Method for analyzing AAV capsid proteins

Abstract

A method is provided for characterizing VP1, VP2, and VPS capsid proteins in adeno-associated virus (AAV) particles using liquid-phase chromatography, mass spectrometry, and / or ultraviolet (UV)-visible spectroscopy. The method generally comprises: (a) applying AAV particles to liquid-phase chromatography to denature them and then separating the VP1, VP2, and VPS capsid proteins; and (b) applying the separated VP1, VP2, and VPS capsid proteins produced in step (a) to UV and mass spectrometry to determine the proportions and masses of the VP1, VP2, and VPS capsid proteins in the AAV particles. In another aspect, the present disclosure provides an AAV composition comprising post-translational modifications. The present disclosure also provides a method for characterizing the purity of an AAV composition using liquid-phase chromatography and mass spectrometry.

Description

Technology Field

[0001] Cross-reference regarding related applications

[0002] This application claims the benefit under 35 USC § 119(e) 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, each of which is incorporated herein by reference in its entirety.

[0003] Technology field

[0004] The present disclosure relates to a method for characterizing the purity of VP1, VP2, and VP3 capsid proteins and AAV compositions in adeno-associated virus (AAV) particles using liquid-phase chromatography and mass spectrometry. Background Technology

[0005] Adeno-associated viruses (AAVs) are becoming one of the most widely used vehicles for delivering gene therapy. An excellent safety profile, along with high transduction efficiency across a wide range of target tissues, has made AAV the most widely used platform for gene therapy. AAV is a small virus belonging to the Parvoviridae family. The virus consists of an unenveloped icosahedral capsid containing a linear single-stranded DNA genome of approximately 4.7 kilobases. AAV is generally expressed recombinantly in suitable host cells. However, recombinant AAV can be contaminated by proteins from host cell lysates.

[0006] The AAV capsid contains a mixture of VP1, VP2, and VP3 proteins, which are generated from a single viral cap gene by alternative splicing and translation and self-assemble to form the capsid. AAV capsid proteins play a significant role in viral infectivity, tissue affinity, and efficacy, and the ability to fully characterize the mass and ratio of capsid proteins is becoming increasingly important for the commercial production of AAVs for gene therapy.

[0007] In particular, the stoichiometry of VP is important for the infectivity of viral vectors. For example, high levels of VP3 capsid were negatively associated with low transduction efficiency and reduced efficacy, even when the VP1 / VP2 ratio was unbalanced (Gene Therapy, volume 25, pp. 415-424 (2018)). Since the ratios of structural proteins VP1, VP2, and VP3 derived from manufacturing can vary within a range, 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 for AAV vector quality control. However, current methods have attempted to measure the mass of capsid proteins but have failed to determine the stoichiometry of each VP (WO 2018 / 035059). Therefore, the gene therapy industry needs a powerful method for more accurate characterization of the ratio and modification of AAV capsid proteins, and the purity of rAAV composition.

[0008] The present disclosure provides a method for characterizing VP1, VP2, and VP3 capsid proteins in adeno-associated virus (AAV) particles using liquid-phase chromatography and mass spectrometry. The method disclosed herein is used to determine the ratio of VP1, VP2, and VP3 capsid proteins in AAV particles, and / or the mass of one or more of the VP1, VP2, and VP3 capsid proteins.

[0009] In some embodiments, the present disclosure provides a method for determining the proportions of VP1, VP2, and VP3 capsid proteins in adeno-associated virus (AAV) particles. The method comprises the step of applying AAV particles to liquid-phase chromatography at about 70°C to about 90°C, wherein the mass and proportion of VP1, VP2, and VP3 capsid proteins are determined by mass spectrometry and / or ultraviolet (UV)-visible spectroscopy. In some embodiments, the individual masses of the capsid proteins are measured by mass spectrometry. In some embodiments, the capsid on the AAV particles is denatured into individual VP1, VP2, and VP3 proteins in a column of liquid-phase chromatography. In some embodiments, the capsid proteins are separated by liquid-phase chromatography.

[0010] In some embodiments, the method further includes the step of determining the mass of one or more of the VPl, VP2 and VP3 capsid proteins in AAV particles using mass spectrometry.

[0011] In some embodiments, the relative amounts of VP1, VP2, and VP3 capsid proteins are determined by analyzing the ultraviolet (UV) chromatograms of VP1, VP2, and VP3 capsid proteins. In some embodiments, the liquid phase chromatography is reverse-phase liquid phase chromatography. In some embodiments, the AAV particle is AAVrh74.

[0012] In some embodiments, chromatography uses a first mobile phase comprising trifluoroacetic acid in water. In some embodiments, chromatography uses a second mobile phase comprising trifluoroacetic acid in a mixture of acetonitrile and water. In some embodiments, the percentage of the second mobile phase in the combination of the first and second mobile phases in chromatography increases over time.

[0013] In some embodiments, mass spectrometry includes a fragmentation voltage of about 125 to 350 V.

[0014] Deamidation is one of the common post-translational modifications (PTMs) observed in proteins, known to have a significant effect on protein activity and stability. Deamidation is generally induced by the hydrolysis of the amide side chain of asparagine, forming a mixture of aspartic acid and isoaspartic acid. In some embodiments, deamidation is a hydrolysis reaction of cytosine to uracil, releasing ammonia in the process. This can occur in vitro through the use of bisulfites, which deaminate cytosine rather than 5-methylcytosine. In some embodiments, the deamination of 5-methylcytosine produces thymine and ammonia. In some embodiments, glutamine residues also undergo deamidation to form a mixture of glutamic acid and isoglutamic acid, but glutamine residues are significantly less sensitive to deamidation compared to asparagine. In some embodiments, the deamination of guanine results in the formation of xanthine. In some embodiments, the deamidation of adenine results in the formation of hypoxanthine. The deamidation of capsid proteins can affect the stability and activity of AAV formulations.

[0015] In some embodiments of the present disclosure, mass spectrometry is used to study post-translational modifications, such as deamidation. In some embodiments, proteins may be denatured using reagents such as guanidine and urea. The denatured protein is reduced using 1,4-dithiothreitol (DTT) or tris(2-carboxyethyl)phosphine (TECP) to break disulfide bonds. Then, the reduced disulfide bonds are alkylated using iodoacetamide. The denaturation and alkylation steps are performed to ensure that the protein unfolds to ensure full access to the protease. Then, the denatured and reduced protein is digested using one of several proteases, such as trypsin. The digested peptides are separated on HPLC / UPLC using RP-HPLC. Then, the separated peptides are detected on a mass spectrometer, typically Q-ToF or Orbitrap, using their m / z ratios. The peptides are identified using appropriate software and databases. Deamidation is identified as an increase of about 1 Da compared to the theoretical value of the peptide.

[0016] In some embodiments, the method further comprises the step of determining a post-translational modification of at least one of the VPl, VP2, and VP3 capsid proteins. In some embodiments, the method further comprises post-translational phosphorylation or acetylation of at least one of the VPl, VP2, and VP3 capsid proteins.

[0017] The present disclosure also provides a method for characterizing host cell proteins in an AAV composition, the method comprising the steps of: immunoprecipitating a viral capsid protein from the composition; degrading a residual host cell protein; and identifying the host cell protein by analyzing the degraded protein using liquid-phase chromatography quadrupole time-of-flight mass spectrometry (LC-QTOF-MS).

[0018] In some embodiments, immunoprecipitation includes the step of incubating an AAV composition with an anti-AAV VP1 antibody, an anti-AAV VP2 antibody, an anti-AAV VP3 antibody, or a combination thereof.

[0019] In some embodiments, the method further includes the step of analyzing host cell proteins degraded by repeated MS / MS.

[0020] In some embodiments, decomposition is carried out in a solution. In some embodiments, decomposition is carried out at a temperature of about 60°C to about 80°C. In some embodiments, decomposition is carried out at about 70°C.

[0021] In some embodiments, the method further comprises the step of spike the AAV composition with a known amount of at least one known protein standard. In some embodiments, the at least one known protein standard is a human or bovine protein standard. In some embodiments, the method further comprises the step of quantifying the amount of host cell protein degraded for at least one protein standard.

[0022] In some embodiments, the liquid phase chromatography is reverse-phase liquid phase chromatography. In some embodiments, the reverse-phase liquid phase chromatography is performed using a C18 column, a C8 column, or a C4 column. In some embodiments, the liquid phase chromatography is performed using a C8 column. In some embodiments, the column contains particles of about 1.2 to 3.5 μm. In some embodiments, the column contains particles of about 1.7 μm or about 1.8 μm. In some embodiments, the column has a length of about 50 mm to about 300 mm and an inner diameter of about 1 mm to about 4.6 mm. In some embodiments, the column has a length of about 150 mm and an inner diameter of about 2.1 mm.

[0023] In some embodiments, liquid-phase chromatography is performed at about 40°C to about 50°C. In some embodiments, liquid-phase chromatography is performed at about 45°C.

[0024] In some embodiments, the liquid phase chromatography comprises a first mobile phase comprising formic acid. In some embodiments, the first mobile phase comprises about 0.05% to about 0.15% of formic acid by volume. In some embodiments, the first mobile phase comprises about 0.1% of formic acid by volume.

[0025] In some embodiments, the liquid-phase chromatography comprises a second mobile phase comprising formic acid in a mixture of acetonitrile and water. In some embodiments, the second mobile phase comprises about 0.05% to about 0.15% by volume of formic acid. In some embodiments, the second mobile phase comprises about 0.1% by volume of formic acid. In some embodiments, the second mobile phase comprises about 80% to 95% by volume of acetonitrile. In some embodiments, the second mobile phase comprises about 90% by volume of acetonitrile and about 10% by volume of water.

[0026] In some embodiments, compared to the combination of the first and second mobile phases in liquid-phase chromatography, the percentage of the second mobile phase increases over time. In some embodiments, the percentage of the second mobile phase increases from about 2% to about 50%. In some embodiments, the percentage of the second mobile phase increases from about 2% to about 50% by volume over about 120 minutes. In some embodiments, the percentage of the second mobile phase subsequently increases to 100% by volume over about 25 minutes. In some embodiments, the percentage of the second mobile phase subsequently remains at 100% by volume for about 1 minute. In some embodiments, the second mobile phase subsequently decreases to about 2% by volume over about 4 minutes. In some embodiments, the percentage of the second mobile phase subsequently increases to 100% by volume over about 5 minutes. In some embodiments, the percentage of the second mobile phase subsequently remains at 100% by volume for about 3 minutes. In some embodiments, the second mobile phase subsequently decreases to about 2% by volume over about 2 minutes.

[0027] In some embodiments, mass spectrometry is performed using a fragmentation voltage of about 125 to 350 V. In some embodiments, mass spectrometry is performed using a fragmentation voltage of about 135 V. In some embodiments, mass spectrometry is performed using a capillary voltage of about 3 to 6 kV. In some embodiments, mass spectrometry is performed using a capillary voltage of about 4 kV.

[0028] Some aspects of the present disclosure relate to recombinant AAVs (rAAVs) comprising heterogroups of capsid proteins containing subgroups having amino acid modifications. In some aspects, the modification is deamidation or oxidation.

[0029] In some embodiments, the heterogenous group comprises deamidated asparagine (N) in one or more of N57, N255, N256, and N263 of AAV.rh74, or in an equivalent residue of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, or AAVrh10, when measured by mass spectrometry and / or ultraviolet (UV)-visible spectrometry. In another embodiment, the heterogenous group comprises deamidated asparagine (N) in any one of the peptide sequences of SEQ ID NOs 1 to 5 or in an equivalent peptide sequence of another AAV serotype.

[0030] In some embodiments, the heterogeneous group comprises a capsid protein having deamidation at N57 of the AAV.rh74 capsid of 70%, 60%, 50%, 40%, 30%, 20%, 15%, 10%, or less than 5%. In some embodiments, the heterogeneous group comprises a capsid protein having deamidation at N57 of the AAV.rh74 capsid of less than 15%. In some embodiments, the heterogeneous group comprises a capsid protein having deamidation at N254 and / or N255 of the AAV.rh74 capsid of 70%, 60%, 50%, 40%, 30%, 20%, 15%, 10%, or less than 5%. In some embodiments, the heterogeneous group comprises a capsid protein having deamidation in N263 of 70%, 60%, 50%, 40%, 30%, 20%, 10%, or less than 5%.

[0031] In some embodiments, the heterogroup comprises methionine oxidized at one or more of M437, M473, M526, M544, M560, and M637 of AAV.Rh74, or at an equivalent residue of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, CAAV11, AAV12, AAV13, or AAVrh10, when measured by mass spectrometry and / or ultraviolet (UV)-visible spectrometry. In some embodiments, the heterogroup comprises a capsid protein having oxidation at M437 of 30%, 20%, 10%, 5%, or less than 1%. In some embodiments, the heterogeneous group comprises a capsid protein having oxidation at M473 in an amount of 30%, 20%, 10%, 5%, or less than 1%. In some embodiments, the heterogeneous group comprises a capsid protein having oxidation at M526 in an amount of 30%, 20%, 10%, 5%, or less than 3%. In some embodiments, the heterogeneous group comprises a capsid protein having oxidation at M544 in an amount of 30%, 20%, 10%, 5%, or less than 2%. In some embodiments, the heterogeneous group comprises a capsid protein having oxidation at M560 in an amount of 30%, 20%, 10%, 5%, or less than 2%. In some embodiments, the heterogeneous group comprises a capsid protein having oxidation at M637 in an amount of 30%, 20%, 10%, 5%, or less than 1%.

[0032] Those skilled in the art will recognize that variations and modifications other than those specifically described herein may be made to the invention described herein. It should be understood that the invention described herein includes all such variations and modifications. The invention also includes all such steps, features, compositions, and compounds referenced or indicated herein, individually or collectively, and any combination of any two or more of any steps or features. Brief explanation of the drawing

[0033] The following drawings form part of this specification and are included to further illustrate aspects of the invention. Figure 1 shows a UV chromatogram for AAVrh74 capsid protein, along with a total confirming the capsid protein ratio. Figure 2 shows the total ion chromatogram for the AAVrh74 capsid protein. Figure 3 shows the deconvolutional MS spectra for each of the capsid proteins VP1 (Fig. 3a), VP2 (Fig. 3b), and VP3 (Fig. 3c), confirming the intact mass of all three capsid proteins and the detection of post-translational modifications of the capsid proteins. Figure 3d shows the deconvolutional MS spectra for VP1 in multiple samples. Figure 3e shows the deconvolutional MS spectra for VP2 in multiple samples. Figure 3f shows the deconvolutional MS spectra for VP3 in multiple samples. Figure 4 illustrates a deamidation analysis method using Tris-HCl as a buffer. Figure 5 shows the results of deamidation of AAV.rh74 using Tris-HCl buffer. Figure 6 shows the oxidation results for AAV.rh74 using Tris-HCl buffer. Specific details for implementing the invention

[0034] A method is provided for characterizing VP1, VP2, and VP3 capsid proteins in adeno-associated virus (AAV) particles using liquid-phase chromatography, mass spectrometry, or ultraviolet (UV)-visible spectroscopy. In some embodiments, a method is provided for determining the proportion of VP1, VP2, and VP3 capsid proteins in AAV particles, and / or the mass of one or more of VP1, VP2, and VP3 capsid proteins. The present disclosure also provides a method for characterizing the purity of an rAAV composition using liquid-phase chromatography and mass spectrometry.

[0035] definition

[0036] For convenience, prior to the further description of the invention, specific terms used in this specification, embodiments, and appended claims are combined herein. These definitions should be read in consideration of the remainder of this disclosure and understood by those skilled in the art. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art. Terms used throughout this specification are defined as follows, unless otherwise limited in specific cases:

[0037] The articles "one," "one of," and "he" are used to refer to one or more (i.e., at least one) grammatical objects of the article.

[0038] As used herein, “about” is used to indicate that the value includes an inherent error range for the device, the method used to determine the value, or a range existing among the subjects of study. In some embodiments, “about” indicates that deviations of 5% to 10% (e.g., at most 5% to 10%) and 5% to less than 10% (e.g., at most 5% to 10%) of a given value or range are maintained within the intended meaning of the cited value or range.

[0039] The term “AAV” or “adeno-associated virus” refers to Dependoparvovirus within the genus Parvovirus of viruses. In this document, AAV may refer to a wild-type virus, or an AAV derived from a naturally occurring wild-type virus, for example, an rAAV genome packed with a capsid derived from a capsid protein encoded by a naturally occurring cap gene, and / or an AAV derived from a rAAV genome packed with a capsid derived from a non-natural capsid cap gene, for example, a capsid protein encoded by AAVrh74.

[0040] AAV may be any serotype, e.g., AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV-10, AAV-11, AAV-12, AAV-13, AAV rh.10, AAV rh.74, or variants and derivatives thereof. In some embodiments, rAAV is serotype AAV rh.74. Generation of similar rAAVs is disclosed, e.g., in WO 01 / 83692, the entirety of which is incorporated by reference. Other types of rAAV variants, e.g., rAAVs with capsid mutations, are also considered. See, for example, the literature by Marsic et al. [Molecular Therapy, 22(11): 1900-1909 (2014)].

[0041] As used herein, the terms “AAV particle,” “AAV vector,” “AAV virion,” “AAV virus particle,” or “AAV vector particle” are used to refer to a viral particle consisting of an AAV capsid and an encapsulated AAV genome. In some embodiments, the AAV particle comprises a heterogeneous polynucleotide (i.e., a polynucleotide other than the wild-type AAV genome, such as a transgene, to be delivered to mammalian cells). In some embodiments, the generation of the AAV vector particle comprises the generation of an AAV vector, which is contained within the AAV vector particle.

[0042] For example, a wild-type (wt) AAV virus particle contains a linear, single-stranded AAV nucleic acid genome associated with an AAV capsid protein coat. The AAV virion may be a single-stranded (ss) AAV or a self-complementary (SC) AAV. In some embodiments, a single-stranded AAV nucleic acid molecule of either the complementary sense, e.g., the "sense" or "antisense" strand, may be packed into the AAV virion, and both strands are equally infectious.

[0043] The terms “recombinant AAV” or “rAAV” are defined herein as an infectious replication-defective virus consisting of an AAV protein shell that encapsulates a heterogeneous nucleotide sequence of interest located on both sides by an AAV ITR. In some embodiments, rAAV is generated in a suitable host cell having an AAV vector, an AAV helper function, and an accessory function introduced therein. In this way, the host cell can encode the AAV polypeptide necessary to package the AAV vector (containing the recombinant nucleotide sequence of interest) into an infectious recombinant virion particle for subsequent gene delivery.

[0044] As used herein, the term “capsid protein” refers to a protein that forms the coat or shell of a virus. The term “AAV capsid protein” refers to a protein that forms the coat of an adeno-associated virus (AAV) consisting of a total of 60 subunits; each subunit is an amino acid sequence, e.g., viral protein 1 (VP1), VP2, or VP3.

[0045] As used herein, the term "liquid phase chromatography (LC)" refers to a technique used to separate, identify, and quantify components within a mixture. In column liquid phase chromatography, a liquid phase mobile phase passes through a column, and components of the mobile phase interact with a solid phase stationary phase. The composition of the mobile phase can be changed during the separation run to alter the interaction strength of the compounds of interest. As the mobile phase continues to flow through the column, the eluent is typically collected in fractions while monitoring the concentration of compounds eluted from the column over time to generate an elution curve or chromatogram.

[0046] As used herein, the term “stationary phase” refers to a material that is maintained in a fixed state on a column. The most commonly used stationary phase columns are carbon-chain-bonded silica, phenyl-bonded silica, and cyano-bonded silica. In some embodiments, the stationary phase may comprise hydrophobic alkyl chains of a specific length, such as C4, C8, or C18. In some embodiments, the reverse phase chromatography is C8 reverse phase chromatography (e.g., reverse phase chromatography using a C8 stationary phase).

[0047] As used herein, the term “mobile phase” refers to water, a solvent, or a mixture of water and a solvent used to elute a compound from a column. The most common mobile phase solvents include, but are not limited to, acetonitrile, methanol, tetrahydrofuran, ethanol, or isopropyl alcohol. In some embodiments, two mobile phases are used. For example, a first mobile phase and a second mobile phase may be mixed in real time to obtain a solvent used to elute a substance from a column. In some embodiments, the volume ratio of the second mobile phase to the first mobile phase is within an increasing gradient during the elution step.

[0048] As used herein, the terms “mass spectrometry” or “MS” refer to an analytical technique for measuring the mass-to-charge ratio (m / z) of ions to identify and quantify molecules in simple and complex mixtures. MS technique generally comprises: (1) a step of ionizing a compound to form a charged compound; and (2) a step of detecting the mass-to-charge ratio of the charged compound and calculating its molecular weight. The compound may be ionized and detected by any suitable means. A “mass spectrometer” generally comprises an ionizer, a mass analyzer, and an ion detector. Generally, one or more molecules of interest are ionized, and the ions are subsequently introduced into a mass spectrometer instrument, where, due to a combination of a magnetic field and an electric field, the ions follow a spatial path dependent on mass (“m”) and charge (“z”). In some mass spectroscopic methods, ions can be separated from each other using, for example, time of flight (TOF), orbitrap, Fourier transform ion cyclotron resonance spectrometer, quadrupole, or ion trap, and then detected using an ion detector.

[0049] As used herein, the terms “ultraviolet-visible spectroscopy,” “ultraviolet-visible spectroscopy,” “UV-Vis,” or “UV / Vis” refer to absorption spectroscopy or reflectance spectroscopy used to determine the optical properties (transmittance, reflectance, and absorbance) of liquids and solids. In some embodiments, ultraviolet-visible spectroscopy is used to characterize the capsid proteins of AAV particles.

[0050] The term “total ion chromatogram (TIC)” as used herein refers to a type of chromatogram generated by summing the intensities of all mass spectrum peaks belonging to the same scan.

[0051] 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 presented in SEQ ID NO. 4 of U.S. Patent No. 9,909,142, which is incorporated herein by reference in its entirety. Additionally, an exemplary variant of the AAVrh74 VP1 capsid protein is presented in U.S. Patent No. 9,909,142.

[0052] As used herein, the term “subgroup” of VP proteins refers to a group of VP proteins that, unless otherwise specified, have at least one defined characteristic in common and consist of at least one group member fewer than all members of the reference group. For example, a “subgroup” of VPl proteins may, unless otherwise specified, be at least one VPl protein and fewer than all VP1 proteins in the assembled AAV capsid. A “subgroup” of VP3 proteins may, unless otherwise specified, be one VP3 protein and fewer than all VP3 proteins in the assembled AAV capsid. For example, VPl proteins may be a subgroup of VP proteins; VP2 proteins may be a separate subgroup of VP proteins; and VP3 is an additional subgroup of VP proteins in the assembled AAV capsid. In another example, VPl, VP2 and VP3 proteins may contain subgroups having different modifications, for example, at least one, two, three, or four highly deamidated asparagines in asparagine-glycine pairs.

[0053] Characterization of AAV VP1, VP2, and VP3 Capsid Proteins

[0054] In some embodiments, the present disclosure provides a method for characterizing VP1, VP2, and VP3 capsid proteins in adeno-associated virus (AAV) particles, the method comprising the step of applying AAV particles to liquid-phase chromatography at about 70°C to about 90°C, wherein the mass and proportion of VP1, VP2, and VP3 capsid proteins are determined by mass spectrometry and / or ultraviolet (UV)-visible spectroscopy. In some embodiments, the mass and proportion of VP1, VP2, and VP3 capsid proteins are determined by mass spectrometry and ultraviolet (UV)-visible spectroscopy. In some embodiments, the capsid on the AAV particles is denatured into individual VP1, VP2, and VP3 proteins in a column of liquid-phase chromatography. In some embodiments, the capsid proteins are separated by liquid-phase chromatography. In some embodiments, the method comprises: (a) applying AAV particles to liquid-phase chromatography to separate VP1, VP2, and VP3 capsid proteins; and (b) determining the ratio of VP1, VP2, and VP3 capsid proteins in AAV particles by applying the separated VP1, VP2, and VP3 capsid proteins produced in step (a) to mass spectrometry and / or ultraviolet-visible spectrometer to determine the relative amounts of VP1, VP2, and VP3 capsid proteins. In some embodiments, the liquid-phase chromatography is performed at about 70°C to about 90°C. In some embodiments, the liquid-phase chromatography is performed at about 70°C, 74°C, 76°C, 78°C, 80°C, 82°C, 84°C, 86°C, 88°C, or 90°C. In some embodiments, liquid-phase chromatography is performed at about 80°C.

[0055] In some embodiments, the method further includes the step of determining the mass of one or more of the VPL, VP2 and VP3 capsid proteins of the AAV particles.

[0056] In some embodiments, the relative amounts of VP1, VP2, and VP3 capsid proteins are determined by comparing the total ion chromatogram (TIC) of VP1, VP2, and VP3 capsid proteins.

[0057] In some embodiments, the liquid phase chromatography is reverse-phase liquid phase chromatography, size exclusion chromatography, hydrophilic interaction liquid phase chromatography, or cation exchange chromatography. In some embodiments, the liquid phase chromatography is reverse-phase liquid phase chromatography.

[0058] In some embodiments, reverse-phase chromatography is performed using a C18 column, a C8 column, or a C4 column. In some embodiments, liquid-phase chromatography is performed using a C8 column.

[0059] In some embodiments, the stationary phase of reverse-phase liquid-phase chromatography is contained within a chromatography column having a length of about 50 to 300 mm and an inner diameter of about 1 to 4.6 mm. In some embodiments, the column is a BEH column. In some embodiments, the column has an inner diameter of 1, 2.1, 3, or 4.6 mm. In some embodiments, the column has a length of 50, 75, 100, 150, or 300 mm. In some embodiments, column sizes are: 1 mm x 50 mm, 2.1 mm x 50 mm, 3 mm x 50 mm, 4.6 mm x 50 mm, 1 mm x 75 mm, 2.1 mm x 75 mm, 3 mm x 75 mm, 4.6 mm x 75 mm, 1 mm x 100 mm, 2.1 mm x 100 mm, 3 mm x 100 mm, 4.6 mm x 100 mm, 1 mm x 150 mm, 2.1 mm x 150 mm, 3 mm x 150 mm, 4.6 mm x 150 mm, 1 mm x 300 mm, 2.1 mm x 300 mm, 3 mm x 300 mm, or 4.6 mm x 300 mm. In some embodiments, the column sizes are 1.6 1.6 x 150 mm, 1.7 x 50 mm, 1.7 x 60, 1.7 x 70 mm, 1.7 x 80 mm, 1.7 x 90 mm, 1.7 x 100 mm, 1.7 x 110 mm, 1.7 x 120 mm, 1.7 x 130 mm, 1.7 x 140 mm, 1.7 x 150 mm, 1.8 x 50 mm, 1.88 x 150 mm, 1.9 x 50 mm, 1.9 x 60 mm, 1.9 x 70 mm, 1.9 x 80 mm, 1.9 x 90 mm, 1.9 x 100 mm, 1.9 x 110 mm, 1.9 x 120 mm, 1.9 x 130 mm, 1.9 x 140 mm, 1.9 x 150 mm, 2.0 x 50 mm, 2.0 x 60 mm, 2.0 x 70 mm, 2.0 x 80 mm, 2.0 x 90 mm, 2.0 x 100 mm, 2.0 x 110 mm, 2.0 x 120 mm, 2.0 x 130 mm, 2.0 x 140 mm, 2.0 x 150 mm, 2.1 x 50 mm, 2.1 x 60 mm, 2.1 x 70 mm, 2.1 x 80 mm, 2.1 x 90 mm, 2.1 x 100 mm, 2.1 x 110 mm, 2.1 x 120 mm, 2.1 x 130 mm, 2.1 x 140 mm, 2.1 x 150 mm, 2.2 x 50 mm, 2.2 x 60 mm, 2.2 x 70 mm, 2.2 x 80 mm, 2.2 x 90 mm, 2.2 x 100 mm, 2.2 x 110 mm, 2.2 x 120 mm, 2.2 x 130 mm, 2.2 x 140 mm, 2.2 x 150 mm, 2.3 x 50 mm, 2.3 x 60 mm, 2.3 x 70 mm, 2.3 x 80 mm, 2.3 x 90 mm, 2.3 x 100 mm, 2.3 x 110 mm, 2.3 x 120 mm, 2.3 x 130 mm, 2.3 x 140 mm, 2.3 x 150 mm, 2.4 x 50 mm, 2.4 x 60 mm, 2.4 x 70 mm, 2.4 x 80 mm, 2.4 x 90 mm, 2.4 x 100 mm, 2.4 x 110 mm, 2.4 x 120 mm, 2.4 x 130 mm, 2.4 x 140 mm, 2.4 x 150 mm, 2.5 x 50 mm, 2.5 x 60, 2.5 x 70 mm, 2.5 x 80 mm, 2.5 x 90 mm, 2.5 x 100 mm, 2.5 x 110 mm, 2.It is 5 x 120 mm, 2.5 x 130 mm, 2.5 x 140 mm, 2.5 x 150 mm, 2.6 x 50 mm, 2.6 x 60 mm, 2.6 x 70 mm, 2.6 x 80 mm, 2.6 x 90 mm, 2.6 x 100 mm, 2.6 x 110 mm, 2.6 x 120 mm, 2.6 x 130 mm, 2.6 x 140 mm, or 2.6 x 150 mm. In some embodiments, the stationary phase of reverse-phase liquid-phase chromatography is contained within a chromatography column having a length of about 100 mm and an inner diameter of about 2.1 mm.

[0060] In some embodiments, the stationary phase of reverse-phase liquid-phase chromatography comprises particles with a size of about 1.2 μm to 2.5 μm. In another embodiment, the stationary phase of reverse-phase liquid-phase chromatography comprises particles with a size of about 1.7 μm, 1.8 μm, or 2.1 μm. In some embodiments, the particle size is about 1.2 μm, 1.3 μm, 1.4 μm, 1.5 μm, 1.6 μm, 1.7 μm, 1.8 μm, 1.9 μm, 2.0 μm, 2.1 μm, 2.2 μm, 2.3 μm, 2.4 μm, or 2.5 μm. In some embodiments, the stationary phase of reverse-phase liquid-phase chromatography consists of particles of about 1.7 μm.

[0061] In some embodiments, chromatography uses a first mobile phase comprising fluoro-substituted acetic acid in water. Fluoro-substituted acetic acid includes monofluoroacetic acid, difluoroacetic acid, and trifluoroacetic acid. In some embodiments, chromatography uses a first mobile phase comprising trifluoroacetic acid in water.

[0062] In some embodiments, the first mobile phase comprises about 0.05% to about 0.15% by volume of fluoro-substituted acetic acid. In some embodiments, the first mobile phase comprises about 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, 0.1%, 0.11%, 0.12%, 0.13%, 0.14%, 0.15%, 0.16%, 0.17%, 0.18%, 0.19%, or 0.2% by volume of fluoro-substituted acetic acid. In some embodiments, the first mobile phase comprises about 0.05% or 0.1% by volume of fluoro-substituted acetic acid. In some embodiments, the first mobile phase comprises about 0.1% by volume of fluoro-substituted acetic acid. In some embodiments, the fluoro-substituted acetic acid is trifluoroacetic acid. In some embodiments, the first mobile phase contains about 0.1% by volume of trifluoroacetic acid.

[0063] In some embodiments, chromatography uses a second mobile phase comprising fluoro-substituted acetic acid in acetonitrile. In some embodiments, chromatography uses a second mobile phase comprising trifluoroacetic acid in acetonitrile. In some embodiments, chromatography uses a second mobile phase comprising fluoro-substituted acetic acid in a mixture of acetonitrile and water. In some embodiments, chromatography uses a second mobile phase comprising trifluoroacetic acid in a mixture of acetonitrile and water.

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

[0065] In some embodiments, the second mobile phase comprises about 75 to 95% by volume of acetonitrile. In some embodiments, the second mobile phase comprises about 75%, 80%, 85%, 90%, or 95% by volume of acetonitrile. In some embodiments, the second mobile phase comprises about 90% by volume of acetonitrile and 10% of water.

[0066] In some embodiments, the percentage of the second mobile phase in the combination of the first mobile phase and the second mobile phase in the chromatography increases over time. In some embodiments, the percentage of the second mobile phase increases from about 10% to about 40% by volume. In some embodiments, the percentage of the second mobile phase increases from about 10% to about 45% by volume. In some embodiments, the percentage of the second mobile phase increases from about 10% to about 100% by volume. In some embodiments, the percentage of the second mobile phase increases from about 10% to about 45% within about 30 to 40 minutes. In some embodiments, the percentage of the second mobile phase increases from about 10% to about 45% within about 35 minutes. In some embodiments, the percentage of the second mobile phase increases from about 10% to about 100% within about 30 to 50 minutes. In some embodiments, the percentage of the second mobile phase increases from about 10% to about 100% within about 36 minutes.

[0067] In some embodiments, the percentage of the second mobile phase increases from about 10% to about 40% within about 5 to 10 minutes, and increases from about 40% to about 45% within about 25 to 35 minutes. In some embodiments, the percentage of the second mobile phase increases from about 45% to about 100% within about 0.5 to 2 minutes. In some embodiments, the percentage of the second mobile phase decreases from about 100% to about 10% within about 0.5 to 2 minutes.

[0068] In some embodiments, the percentage of the second mobile phase increases from about 10% to about 40% within about 6 minutes and from about 40% to about 45% within about 29 minutes. In some embodiments, the percentage of the second mobile phase increases from about 45% to about 100% within about 1 minute. In some embodiments, the percentage of the second mobile phase decreases from about 100% to about 10% within about 1 minute.

[0069] In some embodiments, liquid-phase chromatography is high-pressure liquid-phase chromatography (HPLC). In some embodiments, liquid-phase chromatography is ultra-high-pressure liquid-phase chromatography (UHPLC).

[0070] In some embodiments, mass spectrometry may use any ionization mode, in particular, a mode suitable for analyzing biological molecules, including but not limited to: direct injection-mass spectrometry, electrospray ionization (ESI)-MS, desorption electrospray ionization (DESI)-MS, real-time direct analysis (DART)-MS, atmospheric pressure chemical ionization (APCI)-MS, electron bombardment (El) or chemical ionization (CI), matrix-assisted laser desorption / ionization (MALDI)-MS, and atmospheric pressure ionization-electrospray (API-ES). In some embodiments, mass spectrometry uses the API-ES ionization mode.

[0071] In some embodiments, mass spectrometry scans signals over a range of 400 to 16,000 m / z. In some embodiments, mass spectrometry scans signals over a range of 700 to 13,700 m / z.

[0072] In some embodiments, the scan type of the mass spectrometry is positive polarity. In some embodiments, the data acquisition time of the mass spectrometry is about 10 to 35 minutes. In some embodiments, the data acquisition time of the mass spectrometry is about 17 to 28 minutes.

[0073] In some embodiments, the nozzle voltage of the mass spectrometry is about 400 to 600 V. In some embodiments, the nozzle voltage of the mass spectrometry is about 500 V. In some embodiments, the skimmer voltage of the mass spectrometry is about 60 to 70 V. In some embodiments, the skimmer voltage of the mass spectrometry is about 65 V. In some embodiments, the difference between the nozzle voltage and the skimmer voltage is about 400 to 450 V. In some embodiments, the difference between the nozzle voltage and the skimmer voltage is about 435 V.

[0074] In some embodiments, the dry gas temperature of the mass spectrometry is about 200 to 350°C. In some embodiments, the dry gas temperature of the mass spectrometry is about 300°C. In some embodiments, the dry gas flow rate of the mass spectrometry is about 5 to 13 L / min. In some embodiments, the dry gas flow rate of the mass spectrometry is about 13 L / min.

[0075] In some embodiments, mass spectrometry uses a capillary voltage of about 3 to 6 kV. In some embodiments, mass spectrometry uses a capillary voltage of about 3, 4, 5, or 6 kV. In some embodiments, mass spectrometry uses a capillary voltage of about 5 kV.

[0076] In some embodiments, mass spectrometry uses a fragmentation voltage of about 125 to 350 V. In some embodiments, mass spectrometry uses a fragmentation voltage of approximately 125, 130, 135, 145, 155, 160, 165, 175, 185, 190, 195, 200, 205, 210, 215, 220, 225, 230, 235, 240, 245, 250, 255, 260, 265, 270, 275, 280, 285, 290, 295, 300, 305, 310, 315, 320, 325, 330, 335, 340, 345, or 350 V. In some embodiments, mass spectrometry uses a fragmentation voltage of about 175 V.

[0077] In some embodiments, the AAV particle is AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAVrh10, AAVrh74, or any naturally occurring, recombinant, or synthetic AAV particle. In some embodiments, the AAV particle is a recombinant AAV (rAAV) particle. In some embodiments, the AAV particle is AAVrh74.

[0078] In some of the foregoing embodiments, the present disclosure further comprises the step of determining a post-translational modification of at least one of the VPl, VP2, and VP3 capsid proteins. In some embodiments, the present disclosure further comprises the step of determining a post-translational glycosylation, sialation, acetylation, amino acid loss, amidation, phosphorylation, formylation, hydroxylation, methylation, and / or sulfation of at least one of the VPl, VP2, and VP3 capsid proteins. The post-translational modification includes one or more of loss of N-terminal methionine, loss of threonine, phosphorylation, and acetylation.

[0079] In some embodiments, the present disclosure comprises the step of determining the removal of N-terminal methionine from a VP1, VP2, or VP3 capsid protein. In some embodiments, the present disclosure comprises the step of determining the removal of N-terminal methionine from a VP1, or VP3 capsid protein. In some embodiments, the present disclosure comprises the step of determining N-terminal acetylation after the removal of N-terminal methionine from a VP1, VP2, or VP3 capsid protein. In some embodiments, the present disclosure comprises the step of determining N-terminal acetylation after the removal of N-terminal methionine from a VP1, or VP3 capsid protein.

[0080] In some embodiments, the present disclosure provides a method for characterizing the capsid proteins of AAV particles based at least partially on the ratio of VP1, VP2, and VP3 capsid proteins among the AAV particles and / or the mass of one or more of VPl, VP2, and VP3 capsid proteins.

[0081] In some embodiments, the present disclosure provides a method for determining the serotype of an AAV particle based at least partially on the ratio of VP1, VP2, and VP3 capsid proteins among the AAV particle and / or the mass of one or more of VPl, VP2, and VP3 capsid proteins, wherein the ratio of VP1, VP2, and VP3 capsid proteins and the mass of one or more of VPl, VP2, and VP3 capsid proteins are determined by the method disclosed herein.

[0082] Mass spectrometry is an analytical technique for protein characterization. In some embodiments, a method is provided for characterizing the ratio of AAVrh74 capsid proteins with whole masses for all three capsid proteins by liquid-phase chromatography and mass spectrometry. In some embodiments, the AAVrh74 capsid is denatured into individual capsid proteins VP1, VP2, and VP3 on a column. Denaturation is achieved by heating the column compartment to 80°C (3,4). Subsequently, the capsid proteins are baseline digested on a Waters BEH C8 column with the help of trifluoroacetic acid as an ion-pairing agent in the mobile phase (5). The denatured proteins are first analyzed under UV to provide the capsid ratios, and then analyzed in a mass spectrometer to provide the whole masses for the individual proteins.

[0083] Deamidation is a common post-translational modification that converts asparagine residues into a mixture of isoaspartate and aspartate. Deamidation of glutamine residues also occurs, but at a much slower rate. Oxidation is also a common post-translational modification, resulting from proteins reacting with various free radicals and reactive oxygen species. While methionine oxidation is the most common, oxidation of several other amino acid residues, such as cysteine ​​and tryptophan, has also been observed. Deamidation and oxidation are also common degradation pathways for proteins that occur during manufacturing and storage. Deamidation can affect protein activity and stability. Oxidation can affect protein activity and stability by causing conformational changes. Oxidation can also affect the immunogenicity of proteins. Therefore, it is necessary to carefully monitor critical quality attributes (CQAs) of proteins in relation to post-translational modifications.

[0084] Current methods using ammonium bicarbonate have generated false signals in AAV capsid proteins or overestimated deamidation (Table 7). Herein, the present disclosure provides a method for more accurately measuring post-translational modifications to capsid proteins using Tris-HCl. In some embodiments, the LC-MS method uses a buffer containing Tris-HCl. In some embodiments, the buffer contains acetonitrile. In some embodiments, the buffer contains methionine. In some embodiments, the buffer contains 5 mM to 50 mM Tris-HCl, 5% to 20% acetonitrile, and 1 mM to 50 mM methionine. In some embodiments, the buffer contains 20 mM Tris-HCl, 5% to 10% acetonitrile, and 10 mM methionine.

[0085] In some embodiments, post-translational modification comprises deamidation at one or more of N263, N514, N57, N502, N254, and N94 of AAV8, or at equivalent residues of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV9, AAV10, AAV11, AAV12, AAV13, AAVrh10, or AAVrh74. In some embodiments, post-translational modification comprises deamidation at one or more of N57, N255, N256, and N263 of AAV.Rh74. In some embodiments, post-translational modification comprises oxidation at one or more of M437, M473, M526, M544, M560, and M637 of AAV.Rh74.

[0086] AAV composition

[0087] Some aspects of the present disclosure relate to recombinant AAVs (rAAVs) comprising heterogroups of capsid proteins containing subgroups having amino acid modifications. In some aspects, the modification may be deamidation, acetylation, isomerization, phosphorylation, or oxidation. In some aspects, the modification is deamidation or oxidation.

[0088] In some embodiments, the rAAV capsid may comprise a subgroup of VPL, VP2, and VP3 having at least 1, at least 2, at least 3, at least 4, at least 5 to at least about 25 deamidated amino acid residues, of which at least about 1% to about 10%, at least about 10% to about 25%, at least about 25% to about 50%, at least about 50% to about 70%, at least about 70% to about 100%, at least about 75% to about 100%, about 80% to about 100%, or at least about 90% to about 100% is deamidated compared to the amino acid sequence encoding the VP protein. In some embodiments, most of these may be N residues. In some embodiments, Q residues may be deamidated.

[0089] In some embodiments, the present disclosure provides an AAV composition comprising an AAV capsid comprising deamidation at one or more of N57, N255, N256, and N263 of AAV.Rh74, or at equivalent residues of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, or AAVrh10, when measured by mass spectrometry and / or ultraviolet (UV)-visible spectrometry. In some embodiments, deamidation is measured by any one of the methods disclosed herein.

[0090] In some embodiments, the heterogeneous group is less than approximately 80%, less than approximately 78%, less than approximately 76%, less than approximately 74%, less than approximately 72%, less than approximately 70%, less than approximately 68%, less than approximately 66%, less than approximately 64%, less than approximately 62%, less than approximately 60%, less than approximately 58%, less than approximately 56%, less than approximately 54%, less than approximately 52%, less than approximately 50%, less than approximately 48%, less than approximately 46%, less than approximately 44%, less than approximately 42%, less than approximately 40%, less than approximately 38%, less than approximately 36%, less than approximately 34%, less than approximately 32%, less than approximately 30%, less than approximately 28%, less than approximately 26%, less than approximately 24%, less than approximately 22%, less than approximately 20%, less than approximately 18%, less than approximately 16%, and approximately 15% It includes a capsid protein having deamidation of the AAV.rh74 capsid at N57 of less than, about 14%, about 13%, about 12%, about 11%, or about 10%.

[0091] In some embodiments, the heterogeneous group comprises a capsid protein having deamidation of the AAV.rh74 capsid at N57 of 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%.

[0092] In some embodiments, the heterogeneous group is less than approximately 80%, less than approximately 78%, less than approximately 76%, less than approximately 74%, less than approximately 72%, less than approximately 70%, less than approximately 68%, less than approximately 66%, less than approximately 64%, less than approximately 62%, less than approximately 60%, less than approximately 58%, less than approximately 56%, less than approximately 54%, less than approximately 52%, less than approximately 50%, less than approximately 48%, less than approximately 46%, less than approximately 44%, less than approximately 42%, less than approximately 40%, less than approximately 38%, less than approximately 36%, less than approximately 34%, less than approximately 32%, less than approximately 30%, less than approximately 28%, less than approximately 26%, less than approximately 24%, less than approximately 22%, less than approximately 20%, less than approximately 18%, less than approximately 16%, and approximately 15% It includes a capsid protein having deamidation in N254 and / or N255 of the AAV.rh74 capsid of less than, about 14%, about 13%, about 12%, about 11%, or about 10%, about 8%, about 7%, about 6%, about 5%, about 4%, about 3%, about 2%, or about 1%.

[0093] In some embodiments, the heterogeneous group is less than approximately 80%, less than approximately 78%, less than approximately 76%, less than approximately 74%, less than approximately 72%, less than approximately 70%, less than approximately 68%, less than approximately 66%, less than approximately 64%, less than approximately 62%, less than approximately 60%, less than approximately 58%, less than approximately 56%, less than approximately 54%, less than approximately 52%, less than approximately 50%, less than approximately 48%, less than approximately 46%, less than approximately 44%, less than approximately 42%, less than approximately 40%, less than approximately 38%, less than approximately 36%, less than approximately 34%, less than approximately 32%, less than approximately 30%, less than approximately 28%, less than approximately 26%, less than approximately 24%, less than approximately 22%, less than approximately 20%, less than approximately 18%, less than approximately 16%, and approximately 15% It includes a capsid protein having deamidation in N263 of less than, about 14%, about 13%, about 12%, about 11%, or about 10%.

[0094] In some embodiments, the AAV composition comprises an AAV capsid comprising oxidation at one or more of M437, M473, M526, M544, M560, and M637 of AAV.Rh74, or at an equivalent residue 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 spectrometry. In some embodiments, deamidation is measured by any one of the methods disclosed herein.

[0095] In some embodiments, the heterogeneous group is 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 about It contains a capsid protein having less than 0.5% oxidation in M437.

[0096] In some embodiments, the heterogeneous group is 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 about It contains a capsid protein having less than 0.5% oxidation in M473.

[0097] In some embodiments, the heterogeneous group is 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 about It contains a capsid protein having less than 0.5% oxidation in M526.

[0098] In some embodiments, the heterogeneous group is less than, about 40%, about 48%, about 46%, about 44%, about 42%, about 40%, about 38%, about 36%, about 34%, about 32%, about 30%, about 28%, about 26%, about 24%, about 22%, about 20%, about 18%, about 16%, about 14%, about 12%, about 10%, about 9%, about 8%, about 7%, about 6%, about 5%, about 4%, about 3%, about 2%, about 1%, about 0.9%, about 0.8%, about 0.7%, about 0.6%, or about It contains a capsid protein having less than 0.5% oxidation in M544.

[0099] In some embodiments, the heterogeneous group is 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 about It contains a capsid protein having less than 0.5% oxidation in M560.

[0100] In some embodiments, the heterogeneous group is less than, about 40%, about 48%, about 46%, about 44%, about 42%, about 40%, about 38%, about 36%, about 34%, about 32%, about 30%, about 28%, about 26%, about 24%, about 22%, about 20%, about 18%, about 16%, about 14%, about 12%, about 10%, about 9%, about 8%, about 7%, about 6%, about 5%, about 4%, about 3%, about 2%, about 1%, about 0.9%, about 0.8%, about 0.7%, about 0.6%, or about It contains a capsid protein having less than 0.5% oxidation in M637.

[0101] Characterization of host cell proteins in AAV composition

[0102] In some embodiments, the present disclosure provides a method for characterizing host cell proteins in an AAV composition, such as an AAV-based gene therapy drug product. In some embodiments, the method for characterizing host cell proteins in an AAV composition comprises the steps of immunoprecipitating a viral capsid protein from the composition, degrading residual host cell proteins, and identifying host cell proteins by analyzing the degraded proteins using liquid-phase chromatography quadrupole time-of-flight mass spectrometry (LC-QTOF-MS). As used herein, the terms “residual host cell proteins” or “residual proteins” refer to proteins remaining in the solution after immunoprecipitation. In some embodiments, the method further comprises the step of analyzing the degraded host cell proteins using repeated MS / MS.

[0103] In some embodiments, immunoprecipitation comprises the step of incubating an AAV composition with a VP antibody. In some embodiments, the VP antibody comprises an anti-AAV VP1 antibody, an anti-AAV VP2 antibody, an anti-AAV VP3 antibody, or a combination thereof. In some embodiments, the antibody may be the anti-adeno-associated virus (AAV), VP1 / VP2 / VP3 of American Research Products, Inc. (Catalog #:03-61058).

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

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

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

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

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

[0109] In some embodiments, the stationary phase of reverse-phase liquid-phase chromatography is contained within a chromatography column having a length of about 50 to 300 mm and an inner diameter of about 1 to 4.6 mm. In some embodiments, the column is a BEH column. In some embodiments, the column has an inner diameter of 1, 2.1, 3, or 4.6 mm. In some embodiments, the column has a length of 50, 75, 100, 150, or 300 mm. In some embodiments, column sizes are: 1 mm x 50 mm, 2.1 mm x 50 mm, 3 mm x 50 mm, 4.6 mm x 50 mm, 1 mm x 75 mm, 2.1 mm x 75 mm, 3 mm x 75 mm, 4.6 mm x 75 mm, 1 mm x 100 mm, 2.1 mm x 100 mm, 3 mm x 100 mm, 4.6 mm x 100 mm, 1 mm x 150 mm, 2.1 mm x 150 mm, 3 mm x 150 mm, 4.6 mm x 150 mm, 1 mm x 300 mm, 2.1 mm x 300 mm, 3 mm x 300 mm, or 4.6 mm x 300 mm. In some embodiments, the column sizes are 1.6 1.6 x 150 mm, 1.7 x 50 mm, 1.7 x 60, 1.7 x 70 mm, 1.7 x 80 mm, 1.7 x 90 mm, 1.7 x 100 mm, 1.7 x 110 mm, 1.7 x 120 mm, 1.7 x 130 mm, 1.7 x 140 mm, 1.7 x 150 mm, 1.8 x 50 mm, 1.88 x 150 mm, 1.9 x 50 mm, 1.9 x 60 mm, 1.9 x 70 mm, 1.9 x 80 mm, 1.9 x 90 mm, 1.9 x 100 mm, 1.9 x 110 mm, 1.9 x 120 mm, 1.9 x 130 mm, 1.9 x 140 mm, 1.9 x 150 mm, 2.0 x 50 mm, 2.0 x 60 mm, 2.0 x 70 mm, 2.0 x 80 mm, 2.0 x 90 mm, 2.0 x 100 mm, 2.0 x 110 mm, 2.0 x 120 mm, 2.0 x 130 mm, 2.0 x 140 mm, 2.0 x 150 mm, 2.1 x 50 mm, 2.1 x 60 mm, 2.1 x 70 mm, 2.1 x 80 mm, 2.1 x 90 mm, 2.1 x 100 mm, 2.1 x 110 mm, 2.1 x 120 mm, 2.1 x 130 mm, 2.1 x 140 mm, 2.1 x 150 mm, 2.2 x 50 mm, 2.2 x 60 mm, 2.2 x 70 mm, 2.2 x 80 mm, 2.2 x 90 mm, 2.2 x 100 mm, 2.2 x 110 mm, 2.2 x 120 mm, 2.2 x 130 mm, 2.2 x 140 mm, 2.2 x 150 mm, 2.3 x 50 mm, 2.3 x 60 mm, 2.3 x 70 mm, 2.3 x 80 mm, 2.3 x 90 mm, 2.3 x 100 mm, 2.3 x 110 mm, 2.3 x 120 mm, 2.3 x 130 mm, 2.3 x 140 mm, 2.3 x 150 mm, 2.4 x 50 mm, 2.4 x 60 mm, 2.4 x 70 mm, 2.4 x 80 mm, 2.4 x 90 mm, 2.4 x 100 mm, 2.4 x 110 mm, 2.4 x 120 mm, 2.4 x 130 mm, 2.4 x 140 mm, 2.4 x 150 mm, 2.5 x 50 mm, 2.5 x 60, 2.5 x 70 mm, 2.5 x 80 mm, 2.5 x 90 mm, 2.5 x 100 mm, 2.5 x 110 mm, 2.It is 5 x 120 mm, 2.5 x 130 mm, 2.5 x 140 mm, 2.5 x 150 mm, 2.6 x 50 mm, 2.6 x 60 mm, 2.6 x 70 mm, 2.6 x 80 mm, 2.6 x 90 mm, 2.6 x 100 mm, 2.6 x 110 mm, 2.6 x 120 mm, 2.6 x 130 mm, 2.6 x 140 mm, or 2.6 x 150 mm. In some embodiments, the stationary phase of reverse-phase liquid-phase chromatography is contained within a chromatography column having a length of about 150 mm and an inner diameter of about 2.1 mm.

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

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

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

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

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

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

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

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

[0118] In some embodiments, the percentage of the second mobile phase in the combination of the first mobile phase and the second mobile phase in the chromatography increases over time. In some embodiments, the percentage of the second mobile phase increases from about 2% to about 50% by volume. In some embodiments, the percentage of the second mobile phase increases from about 50% to about 100% by volume. In some embodiments, the percentage of the second mobile phase increases from about 2% to about 50% within about 110 to 130 minutes. In some embodiments, the percentage of the second mobile phase increases from about 2% to about 50% within about 120 minutes. In some embodiments, the percentage of the second mobile phase increases from about 50% to about 100% within about 20 to 30 minutes. In some embodiments, the percentage of the second mobile phase increases from about 50% to about 100% within about 25 minutes. In some embodiments, the percentage of the second mobile phase subsequently increases to 100% by volume over about 5 minutes. In some embodiments, the percentage of the second mobile phase subsequently remains at 100% by volume for about 3 minutes. In some embodiments, the second mobile phase subsequently decreases to about 2% by volume over about 2 minutes.

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

[0120] In some embodiments, the percentage of the second mobile phase decreases from about 100% to about 2% within about 1 to 10 minutes. In some embodiments, the percentage of the second mobile phase decreases from about 100% to about 2% within about 4 minutes.

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

[0122] In some embodiments, mass spectrometry may use any ionization mode, in particular, a mode suitable for analyzing biological molecules, including but not limited to: direct injection-mass spectrometry, electrospray ionization (ESI)-MS, desorption electrospray ionization (DESI)-MS, real-time direct analysis (DART)-MS, atmospheric pressure chemical ionization (APCI)-MS, electron bombardment (El) or chemical ionization (CI), matrix-assisted laser desorption / ionization (MALDI)-MS, and atmospheric pressure ionization-electrospray (API-ES). In some embodiments, mass spectrometry uses the API-ES ionization mode.

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

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

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

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

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

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

[0129] Although the subject matter has been described in considerable detail with reference to its specific modalities, other modalities are also possible. As such, the concept and scope of the appended claims should not be limited to the description of the specific modalities contained therein.

[0130] Examples

[0131] Now, the present disclosure will be illustrated with working examples, which are intended to illustrate the work of the present disclosure rather than to limit any restrictions on the scope of the present disclosure. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which the present disclosure pertains. Although methods and materials similar or equivalent to those described herein may be used to practice the disclosed methods and compositions, exemplary methods, apparatuses, and materials are described herein.

[0132] Example 1: General method and apparatus

[0133] 1.1 reagent

[0134] LCMS water, acetonitrile, and trifluoroacetic acid were obtained from Fisher Scientific, and ammonium bicarbonate was obtained from Sigma Aldrich. Table 1 provides the reagents used for liquid-phase chromatography and solution preparation.

[0135]

[0136] 1.2 device

[0137] The embodiments disclosed herein were performed using the following apparatus.

[0138] a) Waters ACQUITY UPLC BEH C8 column, 2.1 x 100 mm, 1.7 μm; Part No. 186002878

[0139] b) Pierce detergent removal spin column, 0.5 mL; Catalog # 87777

[0140] c) Suitable analytical balance

[0141] d) Automatic pipette

[0142] e) Grade A volumetric measuring glassware

[0143] f) HPLC vial and cap

[0144] g) Spatula and weighing boat

[0145] h) Agilent 1290 Infinity II UHPLC System

[0146] i) Agilent 6545XT AdvanceBio Quadrupole Time of Flight Mass Spectrometer (Q-ToF)

[0147] Example 2: Solution Preparation

[0148] 2.1 Preparation of 100 mM ammonium bicarbonate

[0149] 0.395 ± 0.01 grams of ammonium bicarbonate were weighed into a 50 mL Falcon® tube. Using a measuring cylinder, 50 mL of LCMS water was transferred to the tube, and the ammonium bicarbonate was completely dissolved using a vortex mixer to obtain a 100 mM ammonium bicarbonate solution. The solution is stable for 1 month at 2 to 8°C.

[0150] 2.2 Preparation of the first mobile phase (0.1% aqueous trifluoroacetic acid solution)

[0151] Using a measuring cylinder, 1 L of LCMS water was transferred to a 1 L bottle. Using a pipette, 1000 μL of trifluoroacetic acid was transferred to the bottle. The trifluoroacetic acid and water were mixed well for 5 minutes to obtain the first mobile phase. The first mobile phase is stable for up to 1 month under ambient conditions.

[0152] 2.3 Preparation of the second mobile phase (0.1% trifluoroacetic acid and 10% water in 90% acetonitrile)

[0153] 900 mL of acetonitrile was added to a 1 L measuring cylinder. LCMS water was added to the 1 L measuring cylinder to make a 1 L solution. The solution was transferred to a 1 L bottle. Using a pipette, 1000 μL of trifluoroacetic acid was transferred to the bottle. The trifluoroacetic acid and the solution were mixed thoroughly for 5 minutes to obtain the second mobile phase. The second mobile phase is stable for up to 1 month under ambient conditions.

[0154] Example 3: Sample Preparation

[0155] The bottom plug of the spin column was removed, and the spin column cap was unscrewed. The column was placed in a 2 mL collection tube and centrifuged at 1500 x g for 1 minute. When using a fixed-angle rotor, a mark was placed on the side of the column with the compressed resin tilted upward. Then, the column was placed in the centrifuge with the mark facing outward for all subsequent steps.

[0156] 400 μL of 100 mM ammonium bicarbonate solution was added to the column, and the column was centrifuged at 1500 x g for 1 minute. This step was repeated two more times, and the perfusion was discarded after each step. The column was placed into a new 2 mL collection tube. 5 μg of the sample was slowly spread onto the top of the compressed resin bed and incubated at room temperature for 2 minutes. The column was centrifuged at 1500 x g for 2 minutes, and the polymer-free sample was collected. Then, the sample volume was increased to 100 μL using 100 mM ammonium bicarbonate, and it was transferred to an HPLC vial.

[0157] Example 4: Characterization of VP1, VP2, and VP3 Capsid Proteins in AAV Particles

[0158] The present example describes a method for determining the ratio of VP1, VP2, and VP3 capsid proteins in AAV particles, and the mass of VPl, VP2, and VP3 capsid proteins. Here, AAV particles were denatured and separated into VP1, VP2, and VP3 capsid proteins by liquid-phase chromatography. The separated VP1, VP2, and VP3 capsid proteins were first subjected to UV light to determine the ratio of VP1, VP2, and VP3 capsid proteins in AAV particles, and then subjected to mass spectrometry to obtain the mass of each of the VPl, VP2, and VP3 capsid proteins.

[0159] 4.1 LC operating conditions

[0160] Separation of AAV VP1, VP2, and VP3 capsid proteins was performed on an ACQUITY UPLC® system using an ACQUITY UPLC® BEH 1.7 μm, 2.1 x 100 mm, C8 analytical column (part number 186002878). The mobile phases used were as follows:

[0161] First mobile phase (A): 0.1% trifluoroacetic acid in water; and

[0162] Second mobile phase (B): 0.1% trifluoroacetic acid and 10% water in 90% acetonitrile.

[0163] The column temperature was maintained at approximately 80°C, and separation was performed using mobile phase B increasing from 10% to 40% and from 40% to 45% at a flow rate of 0.4 mL / min, followed by flushing with 100% mobile phase B for 1 minute, and then re-equilibrating with the starting mobile phase composition (10% mobile phase B) for an additional 5 minutes.

[0164] The LC operating conditions are listed in Table 2.

[0165]

[0166] 4.2 Mass Spectrometer (MS) Operating Conditions

[0167] Mass spectrometry was performed using an Agilent 6545XT AdvanceBio Quadrupole Time-of-Flight (Q-ToF) spectrometer with API-ES ionization in survey scans ranging from 700 to 13,700 m / z. The capillary voltage, nozzle voltage, fragmentation voltage, and skimmer voltage were set to 5 kV, 500 V, 175 V, and 65 V, respectively. The dry gas temperature and dry gas flow rate were set to 300°C and 13 L / min, respectively.

[0168] The operating conditions of the mass spectrometer are listed in Table 3.

[0169]

[0170] 4.3 Analysis and Results

[0171] The capsid proteins were first denatured by heating the column compartment to 80°C. Then, three capsid proteins VP1, VP2, and VP3 were baseline separated using a Waters UPLC BEH C8 column (part number 186002878) and eluted by a combination of a first mobile phase containing 0.1% trifluoroacetic acid in water and a second mobile phase containing 0.1% trifluoroacetic acid in a mixture of acetonitrile and water (wherein the percentage of the second mobile phase increases over time). Using 0.1% trifluoroacetic acid as an ion-diffusing agent in the mobile phase helps with baseline resolution.

[0172] The relative amounts of VP1, VP2, and VP3 capsid proteins separated by liquid-phase chromatography were first determined by applying UV light, and then the masses of VP1, VP2, and VP3 capsid proteins were determined by applying mass spectrometry. As shown in Figure 1 and Table 4, a stoichiometry of approximately 1:1:10 for VP1 / VP2 / VP3 was obtained by baseline integration of the UV chromatogram.

[0173]

[0174] Then, the peaks for the three capsid proteins were deconvolved using the parameters listed in Table 5. The total ion chromatograms and deconvolved spectra for all three peaks are shown in Figures 2 and 3a through 3c. The three major masses detected under the VP1 peak (theoretical mass 81587 Da) were 81496 Da, 81578 Da, and 81658 Da. The 81496 Da peak indicates the VP1 protein with a loss of N-terminal methionine and a single acetylation modification. The other two peaks, with a mass shift of +80 Da, indicate phosphorylation. Deconvolution of the VP2 peak (theoretical mass 66381 Da) revealed two major masses: 66282 Da and 66360 Da. The 66282 Da peak represents the VP2 protein with a loss of threonine, and the 66360 Da peak corresponds to a single phosphorylation with a mass shift of +80. The VP3 peak (theoretical mass 59750 Da) represented a single major mass of 59662 Da, which corresponds to the mass of the VP3 protein with a loss of N-terminal methionine and a single acetylation.

[0175]

[0176] Figure 3 shows the detection of post-translational modifications of VP1, VP2, and VP3. Table 6 shows the intact mass spectrometry of AAV.rh74 capsid proteins.

[0177]

[0178] Example 5: Analysis of Deamidation Characteristics of AAVrh74 Using LCMS

[0179] Extensive deamidation in capsid proteins can be determined by mass spectrometry, in which case the deamidation sites and the levels of deamidation in these sites can also be determined. To measure AAV capsid deamidation, the capsid protein was denatured and reduced at 90°C for 10 minutes in the presence of 2 M guanidine hydrochloride and 10 mM DTT. After cooling the sample to room temperature, 30 mM iodoacetamide was added for alkylation, and the sample was incubated in the dark at room temperature for 30 minutes. Then, 1 mL of DTT was added to quench the alkylated mixture. 20 mM ammonium bicarbonate was added to the sample to dilute the guanidine hydrochloride to 200 mM. Then, the sample was digested using trypsin at an enzyme-to-protein ratio of 1:20 and incubated overnight at 37°C. After incubating overnight, trifluoroacetic acid was added to quench the decomposition product to a final concentration of 0.5%, and the sample was analyzed on a Thermo UltiMate 3000 RSLC system coupled to Q Exactive HF using a NanoFlex source.

[0180] Table 7 shows the identified major deamidation sites and the levels of deamidation at these sites (i.e., deamidation percentage). The data for AAV8 in Table 7 are disclosed from a previous publication (Molecular Therapy, Volume 26 No 12, Pages 2848 - 2962 (2018)).

[0181]

[0182] Two buffers (ammonium bicarbonate and Tris-HCl) were used separately to measure the deamidation status of AAV.rh74.

[0183] For ammonium bicarbonate, the sample was denatured by performing a buffer exchange with 100 mM ammonium bicarbonate. The denatured sample was reduced by the addition of 10 mM DTT and incubated at 37°C for 45 minutes. Then, alkylation was performed by adding iodoacetamide to the sample to a final concentration of 30 mM. The denatured, reduced, and alkylated sample was then exchanged again with 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 in which digestion was performed in ammonium bicarbonate, a similar level of deamidation was obtained for AAVrh74 as shown in Table 7.

[0184] For Tris-HCl, a 60 μg sample aliquot was buffer-exchanged with 4 M guanidine and 200 mM Tris pH 7.5 using an Amicon 10 K 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 aliquot. The reaction mixture was incubated at 56°C for 45 minutes, then cooled to room temperature. Iodoacetamide (30 mM) was added, and the mixture was incubated in the dark at room temperature for 60 minutes. Then, Tris buffer (100 mM, pH = 7.5) was added to dilute the guanidine HCl concentration to 0.6 M. Trypsin / Lys-C (60 μg) was added to 60 μg of reduced and alkylated sample (enzyme:protein ratio approximately 1:1 (w:w)). Methionine was added up to 10 mM during degradation to minimize artificial oxidation. Degradation was performed overnight (17 hours) at 37°C. Then, TFA (1%) was added prior to LC-MS / MS analysis.

[0185] As shown in Table 7, when Tris-HCl was used as the buffer, the deamidation state was significantly lower compared to those using ammonium bicarbonate.

[0186] To further optimize the use of Tris-HCl, eight separate AAV capsid samples were measured according to the flowchart in Fig. 4. First, the samples were denatured by performing a buffer exchange with 6 M guanidine hydrochloride, 20 mM Tris-HCl, and pH 7.5. Then, DTT was added to reduce the samples to a final concentration of 10 mM, and the samples were incubated at 37°C for 45 minutes. Alkylation was performed by adding iodoacetamide to a final concentration of 30 mM, and the samples were incubated in the dark at room temperature for 1 hour. The samples were again buffered with 20 mM Tris-HCl and pH 7.5 using a 10 kDa Amicon Ultra filter. Then, acetonitrile was 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. Then, the peptides were isolated on an Agilent 1290 U-HPLC using RP-HPLC. The isolated peptides were then detected using an Agilent 6545XT QToF, and deamidation analysis was performed using MassHunter and Bioconfirm software. The deamidation status is shown in Fig. 5, and the oxidation status is shown in Fig. 6. Of the 52 asparagine residues present, no deamidation was observed in 48 residues (total asparagine residues in VP1: 56). Of the 39 glutamine residues present, no deamidation was observed in any of the 39 residues (total glutamine residues in VP1: 48). When O18-labeled water was used, a small amount of deamidation in N57 was found to be a sample preparation artifact. No oxidation was detected in the remaining 5 methionine residues (total methionine residues in VP1: 11). Of the 14 detected tryptophan residues, no oxidation was observed in any of the 14 residues (total tryptophan residues in VP1: 15).

[0187] The decomposition of ammonium bicarbonate can significantly increase deamidation artifacts because the pH increases over time. Therefore, the observed higher level of deamidation may be deamidation artifacts generated during sample preparation. Deamidation levels were determined by establishing a Tris HCl-based decomposition in Sarepta: 20 mM Tris HCl, pH 7.5 was used as the buffer; 10% acetonitrile (known to reduce deamidation artifacts) was added to the decomposition solution; and 10 mM methionine was added to the decomposition solution to reduce oxidative artifacts.

[0188] Therefore, the method of the present disclosure is more accurate in measuring deamidation, oxidation, or other post-translational modifications with Tris-HCl buffer.

[0189] Example 6: Characterization of host cell proteins using LC-QTOF-MS

[0190] The purity of rAAV-based gene therapy drug products was analyzed by characterizing the host cell proteins remaining in the AAV composition via LC-QTOF-MS.

[0191] 6.1 Preparation of samples

[0192] AAVrh74 samples were spiked with known amounts of human thioredoxin 1 (HTI) protein standards, Invitrogen (Invitrogen, Catalog No. LF-P0001), and bovine carbonic anhydrase II (BCAII) protein standards (Sigma, Catalog No. C7749). Human thioredoxin 1 (HTI) and bovine carbonic anhydrase II (BCAII) were selected as spike protein standards for the quantification of detected human and bovine HCPs, respectively. For the immuno-depletion method, 100 μL of 0.05 mg / mL anti-adeno-associated virus (AAV), VP1 / VP2 / VP3 antibodies, and 100 μL of sample solution were pipetted into 270 μL of IP-MS cell lysis buffer from a Pierce MS-compatible magnetic IP kit along with 20 μL of 0.05 mg / mL BCAII and 10 μL of 0.1 mg / mL HTI. Then, AAV capsid proteins were immunoprecipitated from the samples using the anti-adeno-associated virus (AAV), VP1 / VP2 / VP3 from American Research Products, Inc. (Catalog # 03-6105) and the Pierce MS-compatible magnetic IP kit (Catalog # 90409). Then, the samples were passed through a Pierce desalination spin column (Catalog # 87777).

[0193] Then, the sample was buffer-exchanged with Promega rapid digestion buffer (catalog # VA1060). The sample was reduced, alkylated, and digested using rapid digestion trypsin at 70°C for 60 to 180 minutes.

[0194] 6.2 LC operating conditions

[0195] Separation of degraded residual host cell proteins was performed on an Agilent 1290 HPLC system using a Waters Acquity peptide BEH C18, 1.7 μm, 2.1 x 150 mm column. The mobile phases used are as follows:

[0196] First mobile phase (A): 0.1% formic acid in water; and

[0197] Second mobile phase (B): 0.1% formic acid and 10% water in 90% acetonitrile.

[0198] The column temperature was maintained at approximately 45°C, and separation was performed using mobile phase B increasing from 2% to 50% and from 50% to 100% at a flow rate of 0.3 mL / min, followed by flushing with 100% mobile phase B for 3 minutes, and then re-equilibrating with the starting mobile phase composition (2% mobile phase B) for an additional 5 minutes.

[0199] The LC operating conditions are listed in Table 8.

[0200]

[0201] 6.3 Mass Spectrometer (MS) Operating Conditions

[0202] Mass spectrometry was performed using an Agilent 6545XT AdvanceBio Quadrupole Time-of-Flight (Q-ToF) spectrometer with API-ES ionization in survey scans ranging from 50 to 3000 m / z. The capillary voltage, nozzle voltage, fragmentation voltage, and skimmer voltage were set to 4 kV, 500 V, 135 V, and 65 V, respectively. The dry gas temperature and dry gas flow rate were set to 325°C and 12 L / min, respectively.

[0203] The operating conditions of the mass spectrometer are listed in Table 9.

[0204]

[0205] 6.4 Analysis and Results

[0206] The data generated in Example 6.3 was processed by Protein Metrics' Byos software for searching specific Uniprot protein databases. The identification and relative amount of each residual protein were calculated relative to the amount of spike protein standard. HCP analysis results showed that for three lots of AAV virus particles, only a small number of residual host cell proteins (two small proteins, but no human proteins) were identified by MS (Table 10). The concentrations for the proteins are in units of ng / mL or ppm based on the spike protein standard.

[0207]

[0208] The following references are incorporated herein in their entirety:

[0209] 1. Buller RM, Rose JA. Characterization of adenovirus-associated virus-induced polypeptides in KB cells. J Virol 25: 1978, pages 331-338.

[0210] 2. Johnson FB, Ozer HL, Hoggan MD. Structural proteins of adenovirus-associated viruses. J Virol 8:1971, pages 776-770.

[0211] 3. DW Bauer et al., Exploring the Balance between DNA Pressure and Capsid Stability in Herpesviruses and Phages. J Virol 2015, 9288-98.

[0212] 4. Vamseedhar Rayaprolu 등, Comparative Analysis of Adeno-Associated Virus Capsid Stability and Dynamics. J Virol 2013, 13150-60.

[0213] 5. Xiaoying Jin 등, Direct Liquid Chromatography / Mass Spectrometry Analysis for Complete Characterization of Recombinant Adeno-Associated Virus Capsid Proteins. Human Gene Therapy Methods, Volume 38 Number 5 2017, 255-267.

Claims

Claim 1 A method for characterizing VP1, VP2, and VP3 capsid proteins in adeno-associated virus (AAV) particles, the method comprising the step of applying AAV particles to liquid-phase chromatography at 70°C to 90°C; wherein the ratios of VP1, VP2, and VP3 capsid proteins are determined by mass spectrometry and / or ultraviolet (UV)-visible spectrometry. Claim 2 In claim 1, the method wherein the individual masses of the VP1, VP2, and VP3 capsid proteins are determined by mass spectrometry. Claim 3 A method according to claim 1, wherein the ratio of VP1, VP2, and VP3 capsid proteins is determined by the step of comparing ultraviolet (UV) chromatograms of VP1, VP2, and VP3 capsid proteins. Claim 4 In claim 1, the liquid phase chromatography is reverse phase liquid phase chromatography. Claim 5 In paragraph 4, the reverse phase liquid phase chromatography is performed using a C18 column, a C8 column, or a C4 column. Claim 6 A method according to claim 5, wherein the stationary phase of the column comprises particles having a size of 1.2 to 3.5 μm, and optionally, the stationary phase of the column comprises particles having a size of 1.7 μm or 1.8 μm. Claim 7 In paragraph 5, the method wherein the column has a length of 50 mm to 300 mm and an inner diameter of 1 mm to 4.6 mm. Claim 8 The method of claim 1, wherein the AAV particles are serotypes AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAVrh10, AAVrh74, or any naturally occurring AAV particles, recombinant AAV particles, or synthetic AAV particles. Claim 9 A method according to claim 1, wherein liquid-phase chromatography is performed at 75°C to 85°C. Claim 10 A method according to claim 1, wherein the liquid phase chromatography comprises a first mobile phase comprising trifluoroacetic acid, and optionally, the first mobile phase comprises 0.05 to 0.15% by volume of trifluoroacetic acid. Claim 11 A method according to claim 1, wherein the liquid phase chromatography comprises a second mobile phase comprising trifluoroacetic acid in a mixture of acetonitrile and water, optionally wherein the second mobile phase comprises trifluoroacetic acid in a mixture of acetonitrile and water in an amount of 0.05 to 0.15% by volume. Claim 12 A method according to claim 11, wherein the second mobile phase comprises 80 to 95% by volume of acetonitrile, and optionally, the mixture of acetonitrile and water comprises 90% by volume of acetonitrile and 10% of water. Claim 13 In paragraph 11, the liquid phase chromatography comprises a combination of a first mobile phase and a second mobile phase, and in the liquid phase chromatography, the percentage of the second mobile phase in the combination of the first mobile phase and the second mobile phase increases over time. Claim 14 A method according to paragraph 13, wherein the percentage of the second mobile phase increases from 10% to 40% by volume and then from 40% to 45%, optionally, the percentage of the second mobile phase increases from 10% to 40% by volume over 3 minutes and then from 40% to 45% by volume over 30 minutes. Claim 15 A method according to claim 14, wherein the percentage of the second mobile phase in the combination of the first mobile phase and the second mobile phase subsequently increases to 100% by volume over one minute, and optionally, the percentage of the second mobile phase in the combination of the first mobile phase and the second mobile phase subsequently decreases from 100% by volume to 10% over one minute. Claim 16 A method according to any one of claims 1 to 15, wherein the mass spectrometry is performed using a fragmentor voltage of 125 to 350 V. Claim 17 A method according to any one of claims 1 to 15, wherein the mass spectrometry is performed using a capillary voltage of 3 to 6 kV. Claim 18 The method of claim 1, further comprising the step of determining a post-translation modification of at least one of the VP1, VP2, and VP3 capsid proteins. Claim 19 In paragraph 18, the method wherein the post-translational modification comprises one or more of loss of amino acids, glycosylation, sialation, acetylation, phosphorylation, deamilation, oxidation, formylation, hydroxylation, methylation, and sulfation, and optionally, the post-translational modification comprises one or more of loss of N-terminal methionine, deamilation, loss of threonine, phosphorylation, and acetylation. Claim 20 In any one of claims 1 through 15, 18, and 19, the method is performed by a gradient program comprising: a step of maintaining the percentage of the second mobile phase at 10% by volume for 3 minutes; a step of increasing the percentage of the second mobile phase from 10% by volume to 40% over 3 minutes; a step of increasing the percentage of the second mobile phase from 40% by volume to 45% over 30 minutes; a step of increasing the percentage of the second mobile phase from 45% by volume to 100% over 1 minute; and a step of decreasing the percentage of the second mobile phase from 100% by volume to 10% over 1 minute. Claim 21 A method according to claim 18, wherein a buffer containing Tris-HCl is used in the step of determining post-translational modifications of characterization, and optionally said buffer further contains acetonitrile and / or methionine. Claim 22 The method of claim 21, wherein the buffer comprises 5 mM to 50 mM of Tris-HCl, 5% to 20% by volume of acetonitrile, and 1 mM to 50 mM of methionine, optionally, the buffer comprises 20 mM of Tris-HCl, 5% to 10% by volume of acetonitrile, and 10 mM of methionine. Claim 23 In paragraph 21, the method is that Tris-HCl has a pH of 7.

5. Claim 24 A method according to claim 18, wherein the post-translational modification comprises deamidation in one or more of N263, N514, N57, N502, N254, and N94 of AAV8; or deamidation in equivalent residues of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV9, AAV10, AAV11, AAV12, AAV13, AAVrh10, AAVrh74, or variants thereof. Claim 25 delete Claim 26 delete Claim 27 delete Claim 28 delete Claim 29 delete Claim 30 delete Claim 31 delete Claim 32 delete Claim 33 delete Claim 34 delete Claim 35 delete Claim 36 delete Claim 37 delete Claim 38 delete Claim 39 delete Claim 40 delete Claim 41 delete Claim 42 delete Claim 43 delete Claim 44 delete Claim 45 delete Claim 46 delete Claim 47 delete Claim 48 delete Claim 49 delete Claim 50 delete Claim 51 delete Claim 52 delete Claim 53 delete Claim 54 delete Claim 55 delete Claim 56 delete Claim 57 delete Claim 58 delete Claim 59 delete Claim 60 delete Claim 61 delete Claim 62 delete Claim 63 delete Claim 64 delete Claim 65 delete Claim 66 delete Claim 67 delete Claim 68 delete Claim 69 delete Claim 70 delete Claim 71 delete Claim 72 delete Claim 73 delete Claim 74 delete Claim 75 delete Claim 76 delete Claim 77 delete Claim 78 delete Claim 79 delete Claim 80 delete Claim 81 delete Claim 82 delete Claim 83 delete Claim 84 delete Claim 85 delete Claim 86 delete Claim 87 delete Claim 88 delete Claim 89 delete Claim 90 delete Claim 91 delete Claim 92 delete Claim 93 delete Claim 94 delete Claim 95 delete Claim 96 delete Claim 97 delete

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