Methods for analyzing aav capsid proteins

Abstract

Methods for characterizing VP1 capsid proteins, VP2 capsid proteins, and VPS capsid proteins in adeno-associated virus (AAV) particles using liquid chromatography mass spectrometry and / or ultraviolet (UV)-visible spectroscopy are provided. The methods generally include the steps of: (a) subjecting AAV particles to liquid chromatography to denature and then separate the VP1 capsid proteins, the VP2 capsid proteins, and the VPS capsid proteins; and (b) subjecting the separated VP1 capsid proteins, VP2 capsid proteins, and VPS capsid proteins produced in step (a) to UV and mass spectrometry to determine the ratio and mass of the VP1 capsid proteins, the VP2 capsid proteins, and the VPS capsid proteins in the AAV particles. In another aspect, the disclosure provides an AAV composition comprising a post-translational modification. The disclosure also provides methods for characterizing the purity of an AAV composition using liquid chromatography mass spectrometry.

Description

[0001] Cross-referencing related applications

[0002] This application claims the benefit of U.S. Provisional Application No. 62 / 956,681, filed January 3, 2020; U.S. Provisional Application No. 63 / 073,188, filed September 1, 2020; and U.S. Provisional Application No. 63 / 119,909, filed December 1, 2020, pursuant to 35 U.S. SC §119(e), the contents of which are incorporated herein by reference in their entirety. Technical Field

[0003] This disclosure relates to methods for characterizing the purity of VP1, VP2, and VP3 capsid proteins in adeno-associated virus (AAV) particles and AAV compositions using liquid chromatography and mass spectrometry. Background Technology

[0004] Adeno-associated virus (AAV) is rapidly becoming one of the most widely used delivery media for gene therapy. Its excellent safety profile and high efficiency in transducing across a wide range of target tissues have made AAV 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 typically recombinantly expressed in suitable host cells. However, recombinant AAV can be contaminated with proteins from host cell lysates.

[0005] The AAV capsid comprises a mixture of VP1, VP2, and VP3 proteins, which are produced by a single viral Cap gene through alternating splicing and translation, and self-assemble to form the capsid. AAV capsid proteins play important roles in viral infectivity, tissue tropism, and potency, and the ability to fully characterize the quality and ratios of capsid proteins is increasingly important for the commercial manufacturing of AAVs for gene therapy.

[0006] In particular, the stoichiometry of VPs is crucial for the infectivity of viral vectors. For example, high levels of VP3 capsid are negatively correlated with poor transduction efficiency and reduced potency, even when the VP1 / VP2 ratio is unbalanced (Gene Therapy, Vol. 25, pp. 415-424 (2018)). Since the ratios of the structural proteins VP1, VP2, and VP3 can fluctuate widely from the start of manufacturing, for example, from 1:1:5 to 1:1:20 (Biotechnol Adv., 26(1):73-88 (2008)), accurate measurement of the ratios among the three capsid proteins is important in AAV vector quality control. However, current methods attempt to measure the quality of the capsid proteins but fail to determine the stoichiometry of each VP in the VP composition (WO 2018 / 035059). Therefore, the gene therapy industry needs robust methods for more accurately characterizing the ratios and modifications of AAV capsid proteins and the purity of rAAV compositions. Summary of the Invention

[0007] This 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 method disclosed herein is used to determine the ratio of VP1, VP2, and VP3 capsid proteins and / or the mass of one or more of the VP1, VP2, and VP3 capsid proteins in an AAV particle.

[0008] In some aspects, this disclosure provides a method for determining the ratio of VP1, VP2, and VP3 capsid proteins in adeno-associated virus (AAV) particles. The method comprises the steps of subjecting the AAV particles to liquid chromatography at about 70°C to about 90°C, wherein the mass and ratio of the VP1, VP2, and VP3 capsid proteins are determined by mass spectrometry and / or ultraviolet (UV)-visible spectroscopy. In some aspects, the individual mass of the capsid proteins is determined by mass spectrometry. In some aspects, the capsid on the AAV particles is denatured into individual VP1, VP2, and VP3 proteins on the column of the liquid chromatography. In some aspects, the capsid proteins are separated by the liquid chromatography.

[0009] In some aspects, the method further comprises using mass spectrometry to determine the mass of one or more of the VP1 capsid protein, the VP2 capsid protein, and the VP3 capsid protein in the AAV particle.

[0010] In some aspects, the relative amounts of the VP1, VP2, and VP3 capsid proteins are determined by analyzing their ultraviolet (UV) chromatograms. In some aspects, the liquid chromatography is reversed-phase liquid chromatography. In some aspects, the AAV particles are AAVrh74.

[0011] In some aspects, the chromatography uses a first mobile phase comprising water containing trifluoroacetic acid. In some aspects, the chromatography uses a second mobile phase comprising a mixture of acetonitrile containing trifluoroacetic acid and water. In some aspects, the percentage of the second mobile phase in the chromatogram increases over time in the combination of the first and second mobile phases.

[0012] In some respects, the mass spectrometer contains a fragmentation voltage of approximately 125-350 V.

[0013] Deamidation is one of the common post-translational modifications (PTMs) observed in proteins, known to have a significant impact on protein activity and stability. Deamidation is typically caused by the hydrolysis of the amide side chain of asparagine to form a mixture of aspartic and isoaspartic acid. In some aspects, deamidation is a hydrolysis of cytosine to uracil, thereby releasing ammonia in the process. This can occur in vitro using bisulfite, which deaminates cytosine but not 5-methylcytosine. In some aspects, 5-methylcytosine deamidation yields thymine and ammonia. In some aspects, glutamine residues also undergo deamidation to form a mixture of glutamate and isoglutamate; however, glutamine residues are significantly less prone to deamidation than asparagine. In some aspects, guanine deamidation leads to xanthine formation. In some aspects, adenine deamidation leads to hypoxanthine formation. Capsid protein deamidation may affect the stability and activity of AAV formulations.

[0014] In some aspects of this disclosure, mass spectrometry is used to study post-translational modifications such as deamidation. In some aspects, the protein is denatured using reagents such as guanidine and urea. The denatured protein is reduced to break disulfide bonds using 1,4-dithiothreitol (DTT) or tris(2-carboxyethyl)phosphine (TECP). The reduced disulfide bonds are then alkylated using iodoacetamide. The denaturation and alkylation steps are performed to ensure that the protein is folded and therefore fully usable for proteases. The denatured and reduced protein is then digested using one of several proteases, such as trypsin. The digested peptide is separated by HPLC / UPLC using RP-HPLC. The separated peptide is then detected by mass spectrometry, typically Q-ToF or an orbitrap, using m / z ratio. The peptide is identified using appropriate software and databases. Deamidation is identified as an increase of approximately 1 Da compared to the theoretical value of the peptide.

[0015] In some aspects, the method further comprises determining post-translational modifications of at least one of the VP1 capsid protein, VP2 capsid protein, and VP3 capsid protein. In some aspects, the method further comprises post-translational phosphorylation or acetylation of at least one of the VP1 capsid protein, VP2 capsid protein, and VP3 capsid protein.

[0016] This disclosure also provides a method for characterizing host cell proteins in an AAV composition, the method comprising immunoprecipitation of viral capsid proteins from the composition; digestion of residual host cell proteins; and analysis of the digested proteins using liquid chromatography-quadrupole time-of-flight mass spectrometry (LC-QTOF-MS) to identify the host cell proteins.

[0017] In some aspects, the immunoprecipitation includes incubating the AAV composition with an anti-AAV VP1 antibody, an anti-AAV VP2 antibody, an anti-AAV VP3 antibody, or a combination thereof.

[0018] In some aspects, the method further includes analyzing the digested host cell proteins using iterative MS / MS.

[0019] In some aspects, the digestion is carried out in solution. In some aspects, the digestion is carried out at a temperature of about 60°C to about 80°C. In some aspects, the digestion is carried out at about 70°C.

[0020] In some aspects, the method further includes incorporating a known amount of at least one known protein standard into the AAV composition. In some aspects, the at least one known protein standard is a human or bovine protein standard. In some aspects, the method further includes quantifying the amount of the digested host cell protein relative to the at least one protein standard.

[0021] In some aspects, the liquid chromatography is reversed-phase liquid chromatography. In some aspects, the reversed-phase liquid chromatography is performed using a C18 column, a C8 column, or a C4 column. In some aspects, the liquid chromatography is performed using a C8 column. In some aspects, the column comprises particles of about 1.2-3.5 μm. In some aspects, the column comprises particles of about 1.7 μm or about 1.8 μm. In some aspects, the column is about 50 mm to about 300 mm long and has an inner diameter of about 1 mm to about 4.6 mm. In some aspects, the column is about 150 mm long and has an inner diameter of about 2.1 mm.

[0022] In some aspects, the liquid chromatography is performed at about 40°C to about 50°C. In other aspects, the liquid chromatography is performed at about 45°C.

[0023] In some aspects, the liquid chromatography includes a first mobile phase comprising formic acid. In some aspects, the first mobile phase comprises about 0.05 vol% to about 0.15 vol% formic acid. In some aspects, the first mobile phase comprises about 0.1 vol% formic acid.

[0024] In some aspects, the liquid chromatography includes a second mobile phase comprising a mixture of acetonitrile containing formic acid and water. In some aspects, the second mobile phase comprises about 0.05 vol% to about 0.15 vol% formic acid. In some aspects, the second mobile phase comprises about 0.1 vol% formic acid. In some aspects, the second mobile phase comprises about 80-95 vol% acetonitrile. In some aspects, the second mobile phase comprises about 90 vol% acetonitrile and about 10 vol% water.

[0025] In some aspects, compared to the combination of the first and second mobile phases, the percentage of the second mobile phase in the liquid chromatogram increases over time. In some aspects, the percentage of the second mobile phase increases from about 2% to about 50%. In some aspects, the percentage of the second mobile phase increases from about 2% by volume to about 50% by volume within about 120 minutes. In some aspects, the percentage of the second mobile phase then increases to 100% by volume within about 25 minutes. In some aspects, the percentage of the second mobile phase then remains at 100% by volume for about one minute. In some aspects, the second mobile phase then decreases to about 2% by volume within about 4 minutes. In some aspects, the percentage of the second mobile phase then increases to 100% by volume within about 5 minutes. In some aspects, the percentage of the second mobile phase then remains at 100% by volume for about 3 minutes. In some aspects, the second mobile phase then decreases to about 2% by volume within about 2 minutes.

[0026] In some cases, mass spectrometry is performed using a fragmentation voltage of approximately 125–350 V. In others, it is performed using a fragmentation voltage of approximately 135 V. In still others, it is performed using a capillary voltage of approximately 3–6 kV. In some cases, it is performed using a capillary voltage of approximately 4 kV.

[0027] Some aspects of this disclosure relate to a recombinant AAV (rAAV) comprising a heterogeneous group of capsid proteins containing subgroups with amino acid modifications. In some aspects, the modifications are deamidation or oxidation.

[0028] In some aspects, the heterogeneous group comprises deamidated asparagine (N) located at one or more of the equivalent residues of N57, N255, N256, and N263 of AAV.rh74 or 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 another embodiment, the heterogeneous group comprises deamidated asparagine (N) within the peptide sequence of any one of SEQ ID Nos: 1-5 or the equivalent peptide sequence of another AAV serotype of said peptide sequence.

[0029] In some aspects, the heterogeneous group comprises less than 70%, 60%, 50%, 40%, 30%, 20%, 15%, 10%, or 5% of capsid proteins having deamidated capsid proteins located at N57 of the AAV.rh74 capsid. In some aspects, the heterogeneous group comprises less than 15% of capsid proteins having deamidated capsid proteins located at N57 of the AAV.rh74 capsid. In some aspects, the heterogeneous group comprises less than 70%, 60%, 50%, 40%, 30%, 20%, 15%, 10%, or 5% of capsid proteins having deamidated capsid proteins located at N254 and / or N255 of the AAV.rh74 capsid. In some aspects, the heterogeneous group comprises less than 70%, 60%, 50%, 40%, 30%, 20%, 10%, or 5% of capsid proteins having deamidated capsid proteins located at N263.

[0030] In some aspects, the heterogeneous groups include oxidized methionine residues at one or more of the equivalent residues of AAV.Rh74, such as those measured by mass spectrometry and / or ultraviolet (UV)-visible spectroscopy. In some aspects, the heterogeneous groups include less than 30%, 20%, 10%, 5%, or 1% of capsid proteins with oxidation at M437. In some aspects, the heterogeneous groups include less than 30%, 20%, 10%, 5%, or 1% of capsid proteins with oxidation at M473. In some aspects, the heterogeneous groups comprise less than 30%, 20%, 10%, 5%, or 3% of capsid proteins with oxidation at M526. In some aspects, the heterogeneous groups comprise less than 30%, 20%, 10%, 5%, or 2% of capsid proteins with oxidation at M544. In some aspects, the heterogeneous groups comprise less than 30%, 20%, 10%, 5%, or 2% of capsid proteins with oxidation at M560. In some aspects, the heterogeneous groups comprise less than 30%, 20%, 10%, 5%, or 1% of capsid proteins with oxidation at M637.

[0031] Those skilled in the art will recognize that the invention described herein is subject to variations and modifications other than those specifically described. It should be understood that the invention described herein encompasses all such variations and modifications. The invention also includes all such steps, features, compositions, and compounds individually or collectively referenced or indicated in this specification, as well as any and all combinations of any two or more of these steps or features. Attached Figure Description

[0032] The following figures form part of this specification and are included therein to further illustrate various aspects of the invention.

[0033] Figure 1 A UV chromatogram of the AAVrh74 capsid protein was depicted, and the integral confirmed the capsid protein ratio.

[0034] Figure 2 A total ion chromatogram of AAVrh74 capsid protein was depicted.

[0035] Figure 3 shows VP1( Figure 3A ), VP2 ( Figure 3B ) and VP3 ( Figure 3CMS spectra of the unconvolution of capsid proteins were used to confirm the integrity of all three capsid proteins and to detect post-translational modifications of the capsid proteins. Figure 3D MS spectra of VP1 deconvolution in multiple samples are shown. Figure 3E MS spectra of VP2 deconvolution in multiple samples are shown. Figure 3F MS spectra of VP3 deconvolution in multiple samples are shown.

[0036] Figure 4 The procedure for deamidation analysis using Tris-HCl as buffer is shown.

[0037] Figure 5 The results of deamidation of AAV.rh74 with Tris-HCl buffer are shown.

[0038] Figure 6 The results of oxidation of AAV.rh74 with Tris-HCl buffer are shown. Detailed Implementation

[0039] Methods are provided 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 aspects, methods are provided for determining the ratio of VP1, VP2, and VP3 capsid proteins and / or the mass of one or more of VP1, VP2, and VP3 capsid proteins in AAV particles. This disclosure also provides methods for characterizing the purity of rAAV compositions using liquid chromatography and mass spectrometry.

[0040] definition

[0041] For convenience, certain terms used in the specification, examples, and appended claims are collected herein before further description of the invention. These definitions should be read in accordance with the remainder of this disclosure and understood as those skilled in the art would understand. 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. Unless otherwise limited in specific instances, the terms used throughout this specification are defined as follows.

[0042] The articles “a,” “an,” and “the” are used to refer to one or more (i.e., at least one) grammatical objects of the article.

[0043] As used herein, “about” is used to indicate that the value includes inherent error variations in the apparatus, the method used to determine the value, or variations present among study subjects. In some respects, “about” means that a deviation of 5% to 10% or more (e.g., at most 5% to 10%) and 5% to 10% or less (e.g., at most 5% to 10%) from a given value or range is still within the intended meaning of the value or range.

[0044] The term "AAV" or "adeno-associated virus" refers to a parvovirus-dependent parvovirus within the genus Parvovirus. Here, AAV can refer to a wild-type virus or an AAV derived from a naturally occurring wild-type virus, for example, an AAV derived from an rAAV genome packaged in a capsid encoded by a naturally occurring cap gene and / or an AAV genome packaged in a capsid encoded by a non-natural capsid cap gene, such as AAVrh.74.

[0045] AAV can be any serotype, such as AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV-10, AAV-11, AAV-12, AAV-13, AAV rh.10, AAV rh.74, or variants and derivatives thereof. In some respects, rAAV has the serotype AAVrh.74. The generation of pseudotyped rAAV is disclosed, for example, in WO 01 / 83692, which is incorporated herein by reference in its entirety. Other types of rAAV variants are also envisioned, such as rAAV with capsid mutations. See, for example, Marsic et al., Molecular Therapy, 22(11):1900-1909 (2014).

[0046] As used herein, the terms “AAV particle,” “AAV vector,” “AAV virion,” “AAV viral particle,” or “AAV vector particle” refer to a viral particle consisting of an AAV capsid and an encapsulated AAV genome. In some respects, AAV particles include heterologous polynucleotides (i.e., polynucleotides other than the wild-type AAV genome, such as transgenes intended for delivery to mammalian cells). In some respects, the production of AAV viral particles involves the production of AAV vectors, as AAV vector particles contain such vectors.

[0047] For example, wild-type (wt) AAV viral particles comprise a linear single-stranded AAV nucleic acid genome associated with the capsid coating of AAV capsid proteins. The AAV virion can be single-stranded (ss) AAV or complementary (SC) AAV. In some respects, complementary single-stranded AAV nucleic acid molecules, such as “sense” or “antisense” strands, can be packaged into the AAV virion, and both strands are equally infectious.

[0048] The term "recombinant AAV" or "rAAV" is defined herein as an infectious replication-defective virus consisting of an AAV protein coat encapsulating the heterologous nucleotide sequence of interest flanked by an AAV ITR. In some respects, rAAV is produced in suitable host cells possessing an AAV vector, AAV helper functions, and associated functions. In this manner, the host cell is able to encode an AAV polypeptide required for packaging an AAV vector (containing the recombinant nucleotide sequence of interest) into infectious recombinant virosomal particles for subsequent gene delivery.

[0049] As used herein, the term "capsid protein" refers to the protein that forms the coating or outer shell of a virus. The term "AAV capsid protein" refers to the protein that forms the coating of adeno-associated virus (AAV), which is composed of a total of 60 subunits; each subunit is an amino acid sequence, such as viral protein 1 (VP1), VP2, or VP3.

[0050] 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 flows through a column, and the components of the mobile phase interact with a solid stationary phase. The composition of the mobile phase can be varied during separation runs to alter the strength of the interactions between the compounds of interest. As the mobile phase continues to flow through the column, the eluent is typically collected as fractions, while the concentration of compounds eluted from the column over time is monitored to produce elution profiles or chromatograms.

[0051] As used herein, the term "stationary phase" refers to the substance held stationary in a column. The most commonly used stationary phase columns are carbon-chain-bonded silica, phenyl-bonded silica, and cyano-bonded silica. In some respects, the stationary phase may contain hydrophobic alkyl chains of a specific length, such as C4, C8, or C18. In some respects, reversed-phase chromatography is C8 reversed-phase chromatography (e.g., reversed-phase chromatography using a C8 stationary phase).

[0052] 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. Common mobile phase solvents include, but are not limited to, acetonitrile, methanol, tetrahydrofuran, ethanol, or isopropanol. In some respects, two mobile phases are used. For example, a first and a second mobile phase may be mixed in situ to obtain a solvent for eluting a substance from the column. In some respects, the volume ratio of the second mobile phase to the first mobile phase exhibits a gradient that increases during the elution step.

[0053] 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 involve: (1) ionizing a compound to form a charged compound; and (2) detecting the mass-to-charge ratio of the charged compound and calculating its molecular weight. Compounds can be ionized and detected by any suitable means. A “mass spectrometer” generally comprises an ionizer, a mass analyzer, and an ion detector. Typically, one or more molecules of interest are ionized, and the ions are subsequently introduced into a mass spectrometer, where they follow a path in space due to a combination of magnetic and electric fields, the path depending on the mass (“m”) and charge (“z”). In some mass spectrometry methods, ions can be separated from each other using, for example, time-of-flight (TOF), orbital traps, Fourier transform ion cyclotron resonance spectrometers, quadrupoles, or ion traps, and then detected using an ion detector.

[0054] As used herein, the terms “ultraviolet-visible spectroscopy,” “ultraviolet-visible spectrophotometry,” “UV-Vis,” or “UV / Vis” refer to absorption or reflectance spectra used to determine the optical properties (transmittance, reflectance, and absorbance) of liquids and solids. In some respects, ultraviolet-visible spectroscopy is used to characterize the capsid proteins of AAV particles.

[0055] As used in this article, the term “total ion chromatogram (TIC)” refers to a type of chromatogram produced by summing the intensities of all mass spectrometric peaks belonging to the same scan.

[0056] As used herein, the term "AAVrh74" refers to an AAV particle having AAVrh74 VP1, VP2, and VP3 capsid proteins or variants thereof. An exemplary AAVrh74 VP1 capsid protein sequence is set out in SEQ ID NO:4 of U.S. Patent No. 9,909,142, which is incorporated herein by reference in its entirety. Exemplary variants of the AAVrh74VP1 capsid protein are also set out in U.S. Patent No. 9,909,142.

[0057] As used herein, unless otherwise stated, the term "subgroup" of VP proteins refers to a group of VP proteins that has at least one defined common characteristic and consists of at least one group member to all members of a reference group. For example, unless otherwise stated, a "subgroup" of VP1 proteins may be at least one VP1 protein and may be less than all VP1 proteins in an assembled AAV capsid. Unless otherwise stated, a "subgroup" of VP3 proteins may be at least one VP3 protein to less than all VP3 proteins in an assembled AAV capsid. For example, VP1 proteins may be a subgroup of VP proteins; VP2 proteins may be a separate subgroup of VP proteins; and VP3 may be a further subgroup of VP proteins in an assembled AAV capsid. In another example, VP1, VP2, and VP3 proteins may contain, for example, subgroups of asparagine-glycine with different modifications at the asparagine-glycine pair, for example, at least one, two, three, or four highly deamidinated asparagines.

[0058] Characterization of AAV VP1, VP2 and VP3 capsid proteins

[0059] In some aspects, this 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 about 70°C to about 90°C, wherein the mass and ratio of the VP1, VP2, and VP3 capsid proteins are determined by mass spectrometry and / or ultraviolet (UV)-visible spectroscopy. In some aspects, the mass and ratio of the VP1, VP2, and VP3 capsid proteins are determined by mass spectrometry and ultraviolet (UV)-visible spectroscopy. In some aspects, the capsid on the AAV particles is denatured into individual VP1, VP2, and VP3 proteins on the column of the liquid chromatography. In some aspects, the capsid proteins are separated by the liquid chromatography. In some aspects, the method comprises: (a) subjecting the AAV particles to liquid chromatography to separate the VP1 capsid protein, the VP2 capsid protein, and the VP3 capsid protein; and (b) subjecting the separated VP1, VP2, and VP3 capsid proteins produced in step (a) to mass spectrometry and / or UV-Vis 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 aspects, the liquid chromatography operation is performed at about 70°C to about 90°C. In some aspects, 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 aspects, the liquid chromatography operation is performed at about 80°C.

[0060] In some aspects, the method further comprises determining the mass of one or more of the VP1 capsid protein, the VP2 capsid protein, and the VP3 capsid protein of the AAV particle.

[0061] In some respects, the relative amounts of the VP1 capsid protein, the VP2 capsid protein, and the VP3 capsid protein are determined by comparing the total ion chromatograms (TIC) of the VP1 capsid protein, the VP2 capsid protein, and the VP3 capsid protein.

[0062] In some aspects, the liquid chromatography is reversed-phase liquid chromatography, size exclusion chromatography, hydrophilic interaction liquid chromatography, or cation exchange chromatography. In some aspects, the liquid chromatography is reversed-phase liquid chromatography.

[0063] In some aspects, the reversed-phase chromatography is performed using a C18 column, a C8 column, or a C4 column. In some aspects, the liquid chromatography is performed using a C8 column.

[0064] In some aspects, the stationary phase of the reversed-phase liquid chromatography is contained within a chromatographic column approximately 50-300 mm long and with an inner diameter of approximately 1-4.6 mm. In some aspects, the column is a BEH column. In some aspects, the inner diameter of the column is 1 mm, 2.1 mm, 3 mm, or 4.6 mm. In some aspects, the length of the column is 50 mm, 75 mm, 100 mm, 150 mm, or 300 mm. In some cases, column dimensions are 1mm×50mm, 2.1mm×50mm, 3mm×50mm, 4.6mm×50mm, 1mm×75mm, 2.1mm×75mm, 3mm×75mm, 4.6mm×75mm, 1mm×100mm, 2.1mm×100mm, 3mm×100mm, 4.6mm×100mm, 1mm×150mm, 2.1mm×150mm, 3mm×150mm, 4.6mm×150mm, 1mm×300mm, 2.1mm×300mm, 3mm×300mm, or 4.6mm×300mm. In some aspects, the column dimensions are 1.6×50mm, 1.6×60mm, 1.6×70mm, 1.6×80mm, 1.6×90mm, 1.6×100mm, 1.6×110mm, 1.6×120mm, 1.6×130mm, 1.6×140mm, 1.6×150mm, 1.7×50mm, 1.7×60mm, 1.7×70mm, 1.7×80mm, 1.7×90mm, 1. 7×100mm, 1.7×110mm, 1.7×120mm, 1.7×130mm, 1.7×140mm, 1.7×150mm, 1.8×50mm, 1.8×60mm, 1.8×70mm, 1.8×80mm, 1.8×90mm, 1.8×100mm, 1.8×110mm, 1.8×120mm, 1.8×130mm, 1.8×140mm, 1.8×150mm mm, 1.9×50mm, 1.9×60mm, 1.9×70mm, 1.9×80mm, 1.9×90mm, 1.9×100mm, 1.9×110mm, 1.9×120mm, 1. 9×130mm, 1.9×140mm, 1.9×150mm, 2.0×50mm, 2.0×60mm, 2.0×70mm, 2.0×80mm, 2.0×90mm, 2.0×100 mm, 2.0×110mm, 2.0×120mm, 2.0×130mm, 2.0×140mm, 2.0×150mm, 2.1×50mm, 2.1×60mm, 2.1×70mm, 2.1×80mm, 2.1×90mm, 2.1×100mm, 2.1×110mm, 2.1×120mm, 2.1×130mm, 2.1×140mm, 2.1×150mm, 2.2×50mm, 2.2×60mm, 2.2×70mm, 2.2×80mm, 2.2×90mm, 2.2×100mm, 2.2×110mm, 2.2×120mm, 2.2×130mm, 2.2×140mm, 2.2×150mm, 2.3×50mm, 2.3×60mm, 2.3×70mm, 2.3×80mm, 2.3×90mm, 2.3×100mm, 2.3×110mm, 2.3×120mm, 2.3×130mm, 2.3×140mm, 2.3×150mm, 2.4×50mm, 2.4×60mm, 2.4×70mm, 2.4×80mm, 2.4×90mm, 2.4×100mm mm, 2.4×110mm, 2.4×120mm, 2.4×130mm, 2.4×140mm, 2.4×150mm, 2.5×50mm, 2.5×60mm, 2.5×70mm, 2.5×80mm, 2.5×90mm, 2.5×100mm, 2.5×110mm, 2.5×120mm, 2.5×130mm, 2.5×140mm, 2.5×150mm, 2.6×50mm, 2.6×60mm, 2.6×70mm, 2.6×80mm, 2.6×90mm, 2.6×100mm, 2.6×110mm, 2.6×120mm, 2.6×130mm, 2.6×140mm or 2.6×150mm. In some aspects, the stationary phase of the reversed-phase liquid chromatography is contained within a chromatographic column approximately 100 mm long and approximately 2.1 mm in inner diameter.

[0065] In some aspects, the stationary phase of the reversed-phase liquid chromatography comprises particles with a size set between about 1.2 μm and 2.5 μm. In other aspects, the stationary phase of the reversed-phase liquid chromatography comprises particles with a size of about 1.7 μm, 1.8 μm, or 2.1 μm. In some aspects, 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 aspects, the stationary phase of the reversed-phase liquid chromatography comprises particles of about 1.7 μm.

[0066] In some aspects, the chromatography uses a first mobile phase comprising water containing fluorinated acetic acid. The fluorinated acetic acid includes monofluoroacetic acid, difluoroacetic acid, and trifluoroacetic acid. In some aspects, the chromatography uses a first mobile phase comprising water containing trifluoroacetic acid.

[0067] In some aspects, the first mobile phase comprises about 0.05 vol% to about 0.15 vol% of fluorinated acetic acid. In some aspects, the first mobile phase comprises about 0.05 vol%, 0.06 vol%, 0.07 vol%, 0.08 vol%, 0.09 vol%, 0.1 vol%, 0.11 vol%, 0.12 vol%, 0.13 vol%, 0.14 vol%, 0.15 vol%, 0.16 vol%, 0.17 vol%, 0.18 vol%, 0.19 vol%, or 0.2 vol% of fluorinated acetic acid. In some aspects, the first mobile phase comprises about 0.05 vol% or 0.1 vol% of fluorinated acetic acid. In some aspects, the first mobile phase comprises about 0.1 vol% of fluorinated acetic acid. In some aspects, the fluorinated acetic acid is trifluoroacetic acid. In some aspects, the first mobile phase comprises about 0.1 vol% of trifluoroacetic acid.

[0068] In some aspects, the chromatography uses a second mobile phase comprising acetonitrile containing fluorinated acetic acid. In some aspects, the chromatography uses a second mobile phase comprising acetonitrile containing trifluoroacetic acid. In some aspects, the chromatography uses a second mobile phase comprising a mixture of acetonitrile containing fluorinated acetic acid and water. In some aspects, the chromatography uses a second mobile phase comprising a mixture of acetonitrile containing trifluoroacetic acid and water.

[0069] In some aspects, the second mobile phase comprises about 0.05-0.2 vol% of fluorinated acetic acid. In some aspects, the second mobile phase comprises about 0.05-0.15 vol% of fluorinated acetic acid. In some aspects, the second mobile phase comprises about 0.05 vol%, 0.06 vol%, 0.07 vol%, 0.08 vol%, 0.09 vol%, 0.1 vol%, 0.11 vol%, 0.12 vol%, 0.13 vol%, 0.14 vol%, 0.15 vol%, 0.16 vol%, 0.17 vol%, 0.18 vol%, 0.19 vol%, or 0.2 vol% of fluorinated acetic acid. In some aspects, the second mobile phase comprises about 0.05 vol% or 0.1 vol% of fluorinated acetic acid. In some aspects, the second mobile phase comprises about 0.1% of fluorinated acetic acid. In some aspects, the fluorinated acetic acid is trifluoroacetic acid. In some respects, the second mobile phase contains about 0.1% trifluoroacetic acid.

[0070] In some aspects, the second mobile phase comprises about 75-95 vol% acetonitrile. In some aspects, the second mobile phase comprises about 75 vol%, 80 vol%, 85 vol%, 90 vol%, or 95 vol% acetonitrile. In some aspects, the second mobile phase comprises about 90 vol% acetonitrile and 10% water.

[0071] In some aspects, the percentage of the second mobile phase in the chromatogram increases over time in the combination of the first and second mobile phases. In some aspects, the percentage of the second mobile phase increases from about 10% by volume to about 40% by volume. In some aspects, the percentage of the second mobile phase increases from about 10% by volume to about 45% by volume. In some aspects, the percentage of the second mobile phase increases from about 10% by volume to about 100% by volume. In some aspects, the percentage of the second mobile phase increases from about 10% by volume to about 45% within about 30–40 minutes. In some aspects, the percentage of the second mobile phase increases from about 10% by volume to about 45% within about 35 minutes. In some aspects, the percentage of the second mobile phase increases from about 10% by volume to about 100% by volume within about 30–50 minutes. In some aspects, the percentage of the second mobile phase increases from about 10% by volume to about 100% by volume within about 36 minutes.

[0072] In some respects, the percentage of the second mobile phase increases from about 10% by volume to about 40% by volume in about 5–10 minutes, and from about 40% to about 45% in about 25–35 minutes. In some respects, the percentage of the second mobile phase increases from about 45% to about 100% in about 0.5–2 minutes. In some respects, the percentage of the second mobile phase decreases from about 100% to about 10% in about 0.5–2 minutes.

[0073] In some respects, the percentage of the second mobile phase increases from about 10% by volume to about 40% by volume in about 6 minutes, and from about 40% to about 45% in about 29 minutes. In some respects, the percentage of the second mobile phase increases from about 45% to about 100% in about 1 minute. In some respects, the percentage of the second mobile phase decreases from about 100% to about 10% in about 1 minute.

[0074] In some respects, liquid chromatography is high-performance liquid chromatography (HPLC). In other respects, liquid chromatography is ultra-high-performance liquid chromatography (UHPLC).

[0075] In some respects, mass spectrometry can use any ionization mode, particularly those suitable for the analysis of biomolecules, including, but not limited to: direct infusion mass spectrometry, electrospray ionization (ESI) MS, desorption electrospray ionization (DESI) MS, direct real-time analysis (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 respects, mass spectrometry uses the API-ES ionization mode.

[0076] In some applications, mass spectrometry scans signals in the range of 400–16000 m / z. In other applications, mass spectrometry scans signals in the range of 700–13700 m / z.

[0077] In some cases, mass spectrometry scans are positive polarity. In some cases, data acquisition time is approximately 10–35 minutes. In other cases, data acquisition time is approximately 17–28 minutes.

[0078] In some applications, the nozzle voltage of a mass spectrometer is approximately 400-600 V. In some applications, the nozzle voltage is approximately 500 V. In some applications, the cone voltage is approximately 60-70 V. In some applications, the cone voltage is approximately 65 V. In some applications, the difference between the nozzle voltage and the cone voltage is approximately 400-450 V. In some applications, the difference between the nozzle voltage and the cone voltage is approximately 435 V.

[0079] In some cases, the drying gas temperature for mass spectrometry is approximately 200–350 °C. In others, it is approximately 300 °C. In some cases, the drying gas flow rate is approximately 5–13 L / min. In still others, it is approximately 13 L / min.

[0080] In some applications, mass spectrometry uses capillary voltages of approximately 3-6 kV. In others, it uses capillary voltages of approximately 3 kV, 4 kV, 5 kV, or 6 kV. In still others, it uses a capillary voltage of approximately 5 kV.

[0081] In some applications, mass spectrometry uses fragmentation voltages of approximately 125–350 V. Specifically, mass spectrometry uses fragmentation voltages of approximately 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 respects, mass spectrometry uses a fragmentation voltage of approximately 175V.

[0082] In some aspects, the AAV particles are AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAVrh10, AAVrh74, or any naturally occurring recombinant or synthetic AAV particles. In some aspects, the AAV particles are recombinant AAV (rAAV) particles. In some aspects, the AAV particles are AAVrh74.

[0083] In some of the above aspects, this disclosure further includes determining post-translational modifications of at least one of the VP1 capsid protein, VP2 capsid protein, and VP3 capsid protein. In some aspects, this disclosure further includes determining post-translational glycosylation, sialylation, acetylation, amino acid loss, amidation, phosphorylation, formylation, hydroxylation, methylation, and / or sulfation of at least one of the VP1, VP2, and VP3 capsid proteins. Post-translational modifications include one or more of the following: N-terminal methionine loss, threonine loss, phosphorylation, and acetylation.

[0084] In some aspects, this disclosure includes determining the removal of N-terminal methionine from the VP1, VP2, or VP3 capsid protein. In some aspects, this disclosure includes determining the removal of N-terminal methionine from the VP1 or VP3 capsid protein. In some aspects, this disclosure includes determining N-terminal acetylation after the removal of N-terminal methionine from the VP1, VP2, or VP3 capsid protein. In some aspects, this disclosure includes determining N-terminal acetylation after the removal of N-terminal methionine from the VP1 or VP3 capsid protein.

[0085] In some aspects, this disclosure provides a method for characterizing the capsid proteins of AAV particles based at least in part on the ratio of VP1 capsid protein, VP2 capsid protein and VP3 capsid protein and / or the mass of one or more of VP1 capsid protein, VP2 capsid protein and VP3 capsid protein in AAV particles.

[0086] In some aspects, this disclosure provides a method for determining the serotype of AAV particles based at least in part on the ratio of VP1 capsid protein, VP2 capsid protein and VP3 capsid protein and / or the mass of one or more of VP1 capsid protein, VP2 capsid protein and VP3 capsid protein in AAV particles, wherein the ratio of VP1 capsid protein, VP2 capsid protein and VP3 capsid protein and the mass of one or more of VP1 capsid protein, VP2 capsid protein and VP3 capsid protein are determined by the method disclosed herein.

[0087] Mass spectrometry is an analytical technique used for protein characterization. In some aspects, a method is provided for characterizing the AAVrh74 capsid protein ratio along with the complete mass of all three capsid proteins by liquid chromatography and mass spectrometry. In some aspects, the AAVrh74 capsid is denatured on a column into individual capsid proteins VP1, VP2, and VP3. This denaturation is achieved by heating the column compartment to 80°C (3, 4). The capsid proteins are then baseline resolved on a Waters BEHC8 column using trifluoroacetic acid as an ion-pairing agent in the mobile phase (5). The denatured proteins are first analyzed in UV to achieve the capsid ratio and then analyzed in a mass spectrometer to obtain the complete mass of the individual proteins.

[0088] Deamidation is a common post-translational modification that converts asparagine residues into a mixture of isoaspartic and aspartic acid. Deamidation of glutamine residues also occurs, but at a much slower rate. Oxidation is also a common post-translational modification that causes proteins to react with various free radicals and reactive oxygen species. Methionine oxidation is the most common, however, oxidation of several other amino acid residues, such as cysteine ​​and tryptophan, can also be observed. Deamidation / oxidation is also a common degradation pathway for proteins present during manufacturing and storage. Deamidation can affect protein activity and stability. Oxidation can cause conformational changes in proteins and thus can affect protein activity and stability. Oxidation can also affect protein immunogenicity. Therefore, critical quality attributes (CQAs) of proteins need to be carefully monitored during post-translational modifications.

[0089] Current methods using ammonium bicarbonate produce false signals or overestimate deamidation in AAV capsid proteins (Table 7). This disclosure provides a method for more accurately measuring post-translational modifications on capsid proteins using Tris-HCl. In some aspects, the LC-MS method uses a buffer comprising Tris-HCl. In some aspects, the buffer comprises acetonitrile. In some aspects, the buffer comprises methionine. In some aspects, the buffer comprises 5 mM to 50 mM Tris-HCl, 5%-20% acetonitrile, and 1 mM to 50 mM methionine. In some aspects, the buffer comprises 20 mM Tris-HCl, 5%-10% acetonitrile, and 10 mM methionine.

[0090] In some aspects, the post-translational modification includes deamidation at one or more of the following residues located at N263, N514, N57, N502, N254, and N94 of AAV8, or their equivalent residues at AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV9, AAV10, AAV11, AAV12, AAV13, AAVrh10, or AAVrh74. In some aspects, the post-translational modification includes deamidation at one or more of the following residues located at N57, N255, N256, and N263 of AAV.Rh74. In some aspects, the post-translational modification includes oxidation at one or more of the following residues located at M437, M473, M526, M544, M560, and M637 of AAV.Rh74.

[0091] AAV composition

[0092] Some aspects of this disclosure relate to a recombinant AAV (rAAV) comprising a heterogeneous group of capsid proteins containing subgroups with amino acid modifications. In some aspects, the modifications can be deamidation, acetylation, isomerization, phosphorylation, or oxidation. In some aspects, the modifications are deamidation or oxidation.

[0093] In some aspects, the rAAV capsid may contain subgroups of VP1, VP2, and VP3, said subgroup 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, wherein 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% of said amino acid residues are deamidated compared to the encoded amino acid sequence of the VP protein. In some aspects, most of these amino acid residues may be N residues. In some aspects, Q residues may be deamidated.

[0094] In some aspects, this disclosure provides an AAV composition comprising an AAV capsid comprising deamidation at one or more of the following residues: 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, as measured by mass spectrometry and / or ultraviolet (UV)-visible spectroscopy. In some aspects, deamidation is measured by any of the methods disclosed herein.

[0095] In some respects, heterogeneous groups include 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%, and 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 capsid proteins with deamidation at N57 of the AAV.rh74 capsid.

[0096] In some respects, the heterogeneous groups include less than about 25%, less than about 24%, less than about 23%, less than about 22%, less than about 21%, less than about 20%, less than about 19%, less than about 18%, less than about 17%, less than about 16%, less than about 15%, less than about 14%, less than about 13%, less than about 12%, less than about 11%, or less than about 10% of capsid proteins with deamidation at N57 of the AAV.rh74 capsid.

[0097] In some respects, heterogeneous groups include 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%, and 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 deamidated capsid proteins located at N254 and / or N255 of the AAV.rh74 capsid.

[0098] In some respects, heterogeneous groups include 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%, and 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 capsid proteins with deamidation at N263.

[0099] In some aspects, the AAV composition includes an AAV capsid comprising oxidation at one or more of the equivalent residues of AAV.Rh74, namely M437, M473, M526, M544, M560, and M637, or 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 aspects, deamidation is measured by any of the methods disclosed herein.

[0100] In some respects, heterogeneous groups include 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 about 0.5% of capsid proteins with oxidation at M437.

[0101] In some respects, heterogeneous groups include 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 about 0.5% of capsid proteins with oxidation at M473.

[0102] In some respects, heterogeneous groups include 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 about 0.5% of capsid proteins with oxidation at M526.

[0103] In some respects, heterogeneous groups include 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 about 0.5% of capsid proteins with oxidation at M544.

[0104] In some respects, heterogeneous groups include 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 about 0.5% of capsid proteins with oxidation at M560.

[0105] In some respects, heterogeneous groups include 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 about 0.5% of capsid proteins with oxidation at M637.

[0106] Characterizing host cell proteins in AAV compositions

[0107] In some aspects, this disclosure provides a method for characterizing host cell proteins in AAV compositions, such as AAV-based gene therapy drug products. In some aspects, the method for characterizing host cell proteins in an AAV composition includes immunoprecipitation of viral capsid proteins from the composition; digestion of residual host cell proteins; and analysis of the digested proteins using liquid chromatography-quadrupole time-of-flight mass spectrometry (LC-QTOF-MS) to identify the host cell proteins. As used herein, the terms "residual host cell protein" or "residual protein" refer to proteins that remain in solution after immunoprecipitation. In some aspects, the method further includes analysis of the digested host cell proteins using iterative MS / MS.

[0108] In some aspects, immunoprecipitation includes incubating the AAV composition with a VP antibody. In some aspects, the VP antibody includes anti-AAV VP1 antibody, anti-AAV VP2 antibody, anti-AAV VP3 antibody, or a combination thereof. In some aspects, the antibody may be an anti-adeno-associated virus (AAV) VP1 / VP2 / VP3 antibody (catalog number: 03-61058) from American Research Products, Inc.

[0109] In some respects, residual host cell proteins are digested in solution. In some respects, digestion is rapid digestion. In some respects, rapid digestion is carried out at approximately 60°C to approximately 80°C. In some respects, rapid digestion is carried out at approximately 60°C, approximately 61°C, approximately 62°C, approximately 63°C, approximately 64°C, approximately 65°C, approximately 66°C, approximately 67°C, approximately 68°C, approximately 69°C, approximately 70°C, approximately 71°C, approximately 72°C, approximately 73°C, approximately 74°C, approximately 75°C, approximately 76°C, approximately 77°C, approximately 78°C, approximately 79°C, or approximately 80°C. In some respects, rapid digestion is carried out at approximately 70°C.

[0110] In some aspects, a known amount of at least one known protein standard is incorporated into the AAV composition. In some aspects, said at least one known protein standard is a human or bovine protein standard. In some aspects, the method further includes quantifying the amount of said residual host cell protein relative to said at least one protein standard.

[0111] In some aspects, the liquid chromatography is reversed-phase liquid chromatography, size exclusion chromatography, hydrophilic interaction liquid chromatography, or cation exchange chromatography. In some aspects, the liquid chromatography is reversed-phase liquid chromatography.

[0112] In some aspects, liquid chromatography is performed at temperatures ranging from about 35°C to 55°C. In other aspects, liquid chromatography is performed at temperatures of 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 aspects, liquid chromatography is performed at about 45°C.

[0113] In some aspects, the reversed-phase chromatography is performed using a C18 column, a C8 column, or a C4 column. In some aspects, the liquid chromatography is performed using a C8 column.

[0114] In some aspects, the stationary phase of the reversed-phase liquid chromatography is contained within a chromatographic column approximately 50-300 mm long and with an inner diameter of approximately 1-4.6 mm. In some aspects, the column is a BEH column. In some aspects, the inner diameter of the column is 1 mm, 2.1 mm, 3 mm, or 4.6 mm. In some aspects, the length of the column is 50 mm, 75 mm, 100 mm, 150 mm, or 300 mm. In some cases, column dimensions are 1mm×50mm, 2.1mm×50mm, 3mm×50mm, 4.6mm×50mm, 1mm×75mm, 2.1mm×75mm, 3mm×75mm, 4.6mm×75mm, 1mm×100mm, 2.1mm×100mm, 3mm×100mm, 4.6mm×100mm, 1mm×150mm, 2.1mm×150mm, 3mm×150mm, 4.6mm×150mm, 1mm×300mm, 2.1mm×300mm, 3mm×300mm, or 4.6mm×300mm. In some aspects, the column dimensions are 1.6×50mm, 1.6×60mm, 1.6×70mm, 1.6×80mm, 1.6×90mm, 1.6×100mm, 1.6×110mm, 1.6×120mm, 1.6×130mm, 1.6×140mm, 1.6×150mm, 1.7×50mm, 1.7×60mm, 1.7×70mm, 1.7×80mm, 1.7×90mm, 1. 7×100mm, 1.7×110mm, 1.7×120mm, 1.7×130mm, 1.7×140mm, 1.7×150mm, 1.8×50mm, 1.8×60mm, 1.8×70mm, 1.8×80mm, 1.8×90mm, 1.8×100mm, 1.8×110mm, 1.8×120mm, 1.8×130mm, 1.8×140mm, 1.8×150mm mm, 1.9×50mm, 1.9×60mm, 1.9×70mm, 1.9×80mm, 1.9×90mm, 1.9×100mm, 1.9×110mm, 1.9×120mm, 1. 9×130mm, 1.9×140mm, 1.9×150mm, 2.0×50mm, 2.0×60mm, 2.0×70mm, 2.0×80mm, 2.0×90mm, 2.0×100 mm, 2.0×110mm, 2.0×120mm, 2.0×130mm, 2.0×140mm, 2.0×150mm, 2.1×50mm, 2.1×60mm, 2.1×70mm, 2.1×80mm, 2.1×90mm, 2.1×100mm, 2.1×110mm, 2.1×120mm, 2.1×130mm, 2.1×140mm, 2.1×150mm, 2.2×50mm, 2.2×60mm, 2.2×70mm, 2.2×80mm, 2.2×90mm, 2.2×100mm, 2.2×110mm, 2.2×120mm, 2.2×130mm, 2.2×140mm, 2.2×150mm, 2.3×50mm, 2.3×60mm, 2.3×70mm, 2.3×80mm, 2.3×90mm, 2.3×100mm, 2.3×110mm, 2.3×120mm, 2.3×130mm, 2.3×140mm, 2.3×150mm, 2.4×50mm, 2.4×60mm, 2.4×70mm, 2.4×80mm, 2.4×90mm, 2.4×100mm mm, 2.4×110mm, 2.4×120mm, 2.4×130mm, 2.4×140mm, 2.4×150mm, 2.5×50mm, 2.5×60mm, 2.5×70mm, 2.5×80mm, 2.5×90mm, 2.5×100mm, 2.5×110mm, 2.5×120mm, 2.5×130mm, 2.5×140mm, 2.5×150mm, 2.6×50mm, 2.6×60mm, 2.6×70mm, 2.6×80mm, 2.6×90mm, 2.6×100mm, 2.6×110mm, 2.6×120mm, 2.6×130mm, 2.6×140mm or 2.6×150mm. In some aspects, the stationary phase of the reversed-phase liquid chromatography is contained within a chromatographic column approximately 150 mm long and approximately 2.1 mm in inner diameter.

[0115] In some aspects, the stationary phase of the reversed-phase liquid chromatography comprises particles with a size set between about 1.2 μm and 2.5 μm. In some aspects, the stationary phase of the reversed-phase liquid chromatography comprises particles with a size set between about 1.7 μm, 1.8 μm, or 2.1 μm. In some aspects, 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 aspects, the stationary phase of the reversed-phase liquid chromatography comprises particles of about 1.7 μm.

[0116] In some aspects, the chromatography uses a first mobile phase comprising water containing fluorinated acetic acid. The fluorinated acetic acid includes monofluoroacetic acid, difluoroacetic acid, and trifluoroacetic acid. In some aspects, the chromatography uses a first mobile phase comprising water containing trifluoroacetic acid.

[0117] In some respects, the chromatography uses a first mobile phase containing formic acid.

[0118] In some aspects, the first mobile phase comprises about 0.05 vol% to about 0.15 vol% formic acid. In some aspects, the first mobile phase comprises about 0.05 vol%, 0.06 vol%, 0.07 vol%, 0.08 vol%, 0.09 vol%, 0.1 vol%, 0.11 vol%, 0.12 vol%, 0.13 vol%, 0.14 vol%, 0.15 vol%, 0.16 vol%, 0.17 vol%, 0.18 vol%, 0.19 vol%, or 0.2 vol% formic acid. In some aspects, the first mobile phase comprises about 0.05 vol% or 0.1 vol% formic acid. In some aspects, the first mobile phase comprises about 0.1 vol% formic acid.

[0119] In some aspects, the chromatography uses a second mobile phase comprising acetonitrile containing fluorinated acetic acid. In some aspects, the chromatography uses a second mobile phase comprising acetonitrile containing trifluoroacetic acid. In some aspects, the chromatography uses a second mobile phase comprising a mixture of acetonitrile containing fluorinated acetic acid and water. In some aspects, the chromatography uses a second mobile phase comprising a mixture of acetonitrile containing trifluoroacetic acid and water.

[0120] In some aspects, the chromatography uses a second mobile phase comprising acetonitrile containing formic acid. In other aspects, the chromatography uses a second mobile phase comprising a mixture of acetonitrile containing formic acid and water.

[0121] In some aspects, the second mobile phase comprises about 0.05-0.2 vol% formic acid. In some aspects, the second mobile phase comprises about 0.05-0.15 vol% formic acid. In some aspects, the second mobile phase comprises about 0.05 vol%, 0.06 vol%, 0.07 vol%, 0.08 vol%, 0.09 vol%, 0.1 vol%, 0.11 vol%, 0.12 vol%, 0.13 vol%, 0.14 vol%, 0.15 vol%, 0.16 vol%, 0.17 vol%, 0.18 vol%, 0.19 vol%, or 0.2 vol% formic acid. In some aspects, the second mobile phase comprises about 0.05 vol% or 0.1 vol% formic acid. In some aspects, the second mobile phase comprises about 0.1 vol% formic acid.

[0122] In some aspects, the second mobile phase comprises about 75-95 vol% acetonitrile. In some aspects, the second mobile phase comprises about 75 vol%, 80 vol%, 85 vol%, 90 vol%, or 95 vol% acetonitrile. In some aspects, the second mobile phase comprises about 90 vol% acetonitrile and about 10 vol% water.

[0123] In some aspects, the percentage of the second mobile phase in the chromatogram increases over time in the combination of the first and second mobile phases. In some aspects, the percentage of the second mobile phase increases from about 2% by volume to about 50% by volume. In some aspects, the percentage of the second mobile phase increases from about 50% by volume to about 100% by volume. In some aspects, the percentage of the second mobile phase increases from about 2% to about 50% within about 110–130 minutes. In some aspects, the percentage of the second mobile phase increases from about 2% to about 50% within about 120 minutes. In some aspects, the percentage of the second mobile phase increases from about 50% by volume to about 100% by volume within about 20–30 minutes. In some aspects, the percentage of the second mobile phase increases from about 50% by volume to about 100% by volume within about 25 minutes. In some aspects, the percentage of the second mobile phase then increases to 100% by volume within about 5 minutes. In some aspects, the percentage of the second mobile phase then remains at 100% by volume for about 3 minutes. In some respects, the second mobile phase subsequently decreases to approximately 2% of its volume within approximately 2 minutes.

[0124] In some respects, the percentage of the second mobile phase is maintained at approximately 100% by volume for approximately 0.5–1.5 minutes. In other respects, the percentage of the second mobile phase is maintained at 100% by volume for approximately 1 minute.

[0125] In some respects, the percentage of the second mobile phase decreases from approximately 100% to approximately 2% within approximately 1–10 minutes. In other respects, the percentage of the second mobile phase decreases from approximately 100% to approximately 2% within approximately 4 minutes.

[0126] In some respects, liquid chromatography is high-performance liquid chromatography (HPLC). In other respects, liquid chromatography is ultra-high-performance liquid chromatography (UHPLC).

[0127] In some respects, mass spectrometry can use any ionization mode, particularly those suitable for the analysis of biomolecules, including, but not limited to: direct infusion mass spectrometry, electrospray ionization (ESI) MS, desorption electrospray ionization (DESI) MS, direct real-time analysis (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 respects, mass spectrometry uses the API-ES ionization mode.

[0128] In some applications, mass spectrometry scans signals in the range of 40–5000 m / z. In others, it scans signals in the range of 50–3000 m / z. In still others, it scans signals in the range of 300–3000 m / z.

[0129] In some cases, mass spectrometry scans are positive polarity. In some cases, data acquisition time is approximately 1–130 minutes. In other cases, data acquisition time is approximately 2–120 minutes.

[0130] In some applications, the nozzle voltage of a mass spectrometer is approximately 400-600 V. In some applications, the nozzle voltage is approximately 500 V. In some applications, the cone voltage is approximately 60-70 V. In some applications, the cone voltage is approximately 65 V. In some applications, the difference between the nozzle voltage and the cone voltage is approximately 400-450 V. In some applications, the difference between the nozzle voltage and the cone voltage is approximately 435 V.

[0131] In some cases, the drying gas temperature for mass spectrometry is approximately 200–375 °C. In others, it is approximately 325 °C. In some cases, the drying gas flow rate is approximately 5–13 L / min. In still others, it is approximately 12 L / min.

[0132] In some applications, mass spectrometry uses capillary voltages of approximately 3-6 kV. In others, it uses capillary voltages of approximately 3 kV, 4 kV, 5 kV, or 6 kV. In still others, it uses a capillary voltage of approximately 5 kV.

[0133] In some applications, mass spectrometry uses fragmentation voltages of approximately 125–350 V. Specifically, mass spectrometry uses fragmentation voltages of approximately 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 respects, mass spectrometry uses a fragmentation voltage of approximately 135V.

[0134] While the subject matter of the invention has been described in considerable detail with reference to certain aspects, other aspects are also possible. Therefore, the spirit and scope of the appended claims should not be limited to the specific aspects described herein.

[0135] Example

[0136] This disclosure will now be illustrated using working examples, which are intended to illustrate the work of this disclosure and are not intended to limit the scope of this disclosure in any way. 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 pertains. Although similar or equivalent methods and materials to those described herein may be used to practice the disclosed methods and compositions, exemplary methods, apparatus, and materials are described herein.

[0137] Example 1: General Methods and Devices

[0138] 1.1 Reagents

[0139] Water, acetonitrile, and trifluoroacetic acid for LCMS were obtained from Fisher Scientific, and ammonium bicarbonate was obtained from Sigma Aldrich. Table 1 provides the reagents used for liquid chromatography and solution preparation.

[0140] Table 1: Reagents used in liquid chromatography

[0141] Reagent Name CAS number Required grade, purity, or concentration Suggested source / directory number LCMS water 7732-18-5 LCMS Feishier Company / W6 Acetonitrile 67-63-0 LCMS Feissier Company / A955 Trifluoroacetic acid 76-05-1 LCMS Feissier Company / A11610X1AMP ammonium bicarbonate 1066-33-7 BioUltra Sigma-Aldrich Company / 09830

[0142] 1.2 Equipment

[0143] Use the following devices to execute the instances disclosed herein.

[0144] a) Waters ACQUITY UPLC BEH C8 pillar, 2.1×100mm, 1.7μm; Part number: 186002878

[0145] b) Remove the rotating column with Pierce detergent, 0.5 mL; Catalog No.: 87777

[0146] c) A suitable analytical balance

[0147] d) Automatic pipette

[0148] e) Grade A measuring glassware

[0149] f) HPLC vials and caps

[0150] g) Scraper and weighing vessel

[0151] h) Agilent 1290 Infinity II UHPLC System

[0152] i) Agilent 6545XT AdvanceBio Quadrupole Time-of-Flight Mass Spectrometer (Q-ToF)

[0153] Example 2: Solution Preparation

[0154] 2.1 Preparation of 100mM ammonium bicarbonate

[0155] In 50mL Weigh 0.395 ± 0.01 g of ammonium bicarbonate into the tube. Using a graduated cylinder, transfer 50 mL of LCMS water into the tube and use a vortex mixer to completely dissolve the ammonium bicarbonate to obtain a 100 mM ammonium bicarbonate solution. The solution is stable for one month at 2–8 °C.

[0156] 2.2 Preparation of the first mobile phase (water containing 0.1% trifluoroacetic acid)

[0157] Using a graduated cylinder, transfer 1 L of LCMS water to a 1 L bottle. Using a pipette, transfer 1000 μL of trifluoroacetic acid to the bottle. Mix the trifluoroacetic acid and water thoroughly for 5 minutes to obtain the first mobile phase. The first mobile phase is stable under ambient conditions for up to one month.

[0158] 2.3 Preparation of the second mobile phase (90% acetonitrile containing 0.1% trifluoroacetic acid and 10% water)

[0159] Add 900 mL of acetonitrile to a 1 L graduated cylinder. Add LCMS water to the 1 L graduated cylinder to prepare a 1 L solution. Transfer the solution to a 1 L bottle. Using a pipette, transfer 1000 μL of trifluoroacetic acid to the bottle. Mix the trifluoroacetic acid and solution thoroughly for 5 minutes to obtain a second mobile phase. The second mobile phase is stable under ambient conditions for up to one month.

[0160] Example 3: Sample Preparation

[0161] Remove the bottom seal from the rotating column and loosen the column cap. Place the column into a 2 mL collection tube and centrifuge at 1500 × g for 1 minute. When using a fixed-angle rotor, mark the side of the column where the compacted resin is tilted upwards. Then place the column in the centrifuge with the marked side facing outwards for all subsequent steps.

[0162] Add 400 μL of 100 mM ammonium bicarbonate solution to the column and centrifuge the column at 1500 × g for 1 min. Repeat this step twice more, discarding the runoff after each step. Then transfer the column to a new 2 mL collection tube. Slowly apply 5 μg of sample to the top of the compacted resin bed and incubate at room temperature for 2 min. Centrifuge the column at 1500 × g for 2 min and collect the polymer-free sample. Then bring the sample volume to 100 μL with 100 mM ammonium bicarbonate and transfer to an HPLC vial.

[0163] Example 4: Characterization of VP1, VP2, and VP3 capsid proteins in AAV particles

[0164] This embodiment describes a method for determining the ratio and mass of VP1, VP2, and VP3 capsid proteins in AAV particles. Here, the AAV particles are denatured and separated into VP1, VP2, and VP3 capsid proteins in liquid chromatography. The separated VP1, VP2, and VP3 capsid proteins are first subjected to UV to determine the ratio of VP1, VP2, and VP3 capsid proteins in the AAV particles, and then subjected to mass spectrometry to obtain the mass of each of the VP1, VP2, and VP3 capsid proteins.

[0165] 4.1 LC Operating Conditions

[0166] Using ACQUITY ACQUITY BEH 1.7μm, 2.1×100 mm, C8 analytical column (part number: 186002878) The AAV VP1, VP2, and VP3 capsid proteins were separated using the following mobile phase:

[0167] First mobile phase (A): water containing 0.1% trifluoroacetic acid; and

[0168] Second mobile phase (B): 90% acetonitrile containing 0.1% trifluoroacetic acid and 10% water.

[0169] The column temperature was maintained at approximately 80°C, and separation was achieved by using mobile phase B at a flow rate of 0.4 mL / min, increasing from 10% to 40% and then from 40% to 45%, followed by rinsing with 100% mobile phase B for 1 minute, and then rebalancing for an additional 5 minutes with the starting mobile phase composition (10% mobile phase B).

[0170] The operating conditions for LC are listed in Table 2.

[0171] Table 2: LC Operating Conditions

[0172]

[0173]

[0174] 4.2 Mass Spectrometer (MS) Operating Conditions

[0175] Mass spectrometry was performed using an Agilent 6545XT AdvanceBio quadrupole time-of-flight mass spectrometer (Q-ToF) within the m / z range of 700–13700 m / z during the inspection scan, utilizing API-ES ionization. The capillary voltage, nozzle voltage, fragmentation voltage, and cone voltage were set to 5 kV, 500 V, 175 V, and 65 V, respectively. The drying gas temperature and flow rate were set to 300 °C and 13 L / min, respectively.

[0176] The operating conditions for the mass spectrometer are listed in Table 3.

[0177] Table 3: Mass Spectrometer (MS) Operating Conditions

[0178]

[0179]

[0180] Note: MS parameters are for Agilent 6545XT QToF and may need to be modified depending on the instrument used.

[0181] 4.3 Analysis and Results

[0182] The capsid proteins were first denatured by heating the column compartment to 80°C. The three capsid proteins, VP1, VP2, and VP3, were then baseline separated using a Waters UPLC BEH C8 column (part number: 186002878) by elution with a combination of a first mobile phase comprising water containing 0.1% trifluoroacetic acid and a second mobile phase comprising a mixture of acetonitrile and water containing 0.1% trifluoroacetic acid, wherein the percentage of the second mobile phase increased over time. The use of 0.1% trifluoroacetic acid as an ion-pairing agent in the mobile phase facilitated baseline resolution.

[0183] The VP1, VP2, and VP3 capsid proteins, separated in liquid chromatography, are first subjected to UV to determine their relative amounts, and then subjected to mass spectrometry to determine their masses. This is achieved through methods such as... Figure 1 The baseline integration of the UV chromatograms shown in Table 4 yields the stoichiometry of VP1 / VP2 / VP3, which is approximately 1:1:10.

[0184] Table 4

[0185] AAVrh74 sample VP1 VP2 VP3 Sample 1 1.0 0.9 9.5 Sample 2 1.0 1.7 7.9 Sample 3 1.0 1.4 9.7

[0186] Then, the peaks of the three capsid proteins were deconvolved using the parameters listed in Table 5. The total ion chromatograms and deconvolved spectra of all three peaks are shown in Table 5. Figure 2 and Figures 3A-3CThe results are shown below. Three major masses detected at the VP1 peak (theoretical mass 81587 Da) are 81496 Da, 81578 Da, and 81658 Da. The 81496 Da peak indicates a VP1 protein with N-terminal methionine loss and a single acetylation modification. Two additional peaks with a mass shift of +80 Da indicate phosphorylation. The deconvolution of the VP2 peak (theoretical mass 66381 Da) reveals two major masses: 66282 Da and 66360 Da. The 66282 Da peak indicates a VP2 protein with threonine loss, and the 66360 Da peak matches a single phosphorylation with a mass shift of +80. The VP3 peak (theoretical mass 59750 Da) shows a single major mass of 59662 Da, which matches the mass of a VP3 protein with N-terminal methionine loss and a single acetylation.

[0187] Table 5: Deconvolution Parameters

[0188] parameter set up Deconvolution algorithm Maximum Entropy Quality range 45,000-90,000 Daltons Quality Steps 0.1 Dalton m / z range 700.00-3000.00 Baseline deduction factor 7.00 Isotope width automatic Peak signal-to-noise ratio 30.0 Maximum number of peaks 100 Calculating average mass using 90% of the peak height Minimum continuous charge 5 Minimum protein fit score 8

[0189] Figure 3 shows the detection of post-translational modifications of VP1, VP2, and VP3. Table 6 shows the complete quality analysis of the AAV.rh74 capsid protein.

[0190] Table 6: Quality Analysis

[0191]

[0192] Example 5: Characterization of the deamidation of AAVrh74 using LCMS

[0193] Mass spectrometry can be used to determine extensive deamidation in capsid proteins, including the sites of deamidation and the level of deamidation at these sites. To measure AAV capsid deamidation, the capsid protein was denatured and reduced for 10 min at 90 °C 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 the mixture was incubated in the dark at room temperature for 30 min. Alkylation was then quenched by adding 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 a 1:20 enzyme:protein ratio and incubated overnight at 37 °C. After overnight incubation, consumption 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 with a NanoFlex source and a QExactive HF sensor.

[0194] 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 available from existing publications (Molecular Therapy, Vol. 26, No. 12, pp. 2848-2962 (2018)).

[0195] Table 7: Deamidation analysis results of AAV capsids

[0196]

[0197] The deamidation state of AAV.rh74 was measured using two different buffer solutions (ammonium bicarbonate and Tris-HCl).

[0198] For ammonium bicarbonate, the sample was denatured by exchanging the buffer solution for 100 mM ammonium bicarbonate. The denatured sample was reduced by adding 10 mM MTT and incubated at 37 °C for 45 min. Alkylation was then performed by adding iodoacetamide to the sample to a final concentration of 30 mM. The denatured, reduced, and alkylated sample was then exchanged back into 100 mM ammonium bicarbonate using a 10 kDa Amicon Ultra filter. The sample was then digested with trypsin and incubated overnight at 37 °C. Using this sample preparation with digestion in ammonium bicarbonate, a similar level of deamidation of AAVrh74 was obtained, as shown in Table 7.

[0199] For Tris-HCl, 60 μg aliquots were exchanged with 4 M guanidine and 200 mM Tris at pH 7.5 using an Amicon 10K centrifuge 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 aliquots. The reaction mixture was incubated at 56 °C for 45 min, then cooled to room temperature. Iodoacetamide (30 mM) was added, followed by incubation in the dark at room temperature for 60 min. Tris buffer (100 mM, pH 7.5) was then added to dilute the guanidine hydrochloride concentration to 0.6 M. Trypsin / Lys-C (60 μg) was added to 60 μg of the reduced and alkylated sample (enzyme:protein ratio of approximately 1:1 (w:w)). Methionine was added at 10 mM during digestion to minimize artificial oxidation. Digestion was performed overnight (17 h) at 37 °C. Then add TFA (1%), followed by LC-MS / MS analysis.

[0200] As shown in Table 7, the deamidation state was significantly lower when Tris-HCl was used as the buffer than when ammonium bicarbonate was used.

[0201] To further optimize the method using Tris-HCl, according to Figure 4 The flowchart in the document describes the measurement of eight individual AAV capsid samples. First, the samples were denatured by buffer exchange with 20 mM Tris-HCl at pH 7.5 (6 M guanidine hydrochloride). The samples were then reduced by adding DTT to a final concentration of 10 mM and incubated at 37°C for 45 min. Alkylation was performed by adding iodoacetamide to a final concentration of 30 mM and incubating in the dark at room temperature for 1 h. The samples were then buffer-exchanged again with 20 mM Tris-HCl at pH 7.5 using a 10 kDa Amicon Ultra filter. 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 using RP-HPLC on an Agilent 1290U-HPLC. The separated peptides were then detected using an Agilent 6545XT QToF, and deamidation analysis was performed using MassHunter and Bioconfirm software. The deamidation state was... Figure 5 As shown in the figure, and the oxidation state is in Figure 6 As shown in the figure. Of the 52 asparagine residues present, no deamidation was observed at 48 residues (total asparagine residues in VP1: 56). Of the 39 glutamine residues present, no deamidation was observed at any of the 39 residues (total glutamine residues in VP1: 48). Using water labeled with O18, a small amount of deamidation at N57 was shown as a sample preparation-related artifact. No oxidation was detected in the remaining 5 methionine residues (total methionine residues in VP1: 11). Of the 14 tryptophan residues detected, no oxidation was observed at any of the 14 residues (total tryptophan residues in VP1: 15).

[0202] Digestion in ammonium bicarbonate may significantly increase deamidation artifacts due to the increasing pH over time. Therefore, the observed high level of deamidation is likely a deamidation artifact generated during sample preparation. To confirm the level of deamidation, a Tris HCl-based digestion was performed at Sarepta: 20 mM Tris HCl at pH 7.5 was used as the buffer; 10% acetonitrile was added to the digestion solution, as acetonitrile is known to reduce deamidation artifacts; and 10 mM methionine was also added to the digestion solution to reduce oxidation artifacts.

[0203] Therefore, the method disclosed herein is more accurate when measuring deamidation, oxidation, or other post-translational modifications using Tris-HCl buffer.

[0204] Example 6: Characterizing host cell proteins using LC-QTOF-MS

[0205] The purity of rAAV-based gene therapy drug products was analyzed by characterizing host cell proteins retained in the AAV composition using LC-QTOF-MS.

[0206] 6.1 Sample Preparation

[0207] AAVrh74 samples were incorporated with known amounts of human thioredoxin 1 (HT1) protein standard Invitgen (catalog number: LF-P0001) and bovine carbonic anhydrase II (BCAII) protein standard Sigma (catalog number: C7749). Human thioredoxin 1 (HT1) and bovine carbonic anhydrase II (BCAII) were selected as incorporation protein standards for quantifying human and bovine HCP measurements. For the immune exhaustion process, 100 μL of 0.05 mg / mL anti-adeno-associated virus (AAV), VP1 / VP2 / VP3 antibody, and 100 μL of sample solution, along with 20 μL of 0.05 mg / mL BCAII and 10 μL of 0.1 mg / mL HTI, were pipetted into 270 μL of IP-MS cell lysis buffer from the Pierce MS Compatible Magnetic IP Kit. The AAV capsid protein was then immunoprecipitated from the sample using anti-adeno-associated virus (AAV) VP1 / VP2 / VP3 (catalog number: 03-6105) from Research Products, Inc. (Catalog No.: 90409) and the Pierce MS Compatible Magnetic IP Kit (Catalog No.: 90409). The sample was then passed through a Pierce washing column (Catalog No.: 87777) to remove the AAV capsid protein.

[0208] The sample buffer was then replaced with Promega rapid digestion buffer (catalog number: VA1060). The sample was reduced, alkylated, and digested with rapid digestive trypsin at 70°C for 60–180 minutes.

[0209] 6.2LC Operating Conditions

[0210] Digested residual host cell proteins were separated using a Waters Acquity Peptide BEH C18 column, 1.7 μm, 2.1 × 150 mm, on an Agilent 1290 HPLC system. The mobile phase used was:

[0211] First mobile phase (A): an aqueous solution containing 0.1% formic acid; and

[0212] Second mobile phase (B): 90% acetonitrile containing 0.1% formic acid and 10% water.

[0213] The column temperature was maintained at approximately 45°C, and separation was achieved by using mobile phase B at a flow rate of 0.3 mL / min, increasing from 2% to 50% and then from 50% to 100%, followed by rinsing with 100% mobile phase B for 3 minutes, and then rebalancing with the starting mobile phase composition (2% mobile phase B) for an additional 5 minutes.

[0214] The operating conditions for LC are listed in Table 8.

[0215] Table 8: LC Operating Conditions

[0216]

[0217] 6.3 Mass Spectrometer (MS) Operating Conditions

[0218] Mass spectrometry was performed using an Agilent 6545XT AdvanceBio quadrupole time-of-flight mass spectrometer (Q-ToF) within the m / z range of 50–3000 m / z during the inspection scan, utilizing API-ES ionization. The capillary voltage, nozzle voltage, fragmentation voltage, and cone voltage were set to 4 kV, 500 V, 135 V, and 65 V, respectively. The drying gas temperature and flow rate were set to 325 °C and 12 L / min, respectively.

[0219] The operating conditions for the mass spectrometer are listed in Table 9.

[0220] Table 9: Mass Spectrometer (MS) Operating Conditions

[0221]

[0222]

[0223] 6.4 Analysis and Results

[0224] The data generated in Example 6.3 were processed using Byos software from Protein Metrics to search certain Uniprot protein databases. The identity and relative quantity of each residual protein were calculated based on the amount of incorporated protein standards. HCP analysis showed that for three batches of AAV virus particles, only a few residual host cell proteins (two bovine proteins, but no human proteins) were identified by MS (Table 10). Protein concentrations were listed in order of ng / mL or based on the ppm level of the incorporated protein standards.

[0225] Table 10: Host cell proteins analyzed by LC / MS in three batches

[0226]

[0227] The following references are incorporated herein in their entirety:

[0228] 1. Buller RM, Rose JA. “Characterization of adenovirus-associated virus-induced polypeptides in KB cells” J Virol 25:1978, pp.331-338.

[0229] 2. Johnson FB, Ozer HL, Hoggan MD. “Structural proteins of adenovirus-associated viruses” Journal of Virology 8:1971, pp.776-770.

[0230] 3. DWBauer et al. "Exploring the Balance between DNA Pressure and Capsid Stability in Herpesviruses and Phages." Journal of Virology, 2015, 9288-98.

[0231] 4. Vamseedhar Rayaprolu et al. “Comparative Analysis of Adeno-Associated Virus Capsid Stability and Dynamics.” Journal of Virology, 2013, 13150-60.

[0232] 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, Vol. 38, No. 5, 2017, 255-267.

Claims

1. A method for characterizing VP1, VP2, and VP3 capsid proteins in adeno-associated virus (AAV) particles, the method comprising: The AAV particles are subjected to a column for liquid chromatography, wherein the column is maintained at 70°C to 90°C to denature the capsid protein, wherein the liquid chromatography comprises a first mobile phase comprising 0.05 vol% to 0.15 vol% trifluoroacetic acid. The ratio of VP1 capsid protein, VP2 capsid protein, and VP3 capsid protein was determined by ultraviolet (UV)-visible spectroscopy, and the protein mass was determined by mass spectrometry.

2. The method of claim 1, wherein the individual masses of VP1 capsid protein, VP2 capsid protein and VP3 capsid protein are determined by the mass spectrometry.

3. The method according to claim 1 or 2, wherein the ratio of the VP1 capsid protein, the VP2 capsid protein, and the VP3 capsid protein is determined by comparing the ultraviolet (UV) chromatograms of the VP1 capsid protein, the VP2 capsid protein, and the VP3 capsid protein.

4. The method according to claim 1 or 2, wherein the liquid chromatography is reversed-phase liquid chromatography.

5. The method of claim 4, wherein the reversed-phase liquid chromatography is performed using a C18 column, a C8 column, or a C4 column.

6. The method of claim 5, wherein the column comprises particles of 1.2-3.5 μm.

7. The method of claim 6, wherein the column comprises particles of 1.7 μm or 1.8 μm.

8. The method according to any one of claims 5 and 6, wherein the column is 50 mm to 300 mm long and has an inner diameter of 1 mm to 4.6 mm.

9. The method according to any one of claims 1 to 2 and 5 to 7, wherein the AAV particles have serotypes AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAVrh10, AAVrh74 or any naturally occurring, recombinant or synthetic AAV particles.

10. The method according to any one of claims 1 to 2 and 5 to 7, wherein the liquid chromatography is performed at 75°C to 85°C.

11. The method according to any one of claims 1 to 2 and 5 to 7, wherein the liquid chromatography comprises a second mobile phase comprising a mixture of acetonitrile containing trifluoroacetic acid and water.

12. The method of claim 11, wherein the second mobile phase comprises 0.05 vol% to 0.15 vol% trifluoroacetic acid.

13. The method of claim 11, wherein the second mobile phase comprises 80-95% by volume acetonitrile.

14. The method of claim 13, wherein the second mobile phase comprises 90 vol% acetonitrile and 10 vol% water.

15. The method of claim 11, wherein the percentage of the second mobile phase in the liquid chromatograph increases over time compared to the combination of the first mobile phase and the second mobile phase.

16. The method of claim 15, wherein the percentage of the second mobile phase is increased from 10 vol% to 40 vol%, and then from 40 vol% to 45 vol%.

17. The method of claim 16, wherein the percentage of the second mobile phase is increased from 10 vol% to 40 vol% over 3 minutes, and then from 40 vol% to 45 vol% over 30 minutes.

18. The method of claim 16, wherein the percentage of the second mobile phase in the combination of the first and second mobile phases is subsequently increased to 100% by volume within 1 minute.

19. The method of claim 18, wherein the percentage of the second mobile phase in the combination of the first and second mobile phases is subsequently reduced to 10% by volume within 1 minute.

20. The method according to any one of claims 1 to 2, 5 to 7 and 12 to 19, wherein the mass spectrometry is performed using a fragmentation voltage of 125-350 V.

21. The method according to any one of claims 1 to 2, 5 to 7 and 12 to 19, wherein the mass spectrometry is performed using a capillary voltage of 3-6 kV.

22. The method of claim 1, wherein the characterization further comprises determining post-translational modifications of at least one of VP1 capsid protein, VP2 capsid protein, and VP3 capsid protein.

23. The method of claim 22, wherein the post-translational modification comprises one or more of the following: amino acid loss, glycosylation, sialylation, acetylation, phosphorylation, deamidation, oxidation, formylation, hydroxylation, methylation, and sulfation.

24. The method of claim 23, wherein the post-translational modification comprises one or more of the following: N-terminal methionine loss, deamidation, threonine loss, phosphorylation, and acetylation.

25. The method according to any one of claims 1 to 2, 5 to 7, 12 to 19 and 22 to 24, wherein the method is performed using the gradient procedures listed in Table 2.

26. The method of claim 22, wherein the characterization is performed using a buffer solution comprising Tris-HCl.

27. The method of claim 26, wherein the buffer comprises acetonitrile and / or methionine.

28. The method of claim 26, wherein the buffer comprises 5 mM to 50 mM Tris-HCl, 5% to 20% acetonitrile, and 1 mM to 50 mM methionine.

29. The method of claim 28, wherein the buffer comprises 20 mM Tris-HCl, 5%-10% acetonitrile, and 10 mM methionine.

30. The method of claim 26, wherein the Tris-HCl is at pH 7.

5.

31. The method of claim 22, wherein the post-translational modification comprises deamidation at one or more of N263, N514, N57, N502, N254 and N94 of AAV8 or at equivalent residues in AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV9, AAV10, AAV11, AAV12, AAV13, AAVrh10, AAVrh74 or at recombined or synthetic AAV particles.

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