one of claims 3 to 35, wherein the mass analysis is carried out with a fragmentor voltage of about 135V.

37. The method of any one of claims 3 to 36, wherein the mass spectrometry is carried out using a capillary voltage of about 3 to 6 kV.

38. The method of any one of claims 3 to 37, wherein the mass spectrometry is carried out using a capillary voltage of about 4 kV.

39. A recombinant AAV (rAAV) comprising a heterogeneous population of capsid proteins containing subpopulations having amino acid modifications, wherein the amino acid modifications are post-translational modifications.

40. 40. The rAAV of claim 39, wherein the modification is deamidation or oxidation.

41. 41. The rAAV of claim 39 or 40, wherein the heterogeneous population comprises a deamidated asparagine (N) at one or more of N57, N255, N256, and N263 of AAV.rh74, or the equivalent residues of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, or AAVrhlO, as determined by mass spectrometry and / or ultraviolet (UV) visible spectroscopy.

42. 42. The rAAV of claim 41, wherein the heterogeneous population comprises less than 70%, 60%, 50%, 40%, 30%, 20%, or 15% of capsid protein deamidated at N57 of the AAV.rh74 capsid.

43. 42. The rAAV of claim 41, wherein the heterogeneous group comprises less than 15% of capsid protein deamidated at N57 of the AAV.rh74 capsid.

44. 42. The rAAV of claim 41, wherein the heterogeneous group comprises less than 70%, 60%, 50%, 40%, 30%, 20%, 15%, 10%, or 5% of capsid protein deamidated at N254 and / or N255 of AAV.rh74.

45. 42. The rAAV of claim 41, wherein the heterogeneous population comprises less than 70%, 60%, 50%, 40%, 30%, 20% of capsid protein deamidated at N263.

46. the heterogeneous population comprises an oxidized methionine at one or more of M437, M473, M526, M544, M560, and M637 of AAV.Rh74, or the equivalent residues of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, CAAV11, AAV12, AAV13, or AAVrhlO, as determined by mass spectrometry and / or ultraviolet (UV) visible spectroscopy.

41. The rAAV of claim 39 or 40.

47. 47. The rAAV of claim 46, wherein the heterogeneous population contains less than 30%, 20%, 10%, 5%, or 1% of capsid protein that is M437 oxidized.

48. 47. The rAAV of claim 46, wherein the heterogeneous population contains less than 30%, 20%, 10%, 5%, or 1% of capsid protein that is M473 oxidized.

49. 47. The rAAV of claim 46, wherein the heterogeneous population contains less than 30%, 20%, 10%, 5%, or 3% of capsid protein that is M526 oxidized.

50. 47. The rAAV of claim 46, wherein the heterogeneous population contains less than 30%, 20%, 10%, 5%, or 2% of capsid protein that is oxidized with M544.

51. 47. The rAAV of claim 46, wherein the heterogeneous population contains less than 30%, 20%, 10%, 5%, or 2% of capsid protein oxidized with M560.

52. 47. The rAAV of claim 46, wherein the heterogeneous population contains less than 30%, 20%, 10%, 5%, or 1% of capsid protein that is M637 oxidized.

53. The rAAV of any one of claims 39 to 41, wherein the heterogeneous population comprises a deamidated asparagine (N) within the peptide sequence of any one of SEQ ID NOs: 1-5, or the equivalent peptide sequences of other AAV serotypes.

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