Methods for purifying bispecific antibodies

Abstract

The present disclosure provides a method for purifying asymmetric bispecific antibodies using light chain affinity chromatography.

Description

[Technical field]

[0001] 1. CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of priority to U.S. Provisional Patent Application No. 63 / 269,497, filed March 17, 2022, which is incorporated by reference in its entirety for all purposes.

[0002] The present disclosure relates generally to methods for purifying bispecific antibodies using light chain affinity chromatography. [Background technology]

[0003] Affinity chromatography is commonly used in the large-scale production and purification of many biopharmaceuticals, such as monoclonal antibodies (Non-Patent Document 1; Non-Patent Document 2). Due to its high specificity for the product of interest, affinity chromatography provides process robustness and enables the design of purification platforms that streamline process development and reduce manufacturing risk (Non-Patent Document 3).

[0004] Various therapeutic formats such as multispecific antibodies, fabs and antibody fragments utilize light chain (LC) affinity chromatography to capture the product (Non-Patent Document 4; Non-Patent Document 5; Non-Patent Document 6). Of interest here, asymmetric bispecific antibodies (bsAbs) are attractive therapeutics due to their unique mechanism of action and, importantly, the native antibody format of this asymmetric bispecific antibody that maintains favorable pharmacokinetic, stability, solubility and immunogenicity profiles (Non-Patent Document 7). However, this format has historically faced problems with chain association (Non-Patent Document 8). Protein engineers have controlled the formation of mispaired variants of heavy and light chains by utilizing various strategies such as steric and electrostatic steering, domain crossovers, common light chains and kappa-lambda bodies, among others (Non-Patent Document 9; Non-Patent Document 10; Non-Patent Document 11; Non-Patent Document 12; Non-Patent Document 13). Although these designs improve the fidelity of chain pairing, mispaired variants are still commonly observed after expression. Although optimized expression procedures, in vitro assembly and high-throughput clonal selection may also reduce downstream burden, some level of these strand-mismatched impurities often remains and must be controlled during downstream processing (Non-Patent Document 14; Non-Patent Document 15; Non-Patent Document 16; Non-Patent Document 17; and Non-Patent Document 18). Therefore, product-related impurities remain a significant obstacle to the development of commercial-scale purification processes for asymmetric bsAbs, and additional scalable purification strategies are needed. [Prior art documents] [Non-patent literature]

[0005] [Non-Patent Document 1] KMLacki,et al.,Biotechnol.J.15(2020) [Non-Patent Document 2] GRBolton et al.,Biotechnol.Prog.32(2016)1193-1202 [Non-Patent Document 3] S.Sommerfeld,et al.,Chem.Eng.Process.Process Intensif.44(2005)

Non-licensed Document 4

Non-licensed Document 5

Non-licensed Document 6

Non-licensed Document 7

Non-licensed Document 8

Non-licensed literature 9

Non-licensed literature 10

Non-licensed Document 11

Non-licensed Document 12

Non-licensed Document 13

Non-licensed Document 14

[0006] In some aspects, disclosed herein are methods of purifying an asymmetric bispecific antibody comprising: (a) contacting a composition comprising the bispecific antibody with a lambda light chain affinity matrix; (b) washing the affinity matrix to remove impurities; and (c) eluting the bispecific antibody with an elution buffer comprising about 5 mM to about 45 mM salt.

[0007] In some embodiments, the elution buffer further comprises an aggregation inhibitor, hi some embodiments, the aggregation inhibitor is arginine-HCl.

[0008] In some embodiments, the concentration of the salt is about 20 mM. In some embodiments, the halogen salt is sodium chloride, magnesium chloride, ammonium chloride, sodium acetate, sodium citrate, sodium phosphate, sodium sulfate, arginine-HCl, or histidine-HCl.

[0009] In some embodiments, the pH of the elution buffer is about 3 to about 4.5. In some embodiments, the pH of the elution buffer is about 3.5.

[0010] In some embodiments, the method further comprises contacting the asymmetric bispecific antibody with a kappa light chain affinity matrix and eluting the bound bispecific antibody. In some embodiments, contacting the asymmetric bispecific antibody with the kappa light chain affinity matrix is ​​performed before contacting the bispecific antibody with the lambda light chain affinity matrix. In some embodiments, the eluate obtained after elution is passed directly through a second affinity matrix. In another embodiment, the method is performed in a closed system.

[0011] In some embodiments, the kappa light chain affinity matrix and / or the lambda light chain affinity matrix is ​​coupled to a solid support, hi other embodiments, the solid support is cross-linked agarose.

[0012] In some embodiments, a bispecific antibody comprises a modified heavy chain and / or a modified light chain. In some embodiments, a bispecific antibody comprises (a) a Fab region comprising a modified heavy chain, the CH1 region of which comprises (i) a substitution of a naturally occurring cysteine ​​for a non-cysteine ​​amino acid, and (ii) a substitution of a naturally occurring non-cysteine ​​amino acid for a cysteine ​​amino acid; (b) a corresponding modified light chain, the CL region of which comprises (i) a substitution of a naturally occurring cysteine ​​for a non-cysteine ​​amino acid, and (ii) a substitution of a naturally occurring non-cysteine ​​amino acid for a cysteine ​​amino acid; a light chain; (c) a second Fab region comprising a second heavy chain; and (d) a second corresponding light chain, wherein the modified heavy chain is directly linked to the corresponding modified light chain, and in a separate target binding arm, the second heavy chain is directly linked to the second corresponding light chain, and wherein a substituted cysteine ​​of the modified heavy chain resulting from the substitution of a naturally occurring non-cysteine ​​amino acid with a cysteine ​​amino acid and a substituted cysteine ​​of the corresponding modified light chain resulting from the substitution of a naturally occurring non-cysteine ​​amino acid with a cysteine ​​amino acid are capable of forming a disulfide bond. In some embodiments, (a) the second heavy chain and the second corresponding light chain do not contain any substitutions of naturally occurring non-cysteine ​​amino acids with cysteine ​​amino acids and do not contain any substitutions of naturally occurring cysteines with non-cysteine ​​amino acids; and / or (b) the two light chains each comprise a VL domain and a CL domain, the VL domains having different amino acid sequences, and the CL domains having different amino acid sequences; and / or (c) the two heavy chains each comprise a VH domain, a CH1 domain, and an Fc region, the VL domains having different amino acid sequences, the CH1 domains having different amino acid sequences, and the Fc regions having different amino acid sequences, and optionally one light chain is a kappa light chain and one light chain is a lambda light chain. In some embodiments, the two heavy chains form a heterodimer. In some embodiments, the bispecific antibody specifically binds to two independent antigens or two independent epitopes on the same antigen. In some aspects, the Fc region of either or both heavy chains comprises one or more modifications, optionally which modifications promote heterodimerization of the heavy chains.

[0013] In some embodiments, the methods result in the elimination of mismatched species, removal of aggregates, and / or removal of low molecular weight impurities from the composition. [Brief description of the drawings]

[0014] [Figure 1] Shown are the lambda light chain and corresponding heavy chain in yellow and the kappa light chain in red. The mismatched species shown above would be observed in the Protein A product of the captured cell culture harvest for each bsAb. [Diagram 2] Chromatographic elution profiles of bsAb 1 and 2 on CaptureSelect Kappa XP, CaptureSelect Kappa XL, Capto L and KappaSelect in a pH gradient elution screening experiment. Absorbance at 280 nm (solid line) is referenced on the left y-axis and elution pH (dashed line) is referenced on the right y-axis. [Diagram 3] Chromatographic elution profiles of bsAbs 1 and 3 on Lambda LC affinity media in a pH gradient elution screening experiment. [Figure 4] 1 shows the absorbance traces of the HIC-HPLC analysis of the pH gradient elution screening product. The mismatch composition of the split elution peaks is shown compared to conventionally purified CM, detailing the mismatch impurities present in the load material. [Diagram 5] FIG. 1 shows the elution profile of bsAb 2 with Kappa XP affinity media eluted with a pH gradient and the addition of (a) no modifier, (b) 250 mM sodium chloride, (c) 500 mM sodium citrate, (d) 500 mM MgCl, and (e) 500 mM L-arginine. [Figure 6] Figure 1 shows the elution profile of bsAb 1 from CaptureSelect Lambda XP with pH 3.5 step elution conditions. The first peak appears in the elution step and the second peak appears in the strip step. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0015] In order that this disclosure may be more readily understood, certain terms are first defined. As used herein, unless otherwise expressly indicated herein, each of the following terms shall have the meaning set forth below. Further definitions are set forth throughout this application.

[0016] 6.1 Terminology The methods provided herein are often not limited to particular compositions or process steps, and as such may vary.

[0017] The term "chromatography" refers to any type of technique that separates a protein of interest (e.g., an antibody) from other molecules (e.g., contaminants) present in a mixture. Typically, the protein of interest is separated from other molecules (e.g., contaminants) as a result of differences in the speed at which individual molecules of the mixture move through a stationary medium under the influence of a mobile phase or as a result of differences in binding and elution processes. The terms "matrix", or "chromatographic matrix", or "chromatographic medium", are used interchangeably herein and refer to any type of adsorbent, resin, or solid phase that separates the protein of interest (e.g., an Fc region-containing protein such as an immunoglobulin) from other molecules present in the mixture in a separation process. Non-limiting examples include particulate, monolithic, or fibrous resins and membranes that can be inserted into columns or cartridges. Examples of materials that form matrices include polysaccharides (e.g., agarose and cellulose); and other mechanically stable matrices (e.g., silica (e.g., controlled pore glass), poly(styrenedivinyl)benzene, polyacrylamide, ceramic particles, and derivatives of any of the above). Examples of typical matrix types suitable for the method of the present disclosure are cation exchange resin, affinity resin, anion exchange resin or mixed mode resin. A "ligand" is a functional group that is attached to a chromatography matrix and determines the binding properties of the matrix. Examples of "ligands" include, but are not limited to, ion exchange groups, hydrophobic interaction groups, hydrophilic interaction groups, thiophilic interaction groups, metal affinity groups, affinity groups, bioaffinity groups and mixed mode groups (combinations of the above). Some ligands that can be used herein include, but are not limited to: strong cation exchange groups, such as sulfopropyl, sulfonic acid; strong anion exchange groups, such as trimethylammonium chloride; weak cation exchange groups, such as carboxylic acid; weak anion exchange groups, such as N5N diethylamino or DEAE; hydrophobic interaction groups, such as phenyl, butyl, propyl, hexyl; and affinity groups, such as protein A, protein G and protein L.

[0018] The term "affinity chromatography" refers to a protein separation technique in which a protein of interest (e.g., an Fc region-containing protein or an antibody of interest) specifically binds to a ligand specific for the protein of interest. Such ligands are commonly referred to as biospecific ligands. In some embodiments, the biospecific ligand (e.g., Protein A or a functional variant thereof) is covalently attached to a chromatography matrix material and is accessible to the protein of interest in a solution when the solution contacts the chromatography matrix. The protein of interest generally maintains its specific binding affinity for the biospecific ligand during the chromatography step, while other solutes and / or proteins in the mixture do not appreciably or specifically bind to the ligand. Binding of the protein of interest to the immobilized ligand allows contaminating proteins or protein impurities to pass through the chromatography matrix, while the protein of interest remains specifically bound to the immobilized ligand on the solid phase material. The specifically bound protein of interest is then removed in active form from the immobilized ligand under appropriate conditions (e.g., low pH, high pH, ​​high salt, competing ligands, etc.) and passed through a chromatography column with an elution buffer, which removes contaminating proteins or protein impurities that have already passed through the column. Any component may be used as a ligand to purify its respective specific binding protein (e.g., an antibody).

[0019] The terms "purify," "separate," or "isolate," as used interchangeably herein, refer to increasing the purity of a protein of interest from a composition or sample that contains the protein of interest and one or more impurities. Typically, the purity of the protein of interest is increased by removing (completely or partially) at least one impurity from the composition.

[0020] The term "buffer" as used herein refers to a substance whose presence in a solution increases the amount of acid or alkali that must be added to produce a unit change in pH. Buffers resist changes in pH through the action of their acid-base conjugate components. Buffers used with biological reagents can generally maintain a constant concentration of hydrogen ions so that the pH of the solution is within the physiological range. Typical buffer components include, but are not limited to, organic and inorganic salts, acids and bases.

[0021] The term "chromatography column" or "column" as used herein in reference to chromatography refers to a container, often in the form of a cylindrical or hollow pillar, that is packed with a chromatography matrix or resin, which is a material that imparts physical and / or chemical properties utilized in purification.

[0022] The term "bind and elute" or "bind and elute mode" of purification refers to when the target protein of interest is retained on a column and impurities flow through the column. The process then involves the specific elution of the protein of interest using various column conditions that prevent the binding of the protein of interest to the chromatography medium (usually the resin in the column).

[0023] The term "elution buffer" refers to a buffer that effectively dissociates protein:protein interactions without permanently affecting the structure of the proteins.

[0024] As used herein, the term "contaminants" is used in the broadest sense to cover any undesired components or compounds in a mixture. In cell cultures, cell lysates or clarified bulk (e.g., clarified cell culture supernatants), contaminants include, for example, host cell nucleic acids (e.g., DNA) and host cell proteins present in cell culture medium. Host cell contaminant proteins include, but are not limited to, those naturally or recombinantly produced by host cells and proteins related to or derived from the protein of interest (e.g., proteolytic fragments) as well as other process-related contaminants. In certain embodiments, other means such as centrifugation, sterile filtration, depth filtration and tangential flow filtration are used to separate contaminant precipitates from cell cultures.

[0025] The term "loading buffer" refers to a buffer used to prepare and load a mixture or sample onto a chromatography unit.

[0026] The term "chase buffer" refers to a buffer used after the loading buffer to push a mixture or sample through a chromatographic process.

[0027] The term "HMW species" refers to any one or more undesired proteins present in a mixture. High molecular weight species may include dimers, trimers, tetramers or other multimers. These species are often considered product-related impurities and may be covalently or non-covalently linked, e.g., may consist of misfolded monomers in which hydrophobic amino acid residues are exposed to polar solvents, and may cause aggregation.

[0028] The term "LMW species" refers to any one or more undesirable species present in a mixture. Low molecular weight species are often considered product-related impurities and can include truncated species or half molecules of a compound intended to be a dimer (e.g., a monoclonal antibody).

[0029] The term "host cell protein" or HCP refers to undesirable proteins made by host cells that are unrelated to the production of the intended protein of interest. Undesirable host cell proteins may be secreted into the upstream cell culture supernatant. Undesirable host cell proteins may also be released during cell lysis. Cells used in upstream cell culture require proteins for growth, transcription and protein synthesis, and this unrelated protein is undesirable in the final pharmaceutical product.

[0030] The term "antibody" refers to an immunoglobulin molecule that recognizes and specifically binds to a target (e.g., a protein, polypeptide, peptide, carbohydrate, polynucleotide, lipid, or a combination of the above) through at least one antigen recognition site in the variable region of the immunoglobulin molecule. As used herein, the term "antibody" encompasses intact polyclonal antibodies, intact monoclonal antibodies, chimeric antibodies, humanized antibodies, human antibodies, fusion proteins containing antibodies, and any other modified immunoglobulin molecule, so long as the antibody exhibits the desired biological activity. Antibodies can be any of the five major classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, or subclasses (isotypes) thereof (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2) (based on the identity of the heavy chain constant domains, designated alpha, delta, epsilon, gamma, and mu, respectively). The different classes of immunoglobulins have different known subunit structures and three-dimensional configurations. The antibodies can be naked antibodies or can be conjugated to other molecules (eg, toxins, radioisotopes, etc.).

[0031] Unless expressly stated and unless the context indicates otherwise, the term "antibody" includes monospecific, bispecific or multispecific antibodies and single chain antibodies. In some embodiments, an antibody is a bispecific antibody. The term "bispecific antibody" refers to an antibody that binds to two different epitopes. These epitopes may be on the same target antigen or on different target antigens. The term "bispecific antibody" also refers to an antibody fragment (e.g., Fab) that contains both a kappa light chain and a lambda light chain.

[0032] The term "antibody fragment" refers to a portion of an intact antibody. An "antigen-binding fragment", "antigen-binding domain" or "antigen-binding region" refers to a portion of an intact antibody that binds to an antigen. In the context of a bispecific antibody, an "antigen-binding fragment" binds to two antigens. An antigen-binding fragment may contain an antigen recognition site of an intact antibody (e.g., sufficient complementarity determining regions (CDRs) to specifically bind to an antigen). Examples of antigen-binding fragments of antibodies include, but are not limited to, Fab fragments, Fab' fragments, F(ab')2 fragments and Fv fragments, linear antibodies and single chain antibodies. Antigen-binding fragments of antibodies may be derived from any animal species, such as rodents (e.g., mice, rats or hamsters) and humans, or may be artificially generated.

[0033] A "monoclonal" antibody or antigen-binding fragment thereof refers to a population of homogeneous antibodies or antigen-binding fragments involved in highly specific binding of a single antigenic determinant or epitope. This is in contrast to polyclonal antibodies, which typically contain different antibodies against different antigenic determinants. The term "monoclonal" antibody or antigen-binding fragment thereof encompasses both intact and full-length monoclonal antibodies, as well as antibody fragments (e.g., Fab, Fab', F(ab')2, Fv), single-chain (scFv) variants, fusion proteins containing antibody portions, and any other modified immunoglobulin molecule containing an antigen recognition site. Furthermore, a "monoclonal" antibody or antigen-binding fragment thereof refers to antibodies and antigen-binding fragments thereof as produced by a variety of methods, including, but not limited to, by hybridoma, phage selection, recombinant expression, and transgenic animals.

[0034] As used herein, the terms "variable region" or "variable domain" are used interchangeably and are common in the art. A variable region typically refers to a portion of an antibody, and broadly to a portion of a light or heavy chain, typically about 110-120 or 110-125 amino acids at the amino terminus in a mature heavy chain and about 90-115 amino acids in a mature light chain, which differ in sequence between antibodies and are used for the binding and specificity of a particular antibody to its particular antigen. The sequence variability is concentrated in regions called complementarity determining regions (CDRs), while the more highly conserved regions in the variable domain are called framework regions (FRs). Without wishing to be bound by any particular mechanism or theory, it is believed that the CDRs of the light and heavy chains are primarily responsible for the interaction and specificity of the antibody with the antigen. In some aspects of the present disclosure, the variable region is a human variable region. In some aspects of the present disclosure, the variable region comprises rodent or murine CDRs and human framework regions (FRs). In certain aspects of the disclosure, the variable region is a primate (e.g., non-human primate) variable region. In some aspects of the disclosure, the variable region comprises rodent or murine CDRs and primate (e.g., non-human primate) framework regions (FRs).

[0035] As used herein, the term "heavy chain" or "HC," when used in reference to an antibody, can refer to any of the distinct types (e.g., alpha (α), delta (δ), epsilon (ε), gamma (γ) and mu (μ)) based on the amino acid sequence of the constant domain, which gives rise to the IgA, IgD, IgE, IgG and IgM classes of antibodies, respectively, including subclasses of IgG (e.g., IgG1, IgG2, IgG3 and IgG4). Heavy chain amino acid sequences are known in the art. In some aspects of the disclosure, the heavy chain is a human heavy chain.

[0036] As used herein, the term "light chain" or "LC" when used in reference to an antibody can refer to any distinct type (e.g., κ (kappa) or λ (lambda)) based on the amino acid sequence of the constant domain. Light chain amino acid sequences are known in the art. In some aspects of the disclosure, the light chain is a human light chain.

[0037] Amino acids are referred to herein by either their commonly known three letter symbols or the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Nucleotides are likewise referred to by their commonly accepted one-letter codes.

[0038] As used in this disclosure and claims, the singular forms "a," "an," and "the" include the plural forms unless the context clearly dictates otherwise.

[0039] Whenever an embodiment of the present disclosure is described herein with the word "comprising," it is understood that similar embodiments separately described with the terms "consisting of" and / or "consisting essentially of" are also provided.

[0040] Unless otherwise specified or clear from the context, as used herein, the term "or" is understood to be inclusive. When the term "and / or" is used herein in phrases such as "A and / or B," it is intended to include both "A and B," "A or B," "A" and "B." Similarly, when the term "and / or" is used in phrases such as "A, B and / or C," it is intended to encompass each of the following embodiments: A, B and C; A, B or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).

[0041] As used herein, the terms "about" and "approximately," when used to modify a numerical value or numerical range, indicate deviations of more than 5%-10% and less than 5%-10% from that value or range that remain within the intended meaning of the recited value or range.

[0042] Any composition or method provided herein can be combined with one or more of any of the other compositions and methods provided herein.

[0043] Units, prefixes, and symbols are expressed in their International System of Units (SI) accepted form. Numerical ranges are inclusive of the numbers that define the range. The headings provided herein are not intended to limit the various aspects of the disclosure, which aspects may be obtained by reference to the specification in its entirety. Thus, the terms defined immediately below are more fully defined by reference to the specification in its entirety.

[0044] 6.2 Bispecific antibodies In some aspects, the antibodies provided herein are monovalent bispecific antibodies (MBabs). The monovalent bispecific antibody scaffold described herein, due to its monovalent nature, provides an excellent platform for the creation of bispecific antibodies that realize all the benefits associated with bispecific antibodies while reducing the potential therapeutic risks mentioned above. Furthermore, the MBabs provided herein are likely to be easily expressed, stable, and less immunogenic. As used herein, the term "monovalent bispecific" (which may be abbreviated as "MBab") refers to a bispecific antibody, in which each arm can specifically bind to a different target antigen, and for a given pair of different target antigens (A and B), one MBab can bind to each. In certain aspects, a monovalent bispecific antibody can specifically bind to two independent antigens (or targets) or two independent epitopes on the same antigen. Typically, a monovalent bispecific antibody comprises two different variable regions. In some aspects, the binding affinities for the two independent antigens are approximately the same. In some aspects, the binding affinities for the two independent antigens are different. In some embodiments, the binding affinity for two independent epitopes on the same antigen is approximately the same. In some embodiments, the binding affinity for two independent epitopes on the same antigen is different. In still other embodiments, each arm has the same specificity (e.g., binds to the same or overlapping epitopes) but binds with different affinities. In some embodiments, the affinities can differ by 3-fold or more. When combining variable regions of antibodies with different in vivo potencies, it may be particularly desirable to have one arm with a higher affinity and one arm with a lower affinity to prevent over- or under-dosing one of the arms.

[0045] In certain aspects, MBabs bind to the same or overlapping epitopes on the same antigen (e.g., receptor) with different affinities. In particular, the same or overlapping epitopes are in close proximity when the antigen is dimerized. Such antibodies have dual properties depending on the relative concentration. For example, at high concentrations, when the MBab concentration saturates the antigen concentration, the high affinity binding domain competes with the low affinity binding domain, resulting in little or no affinity effects. That is, the antibody functions mainly as a monovalent binding entity, and little or no antigen cross-linking / signaling occurs. However, at low concentrations, affinity effects come into play, and the MBab can preferably bind to both binding sites on two antigen molecules simultaneously, resulting in antigen cross-linking / signaling. In this way, antigen signaling can be modulated by MBab concentration.

[0046] In certain embodiments, the MBab binds to two different antigens (e.g., different receptors) where homodimerization of these antigens is undesirable and / or where both antigens are present separately on non-target cells / tissues and together on target cells / tissues. Such an antibody binds with only one arm on non-target cells, and this low affinity monovalent binding of only one arm of the MBab to non-target cells / tissues is insufficient to induce homodimerization. In contrast, the MBab can bind to both antigens on the target cells / tissues, and simultaneous binding of both antigens on the target cells results in higher affinity bivalent binding, which can enhance preferential binding to target cells and enhance receptor dimerization.

[0047] In some embodiments, the monovalent bispecific antibody further comprises an additional binding site. This additional binding site may be specific for one or both target antigens (A and B) of the monoclonal bispecific antibody (MBab) and / or may be specific for an additional target antigen. In some embodiments, one or more single chain variable fragments (scFvs) are added to the N-terminus or C-terminus of one or both heavy chains and / or one or both light chains, where the one or more scFvs specifically bind to one or more additional target antigens. For example, a monovalent trispecific antibody may be created by adding an scFv (specific for antigen C) to one chain (e.g., heavy or light chain) of a monovalent bispecific antibody (specific for antigens A and B). In this case, the antibody is monovalent for antigens A, B, and C. If an scFv (specific for antigen C) is added to two chains (e.g., both heavy chains, both light chains, one heavy chain and one light chain), the triabody will be monovalent for antigens A and B and bivalent for antigen C. All possible combinations of additional binding sites are contemplated herein for monovalent bispecific antibodies (see, e.g., Dimasi et al. J. Mol. Biol. (2009) 393:672-692). It is contemplated that the binding affinity of the additional binding site may be approximately the same as one or both arms of the MBab, or may be different from one or both arms of the MBab. As mentioned above, the relative affinities may be selected or adjusted depending on the antigen and the intended use of the molecule.

[0048] Bispecific antibodies can be prepared as full-length antibodies or antibody fragments (e.g., F(ab')2 bispecific antibodies). Traditional production of full-length bispecific antibodies is based on the co-expression of two immunoglobulin heavy-light chain pairs, where the two chains have different specificities. Through random assortment of immunoglobulin heavy and light chains, these hybridomas (quadromas) produce a potential mixture of 10 different antibody molecules, of which only one has the correct bispecific structure. Purification of the correct molecule, which is usually done by affinity chromatography steps, can be rather cumbersome, and product yields can be low.

[0049] Bispecific antibodies include cross-linked or "heteroconjugate" antibodies. For example, one of the antibodies of the heteroconjugate can be conjugated to avidin and the other to biotin. Heteroconjugate antibodies can be made using any convenient cross-linking method. However, this method typically requires the use of non-human proteins, which can have high immunogenic potential. Furthermore, antibody fragments sometimes have little or no effector functions and short half-lives.

[0050] Bispecific antibodies can be prepared using chemical linkage. In one procedure, intact antibodies are proteolytically cleaved to generate F(ab')2 fragments. These fragments are reduced in the presence of the dithiol complexing agent sodium arsenite to stabilize vicinal dithiols and prevent intermolecular disulfide formation. The generated Fab' fragments are then converted to thionitrobenzoate (TNB) derivatives. One of the Fab'-TNB derivatives is then reconverted to a Fab'-thiol by reduction with mercaptoethylamine and mixed with an equimolar amount of the other Fab'-TNB derivative to form the bispecific antibody. However, this method often has low yields, is difficult to control, and the product may have high immunogenic potential. In addition, antibody fragments sometimes have little or no effector function and a short half-life.

[0051] Fab'-SH fragments can be directly recovered from E. coli and chemically coupled to form bispecific antibodies. Fully humanized bispecific antibody F(ab')2 molecules can be produced by secreting each Fab' fragment separately from E. coli and subjecting it to in vitro directed chemical coupling to form bispecific antibodies. However, this method often results in low yields and is difficult to control. Furthermore, antibody fragments sometimes have little or no effector functions and short half-lives.

[0052] Bispecific antibodies can also be produced using leucine zippers. Leucine zipper peptides from Fos and Jun proteins are linked to the Fab' portions of two different antibodies by genetic fusion. The antibody homodimers are reduced at the hinge region to form monomers and then reoxidized to form the antibody heterodimers. This method can also be utilized to generate antibody homodimers. The described "diabody" technology provides an additional mechanism to generate bispecific antibody fragments. The fragments contain a VH linked to a VL by a linker that is too short to allow pairing between the two domains on the same chain. Thus, the VH and VL domains of one fragment are paired with the complementary VL and VH domains of another fragment, thereby forming two antigen-binding sites. Another strategy to generate bispecific antibody fragments is also known in the art by the use of single-chain Fv (sFV) dimers. However, this method often has low yields, is difficult to control, and the products may have high immunogenic potential. Furthermore, antibody fragments sometimes have little or no effector functions and short half-lives.

[0053] Bispecific antibodies can also be generated using heavy chain heterodimerization methods, including the "knobs-in-holes" and strand-exchange engineered domain (SEED) methods, as well as methods to alter the charge polarity across the Fc dimer interface. Such methods are described in further detail herein and in, for example, U.S. Pat. No. 7,183,076; Merchant et al. (1998) Nat. Biotech 16:677-681; Ridgway et al. (1996) Protein Engineering 9:617-621; Davis et al. (2010) Prot. Eng. Design & Selection 23:195-202; WO 2007 / 110205; WO 2007 / 147901; Gunasekaran et al. (2010) JBC 285:19637-46. In these methods, the interface between a pair of antibody molecules may be engineered to maximize the percentage of heterodimers recovered from recombinant cell culture. In the "knobs-in-holes" method, a "protrusion" is created by replacing one or more small amino acid side chains at the interface of a first antibody molecule with a larger one (e.g., tyrosine or tryptophan). A compensatory "cavity" of identical or similar size to the large side chain is created at the interface of a second antibody molecule by replacing amino acids with larger side chains with amino acids with smaller side chains (e.g., alanine or threonine). This provides a mechanism to increase the yield of heterodimers over other unwanted end-products such as homodimers. In the SEED method, Fc homodimers are converted to heterodimers by interlocking beta-strand segments of human IgG and IgA CH3 domains. These derivatives of human IgG and IgA CH3 domains create complementary human SEED CH3 heterodimers composed of alternating segments of human IgA and IgG CH3 sequences. The resulting pair of SEED CH3 domains preferentially associates to form heterodimers when expressed in mammalian cells.Other methods include introducing modifications that change the charge polarity across the Fc dimer interface such that co-expression of electrostatically compatible Fc regions results in heterodimerization. These methods improve heavy chain heterodimerization but do not address the light-heavy chain mispairing that occurs during the formation of bispecific antibodies. In some cases, the use of a common light chain, as explained in WO 98 / 50431, can reduce the number of possible mispairings, but often results in a loss or reduction in binding specificity and / or affinity.

[0054] Dual-specific antibodies are another type of bispecific antibody that can be generated (see, e.g., Bostrum et al. (2009) Science 323:1610-1614). Such antibodies can be generated by creating variants with mutations in the light chain (LC) complementarity determining regions (CDRs) so that they can bind to new antigen targets while maintaining the binding specificity for their natural target antigen. However, the antigen binding sites often overlap, preventing the antibody from binding to both antigens simultaneously. Furthermore, these antibodies are difficult to generate and may not have the desired affinity for each of the two antigens.

[0055] The modified polypeptides provided herein may be useful for making bispecific antibodies and may overcome the limitations and technical difficulties mentioned above. In some embodiments, one heavy chain and one light chain in an antibody are modified, whereby a natural cysteine ​​is replaced with a non-cysteine ​​amino acid, and a natural non-cysteine ​​amino acid is replaced with a cysteine ​​amino acid. Such modifications provided herein are made in the HC and LC domains, and the HC-LC interchain disulfide bridges are rearranged. When making a bispecific antibody from four separate polypeptides, for example, where the modified arm has binding specificity for one target and the unmodified arm has binding specificity for a different target, these four polypeptides are assembled such that the modified heavy chain hybridizes correctly with the modified light chain, and the unmodified heavy chain hybridizes correctly with the unmodified light chain. As used herein, the term "unmodified" refers to heavy and light chains that do not contain the HC-LC modifications introduced for rearrangement of cysteines and / or disulfide bridges, as described herein. Such "unmodified" heavy and light chains may contain other modifications, such as heterodimerization modifications in the CH2 and / or CH3 regions described herein and / or known in the art. The HC-LC modifications provided herein may be combined with further modifications of the heavy chains, particularly in the CH2 and / or CH3 regions, to ensure proper heavy chain heterodimerization and / or enhance purification of the heavy chain heterodimers, as described in more detail below.

[0056] Bispecific antibodies of the invention may be asymmetric and contain one kappa and one lambda light chain. They may also contain a HC and / or LC, such as those described in U.S. Patent No. 9,527,927, which is incorporated herein by reference.

[0057] 6.3 Methods of the Invention The present invention evaluates the ability of commercially available LC affinity chromatography media to separate multiple product-related impurities of a selected asymmetric bsAb construct composed of four unique polypeptide chains (Y. Mazor, et al., MAbs. 7 (2015) 377-389). Currently, several scalable chromatography media options with specificity for kappa and lambda light chains are commercially available. Protein L media provides specificity for the variable region of certain kappa light chain subtypes, while other kappa and lambda LC affinity media provide specificity for the constant regions of kappa and lambda light chains (M. Graille, et al., Structure. 9 (2001) 679-687). With the exception of Protein L, these LC affinity media employ camelid antibody-based ligand technology (C. Hamers-Casterman, et al., Nature. 363 (1993) 446-448; JT Detmers, et al., Bioprocess Int. 8 (2010) 50-54; M. Zandian, et al., J. Chromatogr. A. 1216 (2009) 5548-5556; and TMPabst, et al., Biotechnol. J. 12 (2017) 1600357). Previous publications have detailed the development of one example of these chromatographic media, Lambdafabselect (N. Eifler, et al., Biotechnol. Prog. 30 (2014) 1311-1318). Two separation mechanisms by LC affinity chromatography are described herein, in which impurities lacking a light chain of the affinity media specificity class flow through the column, and in which impurity species with multiple light chain moieties of the same class as the resin specificity are strongly retained relative to the target heterodimer product.

[0058] A growing body of literature describes the application of affinity chromatography media to separate product-related impurities based on binding strength. Notable examples include the use of pH during elution to separate monoclonal antibody monomers from aggregates, polyclonal human IgG subpopulations, and Fc-binding-ablated bsAbs on Protein A and G affinity media (C. Andrade, et al., Biotechnol. Prog. 35 (2019) e2720; J. Weinberg, et al., Biotechnol. Bioeng. 114 (2017) 1803-1812; A.D. Tustian, et al., MAbs. 8 (2016) 828-838; and R. Ollier, et al., MAbs. 11 (2019) 1464-1478). In addition, separation of bsAb mismatched variants containing two light chains of the same class as the resin specificity has been demonstrated for kappa light chains using Protein L and KappaSelect media (C. Chen, et al., MAbs. 11 (2019) 632-638; SW Chen, et al., MAbs. 12 (2020); and T. Qin, et al., Protein Expr. Purif. 171 (2020)). The data of Tustian et al. for Protein A and Chen et al. for Protein L media also outline the benefits of fine tuning affinity-based selectivity with conductivity and various modifiers during elution rather than elution pH alone (AD Tustian, et al., MAbs. 8 (2016) 828-838; and SW Chen, et al., MAbs. 12 (2020)). These findings provide a better understanding of the development space for affinity separations mediated by Proteins A and L, but further understanding of other kappa and lambda LC affinity media is desired due to their specificity for the constant region of the light chain. The LC affinity capture process, particularly one that binds to the constant region of the light chain, is attractive for providing a platformable approach to the purification development of many emerging antibody-based therapeutic formats, such as chain-mispaired bsAbs.

[0059] Thus, the present invention relates to a method of purifying a protein from a solution, the method comprising: (a) contacting a composition comprising a bispecific antibody with a lambda light chain affinity matrix; (b) washing the affinity matrix to remove impurities; and (c) eluting the bispecific antibody with an elution buffer comprising about 5 mM to about 45 mM halogen salt. A variety of lambda light chain affinity matrices are known in the art and are suitable for use in the present invention. Non-limiting examples of commercially available LC resins include Capture Select™, LambdaFabSelect and Capto™ L.

[0060] Binding of bsAbs to an LC matrix is ​​typically accomplished by column chromatography: the LC matrix is ​​formed into a column, a biochemical mixture containing the bsAb is run through the column, the column is subsequently washed by running one or more wash solutions through the column, and then the protein of interest is eluted from the column by running an elution buffer through the column.

[0061] Alternatively, binding of the bsAb to the LC matrix can be accomplished by a batch process in which a biochemical mixture containing the bsAb is incubated with the LC matrix in a vessel to allow binding of the bsAb to the LC matrix, the solid phase medium is removed from the vessel (e.g., by centrifugation), the solid phase medium is washed to remove impurities, recovered again (e.g., by centrifugation), and the bsAb is eluted from the solid phase medium.

[0062] In yet another embodiment, a combination of batch processing and column chromatography may be used, for example, initial binding of the bsAb to the LC matrix may be achieved by batch processing, then the solid phase medium may be packed into a column, followed by washing of the column and elution of the bsAb from the column.

[0063] Most affinity purification procedures involving protein:ligand interactions use a binding buffer at physiological pH and ionic strength, such as phosphate-buffered saline (PBS). Once the binding interaction has occurred, the support is washed with additional buffer to remove unbound components of the sample. Non-specific (e.g., simple ionic) binding interactions can be minimized by adding low levels of detergent or by making appropriate adjustments to salt concentrations in the binding and / or wash buffers.

[0064] Halogen salts (e.g., salts containing chlorine (Cl) and bromine (Br), particularly halogen salts containing alkali metals or alkaline earth metals (e.g., sodium, potassium, calcium, and magnesium)) are referred to as non-buffering salts. In one embodiment, the elution buffer contains a halogen salt (e.g., containing Cl or Br). In another embodiment, the non-buffering salt is a halogen salt containing sodium (Na), potassium (K), calcium (Ca), or magnesium (Mg). In one embodiment, the elution buffer contains Na or Mg. In another embodiment, the halogen salt is NaCl or MgCl. 2 It is.

[0065] The present invention demonstrates that low levels of halogen salts are effective in purifying bsAbs from affinity matrices. Higher levels (e.g., 100 mM) are effective in removing impurities, but these high levels adversely affect the yield of bsAbs. Thus, the concentration of halogen salts in the elution buffer is about 5 mM to 45 mM. In some embodiments, the concentration of halogen salts is about 5 mM, about 10 mM, about 15 mM, about 20 mM, about 25 mM, about 30 mM, about 35 mM, about 40 mM, or about 45 mM.

[0066] The elution buffer may also contain an aggregation inhibitor. In some embodiments, the aggregation inhibitor is arginine or an arginine derivative. The arginine that can be used may be the natural amino acid arginine (e.g., L-arginine), D-arginine or an arginine derivative. Non-limiting examples of arginine derivatives include acetylated arginine, such as acetylarginine and N-alpha-butyroyl-arginine, agmatine, arginic acid and N-alpha-pivaloylarginine. The arginine or arginine derivative may be used in the form of an acid addition salt. Examples of acids that can form acid addition salts include hydrochloric acid and the like.

[0067] The pH of the elution buffer of the present invention is typically acidic. In one embodiment, the pH of the elution buffer is about 3 to about 4.5 depending on the characteristics of the bsAb. In some embodiments, the pH is about 3, about 3.5, about 4, or about 4.5. In some embodiments, the elution buffer has a pH of 3.5.

[0068] The methods of the invention can be carried out in a multi-column chromatography system comprising at least a first chromatography matrix and a second chromatography matrix. The term "multi-column chromatography system" refers to a system of a total of two or more independent, interconnected, or switchable chromatography columns and / or chromatographic membranes. Additional examples of multi-column chromatography systems are known in the art.

[0069] In one embodiment, the second chromatography matrix is ​​a kappa light chain affinity matrix. Commercially available kappa light chain affinity matrices are known in the art, and non-limiting examples include CaptureSelect™ LC-kappa. In one embodiment, the first and second chromatography steps can be performed in any order.

[0070] The chromatography columns and / or chromatographic membranes present in the multi-column chromatography system may be connected or moved relative to each other by a switching mechanism (e.g., a column switching mechanism). A column switching event may be triggered by detection of a level of the protein to be purified, as detected by UV absorbance, a specific amount of liquid (e.g., buffer), or a specific elapsed time, corresponding to a specific level of protein in a fluid passing through the multi-column chromatography system (e.g., inflow to and / or elution from one or more chromatography columns and / or chromatographic membranes in the system). Column switching generally refers to a mechanism that allows at least two different chromatography columns and / or chromatographic membranes in the multi-column chromatography system (e.g., two or more different chromatography columns and / or chromatographic membranes present in the system) to pass through various steps (e.g., equilibration, loading, elution, or washing) substantially simultaneously during at least a portion of the process.

[0071] The method of the invention may be carried out in a continuous mode. In other words, the method of the invention may be a continuous method for purifying bsAb from a solution. The term "continuous method" or "continuous mode method" refers to a method in which a fluid is continuously supplied through at least a portion of a system.

[0072] The following examples are offered by way of illustration and not by way of limitation. EXAMPLES

[0073] The examples in this section are offered by way of illustration and are not intended to be limiting.

[0074] Protein sample preparation. All bsAbs were produced at AstraZeneca from a Chinese Hamster Ovary (CHO) cell expression system. Cell culture supernatants were harvested by centrifugation and depth filtration to generate conditioned cell culture medium (CM). CM was then processed by standard protein A or lambda light chain affinity chromatography methods to generate purified material containing the relevant mismatched species for further experiments.

[0075] Stationary phases, chromatography equipment and reagents. KappaSelect, Capto L and LambdaFabSelect LC affinity media were purchased from Cytiva (formerly GE Healthcare) (Uppsala Sweden). CaptureSelect Kappa XL and XP and CaptureSelect Lambda XP LC affinity media were obtained from Thermo Fisher Scientific (Waltham, MA). Chromatography media attributes were obtained from published vendor literature (Table 1). Chromatography experiments were completed on an AKTA Avant 25 system controlled by Cytiva's Unicorn 7.0 software. Chromatography media was packed into Omnifit column hardware (0.66 cm internal diameter) supplied by Cole-Parmer (Vernon Hills, IL) or Vantage column hardware (1.15 cm internal diameter) supplied by MilliporeSigma (Burlington, MA). All chemicals used in buffer preparation were obtained from JT Baker (Phillipsburg, NJ).

[0076] [Table 1]

[0077] Determination of protein concentration. Protein concentrations in CM were quantified by Protein A and Lambda LC affinity HPLC techniques using a standard curve generated from purified bsAb. CaptureSelect Protein A and CaptureSelect Lambda analytical columns were purchased from Thermo Fisher Scientific (Waltham, MA). Product concentrations in purified samples were determined by absorbance at 280 nanometer wavelength with a Nanodrop 2000c spectrophotometer purchased from Thermo Fisher Scientific.

[0078] Analytical Instruments, Analytes, and Methods. The content of mismatched, aggregated, and fragmented species was quantified by multiple orthogonal analytical techniques. High-performance size-exclusion chromatography (HP-SEC) was performed using a GS3000SWXL column (7.9 × 300 mm) purchased from Tosoh Biosciences (Tokyo, Japan). High-performance hydrophobic interaction chromatography (HIC-HPLC) was performed using a mAb-Pac HIC 10 analytical column purchased from Thermo Fisher Scientific. The HPLC-based method was operated on an Agilent 1260 HPLC system (Palo Alto, CA). Non-reduced capillary gel electrophoresis (NR-CGE) was performed on a LabChip GXII system using the HT Protein Express reagent kit from PerkinElmer (Waltham, MA).

[0079] Determination of dynamic binding capacity. 10% product breakthrough (DBC) of LC affinity chromatography media 10% The approximate dynamic binding capacity at DBC was obtained by overloading the media with purified bsAb to generate a product breakthrough curve. The LC affinity media was loaded with 100 mg of bsAb per mL of packed bed with a residence time of 4 minutes. The approximate DBC 10%Values ​​were calculated from the volume at which the absorbance of the product breakthrough during loading, measured by the AKTA UV detector, was 10% of the absorbance of the load material bypassing the column. The dead volume of the system was subtracted from the volume at 10% breakthrough, and this volume was then converted to a protein load challenge. Previous evaluations have demonstrated excellent linearity between the 280 nm absorbance measurements by the AKTA UV detector and protein concentration up to the maximum protein concentration of the load material used.

[0080] Identification of mismatched and product-related impurity species. HIC-HPLC assays were used to quantify mismatch levels in the chromatographic product and / or load material, HP-SEC to quantify aggregates and monomers, and NR-CGE to quantify half antibodies, LC dimers, and free LC. HIC-HPLC purity was also presented as a general product quality indicator, but notably does not take into account aggregate content.

[0081] pH gradient elution screen. Chromatography was performed with a 4 minute residence time and the LC affinity media was loaded to 10 mg bsAb / mL packed bed. The product was eluted with a 20 column volume (CV) linear gradient of 50 mM sodium citrate / citric acid, pH 6.0-2.5. The elution peak was fractionated and individual peaks were pooled. Protein concentration was determined and then the product pool was pH neutralized and analyzed for aggregate, fragment and mismatch content.

[0082] The resolution between eluting peaks (Rs) was determined by automated peak detection in Unicorn software and using the following formula:

number

[0083] The approximate elution pH of the product peak was calculated by pH measurement with an AKTA pH detector at the peak maximum absorbance. The system dead volume was subtracted from the volume at the peak maximum and the corresponding pH was recorded. The pH electrode was calibrated before each run. The approximate pH values ​​obtained qualitatively matched the overlay of the elution peak profile throughout the experiment.

[0084] Purification of asymmetric bispecific antibody heterodimers Binding affinity may be exploited to separate protein species differing in the number of binding motifs on the affinity media, also described as binding valency. In general, for affinity chromatography media such as Protein A, G, L and camelid antibody-derived media, separation may be achieved by applying a downward pH elution gradient. Commercially available LC affinity media were investigated for the separation of asymmetric bispecific antibody heterodimers containing one lambda light chain and one kappa light chain from mismatched species containing two light chains of the same class as the stationary phase specificity. pH gradient elution experiments were first performed with selected commercially available kappa affinity and lambda LC affinity chromatography media to assess "baseline" selectivity based on product valency. Cell culture harvests containing bsAb1 or 2 were used to assess separation of kappa-kappa mismatched species by kappa LC affinity chromatography, and cell culture harvests containing bsAb1 or 3 were used to separate lambda-lambda mismatched species by lambda affinity chromatography (Figure 1).

[0085] Initial screening results shown in Figures 2, 3 and 4 outline the separation of mismatched impurities in the form of split elution peaks. Of the kappa LC affinity media tested, CaptureSelect Kappa XP showed the best resolution for bsAb1 and Capto L showed the best resolution for bsAb2 (Figure 2).

[0086] [Table 2]

[0087] The difference in chromatographic performance for the two bsAbs may be due to the specificity of Protein L for the variable region of the kappa light chain. In this case, bsAb2 elutes earlier in the pH gradient from Protein L compared to bsAb1. As expected, due to the specificity for the constant region of the kappa light chain, CaptureSelect Kappa XP, CaptureSelect Kappa XL and KappaSelect maintained consistency between bsAb1 and 2, with some imbalance in the size of peaks 1 and 2 for the various bsAbs (Figure 2). This imbalance may be due to the different levels of mismatches and other product-related impurities present in the CM material for bsAb1 and 2. Of the lambda LC affinity media tested, CaptureSelect Lambda XP media provided clearer peak separation compared to LambdaFabSelect for both bsAb1 and 3 (Figure 3). For product quality testing, elution fractions for peaks 1 and 2 were pooled and separated as close to the peak inflection as possible. Product quality data by HIC-HPLC confirm that the observed distinct peaks are mainly due to the separation of heterodimers and light chain mismatch variants (Figure 4). For all bsAbs tested, the second elution peak was also enriched in aggregates and LC dimers (Table 3). Surprisingly, free light chains (by NR-CDE) were enriched in the second peak for all experiments, and the kappa LC affinity medium clearly led to enrichment of half antibodies (by NR-CGE) in the second elution peak for bsAb2 (Table 3). However, enrichment of half antibodies in the second elution peak was less clearly observed with lambda affinity medium for bsAb1 or 3.

[0088] [Table 3]

[0089] The differences in peak resolution observed from pH gradient screening can be attributed to the specific attributes of the various chromatographic matrices. These attributes include the diameter, pore size and pore morphology of the resin particles, which may result in more favorable mass transfer properties, and the density and structure of the ligands, which may result in higher binding capacity and improved likelihood of multiple binding events. With regard to base matrix specificity effects, CaptureSelect Kappa XL and Kappa XP media show significantly different peak resolutions, especially for our bsAb heterodimers and mismatches, although both use the same agarose matrix (Figure 2). In comparison, LambdaFabSelect and Lambda XP use distinct agarose-based matrices, especially with the slightly smaller average particle size of Lambda XP (Table 1). As is evident from the product brochure, Kappa XP uses epoxide conjugation while Kappa XL uses aldehyde conjugation chemistry for ligand conjugation, and Lambda XP also uses epoxide conjugation while LambdaFabSelect uses amide bonds. We hypothesize that single-point ligand conjugation with epoxide chemistry is the primary contributor to this increase in resolution observed with CaptureSelect Kappa XP and Lambda XP media compared to multipoint ligand conjugation with CaptureSelect Kappa XL and Lambda FabSelect. Single-point conjugation may result in more uniform ligand conjugation, increased flexibility, and more favorable orientation compared to multipoint, improving ligand exposure and the opportunity for multiple binding events. Changes to ligand conjugation may also affect binding and displacement kinetics as described by Weinberg et al. (Biotechnol. Bioeng. 114 (2017) 1803-1812). These differences may affect protein migration through the column and therefore resolution. Deeper investigation into the potential contributions of ligand and binding specificity effects and binding and displacement kinetics may provide a clearer understanding of the differences in peak resolution observed here.

[0090] The effect of various elution modifiers on bsAb mismatch separation by Kappa XP and Lambda XP media was then evaluated due to their ability to provide the highest peak resolution in our screening experiments. Previous studies have outlined the ability to improve the resolution of affinity-based affinity separations by incorporating various salts in the elution buffer and / or controlling the elution conductivity (C. Chen, et al., MAbs. 11 (2019) 632-638; SW Chen, et al., MAbs. 12 (2020; and A.D. Tustian, et al., MAbs. 8 (2016) 828-838). For Kappa XP, pH gradient experiments were performed with the addition of 0.5 M sodium citrate (NaCitrate), magnesium chloride (MgCl) or L-arginine-HCl (arginine) or 0.25 M sodium chloride (NaCl) salts. Protein A purified bsAb2 load material was used for this experiment and results were compared to a control experiment with no added salt. Considerable precipitation was observed at the higher concentrations, so the lower 0.25 M NaCl concentrations were used. High concentrations of these salts were necessary to maximize separation because lower concentrations reduced the resolution between the heterodimer and the mismatched impurity (data not shown). In addition, the elution gradient with MgCl was changed to pH 4.3-3.0 due to the buffering properties of the solution, and arginine addition also slightly affected the linearity of the pH gradient.

[0091] Broadening of the elution peaks was observed for all modifiers, with MgCl 2 and arginine increased the separation of impurity peaks (Figure 5). 2 The addition of MgCl provided the greatest improvement in resolution along with unexpected baseline separation. 2 The improvement in resolution with the addition of MgCl and arginine is likely due to preferential enhancement of kappa-kappa mismatches and half-antibody binding to the medium. Under baseline pH gradient conditions without added modifiers, a second peak eluted at approximately pH 3.9, whereas with the addition of MgCl 2In the case of NaCl, no elution of the second peak occurred even at pH 2.8, whereas in the case of arginine elution occurred at pH 3.56 (Table 4). The process performance and product quality with the addition of these modifiers to the Kappa XP medium is shown in Table 4 and shows that the addition of NaCl, MgCl and arginine in particular significantly increased aggregates in the heterodimer peak elution pool. The addition of modifiers maintained separation of the half antibodies and co-elution with the kappa-kappa mismatched species, however the impact of light chain impurities was not evaluated as they are not present in the Protein A purified material. MgCl 2 Despite the high degree of resolution of mismatched impurities observed with ELISA, challenges surrounding high concentrations of modifiers, protein stability, large elution pools, and low product recovery require further optimization to make this method as applicable as possible for large-scale protein purification.

[0092] [Table 4]

[0093] For both Lambda XP and LambdaFabSelect media, it was expected that a much larger effect on elution performance in the presence of salt would occur compared to the Kappa XP results. From the initial screening, incomplete product elution at pH 2.8 was observed with the addition of any test salt at 100 mM concentration to LambdaFabSelect. In addition, for the pH gradient experiments, the basic buffer of 50 mM sodium citrate likely contributed significantly to alteration of the binding interactions between the bsAb and the lambda affinity medium. To minimize baseline buffer conductivity, a 25 mM sodium acetate elution buffer was used, and no 20 mM NaCl, arginine or MgCl 2 A stepwise elution experiment at pH 3.5 was performed to evaluate the effect of modifiers on product bond strength by the addition of 100% glycerol. A control stepwise elution experiment at pH 3.5 was performed without the use of any modifiers.

[0094] As seen in Figures 6a and 6b, increased tailing of the elution peak and a larger 100 mM acetic acid strip peak was observed upon incorporation of all modifiers tested during elution of bsAb1 and 3 from Lambda XP. The peak broadening observed upon incorporation of modifiers was independent of the pH transition, suggesting an effect on bsAb binding strength to the LC affinity medium. Surprisingly, elution with 20 mM of any modifier tested resulted in near complete removal of mismatched species and significant removal of aggregates and low molecular weight (LMW) impurities for both bsAb1 and 3 (Table 5). The lambda half antibodies were also unexpectedly enriched in the strip peak, although in pH gradient screening experiments, bsAb1 half antibodies were found not to substantially co-elute with lambda-lambda mismatches (Table 6). In addition, for bsAb1, the same affinity-binding enhancement was observed with the 20 mM NaCl, pH 3.5 elution method with Lambda FabSelect medium, although it removed less impurities compared to Lambda XP (Table 6 in the supplemental material).

[0095] [Table 5]

[0096] [Table 6]

[0097] For Protein A media, Tustian et al. found that chaotropic salts promoted elution of the bsAb heterodimer product at higher pH (A..D.Tustian, et al., MAbs.8(2016)828-838). For CaptureSelect Kappa XP, no clear trend was observed regarding the chaotropic nature of the modifiers, with the more highly chaotropic salt MgCl 2In the elution, bsAb heterodimers were indeed eluted at a lower pH compared to the control conditions (Table 4). In comparison, Chen et al. found that for Protein L affinity media, the addition of salt during elution preferentially increased the binding strength of mismatched bsAbs (C. Chen, et al., MAbs. 11 (2019) 632-638).

[0098] It is not intuitive that as little as 20 mM NaCl would substantially affect the binding strength between protein and affinity ligand for CaptureSelect Lambda XP. Compared to previous data for Protein L media showing that 50-100 mM NaCl is suitable for enhancing valency-mediated separation, CaptureSelect Lambda XP requires even less salt, 20 mM. Similar to previous data for CaptureSelect Kappa XP and Protein L, valency-mediated separation with CaptureSelect Lambda XP appears to be enhanced by preferential enhancement of impurity binding to the media with the addition of elution modifiers. However, binding strength of bsAbs and impurities is much more sensitive to salt addition / conductivity for CaptureSelect Lambda XP compared to CaptureSelect Kappa XP.

[0099] Significantly, these elution methods on Lambda LC affinity chromatography media are highly suitable for scale-up of large-scale biopharmaceutical manufacturing. The low concentrations of common elution modifiers required for improved impurity separation have minimal impact on subsequent purification or viral inactivation unit operations. In the actual scale-up of this method, strategies can be designed around the loading challenge, elution pH and salt concentration. Tailing of the elution peaks can also pose significant challenges for scale-up due to limitations of intermediate product holding tanks, but strategies around peak recovery criteria and further optimization of mobile phase conditions can mitigate this issue.

[0100] LC affinity chromatography is becoming established as a robust platform for asymmetric bsAb purification. Existing and emerging commercial LC affinity media offer attractive properties that enable manufacturing processes for large-scale antibody therapeutics production. A consistent ability of commercial light chain affinity media to leverage binding affinity to separate mismatched variants, aggregates and LMW impurities of a model asymmetric bsAb was demonstrated. Through pH gradient screening, CaptureSelect Kappa XP and Lambda XP were found to provide the best baseline resolution of product variants with multiple light chains of the same class as the media specificity. The differences in resolution between the various chromatographic media tested are not fully explained by the properties of the media-based matrix alone. It is likely that these effects may also depend on the unique attributes of the ligand and / or conjugation chemistry. The specific running conditions of our experiments, such as residence time, protein loading and gradient slope, also played a role in achieving resolution. Half antibodies and free light chain impurities from several bsAb constructs showed higher binding strengths with the respective LC affinity media compared to bsAb heterodimers. These findings may suggest that affinity-binding alone does not fully explain the increased binding strength observed with affinity media.

[0101] These separations can be enhanced with CaptureSelect Kappa XP and Lambda XP chromatography media by the addition of elution modifiers. CaptureSelect Kappa XP media requires a much higher concentration of elution modifier to significantly improve impurity resolution compared to CaptureSelect Lambda XP media. The addition of 500 mM magnesium chloride surprisingly resulted in baseline separation with CaptureSelect Kappa XP media, with mismatched and half-antibody species still binding at pH 2.8. For CaptureSelect Lambda XP media, the addition of only 20 mM NaCl substantially separated the bsAb heterodimers from associated impurities in isocratic elution conditions. For both CaptureSelect Kappa XP and Lambda XP, the results suggest that improved separation is achieved by preferential enhancement of impurity binding. Comparing these findings with previous literature, some parallels can be seen with Protein A and Protein L binding affinity separations. However, previous conclusions regarding the chaotropic tendencies of the modifiers or the disruption of repulsive forces do not appear to fully explain the phenomena observed with CaptureSelect Kappa XP and Lambda XP media, and further studies are needed to elucidate the mechanism.

[0102] The scalability of these processes can be improved by novel chromatographic media that provide further enhanced mass transfer properties, and perhaps more importantly, by continued ligand optimization. Further ligand optimization, such as increasing ligand density, improving flexibility, decreasing impurity binding, and other modifications, can improve binding capacity and increase accessibility to multiple binding events. Deeper investigations into the mechanisms by which lyotropic salts or other modifiers affect the binding strength of multivalent ligand-protein interactions can expand the operational space for optimizing these separations for various affinity media. Further insight can also be gained by evaluation of potential differences in binding and dissociation kinetics, binding orientation, and protein conformation.

Claims

1. A method for purifying a target asymmetric bispecific antibody, comprising: (a) contacting a composition containing the bispecific antibody with a lambda light chain affinity matrix; (b) washing the affinity matrix to remove impurities; and (c) eluting the bispecific antibody with an elution buffer containing about 5 mM to about 45 mM of salt. The aforementioned asymmetric bispecific antibody comprises a heterodimer containing one lambda light chain and one kappa light chain, and the method results in the removal of mispairs containing two light chains of the same class, removal of aggregates, and removal of low molecular weight impurities from the composition after elution in the single affinity matrix. method.

2. The method according to claim 1, wherein the elution buffer further comprises an agglutination inhibitor containing arginine or an arginine derivative.

3. The method according to claim 2, wherein the aggregation inhibitor is arginine-HCl.

4. The method according to claim 1, wherein the concentration of the salt is approximately 20 mM.

5. The method according to claim 4, wherein the salt is sodium chloride, magnesium chloride, ammonium chloride, sodium acetate, sodium citrate, sodium phosphate, sodium sulfate, arginine-HCl, or histidine-HCl.

6. The method according to claim 1, wherein the pH of the elution buffer is approximately 3 to approximately 4.

5.

7. The method according to claim 6, wherein the pH of the elution buffer is approximately 3.

5.

8. The method according to claim 1, further comprising contacting the asymmetric bispecific antibody with a kappa light chain affinity matrix and eluting the bound bispecific antibody.

9. The method according to claim 8, wherein contacting the asymmetric bispecific antibody with the kappa light chain affinity matrix is ​​performed before contacting the bispecific antibody with the lambda light chain affinity matrix.

10. The method according to claim 8 or 9, wherein the eluate obtained after elution is passed directly through the second affinity matrix.

11. The method according to claim 1, which is performed in a closed system.

12. The method according to claim 1, wherein the lambda light chain affinity matrix is ​​conjugated to a solid support.

13. The method according to claim 8, wherein the kappa light chain affinity matrix and / or the lambda light chain affinity matrix are conjugated to a solid support.

14. The method according to claim 12 or 13, wherein the solid support is crosslinked agarose.

15. The method according to claim 1, wherein the bispecific antibody comprises a modified heavy chain and / or a modified light chain.

16. The bispecific antibody comprises: (a) a Fab region including a modified heavy chain, wherein the CH1 region of the modified heavy chain includes (i) substitution of a natural cysteine ​​with a non-cysteine ​​amino acid, and (ii) substitution of a natural non-cysteine ​​amino acid with a cysteine ​​amino acid; (b) a corresponding modified light chain, wherein the CL region of the modified light chain includes (i) substitution of a natural cysteine ​​with a non-cysteine ​​amino acid, and (ii) substitution of a natural non-cysteine ​​amino acid with a cysteine ​​amino acid; and (c) a second Fab region including a second heavy chain. The method according to claim 15, further comprising (d) a second corresponding light chain, wherein the modified heavy chain is directly coupled to the corresponding modified light chain, and in a separate target binding arm, the second heavy chain is directly coupled to the second corresponding light chain, and the substituted cysteine ​​of the modified heavy chain obtained from the substitution of the natural non-cysteine ​​amino acid to the cysteine ​​amino acid and the substituted cysteine ​​of the corresponding modified light chain obtained from the substitution of the natural non-cysteine ​​amino acid to the cysteine ​​amino acid can form a disulfide bond.

17. (a) The second heavy chain and the second corresponding light chain do not involve substitution of a native non-cysteine ​​amino acid for a cysteine ​​amino acid, and do not involve substitution of a native cysteine ​​for a non-cysteine ​​amino acid; and / or (b) The two light chains each comprise a VL domain and a CL domain, wherein the VL domain has a different amino acid sequence, and the CL domain has a different amino acid sequence; and / or (c) The two heavy chains each comprise a VH domain, a CH1 domain and an Fc region, wherein the VL domain has a different amino acid sequence, the CH1 domain has a different amino acid sequence, and the Fab region has a different amino acid sequence, and optionally one light chain is a kappa light chain and one light chain is a lambda light chain, the method according to claim 16.

18. The method according to claim 17, wherein the two heavy chains form a heterodimer.

19. The method according to any one of claims 15 to 18, wherein the bispecific antibody specifically binds to two independent antigens or two independent epitopes on the same antigen.

20. The method according to claim 19, wherein the Fc region of either or both of the heavy chains comprises one or more modifications, optionally, the modifications promote heterodimerization of the heavy chains.

Images