Method for purifying bispecific antibodies

Abstract

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

Description

[0001] 1. Cross-reference to related applications

[0002] This application claims the benefit of priority to U.S. Provisional Patent Application No. 63 / 269,497, filed on March 17, 2022, which is incorporated herein by reference in its entirety for all purposes. 2. Technical field

[0003] The present disclosure generally relates to methods for purifying bispecific antibodies using light chain affinity chromatography. 3. Background technology

[0004] Affinity chromatography is commonly used for large-scale production and purification of large quantities of biotherapeutics such as monoclonal antibodies. (KM et al., Biotechnol. J., 15 (2020); GR Bolton et al., Biotechnol. Prog, 32 (2016) 1193-1202). Affinity chromatography provides process robustness due to its high specificity for the target product and enables the design of purification platforms that simplify process development and reduce risk manufacturing (S. Sommerfeld et al., Chem. Eng. Process. Process Intensif, 44 (2005)).

[0005] Various therapeutic modalities, including multispecific antibodies, fabs, and antibody fragments, utilize light chain (LC) affinity chromatography for product capture (G. Rodrigo et al., Antibodies [Antibodies], 4 (2015) 259-277; J. Spooner et al., Biotechnol. Bioeng. [Biotechnology and Bioengineering] 112 (2015) 1472-1477; N. Fischer et al., Nat. Commun. [Nature Communications] 6 (2015) 6113). Of interest here is that asymmetric bispecific antibodies (bsAbs) are attractive therapeutic modalities because they have a unique mechanism of action and, importantly, their native antibody form retains good pharmacokinetic, stability, solubility, and immunogenicity characteristics (AF Labrijn et al., Nat. Rev. Drug Discov [Nature Review: Drug Discovery], 18 (2019) 585-608). However, these formats have historically been plagued by chain-related problems (C. Klein et al., MAbs [monoclonal antibodies], 4 (2012) 653-663). Protein engineers have mediated the formation of heavy and light chain mispairing variants by adopting various strategies (such as steric and electrostatic steering, domain crossing, common light chain and kappa-lambda bodies, etc.) (H. Lindhofer et al., J. Immunol. [Journal of Immunology], 155 (1995) 219-25; J. B. B. Ridgway et al., Protein Eng. Des. Sel. [Protein Engineering Design and Selection], 9 (1996) 617-621; K. Gunasekaran et al., J. Biol. Chem. [Journal of Biological Chemistry], 285 (2010) 19637-19646; J. H. Davis et al., Protein Eng. Des. Sel. [Protein Engineering Design and Selection], 23 (2010) 195-202; W. Schaefer et al., Proc. Natl. Acad. Sci. [Proceedings of the National Academy of Sciences of the United States of America], 108 (2011) 11187-11192). Although chain pairing fidelity is improved with these designs, mispairing variants are still commonly observed after expression.Optimized expression programs, in vitro assembly, and high-throughput clonal selection can also reduce the downstream burden, but typically some levels of these chain mispairing impurities remain and must be controlled during downstream processing (AF Labrijn et al., Proc. Natl. Acad. Sci., 110 (2013) 5145-5150; P. Strop et al., J. Mol. Biol., 420 (2012) 204-219; G. Magistrelli et al., MAbs. 9 (2017) 231-239; G. Giese et al., Biotechnol. Prog., 34 (2018) 397-404; and C. Wang et al., MAbs. 10 (2018) 1226-1235). Therefore, product-related impurities remain a major obstacle to the development of commercial-scale purification processes for asymmetric bsAbs, and additional scalable purification strategies are needed. 4. Summary of the invention

[0006] In some aspects, disclosed herein is a method of purifying an asymmetric bispecific antibody, the method 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 between about 5 mM and about 45 mM salt.

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

[0008] In some aspects, the concentration of the salt is about 20 mM. In some aspects, 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 aspects, the pH of the elution buffer is between about 3 and about 4.5. In some aspects, the pH of the elution buffer is about 3.5.

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

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

[0012] In some aspects, the bispecific antibody comprises a modified heavy chain and / or a modified light chain. In some aspects, the bispecific antibody comprises: (a) a Fab region comprising a modified heavy chain, wherein the CH1 region of the modified heavy chain comprises (i) a substitution of a native cysteine ​​to a non-cysteine ​​amino acid, and (ii) a substitution of a native non-cysteine ​​amino acid to a cysteine ​​amino acid; (b) a modified corresponding light chain, wherein the CL region of the modified light chain comprises (i) a substitution of a native cysteine ​​to a non-cysteine ​​amino acid, and (ii) a substitution of a native non-cysteine ​​amino acid to a cysteine ​​amino acid; (c) a second Fc 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 on a separate target binding arm, the second heavy chain is directly linked to the second corresponding light chain, and wherein the substituted cysteine ​​in the modified heavy chain resulting from the substitution of the native non-cysteine ​​amino acid to a cysteine ​​amino acid and the substituted cysteine ​​in the modified corresponding light chain resulting from the substitution of the native non-cysteine ​​amino acid to a cysteine ​​amino acid can form a disulfide bond. In some aspects, (a) the second heavy chain and the second corresponding light chain do not comprise a substitution of a natural non-cysteine ​​amino acid to a cysteine ​​amino acid and do not comprise a substitution of a natural cysteine ​​to a non-cysteine ​​amino acid; and / or (b) each of the two light chains comprises a VL domain and a CL domain, wherein the VL domains have different amino acid sequences and the CL domains have different amino acid sequences; and / or (c) each of the two heavy chains comprises a VH domain, a CH1 domain, and an Fc region, wherein the VH domains have different amino acid sequences, the CH1 domains have different amino acid sequences, and the Fc regions have different amino acid sequences, optionally wherein one light chain is a κ light chain and one light chain is a λ light chain. In some aspects, the two heavy chains form heterodimers. In some aspects, the bispecific antibody specifically binds to two independent epitopes on two independent antigens or the same antigen. In some aspects, the Fc region of any one or both heavy chains comprises one or more modifications, optionally wherein these modifications promote heterodimerization of the heavy chain.

[0013] In some aspects, these methods enable the removal of mispaired species, the removal of aggregates, and / or the removal of low molecular weight impurities from the composition. 5. Description of the drawings

[0014] Figure 1Lambda light chains and corresponding heavy chains are shown in yellow, and kappa light chains are shown in red. For each corresponding bsAb, the mispaired species shown above will be observed in the protein A product of the captured cell culture harvest.

[0015] Figure 2 Chromatographic elution profiles of bsAbs 1 and 2 on CaptureSelect Kappa XP, CaptureSelect Kappa XL, Capto L, and KappaSelect from pH gradient elution screening experiments are shown. Absorbance at 280 nm (solid line) is referenced to the left y-axis, and elution pH (dashed line) is referenced to the right y-axis.

[0016] Figure 3 Shown are the chromatographic elution profiles of bsAbs 1 and 3 on λ LC affinity media from pH gradient elution screening experiments.

[0017] Figure 4 The absorbance trace of the pH gradient elution screening product via HIC-HPLC analysis is shown. The mispaired composition of the separated elution peaks is shown compared to CM purified by conventional methods, detailing the mispaired impurities present in the loaded material.

[0018] Figure 5 Shown are the elution profiles of bsAb2 on KappaXP affinity medium eluted with a pH gradient and supplemented with (a) no modifier, (b) 250 mM sodium chloride, (c) 500 mM sodium citrate, (d) 500 mM MgCl, and (e) 500 mM L-arginine.

[0019] Figure 6 The elution profile of bsAb 1 from CaptureSelectLambdaXP under pH 3.5 step elution conditions is shown. The first peak appears during the elution phase and the second peak appears during the stripping phase. 6. Specific implementation methods

[0020] In order to more easily understand the present disclosure, some terms are first defined. As used in this application, unless otherwise expressly provided herein, each of the following terms shall have the meaning set forth below. Additional definitions are set forth throughout the application.

[0021] 6.1 Terminology

[0022] The methods provided herein are generally not limited to specific compositions or process steps and as such may vary.

[0023] The term "chromatography" refers to any type of technique for separating a target protein (e.g., an antibody) from other molecules present in a mixture (e.g., contaminants). Typically, the target protein is separated from other molecules (e.g., contaminants) due to the different rates at which the individual molecules of the mixture pass through a fixed medium under the influence of a mobile phase or in a binding and elution process. 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 a target protein (e.g., a protein containing an Fc region, such as an immunoglobulin) from other molecules present in a mixture in a separation process. Non-limiting examples include granular resins, monolithic resins or fibrous resins, and membranes that can be placed in a column or cartridge. Examples of materials for forming a matrix include polysaccharides (e.g., agarose and cellulose); and other mechanically stable matrices such as silica (e.g., controlled pore glass), poly(styrenedivinyl) benzene, polyacrylamide, ceramic particles, and derivatives of any of the above substances. Examples of typical matrix types suitable for use in the methods disclosed herein are cation exchange resins, affinity resins, anion exchange resins, or mixed mode resins. "Part " is a functional group attached to the chromatographic matrix and determines the binding properties of the matrix. "Part " examples include but are not limited to ion exchange groups, hydrophobic interaction groups, hydrophilic interaction groups, sulfophilic interaction groups, metal affinity groups, affinity groups, bioaffinity groups and mixed mode groups (combinations of the above). Some ligands that can be used in this article 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.

[0024] The term "affinity chromatography" refers to a protein separation technique in which a target protein (e.g., a target protein or antibody containing an Fc region) is specifically bound to a ligand that is specific to the target protein. Such a ligand is commonly referred to as a biospecific ligand. In some aspects, a biospecific ligand (e.g., protein A or its functional variant) is covalently attached to a chromatographic matrix material, and the target protein in the solution is accessible when the solution contacts the chromatographic matrix. The target protein usually maintains its specific binding affinity to the biospecific ligand in the chromatographic step, while other solutes and / or proteins in the mixture will not be significantly or specifically bound to the ligand. The binding of the target protein to the fixed ligand allows contaminating proteins or protein impurities to pass through the chromatographic matrix, and the target protein maintains specific binding to the fixed ligand on the solid phase material. The target protein of specific binding is then removed from the fixed ligand in an active form under suitable conditions (e.g., low pH, high pH, ​​high salt, competitive ligand, etc.), and is passed through the chromatographic column together with the elution buffer, without allowing the contaminating proteins or protein impurities that pass through the column earlier. Any component can be used as a ligand to purify its corresponding specific binding protein, eg, an antibody.

[0025] The terms "purify", "separate" or "isolate" as used interchangeably herein refer to increasing the degree of purity of a protein of interest from a composition or sample comprising the protein of interest and one or more impurities. Typically, the degree of purity of the protein of interest is increased by removing (completely or partially) at least one impurity from the composition.

[0026] As used herein, the term "buffer" refers to a substance whose presence in a solution increases the amount of acid or base that must be added to cause a unit change in pH. Buffer solutions resist changes in pH through the action of their acid-base conjugate components. Buffer solutions used with biological reagents are generally capable of maintaining a constant hydrogen ion concentration so that the pH of the solution is within the physiological range. Traditional buffer components include, but are not limited to, organic and inorganic salts, acids, and bases.

[0027] As used herein, the term "chromatography column" or "column" in relation to chromatography refers to a container, usually in the form of a cylinder or hollow column, filled with a chromatography matrix or resin. A chromatography matrix or resin is a material that provides physical and / or chemical properties for purification.

[0028] The term "bind-and-elute" or "bind-and-elute mode" of purification refers to when the target protein of interest is retained on the column and the impurities flow through the column. The process then involves specifically eluting the protein of interest using different column conditions that interfere with the binding of the protein of interest to the chromatographic medium (usually the resin in the column).

[0029] The term "elution buffer" refers to a buffer that effectively dissociates protein:protein interactions without permanently affecting the protein structure.

[0030] As used herein, the term "contaminant" is used in its broadest sense to encompass any undesirable component or compound in a mixture. In a cell culture, a cell lysate or a clarified stock solution (e.g., a clarified cell culture supernatant), contaminants include host cell nucleic acids (e.g., DNA) and host cell proteins present in, for example, a cell culture medium. Host cell contaminant proteins include, but are not limited to, those proteins produced naturally or recombinantly by host cells, and proteins (e.g., proteolytic fragments) relevant to or derived from target proteins and other process-related contaminants. In some aspects, another means (such as centrifugation, sterile filtration, depth filtration, and tangential flow filtration) is used to separate the contaminant precipitate from the cell culture.

[0031] The term "loading buffer" refers to the buffer used to prepare the mixture or sample and load it into the chromatography unit.

[0032] The term "wash buffer" refers to the buffer used after the loading buffer in order to drive the mixture or sample through the chromatography process.

[0033] The term "HMW species" refers to any one or more unwanted proteins present in a mixture. High molecular weight species may include dimers, trimers, tetramers or other polymers. These species are generally considered to be impurities associated with the product and may be covalently or non-covalently linked and may also, for example, consist of misfolded monomers in which hydrophobic amino acid residues are exposed to polar solvents and may cause aggregation.

[0034] The term "LMW species" refers to any one or more unwanted species present in a mixture. Low molecular weight species are generally considered to be impurities associated with the product and may include sheared species or half molecules of a compound intended to be a dimer (such as a monoclonal antibody).

[0035] The term "host cell protein" or HCP refers to an undesirable protein produced by the host cell that is unrelated to the production of the intended protein of interest. Undesirable host cell proteins can be secreted into the upstream cell culture supernatant. Undesirable host cell proteins can also be released during cell lysis. Cells used for upstream cell culture require proteins for growth, transcription, and protein synthesis, and these unrelated proteins are undesirable in the final drug product.

[0036] The term "antibody" means an immunoglobulin molecule that recognizes and specifically binds to a target (such as a protein, polypeptide, peptide, carbohydrate, polynucleotide, lipid or a combination of the foregoing) by at least one antigen recognition site in the variable region of an immunoglobulin molecule. As used herein, the term "antibody" encompasses complete polyclonal antibodies, complete monoclonal antibodies, chimeric antibodies, humanized antibodies, human antibodies, fusion proteins comprising antibodies, and any other modified immunoglobulin molecules, as long as these antibodies exhibit the desired biological activity. The antibody can be any of the following five major classes of immunoglobulins: IgA, IgD, IgE, IgG and IgM, or its subclass (isotype) (e.g., IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2), which are referred to as α, δ, ε, γ and μ based on the characteristics of their heavy chain constant domains. Different classes of immunoglobulins have different and well-known subunit structures and three-dimensional configurations. Antibodies can be naked or conjugated to other molecules such as toxins, radioisotopes, etc.

[0037] In the absence of explicit instructions, and unless the context indicates otherwise, the term "antibody" includes monospecific, bispecific or multispecific antibodies and single-chain antibodies. In some aspects, the antibody is a bispecific antibody. The term "bispecific antibody" refers to an antibody that binds to two different epitopes. The epitopes can be on the same target antigen or can be on different target antigens. The term "bispecific antibody" also refers to an antibody fragment comprising both a kappa light chain and a lambda light chain, such as a Fab.

[0038] The term "antibody fragment" refers to a part of a complete antibody. "Antigen binding fragment", "antigen binding domain" or "antigen binding region" refers to a part of a complete antibody that binds to an antigen. In the context of bispecific antibodies, an "antigen binding fragment" binds two antigens. The antigen binding fragment may contain the antigen recognition site of a complete antibody (e.g., a complementary determining region (CDR) sufficient to specifically bind to an antigen). Examples of antigen binding fragments of antibodies include, but are not limited to, Fab, Fab', F(ab')2 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 produced.

[0039] "Monoclonal" antibody or its antigen binding fragment refers to a homologous antibody or its antigen binding fragment group that is related to a single antigenic determinant or epitope with high specificity. This is in contrast to polyclonal antibodies that typically include different antibodies against different antigenic determinants. The term "monoclonal" antibody or its antigen binding fragment encompasses complete and full-length monoclonal antibodies as well as antibody fragments (such as Fab, Fab', F(ab')2, Fv), single-chain (scFv) mutants, fusion proteins comprising antibody portions, and any other modified immunoglobulin molecules comprising antigen recognition sites. In addition, "monoclonal" antibody or its antigen binding fragment refers to such antibodies and their antigen binding fragments prepared in any number of ways (including but not limited to by hybridoma, phage selection, recombinant expression, and transgenic animals).

[0040] 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 part of an antibody, usually a part of a light chain or a heavy chain, typically about 110 to 120 amino acids or 110 to 125 amino acids at the amino terminal in a mature heavy chain and about 90 to 115 amino acids in a mature light chain, which differ in sequence between antibodies and are used for the binding and specificity of a specific antibody to its specific antigen. The variability of the sequence is concentrated in those regions called complementary determining regions (CDRs), while the more conservative regions in the variable domains are called framework regions (FRs). It is not desirable to be bound by any particular mechanism or theory, and it is believed that the CDRs of light and heavy chains are primarily responsible for the interaction and specificity of antibodies with antigens. 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 a particular aspect of the present disclosure, the variable region is a primate (e.g., non-human primate) variable region. In some aspects of the disclosure, the variable regions comprise rodent or murine CDRs and primate (eg, non-human primate) framework regions (FRs).

[0041] As used herein, the term "heavy chain" or "HC" when used to refer to an antibody can refer to any of the different types based on the amino acid sequence of the constant domain, e.g., alpha (α), delta (δ), epsilon (ε), gamma (γ), and mu (μ), which produce IgA, IgD, IgE, IgG, and IgM class antibodies, respectively, including IgG subclasses, e.g., IgG1, IgG2, IgG3, and IgG4. Heavy chain amino acid sequences are well known in the art. In some aspects of the present disclosure, the heavy chain is a human heavy chain.

[0042] As used herein, the term "light chain" or "LC" when used to refer to an antibody can refer to any of the different types, e.g., kappa (κ) or lambda (λ), based on the amino acid sequence of the constant domain. Light chain amino acid sequences are well known in the art. In some aspects of the present disclosure, the light chain is a human light chain.

[0043] Amino acids are referred to herein by either their commonly known three letter symbols or by the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Likewise, nucleotides are referred to by their universally recognized single-letter codes.

[0044] As used in the disclosure and claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

[0045] It should be understood that wherever aspects of the present disclosure are described herein with the language “comprising,” other similar aspects described with respect to “consisting of” and / or “consisting essentially of” are also provided.

[0046] Unless expressly stated or obvious from the context, as used herein, the term "or" is understood to be inclusive. The term "and / or" as used herein in phrases such as "A and / or B" is intended to include "A and B," "A or B," "A," and "B." Similarly, the term "and / or" as used in phrases such as "A, B, and / or C" is intended to cover each of the following: 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).

[0047] As used herein, the terms "about" and "approximately" when used to modify a numerical value or numerical range indicate that deviations of 5% to 10% above and below the numerical value or range are still within the intended meaning of the recited value or range.

[0048] Any composition or method provided herein can be combined with one or more of any other compositions and methods provided herein.

[0049] Units, prefixes and symbols are expressed in their acceptable form of the Système International de Unites (SI). Numerical ranges include numbers that limit the range. The headings provided herein are not limitations of the various aspects of the disclosure, which can be obtained by reference to this specification as a whole. Therefore, the terms defined immediately below are more fully defined by reference to the specification in its entirety.

[0050] 6.2 Bispecific Antibodies

[0051] In some aspects, antibody provided herein is a monovalent bispecific antibody (MBab). The monovalent bispecific antibody scaffold described herein provides an excellent platform for producing bispecific antibodies, and these bispecific antibodies realize all the benefits associated with bispecific antibodies, while reducing the potential treatment risks mentioned above due to their monovalent properties. In addition, MBab provided herein is easy to express, stable and may have low immunogenicity. As used herein, the term "monovalent bispecific" (it can be abbreviated as "MBab") refers to a bispecific antibody (bsAb), wherein each arm can be specifically bound to different target antigens, and for a given pair of different target antigens (A and B), MBab can be combined with each of them. In some aspects, monovalent bispecific antibodies can specifically bind to two independent antigens (or targets) or two independent epitopes on the same antigen. Typically, monovalent bispecific antibodies include two different variable regions. In some aspects, the binding affinity to two independent antigens is approximately the same. In some aspects, the binding affinity to two independent antigens is different. In some respects, the binding affinity to two independent epi-positions on the same antigen is approximately the same. In some respects, the binding affinity to two independent epi-positions on the same antigen is different. In still other respects, each arm has the same specificity (for example, in conjunction with the same or overlapping epi-position), but combines with different affinities. In some respects, the affinities can differ by 3 times or more. When the variable regions with different in vivo effectiveness of the combined antibody, it may be particularly desirable to have an arm with higher affinity and an arm with lower affinity to prevent the dosage in these arms from being excessive or insufficient.

[0052] In some aspects, MBab binds to the same epitope or overlapping epitopes on the same antigen (for example, receptor) with different avidities. Specifically, the same epitope or overlapping epitopes are closely approached when the antigen is dimerized. Depends on relative concentration, such antibody will have dual features. For example, under high concentration, when the MBab concentration makes the antigen concentration saturated, the high-affinity binding domain will compete to eliminate the low-affinity binding domain, and the avidity effect will hardly occur or not occur. That is to say, the antibody will mainly play the role of monovalent binding entity and antigen crosslinking / signaling will hardly occur or not occur. However, under low concentration, the avidity effect will play a role, and MBab can combine two binding sites (preferably on two antigen molecules) simultaneously, thereby causing antigen crosslinking / signaling. In this way, antigen signaling can be regulated by MBab concentration.

[0053] In some aspects, when the homodimerization of antigen is undesirable and / or two antigens are all independently present on non-target cells / tissues and are present on target cells / tissues together, MBab combines two different antigens (for example, different receptors).Such antibody will be combined with only one arm on non-target cells, and only one arm of MBab is combined with this low-affinity unit price of non-target cells / tissues and is not enough to cause homodimerization.By contrast, MBab can be combined to two antigens on target cells / tissues, and being combined to two antigens on target cells simultaneously will cause higher avidity bivalent combination, and this higher avidity bivalent combination can strengthen the preferential combination with target cells and can strengthen receptor dimerization.

[0054] In some aspects, the monovalent bispecific antibody further comprises other binding sites.These other binding sites can be specific to one or two target antigens (A and B) of monoclonal bispecific antibody (MBab) and / or can be specific to other target antigens.In some aspects, one or more single-chain variable fragments (scFv) are added to the N-terminal or C-terminal of one or two heavy chains and / or one or two light chains, wherein one or more scFv specifically binds to one or more other target antigens.For example, monovalent trispecific antibodies can be produced by adding scFv (specific to antigen C) to a chain (for example, heavy chain or light chain) of monovalent bispecific antibodies (specific to antigen A and B).In this case, antibody will be monovalent for antigen A, B and C.If scFv (specific to antigen C) is added to two chains (for example, two heavy chains, two light chains, a heavy chain and a light chain), so trispecific antibodies will be monovalent to antigen A and B and divalent to antigen C. Any possible combination of additional binding sites is contemplated for monovalent bispecific antibodies herein (see, e.g., Dimasi et al., J. Mol. Biol., (2009) 393: 672-692). It is contemplated that the binding affinity of the additional binding sites 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 described above, relative affinity can be selected or customized based on the intended use of the antigen and molecule.

[0055] Bispecific antibodies can be prepared as full-length antibodies or antibody fragments (e.g., F(ab')2 bispecific antibodies). The traditional production of full-length bispecific antibodies is based on the co-expression of two immunoglobulin heavy chain-light chain pairs, where the two chains have different specificities. Due to the random collocation of immunoglobulin heavy and light chains, these hybridomas (quadruplexes) produce a potential mixture of 10 different antibody molecules, only one of which has the correct bispecific structure. The purification of the correct molecule, which is usually performed by an affinity chromatography step, is very cumbersome, and the product yield may be low.

[0056] Bispecific antibodies include cross-linked or "heteroconjugate" antibodies. For example, one of the antibodies in the heteroconjugate can be coupled to avidin and the other to biotin. Heteroconjugate antibodies can be prepared using any convenient cross-linking method. However, this method typically requires the use of non-human proteins, which can carry potential high immunogenicity. In addition, antibody fragments sometimes have little or no effector function and have a shorter half-life.

[0057] Bispecific antibodies can be prepared using chemical connections. In one procedure, complete antibodies are proteolytically cleaved to produce F(ab')2 fragments. These fragments are reduced in the presence of dithiol complexing agents, sodium arsenite, to stabilize adjacent dithiols and prevent the formation of intermolecular disulfides. The generated Fab' fragments are then converted into thionitrobenzoic acid (TNB) derivatives. Then by reducing with mercaptoethylamine, one of the Fab'-TNB derivatives is reconverted into Fab'-thiol and mixed with another Fab'-TNB derivative of equimolar amount to form bispecific antibodies. However, this method often results in poor yields, is difficult to control, and the product can carry potential high immunogenicity. In addition, antibody fragments sometimes have little or no effector function and have a shorter half-life.

[0058] Fab'-SH fragment can be directly recovered from Escherichia coli, and this fragment can be chemically coupled to form bispecific antibodies. Fully humanized bispecific antibody F(ab')2 molecules can be produced by secreting each Fab' fragment from Escherichia coli respectively and carrying out external directed chemical coupling to form bispecific antibodies. However, this method often leads to poor yield and is difficult to control. In addition, antibody fragments sometimes have little or no effector function and have a shorter half-life.

[0059] Bispecific antibodies can also be produced using leucine zippers. Leucine zipper peptides from Fos and Jun proteins are connected to the Fab' parts of two different antibodies by gene fusion. Antibody homodimers are reduced to form monomers at the hinge region, and then oxidized again to form antibody heterodimers. This method can also be used to produce antibody homodimers. The described "diabody" technology provides another mechanism for preparing bispecific antibody fragments. These fragments include VH connected to VL by a linker, and the linker is too short to pair the two domains on the same chain. Therefore, the VH and VL domains of a fragment are forced to pair with the complementary VL and VH domains of another fragment, thereby forming two antigen binding sites. Another strategy for preparing bispecific antibody fragments using single-chain Fv (sFv) dimers is known in the art. However, this method often results in poor yields, is difficult to control, and the product can carry potential high immunogenicity. In addition, antibody fragments sometimes have little or no effector function and have a shorter half-life.

[0060] Bispecific antibodies can also be produced using heavy chain heterodimerization methods. Such methods include "knob inhole" and chain exchange engineered domain (SEED) methods, as well as those that change the charge polarity of the entire Fc dimer interface. Such methods are further described in detail herein and in, for example, the following documents: U.S. Patent No. 7,183,076; Merchant et al. (1998) Nat. Biotech [Natural Biotechnology] 16: 677-681; Ridgway et al. (1996) Protein Engineering [Protein Engineering] 9: 617-621; Davis et al. (2010) Prot. Eng. Design & Selection [Protein Engineering, Design and Selection] 23: 195-202; WO 2007 / 110205; WO2007 / 147901; Gunasekaran et al. (2010) JBC [Journal of Biological Chemistry] 285: 19637-46. In these methods, the interface between a pair of antibody molecules can be engineered to maximize the percentage of heterodimers recovered from recombinant cell culture. In the "knob-in-hole structure" method, "protrusion" is produced by replacing one or more small amino acid side chains from the interface of the first antibody molecule with larger side chains (for example, tyrosine or tryptophan). The compensation "cavity" of size identical or similar to one or more large side chains is produced on the interface of the second antibody molecule by replacing the amino acid with large side chains with amino acids (for example, alanine or threonine) with smaller side chains. This provides a mechanism for increasing the output of heterodimers relative to other undesirable end products (such as homodimers). In the SEED method, Fc homodimers are converted into heterodimers by making the β-chain segments of human IgG and IgA CH3 domains cross each other. These derivatives of human IgG and IgA CH3 domains have produced complementary people SEED CH3 heterodimers consisting of the alternating segments of human IgA and IgG CH3 sequences. When expressed in mammalian cells, the resulting SEED CH3 domain pairs preferentially associate to form heterodimers. Other methods include introducing modifications that change the charge polarity of the entire Fc dimer interface, so that the co-expression of the Fc region of electrostatic matching causes heterodimerization. These methods improve heavy chain heterodimerization, but do not solve the light chain-heavy chain mispairing formed in the bispecific antibody formation process. In some cases, the use of public light chains can reduce the number of possible mispairings, as described in WO 98 / 50431, but often results in the loss or reduction of binding specificity and / or avidity.

[0061] Bispecific antibodies are another type of bispecific antibody that can be produced (see, e.g., Bostrum et al. (2009) Science 323: 1610-1614). Such antibodies can be produced by generating variants with mutations in the light chain (LC) complementary determining region (CDR), so that these antibodies can bind to new antigen targets while maintaining binding specificity to their natural target antigens. However, antigen binding sites often overlap, preventing the antibody from binding to two antigens simultaneously. In addition, these antibodies are difficult to produce and may not have the desired affinity for each of the two antigens.

[0062] The modified polypeptide provided herein can be used to produce bispecific antibodies and overcome the limitations and technical difficulties as described above. In some aspects, a heavy chain and a light chain in an antibody are modified, whereby native cysteine ​​is replaced by non-cysteine ​​amino acids, and native non-cysteine ​​amino acids are replaced by cysteine ​​amino acids. Such modifications provided herein are produced in HC and LC domains and result in the relocation of HC-LC interchain disulfide bridges. When bispecific antibodies are produced from four separate polypeptides, for example, in the case where the modified arm has binding specificity to a target and the unmodified arm has binding specificity to different targets, four polypeptides will be assembled so that the modified heavy chain is appropriately hybridized with the modified light chain and the unmodified heavy chain is appropriately hybridized with the unmodified light chain. As used herein, the term "unmodified" refers to heavy and light chains that do not contain the HC-LC modified heavy chains and light chains introduced for the relocation of cysteine ​​and / or disulfide bridges, as described herein. Such "unmodified" heavy and light chains may contain other modifications, such as, for example, heterodimerization modifications within the CH2 and / or CH3 regions described herein and / or known in the art. The HC-LC modifications provided herein may be combined with additional modifications of the heavy chain, particularly in the CH2 and / or CH3 regions, to ensure proper heavy chain heterodimerization and / or enhance purification of heavy chain heterodimers and are described in detail below.

[0063] The bispecific antibodies of the invention may be asymmetric, containing one kappa light chain and one lambda light chain. The bispecific antibodies may also contain HC and / or LC, such as those described in US Pat. No. 9,527,927, which is incorporated herein by reference.

[0064] 6.3 Method of the present invention

[0065] The present invention evaluates the ability of commercially available LC affinity chromatography media to separate multiple product-related impurities of selected asymmetric bsAb constructs (composed of four unique polypeptide chains) (Y. Mazor et al., MAbs. [monoclonal antibodies] 7 (2015) 377-389). Currently, several scalable chromatography media options with specificity for κ and λ light chains are commercially available. Protein L media provide specificity for the variable regions of certain κ light chain subtypes, while other κ and λ LC affinity media provide specificity for κ and λ light chain constant regions (M. Grile et al., Structure [structure], 9 (2001) 679-687). In addition to protein L, these LC affinity media use camel 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 TM Pabst et al., Biotechnol. J, 12 (2017) 1600357). The development of one example of these chromatographic media, Lambdafabselect, has been described in depth in the literature previously (N. Eifler et al., Biotechnol. Prog., 30 (2014) 1311-1318). Two mechanisms for separation by LC affinity chromatography are described herein, in one of which impurities lacking a light chain of the same class as the affinity medium specificity will flow through the column, and in the second mechanism, impurity species having more than one light chain portion of the same class as the resin specificity will be retained more strongly than the target heterodimeric product.

[0066] A growing body of literature describes the use of affinity chromatography media for separating 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-bound ablated bsAbs on protein A and G affinity media (C. Andrade et al., Biotechnol. Prog. [Progress in Biotechnology], 35 (2019) e2720; J. Weinberg et al., Biotechnol. Bioeng. [Biotechnology and Bioengineering], 114 (2017) 1803-1812; A.D. Tustian et al., MAbs. [Monoclonal Antibodies] 8 (2016) 828-838; and R. Ollier et al., MAbs. [Monoclonal Antibodies] 11 (2019) 1464-1478). In addition, for kappa light chains, separation of bsAb mispaired variants (containing two light chains of the same class as the resin specificity) has been demonstrated using protein L and KappaSelect media (C. Chen et al., MAbs. [monoclonal antibodies] 11 (2019) 632-638; SW Chen et al., MAbs. [monoclonal antibodies] 12 (2020); and T. Qin et al., Protein Expr. Purif. [protein expression and purification], 171 (2020)). Tustian et al. for protein A media and Chen et al. for protein L media also summarize the benefits of fine-tuning affinity-based selectivity during elution by conductivity and different modifiers rather than elution pH alone (AD Tustian et al., MAbs. [monoclonal antibodies] 8 (2016) 828-838; and SW Chen et al., MAbs. [monoclonal antibodies] 12 (2020)). These findings have increased understanding of the development space for protein A versus L-mediated affinity separations, but will hopefully increase understanding of other kappa and lambda LC affinity media because of their specificity for the light chain constant region. LC affinity capture processes, especially those that bind the light chain constant region, are attractive because they provide a platform approach for the purification development of many emerging antibody-based therapeutic modalities, such as chain-mispaired bsAbs.

[0067] Thus, the present invention relates to a method for purifying a protein from a solution, the method comprising 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 a halogen salt between about 5 mM and about 45 mM. A variety of lambda light chain affinity matrices are well known in the art and are suitable for use in the present invention. Non-limiting examples of commercially available LC resins include Capture Select TM, LambdaFabSelect and Capto TM L.

[0068] The binding of bsAb to the LC matrix is ​​usually achieved by column chromatography. That is, the LC matrix is ​​formed into a column, a biochemical mixture containing the bsAb is passed through the column, the column is subsequently washed by passing one or more wash solutions through the column, and the target protein is subsequently eluted from the column by passing an elution buffer through the column.

[0069] Alternatively, binding of the bsAb to the LC matrix can be achieved by a batch process, wherein a biochemical mixture containing the bsAb is incubated with the LC matrix in a container to allow the bsAb to bind to the LC matrix, the solid phase medium is removed from the container (e.g., by centrifugation), the solid phase medium is washed to remove impurities and recovered again (e.g., by centrifugation), and the bsAb is eluted from the solid phase medium.

[0070] In yet another aspect, a combination of batch processing and column chromatography can be used. For example, initial binding of the bsAb to the LC matrix can be achieved by batch processing, and then the solid phase medium can be filled into the column, which is then washed and the bsAb is eluted from the column.

[0071] 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 a binding interaction occurs, the support is washed with additional buffer to remove unbound sample components. Nonspecific (e.g., simple ionic) binding interactions can be minimized by adding low levels of detergent or by appropriately adjusting the salt concentration in the binding and / or washing buffers.

[0072] Halogen salts, including salts containing chlorine (Cl) and bromine (Br), particularly halogen salts containing alkali metals or alkaline earth metals (including sodium, potassium, calcium and magnesium) are referred to as non-buffered salts. In one aspect, the elution buffer comprises a halogen salt (e.g., containing Cl or Br). In another aspect, the non-buffered salt is a halogen salt containing sodium (Na), potassium (K), calcium (Ca) or magnesium (Mg). In one aspect, the elution buffer comprises Na or Mg. In another aspect, the halogen salt is NaCl or MgCl. 2 .

[0073] The present invention proves that low-level halogen salts are effective in purifying bsAb from affinity matrix. Known high levels, such as 100mM, are effective for removing impurities, but these high levels have a negative impact on bsAb productive rate. Therefore, the concentration of halogen salts in elution buffer is between about 5mM and 45mM. In some aspects, the concentration of halogen salts is about 5mM, about 10mM, about 15mM, about 20mM, about 25mM, about 30mM, about 35mM, about 40mM or about 45mM.

[0074] The elution buffer may also include an aggregation inhibitor. In some aspects, the aggregation inhibitor is arginine or an arginine derivative. The arginine that can be used in the present invention may be a natural amino acid arginine (e.g., L-arginine), D-arginine or an arginine derivative. Non-limiting examples of arginine derivatives include acylated arginine, such as acetyl arginine and N-α-butyryl-arginine, agmatine, arginic acid and N-α-pivaloyl arginine. Arginine or arginine derivatives may be used in the form of acid addition salts. Examples of acids that can form acid addition salts include hydrochloric acid, etc.

[0075] The pH of the elution buffer of the present invention is generally acidic. In one aspect, the pH of the elution buffer is between about 3 and about 4.5, depending on the characteristics of the bsAb. In some aspects, the pH is about 3, about 3.5, about 4, or about 4.5. In some aspects, the pH of the elution buffer is 3.5.

[0076] The method of the present invention can be carried out in a multi-column chromatography system, which comprises at least a first and a second chromatography matrix. The term "multi-column chromatography system" means a system having a total of two or more separate, interconnected or switched chromatography columns and / or chromatography membranes. Other examples of multi-column chromatography systems are known in the art.

[0077] In one aspect, the second chromatography matrix is ​​a kappa light chain affinity matrix. Commercially available kappa light chain affinity matrices are known in the art, non-limiting examples include CaptureSelect TM LC-kappa. In one aspect, the first and second chromatography steps can be performed in any order.

[0078] One or more chromatographic columns and / or one or more chromatographic membranes present in the multi-column chromatography system can be connected or moved relative to each other by a switching mechanism (for example, a post switching mechanism). The post switching event can be triggered by detecting the level of protein to be purified, the liquid of a specific volume (for example, buffer) or the specific time passed, and the level of the protein to be purified is detected by the UV absorbance of the protein corresponding to a certain level in the liquid by the multi-column chromatography system (for example, to the input of one or more chromatographic columns and / or chromatographic membranes in the system and / or from the elution of one or more chromatographic columns and / or chromatographic membranes). Post switching generally means such a mechanism, by which, at least two different chromatographic columns and / or chromatographic membranes (for example, two or more different chromatographic columns and / or chromatographic membranes present in the system) in the multi-column chromatography system are allowed to pass through different steps (for example, balance, loading, elution or washing) substantially simultaneously during at least a portion of the process.

[0079] The method of the present invention can be run in a continuous mode. In other words, the method of the present invention can be a continuous method for purifying bsAb from solution. The term "continuous method" or "continuous mode method" means a method in which a liquid is continuously passed through at least a portion of the system.

[0080] The following examples are offered by way of illustration and not by way of limitation.

[0081] 7. Examples

[0082] The examples in this section are offered by way of illustration and not by way of limitation.

[0083] Protein sample preparation. All bsAbs were produced at AstraZeneca using a Chinese hamster ovary (CHO) cell expression system. Cell culture supernatants were harvested by centrifugation and depth filtration to produce conditioned cell culture medium (CM). CM was then processed by standard protein A or lambda light chain affinity chromatography to produce purified material containing relevant mismatched species for further experiments.

[0084] Stationary phase, chromatographic equipment and reagents. KappaSelect, Capto L and LambdaFabSelectLC affinity media were purchased from Cytiva, formerly GE Healthcare (Uppsala, Sweden). CaptureSelectKappaXL and XP, and CaptureSelect Lambda XP LC affinity media were obtained from Thermo Fisher Scientific (Waltham, Massachusetts). Chromatographic media attributes were obtained from publicly available supplier documents, see Table 1. Chromatographic experiments were completed on an AKTA Avant25 system, which was controlled by Unicorn 7.0 software from Cytiva. Chromatographic media were filled into Omnifit column hardware (0.66cmI.D.) provided by Cole-Parmer (Vernon Hills, Illinois) or Vantage column hardware (1.15cm ID) provided by MilliporeSigma (Burnington, Massachusetts). All chemicals used for buffer preparation were obtained from JT Baker (Phillipsburg, NJ).

[0085] Table 1

[0086] Summary of Light Chain Affinity Reagent Properties

[0087]

[0088] a . Chromatographic media for which they have high affinity and specificity for the light chain region of antibodies

[0089] b . Manufacturer not disclosed

[0090] c . Values ​​measured with purified bsAb loading at 4 min residence time

[0091] Protein concentration determination. The product concentration in CM was quantified by Protein A and λLC affinity HPLC techniques using a standard curve generated from the purified bsAb. CaptureSelect Protein A and CaptureSelect Lambda analytical columns were purchased from Thermo Fisher Scientific (Waltham, MA). Product concentration in purified samples was determined by absorbance at 280 nm using a NanoDrop 2000c spectrophotometer purchased from Thermo Fisher Scientific.

[0092] Analytical instruments, materials and methods.The aggregates and fragment species contents of mismatched pairs were quantified by a variety of orthogonal analytical techniques.High performance size exclusion chromatography (HP-SEC) was performed using a GS3000SWXL column (7.9 × 300 mm) purchased from Tosoh Bioscience (Tokyo, Japan).High performance hydrophobic interaction chromatography (HIC-HPLC) was performed using mAb-Pac HIC 10 analytical columns purchased from Thermo Fisher Scientific.HPLC-based methods were operated on Agilent 1260HPLC systems (Palo Alto, California).Non-reducing capillary gel electrophoresis (NR-CGE) was performed on a LabChip GXII system using the HT protein expression kit from PerkinElmer (Waltham, MA, Massachusetts).

[0093] Dynamic Binding Capacity Assay The dynamic binding capacity of the LC affinity chromatography medium at 10% product breakthrough (DBC) was obtained by overloading the medium with purified bsAb to generate a product breakthrough curve. 10％ ) The LC affinity medium was loaded into a packed bed of 100 mg bsAb / mL with a residence time of 4 minutes. The approximate DBC was calculated based on the volume at which the product absorbance breakthrough during loading was measured by the AKTA UV detector. 10％ The value is 10% of the measured absorbance of the loading material bypassing the column. At 10% breakthrough, the system dead volume is subtracted from the volume and this volume is then converted to protein loading. Previous evaluations have demonstrated excellent linearity between the 280nm absorbance measured by the AKTA UV detector and the protein concentration of the loading material used (up to the maximum protein concentration).

[0094] Identification of mispairing and product-related impurity species. The HIC-HPLC assay was used to quantify the level of mispairing in the chromatographic product and / or loading material, HP-SEC was used to quantify aggregates and monomers, and NR-CGE was used to quantify half antibodies, LC dimers, and free LC. HIC-HPLC purity has also been presented as a general product quality metric, but it is worth noting that it does not take into account aggregate content.

[0095] pH gradient elution screening. Chromatography was performed at a residence time of 4 minutes, and the LC affinity medium was loaded into a 10 mg bsAb / mL packed bed. The product was eluted with a 20 column volume (CV) 50 mM sodium citrate / citric acid pH 6.0-2.5 linear gradient. The elution peaks were graded and single peaks were merged. Protein concentration was determined, and the product pool was subsequently pH neutralized, and aggregates, fragments, and mispairing content were analyzed.

[0096] The resolution (Rs) between eluting peaks was determined by the Unicorn software through its automatic peak detection and is given by the following equation:

[0097]

[0098] where t R1 is the peak retention time, and w h is the peak width at half height.

[0099] The approximate elution pH of the product peak is calculated by the pH measurement at the peak maximum absorbance measured by the AKTA pH detector. The system dead volume is subtracted from the volume at the peak maximum, and the corresponding pH is recorded. The pH electrode is calibrated before each run. The approximate pH value obtained is qualitatively consistent with the superposition of the elution peak curves of all experiments.

[0100] Purification of asymmetric bispecific antibody heterodimers

[0101] Binding affinity can be used to separate protein species with different numbers of binding motifs on an affinity medium, also described as binding valency. Typically, for affinity chromatography media (such as protein A, G, L, and camelid antibody-derived media), separation can be achieved by applying a gradient of decreasing pH elution. Commercially available LC affinity media were probed for separation of asymmetric bispecific antibody heterodimers containing one λ light chain and one κ light chain from mispaired species (containing two light chains of the same class as the stationary phase specificity). pH gradient elution experiments were first performed on selected commercially available κ and λ LC affinity chromatography media to assess "baseline" selectivity based on product binding valency. Cell culture harvest material containing bsAb 1 or 2 was used to evaluate the separation of κ-κ mispaired species by κ LC affinity chromatography, and cell culture harvest material containing bsAb 1 or 3 was used to separate λ-λ mispaired species by λ affinity chromatography, see Figure 1 .

[0102] From Figure 2 , 3 The results of the initial screening shown in Figures 4 and 5 summarize the separation of the mispaired impurities as separate elution peaks. Among the kappa LC affinity media tested, CaptureSelect Kappa XP provided the greatest resolution for bsAb 1, and Capto L provided the greatest resolution for bsAb 2, see Table 2.

[0103] Table 2. Summary of LC affinity pH gradient process performance

[0104]

[0105] aResolution between the 1st and 2nd eluting peaks on the chromatogram as calculated by Unicorn software.

[0106] b Not tested.

[0107] The difference in chromatographic performance of the two bsAbs can be attributed to the specificity of Protein L for the kappa light chain variable region. In this case, bsAb2 eluted earlier from Protein L in the pH gradient compared to bsAb 1. As expected, CaptureSelect Kappa XP, CaptureSelect Kappa XL and KappaSelect provided consistent retention between bsAb 1 and 2 due to their specificity for the kappa light chain constant region, with some imbalance in the size of Peaks 1 and 2 for the different bsAbs, see Figure 2. Figure 2 This imbalance can be attributed to the different levels of mispairing and other product-related impurities present in the CM material of bsAbs 1 and 2. Among the tested λLC affinity media, CaptureSelect Lambda XP media provided more distinct peak separation for both bsAbs 1 and 3 compared to LambdaFabSelect, see Figure 3 For product quality testing, the elution fractions of peaks 1 and 2 were combined and separated as close to the peak inflection point as possible. The HIC-HPLC product quality data confirmed that the different peaks observed were mainly due to the separation of heterodimers and light chain mispairing variants, see Figure 4 For all bsAbs tested, the second elution peak was also enriched in aggregates and LC dimers, see Table 3. Unexpectedly, free light chains (by NR-CGE) were enriched in the second peak for all experiments, and the kappa LC affinity medium clearly provided enrichment of half antibodies (by NR-CGE) in the second elution peak for bsAb 2, see Table 3. However, no enrichment of half antibodies in the second elution peak was clearly observed on the lambda affinity medium for bsAb 1 or 3.

[0108] Table 3. Impurity separation by pH gradient elution

[0109]

[0110]

[0111] The differences in peak resolution observed from pH gradient screening can be attributed to the specific properties of the different chromatographic matrices. These properties include resin particle size, pore size, and pore morphology, which can provide more favorable mass transfer properties, and ligand density and architecture, which together can provide higher binding capacity and increased availability of multiple binding events. Of note, in terms of underlying matrix-specific effects, CaptureSelect Kappa XL and Kappa XP media, both of which use the same agarose matrix, showed significantly different peak resolutions for our bsAb heterodimers and mismatches, see Figure 2. Figure 2 . In contrast, LambdaFabSelect and LambdaXP use different agarose base matrices, and notably, LambdaXP has a slightly smaller average particle size, see Table 1. Product literature reveals that KappaXP uses epoxide coupling for ligand attachment, while Kappa XL uses aldehyde coupling chemistry for ligand attachment, and similarly, Lambda XP uses epoxide coupling for ligand attachment, while LambdaFabSelect uses amide bonds for ligand attachment. It is hypothesized that single-point ligand coupling mediated by epoxide chemistry is the primary contributing factor to the increased separations observed with CaptureSelect Kappa XP and Lambda XP media compared to multi-point ligand attachment using CaptureSelect Kappa XL and LambdaFabSelect. Single-point coupling may provide more uniform ligand coupling, increased flexibility, and more favorable orientation compared to multi-point coupling to provide enhanced ligand exposure and opportunity for multiple binding events. Changes in ligand coupling can also affect binding and displacement kinetics, as described by Weinberg et al. (Biotechnol. Bioeng. [Biotechnology and Bioengineering], 114 (2017) 1803-1812). These differences may affect resolution because they can affect protein migration in the column. A more in-depth study of the potential contributions of ligand and attachment specificity effects and binding and displacement kinetics can provide a clearer understanding of the differences in peak resolution observed here.

[0112] Subsequently, the effects of various elution modifiers on the separation of bsAb mispairing on KappaXP and LambdaXP media were evaluated because they were able to provide maximum peak resolution in our screening experiments. Previous work outlined the ability to increase the resolution of affinity-based affinity separations by incorporating various salts into the elution buffer and / or controlling the elution conductivity (C. Chen et al., MAbs. [monoclonal antibodies] 11 (2019) 632-638; SW Chen et al., MAbs. [monoclonal antibodies] 12 (2020); and AD Tustian et al., MAbs. [monoclonal antibodies] 8 (2016) 828-838). For Kappa XP, pH gradient experiments were performed by adding 0.5M sodium citrate (sodium citrate / NaCitrate), magnesium chloride (MgCl), or L-arginine-HCl (arginine), or 0.25M sodium chloride (NaCl) salts. The bsAb 2 of protein A-purification is loaded with material for these experiments, and the result is compared with the control experiment without adding salt. Due to observing a large amount of precipitation at higher concentrations, a lower 0.25MNaCl concentration is used. These high concentrations of salt are needed to maximize separation, because the salt of lower concentrations reduces the resolution between heterodimer and mispairing impurities (data not shown). In addition, due to the solution buffering characteristics, the gradient using MgCl wash-out is modified to pH 4.3-3.0, and similarly, adding arginine slightly affects the pH gradient linearity.

[0113] Elution peak broadening was observed with all modifiers and with MgCl 2 and arginine, see Figure 5 Add MgCl 2 The maximum increase in resolution is achieved with amazing baseline separation. Addition of MgCl 2 The increase in resolution caused by arginine and MgCl appears to be due to preferential enhancement of κ-κ mispairing and half-antibody binding to the matrix. Under baseline pH gradient conditions without added modifiers, the second peak eluted at approximately pH 3.9, whereas for MgCl 2 , no second peak elution occurred even at pH 2.8, and for arginine, elution occurred at pH 3.56, see Table 4. Table 4 provides the process performance and product quality of the addition of these modifiers on KappaXP medium, and notably shows that the addition of NaCl, MgCl, and arginine resulted in a substantial increase in aggregates in the heterodimer peak elution pool. Separation of the half-antibodies and co-elution with kappa-kappa mispaired species was maintained by the addition of modifiers, while the impact of light chain impurities was not evaluated due to their absence in the Protein A purified material. Although the addition of MgCl 2A high degree of separation of the pair-mispair impurities was observed, but difficulties with high modifier concentrations, protein stability, large elution pool volumes, and low product recoveries warrant further optimization to potentially apply this approach to large-scale protein purification.

[0114] Table 4. CaptureSelectKappa XP elution modifier process performance

[0115]

[0116] a The elution pH cannot be determined by experimental conditions.

[0117] For both LambdaXP and LambdaFabSelect media, it is expected that the presence of salt has a much greater impact on elution performance compared to the results of KappaXP. From the initial screening, after adding any test salt at a concentration of 100mM on LambdaFabSelect, incomplete product elution was observed at a pH of 2.8. In addition, for pH gradient experiments, the base buffer of 50mM sodium citrate may more significantly contribute to the changes in the binding interaction between bsAb and the lambda affinity medium. Stepwise elution experiments were performed using 25mM sodium acetate elution buffer at pH 3.5 to minimize the conductivity of the baseline buffer, and 20mM NaCl, arginine or MgCl were added. 2 To evaluate the effect of modifiers on product binding strength, a control step elution experiment was performed at pH 3.5 without adding modifiers.

[0118] As in Figure 6 a and Figure 6 As seen in Fig. 2, during the elution of bsAb 1 and 3 from Lambda XP, an increase in elution peak tailing was observed with the incorporation of all tested modifiers, and a larger 100 mM acetic acid band peak. The peak broadening observed after incorporation of modifiers was independent of the pH shift, suggesting that the modifiers have an effect on the binding strength of the bsAb to the LC affinity medium. Surprisingly, elution with 20 mM of any of the tested modifiers resulted in almost complete removal of the mispaired species for both bsAb 1 and bsAb 3, as well as significant removal of aggregates and low molecular weight (LMW) impurities, see Table 5. The lambda half antibody was also unexpectedly enriched in the band peak, while the bsAb 1 half antibody was not found to co-elute significantly with the lambda-lambda mispair in the pH gradient screening experiment, see Table 6. Additionally, for bsAb 1, the same affinity binding enhancement was observed on LambdaFabSelect medium using an elution method of 20 mM NaCl, pH 3.5, but with less impurity removal compared to Lambda XP, see Table 6 in the Supplementary Material.

[0119] Table 5. Lambda XP purification process performance using elution modifiers

[0120]

[0121] a Not tested

[0122] Table 6. Affinity of λLC with 20 mM NaCl, pH 3.5 Step elution product mass

[0123]

[0124] a Not tested

[0125] For Protein A media, Tustian et al. found that chaotropic salts promoted the elution of bsAb heterodimer products at higher pH values ​​(AD Tustian et al., MAbs. [monoclonal antibodies] 8 (2016) 828-838). For CaptureSelect Kappa XP, there was no clear trend in the chaotropic properties of the modifiers, and the highly chaotropic salt MgCl 2 In fact, bsAb heterodimer elution occurred at a lower pH than the control conditions, see Table 4. In contrast, Chen et al. found that for protein L affinity media, the addition of salt during elution preferentially increased the binding strength of their mispaired bsAbs (C. Chen et al., MAbs. [monoclonal antibodies] 11 (2019) 632-638).

[0126] For CaptureSelect Lambda XP, it is non-intuitive that just 20 mM NaCl would significantly affect the binding strength between the protein and affinity ligand. Compared to previous data on Protein L media, which showed that 50-100 mM NaCl was sufficient to enhance binding titer-mediated separations, CaptureSelect Lambda XP required less salt at 20 mM. Similar to CaptureSelect Kappa XP and previous data on Protein L, on CaptureSelect Lambda XP, binding titer-mediated separations appear to be enhanced by the addition of an elution modifier to preferentially enhance the binding of impurities to the media. However, the binding strength of the bsAb and impurities on CaptureSelect Lambda XP was more sensitive to salt addition / conductivity than on CaptureSelect Kappa XP.

[0127] Notably, these elution methods for λLC affinity chromatography media are more easily scaled up for large-scale biotherapeutic production. The low concentrations of common elution modifiers required to increase impurity separation have minimal impact on subsequent purification or viral inactivation unit operations. For practical expansion of this approach, strategies can be designed around loading amount, elution pH, and salt concentration. Elution peak tailing can also pose significant scale-up challenges due to limitations in intermediate storage tanks, but strategies around peak collection criteria and further optimization of mobile phase conditions may alleviate this issue.

[0128] LC affinity chromatography is consolidating its position as a powerful platform for asymmetric bsAb purification. The characteristics of existing and emerging commercial LC affinity media are attractive, allowing them to be used in large-scale antibody therapeutic manufacturing production processes. The consistent ability of commercial light chain affinity media to separate mispaired variants, aggregates, and LMW impurities of standard asymmetric bsAbs by utilizing binding affinity has been demonstrated. Through pH gradient screening, it was found that CaptureSelect Kappa XP and Lambda XP enable product variants to obtain maximum baseline resolution, and these product variants have more than one light chain of the same category as the medium specificity. The differences between the resolutions of the different chromatographic media tested cannot be fully explained only by the characteristics of the medium base matrix. It seems that these effects may also depend on ligand and / or attachment chemical specific properties. The specific operating conditions of our experiment (such as residence time, protein loading and gradient slope) also play a role in the resolution achieved. Compared with the bsAb heterodimers on the corresponding LC affinity media, the half antibodies and free light chain impurities of some bsAb constructs show higher binding strength. These results may indicate that avidity binding alone cannot fully explain the increase in binding strength observed on the affinity medium.

[0129] These separations can be enhanced on CaptureSelect Kappa XP and Lambda XP chromatographic media by adding elution modifiers. CaptureSelect Kappa XP media required higher concentrations of elution modifiers compared to CaptureSelect Lambda XP media to significantly improve impurity resolution. Surprisingly, the addition of 500 mM magnesium chloride achieved baseline separation on CaptureSelect Kappa XP media, with mispaired and half-antibody species still bound at pH 2.8. For CaptureSelect Lambda XP media, the addition of only 20 mM NaCl resulted in substantial separation of bsAb heterodimers from associated impurities under isocratic elution conditions. For both CaptureSelect Kappa XP and Lambda XP, the results suggest that better separations can be achieved by preferentially enhancing impurity binding. Comparing these findings to previous literature, some similarities to protein A and protein L binding affinity separations can be found. However, previous conclusions regarding the chaotropic properties of the modifiers or the tendency of the repulsive forces to disrupt do not appear to fully explain the phenomena observed with CaptureSelect Kappa XP and Lambda XP media, and additional work is needed to elucidate the mechanism.

[0130] The scalability of these processes could be improved with new chromatographic media that offer further enhanced therapeutic delivery properties and, perhaps more importantly, with continued ligand optimization. Further ligand optimization (such as increased ligand density, increased flexibility, lower impurity binding, and other changes) could improve binding capacity, as well as increase the accessibility of multiple binding events. A deeper investigation of the mechanisms by which lyotropic salts or other modifiers affect the binding strength of multivalent ligand-protein interactions could expand the operating space to optimize these separations with different affinity media. Assessing potential differences in association and dissociation kinetics, binding orientation, and protein conformation could also provide additional insights.

Claims

1. A method for purifying an asymmetric bispecific antibody, the method 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 between about 5 mM and about 45 mM salt.

2. The method of claim 1, wherein the elution buffer further comprises an aggregation inhibitor.

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

4. The method of any one of claims 1-3, wherein the concentration of the salt is about 20 mM.

5. The method of any one of claims 1-4, 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 of any one of claims 1-5, wherein the pH of the elution buffer is between about 3 and about 4.

5.

7. The method of claim 6, wherein the pH of the elution buffer is about 3.

5.

8. The method of any one of claims 1-7, further comprising contacting the asymmetric bispecific antibody with a kappa light chain affinity matrix, and eluting the bound bispecific antibody.

9. The method of claim 8, wherein the asymmetric bispecific antibody is contacted with a kappa light chain affinity matrix prior to contacting the bispecific antibody with a lambda light chain affinity matrix.

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

11. The method of any one of claims 1 to 10, wherein the method is performed in a closed system.

12. The method of any one of claims 1-11, wherein the kappa light chain affinity matrix and / or the lambda light chain affinity matrix is ​​coupled to a solid support.

13. The method of claim 12, wherein the solid support is cross-linked agarose.

14. The method of any one of claims 1-13, wherein the bispecific antibody comprises a modified heavy chain and / or a modified light chain.

15. The method of claim 14, wherein the bispecific antibody comprises: (a) a Fab region comprising a modified heavy chain, wherein the CH1 region of the modified heavy chain comprises (i) a substitution of a native cysteine ​​to a non-cysteine ​​amino acid, and (ii) a substitution of a native non-cysteine ​​amino acid to a cysteine ​​amino acid; (b) a modified corresponding light chain, wherein the CL region of the modified light chain comprises (i) a substitution of a native cysteine ​​to a non-cysteine ​​amino acid, and (ii) a substitution of a native non-cysteine ​​amino acid to a cysteine ​​amino acid; (c) a second Fc 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 on a separate target binding arm, the second heavy chain is directly linked to the second corresponding light chain, and wherein the substituted cysteine ​​in the modified heavy chain resulting from the substitution of the native non-cysteine ​​amino acid for a cysteine ​​amino acid and the substituted cysteine ​​in the modified corresponding light chain resulting from the substitution of the native non-cysteine ​​amino acid for a cysteine ​​amino acid can form a disulfide bond.

16. The method of claim 15, wherein (a) the second heavy chain and the second corresponding light chain do not comprise a native non-cysteine ​​amino acid to a cysteine ​​amino acid substitution and do not comprise a native cysteine ​​to a non-cysteine ​​amino acid substitution; and / or (b) the two light chains each comprise a VL domain and a CL domain, wherein the VL domains have different amino acid sequences and the CL domains have different amino acid sequences; and / or (c) the two heavy chains each comprise a VH domain, a CH1 domain and an Fc region, wherein the VH domains have different amino acid sequences, the CH1 domains have different amino acid sequences, and the Fab regions have different amino acid sequences, optionally wherein one light chain is a kappa light chain and one light chain is a lambda light chain.

17. The method of claim 16, wherein the two heavy chains form a heterodimer.

18. The method of any one of claims 14-17, wherein the bispecific antibody specifically binds to two independent antigens or two independent epitopes on the same antigen.

19. The method of any one of claims 15-18, wherein the Fc region of either or both heavy chains comprises one or more modifications, optionally wherein the modifications promote heterodimerization of the heavy chains.

20. The method of any one of claims 1-19, wherein the method enables removal of mispaired species, removal of aggregates, and / or removal of low molecular weight impurities from the composition.

Images