Systems and methods for analyzing antibody co-formulations

Abstract

The present disclosure provides a system and method for detecting and quantifying two or more different antigen-binding proteins in a sample, which includes donor beads, acceptor beads, and conjugated proteins that can be incubated with the sample and the antigen-binding proteins quantified by a proximity-based homogeneous assay.

Description

[Technical Field]

[0001] The present disclosure relates to systems and methods for detecting and quantifying two or more antigen binding proteins (e.g., antibodies) co-formulated into a single composition. [Background technology]

[0002] Combination therapies using two or more molecules with complementary pharmacological effects have led to increased interest in the development of co-formulated 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 a biologic, or two different biologics, such as a therapeutic antibody) are combined into a single dosage form. These products can often reduce the number and volume of injections, improving patient compliance and reducing discomfort.

[0003] Because co-formulated therapeutics are classified as novel molecular entities (NMEs), they are subject to clinical evaluation by regulatory agencies such as the U.S. Food and Drug Administration (FDA). Thus, even though the efficacy and safety of the individual therapeutics have already been established through independent clinical trials, additional co-formulations of existing therapeutics may be subject to further analysis and evaluation.

[0004] There are also many chemical, manufacturing, and control issues that must be addressed with combination biologics, including analytical challenges in characterizing each molecule in the co-formulation, manufacturing issues in formulating higher concentrations of biologics, and stability issues such as protein-protein interactions, protein aggregation, and the incorporation of particles that are subvisible to the naked eye.

[0005] There remains a need for systems and methods for accurately determining the concentration of co-formulated biological molecules in a pharmaceutical preparation or sample. Summary of the Invention [Means for solving the problem]

[0006] The present disclosure provides a system for detecting and quantifying two or more different antigen-binding proteins in a sample, the system comprising: (a) donor beads (wherein each donor bead comprises a photosensitizer and a first binding pair member); (b) acceptor beads (wherein each acceptor bead comprises a chemiluminescent dye and an anti-idiotype antibody that specifically binds to one of two or more different antigen-binding proteins); and (c) conjugate proteins (wherein each conjugate protein comprises one of two or more different antigen-binding proteins that comprise a second binding pair member), wherein the first binding pair member is configured to specifically bind to the second binding pair member. For example, the donor beads may be conjugated to the photosensitizer and / or the first binding pair member. For example, the acceptor beads may be conjugated to the chemiluminescent dye and / or the anti-idiotype antibody. For example, the antigen-binding protein of the conjugate proteins may be conjugated to the second binding pair member.

[0007] In some embodiments of the system, each of the antigen binding proteins is an antibody, an antibody fragment, or a bispecific T cell engager (BiTE®) molecule.

[0008] In some embodiments of the system, the photosensitizer comprises a phthalocyanine.

[0009] In some embodiments of the system, the first binding pair member comprises streptavidin, glutathione S-transferase (GST), or an affinity tag peptide such as a hexahistidine or FLAG peptide.

[0010] In some embodiments of the system, the second binding pair member comprises biotin, glutathione, or an anti-affinity tag antibody, such as an anti-hexahistidine antibody or an anti-FLAG antibody.

[0011] In some embodiments of the system, the first binding pair member comprises streptavidin and the second binding pair member comprises biotin.

[0012] In some embodiments, the system comprises: (i) a first acceptor bead comprising (e.g., conjugated to) a first chemiluminescent dye and a first anti-idiotypic antibody that binds to a first antigen binding protein in a sample; and (ii) a second acceptor bead comprising (e.g., conjugated to) a second chemiluminescent dye and a second anti-idiotypic antibody that binds to a second antigen binding protein in the sample. In some aspects, the first chemiluminescent dye comprises terbium (Tb) and the second chemiluminescent dye comprises europium (Eu). In some aspects, the first antigen binding protein is a first therapeutic antibody and the second antigen binding protein is a second therapeutic antibody. In some aspects, the system comprises a first conjugated protein comprising a first antigen binding protein and biotin, and a second conjugated protein comprising a second antigen binding protein and biotin. By way of example, a first antigen binding protein may be conjugated to biotin and / or a second antigen binding protein may be conjugated to biotin.

[0013] In some embodiments of the system, the sample is a composition comprising two or more different antigen binding proteins and a pharmaceutically acceptable carrier.

[0014] The present disclosure also provides a method for determining the presence, absence, or concentration of two or more different antigen-binding proteins in a sample, the method comprising: (1) incubating the sample with the above-described system; (2) exposing the incubated sample to light excitation; and (3) detecting at least one chemiluminescent signal after light excitation and comparing the intensity of the detected chemiluminescent signal with a predetermined reference value. The at least one detected chemiluminescent signal is inversely proportional to the respective concentrations of the two or more different antigen-binding proteins. Thus, the presence, absence, or concentration of the two or more different antigen-binding proteins is detected.

[0015] In some aspects of the method, the predetermined reference value comprises a threshold value corresponding to each concentration of the antigen-binding protein, such that a detected chemiluminescent signal greater than the threshold value indicates that the sample lacks or contains less than that concentration of the antigen-binding protein, and a detected chemiluminescent signal equal to or less than the threshold value indicates that the sample contains at least that concentration of the antigen-binding protein.

[0016] In some embodiments of the present methods, the photoexcitation wavelength is from about 500 nm to about 800 nm, for example, about 680 nm.

[0017] In some embodiments of the method, the sample is a composition comprising two or more different antigen binding proteins and a pharmaceutically acceptable carrier.

[0018] In some aspects of the method, each of the antigen binding proteins is an antibody, an antibody fragment, or a bispecific T cell engager (BiTE®) molecule.

[0019] In some embodiments of the method, the system includes (i) a first acceptor bead comprising (e.g., conjugated to) a first chemiluminescent dye and a first anti-idiotypic antibody that binds to a first antigen binding protein in the sample, and (ii) a second acceptor bead comprising (e.g., conjugated to) a second chemiluminescent dye and a second anti-idiotypic antibody that binds to a second antigen binding protein in the sample.

[0020] In some embodiments of the method, the first antigen binding protein comprises a first therapeutic antibody and the second antigen binding protein comprises a second therapeutic antibody.

[0021] In some embodiments, the methods include detecting a first signal emitted from a first chemiluminescent dye and a second signal emitted from a second chemiluminescent dye.

[0022] In some aspects of the method, the first signal and the second signal comprise two different wavelengths.

[0023] In some embodiments of the method, the wavelength of the first signal is from about 520 nm to about 620 nm, and the wavelength of the second signal is from about 520 nm to about 620 nm.

[0024] In some embodiments of the method, the wavelength of the first signal is about 545 nm and the wavelength of the second signal is about 615 nm.

[0025] In some embodiments, the method further comprises quantifying two or more antigen binding proteins in the sample.

[0026] In some aspects of the method, the reference value is from a standard curve, which can be for each acceptor bead in the system.

[0027] In some embodiments, the method further comprises generating a calibration curve for each acceptor bead in the system. [Brief explanation of the drawings]

[0028] [Figure 1] FIG. 1 is a schematic diagram illustrating one embodiment of the method described herein. [Figure 2A] 2A-2B are graphs showing the dose-dependent response of bead-based assays for Ab1 (FIG. 2A) and Ab2 (FIG. 2B) as described in the Examples. [Figure 2B] 2A-2B are graphs showing the dose-dependent response of bead-based assays for Ab1 (FIG. 2A) and Ab2 (FIG. 2B) as described in the Examples. [Figure 3A] 3A and 3B are graphs showing that Ab1 (FIG. 3A) and Ab2 (FIG. 3B) in co-formulations exhibited relative binding rates comparable to their respective controls. [Figure 3B] 3A and 3B are graphs showing that Ab1 (FIG. 3A) and Ab2 (FIG. 3B) in co-formulations exhibited relative binding rates comparable to their respective controls. [Figure 4]Figure 1 shows the linearity of the no-wash assay described in the Examples for Ab1 in co-formulation with Ab2. Various concentrations of Ab1 were co-formulated with 10 mg / mL Ab2. [Figure 5A] 1 is a graph showing that the Ab1- and Ab2-specific reagents used in the co-formulation assays described in the Examples demonstrate high specificity with low potential for reagent cross-reactivity. [Figure 5B] 1 is a graph showing that the Ab1- and Ab2-specific reagents used in the co-formulation assays described in the Examples demonstrate high specificity with low potential for reagent cross-reactivity. [Figure 6A] 6A and 6B are graphs showing terbium (FIG. 6A) and europium (FIG. 6B) signals obtained in the simulated co-formulation assay described in the Examples. [Figure 6B] 6A and 6B are graphs showing terbium (FIG. 6A) and europium (FIG. 6B) signals obtained in the simulated co-formulation assay described in the Examples. DETAILED DESCRIPTION OF THE INVENTION

[0029] The present disclosure is based, at least in part, on the development of a homogeneous multiplex assay for the simultaneous detection and quantification of co-formulated biomolecules (e.g., antibodies) in a single sample. The no-wash assay described herein is adaptable to a variety of co-formulations and scalable to larger formats.

[0030] In some embodiments, the present disclosure provides a bead-based system for detecting and quantifying two or more different antigen-binding proteins in a sample. When biological interactions bring the beads into close proximity (i.e., ≦200 nm), a cascade of chemical reactions occurs, generating a greatly amplified signal. Upon photoexcitation, a photosensitizer (e.g., phthalocyanine) in the "donor" bead converts ambient oxygen to a more excited singlet state at a rate of approximately 60,000 oxygen singlets per second (see, e.g., Eglen, RM et. al., Curr Chem Genomics, 1:2-10 (2008) PMID 20161822). Singlet oxygen molecules diffuse throughout and react first with a thioxene chemical dye in the "acceptor" bead, resulting in resonance energy transfer to an anthracene and then a rubrene chemical dye (these three chemical dyes are referred to as "TAR" (thioxene, anthracene, ruben) complexes) (see, e.g., U.S. Patent Application Publication No. 20040175696A1). In some embodiments, rubrene is the final chemiluminescent dye, emitting signals at distinct wavelengths. In other embodiments, final resonance energy transfer occurs within the same bead to either a terbium (Tb), europium (Eu), or samarium (Sm) chemiluminescent dye, which emits at distinct, detectable wavelengths (e.g., 520-620 nm). Such amplified luminescent proximity homogeneous assay (ALPHA) systems have been used to detect and measure protein and small molecule analytes in biological samples. The ability to have a final chemiluminescent dye in each unique acceptor bead, emitting luminescence signals at different wavelengths with limited spectral overlap, allows complementary acceptor bead pairs (e.g., Tb and Eu acceptor beads) to be used in multiplex assays to analyze different molecules and binding events, including but not limited to, small and large biomolecules or analytes, in a single-plate homogeneous no-wash assay.

[0031] This application describes, for the first time, a no-wash screening system, such as the ALPHA multiplex system, that uses anti-idiotypic antibodies to detect and / or quantify two or more co-formulated antigen-binding proteins. This system includes (a) donor beads, where each donor bead comprises (e.g., conjugated to) a photosensitizer and a first binding pair member; (b) acceptor beads, where each acceptor bead comprises (e.g., conjugated to) a chemiluminescent dye and an anti-idiotypic antibody that specifically binds to one of two or more different antigen-binding proteins; and (c) conjugated proteins, where each conjugated protein comprises one of two or more different antigen-binding proteins and a second binding pair member (e.g., conjugated to an antigen-binding protein). Also described herein are methods for detecting and / or quantifying two or more different antigen-binding proteins in a sample using the aforementioned system.

[0032] antigen-binding proteins The term "antigen-binding protein," as used herein, refers to a protein molecule that specifically binds to an antigen. For example, an antigen-binding protein can include an antibody or antigen-binding fragment thereof (such as a monoclonal antibody, e.g., an IgG1 monoclonal antibody), an antibody protein product, a bispecific T-cell engager (BiTE®) molecule, a bispecific antibody, a trispecific antibody, or an Fc-fusion protein.

[0033] Antigen-binding proteins typically comprise the heavy chain variable region (VH) and / or light chain variable region (VL) of an antibody, or domains derived therefrom. In some embodiments, antigen-binding proteins comprise the structural requirements of an antibody sufficient for immunospecific target binding. This structural requirement can be defined, for example, by 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 all six CDRs. Where (and in what order) these CDRs are located within the antigen-binding protein is within the knowledge of one of ordinary skill in the art.

[0034] As used herein, the term "antibody" refers to an immunoglobulin of any isotype that has specific binding to a target antigen; an antibody can be a polyclonal or monoclonal antibody, a chimeric antibody, a humanized antibody, a human antibody, etc. In a natural antibody, the heavy chain comprises a variable region (VH) and three constant regions (CH1, CH2, and CH3). The VH domain is at the amino terminus of the heavy chain, and the CH3 domain is at the carboxy terminus. In a natural antibody, the light chain comprises a variable region (VL) and a constant region (CL). The variable region of the light chain is located at the amino terminus of the light chain. In a natural antibody, the variable regions of each light / heavy chain pair typically form an antigen-binding site. The constant region is typically responsible for effector function.

[0035] In human antibodies, CH1 refers to the region having the amino acid sequence at positions 118 to 215 of the EU index or EU numbering system, based on the sequential numbering of the first human IgG1 (i.e., "EU antibody") to be sequenced (Edelman et al., Proc Natl Acad Sci USA, 63(1):78-85 (1969)). A highly flexible amino acid region called the "hinge region" is located between CH1 and CH2. CH2 represents the region having the amino acid sequence at positions 231 to 340 of the EU index, and CH3 represents the region having the amino acid sequence at positions 341 to 446 of the EU index.

[0036] "CL" represents the light chain constant region. In the case of a human antibody κ chain, CL represents the region having the amino acid sequence at positions 108 to 214 of the EU index. In the case of a λ chain, CL represents the region having the amino acid sequence at positions 108 to 215 of the EU index.

[0037] In natural antibodies, the variable regions typically exhibit the same general structure, with relatively conserved framework regions (FRs) linked by three hypervariable CDRs. The CDRs from the two chains of each pair are typically aligned by the framework regions, enabling binding to a specific epitope. Both light and heavy chain variable regions typically comprise, from N- to C-terminus, the domains FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. CDR3 is typically the greatest source of molecular diversity within the antigen-binding site. The assignment of amino acids to each domain typically follows 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.); Chothia, C., and Lesk, AM (1987) J. Mol. Biol., 196:901-917; or Chothia et al., Nature, 342:878-883 (1989). In some embodiments, the CDRs of an antigen-binding protein are defined according to the definitions of Kabat or Chothia. In this application, the term "CDR" refers to either the light chain or the heavy chain CDRs, unless otherwise specified.

[0038] Antibodies may include 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 antibody isotypes are defined as IgM, IgD, IgG, IgA, and IgE, respectively. IgG has several subclasses, including, but not limited to, IgG1, IgG2, IgG3, and IgG4. IgM has subclasses, including, but not limited to, IgM1 and IgM2. Embodiments of the present disclosure include all of these classes or isotypes of antibodies. The light chain constant region may be, for example, a kappa- or lambda-type light chain constant region, such as a human kappa- or human 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 region, e.g., a human alpha-, human delta-, human epsilon-, human gamma-, or human mu-type heavy chain constant region. Thus, in exemplary embodiments, the antibody is of the isotype IgA, IgD, IgE, IgG, or IgM, including any one of IgG1, IgG2, IgG3, or IgG4.

[0039] An antibody can be a monoclonal antibody or a polyclonal 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 are typically produced using hybridoma technology, as first described by Kohler and Milstein, Eur. J. Immunol., 5:511-519 (1976). Monoclonal antibodies can also be produced using recombinant DNA methods (see, e.g., U.S. Pat. No. 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 with a fully human immunoglobulin system (see, e.g., XENOMOUSE™ mice, Green et al. (1994) Nature Genetics 7:13-21, U.S. Patent Application Publication No. 2003 / 0070185, WO 96 / 34096, and WO 96 / 33735). In contrast, "polyclonal" antibodies are antibodies secreted by different B-cell lineages within an animal. Polyclonal antibodies are a group of immunoglobulin molecules that recognize multiple epitopes on the same antigen.

[0040] The term "chimeric antibody" refers to an antibody that contains domains from two or more different antibodies. A chimeric antibody may, for example, contain a constant domain from one species and a variable domain from a second species, or more commonly, may contain stretches of amino acid sequence from at least two species. A chimeric antibody may also contain domains from two or more different antibodies within the same species. The term "humanized," when used with respect to antibodies, refers to an antibody with at least the CDR regions from a non-human source that have been modified to have a structure and immune function more similar to that of a true human antibody than the original source antibody. For example, humanization can involve grafting CDRs from a non-human antibody, such as a murine antibody, onto a human antibody. Humanization can also involve selecting amino acid substitutions to make the non-human sequence more similar to human sequences.

[0041] Antibodies can be cleaved into fragments by enzymes, such as papain, pepsin, or other engineered site-specific proteases (e.g., those commercially available from Genovis AB, Lund, Sweden). Papain cleaves antibodies to generate two Fab fragments and one Fc fragment. Pepsin cleaves antibodies to generate an F(ab')2 fragment and a pFc' fragment. In exemplary embodiments, the antigen-binding proteins of the present disclosure comprise antigen-binding antibody fragments. As used herein, the term "antigen-binding antibody fragment" refers to a portion of an antibody molecule capable of binding to the antibody's antigen, and is also known as an "antigen-binding fragment" or "antigen-binding portion." In exemplary examples, the antigen-binding antibody fragment is a Fab fragment or an F(ab')2 fragment.

[0042] Antibody structures span a molecular weight range of at least about 12 to 150 kDa and have been exploited to generate a growing range of alternative formats, ranging from monomers (n = 1) to dimers (n = 2), trimers (n = 3), tetramers (n = 4), and potentially higher valencies (n); such alternative formats are referred to herein as "antibody protein products." Antibody protein products include those based on the complete antibody structure and antibody fragments that retain complete antigen-binding function, such as scFv, Fab (e.g., Fab, Fab', and F(ab')2), and VHH / VH mimics. The smallest antigen-binding antibody fragment that retains an intact antigen-binding site is the Fv fragment, consisting entirely of the variable (V) regions of the light and heavy chains. To stabilize the molecule, soluble and flexible amino acid peptide linkers are used to link the V regions in scFv (single-chain variable fragment) fragments, or constant (C) domains are added to the V regions to generate Fab fragments. Both scFv and Fab fragments can be easily produced in host cells, such as prokaryotic host cells. VHH / VH (or nanobody) is an antigen-binding fragment of a heavy chain-only antibody. Heavy chain-only antibodies (HcAb) are naturally produced in camelids and sharks. Other antibody protein products include dimeric and multimeric antibody formats such as diabodies, triabodies, and tetrabodies, or minibodies (miniAbs), including disulfide-bond-stabilized scFv (ds-scFv), single-chain Fab (scFab), and different formats consisting of scFv linked to an oligomerization domain. Peptibodies, or peptide-Fc fusions, are yet another antibody protein product. The peptibody structure consists of a biologically active peptide grafted onto an Fc domain (see, for example, Shimamoto et al., mAbs 4(5):586-591 (2012)).

[0043] The antigen binding protein of the present disclosure may comprise any one of the antibody protein products described above. 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., diabody, triabody, tetrabody), miniAb, camelid heavy chain antibody peptibody VHH / VH, sdAb, diabody; triabody; tetrabody; bispecific or trispecific antibody, BsIgG, appended IgG, BsAb fragment, bispecific fusion protein, or BsAb conjugate.

[0044] In certain embodiments, antigen-binding proteins of the present disclosure may be "bispecific," meaning that they can specifically bind to two different antigens. In other embodiments, antigen-binding proteins of the present disclosure may be "trispecific," meaning that they can specifically bind to three different antigens. In other embodiments, antigen-binding proteins of the present disclosure may be "tetraspecific," meaning that they can specifically bind to four different antigens.

[0045] In some embodiments, the antigen-binding protein is a BiTE® molecule. BiTE® molecules are engineered bispecific antigen-binding constructs that direct the cytotoxic activity of T cells against cancer cells. They consist of two single-chain variable fragments (scFvs) of different antibodies or amino acid sequences from four different genes fused onto a single peptide chain of approximately 55 kilodaltons. One scFv binds to T cells via the CD3 receptor, while the other binds to tumor cells via a tumor-specific molecule. Blintumomab (BLINCYTO® product) is an example of a CD19-specific BiTE® molecule. Modified BiTE® molecules (e.g., those modified to extend half-life) can also be used in the disclosed methods. These designs make BiTE® molecules uniquely suited to transiently bind T cells to target cells while simultaneously potently activating the intrinsic cytotoxicity of T cells against target cells. See, for example, WO 99 / 54440, WO 2005 / 040220, and WO 2008 / 119567.

[0046] In certain embodiments of the present disclosure, the antigen-binding protein may be multivalent. The valency of a binding protein refers to the number of individual antigen-binding domains within the binding protein. In some embodiments, the bispecific antigen-binding protein may be multivalent. For example, in certain embodiments, the bispecific antigen-binding protein may be tetravalent by comprising four antigen-binding domains: two antigen-binding domains that bind to a first target antigen and two antigen-binding domains that bind to a second target antigen.

[0047] As used herein, the terms "antigen-binding domain" and "binding domain" are used interchangeably and refer to the region of an antigen-binding protein that contains amino acid residues that interact with an antigen and confer specificity and affinity to the antigen-binding protein for that antigen. In some embodiments, the binding domain may be derived from the natural ligand of the target antigen. As used herein, the term "target antigen" refers to the first target antigen and / or the second target antigen of a bispecific molecule, and to the first target antigen, second target antigen, third target antigen, and / or fourth target antigen of a tetraspecific molecule.

[0048] An antigen-binding protein may contain an immunoglobulin domain. As used herein, the term "immunoglobulin domain" refers to a peptide containing approximately 100 amino acid residues that contains an amino acid sequence similar to that of an immunoglobulin and includes at least two cysteine ​​residues. Examples of immunoglobulin domains include the VH, CH1, CH2, and CH3 domains of an antibody heavy chain, and the VL and CL domains of an antibody light chain. Furthermore, immunoglobulin domains are found in proteins other than immunoglobulins. Examples of immunoglobulin domains in proteins other than immunoglobulins include immunoglobulin domains contained in proteins belonging to the immunoglobulin superfamily, such as major histocompatibility complex (MHC), CD1, B7, T cell receptor (TCR), etc.

[0049] In some embodiments, each of the two or more antigen-binding proteins is a therapeutic protein. As used herein, "therapeutic protein" and variations of this root term have their ordinary and accustomed meaning as would be understood by one of skill in the art in light of this disclosure. It refers to a polypeptide for medical use in a subject, typically a human subject. For example, each of the two or more antigen-binding proteins can be a therapeutic antigen-binding protein, such as a therapeutic antibody. By way of example, a therapeutic protein can be a polypeptide approved for medical use by a government regulatory agency, such as the U.S. Food and Drug Administration or the European Medicines Agency.

[0050] Therapeutic antigen binding proteins, such as antibodies, encompassed by the present disclosure can include polypeptides that bind to one or more of the following: (i) CD proteins, such as CD3, CD4, CD8, CD19, CD20, CD22, CD30, and CD34; for example, those that inhibit receptor binding; (ii) HER receptor family proteins, such as HER2, HER3, HER4, and EGF receptor; (iii) cell adhesion molecules, such as LFA-I, MoI, p150, 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, Müllerian inhibitory substance, human macrophage inflammatory protein (MIP-1 alpha), erythropoietin (EPO), nerve growth factors, such as NGF-beta, platelet-derived growth factor (PDGF), fibroblast growth factors, such as aFGF and bFGF, epidermal growth factor (EGF), transforming growth factors (TGF), such as, among others , TGF-α and TGF-β, e.g., TGF-β1, TGF-β2, TGF-β3, TGF-β4, or TGF-β5, insulin-like growth factor-I and -II (IGF-I and IGF-II), des(1-3)-IGF-I (brain IGF-I), and bone morphogenetic factor; (V) insulin and insulin-related proteins, e.g., insulin, insulin A chain, insulin B chain, proinsulin, and insulin-like growth factor binding protein; (vi) coagulation and coagulation-related proteins, e.g., factor VIII, tissue factor, phosphatase, among others. (vii) other blood and serum proteins, such as, but not limited to, albumin, IgE, and blood group antigens; (viii) colony-stimulating factors and their receptors, including, in particular, M-CSF, GM-CSF, and G-CSF, and their receptors (e.g., CSF-1 receptor (c-fms));(ix) receptors and receptor-associated proteins, such as flk2 / flt3 receptor, obesity (OB) receptor, LDL receptor, growth hormone receptor, thrombopoietin receptor ("TPO-R", "c-mpl"), glucagon receptor, interleukin receptor, interferon receptor, T cell receptor, stem cell factor receptor, e.g., c-Kit, and other receptors; (x) receptor ligands, such as OX40L, a ligand for the OX40 receptor; (xi) neurotrophic factors, such as bone-derived neurotrophic factor (BDNF) and neurotrophins. (xii) relaxin A chain, relaxin B chain, and prorelaxin; (xiii) interferons and interferon receptors, such as interferon-α, -β, and -γ, and their receptors; (xiv) interleukins and interleukin receptors, such as, inter alia, IL-1 to IL-33, and IL-1 to IL-33 receptors, such as IL-8 receptor; (xv) viral antigens, such as AIDS envelope virus antigens;(xvi) other proteins, such as lipoproteins, calcitonin, glucagon, atrial natriuretic factor, pulmonary 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 of normal T cells expressed secreted), mouse gonadotropin-related peptide, DNAse, inhibin, integrin, protein A or D, rheumatoid factor, immunotoxin, bone morphogenetic protein (BMP), superoxide dismutase, surface membrane proteins, decay-accelerating factor (D AF), HIV envelope, transport proteins, homing receptors, addressins, regulatory proteins, immunoadhesins, myostatin, TALL proteins such as TALL-I, amyloid proteins such as, but not limited to, amyloid beta protein, thymic stromal lymphopoietin ("TSLP"), RANK ligand ("RANKL" or "OPGL"), c-kit, TNF receptors such as TNF receptor type 1, TRAIL-R2, angiopoietin, delta-like ligand 3 (DLL3), CD112R, prostate-specific membrane antigen (PSMA), and biologically active fragments, or analogs or variants of any of the foregoing;

[0051] Examples of therapeutic antibodies suitable for use in the systems and 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, atrolimumab, tocilizumab, and bapineutide. Ibuprofen, basiliximab, bavituximab, bectumomab, belimumab, bemarituzumab, benralizumab, bertilimumab, besilesomab, bevacizumab, bezlotoxumab, biciromab, bivatuzumab, bivatuzumab mertansine, blinatumomab, brosozumab, brentuximab vedotin, briakinumab, brodalumab, canakinumab, cantuzumab mertansine, caplacizumab, capromab pendetide, carlumab, catumaxomab, cc49, cedelizumab, certolizumab pegol, cetuximab, sitatuzumab bogatox, sixtu Mumab, clazakizumab, clenoliximab, clivatuzumab tetraxetan, conatumumab, crenezumab, cr6261, dacetuzumab, daclizumab, dalotuzumab, daratumumab, demcizumab, denosumab, detumomab, dorlimomab alitox, drozitumab, durigotumab, dupilumab, ecromeximab, eculizumab, edovacomab, edreclomab, efalizumab, efungumab, elotuzumab, elsilimomab, enabatuzumab, enlimomab pegol, enokizumab, enoticumab, enocituximab, epitumomab Tuxetan, epratuzumab, erenumab, erlizumab, ertumaxomab, etaracizumab, etrolizumab, evolocumab, exvilumab, fanolesomab, farimomab, farletuzumab, fasinumab, fbta05, feluvizumab, fezakinumab, ficlatuzumab, figitumumab, framvotumab, fontolizumab, foralumab, foravirumab, fresolimumab, furanumab, futuximab, galiximab, ganitumab, gantenerumab, gavilimomab, gemtuzumab ozogamicin, gevokizumab, girentuximab,Glenbatumumab vedotin, golimumab, gomiliximab, gs6624, ibalizumab, ibritumomab tiuxetan, iclumab, igovomab, imciromab, imgatuzumab, inlacumab, indatuximab ravtansine, infliximab, intetumumab, inolimomab, inotuzumab ozogamicin, ipilimumab, iratumumab, itolizumab, ixekizumab, keliximab, labetuzumab, lebrikizumab, remaresomab, lerdelimumab, lexatumumab, ribivirumab, ligelizumab, lintuzumab, lirilumab, lorvox Ibuprofen mertansine, lucatumumab, rumiliximab, mapatumumab, maslimomab, mavrilimumab, matuzumab, mepolizumab, metelimumab, milatuzumab, minletumomab, mitumomab, mogamulizumab, morolimu- mab, motavizumab, moxetumomab pasudotox, muromonab cd3, nacolomab butafenatox, naptumomab estafenatox, narunatumab, natalizumab, nebacumab, necitumumab, nerelimomab, nesbacumab, nimotuzumab, nivolumab, nofetumomab merpentane, ocaratuzumab, ocrelizumab, Durimomab, ofatumumab, olalaratumab, olokizumab, omalizumab, onartuzumab, oportuzumab monatox, oregovomab, olticumab, otelixizumab, oxelumab, ozanezumab, ozoralizumab, pagibaximab, palivizumab, panitumumab, panobacumab, palsatuzumab, pascolizumab, pateclizumab, patritumab, pemtumomab, perakizumab, pertuzumab, pexelizumab, pidilizumab, pintumomab, placurab, ponezumab, priliximab, pritumumab, PRO140, kiruzumab, Racotumomab, radletumab, rafivirumab, ramucirumab, ranibizumab, raxibacumab, regavirumab, reslizumab, rilotumumab, rituximab, lobatumumab, loredumab, romosozumab, rontalizumab, rovelizumab, ruplizumab, samarizumab, sarilumab, satumomab pendetide, secukinumab, sevirumab, sibrotuzumab, sifalimumab, siltuximab, simtuzumab, siplizumab, sirukumab, solanezumab, solitomab, sonepcizumab, sontuzumab, stamulumab, sulesomab, suvizumab, tabalumab,Takatuzumab tetraxetan, tadocizumab, talizumab, tanezumab, taplitumomab paptox, tarlatamab, tefibazumab, terimomab alitox, tenatumomab, tefibazumab, teneliximab, teplizumab, teprotumumab, tezepelumab, TGN1412, tremelimumab, ticilimumab, tildrakizumab, tigatuzumab, TNX-650, tocilizumab, toralizumab, tositumomab, tralokinumab, trastuzumab, TR BS07, tregalizumab, tucotuzumab celmoleukin, tuvilumab, ublituximab, urelumab, urtoxazumab, ustekinumab, bapaliximab, batelizumab, vedolizumab, veltuzumab, bepalimomab, besencumab, visilizumab, volociximab, borsetuzumab mafodotin, votumumab, zalutumumab, zanolimumab, zatuximab, diralimumab, zolimomab alitox, or a variant of any of the foregoing.

[0052] According to the systems and methods described herein, a sample may contain two or more of any of the aforementioned therapeutic antibodies or antigen-binding fragments thereof. Exemplary combinations of antigen-binding proteins that may be co-formulated in a sample for the analyses described herein include, but are not limited to, a co-formulation of an antibody that specifically binds to PD-1 with at least one additional antibody described herein. In some embodiments, a sample may contain an anti-PD-1 antibody and an antigen-binding protein (e.g., a BiTE® molecule) that specifically binds to DLL3 (e.g., taluratamab), PSMA, CD112R, and / or TIGIT. For example, a sample may contain an anti-PD-1 antibody co-formulated with an antibody that specifically binds to TIGIT and an antibody that specifically binds to CD112R. In other embodiments, a sample may contain an anti-PD-1 antibody co-formulated with a BiTE® molecule that specifically binds to DLL3 or a BiTE® molecule that specifically binds to PSMA. However, the present disclosure is not limited to these particular combinations of antigen-binding proteins.

[0053] System Reagents The disclosed systems include donor and acceptor beads that, when brought into close proximity through biological interactions, undergo a cascade of chemical reactions to generate a detectable signal (e.g., a chemiluminescent signal). The terms "bead" and "particle" are used interchangeably herein and typically refer to a substantially spherical solid support, although other morphologies may also be suitable in some systems. In exemplary embodiments, the donor and acceptor beads may be latex-based and approximately 200 nm to 300 nm in diameter (e.g., approximately 250 nm in diameter). In some embodiments, the donor and acceptor beads may be coated with a hydrogel to minimize nonspecific binding and self-aggregation and to provide reactive aldehyde groups for conjugating biomolecules to the bead surface. Beads that include one or more additional moieties will be understood to encompass beads having one or more additional moieties immobilized thereon. For example, beads that include a photosensitizer and a binding pair member will be understood to encompass beads that are conjugated to both the photosensitizer and the binding pair member.

[0054] The terms "solid phase" and "solid support" are used interchangeably herein and refer to any material that can be used to bind and / or attract and immobilize one or more binding members. The term "immobilized," as used herein, refers to the stable association of a binding member with the surface of a solid support. The terms "binding pair member" and "binding member" are used interchangeably herein and refer to one of two or more distinct molecules that specifically recognizes one molecule relative to substantially less recognition of other molecules. By way of example, when an antibody or other entity (e.g., an 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 to the antigen or epitope with substantially higher affinity than other entities that do not display the antigen or epitope. Thus, two binding pair members bind to each other more tightly than they do to other molecules.

[0055] Energy transfer from donor beads to acceptor beads is mediated in part by the interaction of binding pair members that bind via biological interactions. Thus, in exemplary embodiments, a first binding pair member is configured to specifically bind to a second binding pair member. Suitable binding pairs that can be utilized in the systems described herein include, but are not limited to, streptavidin-biotin, affinity tag peptides and anti-affinity tag antibodies (such as polyhistidine tags and anti-polyhistidine antibodies, e.g., hexahistidine (His6)-anti-His6 antibodies, or FLAG peptide-anti-FLAG antibodies), and GST-glutathione, as well as specific antibodies targeting post-translational modifications such as various histone methylation loci. In some embodiments, the first binding pair member comprises streptavidin, and the second binding pair member comprises biotin. Streptavidin is produced by the bacterium Streptomyces avidinii, and streptavidin homotetramers have a very high affinity for biotin (also known as vitamin B7 or vitamin H). The streptavidin-biotin binding pair is widely used in molecular biology and bionanotechnology due to its resistance to organic solvents, denaturants (e.g., guanidinium chloride), detergents (e.g., SDS, Triton X-100), proteolytic enzymes, and extremes of temperature and pH. Without being limited by theory, biotin has been shown to preferentially bind to free lysine residues. Therefore, it is believed that almost all polypeptides, especially those containing lysine, can be biotinylated. In other exemplary embodiments, the first binding pair member comprises hexahistidine (His6) and the second binding pair member comprises an anti-His6 antibody; the first binding pair member comprises a FLAG peptide and the second binding pair member comprises an anti-FLAG antibody; or the first binding pair member comprises glutathione-S-transferase (GST) and the second binding pair member comprises glutathione.

[0056] In some embodiments, each donor bead may comprise (e.g., be conjugated to) a photosensitizer and a first binding pair member. Thus, for example, the photosensitizer in the "donor" bead converts ambient oxygen to a more excited singlet state upon photoexcitation. The term "photosensitizer," as used herein, refers to a molecule that absorbs light to generate reactive singlet oxygen and transfers energy from the incident light to another nearby molecule. Examples of photosensitizers include porphyrins, phthalocyanines, and bacteriochlorin derivatives. In some embodiments, the photosensitizer comprises a phthalocyanine, which can excite ambient oxygen to a singlet state after high-energy irradiation at about 680 nm.

[0057] The disclosed systems also include acceptor beads, which typically contain a detectable label that reacts with the singlet oxygen generated in the donor beads. The term "detectable label," as used herein, refers to a moiety capable of generating a signal detectable by visual or instrumental means. A detectable label may refer to a signal-producing substance, such as a chromogen, a fluorescent compound, an enzyme, a chemiluminescent compound, or a radioactive compound. In some embodiments, the acceptor beads are conjugated to a chemiluminescent dye. Typically, the acceptor beads are embedded with at least two different chemical dyes, such as thioxene-based dyes, anthracene, and rubrene (TAR). However, in some embodiments, the acceptor beads contain at least a thioxene-based dye and europium (Eu), terbium (Tb), or samarium (Sm) (see, e.g., Eglen et al., supra). In such embodiments, singlet oxygen first reacts with the thioxene to generate light, which is transferred to the Eu, Tb, or Sm, which subsequently emits light at about 520-645 nm.

[0058] In addition to a chemiluminescent dye (e.g., Eu, Tb, Sm), each acceptor bead may contain (e.g., be conjugated to) an anti-idiotypic antibody that binds to any of two or more different antigen-binding proteins. The term "idiotype," as used herein, refers to a set of one or more antigenic determinants ("idiotopes") specific to the variable region of an antibody molecule. Idiotypes are generally highly conformational epitopes that span the CDR regions; however, they may also be specific to the V region. H and V L The idiotype may include non-CDR sequences of the domain. Idiotypes can be subdivided into those present in the antigen-binding site of an antibody molecule (i.e., the "paratope") and those on the regions adjacent to this site (i.e., framework determinants). The term "anti-idiotypic" antibody, as used herein, refers to an antibody directed against an idiotypic determinant.

[0059] Anti-idiotypic antibodies specifically bind to the idiotype of an antigen-binding protein, meaning that they bind more tightly to the idiotype of the antigen-binding protein than to other molecules. Depending on the idiotypic determinant, anti-idiotypic antibodies can be classified as "Ab2 alpha," "Ab2 beta," "Ab2 gamma," or "Ab3." Ab2 alpha antibodies are directed against an idiotype different from the antigen-binding site (paratope) of Ab1. Ab2 alpha anti-idiotypic antibodies recognize Ab1 framework region antigens, and the idiotype / anti-idiotypic interaction cannot be inhibited by haptens that specifically bind to the antigen-binding site, so they are also referred to as "antigen non-inhibitory." Ab2 beta anti-idiotypic antibodies bind to the antigen-binding site of an antibody molecule and compete with the target antigen. Therefore, Ab2 beta antibodies are also referred to as "antigen inhibiting." Ab2 gamma anti-idiotypic antibodies are directed against an idiotype located near, but not within, the antigen-binding site. Ab2 gamma antibodies recognize the paratope-associated idiotype, and their binding induces a change in the three-dimensional structure of the paratope within the antigen-binding site, making them antigen-inhibitory. Anti-idiotypic antibodies are commonly used in the art for preclinical pharmacokinetic (PK) analysis of antibody formulations. Idiotypes and anti-idiotypic antibodies are described in detail, for example, in Mahmoud Abu-Shakra, Yehuda Shoenfeld, 10-Idiotypes And Anti-Idiotypes, Editor(s): Yehuda Shoenfeld, M. Eric Gershwin, Pier Luigi Meroni, Autoantibodies (Second Edition), Elsevier, 2007, pp. 69-76. Methods for screening and selecting anti-idiotypic antibodies specifically specific for variable regions are described, for example, in Salimi-Moosavi et al., Analytical Biochemistry, 470: pages 52-60 (2015).

[0060] In some embodiments, each acceptor bead may comprise (e.g., be conjugated to) a molecule other than an anti-idiotypic antibody that binds to one of two or more different antigen binding proteins. For example, an acceptor bead may comprise a target antigen to which at least one of the antigen binding proteins in the sample specifically binds. In such embodiments, it is desirable that the target antigen be in a format that is compatible with binding to the acceptor bead and does not cross-react with other antigen binding proteins (e.g., therapeutic antibodies) in the co-formulated sample.

[0061] In some embodiments, the systems of the present disclosure comprise a first acceptor bead comprising (e.g., conjugated to) a first anti-idiotypic antibody that binds to one of two or more antigen binding proteins in a sample, and a second, different acceptor bead comprising (e.g., conjugated to) a second anti-idiotypic antibody that binds to the other of the two or more antigen binding proteins in the sample. It will further be understood that in such a "multiplex" system, the first, second, and / or subsequent acceptor beads are conjugated to chemiluminescent dyes that emit signals at different wavelengths for ease of visualization. For example, the system may include (i) a first acceptor bead comprising (e.g., conjugated to) a first chemiluminescent dye (e.g., terbium) and a first anti-idiotypic antibody that binds to a first antigen binding protein in the sample, and (ii) a second acceptor bead comprising (e.g., conjugated to) a second chemiluminescent dye (e.g., europium) and a second anti-idiotypic antibody that binds to a second antigen binding protein in the sample. In embodiments in which the sample includes three or more (e.g., three, four, five, or more) different antigen binding proteins, the system desirably includes acceptor beads comprising (e.g., conjugated to) anti-idiotypic antibodies that bind to each of the three, four, five, or more different antigen binding proteins.

[0062] In some embodiments, the system further includes a conjugated protein, each of which comprises one of two or more different antigen-binding proteins and a second binding pair member. In other words, a conjugated protein in the context of the present disclosure includes different forms of antigen-binding proteins in a sample. For example, the antigen-binding protein of the conjugated protein may be conjugated to a binding pair member. As used herein, the term "conjugate" refers to a molecule consisting of at least two components linked to form a larger biomolecule. For example, a conjugate may include a fusion of two proteins (a "protein conjugate") or a fusion of a small molecule with a protein or peptide. Protein conjugation (also known as "crosslinking") may be achieved by any method known in the art as a suitable method for chemically (e.g., covalently) linking two different peptides or polypeptides. In some embodiments, cross-linkers may be used that contain two or more reactive ends that can chemically bond to specific functional groups (primary amines, sulfhydryls, etc.) on proteins or other molecules (see, e.g., Mattson, G., et al., Molecular Biology Reports, 17:167-183 (1993)).

[0063] By way of example, a sample of interest may contain two different therapeutic antibodies ("Ab1" and "Ab2"). In such embodiments, the system may include a first conjugated protein comprising Ab1 conjugated to a second binding pair member (e.g., biotin) as described herein, and a second conjugated protein comprising Ab2 conjugated to a second binding pair member (e.g., biotin). Thus, in some embodiments, a system of the present disclosure may include a first conjugated protein comprising a first antigen binding protein conjugated to biotin, and a second conjugated protein comprising a second antigen binding protein conjugated to biotin.

[0064] Samples and kits The term "sample" and variations of this root term have their ordinary and accustomed meaning as would be understood by one of skill in the art in light of the present disclosure. It refers to a composition that may contain two or more antigen-binding proteins described herein, such as an in vitro or synthetic sample obtained from the production of the antigen-binding proteins. For example, a sample may be a composition or formulation (also referred to herein as a "pharmaceutical composition") comprising two or more antigen-binding proteins and at least one pharmaceutically acceptable carrier. For example, a sample may be a sample from the manufacturing process of a pharmaceutical composition.

[0065] Acceptable compositions or formulation materials for the therapeutic proteins (e.g., therapeutic antibodies) described herein are preferably non-toxic to recipients at the dosages and concentrations used. In certain embodiments, pharmaceutical compositions may contain formulation materials to alter, maintain, or preserve, 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); antimicrobial agents; antioxidants (such as ascorbic acid, sodium sulfite, or sodium bisulfite); buffers (such as borate, bicarbonate, Tris-HCl, citrate, phosphate, or other organic acids); bulking agents (such as mannitol or glycine); chelating agents (such as ethylenediaminetetraacetic 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 dextrin); proteins (such as serum albumin, gelatin, or immunoglobulins); colorants, flavorings, and diluents; emulsifiers; hydrophilic polymers (such as polyvinylpyrrolidone); These may include 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 pluronic, PEG, sorbitan esters, polysorbates (such as polysorbate 20, polysorbate), Triton, tromethamine, lecithin, cholesterol, tyloxapol, etc.); stability enhancers (such as sucrose or sorbitol); tonicity enhancers (such as alkali metal halides, preferably sodium or potassium chloride, mannitol, sorbitol, etc.); delivery vehicles; diluents; additives and / or pharmaceutical adjuvants.For example, Remington's Pharmaceutical Sciences, 18. th Edition, (AR Genrmo, ed.), 1990, Mack Publishing Company.

[0066] Suitable vehicles or carriers for the compositions can be water for injection, saline, or artificial cerebrospinal fluid, optionally supplemented with other ingredients common in compositions for parenteral administration. Neutral buffered saline or saline mixed with serum albumin are further exemplary vehicles.

[0067] In certain embodiments, a buffer is used to maintain the composition at physiological pH or slightly below, typically within a pH range of about 5 to about 8, e.g., about 6 to about 8. For example, the pH of the formulation can 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.

[0068] The components of the systems described herein can be provided in the form of a kit, i.e., a packaged combination of reagents in predetermined amounts along with instructions for use. In exemplary aspects, the kit can include a sample, donor beads, acceptor beads, and protein conjugates, all of which can be packaged in one or more containers. In some embodiments, the kit further includes a substrate. The substrate can be configured to immobilize the donor beads, acceptor beads, and / or conjugate proteins thereon, or the donor beads, acceptor beads, and / or conjugate proteins of the kit can be immobilized on the substrate. Examples of suitable substrates include tubes, beads, microparticles, nanoparticles, microplates, multiwell plates, gels, colloids, sheets, and chips.

[0069] Assay Method The present disclosure further provides methods for analyzing compositions containing two or more antigen-binding proteins (e.g., therapeutic antibodies). The methods provided herein are desirably homogeneous proximity-based assays. A "homogeneous" assay provides a readout without the need to separate the analytes of interest (e.g., antigen-binding proteins) from the biomolecules used to detect them. In contrast, a "heterogeneous" assay requires one or more separation steps to wash away unbound analytes (e.g., antigen-binding proteins) and / or unbound detection molecules. Proximity-based assays, e.g., no-wash assays such as the ALPHASCREEN® method (e.g., ALPHASCREEN®, ALPHALISA®, and ALPHAPLEX™, PerkinElmer, Inc., Waltham, MA), typically involve bead-based chemistry that examines interactions between molecules in a microplate. ALPHASCREEN® technology is routinely utilized in high-throughput screening assays to quantify analyte accumulation or depletion, bimolecular interactions, and post-translational modifications (see, e.g., Ullman et al., Proc. Natl. Acad. Sci. USA, 91:5426-5430 (1994); and Yasgar et al., Methods Mol Biol., 1439:77-98 (2016) doi:10.1007 / 978-1-4939-3673-1_5).

[0070] In some embodiments, provided herein are methods for determining the presence, absence, or concentration of two or more different antigen-binding proteins in a sample, the methods comprising: (1) incubating the sample with a system described herein; (2) exposing the incubated sample to light excitation; and (3) detecting at least one chemiluminescent signal after light excitation and comparing the detected chemiluminescent signal to a predetermined reference value. The descriptions of donor beads, acceptor beads, protein conjugates, antigen-binding proteins, and samples described herein with respect to the aforementioned systems are also applicable to those same aspects of the disclosed methods.

[0071] The sample may be contacted and incubated with the various components of the system for any suitable period of time and in any suitable order. For example, the sample may be contacted sequentially or simultaneously with the donor beads, acceptor beads, and conjugated protein. In some embodiments, the sample is applied to a substrate (e.g., a well in a microplate), followed by the sequential introduction of the conjugated protein, acceptor beads, and finally the donor beads. In other embodiments, the sample is applied to a substrate, followed by the sequential introduction of the acceptor beads, conjugated protein, and finally the donor beads. In some embodiments, the donor beads, acceptor beads, and / or conjugated protein may be immobilized on the substrate before the sample is introduced. In some applications, it may be appropriate to simultaneously introduce the acceptor beads, conjugated protein, and donor beads to the sample. The appropriate selection of an assay format will be apparent to one of skill in the art.

[0072] The sample and system components may be incubated for a time sufficient to allow interaction between the donor and acceptor beads. In some embodiments, the sample is incubated with the system for at least 15 minutes (e.g., 20, 30, 45, or 60 minutes), but not more than 3 hours (e.g., 2.5, 2, or 1.5 hours). In some embodiments, incubation times of about 30 minutes to about 2 hours (e.g., 35, 50, 75, or 100 minutes) are preferred. An advantage of the disclosed methods is that they can be completed in a time-efficient manner, due in part to the very high signal-to-background ratio. Thus, a wide range of conditions is suitable for generating an adequate signal.

[0073] Whatever format is used, the method involves incubating the sample with the system by converting oxygen (O2) into an excited singlet state ( 1 The method includes exposing the sample to photoexcitation to convert the singlet oxygen to singlet oxygen (O2). The excitation can occur at any wavelength of light capable of generating singlet oxygen. In some embodiments, the photoexcitation wavelength can be about 500 nm to about 800 nm (e.g., about 520 nm, 550 nm, 580 nm, 600 nm, 620 nm, 650 nm, 680 nm, 700 nm, 720 nm, 750 nm, or 780 nm). For example, the photoexcitation wavelength can be about 600 nm to about 700 nm (e.g., about 610 nm, 620 nm, 630 nm, 640 nm, 650 nm, 660 nm, 670 nm, 680 nm, or 690 nm). Desirably, a wavelength of about 680 nm is used for photoexcitation of the incubated sample.

[0074] Chemiluminescent signals are generated within acceptor beads when they receive singlet oxygen within close proximity (e.g., about 200 nm, 150 nm, 100 nm, 50 nm, 25 nm, 5 nm, or less) of donor beads. As described above, depending on the number of acceptor beads used (which in turn depends on the number of antigen-binding proteins in the sample), chemiluminescent signals of various emission wavelengths can be detected. Chemiluminescent dyes (e.g., Tb, Eu, and Sm) that can be used desirably emit signals at wavelengths between about 500 nm and about 700 nm (e.g., about 520 nm, 550 nm, 580 nm, 600 nm, 620 nm, 650 nm, or 680 nm). In some embodiments, the emission wavelength can be between about 520 nm and about 620 nm (e.g., about 530 nm, about 545 nm, about 560 nm, about 575 nm, about 590 nm, or about 615 nm). For example, the terbium emission peak is at 545 nm, the europium emission peak is at 615 nm, and the samarium emission peak is at 645 nm.

[0075] Thus, in some embodiments, the method further comprises assaying the sample for the presence of at least one chemiluminescent signal after light excitation. As described above, systems encompassed by the present disclosure may include (i) a first acceptor bead conjugated to a first chemiluminescent dye and a first anti-idiotypic antibody that binds to a first antigen-binding protein in the sample, and (ii) a second acceptor bead conjugated to a second chemiluminescent dye and a second anti-idiotypic antibody that binds to a second antigen-binding protein in the sample. In such embodiments, for example, the method may include detecting a first signal emitted from the first chemiluminescent dye and a second signal emitted from the second chemiluminescent dye. The first signal and the second signal may comprise two different wavelengths. For example, the wavelength of each of the first and second signals can be about 520 nm to about 620 nm (e.g., about 530 nm, about 540 nm, about 550 nm, about 560 nm, about 570 nm, about 580 nm, about 590 nm, about 600 nm, or about 610 nm). In some embodiments, the wavelength of the first signal is about 545 nm and the wavelength of the second signal is about 615 nm.

[0076] Proximity assays are frequently used in the art to assess the presence of an analyte in a sample by generating a chemiluminescent signal through the interaction of donor beads with acceptor beads and the subsequent transfer of chemical energy from singlet oxygen from the donor beads to the acceptor beads. In such cases, the intensity of the chemiluminescent signal is directly proportional to the concentration of the analyte in the sample. As used herein, the term "intensity" refers to the amount or intensity of electricity, light, heat, or sound per unit area or volume. In other aspects, proximity assays can be designed so that disruption of the donor-acceptor interaction by the analyte of interest results in a decrease in the chemiluminescent signal intensity. In such cases, the intensity of the chemiluminescent signal is inversely proportional to the concentration of the analyte in the sample. For example, in some embodiments of the present methods, detection of one or more chemiluminescent signals indicates that the sample lacks or contains two or more different antigen-binding proteins at low concentrations, while the absence of a chemiluminescent signal or a decrease in the signal intensity indicates that the sample contains two or more different antigen-binding proteins at high concentrations. An exemplary assay format is shown schematically in Figure 1, which shows that in the absence of co-formulated antibody (or low concentrations of co-formulated antibody in the sample), when the anti-idiotypic antibodies on the acceptor beads bind to their respective conjugated proteins (e.g., biotinylated monoclonal antibodies), the acceptor and donor beads come into close proximity, resulting in high signal intensity. When a sample containing a high concentration of co-formulated antibody is used, the anti-idiotypic antibodies on the acceptor beads likely bind to their respective unlabeled antibody targets in the sample, and the donor beads are unable to approach the acceptor beads to transfer energy from singlet oxygen, resulting in a decrease in the luminescence signal. This decrease in signal can occur in a dose-dependent manner.

[0077] According to the methods of the present invention, the intensity of the chemiluminescent signal detected in a sample can be compared to a predetermined reference value, such as a predetermined threshold value. The term "predetermined reference value" refers to an assay value or a predetermined amount of analyte used to evaluate the assay result. For example, the predetermined reference value can be a chemiluminescent signal corresponding to a known amount of a therapeutic protein. The predetermined reference value can be based on a calibration curve as described herein. The reference value can be based on the concentration specification of each therapeutic protein in a pharmaceutical formulation. The terms "predetermined threshold value" and "predetermined cutoff value" are used interchangeably herein and refer to an assay value or a predetermined amount of analyte used to evaluate the concentration of an analyte in an unknown sample by comparing the assay result to a predetermined threshold or cutoff value. While the present disclosure may provide exemplary predetermined threshold levels, it is well known that cutoff values ​​may vary depending on the nature of the assay (e.g., the antibody used, etc.). Furthermore, it is well within the skill of one in the art to adapt the present disclosure to other samples and antigen-binding proteins to obtain assay-specific reference values ​​or cutoff values ​​for such assays based on the present disclosure.

[0078] In some embodiments, the predetermined reference value can be a chemiluminescent signal corresponding to an antigen-binding protein concentration of about 0.05 μg / mL to about 150 μg / mL. Thus, in some embodiments, the predetermined reference value can correspond to an antigen-binding concentration of about 1 μg / mL, about 5 μg / mL, about 10 μg / mL, about 20 μg / mL, about 30 μg / mL, about 40 μg / mL, about 50 μg / mL, about 60 μg / mL, about 70 μg / mL, about 80 μg / mL, about 90 μg / mL, about 100 μg / mL, about 110 μg / mL, about 120 μg / mL, about 130 μg / mL, about 140 μg / mL, or a range defined by any two of the foregoing values, e.g., about 20 μg / mL to about 140 μg / mL.

[0079] In other embodiments, the intensity of the chemiluminescent signal detected in the sample may be compared to a predetermined reference curve for each antigen-binding protein in the sample. For example, the predetermined reference curve may be a dose-response curve established for each antigen-binding protein in the co-formulation sample to determine the acceptable binding range of each antigen-binding protein. Methods for generating dose-response curves in connection with the present disclosure are described in detail in the Examples.

[0080] In some embodiments, the methods described herein further comprise quantifying two or more antigen-binding proteins in a sample. In embodiments in which chemiluminescent signal intensity is detected, spectrophotometric analysis may be used to quantify antigen-binding protein concentrations. As used herein, the terms "spectrophotometric" or "photometric" analysis refer to the quantification of materials in a substance or sample by measuring the amount of light absorbed in the infrared, visible, or ultraviolet region of the spectrum. Spectrophotometric analysis measures changes in the amount of light absorbed by an assay solution (e.g., a sample) after the course of a reaction. Thus, in some embodiments, the concentration of each antigen-binding protein in a sample may be quantified by measuring UV absorbance at 280 nm. It will be appreciated that at this wavelength, the aromatic amino acids tryptophan (Trp) and tyrosine (Tyr) exhibit strong light absorption, and to a lesser extent, cysteine ​​groups that form disulfide bonds (Cys-Cys). Consequently, the absorbance of proteins and peptides at 280 nm is proportional to the content of these amino acids.

[0081] In certain embodiments, the concentration of each of the antigen-binding proteins in a sample can be determined by constructing a calibration curve (also commonly referred to as a standard curve or working curve). A "calibration curve" is a common method for determining the concentration of a substance in an unknown sample by comparing it to a set of standard samples of known concentrations. The responses of the standard samples are used to plot or calculate the standard curve. The absorbance values ​​of the unknown samples are then interpolated into the standard curve plot or equation to determine their concentrations. If the calibration curve has adequate linearity over a wider range in the region of quantitative analysis, the calibration curve can be constructed with a relatively small number of standard samples near the upper, lower, and midpoint of the determination range of the quantitative analysis.

[0082] In a sample containing two or more antigen-binding proteins as described herein, at least two calibration curves can be generated and used to quantify the amount of each antigen-binding protein detected by the methods of the present disclosure. For example, a first calibration curve can be generated at a first wavelength emitted by a first acceptor bead, and a second calibration curve can be generated at a second wavelength emitted by a second acceptor bead.

[0083] In some embodiments, the concentration of antigen binding protein in the sample is measured using the half maximal inhibitory concentration (IC), which is a measure of the effectiveness of the antigen binding protein in inhibiting the interaction between donor beads and acceptor beads. 50 In other embodiments, the concentration of antigen-binding protein in a sample can be characterized by the half maximal effective concentration (EC), which is a measure of the effectiveness of antigen binding resulting in an interaction between donor beads and acceptor beads. 50 )

[0084] Other orthogonal methods for determining protein concentration may be used to cross-check or confirm the results obtained by the methods of the present disclosure. Such methods include, but are not limited to, turbidimetric, nephelometric, and colorimetric assays. In turbidimetric and nephelometric assays, proteins are quantified from the change in turbidity of the reaction mixture based on the aggregation of the protein with its specific binding partner. In colorimetric assays, proteins may be quantified using a coloring reagent. Colorimetric assays are characterized by the formation, change, or disappearance of color in the presence of the protein being quantified. Exemplary colorimetric assays include the Coomassie Blue 250 dye-binding assay (Bradford), the bicinchoninic acid assay (BCA), and the Lowry assay.

[0085] The following examples further illustrate the invention but, of course, should not be construed as in any way limiting its scope. [Example]

[0086] This example describes the generation and use of a system for detecting and quantifying two different antigen binding proteins in a sample.

[0087] Two antibody target-specific reagents, designated "Ab1" and "Ab2," were generated. Specifically, unlabeled Ab1 and Ab1 were biotinylated at Amgen and designated Ab2-biotin and Ab2-biotin, respectively. When glycerol or amines were present in the unlabeled antibody formulation buffer, the unlabeled antibody was desalted using a ZEBA™ spin desalting column (7K MWCO) prior to biotinylation and exchanged into 1x Dulbecco's phosphate-buffered saline (D-PBS, pH 7.0) according to standard protocols. Biotinylation was performed by mixing the unlabeled antibody with EZ-LINK™ sulfo-NHS-LC-LC-biotin reagent in 100 mM borax (pH 9.3) buffer. In this case, a covalent bond is formed between the NHS ester of the biotin reagent and the primary amine on the antibody surface. The molar ratio of biotin to unlabeled antibody (referred to as the "challenge ratio") could be 20:1, 10:1, 5:1, 2:1, 1:1, 1:2, 1:5, 1:10, or 1:20. Samples were mixed constantly (30-60 min, 25°C) and then purified on a ZEBA™ spin desalting column (7K MWCO) and exchanged into 1x Dulbecco's phosphate-buffered saline (D-PBS, pH 7.0) according to standard protocols. The final concentration of biotinylated antibody (Ab1-biotin or Ab2-biotin) was determined by UV-VIS spectrophotometry using a wavelength of 280 nm. The degree of biotinylation was determined by UV-VIS spectrophotometric measurements at 500 nm in triplicate using the Pierce™ Biotin Quantitation Kit according to the manufacturer's protocol. In practice, the degree of biotinylation is reported as a real number with two decimal points, reflecting the average number of biotin molecules per IgG molecule. In this study, Ab1-biotin was determined to have 6.18 biotin molecules per Ab1 molecule, and Ab2-biotin was determined to have 5.62 biotin molecules per Ab2 molecule. In some embodiments, the degree of biotinylation can be any real number that does not exceed the challenge ratio used.For example, in the case of a challenge ratio (molar ratio of biotin to IgG molecules) of 20:1, the degree of biotinylation can be any real ratio ranging from 20:1 to 0:1, the latter being the case when biotinylation fails. However, if the biotinylation reaction efficiency is insufficient, it is not impossible to have a degree of labeling where the ratio of biotin is greater than 0 but less than 1 molecule per IgG molecule.

[0088] Antibody-specific anti-idiotypes for Ab1 and Ab2 were conjugated to acceptor beads containing fluorophores for detection. First, the anti-idiotype antibody specific for Ab1 was directly conjugated to ALPHAPLEX™ 545 (terbium) acceptor beads by reductive amination of the aldehyde groups on the bead surface. Similarly, the anti-idiotype antibody specific for Ab2 was directly conjugated to ALPHALISA® (europium) acceptor beads. The following protocol is available from PerkinElmer as a technical overview titled "Antibody conjugation to ALPHALISA® Acceptor beads." Briefly, acceptor beads were washed by adding 50 μL of 1× PBS (pH 7.4) to a 1-milligram tube of acceptor beads directly from the supplier (PerkinElmer®). The beads were collected by centrifugation (16,000 × g, 15 min) and the supernatant was removed. Ab-specific (Ab1 or Ab2) anti-idiotypic antibodies were added to a buffer mixture (pH 7.4) consisting of 100 mM HEPES, 20 mM sodium cyanoborohydride, and 0.06% Tween-20. This Ab-specific anti-idiotypic antibody mixture was used to resuspend the acceptor bead pellet, and the suspension was transferred to a 0.5 mL screw-cap tube. The conjugation reaction tube was incubated overnight (37 °C, 18–24 h) in a tube rotator. Carboxymethoxylamine (CMO) was added to a concentration of approximately 3 mg / mL to block unreacted aldehyde groups, and the reaction tube was incubated at 37 °C for 1 h. The Ab-specific anti-idiotypic antibody-conjugated acceptor beads were collected in a refrigerated centrifuge (16,000 × g, 15 min, 4°C), the supernatant was removed, and the pellet was washed with 100 mM Tris-HCl (pH 8.0) and subsequently collected using the previous refrigerated centrifuge conditions.After removing the supernatant, the Ab-specific anti-idiotype antibody-conjugated acceptor beads were resuspended at a concentration of 5 mg / mL in 1x PBS, 0.05% PROCLIN™ 300 (pH 7.4) and stored at 2-8°C in amber screw-cap tubes protected from light. Ab1-specific anti-idiotype antibodies or Ab2-specific anti-idiotype antibodies were covalently linked to their respective acceptor bead surfaces via reductive amination between accessible lysine residues on the Ab surface and reactive aldehyde groups on the acceptor bead surface. In some embodiments, Ab1 can be conjugated to ALPHAPLEX™ acceptor beads, and Ab2 can be conjugated to ALPHALISA® acceptor beads. In other embodiments, Ab1 can be conjugated to ALPHALISA® acceptor beads, and Ab2 can be conjugated to ALPHAPLEX™ acceptor beads. Details of the reagents used in the conjugation reaction are shown in Table 1.

[0089] [Table 1]

[0090] Reagents were tested in separate assays specific for each target. Unlabeled antibodies (Ab1 or Ab2) were mixed in 8-point serial dilutions (ranging from 100 μg / mL to 10 pg / mL in well concentrations) with their respective biotinylated antibodies (Ab1-biotin or Ab2-biotin) (0.15 μg / mL in well concentration) and their respective (Ab1-specific or Ab2-specific) anti-idiotypic antibody-conjugated acceptor beads (10 μg / mL in well concentration) in a 96-well plate (Corning®, Cat. No. 3642) and incubated (1 h, 25°C). Streptavidin-coated donor beads (PerkinElmer®, Cat. No. 6760002) were added to each well (40 μg / mL in well concentration), and the reaction was returned to incubation (30 min, 25°C). Replicates of each serial dilution were analyzed on a single assay plate to assess standard deviation and coefficient of variation (CV). Plates were analyzed using a PerkinElmer ENVISION® 2104 plate reader, with excitation at 680 nm, and the emission signal was measured first at 545 nm, followed by the emitted signal at 615 nm, corresponding to the excited terbium and europium fluorophores, respectively. Dose-dependent response curves were generated by analyzing the raw signals with a four-parameter logistic curve fit using SOFTMAX® Pro software v5.4.1 (Molecular Devices, LLC).

[0091] The reagents exhibited high specificity for their respective reagents, as shown in the dose-dependent response curves in Figures 2A and 2B. Small errors between replicates were observed.

[0092] To distinguish the two antibodies in the formulation and establish relative binding percentage values, a mock co-formulation experiment with Ab1 and Ab2 was performed. A "CoForm" sample containing 10 mg / mL of each antibody was prepared in ALPHALISA® Immunoassay Buffer (25 mM HEPES, 0.1% casein, 1 mg / mL Dextra-500, 0.5% Triton X-100, 0.05% Proclin-300, pH 7.4) (PerkinElmer, catalog number AL000F). Other reagents included biotinylated Ab1 and biotinylated Ab2 (Ab1-biotin and Ab2-biotin), terbium-labeled Ab1-specific anti-idiotypic antibody-conjugated acceptor beads, europium-labeled Ab2-specific anti-idiotypic antibody-conjugated acceptor beads, and streptavidin-coated donor beads. In this assay, eight serial dilutions of Ab1 alone, Ab2 alone, and CoForm samples (well concentrations ranging from 100 μg / mL to 6.1 ng / mL) were analyzed in parallel in a 96-well plate (Corning® 3642) in the presence of the same reagents except for the biotinylated antibodies they received. Specifically, Ab1 serial dilution wells received 0.15 μg / mL (well concentration) of Ab1-biotin alone (without Ab2-biotin), Ab2 serial dilution wells received 0.15 μg / mL (well concentration) of Ab2-biotin alone (without Ab1-biotin), and CoForm samples received a mixture containing both Ab1-biotin and Ab2-biotin, each at 0.15 μg / mL (well concentration). A mixture of Ab1-specific anti-idiotypic antibody-conjugated acceptor beads and Ab2-specific anti-idiotypic antibody-conjugated acceptor beads was added to all wells (10 μg / mL in each well), and the plate was then incubated for 1 hour at 25°C. Streptavidin-coated donor beads (PerkinElmer®, catalog no. 6760002) were added to each well (40 μg / mL in each well), and the reaction was returned to incubation for 30 minutes at 25°C. Replicates of each serial dilution were analyzed on a single assay plate to assess standard deviation and coefficient of variation (CV).In analysis using a four-parameter logistic curve fit for the 545 nm (terbium) channel (corresponding to Ab1 binding) in SOFTMAX® Pro software v5.4.1, the signal from Ab1 alone was used as the reference standard to determine the relative binding rate of Ab1 in the CoForm sample, and Ab2 alone was used as a control showing the absence of signal (absence of Ab1 biotin bringing the donor beads and anti-Ab1-specific idiotype acceptor beads into close proximity) (see Figure 3A). Similarly, when analyzing the 615 nm (europium) channel, corresponding to Ab2 binding, the signal from Ab2 alone was used as the reference standard to determine the relative binding rate of Ab2 in the CoForm sample, and Ab1 alone was used as a control showing the absence of signal (see Figure 3B). As shown in Figures 3A and 3B, the antibodies in the co-formulations demonstrated relative binding rates within specification (80-120% binding relative to the control) compared to their respective controls. Furthermore, the results complied with and passed strict internal assay and sample acceptance criteria.

[0093] The linearity of the assay was analyzed with respect to Ab1. In the panel of co-formulation samples, Ab2 was maintained at a constant concentration (10 mg / mL), while Ab1 was included at simulated concentrations expected to achieve relative binding rates of 60%, 80%, 130%, and 160%. Other reagents included biotinylated Ab1 and biotinylated Ab2 (Ab1-biotin and Ab2-biotin), terbium-labeled Ab1-specific anti-idiotypic antibody-conjugated acceptor beads, europium-labeled Ab2-specific anti-idiotypic antibody-conjugated acceptor beads, and streptavidin-coated donor beads. In this assay, eight serial dilutions of simulated co-formulation samples (ranging from 100 μg / mL to 6.1 ng / mL in wells) were analyzed in parallel in a 96-well plate (Corning® 3642) in the presence of the same reagents. Specifically, a mixture containing both Ab1-biotin and Ab2-biotin at 0.15 μg / mL each (well concentration) and a mixture containing both Ab1-specific anti-idiotypic antibody-conjugated acceptor beads (terbium) and Ab2-specific anti-idiotypic antibody-conjugated acceptor beads (europium) at 10 μg / mL each (well concentration) were added to each well of a plate containing simulated co-formulation samples, and the plate was then incubated (1 hour, 25°C). Streptavidin-coated donor beads (PerkinElmer®, catalog number 6760002) were added to each well (well concentration 40 μg / mL), and the reaction was returned to incubation (30 minutes, 25°C). Replicates of each serial dilution were analyzed on a single assay plate to assess standard deviation and coefficient of variation (CV). The raw signals were analyzed using a four-parameter logistic curve fit using SOFTMAX® Pro software v5.4.1, and the relative binding rate for each simulated sample was reported. The results of this analysis are shown in Figure 4. Ab1 exhibited linearity within the normal specification range.

[0094] The specificity of the assay was analyzed to confirm the absence of cross-reactivity between Ab1 and Ab2-specific reagents (i.e., Ab2-specific anti-idiotypic antibody and Ab2-biotin), and between Ab2 and Ab1-specific reagents (i.e., Ab1-specific anti-idiotypic antibody and Ab1-biotin). In this assay, eight serial dilutions of Ab1 were mixed with Ab2-biotin (0.15 mg / mL in well) and Ab2-specific anti-idiotypic antibody-conjugated acceptor beads (10 μg / mL in well) in a 96-well plate (Corning® 3642). These Ab2-specific reagents were also analyzed in the same assay plate in the absence of Ab1, demonstrating that maximal signal was obtained when streptavidin-coated donor beads conjugated with Ab2-biotin were brought into close proximity with Ab2-specific anti-idiotype antibody-conjugated acceptor beads (via binding of Ab2-biotin to the anti-Ab2 idiotype). In the same assay plate, eight serial dilutions of Ab2 were incubated in the presence of Ab1-biotin (0.15 μg / mL in the well) and Ab1-specific anti-idiotype antibody-conjugated acceptor beads (10 μg / mL in the well). These Ab1-specific reagents were also analyzed in the same assay plate in the absence of Ab2, demonstrating that maximal signal was obtained when Ab1-biotin-conjugated donor beads were brought into close proximity with Ab1-specific anti-idiotype antibody-conjugated acceptor beads. The plate was then incubated (1 hour, 25°C), after which streptavidin-coated donor beads (PerkinElmer® catalog no. 6760002) were added to each well (40 μg / mL in-well concentration), and the plate was returned to incubation (30 minutes, 25°C). Replicates of each serial dilution, as well as controls without Ab1 or without Ab2, were analyzed on the same plate to assess standard deviation and coefficient of variation (CV). Raw signals were analyzed using a four-parameter logistic curve fit using SOFTMAX® Pro software v5.4.1. As shown in Figures 5A and 5B, the reagents generated for use in this co-formulation assay exhibit high specificity with low potential for reagent cross-reactivity.

[0095] To confirm that the choice of acceptor bead conjugation partner was optimal for the assay, an anti-Ab specific idiotype was conjugated to the opposite acceptor bead as described above. There were no observable differences in the raw signal or plotted curves between the initial and swapped formats.

[0096] The practical use of the coformulation assay was demonstrated through the analysis of blind mock coformulation samples. The only constraint for sample preparers was that the concentrations of Ab1 and Ab2 must be between 0.5 and 20 mg / mL in each blind mock sample. A CoForm reference standard (Ref Std) was prepared with Ab1 and Ab2 at 10 mg / mL each in ALPHALISA® immunoassay buffer. This mock CoForm Ref Std was used as a reference standard in each assay plate to measure the relative binding rates of the samples and simultaneously used as an assay control to confirm that the assay and operator were performing as expected (measured using assay and sample acceptance criteria). In this assay, eight serial dilutions of the blind mock coformulation samples were analyzed in parallel in a 96-well plate (Corning® 3642) in the presence of the same reagents. Because the samples were blind, they were analyzed using the same dilution factor as the Ref Std and control wells, and all samples were analyzed against 10 mg / mL of unlabeled Ab1 and Ab2, respectively, with the signal proportional to the relative amount of unlabeled Ab1 and Ab2 in the blind mock co-formulation samples. A mixture containing both Ab1-biotin and Ab2-biotin at 0.15 μg / mL each (well concentration) and a mixture containing both Ab1-specific anti-idiotypic antibody-conjugated acceptor beads (terbium) and Ab2-specific anti-idiotypic antibody-conjugated acceptor beads (europium) at 10 μg / mL each (well concentration) was added to all wells, and the plate was then incubated (1 hour, 25°C). Streptavidin-coated donor beads (PerkinElmer®, catalog number 6760002) were added to each well (40 μg / mL in-well concentration), and the reaction was returned to incubation (30 min, 25°C). Replicates of each serial dilution were analyzed in a single assay plate to assess standard deviation and coefficient of variation (CV). Raw signals were analyzed using a four-parameter logistic curve fit using SoftMax® Pro software v5.4.1 (Molecular Devices), and the simulated relative binding percentage for each sample was reported.Three independent assays (i.e., "runs") were performed, and the average relative binding rates for Ab1 and Ab2 in each blind mock co-formulation sample were calculated. Figures 6A and 6B show representative graphs of the resulting 545 nm (terbium) and 615 nm (europium) signals. The results demonstrated that the assay had excellent ability to determine the concentration of Ab1 or Ab2 in blind samples in the range of approximately 1 to 15 mg / mL. At concentrations successively higher or lower than this range, the error rates in the concentration measurements suggest that the assay is unreliable, at least when using the reference standard concentrations tested (10 mg / mL for Ab1 and Ab2, respectively).

[0097] All references, including publications, patent applications, and patents, cited in this specification are herein incorporated by reference to the same extent as if each individual reference was individually and specifically indicated to be incorporated by reference in its entirety and set forth herein.

[0098] In the context of describing the invention (particularly in the context of the claims which follow), the use of the terms "a," "an," "the," and "at least one," and similar referents, should be construed to encompass both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. The term "at least one," when followed by a list of one or more items (e.g., "at least one of A and B"), should be construed to mean one item (A or B) selected from the listed items 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" should be construed as open-ended terms (i.e., meaning "including, but not limited to") unless otherwise noted. The recitation of ranges of values ​​herein is 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 herein by reference as if it were individually set forth herein. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. Any examples provided herein, or the use of illustrative language (e.g., "etc."), are intended merely to further clarify the invention and do not impose limitations on the scope of the invention unless specifically claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention.

[0099] Preferred embodiments of the invention are described herein, including the best mode known to the inventors for carrying out the invention. Variations on those preferred embodiments may become apparent to those of ordinary skill in the art upon reading the foregoing description. The inventors anticipate that skilled artisans will employ such variations as appropriate, and the inventors intend 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. Furthermore, any combination of the above-described elements in all possible variations thereof is encompassed by the invention unless otherwise indicated herein or clearly contradicted by context.

Claims

1. 1. A system for detecting and quantifying two or more different antigen binding proteins in a sample, comprising: (a) donor beads, each donor bead comprising a photosensitizer and a first binding pair member; (b) acceptor beads, each acceptor bead comprising a chemiluminescent dye and an anti-idiotypic antibody that specifically binds to one of the two or more different antigen-binding proteins; and (c) conjugated proteins, each conjugated protein comprising any of the two or more different antigen-binding proteins and a second binding pair member. Including, The system, wherein the first binding pair member is configured to specifically bind to the second binding pair member.

2. 10. The system of claim 1, wherein each of the antigen binding proteins is an antibody, an antibody fragment, or a bispecific T cell engager (BiTE®) molecule.

3. The system of claim 1 or 2, wherein the photosensitizer comprises a phthalocyanine.

4. The system of any one of claims 1 to 3, wherein the first binding pair member comprises streptavidin, glutathione S-transferase (GST), or an affinity tag peptide such as hexahistidine or FLAG peptide.

5. The system of any one of claims 1 to 4, wherein the second binding pair member comprises biotin, glutathione, or an anti-affinity tag antibody, such as an anti-hexahistidine antibody or an anti-FLAG antibody.

6. 6. The system of claim 5, wherein the first binding pair member comprises streptavidin and the second binding pair member comprises biotin.

7. 7. The system of any one of claims 1 to 6, comprising: (i) a first acceptor bead conjugated to a first chemiluminescent dye and a first anti-idiotypic antibody that binds to a first antigen binding protein in the sample; and (ii) a second acceptor bead conjugated to a second chemiluminescent dye and a second anti-idiotypic antibody that binds to a second antigen binding protein in the sample.

8. 8. The system of claim 7, wherein the first chemiluminescent dye comprises terbium (Tb) and the second chemiluminescent dye comprises europium (Eu).

9. 9. The system of claim 7 or 8, wherein the first antigen binding protein is a first therapeutic antibody and the second antigen binding protein is a second therapeutic antibody.

10. 10. The system of any one of claims 6 to 9, comprising a first conjugated protein comprising the first antigen-binding protein and biotin, and a second conjugated protein comprising the second antigen-binding protein and biotin.

11. The system according to any one of claims 1 to 10, wherein the sample is a composition comprising the two or more different antigen-binding proteins and a pharmaceutically acceptable carrier.

12. 1. A method for determining the presence, absence or concentration of two or more different antigen binding proteins in a sample, comprising: (1) incubating the sample with the system according to any one of claims 1 to 11; (2) exposing the incubated sample to light excitation; and (3) detecting at least one chemiluminescent signal after photoexcitation and comparing the detected chemiluminescent signal with a predetermined reference value; the at least one detected chemiluminescent signal is inversely proportional to the concentration of each of the two or more different antigen binding proteins; thereby determining the presence, absence or concentration of two or more different antigen binding proteins in a sample.

13. 13. The method of claim 12, wherein the predetermined reference values ​​include threshold values ​​corresponding to respective concentrations of the antigen-binding proteins, and wherein a detected chemiluminescent signal greater than the threshold value indicates that the sample lacks the antigen-binding protein or contains the antigen-binding protein at a concentration less than the threshold value, and a chemiluminescent signal equal to or less than the threshold value indicates that the sample contains the antigen-binding protein at a concentration equal to or greater than the threshold value.

14. The method of claim 12 or 13, wherein the optical excitation wavelength is from about 500 nm to about 800 nm.

15. 15. The method of claim 14, wherein the optical excitation wavelength is about 680 nm.

16. The method of any one of claims 12 to 15, wherein the sample is a composition comprising the two or more different antigen-binding proteins and a pharmaceutically acceptable carrier.

17. 17. The method of any one of claims 12 to 16, wherein each of the antigen binding proteins is an antibody, an antibody fragment or a bispecific T cell engager (BiTE®) molecule.

18. 18. The method of any one of claims 12 to 17, wherein the system comprises: (i) a first acceptor bead comprising a first chemiluminescent dye and a first anti-idiotypic antibody that binds to a first antigen binding protein in the sample; and (ii) a second acceptor bead comprising a second chemiluminescent dye and a second anti-idiotypic antibody that binds to a second antigen binding protein in the sample.

19. 20. The method of claim 18, wherein the first antigen binding protein comprises a first therapeutic antibody and the second antigen binding protein comprises a second therapeutic antibody.

20. 20. The method of claim 18 or 19, comprising detecting a first signal emitted from the first chemiluminescent dye and a second signal emitted from the second chemiluminescent dye.

21. 21. The method of claim 20, wherein the first signal and the second signal comprise two different wavelengths.

22. 22. The method of claim 21, wherein the wavelength of the first signal is from about 520 nm to about 620 nm and the wavelength of the second signal is from about 520 nm to about 620 nm.

23. 23. The method of claim 22, wherein the wavelength of the first signal is about 545 nm and the wavelength of the second signal is about 615 nm.

24. 24. The method of any one of claims 12 to 23, further comprising quantifying the two or more antigen-binding proteins in the sample.

25. The method of any one of claims 12 to 24, wherein the reference value is from a calibration curve for each acceptor bead in the system.

26. The method of any one of claims 12 to 25, further comprising generating a calibration curve for each acceptor bead in the system.

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