Methods for analyzing co-formulated therapeutic proteins

Abstract

The disclosure provides methods for analyzing two or more different therapeutic proteins co-formulated in a sample mixture. The methods involve intact mass analysis, peptide mapping, and a multi-attribute method (MAM) to detect, quantify, and monitor critical quality attributes (CQAs) of co-formulated therapeutic proteins, such as monoclonal antibodies.

Description

METHODS FOR ANALYZING CO-FORMULATED THERAPEUTIC PROTEINSFIELD

[0001] The disclosure relates to methods for analyzing two or more therapeutic proteins (e.g., antibodies) that are co-formulated in a single composition.CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of U.S. Provisional Application No. 63 / 622,371, filed January 18, 2024, which is hereby incorporated by reference in its entirety.BACKGROUND

[0003] Combination therapy using two or more molecules with complementary pharmacological effects has led to an increased interest in the development of co-formulation products. Co-formulations or fixed-dose combination drugs (FDCs) are therapeutics in which two or more separate drug components (e.g., a small molecule and biologic, or two different biologies such as therapeutic antibodies) are combined in a single dosage form. These products can often reduce the number and volume of injections, improve patient compliance, and reduce discomfort.

[0004] As co-formulated therapeutics are classified as new molecular entities (NMEs), they are subject to clinical evaluation by regulatory bodies, such as the U.S. Food and Drug Administration (FDA). Thus, co-formulating existing therapeutics may be subject to further analysis and evaluation, even though the efficacy and safety of each individual therapeutic has already been established through independent clinical trials. Characterizing co-formulated proteins, such as antibodies, can be challenging due to similar physiochemical properties of the co-formulated proteins, such as molecular weight.

[0005] There are also many chemistry, manufacturing, and control challenges to address for a co-formulated biologic. These include analytical challenges in characterization of each molecule in the co-formulation, manufacturing issues of formulating higher concentration biologies, and stability issues such as protein-protein interactions, protein aggregation, and subvisible particle formulation. For example, structural changes of therapeutic proteins canoccur during the manufacturing, storage, and transportation process steps that lead up to administration to a patient. The change in structure, which is known as an “attribute,” can correspond to different species of the therapeutic proteins that results in a heterogeneous product, with potential impacts to stability, efficacy, and / or safety.

[0006] There remains a need for methods for monitoring critical quality attributes of coformulated therapeutic proteins and accurately determining the concentrations of each therapeutic protein in a pharmaceutical preparation or sample.BRIEF SUMMARY

[0007] In some aspects, disclosure provides a method of analyzing a first therapeutic protein and a second therapeutic protein in a composition, which method includes: (a) optionally applying a stress to a composition including a first therapeutic protein and a second therapeutic protein, wherein the second therapeutic protein is different from the first therapeutic protein; (b) cleaving the first therapeutic protein and the second therapeutic protein into two or more peptides each; (c) separating the two or more peptides of each of the first and second therapeutic proteins;(d) detecting and quantifying one or more structures present on the two or more peptides of either or both of the first and / or second therapeutic proteins, wherein the one or more structures correspond to one or more species of the first and / or second therapeutic protein; and optionally(e) determining a ratio of the first therapeutic protein to the second therapeutic protein in the composition based on an abundance of one or more of the peptides of the first therapeutic protein generated in (b) and an abundance of one or more of the peptides of the second therapeutic protein generated in (b).

[0008] In some aspects, the stress includes an exposure to ultra-violet light, heat, air, freeze / thaw cycle, shaking, long-term storage, change in pH, or change in temperature. In some aspects, the change in pH is greater than about 1.0 or greater than about 2.0, and / or wherein the change in temperature is greater than or about 2°C or greater than or about 5°C.

[0009] In some aspects of the method, the structure includes a chemical modification, optionally, wherein the chemical modification alters the mass-to-charge ratio (m / z) of charged ions of an amino acid of the therapeutic protein.

[0010] In some aspects of the method, the chemical modification is glycosylation, hydroxylation, glycation, deamidation, oxidation, reduction, isomerization, aggregation, degradation, acetylation, or clipping due to hydrolysis proteolysis.

[0011] In some aspects of the method, the stress applied to the composition leads to formation of at least one species of the first therapeutic protein having a unique attribute profile relative to the attribute profile of the first therapeutic protein prior the applied stress.

[0012] In some aspects of the method, the stress applied to the composition leads to formation of at least one species of the second therapeutic protein having a unique attribute profile relative to the attribute profile of the second therapeutic protein prior the applied stress.

[0013] In some aspects of the method, cleaving includes treating the composition with a proteolytic enzyme.

[0014] In some aspects of the method, the proteolytic enzyme is trypsin.

[0015] In some aspects of the method, the separating of (c) and detecting and quantifying of(d) includes using liquid chromatography-mass spectrometry.

[0016] In some aspects of the method, a ratio of the first therapeutic protein to the second therapeutic protein in the composition is determined based on the abundance of one or more of the peptides of the first therapeutic protein generated in (b) and the abundance of one or more of the peptides of the second therapeutic protein generated in (b).

[0017] In some aspects of the method, the ratio of the first therapeutic protein to the second therapeutic protein in the composition is 1 : 1 to 1 : 100, or 1 :2 to 1 : 100, or 1 :2 to 1 :50.

[0018] In some aspects of the method, the ratio of the first therapeutic protein to the second therapeutic protein in the composition is 1:80, 1 :50, 1 :40, 1 :20, 1 : 10, 1 :5, or 1 : 1.

[0019] The disclosure further provides a method of resolving a first therapeutic protein from a second therapeutic protein in a composition, which method includes performing reduced intact mass spectrometry on a composition including a first therapeutic protein and a second therapeutic protein, wherein the second therapeutic protein is different from the first therapeutic protein, and wherein at least one subunit of the first therapeutic protein co-elutes with at least one subunit of the second therapeutic protein, and when the difference in molecular weights of the subunits includes at least about 6 Da, resolving the subunit of the first therapeutic protein from the subunit of the second therapeutic protein by mass spectrometry; when the difference in molecular weights of the subunits includes less than about 6 Da and more than about 1 Da,resolving the subunit of the first therapeutic protein from the subunit of the second therapeutic protein by high resolution mass spectrometry (HRMS); or when the difference in molecular weights of the subunits includes no more than about 1 Da, resolving the subunit of the first therapeutic protein from the subunit of the second therapeutic protein by liquid chromatography including a shallow gradient.

[0020] In some aspects of the method, the difference in molecular weights of the subunits includes at least about 6 Da, and the subunit of the first therapeutic protein is resolved from the subunit of the second therapeutic protein by time-of-flight (TOF) mass spectrometry.

[0021] In some aspects of the method, the HRMS includes a resolving power of greater than 10,000 at m / z 400.

[0022] In some aspects of the method, the HRMS includes a resolving power of 100,000 at m / z 400.

[0023] In some aspects of the method, the liquid chromatography includes reverse phase high performance liquid chromatography (RP-HPLC).

[0024] In some aspects, the shallow gradient includes a mobile phase strength of 32% to 37% over an elution time of 12-20 minutes.

[0025] In some aspects, the method further comprises deconvolution of the individual mass of the subunit of the first therapeutic protein and the individual mass of the subunit of the second therapeutic protein.

[0026] In some aspects of the method, the subunit of the first therapeutic protein is an antibody light chain and the subunit of the second therapeutic protein is an antibody light chain.

[0027] The disclosure also provides a method of analyzing a first therapeutic protein and a second therapeutic protein in a composition via a multi -attribute method (MAM), wherein the MAM includes: (a) providing a composition including a first therapeutic protein and a second therapeutic protein, wherein the second therapeutic protein is different from the first therapeutic protein, and wherein the ratio of first therapeutic protein to second therapeutic protein in the composition ranges from 1 :20 to 20: 1; (b) contacting a sample of the composition with a proteolytic enzyme in a 10: 1 ratio, wherein the sample includes about 200 mg of total protein, and wherein each of the first and second therapeutic proteins is cleaved into two or more peptides by the proteolytic enzyme; and (c) analyzing a sample of the two or more peptides via liquid chromatography-mass spectrometry (LC-MS), whereby one or more structures present onthe two or more peptides of either or both of the first and / or second therapeutic protein are detected and quantified; wherein the total amount of peptides analyzed via LC-MS is about 4.0- 4.5 mg; and wherein the one or more structures correspond to one or more species of the first and / or second therapeutic protein.

[0028] In some aspects of the method, the structure forms as a result of a chemical modification, optionally, wherein the chemical modification alters the mass-to-charge ratio (m / z) of charged ions of an amino acid of the therapeutic protein.

[0029] In some aspects of the method, the chemical modification is glycosylation, hydroxylation, glycation, deamidation, oxidation, reduction, isomerization, aggregation, degradation, acetylation, or clipping due to hydrolysis proteolysis.

[0030] In some aspects of the method, the proteolytic enzyme is trypsin.

[0031] In some aspects of the method, analyzing a sample of the two or more peptides includes liquid chromatography with tandem mass spectrometry (LC-MS / MS).

[0032] In some aspects, each of the first therapeutic protein and the second therapeutic protein is an antigen-binding protein.

[0033] In some aspects, the antigen-binding protein is an antibody, an antibody fragment, or a bispecific T cell engager (BiTE®) molecule.BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 A is a table showing relative quantitation of quality target product profile (QTPP) attributes for Abl formulated individually and co-formulated with other monoclonal antibodies (mAbs) under thermal stress. Figure IB is a table showing relative quantitation of QTPP attributes for Abl formulated individually and co-formulated with other mAbs under photo stress.

[0035] Figures 2A-2D are mirror histogram plots for LC-MS / MS peak area of surrogate peptides for Abl and Ab3 in coformulation under the following conditions: TO (Figure 2A), 40°C for 2 weeks (Figure 2B), 40°C for 4 weeks (Figure 2C), and photo stress (Figure 2D). The ratio of median peak area between Ab 1 and Ab3 was 1.1 for all conditions.

[0036] Figures 3A-3D show results of reduced intact mass analysis of co-formulated Abl+Ab2 (Combo 1). Figure 3 A is a total ion chromatogram for the Combo 1 co-formulation showing baseline-separated heavy chains but co-eluted light chains. Figures 3B and 3C are time-of-flight (TOF) mass spectra for co-eluted light chains that were baseline-resolved with -5.3 Th difference for the highest abundance peak. Figure 3D is a deconvoluted mass spectrum of the co-eluted light chains, which yielded corresponding masses of 23230.04 Da and 23310.07 Da. LC1 and HC1 denote the Abl light chain and heavy chain, and LC2 and HC2 denote the Ab2 light chain and heavy chain.

[0037] Figures 4A-4G show results of reduced intact mass analysis of co-formulated Abl+Ab3 (Combo 2). Figure 4A is a total ion chromatogram for Combo 2 showing baseline- separated heavy chains but co-eluted light chains. Figures 4B and 4C are mass spectra for individual (not co-formulated) light chains showing a ~0.4 Th difference for the peak with highest abundance Figure 4D is a deconvoluted mass spectrum of the co-eluted light chains, which show mass errors of 19 ppm and -28 ppm. Figures 4E-4F are mass spectra of co-eluted ensemble light chains, which yielded one broad and unresolved peak (Figures 4E and 4F), and one deconvoluted mass between the masses of LC1 and LC3. LC1 and HC1 denote the Abl light chain and heavy chain, and LC3 and HC3 denote the Ab3 light chain and heavy chain.

[0038] Figures 5A-5D show results of reduced intact mass analysis of co-formulated Abl+Ab3 (Combo 2) after method optimization. Figure 5 A is a total ion chromatogram for Combo 2 showing baseline-separated heavy chains and light chains when a separation gradient of 32% to 37% in 15 minutes was used. Figures 5B and 5C are mass spectra for individual (not co-formulated) light chains showing a -0.4 Th difference for the peak with highest abundance. Figure 5D is a deconvoluted mass spectrum of the co-eluted light chains, which shows mass errors of 23 ppm (LC1) and 6 ppm (LC3).

[0039] Figure 6 is a mass spectrum showing reference peaks for the MAM analysis of Combo 3.

[0040] Figure 7 is a graph showing attribute levels measured by MAM of Ab4 and Ab5 in co-formulation and formulated individually.DETAILED DESCRIPTION

[0041] The present disclosure is predicated, at least in part, on the development of mass spectrometry (MS)-based assays for detecting and quantifying attributesof different intact therapeutic proteins (e.g., antibodies) co-formulated together in a single formulation. Conventional assays for drug product lot release and stability testing typically include cationexchange high performance liquid chromatography (CEX-HPLC) and capillary electrophoresis (CE), neither of which provides residue-specific critical quality attributes (CQAs). As a result, it is often difficult to assign identified CQAs to a specific therapeutic protein of a co-formulation by CEX-HPLC or CE, as subunits of different proteins may co-elute in such conventional techniques. In addition, the limited resolution and specificity of CEX-HPLC and CE may lead to co-elution of impurities with the main peak, confounding purity analysis. The presently disclosed methods provide significant advantages over these conventional methods. For example, the high resolving power and high mass accuracy of mass spectrometry enables detection of residue-specific attributes and product-specific attribute assignment in a coformulation. The methods described herein also allow for accurate monitoring and control of therapeutic drug products, and the processing thereof, through release and stability testing to ensure accurate dosing.

[0042] In some embodiments, the disclosure provides a method of analyzing a first therapeutic protein and a second therapeutic protein in a composition. The method comprises: (a) optionally applying a stress to a composition comprising a first therapeutic protein and a second therapeutic protein, wherein the second therapeutic protein is different from the first therapeutic protein; (b) cleaving the first therapeutic protein and the second therapeutic protein into two or more peptides each; (c) separating the two or more peptides of each of the first and second therapeutic proteins; (d) detecting and quantifying one or more structures present on the two or more peptides of either or both of the first and / or second therapeutic proteins, wherein the one or more structures correspond to one or more species of the first and / or second therapeutic protein; and optionally (e) determining a ratio of the first therapeutic protein to the second therapeutic protein in the composition based on the abundance of one or more of the peptides of the first therapeutic protein generated in (b) and the abundance of one or more of the peptides of the second therapeutic protein generated in (b).

[0043] In some embodiments, the disclosure provides a method of resolving a first therapeutic protein from a second therapeutic protein in a composition. The method comprises performing reduced intact mass spectrometry on a composition comprising a first therapeutic protein and a second therapeutic protein, wherein the second therapeutic protein is different from the first therapeutic protein, wherein at least one subunit of the first therapeutic protein co-elutes with at least one subunit of the second therapeutic protein (such as co-elution in liquidchromatography or CE), and: when the difference in molecular weights of the subunits comprises at least about 6 Da, resolving the subunit of the first therapeutic protein from the subunit of the second therapeutic protein by mass spectrometry; when the difference in molecular weights of the subunits comprises less than about 6 Da and more than about 1 Da, resolving the subunit of the first therapeutic protein from the subunit of the second therapeutic protein by high resolution mass spectrometry (HRMS); or when the difference in molecular weights of the subunits comprises no more than about 1 Da, resolving the subunit of the first therapeutic protein from the subunit of the second therapeutic protein by liquid chromatography comprising a shallow gradient.

[0044] The disclosure also provides a multi-attribute method (MAM) for analyzing a first therapeutic protein and a second therapeutic protein in a composition. The MAM comprises (a) providing a composition comprising a first therapeutic protein and a second therapeutic protein, wherein the second therapeutic protein is different from the first therapeutic protein, and wherein the ratio of first therapeutic protein to second therapeutic protein in the composition ranges from 1 :20 to 20: 1; (b) contacting a sample of the composition with a proteolytic enzyme in a 10: 1 ratio, wherein the sample comprises about 200 pg of total protein, and wherein each of the first and second therapeutic proteins is cleaved into two or more peptides by the proteolytic enzyme; and (c) analyzing a sample of the two or more peptides via liquid chromatography-mass spectrometry (LC-MS), whereby one or more structures present on the two or more peptides of either or both of the first and / or second therapeutic protein are detected and quantified; wherein the total amount of peptides analyzed via LC-MS is about 4.0-4.5 pg; and wherein the one or more structures correspond to one or more species of the first and / or second therapeutic protein.Definitions

[0045] To facilitate an understanding of the present technology, several terms and phrases are defined below. Additional definitions are set forth throughout the detailed description.

[0046] In some aspects, the term “assaying” means “measuring,” and may be used interchangeably with the terms “testing,” “analyzing,” or “determining.” The level of peptide, with or without a molecular attribute, which is assayed or determined by the presently disclosed methods can be a relative measurement, e.g., a determination that the level is higher, lower, or the same as a reference level. In some aspects, the “assaying” can yield a normalizedmeasurement. For instance, the normalized measurement can be normalized to a reference protein, e.g., serum albumin. The “assaying” in certain instances yields an absolute measurement (e.g., neither normalized nor relative to a reference level).

[0047] In various aspects, a therapeutic protein is placed in a condition that leads to a change in its structure, for example, a change in the structure of an amino acid of the therapeutic protein, leading to the formation of a species of the therapeutic protein. In exemplary aspects, the changed structure of an amino acid is referred to as an “attribute” and may be characterized in terms of its chemical identity or attribute type and location within the amino acid sequence of the therapeutic protein, e.g., the position of the amino acid on which the attribute is present. Nonlimiting examples of molecular attributes for monoclonal antibodies, or antigen-binding fragments thereof, include high molecular weight (BMW) species, charge variants, oxidized species, deamidated species, and glycosylated species.

[0048] The term “attribute” refers to a chemically or physically changed structure on a macromolecule, such as an antigen-binding protein (e.g., an antibody), and may be characterized in terms of its physicochemical identity or attribute type and location within the sequence of the macromolecule, e.g., the position of the amino acid on which the attribute is present. Examples of attributes are shown in Table 1. The term “critical quality attribute (CQA)” as used herein, refers to an attribute within an appropriate limit, range, or distribution to ensure the desired product quality. CQAs are generally associated with a drug product, drug substance, excipients, and intermediates (in process materials). For example, for large polypeptide therapeutic molecules, physical or molecular attributes and modifications of amino acids are important CQAs that are monitored during drug development, manufacture, and storage.

[0049] In some aspects, the therapeutic protein may be an antigen-binding protein. The term “antigen-binding protein,” as used herein, refers to a proteinaceous molecule that specifically binds to an antigen. For example, an antigen-binding protein may comprise an antibody or an antigen-binding fragment thereof, (such as a monoclonal antibody, for example an IgGl or IgG2 monoclonal antibody), an antibody protein product, a bispecific T cell engager (BiTE®) molecule, a bispecific antibody, a trispecific antibody, or an Fc fusion protein.

[0050] An antigen-binding protein typically comprises the heavy chain variable region (VH) and / or the light chain variable region (VL) of an antibody, or comprises domains derived therefrom. In some embodiments, an antigen-binding protein comprises the structuralrequirements of an antibody which are sufficient for immunospecific target binding. This structural requirement may be defined by, for example, the presence of at least three light chain complementarity determining regions (CDRs) (i.e., CDR1, CDR2, and CDR3 of the VL region) and / or three heavy chain CDRs (i.e., CDR1, CDR2, and CDR3 of the VH region), or of all six CDRs. It is within the knowledge of a skilled person where (and in which order) those CDRs are located in the antigen-binding protein.

[0051] As used herein, the term “antibody” refers to an immunoglobulin of any isotype with specific binding to the target antigen; an antibody may be a polyclonal or monoclonal antibody, a chimeric antibody, a humanized antibody, a human antibody, etc. In a native antibody, a heavy chain comprises a variable region, VH, and three constant regions, CHI, CH2, and CH3. The VH domain is at the amino-terminus of the heavy chain, and the CH3 domain is at the carboxyterminus. In a native antibody, a light chain comprises a variable region, VL, and a constant region, CL. The variable region of the light chain is at the amino-terminus of the light chain. In a native antibody, the variable regions of each light / heavy chain pair typically form the antigenbinding site. The constant regions are typically responsible for effector function. A native antibody is a tetramer of two full-length heavy chains and two full-length light chains.

[0052] In a human antibody, CHI means a region having the amino acid sequence at positions 118 to 215 of the EU index or EU numbering system, which is based on the sequential numbering of the first human IgGl sequenced (i.e., the “EU antibody”) (Edelman et al., Proc Natl AcadSci USA, 63(1): 78-85 (1969)). A highly flexible amino acid region called a “hinge region” exists between CHI and CH2. CH2 represents a region having the amino acid sequence at positions 231 to 340 of the EU index, and CH3 represents a region having the amino acid sequence at positions 341 to 446 of the EU index.

[0053] CL” represents a constant region of a light chain. In the case of a kappa (K) chain of a human antibody, CL represents a region having the amino acid sequence at positions 108 to 214 of the EU index. In a lambda (X) chain, CL represents a region having the amino acid sequence at positions 108 to 215.

[0054] In a native antibody, the variable regions typically exhibit the same general structure in which relatively conserved framework regions (FRs) are joined by three hypervariable CDRs. The CDRs from the two chains of each pair typically are aligned by the framework regions, which may enable binding to a specific epitope. From N-terminus to C-terminus, both light andheavy chain variable regions typically comprise the domains FR1, CDR1 , FR2, CDR2, FR3, CDR3 and FR4. Typically, CDR3 is the greatest source of molecular diversity within the antigen binding site. The assignment of amino acids to each domain is typically in accordance with the definitions of Kabat et al. (1991) Sequences of Proteins of Immunological Interest (National Institutes of Health, Publication No. 91-3242, vols. 1-3, Bethesda, Md.); or Chothia, C., and Lesk, A. M. (1987) J. Mol. Biol., 196: 901-917. In some embodiments, the CDRs of an antigen-binding protein are defined according to the definition of Kabat or Chothia. In the present application, the term “CDR” refers to a CDR from either the light or heavy chain, unless otherwise specified.

[0055] Antibodies can comprise any constant region known in the art. Human light chains are classified as kappa and lambda light chains. Heavy chains are classified as mu, delta, gamma, alpha, or epsilon, and define the antibody’s isotype as IgM, IgD, IgG, IgA, and IgE, respectively. IgG has several subclasses, including, but not limited to IgGl, IgG2, IgG3, and IgG4. IgM has subclasses, including, but not limited to, IgMl and IgM2. Embodiments of the present disclosure include all such classes or isotypes of antibodies. The light chain constant region can be, for example, a kappa- or lambda-type light chain constant region, e.g., a human kappa- or lambda-type light chain constant region. The heavy chain constant region can be, for example, an alpha-, delta-, epsilon-, gamma-, or mu-type heavy chain constant regions, e.g., a human alpha-, delta-, epsilon-, gamma-, or mu-type heavy chain constant region. Accordingly, in exemplary embodiments, the antibody is an antibody of isotype IgA, IgD, IgE, IgG, or IgM, including any one of IgGl, IgG2, IgG3 or IgG4.

[0056] The antibody is preferably a monoclonal antibody. The term “monoclonal antibody,” as used herein, refers to an antibody produced by a single clone of B lymphocytes that is directed against a single epitope on an antigen. Monoclonal antibodies typically are produced using hybridoma technology, as first described in Kohler and Milstein, Eur. J. Immunol., 5: 511-519 (1976). Monoclonal antibodies may also be produced using recombinant DNA methods (see, e.g., U.S. Patent 4,816,567), isolated from phage display antibody libraries (see, e.g., Clackson et al. Nature, 352 : 624-628 (1991)); and Marks et al., J. Mol. Biol., 222: 581-597 (1991)), or produced from transgenic mice carrying a fully human immunoglobulin system (see, e.g., XENOMOUSE™ mouse, Green et al., Nature Genetics, 7: 13-21 (1994), US 2003-0070185, WO 96 / 34096, and WO 96 / 33735). In contrast, “polyclonal” antibodies are antibodies that aresecreted by different B cell lineages within an animal. Polyclonal antibodies are a collection of immunoglobulin molecules that recognize multiple epitopes on the same antigen.

[0057] The term “chimeric antibody” refers to an antibody containing domains from two or more different antibodies. A chimeric antibody can, for example, contain the constant domains from one species and the variable domains from a second, or more generally, can contain stretches of amino acid sequence from at least two species. A chimeric antibody also can contain domains of two or more different antibodies within the same species. The term “humanized” when used in relation to antibodies refers to antibodies having at least CDR regions from a nonhuman source which are engineered to have a structure and immunological function more similar to true human antibodies than the original source antibodies. For example, humanizing can involve grafting a CDR from a non-human antibody, such as a mouse antibody, into a human antibody. Humanizing also can involve select amino acid substitutions to make a non-human sequence more similar to a human sequence.

[0058] An antibody can be cleaved into fragments by enzymes, e.g., papain, pepsin, or other engineered site-specific proteases (such as those commercially available from Genovis AB, Lund, Sweden). Papain cleaves an antibody to produce two Fab fragments and a single Fc fragment. Pepsin cleaves an antibody to produce a F(ab’)2 fragment and a pFc’ fragment. In exemplary aspects, the antigen-binding protein of the present disclosure comprises an antigenbinding antibody fragment. As used herein, the term “antigen-binding antibody fragment” refers to a portion of an antibody molecule that is capable of binding to the antigen of the antibody and is also known as “antigen-binding fragment” or “antigen-binding portion.” In exemplary instances, an antigen-binding antibody fragment is a Fab fragment, a F(ab’)2 fragment, or a single chain fragment variable (scFv).

[0059] The architecture of antibodies has been exploited to create a growing range of alternative formats that span a molecular-weight range of at least about 12-150 kDa and has a valency (n) range from monomeric (n = 1), to dimeric (n = 2), to trimeric (n = 3), to tetrameric (n = 4), and potentially higher; such alternative formats are referred to herein as “antibody protein products.” Antibody protein products include those based on the full antibody structure and those that mimic antibody fragments which retain full antigen-binding capacity, e.g., scFvs, Fabs (e.g., Fab, Fab’, and F(ab’)2) and VHH / VH. The smallest antigen-binding antibody fragment that retains its complete antigen binding site is the Fv fragment, which consists entirely ofvariable (V) regions of the light and heavy chains. A soluble, flexible amino acid peptide linker is used to connect the V regions in a scFv fragment for stabilization of the molecule, or the constant (C) domains are added to the V regions to generate a Fab fragment. Both scFv and Fab fragments can be easily produced in host cells, e.g., prokaryotic host cells. A VHH / VH (or nanobody) is the antigen-binding fragment of heavy chain only antibodies. Heavy chain only antibodies (HcAb) are naturally produced by camelids and sharks. Other antibody protein products include bispecific T cell engager (BiTE®) molecules, disulfide-bond stabilized scFv (ds-scFv), single chain Fab (scFab), as well as di- and multimeric antibody formats like dia-, tria- and tetra-bodies, or minibodies (miniAbs) that comprise different formats consisting of scFvs linked to oligomerization domains. A peptibody or peptide-Fc fusion is yet another antibody protein product. The structure of a peptibody consists of a biologically active peptide grafted onto an Fc domain (see, e g., Shimamoto et al., mAbs14(5): 586-591 (2012)).

[0060] The antigen-binding protein of the present disclosure may comprise any one of the antibody protein products described herein. In exemplary aspects, the antigen-binding protein of the present disclosure may comprise any one of an scFv, Fab VHH / VH, Fv fragment, ds-scFv, scFab, dimeric antibody, multimeric antibody (e.g., a diabody, triabody, tetrabody), miniAb, peptibody VHH / VH of camelid heavy chain antibody, sdAb, diabody; a triabody; a tetrabody; a bispecific or trispecific antibody, bispecific T cell engager (BiTE®) molecule, BsIgG, appended IgG, BsAb fragment, bispecific fusion protein, or BsAb conjugate.

[0061] In certain aspects, the antigen-binding proteins of the present disclosure may be “bispecific,” meaning that they are capable of specifically binding to two different antigens. In another aspect, the antigen-binding proteins of the present disclosure may be “trispecific,” meaning that they are capable of specifically binding to three different antigens. In another aspect, the antigen-binding proteins of the present disclosure may be “tetraspecific,” meaning that they are capable of specifically binding to four different antigens.

[0062] In some embodiments, the antigen-binding protein is a BiTE® molecule. BiTE® molecules are engineered bispecific antigen-binding constructs which direct the cytotoxic activity of T cells against cancer cells. They are the fusion of two single-chain variable fragments (scFvs) of different antibodies, or amino acid sequences from four different genes, on a single peptide chain of about 55 kDa. One of the scFvs binds to T cells via the CD3 receptor, and the other to a tumor cell via a tumor specific molecule. Blinatumomab (BLINCYTO®product) is an example of a BiTE® molecule, specific for CD19. BiTE® molecules that are modified, such as those modified to extend their half-lives, can also be used in the disclosed methods. By their design, BiTE® molecules are uniquely suited to transiently connect T cells with target cells and, at the same time, potently activate the inherent cytolytic potential of T cells against target cells. See e.g., WO 99 / 54440, WO 2005 / 040220, and WO 2008 / 119567.

[0063] In certain embodiments of the disclosure, the antigen-binding proteins may be multivalent. The valency of the binding protein denotes the number of individual antigen binding domains within the binding protein. In some embodiments, a bispecific antigen-binding protein may be multivalent. For instance, in certain embodiments, a bispecific antigen-binding protein may be tetravalent by comprising four antigen-binding domains: two antigen-binding domains binding to a first target antigen and two antigen-binding domains binding to a second target antigen.

[0064] As used herein, the terms “antigen-binding domain” and “binding domain” may be used interchangeably to refer to the region of the antigen-binding protein that contains the amino acid residues that interact with the antigen and confer on the antigen-binding protein its specificity and affinity for the antigen. In some embodiments, the binding domain may be derived from the natural ligands of the target antigen(s). As used herein, the term “target antigen(s)” refers to a first target antigen and / or a second target antigen of a bispecific molecule and also refers to a first target antigen, a second target antigen, a third target antigen, and / or a fourth target antigen of a tetraspecific molecule.

[0065] An antigen-binding protein may comprise an immunoglobulin domain. The term “immunoglobulin domain,” as used herein, refers to a peptide comprising an amino acid sequence similar to that of immunoglobulin (i.e., antibody) and comprising approximately 100 amino acid residues including at least two cysteine residues. Examples of immunoglobulin domains include VH, CHI, CH2, and CH3 of an antibody heavy chain, and VL and CL of an antibody light chain. In addition, the immunoglobulin domain is found in proteins other than immunoglobulin. Examples of the immunoglobulin domain in proteins other than immunoglobulin include an immunoglobulin domain included in a protein belonging to an immunoglobulin super family, such as a major histocompatibility complex (MHC), CD1, B7, T cell receptor (TCR), and the like.

[0066] As used herein, the terms “stability” and “stabilizing” are defined as the maintenance of the chemical or physical integrity and / or bioactivity of the antigen-binding polypeptide or protein over a period of time. Stabilizing an antigen-binding polypeptide or protein includes the prevention or delay of degradation or deterioration of the antigen- binding polypeptide or protein from its biologically and / or therapeutically active form to an inactive form. Instability may arise from events such as aggregation, denaturation, fragmentation, or chemical modifications such as oxidation, cross- linking, deamidation and reactions with other components featured in the composition comprising the antigen-binding polypeptide or protein. By way of example, stability may be assessed by size exclusion chromatography to detect the relative abundance of high molecular weight and / or low molecular weight species.

[0067] The terms “binding pair member” and “binding member” are used interchangeably herein and refer to one of two or more different molecules that specifically recognize the other molecule compared to substantially less recognition of other molecules. By way of example, when an antibody or other entity (e.g., antigen-binding protein) “specifically recognizes” or “specifically binds” an antigen or epitope, it preferentially recognizes the antigen in a complex mixture of proteins and / or macromolecules, and binds the antigen or epitope with affinity which is substantially higher than to other entities not displaying the antigen or epitope. Accordingly, the two binding pair members will bind to each other more tightly than to other molecules.

[0068] The term “sample” and variations of this root term has its ordinary and customary meaning as would be understood by a person of ordinary skill in view of this disclosure. It refers to a composition that may contain two or more therapeutic proteins as described herein, such as in vitro or synthetic samples obtained from the manufacturing of the two or more therapeutic proteins. For example, a sample may be a composition or formulation comprising a first therapeutic protein and a second therapeutic protein and at least one pharmaceutically acceptable carrier (also referred to herein as a “pharmaceutical composition”). For example, a sample may be an in-process sample from the manufacture of a pharmaceutical composition.

[0069] Acceptable composition or formulation materials for proteins as described herein (e.g., therapeutic antibodies) preferably are nontoxic to recipients at the dosages and concentrations employed. In certain embodiments, a pharmaceutical composition may contain formulation materials for modifying, maintaining or preserving, for example, the pH, osmolality, viscosity, clarity, color, isotonicity, odor, sterility, stability, rate of dissolution or release,adsorption or penetration of the composition. In such embodiments, suitable formulation materials include, but are not limited to, amino acids (such as glycine, glutamine, asparagine, arginine or lysine); antimicrobials; antioxidants (such as ascorbic acid, sodium sulfite or sodium hydrogen-sulfite); buffers (such as borate, bicarbonate, Tris-HCl, citrates, phosphates or other organic acids); bulking agents (such as mannitol or glycine); chelating agents (such as ethylenediamine tetraacetic acid (EDTA)); complexing agents (such as caffeine, polyvinylpyrrolidone, beta-cyclodextrin or hydroxypropyl-beta-cyclodextrin); fillers; monosaccharides; disaccharides; and other carbohydrates (such as glucose, sucrose, mannose or dextrins); proteins (such as serum albumin, gelatin or immunoglobulins); coloring, flavoring and diluting agents; emulsifying agents; hydrophilic polymers (such as polyvinylpyrrolidone); low molecular weight polypeptides; salt-forming counterions (such as sodium); preservatives (such as benzalkonium chloride, benzoic acid, salicylic acid, thimerosal, phenethyl alcohol, methylparaben, propylparaben, chlorhexidine, sorbic acid or hydrogen peroxide); solvents (such as glycerin, propylene glycol or polyethylene glycol); sugar alcohols (such as mannitol or sorbitol); suspending agents; surfactants or wetting agents (such as pluronics, PEG, sorbitan esters, polysorbates such as polysorbate 20, polysorbate, triton, tromethamine, lecithin, cholesterol, tyloxapal); stability enhancing agents (such as sucrose or sorbitol); tonicity enhancing agents (such as alkali metal halides, preferably sodium or potassium chloride, mannitol sorbitol); delivery vehicles; diluents; excipients and / or pharmaceutical adjuvants. See, e g., Remington, The Science and Practice of Pharmacy, 23rdEdition, Academic Press (2020).

[0070] A suitable vehicle or carrier for the composition may be water for injection, or physiological saline solution, possibly supplemented with other materials common in compositions for parenteral administration. Neutral buffered saline or saline mixed with serum albumin are further exemplary vehicles.

[0071] In certain embodiments, buffers are used to maintain the composition at physiological pH or at a slightly lower pH, typically within a pH range of from about 5 to about 8. For example, the pH of the composition may be about 5.1, about 5.2, about 5.3, about 5.4, about 5.5, about 5.6, about 5.7, about 5.8, about 5.9, about 6.0, about 6.1, about 6.2, about 6.3, about 6.4, about 6.5, about 6.6, about 6.7, about 6.8, about 6.9, about 7.0, about 7.1, about 7.2, about 7.3, about 7.4, about 7.5, about 7.6, about 7.7, about 7.8, about 7.9, or about 8.0, including rangesbetween any two of the listed values, such as 5.1 to 8.0, 5.1 to 7.0, 5.5 to 8.0, 5.5 to 7.0, 6.0 to 8.0, or 6.0 to 70.Peptide Mapping

[0072] To monitor therapeutic protein CQAs in co-eluted peptides, peptide mapping has been widely applied and relies on mass spectrometry to resolve co-eluted peptides. However, compared to individual antibodies, co-formulated therapeutic proteins generate more complex enzymatic peptide mixtures. In addition, co-formulated mAbs of the same isotype generate highly abundant constant region consensus peptides which may interfere with CQA peptide detection. As a result, potentially higher frequency of ion suppression in co-eluted peptides may lead to higher variance in attributes quantitation in a co-formulation compared to individual protein. Unexpectedly, methods comprising peptide mapping as described herein accurately monitored co-formulated therapeutic protein CQAs (See Example 1).

[0073] Provided herein are methods of analyzing co-formulated therapeutic proteins using mass spectrometry. In some aspects, the disclosed methods are used to detect and quantify one or more structures or “attributes” that are present on one or both therapeutic proteins in a coformulation. For example, asparagine and glutamine residues are susceptible to deamidation. A deamidated asparagine at position 10 of a therapeutic protein amino acid sequence is an example of an attribute. A list of exemplary attribute types for particular amino acids is provided in Table 1. As such, a “structure” as used herein can comprise, consist essentially of, or consisting of an attribute type listed in Table 1, or a combination of two or more attribute types listed in Table 1. It will be understood that attributes are examples of structures, and unless stated otherwise, wherever a “structure” is mentioned herein, an attribute is contemplated as an example of the structure.Table 1

[0074] As a therapeutic protein comprises multiple amino acids, a therapeutic protein may have more than one attribute (e.g., more than one amino acid having a changed structure) and may be described in terms of its attribute profile. As used herein, the term “attribute profile” refers to a listing of a therapeutic protein’s attributes. The attribute profile may list the absence, presence, or relative abundance of each of the listed attributes. In various instances, the attribute profile provides the chemical identity or attribute type, e.g., deamidation, optionally, relative to the native structure of the therapeutic protein. In various instances, the attribute profile provides the location of the attribute, e.g., the position of the amino acid on which the attribute is present. An attribute profile, in some aspects, provides a description of all assayed attributes present on the therapeutic protein. In other aspects, an attribute profile provides a description of a subset of attributes present on the therapeutic protein. For example, an attribute profile may provide only those attributes that are present in a particular portion of the therapeutic protein, e.g., the extracellular domain, the variable region, the CDR. A species of a therapeutic protein is characterized by the attribute(s) present on the therapeutic protein. A species of a therapeutic protein may differ from another species of the same therapeutic protein by having a different attribute profile. When two of the same therapeutic protein have differing attribute profiles, each of the two therapeutic proteins represents a different species of the therapeutic protein. When two of the same therapeutic protein have identical attribute profiles, each therapeutic protein is considered to be the same species of the therapeutic protein. As used herein, an attribute that “corresponds” to a species (including grammatical conjugations and variations of this root term) refers to an attribute that is comprised by the species, or that leads to the formation of the species. The attribute, by itself, or in combination with one or more other attributes may define the species. By way of example, the attribute may comprise a structure such as a specified methionine oxidation, that is comprised by the species. The species may be defined by a presence or abundance of that structure, and optionally other attributes as well. By way of example, the attribute may lead to the formation of a structure that is comprised by the species (e.g., a deamidation of a specified aspartic acid, which can lead to the formation of isoaspartic acid).

[0075] In exemplary aspects of the disclosure, the method comprises (a) optionally applying a stress to a composition comprising a first therapeutic protein and a second therapeutic protein, wherein the second therapeutic protein is different from the first therapeutic protein; (b) cleaving the first therapeutic protein and the second therapeutic protein into two or more peptides each;(c) separating the two or more peptides of each of the first and second therapeutic proteins; and(d) detecting and quantifying one or more structures present on the two or more peptides of either or both of the first and / or second therapeutic proteins, wherein the one or more structures lead to formation of one or more species of the first and / or second therapeutic protein. Collectively, the processes of protein cleaving, peptide separation, and peptide detection / quantification are known in the art as “peptide mapping.” The term “peptide mapping,” as used herein, refers to a technique used to identify and characterize the primary structure of a protein (e.g., amino acid sequence and / or chemical modifications). Peptide mapping typically involves the enzymatic digestion of a protein into smaller peptides, separation of the peptides, and analysis of the peptides using mass spectrometry. The technique generates a set of unique peptides, or a ‘fingerprint,’ of the interrogated protein. The masses / spectra of the unique peptides identified may be compared to theoretical masses / spectra from sequences found in a proteomics database.

[0076] In some aspects, the method comprises first applying a stress to the composition comprising a first therapeutic protein and a second therapeutic protein. For example, a stress may be applied to induce the formation of attributes in conjunction with characterizing or developing a therapeutic protein, or to identify CQAs. The stress may be any condition which leads to at least one change in structure of an amino acid of the first therapeutic protein and / or an amino acid of the second therapeutic protein, e.g., the stress may be any condition which leads to the formation of at least one attribute at an amino acid of the first and / or second therapeutic protein. Optionally, the stress leads to a change in structure in more than one amino acid of the first therapeutic protein and / or the second therapeutic protein, e.g., the stress leads to the formation of more than one attribute (e.g., at least or about 2, at least or about 3, at least or about 4, at least or about 5, at least or about 6, at least or about 7, at least or about 8, at least or about 9, at least or about 10, or more attributes). Ideally, the stress leads to a change in structure of at least one amino acid of the first therapeutic protein and at least one amino acid of the second therapeutic protein. An attribute may occur at any location within the first or second therapeuticprotein. In some aspects, the first therapeutic protein and / or second therapeutic protein comprises one or more attributes at locations hypothesized as being a part of a paratope or binding site located within the therapeutic protein, wherein the paratope or binding site is the site at which a target interacts with or binds to the therapeutic protein. For example, when the first and second therapeutic proteins are antibodies, one or more attributes may form within one or more CDRs of the first therapeutic protein and / or the second therapeutic protein. In some aspects, one or more attributes may form within one, two, or all three of the CDRs of the antibody heavy chain. Additionally or alternatively, one or more attributes may form within one, two, or all three of the CDRs of the antibody light chain.

[0077] In some aspects, the stress leads to the formation of one or more attributes that are not present in the first and / or second therapeutic protein prior to the application of the stress. Accordingly, in some aspects, the application of stress leads to the formation of species of the first therapeutic protein and / or second therapeutic protein that were not present prior to the application of stress.

[0078] The stress applied may be (1) exposure to visible light, ultra-violet (UV) light, heat, air / oxygen, freeze / thaw cycle, shaking / agitation, chemicals and materials (e.g., metals, metal ions, chaotropic salts, detergents, preservatives, organic solvents, plastics), (2) change in pH (e.g., a change of greater than 1.0, 1.5, or 2.0), pressure, temperature, osmolality, salinity, or (3) long-term storage.

[0079] In some aspects, the change in temperature is about 1°C, about 2°C, about 3°C, about 4°C, about 5°C, or more. In exemplary aspects, the stress comprises or consists of an exposure to elevated temperatures such as, e.g., 25°C, 40°C, or 50°C. In other aspects, the stress comprises or consists of exposure to one or more of the following conditions that mimic some purification conditions or steps: visible and / or ultra-violet light; oxidizing reagents (e.g., hydrogen peroxide); air / oxygen, freeze / thaw cycle, shaking, long-term storage in formulation under the intended product storage conditions; mildly acidic pH (e.g., pH of 3-4) or elevated pH (e.g., pH of 8-9). In some aspects, the stress comprises or consists of a change in pH of greater than 1.0, 1.5, 2.0 or 3.0. In exemplary aspects, the stress comprises or consists of an exposure to ultra-violet light, heat, air, freeze / thaw cycle, shaking, long-term storage, change in pH, or change in temperature, optionally, wherein the change in pH is greater than about 1.0 or greater than about 2.0, optionally, wherein the change in temperature is greater than or about 2°C orgreater than or about 5°C. The methods of the present disclosure are not limited to any particular types of stresses, however.

[0080] In various aspects, the stress applied to the composition leads to formation of at least one species of the first therapeutic protein and / or at least one species of the second therapeutic protein, each species having a unique attribute profile relative to the attribute profile of the first and / or second therapeutic protein prior the applied stress. In some aspects, the stress applied to the composition leads to formation of at least 2 (e.g., 3, 4, 5, 6, 7, 8, 9, or 10 or more) different species of the first therapeutic protein and / or at least 2 (e.g., 3, 4, 5, 6, 7, 8, 9, or 10 or more) different species of the second therapeutic protein. In other aspects, the stress applied to the composition leads to formation of 10 or more (e.g., 20 or more, 30 or more, 40 or more, or 50 or more) different species of the first therapeutic protein and / or 10 or more (e.g., 20 or more, 30 or more, 40 or more, or 50 or more) different species of the second therapeutic protein. In exemplary aspects, the applied stress leads to formation of at least 2 different species of the first therapeutic protein and at least 2 different species of the second therapeutic protein in the composition.

[0081] In various aspects, the attribute comprises or consists of a chemical modification, such as a chemical modification that alters the mass-to-charge ratio (m / z) of charged ions of an amino acid of the first and / or second therapeutic protein. In other aspects, the chemical modification may be, e.g., glycosylation, hydroxylation, glycation, deamidation, oxidation, reduction, isomerization, aggregation, degradation, acetylation, or clipping due to hydrolysis proteolysis.

[0082] As noted above, peptide mapping involves cleaving a protein into smaller peptides. Thus, in various aspects, the disclosed method comprises cleaving the first therapeutic protein and the second therapeutic protein into two or more peptides each. Protein cleavage or “digestion” for peptide mapping may be accomplished using chemical or enzymatic means. It will be appreciated that different cleavage agents have different amino acid specificities and cleavage types, and the choice of cleavage agent will depend on the particular therapeutic proteins in co-formulation. Exemplary chemical cleavage agents include, but are not limited to, cyanogen bromide, 2-Nitro-5-thio-cyanobenzoic acid, O-Iodosobenzoic acid, dilute acid, and BNPS-skatole. Exemplary enzymatic cleavage agents include, but are not limited to, trypsin, chymotrypsin, pepsin, lysyl endopeptidase (Lys-C endopeptidase), glutamyl endopeptidase,peptidyl-Asp metallo-endopeptidase (endoproteinase Asp-N), and clostripain. In some aspects, cleaving the first and second therapeutic proteins comprises treating the composition with a proteolytic enzyme, such as Lys-C, Asp-N, chymotrypsin, trypsin, or human neutrophil elastase. In various aspects, the proteolytic enzyme is trypsin. Techniques and conditions for optimal proteolytic enzyme digestion of proteins are known in the art and may be used in connection with the disclosed methods (see, e.g., European Pharmacopoiea 6.6, Peptide Mapping 2.2.55 (2009); and United States Pharmacopeia / National Formulary, 1005 (2009)).

[0083] In some aspects, either or both of the first therapeutic protein and second therapeutic protein of the composition are cleaved into two or more (e.g., 3, 4, 5, 6, 7, 8, 9, 10 or more) peptides each. In exemplary aspects, both the first and second therapeutic proteins are cleaved into two or more peptides each. Moreover, it is within the scope of the disclosure that cleavage of the first therapeutic peptide produces a different number of peptides than cleavage of the second therapeutic protein. For example, cleavage of the first therapeutic protein may produce three peptides, while cleavage of the second therapeutic protein may produce seven peptides. In other aspects, the first and second therapeutic proteins are cleaved into peptides of equal number.

[0084] Following cleavage of the first and second therapeutic proteins in the composition, the disclosed method further comprises separating the two or more peptides of each of the first and second therapeutic proteins; and detecting and quantifying one or more structures present on the two or more peptides of either or both of the first and / or second therapeutic proteins.

[0085] Peptide separation may be achieved using any separation technique suitable for peptide mapping. Examples of such methods include, but are not limited to, reversed-phase high performance liquid chromatography (RP-HPLC), Ion-exchange chromatography (IEC), hydrophobic interaction chromatography (HIC), polyacrylamide gel electrophoresis (PAGE), non-denaturing sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE), capillary electrophoresis (CE), paper chromatography-high voltage (PCHV), and high voltagepaper electrophoresis (HVPE). In some embodiments, peptide separation is achieved using a high performance liquid chromatography (HPLC) methodology. HPLC, also referred to as high- pressure liquid chromatography, is a technique used to separate, identify, and quantify each component in a mixture.

[0086] In exemplary aspects, the two or more peptides of the first and second therapeutic proteins are separated by liquid chromatography and analyzed via on-line mass spectrometry.Liquid chromatography-mass spectrometry (LC-MS) is typically used to analyze thermally unstable and nonvolatile molecules (e.g., biological fluids). Protocols for LC-MS peptide mapping are known in the art. See, e.g., Ren et al., Anal.Biochem., 392: 12-21 (2009), and Mouchahoir and Schiel, Anal Bioanl Chem., 410(8): 2111-2126 (2018). Briefly, the therapeutic protein undergoes a digestion process following exposure to one or more stresses, as described herein, to produce peptides with structures or attributes. Once produced, the peptides are loaded onto a column for the subsequent liquid chromatography (e.g., HPLC). Following chromatographic separation, in some aspects, mass spectrometry (MS) is employed to detect and quantify one or more structures present on the two or more peptides of the first and / or second therapeutic proteins. Generally, mass spectrometry is an analytical technique that ionizes chemical species and sorts the ions based on their mass-to-charge ratio (m / z). In this manner, an MS analyzer can measure the mass of a molecule within a sample. In the case of polypeptide attributes, use of mass spectrometry allows for the assessment of more quality attributes using fewer analyses. Following separation, peptides are ionized and sent to a mass analyzer to measure the mass / charge ratio of each peptide. Once ions are separated by a mass analyzer, they reach an ion detector, which generates a current signal from the incident ions. The most commonly used ion detector is an electron multiplier, which converts the arrival of an ion into a cascade of secondary electrons. In some aspects, peptides may be further fragmented and analyzed by tandem mass spectrometry (also known as MS / MS, MS2, or MS11), a two-step technique used to analyze a sample either by using two or more mass spectrometers connected to each other or a single mass spectrometer by several analyzers arranged one after another.

[0087] In some aspects, the mass spectrometry may involve electrospray ionization, matrix assisted laser desorption ionization (MALDI) (e.g., MALDLTOF-MS), or fast-atom bombardment. Mass analyzers that may be used in connection with the disclosed methods include quadrupole, quadrupole ion trap, linear ion trap, time-of-flight (TOF) (e.g., MALDLTOF and quadrupole-TOF), magnetic sector, and Fourier transform mass spectrometry analyzers. In exemplary aspects, an electron multiplier is used to detect separated ions, however, other ion detectors (e.g., Faraday cup, photomultiplier conversion dynode, array detector, or charge detector) are within the scope of the disclosure. The experimentally observed spectra obtained by the disclosed method may be compared to theoretical spectra predicted from sequences in protein databases (e.g., Peptide Atlas and National Institute of Standards and Technology (NIST)MS Search) to aid in identification. Mass spectrometry analysis of proteins is further described in, e.g., Hoffmann DE, Stroobant N. Mass spectrometry: principles and applications. 2nd edition. John Wiley & Sons (2001); Finehout EJ, Lee KH , Biochem Mol Biol Educ, 32: 93-100 (2004); and Bakhtiar R, Tse FL, Mutagenesis, 15: 415-30 (2000).Therapeutic Protein Ratio in Co-Formulation

[0088] In some aspects, one of skill in the art may employ the peptides of the first and second therapeutic proteins generated by the methods described herein to obtain a ratio of the first therapeutic protein to the second therapeutic protein in the composition. Thus, the disclosed method further comprises determining a ratio of the first therapeutic protein to the second therapeutic protein in the composition based on the abundance of one or more peptides characteristic of the first therapeutic protein (but not the second therapeutic protein) and the abundance of one or more peptides characteristic of the second therapeutic protein (but not the first therapeutic protein) generated via cleavage as described herein. It will be appreciated that ratio determination using these “surrogate” peptides requires no additional data acquisition. In some aspects, the surrogate peptides selected for ratio calculation include at least a portion of one or more CDRs of the first and second therapeutic proteins. For example, when the therapeutic proteins are antibodies, the ratio may be calculated based on the abundance of one or more peptides including all or part of CDR1, CDR2, and / or CDR3 of the heavy chain of the first therapeutic protein and the abundance of one or more corresponding peptides of the second therapeutic protein. Additionally or alternatively, the ratio may be calculated based on the abundance of one or more peptides including all or part of CDR1, CDR2, and / or CDR3 of the light chain of the first therapeutic protein and the abundance of one or more corresponding peptides of the second therapeutic protein. In some aspects, peptide abundance may be determined using the area of the mass spectrum peak generated by a particular peptide. In exemplary aspects, the median of surrogate peptide peak areas for each therapeutic protein is used to determine the ratio of first therapeutic protein to second therapeutic protein. In some embodiments, the ratio of first protein to second protein in the composition may be, for example, 1 : 1 to 1: 100, such as 1 :5, 1 :10, 1 :15, 1 :20, 1:30, 1:40, 1 :50, 1 :60, 1 :70, 1 :80, 1 :90, 1 :95, 99:1, 95: 1, 85: 1, 75: 1, 65: 1, 55:1 : 45: 1, 35: 1, 25: 1, 20:1, 10: 1, 5: 1, or a range defined any two of the foregoing ratios.

[0089] Not to be bound by theory, it is believed that determining the ratio of surrogate peptides generated via peptide mapping has the potential to cover a wider ratio range due to high dynamic range compared with purity assay -based methods. In some aspects, determining the ratio of therapeutic proteins in co-formulation by the disclosed methods may be utilized to assess quality and stability of the co-formulation for lot release.

[0090] Other orthogonal methods for determining protein concentration may be employed in order to cross-check or confirm the results obtained by the disclosed methods. Such methods include, but are not limited to, turbidimetric assays, nephelometric assays, and colorimetric assays. In turbidimetric and nephelometric assays, a protein is quantified from the change in the turbidity of the reaction mixture based on the agglutination of the protein and a protein-specific binding partner. In colorimetric assays, a protein may be quantified with the aid of a color reagent. Colorimetric assays are characterized by formation, change, or depletion of color in the presence of the protein to be quantified. Exemplary colorimetric assays include the Coomassie blue G-250 dye-binding (Bradford), bicinchoninic acid (BCA), and Lowry assay.

[0091] In some methods, if the concentration of the first therapeutic protein and the second therapeutic protein are each within a specified range, for example a concentration specification, further processing is performed on the composition in order to manufacture a pharmaceutical composition suitable for medical use. For example, excipients may be added to the composition, or the composition may be disposed in a container suitable for storage or a device suitable for administration such as a syringe or pen.Intact MS Analysis

[0092] The disclosure further provides a method of resolving a first therapeutic protein from a second therapeutic protein in a composition utilizing intact mass spectrometry. As used herein, the term “resolve” refers to distinguishing between ions of different mass-to-charge ratios. Intact mass spectrometry refers to a technique for analyzing and characterizing proteins by mass spectrometry without prior digestion or fragmentation of the protein of interest. Intact mass spectrometry provides a mass measurement for the entire protein, as well as information about its size, structure, and post-translational modifications (PTMs). With respect to therapeutic proteins, intact mass spectrometry is useful for assessing protein purity and gaining insights into the heterogeneity of complex biomolecules. Localizing PTMs after intact mass spectrometrytypically involves tandem mass spectrometry analysis, and the measurement of intact protein mass followed by tandem MS (MS-MS) is sometimes referred to as “top-down” mass spectrometry. Additional details regarding protein structure can be obtained, and the mass spectrum may be simplified, by treating a sample with different enzymes before analysis by intact MS. For example, treatment with glycosidase, such as PNGase F, may be used to remove heterogeneity associated with N-glycans. Similarly, a reducing agent can be utilized to break inter-chain disulfide bonds, such as in the case of antibodies. Indeed, conventional reduced intact MS analysis is used in the art to separate the heavy and light chains of an individually- formulated monoclonal antibody prior to mass spectrometry analysis. In contrast, a coformulation comprising two or more different monoclonal antibodies includes at least two sets of light chains and heavy chains, and this increased complexity may require further resolution of co-eluting species following intact MS.

[0093] Thus, in some aspects, the method comprises performing reduced intact mass spectrometry on a composition comprising a first therapeutic protein and a second therapeutic protein that is different from the first therapeutic protein, wherein at least one subunit of the first therapeutic protein co-elutes with at least one subunit of the second therapeutic protein. A protein “subunit,” as used herein, refers to a polypeptide chain or single protein molecule that assembles (or “coassembles”) with one or more other polypeptide chains or proteins to form a protein complex. In exemplary aspects, as described herein, the first therapeutic protein and the second therapeutic protein may each comprise a monoclonal antibody. In such cases, a subunit of the first or second therapeutic protein may comprise an antibody heavy chain polypeptide or an antibody light chain polypeptide. In some aspects, the subunit of the first therapeutic protein is an antibody light chain and the subunit of the second therapeutic protein is an antibody light chain. In other aspects, the subunit of the first therapeutic protein is an antibody heavy chain and the subunit of the second therapeutic protein also is an antibody heavy chain. The disclosure is not limited to subunits of antibodies, however, and subunits of other therapeutic protein complexes or “multimers” may be separated and resolved in accordance with the above methods.

[0094] Depending on the difference in molecular weights between the subunits, the subunits may be resolved by one of three different methods. In some aspects, when the difference in molecular weights of the subunits comprises at least, or more than about 6 Daltons (Da) (e.g., about 7 Da, 8 Da, 9 Da, 10 Da, 15 Da, 20 Da, 25 Da or more), the subunit of the first therapeuticprotein may be resolved from the subunit of the second therapeutic protein by mass spectrometry (e.g., conventional mass spectrometry). In some aspects, a protein subunit may be fragmented prior to analysis by mass spectrometry, as described herein. Any suitable mass spectrometry technique may be used, such as those described herein. In exemplary aspects, time-of-flight (TOF) mass spectrometry is used. TOF analysis utilizes an electric field to accelerate generated ions through the same electrical potential, and then measures the time each ion takes to reach the detector. Because the ions have the same energy, but a different mass, lighter ions reach the detector first because of their greater velocity, while heavier ions have lower velocity and take longer to reach the detector due. Thus, ion mass is determined from the ion’s time of arrival.

[0095] In other aspects, the difference in molecular weights of the subunits may comprise less than 6 Da. In such cases, the resolving power of conventional mass spectrometry methods may not be sufficient to distinguish between subunits. The “resolving power (R)” of a particular mass analyzer can be defined as the value of the full width at half of the peak’s maximum value (FWHM) at a specific m / z (e.g., m / z 400). Thus, in exemplary aspects, when the difference in molecular weights of the subunits comprises less than about 6 Da and more than about 1 Da (e g., 5 Da, 4 Da, 3 Da, or 2 Da, or 1.5 Da), the subunit of the first therapeutic protein may be resolved from the subunit of the second therapeutic protein by high resolution mass spectrometry (HRMS). HRMS differs from conventional spectrometry (often referred to as low resolution mass spectrometry) by providing both high resolution and high mass accuracy. The degree to which a mass analyzer can accurately determine m / z information is largely a function of an analyzer’s stability and resolution. Commonly used high-resolution mass spectrometers include, but are not limited to, time-of-flight (TOF) mass spectrometers, ion cyclotron resonance (ICR) mass spectrometers, and Orbitrap mass spectrometers. In some aspects, the HRMS comprises a resolving power of greater than 10,000 (e.g., 12,000, 15,000, 16,000, 20,000, 50,000, 60,000 or more) at m / z 400. In other aspects, the HRMS comprises a resolving power of 100,000 at m / z 400. An exemplary mass analyzer with a resolving power of 100,000 at m / z 400 for use in the disclosed methods is an Orbitrap mass analyzer. The Orbitrap is an ion trap mass analyzer that consists of two outer electrodes and a central electrode, which enable it to act as both an analyzer and detector. Orbitrap mass spectrometers can deliver a total possible maximum resolution (FWHM) of 1,000,000 at m / z 200 and a sub-1 ppm mass accuracy (see, e.g., Hu et al., J Mass Spectrom., 40(4): 430-43 (2005)).

[0096] It is also possible that the molecular weights of the subunits of the first therapeutic protein and second therapeutic protein differ by no more than or less than 1 Da. In such instances, the highest resolution mass spectroscopy may not be sufficient to distinguish between the two subunits. Thus, in some aspects, when the difference in molecular weights of the subunits comprises less than (or no more than) about 1 Da, the subunit of the first therapeutic protein may be resolved from the subunit of the second therapeutic protein by liquid chromatography. Any suitable liquid chromatography method may be used, several of which are described herein. In some aspects, the liquid chromatography comprises reverse phase high performance liquid chromatography (RP-HPLC), which involves the separation of molecules on the basis of hydrophobicity. The separation depends on the hydrophobic binding of the solute molecule from the mobile phase to the immobilized hydrophobic ligands attached to the stationary phase. RP-HPLC is used extensively in the art to isolate peptides and proteins from a wide variety of synthetic or biological sources (see, e.g., Aguilar, M-I. (ed.), HPLC of Peptides and Proteins: Methods and Protocols, Human Press (2004)).

[0097] Retention and elution from HPLC columns may be achieved by a gradient elution. The term “gradient elution,” as used herein, refers to a technique used in HPLC where the composition of the mobile phase (the solvent that carries the sample through the chromatographic column) is changed during the course of the analysis. In some HPLC applications, the use of salt gradients or pH gradients can result in a high degree of protein fractionation based on protein charge. In RP-HPLC, however, organic modifiers such as acetonitrile or propanol are typically added to the elution buffer to decrease the water concentration in the mobile phase, instead of using salt gradients to elute hydrophobic species. It will be appreciated that gradient steepness is an important parameter of HPLC which can impact resolution, separation efficiency, and analysis time. The “gradient steepness” refers to how quickly the composition of the mobile phase changes during the gradient elution. Steep gradients change the mobile phase composition rapidly, while shallow gradients change it more gradually. Factors influencing the choice of gradient steepness include the complexity of the sample, the chromatographic column properties, and the desired chromatographic resolution. Steeper gradients are often used for fast analyses, while shallower gradients may be employed for more complex samples requiring higher resolution. Thus, in some aspects, when separating protein species or subunits which are structurally similar, the use of shallow gradients may beadvantageous. Shallow gradients can be obtained either by increasing the gradient time or the flow rate. In some aspects, subunits of the first and second therapeutic protein may be resolved by liquid chromatography comprising a shallow gradient.

[0098] Gradient steepness is often expressed as a rate of change in the composition of the mobile phase per unit of time. Common units for gradient steepness include percentage of organic solvent change per minute (%B / min), where B represents the organic solvent in the mobile phase. The actual determination of the gradient steepness involves setting the rate at which the mobile phase composition changes during the elution. An elution gradient of about l%B / min is commonly used in HPLC protocols. A shallow gradient typically involves a relatively slow change in the composition of the mobile phase over time. In some aspects, an elution gradient of less than l%B / min is considered a shallow gradient. For example, a shallow gradient may comprise a 0.1% to 0.99% (or even lower) change in organic solvent concentration per minute. An exemplary shallow gradient for use in the disclosed method may comprise a mobile phase strength of 32% to 37% over an elution time of 12-20 minutes, which corresponds a gradient of 0.625%B / minute.

[0099] Resolving subunits of the first and second therapeutic protein in co-formulation may further comprise deconvoluting the individual mass of the subunit of the first therapeutic protein and the individual mass of the subunit of the second therapeutic protein. In mass spectrometry, the term “deconvolution” (including grammatical conjugations and variations of this root term) refers to the process of extracting a mass spectrum from a set of raw data that may be convoluted or overlapped. Overlapping of ions with different masses in the recorded spectrum can occur due to factors such as instrumental limitations, resolution, or the presence of isotopes. Deconvolution methods are used to separate these overlapping signals and reconstruct the original spectrum. Deconvolution techniques involve mathematical algorithms to extract the observed mass spectrum into individual components, each corresponding to a distinct molecular species. Any suitable deconvolution strategy or algorithm may be used in connection with the disclosed methods, a variety of which are known in the art (see, e.g., Xu et al., Rapid Communications in Mass Spectrometry, 3 / (10): 763-774 (2018)).Multi-Attribute Methods

[0100] The disclosure further provides a method of analyzing a first therapeutic protein and a second therapeutic protein in a composition via a multi-attribute method (MAM). MAM refers to an analytical approach that can quantify multiple product and process attributes (e.g., quality attributes / CQAs) within a single analysis. For therapeutic proteins, MAM typically involves a large-scale, targeted search of peptide mapping data using commercially available software. MAM may also employ site-specific quantification for a particular amino acid modification. An example conventional MAM workflow for protein therapeutics is described in Rogers et al., AAPS Journal, 20: 7 (2018) and generally involves: (1) protein digestion; (2) LC-MS / MS; (3) interrogation of data with an automated search algorithm; and (4) creation of a processing method to monitor product quality attributes. MAM-based assays are typically targeted towards monitoring downstream processes, but they are increasingly used for quality control assays for lot (e.g., sample) release as well. The MAM for analysis of a co-formulation provided herein is believed to address challenges presented by conventional separation methods (e.g., cationic exchange chromatography (CEX), reduced capillary electrophoresis sodium dodecyl sulfate (rCE-SDS), and ELISA), and is capable of providing specific attribute data for the coformulation.

[0101] In some aspects, the MAM-based analysis of the present disclosure comprises (a) providing a composition comprising a first therapeutic protein and a second therapeutic protein that is different from the first therapeutic protein, wherein the ratio of first therapeutic protein to second therapeutic protein in the composition ranges from 1 :20 to 20:1; (b) contacting a sample of the composition with a proteolytic enzyme in a 10:1 ratio, wherein the sample comprises about 200 pg of total protein, and wherein each of the first and second therapeutic proteins is cleaved into two or more peptides by the proteolytic enzyme; and (c) analyzing a sample of the two or more peptides via liquid chromatography-mass spectrometry (LC-MS), whereby one or more structures present on the two or more peptides of either or both of the first and / or second therapeutic protein are detected and quantified; wherein the total amount of peptides analyzed via LC-MS is about 4.0-4.5 pg; and wherein the one or more structures lead to formation of one or more species of the first and / or second therapeutic protein. Descriptions of the first and second therapeutic proteins, LC-MS, protein structures, protein species, and samples set forth elsewhere herein also are applicable to those same aspects of the disclosed MAM analysis.

[0102] In some aspects, the ratio of first therapeutic protein to second therapeutic protein in the composition ranges from 1 :20 to 20: 1. For example, the ratio of first therapeutic protein to second therapeutic protein in the composition may be 1 :20,1: 15, 1 : 10, 1 :5, 1 :2, 2: 15, 2: 10, 2:5, 2: 1, 3:20, 3: 15, 3: 10, 3:5, 3:2, 4:20, 4: 15, 4: 10, 4:5, 4:3, 5:20, 20: 1, 15: 1, 10:1, 5: 1, 2: 1, 15:2, 10:2, 5:2, 1:2, 20:3, 15:3, 10:3, 5:3, 2:3, 20:4, 15:4, 10:4, 5:4, 3:4, 20:5, or a range defined by any two of the aforementioned ratios. In some aspects, there is a different amount of first therapeutic protein than second therapeutic protein in the composition. As such, in some aspects, the ratio of first therapeutic protein to second therapeutic protein in the composition is not 1 : 1. For example, the composition may comprise first therapeutic protein to second therapeutic protein in the composition ranges from 1 :20 to 20: 1, and in which the ratio of first therapeutic protein to second therapeutic protein is not 1 : 1.

[0103] A sample of the composition may be contacted with any suitable proteolytic enzyme, such as those described herein. For example, the proteolytic enzyme may comprise trypsin, chymotrypsin, human neutrophil elastase, pepsin, lysyl endopeptidase (Lys-C endopeptidase), glutamyl endopeptidase, peptidyl-Asp metallo-endopeptidase (endoproteinase Asp-N), or clostripain. In various aspects, the proteolytic enzyme is trypsin. In some aspects, two or more digests with different proteolytic enzymes are performed. Any suitable amount of proteolytic enzyme may be used to effect cleavage of the first and second therapeutic proteins into two or more peptides. In some aspects, the method comprises contacting a sample of the composition with a proteolytic enzyme: substrate ratio less than about 1 :20. In exemplary aspects, the enzyme: substrate ratio may be 1 : 15, 1 :10, 1 :5, 1 : 1, or less. In some aspects, the enzyme: substrate ratio employed in the MAM analysis is about 1 : 10. In other aspects, the amount of total protein (i.e., first therapeutic protein + second therapeutic protein) in a sample of the composition comprises about 300 pg or less (e.g., 280 pg, 250 pg, 230 pg, 220 pg, 210 pg, 200 pg, 175 pg, 150 pg, 130 pg, 115 pg, 110 pg, 100 pg, 90 pg, 80 pg, 70 pg, 60 pg, 50 pg, 40 pg, 30 pg, 20 pg, 10 pg or less). For example, the sample may comprise about 200 pg of total protein, such as 180 - 220 pg of total protein.

[0104] Following cleavage of the first and second therapeutic proteins, the resulting sample of peptides may be separated and analyzed via liquid chromatography-mass spectrometry (LC- MS). In some aspects, MAM analysis of a co-formulation comprising a first therapeutic protein and a second therapeutic protein may involve loading / injecting twice as much of the total peptideamount on an LC-MS system as compared to a formulation containing a single therapeutic protein. In some aspects, the total amount of peptides analyzed via LC-MS in accordance with the disclosed method is about 4.0-4.5 pg. In some aspects, the total amount of peptides analyzed via LC-MS in accordance with the disclosed method is about 4.4 pg. It will be appreciated that the peptide amount for each respective therapeutic protein will depend on the mixing ratio of the first and second therapeutic proteins in the composition, as described above. For example, the peptide amount for each therapeutic protein analyzed via LC-MS may be about 0.2 pg to about4.3 pg (e.g., 0.3 pg, 0.4 pg, 0.5 pg, 0.6 pg, 0.7 pg, 0.8 pg, 0.9 pg, 1.0 pg, 1.1 pg, 1.2 pg, 1.3 pg,1.4 pg, 1.5 pg, 1.6 pg, 1.7 pg, 1.8 pg, 1.9 pg, 2.0 pg, 2.1 pg, 2.2 pg, 2.3 pg, 2.4 pg, 2.5 pg, 2.6 pg, 2.7 pg, 2.8 pg, 2.9 pg, 3.0 pg, 3.1 pg, 3.2 pg, 3.3 pg, 3.4 pg, 3.5 pg, 3.6 pg, 3.7 pg, 3.8 pg,3.9 pg, 4.0 pg, 4.1 pg, or 4.2 pg), so long as the total peptide amount analyzed is between about4.0 pg and 4.5 pg.

[0105] In some aspects, the sample of the two or more peptides may be analyzed via liquid chromatography with tandem mass spectrometry (LC-MS / MS), which allows for detection and quantification of one or more structures present on the two or more peptides of either or both of the first and / or second therapeutic protein. As discussed herein, LC-MS / MS combines liquid chromatography with two or more mass analyzers in series. The use of liquid chromatography coupled to tandem mass spectrometry aids the separation of the peptide / protein interest from matrix components, improving sensitivity and imprecision and improving specificity by separating interferences, such as isobaric compounds, that cannot be differentiated by the mass spectrometer. It will be appreciated that MS / MS may combine two or more identical types of mass analyzers (e g., TOF / TOF) or two or more different types of mass analyzers (e g., a quadrupole and TOF). For any MS / MS configuration, mass spectrometry analysis is accomplished by performing the first mass analysis or filtering, passing selected ions into a collision cell where fragmentation occurs, followed by a second mass analysis.Therapeutic Proteins

[0106] As used herein “therapeutic protein,” and variations of this root term, has its ordinary and customary meaning as would be understood by one of ordinary skill in the art in view of this disclosure. It refers to a polypeptide for medical use in a subject, typically a human subject. By way of example, a therapeutic protein may be a polypeptide approved for medical use by agovernment regulatory authority, such as the Food and Drug Administration or the European Medicines Agency. In some embodiments, each of the first protein and the second protein is a therapeutic antigen-binding protein, such as a therapeutic antibody.

[0107] Therapeutic antigen-binding proteins, such as antibodies, encompassed by the present disclosure may include polypeptides that bind to one or more of the following: (i) CD proteins, including CD3, CD4, CD8, CD19, CD20, CD22, CD30, CD33, CD34, and CD40; including those that interfere with receptor binding; (ii) HER receptor family proteins, including HER2, HER3, HER4, and the EGF receptor; (iii) cell adhesion molecules, for example, LFA-I, Mol, pl 50, 95, VLA-4, ICAM-I, VCAM, and alpha v / beta 3 integrin; (iv) growth factors, such as vascular endothelial growth factor (“VEGF”), growth hormone, thyroid stimulating hormone, follicle stimulating hormone, luteinizing hormone, growth hormone releasing factor, parathyroid hormone, Mullerian-inhibiting substance, human macrophage inflammatory protein (MIP- 1 alpha), erythropoietin (EPO), nerve growth factor, such as NGF-beta, platelet-derived growth factor (PDGF), fibroblast growth factors, including, for instance, aFGF and bFGF, epidermal growth factor (EGF), transforming growth factors (TGF), including, among others, TGF-a and TGF-0, including TGF-01, TGF-02, TGF-03, TGF- 04, or TGF- 05, insulin-like growth factors-I and -II (IGF-I and IGF-II), des(l-3)-IGF-I (brain IGF-I), and osteoinductive factors; (v) insulins and insulin-related proteins, including insulin, insulin A-chain, insulin B-chain, proinsulin, and insulin-like growth factor binding proteins; (vi) coagulation and coagulation-related proteins, such as, among others, factor VIII, tissue factor, von Willebrand factor, protein C, alpha- 1- antitrypsin, plasminogen activators, such as urokinase and tissue plasminogen activator (“t-PA”), bombazine, thrombin, and thrombopoietin; (vii) other blood and serum proteins, including but not limited to albumin, IgE, and blood group antigens; (viii) colony stimulating factors and receptors thereof, including the following, among others, M-CSF, GM-CSF, and G-CSF, and receptors thereof, such as CSF-1 receptor (c-fms); (ix) receptors and receptor-associated proteins, including, for example, flk2 / flt3 receptor, CD112 receptor (CD112R), obesity (OB) receptor, LDL receptor, growth hormone receptors, thrombopoietin receptors (“TPO-R,” “c- mpl”), glucagon receptors, interleukin receptors, interferon receptors, T-cell receptors, stem cell factor receptors, such as c-Kit, and other receptors; (x) receptor ligands, including, for example, OX40L, the ligand for the 0X40 receptor; (xi) neurotrophic factors, including bone-derived neurotrophic factor (BDNF) and neurotrophin-3, -4, -5, or -6 (NT-3, NT-4, NT-5, or NT-6); (xii)relaxin A-chain, relaxin B-chain, and prorelaxin; (xiii) interferons and interferon receptors, including for example, interferon-a, -P, and -y, and their receptors; (xiv) interleukins and interleukin receptors, including IL-1 to IL-33 and IL-1 to IL-33 receptors, such as the IL-8 receptor or IL-21 receptor, among others; (xv) viral antigens, including an AIDS envelope viral antigen; (xvi) other proteins such as, e.g., lipoproteins, calcitonin, glucagon, atrial natriuretic factor, lung surfactant, tumor necrosis factor-alpha and -beta, enkephalinase, Programmed Cell Death 1 (PD-1), Programmed Cell Death Ligand 1 (PD-L1), T cell immunoreceptor with Ig and ITIM domains (TIGIT), RANTES (regulated on activation normally T-cell expressed and secreted), mouse gonadotropin-associated peptide, DNAse, inhibin, activin, integrin, protein A or D, rheumatoid factors, immunotoxins, bone morphogenetic protein (BMP), superoxide dismutase, surface membrane proteins, decay accelerating factor (DAF), HIV envelope, transport proteins, homing receptors, addressins, regulatory proteins, immunoadhesins, myostatins, TALL proteins, including TALL-I, amyloid proteins, including but not limited to amyloid-beta proteins, thymic stromal lymphopoietins (“TSLP”), RANK ligand (“RANKL” or “OPGL”), c-kit, TNF receptors, including TNF Receptor Type 1, TRAIL-R2, angiopoi etins, calcitonin gene-related peptide receptor (CGRPR), mesothelin, six-transmembrane epithelial antigen of prostate 1 (STEAP1), mucin 17 (MUC17), claudin 6 (CLDN6), claudin 18.2 (CLDN18.2), and biologically active fragments or analogs or variants of any of the foregoing.

[0108] Examples of therapeutic antibodies suitable for the methods described herein include infliximab, bevacizumab, cetuximab, ranibizumab, palivizumab, abagovomab, abciximab, actoxumab, adalimumab, afelimomab, afutuzumab, alacizumab, alacizumab pegol, ald518, alemtuzumab, alirocumab, altumomab, amatuximab, anatumomab mafenatox, anrukinzumab, apolizumab, arcitumomab, aselizumab, altinumab, atlizumab, atorolimiumab, tocilizumab, bapineuzumab, basiliximab, bavituximab, bectumomab, belimumab, bemarituzumab, benralizumab, bertilimumab, besilesomab, bevacizumab, bezlotoxumab, biciromab, bivatuzumab, bivatuzumab mertansine, blinatumomab, blosozumab, brentuximab vedotin, briakinumab, brodalumab, canakinumab, cantuzumab mertansine, cantuzumab mertansine, caplacizumab, capromab pendetide, carlumab, catumaxomab, cc49, cedelizumab, certolizumab pegol, cetuximab, citatuzumab bogatox, cixutumumab, clazakizumab, clenoliximab, clivatuzumab tetraxetan, conatumumab, crenezumab, cr6261, dacetuzumab, daclizumab, dalotuzumab, daratumumab, demcizumab, denosumab, detumomab, dorlimomab aritox,drozitumab, duligotumab, dupilumab, ecromeximab, eculizumab, edobacomab, edrecolomab, efalizumab, efungumab, elotuzumab, elsilimomab, enavatuzumab, enlimomab pegol, enokizumab, enoticumab, ensituximab, epitumomab cituxetan, epratuzumab, erenumab, erlizumab, ertumaxomab, etaracizumab, etrolizumab, evolocumab, exbivirumab, fanolesomab, faralimomab, farletuzumab, fasinumab, fbtaO5, felvizumab, fezakinumab, ficlatuzumab, figitumumab, flanvotumab, fontolizumab, foralumab, foravirumab, fresolimumab, fulranumab, futuximab, galiximab, ganitumab, gantenerumab, gavilimomab, gemtuzumab ozogamicin, gevokizumab, girentuximab, glembatumumab vedotin, golimumab, gomiliximab, gs6624, ibalizumab, ibritumomab tiuxetan, icrucumab, igovomab, imciromab, imgatuzumab, inclacumab, indatuximab ravtansine, infliximab, intetumumab, inolimomab, inotuzumab ozogamicin, ipilimumab, iratumumab, itolizumab, ixekizumab, keliximab, labetuzumab, lebrikizumab, lemalesomab, lerdelimumab, lexatumumab, libivirumab, ligelizumab, lintuzumab, lirilumab, lorvotuzumab mertansine, lucatumumab, lumiliximab, mapatumumab, maslimomab, mavrilimumab, matuzumab, mepolizumab, metelimumab, milatuzumab, minretumomab, mitumomab, mogamulizumab, morolimumab, motavizumab, moxetumomab pasudotox, muromonab-cd3, nacolomab tafenatox, namilumab, naptumomab estafenatox, namatumab, natalizumab, nebacumab, necitumumab, nerelimomab, nesvacumab, nimotuzumab, nivolumab, nofetumomab merpentan, ocaratuzumab, ocrelizumab, odulimomab, ofatumumab, olaratumab, olokizumab, omalizumab, onartuzumab, oportuzumab monatox, oregovomab, orticumab, otelixizumab, oxelumab, ozanezumab, ozoralizumab, pagibaximab, palivizumab, panitumumab, panobacumab, parsatuzumab, pascolizumab, pateclizumab, patritumab, pemtumomab, perakizumab, pertuzumab, pexelizumab, pidilizumab, pintumomab, placulumab, ponezumab, priliximab, pritumumab, PRO 140, quilizumab, racotumomab, radretumab, rafivirumab, ramucirumab, ranibizumab, raxibacumab, regavirumab, reslizumab, rilotumumab, rituximab, robatumumab, roledumab, romosozumab, rontalizumab, rovelizumab, ruplizumab, samalizumab, sarilumab, satumomab pendetide, secukinumab, sevirumab, sibrotuzumab, sifalimumab, siltuximab, simtuzumab, siplizumab, sirukumab, solanezumab, solitomab, sonepcizumab, sontuzumab, stamulumab, sulesomab, suvizumab, tabalumab, tacatuzumab tetraxetan, tadocizumab, talizumab, tanezumab, taplitumomab paptox, tarlatamab, tefibazumab, telimomab aritox, tenatumomab, tefibazumab, teneliximab, teplizumab, teprotumumab, tezepelumab, TGN1412, tremelimumab, ticilimumab, tildrakizumab, tigatuzumab, TNX-650, tocilizumab,toralizumab, tositumomab, tralokinumab, trastuzumab, TRBS07, tregalizumab, tucotuzumab celmoleukin, tuvirumab, ublituximab, urelumab, urtoxazumab, ustekinumab, vapaliximab, vatelizumab, vedolizumab, veltuzumab, vepalimomab, vesencumab, visilizumab, volociximab, vorsetuzumab mafodotin, votumumab, zalutumumab, zanolimumab, zatuximab, ziralimumab, zolimomab aritox, or variants of any of the foregoing.

[0109] In accordance with the methods described herein, a composition may comprise two or more of any of the foregoing therapeutic antibodies, or antigen-binding fragments thereof.

[0110] The following examples further illustrate the invention but, of course, should not be construed as in any way limiting its scope.EXAMPLE 1[0U1] This example describes the development of a mass spectrometry -based method to monitor and quantitate CQAs of two different co-formulated monoclonal antibodies (mAbs).

[0112] Two sets of co-formulated antibodies shown in Table 2 were evaluated using intact mass analysis and peptide mapping to monitor CQAs during release and stability testing at initial condition and at various stressed conditions.Table 2Relative Quantitation of Attributes by Peptide Mapping

[0113] Cleavage of Abl individually generated 321 different tryptic peptides, and peptides containing CQAs were virtually impossible to be completely separated from the complex matrix and quantitated by chromatography alone. Here, Abl CQAs in a protein co-formulation were evaluated and quantitated via a peptide mapping method.

[0114] The CQAs of Abl were quantitated in individual mAb form (140 mg / mL and 70 mg / mL) and co-formulated form (Abl+Ab2 and Abl+Ab3). Besides initial condition (To), thermal stress and photo stress conditions were applied. As shown in Figures 1A and IB, the standard deviation of Abl CQAs in different forms (individual and co-formulated) were wellcontrolled and below 0.6% for all conditions. The low variance in attributes quantitation demonstrated the robustness of peptide mapping in co-formulation.Determination of Co-Formulated mAbs Ratio by Surrogate Peptides Peak Area

[0115] Besides CQAs relative quantitation, peptide mapping was also exploited to determine the ratio of mAbs in the Combo 2 co-formulation. The peak area for mAb surrogate peptides were plotted in mirror histograms shown in Figures 2A-2D, and the peak area medians were extracted for calculation of the Abl : Ab 3 ratio. The ratio of median peak area between Abl and Ab3 was 1 : 1 for all conditions, which demonstrated the consistency of the developed method for ratio determination of co-formulated proteins.Reduced Intact Mass Spectrometry Analysis

[0116] Conventional reduced intact mass spectrometry (intact MS) analysis of individual mAb exploits a chromatogram for desalting and separation between light chain and heavy chain prior to mass spectrometry analysis. Co-formulated mAbs include at least two sets of light chains and heavy chains, however, and this increased complexity requires further resolution of the co-eluted species. As shown in Figure 3 A, Combo 1 (Abl+ Ab2) total ion chromatogram showed baseline-separated heavy chains but co-eluted light chains. The time-of-flight (TOF) mass spectra for co-eluted light chains were baseline-resolved with ~5.3 Th difference for the highest abundance peak (see Figures 3B and 3C). The deconvoluted mass spectrum yielded corresponding masses of 23230.04 Da and 23310.07 Da (see Figure 3D).

[0117] Co-eluting light chains and / or heavy chains with small mass differences pose further challenges to intact MS analysis. As shown in Figure 4A, Combo 2 (Abl+ Ab3) total ion chromatogram showed baseline-separated heavy chains but co-eluted light chains. Individual analysis of two light chains yielded mass spectrum with ~0.4 Th difference for the highest abundance peak, and the mass errors of deconvoluted masses were 19 ppm and -28ppm (see Figures 4B-4D). Because positive systematic error was expected, the -28 ppm mass error indicated interference due to inadequate resolving power. This interference was further confirmed by analysis of ensemble co-eluted Abl light chain (LC1) and Ab3 light chain (LC3). As shown in Figures 4E-4G, one broad and unresolved peak at ~m / z 1660 (highest abundancepeak) was obtained, and its corresponding mass was 23232.71 Da (between masses of LC1 and LC3).

[0118] Two approaches may be used to address the challenge of co-eluting species with similar masses. First, the m / z difference of the highest abundance peak of Combo 2 co-eluted light chains was ~0.4 Th, requiring a mass spectrometer with resolving power of -16,000 (at m / z 400) for baseline-resolution. This resolving power requirement can be satisfied by an instrument with 100,000 resolving power, such as an ORBITRAP™ mass spectrometer. However, if the m / z difference of co-eluted species of a co-formulation is extremely small (e.g., 0.002 Th), even the highest resolving power mass spectrometer cannot distinguish the two species (see, e.g., He et al., Clin Chem., 65, 986-994 (2019)). In such cases, optimized chromatography separation is used. For example, as shown in Figure 5A, a substantially shallower gradient for Combo 2 light chain elution was applied, with 32% to 37% in 15 minutes (as compared to original gradient of 30% to 35 in 10 minutes) to achieve baseline separation between LC1 and LC3. Further mass spectrometry analysis yielded light chain masses of 23230.08 Da (LC1) and 23235.65 Da (LC3) with mass errors of 23 ppm and 6 ppm (see Figures 5B-5D).EXAMPLE 2

[0119] This example describes a MAM-based method for analyzing an antibody coformulation.

[0120] Co-formulations of monospecific and bispecific antigen-binding proteins (e.g., monoclonal antibodies) listed in Table 3 were prepared by mixing each protein at the following ratios: 1 :20, 1 : 10, 1 :5, 1 : 1, 5: 1, 10: 1, to 20: 1.Table 3

[0121] Attribute levels of proteins in co-formulation were measured by MAM and compared to MAM analysis of each individual protein. The MAM parameters that were used are shown in Table 4.Table 4

[0122] The attribute levels measured by MAM for each co-formulation were comparable to those measured for each single therapeutic protein, and sufficient sequence coverage for each molecule was observed. Overall, the co-formulation MAM demonstrated similar - if not superior - performance and better sensitivity than the MAM for each individual therapeutic protein, as the loading and injection amounts of each molecule in the co-formulation MAM was comparatively lower than those in the MAM for single molecule.

[0123] Results of the MAM analysis for “Combo 3” which contained Ab4 co-formulated with Ab5 at ratios 10: 1, 5: 1, 1 : 1, 1 :5, and 1 : 10, are shown in Figures 6 and 7. The structures monitored in Ab4 and Ab5 are shown in Table 5.Table 5

[0124] Reference peaks for the MAM analysis of Combo 3 are show in Figure 6. The MAM for this co-formulation enabled attribute identification at the peptide level, and attribute levels measured for the co-formulation were comparable to those measured for the individually- formulated antibodies, as shown in Figure 7. The developed MAM assay met all qualification criteria on specificity, linearity, repeatability, and precision, as shown in Table 6.Table 6

[0125] All references, including publications, patent applications, and patents, cited herein are hereby incorporated by reference to the same extent as if each reference were individually and specifically indicated to be incorporated by reference and were set forth in its entirety herein.

[0126] As used herein, the term “about” when used as a modifier to a specified numerical value (e.g., pH of “about” 7.0), indicates that variation around the numerical value can occur. These variations can occur by a variety of means, such as typical measuring and handling procedures, inadvertent errors, ingredient purity, and the like. If greater numerical precision is required, in some embodiments, “about” may refer to numerical values withing ±5% of the specified numerical value.

[0127] The use of the terms “a” and “an” and “the” and “at least one” and similar referents in the context of describing the invention (especially in the context of the following claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. The use of the term “at least one” followed by a list of one or more items (for example, “at least one of A and B”) is to be construed to mean one item selected from the listed items (A or B) or any combination of two or more of the listed items (A and B), unless otherwise indicated herein or clearly contradicted by context. The terms “comprising,” “having,” “including,” and “containing” are to be construed as open-ended terms (i.e., meaning “including, but not limited to,”) unless otherwise noted. Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., “such as”) provided herein, is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention unless otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention.

[0128] Preferred embodiments of this invention are described herein, including the best mode known to the inventors for carrying out the invention. Variations of those preferred embodiments may become apparent to those of ordinary skill in the art upon reading the foregoing description. The inventors expect skilled artisans to employ such variations as appropriate, and the inventors intend for the invention to be practiced otherwise than as specifically described herein. Accordingly, this invention includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, any combination of the above-described elements in all possible variations thereof is encompassed by the invention unless otherwise indicated herein or otherwise clearly contradicted by context.

Claims

CLAIM(S):

1. A method of analyzing a first therapeutic protein and a second therapeutic protein in a composition, which method comprises:(a) optionally applying a stress to a composition comprising a first therapeutic protein and a second therapeutic protein, wherein the second therapeutic protein is different from the first therapeutic protein;(b) cleaving the first therapeutic protein and the second therapeutic protein into two or more peptides each;(c) separating the two or more peptides of each of the first and second therapeutic proteins;(d) detecting and quantifying one or more structures present on the two or more peptides of either or both of the first and / or second therapeutic proteins, wherein the one or more structures correspond to one or more species of the first and / or second therapeutic protein; and optionally(e) determining a ratio of the first therapeutic protein to the second therapeutic protein in the composition based on an abundance of one or more of the peptides of the first therapeutic protein generated in (b) and an abundance of one or more of the peptides of the second therapeutic protein generated in (b).

2. The method of claim 1 wherein the stress comprises an exposure to ultra-violet light, heat, air, freeze / thaw cycle, shaking, long-term storage, change in pH, or change in temperature.

3. The method of claim 2, wherein the change in pH is greater than about 1.0 or greater than about 2.0, and / or wherein the change in temperature is greater than or about 2°C or greater than or about 5 °C.

4. The method of any one of claims 1-3, wherein the structure comprises a chemical modification, optionally, wherein the chemical modification alters the mass-to-charge ratio (m / z) of charged ions of an amino acid of the therapeutic protein.

5. The method of claim 4, wherein the chemical modification is glycosylation, hydroxylation, glycation, deamidation, oxidation, reduction, isomerization, aggregation, degradation, acetylation, or clipping due to hydrolysis proteolysis.

6. The method of any one of claims 1-5, wherein the stress applied to the composition leads to formation of at least one species of the first therapeutic protein having a unique attribute profile relative to the attribute profile of the first therapeutic protein prior the applied stress.

7. The method of any one of claims 1-6, wherein the stress applied to the composition leads to formation of at least one species of the second therapeutic protein having a unique attribute profile relative to the attribute profile of the second therapeutic protein prior the applied stress.

8. The method of any one of claims 1-7, wherein cleaving comprises treating the composition with a proteolytic enzyme.

9. The method of claim 8, wherein the proteolytic enzyme is trypsin.

10. The method of any one of claims 1-9, wherein the separating of (c) and detecting and quantifying of (d) comprises using liquid chromatography-mass spectrometry.

11. The method of any one of claims 1-10, which comprises determining a ratio of the first therapeutic protein to the second therapeutic protein in the composition based on the abundance of one or more of the peptides of the first therapeutic protein generated in (b) and the abundance of one or more of the peptides of the second therapeutic protein generated in (b).

12. The method of claim 11, wherein the ratio of the first therapeutic protein to the second therapeutic protein in the composition is 1 : 1 to 1 : 100, or 1:2 to 1 TOO, or 1 :2 to 1 :50.

13. The method of claim 12, wherein the ratio of the first therapeutic protein to the second therapeutic protein in the composition is 1 :80, 1 :50, 1 :40, 1 :20, 1 : 10, 1 :5, or 1 : 1.

14. A method of resolving a first therapeutic protein from a second therapeutic protein in a composition, which method comprises performing reduced intact mass spectrometry on a composition comprising a first therapeutic protein and a second therapeutic protein, wherein the second therapeutic protein is different from the first therapeutic protein, and wherein at least one subunit of the first therapeutic protein co-elutes with at least one subunit of the second therapeutic protein, and when the difference in molecular weights of the subunits comprises at least about 6 Da, resolving the subunit of the first therapeutic protein from the subunit of the second therapeutic protein by mass spectrometry; when the difference in molecular weights of the subunits comprises less than about 6 Da and more than about 1 Da, resolving the subunit of the first therapeutic protein from the subunit of the second therapeutic protein by high resolution mass spectrometry (HRMS); or when the difference in molecular weights of the subunits comprises no more than about 1 Da, resolving the subunit of the first therapeutic protein from the subunit of the second therapeutic protein by liquid chromatography comprising a shallow gradient.

15. The method of claim 14, wherein the difference in molecular weights of the subunits comprises at least about 6 Da, and the subunit of the first therapeutic protein is resolved from the subunit of the second therapeutic protein by time-of-flight (TOF) mass spectrometry.

16. The method of claim 14, wherein the HRMS comprises a resolving power of greater than 10,000 at m / z 400.

17. The method of claim 16, wherein the HRMS comprises a resolving power of 100,000 at m / z 400.

18. The method of claim 14, wherein the liquid chromatography comprises reverse phase high performance liquid chromatography (RP-HPLC).

19. The method of claim 14 or claim 18, wherein the shallow gradient comprises a mobile phase strength of 32% to 37% over an elution time of 12-20 minutes.

20. The method of any one of claims 14-18, further comprising deconvolution of the individual mass of the subunit of the first therapeutic protein and the individual mass of the subunit of the second therapeutic protein.

21. The method of any one of claims 14-20, wherein the subunit of the first therapeutic protein is an antibody light chain and the subunit of the second therapeutic protein is an antibody light chain.

22. A method of analyzing a first therapeutic protein and a second therapeutic protein in a composition via a multi-attribute method (MAM), wherein the MAM comprises:(a) providing a composition comprising a first therapeutic protein and a second therapeutic protein, wherein the second therapeutic protein is different from the first therapeutic protein, and wherein the ratio of first therapeutic protein to second therapeutic protein in the composition ranges from 1 :20 to 20: 1;(b) contacting a sample of the composition with a proteolytic enzyme in a 10:1 ratio, wherein the sample comprises about 200 pg of total protein, and wherein each of the first and second therapeutic proteins is cleaved into two or more peptides by the proteolytic enzyme; and(c) analyzing a sample of the two or more peptides via liquid chromatographymass spectrometry (LC-MS), whereby one or more structures present on the two or more peptides of either or both of the first and / or second therapeutic protein are detected andquantified; wherein the total amount of peptides analyzed via LC-MS is about 4.0-4.5 pg; and wherein the one or more structures correspond to one or more species of the first and / or second therapeutic protein.

23. The method of claim 22, wherein the structure forms as a result of a chemical modification, optionally, wherein the chemical modification alters the mass-to-charge ratio (m / z) of charged ions of an amino acid of the therapeutic protein.

24. The method of claim 23, wherein the chemical modification is glycosylation, hydroxylation, glycation, deamidation, oxidation, reduction, isomerization, aggregation, degradation, acetylation, or clipping due to hydrolysis proteolysis.

25. The method of any one of claims 22-24, wherein the proteolytic enzyme is trypsin.

26. The method of any one of claims 22-25, wherein analyzing a sample of the two or more peptides comprises liquid chromatography with tandem mass spectrometry (LC-MS / MS).

27. The method of any one of claims 1-26, wherein each of the first therapeutic protein and the second therapeutic protein is an antigen-binding protein.

28. The method of claim 27, wherein the antigen-binding protein is an antibody, an antibody fragment, or a bispecific T cell engager (BiTE®) molecule.