Purification of multispecific antibodies

Abstract

The present disclosure provides methods for purifying multispecific antibodies from mismatched variants by performing multimodal chromatography.

Description

[Technical Field]

[0001] Cross-reference of related applications This application claims priority to U.S. Provisional Patent Application No. 63 / 080,950, filed on 21 September 2020, which is incorporated in its entirety by reference, and against which priority is claimed.

[0002] A method is provided for purifying a multispecific antibody from a composition comprising the multispecific antibody and at least one impurity containing at least one product-specific impurity. In some embodiments, the product-specific impurity is, for example, a mispairing variant of the multispecific antibody. Also provided are the multispecific antibody purified according to this method, as well as compositions and formulations containing such a multispecific antibody. [Background technology]

[0003] For recombinant biopharmaceutical proteins acceptable for administration to human patients, it is crucial that residual impurities resulting from the manufacturing and purification processes are removed from the final biological product. These process components include culture medium proteins, immunoglobulin affinity ligands, viruses, endotoxins, DNA, and host cell proteins. The development of new antibody formats, such as multispecific antibodies, presents new challenges because conventional manufacturing and purification processes cannot adequately remove product-specific impurities, including unpaired antibody arms and misassembled antibodies.

[0004] Compared to the purification of standard antibodies, the purification of multispecific antibodies from production media presents unique challenges. While standard monospecific bivalent antibodies arise from the dimerization of identical heavy / light chain subunits, generating multispecific antibodies requires the dimerization of at least two heavy / light chain subunits, each containing a different heavy chain and a different light chain. Ultimately, generating and purifying correctly complete multispecific antibodies with minimal mispaired, misassembled, or incomplete molecules presents various challenges. Chain mispairing (e.g., homodimerization of identical heavy chain peptides, or improper association of heavy / light chains) is frequently observed. Commonly observed product-specific impurities include half (1 / 2) antibodies (containing a single heavy / light chain pair), three-quarter (3 / 4) antibodies (containing a complete antibody lacking a single light chain), and homodimers. Depending on the multispecificity format used, additional product-specific impurities may be observed. For example, if one variable domain of a multispecific antibody is constructed as a single-chain Fab (scFab), a 5 / 4 antibody byproduct (containing an additional heavy or light chain variable domain) may be observed. Such corresponding product-specific impurities would not occur in standard antibody production.

[0005] Conventional purification techniques, designed to remove process-related impurities such as HCP, DNA, endotoxins, and other materials with properties and characteristics entirely different from antibodies, may be insufficient when used to remove impurities that are more closely related to multispecific antibodies. Therefore, there is a need to develop manufacturing and purification schemes that effectively remove product-specific impurities and light-chain mispairing antibodies, thereby obtaining sufficient quantities of correct and complete multispecific antibodies.

[0006] All references cited herein, including patent applications and publications, are incorporated in their entirety by reference for any purpose. [Overview of the project]

[0007] This disclosure relates to a method for purifying a multispecific antibody, a) contacting a composition comprising a multispecific antibody and a mispair variant thereof with a multimode chromatography material under conditions in which the mispair variant preferentially binds to the multimode chromatography material compared to the multispecific antibody, wherein the multispecific antibody comprises a first antigen-binding region that specifically binds to a first antigen and includes the light and heavy chains of the antibody that binds to the first antigen, and a second antigen-binding region that specifically binds to a second antigen and includes the light and heavy chains of the antibody that binds to the second antigen, wherein the variable domains VL and VH are substituted for each other in the second antigen-binding region; The present invention provides a method comprising: a) a) contacting a multimode chromatography material with a composition comprising a multimode chromatography material comprising an anion-exchangeable functional group and a hydrophobic-interacting functional group; b) recovering an elute containing a multispecific antibody and a reduced amount thereof; b) recovering an elute containing a multispecific antibody and a reduced amount thereof; b) recovering an elute containing a multispecific antibody and a reduced amount thereof; b) recovering an elute containing a multispecific antibody and a reduced amount thereof; b) recovering an elute containing a multispecific antibody and a reduced amount thereof; b) a multimode chromatography material comprising a first antigen-binding region comprising a peptide comprising a heavy chain of an antibody that binds to a first antigen and a heavy chain variable domain (VH) and a light chain constant domain (CL) of an antibody that binds to a second antigen; b) recovering a multispecific antibody and a hydrophobic-interacting variant; b) recovering an elute containing a multispecific antibody and a reduced amount thereof; b) recovering a multispecific elute containing a multispecific elute containing a first antigen-binding region comprising a heavy chain of an antibody that binds to a first antigen and a peptide comprising a heavy chain variable domain (VH) and a light chain constant domain (CL) of an antibody that binds to a second antigen; b) recovering a multispecific elute containing-interacting variant; b) recovering a multispecific elute containing a multispecific antibody and a reduced amount thereof; b) recovering a multispecific elute containing a multispecific elute containing a multispecific elute containing a multispecific-interacting variant; b) recovering a multispecific elute containing a multispecific elute containing a multispecific-interacting variant; b) recovering a multispecific elute containing a multispecific elute

[0008] In certain embodiments, the hydrophobic interacting functional group includes alkyl groups, alkenyl groups, alkynyl groups, phenyl groups, benzyl groups, or any combination thereof. In certain embodiments, the functional group includes a benzyl group. In certain embodiments, the anion exchangeable functional group includes a positively charged group. In certain embodiments, the positively charged group is a quaternary ammonium ion. In certain embodiments, the multimode chromatography material includes N-benzyl-N-methylethanolamine. In certain embodiments, the multimode chromatography material is Capto TM Contains adhesive resin. In certain embodiments, the multimode chromatography material is Capto TMIt contains Adhere ImpRes resin.

[0009] In certain embodiments, elution in multimode chromatography is gradient elution. In certain embodiments, gradient elution includes a pH gradient.

[0010] In certain embodiments, the method includes a capture chromatography step. In certain embodiments, the capture chromatography step is an affinity chromatography step. In certain embodiments, the affinity chromatography step is a Protein A chromatography step, a Protein L chromatography step, a Protein G chromatography step, and a Protein A / G chromatography step. In certain embodiments, the affinity chromatography step is a Protein A chromatography step. In certain embodiments, the Protein A chromatography material includes Protein A bound to agarose.

[0011] In certain embodiments, the capture chromatography step and the multimode chromatography step are consecutive. In certain embodiments, the method further includes a purification step after multimode chromatography. In certain embodiments, the method includes concentration of the multispecific antibody.

[0012] In certain embodiments, the multispecific antibody includes a knob-in-hole modification.

[0013] In certain embodiments, the multispecific antibody and its mispaired variants are produced in the same host cell. In certain embodiments, the host cell is a prokaryotic cell or a eukaryotic cell. In certain embodiments, the host cell is a eukaryotic cell. In certain embodiments, the eukaryotic cell is a yeast cell, an insect cell, or a mammalian cell. In certain embodiments, the eukaryotic cell is a CHO cell.

[0014] The present disclosure provides a composition comprising a multispecific antibody purified by the method disclosed herein. In certain embodiments, the composition further includes a pharmaceutically acceptable carrier.

[0015] The present disclosure provides a manufactured article comprising a multispecific antibody purified by the methods disclosed herein. The present disclosure also provides a manufactured article comprising the compositions disclosed herein.

Brief Description of the Drawings

[0016] [Figure 1A-1B] A schematic diagram of a method for producing a multispecific antibody is shown. FIG. 1A shows an overview of the production of a multispecific antibody by using a two-cell approach. FIG. 1B shows an overview of the production of a multispecific antibody by using a single-cell approach. [Figure 2A-2B] Represents variants of a multispecific antibody. FIG. 2A shows a schematic representation of different covalent dimers and light chain mispairing variants. FIG. 2B shows a correctly formed bispecific antibody (left figure) and a crossed light chain mispairing variant (right figure). [Figure 3] A contour plot showing that a common crossed LC mispairing variant binds well to the resin under conditions where the binding of the bispecific antibody is minimized is shown. [Figure 4] A chromatogram showing pH, UV absorbance, elution mixing gradient, and conductivity is shown. [Figure 5] Mass spectrometry data comparing the loaded raw material composition with fractions representing the multispecific antibody and LC mispairing variants is shown. [Figure 6A] A pseudo-chromatogram showing the composition and concentration of the recovered and measured fractions is shown. It is shown that the main peak mainly contains the bispecific antibody. [Figure 6B] A pseudo-chromatogram showing the composition and concentration of the recovered and measured fractions is shown. A normalized pseudo-chromatogram of the bispecific and LC mispairing variants is shown. [Figure 7] An in silico structural analysis of the correct pair of multispecific antibody and LC mispairing variant is shown.

Modes for Carrying Out the Invention

[0017] This disclosure is based, at least in part, on the finding that mispairing variants of multispecific antibodies produced by the same cell can be removed by performing multimode chromatography. Surprisingly, this disclosure shows that multimode chromatography can separate a desired multispecific CrossMab antibody from its unwanted variants.

[0018] Unless otherwise defined, the technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art in the field to which this invention pertains. Singleton et al., Dictionary of Microbiology and Molecular Biology 2nd ed., J. Wiley & Sons (New York, NY 1994), and March, Advanced Organic Chemistry Reactions, Mechanisms and Structure 4th ed., John Wiley & Sons (New York, NY 1992) provide general guidance regarding many of the terms used in this application.

[0019] definition For the purposes of interpreting this specification, the following definitions shall apply, and wherever appropriate, a term used in the singular form shall also include the plural form, and conversely, a term used in the plural form shall also include the singular form. In the event of any conflict between any of the following definitions and any of the references incorporated herein by reference, the following definition shall prevail.

[0020] As used herein and in the appended claims, the singular forms "a," "an," and "the" include plural references unless otherwise explicitly indicated by the context. Thus, for example, a reference to "a protein" or an "antibody" includes multiple proteins or antibodies, a reference to "a cell" includes a mixture of cells, and so on.

[0021] As used herein, the terms “about” or “approximately” mean within an acceptable margin of error of a particular value as determined by those skilled in the art, which in part depends on how that value is measured or determined, i.e., the limits of the measuring system. For example, “about” may mean a standard deviation of 3 or more than 3, according to the practice of the art. Alternatively, “about” may mean a range of up to 20%, preferably 10%, more preferably 5%, and even more preferably 1% of a given value. Or, particularly with respect to biological systems or processes, the term may mean within an order of magnitude of the value, preferably within 5 times, and even more preferably within 2 times. References to “about” values ​​or parameters herein include (and describe) embodiments relating to that value or parameter itself. For example, a statement referring to “about X” includes a statement of “X”.

[0022] The terms “polypeptide” and “protein” are used herein synonymously to refer to polymers of amino acids of any length. The polymers may be linear or branched, may contain modified amino acids, or may be interrupted by non-amino acids. These terms also encompass amino acid polymers that are naturally modified or modified by intervention, such as disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other operation or modification, such as conjugate with a labeling component. Furthermore, polypeptides containing, for example, one or more analogues of amino acids (including, for example, non-natural amino acids), and other modifications known in the art, are also included within the scope of this definition. As used herein, the terms “polypeptide” and “protein” specifically encompass antibodies.

[0023] A “purified” polypeptide (e.g., an antibody or immunoadhesin) means that the polypeptide exists in a purer form than it would in its natural environment, and / or that its purity is increased when it is first synthesized and / or amplified under laboratory conditions. Purity is a relative term and does not necessarily mean absolute purity. The terms “purification,” “separation,” and “isolation,” as used interchangeably herein, refer to increasing the degree of purity of a desired molecule (e.g., a multispecific antibody, or a bispecific antibody) from a composition or sample containing one or more impurities. Typically, the degree of purity of a desired molecule is increased by (completely or partially) removing at least one impurity from the composition.

[0024] A multispecific antibody that "binds to a target antigen" is one that binds to an antigen, such as a protein, with sufficient affinity and does not significantly cross-react with other proteins, making it useful as a diagnostic and / or therapeutic agent targeting a protein or cells or tissues that express that protein. In such embodiments, the degree of binding of the multispecific antibody to "non-target" proteins is less than about 10% of the binding of the multispecific antibody to that particular target protein, as determined, for example, by fluorescence-activated cell classification (FACS) analysis, radioimmunoprecipitation (RIA), or ELISA. With respect to the binding of a multispecific antibody to a target molecule, the terms "specifically binds" or "is specific" to a particular polypeptide or epitope on a particular polypeptide target mean a binding that is clearly different from nonspecific interactions (for example, nonspecific interactions may be binding to bovine serum albumin or casein). Specific binding can be measured, for example, by comparing the binding of the molecule to the binding of a control molecule. For example, specific binding can be determined by competition with a control molecule similar to the target, such as excessive unlabeled targets. In this case, specific binding is indicated when the binding of a labeled target to a probe is competitively inhibited by an excess of unlabeled targets. As used herein, the terms “specific binding,” “specifically binding,” or “specific” to a particular polypeptide or epitope on a particular polypeptide target may be indicated, for example, by a molecule having an affinity for the target with a Kd of at least about 200 nm, or at least about 150 nm, or at least about 100 nm, or at least about 60 nm, or at least about 50 nm, or at least about 40 nm, or at least about 30 nm, or at least about 20 nm, or at least about 10 nm, or at least about 8 nm, or at least about 6 nm, or at least about 4 nm, or at least about 2 nm, or at least about 1 nm or more. In one embodiment, the term “specific binding” refers to binding where a multispecific antigen-binding protein binds to a particular polypeptide or epitope on a particular polypeptide without substantially binding to any other polypeptide or polypeptide epitope.

[0025] "Binding affinity" generally refers to the strength of the sum of non-covalent interactions between a single binding site of a molecule (e.g., a multispecific antibody) and its binding partner (e.g., an antigen). Unless otherwise indicated, "binding affinity" as used herein refers to intrinsic binding affinity, which reflects the 1:1 interaction between members of a binding pair (e.g., an antibody and an antigen). The affinity of molecule X for its partner Y can generally be expressed by a dissociation constant (Kd). For example, Kd may be about 200 nM or less, about 150 nM or less, about 100 nM or less, about 60 nM or less, about 50 nM or less, about 40 nM or less, about 30 nM or less, about 20 nM or less, about 10 nM or less, about 8 nM or less, about 6 nM or less, about 4 nM or less, about 2 nM or less, or about 1 nM or less. Affinity can be measured by common methods known in the art, including the methods described herein. Low-affinity antibodies generally tend to bind slowly to antigens and dissociate easily, while high-affinity antibodies generally tend to bind rapidly to antigens and remain bound for longer periods. Various methods for measuring binding affinity are known in the art, and any of these can be used for the purposes of the methods and compositions provided herein.

[0026] For the purposes of this specification, “active” means a polypeptide (such as a multispecific antibody) that retains the biological and / or immunological activity of a natural or naturally occurring polypeptide; “biological” activity means any biological function (either inhibition or stimulation) caused by a natural or naturally occurring polypeptide other than its ability to induce the production of antibodies against an antigen epitope; and “immunological” activity means the ability of a natural or naturally occurring polypeptide to induce the production of antibodies against an antigen epitope.

[0027] With respect to multispecific antigen-binding proteins provided herein, such as antibodies, fragments, or derivatives thereof, the terms “biologically active,” “biological activity,” and “biological properties” mean the ability to bind to biomolecules, unless otherwise specified.

[0028] The term “antibody” as used herein is used in its broadest sense and specifically includes monoclonal antibodies, polyclonal antibodies, multispecific antibodies formed from at least two intact antibodies (e.g., bispecific antibodies), and antibody fragments, provided they exhibit the desired biological activity. The term “immunoglobulin” (Ig) is used herein interchangeably with “antibody.”

[0029] Antibodies are naturally occurring immunoglobulin molecules with various structures, all based on immunoglobulin folding. For example, an IgG antibody has two "heavy" chains and two "light" chains that are disulfide-bonded to form a functional antibody. Each heavy and light chain contains a "constant" (C) region and a "variable" (V) region. The V region determines the antigen-binding specificity of the antibody, while the C region provides structural support and functions in non-antigen-specific interactions with immunoeffectors. Antigen-binding specificity of an antibody or antigen-binding fragment of an antibody is the ability of the antibody to specifically bind to a particular antigen.

[0030] The antigen-binding specificity of an antibody is determined by the structural characteristics of the V region. The variability is not evenly distributed across the 110-amino acid length of the variable domain. Instead, the V region consists of relatively invariant extensions called framework regions (FRs) of 15-30 amino acids, separated by extremely variable short regions called "hypervariable regions," each 9-12 amino acids long. The variable domains of the natural heavy and light chains each contain four FRs, primarily employing a β-sheet configuration. These are linked by three hypervariable regions, forming loops that connect the β-sheet structure and, in some cases, form part of it. The hypervariable regions within each chain are held in close proximity by the FRs and, together with the hypervariable region from the other chain, contribute to the formation of the antibody's antigen-binding site (see Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991)). The constant domain does not directly participate in antibody binding to antigens, but it exhibits various effector functions, such as contributing to antibody-dependent cytotoxicity (ADCC).

[0031] Each V domain typically contains three complementarity-determining regions ("CDRs"), each containing a "hypervariable loop," and four framework regions. Therefore, the antibody-binding site, the smallest structural unit required to bind to a specific desired antigen with substantial affinity, typically comprises three CDRs and at least three, preferably four, framework regions scattered among them to hold and present the CDRs in a suitable three-dimensional structure. Classical four-chain antibodies have an antigen-binding site determined by the cooperation of the VH and VL domains. Certain antibodies, such as camel and shark antibodies, lack a light chain and rely on a binding site formed solely by the heavy chain. Single-domain manipulated immunoglobulins can be prepared so that, in the absence of cooperation between VH and VL, the binding site is formed solely by the heavy chain or light chain.

[0032] The term "variable" refers to the fact that the sequence of a particular portion of the variable domain differs significantly between antibodies, and this is used in the binding and specificity of each particular antibody to its particular antigen. However, variability is not evenly distributed throughout the variable domain of an antibody. It is concentrated in three segments called hypervariable regions in both the light chain and heavy chain variable domains. The more highly conserved portion of the variable domain is called the framework region (FR). The variable domains of the natural heavy and light chains each contain four FRs, which primarily employ a β3-sheet configuration. These are linked by three hypervariable regions, which link the β-sheet structure and, in some cases, form loops that form part of it. The hypervariable regions within each chain are held together in close proximity by the FRs and, together with the hypervariable region from the other chain, contribute to the formation of the antibody's antigen-binding site (see Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD. (1991)). The constant domain does not directly participate in antibody binding to antigens, but it exhibits various effector functions, such as contributing to antibody-dependent cytotoxicity (ADCC).

[0033] As used herein, the term "hypervariable region" refers to an amino acid residue of an antibody involved in antigen binding. The hypervariable region consists of amino acid residues from the "complementarity-determining region" or "CDR" (e.g., around residues 24-34 (L1), 50-56 (L2), and 89-97 (L3) of VL, and around residues 31-35B (H1), 50-65 (H2), and 95-102 (H3) of VH (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991)) and / or those residues from the "hypervariable loop" (e.g., residues 26-32 (L1), 50-52 (L2), and 91-96 (L3) of VL, and residues 26-32 (H1), 52A-55 (H2), and 96-101 (H3) of VH (Chothia and Lesk J. Mol. This may include Biol. 196:901-917 (1987).

[0034] A “framework” or “FR” residue is a variable domain residue other than a hypervariable region residue as defined herein.

[0035] The "hinge region" in relation to an antibody or semi-antibody is generally defined as the extension of human IgG1 from Glu216 to Pro230 (Burton, Molec. Immunol. 22:161-206 (1985)). Hinge regions of other IgG isotypes may align with the IgG1 sequence by placing the first and last cysteine ​​residues that form the heavy chain disulfide bond in the same position.

[0036] The "lower hinge region" of the Fc domain is typically defined as the elongation of the residue immediately C-terminus of the hinge region, i.e., residues 233-239 of the Fc domain. Prior to this application, FcγR binding was generally attributed to amino acid residues in the lower hinge region of the IgG Fc domain.

[0037] The "CH2 domain" in the human IgG Fc region typically extends from approximately IgG residues 231 to 340. The CH2 domain is unique in that it is not closely paired with other domains. Rather, two N-linked branched carbohydrate chains interpose between the two CH2 domains in an intact, natural IgG molecule. It is hypothesized that the carbohydrates may provide a substitute for domain-domain pairing and help stabilize the CH2 domain. (Burton, Molec. Immunol. 22:161-206 (1985)).

[0038] The "CH3 domain" includes an extension of the residues in the Fc region from the C-terminus to the CH2 domain (i.e., from approximately amino acid residue 341 to approximately amino acid residue 447 of IgG).

[0039] An "antibody fragment" comprises a portion of an intact antibody, preferably including its antigen-binding region. Examples of antibody fragments include Fab, Fab', F(ab')2, and Fv fragments; diabodies; tandem diabodies (taDb); linear antibodies (e.g., U.S. Patent No. 5641870, Example 2; Zapata et al., Protein Eng. 8(10):1057-1062 (1995)); one-arm antibodies, single variable domain antibodies, minibodies, single-chain antibody molecules; multispecific antibodies formed from antibody fragments (e.g., but not limited to Db-Fc, taDb-Fc, taDb-CH3, (scFV)4-Fc, di-scFv, bi-scFv, or tandem (di,tri)-scFv); and bispecific T cell engagers (BiTEs).

[0040] Papain digestion of the antibody yields two identical antigen-binding fragments called "Fab" fragments and a residual "Fc" fragment, named to reflect its ability to readily crystallize. The Fab fragment consists of the entire light chain with a variable region domain (VH) of the heavy chain, and a first constant domain (CH1) of one heavy chain. Pepsin treatment of the antibody yields a single large F(ab')2 fragment, which roughly corresponds to two disulfide-linked Fab fragments with divalent antigen-binding activity and can still crosslink to an antigen. The Fab' fragment differs from the Fab fragment in that it has several additional residues at the carboxyl terminus of the CH1 domain, which contains one or more cysteines derived from the antibody hinge region. Fab'-SH is the herein designation for Fab' fragments in which the cysteine ​​residue(s) of the constant domain have a free thiol group. The F(ab')2 antibody fragment was originally produced as a pair of Fab' fragments with hinge cysteines in between. Other chemical couplings of antibody fragments are also known.

[0041] "Fv" is the minimal antibody fragment containing the complete antigen recognition and binding sites. This region consists of a dimer of one heavy-chain variable domain and one light-chain variable domain, tightly bonded non-covalently. It is in this configuration that the three hypervariable regions of each variable domain interact to define the antigen-binding site on the surface of the VH-VL dimer. In total, the six hypervariable regions confer antigen-binding specificity to the antibody. However, even a single variable domain (or half of Fv containing only the three antigen-specific hypervariable regions) has the ability to recognize and bind to the antigen, but with lower affinity than the full binding site.

[0042] The Fab fragment also includes the constant domain of the light chain and the first constant domain (CH1) of the heavy chain. The Fab' fragment differs from the Fab fragment in that several residues are added to the carboxyl terminus of the heavy chain CH1 domain, which contains one or more cysteines derived from the antibody hinge region. Fab'-SH is the notation used herein for Fab', in which the cysteine ​​residue(s) of the constant domain have at least one free thiol group. The F(ab')2 antibody fragment was originally produced as a pair with the Fab' fragment, which has a hinge cysteine ​​in between. Other chemical couplings of antibody fragments are also known.

[0043] The "light chain" of an antibody (immunoglobulin) derived from any vertebrate species can be assigned to one of two distinctly different types, called kappa(K) and lambda(2), based on the amino acid sequence of its constant domain.

[0044] Antibodies can be assigned to different classes depending on the amino acid sequence of the constant domain of their heavy chain. Intact antibodies have five major classes: IgA, IgD, IgE, IgG, and IgM, some of which can be further divided into "subclasses" (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgA, and IgA2. The constant heavy chain domains corresponding to different classes of antibodies are called a, 6, c, y, and μ, respectively. The subunit structures and three-dimensional arrangements of different classes of immunoglobulins are well known.

[0045] A "single-stranded Fv" or "scFv" antibody fragment contains the VH and VL domains of the antibody, which are located within a single polypeptide chain. In some embodiments, the Fv polypeptide further includes a polypeptide linker between the VH and VL domains, which allows the scFv to form a structure desirable for antigen binding. For an overview of scFv, see, for example, Pliickthun in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds., Springer-Verlag, New York, pp. 269-315 (1994).

[0046] The term "diabody" refers to a small antibody fragment having two antigen-binding sites, each containing a heavy chain variable domain (VH) attached to a light chain variable domain (VL) within the same polypeptide chain (VH-VL). By using a linker that is too short to allow pairing between the two domains on the same chain, these domains are paired with a complementary domain on another chain, generating two antigen-binding sites. Diabodies are described in more detail, for example, EP 404097; International Publication 93 / 11161; and Hollinger et al., Proc. Natl. Acad. Sci. USA, 90:6444-6448 (1993).

[0047] As used herein, the terms “hemiantibody” or “hemimer” refer to a monovalent antigen-binding polypeptide. In certain embodiments, the hemiantibody or hemimer comprises a VH / VL unit and optionally at least a portion of an immunoglobulin constant domain. In certain embodiments, the hemiantibody or hemimer comprises one immunoglobulin heavy chain associated with one immunoglobulin light chain, or its antigen-binding fragment. In certain embodiments, the hemiantibody or hemimer is monospecific, i.e., binds to a single antigen or epitope. Those skilled in the art will readily understand that a hemiantibody may have an antigen-binding domain consisting of a single variable domain derived, for example, from a camelid.

[0048] The term "VH / VL unit" refers to the antigen-binding region of an antibody comprising at least one VH HVR and at least one VL HVR. In certain embodiments, a VH / VL unit comprises at least one, at least two, or all three VH HVRs and at least one, at least two, or all three VL HVRs. In certain embodiments, a VH / VL unit further comprises at least a portion of the framework region (FR). In some embodiments, a VH / VL unit comprises three VH HVRs and three VL HVRs. In some embodiments, a VH / VL unit comprises at least one, at least two, at least three, or all four VH HVRs and at least one, at least two, at least three, or all four VL HVRs.

[0049] The term "multispecific antibody" is used in its broadest sense to specifically encompass antibodies that contain an antigen-binding domain that has multiple epitope specificity (i.e., can specifically bind to two or more different epitopes on one biological molecule, or can specifically bind to epitopes on two or more different biological molecules). In some embodiments, the antigen-binding domain of a multispecific antibody (such as a bispecific antibody or bivalent F(ab')2) contains two VH / VL units, where the first VH / VL unit specifically binds to a first epitope, and the second VH / VL unit specifically binds to a second epitope, and each VH / VL unit contains a heavy chain variable domain (VH) and a light chain variable domain (VL). Examples of such multispecific antibodies include, but are not limited to, full-length antibodies, antibodies having two or more VL and VH domains, antibody fragments (such as Fab, Fv, dsFv, scFv, diabodies, bispecific diabodies, and triabodies), and antibody fragments linked covalently or noncovalently. A VH / VL unit further comprising at least a portion of the heavy chain constant region and / or at least a portion of the light chain constant region may also be referred to as a "hemimer" or "half-antibody." In some embodiments, a half-antibody comprises at least a portion of a single heavy chain variable region and at least a portion of a single light chain variable region. In some such embodiments, a bispecific antibody comprising two half-antibodies and binding to two antigens includes a first half-antibody that binds to a first antigen or first epitope but not to a second antigen or second epitope, and a second half-antibody that binds to a second antigen or second epitope but not to the first antigen or first epitope. According to some embodiments, the multispecific antibody is an IgG antibody that binds to each antigen or epitope with affinity of 5M to 0.001pM, 3M to 0.001pM, 1M to 0.001pM, 0.5M to 0.001pM, or 0.1M to 0.001pM. In some embodiments, the hemimer includes a portion of the heavy chain variable region sufficient to allow the formation of an intramolecular disulfide bond with a second hemimer.In some embodiments, the hemimer includes a knob mutation or a hole mutation that enables heterodimerization with a second hemimer or half-antibody, for example, including a complementary hole mutation or a knob mutation. Knob mutations and hole mutations are discussed further below.

[0050] A "bispecific antibody" is a multispecific antibody that contains an antigen-binding domain capable of specifically binding to two different epitopes on one biological molecule, or to two different epitopes on two different biological molecules. A bispecific antibody may be referred to herein as having "dual specificity" or being "dual specific." Unless otherwise indicated, the order in which the antigens bound by a bispecific antibody are listed by the bispecific antibody name is random. In some embodiments, a bispecific antibody comprises two half-antibodies, each half-antibody comprising a single heavy chain variable region and optionally at least a portion of the heavy chain constant region, and a single light chain variable region and optionally at least a portion of the light chain constant region. In certain embodiments, a bispecific antibody comprises two half-antibodies, each half-antibody comprising a single heavy chain variable region and a single light chain variable region, but not more than one single heavy chain variable region, and not more than one single light chain variable region. In some embodiments, the bispecific antibody comprises two half-antibodies, each half-antibodycete comprising a single heavy chain variable region and a single light chain variable region, the first half-antibodycete binding to the first antigen but not to the second antigen, and the second half-antibodycete binding to the second antigen but not to the first antigen.

[0051] As used herein, the terms “knob-into-hole” or “KiH” technology refer to a technology that aims for in vitro or in vivo pairing of two polypeptides by introducing a bump (knob) into one polypeptide and a cavity (hole) into the other polypeptide at the contact surface where they interact. For example, KiH is introduced at the Fc:Fc binding contact surface, CL:CH1 contact surface, or VH / VL contact surface of an antibody (see, e.g., U.S. Patent Application Publications 2011 / 0287009, 2007 / 0178552, International Publication 96 / 027011, International Publication 98 / 050431, and Zhu et al., 1997, Protein Science 6:781-788). In some embodiments, KiH results in the pairing of two different heavy chains during the production of a multispecific antibody. For example, multispecific antibodies having KiH within their Fc regions may further contain a single variable domain linked to each Fc region, or may further contain different heavy chain variable domains paired with similar or different light chain variable domains. KiH technology can also be used to pair two different receptor extracellular domains together, or with any other polypeptide sequence containing different target recognition sequences (e.g., including affibodies, peptidebodies, and other Fc fusions).

[0052] As used herein, the term “knob mutation” refers to a mutation that introduces a bump (knob) into a polypeptide at a contact surface where one polypeptide interacts with another polypeptide. In some embodiments, the other polypeptide has a whole mutation (see, for example, U.S. Patents 5,731,168, 5,807,706, 5,821,333, 7,695,936, and 8,216,805, each of which is incorporated herein by reference in whole).

[0053] As used herein, the term “hole mutation” refers to a mutation that introduces a cavity (hole) into a polypeptide at a contact surface where one polypeptide interacts with another polypeptide. In some embodiments, the other polypeptide has a knob mutation (see, for example, U.S. Patents 5,731,168, 5,807,706, 5,821,333, 7,695,936, and 8,216,805, each of which is incorporated herein by reference in whole).

[0054] The terms "single-domain antibody" (sdAb) or "single-variable-domain (SVD) antibody" generally refer to antibodies in which a single variable domain (VH or VL) can confer antigen binding. In other words, a single variable domain does not need to interact with other variable domains to recognize the target antigen. Examples of single-domain antibodies include antibodies derived from camelids (llamas and camels) and cartilaginous fish (e.g., nurse sharks), as well as antibodies obtained from recombinant methods using human and mouse antibodies (Nature (1989) 341:544-546; Dev Comp Immunol (2006) 30:43-56; Trend Biochem Sci (2001) 26:230-235; Trends Biotechnol (2003):21:484-490; International Publication No. 2005 / 035572; International Publication No. 03 / 035694; FEBS Lett (1994) 339:285-290; International Publication No. 00 / 29004; International Publication No. 02 / 051870).

[0055] As used herein, the term “monoclonal antibody” refers to an antibody obtained from a substantially homogeneous population of antibodies; that is, the individual antibodies within that population are identical and / or bind to the same epitope, except for any hypothetical variants that may occur during the production of the monoclonal antibody, which are generally present in small amounts. In contrast to polyclonal antibody preparations, which typically contain different antibodies directed to different determinants (epitopes), each monoclonal antibody is directed to a single determinant on an antigen. Monoclonal antibodies are advantageous not only for their specificity but also because they are not contaminated by other immunoglobulins. The modifier “monoclonal” indicates a characteristic of the antibody that it is obtained from a substantially homogeneous population of antibodies and should not be interpreted as requiring the production of the antibody by any particular method. For example, monoclonal antibodies used according to the methods provided herein may be prepared by the hybridoma method first described by Kohler et al., Nature 256:495 (1975), or by the recombinant DNA method (see, for example, U.S. Patent No. 4,816,567). "Monoclonal antibodies" can also be isolated from phage antibody libraries using, for example, the techniques described in Clackson et al., Nature 352:624-628 (1991) and Marks et al., J. Mol. Biol. 222:581-597 (1991).

[0056] Specifically, monoclonal antibodies as used herein include "chimeric" antibodies (immunoglobulins) in which a portion of the heavy chain and / or light chain is identical or homologous to a corresponding sequence in an antibody derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain(s) is identical or homologous to a corresponding sequence in an antibody derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies to the extent that they exhibit the desired biological activity (U.S. Patent No. 4,816,567; Morrison et al., Proc. Natl. Acad. Sci. USA 81:6851-6855 (1984)). The chimeric antibodies of interest as used herein include "primatized" antibodies that include a variable domain antigen-binding sequence derived from a non-human primate (e.g., Old World monkeys such as baboons, rhesus macaques, or cynomolgus macaques) and a human constant region sequence (U.S. Patent No. 5,693,780).

[0057] Humanized non-human (e.g., mouse) antibodies are chimeric antibodies containing the smallest sequence derived from non-human immunoglobulin. In most cases, a humanized antibody is a human immunoglobulin (recipient antibody) in which residues from the recipient's hypervariable region are replaced with residues from the hypervariable region of a non-human species (donor antibody), such as mouse, rat, rabbit, or non-human primate, that have the desired specificity, affinity, and capabilities. In some cases, framework region (FR) residues of human immunoglobulin are replaced with corresponding non-human residues. Furthermore, humanized antibodies may contain residues not found in either the recipient or donor antibody. These modifications are made to further improve the performance of the antibody. Generally, a humanized antibody contains at least one, typically two, substantially all of the variable domains, where, apart from the aforementioned FR substitutions, all or substantially all of the hypervariable loops correspond to the hypervariable loops of non-human immunoglobulin, and all or substantially all of the FRs are FRs of the human immunoglobulin sequence. Humanized antibodies optionally contain the constant region of an immunoglobulin, typically at least a portion of the constant region of a human immunoglobulin. For further details, see Jones et al., Nature 321:522-525 (1986); Riechmann et al., Nature 332:323-329 (1988); and Presta, Curr. Op. Struct. Biol. 2:593-596 (1992).

[0058] In this specification, “intact antibody” includes heavy and light variable domains, as well as an Fc region. The constant domain may be the constant domain of the natural sequence (e.g., the constant domain of the human natural sequence) or an amino acid sequence variant thereof. Preferably, the intact antibody has one or more effector functions.

[0059] "Natural antibodies" are typically heterotetrameric glycoproteins with approximately 150,000 daltons, composed of two identical light (L) chains and two identical heavy (H) chains. Each light chain is linked to a heavy chain by one disulfide covalent bond, while the number of disulfide bonds varies between heavy chains of different immunoglobulin isotypes. Each heavy and light chain also has regularly spaced interchain disulfide bridges. Each heavy chain has a variable domain (VH) at one end, followed by several constant domains. Each light chain has a variable domain (VL) at one end and a constant domain at the other, with the constant domain of the light chain aligned with the first constant domain of the heavy chain, and the variable domain of the light chain aligned with the variable domain of the heavy chain. Certain amino acid residues are thought to form an interface between the light chain variable domain and the heavy chain variable domain.

[0060] A "naked antibody" is an antibody (as defined herein) that is not conjugated with a heterogeneous molecule such as a cytotoxic moiety or radiolabeling.

[0061] As used herein, the term “immunoadhesin” refers to a molecule that combines the binding specificity of a heterologous protein ("adhesin") with the effector function of an immunoglobulin constant domain. Structurally, an immunoadhesin comprises a fusion of an amino acid sequence with desired binding specificity (this amino acid sequence is other than the antigen recognition and binding site of the antibody (i.e., “heterologous” compared to the constant region of the antibody)) and an immunoglobulin constant domain sequence (e.g., the CH2 and / or CH3 sequence of IgG). Exemplary adhesin sequences include adjacent amino acid sequences that include a portion of a receptor or ligand that binds to the protein of interest. An adhesin sequence may also be a sequence that binds to the protein of interest but does not bind to a receptor or ligand sequence (e.g., an adhesin sequence in a peptide body). Such polypeptide sequences can be selected or identified by a variety of methods, including phage display techniques and high-throughput sorting methods. The immunoglobulin constant domain sequence in immunoadhesins can be obtained from any immunoglobulin such as IgG-1, IgG-2, IgG-3, or IgG-4 subtype, IgA (including IgA-1 and IgA-2), IgE, IgD, or IgM.

[0062] The terms “Fc receptor” or “FcR” are used to describe receptors that bind to the Fc region of an antibody. In some embodiments, the FcR is the naturally occurring human FcR. Furthermore, preferred FcRs are those that bind to IgG antibodies (gamma receptors) and include the FcyRI, FcyRII, and FcyRIII subclass receptors, which include alleles and alternative splicing forms of these receptors. FcyRII receptors include FcyRIIA ("activating receptor") and FcyRIIB ("inhibiting receptor"), which have similar amino acid sequences that differ primarily in their cytoplasmic domains. The activating receptor FcyRIIA contains an immunoreceptor tyrosine-based activating motif (ITAM) within its cytoplasmic domain. The inhibiting receptor FcyRIIB contains an immunoreceptor tyrosine-based inhibitory motif (ITIM) within its cytoplasmic domain (see Daeron, Annu. Rev. Immunol. 15:203-234 (1997)). FcRs are outlined in Ravetch and Kinet, Annu. Rev. Immunol 9:457-92 (1991); Capel et al., Immunomethods 4:25£34 (1994); and de Haas et al., J. Lab. Chn. Med. 126:330-41 (1995). Other FcRs, including those to be identified in the future, are encompassed in the term “FcR” herein. This term also includes the neonatal receptor FcRn, which is responsible for the transfer of maternal IgG to the fetus (Guyer et al., J. Immunol. 117:587 (1976) and Kim et al., J. Immunol. 24:249 (1994)).

[0063] The terms “host cell,” “host cell line,” and “host cell culture” are used interchangeably to refer to cells into which exogenous nucleic acids have been introduced, and include the offspring of such cells. Host cells include “transformers” and “transformed cells,” which include primary transformed cells and their offspring, regardless of the number of passages. Offspring may contain mutations, although they may not have the exact same nucleic acid content as the parent cells. In this specification, offspring of mutants having the same function or biological activity as those screened or selected in the initially transformed cells are included.

[0064] "Impurities" refer to substances different from the desired polypeptide product. Impurities may include product-specific polypeptides such as one-arm antibodies and misassembled antibodies, antibody variants including basic and acidic variants, and aggregates. Other impurities include, but are not limited to, host cell material such as host cell proteins (HCPs); leached protein A; nucleic acids; other polypeptides; endotoxins; viral contaminants; and cell culture components. In some cases, impurities may be, but are not limited to, HCPs from bacterial cells, such as E. coli cells, insect cells, prokaryotic cells, eukaryotic cells, yeast cells, mammalian cells, avian cells, and fungal cells. In some cases, impurities may be HCPs from mammalian cells, such as CHO cells, i.e., CHO cell protein (CHOP). Impurities may also refer to accessory proteins used to facilitate the expression, folding, or assembly of multispecific antibodies; for example, prokaryotic chaperones such as FkpA, DsbA, and DsbC.

[0065] As used herein, “complex” or “complexed” refers to the association of two or more molecules that interact with each other by non-peptide bonds and / or forces (e.g., van der Waals, hydrophobic, hydrophilic forces). In one embodiment, the complex is a heteromultimer. As used herein, the terms “protein complex” or “polypeptide complex” should be understood to include complexes having non-protein substances (e.g., chemical molecules such as toxins or detection agents) conjugated to the protein in the protein complex.

[0066] "Isolated" nucleic acids refer to nucleic acid molecules that have been separated from the components of their natural environment. Isolated nucleic acids include nucleic acid molecules that are normally contained within cells that contain nucleic acid molecules, but the nucleic acid molecules are located outside of chromosomes or at chromosomal locations different from their natural chromosomal locations.

[0067] The "amino acid sequence identity percentage (%)" for a reference polypeptide sequence is defined as the percentage of amino acid residues in a candidate sequence that are identical to the amino acid residues of the reference polypeptide, after the sequences have been aligned and gaps introduced where necessary to obtain the maximum percentage of sequence identity, and assuming that no conservative substitutions are considered part of the sequence identity. Alignment for the purpose of determining the amino acid sequence identity percentage can be achieved in various methods within the scope of the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN, or Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for sequence alignment, including any algorithm necessary to achieve the maximum alignment over the entire length of the sequences being compared. In certain embodiments, the amino acid sequence identity % value is generated using the sequence comparison computer program ALIGN-2. The ALIGN-2 sequence comparison computer program was created by Genentech, Inc., and its source code, along with user documentation, has been filed with the U.S. Copyright Office, Washington DC, 20559, and is registered under U.S. Copyright Registration No. TXU510087. The ALIGN-2 program is publicly available from Genentech, Inc. (South San Francisco, California) or can be compiled from its source code. The ALIGN-2 program should be compiled for use with UNIX operating systems, including Digital UNIX V4.0D. All sequence comparison parameters are set by the ALIGN-2 program and remain unchanged.

[0068] In situations where ALIGN-2 is used for amino acid sequence comparison, the amino acid sequence identity percentage of a given amino acid sequence A to, with, or relative to a given amino acid sequence B (or, a given amino acid sequence A may be described as having or containing a certain amino acid sequence identity percentage to, with, or relative to a given amino acid sequence B) is calculated as follows: 100 x fraction X / Y [In the formula, X is the number of amino acid residues scored as a perfect match in the alignment of A and B in the sequence alignment program by the ALIGN-2, and Y is the total number of amino acid residues in B. It is understood that if the length of amino acid sequence A is not equal to the length of amino acid sequence B, the amino acid sequence identity % of A to B is not equal to the amino acid sequence identity % of B to A. Unless otherwise specifically indicated, all amino acid sequence identity % values ​​used herein are obtained using the ALIGN-2 computer program as described in the preceding paragraph.]

[0069] The term "variable region" or "variable domain" refers to a domain of the antibody heavy chain or antibody light chain involved in the binding of an antibody to an antigen. The variable domains of the heavy and light chains of natural antibodies (VH and VL, respectively) generally have a similar structure, with each domain containing four conserved framework regions (FRs) and three hypervariable regions (HVRs). (See, for example, Kindt et al., Kuby Immunology, 6th ed., WH Freeman and Co., p. 91 (2007).) A single VH or VL domain may be sufficient to confer antigen-binding specificity. Furthermore, antibodies that bind to a specific antigen may be isolated by screening a library of complementary VL or VH domains, respectively, using the VH or VL domain of the antibody that binds to the antigen. See, for example, Portolano et al., J. Immunol. 150:880-887 (1993); Clarkson et al., Nature 352:624-628 (1991).

[0070] As used herein, the term “vector” refers to a nucleic acid molecule capable of propagating another nucleic acid to which it is linked. This term includes vectors as self-replicating nucleic acid structures, and vectors incorporated into the genome of a host cell into which they are introduced. Certain vectors can direct the expression of a functionally linked nucleic acid. Such vectors are referred to herein as “expression vectors.”

[0071] As used herein with respect to chromatography, the term “sequential” refers to a specific sequence of chromatographic steps; for example, a first chromatographic step followed by a second chromatographic step, followed by a third chromatographic step, and so on. Additional steps may be included between sequential chromatographic steps.

[0072] As used herein with respect to chromatography, the term “continuous” refers to having a first chromatographic material and a second chromatographic material that are directly connected or connected via some other mechanism that allows for a continuous flow between the two chromatographic materials.

[0073] "Loading density" refers to the amount of composition in contact with the volume of chromatography material (e.g., liters) (e.g., grams). In some cases, loading density is expressed as g / L.

[0074] A “sample” refers to a small portion of a larger quantity of material. Generally, tests performed according to the methods described herein are performed on a sample. Samples are typically obtained, for example, from a cultured recombinant polypeptide-expressing cell line (also referred to herein as a “product cell line”) or from a recombinant polypeptide preparation obtained from a cultured host cell. As used herein, “host cell” does not contain the gene for the expression of the recombinant polypeptide or product of interest. Samples are obtained, for example, from a collected cell culture medium, from a process pool in a specific step of the purification method, or from the final purified product. Samples may include diluents, buffers, washing agents, and contaminants, residues, etc., found when mixed with the desired molecule (multispecific antibodies, e.g., bispecific antibodies).

[0075] The term "pharmaceutical preparation" refers to a preparation in which the biological activity of the active ingredient is effective and which does not contain any further ingredients that are unacceptably toxic to the subject to which the preparation is administered. Such preparations are sterile. A "pharmaceutically acceptable" excipient (vehicle, additive) is one that, when administered appropriately to the target mammal, can provide the effective dose of the active ingredient to be used.

[0076] Method for purifying multispecific antibodies The reliability of antibody production processes has been improved through various strategies, including the expression of "knob-into-hole" multispecific antibodies and the development of CrossMab antibodies. However, when producing multispecific antibodies in a single cell using such strategies, downstream purification steps become necessary to eliminate antibody variants containing mispaired polypeptides.

[0077] In certain non-limiting embodiments, the disclosure provides a method for purifying a multispecific antibody. In certain embodiments, the multispecific antibody is a CrossMab antibody. In certain embodiments, the multispecific antibody is a bispecific antibody. In certain embodiments, the multispecific antibody is a bivalent F(ab')2 comprising a first F(ab) that binds to a first target and a second F(ab) that binds to a second target. In certain embodiments, the multispecific antibody is a bispecific antibody, i.e., an antibody having two antigen-binding arms with identical amino acid sequences, where each Fab arm can recognize two antigens (e.g., a dual-acting Fab antibody).

[0078] In certain embodiments, the purification of multispecific antibodies involves multimode chromatography. In some embodiments, the multispecific antibodies are assembled before capture chromatography.

[0079] In certain embodiments, a multispecific antibody (such as a bispecific antibody or a bivalent F(ab')2) comprises two or more antibody arms, each binding to a different epitope. In certain embodiments, the different epitopes are located on the same antigen. In certain embodiments, the different epitopes are located on different antigens. In certain embodiments, the antibody arms contain VH / VL units. In certain embodiments, the antibody arms contain hemimers, also known as half-antibodies. In certain embodiments, the heavy chain of one antibody arm is modified to contain a “knob,” and the heavy chain of another antibody arm contains a “hole,” such that the knob of the first heavy chain fits into the hole of the second heavy chain.

[0080] In certain embodiments, multispecific antibodies are produced within the same host cell. For example, the following list includes product-associated variants identified in CrossMab bispecific antibody culture harvests where multispecific antibodies are produced in the same host cell and the harvest is purified by protein A affinity chromatography. TIFF0007871252000001.tif45170

[0081] Using an automated liquid handling system, the binding of the feedstock to five different chromatographic resins was tested under various pH and buffer strength conditions. After incubation, the unbound fraction was analyzed, and surprisingly, it was confirmed that depletion of the LC mispaired variants (described in Figures 2A and 2B) occurred only under conditions that simultaneously promoted the anion exchange behavior (high pH) and hydrophobic binding (high salt concentration) shown in Figure 3.

[0082] In certain embodiments, the cell culture medium is recovered and the antibody is subjected to multimode chromatography. In certain embodiments, the multimode material includes functional groups capable of one or more of the following functionalities: anion exchange, cation exchange, hydrogen bonding, π-π bonding interactions, hydrophilic interactions, thiophilic interactions, and hydrophobic interactions. In some embodiments, the multimode material includes functional groups capable of anion exchange and hydrophobic interactions.

[0083] In certain embodiments, the multimode material includes a positively charged group and an aromatic ring structure. In certain embodiments, the positively charged group is an amine or a quaternary ammonium ion. In certain embodiments, the aromatic ring structure is a benzyl group. In certain embodiments, the multimode material includes N-benzyl-N-methylethanolamine, N,N-dimethylbenzylamine, 4-mercapto-ethyl-pyridine, 2-benzamido-4-mercaptobutanoic acid, hexylamine, phenylpropylamine, cross-linked polyallylamine, or a combination thereof. For example, without limitation, the multimode material includes Capto TM Adhere resin, Capto TM MMC resin, MEP HyperCel TM resin, HEA HyperCel TM resin, PPA HyperCel TM resin, Eshmuno® HCX, Capto TM Adhere ImpRes, Capto TMMMC Impres, Nuvia TM cPrime TM A film is included. In certain embodiments, the multimode material is Capto TM Adhere resin. In certain embodiments, the multimode material is Capto TM This is MMC. In certain embodiments, the multimode material is in a column. In certain embodiments, the multimode material is in a membrane. In certain embodiments, multimode chromatography is performed in "binding and elution" mode. In certain embodiments, multimode chromatography is performed in "flow-through" mode.

[0084] In certain embodiments, elution is step elution. In certain embodiments, elution is gradient elution.

[0085] In certain embodiments, the method disclosed herein further includes capture chromatography. In certain embodiments, the capture chromatography is affinity chromatography. In certain embodiments, the affinity chromatography is protein A chromatography. In certain embodiments, the affinity chromatography is protein G chromatography. In certain embodiments, the affinity chromatography is protein A / G chromatography. In certain embodiments, the affinity chromatography is protein L chromatography. Following capture chromatography, the purified antibody arm may be analyzed by, for example, SDS-PAGE, SEC chromatography, mass spectrometry, etc.

[0086] In certain embodiments, the cell culture medium is recovered and subjected to capture chromatography. In certain embodiments, the eluate from the affinity chromatography step is then applied to multimode chromatography as disclosed herein. In certain embodiments, affinity chromatography includes, but is not limited to, protein A chromatography, protein G chromatography, protein A / G chromatography, or protein L chromatography. In certain embodiments, affinity chromatography materials include, for example, ProSep®-vA, ProSep® Ultra Plus, and Protein A Sepharose. TM Fast Flow, ToyopearlTMAF-rProtein A, MabSelect TM MabSelect SuRe TM MabSelect SuRe TM LX, KappaSelect, CaptureSelect TM , and CaptureSelect TM This includes, but is not limited to, FcXL. In certain embodiments, the affinity chromatography material is located in a column. In certain embodiments, affinity chromatography is performed in a "binding and elution mode" (or referred to as a "binding and elution process"). The "binding and elution mode" is a product separation technique in which a product in a sample (such as a multispecific antibody) binds to the affinity chromatography material and is then eluted from the affinity chromatography material. In certain embodiments, elution is a stepwise elution in which the composition of the mobile phase is changed stepwise over one or more occasions during the elution process. In certain embodiments, elution is a gradient elution in which the composition of the mobile phase is continuously changed during the elution process. In certain embodiments, the affinity chromatography material is located in a membrane. In certain embodiments, affinity chromatography is protein A chromatography. In certain embodiments, protein A chromatography is MAbSelect TMThis is SuRe chromatography. In certain embodiments, affinity chromatography is performed using CaptureSelect TM This is chromatography. In certain embodiments, affinity chromatography is performed using CaptureSelect TM This is FcXL chromatography.

[0087] In certain embodiments, capture chromatography and multimode chromatography are continuous, for example, the capture chromatography material and the multimode material are directly connected or connected by some other mechanism that allows for a continuous flow between the capture chromatography material and the multimode material. In certain embodiments, capture chromatography and multimode chromatography are continuous, and multimode chromatography is performed directly after capture chromatography.

[0088] In certain embodiments, the eluate from capture chromatography is subjected to one or more additional chromatographic steps before being applied to the multimode resin. In certain non-limiting embodiments, for example, the eluate from capture chromatography may be subjected to any one or more of the following chromatographic steps in any order and / or any combination before being subjected to multimode chromatography: hydrophobic interaction (HIC) chromatography, anion exchange chromatography, cation exchange chromatography, size exclusion chromatography, affinity chromatography, ceramic hydroxyapatite (CHT) chromatography, hydrophilic interaction liquid chromatography (HILIC), etc.

[0089] Hydrophobic interaction chromatography is a liquid chromatography technique that separates biomolecules according to their hydrophobicity. For example, HIC chromatography materials include, but are not limited to, Toyopearl. TMHexyl-650, Toyopearl™ Butyl-650, Toyopearl™ Phenyl-650, Toyopearl™ Ether-650, HiTrap® Sepharose, Octyl Sepharose®, Phenyl Sepharose TM , or Butyl Sepharose TM Includes. In certain embodiments, the HIC chromatography material includes phenyl Sepharose. In certain embodiments, HIC chromatography is performed in "binding and elution" mode. In certain embodiments, HIC chromatography is performed in "flow-through" mode. In certain embodiments, the HIC chromatography material is in a column. In certain embodiments, the HIC chromatography material is in a membrane.

[0090] Anion exchange chromatography materials are positively charged solid phases having free anions that are exchanged with anions in an aqueous solution (such as a composition containing multispecific antibodies and impurities) passing over or through this solid phase. In certain embodiments, the anion exchange material may be a membrane, a monolith, or a resin. In certain embodiments, the anion exchange material may be a resin. In some embodiments, the anion exchange material may contain primary amines, secondary amines, tertiary amines, or quaternary ammonium ion functional groups, polyamine functional groups, or diethylaminoethyl functional groups. For example, but not limited to, the anion exchange material may be Poros TM HQ 50, Poros TM PI 50, Poros TM D, Mustang TM Q, Q Sepharose TM Fast Flow (QSFF), Accel TM Plus Quaternary Methyl Amine (QMA) resin, Sartobind STIC®, and DEAE-Sepharose TMThis includes: In certain embodiments, anion exchange chromatography is performed in "binding and elution" mode. In certain embodiments, anion exchange chromatography is performed in "flow-through" mode. In certain embodiments, the anion exchange chromatography material is located in a column. In certain embodiments, the anion exchange chromatography material is located in a membrane.

[0091] A cation exchange chromatography material is a negatively charged solid phase having free cations that are exchanged with cations in an aqueous solution (such as a composition containing multispecific antibodies and impurities) passing over or through this solid phase. In certain embodiments, the cation exchange material may be a membrane, a monolith, or a resin. In some embodiments, the cation exchange material may be a resin. The cation exchange material may contain carboxylic acid functional groups or sulfonic acid functional groups. For example, but not limited to, the cation exchange material may contain sulfonates, carboxylic acids, carboxymethylsulfonic acid, sulfisobutyl, sulfoethyl, carboxyl, sulfopropyl, sulfonyl, sulfoxyethyl, or orthophosphates. In some embodiments, the cation exchange chromatography material is a cation exchange chromatography column. In the certain embodiments described above, the cation exchange chromatography material is a cation exchange chromatography membrane. For example, but not limited to, the cation exchange material may be a Mustang TM S, Sartobind®, SO3 Monolith (e.g., CIM®, ClMmultus®, and CIMac®, SO3, etc.), S Ceramic HyperD®, Poros TM XS, Poros TM HS 50, Poros™ HS 20, Sulfopropyl-Sepharose® Fast Flow (SPSFF), SP-Sepharose® XL (SPXL), CM Sepharose TM Fast Flow, Capto TM S, Fractogel TM EMD Se Hicap, FractogelTM EMD S03, or Fractogel TM Includes EMD COO. In certain embodiments, cation exchange chromatography is performed in "binding and elution" mode. In certain embodiments, cation exchange chromatography is performed in "flow-through" mode. In the certain embodiments described above, the cation exchange chromatography material is in a column. In the certain embodiments described above, the cation exchange chromatography material is in a membrane.

[0092] In certain embodiments, the Disclosure provides a method for separating a multispecific antibody, i.e., a bispecific antibody, from a composition comprising subjecting the composition to multimode chromatography and recovering a fraction containing the multispecific antibody, the multispecific antibody being produced by the same host cell. In certain embodiments, the multimode chromatography is performed in "binding and elution" mode.

[0093] In certain embodiments, the method includes subjecting the composition to capture chromatography to produce a first eluate, subjecting the first eluate to multimode chromatography, and recovering a fraction containing a polyspecific antibody. In certain embodiments, the capture chromatography is protein A chromatography.

[0094] In certain embodiments, the eluate from multimode chromatography is subjected to one or more additional chromatographic steps. For example, but are not limited to, the eluate from multimode chromatography can be subjected to any one or more of the following chromatographic steps in any order and / or any combination: hydrophobic interaction (HIC) chromatography, anion exchange chromatography, cation exchange chromatography, size exclusion chromatography, affinity chromatography, ceramic hydroxyapatite (CHT) chromatography, hydrophilic interaction liquid chromatography (HILIC), multimode chromatography, etc.

[0095] In certain embodiments, the method involves using a buffer. Various buffers may be used during the purification of multispecific antibodies. For example, buffers may have different pH and / or conductivity based on the properties of the multispecific antibody. In certain embodiments, the buffer may be a loading buffer, an equilibrium buffer, or a wash buffer. In certain embodiments, one or more of the loading buffer, equilibrium buffer, and / or wash buffers are the same. In certain embodiments, the loading buffer, equilibrium buffer, and / or wash buffers are different. In certain embodiments, the buffer contains a salt. In certain embodiments, the buffer contains sodium chloride, sodium acetate, Tris HC1, Tris acetate, sodium phosphate, potassium phosphate, MES, CHES, MOPS, BisTris, arginine, arginine HC1, or a mixture thereof. In certain embodiments, the buffer is a sodium chloride buffer. In some embodiments, the buffer is a sodium acetate buffer. In certain embodiments, the buffer is a Tris, arginine, phosphate, MES, CHES, or MOPS buffer.

[0096] "Loading" refers to the process of supporting a composition onto a chromatography material. The loading buffer is used to load a composition (e.g., a composition containing multispecific antibodies and impurities) onto a chromatography material (such as one of the chromatography materials described herein). The chromatography material may be equilibrated using an equilibrium buffer before loading the composition to be purified. A washing buffer is used after loading the composition onto the chromatography material. An elution buffer is used to elute the target polypeptide from the solid phase.

[0097] The loading of a composition containing a multispecific antibody (such as a composition containing a multispecific antibody and impurities) onto any of the chromatographic materials described herein may be optimized for the separation of the multispecific antibody from the impurities. In certain embodiments, when chromatography is performed in binding and elution modes (e.g., multimode chromatography), the loading of a composition containing a multispecific antibody (such as a composition containing a multispecific antibody and impurities) onto the chromatographic material is optimized for the binding of the multispecific antibody to the chromatographic material.

[0098] Conductivity refers to the ability of an aqueous solution to conduct electric current between two electrodes. In a solution, electric current flows by ion transport. Therefore, as the amount of ions present in the aqueous solution increases, the solution will have higher conductivity. The basic unit of conductivity is the siemen (mS / cm) or ohm (mho), and it can be measured using a conductivity meter, such as various models of Orion conductivity meters. Since electrolyte conductivity is the ability of ions in a solution to conduct electric current, the conductivity of a solution can be altered by changing the concentration of ions in it. In certain non-limiting embodiments, for example, the concentration of a buffer agent and / or salt (e.g., sodium chloride, sodium acetate, or potassium chloride) in the solution can be changed to achieve a desired conductivity. In certain embodiments, the salt concentrations of various buffers are modified to achieve a desired conductivity.

[0099] In certain non-limiting embodiments, for example, a composition containing a multispecific antibody (such as a composition containing a multispecific antibody and impurities) is loaded onto a chromatographic material in a loading buffer at many different pH values ​​while the conductivity of the loading buffer remains constant. In certain embodiments, a solution containing a multispecific antibody is loaded onto a chromatographic material in a loading buffer at many different conductivity values ​​while the pH of the loading buffer remains constant. Once the loading of the composition containing a multispecific antibody (such as a composition containing a multispecific antibody and impurities) onto the chromatographic material and the elution of the multispecific antibody from the chromatographic material into the pool fraction are complete, the amount of impurities remaining in the pool fraction provides information regarding the separation of the multispecific antibody from the impurities at a given pH or conductivity. Similarly, in chromatography in which a multispecific antibody flows through a chromatographic material, the pH and conductivity of the loading buffer are optimized such that the multispecific antibody flows through the chromatographic material, while the impurities are retained by the chromatographic material or flow through the chromatographic material at a different rate than the multispecific antibody.

[0100] In certain embodiments, the loading density of the solution containing multispecificity is greater than any of the following affinity chromatography material concentrations: approximately 1 g / L, approximately 5 g / L, approximately 10 g / L, approximately 20 g / L, approximately 30 g / L, approximately 40 g / L, approximately 50 g / L, approximately 60 g / L, approximately 70 g / L, approximately 80 g / L, approximately 90 g / L, approximately 100 g / L, approximately 110 g / L, approximately 120 g / L, approximately 130 g / L, approximately 140 g / L, or approximately 150 g / L. In certain embodiments, the loading density of the solution containing the multispecific antibody is one of the following: between approximately 1 g / L and 5 g / L of the capture chromatography material, between approximately 5 g / L and 10 g / L, between approximately 10 g / L and 20 g / L, between approximately 20 g / L and 30 g / L, between approximately 30 g / L and 40 g / L, between approximately 40 g / L and 50 g / L, between approximately 50 g / L and 60 g / L, between approximately 60 g / L and 70 g / L, between approximately 70 g / L and 80 g / L, between approximately 80 g / L and 90 g / L, or between approximately 90 g / L and 100 g / L.

[0101] In certain embodiments, the eluate obtained after capture chromatography is a multimode chromatography material (e.g., Capto TM The antibodies are loaded onto the Adhere. In certain embodiments, the eluate obtained after capture chromatography is loaded onto the multimode chromatography material at a multispecific antibody loading density greater than any of the following: approximately 10 g / L, approximately 20 g / L, approximately 30 g / L, 40 g / L, approximately 50 g / L, approximately 60 g / L, approximately 70 g / L, approximately 80 g / L, approximately 90 g / L, approximately 100 g / L, approximately 110 g / L, approximately 120 g / L, approximately 130 g / L, approximately 140 g / L, or approximately 150 g / L of the multimode chromatography material. In certain embodiments, the eluate obtained after capture chromatography is loaded onto the multimode chromatography material at a loading density of multispecific antibodies between approximately 1 g / L and 5 g / L, between approximately 5 g / L and 10 g / L, between approximately 10 g / L and 20 g / L, between approximately 20 g / L and 30 g / L, between approximately 30 g / L and 40 g / L, between approximately 40 g / L and 50 g / L, between approximately 50 g / L and 60 g / L, between approximately 60 g / L and 70 g / L, between approximately 70 g / L and 80 g / L, between approximately 80 g / L and 90 g / L, and between approximately 90 g / L and 100 g / L.

[0102] In certain embodiments, the eluate obtained after multimode chromatography is loaded onto the subsequent chromatography material (such as hydrophobic interaction (HIC) chromatography material, anion exchange chromatography material, cation exchange chromatography material, size exclusion chromatography material, affinity chromatography material, or additional multimode chromatography material) at a loading density of multiple specific antibodies greater than approximately 30 g / L, approximately 40 g / L, approximately 50 g / L, approximately 60 g / L, approximately 70 g / L, approximately 80 g / L, approximately 90 g / L, approximately 100 g / L, approximately 110 g / L, approximately 120 g / L, approximately 130 g / L, approximately 140 g / L, or approximately 150 g / L of the subsequent chromatography material. In some embodiments, the eluate obtained after multimode chromatography is loaded onto the subsequent chromatography material (such as hydrophobic interaction (HIC) chromatography material, anion exchange chromatography material, cation exchange chromatography material, size exclusion chromatography material, affinity chromatography material, or additional multimode chromatography material) at a loading density of multispecific antibodies between approximately 10 g / L and 20 g / L, between approximately 20 g / L and 30 g / L, between approximately 30 g / L and 40 g / L, between approximately 40 g / L and 50 g / L, between approximately 50 g / L and 60 g / L, between approximately 60 g / L and 70 g / L, between approximately 70 g / L and 80 g / L, between approximately 80 g / L and 90 g / L, or between approximately 90 g / L and 100 g / L.

[0103] As used herein, elution refers to the removal of a product from a chromatographic material, such as a multispecific antibody. The elution buffer is a buffer used to elute a multispecific antibody from a chromatographic material. In certain embodiments, the elution buffer has a lower conductivity than the loading buffer. In certain embodiments, the elution buffer has a higher conductivity than the loading buffer. In certain embodiments, the elution buffer has a lower pH than the loading buffer. In certain embodiments, the elution buffer has a higher pH than the loading buffer. In certain embodiments, the elution buffer has a different conductivity and a different pH than the loading buffer.

[0104] In certain embodiments, the elution of multispecific antibodies from chromatographic material is optimized to minimize impurities and obtain a product yield with the smallest possible elution or pooling volume. In certain non-limiting embodiments, for example, a composition containing multispecific antibodies may be loaded onto the chromatographic material in a loading buffer. Once loading is complete, the multispecific antibodies are eluted using buffers with many different pH values ​​while the conductivity of the elution buffer remains constant. Alternatively, the multispecific antibodies may be eluted from the chromatographic material in elution buffers with many different conductivity values ​​while the pH of the elution buffer remains constant. Once the elution of the multispecific antibodies from the chromatographic material is complete, the amount of impurities in the pooled fraction provides information about the separation of the multispecific antibodies or antibody arms from impurities for a given pH or conductivity. Elution of multispecific antibodies in large column volumes (e.g., 8 column volumes) exhibits "tailing" of the elution profile.

[0105] In certain embodiments, the methods disclosed herein include the use of a buffer. Various buffers may be used based on the desired pH of the buffer, the desired conductivity of the buffer, the properties of the protein of interest, the chromatography material, and the purification process (e.g., “binding and elution” mode or “flow-through” mode). In certain embodiments, the method includes the use of at least one buffer. In certain embodiments, the buffer may be a loading buffer, an equilibrium buffer, an elution buffer, or a wash buffer. In certain embodiments, one or more of the loading buffer, equilibrium buffer, elution buffer, and / or wash buffers are the same. In certain embodiments, the loading buffer, equilibrium buffer, and / or wash buffers are different. In certain embodiments, the buffer contains a salt. In certain embodiments, the loading buffer may contain sodium chloride, sodium acetate, Tris, arginine, phosphate, MOPS, MES, CHES, BisTris, ammonium sulfate, sodium sulfate, citrate, succinate, or a mixture thereof. In certain embodiments, the buffer is a sodium chloride buffer. In certain embodiments, the buffer is a sodium acetate buffer. In certain embodiments, the buffer is Tris, arginine, phosphate, MES, CHES, or MOPS buffer. In certain embodiments, the buffer contains Tris. In certain embodiments, the buffer contains arginine.

[0106] In certain embodiments, the loading buffer has a conductivity greater than any of the following: approximately 1.0 mS / cm, approximately 1.5 mS / cm, approximately 2.0 mS / cm, approximately 2.5 mS / cm, approximately 3.0 mS / cm, approximately 3.5 mS / cm, approximately 4.0 mS / cm, approximately 4.5 mS / cm, approximately 5.0 mS / cm, approximately 5.5 mS / cm, approximately 6.0 mS / cm, approximately 6.5 mS / cm, approximately 7.0 mS / cm, approximately 7.5 mS / cm, approximately 8.0 mS / cm, approximately 8.5 mS / cm, approximately 9.0 mS / cm, approximately 9.5 mS / cm, approximately 10 mS / cm, or approximately 20 mS / cm. In certain embodiments, the conductivity may be between approximately 1 mS / cm and approximately 20 mS / cm, between approximately 4 mS / cm and approximately 10 mS / cm, between approximately 4 mS / cm and approximately 7 mS / cm, between approximately 5 mS / cm and approximately 17 mS / cm, between approximately 5 mS / cm and approximately 10 mS / cm, or between approximately 5 mS / cm and approximately 7 mS / cm. In some embodiments, the conductivity is one of the following: approximately 1.0 mS / cm, approximately 1.5 mS / cm, approximately 2.0 mS / cm, approximately 2.5 mS / cm, approximately 3.0 mS / cm, approximately 3.5 mS / cm, approximately 4 mS / cm, approximately 4.5 mS / cm, approximately 5.0 mS / cm, approximately 5.5 mS / cm, approximately 6.0 mS / cm, approximately 6.5 mS / cm, approximately 7.0 mS / cm, approximately 7.5 mS / cm, approximately 8.0 mS / cm, approximately 8.5 mS / cm, approximately 9.0 mS / cm, approximately 9.5 mS / cm, approximately 10 mS / cm, or approximately 20 mS / cm. In certain embodiments, the conductivity is the conductivity of the loading buffer, equilibrium buffer, and / or wash buffer. In certain embodiments, the conductivity of one or more of the loading buffer, equilibrium buffer, and wash buffer is the same. In some embodiments, the conductivity of the loading buffer is different from that of the washing buffer and / or equilibrium buffer.

[0107] In some embodiments, the elution buffer has a conductivity lower than that of the loading buffer. In certain embodiments, the elution buffer has a conductivity lower than any of the following: about 0 mS / cm, about 0.5 mS / cm, about 1.0 mS / cm, about 1.5 mS / cm, about 2.0 mS / cm, about 2.5 mS / cm, about 3.0 mS / cm, about 3.5 mS / cm, about 4.0 mS / cm, about 4.5 mS / cm, about 5.0 mS / cm, about 5.5 mS / cm, about 6.0 mS / cm, about 6.5 mS / cm, or 7.0 mS / cm. In some embodiments, the conductivity may be between approximately 0 mS / cm and approximately 7 mS / cm, between approximately 1 mS / cm and approximately 7 mS / cm, between approximately 2 mS / cm and approximately 7 mS / cm, between approximately 3 mS / cm and approximately 7 mS / cm, or between approximately 4 mS / cm and approximately 7 mS / cm, between approximately 0 mS / cm and approximately 5.0 mS / cm, between approximately 1 mS / cm and approximately 5 mS / cm, between approximately 2 mS / cm and approximately 5 mS / cm, between approximately 3 mS / cm and approximately 5 mS / cm, or between approximately 4 mS / cm and approximately 5 mS / cm. In some embodiments, the conductivity of the elution buffer is approximately 0 mS / cm, 0.5 mS / cm, 1.0 mS / cm, 1.5 mS / cm, 2.0 mS / cm, 2.5 mS / cm, 3.0 mS / cm, 3.5 mS / cm, 4 mS / cm, 4.5 mS / cm, 5.0 mS / cm, 5.5 mS / cm, 6.0 mS / cm, 6.5 mS / cm, or 7.0 mS / cm.

[0108] In some embodiments, the elution buffer has a conductivity greater than that of the loading buffer. In certain embodiments, the elution buffer has conductivity of approximately 5.5 mS / cm, approximately 6.0 mS / cm, approximately 6.5 mS / cm, approximately 7.0 mS / cm, approximately 7.5 mS / cm, approximately 8.0 mS / cm, approximately 8.5 mS / cm, approximately 9.0 mS / cm, approximately 9.5 mS / cm, approximately 10 mS / cm, approximately 11 mS / cm, approximately 12 mS / cm, approximately 13 mS / cm, approximately 14 mS / cm, approximately 15 mS / cm, approximately 16 mS / cm, approximately It has a conductivity greater than any of the following: 17.0 mS / cm, approximately 18.0 mS / cm, approximately 19.0 mS / cm, approximately 20.0 mS / cm, approximately 21.0 mS / cm, approximately 22.0 mS / cm, approximately 23.0 mS / cm, approximately 24.0 mS / cm, approximately 25.0 mS / cm, approximately 26.0 mS / cm, approximately 27.0 mS / cm, approximately 28.0 mS / cm, approximately 29.0 mS / cm, or approximately 30.0 mS / cm. In certain embodiments, the conductivity may be between approximately 5.5 mS / cm and approximately 30 mS / cm, between approximately 6.0 mS / cm and approximately 30 mS / cm, between approximately 7 mS / cm and approximately 30 mS / cm, between approximately 8 mS / cm and approximately 30 mS / cm, between approximately 9 mS / cm and approximately 30 mS / cm, or between approximately 10 mS / cm and approximately 30 mS / cm. In certain nozzles, the conductivity of the elution buffer is approximately 5.5 mS / cm, approximately 6.0 mS / cm, approximately 6.5 mS / cm, approximately 7.0 mS / cm, approximately 7.5 mS / cm, approximately 8.0 mS / cm, approximately 8.5 mS / cm, approximately 9.0 mS / cm, approximately 9.5 mS / cm, approximately 10 mS / cm, approximately 11 mS / cm, approximately 12 mS / cm, approximately 13 mS / cm, approximately 14 mS / cm, approximately 15 mS / cm, and approximately 1 The conductivity is one of the following: 6 mS / cm, approximately 17.0 mS / cm, 18.0 mS / cm, approximately 19.0 mS / cm, approximately 20.0 mS / cm, approximately 21.0 mS / cm, approximately 22.0 mS / cm, approximately 23.0 mS / cm, approximately 24.0 mS / cm, approximately 25.0 mS / cm, approximately 26.0 mS / cm, approximately 27.0 mS / cm, approximately 28.0 mS / cm, approximately 29.0 mS / cm, or approximately 30.0 mS / cm. In certain embodiments, the conductivity of the elution buffer is varied from the loading buffer and / or washing buffer by a stepped or linear gradient.

[0109] In certain embodiments, a composition containing a multispecific antibody is loaded onto a multimode chromatography material in a loading buffer having a conductivity of less than 100 mS / cm, and the polypeptide is eluted from the mixed chromatography material in an elution buffer having a conductivity of less than 100 mS / cm. In certain embodiments, the loading buffer has a conductivity of less than 100 mS / cm, and the elution buffer has a conductivity of less than 100 mS / cm. In certain embodiments, the loading buffer has a conductivity of less than 100 mS / cm, and the elution buffer has a conductivity of less than 100 mS / cm. In certain embodiments, the loading buffer has a conductivity of less than 100 mS / cm, and the elution buffer has a conductivity of about xxx mS / cm. In certain embodiments, the multimode chromatography material is Capto TM Adhere resin. In certain embodiments, the multimode chromatography material is Capto TM It is MMC resin.

[0110] In certain embodiments, the conductivity of the elution buffer is varied from that of the loading buffer and / or washing buffer by a stepped or linear gradient.

[0111] In certain embodiments, the loading buffer has a pH less than about 10, about 9, about 8, about 7, about 6, or about 5, and includes any range between these values. In certain embodiments, the loading buffer has a pH greater than about 4, about 5, about 6, about 7, about 8, or about 9, and includes any range between these values. In certain embodiments, the loading buffer has a pH between about 4 and about 9, about 4 and about 8, about 4 and about 7, about 5 and about 9, about 5 and about 8, about 5 and about 7, or about 5 and about 6, and includes any range between these values. In certain embodiments, the pH of the loading buffer has a pH of about 4, about 4.5, about 5, about 5.5, about 6, about 6.5, about 7, about 7.5, about 8, or about 8.5, and includes any range between these values.

[0112] In some embodiments, the elution has a pH lower than the pH of the loading buffer. In certain embodiments, the elution buffer has a pH lower than any of about 8, about 7, about 6, about 5, about 4, about 3, or about 2, and includes any range between these values. In certain embodiments, the pH of the elution buffer may be any of the following: about 4 and about 9, about 4 and about 8, about 4 and about 7, about 4 and about 6, about 4 and about 5, about 5 and about 9, about 5 and about 8, about 5 and about 7, about 5 and about 6, about 6 and about 9, about 6 and about 8, or about 6 and about 7, and includes any range between these values. In certain embodiments, the pH of the elution buffer is any of the following: about 4.0, about 4.5, about 5.0, about 5.5, about 6.0, about 6.5, about 7.0, about 7.5, about 8.0, about 8.5, or about 9.0, and includes any range between these values.

[0113] In some embodiments, the elution buffer has a pH greater than that of the loading buffer. In certain embodiments, the elution buffer has a pH greater than any of about 5, about 6, about 7, about 8, or about 9, and includes any range between these values. In certain embodiments, the elution buffer has a pH greater than any of about 2, about 4, or about 4, and includes any range between these values. In certain embodiments, the pH of the elution buffer can be any of the following: about 2 and about 9, about 3 and about 9, about 4 and about 9, about 2 and about 8, about 3 and about 8, about 4 and about 8, about 2 and about 7, about 3 and about 7, about 4 and about 7, about 2 and about 6, about 3 and about 6, and about 4 and about 6, and includes any range between these values. In some embodiments, the pH of the elution buffer is any of the following: about 2.0, about 2.5, about 3.0, about 3.5, or about 4.0, and includes any range between these values.

[0114] In certain embodiments, a solution containing a multispecific antibody is loaded into an affinity chromatograph (e.g., protein A chromatography) at approximately pH 7, and the multispecific antibody or antibody arm is eluted from the affinity chromatograph by a step gradient to pH approximately 2.9.

[0115] In certain embodiments, the pH of the elution buffer is changed from the loading buffer and / or washing buffer by a stepped or linear gradient.

[0116] In certain embodiments, the flow rate is less than approximately 50 CV / hour, less than approximately 40 CV / hour, or less than approximately 30 CV / hour. The flow rate may be between approximately 5 CV / hour and 50 CV / hour, between approximately 10 CV / hour and 40 CV / hour, or between 18 CV / hour and 36 CV / hour. In certain embodiments, the flow rate is one of approximately 9 CV / hour, approximately 18 CV / hour, approximately 25 CV / hour, approximately 30 CV / hour, approximately 36 CV / hour, or approximately 40 CV / hour. In certain embodiments, the flow rate is one of less than approximately 100 cm / hour, less than approximately 75 cm / hour, or less than approximately 50 cm / hour. In certain embodiments, the flow rate may be one of the following: between approximately 25 cm / hour and approximately 150 cm / hour, between approximately 25 cm / hour and approximately 100 cm / hour, between approximately 50 cm / hour and approximately 100 cm / hour, or between approximately 65 cm / hour and approximately 85 cm / hour.

[0117] Floor height refers to the height of the chromatography material being used. In certain embodiments, the floor height is greater than any of the following: approximately 5 cm, approximately 10 cm, approximately 15 cm, approximately 20 cm, approximately 25 cm, approximately 30 cm, approximately 35 cm, approximately 40 cm, approximately 45 cm, or approximately 50 cm. In certain embodiments, the floor height is between approximately 5 cm and approximately 50 cm. In certain embodiments, the floor height is determined based on the amount of polypeptide or contaminant being loaded.

[0118] In a particular embodiment, chromatography is performed in approximately 1 mL, 2 mL, 3 mL, 4 mL, 5 mL, 6 mL, 7 mL, 8 mL, 9 mL, 10 mL, 15 mL, 20 mL, 25 mL, 30 mL, 40 mL, 50 mL, 75 mL, 100 mL, 200 mL, 300 mL, 400 mL, 500 mL, 600 mL, and approximately It is contained in a column or container with a volume greater than 700 mL, approximately 800 mL, approximately 900 mL, approximately 1 L, approximately 2 L, approximately 3 L, approximately 4 L, approximately 5 L, approximately 6 L, approximately 7 L, approximately 8 L, approximately 9 L, approximately 10 L, approximately 25 L, approximately 50 L, approximately 100 L, approximately 200 L, approximately 300 L, approximately 400 L, approximately 500 L, approximately 600 L, approximately 700 L, approximately 800 L, approximately 900 L, or approximately 1000 L.

[0119] In certain embodiments, the fraction is recovered from chromatography. In certain embodiments, the recovered fraction is greater than approximately 0.01 CV, approximately 0.02 CV, approximately 0.03 CV, approximately 0.04 CV, approximately 0.05 CV, approximately 0.06 CV, approximately 0.07 CV, approximately 0.08 CV, approximately 0.09 CV, approximately 0.1 CV, approximately 0.2 CV, approximately 0.3 CV, approximately 0.4 CV, approximately 0.5 CV, approximately 0.6 CV, approximately 0.7 CV, approximately 0.8 CV, 0.9 CV, approximately 1.0 CV, 2.0 CV, approximately 3.0 CV, approximately 4.0 CV, 5.0 CV, approximately 6.0 CV, approximately 7.0 CV, approximately 8.0 CV, approximately 9.0 CV, or 10.0 CV.

[0120] In certain embodiments, fractions containing purified material, such as multispecific antibodies (e.g., bispecific antibodies), are pooled. In certain non-limiting embodiments, the amount of polypeptide in the fraction can be determined by those skilled in the art. For example, but not limited to, the amount of polypeptide in the fraction can be determined by UV spectroscopy. In certain embodiments, fractions are recovered when the OD280 is greater than any of the following: about 0.5, about 0.6, about 0.7, about 0.8, about 0.9, and about 1.0. In certain embodiments, fractions are recovered when the OD280 is between about 0.5 and about 1.0, between about 0.6 and about 1.0, between about 0.7 and about 1.0, between about 0.8 and about 1.0, or between about 0.9 and about 1.0. In certain embodiments, fractions containing detectable multispecific antibodies (e.g., bispecific antibodies) are pooled.

[0121] In certain embodiments, impurities are product-specific impurities. For example, but not limited to, product-specific impurities include unpaired hemiantibodies, unpaired antibody light chains, unpaired heavy chains, mispaired antibodies, antibody fragments, homodimers (e.g., paired hemidimers of bispecific antibodies containing the same heavy and light chains), aggregates, high molecular weight species (MHWS) (e.g., very high molecular weight species (vHMWS)), multispecific antibodies with mispaired disulfides, light chain dimers, heavy chain dimers, low molecular weight species (LMWS), and other variants. Figures 2A and 2B show schematic examples of product-specific impurities.

[0122] In certain embodiments, the Disclosure provides a method for removing or reducing the level of light chain mispairing multispecific antibodies from a composition containing multispecific antibodies (e.g., bispecific antibodies) and impurities. In certain embodiments, the Disclosure provides a method for measuring the presence or level of light chain mispairing antibodies in a composition. For example, but not limited to, light chain mispairing antibodies can be measured by mass spectrometry, CE-SDS, reverse-phase HPLC, HIC HPLC. In certain embodiments, the amount of light chain mispairing antibodies in a composition recovered from one or more purification steps is reduced to less than any of about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 99% (including any range between these values). In certain embodiments, the amount of light chain mispair antibody in the composition recovered from one or more purification steps is reduced to one of the following: between about 10% and about 95%; between about 10% and about 99%; between about 20% and about 95%; between about 20% and about 99%; between about 30% and about 95%; between about 30% and about 99%; between about 40% and about 95%; between about 40% and about 99%; between about 50% and about 95%; between about 50% and about 99%; between about 60% and about 95%; between about 60% and about 99%; between about 70% and about 95%; between about 70% and about 99%; between about 80% and about 95%; between about 80% and about 99%; between about 90% and about 95%; or between about 90% and about 99%.

[0123] In certain embodiments, the multispecific antibody is concentrated after chromatography (e.g., after multimode chromatography). In certain non-limiting embodiments, for example, the concentration method includes ultrafiltration and diafiltration (UFDF). In certain embodiments, the concentration of the concentrated multispecific antibody is one of the following: about 10 mg / mL, about 20 mg / mL, about 30 mg / mL, about 40 mg / mL, about 50 mg / mL, about 60 mg / mL, about 70 mg / mL, about 80 mg / mL, about 90 mg / mL, about 100 mg / mL, about 110 mg / mL, about 120 mg / mL, about 130 mg / mL, about 140 mg / mL, about 150 mg / mL, about 160 mg / mL, about 170 mg / mL, about 180 mg / mL, about 190 mg / mL, about 200 mg / mL, or about 300 mg / mL. In certain embodiments, the concentration of the multispecific antibody is between approximately 10 mg / mL and approximately 20 mg / mL, between approximately 20 mg / mL and approximately 30 mg / mL, between approximately 30 mg / mL and approximately 40 mg / mL, between approximately 40 mg / mL and approximately 50 mg / mL, between approximately 50 mg / mL and approximately 60 mg / mL, between approximately 60 mg / mL and approximately 70 mg / mL, between approximately 70 mg / mL and approximately 80 mg / mL, between approximately 80 mg / mL and approximately 90 mg / mL, between approximately 90 mg / mL and approximately 100 mg / mL, between approximately 100 mg / mL and approximately 110 mg / mL, and approximately 1 The dosage is between 10 mg / mL and approximately 120 mg / mL, between approximately 120 mg / mL and approximately 130 mg / mL, between approximately 130 mg / mL and approximately 140 mg / mL, between approximately 140 mg / mL and approximately 150 mg / mL, between approximately 150 mg / mL and approximately 160 mg / mL, between approximately 160 mg / mL and approximately 170 mg / mL, between approximately 170 mg / mL and approximately 180 mg / mL, between approximately 180 mg / mL and approximately 190 mg / mL, between approximately 190 mg / mL and approximately 200 mg / mL, or between approximately 200 mg / mL or 300 mg / mL.

[0124] In certain embodiments, the methods described herein further include combining a purified polypeptide with a pharmaceutically acceptable carrier. In certain embodiments, the multispecific antibody is formulated into a pharmaceutical product by ultrafiltration / diafiltration.

[0125] In certain embodiments, the methods provided herein generate compositions comprising multispecific antibodies with a purity exceeding approximately 50%, approximately 55%, approximately 60%, approximately 65%, approximately 70%, approximately 75%, approximately 80%, approximately 85%, approximately 90%, or approximately 95%. In certain embodiments, the multispecific antibodies in the composition have a purity higher than approximately 96%, approximately 97%, approximately 98%, or approximately 99%.

[0126] In certain embodiments, the method provided herein generates a composition comprising a multispecific antibody containing mispairing antibodies in amounts of about 0.1% or less, about 0.2% or less, about 0.3% or less, about 0.4% or less, about 0.5% or less, about 0.6% or less, about 0.7% or less, about 0.8% or less, about 0.9% or less, about 1% or less, about 1.5% or less, about 2% or less, about 2.5% or less, about 3% or less, about 3.5% or less, about 4% or less, about 4.5% or less, about 5% or less, about 5.5% or less, about 6% or less, about 6.5% or less, about 7% or less, about 7.5% or less, about 8% or less, about 8.5% or less, about 9% or less, about 9.5% or less, or about 10% or less.

[0127] In certain embodiments, the Disclosure provides a composition comprising a multispecific antibody purified by any one of the methods disclosed herein. In certain embodiments, the multispecific antibody in the composition has a purity higher than any of the following: about 50%, about 55%, about 60%, about 65%, 70%, about 75%, about 80%, about 85%, about 90%, or about 95%. In certain embodiments, the multispecific antibody in the composition has a purity higher than any of the following: about 96%, about 97%, about 98%, or about 99%. In certain embodiments, the composition containing the multispecific antibody contains approximately 0.1%, approximately 0.2%, approximately 0.3%, 0.4%, approximately 0.5% or less, approximately 0.6% or less, approximately 0.7% or less, approximately 0.8% or less, approximately 0.9% or less, approximately 1% or less, approximately 1.5% or less, approximately 2% or less, approximately 2.5% or less, approximately 3% or less, approximately 3.5% or less, approximately 4% or less, approximately 4.5% or less, approximately 5% or less, approximately 5.5% or less, approximately 6% or less, approximately 6.5% or less, approximately 7% or less, approximately 7.5% or less, approximately 8% or less, approximately 8.5% or less, approximately 9% or less, approximately 9.5% or less, or approximately 10% or less of any of the mispaired antibodies.

[0128] In certain embodiments, the Disclosure provides a composition comprising a multispecific antibody purified by any one of the methods disclosed herein. In certain embodiments, the multispecific antibody is a bispecific antibody, such as a knob-in-hole (KiH) antibody, e.g., a KiH bispecific antibody. In certain embodiments, the multispecific antibody is a multispecific CrossMab antibody, e.g., a bispecific CrossMab antibody.

[0129] In certain embodiments, the methods disclosed herein include the removal of host cell proteins, leached protein A, nucleic acids, cell culture components, or viral impurities from a composition.

[0130] multispecific antibodies In certain non-limiting embodiments, the disclosure provides a method for purifying a multispecific antibody, such as a multispecific CrossMab antibody. A multispecific antibody is a monoclonal antibody having binding specificity to at least two different sites. In certain embodiments, the multispecific antibody is produced by the same host cell.

[0131] In certain embodiments, the Disclosure includes methods for producing multispecific antibodies. For example, but not limited to, these techniques include recombinant co-expression of two immunoglobulin heavy-light chain pairs having different specificities (see Milstein and Cuello, Nature 305: 537 (1983), International Publication No. 93 / 08829, and Traunecker et al., EMBO J. 10: 3655 (1991)), the "knob-in-hole" operation (e.g., U.S. Patent No. 5,731,168), and "CrossMab" antibodies (e.g., European Patent No. 3126395B1). Multiple specific antibodies are produced by manipulating the electrostatic steering effect to create Fc-heterodimer molecules of antibodies (International Publication No. 2009 / 089004A1); crosslinking two or more antibodies or fragments (see, e.g., U.S. Patent No. 4676980 and Brennan et al., Science, 229: 81 (1985)); using leucine zippers to produce bispecific antibodies (see, e.g., Kostelny et al., J. Immunol., 148(5):1547-1553 (1992)); using "diabody" techniques to produce bispecific antibody fragments (see, e.g., Hollinger et al., Proc. Natl. Acad. Sci. USA, 90:6444-6448 (1993)); and using single-stranded Fv(sFv) dimers (see, e.g., Gruber et al., J. Immunol., 152:5368) (See 1994); and it can be prepared by preparing a trispecific antibody (see, for example, Tutt et al., J. Immunol. 147: 60 (1991)).

[0132] In certain embodiments, multispecific antibodies are described in International Publications 2009 / 080251, 2009 / 080252, 2009 / 080253, 2009 / 080254, 2010 / 112193, 2010 / 115589, 2010 / 136172, 2010 / 145792, and 2010 / 145793. In certain embodiments, multispecific antibodies include three or more functional antigen-binding sites, such as an "octopus antibody" (see, for example, U.S. Patent Publication 2006 / 0025576A1). In certain embodiments, the multispecific antibody is a "Dual Acting Fab" or "Dual Action Fab" (DAF) that includes an antigen-binding site that binds not only to a first epitope (e.g., on a first antigen) but also to another different epitope (e.g., on the first antigen or a second different antigen) (see, for example, U.S. Patent Publication 2008 / 0069820; Bostrom et al. (2009) Science, 5921, 1610-1614).

[0133] Conventionally, recombinant production of multispecific antibodies (e.g., bispecific antibodies) may be based on the co-expression of two immunoglobulin heavy-chain-light-chain pairs, in which case two or more heavy chains have different specificities (Milstein and Cuello, Nature, 305: 537 (1983)). Due to the random sorting of immunoglobulin heavy and light chains, these hybridomas (quadromas) may produce a mixture of at least 10 different antibody molecules, of which only one has the correct bispecific structure. Purification of the correct molecule, usually performed by affinity chromatography, is cumbersome and results in low product yields. Similar procedures are disclosed in International Publication No. 93 / 08829, published May 13, 1993, and in Traunecker et al., EMBO J., 10: 3655 (1991).

[0134] Furthermore, the production of multispecific antibodies presents unique challenges. For example, the production of bispecific antibodies requires the dimerization of two different heavy / light chain subunits, each containing a different heavy chain and a different light chain. Thus, the production of bispecific antibodies requires the proper interaction of up to four peptide chains. Consequently, chain mispairing (e.g., homodimerization of identical heavy chain peptides, or improper association of heavy / light chains) is frequently observed. Mispairing variants of multispecific antibodies include incorrect pairing of heavy chains, pairing of a light chain with the wrong corresponding heavy chain, or undesirable pairing of light chains.

[0135] CrossMab antibody This disclosure provides a method for purifying a multispecific CrossMab antibody. A CrossMab antibody is a multispecific (i.e., at least bispecific) antibody in which the correct association of the light chain and its equivalent heavy chain is achieved by the exchange of the heavy chain domain and the light chain domain within the Fab of at least one antigen-binding region (Fab) of the multispecific antibody, such that no such exchange occurs in at least one other Fab fragment so as to avoid mispairing in at least two of these Fab fragments. In the case of a bispecific CrossMab antibody, the correct association of the light chain and its equivalent heavy chain is achieved by exchanging the heavy chain and light chain domains within one half of the Fab fragment of the bispecific antibody, while the other half remains unbound or undergoes a different exchange.

[0136] In this specification, the term “CrossMab antibody” refers to a multispecific antibody (or a suitable multispecific fragment thereof) in which either the variable region and / or constant region of the heavy chain and / or light chain are exchanged. For example, a CrossMab antibody may be any of the CrossMab antibodies described or claimed in International Publication Nos. 2009 / 080252, 2009 / 080253, 2009 / 080251, 2009 / 080254, 2010 / 136172, 2010 / 145792, and 2013 / 026831. The term “CrossMab” antibody is generally recognized in the art. For example, see Brinkmann, U. & Kontermann, R., MAbs 9(2): 182-212 (2017); Kontermann, R. & Brinkmann, U., Drug Discovery Today 20(7):838-846 (2015); Schaefer, W. et a, PNAS, 108 (2011) 11187-191; Klein, C. et al., MAbs 8(6):1010-1020 (2016); Klein, C. et al., MAbs 4(6):653-663 (2012).

[0137] In certain embodiments, a multispecific CrossMab antibody is a bispecific bivalent CrossMab antibody. A bispecific bivalent CrossMab antibody comprises a crossover antibody with three different chain compositions. In the first composition, the variable domains of the antibody's heavy and light chains are exchanged. That is, the antibody contains in one Fab region a peptide chain consisting of a light chain variable domain (VL) and a heavy chain constant domain (CH1), and a peptide chain consisting of a heavy chain variable domain (VH) and a light chain constant domain (CL). In the second composition, the constant domains of the antibody's heavy and light chains are exchanged in one Fab region, and the antibody contains in this Fab region a peptide chain consisting of a heavy chain variable domain (VH) and a light chain constant domain (CL), and a peptide chain consisting of a light chain variable domain (VL) and a heavy chain constant domain (CH1). In the third composition, the heavy chain of the antibody, containing a constant domain and a variable domain, is exchanged with the light chain of the antibody, containing a constant domain and a variable domain. In other words, an antibody comprises a peptide chain composed of a light chain variable domain (VH) and a heavy chain constant domain (VL), and a peptide chain composed of a heavy chain variable domain (VL) and a light chain constant domain (CH1).

[0138] In certain embodiments, the CrossMab antibody is a monoclonal antibody. In certain embodiments, the CrossMab antibody comprises its functional fragment, i.e., a fragment that preserves its multispecificity.

[0139] In certain embodiments, the Disclosure provides a method for purifying a multispecific CrossMab antibody from its mispaired variant. As used herein, the term “its mispaired variant” refers to a multispecific CrossMab antibody in which a heavy chain with swapped domains is paired with at least one mispaired light chain, as described above with respect to the CrossMab antibody. For example, but not limited to, at least one light chain of the variant does not pair with its complementary heavy chain. For example, an “unmodified” light chain containing CL and VL may mispair with an “unmodified” heavy chain having CH1 and VL, or an “unmodified” light chain containing CH1 and VL may mispair with an “unmodified” heavy chain having CH1 and VH. As used with respect to the CrossMab antibody, “complementary” domains refer to heavy chain domains and light chain domains that are normally paired. A “non-complementary” domain is a heavy chain domain and light chain domain that are mispaired. For example, but not limited to, a mispaired light chain in a heavy-chain domain-light chain domain pairing may refer to a light chain where the variable domain and / or constant domain of the light chain are exchanged, while the variable domain and / or constant domain of the heavy chain are not exchanged. Another example may refer to a situation where the variable domain and / or constant domain of the light chain are not exchanged, while the variable domain and / or constant domain of the heavy chain are exchanged. As used herein, the term “non-complementary” does not mean an incompletely assembled antibody, such as an antibody lacking one light chain or a fragment thereof (but not limited to this). In certain non-limiting embodiments, for example, the mispaired variant is a variant of a multispecific CrossMab antibody in which one or more light chains are paired with a non-complementary heavy chain.

[0140] In certain embodiments, the multispecific CrossMab antibody is bispecific, triplicate, or quadruplicate. In certain embodiments, the multispecific CrossMab antibody has two, three, or four specific antigen-binding sites. In certain embodiments, the multispecific CrossMab antibody is monovalent. In certain embodiments, the multispecific CrossMab antibody is bivalent.

[0141] In certain embodiments, the multispecific CrossMab antibody contains an Fc fragment. The presence of the Fc fragment is not limiting, but allows for the purification of the multispecific antibody using an Fc binding site such as protein A, protein G, or protein A / G. In certain embodiments, the multispecific CrossMab antibody may be IgG, IgE, IgM, IgA, or IgY. In certain embodiments, the multispecific CrossMab antibody is IgG. In certain embodiments, the Fc fragment of the multispecific antibody contains a modification that facilitates the association of a first Fc fragment subunit and a second Fc fragment subunit. In certain embodiments, the modification is in the first Fc fragment subunit. In certain embodiments, the modification is in the second Fc fragment subunit. In certain embodiments, the modification is in both the first and second Fc fragment subunits. In certain embodiments, the modification is in the CH3 domain of the Fc fragment. In certain non-limiting embodiments, modification of the first and second CH3 domains enables correct heterodimerization of the Fc fragment. In certain embodiments, the modified first CH3 domain heterodimerizes with the modified second CH3 domain by steric complementarity.

[0142] In certain embodiments, the modification is a "knob-into-hole" modification. In certain embodiments, the first Fc fragment contains a knob mutation and the second Fc fragment contains a hole mutation. In certain embodiments, the first Fc fragment contains a hole mutation and the second Fc fragment contains a knob mutation.

[0143] host cell This disclosure provides a method for purifying multispecific antibodies expressed in host cells. In certain embodiments, the host cell is a bacterium, yeast, or other fungal cell, insect cell, plant cell, or mammalian cell. In certain embodiments, the host cell is genetically modified to produce multispecific antibodies.

[0144] In certain embodiments, the host cell is a prokaryote (e.g., a Gram-negative or Gram-positive microorganism). For example, but not limited to, the host cell may be Escherichia coli, Bacillus subtilis, Bacillus licheniformis, or Pseudomonas aeruginosa. In certain embodiments, the host cell secretes a minimal amount of proteolytic enzyme. In certain embodiments, the host cell (e.g., an E. coli host cell) expresses one or more chaperones to facilitate antibody folding and assembly. In certain embodiments, the chaperone is one or more of FkpA, DsbA, or DsbC. In certain embodiments, the chaperone is expressed from an endogenous chaperone gene. In certain embodiments, the chaperone is expressed from an exogenous chaperone gene. In certain embodiments, the chaperone gene is an E. coli chaperone gene (e.g., the E. coli FkpA gene, the E. coli DsbA gene, and / or the E. coli DsbC gene).

[0145] In certain embodiments, prokaryotic host cells are transformed with an expression vector and cultured to promote the expression of multispecific antibodies.

[0146] In certain embodiments, the host cell is a eukaryote. For example, but not limited to, the host cell may be Saccharomyces cerevisiae, Pichia pastoris, Neurospora crassa, or Aspergillus niger. In certain embodiments, the eukaryotic host cell is a mammalian cell. In certain non-limiting embodiments, the mammalian host cell may be CHO cells, COS-7 cells, HEK 293 cells, BHK cells, VERO-76 cells, HELA cells, HepG2 cells, or W138 cells. In certain embodiments, the eukaryotic host cell is transformed with an expression vector and cultured to promote the expression of a multispecific antibody. In certain non-limiting embodiments, the present disclosure provides a method for producing and purifying a multispecific antibody. In certain embodiments, multispecific antibodies are produced by separately producing half-antibodies, each half-antibody containing a VH / VL unit that binds to a specific epitope (e.g., a different epitope on a single target, or different epitopes on two or more targets). In certain embodiments, each half-antibody is produced separately in a host cell. In certain embodiments, each half-antibody is produced in the same host cell. In certain embodiments, each half-antibody is produced together in the same host cell.

[0147] antigen / target molecule This disclosure provides a method for purifying multispecific antibodies capable of targeting various molecules. In certain embodiments, multispecific antibodies purified by the methods disclosed herein can target cytokines, cytokine-related proteins, or cytokine receptors. For example, but are not limited to, multispecific antibodies include 8MPI, 8MP2, 8MP38 (GDFIO), 8MP4, 8MP6, 8MP8, CSFI (M-CSF), CSF2 (GM-CSF), CSF3 (G-CSF), EPO, FGF1 (aFGF), FGF2 ((FGF)), FGF3 (int-2), FGF4 (HST), FGFS, FGF6 (HST-2), FGF7 (KGF), FGF9, FGF1 0, FGF11, FGF12, FGF12B, FGF14, FGF16, FGF17, FGF19, FGF20, FGF21, FGF23, IGF1, IGF2, IFNA1, IFNA2, IFNA4, IFNA5, IFN A6, IFNA7, IFN81, IFNG, IFNWI, FEL1, FEL1(EPSELON), FEL1(ZETA), IL1A, IL1B, IL2, IL3, IL4, IL5, IL6, IL7, IL8, IL9, IL1 0, IL 11, IL 12A, IL 12B, IL 13, IL 14, IL 15, IL 16, IL 17, IL 17B, IL 18, IL 19, IL20, IL22, IL23, IL24, IL25, IL26, IL27, IL28A, IL28B, IL29, IL30, IL33, PDGFA, PDGFB, TGFA, TGFB1, TGFB2, TGFBb3, LTA(TNF- (), LTB, TNF (TNF-a), TNFSF4 (0X40 ligand), TNFSF5 (CD40 ligand), TNFSF6 (FasL), TNFSF7 (CD27 ligand), TNFSF8 (CD30 ligand), TNFSF9 (4-1BB ligand), TNFSF10 (TRAIL), TNFSF11 (TRANCE), TNFSF12 (APO3L), TNFSF13 (April), TNFSF13B, TNFSF14 (HVEM-L), TNFSF15 (VEGI), TNFSF18, HGF (VEGF D), VEGF, VEGFB, VEGFC, IL1R1, IL1R2, IL1RL1, IL1RL2, IL2RA, IL2RB, IL2RG, IL3RA, IL4R, IL5RA, IL6R, IL7R, IL8RA, IL8RB, IL9R, IL10RA, IL10RB, IL 11RA, IL12RB1, IL12RB2, IL13RA1, IL13RA2, IL15RA, It can target IL17R, IL18R1, IL20RA, IL21R, IL22R, IL1HY1, IL1RAP, IL1RAPL1, IL1RAPL2, IL1RN, IL6ST, IL18BP, IL18RAP, IL22RA2, AIF1, HGF, LEP (leptin), PTN, and THPO.

[0148] In certain embodiments, multispecific antibodies purified by the methods disclosed herein can target chemokines, chemokine receptors, or chemokine-related proteins. For example, but not limited to, multispecific antibodies include CCL1(1-309), CCL2(MCP-1 / MCAF), CCL3(MIP-1a), CCL4(MIP-1(3), CCLS(RANTES), CCL7(MCP-3), CCL8(mcp-2), CCL11(eotaxin), CCL13(MCP-4), CCL15(MIP-IS), CCL16(HCC-4), CCL17(TARC), CCL18(PARC), CCL19(MDP-3b), CCL20(MIP-3a), CCL21(SLC / Exodus-2), CCL22(MDC / STC-1), CCL23(MPIF-1), CCL24(MPIF-2) / Eotaxin-2), CCL25(TECK), CCL26(Eotaxin-3), CCL2?(CTACK / ILC), CCL28, CXCLI(GROI), CXCL2(GR02), CXCL3(GR03), CXCLS(ENA-78), CXCL6(GCP-2), CXCL9(MIG), CXCL10(IP 10), CXCL11(1-TAC), CXCL12(SDFI), CXCL13, CXCL14, CXCL16, PF4(CXCL4), PPBP(CXCL7), CX3CL1(SCYDI), SCYEI, XCLI(Lymphotactin), XCL2(SCM-I(3), BLRI(MDR15), CCBP2(D6 / JAB61 ), CCR1(CKRI / HM145), CCR2(mcp-IRB IRA), CCR3(CKR3 / CMKBR3), CCR4, CCRS(CMKBR5 / ChemR13), CCR6(CMKBR6 / CKR-L3 / STRL22 / DRY6), CCRI(CKR7 / EBII), CCR8(CMKBR8 / TERUCKR-L1), CCR9 (GPR-9-6), CCRL1 (VSHK1), CCRL2 (L-CCR), XCR1 (GPR5 / CCXCR1), CMKLR1, CMKOR1 (RDC1), CX3CR1 (V28), CXCR4, GP R2 (CCR10), GPR31, GPR81 (FKSG80), CXCR3 (GPR9 / CKR-L2), CXCR6 (TYMSTR / STRL33 / Bonzo), HM74, IL8RA (IL8Ra), IL8RB (IL8 R(), LTB4R(GPR16), TCP10, CKLFSF2, CKLFSF3, CKLFSF4, CKLFSFS, CKLFSF6, CKLFSF7, CKLFSF8, BDNF, C5R1, CSF3, GRCC10(C10), EPO, FY(DARC), GDFS, HDF1, HDFla, DL8, PRL, RGS3, RGS13, SDF2, SLIT2, TLR2, TLR4, TREM1, TREM2, and VHL can be targeted.

[0149] In certain non-limiting embodiments, for example, the multispecific antibodies purified by the methods disclosed herein include ABCF1, ACVR1, ACVR1B, ACVR2, ACVR2B, ACVRL1, ADORA2A, Aggrecan, AGR2, AICDA, AIF1, AIG1, AKAP1, AKAP2, AMH, AMHR2, ANGPTL, ANGPT2, ANGPTL3, ANGPTL4, ANPEP, APC, APOC1, AR, AZGP1 (sub-). Lead-a-glycoprotein), B7.1, B7.2, BAD, BAFF (BLys), BAG1, BAIl, BCL2, BCL6, BDNF, BLNK, BLRI (MDR15), BMP1, BMP2, BMP3B (GDF10), BMP4, BMP6, BMP8, BMPR1A, BMPR1B, BMPR2, BPAG1 (plectin), BRCAl, Cl9orf10 (IL27w), C3, C4A, C5, C5R1, CA125, CA15-3, CA19- 9, CANT1, CASP1, CASP4, CAV1, CCBP2(D6 / JAB61), CCL1(1-309), CCL11(eotaxin), CCL13(MCP-4), CCL15(MIP18), CCL16(HCC -4), CCL17(TARC), CCL18(PARC), CCL19(MIP-3(3), CCL2(MCP-1), MCAF, CCL20(MIP-3a), CCL21(MTP-2), SLC, Exodus-2, CCL 22(MDC / STC-1), CCL23(MPIF-1), CCL24(MPIF-2 / eotaxin-2), CCL25(TECK), CCL26(eotaxin-3), CCL2?(CTACK / ILC), CCL28, CCL3(MTP-Ia), CCL4(MDP-I(3), CCL5(RANTES), CCL7(MCP-3), CCL8(mcp-2), CCNAl, CCNA2, CCND1, CCNE1, CCNE2, CCR1(CKRI / HM145), CCR2(mcp-IR(3 / RA), CCR3(CKR / CMKBR3), CCR4, CCR5(CMKBR5 / ChemR13), CCR6(CMKBR6 / CKR-L3 / STRL22 / DRY6), CCR7(CKBR7 / EBI1), CCR8(CMKBR8 / TERUCKR-L1), CCR9(GPR-9-6), CCRL1(VSHK1),CCRL2(L-CCR)、CD11a、CD13、CD164、CD19、CD1C、CD20、CD200、CD22、CD23、CD24、CD28、CD3、CD30、CD31、CD33、CD34、CD35、CD37、CD38、CD39、CD3E、CD3G、CD3Z、CD4、CD40、CD40L、CD41、CD44、LCA / CD45、CD45RA、CD45RB、CD45RO、CD5、CD52、CD69、CD7、CD71、CD72、CD74、CD79A、CD79B、CD8、CD80、CD81、CD83、CD86、CD95 / Fas、CD99、CD100、CD106、CDH1(E-カドヘリン)、CD9 / p24、CDH10、CD11a、CD11c、CD13、CD14、CD19、CD20、CDH12、CDH13、CDH18、CDH19、CDH2O、CDH5、CDH7、CDH8、CDH9、CDK2、CDK3、CDK4、CDK5、CDK6、CDK7、CDK9、CDKN1A(p21 / WAF1 / Cipl)、CDKN1B(p27 / Kipl)、CDKN1C、CDKN2A(P16INK4a)、CDKN2B、CDKN2C、CDKN3、CEA、CEBPB、CER1、CHGA、CHGB、Chitinase、CHST10、CKLFSF2、CKLFSF3、CKLFSF4、CKLFSF5、CKLFSF6、CKLFSF7、CKLFSF8、CLDN3、CLDN7(クローディン-7)、CLN3、CLU(クラステリン)、C-MET、CMKLR1、CMKOR1(RDC1)、CNR1、COL 18A1、COL1A1、COL4A3、COL6A1、CR2、CRP、CSFI(M-CSF)、CSF2(GM-CSF)、CSF3(GCSF)、CTLA4、CTNNB1(b-カテニン)、CTSB(カテプシンB)、CTSD(カテプシンD)、CX3CL1(SCYDI)、CX3CR1(V28)、CXCL1(GRO1)、CXCL10(IP-10)、CXCL11(I-TAC / IP-9)、CXCL12(SDF1)、CXCL13、CXCL14、CXCL16、CXCL2(GRO2)、CXCL3(GRO3)、CXCL5(ENA-78 / LIX)、CXCL6(GCP-2)、CXCL9(MIG)、CXCR3(GPR9 / CKR-L2)、CXCR4、CXCR6(TYMSTR / STRL33 / Bonzo), CYB5, CYCl, CYSLTR1, cytokeratin, DAB2IP, DES, DKFZp451J0118, DNCLI, DPP4, E2F1, ECGF1, EDG1, EFNAl, EFNA3, EFNB2, EGF, EGFR, ELAC2, ENG, ENO1, ENO2, ENO3, EPHB4, EPO, ERBB2(Her-2), EREG, ERK8, ESR1, estrogen receptor, progesterone receptor, ESR2, F3(TF), FADD, FasL, FASN, FCER1A, FCER2, FCGR3A, FGF, FGF1(aFGF), FGF10, FGF11, FGF12, FGF12B, FGF13, FGF14, FGF16, FGF17, FGF18, FGF19, FGF2(bFGF), FGF20, FGF21, FGF22, FGF23, FGF3(int-2), FGF4(HST), FGF5, FGF6(HST-2), FGF7(KGF), FGF8, FGF9, FGFR1, FGFR3, FIGF(VEGFD), FELL(EPSILON), Fibrin, FIL1(ZETA), FLJ12584, FLJ 25530, FLRTI (fibronectin), FLT1, FOS, FOSL1 (FRA-1), FY (DARC), GABRP (GABAa), GAGEB1, GAGEC1, GALNAC4S-6ST, GATA3, GDF5, GFIl, GGT1, GM-CSF, GNASI, GNRHI, GPR2 (CCR10), GPR31, GPR44, GPR81 (FKSG80), GRCCIO (C10), GRP, GSN (gelsolin), GSTP1, HAVCR2, HDAC4, HDAC5, HDAC7A, HDAC9, HGF, HIF1A, HOPI Histamine and histamine receptors, HLA-A, HLA-DRA, HM74, HMOXI, HPV protein, HUMCYT2A, ICEBERG, ICOSL, 1D2, IFN-a, IFNA1, IFNA2, IFNA4, IFNA5, IFNA6, IFNA7, IFNB1, IFN-gamma, ITGB7, DFNW1, IBP1, IGF1, IGF1R, IGF2, IGFBP2, IGFBP3, IGFBP6, IL-1, IL1O, IL1ORA, IL1ORB, IL11, IL11RA, IL-12, IL12A, IL12B, IL12RB1,IL12RB2, IL13, IL13RAl, IL13RA2, IL14, IL15, IL15RA, IL16, IL17, IL17B, IL17C, IL17R, IL18, IL18BP, IL18R1, IL18RAP, IL19, ILIA, IL1B, IL 1F1O, IL1F5, IL1F6, IL1F7, IL1F8, IL1F9, IL1HY1, IL1R1, IL1R2, IL1RAP, IL1RAPL1, IL1RAPL2, IL1RL1, IL1RL2, ILIRN, IL2, IL20, IL20RA, IL21 R, IL22, IL22R, IL22RA2, IL23, IL24, IL25, IL26, IL27, IL28A, IL28B, IL29, IL2RA, IL2RB, IL2RG, IL3, IL30, IL3RA, IL33, IL4, IL4R, IL5, IL5RA, IL6, IL6R, IL6ST (glycoprotein 130), P-glycoprotein, EL7, EL7R, EL8, IL8RA , DL8RB, IL8RB, DL9, DL9R, DLK, INHA, INHBA, INSL3, INSL4, IRAK1, ERAK2, ITGA1, ITGA2, ITGA3, ITGA6(a6 integrin), ITGAV, ITGB3, ITGB4 (b4 integrin), JAG1, JAK1, JAK3, JUN, K6HF, KAII, KDR, keratin, KITLG, KLF5 (GC Box BP), KLF6, KLKIO, KLK12, KLK13, KLK14, KLK15, KLK3, KLK4, KLK5, KLK6, KLK9, KRT1, KRT19 (Keratin 19) KRT2A, KHTHB6 (hair-specific H-keratin), kappa light chain, lambda light chain, LAMAS, LEP (leptin), Lingo-p75, Lingo-Troy, LPS, LTA (TNF-b), LTB, LTB4R (GPR16), LTB4R2, LTBR, ​​LEWIS-xMACMARCKS, MAG or Omgp, MAP2K7 (c-Jun), MDK, MIB1, melanosome protein, midkine, MEF, MIP-2, MKI67, (Ki-67), MMP2, MMP9, MS4A1, MSMB, MT3 (metallothionectin-111), MTSS1, MUC1 (mucin), MYC, MY088, NCK2, Neurocan, NFKB1, NFKB2, NGFB (NGF), NGFR,NgR-Lingo, NgR- Nogo66(Nogo), NgR-p75, NgR-Troy, NME1(NM23A), NOX5, NPPB, NR0B1, NROB2, NR1D1, NR1D2, NR1H2, NR1H3, NR1H4, NR112, NR113, NR2C1 , NR2C2, NR2E1, NR2E3, NR2F1, NR2F2, NR2F6, NR3C1, NR3C2, NR4A1, NR4A2, NR4A3, NR5A1, NR5A2, NR6A1, NRP1, NRP2, NT5E, NTN4, ODZI, O PRD1, P2RX7, PAP, PART1, PATE, PAWR, PCA3, PCNA, POGFA, POGFB, PECAM1, PF4(CXCL4), PGF, PGR, phosphacan, PIAS2, PIK3CG, PLAU(uPA), PL G, PLXDC1, PPBP(CXCL7), PPID, PRI, PRKCQ, PRKDI, PRL, PROC, PROK2, PSA, PSAP, PSCA, PTAFR, PTEN, PTGS2(COX-2), PTN, p53, RAC2(p21 Rac2), RAS, Rb, RARB, RGSI, RGS13, RGS3, RNF110 (ZNF144), ROBO2, S100A2, SCGB1D2 (Lipophyllin B), SCGB2A1 (Mammaglobin 2), SCGB2A2 (Mammaglobin 1), SCYEI (Endothelial Monocyte-Activating Cytokine), S-100 SDF2, SERPINAl, SERPINA3, SERP1NB5 (Maspin), SERPINE1 (PAI-1), SERPDMF1, SHBG, SLA2, SLC2A2, SLC33A1, SLC43A1, SLIT2, SPPI, SPRR1B (Sprl), ST6GAL1, STABI, STATE, STEAP, STEAP2, TB4R2, TBX21, TCPIO, TOGFI, TEK, TGFA, TGFBI, transmembrane or cell surface tumor-specific antigens (TAAs), such as TAAs listed in U.S. Patent No. 7,521,541, TAU, TGFB1II, TGFB2, TGFB3, TGFBI, TGFBRI, TGFBR2, TGFBR3, THIL, THBSI (thrombospongin-1), THBS2, THBS4, THPO, TIE (Tie-1) ), TMP3, tissue factor, TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8,TLR9, TLR10, Tn antigen TNF, TNF-a, TNFAEP2 (B94 ), TNFAIP3, TNFRSFIIA, TNFRSF1A, TNFRSF1B, TNFRSF21, TNFRSF5, TNFRSF6 (Fas), TNFRSF7, TNFRSF8, TNFRSF9, TNFSF10 (TRAIL), TNFSF11 (TRANCE), TNFSF12 (AP03L), TNFSF13 (April), TNFSF13B, TNFSF14 (HVEM-L), TNFSF15 (VEGI), TNFSF18, TNFSF4 (0X40 ligand), TNFSF5 (CD40 ligand), TNFSF6 (Fast), TNFSF7 (CD27 ligand), TNFSFS (CD30 ligand), TNFSF9 (4-1 It can target BB ligand, TOLLIP, Toll-like receptor, TOP2A (topoisomerase Ea), TP53, TPM1, TPM2, TRADD, TRAF1, TRAF2, TRAF3, TRAF4, TRAFS, TRAF6, TREM1, TREM2, TRPC6, TSLP, TWEAK, ubiquitin, VEGF, VEGFB, VEGFC, versican, VHL C5, vimentin, VLA-4, XCL1 (lymphotactin), XCL2 (SCM-1b), XCRI (GPRS / CCXCRI), Yy1, and ZFPM2.

[0150] In certain non-limiting embodiments, for example, the multispecific antibodies purified by the methods disclosed herein include CD proteins, e.g., CD3, CD4, CD8, CD16, CD19, CD20, CD34, CD64, CD200 members of the ErbB receptor family such as EGF receptor, HER2, HER3, or HER4 receptor, cell adhesion molecules, e.g., LFA-1, Macl, p150.95, VLA-4, ICAM-1, VCAM, alpha-4 / beta-7 integrands. Glyn and alpha-v / beta-3 integrins (including their alpha or beta subunits) (e.g., anti-CD11, anti-CD18, or anti-CD11b antibodies), growth factors such as VEGF (VEGF-A), FGFR, Angl, KLB, VEGF-C, tissue factor (TF), alpha interferon (alpha-IFN), TNF-alpha, interleukins such as IL-1 beta, IL-3, IL-4, IL-5, IL-S, IL-9, IL-13, IL-17 AF, IL-1S, IL-13, IL-13R alpha-1, IL-13R alpha-2, IL-14, IL-4R, IL-5R, IL-9R, IgE, blood group antigens, flk2 / flt3 receptors, obesity (OB) receptors, mpl receptors, CTLA-4, RANKL, RANK, RSV F protein, protein C, BR3, etc. can be targeted.

[0151] In certain non-limiting embodiments, for example, a multispecific antibody purified by the method disclosed herein can target low-density lipoprotein receptor-associated protein (LRP)-1 or LRP-8 or transferrin receptor, and at least one target selected from the group consisting of 1) beta-secretase (BACE1 or BACE2), 2) alpha-secretase, 3) gamma-secretase, 4) tau-secretase, 5) amyloid precursor protein (APP), 6) death receptor 6 (DR6), 7) amyloid beta peptide, 8) α-synuclein, 9) parkin, 10) huntingtin, 11) p75 NTR, and 12) caspase-6.

[0152] In certain non-limiting embodiments, for example, a multispecific antibody purified by the method disclosed herein can target at least two target molecules selected from the group consisting of: IL-1 alpha and IL-1 beta, IL-12 and IL-1S, IL-13 and IL-9, IL-13 and IL-4, IL-13 and IL-5, IL-5 and IL-4, IL-13 and IL-1 beta, IL-13 and IL-25, IL-13 and TARC, IL-13 and MDC, IL-13 and MEF, IL-13 and TGF, IL-13 and LHR agonist, IL-12 and TWEAK, IL-13 and CL25, IL-13 and SPRR2a, IL-13 and SPRR2b, IL-13 and ADAMS, IL-13 and PED2, IL13 and IL17, IL13 and IL4, IL13 and IL33, IL17A and IL17F, CD3 and CD19, CD138 and CD20, CD138 and CD40, CD19 and CD20, CD20 and CD3, CD3S and CD13S, CD3S and CD20, CD3S and CD40, CD40 and CD20, CD-S and IL-6, CD20 and BR3, TNF Alpha and TGF-Beta, TNF Alpha and IL-1 Beta, TNF Alpha and IL-2, TNF Alpha and IL-3, TNF Alpha and IL-4, TNF Alpha and IL-5, TNF Alpha and IL-6, TNF Alpha and IL-8, TNF Alpha and IL-9, TNF Alpha and IL-10, TNF Alpha and IL-11, TNF Alpha and IL-12, TNF Alpha and IL-13, TNF Alpha and IL-14, TNF Alpha and IL-15, TNF Alpha and IL-16, TNF Alpha and IL-17, TNF Alpha and IL-18, TNF Alpha and IL-19, TNF Alpha and IL-20, TNF Alpha and IL-23, TNF Alpha and IFN Alpha, TNF Alpha and CD4, TNF Alpha and VEGF, TNF Alpha and MIF, TNF Alpha and ICAM-1, TNF Alpha and PGE4, TNF Alpha and PEG2, TNF Alpha and RANK ligand, TNF Alpha and Te38, TNF Alpha and BAFF, TNF Alpha and CD22, TNF Alpha and CTLA-4, TNF Alpha and GP130, TNF a and IL-12p40, FGFR1 and KLB, VEGF and HER2, VEGF-A and HER2, VEGF-A and PDGF, HER1 and HER2, VEGFA and ANG2, VEGF-A and VEGF-C, VEGF-C and VEGF-D, HER2 and DRS, VEGF and IL-8, VEGF and MET, VEGFR and MET receptor, EGFR and MET, VEGFR and EGFR, HER2 and CD64, HER2 and CD3, HER2 and CD16, HER2 and HER3, EGFR(HER1) and HER2, EGFR and HER3, EGFR and HER4, IL-14 and IL-13, IL-13 and CD40L, IL4 and CD40L, TNFR1 and IL-1R, TNFR1 and IL-6R and TNFR1 and IL-18R, EpCAM and CD3, MAPG and CD28, EGFR and CD64, CSPGs and RGM A, CTLA-4 and BTN02, IGF1 and IGF2, IGF1 / 2 and Erb2B, MAG and RGM A, NgR and RGM A, NogoA and RGM A, OMGp and RGM A, POL-1 and CTLA-4, and RGM A and RGM B.

[0153] Pharmaceutical preparation and method for preparing the pharmaceutical preparation This disclosure provides a formulation comprising a multispecific antibody purified by the method described herein, and a method for producing the formulation. For example, the purified polypeptide (e.g., a multispecific antibody) can be combined with a pharmaceutically acceptable carrier.

[0154] In some embodiments, polypeptide formulations can be prepared for storage in the form of lyophilized formulations or aqueous solutions by mixing polypeptides of desired purity with any pharmaceutically acceptable carrier, additive, or stabilizer (Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980)).

[0155] As used herein, “carriers” include pharmaceutically acceptable carriers, additives, or stabilizers that are nontoxic to cells or mammals to which they are exposed at the doses and concentrations used. In many cases, physiologically acceptable carriers are pH-buffered aqueous solutions.

[0156] Permitted carriers, additives, or stabilizers are buffers such as phosphoric acid, citrate, and other organic acids, which are non-toxic to the recipient at the doses and concentrations used; antioxidants including ascorbic acid and methionine; preservatives (e.g., octadecyldimethylbenzylammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl, or benzyl alcohol; alkylparabens such as methyl or propylparaben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol) ); low molecular weight (less than approximately 10 residues) polypeptides; proteins such as serum albumin, gelatin, or immunoglobulin; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrin; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose, or sorbitol; salt-forming counterions such as sodium; metal complexes (e.g., Zn-protein complexes); and / or TWEEN TM PLURONICS TM Alternatively, it may contain nonionic surfactants such as polyethylene glycol (PEG).

[0157] In some embodiments, the polypeptide in the polypeptide formulation maintains its functional activity.

[0158] Formulations used for in vivo administration must be sterile. This can be easily achieved by filtration through a sterile filtration membrane.

[0159] The formulations described herein may contain two or more active compounds necessary for the specific indication being treated, preferably active compounds having complementary activities that do not adversely affect each other. For example, it is desirable to include another polypeptide (e.g., an antibody) in addition to the polypeptide in one formulation. Alternatively or additionally, the composition may further contain chemotherapeutic agents, cytotoxic agents, cytokines, growth inhibitors, antihormonal agents, and / or cardioprotective agents. Such molecules are appropriately present in combination in amounts effective for the intended purpose.

[0160] manufactured goods This disclosure provides a product comprising a formulation containing a multispecific antibody and / or a polypeptide purified by the method described herein. The product may include a container containing the polypeptide and / or polypeptide formulation. In certain embodiments, the product includes (a) a container containing a composition comprising the polypeptide and / or polypeptide formulation described herein, and (b) a package insert having instructions for administering the formulation to a subject.

[0161] In certain embodiments, the product includes a container and a label or accompanying documentation about or relating to the container. Suitable containers include, for example, bottles, vials, syringes, etc. Containers may be formed from a variety of materials, such as glass or plastic. Containers may hold or contain the formulation and have a sterile access port (for example, the container may be an intravenous solution bag or vial with a stopper that can be punctured by a subcutaneous injection needle). At least one active substance in the composition is a polypeptide. The label or accompanying documentation indicates that the composition is intended for use in the target area by providing specific instructions regarding the dosage and interval of the polypeptide provided and any other drugs. The product may further include other materials desirable from a commercial and user perspective, including other buffers, diluents, filters, needles, and syringes. In some embodiments, the container is a syringe. In some embodiments, the syringe is further housed in an infusion device. In some embodiments, the infusion device is a self-injector.

[0162] The term "package insert" is used to refer to the instructions typically included in the marketed packaging of a therapeutic drug, which include information on indications, usage, dosage, administration, contraindications, other therapeutic drugs to be used in combination with the packaged product, and / or warnings regarding the use of such therapeutic drugs.

[0163] Exemplary embodiments of the subject matter disclosed in this book In certain embodiments, the present disclosure relates to a method for purifying a multispecific antibody, comprising: contacting a composition comprising a multispecific antibody and a mispair variant thereof with a multimode chromatography material under conditions in which the mispair variant preferentially binds to the multimode chromatography material compared to the multispecific antibody, wherein the multispecific antibody comprises: a first antigen-binding region that specifically binds to a first antigen, comprising the light and heavy chains of the antibody that binds to the first antigen; and a second antigen-binding region that specifically binds to a second antigen, comprising the light and heavy chains of the antibody that binds to the second antigen, wherein variable domains VL and VH are substituted for each other in the second antigen-binding region. The method involves contacting a multimode chromatography material with a composition comprising: a first antigen-binding region comprising the heavy chain of an antibody that binds to a first antigen and a peptide comprising a heavy chain variable domain (VH) and a light chain constant domain (CL) of an antibody that binds to a second antigen, and a second antigen-binding region comprising the light and heavy chains of an antibody that binds to a second antigen, wherein the variable domains VL and VH are substituted for each other in the second antigen-binding region; and a multimode chromatography material comprising an anion-exchangeable functional group and a hydrophobic-interacting functional group; and recovering an elute containing a multispecific antibody and a reduced amount of the mispair variant.

[0164] In certain embodiments of the methods described herein, the hydrophobic interacting functional groups include alkyl groups, alkenyl groups, alkynyl groups, phenyl groups, benzyl groups, or any combination thereof.

[0165] In certain embodiments of the methods described herein, the functional group capable of hydrophobic interaction includes a benzyl group.

[0166] In certain embodiments of the methods described herein, the anion-exchangeable functional group includes a positively charged group. In certain embodiments of the methods described herein, the positively charged group is a quaternary ammonium ion.

[0167] In certain embodiments of the methods described herein, the multimode chromatography material comprises N-benzyl-N-methylethanolamine.

[0168] In certain embodiments of the methods described herein, the multimode chromatography material comprises a Capto™ Adhere resin.

[0169] In certain embodiments of the methods described herein, the multimode chromatography material is Capto TM Contains Adhere ImpRes resin.

[0170] In certain embodiments of the methods described herein, the elution of multimode chromatography is gradient elution. In certain embodiments of the methods described herein, the gradient elution includes a pH gradient.

[0171] In certain embodiments of the methods described herein, the method includes a capture chromatography step. In certain embodiments of the methods described herein, the capture chromatography step is an affinity chromatography step. In certain embodiments of the methods described herein, the affinity chromatography step is a protein A chromatography step, a protein L chromatography step, a protein G chromatography step, and a protein A / G chromatography step. In certain embodiments of the methods described herein, the affinity chromatography step is a protein A chromatography step. In certain embodiments of the methods described herein, the protein A chromatography step includes a chromatography material containing protein A bound to agarose. In certain embodiments of the methods described herein, the capture chromatography step and the multimode chromatography step are sequential. In certain embodiments of the methods described herein, the method includes a purification step after the multimode chromatography step. In certain embodiments of the methods described herein, a concentration step is included in which a multispecific antibody is concentrated.

[0172] In certain embodiments of the methods described herein, the multispecific antibody includes a knob-in-hole modification.

[0173] In certain embodiments of the methods described herein, the multispecific antibody and its mispair variant are produced in the same host cell culture. In certain embodiments of the methods described herein, the host cells of the host cell culture are prokaryotic or eukaryotic cells. In certain embodiments of the methods described herein, the host cells are eukaryotic cells. In certain embodiments of the methods described herein, the eukaryotic cells are yeast cells, insect cells, or mammalian cells. In certain embodiments of the methods described herein, the eukaryotic cells are CHO cells.

[0174] In certain embodiments, this disclosure relates to compositions comprising multispecific antibodies purified by the methods disclosed herein. In certain embodiments of the compositions described herein, the compositions comprising multispecific antibodies include a pharmaceutically acceptable carrier.

[0175] In certain embodiments, this disclosure relates to a product comprising a multispecific antibody purified by the method disclosed herein.

[0176] From the above description, it should be clear that the subject matter disclosed herein can be modified or altered for use in a variety of applications and conditions. Such embodiments are also within the scope of the following claims.

[0177] Any description of a variable in this specification that includes an enumeration of elements in any definition of a variable includes the definition of that variable as any single element or as a combination (or subcombination) of the enumerated elements. Any description of an embodiment in this specification includes an embodiment as any single embodiment or as an embodiment in combination with any other embodiment or part thereof.

[0178] All patent applications and publications referenced herein are incorporated herein by reference to the same degree as if each individual patent application and publication were specifically and individually indicated as being invoked by reference.

[0179] All features disclosed herein may be combined in any combination. Each feature disclosed herein may be replaced by an alternative feature that serves the same, equivalent, or similar purpose. Thus, unless otherwise expressly indicated, each disclosed feature is merely an example of a general set of equivalent or similar features.

[0180] The above description is considered sufficient to enable those skilled in the art to carry out the methods described herein and / or obtain the compositions described herein. The following examples and detailed description are provided for illustrative purposes only and are not limiting.

[0181] All references disclosed herein are expressly incorporated herein by reference. [Examples]

[0182] The above embodiments are provided for illustrative purposes only and are not intended to limit the scope of the invention in any way. In fact, various modifications other than those presented and described herein will be apparent to those skilled in the art from the above description and are included in the appended claims.

[0183] Given the general description given above, it is understood that various other embodiments may be implemented.

[0184] Example 1 One type of single-cell bispecific design is "CrossMab v2," which improves light-heavy chain pairing through a design utilizing a crossover of Fab domains. One possibility of light chain (LC) mispairing is the proximity of two variable heavy chain (VH) domains. While it is generally understood that in antibodies, the VH domain pairs only with the variable light (VL), the two VH domains in this LC mispairing can denature, potentially causing structural distortion in the LC mispair. Furthermore, the coposition of three negatively charged mutations on the heavy chain (HC, K147E, K213E) and the LC (Q124E) can confer a negatively charged patch to this LC mispairing Fab.

[0185] In this example, we demonstrate that the anion exchange component interacts with the negatively charged patch of the constant domain, and the hydrophobic interaction component binds to the hydrophobic residue revealed by the structural modification of the variable domain, allowing a multimode chromatography resin (e.g., anion exchange and hydrophobic interaction chromatography (MMAEX)) to bind to this LC mispair and clear it in downstream processes. Overall, this multimode chromatography improves the purification of multispecific antibodies.

[0186] raw material A bispecific antibody against antigen A / antigen B (aAgA / aAgB) was expressed in Chinese hamster ovary (CHO) cells as CrossMab v2, which has a domain crossover in the aAgB arm. The resulting harvested cell culture was purified by protein A affinity chromatography to capture the bispecific antibody and its product-related variants (e.g., unassembled half-antibodies, homodimers, and LC mispairs). The composition of the mixture was analyzed by reverse-phase HPLC and mass spectrometry and determined as shown in the table below. TIFF0007871252000002.tif45170

[0187] High-throughput screening Using an automated liquid processing system, the binding of raw materials to five different chromatography resins (including Capto Adhere (MMAEX resin)) was tested under various pH and buffer strength conditions. After incubation, analysis of the unbound fraction revealed the depletion of LC mispair variants under conditions that promoted anion exchange behavior (high pH) and hydrophobic binding (high salt concentration), as shown in Figure 3. Surprisingly, only the anion exchange resin and the hydrophobic interaction multimode chromatography resin were able to bind these LC mispair species.

[0188] Execution of confirmation column chromatography Using an Aekta chromatography system connected to a chromatography column with a Capto Adhere resin-packed bed, pH-adjusted raw materials were loaded onto the resin under strong binding conditions, and then eluted using a pH gradient from high pH (pH 8.6) to low pH (pH 5.5). Protein elution was observed as a main peak with a long tail (Figure 4). The peak and tail were collected as fractions and their compositions were analyzed.

[0189] Analysis of the recovered fraction's composition revealed that the main elution peak was rich in bispecific antibodies, while the post-peak tail was rich in LC mispairs. Figure 5 shows the mass spectra comparing the load material composition (load) with the fraction representing the bispecific main peak (Fraction 3) and the fraction representing the LC mispair-rich post-peak tail (Fraction 9).

[0190] To further evaluate the role of the method disclosed herein, pseudochromatograms showing the composition and concentration of the recovered and measured fractions were analyzed. As shown in Figure 6A, the main peak mainly contained the bispecific antibody, while the post-peak tail mainly contained the LC mispair variant. Other product-related variants were present at minor levels. When the pseudochromatograms of bispecificity and LC mispair were normalized (e.g., scaled to the same height) and superimposed, it became clearer that the method disclosed herein separates the bispecific antibody from the LC mispair variant (Figure 6B).

[0191] molecular structure research Next, to support the experimental results, 3D homology models of the Fab of this molecule were created for both correctly paired HC-LC combinations and mispaired HC-LC combinations. It was confirmed that the LC mispaired Fab exhibits a loosely denatured variable domain structure (since the VH domain is not considered to have affinity with other VH domains). Furthermore, since the three negatively charged amino acids are located on the protein surface, the constant domain can create a negatively charged patch on the protein surface.

[0192] Simulation structures of correctly paired species (Figures 7A and 7B) and LC mispaired species (Figures 7C and 7D). The LC mispaired species exhibiting the highest structural strain and negative charge clusters was removed (Figure 7C).

[0193] conclusion In single-cell bispecific design, some degree of LC mispairing is unavoidable. While it is extremely difficult to remove LC mispairing from correctly formed bispecificity, it is possible to design single-cell bispecificity in a way that improves the ability to remove specific LC mispairings when certain combinations of LC and HC result in product-related variants suspected of exhibiting risk (e.g., patient risk). The method disclosed herein can remove LC mispairing from Crossmab v2 bispecificity, as long as the mispairing is between crossed LC and non-crossed HC.

Claims

1. A method for purifying multispecific antibodies, a) Contacting a composition comprising a multispecific antibody and its mispair variant with a multimode chromatography material under conditions in which the mispair variant preferentially binds to the multimode chromatography material compared to the multispecific antibody, i) Multispecific antibodies, 1) A first antigen-binding region that specifically binds to a first antigen, comprising the light chain and heavy chain of an antibody that binds to the first antigen, 2) A second antigen-binding region that specifically binds to a second antigen, comprising the light chain and heavy chain of an antibody that binds to the second antigen, wherein the variable domains VL and VH are substituted for each other in the second antigen-binding region. Includes, ii) That mismatch variant is, 1) A first antigen-binding region comprising the heavy chain of an antibody that binds to a first antigen, and a peptide comprising the heavy chain variable domain (VH) and light chain constant domain (CL) of an antibody that binds to a second antigen, 2) A second antigen-binding region comprising the light and heavy chains of an antibody that binds to a second antigen, wherein the variable domains VL and VH in the second antigen-binding region are substituted for each other. Includes, iii) Multimode chromatography materials, 1) Functional groups capable of anion exchange, 2) Functional groups capable of hydrophobic interactions and including, Contacting the composition with a multimode chromatography material, b) Recover the elute containing the multispecific antibody and the reduced amount of its mispair variant. Methods that include...

2. The method according to claim 1, wherein the functional group capable of hydrophobic interaction includes an alkyl group, an alkenyl group, an alkynyl group, a phenyl group, a benzyl group, or any combination thereof.

3. The method according to claim 2, wherein the functional group capable of hydrophobic interaction includes a benzyl group.

4. The method according to any one of claims 1 to 3, wherein the functional group capable of anion exchange is a positively charged group.

5. The method according to claim 4, wherein the positively charged group is a quaternary ammonium ion.

6. The method according to any one of claims 1 to 5, wherein the multimode chromatography material comprises N-benzyl-N-methylethanolamine.

7. The method according to any one of claims 1 to 6, wherein the elution by multimode chromatography is gradient elution.

8. The method according to claim 7, wherein the gradient elution includes a pH gradient.

9. The method according to any one of claims 1 to 8, comprising a capture chromatography step.

10. The method according to claim 9, wherein the capture chromatography step is an affinity chromatography step.

11. The method according to claim 10, wherein the affinity chromatography step is selected from the group consisting of a protein A chromatography step, a protein L chromatography step, a protein G chromatography step, and a protein A / G chromatography step.

12. The method according to claim 10 or 11, wherein the affinity chromatography step is a protein A chromatography step.

13. The method according to claim 12, wherein the protein A chromatography step includes a chromatography material containing protein A bound to agarose.

14. The method according to any one of claims 9 to 13, wherein the capture chromatography step and the multimode chromatography step are continuous.

15. The method according to any one of claims 1 to 14, comprising a purification step after a multimode chromatography step.

16. The method according to any one of claims 1 to 15, comprising enrichment of a multispecific antibody.

17. The method according to any one of claims 1 to 16, wherein the multispecific antibody comprises a knob-into-hole modification.

18. The method according to any one of claims 1 to 17, wherein a multispecific antibody and its mispair variant are produced in the same host cell culture.

19. The method according to claim 18, wherein the host cells of the host cell culture are prokaryotic cells or eukaryotic cells.

20. The method according to claim 18 or 19, wherein the host cell is a eukaryotic cell.

21. The method according to claim 20, wherein the eukaryotic cell is a yeast cell, an insect cell, or a mammalian cell.

22. The method according to claim 20 or 21, wherein the eukaryotic cell is a CHO cell.

23. A method for producing a composition containing a multispecific antibody, comprising purifying the multispecific antibody using the method according to any one of claims 1 to 22.

24. The method according to claim 23, wherein the composition further comprises a pharmaceutically acceptable carrier.

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