Methods for analyzing antibody co-formulations

Abstract

The disclosure provides methods for analyzing two or more different proteins co-formulated in a sample mixture. The methods involve cation exchange-high performance liquid chromatography (CEX-HPLC), size exclusion ultra high performance liquid chromatography (SE-UHPLC), and / or capillary electrophoresis (CE) optimized to accurately determine the concentration, and assess certain critical quality attributes of, each protein in the sample.

Description

Attorney Docket No.: 10501-WO01-SEC METHODS FOR ANALYZING ANTIBODY CO-FORMULATIONS FIELD

[0001] The disclosure relates to methods for detecting and quantifying two or more antigen- binding proteins (e.g., antibodies) that are co-formulated in a single composition. BACKGROUND

[0002] Combination therapy using two or more molecules with complimentary pharmacological effects has led to an increased interest in the development of co-formulation products. Co-formulations or fixed-dose combination drugs (FDCs) are therapeutics in which two or more separate drug components (e.g., a small molecule and biologic, or two different biologics such as therapeutic antibodies) are combined in a single dosage form. These products can often reduce the number and volume of injections, improve patient compliance, and reduce discomfort.

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

[0004] There are also many chemistry, manufacturing, and control issues to address for a co- formulated biologic. These include analytical challenges in characterization of each molecule in the co-formulation, manufacturing issues of formulating higher concentration biologics, and stability problems such as protein-protein interactions, protein aggregation, and subvisible particle formulation.

[0005] There remains a need for methods for monitoring critical quality attributes of co- formulated biologic molecules (e.g., high molecular weight species) and accurately determining the concentrations of each biologic in a pharmaceutical preparation or sample.Attorney Docket No.: 10501-WO01-SEC BRIEF SUMMARY

[0006] The disclosure provides a method of determining the concentration of each of a first protein and a second protein that is different from the first protein in a composition, comprising: providing said composition comprising the first protein and the second protein, wherein the first protein has a first extinction coefficient and the second protein has a second extinction coefficient; resolving the first protein from the second protein of said composition by cation exchange chromatography (CEX), whereby a ratio of the first protein to the second protein in said composition is obtained; measuring a total absorbance of the composition for a path length; and calculating a concentration of said first protein and said second protein based on the ratio of the total absorbance to said path length and said extinction coefficients, prorated by the ratio.

[0007] In some aspects of the method, calculating the concentration of the second protein comprises Equation 1: ^^ଶ= ^^௧^௧^^ / (ε1b + ε2b ^^) [Equation 1] wherein C2 is the concentration of the second protein, ^^௧^௧^^is the total absorbance, ε1 is the extinction coefficient of the first protein, ε2 is the extinction coefficient of the second protein, b is the path length, and k is the is the ratio of the first protein to the second protein in the composition. In some aspects of the method, calculating the concentration of said first protein further comprises Equation 2: ^^^= ^^ଶ× ^^ [Equation 2], wherein ^^^is the concentration of the first protein, C2 is the concentration of the second protein, and k is the is the ratio of the first protein to the second protein in the composition.

[0008] In some aspects of the method, the ratio of the first protein to the second protein is 1:1 to 1:100, such as, for example, 1:1 to 1:80, 1:1 to 1:40, 1:1 to 1:20, 1:1.1 to 1:100, 1:1.1 to 1:80, 1:1.1 to 1:40, 1:1.1 to 1:20, 1:2 to 1:100, 1:2 to 1:80, 1:2 to 1:40, or 1:2 to 1:20.

[0009] In some aspects of the method, the first protein has an isoelectric point (pI) that differs from the isoelectric point of the second protein by at least 0.2.

[0010] In some aspects, the method further comprises resolving the first protein from the second protein in the composition by CEX, hydrophobic interaction chromatography (HIC), or reverse phase high performance liquid chromatography (RP-HPLC), and determining theAttorney Docket No.: 10501-WO01-SEC concentration of the first protein and the second protein based on a calibration curve for each protein.

[0011] The disclosure also provides a chromatography method of analyzing a composition comprising a first protein and a second protein that is different from the first protein, which method comprises: providing said composition comprising the first protein and the second protein, and at least one of: a) determining a level of high molecular weight (HMW) species of the first protein and / or second protein in the composition by size exclusion ultra high performance liquid chromatography (SE-UHPLC), wherein the SE-UHPLC is performed with a buffer comprising about 100-200 mM KCl, about 1%-5% isopropyl alcohol (IPA), and about 40- 60 mM K3PO4; or b) separating charge variants of the first protein and / or the second protein in the composition by pH-gradient cation exchange-high performance liquid chromatography (CEX-HPLC), wherein the CEX-HPLC comprises a first mobile phase at pH 5-6 and a second mobile phase at pH 10-11.

[0012] In some aspects of the chromatography method, the HMW species comprise one or more of dimers, trimers, tetramers, pentamers, hexamers, heptamers, and octamers.

[0013] In some aspects of the chromatography method, the SE-UHPLC is performed with a buffer comprising about 150 mM KCl, about 2% IPA, and about 50 mM K3PO4.

[0014] In some aspects of the chromatography method, the SE-UHPLC comprises a mobile phase having a flow rate of about 0.2-0.5 mL / min. For example, the mobile phase flow rate may be about 0.4 mL / min.

[0015] In some aspects of the chromatography method, the CEX-HPLC comprises a first mobile phase of pH of about 5.6, such as pH 5.6. In some aspects of the chromatography method, the CEX-HPLC comprises a first mobile phase of pH of 5.4-5.8.

[0016] In some aspects of the chromatography method, the CEX-HPLC comprises a second mobile phase of pH of about 10.2, such as pH 10.2. In some aspects of the chromatography method, the CEX-HPLC comprises a second mobile phase of pH of 10.0-10.4.

[0017] In some aspects of the chromatography method, the CEX-HPLC comprises a gradient from 100% of the first mobile phase + 0% of the second mobile phase to no more than 20% of the first mobile phase + at least 80% of the second mobile phase, over at least 40, 5060, or 70 minutes.Attorney Docket No.: 10501-WO01-SEC

[0018] In some aspects of the chromatography method, the CEX-HPLC comprises a column that is at least 50 mm, 75 mm, or 100 mm. Optionally, the CEX-HPLC column may comprise a matrix of hydrophilic porous polymer beads having an average diameter of about 3 µm to about 7 µm, such as about 5 µm. The CEX-HPLC column may comprise a sulfopropyl ion exchanger chemistry.

[0019] In some aspects, the chromatography method further comprises, after separating said charge variants by CEX-HPLC, determining the concentration of the first protein and the second protein in the composition based on a calibration curve for each protein.

[0020] The disclosure further provides a capillary electrophoresis method of determining concentration of each of a first protein and a second protein in a composition, wherein the first protein is different from the first protein, which method comprises: analyzing the composition comprising the first protein and the second protein by capillary electrophoresis sodium dodecyl sulfate (CE-SDS), wherein the CE-SDS comprises a first peak of the first protein, a second peak of the second protein, and a comigration peak of the first protein and the second protein; and determining the concentration of each of the first protein and second protein based on a ratio of the first peak to the second peak.

[0021] In some aspects of the capillary electrophoresis method, the CE-SDS is reduced (rCE- SDS) or non-reduced (nrCE-SDS).

[0022] In some aspects of the above methods, each of the first protein and the second protein is an antigen-binding protein.

[0023] In some aspects of the above methods, the antigen-binding protein is an antibody, an antibody fragment, or a bispecific T cell engager (BiTE®) molecule.

[0024] In some aspects of the above methods, each of the first protein and the second protein is a therapeutic antibody.

[0025] In some aspects of the above methods, the therapeutic antibody specifically binds to TIGIT, CD112R, PD-1, or VEGF.

[0026] In some aspects of the above methods, the first protein is an antibody that specifically binds to CD112R and the second protein is an antibody that specifically binds to TIGIT.

[0027] In some aspects of the above methods the first protein is an antibody that specifically binds to PD-1 and the second protein is an antibody that specifically binds to VEGF.Attorney Docket No.: 10501-WO01-SEC BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 is a plot showing results of high molecular weight (HMW) species analysis for the Ab1 / Ab2 co-formulation using the optimized SE-UHPLC method described in Example 1.

[0029] Figure 2 is a plot showing the results of an analysis of the Ab1 / Ab2 co-formulation with the developed CEX-HPLC method described in Example 2.

[0030] Figures 3A and 3B are graphs of the measured / experimental ratio of Ab1:Ab2 compared to the theoretical ratio in unstressed (To) (Fig.3A) and stressed (four weeks at 40 °C) (Fig.3B) conditions as determined by the methods described in Example 3.

[0031] Figures 4A and 4B are graphs of the measured / experimental ratio of Ab1:Ab3 compared to the theoretical ratio in unstressed (To) (Fig.4A) and stressed (four weeks at 40 °C) (Fig.4B) conditions as determined by the methods described in Example 3.

[0032] Figures 5A and 5B are graphs of the measured / experimental ratio of Ab2:Ab4 compared to the theoretical ratio in unstressed (To) (Fig.5A) and stressed (four weeks at 40 °C) (Fig.5B) conditions as determined by the methods described in Example 3.

[0033] Figures 6A and 6B are graphs of the measured / experimental ratio of Ab1:Ab4 compared to the theoretical ratio in unstressed (To) (Fig.6A) and stressed (four weeks at 40 °C) (Fig.6B) conditions as determined by the methods described in Example 3.

[0034] Figures 7A and 7B show results of direct concentration measurement of Ab1 / Ab2 (Fig.7A) and Ab1 / Ab3 (Fig.7B) using a RP-HPLC-based titer method.

[0035] Figure 8 shows results of direct concentration measurement of Ab1 / Ab2 and Ab1 / Ab3 using an HIC-based titer method.

[0036] Figure 9A is a plot of HMW and main peak species for different ratios of the co- formulated Ab4 / Ab5 mixture as measured by SE-UHPLC. Figure 9B is a graph showing % Ab5 vs. % total HMW species.

[0037] Figure 10 is a rCE-SDS profile of the Ab4 / Ab5 co-formulated mixture.

[0038] Figure 11 includes graphs showing the concentration of Ab4 and Ab5 after mixing at different ratios as measured using rCE-SDS.

[0039] Figure 12 is a nrCE-SDS profile of the Ab4 / Ab5 co-formulated mixture.

[0040] Figure 13 includes CEX profiles for different mixing ratios of the Ab4 / Ab5 co- formulation.Attorney Docket No.: 10501-WO01-SEC DETAILED DESCRIPTION

[0041] The present disclosure is predicated, at least in part, on the development of assays for resolving and quantifying different intact proteins (e.g., antibodies) co-formulated together in a single sample, as well as monitoring critical quality attributes (CQAs) of each protein in the co- formulation. The methods described herein allow for accurate monitoring and control of therapeutic drug products, and the processing thereof, through release and stability testing to ensure accurate dosing. Monitoring the concentrations of each component in a co-formulated drug product also is critical for assessing combined therapeutic efficacy, as well as other more specific applications, such as IV bag stability for co-administered therapeutic.

[0042] In some embodiments, the disclosure provides method of determining the concentration of each of a first protein and a second protein that is different from the first protein in a composition. The method comprises providing a composition comprising the first protein and the second protein, wherein the first protein has a first extinction coefficient and the second protein has a second extinction coefficient; resolving the first protein from the second protein of said composition by cation exchange chromatography (CEX), whereby a ratio of the first protein to the second protein in said composition is obtained; measuring a total absorbance of the composition for a path length; and calculating a concentration of said first protein and said second protein based on the ratio of the total absorbance to said path length and said extinction coefficients, prorated by the ratio.

[0043] The disclosure also provides a chromatography method of analyzing a composition comprising a first protein and a second protein that is different from the first protein. The method comprises: providing said composition comprising the first protein and the second protein, and at least one of: a) determining a level of high molecular weight (HMW) species of the first protein and / or second protein in the composition by size exclusion ultra high performance liquid chromatography (SE-UHPLC), wherein the SE-UHPLC is performed with a buffer comprising about 100-200 mM KCl, about 1%-5% isopropyl alcohol (IPA), and about 40- 60 mM K3PO4; or b) separating charge variants of the first protein and / or the second protein in the composition by pH-gradient cation exchange-high performance liquid chromatography (CEX-HPLC), wherein the CEX-HPLC comprises a first mobile phase at pH 5-6 and a second mobile phase at pH 10-11.Attorney Docket No.: 10501-WO01-SEC

[0044] In further embodiments, the disclosure provides a capillary electrophoresis method of determining concentration of each of a first protein and a second protein in a composition, wherein the first protein is different from the first protein. The method comprises analyzing the composition comprising the first protein and the second protein by capillary electrophoresis sodium dodecyl sulfate (CE-SDS), wherein the CE-SDS comprises a first peak of the first protein, a second peak of the second protein, and a comigration peak of the first protein and the second protein; and determining the concentration of each of the first protein and second protein based on a ratio of the first peak to the second peak. Definitions

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

[0046] In some aspects, the term “assaying” means “measuring,” and may be used interchangeably with the terms “testing,” “analyzing,” or “determining.” The level of protein or protein attribute that is assayed or determined by the presently disclosed methods can be a relative measurement, e.g., a determination that the level is higher or lower or the same as a reference level. For example, in some aspects, the method of the present disclosure can assay the level of HMW species of a the first protein and / or second protein in a composition. The “assaying” in some aspects can yield a normalized measurement. For instance, the normalized measurement can be normalized to a reference protein, e.g., serum albumin. The “assaying” in certain instances yields an absolute measurement (e.g., neither normalized nor relative to a reference level).

[0047] A “critical quality attribute (CQA)” refers to a physical, chemical, biological, or microbiological property or characteristic that should be within an appropriate limit, range, or distribution to ensure the desired product quality. CQAs are generally associated with a drug product, drug substance, excipients, and intermediates (in-process materials). For example, for large polypeptide therapeutic molecules, physical attributes and modifications of amino acids are important CQAs that are monitored during and after manufacturing, as well as during drug development. Non-limiting examples of CQAs for monoclonal antibodies, or antigen-binding fragments thereof, include high molecular weight (HMW) species, charge variants, oxidized species, deamidated species, and glycosylation.Attorney Docket No.: 10501-WO01-SEC

[0048] The term “antigen-binding protein,” as used herein, refers to a proteinaceous molecule that specifically binds to an antigen. For example, an antigen-binding protein may comprise an antibody or an antigen-binding fragment thereof, (such as a monoclonal antibody, for example an IgG1 or IgG2 monoclonal antibody), an antibody protein product, a bi-specific T cell engager (BiTE®) molecule, a bispecific antibody, a trispecific antibody, or an Fc fusion protein.

[0049] An antigen-binding protein typically comprises the heavy chain variable region (VH) and / or the light chain variable region (VL) of an antibody, or comprises domains derived therefrom. In some embodiments, an antigen-binding protein comprises the structural requirements of an antibody which are sufficient for immunospecific target binding. This structural requirement may be defined by, for example, the presence of at least three light chain complementarity determining regions (CDRs) (i.e., CDR1, CDR2 and CDR3 of the VL region) and / or three heavy chain CDRs (i.e., CDR1, CDR2 and CDR3 of the VH region), or of all six CDRs. It is within the knowledge of a skilled person where (and in which order) those CDRs are located in the antigen-binding protein.

[0050] As used herein, the term “antibody” refers to an immunoglobulin of any isotype with specific binding to the target antigen; an antibody may be a polyclonal or monoclonal antibody, a chimeric antibody, a humanized antibody, a human antibody, etc. In a native antibody, a heavy chain comprises a variable region, VH, and three constant regions, 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 native antibody, a light chain comprises a variable region, VL, and a constant region, CL. The variable region of the light chain is at the amino-terminus of the light chain. In a native antibody, the variable regions of each light / heavy chain pair typically form the antigen- binding site. The constant regions are typically responsible for effector function. A native antibody is a tetramer of two full-length heavy chains and two full-length light chains.

[0051] In a human antibody, CH1 means a region having the amino acid sequence at positions 118 to 215 of the EU index or EU numbering system, which is based on the sequential numbering of the first human IgG1 sequenced (i.e., the “EU antibody”) (Edelman et al., Proc Natl Acad Sci USA, 63(1): 78-85 (1969)). A highly flexible amino acid region called a “hinge region” exists between CH1 and CH2. CH2 represents a region having the amino acid sequenceAttorney Docket No.: 10501-WO01-SEC at positions 231 to 340 of the EU index, and CH3 represents a region having the amino acid sequence at positions 341 to 446 of the EU index.

[0052] “CL” represents a constant region of a light chain. In the case of a κ chain of a human antibody, CL represents a region having the amino acid sequence at positions 108 to 214 of the EU index. In a λ chain, CL represents a region having the amino acid sequence at positions 108 to 215.

[0053] In a native antibody, the variable regions typically exhibit the same general structure in which relatively conserved framework regions (FRs) are joined by three hypervariable CDRs. The CDRs from the two chains of each pair typically are aligned by the framework regions, which may enable binding to a specific epitope. From N-terminus to C-terminus, both light and heavy chain variable regions typically comprise the domains FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4. Typically, CDR3 is the greatest source of molecular diversity within the antigen binding site. The assignment of amino acids to each domain is typically in accordance with the definitions of Kabat et al. (1991) Sequences of Proteins of Immunological Interest (National Institutes of Health, Publication No.91-3242, vols.1-3, Bethesda, Md.); Chothia, C., and Lesk, A. M. (1987) J. Mol. Biol., 196: 901-917. In some embodiments, the CDRs of an antigen binding protein are defined according to the definition of Kabat or Chothia. In the present application, the term “CDR” refers to a CDR from either the light or heavy chain, unless otherwise specified.

[0054] Antibodies can comprise any constant region known in the art. Human light chains are classified as kappa and lambda light chains. Heavy chains are classified as mu, delta, gamma, alpha, or epsilon, and define the antibody’s isotype as IgM, IgD, IgG, IgA, and IgE, respectively. IgG has several subclasses, including, but not limited to IgG1, IgG2, IgG3, and IgG4. IgM has subclasses, including, but not limited to, IgM1 and IgM2. Embodiments of the present disclosure include all such classes or isotypes of antibodies. The light chain constant region can be, for example, a kappa- or lambda-type light chain constant region, e.g., a human kappa- or lambda-type light chain constant region. The heavy chain constant region can be, for example, an alpha-, delta-, epsilon-, gamma-, or mu-type heavy chain constant regions, e.g., a human alpha-, delta-, epsilon-, gamma-, or mu-type heavy chain constant region. Accordingly, in exemplary embodiments, the antibody is an antibody of isotype IgA, IgD, IgE, IgG, or IgM, including any one of IgG1, IgG2, IgG3 or IgG4.Attorney Docket No.: 10501-WO01-SEC

[0055] The 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 typically are produced using hybridoma technology, as first described in Kohler and Milstein, Eur. J. Immunol., 5: 511-519 (1976). Monoclonal antibodies may also be produced using recombinant DNA methods (see, e.g., U.S. Patent 4,816,567), isolated from phage display antibody libraries (see, e.g., Clackson et al. Nature, 352 : 624-628 (1991)); and Marks et al., J. Mol. Biol., 222: 581-597 (1991)), or produced from transgenic mice carrying a fully human immunoglobulin system (see, e.g., XENOMOUSE™ mouse, Green et al. (1994) Nature Genetics 7:13-21, US 2003-0070185, WO 96 / 34096, and WO 96 / 33735). In contrast, “polyclonal” antibodies are antibodies that are secreted by different B cell lineages within an animal. Polyclonal antibodies are a collection of immunoglobulin molecules that recognize multiple epitopes on the same antigen.

[0056] The term “chimeric antibody” refers to an antibody containing domains from two or more different antibodies. A chimeric antibody can, for example, contain the constant domains from one species and the variable domains from a second, or more generally, can contain stretches of amino acid sequence from at least two species. A chimeric antibody also can contain domains of two or more different antibodies within the same species. The term “humanized” when used in relation to antibodies refers to antibodies having at least CDR regions from a non- human source which are engineered to have a structure and immunological function more similar to true human antibodies than the original source antibodies. For example, humanizing can involve grafting a CDR from a non-human antibody, such as a mouse antibody, into a human antibody. Humanizing also can involve select amino acid substitutions to make a non-human sequence more similar to a human sequence.

[0057] An antibody can be cleaved into fragments by enzymes, e.g., papain, pepsin, or other engineered site-specific proteases (such as those commercially available from Genovis AB, Lund, Sweden). Papain cleaves an antibody to produce two Fab fragments and a single Fc fragment. Pepsin cleaves an antibody to produce a F(ab’)2 fragment and a pFc’ fragment. In exemplary aspects, the antigen-binding protein of the present disclosure comprises an antigen binding antibody fragment. As used herein, the term “antigen binding antibody fragment” refers to a portion of an antibody molecule that is capable of binding to the antigen of the antibody andAttorney Docket No.: 10501-WO01-SEC is also known as “antigen-binding fragment” or “antigen-binding portion.” In exemplary instances, the antigen binding antibody fragment is a Fab fragment or a F(ab’)2 fragment.

[0058] The architecture of antibodies has been exploited to create a growing range of alternative formats that span a molecular-weight range of at least about 12–150 kDa and has a valency (n) range from monomeric (n = 1), to dimeric (n = 2), to trimeric (n = 3), to tetrameric (n = 4), and potentially higher; such alternative formats are referred to herein as “antibody protein products.” Antibody protein products include those based on the full antibody structure and those that mimic antibody fragments which retain full antigen-binding capacity, e.g., scFvs, Fabs (e.g., Fab, Fab’, and F(ab’)2) and VHH / VH. The smallest antigen binding antibody fragment that retains its complete antigen binding site is the Fv fragment, which consists entirely of variable (V) regions of the light and heavy chains. A soluble, flexible amino acid peptide linker is used to connect the V regions in a scFv (single chain fragment variable) fragment for stabilization of the molecule, or the constant (C) domains are added to the V regions to generate a Fab fragment. Both scFv and Fab fragments can be easily produced in host cells, e.g., prokaryotic host cells. A VHH / VH (or nanobody) is the antigen binding fragment of heavy chain only antibodies. Heavy chain only antibodies (HcAb) are naturally produced by camelids and sharks. Other antibody protein products include bispecific T-cell engager (BiTE®) molecules, disulfide-bond stabilized scFv (ds-scFv), single chain Fab (scFab), as well as di- and multimeric antibody formats like dia- , tria- and tetra-bodies, or minibodies (miniAbs) that comprise different formats consisting of scFvs linked to oligomerization domains. A peptibody or peptide-Fc fusion is yet another antibody protein product. The structure of a peptibody consists of a biologically active peptide grafted onto an Fc domain (see, e.g., Shimamoto et al., mAbs 4(5): 586-591 (2012)).

[0059] The antigen-binding protein of the present disclosure may comprise any one of the above-described antibody protein products. In exemplary aspects, the antigen-binding protein of the present disclosure may comprise any one of an scFv, Fab VHH / VH, Fv fragment, ds-scFv, scFab, dimeric antibody, multimeric antibody (e.g., a diabody, triabody, tetrabody), miniAb, peptibody VHH / VH of camelid heavy chain antibody, sdAb, diabody; a triabody; a tetrabody; a bispecific or trispecific antibody, bispecific T-cell engager (BiTE®) molecule, BsIgG, appended IgG, BsAb fragment, bispecific fusion protein, or BsAb conjugate.

[0060] In certain aspects, the antigen-binding proteins of the present disclosure may be “bispecific,” meaning that they are capable of specifically binding to two different antigens. InAttorney Docket No.: 10501-WO01-SEC another aspect, the antigen-binding proteins of the present disclosure may be “trispecific,” meaning that they are capable of specifically binding to three different antigens. In another aspect, the antigen-binding proteins of the present disclosure may be “tetraspecific,” meaning that they are capable of specifically binding to four different antigens.

[0061] In some embodiments, the antigen-binding protein is a BiTE® molecule. BiTE® molecules are engineered bispecific antigen binding constructs which direct the cytotoxic activity of T cells against cancer cells. They are the fusion of two single-chain variable fragments (scFvs) of different antibodies, or amino acid sequences from four different genes, on a single peptide chain of about 55 kilodaltons. One of the scFvs binds to T cells via the CD3 receptor, and the other to a tumor cell via a tumor specific molecule. Blinatumomab (BLINCYTO® product) is an example of a BiTE® molecule, specific for CD19. BiTE® molecules that are modified, such as those modified to extend their half-lives, can also be used in the disclosed methods. By their design, BiTE® molecules are uniquely suited to transiently connect T cells with target cells and, at the same time, potently activate the inherent cytolytic potential of T cells against target cells. See e.g., WO 99 / 54440, WO 2005 / 040220, and WO 2008 / 119567.

[0062] In certain embodiments of the disclosure, the antigen-binding proteins may be multivalent. The valency of the binding protein denotes the number of individual antigen binding domains within the binding protein. In some embodiments, a bispecific antigen binding protein may be multivalent. For instance, in certain embodiments, a bispecific antigen binding protein may be tetravalent by comprising four antigen-binding domains: two antigen-binding domains binding to a first target antigen and two antigen-binding domains binding to a second target antigen.

[0063] As used herein, the terms “antigen binding domain” and “binding domain” may be used interchangeably to refer to the region of the antigen-binding protein that contains the amino acid residues that interact with the antigen and confer on the antigen-binding protein its specificity and affinity for the antigen. In some embodiments, the binding domain may be derived from the natural ligands of the target antigen(s). As used herein, the term “target antigen(s)” refers to a first target antigen and / or a second target antigen of a bispecific molecule and also refers to a first target antigen, a second target antigen, a third target antigen, and / or a fourth target antigen of a tetraspecific molecule.Attorney Docket No.: 10501-WO01-SEC

[0064] An antigen-binding protein may comprise an immunoglobulin domain. The term “immunoglobulin domain,” as used herein, refers to a peptide comprising an amino acid sequence similar to that of immunoglobulin (i.e., antibody) and comprising approximately 100 amino acid residues including at least two cysteine residues. Examples of immunoglobulin domains include VH, CH1, CH2, and CH3 of an antibody heavy chain, and VL and CL of an antibody light chain. In addition, the immunoglobulin domain is found in proteins other than immunoglobulin. Examples of the immunoglobulin domain in proteins other than immunoglobulin include an immunoglobulin domain included in a protein belonging to an immunoglobulin super family, such as a major histocompatibility complex (MHC), CD1, B7, T- cell receptor (TCR), and the like.

[0065] As used herein, the terms “stability” and “stabilizing” are defined as the maintenance of the chemical or physical integrity and / or bioactivity of the antigen-binding polypeptide or protein over a period of time. Stabilizing an antigen-binding polypeptide or protein includes the prevention or delay of degradation or deterioration of the antigen- binding polypeptide or protein from its biologically and / or therapeutically active form to an inactive form. Instability may arise from events such as aggregation, denaturation, fragmentation, or chemical modifications such as oxidation, cross- linking, deamidation and reactions with other components featured in the composition comprising the antigen-binding polypeptide or protein.

[0066] The terms “binding pair member” and “binding member” are used interchangeably herein and refer to one of two or more different molecules that specifically recognize the other molecule compared to substantially less recognition of other molecules. By way of example, when an antibody or other entity (e.g., antigen-binding protein) “specifically recognizes” or “specifically binds” an antigen or epitope, it preferentially recognizes the antigen in a complex mixture of proteins and / or macromolecules, and binds the antigen or epitope with affinity which is substantially higher than to other entities not displaying the antigen or epitope. Accordingly, the two binding pair members will bind to each other more tightly than to other molecules. Methods of Determining Protein Concentration

[0067] Provided herein are methods of determining the concentration each of a first protein and a second protein that is different from the first protein in a composition. In some embodiments, the disclosure provides a method comprising providing a composition comprisingAttorney Docket No.: 10501-WO01-SEC the first protein and the second protein, wherein the first protein has a first extinction coefficient and the second protein has a second extinction coefficient; resolving the first protein from the second protein of the composition by cation exchange chromatography (CEX), whereby a ratio of the first protein to the second protein in the composition is obtained; measuring a total absorbance of the composition for a path length; and calculating a concentration of the first protein and the second protein based on the ratio of the total absorbance to the path length and the extinction coefficients, prorated by the ratio.

[0068] The terms “extinction coefficient” and “molar extinction coefficient,” may be used interchangeably herein to refer to a measurement of how strongly a chemical species or substance absorbs, and thereby attenuates, light at a given wavelength. The extinction coefficient (ε) is an intrinsic property of a chemical species that is dependent upon its chemical composition and structure. It will be appreciated that there is no single extinction coefficient value for a peptide or protein, as minor differences in buffer type, ionic strength and pH affects absorptivity values at least slightly. Thus, extinction coefficients typically are determined empirically using a solution of a protein of known concentration dissolved in the same buffer as a sample of interest. Alternatively, extinction coefficients for many proteins have been compiled and reported in, e.g., Fasman, D.G., Ed., “The Practical Handbook of Biochemistry and Molecular Biology,” CRC Press, Boston (1992).

[0069] The first protein may be resolved or separated from the second protein using any suitable protein separation method, a variety of which are known in the art. For example, the first protein may be resolved from the second protein using one or more chromatography techniques, such as affinity chromatography, anion exchange chromatography, cation exchange chromatography, gel-permeation chromatography, paper chromatography, thin-layer chromatography, gas chromatography, size exclusion chromatography (SEC), hydrophobic interaction chromatography (HIC), reverse phase high performance liquid chromatography (RP- HPLC), ultracentrifugation (UC), etc. (see, e.g., Coskun, North Clin Istanb 3(2): 156-160 (2016)). In some embodiments, separation of the first protein from the second protein is achieved using a high performance liquid chromatography (HPLC) methodology. HPLC, also referred to as high-pressure liquid chromatography, is a technique used to separate, identify, and quantify each component in a mixture. HPLC relies on pumps to pass a pressurized liquid solvent containing the sample mixture through a column filled with a solid adsorbent material.Attorney Docket No.: 10501-WO01-SEC Each component in the sample interacts slightly differently with the adsorbent material, causing different flow rates for the different components and leading to the separation of the components as they flow out of the column.

[0070] In some embodiments, cation exchange chromatography (CEX) is used to resolve the first protein from the second protein of the composition. CEX is a type of ion-exchange chromatography, which is based on electrostatic interactions between charged protein groups and a solid support or matrix. Cation exchange chromatography, more specifically, uses a negatively charged ion exchange resin with an affinity for molecules having net positive surface charges. Proteins are separated from the column either by changing pH, concentration of ion salts, or ionic strength of the buffer solution. In some embodiments, cation exchange high performance liquid chromatography (CEX-HPLC) may be employed. CEX systems and reagents are commercially available from a variety of sources, any of which may be employed in the disclosed methods.

[0071] Proteins with different isoelectric points (pI) may have varying degrees of charge at a given pH and thereby have different affinities for the negatively charged surface groups on the particles of the cation exchange media; therefore, different proteins may bind to a cationic exchange resin with different strengths, facilitating their separation. Not to be bound by theory, it is believed that the retention time of different co-formulated proteins on a cation exchange resin is directly proportional to the pI of each protein, and that a larger pI difference between co- formulated proteins facilitates chromatographic separation. In some embodiments, the first protein of the composition has an isoelectric point (pI) that differs from the isoelectric point of the second protein by at least 0.2. For example, the first protein has a pI that differs from the pI of the second protein by about 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, or more. In some embodiments, the pIs of the first protein and second protein do not differ by more than about 14 (e.g., about 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or less).

[0072] Other liquid chromatography methods may be used to resolve the first protein from the second protein in the composition. For example, in some embodiments, hydrophobic interaction chromatography (HIC) HIC is a technique which separates molecules based on their hydrophobicity and is a useful for purifying proteins while maintaining biological activity due to the use of conditions and matrices that operate under less denaturing conditions. In other embodiments, reverse phase high performance liquid chromatography (RP-HPLC) may be used.Attorney Docket No.: 10501-WO01-SEC RP-HPLC involves the separation of molecules on the basis of hydrophobicity. The separation depends on the hydrophobic binding of the solute molecule from the mobile phase to the immobilized hydrophobic ligands attached to the stationary phase, i.e., the sorbent. RP-HPLC is used extensively in the art for the isolation of peptides and proteins from a wide variety of synthetic or biological sources and is used for both analytical and preparative applications (see, e.g., Aguilar, M-I. (ed.), HPLC of Peptides and Proteins: Methods and Protocols, Human Press (2004)).

[0073] Resolving the first and second proteins in the composition by CEX allows one of skill in the art to obtain a ratio of the first protein to the second protein in the composition. In some embodiments, the ratio of first protein to second protein in the composition may be, for example, 1:1 to 1:100, such as 1:1 to 1:80, 1:1 to 1:40, 1:1 to 1:20, 1:1.1 to 1:100, 1:1.1 to 1:80, 1:1.1 to 1:40, 1:1.1 to 1:20, 1:2 to 1:100, 1:2 to 1:80, 1:2 to 1:40, 1:2 to 1:20, or any suitable ratio falling within the aforementioned ranges.

[0074] Once the ratio of the first protein to the second protein is determined, the method comprises determining the total absorbance of the composition for a particular path length. In some embodiments, the method comprises measuring the amount of light the composition absorbs in the infrared, visible, or ultraviolet region of the spectrum. For example, in some embodiments, the total absorbance of the composition may be quantified by measuring the UV absorbance at 280 nm. It will be appreciated that at 280 nm wavelength, the aromatic amino acids tryptophan (Trp) and tyrosine (Tyr) exhibit strong light absorption, and to a lesser extent cysteine groups forming disulfide bonds (Cys–Cys) also absorb. Consequently, absorption of proteins and peptides at 280 nm is proportional to the content of these amino acids, and total absorption is directly proportional to total protein content in the composition. The term “path length,” as used herein, generally refers to the distance that light (e.g., UV or visible) travels through a sample in an analytical cell. Path length also is directly proportional to absorbance, as a longer pathlength accommodates more protein molecules in the path of the light source.

[0075] The total absorbance of the composition for a particular pathlength can be determined by spectrophotometric methods that utilize a fixed path length or a variable pathlength. Conventional spectrophotometric assays are based on a fixed pathlength depending on the analytical cell (e.g., cuvette) used to hold the sample. In contrast, variable pathlength spectrophotometry systems (e.g., SOLOVPE®, Repligen Corp, Bridgewater, NJ) automaticallyAttorney Docket No.: 10501-WO01-SEC adjust the optical pathlength from, e.g., 0 mm to 15 mm in 5‑µm increments. Variable pathlength spectrophotometers can determine appropriate pathlength and linearity at significantly higher sample concentrations than those determined by fixed‑pathlength spectrophotometers. In some instances, variable pathlength spectrophotometry uses pathlength as the variable, unlike conventional fixed pathlength methods where the concentration is variable, allowing the concentration to remain constant. This eliminates the need for dilution of the composition and decreases the chances of contamination.

[0076] Using the ratio of the first and second proteins and the total absorbance, the Beer- Lambert Law (or “Beer’s Law”) may be used to determine the individual concentrations of each of the first protein and the second protein in the composition. According to the Beer-Lambert law, the absorbance of a solution is directly proportional to the concentration of the absorbing material present in the solution and pathlength. Thus, the concentration of a solution can be calculated by measuring its absorbance. The Beer-Lambert law is commonly expressed by the formula: ^^ = ^^ ^^ ^^ where A is absorbance, ^^ is the extinction coefficient, b is the pathlength, and C is the concentration. The linear relationship of absorbance, extinction coefficient, pathlength, and concentration provided by the Beer-Lambert law can be applied to a composition in which two or more antigen-binding proteins (e.g., antibodies) are co-formulated by incorporating the ratio of the first protein to the second protein determined as described above. Thus, in some embodiments, the concentration of the first protein and the second protein may be calculated based on the ratio of the total absorbance to the pathlength and the extinction coefficients, prorated by the ratio of first protein to second protein. For example, calculating a concentration of the first protein and the second protein in the composition may comprise Equation 1 and Equation 2: ^^ଶ= ^^௧^௧^^ / (ε1b + ε2b ^^) [Equation 1] ^^^= ^^ଶ× ^^ [Equation 2] where C2 is the concentration of the second protein, ^^௧^௧^^is the total absorbance, ε1 is the extinction concentration of the first protein, ε2 is the extinction concentration of the secondAttorney Docket No.: 10501-WO01-SEC protein, b is the path length, k is the ratio of the first protein to the second protein, and C1 is the concentration of the first protein.

[0077] In other embodiments, the concentration of the first protein and the second protein in the composition may be determined by generating a calibration curve (also commonly referred to as standard curve or working curve). A “calibration curve” is a general method for determining the concentration of a substance in an unknown sample by comparing the unknown to a set of standard samples of known concentration. The responses of the standards are used to plot or calculate a standard curve. Absorbance values of unknown samples are then interpolated onto the plot or formula for the standard curve to determine their concentrations. When the calibration curve has an adequate linearity over a wider range in the region of quantitative analysis, the calibration curve can be prepared with a relatively smaller number of standard samples, which are near the upper limit, lower limit, and in the intermediate point in the determination range of the quantitative analysis. When the composition comprises two or more antigen-binding proteins, as described herein, at least two calibration curves may be generated and utilized to quantify the amount of each antigen-binding protein detected in the disclosed methods.

[0078] In some aspects, the first protein may be resolved from the second protein in the composition by a chromatography-based titer method to measure protein concentration directly. For example, CEX, hydrophobic interaction chromatography (HIC), or reverse phase high performance liquid chromatography (RP-HPLC) may be used to separate the first protein from the second protein. The concentration of each protein can then be calculated based on calibration curve established for each protein, as well as the peak area for each protein.

[0079] Other orthogonal methods for determining protein concentration may be employed in order to cross-check or confirm the results obtained by the disclosed methods. Such methods include, but are not limited to, turbidimetric assays, nephelometric assays, and colorimetric assays. In turbidimetric and nephelometric assays, a protein is quantified from the change in the turbidity of the reaction mixture based on the agglutination of the protein and a protein-specific binding partner. In colorimetric assays, a protein may be quantified with the aid of a color reagent. Colorimetric assays are characterized by formation, change, or depletion of color in the presence of the protein to be quantified. Exemplary colorimetric assays include the Coomassie blue G-250 dye-binding (Bradford), bicinchoninic acid (BCA), and Lowry assay.Attorney Docket No.: 10501-WO01-SEC

[0080] In some methods, if the concentration of the first protein and the second protein are each within a specified range, for example a concentration specification, further processing is performed on the composition in order to manufacture a pharmaceutical composition suitable for medical use. For example, excipients may be added to the composition, or the composition may be disposed in a container suitable for storage or a device suitable for administration such as a syringe or pen. Analysis of High Molecular Weight Species and Charge Variants

[0081] The disclosure also provides a chromatography method of analyzing a composition comprising a first protein and a second protein that is different from the first protein, which comprises providing a composition comprising the first protein and the second protein, and at least one of: a) determining a level of high molecular weight (HMW) species of the first protein and / or second protein in the composition by size exclusion ultra high performance liquid chromatography (SE-UHPLC), wherein the SE-UHPLC is performed with a buffer comprising about 100-200 mM KCl, about 1%-5% isopropyl alcohol (IPA), and about 40-60 mM K3PO4; or b) separating charge variants of the first protein and / or the second protein in the composition by pH-gradient cation exchange-high performance liquid chromatography (CEX-HPLC), wherein the CEX-HPLC comprises a first mobile phase at pH 5-6 and a second mobile phase at pH 10- 11.

[0082] The term “HMW species,” as used herein, in reference to a therapeutic protein, refers to a formed aggregate of two or more molecules (e.g., therapeutic proteins) linked by non- covalent bonds. HMW species include, but are not limited to, dimers (comprising two therapeutic proteins), trimers (comprising three therapeutic proteins), tetramers (comprising four therapeutic proteins), pentamers (comprising five therapeutic proteins), hexamers (comprising six therapeutic proteins), heptamers (comprising seven therapeutic proteins), and octamers (comprising eight therapeutic proteins), of a therapeutic protein. In exemplary aspects, a HMW species can be of higher order, e.g., can comprise more than eight therapeutic proteins. For instance, the HMW species can be a enneamer (comprising nine therapeutic proteins), decamer (comprising 10 therapeutic proteins), hendecamer (comprising 11 therapeutic proteins), dodecamer (comprising 12 therapeutic proteins), triadecamer (comprising 13 therapeutic proteins), quatrodecamer (comprising 14 therapeutic proteins), quindecamer (comprising 15Attorney Docket No.: 10501-WO01-SEC therapeutic proteins), sexdecamer (comprising 16 therapeutic proteins), septendecamer (comprising 17 therapeutic proteins), octodecamer (comprising 18 therapeutic proteins), or a novendecamer (comprising 19 therapeutic proteins). In various embodiments, the HMW species analyzed by the presently disclosed methods can comprise one or more of dimers, trimers, tetramers, pentamers, hexamers, heptamers, and octamers of the therapeutic protein.

[0083] In exemplary aspects, the size of the HMW species analyzed by the presently disclosed methods is less than about 0.1 microns (100 nm). Optionally, the size of the HMW species is about 99 nm or less. In exemplary aspects, the size of the HMW species is greater than about 10 nm and less than about 99 nm. In exemplary aspects, the size of the HMW species is greater than about 15 nm and less than about 99 nm. In exemplary aspects, the size of the HMW species is about 15 nm to about 99 nm, about 20 nm to about 99 nm, about 30 nm to about 99 nm, about 40 nm to about 99 nm, about 50 nm to about 99 nm, about 60 nm to about 99 nm, about 70 nm to about 99 nm, about 80 nm to about 99 nm, about 90 nm to about 99 nm. In exemplary instances, the size of the HMW species is about 15 nm to about 90 nm, about 15 nm to about 80 nm, about 15 nm to about 70 nm, about 15 nm to about 60 nm, about 15 nm to about 50 nm, about 15 nm to about 40 nm, about 15 nm to about 30 nm, or about 15 nm to about 20 nm. In various aspects, the size of the HMW species is less than about 15 nm. Optionally, the size of the HMW species is less than about 10 nm or less than about 5 nm.

[0084] The level of HMW species may be determined by any suitable method known in the art. In some aspects, the level of HMW species may be determined by size exclusion chromatography (SEC). In exemplary aspects, the SEC is SEC-high performance liquid chromatography (SEC-HPLC) or SEC Fluorescence (SEC-Fluor) or SEC-UV. It will be appreciated that SEC, also referred to as gel-filtration, steric exclusion, or gel chromatography, is a partition chromatography that separates molecules according to their molecular sizes. For example, SEC can separate monoclonal antibodies into three major species: high molecular weight species, main peak (predominantly monomer), and low molecular weight species. Additionally or alternatively, other techniques may be employed to determine the level of HMW species of the first protein and / or the second protein in the composition. For example, the method can include one or more of: mass spectrometry (MS) and / or SEC coupled to ultra-high performance liquid chromatography (UHPLC). It is believed that SE-UHPLC results in more accurate resolution of size variants in a shorter period of time than conventional SEC columns.Attorney Docket No.: 10501-WO01-SEC

[0085] In some embodiments, a level of high molecular weight (HMW) species of the first protein and / or second protein in the composition is determined by size exclusion ultra high performance liquid chromatography (SE-UHPLC). The SE-UHPLC may be performed under conditions which optimize separation of the first protein and the second protein in the composition. For example, the SE-UHPLC may be performed using any suitable column known in the art, such as those commercially available from Waters Corporation (e.g., XBRIDGE™ columns), Phenominex (BIOZEN™ columns), and / or Sepax Technologies (e.g., Unix-C SEC- 300 column). Buffer components also may be adjusted to optimize quantitation of HMW species. Such components include, for example, salts, acidic and / or basic components (to achieve a desired pH), and organic solvents. Exemplary salts include NaCl or KCl, which may be included at concentrations up to about 500 mM (e.g., 0 mM, 50 mM, 100 mM, 150 mM, 200 mM, 250 mM, 300 mM, 350 mM, 400 mM, 450 mM, or a range defined by any two of the foregoing values). Furthermore, in some aspects, the buffer may comprise potassium phosphate (K3PO4; monobasic or dibasic), sodium phosphate (NA3PO4), isopropyl alcohol (IPA), ammonium acetate, acetonitrile, and / or ammonium chloride. For example, the SE-UHPLC may be performed with a buffer containing KCl, isopropyl alcohol, and K3PO4. In some embodiments, the buffer comprises about 100-200 mM KCl, about 1%-5% isopropyl alcohol (IPA), and about 40-60 mM K3PO4.

[0086] A further parameter of SEC-UHPLC that may be optimized for determining HMW species in an antibody co-formulation is the flow rate of sample fluid (i.e., “mobile phase”) through the SEC-UHPLC column. In some embodiments, the SE-UHPLC comprises a mobile phase having a flow rate of about 0.2-0.5 mL / min (e.g., about 0.25 mL / min, 0.3 mL / min, 0.35 min / mL, 0.4 mL / min, or 0.45 mL / min). For example, the mobile phase flow rate may be about 0.4 mL / min.

[0087] It will be appreciated that numerous post-translational modifications may induce variations of charge distribution on monoclonal antibodies that can potentially affect their biological activity. Indeed, monoclonal antibodies can undergo chemical degradation via several different mechanisms, including oxidation, deamidation, isomerization and fragmentation, that result in the formation of various charge variants and heterogeneity, thus modifying their isoelectric pH (pI) values (Khwali et al., MAbs., 2(6): 613-624 (2010)). Such charge variants typically are separated and characterized via charged based-separation techniques such asAttorney Docket No.: 10501-WO01-SEC isoelectric focusing (IEF) gel electrophoresis, capillary isoelectric focusing (cIEF) gel electrophoresis, cation exchange chromatography (CEX) and anion exchange chromatography (AEX). In some embodiments, the chromatography method of analyzing a composition comprising a first protein and a second different protein comprises separating charge variants of the first protein and / or the second protein in the composition by pH-gradient cation exchange- high performance liquid chromatography (CEX-HPLC). As discussed herein, CEX-HPLC separates proteins based on differences in their surface charges using a negatively charged ion exchange resin with an affinity for molecules having net positive surface charges.

[0088] CEX-HPLC may be performed under conditions which optimize separation of the charge variants of each protein in the composition. For example, the CEX-HPLC may be performed using any suitable cationic exchange resin or column known in the art, such PROPAC™ columns (ThermoFisher Scientific, Inc.), BIOSUITE™ columns (Waters Corporation), and BIOPRO™ IEX columns (YMC America, Inc.). Appropriate buffer systems may be chosen based on the first and second proteins in a co-formulation. Retention and elution from CEX columns may achieved by a linear, gradient elution or using a step isocratic elution. In some embodiments, a gradient elution is used. For example, a continuous salt (ionic-strength) gradient or a pH gradient results in a high degree of protein fractionation based on protein charge. In salt-gradient-based ionic exchange chromatography (IEC), the pH of the buffer system is fixed. In addition to choosing the appropriate pH of the starting buffer, its ionic strength is kept low since the affinity of proteins for IEC resins decreases as ionic strength increases. The proteins are then eluted by increasing the ionic strength (salt concentration) of the buffer to increase the competition between the buffer ions and proteins for charged groups on the IEC resin. As a result, the interaction between the IEC resin and proteins is reduced, causing the proteins to elute. In pH-gradient-based IEC, the pH of the starting buffer is maintained at a constant level to ensure the proteins obtain the opposite charge of the stationary phase and bind to it. The proteins are eluted by changing the buffer pH so the proteins transition to a net zero charge (ultimately the same charge as the resin) and elute from the column.

[0089] In exemplary aspects, the disclosed method utilizes pH gradient-based CEX-HPLC to separate and elute charge variants of first and second proteins in the composition. In some embodiments, the pH gradient-based CEX-HPLC comprises the use of two buffers (or “mobile phases”) with different pHs. For example, the CEX-HPLC comprises a first mobile phase orAttorney Docket No.: 10501-WO01-SEC buffer at pH 5-6 (e.g., pH 5.1, 5.2, 5.3, 5.4, 5.5., 5.6, 5.7, 5.8, or 5.9) and a second mobile phase or buffer at pH 10-11 (e.g., pH 10.1, 10.2, 10.3, 10.4, 10.5, 10.6, 10.7, 10.8, or 10.9). For example, the first mobile phase may comprise a pH of about 5.6, while the second mobile phase may comprise a pH of about 10.2. In some embodiments, the CEX-HPLC comprises a column that is at least 50 mm, 75 mm, or 100 mm. In other embodiments, the CEX-HPLC comprises a gradient from 100% of the first mobile phase + 0% of the second mobile phase to no more than 20% of the first mobile phase + at least 80% of the second mobile phase, over at least 40, 5060, or 70 minutes (e.g., 45, 55, or 65 minutes).

[0090] In further exemplary aspects, the disclosed method may utilize a salt gradient with optimal mobile phase pH. Without being bound by theory, a salt gradient with optimal mobile phase pH can improve the separation of charge variants in co-formulated drug products with similar pI values, resulting in minimal overlapping basic Peaks and acidic Peaks. In such embodiments, the mobile phase or buffer pH is between about 5.0 and about 8.0 (e.g., pH 5.5., 6.0, 6.5, 7.0, or 7.5), such as between about 6.0 and 7.5 (e.g., pH 6.1, 6.2, 6.3, 6.4, 6.5, 6.6., 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, or 7.4). For example, the mobile phase pH may be about 6.0, 6.5, 7.8, 7.0, or 7.5. Any suitable buffer may be employed as the mobile phase. Buffer characteristics to be considered include, for example, pKa, buffer type, cost, ease of use, and buffers used in previous and subsequent steps. Buffers typically used for CEX equilibration, binding, and / or elution of monoclonal antibodies include, but are not limited to, acetate, citrate, or phosphate. An exemplary buffer that may be employed in the disclosed methods is sodium phosphate.

[0091] Once charge variants of the first and second proteins in co-formulation have been separated and eluted by CEX-HPLC, the method may further comprise determining the concentration of the first protein and the second protein in the composition. Protein concentration may be determined. Examples of suitable methods of determining protein concentration are described herein. In some embodiments, the concentration of each of the first protein and the second protein is determined based on a calibration curve for each protein, as described herein.Attorney Docket No.: 10501-WO01-SEC Capillary Electrophoresis

[0092] In other aspects of the disclosure, a capillary electrophoresis (CE) method may be employed to determine the concentration of each of the first protein and the second different protein in a composition. The term “capillary electrophoresis,” as used herein, refers to a liquid phase micro-separation analytical technique that separates ions based on their electrophoretic mobility using a high-voltage direct current in a capillary tube or channel. CE can be used to analyze substances ranging from organic ions to biological macromolecules, such as proteins and nucleic acids. Various forms of CE are known in the art and may be used in connection with the disclosed methods, including, for example, capillary zone electrophoresis (CZE); capillary gel electrophoresis (CGE); micellar electrokinetic capillary chromatography (MEKC), and capillary isoelectric focusing (CIF or CIEF) (see, e.g., Capillary Electrophoresis; retrieved from chem.libretexts.org / @go / page / 294 (July 8, 2022); and Dawod et al., Analyst.2017 May 30; 142(11): 1847–1866. doi:10.1039 / c7an00198c).

[0093] In some embodiments, the concentration of the first protein and the second different protein is determined using capillary gel electrophoresis (CGE; also referred to “CGE-SDS” or “CE-SDS”), which separates molecules based on the difference in solute size as the molecules migrate through a gel or matrix. CGE has many advantages over conventional sodium dodecyl sulfate polyacrylamide slab gel electrophoresis (SDS-PAGE), including but not limited to, fast separation times, high capability of recovery and regeneration, on-line detection, increased analysis throughput, and ease of operation (Zhu et al., Anal Chim Acta.2012;709:21-31; and Wu D, Regnier FE. Journal of Chromatography.1992;608:349-356). CGE methods also allow for more automated analysis at higher voltages which leads to faster and more efficient separations. With respect to antibody analysis, CE-SDS may be used as an orthogonal and alternative technique to size exclusion chromatography (SEC) in purity assessment of monoclonal antibodies and profiling of molecular size variants.

[0094] Any suitable gel or matrix can be used for CE-SDS. Such gels or matrices include, for example, polyacrylamide (linear or cross-linked), agarose, poly(ethylene glycol), poly(ethylene oxide), dextran, and pullulan. CE-SDS may be performed under reduced (rCE- SDS) or non-reduced (nrCE-SDS) conditions. In reducing conditions, a sample is incubated with a reducing reagent (e.g., β-mercaptoethanol) to break up inter- and intra-chain disulfide bonds. rCE-SDS is commonly used to obtain the relative percentage of antibody light chain (LC),Attorney Docket No.: 10501-WO01-SEC heavy-chain (HC), and non-glycosylated heavy chain (NGHC), especially (LC^+^HC)% as purity of the analyzed sample under reduced conditions. nrCE-SDS may be used to analyze purity of an intact antibody as well as product related impurities such as fragments.

[0095] Separated molecules may be detected by a variety of methods, including UV absorbance, capacitively coupled contactless conductivity, mass spectrometry (MS), and laser- induced fluorescence (LIF). A chromatogram may then be generated, which graphically displays in real time the peaks generated as the separated components pass through a detector. It will be appreciated that the area under the peak is considered as a measure of component concentration. When a sample contains multiple analytes or components, each component may be eluted from the CE-SDS at different retention times depending upon solute-stationary phase interactions and mobile phase flow characteristics.

[0096] In embodiments where the first and second proteins are each antibodies (or other multi-chain antigen binding proteins), the CE-SDS may result in in multiple peaks for each protein (e.g., a heavy chain peak and a light chain peak). In such cases, the heavy chains and / or light chains of both proteins may have similar retention times in the CE-SDS (referred to as “co- migrating”), which may result overlapping peaks. In the event of overlapping peaks, the concentrations of each antibody may be determined based on the non-overlapping peaks. Thus, in some embodiments, the CE-SDS results in a first peak of the first protein, a second peak of the second protein, and a co-migration peak of the first protein and the second protein, and the concentration of each of the first protein and second protein is determined based on a ratio of the first peak to the second peak. For example, if a first antibody has a light chain peak that overlaps with the light chain peak of a second antibody, a ratio of the peak areas for the respective non- overlapping heavy chains may be used to determine the concentration of each antibody in the composition. In other words, a select peak or group of peaks may provide a reasonable estimate of the relative concentration of each protein in a co-formulated composition. Therapeutic Proteins

[0097] In some embodiments, each of the first protein and the second protein is a therapeutic protein. As used herein “therapeutic protein,” and variations of this root term, has its ordinary and customary meaning as would be understood by one of ordinary skill in the art in view of this disclosure. It refers to a polypeptide for medical use in a subject, typically a human subject. ByAttorney Docket No.: 10501-WO01-SEC way of example, a therapeutic protein may be a polypeptide approved for medical use by a government regulatory authority, such as the Food and Drug Administration or the European Medicines Agency. In some embodiments, each of the first protein and the second protein is a therapeutic antigen-binding protein, such as a therapeutic antibody.

[0098] Therapeutic antigen-binding proteins, such as antibodies, encompassed by the present disclosure may include polypeptides that bind to one or more of the following: (i) CD proteins, including CD3, CD4, CD8, CD19, CD20, CD22, CD30, and CD34; including those that interfere with receptor binding; (ii) HER receptor family proteins, including HER2, HER3, HER4, and the EGF receptor; (iii) cell adhesion molecules, for example, LFA-I, MoI, pl50, 95, VLA-4, ICAM- I, VCAM, and alpha v / beta 3 integrin; (iv) growth factors, such as vascular endothelial growth factor (“VEGF”), growth hormone, thyroid stimulating hormone, follicle stimulating hormone, luteinizing hormone, growth hormone releasing factor, parathyroid hormone, Mullerian- inhibiting substance, human macrophage inflammatory protein (MIP-1alpha), erythropoietin (EPO), nerve growth factor, such as NGF-beta, platelet-derived growth factor (PDGF), fibroblast growth factors, including, for instance, aFGF and bFGF, epidermal growth factor (EGF), transforming growth factors (TGF), including, among others, TGF-α and TGF-β, including TGF- βl, TGF-β2, TGF-β3, TGF- β4, or TGF- β5, insulin-like growth factors-I and -II (IGF-I and IGF- II), des(l-3)-IGF-I (brain IGF-I), and osteoinductive factors; (v) insulins and insulin-related proteins, including insulin, insulin A-chain, insulin B-chain, proinsulin, and insulin-like growth factor binding proteins; (vi) coagulation and coagulation-related proteins, such as, among others, factor VIII, tissue factor, von Willebrand factor, protein C, alpha-1-antitrypsin, plasminogen activators, such as urokinase and tissue plasminogen activator (“t-PA”), bombazine, thrombin, and thrombopoietin; (vii) other blood and serum proteins, including but not limited to albumin, IgE, and blood group antigens; (viii) colony stimulating factors and receptors thereof, including the following, among others, M-CSF, GM-CSF, and G-CSF, and receptors thereof, such as CSF- 1 receptor (c-fms); (ix) receptors and receptor-associated proteins, including, for example, flk2 / flt3 receptor, CD112 receptor (CD112R), obesity (OB) receptor, LDL receptor, growth hormone receptors, thrombopoietin receptors (“TPO-R,” “c-mpl”), glucagon receptors, interleukin receptors, interferon receptors, T-cell receptors, stem cell factor receptors, such as c- Kit, and other receptors; (x) receptor ligands, including, for example, OX40L, the ligand for the OX40 receptor; (xi) neurotrophic factors, including bone-derived neurotrophic factor (BDNF)Attorney Docket No.: 10501-WO01-SEC and neurotrophin-3, -4, -5, or -6 (NT-3, NT-4, NT-5, or NT-6); (xii) relaxin A-chain, relaxin B- chain, and prorelaxin; (xiii) interferons and interferon receptors, including for example, interferon-α, -β, and -γ, and their receptors; (xiv) interleukins and interleukin receptors, including IL-1 to IL-33 and IL-1 to IL-33 receptors, such as the IL-8 receptor, among others; (xv) viral antigens, including an AIDS envelope viral antigen; (xvi) other proteins such as, e.g., lipoproteins, calcitonin, glucagon, atrial natriuretic factor, lung surfactant, tumor necrosis factor- alpha and -beta, enkephalinase, Programmed Cell Death 1 (PD-1), Programmed Cell Death Ligand 1 (PD-L1), T cell immunoreceptor with Ig and ITIM domains (TIGIT), RANTES (regulated on activation normally T-cell expressed and secreted), mouse gonadotropin-associated peptide, DNAse, inhibin, activin, integrin, protein A or D, rheumatoid factors, immunotoxins, bone morphogenetic protein (BMP), superoxide dismutase, surface membrane proteins, decay accelerating factor (DAF), HIV envelope, transport proteins, homing receptors, addressins, regulatory proteins, immunoadhesins, myostatins, TALL proteins, including TALL-I, amyloid proteins, including but not limited to amyloid-beta proteins, thymic stromal lymphopoietins (“TSLP”), RANK ligand (“RANKL” or “OPGL”), c-kit, TNF receptors, including TNF Receptor Type 1, TRAIL-R2, angiopoietins, and biologically active fragments or analogs or variants of any of the foregoing.

[0099] Examples of therapeutic antibodies suitable for the methods described herein include infliximab, bevacizumab, cetuximab, ranibizumab, palivizumab, abagovomab, abciximab, actoxumab, adalimumab, afelimomab, afutuzumab, alacizumab, alacizumab pegol, ald518, alemtuzumab, alirocumab, altumomab, amatuximab, anatumomab mafenatox, anrukinzumab, apolizumab, arcitumomab, aselizumab, altinumab, atlizumab, atorolimiumab, tocilizumab, bapineuzumab, basiliximab, bavituximab, bectumomab, belimumab, bemarituzumab, benralizumab, bertilimumab, besilesomab, bevacizumab, bezlotoxumab, biciromab, bivatuzumab, bivatuzumab mertansine, blinatumomab, blosozumab, brentuximab vedotin, briakinumab, brodalumab, canakinumab, cantuzumab mertansine, cantuzumab mertansine, caplacizumab, capromab pendetide, carlumab, catumaxomab, cc49, cedelizumab, certolizumab pegol, cetuximab, citatuzumab bogatox, cixutumumab, clazakizumab, clenoliximab, clivatuzumab tetraxetan, conatumumab, crenezumab, cr6261, dacetuzumab, daclizumab, dalotuzumab, daratumumab, demcizumab, denosumab, detumomab, dorlimomab aritox, drozitumab, duligotumab, dupilumab, ecromeximab, eculizumab, edobacomab, edrecolomab,Attorney Docket No.: 10501-WO01-SEC efalizumab, efungumab, elotuzumab, elsilimomab, enavatuzumab, enlimomab pegol, enokizumab, enoticumab, ensituximab, epitumomab cituxetan, epratuzumab, erenumab, erlizumab, ertumaxomab, etaracizumab, etrolizumab, evolocumab, exbivirumab, fanolesomab, faralimomab, farletuzumab, fasinumab, fbta05, felvizumab, fezakinumab, ficlatuzumab, figitumumab, flanvotumab, fontolizumab, foralumab, foravirumab, fresolimumab, fulranumab, futuximab, galiximab, ganitumab, gantenerumab, gavilimomab, gemtuzumab ozogamicin, gevokizumab, girentuximab, glembatumumab vedotin, golimumab, gomiliximab, gs6624, ibalizumab, ibritumomab tiuxetan, icrucumab, igovomab, imciromab, imgatuzumab, inclacumab, indatuximab ravtansine, infliximab, intetumumab, inolimomab, inotuzumab ozogamicin, ipilimumab, iratumumab, itolizumab, ixekizumab, keliximab, labetuzumab, lebrikizumab, lemalesomab, lerdelimumab, lexatumumab, libivirumab, ligelizumab, lintuzumab, lirilumab, lorvotuzumab mertansine, lucatumumab, lumiliximab, mapatumumab, maslimomab, mavrilimumab, matuzumab, mepolizumab, metelimumab, milatuzumab, minretumomab, mitumomab, mogamulizumab, morolimumab, motavizumab, moxetumomab pasudotox, muromonab-cd3, nacolomab tafenatox, namilumab, naptumomab estafenatox, narnatumab, natalizumab, nebacumab, necitumumab, nerelimomab, nesvacumab, nimotuzumab, nivolumab, nofetumomab merpentan, ocaratuzumab, ocrelizumab, odulimomab, ofatumumab, olaratumab, olokizumab, omalizumab, onartuzumab, oportuzumab monatox, oregovomab, orticumab, otelixizumab, oxelumab, ozanezumab, ozoralizumab, pagibaximab, palivizumab, panitumumab, panobacumab, parsatuzumab, pascolizumab, pateclizumab, patritumab, pemtumomab, perakizumab, pertuzumab, pexelizumab, pidilizumab, pintumomab, placulumab, ponezumab, priliximab, pritumumab, PRO 140, quilizumab, racotumomab, radretumab, rafivirumab, ramucirumab, ranibizumab, raxibacumab, regavirumab, reslizumab, rilotumumab, rituximab, robatumumab, roledumab, romosozumab, rontalizumab, rovelizumab, ruplizumab, samalizumab, sarilumab, satumomab pendetide, secukinumab, sevirumab, sibrotuzumab, sifalimumab, siltuximab, simtuzumab, siplizumab, sirukumab, solanezumab, solitomab, sonepcizumab, sontuzumab, stamulumab, sulesomab, suvizumab, tabalumab, tacatuzumab tetraxetan, tadocizumab, talizumab, tanezumab, taplitumomab paptox, tarlatamab, tefibazumab, telimomab aritox, tenatumomab, tefibazumab, teneliximab, teplizumab, teprotumumab, tezepelumab, TGN1412, tremelimumab, ticilimumab, tildrakizumab, tigatuzumab, TNX-650, tocilizumab, toralizumab, tositumomab, tralokinumab, trastuzumab, TRBS07, tregalizumab, tucotuzumabAttorney Docket No.: 10501-WO01-SEC celmoleukin, tuvirumab, ublituximab, urelumab, urtoxazumab, ustekinumab, vapaliximab, vatelizumab, vedolizumab, veltuzumab, vepalimomab, vesencumab, visilizumab, volociximab, vorsetuzumab mafodotin, votumumab, zalutumumab, zanolimumab, zatuximab, ziralimumab, zolimomab aritox, or variants of any of the foregoing.

[0100] In accordance with the methods described herein, a composition may comprise two or more of any of the foregoing therapeutic antibodies, or antigen-binding fragments thereof. Exemplary combinations of antibodies that may be co-formulated in a composition for analysis as described herein include, but are not limited to, an antibody that binds to specifically to PD-1 and at least one additional antibody described herein. In some embodiments, the composition may comprise an anti-PD-1 antibody and an antigen-binding protein (e.g., a BiTE® molecule) that specifically binds to vascular endothelial growth factor (VEGF), DLL3 (e.g., tarlatamab), PSMA, CD112R, and / or TIGIT. For example, the composition may comprise an anti-PD-1 antibody co-formulated with an antibody that specifically binds to VEGF. In other embodiments, the composition may comprise an antibody that specifically binds to CD112R and an antibody that specifically binds to TIGIT. In other embodiments, the composition may comprise 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, the composition may comprise 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. The present disclosure is not limited to these particular combinations of antigen-binding proteins, however. Samples and Compositions

[0101] The term “sample” and variations of this root term has its ordinary and customary meaning as would be understood by a person of ordinary skill in view of this disclosure. It refers to a composition that may contain two or more antigen-binding proteins as described herein, such as in vitro or synthetic samples obtained from the manufacturing of an antigen-binding protein. For example, a sample may be a composition or formulation comprising the first protein and the second protein and at least one pharmaceutically acceptable carrier (also referred to herein as a “pharmaceutical composition”). For example, a sample may be an in-process sample from the manufacture of a pharmaceutical composition.Attorney Docket No.: 10501-WO01-SEC

[0102] Acceptable composition or formulation materials for proteins as described herein (e.g., therapeutic antibodies) preferably are nontoxic to recipients at the dosages and concentrations employed. In certain embodiments, a pharmaceutical composition may contain formulation materials for modifying, maintaining or preserving, for example, the pH, osmolality, viscosity, clarity, color, isotonicity, odor, sterility, stability, rate of dissolution or release, adsorption or penetration of the composition. In such embodiments, suitable formulation materials include, but are not limited to, amino acids (such as glycine, glutamine, asparagine, arginine or lysine); antimicrobials; antioxidants (such as ascorbic acid, sodium sulfite or sodium hydrogen-sulfite); buffers (such as borate, bicarbonate, Tris-HCl, citrates, phosphates or other organic acids); bulking agents (such as mannitol or glycine); chelating agents (such as ethylenediamine tetraacetic acid (EDTA)); complexing agents (such as caffeine, polyvinylpyrrolidone, beta-cyclodextrin or hydroxypropyl-beta-cyclodextrin); fillers; monosaccharides; disaccharides; and other carbohydrates (such as glucose, sucrose, mannose or dextrins); proteins (such as serum albumin, gelatin or immunoglobulins); coloring, flavoring and diluting agents; emulsifying agents; hydrophilic polymers (such as polyvinylpyrrolidone); low molecular weight polypeptides; salt-forming counterions (such as sodium); preservatives (such as benzalkonium chloride, benzoic acid, salicylic acid, thimerosal, phenethyl alcohol, methylparaben, propylparaben, chlorhexidine, sorbic acid or hydrogen peroxide); solvents (such as glycerin, propylene glycol or polyethylene glycol); sugar alcohols (such as mannitol or sorbitol); suspending agents; surfactants or wetting agents (such as pluronics, PEG, sorbitan esters, polysorbates such as polysorbate 20, polysorbate, triton, tromethamine, lecithin, cholesterol, tyloxapal); stability enhancing agents (such as sucrose or sorbitol); tonicity enhancing agents (such as alkali metal halides, preferably sodium or potassium chloride, mannitol sorbitol); delivery vehicles; diluents; excipients and / or pharmaceutical adjuvants. See, e.g., Remington, The Science and Practice of Pharmacy, 23rdEdition, Academic Press (2020).

[0103] A suitable vehicle or carrier for the composition may be water for injection, physiological saline solution, or artificial cerebrospinal fluid, possibly supplemented with other materials common in compositions for parenteral administration. Neutral buffered saline or saline mixed with serum albumin are further exemplary vehicles.

[0104] In certain embodiments, buffers are used to maintain the composition at physiological pH or at a slightly lower pH, typically within a pH range of from about 5 to about 8. ForAttorney Docket No.: 10501-WO01-SEC example, the pH of the composition may be about 5.1, about 5.2, about 5.3, about 5.4, about 5.5, about 5.6, about 5.7, about 5.8, about 5.9, about 6.0, about 6.1, about 6.2, about 6.3, about 6.4, about 6.5, about 6.6, about 6.7, about 6.8, about 6.9, about 7.0, about 7.1, about 7.2, about 7.3, about 7.4, about 7.5, about 7.6, about 7.7, about 7.8, about 7.9, or about 8.0, including ranges between any two of the listed values, such as 5.1 to 8.0, 5.1 to 7.0, 5.5 to 8.0, 5.5 to 7.0, 6.0 to 8.0, or 6.0 to 70.

[0105] The following examples further illustrate the invention but, of course, should not be construed as in any way limiting its scope. EXAMPLE 1

[0106] This example describes a method for monitoring high molecule weight species in co- formulation protein therapeutics.

[0107] A size exclusion ultra high performance liquid chromatography (SE-UHPLC) method was developed to monitor total high molecular weight (HMW) species, and a cation exchange high performance liquid chromatography (CEX-HPLC) method was developed to separate individual charge variants in co-formulated monoclonal antibodies mixtures containing antibody 1 “Ab1” and antibody 2 “Ab2.”

[0108] HMW is a critical product quality attribute to monitor and control due to its potential impact on efficacy and safety. Assessment of the HMW in certain co-formulated antibody drug products with a platform SE-UHPLC method is challenging due to overlapping main peaks, which interferes with quantitation of the individual HMW species. Thus, a SE-UHPLC method was developed for quantitation of total HMW in co-formulated Ab1 / Ab2 drug products. The method monitors all HMWs in a co-formulation drug product that are observed in the drug products for Ab1 and Ab2 individually. To optimize the SE-UHPLC method for quantitation of HMW in a co-formulation drug product, focus was placed on minimizing secondary interaction to allow all HMW species elute within a similar elution window without interference from monomers. The parameters evaluated during development of the method are summarized in Table 1, and the optimized SE-UHPLC conditions are summarized in Table 2. Figure 1 shows results of HMW analysis for the Ab1 / Ab2 co-formulation using the optimized SE-UHPLC method.Attorney Docket No.: 10501-WO01-SEC Table 1. Method parameters evaluated for optimization of SE-UHPLC method for co- formulation Vendor Column details Conditions Tested Notes Platform BEH200 SEC Salt: Best condition: BEH / C l 1 Cl 0 / 50 / 100 / 150 / 250 / 350 / 450 M 50 M KPO 150 M yTable 2. Optimized SE-UHPLC Method Conditions for Co-Formulation SEC Column Flow Rate (mL / min) BEH20017 il i 46 04 dAttorney Docket No.: 10501-WO01-SEC EXAMPLE 2

[0109] This example describes a method for separating individual charge variants in co- formulation protein therapeutics.

[0110] A cation exchange high performance liquid chromatography (CEX-HPLC) method was developed to separate individual charge variants in co-formulated mixtures of monoclonal antibodies Ab1 and Ab2.

[0111] Charge variants are monitored during production of protein therapeutics, since chemical modifications such as deamidation may impact product quality. Assessment of the charge variants in certain co-formulated drug products with a platform pH gradient CEX-HPLC method is challenging due to overlapping basic peaks and acidic peaks from each molecule, which can interfere with quantitation of charge variants using conventional methods. In addition, stress conditions may pose additional challenges to monitor charge variants for basic peaks and acidic peaks of co-formulated antibody mixtures.

[0112] A pH gradient- and a salt gradient-based CEX-HPLC method were developed and optimized for separating and quantifying charge variants in an Ab1 / Ab2 co-formulation. The method maintains and monitors all charge variants in a co-formulation drug product that are observed in the Ab1 and Ab2 drug substances individually.

[0113] To optimize the CEX method for quantitation of charge variants in a co-formulation drug product, focus was placed on improving separation of the overlapping basic peaks and acidic peaks of Ab1 and Ab2. Both pH gradient and salt gradient CEX-HPLC methods were evaluated. Parameters evaluated during method development are summarized in Table 3 and Table 4. Table 3. Method parameters evaluated for optimization of pH gradient CEX-HPLC method Method Parameter ConditionsAttorney Docket No.: 10501-WO01-SEC Table 4. Method parameters evaluated for optimization of salt gradient CEX-HPLC method Method Parameter Conditions Column (single and tandem)• YMC BioPro SP-F 4.6 x 100 mm 5 μm (YMC Co.method are shown in Table 5. Results of an analysis of the Ab1 / Ab2 co-formulation with the developed CEX-HPLC method are shown in Figure 2. Table 5. Developed CEX-HPLC method conditions for co-formulationAttorney Docket No.: 10501-WO01-SEC EXAMPLE 3

[0115] This example describes a method of determining the concentration of a first protein and a second protein that is different from the first protein in a composition.

[0116] A strategy for measuring the concentration of two different proteins in a co- formulated mixture was developed based on an ultraviolet absorbance assay. The general strategy involves determining the ratio (k) of Ab1 to Ab2 in a composition using cationic exchange chromatography (CEX) according to the conditions set forth in Table 3. The total absorbance of the composition is then determined using the variable pathlength spectrophotometry system (e.g., SOLOVPE®, Repligen Corp, Bridgewater, NJ) or a fixed pathlength method. Using Beer’s law, the concentration of the first protein and the second protein is then calculated: ^^ଶ= ^^௧^௧^^ / (ε1b + ε2bk) [Equation 1] ^^^= ^^ଶ× ^^ [Equation 2] wherein ^^^is the concentration of the first protein, C2 is the concentration of the second protein, ^^௧^௧^^is the total absorbance, ε1 is the extinction coefficient of the first protein, ε2 is the extinction coefficient of the second protein, b is the path length, and k is the ratio of the first protein to the second protein in the composition.

[0117] Ab1 was co-formulated with Ab2 at a ratio (Ab1:Ab2) of 1:1, 1:2, 1:3, 2:1, or 3:1. The ratio (k) of Ab1:Ab2 in the co-formulation was determined by CEX, and the ^^௧^௧^^was measured using the extinction coefficients of 1.49 and 1.41 of Ab 1 and Ab2, respectively: ^^^ୠଶ= ^^௧^௧^^ / (1.49b + 1.41bk) ^^^ୠ^= ^^^ୠଶ× ^^.

[0118] The experimental ratio ofto the theoretical ratio (based on initial mixing) in unstressed (To) and stressed (four weeks at 40 °C) conditions. The results of this analysis are shown in Figures 3A and 3B. A positive linear correlation (R2~>0.99) was observed between the measured experimental ratio and theoretical ratio for the Ab1 / Ab2 co- formulation at both To and stressed conditions.

[0119] Ab1 was then co-formulated with a third monoclonal antibody (“Ab3”) at a ratio (Ab1:Ab3) of 1:1, 1:2, 1:3, 2:1, or 3:1. Beer’s law was used as described above to measure the experimental concentrations of each of Ab1 and Ab3 in unstressed (T0) and stressed (four weeks at 40 °C) conditions. The experimental ratios of Ab1:Ab3 in the co-formulation were comparedAttorney Docket No.: 10501-WO01-SEC to the theoretical ratio (based on initial mixing) under the tested conditions. The results of this analysis are shown in Figures 4A and 4B. A positive linear correlation (R2~>0.99) was observed between the measured experimental ratio and theoretical ratio for the Ab1 / Ab3 co-formulation at both T0 and stressed conditions.

[0120] Ab2 was co-formulated with a fourth monoclonal antibody (“Ab4”) at a ratio (Ab2:Ab4) of 1:1, 1:2, 1:3, 2:1, 3:1, 10:1, 20:1, 1:10, 1:20, 40:1, 80:1, 1:40, or 1:80. Ab1 also was co-formulated with Ab4 at the following ratios: 1:1, 1:2, 1:3, 1:10, 1:20, 2:1, 3:1, 10:1, 20:1. Beer’s law was used as described above to measure the experimental concentrations of each of Ab2 and Ab4 and each of Ab1 and Ab4 in unstressed (T0) and stressed (four weeks at 40 °C) conditions. The experimental ratios of Ab2:Ab4 and Ab1:Ab4 in each co-formulation were compared to the theoretical ratio (based on initial mixing) under the tested conditions. The results of this analysis are shown in Figures 5A, 5B, 6A, and 6B.

[0121] An alternative strategy involving direct concentration measurement also was tested using a RP-HPLC-based titer or HIC method for co-formulations of Ab1 / Ab2 and Ab1 / Ab3. The results of the RP-HPLC analysis are shown in Figures 7A and 7B, and results of the HIC analysis are shown in Figure 8. EXAMPLE 4

[0122] This example describes the characterization and release / stability testing of a composition comprising two different antibodies by various analytical methods.

[0123] SE-UPLC, direct protein concentration, reduced and non-reduced capillary gel electrophoresis (rCE-SDS and nrCE-SDS), and CEX-HPLC were used to analyze monoclonal antibodies Ab4 and “Ab5” individually and as a co-formulated mixture.

[0124] Attribute levels in the co-formulated mixture measured by SE-UHPLC (% total HMW) were comparable to expected values (Table 6), as shown in Figures 9A and 9B. Total protein concentration as measured by SOLOVPE® also was comparable to expected values, as shown in Table 7.Attorney Docket No.: 10501-WO01-SEC Table 6 % Diff luted Ab5 D between Ab4 Di iluted *Expected % Experimental expected and C n (m / ml) Conc. T t l HMW %T t l HMW x rim nt l % Comments d% Difference Experimental between s g .

[0125] Partial co-migration of each antibody was observed for rCE-SDS and nrCE-SDS profiles of the co-formulated mixture, as shown in Figures 10 and 12, respectively. A selected group of peaks gave a good estimate of the mixing ratio of proteins in the co-formulation. Ab4 and Ab5 also were mixed at different ratios and the concentration of each protein was quantified using rCE-SDS, the results of which are shown in Figure 11 and Table 8. A CEX-HPLC method also was applied to the different co-formulations of Ab4 and Ab5, and was able to monitor stability of the co-formulations despite co-migration (see Figure 13).Attorney Docket No.: 10501-WO01-SEC Table 8 Ab4 HC : Ab5 Th Ab4 HC Ab5 HC ombo Ab4eoretical Ab5 HC (m / ml)(m / ml) mixin r ti Area Area ( r / r ) Comments g ., , , , are hereby incorporated by reference to the same extent as if each reference were individually and specifically indicated to be incorporated by reference and were set forth in its entirety herein.

[0127] As used herein, the term “about” when used as a modifier to a specified numerical value (e.g., pH of “about” 7.0), indicates that variation around the numerical value can occur. These variations can occur by a variety of means, such as typical measuring and handling procedures, inadvertent errors, ingredient purity, and the like. If greater numerical precision is required, in some embodiments, “about” may refer to numerical values withing ±5% of the specified numerical value.

[0128] The use of the terms “a” and “an” and “the” and “at least one” and similar referents in the context of describing the invention (especially in the context of the following claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. The use of the term “at least one” followed by a list of one or more items (for example, “at least one of A and B”) is to be construed to mean one item selected from the listed items (A or B) or any combination of two or more of the listed items (A and B), unless otherwise indicated herein or clearly contradicted by context. The terms “comprising,” “having,” “including,” and “containing” are to be construed as open-ended terms (i.e., meaning “including, but not limited to,”) unless otherwise noted. Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., “such as”) provided herein, is intended merely to better illuminate the inventionAttorney Docket No.: 10501-WO01-SEC and does not pose a limitation on the scope of the invention unless otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention.

[0129] Preferred embodiments of this invention are described herein, including the best mode known to the inventors for carrying out the invention. Variations of those preferred embodiments may become apparent to those of ordinary skill in the art upon reading the foregoing description. The inventors expect skilled artisans to employ such variations as appropriate, and the inventors intend for the invention to be practiced otherwise than as specifically described herein. Accordingly, this invention includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, any combination of the above-described elements in all possible variations thereof is encompassed by the invention unless otherwise indicated herein or otherwise clearly contradicted by context.

Claims

Attorney Docket No.: 10501-WO01-SEC CLAIM(S):

1. A method of determining the concentration of each of a first protein and a second protein that is different from the first protein in a composition, comprising: providing said composition comprising the first protein and the second protein, wherein the first protein has a first extinction coefficient and the second protein has a second extinction coefficient; resolving the first protein from the second protein of said composition by cation exchange chromatography (CEX), whereby a ratio of the first protein to the second protein in said composition is obtained; measuring a total absorbance of the composition for a path length; and calculating a concentration of said first protein and said second protein based on the ratio of the total absorbance to said path length and said extinction coefficients, prorated by the ratio of the first protein to the second protein in the composition.

2. The method of claim 1, wherein said calculating comprises Equation 1: ^^ଶ= ^^௧^௧^^ / (ε1b + ε2bk) [Equation 1] wherein C2 is the concentration of the second protein, ^^௧^௧^^is the total absorbance, ε1 is the extinction coefficient of the first protein, ε2 is the extinction coefficient of the second protein, b is the path length, and k is the ratio of the first protein to the second protein in the composition.

3. The method of claim 2, wherein said calculating further comprises Equation 2: ^^^= ^^ଶ× ^^ [Equation 2], wherein ^^^is the concentration of the first protein and k is the ratio of the first protein to the second protein in the composition.Attorney Docket No.: 10501-WO01-SEC 4. The method of any one of claims 1-3, wherein the ratio of the first protein to the second protein is 1:1 to 1:100, such as 1:1 to 1:80, 1:1 to 1:40, 1:1 to 1:20, 1:1.1 to 1:100, 1:1.1 to 1:80, 1:1.1 to 1:40, 1:1.1 to 1:20, 1:2 to 1:100, 1:2 to 1:80, 1:2 to 1:40, or 1:2 to 1:

20.

5. The method of any one of claims 1-4, wherein the first protein has an isoelectric point (pI) that differs from the isoelectric point of the second protein by at least 0.

2.

6. The method of any one of claims 1-5, which further comprises: resolving the first protein from the second protein in the composition by CEX, hydrophobic interaction chromatography (HIC), or reverse phase high performance liquid chromatography (RP-HPLC), determining the concentration of the first protein and the second protein based on a calibration curve for each protein.

7. A chromatography method of analyzing a composition comprising a first protein and a second protein that is different from the first protein, which method comprises: providing said composition comprising the first protein and the second protein, and at least one of: a) determining a level of high molecular weight (HMW) species of the first protein and / or second protein in the composition by size exclusion ultra high performance liquid chromatography (SE-UHPLC), wherein the SE-UHPLC is performed with a buffer comprising about 100-200 mM KCl, about 1%-5% isopropyl alcohol (IPA), and about 40-60 mM K3PO4; or b) separating charge variants of the first protein and / or the second protein in the composition by pH-gradient cation exchange-high performance liquid chromatography (CEX-HPLC), wherein the CEX-HPLC comprises a first mobile phase at pH 5-6 and a second mobile phase at pH 10-11.

8. The method of claim 7, wherein the HMW species comprise one or more of dimers, trimers, tetramers, pentamers, hexamers, heptamers, and octamers.Attorney Docket No.: 10501-WO01-SEC 9. The method of claim 7 or claim 8, wherein the SE-UHPLC is performed with a buffer comprising about 150 mM KCl, about 2% IPA, and about 50 mM K3PO4.

10. The method of any one of claims 7-9, wherein the SE-UHPLC comprises a mobile phase having a flow rate of about 0.2-0.5 mL / min.

11. The method of claim 10, wherein the mobile phase flow rate is about 0.4 mL / min.

12. The method of any one of claims 7-11, wherein the CEX-HPLC comprises a first mobile phase of pH of about 5.

6.

13. The method of any one of claims 7-12, wherein the CEX-HPLC comprises a second mobile phase of pH of about 10.

2.

14. The method of any one of claims 7-13, wherein the CEX-HPLC comprises a gradient from 100% of the first mobile phase + 0% of the second mobile phase to no more than 20% of the first mobile phase + at least 80% of the second mobile phase, over at least 40, 5060, or 70 minutes.

15. The method of any one of claims 7-14, wherein the CEX-HPLC comprises a column that is at least 50 mm, 75 mm, or 100 mm.

16. The method of any one of claims 7-15, which further comprises, after separating said charge variants by CEX-HPLC, determining the concentration of the first protein and the second protein in the composition based on a calibration curve for each protein.

17. A capillary electrophoresis (CE) method of determining concentration of each of a first protein and a second protein in a composition, wherein the first protein is different from the second protein, which method comprises: analyzing the composition comprising the first protein and the second protein by capillary electrophoresis SDS (CE-SDS), wherein the CE-SDS comprises a first peak ofAttorney Docket No.: 10501-WO01-SEC the first protein, a second peak of the second protein, and a comigration peak of the first protein and the second protein; and determining the concentration of each of the first protein and second protein based on a ratio of the first peak to the second peak.

18. The method of claim 17, wherein the CE-SDS is reduced (rCE-SDS) or non- reduced (nrCE-SDS).

19. The method of any one of claims 1-18, wherein each of the first protein and the second protein is an antigen-binding protein.

20. The method of claim 19, wherein the antigen-binding protein is an antibody, an antibody fragment, or a bispecific T cell engager (BiTE®) molecule.

21. The method of claim 20, wherein each of the first protein and the second protein is a therapeutic antibody.

22. The method of claim 21, wherein the therapeutic antibody specifically binds to TIGIT, CD112R, PD-1, or VEGF.

23. The method of claim 22, wherein the first protein is an antibody that specifically binds to CD112R and the second protein is an antibody that specifically binds to TIGIT.

24. The method of claim 22, wherein the first protein is an antibody that specifically binds to PD-1 and the second protein is an antibody that specifically binds to VEGF.

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