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 on January 3, 2020, U.S. Provisional Patent Application No. 63 / 073,188, filed on September 1, 2020, and U.S. Provisional Patent Application No. 63 / 119,909, filed on December 1, 2020, the entire contents of each of which are incorporated herein by reference in their entirety.

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

Background Art

[0003] Adeno - associated virus (AAV) is rapidly becoming one of the most widely used vectors for delivering gene therapy. With high efficiency of transduction of a wide range of target tissues and an excellent safety profile, AAV has been the most widely used platform for gene therapy. AAV is a small virus belonging 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 recombinantly expressed in appropriate host cells. However, recombinant AAV can be contaminated by proteins derived 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 efficacy, and the ability to fully characterize the mass and ratio of capsid proteins is becoming increasingly important for the commercial production of AAV for gene therapy.

[0005] Specifically, the stoichiometry of VP is extremely important for the infectivity of viral vectors. For example, even when the balance of the VP1 / VP2 ratio was not achieved, a high level of VP3 capsid was negatively related to poor transduction efficiency and reduced efficacy (Gene Therapy, volume 25, pages 415-424 (2018)). Since the ratios of the structural proteins VP1, VP2, and VP3 obtained in production can vary over a wide range, for example, from 1:1:5 to 1:1:20 (Biotechnol Adv., 26(1):73-88 (2008)), accurate measurement of the ratios between the three capsid proteins is important in the quality control of AAV vectors. However, current methods have attempted to measure the mass of the capsid proteins but have not been able to determine the stoichiometry of each VP (WO2018 / 035059). Therefore, a robust method for more accurately characterizing the ratios and modifications of AAV capsid proteins and the purity of rAAV compositions is sought in the gene therapy industry. Summary of the Invention Means for Solving the Problems

[0006] The present disclosure provides a method for characterizing VP1, VP2, and VP3 capsid proteins in adeno-associated virus (AAV) particles using liquid chromatography and mass spectrometry. The methods disclosed herein are used to determine the ratios of VP1, VP2, and VP3 capsid proteins in AAV particles and / or the mass of one or more of the VPl, VP2, and VP3 capsid proteins.

[0007] In some embodiments, the present disclosure provides a method for determining the ratio of VP1, VP2, and VP3 capsid proteins in adeno-associated virus (AAV) particles. The method includes subjecting the AAV particles to liquid chromatography at a temperature of about 70 °C to about 90 °C, wherein the masses and ratios of the VP1, VP2, and VP3 capsid proteins are determined by mass spectrometry and / or ultraviolet (UV) visible spectroscopy. In some embodiments, the individual masses of the capsid proteins are measured by mass spectrometry. In some embodiments, the capsid on the AAV particles is denatured into each of the VP1, VP2, and VP3 proteins within the column of the liquid chromatography. In some embodiments, the capsid proteins are separated by liquid chromatography.

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

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

[0010] In some embodiments, the chromatography uses a first mobile phase containing trifluoroacetic acid in water. In some embodiments, the chromatography uses a second mobile phase containing 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 mobile phase and the second mobile phase increases over time.

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

[0012] Deamidation is one of the common post-translational modifications (PTMs) observed in proteins, which is known to have a significant impact on protein activity and stability. Deamidation is usually caused by the hydrolysis of the amide side chain of asparagine, forming a mixture of aspartic acid and isoaspartic acid. In some embodiments, deamidation is the 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 embodiments, the deamination of 5-methylcytosine results in thymine and ammonia. In some embodiments, glutamine residues also undergo deamidation to form a mixture of glutamic acid and isoglutamic acid, but glutamine residues are significantly less sensitive to deamidation compared to asparagine. In some embodiments, the deamination of guanine results in the formation of xanthine. In some embodiments, the deamination of adenine results in the formation of hypoxanthine. Deamidation of the capsid protein can potentially affect the stability and activity of AAV formulations.

[0013] In some aspects of the present disclosure, mass spectrometry is used to study post-translational modifications such as deamidation. In some aspects, proteins can be denatured using reagents such as guanidine and urea. The denatured protein is 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 thus fully accessible to proteases. The denatured and reduced protein is 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 on a mass spectrometer, typically a Q-ToF or Orbitrap, using their m / z ratio. 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 aspects, the method further comprises determining the post-translational modification of at least one of the VP1, VP2, and VP3 capsid proteins. In some aspects, the method further comprises post-translational phosphorylation or acetylation of at least one of the VP1, VP2, and VP3 capsid proteins.

[0015] The present 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 host cell proteins.

[0016] In some embodiments, 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 repetitive MS / MS.

[0018] In some embodiments, the digestion is performed 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 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 digested host cell proteins as compared to at least one protein standard.

[0020] In some embodiments, the liquid chromatography is reverse-phase liquid chromatography. In some embodiments, the reverse-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 - 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 in length and has an inner diameter of about 1 mm to about 4.6 mm. In some embodiments, the column is about 150 mm in length 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 includes a first mobile phase containing formic acid. In some embodiments, the first mobile phase contains formic acid at about 0.05% to about 0.15% by volume. In some embodiments, the first mobile phase contains formic acid at about 0.1% by volume.

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

[0024] In some embodiments, in the liquid chromatography, compared to the combination of the first mobile phase and the second mobile phase, the percentage of 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%. 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 spectrometry is performed using a fragmenter voltage of about 125 - 350 V. In some embodiments, mass spectrometry is performed using a fragmenter voltage of about 135 V. In some embodiments, mass spectrometry is performed using a capillary voltage of about 3 - 6 kV. In some embodiments, mass spectrometry is performed using a capillary voltage of about 4 kV.

[0026] Some embodiments of the present disclosure are directed to recombinant AAV (rAAV) containing a heterogeneous group of capsid proteins containing subpopulations with amino acid modifications. In some embodiments, the modification is deamidation or oxidation.

[0027] In some embodiments, when measured by mass spectrometry and / or ultraviolet (UV) visible spectroscopy, the heterogeneous group contains deamidated asparagine (N) at one or more of N57, N255, N256, and N263 of AAV.rh74, or at equivalent residues of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, or AAVrh10. In another embodiment, the heterogeneous group contains deamidated asparagine (N) within the peptide sequence of any one of SEQ ID NOs: 1 - 5, or its equivalent peptide sequence of other AAV serotypes.

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

[0029] In some embodiments, when measured by mass spectrometry and / or ultraviolet (UV) - visible spectroscopy, the heterogeneous population comprises methionine - oxide at one or more of M437, M473, M526, M544, M560, and M637 of AAV.Rh74, or at equivalent residues of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, CAAV11, AAV12, AAV13, or AAVrh10. In some embodiments, the heterogeneous population comprises less than 30%, 20%, 10%, 5%, or 1% of the capsid protein oxidized at M437. In some embodiments, the heterogeneous population comprises less than 30%, 20%, 10%, 5%, or 1% of the capsid protein oxidized at M473. In some embodiments, the heterogeneous population comprises less than 30%, 20%, 10%, 5%, or 3% of the capsid protein oxidized at M526. In some embodiments, the heterogeneous population comprises less than 30%, 20%, 10%, 5%, or 2% of the capsid protein oxidized at M544. In some embodiments, the heterogeneous population comprises less than 30%, 20%, 10%, 5%, or 2% of the capsid protein oxidized at M560. In some embodiments, the heterogeneous population comprises less than 30%, 20%, 10%, 5%, or 1% of the capsid protein oxidized at 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, individually or collectively, all such steps, features, compositions, and compounds referred to or indicated in this specification, and any and all combinations of any two or more of these steps or features. In embodiments of the present invention, for example, the following items are provided. (Item 1) A method for characterizing VP1, VP2, and VP3 capsid proteins in adeno - associated virus (AAV) particles, comprising subjecting the AAV particles to liquid chromatography at about 70 °C to about 90 °C, a method wherein the ratio of the VP1, VP2, and VP3 capsid proteins is determined by mass spectrometry and / or ultraviolet (UV) visible spectroscopy. (Item 2) The method according to item 1, wherein the individual masses of the VPl, VP2, and VP3 capsid proteins are determined by the mass spectrometry. (Item 3) The method according to item 1 or 2, wherein the ratio of the VP1, VP2, and VP3 capsid proteins is determined by comparing the 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) The method according to item 4, wherein the reverse-phase liquid chromatography is performed using a C18 column, a C8 column, or a C4 column. (Item 6) The method according to item 5, wherein the liquid chromatography is performed using a C8 column. (Item 7) The method according to item 5 or 6, wherein the column contains particles of about 1.2 to 3.5 μm. (Item 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) The method according to any one of items 5 to 8, wherein the column has a length of about 50 mm to about 300 mm and an inner diameter of about 1 mm to about 4.6 mm. (Item 10) The method according to item 9, wherein the chromatography column has a length of about 100 mm and an inner diameter of about 2.1 mm. (Item 11) The method according to any one of items 1 to 10, wherein the AAV particles are of serotype AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAVrh10, AAVrh74, or are any natural, recombinant, or synthetic AAV particles. (Item 12) The method according to any one of items 1 to 11, wherein the AAV is of serotype AAVrh74. (Item 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) The method according to any one of items 1 to 13, wherein the liquid chromatography is carried out at about 80 °C. (Item 15) The method according to any one of items 1 to 14, wherein the liquid chromatography comprises a first mobile phase containing trifluoroacetic acid. (Item 16) The method according to item 15, wherein the first mobile phase contains about 0.05 to about 0.15% trifluoroacetic acid by volume. (Item 17) The method according to item 15 or 16, wherein the first mobile phase contains about 0.1% trifluoroacetic acid by volume. (Item 18) The method according to any one of items 1 to 17, wherein the liquid chromatography comprises a second mobile phase containing trifluoroacetic acid in the mixture of acetonitrile and water. (Item 19) The method according to item 18, wherein the second mobile phase contains about 0.05 to about 0.15% trifluoroacetic acid by volume. (Item 20) The method according to item 18 or 19, wherein the second mobile phase contains about 0.1% trifluoroacetic acid by volume. (Item 21) The method according to any one of items 18 to 20, wherein the second mobile phase contains about 80 to 95% acetonitrile by volume. (Item 22) The method according to any one of items 18 to 21, wherein the second mobile phase contains about 90% acetonitrile and 10% water by volume. (Item 23) The method according to any one of items 18 to 22, wherein in the liquid chromatography, the percentage of the second mobile phase is increased over time as compared with the combination of the first mobile phase and the second mobile phase. (Item 24) The method according to item 23, wherein the percentage of the second mobile phase is increased from about 10% to about 40% by volume, and then from about 40% to about 45%. (Item 25) The method according to item 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) The method according to item 25, wherein the percentage of the second mobile phase is increased to 100% by volume over about 1 minute in the combination of the first mobile phase and the second mobile phase. (Item 27) The method according to item 26, wherein the percentage of the second mobile phase is reduced to 10% by volume over about 1 minute in the combination of the first mobile phase and the second mobile phase. (Item 28) The method according to any one of items 1 to 27, wherein the mass spectrometry is performed using a fragmenter voltage of about 125 to 350 V. (Item 29) The method according to any one of items 1 to 28, wherein the mass spectrometry is performed using a fragmenter voltage of about 175 V. (Item 30) The method according to any one of items 1 to 29, wherein the mass spectrometry is performed using a capillary voltage of about 3 to 6 kV. (Item 31) The method according to any one of items 1 to 30, wherein the mass spectrometry is performed using a capillary voltage of about 5 kV. (Item 32) The method according to item 1, wherein the characterization further includes determining at least one post-translational modification of VPl, VP2, and VP3 capsid proteins. (Item 33) The method according to item 32, wherein the post-translational modification includes one or more of amino acid deletion, glycosylation, sialylation, acetylation, phosphorylation, deamidation, oxidation, formylation, hydroxylation, methylation, and sulfation. (Item 34) The method according to item 32, wherein the post-translational modification includes 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 performed using the gradient program listed in Table 2. (Item 36) The method according to item 32, wherein the characterization uses a buffer containing Tris-HCl. (Item 37) The method according to item 36, wherein the buffer contains acetonitrile. (Item 38) The method according to item 36, wherein the buffer contains methionine. (Item 39) The method according to any one of items 36 to 38, wherein the buffer contains 5 mM to 50 mM Tris-HCl, 5% to 20% acetonitrile, and 1 mM to 50 mM methionine. (Item 40) The method according to item 39, wherein the buffer contains 20 mM Tris-HCl, 5% to 10% acetonitrile, and 10 mM methionine. (Item 41) The method according to item 36, wherein the Tris-HCl has a pH of 7.5. (Item 42) The method according to item 32, wherein the post-translational modification comprises deamidation of one or more of N263, N514, N57, N502, N254, and N94 of AAV8, or their equivalent residues in AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV9, AAV10, AAV11, AAV12, AAV13, AAVrh10, or AAVrh74. (Item 43) The method according to item 32, wherein the post-translational modification comprises deamidation of one or more of N57, N255, N256, and N263 of AAV.Rh74. (Item 44) The method according to item 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 of one or more of N57, N255, N256, and N263 of AAV.Rh74, or their equivalent residues in AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, or AAVrh10. (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 their equivalent residues in AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, CAAV11, AAV12, AAV13, or AAVrh10. (Item 47) A method for characterizing host cell proteins in an AAV composition, comprising: immunoprecipitating viral capsid proteins from the composition; digesting residual host cell proteins; A method comprising identifying host cell proteins by analyzing the digested proteins using liquid chromatography quadrupole time-of-flight mass spectrometry (LC-QTOF-MS). (Item 48) The method according to item 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) The method according to item 47 or 48, further comprising analyzing the digested host cell proteins by iterative MS / MS. (Item 50) The method according to any one of items 47 to 49, wherein the digestion is performed in solution. (Item 51) The method according to item 50, wherein the digestion comprises rapid digestion performed at a temperature of about 60°C to about 80°C. (Item 52) The method according to item 51, wherein the temperature is about 70°C. (Item 53) The method according to 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) The method according to item 53, wherein the at least one known protein standard is a human or bovine protein standard. (Item 55) The method according to item 53 or 54, further comprising quantifying the amount of the digested host cell proteins as compared to the at least one protein standard. (Item 56) The method according to any one of items 47 to 55, wherein the liquid chromatography is reverse-phase liquid chromatography. (Item 57) The method according to item 56, wherein the reverse-phase liquid chromatography is performed using a C18 column, a C8 column, or a C4 column. (Item 58) The method according to item 57, wherein the liquid chromatography is carried out using a C8 column. (Item 59) The method according to item 57 or 58, wherein the column contains particles of about 1.2 to 3.5 μm. (Item 60) The method according to any one 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 the preceding items. (Item 61) The method according to any one of items 57 to 60, wherein the column has a length of about 50 mm to about 300 mm and an inner diameter of about 1 mm to about 4.6 mm. (Item 62) The method according to item 61, wherein the column has a length of about 150 mm and an inner diameter of about 2.1 mm. (Item 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) The method according to any one of items 47 to 63, wherein the liquid chromatography is carried out at about 45 °C. (Item 65) The method according to any one of items 47 to 64, wherein the liquid chromatography comprises a first mobile phase containing formic acid. (Item 66) The method according to item 65, wherein the first mobile phase contains formic acid in an amount of about 0.05% to about 0.15% by volume. (Item 67) The method according to item 65 or 66, wherein the first mobile phase contains formic acid in an amount of about 0.1% by volume. (Item 68) The method according to any one of items 47 to 67, wherein the liquid chromatography comprises a second mobile phase containing formic acid in a mixture of acetonitrile and water. (Item 69) The method according to item 68, wherein the second mobile phase contains formic acid in an amount of about 0.05% to about 0.15% by volume. (Item 70) The method according to item 68 or 69, wherein the second mobile phase contains formic acid at about 0.1% by volume. (Item 71) The method according to any one of items 68 to 70, wherein the second mobile phase contains acetonitrile at about 80 to 95% by volume. (Item 72) The method according to item 71, wherein the second mobile phase contains acetonitrile at about 90% by volume and water at 10% by volume. (Item 73) The method according to any one of items 68 to 72, wherein in the liquid chromatography, the percentage of the second mobile phase is increased over time as compared with the combination of the first mobile phase and the second mobile phase. (Item 74) The method according to item 73, wherein the percentage of the second mobile phase is increased from about 2% to about 50%. (Item 75) The method according to item 74, wherein the percentage of the second mobile phase is increased from about 2% to about 50% by volume over about 120 minutes. (Item 76) The method according to item 75, wherein the percentage of the second mobile phase is then increased to 100% by volume in about 5 minutes. (Item 77) The method according to item 76, wherein the percentage of the second mobile phase is then maintained at 100% by volume for about 3 minutes. (Item 78) The method according to item 77, wherein the percentage of the second mobile phase is then decreased to about 2% by volume in about 2 minutes. (Item 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) The method according to any one of items 47 to 79, wherein the mass spectrometry is carried out using a fragmentor voltage of about 135 V. (Item 81) The method according to any one of items 47 to 80, wherein the mass spectrometry is performed using a capillary voltage of about 3 to 6 kV. (Item 82) The method according to any one of items 47 to 81, wherein the mass spectrometry is performed using a capillary voltage of about 4 kV. (Item 83) A recombinant AAV (rAAV) comprising a heterogeneous group of capsid proteins containing a subpopulation having an amino acid modification, wherein the amino acid modification is a post-translational modification. (Item 84) The rAAV according to item 83, wherein the modification is deamidation or oxidation. (Item 85) When measured by mass spectrometry and / or ultraviolet (UV) visible spectroscopy, the heterogeneous group is one or more of N57, N255, N256, and N263 of AAV.rh74, or equivalent residues of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, or AAVrh10, and contains deamidated asparagine (N). The rAAV according to item 83 or 84. (Item 86) The rAAV according to item 85, wherein the heterogeneous group contains less than 70%, 60%, 50%, 40%, 30%, 20%, or 15% of the capsid protein deamidated at N57 of the AAV.rh74 capsid. (Item 87) The rAAV according to item 85, wherein the heterogeneous group contains less than 15% of the capsid protein deamidated at N57 of the AAV.rh74 capsid. (Item 88) The rAAV according to item 85, wherein the heterogeneous group contains less than 70%, 60%, 50%, 40%, 30%, 20%, 15%, 10%, or 5% of the capsid protein deamidated at N254 and / or N255 of AAV.rh74. (Item 89) The rAAV according to item 85, wherein the heterogeneous group contains 70%, 60%, 50%, 40%, 30%, less than 20% of the capsid protein deamidated with N263. (Item 90) (Item 91) The rAAV according to item 83 or 84, wherein when measured by mass spectrometry and / or ultraviolet (UV) visible spectroscopy, the heterogeneous group contains methionine oxide at one or more of M437, M473, M526, M544, M560, and M637 of AAV.Rh74, or at equivalent residues of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, CAAV11, AAV12, AAV13, or AAVrh10. (Item 92) The rAAV according to item 90, wherein the heterogeneous group contains less than 30%, 20%, 10%, 5%, or 1% of the capsid protein oxidized at M437. (Item 93) The rAAV according to item 90, wherein the heterogeneous group contains less than 30%, 20%, 10%, 5%, or 1% of the capsid protein oxidized at M473. (Item 94) The rAAV according to item 90, wherein the heterogeneous group contains less than 30%, 20%, 10%, 5%, or 3% of the capsid protein oxidized at M526. (Item 95) The rAAV according to item 90, wherein the heterogeneous group contains less than 30%, 20%, 10%, 5%, or 2% of the capsid protein oxidized at M544. (Item 96) The rAAV according to item 90, wherein the heterogeneous group contains less than 30%, 20%, 10%, 5%, or 2% of the capsid protein oxidized at M560. (Item 97) The rAAV according to item 90, wherein the heterogeneous group contains less than 30%, 20%, 10%, 5%, or 1% of the capsid protein oxidized at M637. (Item 98) Item 83, wherein the heterogeneous group comprises deamidated asparagine (N) within one of the peptide sequences of SEQ ID NOs: 1-5, or its equivalent peptide sequence of another AAV serotype The rAAV according to any one of items 83

Brief Description of the Drawings

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

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[0038] Provided are methods 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 are methods for determining the ratios of VP1, VP2, and VP3 capsid proteins in AAV particles and / or the mass of one or more of the VP1, VP2, and VP3 capsid proteins. The present disclosure also provides methods for characterizing the purity of rAAV compositions using liquid chromatography and mass spectrometry. Definition

[0039] For convenience, before further describing the invention, certain terms used herein, in the examples, and in the appended claims are grouped here. These definitions are to be read and understood in view of the remainder of the disclosure by those 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. The terms used throughout this specification are defined as follows, unless otherwise limited in a particular instance.

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

[0041] As used herein, "about" is used to indicate that a value includes the error variation inherent in the variations that exist between devices, the methods employed to determine the value, or the test subjects. In some embodiments, "about" indicates that a deviation of from 5% to more than 10% (e.g., from 5% to up to 10% more) and from 5% to less than 10% (e.g., from 5% to up to 10% less) 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 family Parvoviridae. As used herein, AAV can refer to wild-type virus, or AAV derived from a natural wild-type virus, e.g., AAV derived from an rAAV genome packaged within a capsid derived from a capsid protein encoded by a native cap gene, and / or AAV derived from an rAAV genome packaged within a capsid derived from a capsid protein encoded by a non-native cap gene, e.g., AAVrh.74.

[0043] AAV can be of any serotype, e.g., 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, e.g., in WO01 / 83692, which is incorporated by reference in its entirety. Other types of rAAV variants, e.g., rAAV having capsid mutations, are also contemplated. See, e.g., Marsic et al., Molecular Therapy, 22(11):1900-1909(2014).

[0044] As used herein, the terms "AAV particle", "AAV vector", "AAV virion", "AAV virus particle", or "AAV vector particle" are used to refer to virus particles composed of an AAV capsid and a packaged AAV genome. In some embodiments, AAV particles contain a heterologous polynucleotide (i.e., a polynucleotide other than the wild-type AAV genome, such as a transgene to be delivered to mammalian cells). Production of AAV vector particles, in some embodiments, includes production of an AAV vector, where such a vector is contained within the AAV vector particles.

[0045] For example, wild-type (wt) AAV virus particles containing a linear single-stranded AAV nucleic acid genome that associates with the AAV capsid protein coat. The AAV virion can be either single-stranded (ss) AAV or self-complementary (SC) AAV. In some embodiments, either complementary sense strand, e.g., the "sense" strand or the "antisense" strand, of a single-stranded AAV nucleic acid molecule 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-deficient virus composed of an AAV protein shell encapsulating 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 an AAV vector (containing the recombinant nucleotide sequence of interest) into infectious recombinant virion particles 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) composed of a total of 60 subunits, each subunit being an amino acid sequence, e.g., 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 passes through a column, and the components of the mobile phase interact with a solid stationary phase. The composition of the mobile phase can be changed during a separation run to vary the strength of the interaction of the compound of interest. As the mobile phase flows through the column, typically, the concentration of the compounds eluting from the column over time is monitored to generate an elution curve or chromatogram while collecting the eluent in fractions.

[0049] As used herein, the term "stationary phase" refers to a substance that remains fixed in a column. The most commonly used stationary phase columns are carbon chain-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, reverse-phase chromatography is C8 reverse-phase chromatography (e.g., reverse-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 a solvent used to elute a substance from a column. In some embodiments, the volume ratio of the second mobile phase to the first mobile phase is in a gradient that increases 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 a compound to form a charged compound, and (2) detecting the mass-to-charge ratio of the charged compound to calculate the molecular weight. The compound can be ionized and detected by any suitable means. A "mass spectrometer" generally includes an ionization device, a mass analyzer, and an ion detector. Generally, one or more target molecules are ionized, and then the ions are introduced into a mass spectrometry device where, by a combination of a magnetic field and an electric field, the ions follow a path in space that depends on their mass ("m") and charge ("z"). In some mass spectrometry methods, the ions can be separated from each other using, for example, time-of-flight (TOF), an orbitrap, a Fourier transform ion cyclotron resonance spectrometer, a quadrupole, or an ion trap, and then detected using an ion detector.

[0052] As used herein, the term "ultraviolet-visible spectroscopy", "ultraviolet-visible spectrophotometry", "UV-Vis", or "UV / Vis" refers to an absorption spectroscopy 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 proteins 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 AAV particles having the VP1, VP2, and VP3 capsid proteins of AAVrh74 or variants thereof. An exemplary VP1 capsid protein sequence of AAVrh74 is set forth in SEQ ID NO: 4 of U.S. Patent No. 9,909,142, which is hereby incorporated by reference in its entirety. Examples of variants of the VP1 capsid protein of AAVrh74 are also described in U.S. Patent No. 9,909,142.

[0055] As used herein, the term "subpopulation" of VP proteins, unless otherwise specified, refers to a group of VP proteins that commonly have at least one defined characteristic and that do not meet for all members from at least one group member of a reference group. For example, unless otherwise specified, a "subpopulation" of VPl proteins can be VP1 proteins that do not meet for all from at least one VPl protein in an assembled AAV capsid. A "subpopulation" of VP3 proteins can be VP3 proteins that do not meet for all from one VP3 protein in an assembled AAV capsid. For example, the VPl protein may be a subpopulation of VP proteins in an assembled AAV capsid, the VP2 protein may be another subpopulation of VP proteins, and VP3 may be yet another subpopulation of VP proteins. In another example, the VPl, VP2, and VP3 proteins may contain subpopulations having different modifications, such as at least one, two, three, or four highly deamidated asparagines, for example, in an asparagine-glycine pair. Characterization of AAV VP1, VP2, and VP3 Capsid Proteins

[0056] In some embodiments, the present disclosure provides a method for characterizing VP1, VP2, and VP3 capsid proteins in adeno-associated virus (AAV) particles, the method comprising subjecting the AAV particles to liquid chromatography at from about 70 °C to about 90 °C, wherein the masses and ratios of the VP1, VP2, and VP3 capsid proteins are determined by mass spectrometry and / or ultraviolet (UV) - visible spectroscopy. In some embodiments, the masses and ratios of the VP1, VP2, and VP3 capsid proteins are determined by mass spectrometry and ultraviolet (UV) - visible spectroscopy. In some embodiments, the capsid on the AAV particles is denatured to each of the VP1, VP2, and VP3 proteins within the column of the liquid chromatography. In some embodiments, the capsid proteins are separated by liquid chromatography. In some embodiments, the method comprises: (a) subjecting the AAV particles to liquid chromatography to separate the 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 ultraviolet - visible spectroscopy to determine the relative amounts of the VP1, VP2, and VP3 capsid proteins, thereby determining the ratio of the VP1, VP2, and VP3 capsid proteins in the AAV particles. In some embodiments, the liquid chromatography is performed at from 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 comprises determining the mass of one or more of the VPl, VP2, and VP3 capsid proteins of the AAV particles.

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

[0059] In some embodiments, liquid chromatography is reverse-phase liquid chromatography, size-exclusion chromatography, hydrophilic interaction liquid chromatography, or cation-exchange chromatography. In some embodiments, liquid chromatography is reverse-phase liquid chromatography.

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

[0061] In some embodiments, the stationary phase of the reverse-phase liquid chromatography is contained within a chromatography column having a length of about 50 to 300 mm and an inner diameter of about 1 to 4.6 mm. In some embodiments, the column is a BEH column. In some embodiments, the column has an inner 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 × 50 mm, 2.1 mm × 50 mm, 3 mm × 50 mm, 4.6 mm × 50 mm, 1 mm × 75 mm, 2.1 mm × 75 mm, 3 mm × 75 mm, 4.6 mm × 75 mm, 1 mm × 100 mm, 2.1 mm × 100 mm, 3 mm × 100 mm, 4.6 mm × 100 mm, 1 mm × 150 mm, 2.1 mm × 150 mm, 3 mm × 150 mm, 4.6 mm × 150 mm, 1 mm × 300 mm, 2.1 mm × 300 mm, 3 mm × 300 mm, or 4.6 mm × 300 mm. In some embodiments, the column size is 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 × 80 mm, 1.7 × 90 mm, 1.7 × 100 mm, 1.7 × 110 mm, 1.7 × 120 mm, 1.7 × 130 mm, 1.7 × 140 mm, 1.7 × 150 mm, 1.8 × 50 mm, 1.8 × 60, 1.8 × 70 mm, 1.8 × 80 mm, 1.8 × 90 mm, 1.8 × 100 mm, 1.8 × 110 mm, 1.8 × 120 mm, 1.8 × 130 mm, 1.8 × 140 mm, 1.8 × 150 mm, 1.9 × 50 mm, 1.9 × 60 mm, 1.9 × 70 mm, 1.9 × 80 mm, 1.9 × 90 mm, 1.9 × 100 mm, 1.9 × 110 mm, 1.9 × 120 mm, 1.9 × 130 mm, 1.9 × 140 mm, 1.9 × 150 mm, 2.0 × 50 mm, 2.0 × 60 mm, 2.0 × 70 mm, 2.0 × 80 mm, 2.0 × 90 mm, 2.0 × 100 mm, 2.0 × 110 mm, 2.0 × 120 mm, 2.0 × 130 mm, 2.0 × 140 mm, 2.0 × 150 mm, 2.1 × 50 mm, 2.1 × 60 mm, 2.1 × 70 mm, 2.1 × 80 mm, 2.1×90 mm, 2.1×100 mm, 2.1×110 mm, 2.1×120 mm, 2.1×130 mm, 2.1×140 mm, 2.1×150 mm, 2.2×50 mm, 2.2×60 mm, 2.2×70 mm, 2.2×80 mm, 2.2×90 mm, 2.2×100 mm, 2.2×110 mm, 2.2×120 mm, 2.2×130 mm, 2.2×140 mm, 2.2×150 mm, 2.3×50 mm, 2.3×60 mm, 2.3×70 mm, 2.3×80 mm, 2.3×90 mm, 2.3×100 mm, 2.3×110 mm, 2.3×120 mm, 2.3×130 mm, 2.3×140 mm, 2.3×150 mm, 2.4×50 mm, 2.4×60 mm, 2.4×70 mm, 2.4×80 mm, 2.4×90 mm, 2.4×100 mm, 2.4×110 mm, 2.4×120 mm, 2.4×130 mm, 2.4×140 mm, 2.4×150 mm, 2.5×50 mm, 2.5×60, 2.5×70 mm, 2.5×80 mm, 2.5×90 mm, 2.5×100 mm, 2.5×110 mm, 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 stationary phase of the reversed-phase liquid chromatography is contained within a chromatography column having a length of about 100 mm and an inner diameter of about 2.1 mm.

[0062] In some embodiments, the stationary phase of the reversed-phase liquid chromatography comprises particles sized between about 1.2 μm and 2.5 μm. In another embodiment, the stationary phase of the reversed-phase liquid chromatography comprises particles sized 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.

[0063] In some embodiments, chromatography uses a first mobile phase that contains a fluorinated acetic acid in water. Examples of the fluorinated acetic acid include monofluoroacetic acid, difluoroacetic acid, and trifluoroacetic acid. In some embodiments, chromatography uses a first mobile phase that contains trifluoroacetic acid in water.

[0064] In some embodiments, the first mobile phase contains from about 0.05 to about 0.15% fluorinated acetic acid by volume. In some embodiments, the first mobile phase contains 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% fluorinated acetic acid by volume. In some embodiments, the first mobile phase contains about 0.05% or 0.1% fluorinated acetic acid by volume. In some embodiments, the first mobile phase contains about 0.1% fluorinated acetic acid by volume. In some embodiments, the fluorinated acetic acid is trifluoroacetic acid. In some embodiments, the first mobile phase contains about 0.1% trifluoroacetic acid by volume.

[0065] In some embodiments, chromatography uses a second mobile phase that contains a fluorinated acetic acid in acetonitrile. In some embodiments, chromatography uses a second mobile phase that contains trifluoroacetic acid in acetonitrile. In some embodiments, chromatography uses a second mobile phase that contains a fluorinated acetic acid in a mixture of acetonitrile and water. In some embodiments, chromatography uses a second mobile phase that contains trifluoroacetic acid in a mixture of acetonitrile and water.

[0066] In some embodiments, the second mobile phase comprises from about 0.05 to 0.2% by volume of a fluorinated acetic acid. In some embodiments, the second mobile phase comprises from about 0.05 to 0.15% by volume of a fluorinated acetic acid. In some embodiments, the second mobile phase comprises from 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 a fluorinated acetic acid. In some embodiments, the second mobile phase comprises from about 0.05% or 0.1% by volume of a fluorinated acetic acid. In some embodiments, the second mobile phase comprises about 0.1% by volume of a fluorinated acetic acid. In some embodiments, the fluorinated acetic acid is trifluoroacetic acid. In some embodiments, the second mobile phase comprises about 0.1% by volume of trifluoroacetic acid.

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

[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 increases 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% by volume in about 30 - 40 minutes. In some embodiments, the percentage of the second mobile phase is increased from about 10% to about 45% by volume 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 decreased 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 decreased from about 100% to about 10% in about 1 minute.

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

[0072] In some embodiments, the mass spectrometry may use any ionization mode, specifically a mode suitable for the analysis of biomolecules, such as, but not limited to, direct injection 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 (EI) or chemical ionization (CI), matrix-assisted laser desorption / ionization (MALDI)-MS, and atmospheric pressure ionization-electrospray (API-ES). In some embodiments, the mass spectrometry uses the API-ES ionization mode.

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

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

[0075] In some embodiments, the nozzle voltage of mass spectrometry is about 400 - 600V. In some embodiments, the nozzle voltage of mass spectrometry is about 500V. In some embodiments, the skimmer voltage of mass spectrometry is about 60 - 70V. In some embodiments, the skimmer voltage of mass spectrometry is about 65V. In some embodiments, the difference between the nozzle voltage and the skimmer voltage is about 400 - 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 of mass spectrometry is about 200 - 350°C. In some embodiments, the drying gas temperature of mass spectrometry is about 300°C. In some embodiments, the drying gas flow rate of mass spectrometry is about 5 - 13 L / min. In some embodiments, the drying gas flow rate of mass spectrometry is about 13 L / min.

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

[0078] In some embodiments, mass spectrometry uses a fragmenter voltage of about 125 - 350V. In some embodiments, mass spectrometry uses a fragmenter 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, mass spectrometry uses a fragmenter voltage of about 175V.

[0079] In some embodiments, the AAV particle is an AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAVrh10, 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 AAVrh74.

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

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

[0082] In some embodiments, the disclosure provides a method for characterizing the capsid protein of an AAV particle, based at least in part on the ratio of the 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 embodiments, the present disclosure provides a method for determining the serotype of an AAV particle based at least in part on the ratio of the VP1, VP2, and VP3 capsid proteins in the AAV particle and / or the mass of one or more of the VPl, VP2, and VP3 capsid proteins, wherein the ratio of the VP1, VP2, and VP3 capsid proteins and the mass of one or more of the VPl, VP2, and VP3 capsid proteins are determined by the methods disclosed herein.

[0084] Mass spectrometry is an analytical technique for characterizing proteins. In some embodiments, liquid chromatography and mass spectrometry are used to provide a method for characterizing the ratio of the AAVrh74 capsid proteins, along with the intact mass of all three capsid proteins. In some embodiments, the AAVrh74 capsid is denatured on-column into the individual capsid proteins VP1, VP2, and VP3. Denaturation is achieved by heating the column compartment to 80° C. (3, 4). Next, trifluoroacetic acid is utilized as an ion-pairing agent in the mobile phase to baseline separate the capsid proteins on a Waters BEH C8 column (5). The denatured proteins are first analyzed by UV to obtain the capsid ratio and then by mass spectrometry to obtain the intact mass 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 acid and aspartic acid. Deamidation of glutamine residues also occurs, but at a much slower rate. Oxidation is also a common post-translational modification that results from the reaction of proteins with various free radicals and reactive oxygen species. Methionine oxidation is the most common, but oxidation of some 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 the activity and stability of proteins. Oxidation can cause conformational changes in proteins and thus may affect the activity and stability of proteins. Oxidation can also affect the immunogenicity of proteins. Therefore, it is necessary to carefully monitor post-translational modifications for very important quality characteristics (CQAs) of proteins.

[0086] The current method using ammonium bicarbonate either generated false signals or overestimated deamidation in the AAV capsid protein (Table 7). Therefore, the present disclosure provides a method for more accurately measuring post-translational modifications on the capsid protein 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 at the equivalent residues in AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV9, AAV10, AAV11, AAV12, AAV13, AAVrh10, 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 embodiments of the present disclosure are directed to recombinant AAV (rAAV) comprising a heterogeneous group of capsid proteins containing a subpopulation having an amino acid modification. In some embodiments, the modification can be deamidation, acetylation, isomerization, phosphorylation, or oxidation. In some embodiments, the modification is deamidation or oxidation.

[0089] In some embodiments, the rAAV capsid can contain a subpopulation of VPl, VP2, and VP3 having at least 1, at least 2, at least 3, at least 4, at least 5 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, most of these can be N residues. In some embodiments, Q residues can be deamidated.

[0090] In some embodiments, the present disclosure provides an AAV composition comprising an AAV capsid that contains deamidation at one or more of N57, N255, N256, and N263 of AAV.Rh74, or at equivalent residues of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, or AAVrh10, as measured by mass spectrometry and / or ultraviolet (UV) visible spectroscopy. In some embodiments, the 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 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% of the capsid protein deamidated at N57 of the AAV.rh74 capsid.

[0092] In some embodiments, 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 the 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%, 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%, or less than about 1% of the capsid protein deamidated at N254 and / or N255 of the AAV.rh74 capsid.

[0094] 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%, 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% of the capsid protein deamidated at N263.

[0095] In some embodiments, the AAV composition comprises an AAV capsid that contains oxidation in one or more of M437, M473, M526, M544, M560, and M637 of AAV.Rh74, or in equivalent residues of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, or AAVrh10, 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% of the capsid protein oxidized at 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% of the capsid protein oxidized at 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 oxidized by 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 oxidized by 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 oxidized by M560.

[0101] In some embodiments, the heterogeneous group 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 oxidized by M637. Characterization of Host Cell Proteins in AAV Compositions

[0102] In some embodiments, 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 embodiments, the method for characterizing host cell proteins in an AAV composition comprises 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 protein" or "residual protein" means the protein remaining in solution after immunoprecipitation. In some embodiments, the method further comprises analyzing the digested host cell proteins by tandem MS / MS.

[0103] In some embodiments, 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 anti-adeno-associated virus (AAV) VP1 / VP2 / VP3 (catalog number: 03-61058) obtained from American Research Products.

[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 carried out at about 60 °C to about 80 °C. In some embodiments, the rapid digestion is carried out 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 carried out at about 70 °C.

[0105] In some embodiments, the AAV composition is spiked with at least one known protein standard of known amount. 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 compared to at least one known protein standard.

[0106] In some embodiments, the liquid chromatography is reverse phase liquid chromatography, size exclusion chromatography, hydrophilic interaction liquid chromatography, or cation exchange chromatography. In some embodiments, the liquid chromatography is reverse phase liquid chromatography.

[0107] In some embodiments, the liquid chromatography is carried out at about 35 °C to about 55 °C. In some embodiments, the liquid chromatography is carried out 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, the liquid chromatography is carried out at about 45 °C.

[0108] In some embodiments, the reverse phase chromatography is carried out using a C18 column, a C8 column, or a C4 column. In some embodiments, the liquid chromatography is carried out 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 to 300 mm and an inner diameter of about 1 to 4.6 mm. In some embodiments, the column is a BEH column. In some embodiments, the column has an inner 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 × 50 mm, 2.1 mm × 50 mm, 3 mm × 50 mm, 4.6 mm × 50 mm, 1 mm × 75 mm, 2.1 mm × 75 mm, 3 mm × 75 mm, 4.6 mm × 75 mm, 1 mm × 100 mm, 2.1 mm × 100 mm, 3 mm × 100 mm, 4.6 mm × 100 mm, 1 mm × 150 mm, 2.1 mm × 150 mm, 3 mm × 150 mm, 4.6 mm × 150 mm, 1 mm × 300 mm, 2.1 mm × 300 mm, 3 mm × 300 mm, or 4.6 mm × 300 mm. In some embodiments, the column size is 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 × 80 mm, 1.7 × 90 mm, 1.7 × 100 mm, 1.7 × 110 mm, 1.7 × 120 mm, 1.7 × 130 mm, 1.7 × 140 mm, 1.7 × 150 mm, 1.8 × 50 mm, 1.8 × 60, 1.8 × 70 mm, 1.8 × 80 mm, 1.8 × 90 mm, 1.8 × 100 mm, 1.8 × 110 mm, 1.8 × 120 mm, 1.8 × 130 mm, 1.8 × 140 mm, 1.8 × 150 mm, 1.9 × 50 mm, 1.9 × 60 mm, 1.9 × 70 mm, 1.9 × 80 mm, 1.9 × 90 mm, 1.9 × 100 mm, 1.9 × 110 mm, 1.9 × 120 mm, 1.9 × 130 mm, 1.9 × 140 mm, 1.9 × 150 mm, 2.0 × 50 mm, 2.0 × 60 mm, 2.0 × 70 mm, 2.0 × 80 mm, 2.0 × 90 mm, 2.0 × 100 mm, 2.0 × 110 mm, 2.0 × 120 mm, 2.0 × 130 mm, 2.0 × 140 mm, 2.0 × 150 mm, 2.1 × 50 mm, 2.1 × 60 mm, 2.1 × 70 mm, 2.1 × 80 mm, 2.1×90 mm, 2.1×100 mm, 2.1×110 mm, 2.1×120 mm, 2.1×130 mm, 2.1×140 mm, 2.1×150 mm, 2.2×50 mm, 2.2×60 mm, 2.2×70 mm, 2.2×80 mm, 2.2×90 mm, 2.2×100 mm, 2.2×110 mm, 2.2×120 mm, 2.2×130 mm, 2.2×140 mm, 2.2×150 mm, 2.3×50 mm, 2.3×60 mm, 2.3×70 mm, 2.3×80 mm, 2.3×90 mm, 2.3×100 mm, 2.3×110 mm, 2.3×120 mm, 2.3×130 mm, 2.3×140 mm, 2.3×150 mm, 2.4×50 mm, 2.4×60 mm, 2.4×70 mm, 2.4×80 mm, 2.4×90 mm, 2.4×100 mm, 2.4×110 mm, 2.4×120 mm, 2.4×130 mm, 2.4×140 mm, 2.4×150 mm, 2.5×50 mm, 2.5×60, 2.5×70 mm, 2.5×80 mm, 2.5×90 mm, 2.5×100 mm, 2.5×110 mm, 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 stationary phase of the reversed-phase liquid chromatography is contained within a chromatography column having a length of about 150 mm and an inner diameter of about 2.1 mm.

[0110] In some embodiments, the stationary phase of the reversed-phase liquid chromatography comprises particles sized between about 1.2 μm and 2.5 μm. In some embodiments, the stationary phase of the reversed-phase liquid chromatography comprises particles sized 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 about 1.7 μm in size.

[0111] In some embodiments, chromatography uses a first mobile phase that contains fluorinated acetic acid in water. Examples of fluorinated acetic acid include monofluoroacetic acid, difluoroacetic acid, and trifluoroacetic acid. In some embodiments, chromatography uses a first mobile phase that contains trifluoroacetic acid in water.

[0112] In some embodiments, chromatography uses a first mobile phase that contains formic acid.

[0113] In some embodiments, the first mobile phase contains from about 0.05 to about 0.15% formic acid by volume. In some embodiments, the first mobile phase contains 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 contains about 0.05% or 0.1% formic acid by volume. In some embodiments, the first mobile phase contains about 0.1% formic acid by volume.

[0114] In some embodiments, chromatography uses a second mobile phase that contains fluorinated acetic acid in acetonitrile. In some embodiments, chromatography uses a second mobile phase that contains trifluoroacetic acid in acetonitrile. In some embodiments, chromatography uses a second mobile phase that contains fluorinated acetic acid in a mixture of acetonitrile and water. In some embodiments, chromatography uses a second mobile phase that contains trifluoroacetic acid in a mixture of acetonitrile and water.

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

[0116] In some embodiments, the second mobile phase contains formic acid in an amount of about 0.05 to 0.2% by volume. In some embodiments, the second mobile phase contains formic acid in an amount of about 0.05 to 0.15% by volume. In some embodiments, the second mobile phase contains formic acid in an amount of 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. In some embodiments, the second mobile phase contains formic acid in an amount of about 0.05% or 0.1% by volume. In some embodiments, the second mobile phase contains formic acid in an amount of about 0.1% by volume.

[0117] In some embodiments, the second mobile phase contains acetonitrile in an amount of about 75 to 95% by volume. In some embodiments, the second mobile phase contains acetonitrile in an amount of about 75%, 80%, 85%, 90%, or 95% by volume. In some embodiments, the second mobile phase contains 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 increases 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 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.

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

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

[0122] In some embodiments, the mass spectrometry may use any ionization mode, specifically a mode suitable for the analysis of biomolecules, such as, but not limited to, direct injection 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 (EI) or chemical ionization (CI), matrix-assisted laser desorption / ionization (MALDI)-MS, and atmospheric pressure ionization-electrospray (API-ES). In some embodiments, the mass spectrometry uses the API-ES ionization mode.

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

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

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

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

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

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

[0129] The subject matter has been described in considerable detail with reference to certain specific embodiments, but other embodiments are possible. Accordingly, the spirit and scope of the appended claims should not be limited to the description of the specific embodiments contained herein.

Examples

[0130] The present disclosure will now be described with reference to examples, which are intended to illustrate the work of the present disclosure and are not intended to impose 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 one of ordinary skill in the art to which this 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 in liquid chromatography and solution preparation.

[0134]

Table 1-1

Table 1-2

[0135] 1.2 Equipment

[0136] The following equipment was used to implement the examples disclosed herein. a) Waters ACQUITY UPLC BEH C8 column, 2.1×100 mm, 1.7 μm; part number 186002878 b) Pierce surfactant removal spin column, 0.5 mL; catalog number 87777 c) 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 at 2 - 8 °C for 1 month.

[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 a 1 L graduated cylinder to make a 1 L 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 the solution were mixed well for 5 minutes to obtain a second mobile phase. The second mobile phase is stable for up to 1 month at ambient temperature.

[0144] Example 3: Preparation of Samples

[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 in a 2 mL collection tube and centrifuged at 1500 × g for 1 minute. When using a fixed-angle rotor, the packed resin was tilted upward and a mark was made on the side of the column. Then, in all subsequent steps, the column was placed in the centrifuge with this mark facing outward.

[0146] 400 μL of 100 mM ammonium bicarbonate solution was added to the column and the column was centrifuged at 1500 × g for 1 minute. This step was repeated two more times, and after each step, the flow-through was discarded. The column was placed in a new 2 mL collection tube. 5 μg of the sample was slowly applied on top of the packed resin bed and incubated at room temperature for 2 minutes. The column was centrifuged at 1500 × g for 2 minutes to collect the sample without polymer. Then, after adjusting the sample volume to a maximum of 100 μL with 100 mM ammonium bicarbonate, it was 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 for determining the ratios of the VP1, VP2, and VP3 capsid proteins in AAV particles, as well as the masses of the VPl, VP2, and VP3 capsid proteins. Here, the 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 ratios of the VP1, VP2, and VP3 capsid proteins in the AAV particles, and then subjected to mass spectrometry to obtain the respective masses of the VPl, VP2, and VP3 capsid proteins.

[0149] 4.1 LC operating conditions

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

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

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

Table 2-1

Table 2-2

[0153] 4.2 Operating Conditions of Mass Spectrometer (MS)

[0154] Mass spectrometry was performed using an Agilent 6545XT AdvanceBio quadrupole time-of-flight mass spectrometer (Q-ToF) with API-ES ionization in a survey scan in the range of m / z values from 700 to 13700 m / z. The capillary voltage, nozzle voltage, fragmentor voltage, and skimmer voltage were set to 5 kV, 500 V, 175 V, and 65 V, respectively. The dry gas temperature and dry gas flow rate were set to 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 protein was first denatured by heating the column compartment to 80 °C. Then, the three capsid proteins VP1, VP2, and VP3 were baseline separated 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 being increased 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 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 around 1:1:10 for VP1 / VP2 / VP3 was obtained by baseline integration of the UV chromatogram.

Table 4

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

Table 5

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

Table 6 - 1

Table 6 - 2

[0161] Example 5: Characterization of Deamidation of AAVrh74 Using LCMS

[0162] Widespread deamidation in the capsid protein can be determined by mass spectrometry, which can also determine the sites of deamidation in the capsid protein and the level of deamidation at these sites. To measure the deamidation of the AAV capsid, the capsid protein was denatured and reduced at 90 °C for 10 minutes in the presence of 2 M guanidine hydrochloride and 10 mM DTT. After cooling the sample to room temperature, 30 mM iodoacetamide was added for alkylation and incubated in the dark at room temperature for 30 minutes. Then, alkylation was quenched by the addition of 1 mL of DTT. 20 mM ammonium bicarbonate was added to the sample to dilute the guanidine hydrochloride to 200 mM. The sample was 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 the sample was analyzed on a Thermo UltiMate 3000 RSLC system coupled to a Q Exactive HF using a NanoFlex source.

[0163] Table 7 shows the identified major deamidation sites and the level of deamidation (i.e., percentage of deamidation) at these sites. The data for AAV8 in Table 7 are disclosed from 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, the sample was denatured by buffer exchange to 100 mM ammonium bicarbonate. The denatured sample was reduced by the addition of 10 mM DTT and incubated at 37 °C for 45 minutes. Subsequently, alkylation was carried out by adding iodoacetamide in the sample to a final concentration of 30 mM. Then, the denatured, reduced, and alkylated sample was buffer-exchanged back to 100 mM ammonium bicarbonate using a 10 kDa Amicon ultrafilter. Next, the sample was digested with trypsin and incubated at 37 °C overnight. Using this sample preparation digested in ammonium bicarbonate, a similar level of deamidation as that for AAVrh74 was obtained as shown in Table 7.

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

[0167] As shown in Table 7, when using Tris-HCl as the buffer, the deamidated state was significantly lower compared to that using ammonium bicarbonate.

[0168] To further optimize the method using Tris-HCl, eight separate AAV capsid samples were measured according to the flowchart in Figure 4. The samples were first denatured by performing buffer exchange to 6 M guanidine hydrochloride, 20 mM Tris-HCl, pH 7.5. The samples were then reduced by adding DTT to a final concentration of 10 mM and incubated at 37 °C for 45 minutes. Alkylation was performed by adding iodoacetamide to a final concentration of 30 mM and incubating in the dark at room temperature for 1 hour. The samples were buffer-exchanged again to 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. The samples were digested overnight at 37 °C using trypsin. The peptides were then separated on an Agilent 1290 U-HPLC using RP-HPLC. Next, the separated peptides were detected using an Agilent 6545XT QToF, and deamidation analysis was performed using MassHunter and Bioconfirm software. The state of deamidation is shown in Figure 5, and the state of oxidation is shown in Figure 6. Among the 52 asparagine residues present, deamidation was not observed in 48 residues (total asparagine residues in VP1: 56). Among the 39 glutamine residues present, deamidation was not observed in all 39 residues (total glutamine residues in VP1: 48). Using O18-labeled water, minor deamidation at N57 was shown to be an artifact related to sample preparation. Oxidation was not detected in the remaining five methionine residues (total methionine residues in VP1: 11). Among the 14 tryptophan residues detected, oxidation was not observed in all 14 residues (total tryptophan residues in VP1: 15).

[0169] The digestion of ammonium bicarbonate can significantly increase deamidation artifacts because the pH increases over time. Therefore, the higher levels of deamidation observed may have been deamidation artifacts generated during sample preparation. To confirm the deamidation levels, a Tris HCl-based digestion was set up at Sarepta. 20 mM Tris HCl, pH 7.5 was used as the buffer. Since it is known to reduce deamidation artifacts, 10% acetonitrile was added to the digestion solution. Also, to reduce oxidation artifacts, 10 mM methionine was added to the digestion solution.

[0170] Therefore, the method of the present disclosure is more accurate when measuring deamidation, oxidation, or other post-translational modifications with a Tris-HCl buffer.

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

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

[0173] 6.1 Sample preparation

[0174] To the AAVrh74 sample, known amounts of Invitrogen's human thioredoxin 1 (HTI) protein standard (catalog number LF-P0001) and Sigma's bovine carbonic anhydrase II (BCAII) protein standard (catalog number C7749) were added. Human thioredoxin 1 (HTI) and bovine carbonic anhydrase II (BCAII) were selected as spiking protein standards for quantifying the found human HCP and bovine HCP, 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 the sample solution were pipetted into 270 μL of the IP-MS cell lysis buffer of the Pierce MS-compatible magnetic IP kit together with 20 μL of 0.05 mg / mL BCAII and 10 μL of 0.1 mg / mL HTI. Subsequently, the AAV capsid protein was immunoprecipitated from the sample using anti-adeno-associated virus (AAV) VP1 / VP2 / VP3 (catalog number 03-6105) obtained from American Research Products, Inc. and the Pierce MS-compatible magnetic IP kit (catalog number 90409). Then, the sample was passed through a Pierce surfactant removal spin column (catalog number 87777).

[0175] Subsequently, the sample was buffer-exchanged into Promega rapid digestion buffer (catalog number VA1060). The sample was reduced, alkylated, and digested with rapid digestion trypsin at 70 °C for 60 - 180 minutes.

[0176] 6.2 LC operating conditions

[0177] Separation of the digested 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 phases used were as follows:

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

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

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

[0181] The operating conditions of the LC are listed in Table 8.

Table 8

[0182] Mass spectrometry was performed using an Agilent 6545XT AdvanceBio quadrupole time-of-flight mass spectrometer (Q-ToF) with API-ES ionization in a survey scan in the range of m / z values from 50 to 3000 m / z. The capillary voltage, nozzle voltage, fragmentor voltage, and skimmer voltage were set to 4 kV, 500 V, 135 V, and 65 V, respectively. The dry gas temperature and dry gas flow rate were set to 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 obtained from Protein Metrics and searched against a certain Uniprot protein database. The identity and relative amount of each residual protein were calculated relative to the amount of the spiked protein standard. In the HCP analysis, it was indicated that for 3 lots of AAV virus particles, there were very few host cell proteins identified by MS (only two bovine proteins and no human proteins) (Table 10). The protein concentration is on the order of ng / mL or at the ppm level based on the spiked protein standard.

Table 10

[0186] The following references are hereby incorporated by reference 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. D.W. Bauer, 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

【Claim 1】 The invention described in this specification.

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