CN117795334A - Method for virus particle characterization using two-dimensional liquid chromatography-mass spectrometry - Google Patents

Abstract

Methods for identifying viral protein components in a sample of viral particles and quantifying the relative abundance of such viral protein components are disclosed. In embodiments, the method comprises separating a first dimension chromatograph of an intact viral capsid component of the sample, online denaturation of a viral capsid component that produces an intact viral protein, separating a second dimension chromatograph of the viral protein, and determining the mass of the viral protein and identifying a mass spectrum of the viral protein component of the sample.

Description

Method for virus particle characterization using two-dimensional liquid chromatography-mass spectrometry

Technical Field

The present invention relates to methods for characterizing the mass properties of viral particles (e.g., AAV capsids) using a two-dimensional liquid chromatography-mass spectrometry platform.

Background

Adeno-associated virus (AAV) is a non-enveloped, single-stranded DNA virus that has become an attractive therapeutic agent for delivering genetic material to host cells for gene therapy due to its ability to transduce a variety of substances and tissues in vivo, low risk of immune toxicity, and mild innate and adaptive immune responses. The complex nature of viral vectors (such as AAV) requires specific analytical methods for product testing and characterization.

Existing analytical techniques often do not provide sufficient separation to quantify the homogeneity of clinical-grade viral vector preparations. To ensure product quality and consistency, complete characterization of constitutive viral capsid proteins, such as those of AAV vectors, including their sequences and post-translational modifications (PTMs), is required. Thus, there is a need for methods of determining the homogeneity of a viral particle and identifying various viral proteins within a viral particle.

Disclosure of Invention

The present disclosure relates to an online two-dimensional liquid chromatography-mass spectrometry (2 DLC-MS) platform for the characterization of viral particles (e.g., AAV) that can be simultaneously characterized by chromatographic separation of viral particles and viral proteins in combination with mass spectrometry for empty and full ratios and viral proteins. In exemplary embodiments, the characterization of empty to full ratio and viral proteins is performed by anion exchange chromatography (AEX) and Reverse Phase Liquid Chromatography (RPLC) in combination with Mass Spectrometry (MS), respectively.

In one aspect, the present disclosure provides a method for identifying a viral protein component of a sample of viral particles, the method comprising: (a) Subjecting a sample of the viral particles to a first dimension chromatography to separate the intact viral capsid components of the sample; (b) Subjecting at least a portion of the intact viral capsid component to in-line denaturation to produce individual intact viral proteins; (c) Subjecting the intact viral proteins to a second dimension chromatography to isolate the intact viral proteins; and (d) determining the mass of the isolated intact viral protein to identify the viral protein component of the sample of viral particles.

In some embodiments, the method further comprises selecting a portion of the isolated whole viral capsid component, wherein subjecting at least a portion of the whole viral capsid component to in-line denaturation to produce individual viral proteins comprises subjecting the selected portion of the isolated whole viral capsid component to in-line denaturation.

In some embodiments, the sample of viral particles comprises adeno-associated virus (AAV) particles. In some cases, the AAV particle has serotypes AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV-DJ/8, AAV-Rh10, AAV-retro, AAV-PHP.B, AAV8-PHP.eB, or AAV-PHP.S. In some cases, the AAV particle has serotype AAV1. In some cases, the AAV particle has serotype AAV5. In some cases, the AAV particle has serotype AAV8.

In some embodiments, the complete viral capsid component comprises an empty viral capsid and a complete viral capsid.

In some embodiments, the first dimension chromatography comprises ion exchange chromatography. In some cases, the ion exchange chromatography is anion exchange chromatography.

In some embodiments, the second dimension chromatography comprises reverse phase chromatography. In some embodiments, the second dimension chromatography comprises hydrophilic interaction liquid chromatography.

In some embodiments, determining the mass of the isolated intact viral protein comprises subjecting the isolated intact viral protein to electrospray ionization mass spectrometry.

In some embodiments, the viral protein component comprises VP1, VP2, and/or VP3 of the AAV particle. In some cases, the viral protein component comprises a post-translational variant of VP1, VP2, and/or VP3. In some cases, the post-translational variants of VP1, VP2, and/or VP3 include acetylated, phosphorylated, and/or oxidized variants of VP1, VP2, and/or VP3. In some cases, the post-translational variants of VP1, VP2, and/or VP3 comprise fragments of VP1, VP2, and/or VP3 resulting from cleavage of an aspartate-proline bond and/or cleavage of an aspartate-glycine bond.

In some embodiments, the method further comprises detecting the intact viral capsid component separated by the first dimension chromatography, and identifying the ratio of empty viral capsids to complete and partially complete viral capsids.

In some embodiments, the method further comprises detecting the intact viral protein separated by a second dimension chromatograph, and quantifying the relative abundance of the viral protein component of the sample of viral particles.

In some cases, the intact viral capsid components and/or the intact viral proteins are detected using ultraviolet or fluorescence detectors.

In one aspect, the present disclosure provides a method for identifying a viral protein component of a sample of adeno-associated virus (AAV) particles, the method comprising: (a) Subjecting a sample of AAV particles to anion exchange chromatography to isolate an intact viral capsid component in the sample, wherein the intact viral capsid component comprises an intact empty viral capsid and an intact complete viral capsid, the intact complete viral capsid comprising a heterologous nucleic acid molecule; (b) Selecting a portion of the complete viral capsid component for online desalting and denaturation; (c) Subjecting selected portions of the intact viral capsid component to online desalting and denaturing to produce individual intact viral proteins, wherein the intact individual viral proteins comprise VP1, VP2, VP3 and at least one variant of VP1, VP2 or VP 3; (d) Subjecting the intact viral proteins to reverse phase liquid chromatography or hydrophilic interaction liquid chromatography to isolate the intact viral proteins; and (e) determining the mass of the isolated intact viral protein to identify the viral protein component of the sample of AAV particles.

In some embodiments, the intact viral proteins are subjected to reverse phase liquid chromatography. In some embodiments, the intact viral proteins are subjected to hydrophilic interaction liquid chromatography.

In some embodiments, the method further comprises detecting the intact viral capsid component separated by anion exchange chromatography, and identifying the ratio of empty viral capsids to complete and partially complete viral capsids.

In some embodiments, the method further comprises detecting intact viral proteins separated by reverse phase liquid chromatography or hydrophilic interaction liquid chromatography, and quantifying the relative abundance of viral protein components of the sample of AAV particles.

In some embodiments, the AAV particle has serotypes AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV-DJ/8, AAV-Rh10, AAV-retro, AAV-PHP.B, AAV8-PHP.eB, or AAV-PHP.S. In some cases, the AAV particle has serotype AAV1. In some cases, the AAV particle has serotype AAV5. In some cases, the AAV particle has serotype AAV8.

In some embodiments, at least one variant of VP1, VP2, or VP3 comprises a post-translational variant of VP1, VP2, or VP 3. In some cases, the post-translational variants of VP1, VP2, or VP3 include acetylated variants of VP1, VP2, or VP 3. In some cases, the post-translational variants of VP1, VP2, or VP3 include phosphorylated variants of VP1, VP2, or VP 3. In some cases, the post-translational variants of VP1, VP2, or VP3 include oxidized variants of VP1, VP2, or VP 3. In some cases, the post-translational variant of VP1, VP2, or VP3 comprises a fragment of VP1, VP2, or VP3 resulting from cleavage of an aspartate-proline bond. In some cases, the post-translational variant of VP1, VP2, or VP3 comprises a fragment of VP1, VP2, or VP3 resulting from cleavage of an aspartate-glycine bond.

In some embodiments, the intact viral capsid components and/or intact viral proteins are detected using an ultraviolet or fluorescence detector.

In some embodiments, determining the mass of the isolated intact viral protein comprises subjecting the isolated intact viral protein to electrospray ionization mass spectrometry.

In some embodiments, the intact viral capsid components of a sample that has been subjected to anion exchange chromatography are separated using a first mobile phase comprising 15mM to 25mM bis-tri-propane (BTP), 250mM to 1M tetramethyl ammonium chloride (TMAC), and 1mM to 3mM magnesium chloride (pH 8 to 9). In some cases, the first mobile phase comprises 20 mM+ -2 mM BTP, 500 mM+ -50 mM TMAC, and 2 mM+ -0.2 mM MgCl ₂ (pH 8.5.+ -. 0.1). In some embodiments, the intact viral capsid components of a sample that has been subjected to anion exchange chromatography are separated using a first mobile phase and a second mobile phase, the second mobile phase comprising 15mM to 25mM bis-tri-propane (BTP) and 1mM to 3mM magnesium chloride (pH 8 to 9). In some cases, the second mobile phase comprises 20 mM+ -2 mM BTP and 2 mM+ -0.2 mM MgCl ₂ (pH 8.5.+ -. 0.1). In some embodiments, the intact viral capsid components of the sample that has been subjected to anion exchange chromatography are separated using a first mobile phase, a second mobile phase, and a third mobile phase comprising 1.5M to 2.5M sodium chloride. In some cases, the third flow The phase contained 2m±0.1M sodium chloride. In some embodiments, the separation of intact viral capsid components is performed using a mobile phase gradient. In some cases, the mobile phase gradient comprises, in order: 10% of the first mobile phase and 90% of the second mobile phase for 1 minute; increasing the first mobile phase from 10% to 42% and decreasing the second mobile phase from 90% to 58% over a period of 20 minutes; 100% of a third mobile phase, for 5 minutes; and 10% of the first mobile phase and 90% of the second mobile phase for 10 minutes.

In some embodiments, the method further comprises identifying the amount of intact empty viral capsids and the amount of intact viral capsids in the sample, and determining the relative abundance of intact empty viral capsids and intact complete viral capsids in the sample.

In various embodiments, any features or components of the embodiments discussed above or herein may be combined, and such combinations are contemplated as being within the scope of the present disclosure. Any of the specified values discussed above or herein may be combined with another related value discussed above or herein to list a range, the values representing the upper and lower limits of the range, and such ranges are encompassed within the scope of the disclosure.

Other embodiments will become apparent upon reading the detailed description that follows.

Drawings

FIGS. 1A, 1B and 1C illustrate AAV capsids (FIG. 1A) comprising a heterologous nucleic acid molecule (e.g., a therapeutic gene or a gene of interest (GOI)); empty, partially complete and complete capsids (fig. 1B); and an AAV capsid consisting of 60 copies of the three viral proteins (VP 1, VP2 and VP 3) (yielding a range of theoretical capsid stoichiometries) (fig. 1C).

Fig. 2A and 2B illustrate an exemplary two-dimensional liquid chromatography-mass spectrometry system (2 DLC-MS) in which, for mass spectrometry analysis, viral capsids are separated in a first dimension and viral proteins are separated in a second dimension, according to embodiments of the disclosure.

Fig. 2C illustrates a valve setting for a 2DLC-MS system according to an embodiment of the disclosure. Part (a) shows a first position of the valve arrangement wherein the second liquid chromatography stream is used to maintain the RPLC column temperature and one fraction from the capture loop enters the capture column for desalting and denaturing. Part (a) also shows a second position of the valve arrangement, wherein viral proteins from the denatured viral capsid (e.g., AAV) migrate from the capture column to the analytical column (e.g., RPLC) for separation, followed by mass spectrometry. Parts (b) (c) (d) show the separation of viral proteins with (part (c)) or without (part (b)) a capture column and with different flow rates (0.2 mL/min in part (c) and 0.1mL/min in part (d). As shown, separation of viral proteins can be better achieved using a trap column, and the peak variation is insignificant with variation in flow rate. Part (e) shows that no salt adducts associated with the three AAV viral proteins (from deconvolution spectra) were observed using the exemplary valve settings.

Figure 2D shows a pair of chromatograms showing on-line denaturation (bottom chromatogram) that provides efficient dissociation of AAV viral proteins without denaturation prior to sample injection.

FIG. 2E shows the raw deconvolution spectra representing the high molecular weight species obtained from the 2DLC-MS system (for AAV8-GOI 1), and confirming that the identity of the high molecular weight species is the peak of VP3 dimer and VP2+VP3 heterodimer. The use of a trap column for in-line denaturation eliminates the high molecular weight species (compare parts (b) and (C) of fig. 2C, which show the presence of high molecular weight species without the use of a trap column and the absence of high molecular weight species with the use of a trap column, respectively).

Fig. 3A and 3B represent chromatograms obtained from a first dimension chromatogram (e.g., AEX) and show the separation of viral capsids (empty or containing GOI) using tetramethyl ammonium chloride or tetraethyl ammonium chloride (fig. 3A) and various AAV serotypes (fig. 3B), respectively.

Figures 3C and 3D represent chromatograms and mass spectra obtained from a second dimension chromatograph (e.g., RPLC) and mass spectrum and show that viral proteins in AAV samples can be efficiently separated in both AAV8 and AAV1 samples in the presence or absence of GOI, and that GOI does not interfere with separation (figure 3C), and that separation of viral proteins in combination with mass spectrometry can be used to identify viral proteins (figure 3D).

Fig. 4A shows a process according to an embodiment of the present disclosure, showing (i) a chromatogram in which the hollow capsid (AAV 8-Em) has been separated from the capsid containing the gene of interest (AAV 8-GOI), (ii) a selection of a portion of the separated capsid for denaturation ("centre cut"), (iii) a chromatogram in which the viral proteins (VP 1, VP2, VP3, etc.) have been separated from each other, and (iv) a mass spectrum corresponding to the separated viral proteins.

FIG. 4B shows a plurality of "center cuts" of peaks after a first dimension of chromatography (e.g., AEX), followed by a second dimension of chromatography (e.g., RPLC) and mass spectrometry that identify and characterize viral protein components of peaks in AAV8-GOI samples.

FIG. 5 shows the effectiveness of a first dimension chromatography in separating empty viral capsids from capsids containing heterologous nucleic acid molecules (e.g., genes of interest or GOI). Chromatographic separation yields a ratio of viral capsids, wherein (i) empty capsids and (ii) partially complete and complete capsids are consistent with data generated by Analytical Ultracentrifugation (AUC) techniques.

Figure 6 shows chromatographic separation of viral proteins by a second dimension chromatograph, and the relative numbers of each of VP1, VP2 and VP3 (of AAV).

Figures 7A and 7B show chromatographic separation of viral proteins (VP 1, VP2 and VP3 of AAV) and post-translational variants of viral proteins by a second dimension of chromatography. Fig. 7A shows labeling of abundant substances, and fig. 7B shows labeling of low abundant substances.

For AAV8 samples of viral particles, fig. 7C shows the identities of the labeled substances in fig. 7A and 7B, as well as the observed and theoretical mass of each substance. "Ac" refers to acetylation, "P" refers to phosphorylation, "scissoring (DP)" refers to cleavage of an aspartic acid-proline bond resulting in a fragment, "Ox" refers to oxidation, "scissoring (DG)" refers to cleavage of an aspartic acid-glycine bond resulting in a fragment.

Fig. 7D shows the identity of the substance of the AAV1 sample of viral particles in the same manner as fig. 7C.

Fig. 8A and 8B show mass spectra of viral protein components corresponding to AAV8 or AAV1 capsids that are empty or contain a gene of interest (GOI).

Detailed Description

Before describing the present invention, it is to be understood that this invention is not limited to the particular methodology and experimental conditions described, as such methods and conditions may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present invention will be limited only by the appended claims.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. As used herein, the term "about" when used in reference to a specifically recited value means that the value may vary by no more than 1% from the recited value. For example, as used herein, the expression "about 100" includes 99 and 101 and all values therebetween (e.g., 99.1, 99.2, 99.3, 99.4, etc.).

As used herein, the terms "include" and "comprising" are intended to be non-limiting and may be understood to mean "comprising" and "including", respectively.

Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials are now described. All patents, applications, and non-patent publications mentioned in this specification are herein incorporated by reference in their entirety.

Selected abbreviations

2 DLC-MS-two-dimensional liquid chromatography-Mass Spectrometry

LC-MS-liquid chromatography-mass spectrometry

MS: mass spectrum or mass spectrometer

ESI: electrospray ionization

rAAV: recombinant AAV particles or capsids

AAV: adeno-associated virus

LC (liquid crystal): liquid chromatography

RPLC: reversed phase liquid chromatography

HILIC-hydrophilic interaction liquid chromatography

AEX-anion exchange chromatography

IEX-ion exchange chromatography

GOI-target gene

VP1-AAV viral protein 1 subunit

VP2-AAV viral protein 2 subunit

VP3-AAV viral protein 3 subunit

UV-ultraviolet

FLR-fluorescence

Definition of the definition

"intact viral capsid component" refers to viral capsids (e.g., empty viral capsids, partially complete viral capsids, and/or complete viral capsids) that are intact (i.e., have not been denatured or broken down or disintegrated into their constituent parts (e.g., different viral proteins) and retain the structural characteristics of the viral capsids (e.g., the icosahedral conformation of the AAV capsids).

The term "empty viral capsid" or "empty capsid" refers to a capsid that does not contain a heterologous nucleic acid molecule (e.g., a therapeutic gene), as shown in fig. 1B.

The term "partially complete viral capsid" or "partially complete capsid" refers to a capsid that contains only a portion of a heterologous nucleic acid molecule (e.g., a therapeutic gene), as shown in fig. 1B.

The term "complete viral capsid" or "complete capsid" refers to a capsid containing all heterologous nucleic acid molecules (e.g., therapeutic genes or genes of interest), as shown in fig. 1B.

As used herein, the term "sample" refers to a mixture of viral particles (e.g., AAV particles) comprising at least one viral capsid component (i.e., empty capsid, partially complete capsid, and/or complete capsid) that is manipulated according to the methods of the invention (including, e.g., isolation and analysis).

The term "analysis" or "analysis" is used interchangeably and refers to any of a variety of methods of isolating, detecting, isolating, purifying, and/or characterizing a viral particle or viral protein of interest (e.g., an AAV protein). Examples include, but are not limited to, mass spectrometry (e.g., ESI-MS), liquid chromatography (e.g., AEX, RPLC, or HIlic), and combinations thereof.

As used herein, "contacting" includes bringing together at least two substances in solution or solid phase, e.g., contacting a stationary phase of a chromatographic material with a sample (such as a sample containing viral particles or viral proteins).

As used herein, "complete mass analysis" includes experiments in which viral proteins are characterized as complete proteins. Complete mass analysis can minimize sample preparation.

As used herein, the term "liquid chromatography" refers to a process in which a chemical mixture carried by a liquid can be separated into components due to the differential distribution of chemical entities as they flow around or over a stationary liquid or solid phase. Non-limiting examples of liquid chromatography include reversed phase liquid chromatography, ion exchange chromatography, size exclusion chromatography, affinity chromatography, and hydrophobic interaction chromatography.

As used herein, the term "mass spectrometer" refers to a device capable of detecting specific molecular species and accurately measuring their mass. The term may be meant to include any molecular detector that: viral proteins (e.g., AAV proteins) may be eluted therein for detection and/or characterization. Mass spectrometers consist of three main parts: an ion source, a mass analyzer, and a detector. The ion source functions to generate gas phase ions. The analyte atoms, molecules or clusters may be converted to gas phase or simultaneous ionization (e.g., electrospray ionization). The choice of ion source depends on the application. As used herein, the term "electrospray ionization" or "ESI" refers to a process of spray ionization in which cations or anions in a solution are converted to the gas phase by formation and desolvation at the atmospheric pressure of a stream of highly charged droplets that are generated by applying a potential difference between the tip of an electrospray emitter needle containing the solution and a counter electrode. The process of generating gas phase ions from electrolyte ions in solution is divided into three main steps. The steps are as follows: (a) generating charged droplets at the ES infusion tip; (b) Shrinkage of the charged droplets due to solvent evaporation and repeated droplet splitting, resulting in small highly charged droplets capable of generating gas phase ions; and (c) a mechanism to generate gas phase ions from very small and highly charged droplets. Stages (a) - (c) typically occur in the atmospheric region of the apparatus.

As used herein, the term "electrospray ionization source" refers to an electrospray ionization system that is compatible with mass spectrometers for mass analysis of viral particles.

Non-denaturing MS is a particular method based on electrospray ionization in which the biological analyte is sprayed from a non-denaturing solvent. It is defined as a process by which biomolecules, such as large biomolecules, and their complexes can be converted from the three-dimensional functional presence of a condensed liquid phase to a gas phase by the process of electrospray ionization mass spectrometry (ESI-MS).

As used herein, the term "nanoelectrospray" or "nanospray" refers to electrospray ionization at very low solvent flow rates (typically hundreds of nanoliters per minute or less of sample solution, typically without the use of external solvent delivery).

As used herein, "mass analyzer" refers to a device that can separate substances (i.e., atoms, molecules, or clusters) according to their mass. Non-limiting examples of mass analyzers that can be used for rapid protein sequencing are time of flight (TOF), magnetic/electrical sector, quadrupole mass filter (Q), quadrupole Ion Trap (QIT), orbitrap, fourier Transform Ion Cyclotron Resonance (FTICR), and Accelerator Mass Spectrometry (AMS) techniques.

As used herein, "mass-to-charge ratio" or "m/z" is used to denote a dimensionless quantity (regardless of sign) formed by dividing the mass of an ion of a uniform atomic mass unit by its charge number.

As used herein, the term "quadrupole-orbitrap hybrid mass spectrometer" refers to a hybrid system made by coupling a quadrupole mass spectrometer to an orbitrap mass analyzer. Tandem real-time experiments using quadrupole-orbitrap hybrid mass spectrometers first eject all ions from the quadrupole mass spectrometer except for ions in a selected narrow m/z range. Selected ions may be inserted into the orbitrap and most often fragmented by a low energy CID. Fragments within the m/z acceptance range of the trap should remain in the trap and an MS-MS spectrum can be obtained.

An "adeno-associated virus" or "AAV" is a nonpathogenic parvovirus having single-stranded DNA, a genome of about 4.7kb, non-enveloped, and an icosahedral conformation. AAV was first discovered in 1965 as a contaminant of adenovirus preparations. AAV belongs to the genus dependoviridae and parvoviridae, and requires helper functions of either the herpes virus or adenovirus for replication. In the absence of helper virus, AAV can set latency by integrating into the 19q13.4 position of human chromosome 19. The AAV genome consists of two Open Reading Frames (ORFs), one open reading frame corresponding to each of the two AAV genes Rep and Cap. AAV DNA ends have an Inverted Terminal Repeat (ITR) of 145bp and 125 terminal bases are palindromic, yielding a characteristic T-shaped hairpin structure.

As used herein, the term "polynucleotide" or "nucleic acid" refers to a polymeric form of nucleotides of any length, i.e., ribonucleotides or deoxyribonucleotides. Thus, the term includes, but is not limited to, single-stranded, double-stranded or multi-stranded DNA or RNA, genomic DNA, cDNA, DNA-RNA hybrids, or polymers comprising purine and pyrimidine bases or other natural, chemically or biochemically modified, non-natural or derivatized nucleotide bases. The backbone of the nucleic acid may comprise sugar and phosphate groups (as may be typically present in RNA or DNA), or modified or substituted sugar or phosphate groups.

"recombinant viral particle" refers to a viral particle comprising one or more heterologous sequences (e.g., nucleic acid sequences of non-viral origin) that may flank at least one viral nucleotide sequence.

"recombinant AAV particle" refers to an adeno-associated viral particle comprising one or more heterologous sequences (e.g., non-AAV-derived nucleic acid sequences) that may flank at least one (e.g., two) AAV Inverted Terminal Repeats (ITRs). Such rAAV particles can replicate and package when present in host cells that have been infected with a suitable helper virus (or express a suitable helper function) and express AAV Rep and Cap gene products (i.e., AAV Rep and Cap proteins).

By "viral particle" is meant a viral particle consisting of at least one viral capsid protein and an encapsulated viral genome.

"heterologous" means derived from an entity that is genotype-wise different than the remainder of the entity being compared or introduced or incorporated. For example, nucleic acids introduced into different cell types by genetic engineering techniques are heterologous nucleic acids (and may encode heterologous polypeptides when expressed). Similarly, a cellular sequence (e.g., a gene or portion thereof) incorporated into a viral particle is a heterologous nucleotide sequence with respect to the viral particle.

An "inverted terminal repeat" or "ITR" sequence is a relatively short sequence at the end of the viral genome that is in the opposite orientation. An "AAV Inverted Terminal Repeat (ITR)" sequence is a sequence of about 145 nucleotides present at both ends of a single stranded AAV genome.

The term "corresponding" is a relative term, indicating a similarity in position, purpose or structure. The mass spectrum signal due to a particular peptide or protein is also referred to as a signal corresponding to the peptide or protein. In certain embodiments, a particular peptide sequence or collection of amino acids (such as a protein) may be assigned to a corresponding peptide mass.

As used herein, the term "isolated" refers to a biological component (such as a nucleic acid, peptide, protein, lipid, viral particle, or metabolite) that has been substantially separated from, produced from, or purified from other biological components in the cells of an organism in which the component naturally occurs or is expressed in the cells of the organism.

The terms "peptide," "protein," and "polypeptide" are used interchangeably to refer to a polymer of amino acids and/or amino acid analogs linked by peptide bonds or peptidomimetics. Twenty naturally occurring amino acids and their single and three letter designations are as follows: alanine a Ala; cysteine C Cys; aspartic acid D Asp; glutamic acid E Glu; phenylalanine F Phe; glycine G Gly; histidine hhis; isoleucine I He; lysine K Lys; leucine L Leu; methionine M Met; asparagine N Asn; proline P Pro; glutamine Q Gln; arginine R Arg; serine S Ser; threonine T Thr; valine V Val; tryptophan w Trp; and tyrosine ytyr.

References to the mass of an amino acid mean the monoisotopic mass or average mass of the amino acid at a given isotopic abundance (such as natural abundance). In some examples, the mass of the amino acid may be offset, for example, by labeling the amino acid with an isotope. Based on the exact isotopic composition of the amino acids, a certain degree of variation in the average mass of the amino acids of the individual amino acids is expected. The mass of an amino acid, including monoisotopic mass and average mass, can be readily obtained by one of ordinary skill in the art.

Similarly, reference to the mass of a peptide or protein means the monoisotopic mass or average mass of the peptide or protein at a given isotopic abundance (such as natural abundance). In some examples, the mass of the peptide may be offset, for example, by labeling one or more amino acids in the peptide or protein with an isotope. Based on the exact isotopic composition of the peptides, a certain degree of variation in the average peptide mass of the individual peptides is expected. The mass of a particular peptide can be determined by one of ordinary skill in the art.

As used herein, "vector" refers to a recombinant plasmid or virus comprising a nucleic acid to be delivered to a host cell in vitro or in vivo.

"recombinant viral vector" refers to a recombinant polynucleotide vector comprising one or more heterologous sequences (i.e., nucleic acid sequences of non-viral origin).

The term "hydrophilic interaction chromatography" or HILIC is intended to include processes employing a hydrophilic stationary phase and a hydrophobic organic mobile phase, wherein the hydrophilic compounds remain longer than the hydrophobic compounds. In certain embodiments, the process utilizes a water miscible solvent mobile phase.

The term "reverse phase liquid chromatography" or RPLC is intended to include the following processes: the analytes are separated based on nonpolar interactions between the analytes and a stationary phase (e.g., a matrix). The non-polar analyte is associated with the non-polar stationary phase and is retained by the non-polar stationary phase. The adsorption strength increases with the non-polarity of the analyte and the interaction between the non-polar analyte and the non-polar stationary phase (relative to the mobile phase) increases the elution time. The use of more nonpolar solvents in the mobile phase will shorten the retention time of the analyte, while more polar solvents tend to increase the retention time.

The term "anion exchange chromatography" or AEX is intended to include a process of separating a substance based on the charge of the substance using an ion exchange resin containing positively charged groups, such as diethyl-aminoethyl groups. In solution, the resin is coated with positively charged counterions.

General description

The present disclosure provides two-dimensional liquid chromatography and non-denaturing Mass Spectrometry (MS) methods that provide sensitive, rapid identification and quantitative characterization of viral protein components of samples of viral particles (e.g., AAV particles). Complete characterization of the viral protein component of the viral particle composition (such as the viral protein component of the viral capsid component of a sample of AAV particles) is necessary to ensure product quality and consistency to maintain the safety and efficacy of the composition.

Recombinant viral vector compositions (e.g., AAV vector compositions) can contain varying levels of viral proteins as well as post-translational modifications of such viral proteins resulting from different production, purification, and storage conditions. The methods of the invention provide analytical techniques for identifying and quantifying the ratio of viral capsid components in a sample of viral particles, as well as identifying and quantifying viral protein components of viral particles (including low abundance viral protein components, including acetylated, phosphorylated, oxidized, and fragmented variants of viral proteins).

Methods for identifying and quantifying viral protein components

Aspects of the present disclosure relate to methods for identifying and quantifying viral protein components in a sample of viral particles (e.g., recombinant AAV particles) in a two-dimensional liquid chromatography-mass spectrometry (2 DLC-MS) system.

In some cases, the method comprises: (a) Subjecting a sample of the viral particles to a first dimension chromatography to separate the intact viral capsid components of the sample; (b) Subjecting at least a portion of the intact viral capsid component to in-line denaturation to produce individual intact viral proteins; (c) Subjecting the intact viral proteins to a second dimension chromatography to isolate the intact viral proteins; and (d) determining the mass of the isolated intact viral protein to identify the viral protein component of the sample of viral particles.

In some cases, the method comprises: (a) Subjecting a sample of AAV particles to anion exchange chromatography to isolate an intact viral capsid component in the sample, wherein the intact viral capsid component comprises an intact empty viral capsid and an intact complete viral capsid, the intact complete viral capsid comprising a heterologous nucleic acid molecule; (b) Selecting a portion of the complete viral capsid component for online desalting and denaturation; (c) Subjecting selected portions of the intact viral capsid component to online desalting and denaturing to produce individual intact viral proteins, wherein the intact individual viral proteins comprise VP1, VP2, VP3 and at least one variant of VP1, VP2 or VP 3; (d) Subjecting the intact viral proteins to reverse phase liquid chromatography or hydrophilic interaction liquid chromatography to isolate the intact viral proteins; and (e) determining the mass of the isolated intact viral protein to identify the viral protein component of the sample of AAV particles.

In various embodiments of the methods, the viral protein component comprises a viral protein and a post-translational variant of the viral protein. For example, in a composition of AAV particles, the viral protein component comprises viral proteins VP1, VP2, and VP3, as well as post-translational variants of VP1, VP2, and/or VP3, in some cases including acetylated, phosphorylated, and/or oxidized variants of VP1, VP2, and/or VP3, and/or fragments of VP1, VP2, and/or VP3 resulting from cleavage of peptide bonds (e.g., cleavage of an aspartate-proline bond and/or cleavage of an aspartate-glycine bond).

In the methods disclosed herein, a 2DLC-MS system is illustrated by the schematic diagrams shown in fig. 2A and 2B. In the example shown in fig. 2B, the 2DLC-MS system 100 includes a first dimension liquid chromatography column 102 (e.g., AEX column), into which a sample of viral particles 101 (e.g., AAV particles) is introduced to separate viral capsid components of the sample from each other, a detector 104 (e.g., FLR detector) for detecting an eluate from the first dimension column 102, peak picking or center cutting software 106 capable of selecting a portion of the eluted and separated viral capsid components of the sample, a capture loop 108 for on-line desalting and denaturing, and for temporarily storing the selected viral capsid components to be transferred to the second dimension chromatography column 110 (e.g., RPLC column), the selected viral capsid components being transferred to the second dimension chromatography column to produce complete viral proteins from the viral capsids (e.g., via a starting mobile phase), a detector 112 (e.g., FLR detector) for detecting an eluate from the second dimension column 110, and a mass spectrometer (e.g., ESI-114) for determining mass fractions of the viral proteins in the sample 101, thereby identifying the mass fractions of the separated viral particles in the mass spectrum 116. One advantage of the 2DLC-MS system discussed herein is the ability to incorporate salts incompatible with MS in a first dimension for high resolution separation, and then use MS compatible reagents in a second dimension for MS characterization. For example, this advantage may be achieved using the valve arrangement illustrated in FIG. 2C. As shown in FIG. 2D, online denaturation allows for efficient separation of viral proteins.

In various embodiments of the methods discussed herein, the separation of viral capsid components (e.g., empty and complete capsids) in a first dimension chromatograph can be used to determine the relative number of capsid components within a sample of a viral particle. For example, in the context of AEX chromatography, empty viral capsids (i.e., those that do not contain heterologous nucleic acid molecules) will be eluted prior to the partially complete or complete viral capsids because negatively charged nucleic acids (e.g., DNA) encapsulated within the partially complete and complete viral capsids produce lower isoelectric point (pI) values and have higher affinity for positively charged AEX resins. An example of such an isolation is shown in FIG. 3B, where AAV1, AAV5 and AAV8 empty capsid elution are shown prior to the capsid comprising the gene of interest (GOI). Such separation may be accomplished using, for example, tetramethyl ammonium chloride or tetraethyl ammonium chloride, as shown in fig. 3A. As shown in fig. 5, detection of eluted viral capsid components (e.g., by fluorescence detector) can then be used to determine the ratio of empty capsids to capsids containing GOI. These data are consistent with the data generated by AUC measurements, which are generally considered the most advanced technique for determining the relative amounts of empty, partially and fully viral capsid components (note that partially and fully viral capsid components are incorporated in the AEX column of the table shown in fig. 5).

In various embodiments, the methods of the present disclosure may be used to determine the identity and stoichiometry of various viral protein components contained within the viral particles of a sample that has undergone a 2DLC-MS system. In embodiments, the viral capsid component isolated from the first dimension chromatography is denatured to produce the complete viral proteins previously comprising the viral capsids (e.g., VP1, VP2, and VP3 of AAV capsids). The intact viral protein is then subjected to a second dimension chromatography to isolate the viral protein, which may comprise a modified variant of the viral protein (e.g., a native or post-translational variant resulting from the conditions of production, purification, or storage). An example of such isolation is shown in fig. 3C, which shows that the presence of GOI does not affect the isolation of viral proteins, and fig. 3D (top) shows viral proteins of empty AAV8 capsids that have been isolated on RPLC columns. The chromatogram in fig. 3D shows peaks of three native viral proteins (VP 1, VP2 and VP 3) of the AAV capsid and variants of VP3 resulting from cleavage of peptide bonds (non-specific).

The isolated viral proteins are then subjected to mass spectrometry to determine the identity of the various viral proteins. FIG. 3D (bottom) shows an example property profile showing the identification of VP2 viral proteins and phosphorylated VP2 viral proteins (from AAV capsids). Further characterization of the relative ratios of viral proteins and the characterization of viral proteins and variants are shown in figures 6, 7A, 7B, 7C and 7D.

In the embodiments of the methods discussed herein, a subset of the viral capsid components separated in the first dimension chromatography may also be selected for denaturation and separation/analysis in the second dimension chromatography and mass spectrometry portions of the 2DLC-MS system. As shown in fig. 4A, a "centre cut" may be performed to select a designated portion of the eluate from the first dimension chromatograph for further processing in the second dimension chromatograph, and subsequent mass spectrometry. This technique can improve the separation and analysis of specific components, such as low abundance viral protein materials that may be present in the sample under investigation. Multiple "center cuts" can also be made to analyze various peaks from the first dimension chromatogram, as shown in fig. 4B.

The methods discussed herein include subjecting a sample of viral proteins to Reverse Phase Liquid Chromatography (RPLC) or hydrophilic interaction liquid chromatography (HILIC) to isolate the protein component of the viral capsid of a viral particle, such as a viral particle of interest, wherein information about the capsid is desired. In embodiments, the RPLC or HILIC column is contacted with the intact viral protein after first dimension chromatography and denaturation. In certain embodiments, the methods comprise determining the mass of the protein component of the viral capsid, for example, using mass spectrometry techniques such as those described herein, to identify the protein component separated by a second dimension chromatography (e.g., RPLC or HILIC). In embodiments, the method includes calculating the relative abundance of a protein component from an isolated viral capsid to determine the stoichiometry of the protein component of the viral capsid of the viral particle, e.g., using Ultraviolet (UV) detection or Fluorescence (FLR) detection of the protein component of the viral capsid eluted from an RPLC or HILIC column. For example, the area of the UV or FLR peak can be used to determine the relative abundance of the capsid protein and to calculate the stoichiometry of the capsid protein in the viral capsid. In another example, peak heights and/or peak UV or FLR intensities are used to determine relative abundance. In some embodiments, the retention time of the different proteins on the second dimension chromatographic column (e.g., RPLC or HILIC) is determined as a function of the mobile phase used, and in subsequent analysis, this retention time can be used to determine the relative abundance of proteins from the viral particles and proteins without the need to determine the mass and/or identity of the proteins each time the stoichiometry is determined, e.g., one or more standard values can be formulated. In some cases, a second dimension chromatography column may be used for denaturation and separation of viral protein components. In some cases, the methods discussed herein can be used to determine the serotype of a viral particle. For example, the mass of VP1, VP2, and VP3 for each AAV serotype is unique and can be used to identify or distinguish AAV capsid serotypes. In addition, the isolated capsid protein can be subjected to downstream analysis, such as determination of protein sequence and post-translational modification of the capsid protein, e.g., accurate mass measurement at the intact protein level.

In some embodiments of the methods discussed herein, the methods can be used to determine heterogeneity of protein components in the capsids of viral particles. In embodiments, the method comprises subjecting the viral particle to a first dimension chromatography to separate the viral capsid component, and subjecting at least a portion of the viral capsid component to a second dimension chromatography to separate the protein component of the viral particle capsid. In embodiments, the method comprises determining the mass of the protein component of the viral capsid. In some cases, the mass of the protein component of the viral capsid is compared to the theoretical mass of the viral capsid. Deviations in one or more of the masses of the protein components of the viral capsid indicate that the protein or proteins of the capsid are heterogeneous. In contrast, no deviation indicated that the capsid protein was homogenous. In embodiments, the heterogeneity is due to one or more of a mixed serotype, variant capsid, capsid amino acid substitution, truncated capsid, or modified capsid. In some embodiments, the determination of the stoichiometry of the protein component of the viral capsid of the viral particle and the determination of the heterogeneity of protein components in the capsid of the viral particle are performed on the same sample.

In certain embodiments, the viral particles are adeno-associated viral (AAV) particles and the disclosed methods can be used to determine the identity (and optionally stoichiometry) of the protein component in the capsid of the AAV particles and/or the heterogeneity of the protein component in the capsid of the AAV particles. In embodiments, the protein component of the protein capsid comprises VP1, VP2, and VP3 of the AAV particle, and one or more variants of VP1, VP2, or VP 3. In embodiments, the AAV particle is a recombinant AAV (rAAV) particle. In embodiments, the AAV particle comprises an AAV vector encoding a heterologous transgene. In some embodiments, the measured or calculated mass of the disclosure (e.g., the measured or calculated mass of VP1, VP2, and/or VP3, or variants thereof, of an AAV particle) can be compared to a reference (e.g., the theoretical mass of VP1, VP2, and/or VP3, or variants thereof, of one or more AAV serotypes). References may include theoretical masses of VP1, VP2, and/or VP3, or variants thereof, of one or more of any of the AAV serotypes. For example, in some embodiments, the mass of VP1, VP2, and/or VP3, or variants thereof, is compared to the theoretical mass of one or more of an AAV1 capsid, an AAV2 capsid, an AAV3 capsid, an AAV4 capsid, an AAV5 capsid, an AAV6 capsid, an AAV7 capsid, an AAV8 capsid, an AAVrh8 capsid, an AAV9 capsid, an AAV10 capsid, an AAV11 capsid, an AAV12 capsid, or variants thereof. In some embodiments, the measured or calculated mass (e.g., of VP1, VP2, and/or VP3 of an AAV particle) can be compared to the theoretical mass of VP1, VP2, and/or VP3 of the corresponding AAV serotype.

Virus particles

In certain aspects, the viral particles are AAV particles, and the disclosed methods can be used to determine the relative abundance of viral capsid components, as well as the identity and stoichiometry of the viral protein components of the viral capsids in a sample of AAV particles. The AAV particles may be recombinant AAV (rAAV) particles. The rAAV particles comprise AAV vectors encoding heterologous transgenes or heterologous nucleic acid molecules.

In certain aspects, the AAV particle comprises an AAV1 capsid, an AAV2 capsid, an AAV3 capsid, an AAV4 capsid, an AAV5 capsid, an AAV6 capsid, an AAV7 capsid, an AAV8 capsid, an AAVrh8 capsid, an AAV9 capsid, an AAV10 capsid, an AAV11 capsid, an AAV12 capsid, or variants thereof. In certain aspects, the AAV particle has serotypes AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV-DJ/8, AAV-Rh10, AAV-retro, AAV-PHP.B, AAV8-PHP.eB, or AAV-PHP.S. In some embodiments, the AAV particle has serotype AAV1 or AAV8.

While AAV is a model viral particle of the present disclosure, it is contemplated that the disclosed methods can be applied to characterize a variety of viruses, such as the viridae, subfamilies, and genera. The methods of the present disclosure can be used, for example, to characterize viral particles to monitor or detect the relative abundance of viral capsid components and the identity and stoichiometry of viral protein components of viral capsids in compositions of the viral particles during production, purification, or storage of such compositions.

In an exemplary embodiment, the viral particle belongs to the family of viruses selected from the group consisting of: adenoviridae (Adenoviridae), parvoviridae (Parvoviridae), retroviridae (Retroviridae), baculovirusae (Baculoviridae) and Herpesviridae (Herpesviridae).

In certain aspects, the viral particle belongs to a genus of virus selected from the group consisting of: aviruses (Atadenovirus), aviruses (aviadenvirus), fish adenoviruses (Ichtadenovirus), mammalian adenoviruses (castanovirus), sialidase adenoviruses (siadenvirus), double sense synucleviruses (ambidenvirus), short-term synucleviruses (brev idenvirus), hepatopancreatic synucleviruses (hepadnavirus), itaconviruses (iterprenavirus), prawn-synnumovirus (pensteldenvirus), aviruses (amblyvidivovirus), aviruses (amblyparvovirus), boparvoviruses (boparvoviruses), family parvoviruses (copparvoviruses), dependsubparvoviruses (ependparvoviruses), erythroviruses (erythroparvoviruses), tetraviruses (provirus) alpha retrovirus (alpha retrovirus), beta retrovirus (beta retrovirus), delta retrovirus (delta retrovirus), epsilon retrovirus (epsilon retrovirus), marlivirus (gamma retrovirus), herpes simplex virus (varicella), virus cell (Cytomevirus), virus cell (Mukovirus), virus cell (Programme virus cell), rosewood fever virus (roseola), lymphokines (Lymphocryptovirus), macadamia (macavir), equine herpes virus (Percavirus), and arachnoviruses (Rhadinovirus).

In certain aspects, the retrovirus family is moloney murine sarcoma virus (MoMSV), haven murine sarcoma virus (hamus v), murine mammary tumor virus (MuMTV), gibbon ape leukemia virus (GaLV), feline Leukemia Virus (FLV), foamy virus, friedel virus, murine Stem Cell Virus (MSCV), rous Sarcoma Virus (RSV), human T cell leukemia virus, human Immunodeficiency Virus (HIV), feline Immunodeficiency Virus (FIV), equine Immunodeficiency Virus (EIV), meidi-vesnaria virus; goat arthritis-encephalitis virus; equine infectious anemia virus; feline Immunodeficiency Virus (FIV); bovine Immunodeficiency Virus (BIV); or Simian Immunodeficiency Virus (SIV).

In some aspects, the viral particles (e.g., AAV particles) contain a heterologous nucleic acid molecule (e.g., a therapeutic gene or gene of interest). In some aspects, the heterologous nucleic acid molecule is operably linked to a promoter. Exemplary promoters include, but are not limited to, the Cytomegalovirus (CMV) immediate early promoter, the RSV LTR, the MoMLV LTR, the phosphoglycerate kinase-1 (PGK) promoter, the simian virus 40 (SV 40) promoter, and the CK6 promoter, transthyretin promoter (TTR), TK promoter, tetracycline-responsive promoter (TRE), HBV promoter, hAAT promoter, LSP promoter, chimeric liver-specific promoter (LSP), E2F promoter, telomerase (hTERT) promoter; a cytomegalovirus enhancer/chicken beta-actin/rabbit beta-globin promoter and an elongation factor 1-alpha promoter (EF 1-alpha) promoter. In some aspects, the promoter comprises a human β -glucuronidase promoter or a cytomegalovirus enhancer linked to a chicken β -actin (CBA) promoter. The promoter may be a constitutive, inducible or repressible promoter. In some aspects, the invention provides recombinant vectors comprising a nucleic acid encoding a heterologous transgene of the present disclosure operably linked to a CBA promoter. In some cases, a native promoter of the transgene, or a fragment thereof, will be used. Where expression of the transgene is desired to mimic natural expression, a natural promoter may be used. A natural promoter may be used when expression of the transgene must be regulated in time or developmentally, or in a tissue-specific manner, or in response to specific transcriptional stimuli. In another aspect, other natural expression control elements (such as enhancer elements, polyadenylation sites, or Kozak consensus sequences) may also be used to mimic natural expression.

Two-dimensional liquid chromatography-mass spectrometry (2 DLC-MS) system

The methods disclosed herein include subjecting the viral particles to two-dimensional liquid chromatography/mass spectrometry (2 DLC-MS). LC/MS utilizes liquid chromatography to physically separate ions and mass spectrometry to generate mass spectrometry data from ions, as is known in the art. Such mass spectral data can be used to determine, for example, molecular weight or structure, identify particles by mass, quantity, purity, and the like. These data may represent properties of the detected ions, such as signal strength (e.g., abundance) over time (e.g., retention time), or relative abundance to mass-to-charge ratio. The exemplary 2DLC-MS system shown in fig. 2B can be used to determine the relative abundance of viral capsid components in a sample of viral particles and to identify and quantify the viral protein components of the viral capsids (or a portion thereof). However, modifications to the exemplary system shown can also be used to determine the relative abundance of intact viral capsid components, and to identify and quantify viral protein components of viral capsids.

Non-limiting examples of the first and second dimension liquid chromatography columns 102 and 110 (see fig. 2B) include reversed phase liquid chromatography, ion exchange chromatography, size exclusion chromatography, affinity chromatography, hydrophilic interaction chromatography, and hydrophobic chromatography. Liquid chromatography (including HPLC) can be used to separate components of a sample of viral particles into viral capsid components and to separate the viral protein components of the viral capsids for further analysis. In some embodiments, the first dimension chromatography comprises anion exchange chromatography and the second dimension chromatography comprises reversed phase liquid chromatography. In some embodiments, the first dimension chromatography comprises anion exchange chromatography and the second dimension chromatography comprises hydrophilic interaction liquid chromatography.

In various embodiments, the first dimension chromatography comprises anion exchange chromatography employing mobile phase a containing 20mM bis-tri-propane/water and mobile phase B containing 20mM bis-tri-propane and 1M tetraalkylammonium salt (e.g., tetramethylammonium chloride or tetraethylammonium chloride). In some cases, the tetraalkylammonium salt is present in a concentration of about 0.1M to about 10M. In various embodiments, the tetraalkylammonium salt is present at a concentration of about 0.5M, about 0.6M, about 0.7M, about 0.8M, about 0.9M, about 1M, about 1.1M, about 1.2M, about 1.3M, about 1.4M, about 1.5M, about 1.6M, about 1.7M, about 1.8M, about 1.9M, about 2M, about 2.5M, about 3M, about 3.5M, about 4M, about 4.5M, about 5M, about 6M, about 7M, about 8M, about 9M, or about 10M. In some embodiments, mobile phase a, mobile phase B, or both mobile phase a and mobile phase B comprise about 1M sodium chloride. In various embodiments, sodium chloride is present at a concentration of about 0.1M to about 10M. In various embodiments, sodium chloride is present at a concentration of about 0.5M, about 0.6M, about 0.7M, about 0.8M, about 0.9M, about 1M, about 1.1M, about 1.2M, about 1.3M, about 1.4M, about 1.5M, about 1.6M, about 1.7M, about 1.8M, about 1.9M, about 2M, about 2.5M, about 3M, about 3.5M, about 4M, about 4.5M, about 5M, about 6M, about 7M, about 8M, about 9M, or about 10M. In some embodiments, mobile phase a or mobile phase B, or both, contain an alkali or alkaline earth metal halide salt (e.g., a chloride, bromide, or iodide salt of sodium, potassium, lithium, calcium, or magnesium) at any of the above concentrations. In some cases, the pH of the mobile phase is from about 7 to about 12. In some cases, the pH of the mobile phase is from about 8 to about 11. In some cases, the pH of the mobile phase is about 9, about 9.1, about 9.2, about 9.3, about 9.4, about 9.5, about 9.6, about 9.7, about 9.8, about 9.9, or about 10. In some embodiments, the flow rate is about 0.1mL/min or about 0.2mL/min or about 0.3mL/min.

In various embodiments, the second dimension chromatography comprises reverse phase liquid chromatography or hydrophilic interaction liquid chromatography. In some cases, the second dimension chromatography comprises a reverse phase liquid chromatography employing mobile phase a containing 0.1% to 0.5% difluoroacetic acid (DFA)/water and mobile phase B containing 0.1% to 0.5% DFA/Acetonitrile (ACN). In various embodiments, the DFA concentration is about 0.1%, about 0.15%, about 0.2%, about 0.25%, about 0.3%, about 0.35%, about 0.4%, about 0.45%, or about 0.5%. In some embodiments, the flow rate is about 0.1mL/min or about 0.2mL/min or about 0.3mL/min.

In various embodiments, the UV from the FLR detector is used to detect the eluate from the first and/or second dimension chromatography. In some cases, the FLR detector utilizes an excitation wavelength of about 260nm to about 300nm (e.g., about 280 nm) and an emission wavelength of about 310 to about 370nm (e.g., about 330nm or about 350 nm).

In various embodiments, denaturation of the viral capsid components (or portions thereof) is performed with about 10% acetic acid. In some embodiments, denaturation is achieved in the second dimension chromatography column by applying the initial mobile phase for a period of time (e.g., about 10 minutes) followed by a gradient to separate the intact viral proteins produced during denaturation. In some embodiments, the starting mobile phase comprises 80% mobile phase a and 20% mobile phase B, wherein mobile phase a comprises 0.1% to 0.5% difluoroacetic acid (DFA)/water and mobile phase B comprises 0.1% to 0.5% DFA/acetonitrile.

In some embodiments, the mobile phase of the first dimension chromatography and/or the second dimension chromatography is an aqueous mobile phase. In an exemplary embodiment, the mobile phase used to elute the viral protein from the second dimension chromatograph is a mobile phase compatible with a mass spectrometer. In some exemplary embodiments, the mobile phase used in the first or second dimension liquid chromatography column may include water, acetonitrile, difluoroacetic acid, or a combination thereof. The mobile phase may include a buffer with or without ion pairing agents (e.g., acetonitrile and water). Ion pairing agents include acetates, difluoroacetic acid and salts. A gradient of buffers may be used, for example, if two buffers are used, the concentration or percentage of the first buffer may decrease and the concentration or percentage of the second buffer increases during a chromatographic run. For example, the percentage of the first buffer may be reduced from about 100%, about 99%, about 95%, about 90%, about 85%, about 80%, about 75%, about 70%, about 65%, about 60%, about 50%, about 45%, or about 40% to about 0%, about 1%, about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, or about 40% during chromatographic running. As another example, the percentage of the second buffer may be increased from about 0%, about 1%, about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, or about 40% to about 100%, about 99%, about 95%, about 90%, about 85%, about 80%, about 75%, about 70%, about 65%, about 60%, about 50%, about 45%, or about 40% during the same run. In certain aspects, the proportion of mobile phase a in the chromatograph increases over time. Optionally, the concentrations or percentages of the first buffer and the second buffer may be returned to their starting values at the end of the chromatographic run. The percentages may be varied gradually in a linear gradient or in a non-linear (e.g., stepwise) manner. For example, the gradient may be multi-phase, e.g., biphasic, triphasic, etc.

In some exemplary embodiments, the mobile phase may have a flow rate through the liquid chromatography column of about 0.1 μl/min to about 100mL/min or about 0.05mL/min to about 5 mL/min. In some cases, the flow rate is about 0.05mL/min, about 0.06mL/min, about 0.07mL/min, about 0.08mL/min, about 0.09mL/min, about 0.1mL/min, about 0.11mL/min, about 0.12mL/min, about 0.13mL/min, about 0.14mL/min, about 0.15mL/min, about 0.16mL/min, about 0.17mL/min, about 0.18mL/min, about 0.19mL/min, about 0.2mL/min, about 0.21mL/min, about 0.22mL/min, about 0.23mL/min, about 0.24mL/min, about 0.25mL/min, about 0.26mL/min, about 0.27mL/min, about 0.28mL/min, about 0.29mL/min, about 0.3mL/min, about 0.4mL/min, about 0.5mL/min, about 6.7 mL/min, about 7mL/min, about 8mL/min, about 7.8 mL/min, about 7mL/min, about 8mL/min, about 5 mL/min. In some cases, the flow rate is 0.1mL/min. In some cases, the flow rate is 0.2mL/min.

In some aspects, mass spectrometry (e.g., used in 2DLC-MS as described herein) may refer to electrospray ionization mass spectrometry (ESI-MS). ESI-MS is known in the art as a technique that uses electrical energy to analyze ions derived from a solution by mass spectrometry. Ionic species (including neutral species ionized in a solution or gas phase) are converted from a solution to a gas phase by dispersion in an aerosol of charged droplets. Subsequently, solvent evaporation is performed to reduce the size of the charged droplets. Then, as the solution passes through the small capillary at a voltage relative to ground, sample ions are ejected from the charged droplet. For example, the walls of the surrounding ESI chamber are made by: the sample is mixed with a volatile acid and an organic solvent and infused through a conductive needle with high pressure. Charged droplets sprayed (or jetted) from the needle end are directed into a mass spectrometer and dried by heat and vacuum as they fly in. After the droplet is dried, the remaining charged molecules are directed by an electromagnetic lens into a mass detector and mass analyzed. In one aspect, the eluted sample is deposited directly from the capillary into the electrospray nozzle, e.g., the capillary acts as a sample loader. In another aspect, the capillary itself acts as both an extraction device and an electronozzle.

In some exemplary embodiments, the electrospray ionization emitter comprises a plurality of emitter nozzles, such as at least two, at least three, at least four, at least five, at least six, at least seven, at least eight emitter nozzles, such as two, three, four, five, six, seven, or eight emitter nozzles. In some exemplary embodiments, the electrospray ionization emitter is an M3 emitter from newmics (Berkeley, CA) that includes 8 emitter nozzles.

In some exemplary embodiments, other ionization modes are used, such as turbo spray ionization mass spectrometry, nano spray ionization mass spectrometry, thermal spray ionization mass spectrometry, sonic spray ionization mass spectrometry, SELDI-MS, and MALDI-MS. In general, these methods (e.g., ESI-MS) have the advantage that they can purify the sample "in time" and introduce the sample directly into the ionization environment. Notably, the various ionization and detection modes impose respective limitations on the nature of the desorption solution used, and it is important that the desorption solution be compatible with both. For example, sample matrices in many applications must have low ionic strength, or lie within a specific pH range, etc. In ESI, salts in the sample can reduce ionization or clog the nozzle, thereby preventing detection. This problem may be solved by providing the analyte in a low salt form and/or by using a volatile salt. In the case of MALDI, the analyte should be in a solvent compatible with the spotting of the target and the ionization matrix used.

In some exemplary embodiments, the electrospray ionization source provides electrospray with a solvent flow rate of about 1 μL/min to about 20 μL/min. In various embodiments, the flow rate into the ESI emitter is about 1 μL/min, about 2 μL/min, about 3 μL/min, about 4 μL/min, about 5 μL/min, about 6 μL/min, about 7 μL/min, about 8 μL/min, about 9 μL/min, about 10 μL/min, about 11 μL/min, about 12 μL/min, about 13 μL/min, about 14 μL/min, about 15 μL/min, about 16 μL/min, about 17 μL/min, about 18 μL/min, about 19 μL/min, or about 20 μL/min.

The mass spectrometer may be a non-denaturing ESI mass spectrometry system. In some exemplary embodiments, the mass spectrometer may be a quadrupole-orbitrap hybrid mass spectrometer. The quadrupole-orbitrap hybrid mass spectrometer may be a QExactive ^TM Focus mixing Quadripole-Orbitrap ^TM Mass spectrometer, Q exact ^TM Plus mix Quadrupole-Orbitrap ^TM Mass spectrometer, Q exact ^TM BioPharma platform, Q exact ^TM UHMR mixed Quadrapole-Orbitrap ^TM Mass spectrometer, Q exact ^TM HF mixed Quadrapole-Orbitrap ^TM Mass spectrometer, Q exact ^TM HF-X mixed Quadrupole-Orbitrap ^TM Mass spectrometer and Qexact ^TM Mixed Quadrapole-Orbitrap ^TM A mass spectrometer. In some exemplary embodiments, the mass spectrometry system is a Thermo Exactive EMR mass spectrometer. The mass spectrometry system can also include an ultraviolet light detector.

A variety of mass analyzers suitable for LC/MS are known in the art, including but not limited to time of flight (TOF) analyzers, quadrupole mass filters, quadrupole TOF (QTOF) and ion traps (e.g., fourier transform based mass spectrometers or orbitraps). In the orbitrap, barrel-shaped outer electrodes and spindle-shaped center electrodes at ground potential are used to trap ions in trajectories that are elliptical in rotation about the center electrode and oscillate along the central axis, and are limited by the balance of centrifugal and electrostatic forces. The use of such instruments employs fourier transform operations to convert the time domain signal (e.g., frequency) from the detection of the image current to a high separation quality measurement.

Examples

The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the methods and compositions of the present invention, and are not intended to limit the scope of what the inventors regard as their invention. Efforts have been made to ensure accuracy with respect to numbers used (e.g., amounts, temperature, etc.), but some experimental errors and deviations should be accounted for. Unless otherwise indicated, parts are parts by weight, molecular weight is average molecular weight, temperature is in degrees celsius and pressure is at or near atmospheric pressure.

Example 1: characterization of viral protein component of AAV capsids

AAV empty samples and complete samples of different serotypes were prepared internally. Empty and full AAV samples were directly mixed and analyzed using the Agilent1290 information II 2D-LC system. In the first dimension, empty and full AAV capsids are isolated by ProPac SAX-10 column (Thermo Scientific). In the second dimension, AAV capsids are first denatured and desalted, and viral proteins are separated by ACQUITY UPLC Protein BEH C column (Waters Corporation). At Q exact ^TM MS analysis of viral proteins was performed on Plus hybrid quadrupole-orbitrap Mass spectrometer (Thermo Scientific) and Xcalibur (Thermo Scientific) and complete Mass were used ^TM (Protein Metrics inc.) to analyze MS data.

Chemical and reagent

Unless otherwise indicated, all chemicals and reagents were obtained from millipore sigma (Burlington, MA, USA). Empty and full capsids of the three AAV serotypes (AAV 8, AAV5 and AAV 1) were produced internally by Regeneron Pharmaceuticals inc. (Tarrytown, NY, USA), and detailed sample information and concentrations are shown in table 1 below. Acetonitrile (ACN) was obtained from Thermo Fisher Scientific (Waltham, MA, USA). Difluoroacetic acid (DFA) was obtained from Waters Corporations (Milford, MA, USA). Deionized water (Milli-Q water) was obtained from the Milli-Q integral water purification system (Millipore Sigma).

Table 1: concentration of AAV samples

Sample of	Concentration of
		AAV 8-null	3.07×10 13 Individual capsids/mL
AAV8–GOI1	2.70×10 13 vg/mL
		AAV8–GOI2	2.70×10 13 vg/mL
AAV 5-null	3.32×10 13 Individual capsids/mL
		AAV5–GOI1	4.61×10 13 vg/mL
AAV 1-null	2.54×10 13 Individual capsids/mL
		AAV1–GOI1	1.82×10 13 vg/mL

vg/mL-viral genome/mL

Anion exchange chromatography (AEX) experiments

AAV samples were directly analyzed by AEX without any sample pretreatment. AEX separation was performed on an ACQUITY UPLC I-type system (Waters Corporation) equipped with a fluorescence detector using a Thermo ProPac SAX-10 column (10 μm,2 mm. Times.250 mm) (Thermo Fisher Scientific). Mobile Phase A (MPA) contained 20mM bis-tri-propane/Milli-Q water and Mobile Phase B (MPB) contained 20mM bis-tri-propane and 1M tetramethylammonium chloride (TMAC) or tetraethylammonium chloride (TEAC)/Milli-Q water. MPA and MPB were adjusted to pH 9.5 using hydrochloric acid. The AEX flow rate was 0.2mL/min, gradient consisted of 0 to 10 min 10% to 30% MPB, 10 to 10.1 min 30% to 90% MPB and up to 12 min 90% MPB. MPB was reduced to 10% for 12 to 12.1 minutes and then maintained at 10% until the 20 minute gradient ended. For all AEX analyses, 1 μl of sample was injected. Data were recorded using fluorescence detectors with excitation (Ex) and emission (Em) wavelengths of 280nm and 350nm, respectively.

Reversed Phase Liquid Chromatography (RPLC) experiments

AAV samples were denatured with 10% acetic acid for 10 minutes prior to RPLC analysis. The ACQUITY UPLC protein BEH C4 column (1.7 μm, 2.1 mm. Times.150 mm) (Waters Corporation) RPLC experiments were performed on an ACQUITY UPLC I-Class system (Waters Corporation) equipped with a fluorescence detector. MPA was prepared using 0.1% DFA/Milli-Q water and MPB was prepared using 0.1% DFA/acetonitrile. The gradient was run at 0.2mL/min, starting at 0 to 1 min 20% to 32% MPB, then 1 to 16 min 32% to 36% MPB,20 to 21.5 min 36% to 80% MPB,21.5 to 22 min 80% to 20% MPB, then 20% MPB until the 30 min gradient ended. For all RPLC assays, 1 μl of sample was injected. Data were collected using fluorescence detectors with an Ex wavelength of 280nm and an Em wavelength of 350 nm.

Two-dimensional liquid chromatography (2 DLC) conditions

A2 DLC experiment was performed on an Agilent 1290Infinity II 2D-LC system (Agilent Technologies, santa Clara, calif., USA). The AEX gradient was applied to the first dimension at a flow rate of 0.1mL/min (instead of 0.2 mL/min). At this lower flow rate, the 40 μl capture loop can capture 0.4 seconds of sample. 6. Mu.L of AAV8 samples containing the gene of interest 1 (GOI 1) were injected. The center cut was performed using a time-based high-resolution sampling mode, in which peaks were selected according to the UV spectrum at a wavelength of 280 nm. The sample is then transferred from the capture loop to a second dimension RP column. An RPLC gradient was also applied to the second dimension and a 10 minute hold time was added at 80% MPA to remove MS-incompatible salts for fpr AEX isolation and online denaturation of intact viral capsids. ACQUITY UPLC protein BEH The C4 column (1.7 μm,2.1 mm. Times.50 mm) (Waters Corporation) was used as a capture column before an analytical column of 150mm length. During denaturation and desalting, the flow was directed to the waste and analytical column (for downstream analysis) using a diverter valve. To maintain the temperature of the analytical column, the initial RPLC mobile phase was maintained at 0.05mL/min using an additional LC pump.

Mass Spectrometry (MS) data acquisition

RPLC-MS data usage Thermo Scientific Q Exactive ^TM Plus hybrid quadrupole-orbitrap mass spectrometer (Bremen, germany). For data acquisition, the separation was set to 17,500, the agc target was set to 3e6, and the maximum injection time was set to 500ms. The spray voltage was set at 3.8kV and the S-lens RF level was set at 50. The sheath flow and the auxiliary gas flow were 40 and 15, respectively, and the capillary temperature and the auxiliary gas heater temperature were set at 250 ℃. Mass spectra were acquired at 1,000 to 3,000 m/z.

All 2DLC-MS data were collected on a Thermo Scientific orbitrap exporis 480 mass spectrometer (Bremen, germany) equipped with a Thermo Scientific NanoSpray Flexx ion source. A nanoshunt from Analytical Scientific Instruments (Richmond, CA, USA) WAs set to 50 and electrospray ionization emitter tips (CoAnn Technologies, richland, WA, USA) were used for electrospray. For data acquisition, the degree of separation, AGC target, maximum injection time and number of microscans were set to 15,000, 3e6, auto and 3, respectively. The spray voltage was set to 2,200v, the rf lens level was set to 50% and the ion transport tube temperature was set to 275 ℃. Mass spectra were acquired at 1,000 to 3,000 m/z.

Data analysis

For data collected on Waters instrument, analysis was performed on the Empower 3 version 1.65. Mass spectral data were analyzed using Xcalibur 4.3.73.11. Data collected on the Agilent instrument was analyzed using OpenLAB CDS ChemStation version rev.c.01.07 sr2. Using an input Mass ^TM Version 3.11-1 (Protein Metrics inc., cupertino, CA, USA) for completionAnd (5) mass analysis.

Results and discussion

A 2DLC-MS platform (as schematically shown in fig. 2B) was used for AAV characterization. The method implements a high degree of separation AEX in the first dimension for empty and complete viral capsid separation (fig. 3A, 3B and 5). After on-line denaturation and desalting of MS-incompatible salts, the viral proteins were subjected to complete protein separation in the second RPLC dimension and complete protein characterization by MS (fig. 3C, 3D, 6, 7A, 7B, 7C, 7D, 8A and 8B).

In the first dimension AEX was used to perform empty and full AAV capsid isolation. The empty and full AAV capsids of all test samples were baseline resolved using a salt gradient of tetramethylammonium chloride and tetraethylammonium chloride as compared to conventionally used sodium chloride. For each of AAV1, AAV5 and AAV8 serotypes, empty capsids and capsids containing the gene of interest (GOI) were isolated (fig. 3A and 3B). The separation of empty and full capsids allows for the quantification of the relative percentages of empty and full capsids in the sample (e.g., fig. 5), consistent with the results of AUC determinations. After high degree of separation of the first dimension void and complete capsid, online capture is performed to select peaks of interest. Prior to the second dimension RPLC analysis, the intact AAV capsids were denatured into individual viral proteins. The initial mobile phase composition of RPLC was used to denature AAV capsids by acidification and to remove MS-incompatible salts used in AEX isolation. In the second dimension, viral proteins were isolated by RPLC using difluoroacetic acid as the ion pairing reagent. MS analysis of viral proteins shows that low abundance species include unmodified, phosphorylated and oxidized protein forms. Furthermore, differences in the phosphorylation levels of VP2 were observed in AAV samples (see, e.g., fig. 7A, 7B, 7C, 7D, 8A and 8B).

AAV capsids contain three types of Viral Protein (VP) subunits VP1, VP2 and VP3, totaling 60 copies, in a 1:1:10 ratio (VP 1: VP2: VP 3). These capsid proteins are alternatively spliced from one mRNA and thus share a common sequence.

In reverse phase liquid chromatography-mass spectrometry (RPLC-MS) analysis, the major protein forms include acetylated VP1 and its phosphorylated form, VP2 and its phosphorylated form, acetylated VP3 and VP3 sheared matter. Minor protein forms include protein forms resulting from cleavage of aspartate-proline (DP) bonds. This DP bond cleavage yields materials that include acetylated VP1 cleavage, VP2 cleavage and its phosphorylated forms, acetylated VP3 cleavage and DP cleavage fragments. The scissoring material results from cleavage of an aspartate-proline (DP) bond, which may be introduced during denaturation and isolation. In addition, unmodified and oxidized VP3 species were also observed, as well as additional acetylated VP3 scission species with cleavage of aspartate-glycine (DG) bonds (fig. 8A).

Similarly, RPLC-MS analysis of AAV 1-empty capsid samples showed that the major protein forms included acetylated VP1 and its phosphorylated form, VP2 and its phosphorylated form, acetylated VP3, and VP cleavage resulting from DP bond cleavage. DP bond cleavage also generates protein forms such as VP2 cleavage and its phosphorylated forms, acetylated VP3 cleavage and cleavage fragments. For all three VPs, a low abundance oxidized protein form was detected. DG bond cleavage was also observed, which provided additional acetylated VP3 scissoring material and DG scissoring fragments (fig. 8B).

In addition to protein formal identification, complete mass analysis also showed differences in post-translational modification (PTM) levels. Previous studies have shown that Ser149 is the primary phosphorylation site in AAV8 sequences. Ser149 is not included in the VP3 sequence since the three viral proteins are alternately cleaved. Although the phosphorylation levels of VP1 remained similar in the three AAV8 samples, there was a significant difference in the phosphorylation levels of VP2 (fig. 8A). Both AAV8 samples comprising GOI showed increased levels of VP2 phosphorylation compared to AAV8 samples without GOI. For AAV1, no difference in phosphorylation was observed in viral proteins of empty and full capsids (fig. 8B).

The 2DLC-MS method presented herein can enable high-throughput and multi-attribute AAV characterization in a single system. In the first dimension, AEX provides high degree of separation of empty and complete capsids using TMAC or TEAC. On-line denaturation and desalting was achieved to dissociate AAV capsids into viral proteins. In the second dimension, the coupling of RPLC to MS is used to characterize viral proteins. With this method, AAV samples can be directly analyzed without sample pretreatment, thereby minimizing sample processing and avoiding sample loss. The platform combines two characterization techniques in one analysis and provides good separation and high sensitivity, enabling detection of protein forms and fragments of primary and secondary viral proteins.

The scope of the invention is not limited by the specific embodiments described herein. Indeed, various modifications of the invention in addition to those described herein will become apparent to those skilled in the art from the foregoing description. Such modifications are intended to fall within the scope of the appended claims.

Claims (42)

1. A method for identifying viral protein components of a sample of viral particles, the method comprising:

(a) Subjecting a sample of said viral particles to a first dimension chromatography to separate the intact viral capsid components of said sample;

(b) Subjecting at least a portion of the intact viral capsid component to in-line denaturation to produce individual intact viral proteins;

(c) Subjecting the intact viral protein to a second dimension chromatography to isolate the intact viral protein; and

(d) The mass of the isolated intact viral protein is determined to identify the viral protein component of a sample of the viral particle.

2. The method of claim 1, further comprising selecting a portion of an isolated whole viral capsid component, wherein subjecting at least a portion of the whole viral capsid component to in-line denaturation to produce individual viral proteins comprises subjecting the selected portion of the isolated whole viral capsid component to in-line denaturation.

3. The method of claim 1 or 2, wherein the sample of viral particles comprises adeno-associated virus (AAV) particles.

4. The method of claim 3, wherein the AAV particle has serotypes AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV-DJ/8, AAV-Rh10, AAV-retro, AAV-php.b, AAV8-php.eb, or AAV-php.s.

5. The method of claim 4, wherein the AAV particle has serotype AAV1 or AAV8.

6. The method of any one of claims 1 to 5, wherein the complete viral capsid component comprises an empty viral capsid and a complete viral capsid.

7. The method of any one of claims 1 to 6, wherein the first dimension chromatography comprises ion exchange chromatography.

8. The method of claim 7, wherein the ion exchange chromatography is anion exchange chromatography.

9. The method of any one of claims 1 to 8, wherein the second dimension chromatography comprises reverse phase chromatography.

10. The method of any one of claims 1 to 8, wherein the second dimension chromatography comprises hydrophilic interaction liquid chromatography.

11. The method of any one of claims 1 to 10, wherein determining the mass of the isolated whole viral protein comprises subjecting the isolated whole viral protein to electrospray ionization mass spectrometry.

12. The method of any one of claims 3 to 11, wherein the viral protein component comprises VP1, VP2 and/or VP3 of an AAV particle.

13. The method of claim 12, wherein the viral protein component comprises a post-translational variant of VP1, VP2, and/or VP3.

14. The method of claim 13, wherein the post-translational variants of VP1, VP2, and/or VP3 comprise acetylated, phosphorylated, and/or oxidized variants of VP1, VP2, and/or VP3.

15. The method of claim 13 or 14, wherein the post-translational variant of VP1, VP2 and/or VP3 comprises a fragment of VP1, VP2 and/or VP3 resulting from cleavage of an aspartate-proline bond and/or cleavage of an aspartate-glycine bond.

16. The method of any one of claims 1 to 15, further comprising detecting the intact viral capsid component separated by the first dimension chromatography, and identifying the ratio of empty viral capsids to full and partial full viral capsids.

17. The method of any one of claims 1 to 16, further comprising detecting the intact viral protein separated by the second dimension chromatography, and quantifying the relative abundance of the viral protein component of a sample of the viral particles.

18. The method of claim 16 or 17, wherein the intact viral capsid component and/or the intact viral protein is detected using an ultraviolet or fluorescence detector.

19. A method for identifying a viral protein component of a sample of adeno-associated virus (AAV) particles, the method comprising:

(a) Subjecting a sample of the AAV particles to anion exchange chromatography to isolate an intact viral capsid component in the sample, wherein the intact viral capsid component comprises an intact empty viral capsid and an intact complete viral capsid, the intact complete viral capsid comprising a heterologous nucleic acid molecule;

(b) Selecting a portion of the intact viral capsid component for online desalting and denaturation;

(c) Subjecting selected portions of the intact viral capsid component to online desalting and denaturing to produce individual intact viral proteins, wherein the intact individual viral proteins comprise VP1, VP2, VP3 and at least one variant of VP1, VP2 or VP 3;

(d) Subjecting the intact viral protein to reverse phase liquid chromatography or hydrophilic interaction liquid chromatography to isolate the intact viral protein; and

(e) Determining the mass of the isolated intact viral protein to identify the viral protein component of a sample of the AAV particle.

20. The method of claim 19, further comprising detecting the intact viral capsid component separated by the anion exchange chromatography and identifying the ratio of empty viral capsids to full and partial full viral capsids.

21. The method of claim 19 or 20, further comprising detecting the intact viral protein separated by the reverse phase liquid chromatography or hydrophilic interaction liquid chromatography, and quantifying the relative abundance of the viral protein component of a sample of the AAV particle.

22. The method of any one of claims 19-21, wherein the AAV particle has serotype AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV-DJ/8, AAV-Rh10, AAV-retro, AAV-php.b, AAV8-php.eb, or AAV-php.s.

23. The method of claim 22, wherein the AAV particle has serotype AAV1 or AAV8.

24. The method of any one of claims 19 to 23, wherein the at least one variant of VP1, VP2, or VP3 comprises a post-translational variant of VP1, VP2, or VP 3.

25. The method of claim 24, wherein the post-translational variant of VP1, VP2, or VP3 comprises an acetylated variant of VP1, VP2, or VP 3.

26. The method of claim 24, wherein the post-translational variant of VP1, VP2, or VP3 comprises a phosphorylated variant of VP1, VP2, or VP 3.

27. The method of claim 24, wherein the post-translational variant of VP1, VP2, or VP3 comprises an oxidized variant of VP1, VP2, or VP 3.

28. The method of claim 24, wherein the post-translational variant of the VP1, VP2, or VP3 comprises a fragment of VP1, VP2, or VP3 resulting from cleavage of an aspartate-proline bond.

29. The method of claim 24, wherein the post-translational variant of the VP1, VP2, or VP3 comprises a fragment of VP1, VP2, or VP3 resulting from cleavage of an aspartate-glycine bond.

30. The method of any one of claims 20 to 29, wherein the intact viral capsid components and/or the intact viral proteins are detected using an ultraviolet or fluorescence detector.

31. The method of any one of claims 19 to 30, wherein determining the mass of the isolated whole viral protein comprises subjecting the isolated whole viral protein to electrospray ionization mass spectrometry.

32. The method of any one of claims 19 to 31, wherein the whole viral protein is subjected to reverse phase liquid chromatography.

33. The method of any one of claims 19 to 31, wherein the intact viral protein is subjected to hydrophilic interaction liquid chromatography.

34. The method of any one of claims 8 or 19-31, wherein the intact viral capsid components of the sample that has been subjected to anion exchange chromatography are separated using a first mobile phase comprising 15mM to 25mM bis-tri-propane (BTP), 250mM to 1M tetramethyl ammonium chloride (TMAC), and 1mM to 3mM magnesium chloride at a pH of 8 to 9.

35. The method of claim 34, wherein the first mobile phase comprises 20mM ± 2mM BTP, 500mM ± 50mM TMAC, and 2mM ± 0.2mM MgCl at pH 8.5 ± 0.1 ₂ 。

36. The method of claim 34 or 35, wherein the intact viral capsid components of the sample that has been subjected to anion exchange chromatography are separated using the first mobile phase and a second mobile phase comprising 15mM to 25mM bis-tri-propane (BTP) and 1mM to 3mM magnesium chloride at a pH of 8 to 9.

37. The method of claim 36, wherein the second mobile phase comprises 20mM ± 2mM BTP and 2mM ± 0.2mM MgCl at pH 8.5 ± 0.1 ₂ 。

38. The method of claim 36 or 37, wherein the intact viral capsid components of the sample that has been subjected to anion exchange chromatography are separated using the first mobile phase, the second mobile phase and a third mobile phase, the third mobile phase comprising 1.5M to 2.5M sodium chloride.

39. The method of claim 38, wherein the third mobile phase comprises 2M ± 0.1M sodium chloride.

40. The method of claim 38 or 39, wherein the separation of the intact viral capsid components is performed using a mobile phase gradient.

41. The method of claim 40, wherein the mobile phase gradient comprises, in order: 10% of the first mobile phase and 90% of the second mobile phase for 1 minute; increasing the first mobile phase from 10% to 42% and decreasing the second mobile phase from 90% to 58% over a period of 20 minutes; 100% of a third mobile phase, for 5 minutes; and 10% of the first mobile phase and 90% of the second mobile phase for 10 minutes.

42. The method of any one of claims 34 to 41, further comprising identifying the amount of intact empty viral capsids and the amount of intact viral capsids in the sample, and determining the relative abundance of the intact empty viral capsids and intact complete viral capsids in the sample.