WO2025166285A1

WO2025166285A1 - Methods for characterizing anti-drug antibodies in samples

Info

Publication number: WO2025166285A1
Application number: PCT/US2025/014188
Authority: WO
Inventors: Xiaoxiao HUANG; Yuetian Yan; Shunhai WANG; Ning Li
Original assignee: Regeneron Pharmaceuticals Inc
Current assignee: Regeneron Pharmaceuticals Inc
Priority date: 2024-02-01
Filing date: 2025-01-31
Publication date: 2025-08-07
Anticipated expiration: 2026-08-01

Abstract

Described herein are methods for characterizing a peptide or protein in a sample, involving forming a complex between the peptide or protein and a binding partner, binding the complex to an affinity ligand conjugated to a solid support, washing to remove unbound material, eluting and digesting the peptide or protein, and using liquid chromatography-mass spectrometry (LC-MS) analysis to characterize the peptide or protein. For example, the methods described herein could be used to characterize an anti-drug antibody in a biological sample, such as a serum sample.

Description

METHODS FOR CHARACTERIZING ANTI-DRUG ANTIBODIES IN SAMPLES CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional Patent Application No. 63/627,867 filed on February 1, 2024, which is hereby incorporated by reference in its entirety.

TECHNICAL FIELD

[0002] This disclosure generally relates to methods for characterizing a peptide or protein, such as an anti-drug antibody, in a sample.

INTRODUCTION

[0003] Viral vectors, such as adeno-associated viral (AAV) vectors, is a promising platform for therapeutic gene therapy. However, immune responses against viral vectors are of great concern, emphasizing the importance of assessing immunogenicity of such vectors. For example, anti-drug antibodies (AD As) may develop in response to the presence of therapeutic AAV vectors. Human antibodies (immunoglobulins) may comprise various isotypes which operate in different locations in the body and which have different functions in the immune response. Characterizing the isotypes of anti-AAV antibodies and their corresponding levels in patient sera could provide important information in clinical development.

[0004] Several methods have been developed to characterize AD As, including cell neutralizing assays and ligand basing assays such as bridging assays, bead-based assays, and surface plasmon resonance. Ligand binding assays have been conventionally used to quantify peptides and proteins. However, ligand binding assays are time-consuming to develop, highly variable in their efficacy, and generally lack specificity.

[0005] Liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) is preferable for the analysis of biomolecules and biopharmaceuticals, as it provides a number of advantages that provide for a significantly faster method development process than conventional methods. For example, LC-MS/MS may be used to analyze a protein through quantification of a surrogate peptide derived from proteolytic digestion, which can be used as a unique identifier for the protein.

However, sensitive and specific identification of the surrogate peptide from a complex matrix using LC-MS/MS may require ongoing method development and optimization when using a direct digestion method. Alternatively, it could require assay-specific reagents when using an immunoprecipitation method (assuming such reagents are available, or affordable). Therefore, there exists a need for methods that can identify, quantify, and characterize anti-AAV antibodies from biological samples SUMMARY OF THE DISCLOSURE

[0006] Provided herein are methods for characterizing a peptide or protein in a sample. In some embodiments, the method may comprise: contacting the sample including the peptide or protein to a binding partner to form a complex; contacting the complex to a solid support conjugated to an affinity ligand capable of binding the complex to form an immobilized complex; washing the immobilized complex to remove unbound material; eluting the peptide or protein from the immobilized complex to produce an enriched peptide or protein sample; subjecting the enriched peptide or protein sample to enzymatic digestion conditions to produce a peptide digest; and subjecting the peptide digest to liquid chromatography-mass spectrometry (LC-MS) analysis to characterize the peptide or protein.

[0007] In some aspects, the binding partner may comprise a viral vector. In some aspects, the binding partner may comprise an adeno-associated viral (AAV) vector. In some aspects, the AAV vector may comprise a serotype selected from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, or a combination thereof

[0008] In some aspects, the solid support may comprise streptavidin. In some aspects, the affinity ligand may bind to the binding partner. In some aspects, the affinity ligand may comprise an antibody or a fragment thereof. In some aspects, the affinity ligand may comprise an anti-AAVX antibody or a fragment thereof. In some aspects, the anti-AAVX antibody may be conjugated to biotin.

[0009] In some aspects, the enzymatic digestion may comprise contacting the peptide or protein to a digestive enzyme mixture comprising one or more of pepsin, trypsin, Tryp-N, chymotrypsin, Lys-N, Lys-C, Asp-N, Arg-C, Glu-C, papain, IdeS, IdeZ, igdE, glyserias, or a variant thereof.

[0010] In some aspects, the sample may comprise a biological sample. In some aspects, the sample may comprise a serum sample. In some aspects, the sample may comprise a human serum sample. [0011] In some aspects, an amount of the affinity ligand used may be about 1 pg to about 15 pg. In some aspects, an amount of affinity ligand used may be about 10 pg.

[0012] In some aspects, subjecting the peptide digest to LC-MS analysis may comprise subjecting the peptide digest to a mass spectrometer capable of performing LC-MS or liquid chromatographymultiple reaction monitoring-mass spectrometry (LC-MRM-MS). In some aspects, subjecting the peptide digest to LC-MS analysis may comprise subjecting the peptide digest to an electrospray ionization mass spectrometer, a nano-electrospray ionization mass spectrometer, a triple quadrupole mass spectrometer, or a quadrupole time-of-flight. In some aspects, the mass spectrometer may be a triple quadrupole mass spectrometer. In some aspects, the LC-MS analysis may comprise multiple reaction monitoring (MRM).

[0013] In some aspects, the peptide or protein may be an anti-drug antibody (ADA) or a fragment thereof. In some aspects, the ADA may be a monoclonal antibody. In some aspects, the ADA may be a bispecific antibody. In some aspects, the ADA may be a human antibody. In some aspects, an isotype of the human antibody may be IgGl, IgG2, IgG3, IgG4, IgAl, IgA2, IgM, or IgE.

BRIEF DESCRIPTION OF THE DRAWINGS

[0014] The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate various examples and together with the description, serve to explain the principles of the disclosed examples and embodiments.

[0015] Aspects of the disclosure may be implemented in connection with embodiments illustrated in the attached drawings. These drawings show different aspects of the present disclosure and, where appropriate, reference numerals illustrating like structures, components, materials, and/or elements in different figures are labeled similarly. It is understood that various combinations of the structures, components, and/or elements, other than those specifically shown, are contemplated and are within the scope of the present disclosure.

[0016] Moreover, there are many embodiments described and illustrated herein. The present disclosure is neither limited to any single aspect nor embodiment thereof, nor to any combinations and/or permutations of such aspects and/or embodiments. Moreover, each of the aspects of the present disclosure, and/or embodiments thereof, may be employed alone or in combination with one or more of the other aspects of the present disclosure and/or embodiments thereof. For the sake of brevity, certain permutations and combinations are not discussed and/or illustrated separately herein. Notably, an embodiment or implementation described herein as “exemplary” is not to be construed as preferred or advantageous, for example, over other embodiments or implementations; rather, it is intended reflect or indicate the embodiment(s) is/are “example” embodiment s).

[0017] FIG. 1 is an illustration depicting an exemplary complex of binding partner, affinity ligand, and anti-drug antibody, according to aspects of the present disclosure.

[0018] FIG. 2 is an illustration depicting an exemplary method for characterizing anti-drug antibodies, according to aspects of the present disclosure.

[0019] FIG. 3 is a bar graph depicting antibody recovery as a function of an exemplary antibody concentration in serum, according to aspects of the present disclosure.

[0020] FIGS. 4A-4C are graphs depicting antibody recovery as a function of automated immunoprecipitation and manual immunoprecipitation across three exemplary antibody isotype classes, according to aspects of the present disclosure. FIG. 4A is a bar graph depicting automated and manual immunoprecipitation results for an IgGl antibody. FIG. 4B is a bar graph depicting automated and manual immunoprecipitation results for an IgG2 antibody. FIG. 4C is a bar graph depicting automated and manual immunoprecipitation results for an IgG4 antibody.

[0021] FIGS. 5A-5B are graphs depicting detection limits of an exemplary IgGl antibody immunoprecipitation as a function of concentration of the antibody in serum (antibody serum concentrations), according to aspects of the present disclosure. FIG. 5A is a bar graph depicting peak area as a function of antibody serum concentration. FIG. 5B is a line graph depicting a positive linear correlation between peak area and antibody serum concentration.

[0022] FIG. 6 is a bar graph depicting results of an exemplary method for characterizing an IgGl isotype of anti-AAV8 antibodies in patient sera, according to aspects of the present disclosure. [0023] FIG. 7 is a bar graph depicting results of an exemplary method for characterizing an IgG4 isotype of anti-AAV8 antibodies in patient sera, according to aspects of the present disclosure. [0024] FIG. 8 is a bar graph depicting results of an exemplary method for characterizing an IgM isotype of anti-AAV8 antibodies in patient sera, according to aspects of the present disclosure.

[0025] FIG. 9 is a bar graph depicting results of an exemplary method for characterizing an IgAl isotype of anti-AAV8 antibodies in patient sera, according to aspects of the present disclosure. [0026] FIG. 10 is a bar graph depicting results of an exemplary method for characterizing an IgG2 isotype of anti-AAV8 antibodies in patient sera, according to aspects of the present disclosure. [0027] FIG. 11 is a bar graph depicting results of an exemplary method for characterizing an IgG3 isotype of anti-AAV8 antibodies in patient sera, according to aspects of the present disclosure.

[0028] FIG. 12 is a bar graph depicting results of an exemplary method for characterizing an IgE isotype of anti-AAV8 antibodies in patient sera, according to aspects of the present disclosure. [0029] FIG. 13 is a bar graph depicting results of an exemplary method for characterizing an IgAl isotype of anti-AAV8 antibodies in patient sera, according to aspects of the present disclosure.

[0030] FIG. 14 is a bar graph depicting results of an exemplary method for characterizing an IgGl isotype of anti-AAV2 antibodies in patient sera, according to aspects of the present disclosure. [0031] FIG. 15 is a bar graph depicting results of an exemplary method for characterizing an IgG4 isotype of anti-AAV2 antibodies in patient sera, according to aspects of the present disclosure. [0032] FIG. 16 is a bar graph depicting results of an exemplary method for characterizing an IgM isotype of anti-AAV2 antibodies in patient sera, according to aspects of the present disclosure.

[0033] FIG. 17 is a bar graph depicting results of an exemplary method for characterizing an IgAl isotype of anti-AAV2 antibodies in patient sera, according to aspects of the present disclosure. [0034] FIG. 18 is a bar graph depicting results of an exemplary method for characterizing an IgG2 isotype of anti-AAV2 antibodies in patient sera, according to aspects of the present disclosure.

[0035] FIG. 19 is a bar graph depicting results of an exemplary method for characterizing an IgG3 isotype of anti-AAV2 antibodies in patient sera, according to aspects of the present disclosure.

[0036] FIG. 20 is a bar graph depicting results of an exemplary method for characterizing an IgE isotype of anti-AAV2 antibodies in patient sera, according to aspects of the present disclosure. [0037] FIG. 21 is a bar graph depicting results of an exemplary method for characterizing an IgA2 isotype of anti-AAV2 antibodies in patient sera, according to aspects of the present disclosure.

[0038] FIG. 22 is a line graph depicting recovery of AAV1 bound to an affinity ligand as a function of loading volume using an exemplary immunoprecipitation method, according to aspects of the present disclosure.

[0039] FIG. 23 is a bar graph depicting results of an exemplary method for characterizing an IgGl isotype of anti-AAVl antibodies in patient sera, according to aspects of the present disclosure.

[0040] FIG. 24 is a bar graph depicting results of an exemplary method for characterizing an IgG4 isotype of anti-AAVl antibodies in patient sera, according to aspects of the present disclosure.

[0041] FIG. 25 is a bar graph depicting results of an exemplary method for characterizing an IgM isotype of anti-AAVl antibodies in patient sera, according to aspects of the present disclosure.

[0042] FIG. 26 is a bar graph depicting results of an exemplary method for characterizing an IgAl isotype of anti-AAVl antibodies in patient sera, according to aspects of the present disclosure.

[0043] FIG. 27 is a bar graph depicting results of an exemplary method for characterizing an IgG2 isotype of anti-AAVl antibodies in patient sera, according to aspects of the present disclosure.

[0044] FIG. 28 is a bar graph depicting results of an exemplary method for characterizing an IgG3 isotype of anti-AAVl antibodies in patient sera, according to aspects of the present disclosure. [0045] FIG. 29 is a bar graph depicting results of an exemplary method for characterizing an IgE isotype of anti-AAVl antibodies in patient sera, according to aspects of the present disclosure.

[0046] FIG. 30 is a bar graph depicting results of an exemplary method for characterizing an IgA2 isotype of anti-AAVl antibodies in patient sera, according to aspects of the present disclosure.

[0047] FIGS. 31 A-3 IB are activity maps depicting results of an exemplary neutralizing assay to determine cell-based neutralization of activity of AAV2 vectors (top) and AAV8 vectors (bottom) in several sample types, according to aspects of the present disclosure.

[0048] FIGS. 32A-32F are bar graphs depicting an exemplary method for characterizing peptides identified after neutralizing assays for AAV8 and AAV2 vectors with several different isotypes of antibodies, according to aspects of the present disclosure. FIG. 32A depicts IgGl antibodies and AAV8 vectors. FIG. 32B depicts IgGl antibodies and AAV2 vectors. FIG. 32C depicts IgG4 antibodies and AAV8 vectors. FIG. 32D depicts IgG4 antibodies and AAV2 vectors. FIG. 32E depicts IgM antibodies and AAV8 vectors. FIG. 32F depicts IgM antibodies and AAV2 vectors. [0049] FIGS. 33A-33C are bar graphs depicting results of an exemplary method for characterizing peptides identified after a neutralizing assay for AAV1 vectors with several different isotypes of antibodies, according to aspects of the present disclosure. FIG. 33A depicts IgGl antibodies with AAV1 vectors. FIG. 33B depicts IgG4 antibodies with AAV1 vectors. FIG. 33C depicts IgM antibodies with AAV1 vectors.

[0050] FIGS. 34A-34D are graphs depicting results of evaluating an exemplary method for characterizing peptides identified after neutralizing assays for AAV8 and AAV1 vectors with IgGl antibodies, according to aspects of the present disclosure. FIG. 34A is a calibration curve depicting a relationship between concentration of an IgGl antibody and detected presence of the antibody in a sample with AAV8 vectors. FIG. 34B is a bar graph depicting concentration of the IgGl antibody in various types of samples with AAV8 vectors. FIG. 34C is a calibration curve depicting a relationship between concentration of an IgGl antibody and detected presence of the antibody in a sample with AAV1 vectors. FIG. 34D is a bar graph depicting concentration of the IgGl antibody with various types of samples with AAV1 vectors.

[0051] FIGS. 35A-35D are line graphs depicting correction of the calibration curves of FIGS. 34A and 34C, according to aspects of the present disclosure. FIG. 35A depicts the original calibration curve of FIG. 34A, and FIG. 35B depicts the corrected calibration curve. FIG. 35C depicts the original calibration curve of FIG. 34C, and FIG. 35D depicts the corrected calibration curve.

[0052] FIGS. 36A-36C are bar graphs depicting the results of an exemplary method for identifying and quantifying anti-drug antibodies based on isotype in samples with AAV vectors, according to aspects of the present disclosure. FIG. 36A depicts IgGl antibodies (top), IgG4 antibodies (middle) and IgM antibodies (bottom) in samples with AAV8 vectors. FIG. 36A depicts IgGl antibodies (top) and IgM antibodies (bottom) in samples with AAV2 vectors. FIG. 36A depicts IgGl antibodies (top), IgG4 antibodies (middle) and IgM antibodies (bottom) in samples with AAV1 vectors.

[0053] Again, there are many embodiments described and illustrated herein. The present disclosure is neither limited to any single aspect nor embodiment thereof, nor to any combinations and/or permutations of such aspects and/or embodiments. Each of the aspects of the present disclosure, and/or embodiments thereof, may be employed alone or in combination with one or more of the other aspects of the present disclosure and/or embodiments thereof. For the sake of brevity, many of those combinations and permutations are not discussed separately herein.

[0054] Notably, for simplicity and clarity of illustration, certain aspects of the figures depict the general structure and/or manner of construction of the various embodiments. Descriptions and details of well-known features and techniques may be omitted to avoid unnecessarily obscuring other features. Elements in the figures are not necessarily drawn to scale; the dimensions of some features may be exaggerated relative to other elements to improve understanding of the example embodiments.

DETAILED DESCRIPTION

[0055] Reference will now be made in detail to examples of the present disclosure, which are illustrated in the accompanying drawings. Wherever possible, the same reference numbers will be used throughout the drawings to refer to the same or like parts. In the discussion that follows, relative terms such as “about,” “substantially,” “approximately,” etc. are used to indicate a possible variation of ±10% in a stated numeric value.

[0056] Immunogenicity refers to the propensity of a therapeutic drug product to induce an immune response to the drug product and/or related proteins, such as inducing an immunologically-related adverse clinical event. Increasing concerns about drug efficacy and patient safety due to immunogenicity incidents of biopharmaceutical drug products have led to an increasing demand to characterize anti-drug antibodies (AD As). The demands of characterizing AD As are driven by, for example, the impact of AD As on drug efficacy, cross-reaction to endogenous proteins, and alteration of pharmacokinetics of pharmaceutical products. Characterization data of AD As can enhance safety of drug administration by providing valuable information regarding immunogenicity. [0057] One example of a biopharmaceutical drug product is adeno-associated viral (AAV) vectors. AAV vectors have been widely used to deliver genetic material, such as delivering nucleic acid molecules for gene therapy. Advantageously, AAV vectors are non-pathogenic and have low immunogenicity. AAV vectors are nonpathogenic members of the Dependovirus genus of the Parvoviridae family. AAV vectors require helpers, such as adenovirus or herpesvirus, to perform infection. See, e.g., Venkatakrishnan et al., “Structure and Dynamics of Adeno-Associated Virus Serotype 1 VP1- Unique N-Terminal Domain and Its Role in Capsid Trafficking”, Journal of Virology, 2013, Volume 87, Issue 9, Pages 4974-4984, the entire contents of which are hereby incorporated by reference. An AAV vector encapsulates a single-stranded DNA genome of about 4.8 kilobases (kb) in an icosahedral capsid, which is made of a shell of capsid viral proteins. Recombinant AAV genomes are nonpathogenic and do not integrate into the genome of a host cell, but instead, exist as stable episomes providing long-term expression. AAV vectors comprise serotypes, which make them very useful for preferentially transducing specific cell types. Due to these properties of AAV vectors, AAV-based therapy has the advantages of being non-pathogenic, being non-toxic, having cell type-specific infection, and offering different serotypes with varying cell transduction efficiencies. However, one disadvantage of AAV-based therapy is that producing, purifying, and characterizing AAV-based therapeutics is more complex than antibody-based therapeutics. For example, fully packaged AAV vectors consist of an icosahedral capsid contain about a single-stranded, about 4.8 kb genome. An empty capsid has a molecular weight of about 3750 kDa. The purity of AAV vectors is defined by several product-related impurities, including empty capsids, capsids containing partial or incorrect genomes, aggregated capsids, degraded capsids, and residual host cell proteins (HCPs).

[0058] Drug product purity is one of a number of factors which influence the formation of AD As. Other factors include, but are not limited to, product- or process-related issues such as molecular structure, and post-translational modifications. AD As may cause a decrease in drug concentration in a patient’s body, which may contribute to reduced drug efficacy and may pose a problem for patient safety.

[0059] AD As are capable of binding to different sites of a drug product, yielding different effects. Neutralizing AD As are capable of binding to an active site of the drug product, such as to a variable region of an antibody drug product, rendering inactive the drug product. Non-neutralizing AD As are capable of binding to a non-active site of the drug product, such as a constant region or a scaffold of an antibody drug product. Non-neutralizing AD As allow a drug product to maintain its activity, but they may contribute to changes in clinical pharmacology of the drug product.

[0060] Relevant immunogenicity information includes the induction of binding antibodies, the induction of neutralizing antibodies, altered pharmacokinetics, reduced efficacy, and other safety concerns. However, the clinical significance of AD As is unknown prior to extensive research. In addition, the limited available data may preclude a determination of the effect of AD As. AD As may associate with a concordance between an increase in systemic clearance of pharmaceutical products and a reduction of efficacy. Some drug products may produce drug-sustaining AD As which result in a reduced clearance, possibly due to the formation of an ADA-drug complex. See, e.g., Wang et al., “Evaluating and Reporting the Immunogenicity Impacts for Biological Products-a Clinical Pharmacology Perspective”, The AAPS Journal, 2016, Volume 18, Number 2, Pages 395 403, the entire contents of which are hereby incorporated by reference. [0061] AD As are immunoglobulins (antibodies), which are heterodimeric proteins composed of two heavy chains and two light chains. Immunoglobulins have variable domains that bind antigens, and constant domains that specify effector functions. Immunoglobulins can be categorized based on heavy chain constant domain into five main classes (isotypes): IgM, IgG, IgA, IgD, and IgE. Immunoglobulins can be further categorized into subclasses of each of the five main classes. For example, IgG immunoglobulins can be categorized into four subclasses: IgGl, IgG2, IgG3, and IgG4. As another example, IgA immunoglobulins can be categorized into two subclasses: IgAl and IgA2. Different isotypes are associated with different immune responses. For example, an ADA having an IgM isotype may be generated by a first drug exposure at 7 days with a concentration of 1.5 mg/mL in serum. The function of an IgM antibody includes primary response and fixed complements. The monomer of IgM can serve as a B cell receptor. By contrast, an ADA having an IgG isotype may be generated by a second drug exposure at 25-35 days with a concentration of 0.5- 9 mg/mL in serum. The function of the IgG antibody includes providing main blood antibody, neutralizing toxin, and opsonization. AD As having an IgA isotype can have a concentration of 0.5- 3 mg/mL in serum. AD As having an IgA isotype can be secreted into mucus, tears, and saliva. AD As having an IgE isotype have a concentration of 0.05 mg/mL in serum. AD As having an IgE isotype provide allergy and anti-parasitic activities. ADAs having an IgD isotype can serve as a B cell receptor. See, e.g., Schroeder et al., “Structure and function of immunoglobulins”, Journal of Allergy and Clinical Immunology, 2010, Volume 125, Issue 202, Supplement 2, Pages S41-S52, the entire contents of which are hereby incorporated by reference.

[0062] Based on these varied effects, the U.S. Food and Drug Administration recommends characterizing ADAs to understand potential patient immune responses, particularly for understanding the incidence of induction of ADAs and the implications of ADA-facilitated immune responses on biopharmaceutical drug product safety and efficacy. Useful characterization assays include isotyping, epitope mapping, and assessing cross-reactivity to discriminate between antibody isotypes. See, e.g., U.S. Food and Drug Administration, “Assay Development and Validation for Immunogenicity Testing of Therapeutic Protein Products: Draft Guidance”, U.S. Department of Health and Human Services, April 2016, Revision 1, Pages 1-31, the entire contents of which are hereby incorporated by reference.

[0063] Standard methods for detecting ADAs include ligand binding assays, including, but not limited to, bridging assays, radioligand binding assays, and surface plasmon resonance (SPR) assays. In a bridging assay, the ADA forms a bridge between two differently-labeling drugs and is captured on a plate, resulting in fluorescence. In bead-based radioligand binding assays, the ADA binds a drug linked to a bead and to a labeled drug, resulting in fluorescence. Both bridging assays and bead-based assays exhibit high sensitivity, good specificity, and high throughput. However, these assays are not capable of characterizing isotype, and both assays have the disadvantages of interference from other drugs or serum components, which may cause false positive or false negative results. SPR assays utilize changes in light refraction to measure ADA-drug interactions. SPR assays can detect low affinity antibodies for early immune responses, characterize isotype, and perform quantification. However, SPR assays exhibit low sensitivity, low throughput, and are expensive to perform.

[0064] The present disclosure satisfies the aforementioned demands by providing methods to identify, quantify, and characterize anti-drug antibodies in samples, such as serum samples, induced by the administration of therapeutic AAV vectors. The methods may include using techniques such as immunocapture and liquid chromatography-mass spectrometry (LC-MS). These methods can be applied in preclinical toxicology or pharmacokinetic studies to monitor and characterize AD As over time after the administration of a drug product.

[0065] Unless described 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. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing, particular methods and materials are now described.

[0066] The term “a” should be understood to mean “at least one” and the terms “about” and “approximately” should be understood to permit standard variation as would be understood by those of ordinary skill in the art, and where ranges are provided, endpoints are included. As used herein, the terms “include,” “includes,” and “including” are meant to be non-limiting and are understood to mean “comprise,” “comprises,” and “comprising” respectively.

[0067] As used herein, the terms “vector” or “viral vector” refer to a recombinant plasmid or virus that comprises a nucleic acid to be delivered into a host cell, either in vitro or in vivo. Vectors derived from AAV are particularly attractive for delivering genetic material because (i) they are able to infect (transduce) a wide variety of non-dividing and dividing cell types including muscle fibers and neurons; (ii) they are devoid of the virus structural genes, thereby eliminating the natural host cell responses to virus infection, for example, interferon-mediated responses; (iii) wild type AAVs have never been associated with any pathology in humans; (iv) in contrast to wild type AAVs, which are capable of integrating into the host cell genome, replication-deficient AAV vectors generally persist as episomes, thus limiting the risk of insertional mutagenesis or activation of oncogenes; and (v) in contrast to other vector systems, AAV vectors do not trigger a significant immune response (see ii), thus granting long-term expression of the therapeutic transgenes (provided their gene products are not rejected). A “recombinant viral vector” refers to a recombinant polynucleotide vector including one or more heterologous sequences (e.g., nucleic acid sequence not of viral origin). A “recombinant AAV vector (rAAV vector)” refers to a polynucleotide vector including one or more heterologous sequences (e.g., nucleic acid sequence not of AAV origin) that may be flanked by at least one, for example, two, AAV inverted terminal repeat sequences (ITRs). Such rAAV vectors can be replicated and packaged into infectious viral particles when present in a host cell that has been infected with a suitable helper virus (or that is expressing suitable helper functions) and that is expressing AAV rep and cap gene products (e.g., AAV Rep and Cap proteins). [0068] As used herein, the term “capsid” refers to the protein shell of a virus, which encloses the genetic material. Three viral capsid proteins, VP1, VP1 and VP3, form the viral icosahedral capsid of 60 subunits in a ratio of 1 : 1 : 10. A full capsid contains genetic material and is required to provide therapeutic benefit. An empty capsid lacks the genome and therefore lacks the ability to provide therapeutic benefit to the patient.

[0069] As used herein, the term “viral particle” refers to a particle composed of at least one viral capsid protein and an encapsulated viral genome. While AAV is described in this disclosure as a model virus or viral particle, it is contemplated that the disclosed methods can be applied to profile a variety of viruses, e.g., the viral families, subfamilies, and genera. In some aspects, the viral capsid, virus, or viral particle belongs to a viral family selected from the group consisting of Adenoviridae, Parvoviridae, Retroviridae, Baculoviridae, and Herpesviridae. In some aspects, the viral capsid, virus, or viral particle belongs to a viral genus selected from the group consisting of Atadenovirus, Aviadenovirus, Ichtadenovirus, Mastadenovirus, Siadenovirus, Ambidensovirus, Brevidensovirus, Hepandensovirus, Iteradensovirus, Penstyldensovirus, Amdoparvovirus, Aveparvovirus, Bocaparvovirus, Copiparvovirus, Dependoparvovirus, Erythroparvovirus, Protoparvovirus, Tetraparvovirus, Alpharetrovirus, Betaretrovirus, Deltaretrovirus, Epsilonretrovirus, Gammaretrovirus, Lentivirus, Spumavirus, Alphabaculovirus, Betabaculovirus, Deltabaculovirus, Gammabaculovirus, Iltovirus, Mardivirus, Simplexvirus, Varicellovirus, Cytomegalovirus, Muromegalovirus, Proboscivirus, Roseolovirus, Lymphocryptovirus, Macavirus, Percavirus, and Rhadinovirus.

[0070] As used herein, the terms “peptide”, “protein”, and “protein of interest” can include any amino acid polymer having covalently linked amide bonds. Proteins comprise one or more amino acid polymer chains, generally known in the art as “polypeptides.” A polypeptide may be composed of amino acid residues, related naturally occurring structural variants, and synthetic non-naturally occurring analogs thereof, linked via peptide bonds. The terms “synthetic peptide” and “synthetic polypeptide” refer to a non-naturally occurring peptide or polypeptide. Synthetic peptides or polypeptides can be synthesized, for example, using an automated polypeptide synthesizer. Various solid phase peptide synthesis methods are known to those of skill in the art. A protein may comprise one or multiple polypeptides to form a single functioning biomolecule. The twenty naturally- occurring amino acids and their single-letter 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 H His; Isoleucine I He; Lysine K Lys; Leucine L Leu; Methionine M Met; Asparagine N Asn; Proline P Pro; Glutamine Q Gin; Arginine R Arg; Serine S Ser; Threonine T Thr; Valine V Vai; Tryptophan w Trp; and Tyrosine Y Tyr.

[0071] As used herein, the term “antibody” includes an immunoglobulin molecule comprising four polypeptide chains, two heavy (H) chains and two light (L) chains, interconnected by disulfide bonds, as well as multimers thereof (e.g., IgM). Each heavy chain comprises a heavy chain variable region (HCVR or VH) and a heavy chain constant region. The heavy chain constant region comprises three domains, CHI, CH2, and CH3. Each light chain comprises a light chain variable region (LCVR or VL) and a light chain constant region. The light chain constant region comprises one domain (CL1). The VH and VL regions can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDRs), interspersed with regions that are more conserved, termed framework regions (FR). Each VH and VL is composed of three CDRs and four FRs, arranged from amino-terminus to carboxy -terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. In different embodiments of the present invention, the FRs of the anti-big-ET-1 antibody (or antigen-binding portion thereof) may be identical to the human germline sequences or may be naturally or artificially modified. An amino acid consensus sequence may be defined based on a side-by-side analysis of two or more CDRs.

[0072] The term “antibody” also includes antigen-binding fragments of full antibody molecules. The terms “antigen-binding portion” and “antigen-binding fragment” include any naturally occurring, enzymatically obtainable, synthetic or genetically engineered polypeptide or glycoprotein that specifically binds an antigen to form a complex. Antigen-binding fragments of an antibody may be derived, for example, from full antibody molecules using any suitable standard techniques such as proteolytic digestion or recombinant genetic engineering techniques involving the manipulation and expression of DNA molecules encoding antibody variable domains, and optionally encoding antibody constant domains. Sequences of such DNA molecules are known, and/or are readily available from, for example, commercial sources, DNA libraries (including, e.g., phage-antibody libraries), or can be synthesized. The DNA molecules may be sequenced and manipulated chemically or by using molecular biology techniques, for example, to arrange one or more variable and/or constant domains into a suitable configuration, or to introduce codons, create cysteine residues, modify, add or delete amino acids, etc.

[0073] As used herein, the term “antibody fragment” includes a portion of an intact antibody, such as, for example, the antigen-binding portion of antibody or a variable region of an antibody.

Examples of antibody fragments include, but are not limited to, a Fab fragment, a Fab’ fragment, a F(ab’)2 fragment, a scFv fragment, a Fv fragment, a dsFv diabody, a dAb fragment, a Fd’ fragment, a Fd fragment, and an isolated complementarity determining region (CDR) region, as well as triabodies, tetrabodies, linear antibodies, single-chain antibody molecules, and multi specific antibodies formed from antibody fragments. Fv fragments are the combination of the variable regions of the immunoglobulin heavy and light chains, and ScFv proteins are recombinant single chain polypeptide molecules in which immunoglobulin light and heavy chain variable regions are connected by a peptide linker. In some embodiments, an antibody fragment comprises a sufficient amino acid sequence of the parent antibody of which it is a fragment, such that the fragment binds to the same antigen as the parent antibody. In some embodiments, a fragment binds to the antigen with a comparable affinity to that of the parent antibody and/or competes with the parent antibody for binding to the antigen. An antibody fragment may be produced by any means known to one of skill in the art. For example, an antibody fragment may be enzymatically or chemically produced by fragmentation of an intact antibody and/or it may be recombinantly produced from a gene encoding the partial antibody sequence. Alternatively, or additionally, an antibody fragment may be wholly or partially synthetically produced. An antibody fragment may optionally comprise a single chain antibody fragment. Alternatively, or additionally, an antibody fragment may comprise multiple chains that are linked together, for example, by disulfide linkages. An antibody fragment may optionally comprise a multi-molecular complex. A functional antibody fragment typically comprises at least about 50 amino acids and more typically comprises at least about 200 amino acids.

[0074] As used herein, the term “bispecific antibody” or “bsAb” refers to an antibody capable of selectively binding two antigens or two epitopes of an antigen. Bispecific antibodies generally comprise two different heavy chains with each heavy chain specifically binding a different epitope — either on two different molecules (e.g., antigens) or on the same molecule (e.g., on the same antigen). If a bispecific antibody is capable of selectively binding two different epitopes (a first epitope and a second epitope), the affinity of the first heavy chain for the first epitope may be one to four orders of magnitude lower than the affinity of the first heavy chain for the second epitope, and vice versa. The epitopes recognized by the bispecific antibody can be on the same or a different target (e g., on the same or a different protein or antigen). Bispecific antibodies can be made, for example, by combining heavy chains that recognize different epitopes of the same antigen. For example, nucleic acid sequences encoding heavy chain variable sequences that recognize different epitopes of the same antigen can be fused to nucleic acid sequences encoding different heavy chain constant regions, and such sequences can be expressed in a cell that expresses an immunoglobulin light chain.

[0075] A typical bispecific antibody has two heavy chains each having three heavy chain CDRs, followed by a CHI domain, a hinge, a CH2 domain, and a CH3 domain, and an immunoglobulin light chain that either does not confer antigen-binding specificity but that can associate with each heavy chain, or that can associate with each heavy chain and that can bind one or more of the epitopes bound by the heavy chain antigen-binding regions, or that can associate with each heavy chain and enable binding of one or both of the heavy chains to one or both epitopes. Bispecific antibodies can be divided into two major classes, those bearing an Fc region (IgG-like) and those lacking an Fc region, the latter normally being smaller than the IgG and IgG-like bispecific molecules comprising an Fc. The IgG-like bsAbs can have different formats such as, but not limited to, triomab, knobs into holes IgG (kih IgG), crossMab, orth-Fab IgG, Dual-variable domains Ig (DVD-Ig), two-in-one or dual action Fab (DAF), IgG-single-chain Fv (IgG-scFv), or Kk-bodies. The non-IgG-like different formats include tandem scFvs, diabody format, single-chain diabody, tandem diabodies (TandAbs), dual-affinity retargeting molecule (DART), DART-Fc, nanobodies, or antibodies produced by the dock-and-lock (DNL) method. See, e.g., Fan et al., “Bispecific antibodies and their applications”, Journal of Hematology & Oncology, Volume 8, Issue 130, Pages 1-14; and see Muller et al., “Chapter 11 : Bispecific Antibodies”, Handbook of Therapeutic Antibodies, 2014, Pages 265-310, each of which is hereby incorporated by reference in its entirety. Methods of producing bispecific antibodies include, but are not limited to, quadroma technology based on the somatic fusion of two different hybridoma cell lines, chemical conjugation using chemical cross-linkers, and genetic approaches using recombinant DNA technology.

[0076] As used herein, the term “multispecific antibody” refers to an antibody capable of selectively binding two or more antigens or two or more epitopes of an antigen. A bispecific antibody is an example of a multispecific antibody. Multispecific antibodies also encompass antibodies with additional binding capabilities, such as trispecific antibodies and KIH trispecific antibodies. [0077] As used herein, the term “monoclonal antibody” refers to an antibody produced through hybridoma technology. A monoclonal antibody can be derived from a single clone, including any eukaryotic, prokaryotic, or phage clone, by any means available or known in the art. Monoclonal antibodies can be prepared using a wide variety of techniques known in the art including the use of hybridoma, recombinant, and phage display technologies, or a combination thereof.

[0078] As used herein, the terms “anti-drug antibody” or “ADA” refer to an antibody produced by a subject’s immune system, and which targets an epitope of a drug. The term “drug” includes monomeric proteins, multimeric proteins, small molecules, viral vectors, or any other such chemical entity produced for therapeutic or diagnostic purposes. Anti-drug antibodies may form during therapy as part of an immunogenic reaction of the subject. In the case of viral vector therapies, such as AAV vector-based therapy, a subject may have ADAs due to prior exposure to a related virus. It should be understood that an ADA is not limited to an antibody produced as a response to a viral vector therapy, but includes antibodies that may occur in response to wild-type virus of the same or similar virus type or serotype as a viral vector of interest. For example, an antibody that may have been produced in response to a wild-type AAV8 infection would be considered an ADA in the context of an AAV8 viral vector, or any viral vector that could be bound by the antibody.

[0079] As used herein, the terms “neutralizing antibody” or “Nab” refers to a type of anti-drug antibody which binds to a drug in a manner which inhibits or neutralizes the pharmacological activity of the drug. For example, a neutralizing antibody may bind to a viral vector and inhibit or prevent transduction performed by the viral vector. Neutralizing antibodies may affect clinical efficacy of a drug, and as such, must be monitored when administering the drug to a subject.

[0080] As used herein, the terms “immunoassay” or “ADA immunoassays” include immunoassays known to the skilled artisan. Method for carrying out immunoassays, as well as practical applications and procedures therefor, are well-known in the art. See, e.g., Colowick et al. (eds.), METHODS IN ENZYMOLOGY, Academic Press, Volumes 70, 73, 74, 84, 92, and 121. Principles of various immunoassays are also well-known in the art. See, e.g., Hage, “Immunoassays”, Analytical Chemistry, 1999, Volume 71, Issue 12, Pages 294R-304R, the entire contents of which are hereby incorporated by reference. Oriented immobilization of antibodies for use in immunoassays is described in, e.g., Lu et al., “Tutorial review. Oriented immobilization of antibodies and its applications in immunoassays and immunosensors”, Analyst, 1996, Volume 121, Issue 3, Pages 29R-32R. Avidin-biotin-mediated immunoassays are described, e.g., Wilchek et al., “[54]: Avidinbiotin mediated immunoassays: Overview”, Methods in Enzymology, 1990, Volume 184, Pages 467-469. [0081] One commonly used ADA immunoassay is a bridging immunoassay. See, e.g., Liao et al., “Inhibition of interleukin-5 induced false positive anti-drug antibody responses against mepolizumab through the use of a competitive booking antibody”, Journal of Immunological Methods, 2017, Volume 441, Pages 15-23; Dai et al., “Development of a method that eliminates false-positive results due to nerve growth factor interference in the assessment of fulranumab immunogenicity”, The AAPS Journal, 2014, Volume 16, Issue 3, Pages 464-477; and Zhong et al., “Drug target interference in immunogenicity assays: recommendations and mitigation strategies”, The AAPS Journal, 2017, Volume 19, Issue 6, Pages 1564-1575, the entire contents of each being incorporated herein by reference. An ADA bridging immunoassay is a sandwich-type immunoassay in which a multi-valent ADA is bound by a capture reagent and a detection reagent, optionally wherein one or both of the reagents are the drug of interest, each binding to a different, not overlapping or interfering epitope of the ADA. The capture reagent and/or the detection reagent may be a drug or therapeutic protein or vector of interest, or may be an antibody targeted against the ADA. In particular, in this assay, a sample is incubated with a capture reagent and a detection reagent, comprising a detectable label. After sample incubation, a sandwich comprising the capture reagent, the ADA, and the detection reagent is formed and, thus, the ADA bridges two reagents binding to it and the bound ADA can be detected. The ADA bridging immunoassay is a high- throughput assay. The ADA bridging immunoassay further determines a presence and/or an amount of an ADA. Thus, the present disclosure provides a detection reagent, for example an antibody, an AAV vector or AAV capsid, conjugated to a detectable label.

[0082] Non-limiting examples of detectable labels for any of the methods of the disclosure include ruthenium, a radiologic label, a photoluminescent label, a chemiluminescent label, a fluorescent label, a fluorophore, a hapten, an electrochemiluminescent label, or an enzyme label. The detectable label can be measured using instruments and devices known to those skilled in the art.

[0083] This disclosure generally describes assays wherein a signal is generated by binding of an ADA to a drug, for example a viral vector. Assays wherein a signal is inhibited or quenched by binding of an ADA to a drug are also contemplated. For simplicity, this disclosure discusses assays wherein a signal results from binding of an ADA to a drug or other reagent, although the methods and compositions described herein may equally be applied to assays wherein a signal is inhibited or quenched by binding of an ADA to a drug or other reagent.

[0084] As used herein, the term “database” refers to a compiled collection of protein sequences that may possibly exist in a sample, for example in the form of a file in a FASTA format. Relevant protein sequences may be derived from cDNA sequences of a species being studied. Public databases that may be used to search for relevant protein sequences included databases hosted by, for example, Uniprot or Swiss-prot. Databases may be searched using what are herein referred to as “bioinformatics tools”. Bioinformatics tools provide the capacity to search uninterpreted MS/MS spectra against all possible sequences in the database(s), and provide interpreted (annotated) MS/MS spectra as an output. Non-limiting examples of such tools are Mascot (matrixscience.com), Spectrum Mill (chem.agilent.com), PLGS (waters.com), PEAKS (bioinformaticssolutions.com), Proteinpilot (download.appliedbiosystems.com/proteinpilot), Phenyx (phenyx-ms.com), Sorcerer (sagenresearch.com), OMSSA (pubchem.ncbi.nlm.nih.gov/omssa/), XITandem (thegpm.org/TANDEM/), Protein Prospector (prospector.ucsf.edu/prospector/mshome.htm), Byonic (proteinmetrics.com/products/byonic) or Sequest (fields.scripps.edu/sequest).

[0085] It is understood that the present invention is not limited to any of the aforesaid protein(s), antibody(s), sample(s), AAV(s), virus(es), serotype(s), vector(s), protein denaturing agent(s), digestive enzyme(s), chromatographic method(s), mass spectrometer(s), database(s), bioinformatics tool(s), pH range(s) or value(s), temperature(s), or concentration(s), and any protein(s), antibody(s), sample(s), AAV(s), virus(es), serotype(s), vector(s), protein alkylating agent(s), protein denaturing agent(s), digestive enzyme(s), chromatographic method(s), mass spectrometer(s), database(s), bioinformatics tool(s), pH, temperature(s), or concentration(s) can be selected by any suitable means.

Methods

[0086] Provided herein are methods for identifying the presence of a peptide or protein in a sample. The peptide or protein may comprise a polymer of amino acids and/or amino acid analogs joined by peptide bonds or peptide bond mimetics. In some embodiments, the peptide or protein may be or comprise an anti-drug antibody (ADA) or a fragment thereof. The ADA may be or comprise a human antibody.

[0087] In some embodiments, the method may comprise contacting the sample including the peptide or protein to a binding partner to form a complex.

[0088] The binding partner may be a viral vector, a peptide, or a protein. For example, the binding partner may be an AAV vector. The AAV vector may comprise a serotype, a variation of a serotype, or a combination of serotypes. The serotype may be AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV 10, AAV 11 , AAV 12, or a variation thereof, or a combination thereof. In some embodiments, the binding partner may be a drug, such as a therapeutic peptide, protein, or viral vector. In some embodiments, the drug may be a therapeutic AAV vector. [0089] The sample may be or comprise a biological sample, such as a serum sample. A biological sample may be a sample taken from a living organism, such as a human or anon-human mammal. The biological sample may comprise or consist of whole blood, plasma, serum, saliva, tears, semen, cheek tissue, organ tissue, urine, feces, skin, or hair. The biological sample may be taken from a patient, for example, a clinical sample. In some embodiments, a sample may be taken from a nonhuman animal, for example, a preclinical sample. In some embodiments, a sample may be further processed form of any of the aforementioned examples of samples.

[0090] The sample may comprise a mixture of molecules including a viral particle or vector, such as an AAV particle, or an empty viral capsid, that is subjected to manipulation in accordance with the methods of the invention, including, for example, separating, analyzing, extracting, concentrating, profiling and the like.

[0091] The sample may comprise an antibody against a drug of interest, for example a viral vector, an AAV vector, or specifically the capsid thereof. The antibody may be an antibody produced by the immune system of a patient or non-human animal, or it may be a recombinant antibody.

[0092] The method may further comprise contacting the complex to a solid support conjugated to an affinity ligand capable of binding the complex to form an immobilized complex.

[0093] The affinity ligand may be capable of binding to the binding partner in the complex, or the affinity ligand may be capable of binding to the peptide or protein of the complex. In some embodiments, the affinity ligand may be an antibody or antibody fragment capable of binding to the binding partner. In some embodiments, the binding partner may be an AAV vector, and the affinity ligand may be an antibody capable of binding multiple serotypes of AAV vectors, such as an anti- AAVX antibody. In some embodiments, the affinity ligand may be conjugated to a biotin. In some embodiments, the affinity ligand may be an anti-AAVX antibody conjugated to biotin, such as CAPTURESELECT™ Biotin Anti-AAVX. In some embodiments, the solid support may be or comprise streptavidin. In some embodiments, the amount of affinity ligand used may be about 5 pg to about 15 pg, about 5 pg, about 6 pg, about 7 pg, about 8 pg, about 9 pg, about 10 pg, about 11 pg, about 12 pg, about 13 pg, about 14 pg, or about 15 pg. In some embodiments, the amount of affinity ligand used is about 10 pg.

[0094] The solid support may be or comprise any surface with an ability to bind a protein or peptide. Non-limiting examples of solid supports can include affinity resins, beads and coated plates or microplates. The solid support may be attached to a molecule capable of binding to a protein or peptide, including an affinity ligand, an affinity reagent, an antigen-binding molecule, and an interacting peptide ligand. [0095] By way of example only, formation of an exemplary complex is illustrated in FIG. 1, including a binding partner 10, a peptide or protein 20, an affinity ligand 30, and a solid support 40, joining together to form a complex. As shown, binding partner 10 is bound to peptide or protein 20 and to affinity ligand 30. Affinity ligand 30 is further bound to solid support 40. Other binding arrangements are also contemplated as part of this disclosure.

[0096] The method may further comprise washing the immobilized complex to remove unbound material. For example, the washing may comprise contacting one or more washing solutions to the immobilized complex. Each washing solution of the one or more washing solutions may be contacted to the immobilized complex one or more times, such as one time, two times, three times, four times, or more. Washing solution may comprise bovine serum albumin (BSA) and a buffer. In some embodiments, the buffer may comprise or be HBS-T (0.01M HEPES, 0.15MNaCl, 0.1% T20, pH 7.4). In some embodiments, the washing solution may comprise about 1% to about 5%, about 1%, about 2%, about 3%, about 4%, about 5%, 1%, 2%, 3%, 4%, or 5% BSA in HBS-T. In some embodiments, the washing may comprise contacting the immobilized complex with a washing solution comprising 3% BSA in HBS-T. In some embodiments, washing may comprise contacting the immobilized complex with a first washing solution including 3% BSA in HBS-T, and then contacting the immobilized complex with a second washing solution including HBS-T without BSA. In some embodiments, washing may comprise contacting the immobilized complex with a first washing solution and then contacting the immobilized complex with a second washing solution one or more times. In some embodiments, washing may comprise contacting the immobilized complex with a first washing solution including 3% BSA in HBS-T, contacting the immobilized complex with a second washing solution including HBS-T without BSA a first time, and contacting the immobilized complex with the second washing solution a second time.

[0097] The method may further comprise eluting the peptide or protein from the immobilized complex to produce an enriched peptide or protein sample. For example, eluting may comprise contacting the immobilized complex to an elution buffer. Elution buffer may comprise acetonitrile (ACN), formic acid (FA), or a combination thereof. In some embodiments, the elution buffer may comprise about 5% to about 30% CAN, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 11%, about 12%, about 13%, about 14%, about 15%, about 16%, about 17%, about 18%, about 19%, about 20%, about 25%, about 30%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 25%, or 30% ACN. In some embodiments, elution buffer may comprise about 0.1% to about 2.0%, about 0.1%, about 0.2%, about 0.3%, about 0.4%, about 0.5%, about 0.6%, about 0.7%, about 0.8%, about 0.9%, about 1.0%, about 1.1%, about 1.2%, about 1.3%, about 1.4%, about 1.5%, about 1.6%, about 1.7%, about 1.8%, about 1.9%, about 2.0%, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1.0%, 1.1%, 1.2%, 1.3%, 1.4%, 1.5%, 1.6%, 1.7%, 1.8%, 1.9%, or 2.0% FA. In some embodiments, eluting may comprise contacting the immobilized complex with an elution buffer comprising about 20% ACN and about 1% FA. In some embodiments, eluting may comprise contacting the immobilized complex with an elution buffer comprising 20% ACN and 1% FA.

[0098] The method may further comprise subjecting the enriched peptide or protein to liquid chromatography-mass spectrometry (LC-MS) analysis to characterize the peptide or protein.

[0099] The peptide or protein may comprise an antibody or a fragment thereof, and characterizing the peptide or protein may comprise determining an isotype or subclass of the antibody. The antibody may be a human antibody, having any of the following isotypes described herein, such as IgGl, IgG2, IgG3, IgG4, IgAl, IgA2, IgM, or IgE. The antibody may be a monkey antibody, having any of the following isotypes described herein, such as IgGl, IgG2, IgG3, IgG4, IgM, or IgA.

[0100] Liquid chromatography (LC) may comprise a process in which a biological and/or chemical mixture carried by a liquid is separated into components as a result of differential distribution of the components as they flow through (or into) a stationary liquid or solid phase. Non-limiting examples of liquid chromatography may include reverse phase liquid chromatography, ion-exchange chromatography, size exclusion chromatography, affinity chromatography, hydrophobic interaction chromatography, hydrophilic interaction chromatography, or mixed-mode chromatography. In some aspects, the sample, eluate, or enriched peptide or protein sample may be subjected to any one of the aforementioned chromatographic methods or a combination thereof.

[0101] Mass spectrometry (MS) may comprise using a device (mass spectrometer) capable of identifying specific molecular species and measuring their accurate masses, including any molecular detector into which a polypeptide or peptide may be characterized. The mass spectrometer may include three major parts: an ion source, a mass analyzer, and a detector. The role of the ion source is to create gas phase ions. Analyte atoms, molecules, or clusters can be transferred into gas phase and ionized either concurrently (as in electrospray ionization) or through separate processes. The choice of ion source depends on the application.

[0102] The mass spectrometer may be coupled to a liquid chromatography-multiple reaction monitoring system. Multiple reaction monitoring (MRM) comprises a mass spectrometry-based technique that can precisely quantify small molecules, peptides, and proteins within complex matrices with high sensitivity, specificity and a wide dynamic range. See, e.g., Picotti et al., “Selected reaction monitoring-based proteomics: workflows, potential, pitfalls and future directions”, Nature Methods, 2012, Volume 9, Pages 555-566, the entire contents of which are hereby incorporated by reference. MRM may be performed with a triple quadrupole mass spectrometer, wherein a precursor ion corresponding to the selected small molecules/peptides is selected in the first quadrupole and a fragment ion of the precursor ion was selected for monitoring in the third quadrupole. See, e.g., Choi et al., “Targeted human cerebrospinal fluid proteomics for the validation of multiple Alzheimer’s disease biomarker candidates”, Journal of Chromatography B, 2013, Volume 930, Pages 129-135.

[0103] In some embodiments, the mass spectrometer may be capable of analysis by selected reaction monitoring (SRM), including consecutive reaction monitoring (CRM) and parallel reaction monitoring (PRM).

[0104] SRM/MRM/Selected-ion monitoring (SIM) is a method that may be used in tandem mass spectrometry, in which an ion of a particular mass is selected in the first stage of a tandem mass spectrometer and an ion product of a fragmentation reaction of the precursor ion is selected in the second mass spectrometer stage for detection. Examples of triple quadrupole mass spectrometers (TQMS) that can perform MRM/SRM/SIM include, but are not limited to, QTRAP® 6500 System (Sciex), QTRAP® 5500 System (Sciex), Triple QTriple Quad 6500 System (Sciex), Agilent 6400 Series Triple Quadrupole LC/MS systems, and Thermo Scientific™ TSQ™ Triple Quadrupole system.

[0105] In addition to MRM, the choice of peptides may also be quantified through Parallel-Reaction Monitoring (PRM). PRM is the application of SRM with parallel detection of all transitions in a single analysis using a high-resolution mass spectrometer. PRM provides high selectivity, high sensitivity and high-throughput to quantify selected peptides (QI), and hence quantify proteins. Multiple peptides can be specifically selected for each protein. PRM methodology can use the quadrupole of a mass spectrometer to isolate a target precursor ion, fragment the targeted precursor ion in the collision cell, and then detect the resulting product ions in the Orbitrap mass analyzer. PRM may be performed with a quadrupole time-of-flight (QTOF) or a hybrid quadrupole-orbitrap (QOrbitrap) mass spectrometer to carry out the identification of peptides and/or proteins. Examples of QTOF include but are not limited to TRIPLETOF® 6600 System (Sciex), TRIPLETOF® 5600 System (Sciex), X500R QTOF System (Sciex), 6500 Series Accurate-Mass Quadrupole Time-of- Flight (Q-TOF) (Agilent) and Xevo G2-XS QT of Quadrupole Time-of-Flight Mass Spectrometry (Waters). Examples of QObitrap include but are not limited to Q EXACTIVE™ Hybrid Quadrupole-Orbitrap Mass Spectrometer (Thermo Scientific) and ORBITRAP FUSION™ TRIBRID™ (Thermo Scientific). Advantages of PRM include, but are not limited to, elimination of most interferences; providing more accuracy and attomole-level limits of detection and quantification; enabling the confident confirmation of the peptide identity with spectral library matching; reducing assay development time since no target transitions need to be preselected; and ensuring UHPLC-compatible data acquisition speeds with spectrum multiplexing and advanced signal processing.

[0106] In some embodiments, the mass spectrometer may be or comprise an electrospray ionization mass spectrometer, a nano-electrospray ionization mass spectrometer, or a triple quadrupole mass spectrometer. In some embodiments, the mass spectrometer may be coupled to a liquid chromatography system. In some embodiments, the mass spectrometer is capable of performing liquid chromatography-mass spectrometry (LC-MS) analysis or liquid chromatography-parallel reaction monitoring-mass spectrometry (LC-PRM-MS) analysis. In some exemplary embodiments, the identification of peptides is performed using PRM-MS.

[0107] In some embodiments, the mass spectrometer may be or comprise a tandem mass spectrometer. Tandem mass spectrometry includes a technique where structural information on sample molecules is obtained by using multiple stages of mass selection and mass separation. A prerequisite is that the sample molecules be transformed into a gas phase and ionized so that fragments are formed in a predictable and controllable fashion after the first mass selection step. MS/MS, or MS², can be performed by first selecting and isolating a precursor ion (MS¹), and fragmenting it to obtain meaningful information. Tandem MS has been successfully performed with a wide variety of analyzer combinations. Which analyzers to combine for a certain application can be determined by many different factors, such as sensitivity, selectivity, and speed, but also size, cost, and availability. Two major categories of tandem MS methods are tandem-in-space and tandem-in-time, but there are also hybrids where tandem-in-time analyzers are coupled in space or with tandem-in-space analyzers. A tandem-in-space mass spectrometer comprises an ion source, a precursor ion activation device, and at least two non-trapping mass analyzers. Specific m/z separation functions can be designed so that in one section of the instrument ions are selected, dissociated in an intermediate region, and the product ions are then transmitted to another analyzer for m/z separation and data acquisition. In tandem-in-time, mass spectrometer ions produced in the ion source can be trapped, isolated, fragmented, and m/z separated in the same physical device. [0108] The peptide or protein identified by the mass spectrometer may be used as a surrogate representative of an intact protein and its post-translational modifications. The peptide or protein may be used for protein characterization by correlating experimental and theoretical MS/MS data, the latter generated from possible peptides in a protein sequence database. Characterization may include, but is not limited to, identifying the protein, sequencing amino acids of the protein fragments, determining protein sequencing, quantifying the protein, locating post-translational modifications, identifying post translational modifications, or comparability analysis, or combinations thereof.

[0109] In some embodiments, the mass spectrometer may use nanoelectrospray or nanospray ionization. The term “nanoelectrospray” or “nanospray” as used herein refers to electrospray ionization at a very low solvent flow rate, typically hundreds of nanoliters per minute of sample solution or lower, often without the use of an external solvent delivery. The electrospray infusion setup forming a nanoelectrospray can use a static nanoelectrospray emitter or a dynamic nanoelectrospray emitter. A static nanoelectrospray emitter performs a continuous analysis of small sample (analyte) solution volumes over an extended period of time. A dynamic nanoelectrospray emitter uses a capillary column and a solvent delivery system to perform chromatographic separations on mixtures prior to analysis by the mass spectrometer.

[0110] A triple quadruple mass spectrometer is a tandem mass spectrometer consisting of two quadrupole mass analyzers in series, with a (non-mass-resolving) radio frequency (RF), only quadrupole between them to act as a cell for collision-induced dissociation. In a triple quadrupole mass spectrometer, a peptide sample is injected onto an LC coupled with a MS instrument. The first quadrupole can be used as a mass filter to isolate peptides with a targeted m/z. The second quadrupole serves as a collision cell to break the peptide into fragments. The third quadrupole serves as a second mass filter for specified m/z fragments from the initial parent peptide. Tandem mass spectrometry may include a technique where structural information on sample molecules can be obtained by using multiple stages of mass selection and mass separation. A prerequisite is that the sample molecules can be transferred into gas phase and ionized intact and that they can be induced to fall apart in some predictable and controllable fashion after the first mass selection step. Multistage MS/MS, or MSn, can be performed by first selecting and isolating a precursor ion (MS²), fragmenting it, isolating a primary fragment ion (MS³), fragmenting it, isolating a secondary fragment (MS⁴), and so on as long as one can obtain meaningful information or the fragment ion signal can be detectable. Tandem MS have been successfully performed with a wide variety of analyzer combinations. What analyzers to combine for a certain application can be determined by many different factors, such as sensitivity, selectivity, and speed, but also size, cost, and availability. The two major categories of tandem MS methods are tandem-in-space and tandem -in-time, but there are also hybrids where tandem-in-time analyzers are coupled in space or with tandem-in-space analyzers. A tandem-in-space mass spectrometer comprises an ion source, a precursor ion activation device, and at least two non-trapping mass analyzers. Specific m/z separation functions can be designed so that in one section of the instrument ions are selected, dissociated in an intermediate region, and the product ions are then transmitted to another analyzer for m/z separation and data acquisition. In tandem-in-time mass spectrometer ions produced in the ion source can be trapped, isolated, fragmented, and m/z separated in the same physical device.

[oni] The method may further comprise, prior to LC-MS analysis, additional preparation steps. Such preparation steps may include reduction, denaturation, alkylation, dilution, digestion, and/or separation (for example, centrifugation).

[0112] For example, the methods described here may further comprise, prior to LC-MS analysis, subjecting the peptide or protein sample to denaturation conditions. Denaturation refers to a process in which the three-dimensional shape of a molecule is changed from its native state. Protein denaturation may be carried out using a protein denaturing agent (e.g., denaturant). In some embodiments, the protein denaturing agent may be or comprise heat, high pH, low pH, a reducing agent (e.g., DTT), or exposure to a chaotropic agent. Chaotropic agents increase the entropy of the system by interfering with intramolecular interactions mediated by non-covalent forces such as hydrogen bonds, van der Waals forces, and hydrophobic effects. In some embodiments, Nonlimiting examples of chaotropic agents include butanol, ethanol, guanidinium chloride, lithium perchlorate, lithium acetate, magnesium chloride, phenol, propanol, sodium dodecyl sulfate, thiourea, N-lauroylsarcosine, urea, and salts thereof.

[0113] As another example, the methods described herein may further comprise, prior to LC-MS analysis, subjecting the peptide or protein sample to reduction conditions. Subjecting the peptide or protein sample to reduction condition may comprise contacting the peptide or protein sample to a reducing agent. Non-limiting examples of a protein denaturing agent include heat, high or low pH, reducing agents like DTT, or exposure to chaotropic agents. Several chaotropic agents can be used as protein denaturing agents. Chaotropic solutes increase the entropy of the system by interfering with intramolecular interactions mediated by non-covalent forces such as hydrogen bonds, van der Waals forces, and hydrophobic effects. Non-limiting examples of chaotropic agents include butanol, ethanol, guanidinium chloride, lithium perchlorate, lithium acetate, magnesium chloride, phenol, propanol, sodium dodecyl sulfate, thiourea, N-lauroylsarcosine, urea, and salts thereof.

[0114] As another example, the methods described herein may further comprise, prior to LC-MS analysis, subjecting the peptide or protein sample to enzymatic digestion conditions to produce a peptide digest; and subjecting the peptide digest to liquid chromatography-mass spectrometry (LC- MS) analysis to characterize the peptide or protein. The peptide digest may comprise constituent peptides of the peptide or protein, which can be further analyzed using, for example, peptide mapping analysis. Enzymatic digestion may comprise hydrolysis of one or more peptide bonds of the peptide or protein.

[0115] In some embodiments, subjecting the enriched peptide or protein sample to enzymatic digestion condition may comprise contacting the enriched peptide or protein sample to a hydrolyzing agent. Non-limiting examples of hydrolyzing agents that can carry out enzymatic digestion include protease from Aspergillus Saitoi, elastase, subtilisin, protease Xin, pepsin, trypsin, Tryp-N, chymotrypsin, aspergillopepsin I, LysN protease (Lys-N), LysC endoproteinase (Lys-C), endoproteinase Asp-N (Asp-N), endoproteinase Arg-C (Arg-C), endoproteinase Glu-C (Glu-C) or outer membrane protein T (OmpT), immunoglobulin-degrading enzyme of Streptococcus pyogenes (IdeS), IdeZ, igdE, glyserias, thermolysin, papain, pronase, V8 protease, or biologically active fragments or homologs thereof or combinations thereof.

[0116] In some embodiments, subjecting the enriched peptide or protein sample to enzymatic digestion condition may comprise contacting the enriched peptide or protein sample to at least one digestive enzyme. In some embodiments, the at least one digestive enzyme comprises pepsin, trypsin, Tryp-N, chymotrypsin, Lys-N, Lys-C, Asp-N, Arg-C, Glu-C, papain, IdeS, IdeZ, igdE, glyserias, variants thereof, or any combination thereof. In some embodiments, the at least one digestive enzyme comprises trypsin.

[0117] In some embodiments, enzymatic digestion may comprise any technique known in the art, such as described in Switzar et al., “Protein Digestion: An Overview of the Available Techniques and Recent Developments”, Journal of Proteome Research, 2013, Volume 12, Pages 1067-1077, the entire contents of which are hereby incorporated by reference.

[0118] The present invention will be more fully understood by reference to the following examples. They should not, however, be construed as limiting the scope of this disclosure.

EXAMPLES

[0119] General Materials and Methods

[0120] For immunoprecipitation, 10 pL of streptavidin magnetic beads (Thermo Streptavidin Tl) were first blocked with 50 pL of pooled mouse serum at room temperature for 30 minutes, and then washed with 3% BSA in HBST. Next, the beads were conjugated with 10 pL of biotinylated affinity ligand capable of binding multiple AAV serotypes (Thermo CaptureSelect Biotin Anti-AAVX) at room temperature for 30 minutes. 20 pL of recombinant AAV vectors (concentrated at 4E11) were introduced into 50 pL serum samples with 230 pL of HBST, followed by incubation at room temperature for one hour. The incubated serum samples were then added to the conjugated magnetic beads and incubated at room temperature for one hour to form ADA-AAV-affinity ligand complexes. The complexed beads were washed with 3% BSA in HBST, then washed twice with HBST, followed by elution with 50 pL of 20% acetonitrile (ACN) and 1% FA. Next, the eluate was digested with trypsin before being subjected to LC/MS analysis on a liquid chromatography system (Agilent 1290 Infinity II LC system) paired with a triple quadrupole mass spectrometer (Agilent 6495). One surrogate peptide was chosen for each isotype. The top two MRM transitions with optimized collision energy were used for each peptide. The MRM-MS data was analyzed using Skyline, with total peak areas used for quantification.

[0121] Example 1: Optimization of Immunocapture of Anti-AAV Antibodies

[0122] The experimental workflow utilized an affinity ligand conjugated to biotin, CAPTURESELECT™ Biotin Anti-AAVX, which binds to a large set of different AAV vector serotypes. AAV bound to the affinity ligand is further capable of interacting with anti-AAV antibodies or anti-drug antibodies present in a sample. A complex of anti-drug antibodies (ADAs) bound to AAV vectors were bound to the biotinylated affinity ligand, which was then bound to a bead comprising streptavidin. The bound complex was then eluted, digested, and subjected to LC- MS. LC-MS was carried out as illustrated in FIG. 2.

[0123] Effectiveness of AAV vector binding to the affinity ligand was evaluated using varying concentrations of stock material listed in Table 1, below. A total of 10 pg of the affinity ligand adequately bound 4E¹¹ viral genomes, constituting an 88% recovery.

Table 1

[0124] Binding capacity of the bound AAV vector was evaluated using a range of binding to a positive control. A known human IgGl antibody was used as a positive control, diluted in monkey serum to the concentrations listed in Table 2, below. As shown in FIG. 3, peptide recovery (measured in peak area) increased as the amount of the antibody increased, effectively capturing 1 pg of the antibody. For comparison, three negative control conditions were used: control #1 (no immunoprecitation, no antibody); control #2 (no immunoprecipitation, 100 ng antibody); and control #3 (no AAV vector).

Table 2

[0125] Since certain components in monkey serum may cause nonspecific interactions which lead to high background noises, optimization of immunocapture was further optimized by comparing automated and manual immunocapture methods. Magnetic beads were prepared by first blocking the beads, followed by incubation with the affinity ligand for binding. Next, the beads bound with the affinity ligand were incubated with AAV vectors in mouse serum, forming a bead-affinity ligand- AAV complex, and washed. Automated and manual immunocapture methods were performed with monkey IgGl antibodies (FIG. 4A), monkey IgG2 antibodies (FIG. 4B), and monkey IgG4 antibodies (FIG. 4C) to evaluate any nonspecific (background) interactions. As shown in FIGS. 4A-4C, the manual immunocapture method demonstrated significantly reduced background interactions as compared to the automated method.

[0126] Detection limit of the optimized immunocapture method was determined using a known human IgGl antibody diluted in naive human serum samples at the concentrations ranging from 78 ng/mL to 10 pg/mL, as shown in FIGS. 5A-5B. As shown in FIG. 5B, the calibration curve demonstrated linearity.

[0127] Example 2: Characterization of Pre-Existing Anti-AAV Antibodies in Patient Serum Samples

[0128] The immunocapture and LC-MS methods described above were used to successfully characterize isotypes of potential pre-existing anti-AAV antibodies in patient serum samples. [0129] Multiple patient serum samples were found to include pre-existing anti-AAV8 antibodies. As shown in FIG. 6, several patient samples demonstrated the presence of pre-existing anti-AAV8 antibodies having an IgGl isotype (positive controls of 41 ng/mL to 10 pg/mL were used for comparison). As shown in FIG. 7, several patient samples demonstrated the presence of pre-existing anti-AAV8 antibodies having an IgG4 isotype (negative controls used for comparison). Only one patient sample (patient 6) demonstrated the presence of pre-existing anti-AAV8 antibodies having both IgGl and IgG4 isotypes. Despite high background interactions, several patient samples demonstrate the presence of anti-AAV8 antibodies having IgM (FIG. 8) and IgAl (FIG. 9) isotypes (negative controls used for comparison). Most patient samples were mostly negative for the presence of anti-AAV8 antibodies having IgG2 (FIG. 10) and IgG3 (FIG. 11) isotypes, and completely negative for anti-AAV8 antibodies having IgE (FIG. 12) and IgA2 (FIG. 13) isotypes (negative controls used for comparison).

[0130] Multiple patient samples were also found to include pre-existing anti-AAV2 antibodies. As shown in FIG. 14, multiple patient samples demonstrated the presence of pre-existing anti-AAV2 antibodies having an IgGl isotype (positive controls used for comparison). As shown in FIG. 15, multiple patient samples demonstrated the presence of pre-existing anti-AAV2 antibodies having an IgG4 isotype (negative controls used for comparison). Consistent with the tests for anti-AAV8 antibodies, only one patient sample (patient 6) demonstrated the presence of pre-existing anti-AAV2 antibodies having both IgGl and IgG4 isotypes. As shown in FIG. 16, despite high background interactions, several patient samples demonstrated the presence of anti-AAV2 antibodies having an IgM isotype (negative controls used for comparison). However, as shown in FIG. 17, most patient samples did not demonstrate a presence of anti-AAV2 antibodies having an IgAl isotype (negative controls used for comparison). Multiple patient samples demonstrated the presence of anti-AAV2 antibodies having an IgG2 isotype (FIG. 18). Most patient samples were mostly negative for anti- AAV2 antibodies having an IgG3 isotype (FIG. 19), and were completely negative for anti-AAV2 antibodies having IgE (FIG. 20) or IgA2 (FIG. 21) isotypes.

[0131] Effectiveness of AAV vector binding to the affinity ligand (anti-AAVX antibody, as described above) was evaluated using 10 pg of the affinity ligand, AAV1 vectors, and varying concentrations of stock material, as listed in Table 3, below. As shown in FIG. 22, efficiency of binding to AAV1 was comparable across load volumes.

Table 3

[0132] Immunocapture and LC-MS methods as described above were used to characterize isotypes of potential pre-existing anti-AAVl antibodies in patient serum samples. As shown in FIG. 23, multiple patient samples were positive for anti -AAV 1 antibodies having an IgGl isotype (positive controls of 41 ng/mL to 10 pg/mL were used for comparison). As shown in FIG. 24, multiple patient samples were positive for anti-AAVl antibodies having an IgG4 isotype (negative controls used for comparison). Several patient samples (patient_2, patient_6, patient_9, and patient_22) demonstrated the presence of pre-existing anti-AAVl antibodies having both IgGl and IgG4 isotypes. Consistent with the tests of anti-AAV8 antibodies, background interactions for anti-AAVl antibodies having an IgM isotype were relatively high, however, multiple patient samples were positive for anti-AAVl antibodies having an IgM isotype (FIG. 25). In contrast to the tests of anti- AAV8 antibodies, patient samples were negative for anti-AAVl antibodies having an IgAl isotype (FIG. 26). Patient samples were mostly negative for anti-AAVl antibodies having an IgG2 (FIG. 27) or IgG3 (FIG. 28) isotype, and completely negative for anti-AAVl antibodies having an IgE (FIG. 29) or IgA2 (FIG. 30) isotype.

[0133] In comparing the results of the anti-AAV8 antibody characterization to the anti-AAVl antibody characterization, both types of antibodies demonstrated similar levels of IgGl, IgG3, IgA2, and IgE isotypes. Both types of antibodies also demonstrated low levels of IgM isotype, associated with the innate immune response. However, the two types of antibodies displayed different levels of IgG2, IgG4, and IgAl isotypes.

[0134] Example 3: Characterization of Anti-AAV Antibodies in Neutralizing Assay

[0135] To demonstrate characterization of neutralizing activity and isotype of anti-drug antibodies, a neutralizing assay was performed using the twenty-four patient serum samples tested and characterized in Example 2.

[0136] To detect the presence of neutralizing antibodies in each sample, a cell-based assay was performed to test for luciferase expression after infection with either AAV8 vectors, AAV1 vectors, or AAV2 vectors. After dilution of the titer, a color change from red (low fluorescence) to green (high fluorescence) indicated that the concentration of neutralizing antibody was low enough to no longer neutralize the AAV vector. No color change indicated that the concentration of neutralizing antibody was high enough to continue neutralizing the AAV vector.

[0137] FIGS. 31A and 3 IB demonstrate luciferase expression for AAV2 and AAV8 vectors, respectively. For both types of AAV vectors, patient samples 13, 50, 55, 4, 16, and 21 (“Group 1”) demonstrated high levels of neutralization, while patient samples 7, 22, 26, 39, 47, and 48 (“Group 3”) demonstrated none or practically no neutralization. Patient samples 34, 70, 6, 12, 15, 3, 23, and 25 (“Group 2”) demonstrated some neutralization, but less than Group 1. Patient samples 19, 29, 31, and 33 (“Group 4”) demonstrated some neutralization for AAV8 vectors but not AAV2 vectors. [0138] FIGS. 32A-32F demonstrate levels of various isotypes for the patient samples, grouped as described above. IgGl isotype levels are shown for anti-AAV8 antibodies (FIG. 32A), anti-AAV2 antibodies (FIG. 32B), and anti-AAVl antibodies (FIG. 33A). IgG4 isotype levels are shown for anti-AAV8 antibodies (FIG. 32C), anti-AAV2 antibodies (FIG. 32D), and anti-AAVl antibodies (FIG. 33B). IgM isotype levels are shown for anti-AAV8 antibodies (FIG. 32E), anti-AAV2 antibodies (FIG. 32F), and anti-AAVl antibodies (FIG. 33C).

[0139] Overall, for all isotypes tested, there was a strong correlation between IgGl isotype levels and neutralizing activity of antibodies, with no such correlation observed for the other isotypes.

[0140] Example 4: Characterization of Isotype and Quantity of IgGl Anti-AAV Antibodies [0141] The methods described above were further shown to accurately and precisely identify and quantify the IgGl isotype of anti-drug antibodies, including both anti-AAVl and anti-AAV8 antibodies, using the twenty-four patient samples tested in Examples 2 and 3.

[0142] As shown in FIG. 34 A, a calibration curve utilizing peak area and concentration of an IgGl anti-AAV8 antibody was generated demonstrated good linearity (R² = 0.9831). IgGl anti-AAV8 concentration was shown to be higher for the Group 1 samples, and practically nonexistent for the rest of the samples (FIG. 34B). As shown in FIG. 34C, a similar calibration curve was generated for an IgGl anti-AAVl antibody, and also demonstrated good linearity (R² = 0.9919). Similarly, IgGl anti-AAVl concentration was shown to be higher for the Group 1 samples, and nonexistent for the rest of the samples (FIG. 34D).

[0143] To account for the background signal due to nonspecific precipitation of other human Ig from serum, baseline levels were calculated and used to generate corrected calibration curves. FIG. 35A demonstrates the original calibration curve for the IgGl anti-AAV8 antibody, and FIG. 35B demonstrates the corrected calibration curve for the IgGl anti-AAV8 antibody. FIG. 35C demonstrates the original calibration curve for the IgGl anti-AAVl antibody, and FIG. 35D demonstrates the corrected calibration curve for the IgGl anti-AAVl antibody.

[0144] The corrected calibration curves were used to determine how much of each isotype (IgGl, IgG4, and IgM) was present among the samples. FIG. 36A demonstrates isotype distribution of the anti-AAV8 antibodies for IgGl (top), IgG4 (middle), and IgM (bottom). FIG. 36B demonstrates isotype distribution of anti-AAV2 antibodies for IgGl (top) and IgM (bottom). FIG. 36C demonstrates isotype distribution for anti-AAVl antibodies for IgGl (top), IgG4 (middle), and IgM (bottom). For all types, Group 1 samples demonstrate high levels of IgGl isotype antibodies.

[0145] While preferred embodiments of the present invention have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the invention. It should be understood that various alternatives to the embodiments of the invention described herein may be employed in practicing the invention. It is intended that the following claims define the scope of the invention and that methods and structures within the scope of these claims and their equivalents be covered thereby.

[0146] The present disclosure is further described by the following non-limiting items.

[0147] Item 1. A method of identifying the presence of a peptide or protein in a sample, comprising:

(a) contacting the sample including the peptide or protein to a binding partner to form a complex;

(b) contacting the complex to a solid support conjugated to an affinity ligand capable of binding the complex to form an immobilized complex;

(c) washing said immobilized complex to remove unbound material;

(d) eluting said peptide or protein from the immobilized complex to produce an enriched peptide or protein sample;

(e) subjecting said enriched peptide or protein sample to enzymatic digestion conditions to produce a peptide digest; and

(f) subjecting said peptide digest to liquid chromatography -mass spectrometry (LC-MS) analysis to identify said peptide or protein.

[0148] Item 2. The method of item 1, wherein said binding partner is a viral vector.

[0149] Item 3. The method of item 2, wherein said viral vector is an AAV vector.

[0150] Item 4. The method of item 3, wherein said at least one AAV vector comprises a serotype selected from the group comprising AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, variations thereof and combinations thereof.

[0151] Item 5. The method of item 1, wherein said solid support is streptavidin.

[0152] Item 6. The method of item 1, wherein said affinity ligand is an antibody or antibody fragment to the binding partner.

[0153] Item 7. The method of item 6, wherein antibody or antibody fragment is an anti-AAVX.

[0154] Item 8. The method of item 7, wherein the anti-AAVX is conjugated to biotin.

[0155] Item 9. The method of item 6, wherein the affinity ligand is CaptureSelect™ Biotin Anti- AAVX.

[0156] Item 10. The method of item 1, wherein the enzymatic digestion comprises contacting said peptide or protein to at least one digestive enzyme selected from a group consisting of pepsin, trypsin, Tryp-N, chymotrypsin, Lys-N, Lys-C, Asp-N, Arg-C, Glu-C, papain, IdeS, IdeZ, igdE, glyserias, variants thereof, and combinations thereof.

[0157] Item 11. The method of item 10, wherein the enzymatic digestion comprises trypsin.

[0158] Item 12. The method of item 1, wherein said sample comprises a biological sample. [0159] Item 13. The method of item 12, wherein said biological sample is a serum sample.

[0160] Item 14. The method of item 1, wherein said amount of affinity ligand used is about 1 pg to about 15 pg.

[0161] Item 15. The method of item 14, wherein said amount of affinity ligand used is about 10 pg. [0162] Item 16. The method of item 1, wherein the mass spectrometer is capable of performing LC- MS (liquid chromatography -mass spectrometry) or a LC-MRM-MS (liquid chromatographymultiple reaction monitoring-mass spectrometry) analyses.

[0163] Item 17. The method of item 1, wherein said mass spectrometer is an electrospray ionization mass spectrometer, nano-electrospray ionization mass spectrometer, triple quadrupole mass spectrometer or a quadrupole time-of-flight.

[0164] Item 18. The method of item 17, wherein the mass spectrometer is a triple quadrupole mass spectrometer.

[0165] Item 19. The method of item 1, wherein said LC-MS analysis comprises multiple reaction monitoring (MRM).

[0166] Item 20. The method of item 1, wherein the peptide or protein is an anti-drug antibody (ADA).

[0167] Item 21. The method of item 20, wherein an isotype or subclass of the isolated ADA is determined.

[0168] Item 22. The method of item 20, wherein the ADA in the sample is a human antibody.

[0169] Item 23. The method of item 22, wherein an isotype of the human antibody is IgGl, IgG2, IgG3, IgG4, IgAl, IgA2, IgM, or IgE,

[0170] Those skilled in the art will appreciate that the conception upon which this disclosure is based may readily be used as a basis for designing other devices and systems for carrying out the several purposes of the present disclosure. Accordingly, the claims are not to be considered as limited by the foregoing description.

Claims

1. A method for characterizing a peptide or protein in a sample, comprising: contacting the sample including the peptide or protein to a binding partner to form a complex; contacting the complex to a solid support conjugated to an affinity ligand capable of binding the complex to form an immobilized complex; washing the immobilized complex to remove unbound material; eluting the peptide or protein from the immobilized complex to produce an enriched peptide or protein sample; subjecting the enriched peptide or protein sample to enzymatic digestion conditions to produce a peptide digest; and subjecting the peptide digest to liquid chromatography-mass spectrometry (LC-MS) analysis to characterize the peptide or protein.

2. The method of claim 1, wherein the binding partner comprises a viral vector.

3. The method of claim 1, wherein the binding partner comprises an adeno-associated viral (AAV) vector.

4. The method of claim 3, wherein the AAV vector comprises a serotype selected from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, or a combination thereof.

5. The method of claim 1, wherein the solid support comprises streptavidin.

6. The method of claim 1, wherein the affinity ligand binds to the binding partner.

7. The method of claim 6, wherein the affinity ligand comprises an anti-AAVX antibody or a fragment thereof.

8. The method of claim 7, wherein the anti-AAVX antibody is conjugated to biotin.

9. The method of claim 1, wherein the enzymatic digestion comprises: contacting the peptide or protein to a digestive enzyme mixture comprising one or more of pepsin, trypsin, Tryp-N, chymotrypsin, Lys-N, Lys-C, Asp-N, Arg-C, Glu-C, papain, IdeS, IdeZ, igdE, glyserias, or a variant thereof.

10. The method of claim 1, wherein the sample comprises a biological sample.

11. The method of claim 1, wherein the sample comprises a serum sample.

12. The method of claim 1, wherein an amount of the affinity ligand used is about 1 pg to about

15 pg-

13. The method of claim 12, wherein the amount of affinity ligand used is about 10 pg.

14. The method of claim 1, wherein subjecting the peptide digest to LC-MS analysis comprises: subjecting the peptide digest to a mass spectrometer capable of performing LC-MS or liquid chromatography-multiple reaction monitoring-mass spectrometry (LC-MRM-MS).

15. The method of claim 1, wherein subjecting the peptide digest to LC-MS analysis comprises: subjecting the peptide digest to an electrospray ionization mass spectrometer, a nanoelectrospray ionization mass spectrometer, a triple quadrupole mass spectrometer, or a quadrupole time-of-flight.

16. The method of claim 15, wherein the mass spectrometer is a triple quadrupole mass spectrometer.

17. The method of claim 1, wherein the LC-MS analysis comprises multiple reaction monitoring (MRM).

18. The method of claim 1, wherein the peptide or protein is an anti-drug antibody (ADA) or a fragment thereof.

19. The method of claim 18, wherein the ADA is a human antibody.

20. The method of claim 19, wherein an isotype of the human antibody is IgGl, IgG2, IgG3, IgG4, IgAl, IgA2, IgM, or IgE.