A method for purifying a biologically active peptide using protein a affinity chromatography

Abstract

The present invention provides a method for purifying a mixture of Fc-containing bioactive peptides using an affinity chromatography column comprising an affinity matrix containing a Protein A ligand, wherein the Fc-containing bioactive peptides comprise a first Fc-containing bioactive peptide and a second Fc-containing bioactive peptide, wherein the second Fc-containing bioactive peptide further comprises at least one VH3 domain compared to the first Fc-containing bioactive peptide. According to the purification method, bioactive peptides having the same or similar structure can be accurately separated in high purity while achieving a simplified process.

Description

Technical Field

[0001] This invention relates to a method for purifying Fc-containing bioactive peptides using protein A affinity chromatography, and more specifically, to a method for purifying specific peptides with high purity from a mixture of peptides 20 by utilizing differences in binding affinity to protein A caused by differences in the number of human VH3 domains contained in Fc-containing bioactive peptides. Background Technology

[0002] Various purification methods are used to isolate specific peptides. For example, ion exchange chromatography, hydrophobic chromatography, size exclusion chromatography, affinity chromatography, etc.

[0003] However, for peptides with similar structures, with minimal differences in isoelectric point, hydrophobicity, size, and affinity, obtaining high-purity specific peptides using conventional purification methods is difficult. In such cases, numerous methods are known to utilize the differences in isoelectric point and affinity between the desired specific peptide and other possible peptides by artificially altering its sequence. In both cases, the physicochemical properties of the specific peptide are altered based on the artificial sequence alteration to increase the isoelectric point or affinity, potentially inducing unpredictable immunogenicity, etc. Summary of the Invention

[0004] Technical issues

[0005] One object of the present invention is to provide a method for high-purity purification of Fc-containing bioactive peptides or antibodies from peptide mixtures or antibody mixtures by affinity chromatography, which utilizes the difference in binding affinity to protein A caused by the difference in the number of human VH3 domains of Fc-containing bioactive peptides contained in the bioactive peptide mixture. More specifically, when peptides with small differences in affinity or isoelectric point properties but differences in the number of VH3 domains are prepared together, the affinity with full-domain protein A will differ depending on the number of VH3 domains contained in the peptide. Therefore, one object of the present invention is to provide a method for obtaining desired peptides with high purity, which separates the peptides according to the number of VH3 domains by affinity chromatography, wherein the affinity chromatography utilizes the difference in affinity between peptides with different numbers of VH3 domains and full-domain protein A.

[0006] In particular, an object of the present invention is to provide a method for separating and purifying asymmetric heterodimers and symmetric homodimers based on the difference in the number of VH3 domains contained in each protein when unwanted symmetric homodimers are generated simultaneously during the preparation of asymmetric heterodimers.

[0007] Technical solution

[0008] Each description and embodiment disclosed in this invention is also applicable to various different descriptions and embodiments. That is, all combinations of the various elements disclosed in this invention are within the scope of this invention.

[0009] Furthermore, the scope of this invention should not be considered limited by the following specific description.

[0010] The terms "first," "second," etc., used in this specification may be used to describe various component elements, but the component elements should not be limited by the terms. Each term is used only to distinguish one component element from others. For example, a first component element may be named a second component element without departing from the scope of the invention, and similarly, a second component element may be named a first component element. Unless the context clearly specifies otherwise, singular expressions include plural expressions.

[0011] Unless otherwise defined, all terms used in this specification (including technical and scientific terms) have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Furthermore, terms such as those defined in common dictionaries should be interpreted as having the same meaning as they have in the context of the relevant technical field, and are expressly so defined herein, unless interpreted in an idealized or overly formal sense.

[0012] It should be understood that terms such as “comprising” or “containing” are intended to specify the presence of features, numbers, steps, operations, components or combinations thereof described in the specification, without precluding the possibility of the presence or addition of one or more other features or numbers, steps, operations, components or combinations thereof.

[0013] "And / or" includes one or more combinations of any relevant components that can be defined.

[0014] The term "bioactive peptide" as used in this invention is a concept that includes peptides, polypeptides, proteins, portions of proteins, and fusion proteins that exhibit one or more biological activities, and is not limited to specific molecular weights, amino acid lengths, or types.

[0015] As used in this invention, the term "Fc-containing bioactive peptide" refers to a bioactive peptide that contains the Fc region (fragment crystallizable region) found in antibodies. For example, the Fc-containing bioactive peptide is a substance containing an antibody containing an Fc region.

[0016] The present invention can provide a method for isolating and / or purifying Fc-containing bioactive peptides from a mixture of Fc-containing bioactive peptides.

[0017] As used herein, a bioactive peptide is a peptide that is delivered to an organism and regulates the organism's functions. For example, peptides that regulate the functions of an organism, such as hormones, cytokines, enzymes, antibodies, growth factors, transcription factors, blood factors, vaccines, structural proteins, ligand proteins, or receptors, can be defined as bioactive substances. For example, the aforementioned peptides can be in the form of being attached to the N-terminus or C-terminus of an Fc.

[0018] According to an embodiment of the present invention, a method for purifying Fc-containing bioactive peptides is provided, comprising the following steps: (a) loading a mixture of Fc-containing bioactive peptides onto a chromatographic column containing an affinity matrix containing a protein A ligand, the mixture comprising a first Fc-containing bioactive peptide and a second Fc-containing bioactive peptide, wherein the second Fc-containing bioactive peptide further comprises at least one human VH3 domain compared to the first Fc-containing bioactive peptide; and (b) loading an eluent onto the chromatographic column, separating and eluting the Fc-containing bioactive peptides at different pH values ​​according to the number of human VH3 domains contained in each of the Fc-containing bioactive peptides in the mixture.

[0019] In one embodiment, compared to the first Fc-containing bioactive peptide, the second Fc-containing bioactive peptide further comprises at least one human VH3 domain, or may further comprise at least one amino acid sequence having greater than or equal to 95%, greater than or equal to 90%, greater than or equal to 80%, or greater than or equal to 70% homology with the human VH3 domain. The human VH3 domain is an amino acid sequence located between CDR2 and CDR3 in the variable region of the heavy chain of a VH3 family human antibody, and may comprise any of the sequences in SEQ ID No. 23 to SEQ ID No. 37, but is not limited thereto, and is any sequence of the human VH3 domain contained in a protein that can be readily determined by a person skilled in the art. The human VH3 domain is primarily located in VH3 family human antibodies, but rarely, it can be found in mammalian (or chimeric) antibody frames such as mice. For example, hu11F11 used in the embodiments of the present invention is an antibody containing a human VH3 domain, while ch1E4 and anti-BACE antibodies are antibodies that do not contain a human VH3 domain. However, ch11F11 is an exception. Although it is a chimeric antibody, it is still an antibody that contains the human VH3 domain.

[0020] The protein A ligand may be a global protein A ligand. That is, the protein A ligand may contain all global domains (E, D, A, B, and C domains). However, the embodiments are not limited thereto, and the protein A ligand may contain one or more domains selected from the E and D domains. For example, the protein A ligand may contain both the E and D domains.

[0021] The support for the protein A ligand in the affinity matrix can be a particle with a spherical or bead-like morphology. The average particle size of the support in the affinity matrix can be, for example, less than about 90 μm, or less than or equal to about 80 μm or less than or equal to 75 μm. Furthermore, the average particle size of the support in the affinity matrix can be, for example, greater than or equal to about 1 μm or greater than or equal to 5 μm. When the size of the support in the affinity matrix meets the above ranges, the resolution of the bioactive peptide may be better.

[0022] Step (b) may include the following steps: (b1) loading an eluent having a first pH range onto the chromatographic column to elute the first Fc-containing bioactive peptide; and (b2) loading an eluent having a second pH range lower than the first pH range onto the chromatographic column to elute the second Fc-containing bioactive peptide. The eluent having the first pH range and the eluent having the second pH range may be loaded onto the chromatographic column individually or sequentially, or the eluent may be loaded continuously onto the chromatographic column, changing the concentration gradient from the first pH range to the second pH range according to the loading time.

[0023] The bioactive peptide mixture may further include a third Fc-containing bioactive peptide, which, compared with the second Fc-containing bioactive peptide, also contains at least one human VH3 domain.

[0024] According to the purification method of the present invention, a first Fc-containing bioactive peptide, a second Fc-containing bioactive peptide, and a third Fc-containing bioactive peptide with different amounts of VH3 domains can be separated and purified from each other, and each of them has the advantage of being able to be purified to a high degree of purity, so as to achieve the purpose of purification.

[0025] Furthermore, this invention does not exclude Fc from the composition of the substance to be purified, and also considers the binding between Fc and protein A in the purification process. Since Fc itself has an affinity for protein A, purification methods utilizing the affinity between Fc and protein A typically exclude the affinity of other constituent elements besides Fc, and vice versa. On the other hand, this invention aims to provide a method for purifying Fc-containing bioactive peptides, where both heavy chain Fc and VH3 domains are constituent elements, based on the number of VH3 domains.

[0026] The bioactive peptide mixture may comprise: a first Fc-containing bioactive peptide containing n (n is an integer greater than or equal to 0) human VH3 domains; a second Fc-containing bioactive peptide containing n+1 to n+9 human VH3 domains; and a third Fc-containing bioactive peptide containing n+2 to n+10 human VH3 domains. For example, the first Fc-containing bioactive peptide contains n (n is an integer greater than or equal to 0) human VH3 domains, the second Fc-containing bioactive peptide contains n+1 human VH3 domains, and the third Fc-containing bioactive peptide contains n+2 human VH3 domains.

[0027] n can be, for example, greater than or equal to 0 and less than or equal to 10, greater than or equal to 0 and less than or equal to 8, greater than or equal to 0 and less than or equal to 6, greater than or equal to 0 and less than or equal to 5, greater than or equal to 0 and less than or equal to 3, greater than or equal to 1 and less than or equal to 10, greater than or equal to 1 and less than or equal to 8, greater than or equal to 1 and less than or equal to 6, greater than or equal to 1 and less than or equal to 5, greater than or equal to 1 and less than or equal to 3, greater than or equal to 2 and less than or equal to 10, greater than or equal to 2 and less than or equal to 8, greater than or equal to 2 and less than or equal to 6, or greater than or equal to 2 and less than or equal to 5. n can be, for example, 0, 1, 2, or 3.

[0028] Step (b) may further include the following steps: (b1) loading an eluent having a first pH range onto the column to elute the first Fc-containing bioactive peptide; (b2) loading an eluent having a second pH range lower than the first pH range onto the column to elute the second Fc-containing bioactive peptide; and (b3) eluting the third Fc-containing bioactive peptide using an eluent having a third pH range lower than the second pH range. Steps (b1) to (b3) may be performed sequentially (linear gradient). That is, the first to third pH ranges are contained within a continuous pH range between the starting value of the first pH range and the ending value of the third pH range, and the respective pH ranges are not discontinuous. Steps (b1) to (b3) are performed by initially loading an eluent having the first pH range onto the column and then gradually decreasing the pH of the continuously loaded eluent, thereby gradually transitioning the pH of the eluent to the second and third pH ranges. In addition, the pH range used for elution may vary depending on the type of bioactive peptide to be purified.

[0029] Alternatively, steps (b1) to (b3) can be performed step-by-step (step gradient).

[0030] The Fc can be an Fc without a mutation affecting the binding affinity to wild-type protein A. The mutation refers to a polypeptide with insertion, deletion, addition, and / or substitution at one or more amino acid residues compared to the polypeptide sequence of the Fc. That is, in one embodiment, the Fc can be an Fc without a mutation that hinders binding affinity to protein A or a mutation that enhances binding affinity to protein A.

[0031] The first, second, and / or third Fc-containing bioactive peptides may include peptide drugs bound to the Fc. Here, "peptide drug" refers to an independent peptide having the same or different biological activity as the bioactive peptide and bound to the bioactive peptide.

[0032] In the bioactive peptide containing Fc, the peptide drug may bind to the N- or C-terminus of the Fc. For example, in the bioactive peptide containing Fc, the peptide drug may bind to one or more of the two N-termini and two C-termini selected from the two heavy chains of the Fc.

[0033] As used herein, the term "binding" is defined to include both direct physical or chemical binding of two objects and binding or connection via a linker. In one embodiment, where Fc is linked to a peptide drug via a linker, the linker may be, for example, a peptide, but there are no particular limitations as long as it is a linker peptide material commonly used in the art.

[0034] The peptide drug may be at least one selected from the group consisting of hormones, cytokines, enzymes, antibodies, growth factors, transcriptional regulatory factors, blood factors, vaccines, structural proteins, ligand proteins, and receptors. For example, the peptide drug may be selected from human growth hormone, growth hormone-releasing hormone, growth hormone-releasing peptide, interferon, interferon receptor, colony-stimulating factors, such as glucagon-like peptide-1 (GLP-1), or G protein-coupled receptors (G-protein-coupled receptors). Interleukins, interleukin receptors, enzymes, interleukin-binding proteins, cytokine-binding proteins, macrophage activating factors, macrophage peptides, B cytokines, T cytokines, allergy inhibitors, cell necrosis glycoproteins, immunotoxins, lymphotoxins, tumor necrosis factor, tumor suppressor factors, metastatic growth factor, α-1 antitrypsin, albumin, α-lactalbumin, apolipoprotein-E, erythropoietin, hyperglycated erythropoietin, angiopoietin, hemoglobin, thrombin, thrombin receptor activating peptide, thrombomodulin, blood factors VII, VIIa, VIII, IX and XIII, plasminogen activator, fibrin-binding peptide, urokinase, streptokinase, hirudin, protein C, C-reactive protein, renin inhibitors, collagenase inhibitors, superoxide dismutase. The group consisting of at least one of the following: leptin, platelet-derived growth factor, epidermal growth factor, epidermal growth factor, angiostatin, angiotensin, bone growth factor, bone-stimulating protein, calcitonin, insulin, atrial peptide hormone, chondroitin inducible factor, calcitonin-dependent, connective tissue activating factor, tissue factor pathway inhibitor, follicle-stimulating hormone, luteinizing hormone, luteinizing hormone-releasing hormone, nerve growth factor, parathyroid hormone, relaxin, secretin, somatostatin, insulin-like growth factor, adrenocortical hormone, glucagon, cholecystokinin, pancreatic polypeptide, gastrin-releasing peptide, corticotropin-releasing factor, thyroid-stimulating hormone, autocrine motor factor, lactoferrin, myostatin, receptor, receptor antagonist, cell surface antigen, viral vaccine antigen, monoclonal antibody, polyclonal antibody, and antibody fragment.

[0035] The glucagon-like peptide may be selected from at least one of exenatide, liraglutide, taspoglutide, albiglutide, lixisenatide and glucagon-like peptide-1 (GLP-1), such as glucagon-like peptide-1 (GLP-1).

[0036] When the first or second Fc-containing bioactive peptide contains a VH3 domain, the first and second Fc-containing bioactive peptides may each independently contain any one of the following variable region sequences containing a VH domain: the heavy chain variable sequence of SEQ ID No. 1 and the light chain variable sequence of SEQ ID No. 2; the heavy chain variable sequence of SEQ ID No. 3 and the light chain variable sequence of SEQ ID No. 4; the heavy chain variable sequence of SEQ ID No. 5 and the light chain variable sequence of SEQ ID No. 6; the heavy chain variable sequence of SEQ ID No. 7 and the light chain variable sequence of SEQ ID No. 8; or the heavy chain variable sequence of SEQ ID No. 9 and the light chain variable sequence of SEQ ID No. 10. For example, the heavy chain variable sequence and the light chain variable sequence may be the heavy chain variable sequence and the light chain variable sequence of scFv.

[0037] The third Fc-containing bioactive peptide may comprise any of the following variable region sequences containing a VH domain: the heavy chain variable sequence of SEQ ID No. 1 and the light chain variable sequence of SEQ ID No. 2; the heavy chain variable sequence of SEQ ID No. 3 and the light chain variable sequence of SEQ ID No. 4; the heavy chain variable sequence of SEQ ID No. 5 and the light chain variable sequence of SEQ ID No. 6; the heavy chain variable sequence of SEQ ID No. 7 and the light chain variable sequence of SEQ ID No. 8; or the heavy chain variable sequence of SEQ ID No. 9 and the light chain variable sequence of SEQ ID No. 10. For example, the heavy chain variable sequence and the light chain variable sequence may be the heavy chain variable sequence and the light chain variable sequence of scFv.

[0038] However, since the VH3 domain is contained in the heavy chain variable sequence, scFv composed of heavy chain variable sequences selected from the above sequences and other light chain variable sequences, or single-chain antibodies composed only of heavy chain variable sequences selected from the above sequences, can also be contained in the bioactive peptide containing Fc.

[0039] The bioactive peptide containing Fc can be an antibody containing immunoglobulin G (hereinafter referred to as IgG).

[0040] As used herein, "antibody" can refer to any intact immunoglobulin of the same type, an antigen-binding fragment that can compete with an intact antibody for binding to a target antigen, or a combination thereof. For example, it can include mouse, chimeric, humanized, fully human antibodies, their antigen-binding fragments, or combinations thereof. An antibody itself can be a type of antigen-binding protein. Antibodies typically contain at least two full-length heavy chains and two full-length light chains, but in some cases, antibodies may contain only heavy chains. The antibodies include monospecific antibodies that specifically bind to one target and multispecific antibodies (e.g., bispecific and trispecific antibodies) that specifically bind to multiple targets.

[0041] The antibody further comprises monoclonal antibodies and polyclonal antibodies, and the monoclonal antibody may be a separable antibody that specifically binds to IGF1R, which may be a human antibody, a humanized antibody, or a chimeric antibody. The monoclonal antibody may be a separable antibody that specifically binds to IGF1R, which may be of the IgG1, IgG2, IgG3, or IgG4 type.

[0042] In this application, a "light chain" may comprise a full-length light chain and fragments thereof, having a variable region sequence sufficient to provide specific binding to an antigen or epitope. The full-length light chain may comprise a variable region domain (VL) and a constant region domain (CL). The variable region domain of the light chain may be located at the amino terminus of the light chain polypeptide. Light chains may be of the type comprising kappa (κ) and lambda (λ) chains.

[0043] As used in this article, “complementarity-determining regions (CDRs)” can refer to regions in the variable region of an antibody that confer specific binding to an antigen.

[0044] In this application, the "heavy chain" may comprise a full-length heavy chain and fragments thereof, having a variable region sequence sufficient to provide specific binding to an antigen or epitope. The full-length heavy chain may comprise a variable region domain and three constant region domains CH1, CH2, and CH3. The variable region (VH) domain is located at the amino terminus of the heavy chain polypeptide, the constant region (CH) domain is located at the carboxyl terminus, and CH3 may be located closest to the carboxyl terminus. The heavy chain may comprise isotypes of IgG (including IgG1, IgG2, IgG3, and IgG4 isotypes), IgA (including IgA1 and IgA2 isotypes), IgM, and IgE.

[0045] The antibody can be selected from all immunoglobulin subtypes (e.g., IgA, IgD, IgE, IgG (IgG1, IgG2, IgG3, IgG4), IgM, etc.). The IgG antibody can be an IgG1, IgG2, IgG3, or IgG4 subtype, for example, an IgG1 or IgG2 subtype. The IgG antibody comprises two heavy chains and two light chains, each heavy chain and light chain being bound together by disulfide bonds to form two heavy chain-light chain constructs (dimers). The two formed heavy chain-light chains can be linked by disulfide bonds in the Fc region of the heavy chain. The IgG antibody can be a single-target antibody, which contains antigen-binding sites against the same antigen in both heavy chain-light chain constructs to target one antigen, or it can be a bispecific antibody, which contains antigen-binding sites against different antigens in both heavy chain-light chain constructs to target two antigens.

[0046] The antibodies according to the present invention include, but are not limited to, bispecific antibodies, whole antibodies, microantibodies, domain antibodies, antibody mimics (or synthetic antibodies), antibody fusions (or antibody conjugates), fragments thereof, and combinations thereof. The structures of various antibodies are further disclosed below.

[0047] In this invention, for example, compared to other peptide sequences, "variants" of peptides such as antigen-binding fragments, proteins, or antibodies are peptides in which insertions, deletions, additions, and / or substitutions have occurred at one or more amino acid residues, and may include fusion peptides. For example, a portion of an antibody may contain conserved amino acid substitutions at one or more residues of the heavy or light chain, variable region, or CDR sequence.

[0048] The antibodies according to the invention can be generated from transgenic animals (e.g., transgenic mice) and target antibodies can be selected using hybridoma technology. Such antibodies can be cloned and expressed using a suitable vector and host cells, or they can be obtained from cultured hybridoma cells. Furthermore, the antibodies can be derived from a phage-display library. Phage display technology is a method that mimics immune selection, displaying an antibody library on the surface of filamentous phages to screen for phages that bind to target antigens. One such technique can be found in embodiments of the invention or PCT publication WO99 / 10494.

[0049] Antibodies or their antigen-binding fragments may be derived from a single source or may be chimeric. Chimeric antibodies comprise portions derived from two different types of antibodies, which will be described in more detail below. Antibodies or their antigen-binding fragments may be produced by hybridoma, recombinant DNA technology, or enzymatic or chemical cleavage of intact antibodies. Unless otherwise stated, the term "antibody" as used herein includes not only antibodies comprising two full-length heavy chains and two full-length light chains, but also their derivatives, variants, immunofunctional immunoglobulin fragments, mutants, and combinations thereof. For example, an "antibody" may, in addition to two full-length heavy chains and two full-length light chains, contain one or two scFvs, as described below.

[0050] As used herein, an "antigen-binding fragment" can refer to a portion of an antibody or a polypeptide containing an antigen that has the specific binding ability to bind to an antigen. For example, an antigen-binding fragment can be a portion of an antibody containing amino acid residues that interact with an antigen (e.g., an epitope) to confer specificity and / or affinity to the antigen to the antibody, or it can be a polypeptide containing such residues. Such fragments contain at least one CDR located in the full-length light or heavy chain, and in some embodiments may contain a single heavy chain and / or a portion thereof. Such bioactive fragments can be generated by recombinant DNA technology or by, for example, enzymatic or chemical cleavage of an intact antibody.

[0051] Immunofunctional immunoglobulin fragments include, but are not limited to, Fab, Fab', F(ab')2, Fv, domain antibodies, and single-chain antibodies (e.g., scFv, Fc-scFv, etc.). Furthermore, immunofunctional immunoglobulin fragments can be derived from any mammal, including, but not limited to, humans, mice, rats, camels, or rabbits. The functional portions of antibodies disclosed in this specification, such as one or more CDRs, can be covalently linked to a second protein or small molecule compound for use as a targeted therapeutic agent against a specific target.

[0052] As used in this application, a "single-chain antibody" is a single polypeptide chain with an antigen-binding region, wherein the heavy and light chain variable regions are linked by flexible linkers. For example, the single-chain antibody may be one or more selected from the group consisting of scFv with heavy and light chain variable regions linked in single-chain form, Fc-scFv with heavy chain variable region, light chain variable region, and Fc linked in single-chain form. See, for example, U.S. Patent No. 5,260,203 for examples of single-chain antibodies.

[0053] As used herein, “affinity” refers to the strength of the interaction between an antibody or its antigen-binding fragment and an antigen, and can depend on the CDR sequence of the antibody or antigen-binding fragment and / or the physicochemical properties (hydrophilicity / hydrophobicity, electrostatic properties, etc.) of the antibody or antigen-binding fragment, as well as the characteristics of the antigen, such as its size, shape, and / or charge. Methods for determining this affinity are known in the art and can typically be expressed as the dissociation constant (KD), but are not limited thereto.

[0054] In this application, the "multispecific antigen-binding protein" or "multispecific antibody" targets two or more antigens or epitopes.

[0055] In this application, a “bispecific” antigen-binding protein or antibody is a hybrid antigen-binding protein or antibody having two distinct antigen-binding sites. Such a bispecific antibody is a multispecific antigen-binding protein or antibody that can be produced by various known methods, such as hybridoma fusion or linking of Fab' fragments or scFv fragments. See, for example, Songsivilai and Lachmann, Clin. Exp. Immunol. 79:315-321 (1990); Kostelny et al., J. Immunol. 148:1547-1553 (1992), etc. The two distinct epitopes bound to the two antigen-binding sites of a bispecific antigen-binding protein or antibody can be located on the same or different protein targets.

[0056] The bioactive peptide containing Fc may comprise an antibody protein that binds a heavy chain Fc and an antigen-binding fragment. For example, the heavy chain Fc may be contained in IgG, and the antigen-binding fragment, as an scFv containing a heavy chain variable region or a single-chain antibody, may be attached to the C-terminus of the heavy chain Fc.

[0057] For example, the bioactive peptide containing Fc can be an IgG-scFv biantibody that binds IgG and scFv. The IgG-scFv biantibody can be a monovalent bispecific antibody that binds one scFv to IgG or a bivalent bispecific antibody that binds two scFvs to IgG.

[0058] The bioactive peptide containing Fc may include a peptide drug that binds to the Fc. Here, the definition of the peptide drug may be the same as that described above. The peptide drug may bind to the N-terminus or C-terminus of the Fc of the bioactive peptide containing Fc. The peptide drug may bind to the N-terminus of at least one of the two heavy chains of the Fc of the bioactive peptide containing Fc.

[0059] The first Fc-containing bioactive peptide is an antibody containing immunoglobulin G (hereinafter referred to as IgG), and the second Fc-containing bioactive peptide may be an IgG-scFv biantibody. The first Fc-containing bioactive peptide does not contain scFv, and the second Fc-containing bioactive peptide may contain an scFv composed of SEQ ID No. 11, which is linked to the C-terminus of either of the two heavy chains of the Fc.

[0060] The first Fc-containing bioactive peptide may be an antibody containing immunoglobulin G (hereinafter referred to as IgG), the second Fc-containing bioactive peptide is an IgG-scFv monovalent bispecific antibody, and the third Fc-containing bioactive peptide may be an IgG-scFv bivalent bispecific antibody. The first Fc-containing bioactive peptide does not contain scFv, the second Fc-containing bioactive peptide may contain one scFv composed of SEQ ID No. 11, which is attached to the C-terminus of either of the two heavy chains of the Fc, and the third Fc-containing bioactive peptide may contain two scFvs composed of SEQ ID No. 11, each attached to the C-terminus of one of the two heavy chains of the Fc.

[0061] The first to third Fc-containing bioactive peptides are IgG, and the variable region of the IgG antibody may include a human VH3 domain. For example, in this case, each of the first to third Fc-containing bioactive peptides has two VH3 domains in the IgG (in the heavy chain of the variable region), but in the region outside the IgG portion, there may be different numbers of VH3 domains.

[0062] The first to third Fc-containing bioactive peptides are IgG, and the variable region of the IgG antibody may not contain a human VH3 domain. For example, in this case, none of the first to third Fc-containing bioactive peptides have a VH3 domain in the IgG, but different numbers of VH3 domains may be present in the region outside the IgG portion.

[0063] In the case where the Fc-containing bioactive peptide includes a human VH3 domain, the human VH3 domain may be contained in the heavy chain variable region of the Fc-containing bioactive peptide.

[0064] When the Fc-containing bioactive peptide includes a human VH3 domain, at least one or more antibodies or fragments thereof containing the human VH3 domain can bind to the Fc-containing bioactive peptide. For example, scFv containing the human VH3 domain can bind to the Fc-containing bioactive peptide.

[0065] In the case where the first or second Fc-containing bioactive peptide contains a human VH3 domain, the human VH3 domain is contained in the heavy chain variable region of the first or second Fc-containing bioactive peptide, and at least one or more antibodies or fragments thereof containing the human VH3 domain can bind to the bioactive peptide.

[0066] In the aforementioned IgG-scFv biantibody, at least one of IgG and scFv may contain a human VH3 domain. When both IgG and scFv contain a human VH3 domain, the sequences of the human VH3 domains of IgG and scFv may be identical or different. The aforementioned IgG-scFv biantibody may possess one or more biological activities.

[0067] When the bioactive peptide containing Fc includes an IgG antibody, the IgG antibody may contain one or more components selected from a first heavy chain and a second heavy chain. Here, the first heavy chain may consist of VH-CH1-CH2-CH3-scFv, and the second heavy chain may consist of VH-CH1-CH2-CH3.

[0068] To increase the dimerization probability of the first and second heavy chains for easier preparation of monovalent bispecific antibodies, a specific position in the heavy chain constant region of one heavy chain is subjected to a hole mutation (e.g., T366S, L368A, or Y406V), and a specific position in the heavy chain constant region of the other heavy chain may be subjected to a knot mutation (e.g., T366W). The Fc-containing bioactive peptide may contain anti-α-synuclein IgG antibody or anti-BACE antibody.

[0069] The anti-α-synuclein antibody may contain at least one of a first heavy chain or a second heavy chain. For example, the first heavy chain may contain a heavy chain variable region of the anti-α-synuclein antibody, a human heavy chain constant region, and an anti-IGF1R antibody or a fragment thereof (e.g., anti-IGF1RscFv) bound to the C-terminus of the heavy chain constant region. The second heavy chain contains the heavy chain variable region of the anti-α-synuclein antibody and the human heavy chain constant region, and may not contain the aforementioned anti-IGF1R antibody.

[0070] The anti-α-synuclein antibody may contain two of the first heavy chains or two of the second heavy chains. Alternatively, the α-synuclein antibody may contain one of the first heavy chains and one of the second heavy chains.

[0071] The second Fc-containing bioactive peptide comprises one first heavy chain and one second heavy chain, respectively. The third Fc-containing bioactive peptide may comprise two first heavy chains, and the first Fc-containing bioactive peptide may comprise two second heavy chains.

[0072] The Fc-containing bioactive peptide mixture may comprise: a first Fc-containing bioactive peptide that does not bind to the anti-IGF1R antibody or a fragment thereof at the C-terminus of each of the two heavy chains of the anti-α-synuclein antibody; a second Fc-containing bioactive peptide that binds to the anti-IGF1R antibody or a fragment thereof at the C-terminus of either of the two heavy chains of the anti-α-synuclein antibody; and a third Fc-containing bioactive peptide that binds to the anti-IGF1R antibody or a fragment thereof at the C-terminus of each of the two heavy chains of the anti-α-synuclein antibody.

[0073] As used herein, the description of ch11F11 in PCT Publication WO2019-117684 applies to ch11F11 herein unless there is a contradiction, and the description of hu11F11 in PCT Publication WO2019 / 117684 applies to hu11F11 herein unless there is a contradiction. Sequences for ch11F11 and hu11F11 can be found in the aforementioned PCT Publications.

[0074] The bioactive peptide may be an anti-α-synuclein antibody comprising a heavy chain variable region of any anti-α-synuclein antibody selected from the group consisting of ch11F11, ch1E4, anti-BACE, and hu11F11. The anti-IGF1R antibody or a fragment thereof may comprise an amino acid sequence represented by the heavy chain variable sequence of SEQ ID No. 1 and the light chain variable sequence of SEQ ID No. 2; the heavy chain variable sequence of SEQ ID No. 3 and the light chain variable sequence of SEQ ID No. 4; the heavy chain variable sequence of SEQ ID No. 5 and the light chain variable sequence of SEQ ID No. 6; the heavy chain variable sequence of SEQ ID No. 7 and the light chain variable sequence of SEQ ID No. 8; or the heavy chain variable sequence of SEQ ID No. 9 and the light chain variable sequence of SEQ ID No. 10.

[0075] According to one embodiment of the method for separating Fc-containing bioactive peptides, affinity chromatography can be used to easily separate the desired Fc-containing bioactive peptides from a mixture of Fc-containing bioactive peptides. In particular, the purification method for Fc-containing bioactive peptides according to one embodiment simplifies the process and allows for the precise separation of bioactive peptides with the same or similar structures with high purity.

[0076] Furthermore, the method for isolating Fc-containing bioactive peptides according to one embodiment allows for the high-purity and precise isolation of bioactive peptides with the same or similar structures without the need to artificially control affinity for protein A ligands by introducing specific mutations into the Fc region. Moreover, the method for isolating Fc-containing bioactive peptides according to one embodiment allows for the simultaneous isolation of bioactive peptides using wild-type protein A ligands without introduced mutations or existing protein A ligands. Therefore, the preparation method according to one embodiment allows for the high-purity and precise isolation of Fc-containing bioactive peptides without the need for expensive protein A ligands that introduce mutations to enhance affinity for the Fc region.

[0077] According to another embodiment of the present invention, a method for purifying antibodies from an antibody mixture can be provided. Of course, without contradiction, the description of the embodiments for the purification method of bioactive peptides can also be applied to the embodiments of the antibody purification method described below.

[0078] According to an embodiment of the present invention, a method for purifying antibodies is provided, comprising the following steps: (a-1) loading an antibody mixture onto a chromatographic column containing an affinity matrix containing a protein A ligand, wherein the antibody mixture comprises a monospecific antibody, a monovalent bispecific antibody, and a bivalent bispecific antibody, wherein the monovalent bispecific antibody is an antigen-binding fragment containing a human VH3 domain bound to the C-terminus of one of the two heavy chain constant regions of the monospecific antibody, and the bivalent bispecific antibody is an antigen-binding fragment each bound to the C-terminus of one of the two heavy chain constant regions of the monospecific antibody; (b-1) loading an eluent having a first pH range onto the chromatographic column to elute the monospecific antibody; (c-1) loading an eluent having a second pH range lower than the first pH range onto the chromatographic column to elute the monovalent bispecific antibody; and (d-1) loading an eluent having a third pH range lower than the second pH range onto the chromatographic column to elute the bivalent bispecific antibody.

[0079] The variable region of the monospecific antibody may or may not contain a human VH3 domain.

[0080] The antigen-binding fragment may be an scFv. The antigen-binding fragment may be an scFv containing the amino acid sequence of SEQ ID No. 11.

[0081] That is, the exemplary bispecific antibody according to the present invention may comprise a heavy chain composition and a light chain of an anti-α-synuclein antibody, wherein the heavy chain composition may comprise (1) a heavy chain of an anti-α-synuclein antibody and (2) a heavy chain and a light chain of an anti-IGF1R antibody. In other words, the bispecific antibody according to the present invention may be in the form of an anti-IGF1R antibody in scFv form bound to one or both C-termini (referred to as monovalent or bivalent, respectively) of an anti-α-synuclein antibody in IgG form.

[0082] The first pH range can be greater than or equal to 3.4 and less than or equal to 5.0, the second pH range can be greater than or equal to 3.3 and less than or equal to 4.1, and the third pH range can be greater than or equal to 3.0 and less than or equal to 4.0. For example, the first pH range can be greater than or equal to 3.4 and less than or equal to 4.5, the second pH range can be greater than or equal to 3.3 and less than or equal to 4.1, and the third pH range can be greater than or equal to 3.0 and less than or equal to 4.0.

[0083] However, the first to third pH ranges are merely exemplary, and anyone skilled in the art can easily deduce the first to third pH ranges that vary depending on the type of bioactive peptide by referring to the description in this application.

[0084] In particular, for proteins in the form of asymmetric heterodimers, it is difficult to remove homodimers that occur during the preparation process during purification. This is because the difference in affinity and isoelectric point (pI) between heterodimers and homodimers is not significant, making them unsuitable for purification using affinity chromatography or ion exchange chromatography columns commonly used in protein purification.

[0085] Regarding the separation of heterodimeric antibodies, US Patent Application Publication No. US8586713 B2, among others, has attempted a method of artificially substituting the Fc sequence to improve affinity and isoelectric point differences. However, with the artificial substitution of amino acids, immunogenicity issues arise. Furthermore, when the VH3 sequence is also present, two affinity resins must be used, and the monovalent bispecific antibody and monospecific antibody can only be separated after the bivalent bispecific antibody is removed first. Therefore, the process is complex, and the separation requires significant time and expense.

[0086] However, according to one embodiment of the antibody purification method, since the isoelectric point difference is very small (e.g., less than or equal to 0.1), similar to that of heterodimers and homodimers, it is not necessary to perform artificial amino acid substitution on peptides that are difficult to purify by conventional affinity chromatography or ion exchange chromatography, and heterodimers can be purified to a purity of 85% to 90% or higher. Furthermore, according to one embodiment of the separation method, not only antibody fragments can be effectively separated, but also the entire antibody containing the Fc region can be separated, and precise separation can be achieved based on the amount of VH3.

[0087] Beneficial effects

[0088] A method for purifying Fc-containing bioactive peptides or antibodies according to one embodiment can purify peptides or antibodies with high purity by utilizing the difference in binding affinity of global protein A ligands caused by the difference in the number of VH3 domains of the peptide, wherein the peptide is contained in a mixture of Fc-containing bioactive peptides or antibodies. Furthermore, the method for purifying Fc-containing bioactive peptides or antibodies according to one embodiment eliminates the need to introduce separate mutations into the Fc or protein A ligand to control the binding affinity between the Fc and protein A ligands; therefore, it can reduce process costs and simplify the process while purifying proteins with high purity. Attached Figure Description

[0089] Figure 1 The purification curves of a mixture of bioactive peptides containing monospecific antibodies, monovalent bispecific antibodies, and bivalent bispecific antibodies are shown by affinity chromatography using monoclonal antibody purification gel (MabSelet Sure) resin.

[0090] Figure 2 The purification curves of a mixture of bioactive peptides containing monospecific antibodies, monovalent bispecific antibodies, and bivalent bispecific antibodies are shown by affinity chromatography using antibody purification gel (MabSelect PrismA) resin.

[0091] Figure 3 The purification curves of a mixture of bioactive peptides containing monospecific antibodies, monovalent bispecific antibodies, and bivalent bispecific antibodies are shown by affinity chromatography using Protein AFF resin.

[0092] Figure 4 The purification curves of a mixture of bioactive peptides containing monospecific antibodies, monovalent bispecific antibodies, and bivalent bispecific antibodies using affinity chromatography with MabSelect Xtra resin are shown.

[0093] Figure 5The results show the purification of a mixture of bioactive peptides containing monospecific antibodies, monovalent bispecific antibodies, and bivalent bispecific antibodies using MabSelect Xtra resin, as confirmed by size exclusion high-performance liquid chromatography (SE-HPLC).

[0094] Figure 6 The results show the purification of a mixture of bioactive peptides containing monospecific antibodies, monovalent bispecific antibodies, and bivalent bispecific antibodies using MabSelect Xtra resin, as confirmed by polyacrylamide gel electrophoresis (SDS-PAGE).

[0095] Figure 7 The purification curves of a mixture of bioactive peptides containing monospecific antibodies, monovalent bispecific antibodies, and bivalent bispecific antibodies are shown using affinity chromatography with Protein AHP resin.

[0096] Figure 8 The results show the purification of a mixture of bioactive peptides containing monospecific antibodies, monovalent bispecific antibodies, and bivalent bispecific antibodies using Protein A HP resin, as confirmed by size exclusion high-performance liquid chromatography (SE-HPLC).

[0097] Figure 9 The results show the purification of a mixture of bioactive peptides containing monospecific antibodies, monovalent bispecific antibodies, and bivalent bispecific antibodies using Protein A HP resin, as confirmed by polyacrylamide gel electrophoresis (SDS-PAGE).

[0098] Figure 10 The purification curves of a mixture of bioactive peptides containing monospecific antibodies, monovalent bispecific antibodies, and bivalent bispecific antibodies are shown using affinity chromatography with POROS MabCapture A Select resin.

[0099] Figure 11 The results show the purification of a mixture of bioactive peptides containing monospecific antibodies, monovalent bispecific antibodies, and bivalent bispecific antibodies using POROS MabCapture ASelect resin, as confirmed by size exclusion high-performance liquid chromatography (SE-HPLC).

[0100] Figure 12 The results show the purification of a mixture of bioactive peptides containing monospecific antibodies, monovalent bispecific antibodies, and bivalent bispecific antibodies using POROS MabCapture ASelect resin, as confirmed by polyacrylamide gel electrophoresis (SDS-PAGE).

[0101] Figure 13 The purification curves of a mixture of bioactive peptides containing monospecific antibodies, monovalent bispecific antibodies, and bivalent bispecific antibodies using affinity chromatography with AbSolute High Cap resin are shown.

[0102] Figure 14 The results show the purification of a mixture of bioactive peptides containing monospecific antibodies, monovalent bispecific antibodies, and bivalent bispecific antibodies using AbSolute High Cap resin, as confirmed by size exclusion high-performance liquid chromatography (SE-HPLC).

[0103] Figure 15 The results show the purification of a mixture of bioactive peptides containing monospecific antibodies, monovalent bispecific antibodies, and bivalent bispecific antibodies using AbSolute High Cap resin, as confirmed by polyacrylamide gel electrophoresis (SDS-PAGE).

[0104] Figure 16 The graphs show the analysis of culture medium prepared by the transient method (harvested cell culture fluid) and samples obtained by purifying the culture medium with protein A using a POROS A 20 μm analytical column. Peak 1, peak 2, and peak 3 represent the retention time of each substance when the purified monospecific antibody, monovalent bispecific antibody, and bivalent bispecific antibody were loaded onto the POROS A 20 μm analytical column.

[0105] Figure 17 The purification curves of a mixture of bioactive peptides containing hu11F11 and hu11F11-F06 are shown by affinity chromatography using POROSMabCapture A Select resin.

[0106] Figure 18 The results show the purification of a mixture of bioactive peptides containing hu11F11 and hu11F11-F06 using POROS MabCapture A Select resin, as confirmed by size exclusion high performance liquid chromatography (SE-HPLC).

[0107] Figure 19The results show the purification of a mixture of bioactive peptides containing hu11F11 and hu11F11-F06 using POROS MabCapture A Select resin, as confirmed by polyacrylamide gel electrophoresis (SDS-PAGE).

[0108] Figure 20 The purification curves of a mixture of bioactive peptides containing ch1E4 and ch1E4-F06 using affinity chromatography with POROSMabCapture A Select resin are shown.

[0109] Figure 21 The purification curves of a mixture of bioactive peptides containing anti-BACE and anti-BACE-F06 are shown using affinity chromatography with POROS MabCapture A Select resin.

[0110] Figure 22 The results show the purification of a mixture of bioactive peptides containing ch1E4 and ch1E4-F06 using POROS MabCapture A Select resin, as confirmed by size exclusion high performance liquid chromatography (SE-HPLC).

[0111] Figure 23 The results show the purification of a mixture of bioactive peptides containing anti-BACE and anti-BACE-F06 using POROS MabCapture ASelect resin, as confirmed by size exclusion high-performance liquid chromatography (SE-HPLC).

[0112] Figure 24 The results show the purification of a mixture of bioactive peptides containing ch1E4 and ch1E4-F06 using POROS MabCapture A Select resin, as confirmed by polyacrylamide gel electrophoresis (SDS-PAGE).

[0113] Figure 25 The results show the purification of a mixture of bioactive peptides containing anti-BACE and anti-BACE-F06 using POROS MabCapture ASelect resin, as confirmed by polyacrylamide gel electrophoresis (SDS-PAGE).

[0114] Figure 26The purification curves of a mixture of bioactive peptides containing Fc and Fc-F06 using affinity chromatography with POROS MabCapture ASelect resin are shown.

[0115] Figure 27 The results show the purification of a mixture of bioactive peptides containing Fc and Fc-F06 using POROS MabCapture A Select resin, as confirmed by size exclusion high performance liquid chromatography (SE-HPLC).

[0116] Figure 28 The results show the purification of a mixture of bioactive peptides containing Fc and Fc-F06 using POROS MabCapture A Select resin, as confirmed by polyacrylamide gel electrophoresis (SDS-PAGE).

[0117] Figure 29 The purification curves of a mixture of bioactive peptides containing GLP-1-Fc and GLP-1-Fc-F06 by affinity chromatography using POROSMabCapture A Select resin are shown.

[0118] Figure 30 The results show the purification of a mixture of bioactive peptides containing GLP-1-Fc and GLP-1-Fc-F06 using POROS MabCapture A Select resin, as confirmed by size exclusion high performance liquid chromatography (SE-HPLC).

[0119] Figure 31 The purification curves of samples containing anti-BACE, anti-BACE-F06, hu11F11, and hu11F11-F06 by affinity chromatography using POROS MabCapture A Select resin are shown.

[0120] Figure 32 The results show the purification of samples containing anti-BACE, anti-BACE-F06, hu11F11, and hu11F11-F06 using POROS AbCapture A Select resin, as confirmed by size exclusion high performance liquid chromatography (SE-HPLC).

[0121] Figure 33The results show the purification of samples containing anti-BACE, anti-BACE-F06, hu11F11, and hu11F11-F06 using POROS A AbCapture A Select resin, as confirmed by polyacrylamide gel electrophoresis (SDS-PAGE).

[0122] Figure 34 The results of purification of samples containing anti-BACE, anti-BACE-F06, hu11F11, and hu11F11-F06 using POROSMabCapture A Select resin are shown, as confirmed by liquid chromatography-mass spectrometry (LC / MS).

[0123] Figure 35 The purification curves of the sample containing ch1E4, ch1E4-F06, hu11F11, and hu11F11-F06 by affinity chromatography using POROSMabCapture A Select resin are shown. Detailed Implementation

[0124] The advantages and features of the present invention, as well as methods for implementing it, will be described in detail below with reference to the following embodiments. However, the present invention is not limited to the embodiments disclosed below and can be implemented in various different forms. These embodiments are provided merely to make the disclosure of the present invention more complete and to provide a full scope of the invention to those skilled in the art, and are defined based on the claims of the present invention.

[0125] Example 1: Preparation of monovalent bispecific antibodies

[0126] (1) Construction of monovalent bispecific antibody expression vector

[0127] To construct a monovalent bispecific antibody expression vector, the nucleotide sequence of the antibody, containing the signal sequence, was inserted into the multiple cloning site (MCS) of the pcDNA3.4 (Invitrogen) vector. The expression vector used was a monocistronic vector, and heavy chain and light chain expression vectors were prepared separately.

[0128] As heavy chain expression vectors, heavy chain expression vector 1 having a first heavy chain sequence and heavy chain expression vector 2 having a second heavy chain sequence were prepared.

[0129] Heavy chain expression vector 1 is a form in which anti-IGF1R scFv (1564, F06, VH5, VH9, VH16, see Table 2) is linked to the C-terminus of an immunoglobulin via a linker. The C-terminus of the immunoglobulin is a heavy chain variable region linked to the human heavy chain constant region of an anti-α-synuclein antibody, specifically ch11F11 (see the sequence listing in International Patent Publication No. WO2019 / 117684), ch1E4 (see heavy chain SEQ ID NO. 18 and light chain SEQ ID NO. 19), or hu11F11 (see the sequence listing in International Patent Publication No. WO2019 / 117684), anti-BACE (see the sequence of hu2H8v29 in International Patent Publication No. WO2019 / 094608), or anti-BACE (see the sequence of hu2H8v29 in International Patent Publication No. WO2019 / 094608). Sequence substitution (hole) occurs at a specific position within the heavy chain constant region. mutations, pore mutations: T366S, L368A, Y406V).

[0130] In heavy chain expression vector 2, the variable region of the heavy chain encoding anti-α-synuclein antibody or anti-BACE antibody is linked to the constant region of the human heavy chain, and a sequence substitution (knob mutation: T366W) occurs at a specific position in the constant region of the heavy chain.

[0131] When a light chain sequence is inserted into a light chain expression vector, the variable region of the light chain encoding anti-α-synuclein antibody or anti-BACE antibody is linked to the constant region of the human light chain.

[0132] In Example 1, the nucleic acid sequences encoding antibodies in heavy chain expression vector 1, heavy chain expression vector 2 and light chain expression vector are shown in SEQ ID NO.20 to SEQ ID NO.22.

[0133] The amino acid sequence of an example hu11F11 x F06, which constitutes a monovalent bispecific antibody according to an embodiment of this application, is shown in Table 1 below.

[0134] [Table 1]

[0135]

[0136]

[0137] Monovalent bispecific antibodies are heterodimers in which the scFv is attached to the C-terminus of one of the heavy chain Fcs of an anti-α-synuclein antibody or an anti-BACE antibody, but the scFv is not attached to the other heavy chain Fc.

[0138] Generally, combinations of button-knob dimers, button-hole dimers, and hole-hole dimers are generated during the preparation of monovalent bispecific antibodies. In the purification process to isolate pure heterodimers (button-hole dimers), monospecific antibodies (150 kDa, the same size as button-knob dimers) and bivalent bispecific antibodies (200 kDa, the same size as hole-hole dimers) are cloned separately to compare purification resolution and as controls.

[0139] In the case of preparing the heavy chain sequence of monospecific antibodies for comparison and analysis of purification resolution control substances, only the heavy chain variable region encoding anti-α-synuclein antibody or anti-BACE antibody and the human immunoglobulin heavy chain constant region are present. In the case of the heavy chain sequence of bivalent bispecific antibodies, the scFv is linked to the C-terminus of the same heavy chain sequence as the monospecific antibody via a linker, but no sequence substitution is performed in the heavy chain constant region. Monospecific antibodies, bivalent bispecific antibodies, and monovalent bispecific antibodies all have the same light chain sequence region.

[0140] The heavy chain and light chain variable sequences of the anti-IGF1R scFv (1564, F06, VH5, VH9, VH16) used in this embodiment are shown in Table 2 below.

[0141] [Table 2]

[0142]

[0143]

[0144] (2) Transient expression

[0145] The prepared vector was subjected to maxi-prep (Qiagen) to ensure a large amount of plasmid DNA, and then introduced into cells as follows. In the case of preparing monovalent bispecific antibodies, hole-type heavy chain expression vector DNA, knot-type heavy chain expression vector DNA, and light chain expression vector DNA were transduced in a ratio of 0.5:0.5:1. In the case of preparing monospecific or bispecific antibodies, heavy chain expression vector DNA and light chain expression vector DNA were transduced in a 1:1 ratio.

[0146] The day before transfection, ExpiCHO TM (Gibco, Cat: A29127) cells in ExpiCHO TM The expression medium (Gibco, Cat: A29100-01) was adjusted to a concentration of 3x10E6 to 4x10E6 viable cells / mL and cultured for 1 day at 8% CO2, 37°C, and 120 rpm. On the day of DNA transfection, cells that had grown to 7x10E6 to 10x10E6 viable cells / mL with a viability of 95% or higher were diluted to 6x106 viable cells / mL using fresh medium.

[0147] To transfect into prepared parental cells, ExpiFectamine was used. TM CHO transfection kit (Gibco, Cat: A29129) - Preparation of ExpiFectamine TM CHO & plasmid DNA complex. Dispense cold OptiPRO. TM After inoculating with (Gibco, Cat: 12309019) culture medium, inoculate with appropriate concentrations of prepared DNA and ExpiFectamine, respectively. TM After mixing the CHO reagent, let it stand at room temperature for 5 minutes, then seed it into parental cells and transfect them before incubating. The day after transfection, use ExpiFectamine... TM The enhancer and feed contained in the CHO transfection kit are seeded into transfected cells. Five days later, an additional feed is seeded, and the cells are then cultured for 10 days at 8% CO2, 37°C, and 120 rpm to complete the preparation.

[0148] (3) Culture medium harvest

[0149] To obtain the prepared culture medium, the culture medium was transferred to a centrifuge bottle and centrifuged at 4°C and 6500 rpm for 30 minutes. The mixture was then filtered through a 0.2 μm filter, and the culture medium with suspended solids removed was harvested as a sample. Purification was then performed.

[0150] Example 2: Experimental preparation for confirming the process conditions for purifying monovalent bispecific antibodies by affinity chromatography. Preparation

[0151] Monovalent bispecific antibodies were prepared according to the method of Example 1. In this case, the ch11F11 or hu11F11 clone was used as an anti-α-synuclein antibody, and the 1564, VH5, VH9, VH16 or F06 clone was used as an anti-IGF-1R antibody. Both of these belong to the VH3 family of antibodies, therefore, each heavy chain variable region contains a VH3 domain.

[0152] In actual preparation, the monovalent bispecific antibody, serving as the target antibody, is mainly produced via a knife-in-hole method, and the amount of the bivalent bispecific antibody and monospecific antibody, which are secondary products, is relatively small. However, in this embodiment, to more clearly demonstrate the fact that the purification process according to the present invention can separate antibodies based on the affinity differences caused by the number of VH3 domains, a composition that increases the concentration of the bivalent bispecific antibody and monospecific antibody, which are considered secondary products, i.e., impurities, in this purification process (i.e., providing more unfavorable conditions for purification), was prepared, and this composition was purified.

[0153] This composition contains monospecific antibodies, monovalent bispecific antibodies, and bivalent bispecific antibodies prepared in the same manner as in Example 1, in a 1:3:1 ratio, but unlike Example 1, it does not contain a knob or hole. This composition contains antibodies isolated through the following purification process:

[0154] Purification was performed using affinity chromatography with only B(Z) domains as the resin to separate the Fc-containing substances.

[0155] After equilibration with equilibration buffer (50 mM Tris-HCl (pH 7.2), 100 mM sodium chloride (NaCl)), the recovered culture medium was loaded onto the chromatographic column.

[0156] After loading, wash with 3 column volumes of equilibration buffer, then elute with 50 mM sodium citrate (pH 3.4). Neutralize the eluent to pH 7.0 with 1 M Tris-HCl (pH 9.0), and then sterile filter through a 0.2 μm filter.

[0157] Example 3: Comparison of antibody purification effects using various Protein A resins

[0158] To select a suitable Protein A resin for purifying monovalent bispecific antibodies, commercially available Protein A columns with various specifications and domains, as shown in Table 3 below, were used to purify samples containing ch11F11 monospecific antibody, ch11F11-F06 monovalent bispecific antibody, and ch11F11-F06 bispecific antibody, and their performance was compared.

[0159] [Table 3]

[0160]

[0161] The purification methods for each resin are described in detail in Examples 3-1 to 3-7 below.

[0162] The purified samples were analyzed by size exclusion-high performance liquid chromatography (SE-HPLC), sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), or liquid chromatography-mass spectrometry (LC / MS) to confirm the purification results.

[0163] For size exclusion high-performance liquid chromatography (SE-HPLC), 40 mM sodium phosphate and 400 mM sodium perchlorate (pH 6.8) were used as the mobile phase. A 20 μL sample volume was injected at a sample concentration of 1 mg / mL, and analysis was performed at a flow rate of 0.8 mL / min. The analytical peak was determined by absorbance at 280 nm. For polyacrylamide gel electrophoresis (SDS-PAGE), NuPAGE 4-12% Bis-Tris gel and MOPS electrophoresis buffer were used under both non-reducing and reducing conditions. Under reducing conditions, 5% 2-mercaptoethanol was added to the sample, and the mixture was heated at 70 °C for 10 min before analysis. Analysis using liquid chromatography-mass spectrometry (LC / MS) consisted of a Waters Acquity Ultra High Performance Liquid Chromatography (UPLC) system and a Waters Synapt G2-S Quadrupole-Time of Flight (Q-TOF) mass spectrometer, and utilized a PLRP-S... Analysis was performed using a BEH S-200 column. Data were analyzed using Waters BiopharmaLynx V.1.3 software.

[0164] Example 3-1: Simple Antioxidant Gel (MabSelect Sure)

[0165] The resolution of the samples was confirmed using HiTrap anti-chemical gel (MabSelect Sure) (GE Healthcare, Cat. No. 29-0486-84). After equilibration with equilibration buffer (25 mM citrate / 25 mM sodium phosphate (pH 7.0)), the samples prepared in Example 1 were loaded onto the column. Elution buffer (25 mM citrate / 25 mM sodium phosphate (pH 2.5)) was used with a residence time of 5 minutes, and wash conditions included 0% > 50% elution buffer per column volume at a pH range of 7.0 to 5.0. Gradient conditions were performed using 50% > 100% elution buffer per 30 column volumes.

[0166] Figure 1 The purification curves according to the affinity chromatography method of Example 3-1 are shown. Figure 1 In the diagram, the x-axis represents the sample volume in mL, and the y-axis represents the ultraviolet (UV) wavelength at 280 nm in mA. For example... Figure 1 As shown, in Example 3-1, only one elution peak appeared. This confirms that the HiTrap anti-oxidative gel (MabSelect Sure) resin does not exhibit differences in affinity based on the number of VH3 domains of peptides present in the sample.

[0167] Example 3-2: Antibody purification gel (MabSelect PrismA)

[0168] The sample resolution was confirmed using an antibody purification gel (MabSelect PrismA) (GE Healthcare, Cat. No. 17-5199-01). After equilibration with equilibration buffer (25 mM citrate / 25 mM sodium phosphate (pH 7.0)), the sample prepared in Example 1 was loaded onto the column. Elution buffer (25 mM citrate / 25 mM sodium phosphate (pH 2.5)) was used with a residence time of 5 minutes, and wash conditions included using elution buffer at 0% > 50% / 1 column volume at a pH range of 7.0 to 5.0. Gradient conditions were performed using elution buffer at 50% > 100% / 30 column volumes.

[0169] Figure 2 The purification curves according to the affinity chromatography method in Examples 3-2 are shown. Figure 2 As shown, in Example 3-2, only one elution peak appeared. This confirms that the antibody purification gel (MabSelect PrismA) resin did not exhibit differences in affinity based on the number of VH3 domains of peptides present in the sample.

[0170] Example 3-3: Protein A FF

[0171] The sample resolution was confirmed using HiTrap Protein A FF (GE Healthcare, Cat. No. 17-5079-01). After equilibration with equilibration buffer (25 mM citrate / 25 mM sodium phosphate (pH 7.0)), the sample prepared in Example 1 was loaded onto the column. Elution buffer (25 mM citrate / 25 mM sodium phosphate (pH 2.5)) was used with a residence time of 5 minutes, and wash conditions included using elution buffer at 0% > 50% / 1 column volume, with a pH range of 7.0 to 5.0. Gradient conditions were performed using elution buffer at 50% > 100% / 30 column volumes.

[0172] Figure 3The purification curves according to the affinity chromatography method of Examples 3-3 are shown. Figure 3 As shown, in Examples 3-3, no significant separation was observed in the elution peaks. This confirms that the HiTrap protein AFF resin did not exhibit significant differences in affinity due to variations in the number of VH3 domains of peptides present in the samples.

[0173] Examples 3-4: MabSelect Xtra

[0174] The sample resolution was confirmed using MabSelect Xtra (GE Healthcare, Cat. No. 17-5269-07). After equilibration with equilibration buffer (25 mM citrate / 25 mM sodium phosphate (pH 7.0)), the sample prepared in Example 1 was loaded onto the column. Elution buffer (25 mM citrate / 25 mM sodium phosphate (pH 2.5)) was used with a residence time of 5 minutes, and wash conditions included using elution buffer at 0% > 50% / 1 column volume at a pH range of 7.0 to 5.0. Gradient conditions were performed using elution buffer at 50% > 100% / 30 column volumes.

[0175] Figure 4 The purification curves according to the affinity chromatography method in Examples 3-4 are shown. Figure 5 and Figure 6 The results of size exclusion high-performance liquid chromatography (SE-HPLC) and polyacrylamide gel electrophoresis (SDS-PAGE) are shown respectively. Figure 4 As shown, three peaks were confirmed within the pH range of the elution buffer. Specifically, at relatively high pH, ​​monospecific antibodies with a small number (two) VH3 domains were eluted first (Peak 1). As the pH decreased, monovalent bispecific antibodies with three VH3 domains were eluted second, and then, at even lower pH, bivalent bispecific antibodies with up to four VH3 domains were eluted last. This confirms that the MabSelect Xtra resin exhibits differences in affinity based on the number of VH3 domains present in the peptides in the sample, and that ch11F11-F06 can be selectively purified based on these affinity differences. Similar results were observed with other cloning combinations.

[0176] In addition, by means of Figure 5 and Figure 6 The results of size exclusion high performance liquid chromatography (SE-HPLC) and polyacrylamide gel electrophoresis (SDS-PAGE) further confirmed that the peptides separated by affinity chromatography corresponded to monospecific antibodies, monovalent bispecific antibodies, and bivalent bispecific antibodies, respectively.

[0177] Examples 3-5: Protein A HP

[0178] The sample resolution was confirmed using HiTrap Protein A HP (GE Healthcare, Cat. No. 17-0402-01). After equilibration with equilibration buffer (25 mM citrate / 25 mM sodium phosphate (pH 7.0)), the sample prepared in Example 1 was loaded onto the column. Elution buffer (25 mM citrate / 25 mM sodium phosphate (pH 2.5)) was used with a residence time of 5 minutes, and wash conditions included using elution buffer at 0% > 50% / 1 column volume, with a pH range of 7.0 to 5.0. Gradient conditions were performed using elution buffer at 50% > 100% / 30 column volumes.

[0179] Figure 7 The purification curves according to the affinity chromatography method in Examples 3-5 are shown. Figure 8 and Figure 9 The results of size exclusion high-performance liquid chromatography (SE-HPLC) and polyacrylamide gel electrophoresis (SDS-PAGE) are shown respectively. Figure 7 As shown, three peaks were confirmed within the pH range of the elution buffer. Specifically, at relatively high pH, ​​monospecific antibodies with a small number (two) VH3 domains were eluted first (Peak 1). As the pH decreased, monovalent bispecific antibodies with three VH3 domains were eluted second, and then, at even lower pH, bivalent bispecific antibodies with up to four VH3 domains were eluted last. This confirms that HiTrap Protein A HP resin exhibits differences in affinity based on the number of VH3 domains present in the peptides in the sample, and that ch11F11-F06 can be selectively purified based on these affinity differences. Similar results were observed with other cloning combinations.

[0180] In addition, by means of Figure 8 and Figure 9The results of size exclusion high performance liquid chromatography (SE-HPLC) and polyacrylamide gel electrophoresis (SDS-PAGE) further confirmed that the peptides separated by affinity chromatography corresponded to monospecific antibodies, monovalent bispecific antibodies, and bivalent bispecific antibodies, respectively.

[0181] Examples 3-6: POROS MabCapture A Selection

[0182] The sample resolution was confirmed using POROS MabCapture A Select (Thermo Fisher SCIENTIFIC, Cat. No. A26458). After equilibration with equilibration buffer (25 mM citrate / 25 mM sodium phosphate (pH 7.0)), the sample prepared in Example 1 was loaded onto the column. Elution buffer (25 mM citrate / 25 mM sodium phosphate (pH 2.5)) was used with a residence time of 5 minutes, and wash conditions included using elution buffer at 0% > 50% / 1 column volume, with a pH range of 7.0 to 5.0. Gradient conditions were performed using elution buffer at 50% > 100% / 30 column volumes.

[0183] Figure 10 The purification curves according to the affinity chromatography method of Examples 3-6 are shown. Figure 11 and Figure 13 The results of size exclusion high-performance liquid chromatography (SE-HPLC) and polyacrylamide gel electrophoresis (SDS-PAGE) are shown respectively. Figure 10 As shown, three peaks were confirmed within the pH range of the elution buffer. Specifically, at relatively high pH, ​​monospecific antibodies with a small number (two) VH3 domains were eluted first (Peak 1). As the pH decreased, monovalent bispecific antibodies with three VH3 domains were eluted second, and then, at even lower pH, bivalent bispecific antibodies with up to four VH3 domains were eluted last. This confirms that the POROS MabCapture A Select resin exhibits differences in affinity based on the number of VH3 domains present in the peptides in the sample, and that ch11F11-F06 can be selectively purified based on these affinity differences. Similar results were observed with other cloning combinations.

[0184] In addition, by means of Figure 11 and Figure 12The results of size exclusion high performance liquid chromatography (SE-HPLC) and polyacrylamide gel electrophoresis (SDS-PAGE) further confirmed that the peptides separated by affinity chromatography corresponded to monospecific antibodies, monovalent bispecific antibodies, and bivalent bispecific antibodies, respectively.

[0185] Examples 3-7: Absolute High Cap

[0186] The sample resolution was confirmed using AbSolute High Cap (AGC). After equilibration with equilibration buffer (25 mM citrate / 25 mM sodium phosphate (pH 7.0)), the sample prepared in Example 1 was loaded onto the column. Elution buffer (25 mM citrate / 25 mM sodium phosphate (pH 2.5)) was used with a residence time of 5 minutes, and wash conditions included using elution buffer at 0% > 50% / 1 column volume at a pH range of 7.0 to 5.0. Gradient conditions were performed using elution buffer at 50% > 100% / 30 column volumes.

[0187] Figure 13 The purification curves according to the affinity chromatography method of Examples 3-6 are shown. Figure 14 and Figure 15 The results of size exclusion high-performance liquid chromatography (SE-HPLC) and polyacrylamide gel electrophoresis (SDS-PAGE) are shown respectively. Figure 13 As shown, three peaks were confirmed within the pH range of the elution buffer. Specifically, at relatively high pH, ​​monospecific antibodies with a small number (two) VH3 domains were eluted first (Peak 1). As the pH decreased, monovalent bispecific antibodies with three VH3 domains were eluted second, and then, at even lower pH, bivalent bispecific antibodies with up to four VH3 domains were eluted last. This confirms that AbSolute High Cap resin exhibits differences in affinity based on the number of VH3 domains present in the peptides in the sample, and that ch11F11-F06 can be selectively purified based on these affinity differences. Similar results were observed with other cloning combinations.

[0188] In addition, by means of Figure 14 and Figure 15The results of size exclusion high performance liquid chromatography (SE-HPLC) and polyacrylamide gel electrophoresis (SDS-PAGE) further confirmed that the peptides separated by affinity chromatography corresponded to monospecific antibodies, monovalent bispecific antibodies, and bivalent bispecific antibodies, respectively.

[0189] Summary of purification results in Examples 3-1 to 3-7

[0190] The pH concentration gradient of the elution buffer for which peaks were detected in Examples 3-4 to 3-7 is shown in Table 4 below.

[0191] [Table 4]

[0192]

[0193] The purification results of Examples 3-1 to 3-7 are summarized and described in detail in Table 5 below.

[0194] [Table 5]

[0195]

[0196] The resolution (Rs), recovery yield, and monovalent antibody yield for each embodiment were calculated using the following formula.

[0197]

[0198] tR1: Retention time of peak 1.

[0199] tR2: Retention time of peak 2

[0200] W1 / 21: The width between peak 1 and half height

[0201] W1 / 22: The width between peak 2 and half height

[0202] **Recovery (%) = Output (mg) / Input (mg) x 100

[0203] ***Monovalent antibody yield (%) = Monovalent antibody output (mg) x Purity (%) / Monovalent antibody input (mg) x Purity (%) x 100

[0204] As a result of the experiments, only the chromatographic columns used in Examples 3-4 to 3-7 showed differences in affinity based on the number of VH3 domains contained in the peptides. These results confirmed that monospecific antibodies, monovalent bispecific antibodies, and bivalent bispecific antibodies were effectively separated according to pH concentration gradients. More specifically, the antibodies separated in Examples 3-4 to 3-7 had two, three, and four different numbers of VH3 domains depending on the presence and quantity of anti-IGF1R scFv, wherein the anti-IGF1R scFv is linked to the C-terminus of the Fc of the monospecific antibody containing a VH3 domain in the variable region. Furthermore, the chromatographic columns used in Examples 3-4 to 3-7, due to the full-domain nature of the protein A resin, could accurately identify the differences in the number of VH3 domains in the antibodies, thus enabling effective antibody separation. Generally, it is more preferable to use improved chromatographic columns, such as MabSelect Sure and MabSelect PrismA, which use only specific domain B (Z) to improve Fc binding affinity and alkaline stability. In contrast, according to this disclosure, resins containing conventional (without introducing specific mutant) protein A can be used more effectively to separate peptides from a mixture of peptides containing VH3 domains based on the number of VH3 domains.

[0205] Example 4: Antibody purity analysis from culture medium

[0206] Using POROS A 20 μm (Thermo Fisher, Cat. No. 1-5022-26), it was confirmed that the desired Fc-containing bioactive peptides could be analyzed even when culture medium was used directly without purifying protein A. After equilibration with equilibration buffer (25 mM citrate / 25 mM sodium phosphate (pH 7.0)), the culture medium (harvested cell culture fluid) was directly loaded. An unbound wash was performed using 2 column volumes of equilibration buffer, followed by elution buffer (25 mM citrate / 25 mM sodium phosphate (pH 2.5)) with a residence time of 1 minute. The gradient conditions were 50% > 100% elution buffer / 30 column volumes. Samples pre-purified using protein A were analyzed using the same method, and the results were compared.

[0207] Figure 16 The analytical curves according to the affinity chromatography method of Example 4 are shown. Figure 16 As shown, the results of direct analysis of the culture medium without passing through the protein A column were confirmed to be very similar to those of analysis of samples pre-purified with protein A, at 90.3% and 90.5%, respectively, and the elution curves and resolutions were also similar.

[0208] The above results confirm that, according to the separation method of one embodiment, peptides can be separated and analyzed with excellent resolution even without using the transient culture medium for primary purification of protein A. Therefore, the separation method according to one embodiment can be used as a very efficient analytical method, allowing for direct confirmation of antibody proportions and purity using the culture medium even when there are many candidate cell lines in the early stages of cell line development, without the need for a primary purification process.

[0209] Example 5: Purification of various asymmetric forms of peptides

[0210] Example 5-1: IgG(VH3(+))-scFv(VH3+)

[0211] The resolution of the sample was confirmed using the protein A resin used in Example 3. All protein A resins shown in Table 5 that confirmed resolution can be used as protein A resins, but as representative, POROS MabCapture ASelect (Thremo Fisher SCIENTIFIC, Cat. No. A26458) was used for purification. The sample used for purification was a mixture of a monospecific antibody (hu11F11) with two VH3 domains and a monovalent bispecific antibody (hu11F11-F06) with three VH3 domains, prepared according to the method in Example 1.

[0212] After equilibration with equilibration buffer (25 mM citrate / 25 mM sodium phosphate (pH 7.0)), the prepared sample is loaded onto the column. Elution buffer (25 mM citrate / 25 mM sodium phosphate (pH 2.5)) is used with a residence time of 5 minutes. Washing conditions include using elution buffer at 0% > 50% / 1 column volume at a pH range of 7.0 to 5.0. Gradient conditions are then performed using elution buffer at 50% > 100% / 30 column volumes. Figure 17The purification curves of affinity chromatography are shown. Figure 18 and Figure 19 The results of size exclusion high performance liquid chromatography (SE-HPLC) and polyacrylamide gel electrophoresis (SDS-PAGE) are shown respectively.

[0213] Results of the applicable pH concentration gradient of the elution buffer are as follows Figure 17 As shown, two peaks were separated. Specifically, at a relatively high pH, ​​the monospecific antibody with a small number (two) VH3 domains was eluted first (Peak 1), and as the pH decreased, the monovalent bispecific antibody with three VH3 domains was eluted second (Peak 2). Figure 18 and Figure 19 These results were further confirmed by size exclusion high performance liquid chromatography (SE-HPLC) and polyacrylamide gel electrophoresis (SDS-PAGE).

[0214] Example 5-2. IgG(VH3(-))-scFv(VH3+)

[0215] The resolution of the sample was confirmed using the protein A resin used in Example 3. All protein A resins shown in Table 5 that confirmed resolution can be used as protein A resins, but POROS MabCapture ASelect (Thremo Fisher SCIENTIFIC, Cat. No. A26458) was used for purification as representative. The sample used for purification was a mixture of a monospecific antibody (ch1E4 or anti-BACE) without a VH3 domain and a monovalent bispecific antibody (ch1E4-F06 or anti-BACE-F06) with one VH3 domain, produced according to the same method as in Example 1.

[0216] After equilibration with equilibration buffer (25 mM citrate / 25 mM sodium phosphate (pH 7.0)), the prepared sample is loaded onto the column. Elution buffer (25 mM citrate / 25 mM sodium phosphate (pH 2.5)) is used with a residence time of 5 minutes. Washing conditions include using elution buffer at 0% > 50% / 1 column volume at a pH range of 7.0 to 5.0. Gradient conditions are then performed using elution buffer at 50% > 100% / 30 column volumes. Figure 20 and Figure 21 The elution peaks of affinity chromatography are shown separately. Figure 22 and Figure 23 The results of size exclusion high-performance liquid chromatography (SE-HPLC) are shown separately, and Figure 24 and Figure 25 The results of polyacrylamide gel electrophoresis (SDS-PAGE) are shown.

[0217] Results of the applicable pH concentration gradient of the elution buffer are as follows Figure 20 and Figure 21 As shown, two peaks were separated. Specifically, at relatively high pH, ​​the monospecific antibody without (0) VH3 domains was eluted first (Peak 1), and as the pH decreased, the monovalent bispecific antibody with one VH3 domain was eluted second (Peak 2). Figures 22 to 25 These results were further confirmed by size exclusion high performance liquid chromatography (SE-HPLC) and polyacrylamide gel electrophoresis (SDS-PAGE).

[0218] Example 5-3. Fc-scFv(VH3+)

[0219] The resolution of the sample was confirmed using the protein A resin used in Example 3. All protein A resins shown in Table 5 that confirmed resolution can be used as protein A resins, but as representative, POROS MabCapture ASelect (Thremo Fisher SCIENTIFIC, Cat. No. A26458) was used for purification. As the sample used for purification, the monospecific antibody (hu11F11) and the monovalent bispecific antibody (hu11F11-F06) were treated with papain to prepare Fab-free Fc and Fc-scFv (F06). Specifically, the papain was diluted with digestion buffer (10 mM phosphate-buffered saline (PBS), 20 mM EDTA, 10 mM cysteine-HCl (pH 7.4)) and mixed with the prepared sample at a ratio of 1:100 (papain:antibody), and reacted at 37°C for 4 hours.

[0220] After equilibration with equilibration buffer (25 mM citrate / 25 mM sodium phosphate (pH 7.0)), the prepared sample was loaded onto the column. Elution buffer (25 mM citrate / 25 mM sodium phosphate (pH 2.5)) was used with a residence time of 5 minutes, and wash conditions included elution buffer of 0% > 50% / 1 column volume at a pH range of 7.0 to 5.0. Gradient conditions were then performed using elution buffer of 50% > 100% / 30 column volumes. Figure 26 The purification curves of affinity chromatography are shown. Figure 27 and Figure 28 The results of size exclusion high performance liquid chromatography (SE-HPLC) and polyacrylamide gel electrophoresis (SDS-PAGE) are shown respectively.

[0221] Results of the applicable pH concentration gradient of the elution buffer are as follows Figure 26 As shown, two peaks were separated. In conventional antibodies, the Fab is linked to the N-terminus of the Fc, while in this embodiment, the N-terminus of the Fc of the separated sample does not contain Fab. Even so, according to... Figure 26At relatively high pH, ​​Fc without (0) VH3 domains was eluted first (Peak 1). As pH decreased, Fc-scFv (F06) with one VH3 domain was eluted second (Peak 2). Figure 27 and Figure 28 Size exclusion high-performance liquid chromatography (SE-HPLC) and polyacrylamide gel electrophoresis (SDS-PAGE) results further confirmed these findings. These results indicate that the purification method according to the invention is not only applicable to simple antibody purification, but also broadly applicable to various Fc-containing peptides with different numbers of VH3 domains, for example, various peptides containing Fc-scFv (e.g., Fc-scFv linked to drugs or functional peptides).

[0222] Example 5-4. GLP-1-Fc-scFv(VH3+)

[0223] The resolution of the sample was confirmed using the protein A resin used in Example 3. All protein A resins shown in Table 5 that confirmed resolution can be used as protein A resins, but as representative, POROS MabCapture ASelect (Thremo Fisher SCIENTIFIC, Cat. No. A26458) was used for purification. The sample used for purification was a mixture of GLP-1-Fc (F06) and GLP-1-Fc-scFv (F06) having a VH3 domain, produced using the same method as in Example 1, except that the known sequence of GLP-1 (Glucagon-like Peptide-1) was linked at Fc instead of Fab. GLP-1, used in this example, is a peptide drug whose research and clinical trials related to diseases such as diabetes and Alzheimer's disease are actively underway. This example tested whether separation based on the number of VH3 domains was possible even when such a peptide drug was present.

[0224] After equilibration with equilibration buffer (25 mM citrate / 25 mM sodium phosphate (pH 7.0)), the prepared sample was loaded onto the column. Elution buffer (25 mM citrate / 25 mM sodium phosphate (pH 2.5)) was used with a residence time of 5 minutes, and wash conditions included elution buffer of 0% > 50% / 1 column volume at a pH range of 7.0 to 5.0. Gradient conditions were then performed using elution buffer of 50% > 100% / 30 column volumes. Figure 29 The purification curves of affinity chromatography are shown. Figure 30 The results of size exclusion high performance liquid chromatography (SE-HPLC) are shown.

[0225] Results of the applicable pH concentration gradient of the elution buffer are as follows Figure 29 As shown, two peaks were separated. In conventional antibodies, the Fab is linked to the N-terminus of the Fc, while in this embodiment, the separated sample contains a GLP-1 fusion peptide at the N-terminus of the Fc instead of the Fab. Even so, according to... Figure 29 At relatively high pH, ​​GLP-1-Fc without (0) VH3 domains was eluted first (Peak 1). As pH decreased, GLP-1-Fc-scFv (F06) with one VH3 domain was eluted second (Peak 2). Figure 30 Size exclusion high-performance liquid chromatography (SE-HPLC) results reconfirmed these findings. These results indicate that the purification method according to the invention is not only applicable to simple antibody purification, but also widely applicable to the separation of various Fc-containing peptides with different numbers of VH3 domains, particularly bioactive peptides containing other active components besides antibody constituents.

[0226] The pH concentration gradient of the elution buffer in Examples 5-1 to 5-4 is shown in Table 6 below.

[0227] [Table 6]

[0228]

[0229]

[0230] Example 5-5. IgG(VH3(±))-scFv(VH3+)

[0231] The resolution of the sample was confirmed using the protein A resin used in Example 3. All protein A resins shown in Table 5 that confirmed resolution can be used as protein A resins, but POROS MabCapture ASelect (Thremo Fisher SCIENTIFIC, Cat. No. A26458) was used for purification as representative. The sample used for purification was a mixture containing four types of antibodies, namely: a monospecific antibody (hu11F11) with two VH3 domains and a monovalent bispecific antibody (hu11F11-F06) with three VH3 domains, generated according to the method of Example 1; and a monospecific antibody (anti-BACE, anti-BACE) without a VH3 domain and a monovalent bispecific antibody (anti-BACE-F06, anti-BACE-F06) with one VH3 domain, generated according to the same method as in Example 1, except that an anti-BACE antibody without a VH3 domain in the IgG variable region was used.

[0232] After equilibration with equilibration buffer (25 mM citrate / 25 mM sodium phosphate (pH 7.0)), the prepared sample was loaded onto the column. Elution buffer (25 mM citrate / 25 mM sodium phosphate (pH 2.5)) was used with a residence time of 5 minutes, and wash conditions included elution buffer of 0% > 50% / 1 column volume at a pH range of 7.0 to 5.0. Gradient conditions were then performed using elution buffer of 50% > 100% / 30 column volumes. Figure 31 The purification curves of affinity chromatography are shown. Figures 32 to 34 The results are shown by size exclusion high performance liquid chromatography (SE-HPLC), polyacrylamide gel electrophoresis (SDS-PAGE), and liquid chromatography-mass spectrometry (LC / MS).

[0233] As a result of the applicable elution buffer pH concentration gradient, such as Figure 31 As shown, four peaks were separated, and based on the number of VH3 domains, the peaks of the peptides (anti-BACE antibody: 0, anti-BACE-F06: 1, hu11F11 antibody: 2, hu11F11-F06: 3) were significantly separated, confirming the selective purification of the desired peptides. Furthermore, Figures 32 to 34 These results were further confirmed by size exclusion high-performance liquid chromatography (SE-HPLC), polyacrylamide gel electrophoresis (SDS-PAGE), and liquid chromatography-mass spectrometry (LC / MS). This demonstrates that the separation method of this application can purify peptides very precisely based on the number of VH3 domains contained in each peptide.

[0234] Examples 5-6. IgG(VH3(±))-scFv(VH3+)

[0235] The resolution of the sample was confirmed using the protein A resin used in Example 3. All protein A resins shown in 5 that confirmed the resolution could be used as protein A resins, but as representative, POROS MabCapture A Select (Thremo Fisher SCIENTIFIC, Cat. No. A26458) was used for purification. As the sample used for purification, a mixture containing four types of antibodies was used, wherein the four types of antibodies included: a monospecific antibody (hu11F11) with two VH3 domains and a monovalent bispecific antibody (hu11F11-F06) with three VH3 domains generated according to the method of Example 1; and a monospecific antibody (ch1E4) without a VH3 domain and a monovalent bispecific antibody (ch1E4-F06) with one VH3 domain generated according to the same method as in Example 1, except that the ch1E4 antibody, which does not contain a VH3 domain in the IgG variable region, was used as an antibody clone.

[0236] After equilibration with equilibration buffer (25 mM citrate / 25 mM sodium phosphate (pH 7.0)), load the prepared sample onto the column. Use elution buffer (25 mM citrate / 25 mM sodium phosphate (pH 2.5)) with a residence time of 5 minutes. Wash conditions include using elution buffer at 0% > 50% / 1 column volume at a pH range of 7.0 to 5.0. Gradient conditions are then performed using elution buffer at 50% > 100% / 30 column volumes. Figure 35 The purification curves of affinity chromatography are shown.

[0237] As a result of the applicable elution buffer pH concentration gradient, such as Figure 35As shown, four peaks were separated, and the peaks of the peptides (ch1E4: 0, ch1E4-F06: 1; hu11F11: 2, hu11F11-F06: 3) were significantly separated according to the number of VH3 domains, confirming that the desired peptides can be selectively purified according to the number of VH3 domains. <110> Albile Biotechnology Co., Ltd. <120> A method for purifying bioactive peptides using protein A affinity chromatography <130> PX210081PCT <150> KR 10-2020-0173808 <151> 2020-12-11 <150> US 62 / 953,687 <151> 2019-12-26 <150> US 62 / 953,685 <151> 2019-12-26 <160> 37 <170> KoPatentIn 3.0 <210> 1 <211> 121 <212> PRT <213> Artificial sequence <220> <223> H variable region 1564 <400> 1 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Asp Met Ser Trp Val Arg Gln Ala Pro Gly Lys Cys Leu Glu Trp Val 35 40 45 Ser Ala Ile Ser Tyr Asp Asn Ala Asn Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Gly Val Leu Thr Thr Leu Met Asn Trp Phe Asp Tyr Trp Gly 100 105 110 Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 2 <211> 110 <212> PRT <213> Artificial sequence <220> <223> L variable region 1564 <400> 2 Gln Ser Val Leu Thr Gln Pro Pro Ser Ala Ser Gly Thr Pro Gly Gln 1 5 10 15 Arg Val Thr Ile Ser Cys Thr Gly Ser Ser Ser Asn Ile Gly Ser Asn 20 25 30 Asp Val Ser Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu Leu 35 40 45 Ile Tyr Ala Asn Val Asn Arg Pro Ser Gly Val Pro Asp Arg Phe Ser 50 55 60 Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Ser Gly Leu Arg 65 70 75 80 Ser Glu Asp Glu Ala Asp Tyr Tyr Cys Gly Ala Trp Asp Asp Ser Leu 85 90 95 Asn Ala Tyr Val Phe Gly Cys Gly Thr Lys Leu Thr Val Leu 100 105 110 <210> 3 <211> 121 <212> PRT <213> Artificial Sequence <220> <223> H variable region F06 <400> 3 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Asp Met Ser Trp Val Arg Gln Ala Pro Gly Lys Cys Leu Glu Trp Val 35 40 45 Ser Ala Ile Ser Tyr Asp Asn Ala Asn Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Gly Val Leu Thr Thr Leu Met Asn Trp Phe Asp Tyr Trp Gly 100 105 110 Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 4 <211> 110 <212> PRT <213> Artificial Sequence <220> <223> L variable region F06 <400> 4 Gln Ser Val Leu Thr Gln Pro Pro Ser Ala Ser Gly Thr Pro Gly Gln 1 5 10 15 Arg Val Thr Ile Ser Cys Thr Gly Ser Ser Ser Asn Ile Gly Ser Asn 20 25 30 Asp Val Ser Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu Leu 35 40 45 Ile Tyr Ala Asn Val Asn Arg Pro Ser Gly Val Pro Asp Arg Phe Ser 50 55 60 Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Ser Gly Leu Arg 65 70 75 80 Ser Glu Asp Glu Ala Asp Tyr Tyr Cys Gly Thr Trp Ala Gly Ser Leu 85 90 95 Asn Ala Tyr Val Phe Gly Cys Gly Thr Lys Leu Thr Val Leu 100 105 110 <210> 5 <211> 121 <212> PRT <213> Artificial sequence <220> <223> H variable area VH5 <400> 5 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Asp Met Ser Trp Val Arg Gln Ala Pro Gly Lys Cys Leu Glu Trp Val 35 40 45 Ser Ala Ile Ser Gly Asp Asn Ala Ser Thr Tyr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Gly Val Leu Thr Thr Leu Met Asn Trp Phe Asp Ser Trp Gly 100 105 110 Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 6 <211> 110 <212> PRT <213> Artificial sequence <220> <223> L variable area VH5 <400> 6 Gln Ser Val Leu Thr Gln Pro Pro Ser Ala Ser Gly Thr Pro Gly Gln 1 5 10 15 Arg Val Thr Ile Ser Cys Thr Gly Ser Ser Ser Asn Ile Gly Ser Asn 20 25 30 Asp Val Ser Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu Leu 35 40 45 Ile Tyr Ala Asn Val Asn Arg Pro Ser Gly Val Pro Asp Arg Phe Ser 50 55 60 Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Ser Gly Leu Arg 65 70 75 80 Ser Glu Asp Glu Ala Asp Tyr Tyr Cys Gly Ala Trp Asp Asp Ser Leu 85 90 95 Asn Ala Tyr Val Phe Gly Cys Gly Thr Lys Leu Thr Val Leu 100 105 110 <210> 7 <211> 121 <212> PRT <213> artificial sequence <220> <223> VH16 can be changed <400> 7 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Asp Met Ser Trp Val Arg Gln Ala Pro Gly Lys Cys Leu Glu Trp Val 35 40 45 Ser Ala Ile Ser Gly Ser Asn Ala Asn Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Gly Val Leu Thr Thr Leu Met Asn Trp Phe Asp Tyr Trp Gly 100 105 110 Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 8 <211> 110 <212> PRT <213> Artificial Sequence <220> <223> L variable region VH16 <400> 8 Gln Ser Val Leu Thr Gln Pro Pro Ser Ala Ser Gly Thr Pro Gly Gln 1 5 10 15 Arg Val Thr Ile Ser Cys Thr Gly Ser Ser Ser Asn Ile Gly Ser Asn 20 25 30 Asp Val Ser Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu Leu 35 40 45 Ile Tyr Ala Asn Val Asn Arg Pro Ser Gly Val Pro Asp Arg Phe Ser 50 55 60 Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Ser Gly Leu Arg 65 70 75 80 Ser Glu Asp Glu Ala Asp Tyr Tyr Cys Gly Ala Trp Asp Asp Ser Leu 85 90 95 Asn Ala Tyr Val Phe Gly Cys Gly Thr Lys Leu Thr Val Leu 100 105 110 <210> 9 <211> 121 <212> PRT <213> Artificial Sequence <220> <223> H variable region VH9 <400> 9 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Asp Met Ser Trp Val Arg Gln Ala Pro Gly Lys Cys Leu Glu Trp Val 35 40 45 Ser Ala Ile Ser Gly Asp Asn Gly Ser Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Gly Val Leu Thr Thr Leu Met Asn Trp Phe Asp Ser Trp Gly 100 105 110 Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 10 <211> 110 <212> PRT <213> artificial sequence <220> <223> L can change district VH9 <400> 10 Gln Ser Val Leu Thr Gln Pro Pro Ser Ala Ser Gly Thr Pro Gly Gln 1 5 10 15 Arg Val Thr Ile Ser Cys Thr Gly Ser Ser Ser Asn Ile Gly Ser Asn 20 25 30 Asp Val Ser Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu Leu 35 40 45 Ile Tyr Ala Asn Ser Asn Arg Pro Ser Gly Val Ser Asp Arg Phe Ser 50 55 60 Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Ser Gly Leu Arg 65 70 75 80 Ser Glu Asp Glu Ala Asp Tyr Tyr Cys Gly Ala Trp Asp Asp Ser Leu 85 90 95 Asn Gly Tyr Val Phe Gly Cys Gly Thr Lys Leu Thr Val Leu 100 105 110 <210> 11 <211> 251 <212> PRT <213> Artificial Sequence <220> <223> scFv 3_4 <400> 11 Gln Ser Val Leu Thr Gln Pro Pro Ser Ala Ser Gly Thr Pro Gly Gln 1 5 10 15 Arg Val Thr Ile Ser Cys Thr Gly Ser Ser Ser Asn Ile Gly Ser Asn 20 25 30 Asp Val Ser Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu Leu 35 40 45 Ile Tyr Ala Asn Val Asn Arg Pro Ser Gly Val Pro Asp Arg Phe Ser 50 55 60 Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Ser Gly Leu Arg 65 70 75 80 Ser Glu Asp Glu Ala Asp Tyr Tyr Cys Gly Thr Trp Ala Gly Ser Leu 85 90 95 Asn Ala Tyr Val Phe Gly Cys Gly Thr Lys Leu Thr Val Leu Gly Gly 100 105 110 Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly 115 120 125 Gly Ser Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro 130 135 140 Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser 145 150 155 160 Ser Tyr Asp Met Ser Trp Val Arg Gln Ala Pro Gly Lys Cys Leu Glu 165 170 175 Trp Val Ser Ala Ile Ser Tyr Asp Asn Ala Asn Thr Tyr Tyr Ala Asp 180 185 190 Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr 195 200 205 Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr 210 215 220 Tyr Cys Ala Lys Gly Val Leu Thr Thr Leu Met Asn Trp Phe Asp Tyr 225 230 235 240 Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 245 250 <210> 12 <211> 716 <212> PRT <213> Artificial Sequence <220> <223> hu11F11(ver.2)(M428L)-F06(de2)(StoP) <400> 12 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Thr Ser Gly Phe Thr Phe Ser Asp Phe 20 25 30 Tyr Met Glu Trp Val Arg Gln Pro Pro Gly Lys Arg Leu Glu Trp Ile 35 40 45 Ala Ala Ser Arg Asn Lys Ala Asn Asp Tyr Thr Thr Glu Tyr Ser Ala 50 55 60 Ser Val Lys Gly Arg Phe Thr Val Ser Arg Asp Asp Ser Lys Ser Ser 65 70 75 80 Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Ile Tyr 85 90 95 Tyr Cys Ala Arg Asp Ala His Gly Lys Pro Phe Ala Tyr Trp Gly Gln 100 105 110 Gly Thr Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val 115 120 125 Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala 130 135 140 Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser 145 150 155 160 Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val 165 170 175 Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro 180 185 190 Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys 195 200 205 Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp 210 215 220 Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly 225 230 235 240 Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile 245 250 255 Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu 260 265 270 Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His 275 280 285 Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg 290 295 300 Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys 305 310 315 320 Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu 325 330 335 Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr 340 345 350 Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu 355 360 365 Ser Cys Ala Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp 370 375 380 Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val 385 390 395 400 Leu Asp Ser Asp Gly Ser Phe Phe Leu Val Ser Lys Leu Thr Val Asp 405 410 415 Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Leu His 420 425 430 Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro 435 440 445 Gly Lys Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly 450 455 460 Ser Gln Ser Val Leu Thr Gln Pro Pro Ser Ala Ser Gly Thr Pro Gly 465 470 475 480 Gln Arg Val Thr Ile Ser Cys Thr Gly Ser Ser Ser Asn Ile Gly Ser 485 490 495 Asn Asp Val Ser Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu 500 505 510 Leu Ile Tyr Ala Asn Val Asn Arg Pro Ser Gly Val Pro Asp Arg Phe 515 520 525 Ser Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Ser Gly Leu 530 535 540 Arg Ser Glu Asp Glu Ala Asp Tyr Tyr Cys Gly Thr Trp Ala Gly Ser 545 550 555 560 Leu Asn Ala Tyr Val Phe Gly Cys Gly Thr Lys Leu Thr Val Leu Gly 565 570 575 Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly 580 585 590 Gly Gly Ser Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln 595 600 605 Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe 610 615 620 Ser Ser Tyr Asp Met Ser Trp Val Arg Gln Ala Pro Gly Lys Cys Leu 625 630 635 640 Glu Trp Val Ser Ala Ile Ser Tyr Asp Asn Ala Asn Thr Tyr Tyr Ala 645 650 655 Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn 660 665 670 Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val 675 680 685 Tyr Tyr Cys Ala Lys Gly Val Leu Thr Thr Leu Met Asn Trp Phe Asp 690 695 700 Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 705 710 715 <210> 13 <211> 450 <212> PRT <213> artificial sequence <220> <223> hu11F11(ver.2)(M428L) <400> 13 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Thr Ser Gly Phe Thr Phe Ser Asp Phe 20 25 30 Tyr Met Glu Trp Val Arg Gln Pro Pro Gly Lys Arg Leu Glu Trp Ile 35 40 45 Ala Ala Ser Arg Asn Lys Ala Asn Asp Tyr Thr Thr Glu Tyr Ser Ala 50 55 60 Ser Val Lys Gly Arg Phe Thr Val Ser Arg Asp Asp Ser Lys Ser Ser 65 70 75 80 Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Ile Tyr 85 90 95 Tyr Cys Ala Arg Asp Ala His Gly Lys Pro Phe Ala Tyr Trp Gly Gln 100 105 110 Gly Thr Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val 115 120 125 Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala 130 135 140 Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser 145 150 155 160 Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val 165 170 175 Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro 180 185 190 Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys 195 200 205 Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp 210 215 220 Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly 225 230 235 240 Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile 245 250 255 Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu 260 265 270 Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His 275 280 285 Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg 290 295 300 Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys 305 310 315 320 Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu 325 330 335 Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr 340 345 350 Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu 355 360 365 Trp Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp 370 375 380 Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val 385 390 395 400 Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp 405 410 415 Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Leu His 420 425 430 Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro 435 440 445 Gly Lys 450 <210> 14 <211> 15 <212> PRT Artificial Sequence <220> (G4S)3 <400> 14 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 1 5 10 15 <210> 15 <211> 110 <212> PRT <213> Artificial Sequence <220> <223> F06(de2)(StoP) VL <400> 15 Gln Ser Val Leu Thr Gln Pro Pro Ser Ala Ser Gly Thr Pro Gly Gln 1 5 10 15 Arg Val Thr Ile Ser Cys Thr Gly Ser Ser Ser Asn Ile Gly Ser Asn 20 25 30 Asp Val Ser Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu Leu 35 40 45 Ile Tyr Ala Asn Val Asn Arg Pro Ser Gly Val Pro Asp Arg Phe Ser 50 55 60 Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Ser Gly Leu Arg 65 70 75 80 Ser Glu Asp Glu Ala Asp Tyr Tyr Cys Gly Thr Trp Ala Gly Ser Leu 85 90 95 Asn Ala Tyr Val Phe Gly Cys Gly Thr Lys Leu Thr Val Leu 100 105 110 <210> 16 <211> 20 <212> PRT <213> Artificial Sequence <220> <223> (G4S)4 <400> 16 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly 1 5 10 15 Gly Gly Gly Ser 20 <210> 17 <211> 121 <212> PRT <213> Artificial Sequence <220> <223> F06(de2)(StoP) VH <400> 17 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Asp Met Ser Trp Val Arg Gln Ala Pro Gly Lys Cys Leu Glu Trp Val 35 40 45 Ser Ala Ile Ser Tyr Asp Asn Ala Asn Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Gly Val Leu Thr Thr Leu Met Asn Trp Phe Asp Tyr Trp Gly 100 105 110 Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 18 <211> 115 <212> PRT <213> Artificial Sequence <220> <223> ch1E4 VH <400> 18 Gln Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Val Arg Pro Gly Thr 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Ala Phe Thr Asn Tyr 20 25 30 Leu Ile Glu Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Val Ile Asn Pro Gly Ser Gly Gly Thr Asn Tyr Asn Glu Lys Phe 50 55 60 Lys Gly Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Ser Thr Ala Tyr 65 70 75 80 Met Gln Leu Ser Ser Leu Thr Ser Asp Asp Ser Ala Val Tyr Phe Cys 85 90 95 Ala Ser Gly Asn Tyr Asp Thr Tyr Trp Gly Gln Gly Thr Leu Val Thr 100 105 110 Val Ser Ala 115 <210> 19 <211> 112 <212> PRT <213> Artificial sequence <220> <223> ch1E4 VL <400> 19 Asp Val Val Met Thr Gln Thr Pro Leu Ser Leu Pro Val Ser Leu Gly 1 5 10 15 Asp Gln Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Val His Ser 20 25 30 Asn Gly Asn Thr Tyr Leu His Trp Tyr Leu Gln Lys Pro Gly Gln Ser 35 40 45 Pro Lys Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro 50 55 60 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile 65 70 75 80 Ser Arg Val Glu Ala Glu Asp Leu Gly Val Tyr Phe Cys Ser Gln Ser 85 90 95 Thr His Val Pro Arg Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 100 105 110 <210> 20 <211> 2148 <212> DNA <213> Artificial sequence <220> <223> hu11F11 F06 Constant Unit Price Carrier 1 <400> 20 gaggtgcagc tcgtagagtc aggcggggga ctcgtccaac ccggtggttc tcttagattg 60 tcatgtgcca caagcggttt taccttttcc gatttttaca tggagtgggt tcgtcagcca 120 ccagggaagc gacttgagtg gatcgcagca tcccgtaata aggccaatga ctatactacc 180 gagtattccg cttcagtaaa gggcagattc accgtcagta gggatgacag caagagcagc 240 ctctatctcc agatgaatag tctgagggct gaagataccg ccatttacta ctgtgcacgg 300 gatgctcatg ggaagccctt cgcttactgg ggccaaggca ccactgtaac cgtatcctct 360 gcctccacca agggcccctc cgtgttcccc ctggccccct cctccaagtc cacctccggc 420 ggcaccgccg ccctgggctg cctggtgaag gactacttcc ccgagcccgt gaccgtgtcc 480 tggaactccg gcgccctgac ctccggcgtg cacaccttcc ccgccgtgct gcagtcctcc 540 ggcctgtact ccctgtcctc cgtcgtgacc gtgccctcct cctccctggg caccagacc 600 tacatctgca acgtgaacca caagccctcc aacaccaagg tggacaagaa ggtggagccc 660 aagtcctgcg aaagaccca cacctgccct ccctgccccg cccccgagct gctgggcggc 720 ccctccgtgt tcctgttccc tcctaagccc aaggacaccc tgatgatctc ccggaccccc 780 gaggtgactt gcgtggtggt ggacgtgtcc cacgaggacc ccgaggtgaa gttcaactgg 840 tacgtggacg gcgtggaggt gcacaacgcc aagaccaagc cccgggagga gcagtacaac 900 tccacctacc gggtggtgtc cgtgctgacc gtgctgcacc aggactggct gaacggcaag 960 gagtacaagt gcaaggtgtc caacaaggcc ctgcccgccc ccatcgagaa gaccatctcc 1020 aaggccaagg gccagccccg ggagccccag gtgtacaccc tgcccccctc ccgggatgaa 1080 cttactaaga accaggtctc tctgtcatgc gccgtcaaag gtttttatcc atccgacatt 1140 gccgtcgagt gggagtcaaa tggtcagcca gagaataact acaagacaac tccacccgtg 1200 ctcgactccg acggcagctt ctttttggtg tccaagttga ccgttgacaa gtctcgttgg 1260 caacagggta acgtattttc ctgtagtgtc cttcacgagg ccctgcataa ccactatact 1320 caaaaatctc tgtccctgag ccctggaaaa ggtggcggag gatctggcgg tggtggttct 1380 ggcggaggcg gatctcagtc tgttctgacc cagcctcctt ctgcttctgg cacccctggc 1440 cagagagtga ccatctcttg taccggctcc tcctccaaca tcggctccaa cgacgtgtcc 1500 tggtatcagc agctgcctgg cacagcccct aaactgctga tctacgccaa cgtaaaccgg 1560 ccttctggcg tgcctgacag attctccggc tccaagtctg gcacctctgc ctccctggct 1620 atctccggcc tgagatctga ggacgaggcc gattactact gcggcacctg ggccggatct 1680 ctgaacgcct atgtgttcgg ctgcggcaca aagctgacag tgttgggagg cggaggaagt 1740 ggtggcggcg gttcaggcgg cggtggcagc ggaggtggtg gatctgaagt tcagctgctg 1800 gaatctggcg gcggattggt tcagcctggt ggctctctga gactgtcttg tgccgcctct 1860 ggcttcacct tctccagcta cgatatgtcc tgggtccgac aggcccctgg caagtgtttg 1920 gagtgggtgt ccgccatctc ctacgacaac gccaacacct actacgccga ctccgtgaag 1980 ggccggttca ccatctctcg ggacaacagc aagaacaccc tgtacctgca gatgaactcc 2040 ctgagagccg aggacaccgc cgtgtactat tgtgctaagg gcgtgctgac caccctgatg 2100 aattggttcg attactgggg ccagggcacc ctggtcaccg tgtcatct 2148 <210> 21 <211> 1350 <212> DNA <213> Artificial sequence <220> <223> hu11F11 F06 monovalent constant vector 2 <400> 21 gaggtgcagc tcgtagagtc aggcggggga ctcgtccaac ccggtggttc tcttagattg 60 tcatgtgcca caagcggttt taccttttcc gatttttaca tggagtgggt tcgtcagcca 120 ccagggaagc gacttgagtg gatcgcagca tcccgtaata aggccaatga ctatactacc 180 gagtattccg cttcagtaaa gggcagattc accgtcagta gggatgacag caagagcagc 240 ctctatctcc agatgaatag tctgagggct gaagataccg ccatttacta ctgtgcacgg 300 gatgctcatg ggaagccctt cgcttactgg ggccaaggca ccactgtaac cgtatcctct 360 gcctccacca agggcccctc cgtgttcccc ctggccccct cctccaagtc cacctccggc 420 ggcaccgccg ccctgggctg cctggtgaag gactacttcc ccgagcccgt gaccgtgtcc 480 tggaactccg gcgccctgac ctccggcgtg cacaccttcc ccgccgtgct gcagtcctcc 540 ggcctgtact ccctgtcctc cgtcgtgacc gtgccctcct cctccctggg cacccagacc 600 tacatctgca acgtgaacca caagccctcc aacaccaagg tggacaagaa ggtggagccc 660 aagtcctgcg acaagaccca cacctgccct ccctgccccg cccccgagct gctgggcggc 720 ccctccgtgt tcctgttccc tcctaagccc aaggacaccc tgatgatctc ccggaccccc 780 gaggtgactt gcgtggtggt ggacgtgtcc cacgaggacc ccgaggtgaa gttcaactgg 840 tacgtggacg gcgtggaggt gcacaacgcc aagaccaagc cccgggagga gcagtacaac 900 tccacctacc gggtggtgtc cgtgctgacc gtgctgcacc aggactggct gaacggcaag 960 gagtacaagt gcaaggtgtc caacaaggcc ctgcccgccc ccatcgagaa gaccatctcc 1020 aaggccaagg gccagccccg ggagccccag gtgtacaccc tgcccccctc ccgtgatgaa 1080 ctcactaaga atcaagtcag tctctggtgt ttggtcaaag gcttctatcc atctgatatt 1140 gctgttgaat gggagagtaa cgggcaacct gaaaataatt acaagactac acctcccgtg 1200 cttgattccg acggttcctt ctttctgtat tccaagctga cagttgataa gagtcgctgg 1260 cagcagggga acgtatttag ttgttctgta cttcacgaag ctctccataa ccattacact 1320 caaaagtccc tgtccctgtc ccccggcaag 1350 <210> 22 <211> 660 <212> DNA <213> Artificial sequence <220> <223> hu11F11 F06 monovalent variable vector <400> 22 gatatcgtga tgacccagtc tccttcctct ctggctgtgt ccctgggcga gcgcgtcacc 60 atgtcctgca agtcctctca gtccctgctg tactcctcca accagaagaa ctacctggcc 120 tggtatcagc agaagcccgg ccagtctcca aagctgctga tctactgggc ctccaccaga 180 gaatctggcg tgccagatag attcaccggc tctggctctg gcaccgactt caccctgaca 240 atctcttccg tgaaggccga ggacgtggcc gtgtactact gccagcagta ctacagctac 300 ccctggacct ttggcggagg caccaagctg gaaatcaagc ggaccgtggc cgctccctcc 360 gtgttcatct tcccaccctc cgacgagcag ctgaagtccg gcaccgcttc cgtcgtgtgc 420 ctgctgaaca acttctaccc ccgcgaggcc aaggtgcagt ggaaggtgga caacgccctg 480 cagtccggca actcccagga atccgtcacc gagcaggact ccaaggacag cacctacagc 540 ctgtcctcca ccctgaccct gtccaaggcc gactacgaga agcacaaggt gtacgcctgc 600 gaagtgaccc accagggcct gtccagcccc gtgaccaagt ccttcaaccg gggcgagtgc 660 660 <210> twenty three <211> 8 <212> PRT <213> Artificial sequence <220> <223> Human VH3 domain sequence <400> twenty three Leu Tyr Leu Gln Met Asn Ser Leu 1 5 <210> twenty four <211> 8 <212> PRT <213> Artificial sequence <220> <223> Human VH3 domain sequence <400> twenty four Asn Tyr Leu Gln Met Asn Ser Leu 1 5 <210> 25 <211> 8 <212> PRT <213> Artificial sequence <220> <223> Human VH3 domain sequence <400> 25 Leu His Leu Gln Met Asn Ser Leu 1 5 <210> 26 <211> 8 <212> PRT <213> Artificial sequence <220> <223> Human VH3 domain sequence <400> 26 Leu Tyr Leu Gln Met Asp Ser Leu 1 5 <210> 27 <211> 8 <212> PRT <213> Artificial sequence <220> <223> Human VH3 domain sequence <400> 27 Ala Tyr Leu Gln Met Asn Ser Leu 1 5 <210> 28 <211> 8 <212> PRT <213> Artificial sequence <220> <223> Human VH3 domain sequence <400> 28 Thr Tyr Leu Gln Met Asn Ser Leu 1 5 <210> 29 <211> 8 <212> PRT <213> Artificial sequence <220> <223> Human VH3 domain sequence <400> 29 Met Tyr Leu Gln Met Ser Asn Leu 1 5 <210> 30 <211> 8 <212> PRT <213> Artificial sequence <220> <223> Human VH3 domain sequence <400> 30 Leu Tyr Leu Gln Met Ser Ser Leu 1 5 <210> 31 <211> 8 <212> PRT <213> Artificial sequence <220> <223> Human VH3 domain sequence <400> 31 Met Tyr Leu Gln Met Asn Asn Leu 1 5 <210> 32 <211> 8 <212> PRT <213> Artificial sequence <220> <223> Human VH3 domain sequence <400> 32 Leu Tyr Leu Gln Val Asn Ser Leu 1 5 <210> 33 <211> 8 <212> PRT <213> Artificial sequence <220> <223> Human VH3 domain sequence <400> 33 Leu Tyr Leu Gln Thr Asn Ser Leu 1 5 <210> 34 <211> 8 <212> PRT <213> Artificial sequence <220> <223> Human VH3 domain sequence <400> 34 Leu Phe Leu Gln Met Asn Ser Leu 1 5 <210> 35 <211> 8 <212> PRT <213> Artificial sequence <220> <223> Human VH3 domain sequence <400> 35 Leu Tyr Val Gln Met Ser Ser Leu 1 5 <210> 36 <211> 8 <212> PRT <213> Artificial sequence <220> <223> Human VH3 domain sequence <400> 36 Leu Tyr Leu Gln Met Gly Ser Leu 1 5 <210> 37 <211> 8 <212> PRT <213> Artificial sequence <220> <223> Human VH3 domain sequence <400> 37 Met Tyr Leu Gln Met Asn Ser Leu 1 5

Claims

1. A method for purifying Fc-containing bioactive peptides, comprising the following steps: (a) Loading a mixture of Fc-containing bioactive peptides onto a chromatographic column containing an affinity matrix containing a global protein A ligand, the mixture of bioactive peptides comprising a first Fc-containing bioactive peptide and a second Fc-containing bioactive peptide. The affinity matrix comprises a support for supporting the global protein A ligand, and the average particle size of the support is less than 90 µm. The number of human VH3 domains in the second Fc-containing bioactive peptide differs from the number of human VH3 domains in the first Fc-containing bioactive peptide. Furthermore, each of the human VH3 domains contained in the Fc-containing bioactive peptide binds to the protein A ligand; and (b) The eluent is loaded onto the chromatographic column, and the Fc-containing bioactive peptides are separated and eluted at different pH values ​​depending on the number of human VH3 domains contained in each of the Fc-containing bioactive peptides in the mixture.

2. The purification method according to claim 1, wherein the bioactive peptide mixture further comprises a third Fc-containing bioactive peptide, wherein the number of human VH3 domains in the third Fc-containing bioactive peptide is different from the number of human VH3 domains in the second Fc-containing bioactive peptide and different from the number of human VH3 domains in the first Fc-containing bioactive peptide, and wherein each of the human VH3 domains contained in the Fc-containing bioactive peptide binds to the protein A ligand.

3. The purification method according to claim 1, wherein the bioactive peptide mixture comprises: a first Fc-containing bioactive peptide containing n individual VH3 domains; and a second Fc-containing bioactive peptide containing n+1 individual VH3 domains, wherein n is an integer greater than or equal to 0.

4. The purification method according to claim 3, wherein step (b) comprises the following steps: (b1) Loading an eluent having a first pH range onto the chromatographic column to elute the first Fc-containing bioactive peptide; and (b2) Load the eluent having a second pH range lower than the first pH range onto the chromatographic column to elute the second Fc-containing bioactive peptide.

5. The purification method according to claim 2, wherein the bioactive peptide mixture comprises: a first Fc-containing bioactive peptide containing n individual VH3 domains; a second Fc-containing bioactive peptide containing n+1 individual VH3 domains; and a third Fc-containing bioactive peptide containing n+2 individual VH3 domains, wherein n is an integer greater than or equal to 0.

6. The purification method according to claim 5, wherein step (b) further comprises the following steps: (b1) Load the eluent having a first pH range onto the chromatographic column to elute the first Fc-containing bioactive peptide; (b2) Loading an eluent having a second pH range lower than the first pH range onto the chromatographic column to elute the second Fc-containing bioactive peptide; as well as (b3) Elute the third Fc-containing bioactive peptide using an elution buffer having a third pH range that is lower than the second pH range.

7. The purification method according to claim 1, wherein the Fc is a mutation that has not been introduced that affects the binding affinity with wild-type protein A.

8. The purification method according to claim 1, In the case where the first or second Fc-containing bioactive peptide includes a VH3 domain, the first or second Fc-containing bioactive peptide includes a variable region sequence containing a VH domain selected from the following sequences: The heavy chain variable sequence of SEQ ID No. 1 and the light chain variable sequence of SEQ ID No. 2; The heavy chain variable sequence of SEQ ID No. 3 and the light chain variable sequence of SEQ ID No. 4; The heavy chain variable sequence of SEQ ID No. 5 and the light chain variable sequence of SEQ ID No. 6; The heavy chain variable sequence of SEQ ID No. 7 and the light chain variable sequence of SEQ ID No. 8; or The heavy chain variable sequence of SEQ ID No. 9 and the light chain variable sequence of SEQ ID No.

10.

9. The purification method according to claim 2, The third Fc-containing bioactive peptide comprises a variable region sequence containing a VH domain selected from the following sequences: The heavy chain variable sequence of SEQ ID No. 1 and the light chain variable sequence of SEQ ID No. 2; The heavy chain variable sequence of SEQ ID No. 3 and the light chain variable sequence of SEQ ID No. 4; The heavy chain variable sequence of SEQ ID No. 5 and the light chain variable sequence of SEQ ID No. 6; The heavy chain variable sequence of SEQ ID No. 7 and the light chain variable sequence of SEQ ID No. 8; or The heavy chain variable sequence of SEQ ID No. 9 and the light chain variable sequence of SEQ ID No.

10.

10. The purification method according to claim 1, wherein the Fc-containing bioactive peptide is an antibody containing IgG.

11. The purification method according to claim 1, wherein the Fc-containing bioactive peptide is an IgG-scFv double antibody.

12. The purification method according to claim 1, wherein the bioactive peptide containing Fc comprises a peptide drug bound to Fc.

13. The purification method according to claim 12, wherein the peptide drug is selected from the group consisting of hormones, cytokines, enzymes, antibodies, growth factors, transcriptional regulatory factors, blood factors, vaccines, structural proteins, ligand proteins, and receptors.

14. The purification method according to claim 2, The first bioactive peptide containing Fc does not contain scFv; The second Fc-containing bioactive peptide comprises an scFv consisting of SEQ ID No. 11, which is attached to the C-terminus of either of the two heavy chains of Fc; and The third Fc-containing bioactive peptide comprises two scFvs consisting of SEQ ID No. 11, each attached to the C-terminus of the two heavy chains of Fc.

15. The purification method according to claim 1, The bioactive peptide containing Fc is IgG. The variable region of the IgG antibody includes a human VH3 domain.

16. A purification method comprising the following steps: (a-1) An antibody mixture containing Fc is loaded onto a chromatographic column containing an affinity matrix containing a global protein A ligand, wherein, The Fc-containing antibody mixture comprises: a monospecific antibody; a monovalent bispecific antibody comprising an antigen-binding fragment containing a human VH3 domain bound to the C-terminus of either of the two heavy chain constant regions of the monospecific antibody, wherein the human VH3 domain is bound to the protein A ligand; and a bivalent bispecific antibody comprising the antigen-binding fragment containing the human VH3 domain bound to the C-terminus of each of the two heavy chain constant regions of the monospecific antibody, wherein the human VH3 domain is bound to the protein A ligand. The affinity matrix contains a support for supporting the global protein A ligand, and the average particle size of the support is less than 90 µm; (b-1) Load the eluent having a first pH range onto the chromatographic column to elute the monospecific antibody; (c-1) Loading an eluent having a second pH range lower than the first pH range onto the chromatographic column to elute the monovalent bispecific antibody; and (d-1) An eluent having a third pH range lower than the second pH range is loaded onto the chromatographic column to elute the bivalent bispecific antibody.

17. The purification method according to claim 16, wherein the variable region of the monospecific antibody comprises a human VH3 domain.

18. The purification method according to claim 16, wherein the variable region of the monospecific antibody does not contain a human VH3 domain.

19. The purification method according to claim 16, wherein the antigen-binding fragment is an scFv containing the amino acid sequence of SEQ ID No.

11.

20. The purification method according to claim 16, The first pH range is greater than or equal to 3.4 and less than or equal to 5.

0. The second pH range is greater than or equal to 3.3 and less than or equal to 4.

1. The third pH range is greater than or equal to 3.0 and less than or equal to 4.0.

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