Method for analyzing antibody co-formulations

Abstract

The present disclosure provides methods for analyzing two or more different proteins co-formulated in a sample mixture. These methods relate to 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 of each protein in the sample and assess certain key quality attributes thereof.

Description

Technical Field

[0001] This disclosure relates to a method for detecting and quantifying two or more antigen-binding proteins (e.g., antibodies) co-formulated in a single composition. Background Technology

[0002] The use of combination therapies involving two or more molecules with complementary pharmacological effects has spurred increasing interest in the development of co-formulated products. Co-formulated products, or fixed-dose combination drugs (FDCs), are therapeutic agents that combine two or more individual drug components (e.g., small molecules and biologics, or two different biologics such as therapeutic antibodies) in a single dosage form. These products typically reduce the number and volume of injections, improve patient compliance, and reduce discomfort.

[0003] Because co-formulated therapeutics are classified as new molecular entities (NMEs), they are subject to clinical evaluation by regulatory agencies such as the U.S. Food and Drug Administration (FDA). Therefore, existing therapeutics that are co-formulated separately may require further analysis and evaluation, even if the efficacy and safety of each individual therapeutic have been established through independent clinical trials.

[0004] For co-formulated biologics, there are still many chemical, manufacturing, and control issues to be addressed. These issues include the analytical challenges of characterizing the various molecules in the co-formulated product, the manufacturing challenges of formulating high-concentration biologics, and stability issues (such as protein-protein interactions, protein aggregation, and subvisible particulate formulations).

[0005] There is still a need for methods to monitor key quality properties of co-formulated biomolecules (e.g., high molecular weight substances) and to accurately determine the concentration of each biomolecule in a pharmaceutical formulation or sample. Summary of the Invention

[0006] This disclosure provides a method for determining the concentrations of a first protein and a second protein, distinct from the first protein, in a composition. The method includes: providing the 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; separating the first protein and the second protein in the composition by cation exchange chromatography (CEX) to obtain a ratio of the first protein to the second protein in the composition; measuring the total absorbance of the composition over a path length; and calculating the concentrations of the first protein and the second protein by distributing them according to the ratio of the total absorbance to the path length and the extinction coefficient.

[0007] In some aspects of this method, calculating the concentration of the second protein includes Equation 1:

[0008]

[0009] Where C2 is the concentration of the second protein, A 总 ε 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. In some aspects of this method, calculating the concentration of the first protein further includes Equation 2:

[0010] ,

[0011] Where C1 is the concentration of the first protein, C2 is the concentration of the second protein, and k is the ratio of the first protein to the second protein in the composition.

[0012] In some aspects of this method, 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.

[0013] In some aspects of this method, the isoelectric point (pI) of the first protein differs from that of the second protein by at least 0.2.

[0014] In some aspects, the method further includes separating the first protein from the second protein in the composition by CEX, hydrophobic interaction chromatography (HIC), or reversed-phase high-performance liquid chromatography (RP-HPLC), and determining the concentrations of the first protein and the second protein based on calibration curves for each protein.

[0015] This disclosure also provides a chromatographic method for analyzing compositions comprising a first protein and a second protein different from the first protein, the method comprising: providing said composition comprising the first protein and the second protein; and at least one of the following: a) determining the level of high molecular weight (HMW) substances of the first protein and / or the 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% isopropanol (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 of pH 5-6 and a second mobile phase of pH 10-11.

[0016] In some aspects of chromatographic methods, HMW substances comprise one or more of the following: dimer, trimer, tetramer, pentamer, hexamer, heptamer, and octamer.

[0017] In some aspects of the chromatographic method, SE-UHPLC is performed using a buffer solution containing approximately 150 mM KCl, approximately 2% IPA, and approximately 50 mM K3PO4.

[0018] In some aspects of the chromatographic method, SE-UHPLC includes a mobile phase with a flow rate of approximately 0.2–0.5 mL / min. For example, the flow rate of the mobile phase can be approximately 0.4 mL / min.

[0019] In some aspects of the chromatographic method, CEX-HPLC includes a first mobile phase with a pH of approximately 5.6 (e.g., pH 5.6). In other aspects, CEX-HPLC includes a first mobile phase with a pH of 5.4–5.8.

[0020] In some aspects of the chromatographic method, CEX-HPLC includes a second mobile phase with a pH of approximately 10.2 (e.g., pH 10.2). In other aspects of the chromatographic method, CEX-HPLC includes a second mobile phase with a pH of 10.0–10.4.

[0021] In some aspects of the chromatographic method, CEX-HPLC includes a gradient from 100% first mobile phase + 0% second mobile phase to no more than 20% first mobile phase + at least 80% second mobile phase over a period of at least 40, 50, 60 or 70 minutes.

[0022] In some aspects of the chromatographic method, CEX-HPLC comprises a column of at least 50 mm, 75 mm, or 100 mm. Optionally, the CEX-HPLC column may comprise a matrix of hydrophilic porous polymer beads with an average diameter of about 3 μm to about 7 μm (e.g., about 5 μm). The CEX-HPLC column may include sulfopropyl ion exchanger chemicals.

[0023] In some aspects, the chromatographic method further includes determining the concentrations of the first and second proteins in the composition based on calibration curves for each protein after separating the charge variants by CEX-HPLC.

[0024] This disclosure further provides a capillary electrophoresis method for determining the concentrations of a first protein and a second protein in a composition, wherein the first protein is different from the first protein. The method includes: analyzing the composition containing the first protein and the second protein by capillary electrophoresis with sodium dodecyl sulfate (CE-SDS), wherein the CE-SDS contains 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 determining the concentrations of the first protein and the second protein based on the ratio of the first peak to the second peak.

[0025] In some aspects of capillary electrophoresis methods, CE-SDS is either reducing (rCE-SDS) or non-reducing (nrCE-SDS).

[0026] In some aspects of the above methods, the first protein and the second protein are each antigen-binding proteins.

[0027] In some aspects of the above methods, the antigen-binding protein is an antibody, an antibody fragment, or a bispecific T-cell binding agent (BiTE®) molecule.

[0028] In some aspects of the above methods, the first protein and the second protein are each a therapeutic antibody.

[0029] In some aspects of the above methods, therapeutic antibodies specifically bind to TIGIT, CD112R, PD-1, or VEGF.

[0030] In some aspects of the above method, the first protein is an antibody that specifically binds to CD112R, and the second protein is an antibody that specifically binds to TIGIT.

[0031] In some aspects of the above method, the first protein is an antibody that specifically binds to PD-1, and the second protein is an antibody that specifically binds to VEGF. Attached Figure Description

[0032] Figure 1This is a graph showing the results of high molecular weight (HMW) analysis of the Ab1 / Ab2 co-formulation using the optimized SE-UHPLC method described in Example 1.

[0033] Figure 2 This is a graph showing the results of analyzing the Ab1 / Ab2 co-formulation using the CEX-HPLC method developed in Example 2.

[0034] Figure 3A and Figure 3B As determined by the method described in Example 3, under non-stress conditions (To) Figure 3A ) and stress conditions (four weeks at 40°C) Figure 3B The graph below shows a comparison between the measured / experimental ratio and the theoretical ratio of Ab1:Ab2.

[0035] Figure 4A and Figure 4B As determined by the method described in Example 3, under non-stress conditions (To) Figure 4A ) and stress conditions (four weeks at 40°C) Figure 4B The graph below shows the comparison between the measured / experimental ratio and the theoretical ratio of Ab1:Ab3.

[0036] Figure 5A and Figure 5B As determined by the method described in Example 3, under non-stress conditions (To) Figure 5A ) and stress conditions (four weeks at 40°C) Figure 5B The graph below shows the comparison between the measured / experimental ratio and the theoretical ratio of Ab2:Ab4.

[0037] Figure 6A and Figure 6B As determined by the method described in Example 3, under non-stress conditions (To) Figure 6A ) and stress conditions (four weeks at 40°C) Figure 6B The graph below shows the comparison between the measured / experimental ratio and the theoretical ratio of Ab1:Ab4.

[0038] Figure 7A and Figure 7B This demonstrates the direct measurement of Ab1 / Ab2 using an RP-HPLC-based titration method. Figure 7A ) and Ab1 / Ab3 ( Figure 7B The result of the concentration of ).

[0039] Figure 8 The results show the results of directly measuring the concentrations of Ab1 / Ab2 and Ab1 / Ab3 using a HIC-based titration method.

[0040] Figure 9AThe graph shows the HMW and main peak of co-prepared Ab4 / Ab5 mixtures at different ratios, as measured by SE-U HPLC. Figure 9B This is a graph showing Ab5% compared to the total HMW percentage.

[0041] Figure 10 This is the rCE-SDS spectrum of the mixture prepared by co-concentration of Ab4 / Ab5.

[0042] Figure 11 This includes graphs showing the concentrations of Ab4 and Ab5 mixed at different ratios, as measured using rCE-SDS.

[0043] Figure 12 This is the nrCE-SDS spectrum of the mixture prepared by co-concentration of Ab4 / Ab5.

[0044] Figure 13 CEX spectra of Ab4 / Ab5 co-preparations with different mixing ratios. Detailed Implementation

[0045] This disclosure is based, at least in part, on the development of assays for the isolation and quantification of different intact proteins (e.g., antibodies) co-formulated in a single sample, and for monitoring critical quality attributes (CQA) of each protein in a co-formulated product. The methods described herein allow for the accurate monitoring and control of therapeutic drugs and their processing through release and stability testing to ensure accurate dosing. Monitoring the concentration of each component in a co-formulated drug is also crucial for assessing the efficacy of combined therapeutics, as well as for other more specific applications, such as the stability of IV bags for co-administered therapeutic agents.

[0046] In some embodiments, this disclosure provides a method for determining the concentrations of a first protein and a second protein, different from the first protein, in a composition. The method includes providing a composition comprising a first protein and a second protein, wherein the first protein has a first extinction coefficient and the second protein has a second extinction coefficient; separating the first protein and the second protein in the composition by cation exchange chromatography (CEX) to obtain a ratio of the first protein to the second protein in the composition; measuring the total absorbance of the composition over a path length; and calculating the concentrations of the first protein and the second protein by distributing them according to the ratio of the total absorbance to the path length and the extinction coefficient.

[0047] This disclosure also provides a chromatographic method for analyzing compositions comprising a first protein and a second protein different from the first protein. The method comprises: providing said composition comprising a first protein and a second protein; and at least one of the following: a) determining the level of high molecular weight (HMW) substances of the first protein and / or the 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% isopropanol (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 of pH 5-6 and a second mobile phase of pH 10-11.

[0048] In another embodiment, this disclosure provides a capillary electrophoresis method for determining the concentrations of a first protein and a second protein in a composition, wherein the first protein is different from the second protein. The method includes analyzing a composition comprising the first protein and the second protein by capillary electrophoresis with sodium dodecyl sulfate (CE-SDS), wherein the CE-SDS contains a first peak of the first protein, a second peak of the second protein, and co-migration peaks of the first and second proteins; and determining the concentrations of the first protein and the second protein based on the ratio of the first peak to the second peak.

[0049] definition

[0050] To facilitate understanding of the technology of this invention, several terms and phrases are defined below. Further definitions are set forth throughout the specific embodiments.

[0051] In some respects, the term “determination” means “measurement” and may be used interchangeably with the terms “test,” “analysis,” or “determination.” The level of a protein or protein property determined or determined by the methods of this disclosure may be a relative measurement, for example, a determination of a level higher than or lower than a reference level, or the same as a reference level. For example, in some respects, the methods of this disclosure can determine the level of HMW substances of a first protein and / or a second protein in a composition. In some respects, “determination” may yield a normalized measurement. For example, a normalized measurement may be normalized relative to a reference protein (e.g., serum albumin). In some cases, “determination” yields an absolute measurement (e.g., neither normalized nor relative to a reference level).

[0052] "Critical Quality Attributes (CQAs)" refer to physical, chemical, biological, or microbiological characteristics or features that should be within appropriate limits, ranges, or distributions to ensure desired product quality. CQAs are typically associated with pharmaceutical products, active pharmaceutical ingredients (APIs), excipients, and intermediates (process materials). For example, for large therapeutic peptide molecules, the physical properties and modifications of amino acids are important CQAs to monitor during and after manufacturing, as well as during drug development. Non-limiting examples of CQAs for monoclonal antibodies or their antigen-binding fragments include high molecular weight (HMW) substances, charge variants, oxidized substances, deamidated substances, and glycosylation.

[0053] As used herein, the term "antigen-binding protein" refers to a protein molecule that binds specifically to an antigen. For example, antigen-binding proteins may include antibodies or their antigen-binding fragments (such as monoclonal antibodies, such as IgG1 or IgG2 monoclonal antibodies), antibody protein products, bispecific T-cell conjugates (BiTE®) molecules, bispecific antibodies, trispecific antibodies, or Fc fusion proteins.

[0054] Antigen-binding proteins typically comprise heavy chain variable regions (VH) and / or light chain variable regions (VL) of an antibody, or domains derived therefrom. In some embodiments, antigen-binding proteins contain structural requirements sufficient for the antibody to bind to an immune-specific target. Such structural requirements can 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 all six CDRs. The location (and order) of these CDRs in the antigen-binding protein is within the knowledge of a person skilled in the art.

[0055] As used herein, the term "antibody" refers to any isotype of immunoglobulin that specifically binds to a target antigen; antibodies can be polyclonal or monoclonal antibodies, chimeric antibodies, humanized antibodies, human antibodies, etc. In natural antibodies, the heavy chain contains a variable region VH and three constant regions CH1, CH2, and CH3. The VH domain is located at the amino terminus of the heavy chain, and the CH3 domain is located at the carboxyl terminus. In natural antibodies, the light chain contains a variable region VL and a constant region CL. The variable region of the light chain is located at the amino terminus of that light chain. In natural antibodies, the variable regions of each light chain / heavy chain pair typically form antigen-binding sites. The constant regions typically perform effector functions. Natural antibodies are tetramers of two full-length heavy chains and two full-length light chains.

[0056] In human antibodies, CH1 refers to the region containing the amino acid sequence at positions 118 to 215 of the EU index or EU numbering system, which is based on the sequence number of the first human IgG1 (i.e., the "EU antibody") sequenced (Edelman et al., Proc Natl Acad Sci USA [Proceedings of the National Academy of Sciences], 63(1): 78-85(1969)). A highly flexible amino acid region, called the "hinge region," exists between CH1 and CH2. CH2 represents the region containing the amino acid sequence at positions 231 to 340 of the EU index, and CH3 represents the region containing the amino acid sequence at positions 341 to 446 of the EU index.

[0057] “CL” indicates the constant region of the light chain. In the case of the κ chain of human antibodies, CL represents the region containing the amino acid sequence at positions 108 to 214 of the EU index. In the λ chain, CL represents the region containing the amino acid sequence at positions 108 to 215.

[0058] In natural antibodies, the variable region typically exhibits the same general structure, where the relatively conserved framework region (FR) is linked by three hypervariable CDRs. The CDRs from the two chains of each pair are typically aligned through the framework region, which allows for the binding of specific epitopes. From the N-terminus to the C-terminus, both the light chain and heavy chain variable regions typically contain the domains FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. Typically, CDR3 is the largest source of molecular diversity within the antigen-binding site. The amino acid assignment of each domain is typically defined according to the following definitions: Kabat et al. (1991) Sequences of Proteins of Immunological Interest (National Institutes of Health, Publication No. 91-3242, Vol. 1-3, Bethesda, MD); Chothia, C. and Lesk, AM (1987) J. Mol. Biol., 196: 901-917. In some embodiments, the CDR of an antigen-binding protein is defined according to the definition of Kabat or Chothia. In this application, unless otherwise stated, the term “CDR” refers to a CDR derived from the light chain or heavy chain.

[0059] Antibodies may contain any constant region known in the art. Human light chains are classified as κ light chains and λ light chains. Heavy chains are classified as μ, δ, γ, α, or ε, and antibody isotypes are defined as IgM, IgD, IgG, IgA, and IgE, respectively. IgG has several subclasses, including but not limited to IgG1, IgG2, IgG3, and IgG4. IgM has subclasses, including but not limited to IgM1 and IgM2. The embodiments disclosed herein include all such antibody classes or isotypes. Light chain constant regions may be, for example, κ-type or λ-type light chain constant regions, such as human κ-type or λ-type light chain constant regions. Heavy chain constant regions may be, for example, α-type, δ-type, ε-type, γ-type, or μ-type heavy chain constant regions, such as human α-type, δ-type, ε-type, γ-type, or μ-type heavy chain constant regions. Therefore, 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.

[0060] Antibodies can be monoclonal or polyclonal. As used herein, the term “monoclonal antibody” refers to an antibody produced by a single clone of B lymphocytes that targets a single epitope on an antigen. Monoclonal antibodies are typically produced using hybridoma technology, as first described in Kohler and Milstein, Eur. J. Immunol. [European Journal of Immunology], 5: 511-519 (1976). Monoclonal antibodies can also be produced using recombinant DNA methods (see, for example, U.S. Patent 4,816,567), isolated from phage display antibody libraries (see, for example, Clackson et al., Nature, 352:624-628 (1991); and Marks et al., J. Mol. Biol., 222:581-597 (1991)), or produced by transgenic mice carrying a fully human immunoglobulin system (see, for example, XENOMOUSE™ mice, Green et al. (1994), Nature Genetics 7:13-21, US 2003-0070185, WO96 / 34096, and WO 96 / 33735). In contrast, “polyclonal” antibodies are antibodies 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.

[0061] The term "chimeric antibody" refers to an antibody containing domains from two or more different antibodies. A chimeric antibody may, for example, contain a constant domain from one species and a variable domain from a second species, or more generally, it may contain segments of amino acid sequences from at least two species. Chimeric antibodies may also contain domains from two or more different antibodies within the same species. The term "humanization," in relation to antibody use, refers to an antibody having at least a non-human source CDR region engineered to have a structure and immunological function more similar to a real human antibody than the original source antibody. For example, humanization may involve transplanting a CDR from a non-human antibody (such as a mouse antibody) into a human antibody. Humanization may also involve selected amino acid substitutions to make the non-human sequence more similar to a human sequence.

[0062] Antibodies can be cleaved into fragments by enzymes such as papain, pepsin, or other engineered site-specific proteases, such as those commercially available from Genovis AB in Lund, Sweden. Papain cleaves the antibody to produce two Fab fragments and a single Fc fragment. Pepsin cleaves the antibody to produce an F(ab')2 fragment and a pFc' fragment. In an exemplary aspect, the antigen-binding protein disclosed herein comprises an antigen-binding antibody fragment. As used herein, the term "antigen-binding antibody fragment" refers to a portion of an antibody molecule capable of binding to an antigen of the antibody, also referred to as an "antigen-binding fragment" or "antigen-binding moiety." In an exemplary case, the antigen-binding antibody fragment is a Fab fragment or an F(ab')2 fragment.

[0063] Antibody architectures have been used to generate an increasing number of alternative forms, spanning a molecular weight range of at least about 12–150 kDa and having valences (n) ranging from monomers (n = 1), to dimers (n = 2), to trimers (n = 3), to tetramers (n = 4), and possibly higher; such alternative forms are referred to herein as “antibody protein products.” Antibody protein products include those based on the intact antibody structure and those mimicking antibody fragments that retain the intact antigen-binding capability, such as scFv, Fab (e.g., Fab, Fab', and F(ab')2), and VHH / VH. The smallest antigen-binding antibody fragment that retains its intact antigen-binding site is the Fv fragment, which consists entirely of variable (V) regions of light and heavy chains. V regions are linked into scFv (single-chain variable fragment) fragments using soluble, flexible amino acid peptide linkers to stabilize the molecule, or constant (C) domains are added to the V regions to generate Fab fragments. Both scFv and Fab fragments can be readily generated in host cells (e.g., prokaryotic host cells). VHH / VH (or nanobodies) are antigen-binding fragments of heavy-chain-only antibodies. Heavy-chain-only antibodies (HcAbs) are naturally produced by camels and sharks. Other antibody protein products include bispecific T-cell conjugates (BiTEs). ® Antibody proteins include molecule-based antibodies, disulfide-bonded scFvs (ds-scFvs), single-chain Fabs (scFabs), and dimer and polymeric antibody forms (such as biantibodies, triantibodies, and tetraantibodies, or miniantibodies in various forms consisting of scFvs linked to oligomeric domains). Peptibody or peptide-Fc fusion is another type of antibody protein product. The structure of a peptibody consists of a biologically active peptide grafted onto the Fc domain (see, for example, Shimamoto et al., mAbs [Monoclonal Antibodies] 4(5): 586-591(2012)).

[0064] The antigen-binding protein disclosed herein may comprise any of the antibody protein products described above. In an exemplary aspect, the antigen-binding protein disclosed herein may comprise any of the following: scFv, Fab, VHH / VH, Fv fragment, ds-scFv, scFab, dimeric antibody, polyantibody (e.g., biantibody, triantibody, tetraantibody), miniAb, peptide of camelid heavy chain antibody VHH / VH, sdAb, biantibody; triantibody; tetraantibody; bispecific antibody or trispecific antibody, bispecific T-cell conjugate (BiTE®) molecule, BsIgG, attached IgG, BsAb fragment, bispecific fusion protein, or BsAb conjugate.

[0065] In some respects, the antigen-binding proteins disclosed herein may be "bispecific," meaning they can bind to two different antigens. In other respects, the antigen-binding proteins disclosed herein may be "trispecific," meaning they can bind to three different antigens. In yet another respect, the antigen-binding proteins disclosed herein may be "tetraspecific," meaning they can bind to four different antigens.

[0066] In some embodiments, the antigen-binding protein is a BiTE® molecule. BiTE® molecules are engineered bispecific antigen-binding constructs that guide the cytotoxic activity of T cells against cancer cells. They are fusions of two single-chain variable fragments (scFvs) of different antibodies on a single peptide chain of approximately 55 kDa, or amino acid sequences from four different genes. One scFv binds to T cells via the CD3 receptor, while the other binds to tumor cells via a tumor-specific molecule. Bonatumab (a BLINCYTO® product) is an example of a BiTE® molecule specific to CD19. Modified BiTE® molecules (such as those modified to extend their half-life) may also be used in the disclosed methods. By design, BiTE® molecules are uniquely suited to transiently connect T cells to target cells while simultaneously and potently activating the inherent cytolytic potential of T cells against target cells. See, for example, WO 99 / 54440, WO 2005 / 040220, and WO 2008 / 119567.

[0067] In some embodiments disclosed herein, the antigen-binding protein may be multivalent. The valence of a binding protein indicates the number of individual antigen-binding domains within that binding protein. In some embodiments, a bispecific antigen-binding protein may be multivalent. For example, in some embodiments, a bispecific antigen-binding protein may be tetravalent, 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.

[0068] As used herein, the terms "antigen-binding domain" and "binding domain" are used interchangeably and refer to a region of an antigen-binding protein containing amino acid residues that interact with the antigen and confer specificity and affinity to the antigen. In some embodiments, the binding domain may be derived from a natural ligand of one or more target antigens. As used herein, the term "one or more target antigens" refers to the first and / or second target antigen of a bispecific molecule, and also to the first, second, third, and / or fourth target antigen of a tetraspecific molecule.

[0069] Antigen-binding proteins can contain immunoglobulin domains. As used herein, the term "immunoglobulin domain" refers to a peptide containing an amino acid sequence similar to that of an immunoglobulin (i.e., an 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 the antibody heavy chain, and VL and CL of the antibody light chain. Additionally, immunoglobulin domains are found in proteins other than immunoglobulins. Examples of immunoglobulin domains in proteins other than immunoglobulins include those contained in proteins belonging to the immunoglobulin superfamily, such as the major histocompatibility complex (MHC), CD1, B7, and the T-cell receptor (TCR).

[0070] As used herein, the terms “stability” and “steady” are defined as maintaining the chemical or physical integrity and / or biological activity of an antigen-binding polypeptide or protein over a period of time. Stabilizing an antigen-binding polypeptide or protein includes preventing or delaying the degradation or degeneration of the antigen-binding polypeptide or protein from its biologically and / or therapeutically active form to an inactive form. Instability can result from events such as aggregation, denaturation, fragmentation, or chemical modification (e.g., oxidation, cross-linking, deamidation), as well as reactions with other components characteristic of compositions containing antigen-binding polypeptides or proteins.

[0071] The terms “binding pair member” and “binding member” are used interchangeably herein and refer to one of two or more different molecules that specifically recognizes another molecule (compared to a substantially lower recognition of other molecules). For example, when an antibody or other entity (e.g., an antigen-binding protein) “specifically recognizes” or “specifically binds” to an antigen or epitope, it preferentially recognizes that antigen in a complex mixture of proteins and / or macromolecules and binds to that antigen or epitope with a substantially higher affinity than to other entities that do not exhibit that antigen or epitope. Therefore, the binding of two binding pair members to each other will be more robust than their binding to other molecules.

[0072] Methods for determining protein concentration

[0073] This document provides a method for determining the concentrations of a first protein and a second protein, different from the first protein, in a composition. In some embodiments, this disclosure provides a method comprising providing a composition comprising a first protein and a second protein, wherein the first protein has a first extinction coefficient and the second protein has a second extinction coefficient; separating the first protein and the second protein in the composition by cation exchange chromatography (CEX) to obtain a ratio of the first protein to the second protein in the composition; measuring the total absorbance of the composition over a path length; and calculating the concentrations of the first protein and the second protein by dispensing them according to the ratio of the total absorbance to the path length and the extinction coefficient.

[0074] The terms “extinction coefficient” and “molar extinction coefficient” are used interchangeably in this document and refer to a measure of the degree to which a chemical substance absorbs light at a given wavelength and thus attenuates the intensity of light. The extinction coefficient (ε) is an inherent property of a chemical substance, depending on its chemical composition and structure. It should be understood that peptides or proteins do not have a single extinction coefficient value, as small differences in buffer type, ionic strength, and pH at least slightly affect the extinction coefficient value. Therefore, the extinction coefficient is typically determined empirically using a protein solution of known concentration dissolved in the same buffer as the target sample. Alternatively, the extinction coefficients of many proteins have been compiled and reported, for example, in Fasman, DG, ed., “The Practical Handbook of Biochemistry and Molecular Biology,” CRC Press, Boston (1992).

[0075] The first protein can be separated from the second protein using any suitable protein separation method, many of which are known in the art. For example, the first protein can be separated from the second protein using one or more chromatographic 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), reversed-phase high-performance liquid chromatography (RP-HPLC), ultracentrifugation (UC), etc. (see, for example, Coskun, North Clin Istanb [Istanbul North Clinic] 3(2): 156-160 (2016)). In some embodiments, high-performance liquid chromatography (HPLC) is used to separate the first protein from the second protein. HPLC (also known as high-pressure liquid chromatography) is a technique used to separate, identify, and quantify various components in a mixture. HPLC relies on a pump to pass a pressurized liquid solvent containing a sample mixture through a column packed with a solid adsorbent material. The different components in the sample interact slightly differently with the adsorbent material, resulting in different flow rates for different components and causing separation of the components when they flow out of the column.

[0076] In some embodiments, cation exchange chromatography (CEX) is used to separate a first protein from a second protein in the composition. CEX is a type of ion exchange chromatography based on the electrostatic interaction between charged protein groups and a solid support or matrix. More specifically, cation exchange chromatography uses a negatively charged ion exchange resin that has an affinity for molecules with a net positive surface charge. Proteins are separated from the column by varying the pH, the concentration of the ion salt, or the 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 can be used in the disclosed methods.

[0077] Proteins with different isoelectric points (pI) may have different degrees of charge at a given pH, and thus different affinities for negatively charged surface groups on cation exchange medium particles; therefore, different proteins can bind to cation exchange resins with varying strengths, thereby facilitating their separation. Without being bound by theory, it is believed that the retention times of different co-formulated proteins on cation exchange resins are proportional to the pI of each protein, and that larger pI differences between co-formulated proteins contribute to chromatographic separation. In some embodiments, the isoelectric point (pI) difference between the first protein and the second protein in the composition is at least 0.2. For example, the pI difference between the first protein and the second protein is 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 greater. In some embodiments, the pI difference between the first protein and the second protein is no more than about 14 (e.g., about 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2 or less).

[0078] Other liquid chromatography methods can be used to separate the first protein from the second protein in a composition. For example, in some embodiments, hydrophobic interaction chromatography (HIC) is used. HIC is a technique for separating molecules based on their hydrophobicity and can be used to purify proteins while maintaining their biological activity due to the use of conditions and matrices operated under relatively weak denaturing conditions. In other embodiments, reversed-phase high-performance liquid chromatography (RP-HPLC) can be used. RP-HPLC involves separating molecules based on their hydrophobicity. The separation depends on the hydrophobic binding of solute molecules from the mobile phase to immobilized hydrophobic ligands attached to the stationary phase (i.e., the adsorbent). RP-HPLC is widely used in the art to separate peptides and proteins from a variety of synthetic or biological sources and for both analytical and preparative applications (see, for example, Aguilar, MI. (ed.), HPLC of Peptides and Proteins: Methods and Protocols, Human Press (2004)).

[0079] Separating the first protein from the second protein in the composition by CEX allows those skilled in the art to obtain the ratio of the first protein to the second protein in the composition. In some embodiments, the ratio of the first protein to the 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 above ranges.

[0080] Once the ratio of the first protein to the second protein is determined, the method includes determining the total absorbance of the composition at a specific path length. In some embodiments, the method includes measuring the amount of light absorbed by the composition in the infrared, visible, or ultraviolet regions of the spectrum. For example, in some embodiments, the total absorbance of the composition can be quantified by measuring the UV absorbance at 280 nm. It should be understood that at a wavelength of 280 nm, the aromatic amino acids tryptophan (Trp) and tyrosine (Tyr) exhibit strong light absorption, and the cysteine ​​group (Cys-Cys) that forms disulfide bonds also exhibits a lower degree of absorption. Therefore, the absorption of proteins and peptides at 280 nm is proportional to the amount of these amino acids, and the total absorbance is proportional to the total protein content in the composition. As used herein, the term "path length" generally refers to the distance that light (e.g., UV or visible light) travels through the sample in the analytical cell. Path length is also proportional to absorbance because the longer the path length, the more protein molecules can be accommodated in the path of the light source.

[0081] The total absorbance of a composition at a specific path length can be determined using spectrophotometry with either a fixed or variable path length. Conventional spectrophotometric determinations are based on a fixed path length, which depends on the analytical cell (e.g., cuvette) used to contain the sample. In contrast, variable path length spectrophotometric systems (e.g., SOLOVPE®, Repligen Corp, Bridgewater, NJ) automatically adjust the optical path length from, for example, 0 mm to 15 mm in 5 μm increments. Variable path length spectrophotometers can determine the appropriate path length and linearity at sample concentrations significantly higher than those determined by fixed path length spectrophotometers. In some cases, unlike conventional fixed path length methods with variable concentrations, variable path length spectrophotometry uses the path length as a variable, thus allowing the concentration to remain constant. This eliminates the need for composition dilution and reduces the possibility of contamination.

[0082] By using the ratio of the first protein to the second protein and the total absorbance, Beer-Lambert's Law (or "Beer's Law") can be used to determine the individual concentrations of the first and second proteins in a composition. According to Beer-Lambert's Law, the absorbance of a solution is directly proportional to the concentration of the absorbing material present in the solution and the path length. Therefore, the concentration of a solution can be calculated by measuring its absorbance. Beer-Lambert's Law is typically expressed by the following formula:

[0083]

[0084] Where A is absorbance, ε is extinction coefficient, b is path length, and C is concentration. The linear relationship between absorbance, extinction coefficient, path length, and concentration provided by the Beer-Lambert law can be applied to compositions 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 as determined above. Therefore, in some embodiments, the concentrations of the first and second proteins can be calculated based on the ratio of total absorbance to path length and extinction coefficient, according to the ratio of the first protein to the second protein. For example, calculating the concentrations of the first and second proteins in the composition can include Equations 1 and 2:

[0085]

[0086] Where C2 is the concentration of the second protein, A 总 ε 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, k is the ratio of the first protein to the second protein, and C1 is the concentration of the first protein.

[0087] In other embodiments, the concentrations of the first and second proteins in the composition can be determined by generating calibration curves (often also referred to as standard curves or working curves). A “calibration curve” is a general method for determining the concentration of a substance in an unknown sample by comparing it to a set of standard samples of known concentrations. A standard curve is plotted or calculated using the response values ​​of the standards. The absorbance value of the unknown sample is then interpolated into a graph or formula of the standard curve to determine its concentration. When the calibration curve has sufficient linearity over a wide range of the quantification region, it can be prepared with a relatively small number of standard samples (approaching the upper and lower limits of the quantification range and the midpoint of that range). As described herein, when the composition contains two or more antigen-binding proteins, at least two calibration curves can be generated and used to quantify the amount of each antigen-binding protein detected in the disclosed method.

[0088] In some respects, the first protein and the second protein in a composition can be separated using chromatographic titration methods to directly measure protein concentration. For example, the first protein and the second protein can be separated using CEX, hydrophobic interaction chromatography (HIC), or reversed-phase high-performance liquid chromatography (RP-HPLC). The concentration of each protein can then be calculated based on calibration curves established for each protein and the peak area of ​​each protein.

[0089] Other orthogonal methods can be used to determine protein concentrations to cross-check or confirm results obtained through the disclosed methods. Such methods include, but are not limited to, turbidimetric assays, scattering assays, and colorimetric assays. In turbidimetric and scattering assays, proteins are quantified based on changes in the turbidity of the reaction mixture, according to the aggregation of the protein with its specific binding pair. In colorimetric assays, proteins can be quantified using a chromogenic reagent. Colorimetric assays are characterized by the formation, change, or reduction of color in the presence of the protein to be quantified. Exemplary colorimetric assays include Coomassie Blue G-250 dye binding (Bradford), dioctanic acid (BCA), and the Lowryassay.

[0090] In some methods, if the concentrations of the first and second proteins are each within a specified range (e.g., concentration specifications), the composition is further processed to manufacture a pharmaceutical composition suitable for medical use. For example, excipients may be added to the composition, or the composition may be placed in a container suitable for storage or a device suitable for administration (such as a syringe or pen).

[0091] Analysis of high molecular weight substances and charge variants

[0092] This disclosure also provides a chromatographic method for analyzing compositions comprising a first protein and a second protein different from the first protein, the method comprising providing a composition comprising the first protein and the second protein; and at least one of the following: a) determining the level of high molecular weight (HMW) substances of the first protein and / or the 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% isopropanol (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 of pH 5-6 and a second mobile phase of pH 10-11.

[0093] As used herein, the term "HMW substance" in relation to therapeutic proteins refers to an aggregate of two or more molecules (e.g., therapeutic proteins) formed by non-covalent bonds. HMW substances include, but are not limited to, dimers (containing two therapeutic proteins), trimers (containing three therapeutic proteins), tetramers (containing four therapeutic proteins), pentamers (containing five therapeutic proteins), hexamers (containing six therapeutic proteins), heptamers (containing seven therapeutic proteins), and octamers (containing eight therapeutic proteins). In an exemplary aspect, HMW substances can be more advanced, for example, they can contain more than eight therapeutic proteins. For example, the HMW substance can be a nonamer (containing nine therapeutic proteins), a decamer (containing 10 therapeutic proteins), an undecamer (containing 11 therapeutic proteins), a dodecamer (containing 12 therapeutic proteins), a tridecamer (containing 13 therapeutic proteins), a tetradecamer (containing 14 therapeutic proteins), a decamer (containing 15 therapeutic proteins), a hexadecameron (containing 16 therapeutic proteins), a heptadecameron (containing 17 therapeutic proteins), an octadecameron (containing 18 therapeutic proteins), or a nonademmer (containing 19 therapeutic proteins). In several embodiments, the HMW substance analyzed by the methods disclosed herein can contain one or more of the following: dimers, trimers, tetramers, pentamers, hexamers, heptamers, and octamers of therapeutic proteins.

[0094] In an exemplary aspect, the size of the HMW material analyzed by the method disclosed herein is less than about 0.1 micrometers (100 nm). Optionally, the size of the HMW material is about 99 nm or less. In an exemplary aspect, the size of the HMW material is greater than about 10 nm and less than about 99 nm. In an exemplary aspect, the size of the HMW material is greater than about 15 nm and less than about 99 nm. In an exemplary aspect, the size of the HMW material 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, or about 90 nm to about 99 nm. In exemplary cases, the size of the HMW material 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 several aspects, the size of the HMW material is less than about 15 nm. Optionally, the size of the HMW material is less than about 10 nm or less than about 5 nm.

[0095] The level of HMW substances can be determined by any suitable method known in the art. In some aspects, the level of HMW substances can be determined by size exclusion chromatography (SEC). In exemplary aspects, SEC is SEC-high performance liquid chromatography (SEC-HPLC), SEC-Fluor, or SEC-UV. It should be understood that SEC (also known as gel filtration, size exclusion, or gel chromatography) is a partition chromatography that separates molecules according to their molecular size. For example, SEC can separate monoclonal antibodies into three main substances: a high molecular weight substance, a main peak (primarily monomer), and a low molecular weight substance. Alternatively or alternatively, other techniques can be used to determine the level of HMW substances of the first and / or second proteins in the composition. For example, the method may include one or more of the following: mass spectrometry (MS) and / or SEC coupled with ultra-high performance liquid chromatography (UHPLC). It is believed that SE-UHPLC achieves more accurate size variant separation in a shorter time period than conventional SEC columns.

[0096] In some embodiments, the levels of high molecular weight (HMW) substances of the first and / or second proteins in the composition are determined by size exclusion ultra-high performance liquid chromatography (SE-UHPLC). SE-UHPLC can be performed under conditions optimized for the separation of the first and second proteins in the composition. For example, SE-UHPLC can 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 columns). The buffer composition can also be adjusted to optimize the quantification of HMW substances. Such components include, for example, salts, acidic and / or basic components (to achieve the desired pH), and organic solvents. Exemplary salts include NaCl or KCl, which may be included at concentrations of 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 include potassium phosphate (K3PO4; potassium dihydrogen phosphate or dipotassium hydrogen phosphate), sodium phosphate (NA3PO4), isopropanol (IPA), ammonium acetate, acetonitrile, and / or ammonium chloride. For example, SE-UHPLC can be performed using a buffer containing KCl, isopropanol, and K3PO4. In some embodiments, the buffer contains about 100-200 mM KCl, about 1%-5% isopropanol (IPA), and about 40-60 mM K3PO4.

[0097] Another SEC-UHPLC parameter that can be optimized to determine the HMW substance in the antibody co-prepared formulation is the flow rate of the sample fluid (i.e., the "mobile phase") through the SEC-UHPLC column. In some embodiments, the SE-UHPLC comprises a mobile phase with 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 flow rate of the mobile phase can be about 0.4 mL / min.

[0098] It should be understood that many post-translational modifications can induce changes in the charge distribution on monoclonal antibodies, which may potentially affect their biological activity. Indeed, monoclonal antibodies can undergo chemical degradation via several different mechanisms, including oxidation, deamidation, isomerization, and fragmentation, resulting in multiple charge variants and heterogeneity, thereby altering their isoelectric pH (pI) values ​​(Khwali et al., MAbs. [Monoclonal Antibodies], 2(6): 613-624 (2010)). Such charge variants are typically separated and characterized by charge-based separation techniques, such as isoelectric focusing (IEF) gel electrophoresis, capillary isoelectric focusing (cIEF) gel electrophoresis, cation exchange chromatography (CEX), and anion exchange chromatography (AEX). In some embodiments, chromatographic methods for analyzing compositions comprising a first protein and various second proteins include separating the charge variants of the first and / or second proteins in the composition by pH gradient cation exchange high-performance liquid chromatography (CEX-HPLC). As discussed in this paper, CEX-HPLC uses negatively charged ion-exchange resins that have an affinity for molecules with a net positive surface charge to separate proteins based on differences in protein surface charge.

[0099] CEX-HPLC can be performed under conditions optimized for the separation of charge variants of various proteins in a composition. For example, CEX-HPLC can be performed using any suitable cation exchange resin or column known in the art, such as the PROPAC™ column (ThermoFisher Scientific, Inc.), the BIOSUITE™ column (Waters Corporation), and the BIOPRO™ IEX column (YMC America, Inc.). (Inc.). An appropriate buffer system can be selected based on the first and second proteins in the co-prepared preparation. Retention and elution from the CEX column can be achieved by linear gradient elution or by using stepwise isocratic elution. In some embodiments, gradient elution is used. For example, a continuous salt (ionic strength) gradient or pH gradient results in highly fractionated protein separation based on protein charge. In salt gradient-based ion exchange chromatography (IEC), the pH of the buffer system is constant. Its ionic strength is kept low, except for selecting an appropriate pH for the starting buffer, because the affinity of proteins for IEC resin decreases with increasing ionic strength. Proteins are then eluted by increasing the ionic strength (salt concentration) of the buffer to increase competition between the buffer ions and the protein for charged groups on the IEC resin. Therefore, the interaction between the IEC resin and the protein decreases, resulting in protein elution. In pH gradient-based IEC, the pH of the starting buffer is maintained at a constant level to ensure that the protein acquires and binds to the stationary phase with a charge opposite to that of the stationary phase. Proteins are eluted by changing the pH of the buffer, causing the protein to become net-zero charged (ultimately the same charge as the resin) and elute from the column.

[0100] In an exemplary aspect, the disclosed method utilizes pH gradient-based CEX-HPLC to separate and elute charge variants of a first and second protein in a composition. In some embodiments, pH gradient-based CEX-HPLC includes the use of two buffers (or “mobile phases”) with different pH values. For example, CEX-HPLC comprises a first mobile phase or 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 contain a pH of about 5.6, while the second mobile phase may contain a pH of about 10.2. In some embodiments, CEX-HPLC comprises a column of at least 50 mm, 75 mm, or 100 mm. In other embodiments, CEX-HPLC comprises a gradient from 100% first mobile phase + 0% second mobile phase to no more than 20% first mobile phase + at least 80% second mobile phase over a period of at least 40, 50, 60 or 70 minutes (e.g. 45, 55 or 65 minutes).

[0101] In another exemplary aspect, the disclosed method can utilize a salt gradient with an optimal mobile phase pH. Without being bound by theory, a salt gradient with an optimal mobile phase pH can improve the separation of charge variants in co-formulated pharmaceutical products with similar pI values, resulting in minimal overlap of basic and acidic peaks. In such embodiments, the mobile phase or buffer pH is from about 5.0 to about 8.0 (e.g., pH 5.5, 6.0, 6.5, 7.0, or 7.5), such as from about 6.0 to 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 can be about 6.0, 6.5, 7.8, 7.0, or 7.5. Any suitable buffer can be used as the mobile phase. Buffer characteristics to consider include, for example, pKa, buffer type, cost, ease of use, and the buffer used in preceding 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 used in the disclosed methods is sodium phosphate.

[0102] Once the charge variants of the first and second proteins in the co-prepared article have been separated and eluted by CEX-HPLC, the method may further include determining the concentrations of the first and second proteins in the composition. The protein concentrations can be determined. Examples of suitable methods for determining protein concentrations are described herein. In some embodiments, the respective concentrations of the first and second proteins are determined based on calibration curves for each protein, as described herein.

[0103] Capillary electrophoresis

[0104] In other aspects of this disclosure, capillary electrophoresis (CE) can be used to determine the concentrations of a first protein and various second proteins in a composition. As used herein, the term "capillary electrophoresis" refers to a liquid-phase microseparation analysis technique that uses high-voltage direct current to separate ions based on their electrophoretic mobility in a capillary 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 can be used in conjunction 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, for example, Capillary Electrophoresis [capillary electrophoresis]; retrieved from chem.libretexts.org / @go / page / 294 (July 8, 2022); and Dawod et al., Analyst [analytes]. May 30, 2017; 142(11): 1847-1866. doi:10.1039 / c7an00198c).

[0105] In some embodiments, capillary gel electrophoresis (CGE; also known as “CGE-SDS” or “CE-SDS”) is used to determine the concentrations of a first protein and distinct second proteins. This capillary gel electrophoresis separates molecules based on differences in solute size as they migrate through a gel or matrix. Compared to conventional sodium dodecyl sulfate polyacrylamide plate gel electrophoresis (SDS-PAGE), CGE offers numerous advantages, including but not limited to rapid separation times, high recovery and regeneration capabilities, online detection, increased analytical throughput, and ease of operation (Zhu et al., Anal Chim Acta [Chinese Journal of Analytical Chemistry]. 2012;709:21-31; and Wu D, Regnier FE. Journal of Chromatography [Journal of Chromatography]. 1992;608:349-356). The CGE method also allows for more automated analysis at higher voltages, resulting in faster and more efficient separations. Regarding antibody analysis, CE-SDS can be used as an orthogonal and alternative technique to size exclusion chromatography (SEC) for the purity assessment of monoclonal antibodies and the analysis of molecular size variants.

[0106] 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 pachyman. CE-SDS can be performed under reducing (rCE-SDS) or non-reducing (nrCE-SDS) conditions. Under reducing conditions, the sample is incubated with a reducing agent (e.g., β-mercaptoethanol) to break inter- and intra-chain disulfide bonds. rCE-SDS is commonly used to obtain the relative percentages of the antibody light chain (LC), heavy chain (HC), and non-glycosylated heavy chain (NGHC), especially (LC + HC)% (as a measure of the purity of the sample analyzed under reducing conditions). nrCE-SDS can be used to analyze the purity of intact antibodies as well as product-related impurities such as fragments.

[0107] The separated molecules can be detected using a variety of methods, including UV absorbance, capacitively coupled non-contact conductivity, mass spectrometry (MS), and laser-induced fluorescence (LIF). A chromatogram can then be generated, graphically displaying the peaks formed as the separated components pass through the detector in real time. It should be understood that the area under the peak is considered a measure of the component concentration. When the sample contains multiple analytes or components, depending on the solute-stationary phase interactions and the flow characteristics of the mobile phase, various components may elute from CE-SDS at different retention times.

[0108] In embodiments where the first and second proteins are each antibodies (or other multi-chain antigen-binding proteins), CE-SDS can generate multiple peaks (e.g., heavy chain peaks and light chain peaks) for each protein. In such cases, the heavy and / or light chains of the two proteins may have similar retention times in the CE-SDS (referred to as "co-migration"), which can lead to overlapping peaks. In the case of overlapping peaks, the concentration of each antibody can be determined based on the non-overlapping peaks. Therefore, in some embodiments, CE-SDS generates a first peak for the first protein, a second peak for the second protein, and a co-migration peak for the first and second proteins, and the respective concentrations of the first and second proteins are determined based on the ratio of the first peak to the second peak. For example, if the light chain peak of the first antibody overlaps with the light chain peak of the second antibody, the concentration of each antibody in the composition can be determined using the ratio of the peak areas of the corresponding non-overlapping heavy chains. In other words, the selected peaks or groups of peaks can provide a reasonable estimate of the relative concentration of each protein in the co-formulated composition.

[0109] Therapeutic protein

[0110] In some embodiments, the first protein and the second protein are each a therapeutic protein. As used herein, “therapeutic protein” and variations of this root term have their common and conventional meaning as will be understood by one of ordinary skill in the art in light of this disclosure. It refers to a polypeptide intended for medical use in a subject, typically a human subject. For example, a therapeutic protein may be a polypeptide approved for medical use by a government regulatory agency, such as the U.S. Food and Drug Administration (FDA) or the European Medicines Agency (EMA). In some embodiments, the first protein and the second protein are each a therapeutic antigen-binding protein, such as a therapeutic antibody.

[0111] The therapeutic antigen-binding proteins (such as antibodies) covered by this disclosure may include peptides 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 EGF receptors; (iii) cell adhesion molecules, such as LFA-I, MoI, p150, 95, VLA-4, ICAM-I, VCAM, and αv / β3 integrins; (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, and Müllerian-inhibiting substances. (v) Insulin and insulin-related proteins, including insulin, insulin A chain, insulin B chain, proinsulin, and insulin-like growth factor binding protein; (vi) Insulin and insulin-related proteins, including insulin, insulin A chain, insulin B chain, proinsulin, and insulin-like growth factor binding protein; Coagulation proteins and coagulation-related proteins, especially factor VIII, tissue factor, von Willebrand factor, protein C, α-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 their receptors, especially including M-CSF, GM-CSF, and G-CSF, and their receptors, such as CSF-1 receptor (c-fms); (ix) Receptors and receptor-associated proteins, including, for example, flk2 / flt3 receptors, CD112 receptor (CD112R), obesity (OB) receptor, LDL receptor, growth hormone receptor, thrombopoietin receptor (“TPO-R”, “c-mpl”), glucagon receptor, interleukin receptor, interferon receptor, T cell receptor, stem cell factor receptor (e.g., c-Kit) and other receptors; (x) receptor ligands, including, for example, OX40L (ligand of OX40 receptor);(xi) Neurotrophic factors, including bone-derived neurotrophic factor (BDNF) and neurotrophin-3, neurotrophin-4, neurotrophin-5, or neurotrophin-6 (NT-3, NT-4, NT-5, or NT-6); (xii) relaxin A chain, relaxin B chain, and pro-relaxin; (xiii) interferons and interferon receptors, including, for example, interferon-α, interferon-β, and interferon-γ, and their receptors; (xiv) interleukins and interleukin receptors, particularly IL-1 to IL-33 and IL-1 to IL-33 receptors, such as IL-8 receptors; (xv) viral antigens, including AIDS enveloped virus antigens; (xvi) Other proteins, such as lipoproteins, calcitonin, glucagon, atrial diuretic natriuretic factor, pulmonary surfactant, tumor necrosis factor-α and tumor necrosis factor-β, enkephalin, programmed cell death 1 (PD-1), programmed cell death ligand 1 (PD-L1), T cell immune receptor with Ig and ITIM domains (TIGIT), RANTES (which regulate the expression of secretory factors activated by normal T cells), mouse gonadotropin-related peptide, DNase, inhibin, activin, integrin, protein A or D, rheumatoid factor, immunotoxins, bone morphogenetic protein (BMP), and super... Superoxide dismutase, surface membrane proteins, decay accelerator factor (DAF), HIV envelope, transport proteins, homing receptors, addressins, regulatory proteins, immunoadhesins, myoinhibitory proteins, TALL proteins (including TALL-I), amyloid proteins (including but not limited to β-amyloid), thymic stromal lymphopoietin (“TSLP”), RANK ligands (“RANKL” or “OPGL”), c-kit, TNF receptors (including type 1 TNF receptors), TRAIL-R2, angiopoietin, and any biologically active fragments or analogues or variants of the foregoing.

[0112] Examples of therapeutic antibodies suitable for the methods described above include infliximab, bevacizumab, cetuximab, ranibizumab, palizumab, abavoximab, abciximab, actosulamab, adalimumab, afimomab, avtocilizumab, araciizumab, pegoa-araciizumab, ald518, alenmab, alikumab, atormomab, and anatumomab. Mafenatox, Amluzometab, Apizumab, Asimumab, Aserizumab, Altinumab, Atlizumab, Atorlimumab, Tocilizumab, Bapicillin, Baliximab, Baviximab, Betomo, Belimumab, Bemaritocilizumab, Benaliximab, Bertilimab, Besoxumab, Bevacizumab, Belottosumab, Bisimab, Bivatuzumab (Bivutuzumab) Mertansine, bonatomumab, butozymec, brentuximab, brenumab, brodatumab, cannaginumab, mocantuzumab, mertansine, mocantuzumab, carracizumab, capromabPendetide, Carruzumab, Caputoxumab, CC49, Cedelizumab, Sertolizumab, Cetuximab, Pocitazone, Cetuximab, Krazazumab, Crizotinib, Clivatuzumab, Tetraxetan, Kronumab, Krejuvenumab, CR6261, Darcyzone, Daciumib, Darotoxzone, Darelimumab, Desaizumab, Desaizumab, Desaizumab, Desaizumab, Desaizumab, Desaizumab Triggetuzumab, Duligotuzumab, Dupilumab, Emexici, Iculizumab, Ebazumab, Ezocurumab, Fallizumab, Efengumab, Elotuzumab, Erotozumab, Esimerizumab, Enartoxumab, Pegrelimorizumab, Enoxacumab, Enoxuzumab, Entoxici, Ciepimorizumab, Ipatizumab, Erenurumab, Erizumab, Erlinumab, Erituximab, Idazumab, Etralizumab, Evolomab, Avirazumab, Fanoles OMAB, Falamomumab, Fatozumab, Fasinumab, FBTA05, Flavizumab, Fizanumbumab, Flavizumab, Fentuximab, Frantuximab, Frantuximab, Forlanumab, Foravirimab, Femtozumab, Forlanumab, Vortoximab, Garliximab, Gantrub, Garliximab, Gejutuzumab, Ozolmicin, Gejutuzumab, Gejutuzumab, Vitingagrentoximab, Golimumab, Golimumab (gomiliximab), GS662 4. Ibalizumab, Imomumab, Irukumab, Igovomarab, Incemarab, Imtracumab, Inxiocumab, Reintoxicumab, Inflixicumab, Intolimab, Innomumab, Oga-Itozumab, Ipilimumab, Itolimumab, Ilizumab, Ixizumab, Keliximab, Labetizumab, Lejinzumab, Lemasomumab, Ledemarab, Lesamumab, Riviremab, Ligizumab, Lintozumab, Lirerutab, Lovotozumab (Lorvotuzumab) Mertansine, Lucarumumab, Lucizimab, Mapamumumab, Massomumab, Mafulimumab, Matozumab, Meperizumab, Meperizumab, Milazumab, Minremumab, Mitomumab, Moglizazumab, Moromumab, Movizumab, Pasutomumab, Moromumab-CD3, Tanacomab, Namerumab, Eto-Naptumomab (estafenatox), Nanatuzumab, Natazumab, Nebakumab, Nexitozumab, Neremumab, Nevasumab, Nitozumab, Navonab, Nofetumomab-MerpentanMerpentan, Ocalatuzumab, Ocalatuzumab, Ocaltumab, Ocalatuzumab, Ocalatuzumab, Ocalatuzumab, Ocalatuzumab, Ocalatuzumab, Ocalatuzumab, Ocalatuzumab, Ocalatuzumab, Ocalatuzumab, Ocalatuzumab, Ocalatuzumab, Ocalatuzumab, Ocalatuzumab, Ocalatuzumab, Ocalatuzumab, Ocalatuzumab, Pajixici, Palizumab, Pabucubitzumab, Pasatuzumab, Pacozumab, Pattizumab, Patratuzumab, Pemtumomab, Pelacaizumab, Pertuzumab, Pecalatuzumab, Pidtizumab, Pintomorab, Pralucumab, Ponaizumab, Priliximab, Putopilumab, PRO 140. Quinolizumab, Raltomumab, Ratrastuzumab, Raviolizumab, Ramucirumab, Ranibizumab, Raxicurumab, Regavir, Relizumab, Rituximab, Rituximab, Rostrumab, Roledoumab, Romosuzumab, Longlizumab, Roveizumab, Lulizumab, Shamazoliumab, Saliruzumab, Satumomab, Pendivitide, Secukinumab, Sevirumab, Sirozizumab, Siflomumab, Sifamumab, Secuximab, Simutuzumab, Siplizumab, Sirukumab, Sorapizumab, Solitoumab, Sonepizumab, Sonetizumab, Staluronumab, Thioxalumab, Suvetuzumab, Tabelucirumab, Titanituzumab Tetraxetan, Tadalafil, Talizumab, Talizumab, Taplitumomab Paptox, Talataumab, Terfenadine, Atemolimumab, Tetomolimumab, Terfenadine, Teneriximab, Tetomolimumab, Tetomolimumab, Tzerulimumab, TGN1412, Trimelimumab, Tetomolimumab, Tetomolimumab, Tetomolimumab, TNX-650, Tocilizumab, Tolizumab, Tosimolimumab, Trastuzumab, TRBS07, Trastuzumab The following are variants of the following: tucotuzumab celmoleukin, tuvirumab, ublituximab, urinumab, utzinumab, ustekinumab, varricizumab, varricizumab, vedalizumab, vetozumab, vepamozumab, vesenokumab, vexizumab, voloxizumab, vorsetuzumab mafodotin, vortozumab, zatuximab, zamumab, zatuximab, ziralimumab, zolimomab aritox, or any of the foregoing.

[0113] According to the methods described herein, the composition may comprise two or more of any of the aforementioned therapeutic antibodies or their antigen-binding fragments. Exemplary combinations of antibodies co-formulated in the composition that can be used for analysis as described herein include, but are not limited to, an antibody that specifically binds 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., talatumab), 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. However, this disclosure is not limited to these specific combinations of antigen-binding proteins.

[0114] Samples and compositions

[0115] The term "sample" and variations thereof have their common and conventional meaning as will be understood by one of ordinary skill in the art in light of this disclosure. It refers to a composition that may contain two or more antigen-binding proteins as described herein, such as an in vitro or synthetic sample obtained from the manufacture of antigen-binding proteins. For example, a sample may be a composition or formulation comprising a first protein and a second protein, and at least one pharmaceutically acceptable carrier (also referred to herein as a "pharmaceutical composition"). For example, a sample may be derived from a sample produced during the manufacture of a pharmaceutical composition.

[0116] Acceptable compositions or formulation materials for proteins (e.g., therapeutic antibodies) as described herein are preferably non-toxic to the recipient at the doses and concentrations used. In some embodiments, the pharmaceutical composition may contain formulation materials for altering, maintaining, or preserving, for example, the composition's pH, osmotic pressure, viscosity, clarity, color, isotonicity, odor, sterility, stability, dissolution or release rate, adsorption, or penetration. In such embodiments, suitable formulation materials include, but are not limited to, amino acids (such as glycine, glutamine, asparagine, arginine, or lysine); antimicrobial agents; antioxidants (such as ascorbic acid, sodium sulfite, or sodium bisulfite); buffers (such as borates, bicarbonates, Tris-HCl, citrates, phosphates, or other organic acids); leavening agents (such as mannitol or glycine); chelating agents (such as ethylenediaminetetraacetic acid (EDTA)); complexing agents (such as caffeine, polyvinylpyrrolidone, β-cyclodextrin, or hydroxypropyl-β-cyclodextrin); fillers; monosaccharides; disaccharides; and other carbohydrates (such as glucose, sucrose, mannose, or dextrin); proteins (such as serum albumin, gelatin, or immunoglobulins); colorants, flavorings, and diluents; emulsifiers; hydrophilic polymers (such as polyvinylpyrrolidone); low molecular weight peptides; and other formulation materials. Salt counterions (e.g., sodium); preservatives (e.g., benzalkonium chloride, benzoic acid, salicylic acid, thimerosal, phenethyl alcohol, methylparaben, propylparaben, chlorhexidine, sorbic acid, or hydrogen peroxide); solvents (e.g., glycerol, propylene glycol, or polyethylene glycol); sugar alcohols (e.g., mannitol or sorbitol); suspending agents; surfactants or wetting agents (e.g., pluronics, PEG, dehydrated sorbitol esters, polysorbate esters (e.g., polysorbate 20, polysorbate esters), triton, tromethamine, lecithin, cholesterol, tyloxacin); stability enhancers (e.g., sucrose or sorbitol); tension enhancers (e.g., alkali metal halides (preferably sodium chloride or potassium chloride), mannitol, sorbitol); delivery media; diluents; excipients and / or pharmaceutical adjuvants. See, for example, Remington, The Science and Practice of Pharmacy, 23rd edition, Academic Press (2020).

[0117] Suitable mediators or carriers for the composition may be water for injection, physiological saline solution, or artificial cerebrospinal fluid, possibly supplemented with other materials commonly found in compositions for parenteral administration. Neutral buffered saline or saline mixed with serum albumin are other exemplary mediators.

[0118] In some embodiments, a buffer solution is used to maintain the composition at a physiological pH or slightly lower, typically in the pH range of about 5 to about 8. For example, the pH of the composition can be about 5.1, about 5.2, about 5.3, about 5.4, about 5.5, about 5.6, about 5.7, about 5.8, about 5.9, about 6.0, about 6.1, about 6.2, about 6.3, about 6.4, about 6.5, about 6.6, about 6.7, about 6.8, about 6.9, about 7.0, about 7.1, about 7.2, about 7.3, about 7.4, about 7.5, about 7.6, about 7.7, about 7.8, about 7.9, or about 8.0, including the range 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.

[0119] The following examples further illustrate the invention, but should not be construed as limiting its scope in any way.

[0120] Example 1

[0121] This example describes a method for monitoring high molecular weight substances in co-formulated protein therapeutic agents.

[0122] Size exclusion ultra-high performance liquid chromatography (SE-UHPLC) was developed to monitor total high molecular weight (HMW) substances, and cation exchange high performance liquid chromatography (CEX-HPLC) was developed to separate single charge variants in a co-formulated monoclonal antibody mixture containing antibody 1 (“Ab1”) and antibody 2 (“Ab2”).

[0123] High molecular weight molecular weight (HMW) is a critical product quality attribute for monitoring and control due to its potential impact on efficacy and safety. Assessing HMW in certain co-formulated antibody drugs using platform-based SE-UHPLC methods is challenging because peak overlap can interfere with the quantification of individual HMW substances. Therefore, an SE-UHPLC method was developed for the quantification of total HMW in co-formulated Ab1 / Ab2 drugs. This method monitors all HMWs observed individually in both Ab1 and Ab2 drugs within the co-formulated drug. To optimize the SE-UHPLC method for the quantification of HMW in co-formulated drugs, emphasis was placed on minimizing secondary interactions to allow all HMW substances to elute within similar elution windows without monomer interference. The parameters evaluated during the development of this method are summarized in Table 1, and the optimized SE-UHPLC conditions are summarized in Table 2. Figure 1 The results of HMW analysis of the Ab1 / Ab2 co-formulation using the optimized SE-UHPLC method are shown.

[0124] Table 1. Method parameters evaluated for optimizing the SE-UHPLC method for co-formulated products.

[0125]

[0126] Table 2. Optimized SE-UHPLC method conditions for co-formulated products

[0127]

[0128] Example 2

[0129] This example describes a method for isolating a single charge variant in a co-formulated protein therapeutic agent.

[0130] A cation exchange high-performance liquid chromatography (CEX-HPLC) method was developed to separate single charge variants in a mixture of co-formulated monoclonal antibodies Ab1 and Ab2.

[0131] Monitoring charge variants during the production of protein therapeutics is crucial because chemical modifications (such as deamidation) can affect product quality. Assessing charge variants in certain co-formulated pharmaceuticals using a platform pH gradient CEX-HPLC method is challenging because the basic and acidic peaks of various molecules overlap, which can interfere with quantification of charge variants using conventional methods. Furthermore, stress conditions can pose additional challenges to monitoring charge variants in the basic and acidic peaks of co-formulated antibody mixtures.

[0132] CEX-HPLC methods based on pH gradients and salt gradients were developed and optimized for the separation and quantification of charge variants in Ab1 / Ab2 co-formulated pharmaceutical products. This method maintains and monitors all charge variants in the co-formulated pharmaceutical products that are individually observed in the Ab1 and Ab2 active pharmaceutical ingredients.

[0133] To optimize the CEX method for quantifying charge variants in co-formulated pharmaceutical products, the focus was on improving the separation of overlapping basic and acidic peaks from Ab1 and Ab2. Both pH gradient CEX-HPLC and salt gradient CEX-HPLC methods were evaluated. The parameters evaluated during method development are summarized in Tables 3 and 4.

[0134] Table 3. Method parameters evaluated for optimizing the pH gradient CEX-HPLC method

[0135]

[0136] Table 4. Method parameters evaluated for optimizing the salt gradient CEX-HPLC method

[0137]

[0138] The final parameters and conditions selected for the salt gradient CEX-HPLC method are shown in Table 5. The results of the analysis of the Ab1 / Ab2 co-formulation using the developed CEX-HPLC method are shown in... Figure 2 middle.

[0139] Table 5. CEX-HPLC method conditions developed for co-formulated products

[0140]

[0141] Example 3

[0142] This example describes a method for determining the concentrations of a first protein and a second protein different from the first protein in a composition.

[0143] A strategy for measuring the concentrations of two different proteins in a co-prepared mixture was developed based on UV absorbance determination. The general strategy involves determining the ratio (k) of Ab1 to Ab2 in the composition using cation exchange chromatography (CEX) under the conditions described in Table 3. The total absorbance of the composition is then determined using a variable path length spectrophotometric system (e.g., SOLOVPE®, Ripleykin, Bridgewater, NJ) or a fixed path length method. The concentrations of the first and second proteins are then calculated using Beer's Law.

[0144]

[0145] Where C1 is the concentration of the first protein, C2 is the concentration of the second protein, and A 总 ε 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.

[0146] Ab1 and Ab2 were co-prepared in ratios of 1:1, 1:2, 1:3, 2:1, or 3:1 (Ab1:Ab2). The Ab1:Ab2 ratio (k) in the co-prepared article was determined by CEX, and A was measured using the extinction coefficients of Ab1 and Ab2, which were 1.49 and 1.41, respectively. 总 :

[0147]

[0148] .

[0149] The experimental ratio of Ab1:Ab2 under non-stress (To) and stress (four weeks at 40°C) conditions was compared with the theoretical ratio (based on the initial mixture). The results of this analysis show that... Figure 3A and Figure 3BIn both To and stress conditions, a positive linear correlation (R0) was observed between the measured experimental ratio and the theoretical ratio of the Ab1 / Ab2 co-formulated product. 2 (Approximately > 0.99).

[0150] Then, Ab1 was co-prepared with the third monoclonal antibody (“Ab3”) at ratios of 1:1, 1:2, 1:3, 2:1, or 3:1 (Ab1:Ab3). The experimental concentrations of Ab1 and Ab3 under non-stress (T0) and stress (four weeks at 40°C) conditions were measured using Beer's Law as described above. The experimental ratio of Ab1:Ab3 in the co-prepared formulations under the test conditions was compared with the theoretical ratio (based on the initial mixing). The results of this analysis showed… Figure 4A and Figure 4B In both T0 and stress conditions, a positive linear correlation (R0) was observed between the measured experimental ratio and the theoretical ratio of the Ab1 / Ab3 co-formulated product. 2 (Approximately > 0.99).

[0151] Ab2 was co-prepared with the fourth monoclonal antibody (“Ab4”) at ratios 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 (Ab2:Ab4). Ab1 was also co-prepared with Ab4 at ratios of 1:1, 1:2, 1:3, 1:10, 1:20, 2:1, 3:1, 10:1, and 20:1. The experimental concentrations of Ab2 and Ab4, as well as the experimental concentrations of Ab1 and Ab4, were measured using Beer’s Law under non-stress (T0) and stress (four weeks at 40°C) conditions, as described above. The experimental ratios of Ab2:Ab4 and Ab1:Ab4 in various co-prepared products under test conditions were compared with the theoretical ratios (based on initial mixing). The results of this analysis show... Figure 5A , Figure 5B , Figure 6A and Figure 6B middle.

[0152] Alternative strategies for direct concentration measurement involving Ab1 / Ab2 and Ab1 / Ab3 co-formulations were also tested using RP-HPLC-based titer or HIC methods. Results of RP-HPLC analysis showed... Figure 7A and Figure 7B In, and the results of HIC analysis showed Figure 8 middle.

[0153] Example 4

[0154] This example describes the characterization and release / stability testing of a composition containing two different antibodies using a variety of analytical methods.

[0155] Monoclonal antibodies Ab4 and “Ab5” were analyzed individually and as co-formulated mixtures using SE-UPLC, direct protein concentration, reduced and non-reduced capillary gel electrophoresis (rCE-SDS and nrCE-SDS), and CEX-HPLC.

[0156] The property levels (total HMW%) of the co-prepared mixtures, measured by SE-UHPLC, were comparable to the expected values ​​(Table 6). Figure 9A and Figure 9B As shown in Table 7, the total protein concentration measured by SOLOVPE® was also in line with expectations.

[0157] Table 6

[0158]

[0159] Table 7

[0160]

[0161] For the rCE-SDS and nrCE-SDS spectra of the co-prepared mixture, partial co-migration of each antibody was observed, as shown below. Figure 10 and Figure 12 As shown. A selected set of peaks provides a good estimate of the mixing ratio of proteins in the co-prepared formulation. Ab4 and Ab5 were also mixed at different ratios, and the concentration of each protein was quantified using rCE-SDS, with results shown in... Figure 11 And as shown in Table 8. The CEX-HPLC method was also applied to different co-formulations of Ab4 and Ab5, and it was able to monitor the stability of the co-formulations even in cases of co-migration (see Table 8). Figure 13 ).

[0162] Table 8

[0163]

[0164] All references cited in this article (including publications, patent applications and patents) are hereby incorporated by reference as if each reference were individually and explicitly indicated to be incorporated by reference and presented in its entirety in this article.

[0165] As used herein, the term “about” when used as a modifier of a specified value (e.g., “about” pH 7.0) indicates the variation that can occur around that value. These variations can occur in a variety of ways, such as typical measurement and handling procedures, errors due to negligence, component purity, etc. In some embodiments, where higher numerical accuracy is required, “about” may refer to a value within ± 5% of the specified value.

[0166] Unless otherwise indicated herein or clearly contradicted by the context, in the context of describing the invention (especially in the context of the appended claims), the terms "a / an" and "the," "at least one / a," and similar designations shall be construed as encompassing both the singular and plural. Unless otherwise indicated herein or clearly contradicted by the context, the use of the term "at least one" followed by a list of one or more items (e.g., "at least one of A and B") shall be construed as meaning one item selected from the list (A or B), or any combination of two or more of the list (A and B). Unless otherwise noted, the terms "comprising," "having," "including," and "containing" shall be understood as open-ended terms (i.e., meaning "including but not limited to"). Unless otherwise indicated herein, statements of ranges of values ​​herein are intended only as a way of individually referring to each independent value within that range, and each independent value is incorporated into the specification as if it were individually stated herein. Unless otherwise indicated herein or clearly contradicted by the context, all methods described herein may be performed in any suitable order. Unless otherwise claimed, the use of any and all instances or exemplary language (e.g., "such") provided herein is intended only to better describe the invention and not to limit its scope. No language in this specification should be construed as indicating that any non-claimed element is essential to the practice of the invention.

[0167] This document describes preferred embodiments of the invention, including the best modes known to the inventors for carrying out the invention. Variations of those preferred embodiments will become apparent to those skilled in the art upon reading the above description. The inventors expect those skilled in the art to adopt such variations as appropriate, and the inventors intend that the invention be practiced in a manner different from that specifically described herein. Therefore, the invention includes all modifications and equivalents to the subject matter set forth in the appended claims as permitted by applicable law. Furthermore, unless otherwise indicated herein or otherwise clearly contradicted by the context, the invention covers any combination of the foregoing elements in all their possible variations.

Claims

1. A method for determining the concentrations of a first protein and a second protein different from the first protein in a composition, the method comprising: The composition comprising the first protein and the second protein is provided, wherein the first protein has a first extinction coefficient and the second protein has a second extinction coefficient; The first protein and the second protein in the composition are separated by cation exchange chromatography (CEX), thereby obtaining the ratio of the first protein to the second protein in the composition; The total absorbance of the composition was measured over a given path length; and Based on the ratio of the total absorbance to the path length and the extinction coefficient, the concentrations of the first protein and the second protein are calculated according to the ratio of the first protein to the second protein in the composition.

2. The method of claim 1, wherein the calculation comprises Equation 1: where C2 is the concentration of the second protein, A 总 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 the calculation further comprises Equation 2: ， Where C1 is the concentration of the first protein, and k is the ratio of the first protein to the second protein in the composition.

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

2.

6. The method of any one of claims 1-5, further comprising: The first protein and the second protein in the composition were separated by CEX, hydrophobic interaction chromatography (HIC), or reversed-phase high-performance liquid chromatography (RP-HPLC). The concentrations of the first and second proteins were determined based on the calibration curves for each protein.

7. A chromatographic method for analyzing a composition comprising a first protein and a second protein different from the first protein, the method comprising: The composition comprising the first protein and the second protein is provided; and at least one of the following: a) Determine the level of high molecular weight (HMW) substances of the first protein and / or the second protein in the composition by size exclusion ultra-high performance liquid chromatography (SE-UHPLC), wherein the SE-UHPLC is performed with a buffer containing about 100-200 mM KCl, about 1%-5% isopropanol (IPA) and about 40-60 mM K3PO4; or b) Separate the 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 contains a first mobile phase of pH 5-6 and a second mobile phase of pH 10-11.

8. The method of claim 7, wherein the HMW substances comprise one or more of dimers, trimers, tetramers, pentamers, hexamers, heptamers, and octamers.

9. The method of claim 7 or claim 8, wherein the SE-UHPLC is performed using a buffer containing 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 with a flow rate of about 0.2-0.5 mL / min.

11. The method of claim 10, wherein the flow rate of the mobile phase 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 having a pH of about 5.

6.

13. The method of any one of claims 7-12, wherein the CEX-HPLC comprises a second mobile phase having a 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 a period of at least 40, 50, 60 or 70 minutes.

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

16. The method of any one of claims 7-15, further comprising determining the concentrations of the first protein and the second protein in the composition based on calibration curves for each protein after separating the charge variant by CEX-HPLC.

17. A capillary electrophoresis (CE) method for determining the concentrations of a first protein and a second protein in a composition, wherein the first protein is different from the second protein, the method comprising: The composition comprising the first protein and the second protein was analyzed by capillary electrophoresis SDS (CE-SDS), wherein the CE-SDS contained 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 concentrations of the first protein and the second protein are determined based on the ratio of the first peak to the second peak.

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

19. The method of any one of claims 1-18, wherein the first protein and the second protein are each 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 binding agent (BiTE®) molecule.

21. The method of claim 20, wherein the first protein and the second protein are each 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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