Method and device for purifying polypeptide

Abstract

Provided is a method for purifying a polypeptide, the method including: a membrane separation step for separating a liquid containing complexes of a polypeptide and a ligand into a concentrate containing the complexes and a permeate using a separation membrane installed in a membrane module; and, after the membrane separation step, a dissociation step for supplying to the membrane module a dissociation solution for dissociating the complexes, and dissociating the complexes into the polypeptide and the ligand.

Description

Polypeptide purification method and polypeptide purification device

[0001] The present invention relates to a method for purifying a polypeptide and an apparatus for purifying a polypeptide. This application claims priority to Japanese Patent Application No. 2024-049627, filed on March 26, 2024, the contents of which are incorporated herein by reference.

[0002] In recent years, with the development of biopharmaceuticals, numerous antibody drugs have begun to be marketed. Typically, the active ingredient of an antibody drug is produced by culturing cells to produce antibodies, and then purifying the antibodies from the resulting culture medium using affinity chromatography. Although affinity chromatography has excellent separation performance, the affinity resin used in the separation process is expensive, necessitating repeated use of the affinity resin. However, repeated use of the affinity resin poses problems such as the cost and time required for validation management and storage and management of the affinity resin. As a method for solving the above problems, Patent Document 1 discloses a method in which a complex is formed between a polypeptide (i.e., an antibody) and a specific ligand, and a liquid containing the resulting complex is filtered through a separation membrane.

[0003] International Publication No. 2020 / 004583

[0004] However, in the case of the method using a separation membrane as described in Patent Document 1, there is room for improvement in the recovery rate of the polypeptide. An object of the present invention is to provide a method for purifying a polypeptide and an apparatus for purifying a polypeptide, which are capable of recovering a polypeptide with a high recovery rate.

[0005] As a result of intensive research to solve the above-mentioned problems, the present inventors discovered that, when purifying a polypeptide, after membrane separation of a liquid containing a complex of a polypeptide and a ligand, supplying a dissociation liquid to the membrane module used for membrane separation dissociates the complex into the polypeptide and the ligand, turning them into a solution, thereby improving the recovery rate of the polypeptide, and thus completed the present invention.

[0006] That is, the present invention has the following aspects. [1] A method for purifying a polypeptide, comprising: a membrane separation step of separating a liquid containing a polypeptide-ligand complex into a concentrate containing the complex and a permeate using a separation membrane provided in a membrane module; and a dissociation step of supplying a dissociation liquid that dissociates the complex to the membrane module after the membrane separation step, thereby dissociating the complex into the polypeptide and the ligand. [2] The method for purifying a polypeptide according to [1], wherein the polypeptide is one or more selected from an antibody, an antibody fragment, an antibody derivative, and an antibody fragment derivative. [3] The method for purifying a polypeptide according to [1] or [2], further comprising, before the membrane separation step, a complex formation step of mixing the polypeptide and the ligand in a liquid to form a complex between the polypeptide and the ligand, thereby obtaining a liquid containing the complex. [4] The method for purifying a polypeptide according to any one of [1] to [3], wherein the polypeptide and the ligand dissociated in the dissociation step are recovered from the secondary side of the membrane module. [5] The method for purifying a polypeptide according to any one of [1] to [4], wherein, in the dissociation step, the dissociation solution is supplied to a concentrate containing the complex to adjust the pH of the concentrate containing the complex. [6] The method for purifying a polypeptide according to [5], wherein, in the dissociation step, the pH of the concentrate containing the complex is adjusted to less than 7. [7] The method for purifying a polypeptide according to any one of [1] to [6], wherein the pore size of the micropores formed in the separation membrane is 0.05 to 3 μm. [8] The method for purifying a polypeptide according to any one of [1] to [7], wherein the membrane separation step is performed by cross-flow filtration or dead-end filtration. [9] The method for purifying a polypeptide according to any one of [1] to [8], wherein the separation membrane comprises a polyolefin resin.

[10] The method for purifying a polypeptide according to any one of [1] to [8], wherein the separation membrane comprises polyethylene.

[11] The method for purifying a polypeptide according to any one of [1] to [8], wherein the separation membrane comprises high-density polyethylene.

[12] The method for purifying the polypeptide according to any one of [1] to

[11] , further comprising a buffer supplying step of supplying a buffer to a concentrated solution containing the complex.

[13] A polypeptide purification apparatus comprising: a membrane separation device including a membrane module equipped with a separation membrane, which separates a liquid containing a polypeptide-ligand complex into a concentrate containing the complex and a permeate using the separation membrane; and a dissociation liquid supply device that supplies a dissociation liquid to the membrane module to dissociate the complex.

[14] The polypeptide purification apparatus according to

[13] , wherein the polypeptide is one or more selected from an antibody, an antibody fragment, an antibody derivative, and an antibody fragment derivative.

[15] The polypeptide purification apparatus according to

[13] or

[14] , further comprising, upstream of the membrane separation device, a complex formation device that mixes the polypeptide and the ligand in a liquid to form the complex of the polypeptide and the ligand and obtain a liquid containing the complex.

[16] The polypeptide purification apparatus according to any one of

[13] to

[15] , further comprising a recovery device that recovers the polypeptide and the ligand dissociated by the dissociation liquid supply device from the secondary side of the membrane module.

[17] The polypeptide purification device according to any one of

[13] to

[16] , wherein the dissociation liquid supplying device is provided with a dissociation liquid supply pipe for supplying the dissociation liquid to a concentrated solution containing the complex.

[18] The polypeptide purification device according to any one of

[13] to

[17] , wherein the dissociation liquid supplying device adjusts the pH of the concentrated solution containing the complex to less than 7.

[19] The polypeptide purification device according to any one of

[13] to

[18] , wherein the pore size of the micropores formed in the separation membrane is 0.05 to 3 μm.

[20] The polypeptide purification device according to any one of

[13] to

[19] , wherein the membrane separation in the membrane separation device is a cross-flow filtration method or a dead-end filtration method.

[21] The polypeptide purification device according to any one of

[13] to

[20] , wherein the separation membrane comprises a polyolefin resin.

[22] The polypeptide purification device according to any one of

[13] to

[20] , wherein the separation membrane comprises polyethylene.

[23] The polypeptide purification device according to any one of

[13] to

[20] , further comprising a buffer supplying device for supplying a buffer to the concentrated solution containing the complex.

[0007] According to the present invention, a method for purifying a polypeptide and an apparatus for purifying a polypeptide, which are capable of recovering a polypeptide at a high recovery rate, can be provided.

[0008] 1 is a table comparing the amino acid sequences of the E, D, A, B, C, and Z domains of Staphylococcus protein A. A "- (hyphen)" indicates that the amino acid residue is the same as that of the C domain, and a " / (diagonal line)" indicates an amino acid deletion. FIG. 2 is a schematic diagram showing an example of an apparatus for purifying a polypeptide according to a first aspect of the present invention. FIG. 3 is a schematic diagram showing an example of an apparatus for purifying a polypeptide according to a second aspect of the present invention.

[0009] The present invention will be described in more detail below with reference to preferred embodiments of the invention. The following description is an example of an embodiment of the present invention, and the present invention is not limited to the following description and various modifications or changes are possible within the scope of the gist of the claims. In this specification, the term "to" indicating a range of numerical values ​​means that the numerical values ​​before and after it are included as the lower and upper limits.

[0010] [Polypeptide Purification Method] One embodiment of the polypeptide purification method of the present invention is described below. The polypeptide purification method of this embodiment includes the membrane separation step and dissociation step described below. The polypeptide purification method of this embodiment may further include a complex formation step, a buffer supply step, and a recovery step.

[0011] <Complex Formation Step> The complex formation step is a step of mixing a polypeptide and a ligand in a liquid to form a polypeptide-ligand complex and obtain a liquid containing the complex. Here, the "liquid containing the complex" means one or more liquids selected from a solution in which the complex is dissolved, a liquid in which the complex is dispersed, and a liquid in which the complex is precipitated.

[0012] (Polypeptide) The polypeptide to be purified in this embodiment is one or more selected from antibodies, antibody fragments, antibody derivatives, and antibody fragment derivatives. Antibodies have the function of specifically binding to antigens and the function of detoxifying or removing antigenic factors in cooperation with other biological molecules or cells. The term "antibody" emphasizes the antigen-binding function, and the substance is a protein called "immunoglobulin (IG)." Human antibodies are preferred. The light chains constituting human antibodies may be either λ chains or κ chains. Furthermore, the heavy chains constituting human antibodies include γ chains, μ chains, α chains, σ chains, and ε chains, which correspond to IgG, IgM, IgA, IgD, and IgE, respectively. Specifically, antibodies include IgG, IgA, IgM, IgD, and IgE. The heavy chains constituting antibodies may be either γ chains, μ chains, α chains, σ chains, or ε chains. Furthermore, the γ-chain of an antibody heavy chain includes γ1 chain, γ2 chain, γ3 chain, and γ4 chain, which correspond to IgG1, IgG2, IgG3, and IgG4, respectively.

[0013] An antibody fragment is an antibody fragment obtained by fragmenting the antibody using enzymes, genetic engineering, or the like. An example of an antibody fragment is a protein consisting only of the Fab region of human IgG. An antibody derivative is an antibody modified using enzymes, genetic engineering, chemical treatment, or the like. Examples of antibody derivatives include chimeric antibodies in which a partial domain of human IgG is replaced with a domain of an IgG antibody from a species other than human and fused, and humanized antibodies in which the CDR portions of human IgG are replaced with the CDR portions of an antibody from a species other than human and fused. An example of an antibody fragment derivative is an artificial antibody in which the Fv and Fc regions of human IgG are fused. Here, a "domain" is a unit of a protein's higher-order structure, consisting of a sequence of several tens to several hundreds of amino acid residues, and refers to a partial region of a protein sufficient to express some physicochemical or biochemical function.

[0014] Among polypeptides, antibodies and antibody derivatives are preferred, more preferably antibodies, and even more preferably IgG, because of their excellent ligand binding and precipitability. These polypeptides may be used alone or in combination of two or more.

[0015] (Ligand) A ligand is a substance that has an adsorptive interaction with a polypeptide. Here, having an adsorptive interaction refers to a state in which there is an attractive force between molecules. The ligand is preferably a protein that specifically binds to the polypeptide to be purified and forms a complex, and examples of such a ligand include one or more selected from protein A, protein G, protein L, and variants thereof. Among these ligands, one or more selected from variants of protein A, protein G, and protein L are more preferred, and variants of protein A are even more preferred, because of their excellent molecular recognition selectivity and binding strength. These ligands may be used alone or in combination of two or more.

[0016] Protein A is a known protein A present in the cell wall of Staphylococcus aureus and is a protein composed of five linked immunoglobulin-binding domains. Protein A specifically binds to the Fc region of immunoglobulins (especially human antibodies of IgG1, IgG2, and IgG4 types). Several microorganisms express Protein A, including Staphylococcus. The E domain (SEQ ID NO: 1), D domain (SEQ ID NO: 2), A domain (SEQ ID NO: 3), B domain (SEQ ID NO: 4), C domain (SEQ ID NO: 5), and Z domain (SEQ ID NO: 6) of Protein A are immunoglobulin-binding proteins capable of binding to regions other than the complementarity-determining regions (CDRs) of immunoglobulins. Each domain binds to the Fc region, Fab region, and, particularly, the Fv region of the Fab region of immunoglobulins, respectively.

[0017] As shown in the sequence comparison table in Figure 1, the E, D, A, B, C, and Z domains derived from Protein A have highly homologous amino acid sequences, with an amino acid sequence identity of 60% or more. A hyphen (-) indicates that the amino acid residue is the same as that of the C domain. A diagonal line ( / ) indicates an amino acid deletion.

[0018] The "domain derived from any of domains E, D, A, B, C, and Z of Protein A" described below refers to a domain having an amino acid sequence derived from the amino acid sequence of each wild-type domain, and the amino acid sequence of each wild-type domain may contain a mutation other than the mutated linkage site sequence (also referred to as the mutated first linkage element) described below, as long as it encodes a protein that has the ability to bind to an Fc region.

[0019] A modified form of Protein A is obtained by modifying Protein A through partial amino acid substitution (e.g., substitution of 1 to 10 amino acids in the amino acid sequence), insertion, deletion, chemical modification, etc. Examples of modified forms of Protein A include modified forms of Protein A according to the first and second embodiments shown below.

[0020] A first embodiment of the variant of Protein A is a multidomain protein (also referred to as a multi-domain protein) having two or more single domains derived from any of the E, D, A, B, C, and Z domains of Protein A. The number of domains is preferably three or more, more preferably four or more. The number of domains is preferably 10 or less, more preferably eight or less, and even more preferably six or less.

[0021] These multidomain proteins may be homopolymers such as homodimers and homotrimers, which are linkages of single immunoglobulin-binding domains, or heteropolymers such as heterodimers and heterotrimers, which are linkages of different types of immunoglobulin-binding domains. Preferably, all of the domains contained in the protein are homopolymers derived from any one of the E, D, A, B, C, and Z domains of Protein A set forth in SEQ ID NOs: 1 to 6.

[0022] A second embodiment of the modified protein A is a multidomain protein (also called a multi-domain protein) having two or more single domains derived from any of the A, B, C, and Z domains of the above-mentioned protein A. The number of domains is preferably three or more, more preferably four or more. The number is also preferably ten or less, more preferably eight or less, and even more preferably six or less.

[0023] A specific aspect of the second embodiment is a protein having two or more domains derived from any of the A, B, C, and Z domains of Protein A set forth in SEQ ID NOs: 3 to 6, and at least one of the linking elements (also referred to as second linking elements) linking the domains is a mutated linking element (also referred to as mutated second linking element) consisting of one or more amino acids and composed of amino acids other than hydrophobic amino acids.

[0024] Protein G is a known protein G, and is a protein present in the cell wall of group G hemolytic streptococcus. Protein G specifically binds to the Fc region of immunoglobulins (especially human antibodies of IgG1, IgG2, IgG3, and IgG4 types), and also weakly binds to Fab fragments. Modified forms of Protein G are modified by partial amino acid substitution (e.g., substitution of 1 to 10 amino acids in the amino acid sequence), insertion, deletion, chemical modification, etc.

[0025] Protein L is a known protein L, and is a protein derived from Peptostreptococcus magnus. Protein L has the property of specifically binding to the κ light chain of immunoglobulin (particularly IgG). Modified forms of Protein L are modified versions of Protein L by partial amino acid substitution (e.g., substitution of 1 to 10 amino acids in the amino acid sequence), insertion, deletion, chemical modification, or the like.

[0026] The weight-average molecular weight of the ligand is preferably 10,000 or more, more preferably 20,000 or more. The weight-average molecular weight of the ligand is preferably 100,000 or less, more preferably 80,000 or less. When the weight-average molecular weight of the ligand is equal to or greater than the lower limit, the formation of a complex between the polypeptide and the ligand is favorable. When the weight-average molecular weight of the ligand is equal to or less than the upper limit, the polypeptide and the ligand are easily separated.

[0027] In this specification, the weight-average molecular weight of a ligand is a value measured by size exclusion chromatography. However, when it is difficult to measure the weight-average molecular weight by size exclusion chromatography due to the type of ligand or the like, a value measured by fractionation gel electrophoresis or a method combining high performance liquid chromatography and mass spectrometry may also be used.

[0028] (Complex) The complex is a polypeptide-ligand complex (hereinafter also referred to as "complex (C)") formed by binding between a polypeptide and a ligand. Since the larger the particle size of the complex (C), the easier the membrane separation operation in the membrane separation step becomes, it is preferable that 1 to 6 polypeptide molecules are bound to 1 ligand molecule, more preferably 3 to 6 polypeptide molecules are bound, and even more preferably 3 to 4 polypeptide molecules are bound. In other words, it is preferable that the complex (C) is one in which 1 to 6 polypeptide molecules are bound to 1 ligand molecule.

[0029] (Method for forming a complex) The complex (C) can be obtained by mixing the polypeptide and the ligand in a liquid. Examples of mixing methods include a method of adding the ligand to a solution containing the polypeptide and mixing them, a method of adding the polypeptide to a solution containing the ligand and mixing them, a method of mixing a solution containing the polypeptide with a solution containing the ligand, and a method of adding the polypeptide and the ligand to a solvent and mixing them. Among these, the method of adding the ligand to a solution containing the polypeptide and mixing them, and the method of mixing a solution containing the polypeptide with a solution containing the ligand are preferred. Examples of solvents contained in the solution include water, phosphate buffer, acetate buffer, and citrate buffer.

[0030] Here, a "solution containing a polypeptide" refers to a solution in which the polypeptide is dissolved and / or a liquid in which the polypeptide is dispersed. Similarly, a "solution containing a ligand" refers to a solution in which the ligand is dissolved and / or a liquid in which the ligand is dispersed.

[0031] The solution containing the polypeptide is not particularly limited as long as it contains the polypeptide, but a solution containing the polypeptide and impurities is preferred, as the effects of the present invention are significantly superior. The impurities have a molecular size smaller than that of the complex (C) and smaller than the pore size of the micropores formed in the separation membrane used in the membrane separation step described below. Examples of impurities include host cell-derived proteins (HCPs) and host cell-derived DNA.

[0032] The mixing ratio of polypeptide to ligand is preferably 0.3 mol or more of polypeptide per 1 mol of ligand, more preferably 0.6 mol or more, and even more preferably 1.2 mol or more. Furthermore, per 1 mol of ligand, the ratio of polypeptide is preferably 3.0 mol or less, more preferably 2.7 mol or less, and even more preferably 2.4 mol or less. If the ratio of polypeptide is above the lower limit, it becomes easier to obtain a complex (C) in which one or more polypeptide molecules are bound to one molecule of ligand. Therefore, the particle size of complex (C) becomes larger, making the membrane separation operation in the membrane separation step easier. If the ratio of polypeptide is below the upper limit, the ratio of polypeptides that do not bind to the ligand can be reduced.

[0033] <Membrane Separation Step> The membrane separation step is a step in which a liquid containing a complex (C) of a polypeptide and a ligand is supplied to a separation membrane provided in a membrane module, and the liquid is separated into a concentrate containing the complex (C) and a permeate. The membrane separation step allows the complex (C) to be separated from the liquid containing the complex (C). The complex (C) may be the complex obtained in the previous complex formation step. The complex (C) thus obtained has the above-mentioned impurities and the like sufficiently removed.

[0034] Here, membrane separation is a method of separating a liquid containing the complex (C) and impurities into a concentrated liquid in which the complex (C) is concentrated and does not permeate the separation membrane, and a permeated liquid that permeates the separation membrane. Impurities in the liquid containing the complex (C) permeate the separation membrane and are discharged as the permeated liquid.

[0035] The membrane module may be a type in which a separation membrane is housed in a casing (container) such as a cylindrical one, i.e., a casing-type membrane module, or a type in which a separation membrane is not housed in a casing, i.e., a non-casing-type membrane module.

[0036] Examples of materials for the separation membrane include polystyrene resin, polyolefin resin, and polyvinylidene fluoride resin. Among these, polyolefin resin and polyvinylidene fluoride resin are preferred, and polyolefin resin is more preferred, from the viewpoint of improving the mechanical strength of the membrane while ensuring sufficient liquid permeability. That is, it is preferable that the separation membrane contains a polyolefin resin, in other words, the separation membrane is formed from a material containing a polyolefin resin. Examples of polyolefin resins include olefin homopolymers or copolymers, and copolymers of olefins and other monomers copolymerizable with olefins. Among these, polyethylene is preferred from the viewpoints of easy availability and ease of forming a separation membrane at low cost. Among these, high-density polyethylene (HDPE) is more preferred from the viewpoint of ease of film formation. The density of high-density polyethylene is usually 0.910 g / cm ３ 0.965g / cm or more ３ is defined as less than 0.942 g / cm ３ More preferably, 0.960 g / cm ３ The above is more preferable. From the viewpoint of easy permeation of impurities, the separation membrane preferably has a structure in which micropores communicate with each other in the thickness direction of the membrane.

[0037] The separation membrane is preferably a porous membrane. Examples of porous membranes include microfiltration membranes (MF membranes) and ultrafiltration membranes (UF membranes). Among these, microfiltration membranes are preferred because they can separate liquids containing the complex (C) with low energy. The pore size of the micropores formed in the separation membrane is preferably 0.05 to 3 μm, more preferably 0.08 to 2 μm, and even more preferably 0.1 to 0.5 μm. Membranes with a pore size of 0.05 μm or more are classified as microfiltration membranes and can separate the complex (C) with low energy. A pore size of 3 μm or less can sufficiently capture the complex (C). In the case of a microfiltration membrane, the pore size refers to the particle size at which the rejection rate of latex particles of each particle size is measured and calculated from the approximation curve of particle size and rejection rate, resulting in a rejection rate of 90%. When a commercially available separation membrane is used, the catalog value may be used as the pore size.

[0038] Examples of the form of the separation membrane include hollow fiber membranes, tubular membranes (tubular membranes, single-bore type, multi-bore type, etc.), spiral membranes, flat membranes, monolithic membranes, etc. Among these, hollow fiber membranes and tubular membranes are preferred, and hollow fiber membranes are more preferred, since they can ensure a sufficient membrane area.

[0039] The outer diameter of the hollow fiber membrane is preferably 0.01 to 3 mm, more preferably 0.05 to 1 mm. The inner diameter of the hollow fiber membrane is preferably 0.005 mm or more and less than 1 mm, more preferably 0.03 to 0.8 mm. The outer diameter of the hollow fiber membrane refers to the diameter of the smallest circle inscribed on the outer edge of the cut surface when the hollow fiber membrane is cut along any plane perpendicular to the longitudinal direction, and is calculated as the average value measured at any two or more and ten or less locations. The inner diameter of the hollow fiber membrane refers to the diameter of the smallest circle inscribed on the inner edge of the cut surface when the hollow fiber membrane is cut along any plane perpendicular to the longitudinal direction, and is calculated as the average value measured at any two or more and ten or less locations.

[0040] The membrane separation method (also referred to as a filtration method) using a membrane module may be a cross-flow filtration method or a dead-end filtration method (also referred to as a total-amount filtration method). From the viewpoint of being able to suppress clogging, the cross-flow filtration method is preferable. Here, the cross-flow filtration method is a method in which filtration is performed by flowing a supply liquid parallel to the filtration surface, thereby suppressing the deposition of suspended matter, colloids, etc. in the supply liquid (in this embodiment, the composite (C) which is the filtration residue) on the separation membrane. On the other hand, the dead-end filtration method is a method in which filtration is performed by flowing the entire amount of the supply liquid through the separation membrane, and filtration is performed while cake, etc. (in this embodiment, the composite (C) which is the filtration residue) adheres to the separation membrane.

[0041] The membrane separation step may be performed two or more times. When the membrane separation step is performed two or more times, for example, the concentrated liquid obtained by membrane separation may be subjected to the membrane separation step again. In the membrane separation step, the liquid containing the composite (C) is supplied to the separation membrane while the separation membrane is in contact with the liquid containing the composite (C). For example, the separation membrane may be immersed in the liquid containing the composite (C) (dead-end filtration method), or the liquid containing the composite (C) may be directly supplied to the membrane module (cross-flow filtration method).

[0042] <Buffer Solution Supplying Step> The buffer solution supplying step is a step of supplying a buffer solution to a concentrate containing the complex (C). When membrane separation is performed in the membrane separation step, a concentrate in which the complex (C) is concentrated without permeating the separation membrane, i.e., a liquid containing the complex (C) after membrane separation, is obtained. The volume of the concentrate is reduced by the amount of permeate that permeates through the separation membrane compared to the liquid containing the complex (C) before membrane separation. In particular, as the number of membrane separation steps increases, the complex (C) is further concentrated accordingly, and the volume of the concentrate decreases. Therefore, it is preferable to supply a buffer solution in an amount approximately equal to the permeate to the concentrate, i.e., the liquid containing the complex (C) after membrane separation, to maintain the volume of the concentrate constant.

[0043] When membrane separation is performed by cross-flow filtration, it is preferable to supply a buffer solution to the concentrated liquid (liquid containing the composite (C) after membrane separation) discharged from the membrane module. When membrane separation is performed by dead-end filtration, it is preferable to supply a buffer solution to the concentrated liquid (liquid containing the composite (C) after membrane separation) in the membrane module. Examples of buffer solutions include phosphate buffer solutions, acetate buffer solutions, and citrate buffer solutions. Among these, phosphate buffer solutions are preferred.

[0044] <Dissociation Step> The dissociation step is a step in which, after the membrane separation step, a dissociation liquid that dissociates complex (C) is supplied to the membrane module to dissociate complex (C) into a polypeptide and a ligand. The pH of the concentrate containing complex (C) is adjusted by supplying the dissociation liquid to the concentrate containing complex (C). The "concentrate containing complex (C)" in the dissociation step refers to the liquid containing complex (C) after membrane separation from which impurities have been removed.

[0045] The pH of the concentrate containing complex (C) is not particularly limited as long as it is unlikely to adversely affect the structural stability of the polypeptide and the ligand and allows dissociation of the polypeptide and the ligand, i.e., the bond between the polypeptide and the ligand is cleaved. For example, the pH of the concentrate containing complex (C) after adjustment is preferably less than 7, more preferably 3 or more but less than 7, even more preferably 3 to 5, and particularly preferably 4 to 5. By adjusting the pH of the concentrate containing complex (C) to less than 7, complex (C) can be easily dissociated into the polypeptide and the ligand. By adjusting the pH of the concentrate containing complex (C) to 3 or more, adverse effects on the structural stability of the polypeptide and the ligand can be reduced. Note that the pH of the concentrate containing complex (C) is measured at 20°C.

[0046] The dissociation solution is a solution containing one or more acids or acid salts, or one or more alkalis. Specific examples of the acids, acid salts, and alkalis contained in the dissociation solution include organic acids such as acetic acid, citric acid, formic acid, gluconic acid, lactic acid, oxalic acid, and tartaric acid; inorganic acids such as phosphoric acid, hydrochloric acid, sulfuric acid, and nitric acid; alkalis such as sodium hydroxide and calcium hydroxide; and acid salts such as sodium acetate, sodium citrate, and glycine hydrochloride. Among these, acids or acid salts are preferred, and acetic acid, citric acid, sodium acetate, sodium citrate, and glycine hydrochloride are more preferred, from the viewpoint of minimizing adverse effects on the structural stability of the polypeptide and ligand when adjusting the pH of the concentrated solution containing complex (C) to the acidic side.

[0047] From the viewpoint that the composite (C) easily adheres to the separation membrane, it is preferable to supply the above-mentioned dissociation liquid to the membrane module. Here, "supplying the dissociation liquid to the membrane module" means, for example, when a casing-type membrane module is used as the membrane module, supplying the dissociation liquid into the casing (container) containing the separation membrane. Alternatively, it means adding the dissociation liquid to the mixing container 31 and supplying it to the membrane module 41 through the third pipe 33. Furthermore, when a non-casing-type membrane module is used as the membrane module, it means supplying the dissociation liquid to a container in which the non-casing-type membrane module is immersed. In other words, the dissociation liquid is supplied to the membrane module by supplying the dissociation liquid to the concentrated solution containing the composite.

[0048] <Recovery Step> The concentrated solution containing the complex (C) is converted into a solution containing the polypeptide and the ligand in a dissociated state by the dissociation step. Typically, the solution containing the polypeptide and the ligand in a dissociated state obtained in this manner is discharged and recovered from the primary side of the membrane module. Furthermore, from the viewpoint of removing impurities larger than the pore size of the micropores formed in the separation membrane, it is preferable to discharge and recover the solution containing the polypeptide and the ligand in a dissociated state from the secondary side of the membrane module. When recovering from the secondary side of the membrane module, the fourth pump 43 is operated, and the solution containing the polypeptide and the ligand in a dissociated state is permeated and discharged through the fourth pipe 42. Any impurities larger than those in the membrane step remain on the primary side of the membrane and are not mixed into the discharged dissociated solution. Here, the "primary side" refers to the side where the fluid (raw solution containing fine particles and solutes) before filtration or separation is present in a separation process using a membrane module, i.e., the feed side. The "secondary side" refers to the side where the fluid is present after passing through the membrane module, i.e., the permeation side. The polypeptide and the ligand are then separated, allowing the purified polypeptide to be recovered independently.

[0049] <Effects> According to the polypeptide purification method of this embodiment described above, after membrane separation of a liquid containing a complex (C) of a polypeptide and a ligand, a dissociation liquid that dissociates the complex is supplied to a membrane module, dissociating the complex into the polypeptide and the ligand. Because the complex (C) is in a solid state before pH adjustment, the solid-state complex (C) attached to the separation membrane is difficult to recover. This tends to reduce the recovery rate of the polypeptide. However, in the polypeptide purification method of this embodiment, the complex (C) after membrane separation is dissociated into the polypeptide and the ligand by pH adjustment, and the polypeptide and the ligand are dissolved in solution. Therefore, even if the complex (C) is attached to the separation membrane, the complex (C) can be dissociated into the polypeptide and the ligand and recovered in a dissolved state in solution, allowing the polypeptide to be recovered with a high recovery rate.

[0050] <Applications> The polypeptide purification method of this embodiment can be applied even on an industrial scale and is excellent in polypeptide productivity. The polypeptide obtained by the polypeptide purification method of this embodiment can be suitably used, for example, in prescription drugs, functional foods, and intermediates for the synthesis of high-value-added compounds, and is particularly suitable for use in prescription drugs because of its excellent health-improving effects.

[0051] [Polypeptide Purification Apparatus] The polypeptide purification apparatus of this embodiment includes a membrane module equipped with a separation membrane, and includes a membrane separation device that supplies a liquid containing a polypeptide-ligand complex to the separation membrane and separates the liquid into a concentrate containing the complex and a permeate, and a dissociation liquid supply device that supplies a dissociation liquid to the membrane module to dissociate the complex. The polypeptide purification apparatus of the present invention may have, for example, the following embodiments. <First Embodiment> Figure 2 shows an example of a polypeptide purification apparatus of the first embodiment of the present invention. The polypeptide purification apparatus 1 shown in Figure 2 includes a storage tank 10 that contains a solution containing the polypeptide, a first tank 20 that stores the ligand, a complex formation device 30, a membrane separation device 40, a buffer solution supply device 50, and a dissociation liquid supply device 60. Note that, for convenience, the drawings used in the following description may show characteristic parts enlarged to make the features easier to understand, and the dimensional proportions of each component may differ from the actual ones. Furthermore, the materials, dimensions, etc. exemplified in the following description are merely examples, and the present invention is not limited to them, and can be implemented by making appropriate changes within the scope that does not change the gist of the present invention.

[0052] (Storage Tank) The storage tank 10 is a tank that stores a solution containing a polypeptide. Examples of the storage tank 10 include a culture tank for cell culture of the polypeptide. One end of a first pipe 11 that supplies the solution containing the polypeptide to the complex formation device 30 is connected to the storage tank 10. The other end of the first pipe 11 is connected to a mixing container 31 of the complex formation device 30, which will be described later. The solution containing the polypeptide is supplied to the complex formation device 30 via the first pipe 11. In addition, a first pump 12 is installed midway through the first pipe 11.

[0053] (First Tank) The first tank 20 is a tank for storing a ligand. The first tank 20 may store only the ligand, or may store a solution containing the ligand. One end of a second pipe 21 that supplies the ligand or a solution containing the ligand to the complex formation device 30 is connected to the first tank 20. The other end of the second pipe 21 is connected to a mixing container 31 of the complex formation device 30. The ligand or a solution containing the ligand is supplied to the complex formation device 30 via the second pipe 21. In addition, a second pump 22 is installed midway through the second pipe 21.

[0054] (Complex Formation Apparatus) The complex formation apparatus 30 is an apparatus that mixes a polypeptide and a ligand in a liquid to form a complex (C) between the polypeptide and the ligand, and obtains a liquid containing the complex (C). The complex formation apparatus 30 is not particularly limited as long as it has a mechanism for complexing the polypeptide and the ligand. The complex formation apparatus 30 shown in the illustrated example includes a mixing container 31, an agitator 32, and a third pipe 33 that supplies the liquid containing the complex (C) to the membrane separation apparatus 40.

[0055] The other end of the first pipe 11 and the other end of the second pipe 21 are connected to the mixing vessel 31. A solution containing the polypeptide and a ligand or a solution containing the ligand are supplied to the mixing vessel 31 and mixed in the mixing vessel 31 to obtain a liquid containing the complex (C).

[0056] The third pipe 33 is a pipe that supplies the liquid containing the complex (C) obtained in the mixing vessel 31 to the membrane separation device 40. One end of the third pipe 33 is connected to the mixing vessel 31, and the other end is connected to a membrane module 41 of the membrane separation device 40, which will be described later. The liquid containing the complex (C) is supplied to the membrane separation device 40 via the third pipe 33. Furthermore, a third pump 34 is installed midway along the third pipe 33.

[0057] (Membrane Separation Apparatus) The membrane separation apparatus 40 is an apparatus that supplies a liquid containing a complex (C) between a polypeptide and a ligand to a separation membrane and separates the liquid into a concentrated liquid containing the complex (C) and a permeate. The membrane separation apparatus 40 is not particularly limited as long as it has a mechanism for membrane separation of the liquid containing the complex (C) obtained in the complex formation apparatus 30. The membrane separation apparatus 40 in the illustrated example includes a casing-type membrane module 41 equipped with a separation membrane (not shown), a fourth pipe 42 for discharging the permeate from the membrane module 41, and a fifth pipe 44 for discharging a solution containing the polypeptide and the ligand in a dissociated state from the membrane module 41.

[0058] The other end of the third pipe 33 is connected to the membrane module 41. A liquid containing the complex (C) is supplied to the membrane module 41. The membrane module 41 is an apparatus that separates a liquid containing the complex (C) introduced into a separation membrane through membrane separation into a concentrated liquid (liquid containing the complex (C) after membrane separation) that does not permeate the separation membrane and is concentrated with the complex (C), and a permeate that permeates the separation membrane. The permeate contains impurities. The membrane separation method (filtration method) using the membrane module 41 may be a cross-flow filtration method or a dead-end filtration method (dead-end filtration method). Note that the membrane separation method using the membrane module 41 shown in FIG. 2 is a dead-end filtration method. Examples of separation membranes provided in the membrane module 41 include the separation membranes exemplified above in the description of the separation step in the polypeptide purification method of the present invention. Porous membranes are particularly preferred. A hollow fiber membrane is preferred as the form of the separation membrane.

[0059] The membrane module 41 in the illustrated example is a pressurized type for filtration. The pressurization method is not particularly limited, and may be an external pressure type or an internal pressure type. Here, the external pressure type refers to a filtration method (out-in type) in which, when the separation membrane has a hollow portion, such as a hollow fiber membrane or a tubular membrane, feed water (in this embodiment, a liquid containing the composite (C)) is passed outside the hollow fiber membrane or tubular membrane, and permeated water is obtained on the inside. The internal pressure type refers to a filtration method (in-out type) in which feed water (in this embodiment, a liquid containing the composite (C)) is passed inside the hollow fiber membrane or tubular membrane, and permeated water is obtained on the outside.

[0060] The fourth pipe 42 is a pipe for discharging the permeate from the membrane module 41. One end of the fourth pipe 42 is connected to the membrane module 41. The permeate is discharged from the membrane module 41 via the fourth pipe 42. A fourth pump 43 is installed midway along the fourth pipe 42, and a pressure gauge 49 is installed between the fourth pump 43 and the valve 43B.

[0061] The fifth pipe 44 is a pipe that discharges the solution containing the polypeptide and the ligand in a dissociated state from the membrane module 41. One end of the fifth pipe 44 is connected to the membrane module 41. The solution containing the polypeptide and the ligand in a dissociated state is discharged from the membrane module 41 via the fifth pipe 44. A fifth pump 45 is installed midway along the fifth pipe 44.

[0062] (Buffer Solution Supplying Device) The buffer solution supplying device 50 is a device that supplies a buffer solution to a concentrated solution containing the complex (C). The buffer solution supplying device 50 is not particularly limited as long as it has a mechanism for supplying a buffer solution to a concentrated solution in which the complex (C) has not permeated the separation membrane, i.e., a liquid containing the complex (C) after membrane separation. The buffer solution supplying device 50 in the illustrated example includes a second tank 51 that stores a buffer solution and a buffer solution supply pipe 52 that supplies the buffer solution to the concentrated solution containing the complex (C).

[0063] The second tank 51 is a tank for storing a buffer solution, and examples of the buffer solution include the buffer solutions exemplified above in the description of the buffer solution supply step in the method for purifying a polypeptide of the present invention.

[0064] The buffer solution supply pipe 52 is a pipe that supplies a buffer solution to the concentrated solution containing the complex (C). In the following description, the buffer solution supply pipe 52 is also referred to as the "sixth pipe." One end of the buffer solution supply pipe 52 is connected to the second tank 51, and the other end is connected to the membrane module 41. The buffer solution is supplied to the concentrated solution containing the complex (C) after membrane separation in the membrane module 41 via the buffer solution supply pipe 52. A sixth pump 53 is also installed midway along the buffer solution supply pipe 52.

[0065] (Dissociation Liquid Supply Device) The dissociation liquid supply device 60 is a device that supplies a dissociation liquid that dissociates complex (C) to the membrane module 41, thereby dissociating the complex (C) into a polypeptide and a ligand. By supplying the dissociation liquid, the pH of a concentrated solution containing complex (C) (i.e., a liquid in which complex (C) is concentrated and does not pass through the separation membrane provided in the membrane module 41) is adjusted. The dissociation liquid supply device 60 in the illustrated example includes a third tank 61 that stores the dissociation liquid, and a dissociation liquid supply pipe 62 that supplies the dissociation liquid to the concentrated solution containing complex (C).

[0066] The third tank 61 is a tank for storing a dissociation liquid. Examples of the dissociation liquid include the dissociation liquids exemplified above in the description of the dissociation step in the method for purifying the polypeptide of the present invention.

[0067] The dissociation liquid supply pipe 62 is a pipe that supplies the dissociation liquid to the concentrated liquid containing the complex (C). In the following description, the dissociation liquid supply pipe 62 is also referred to as the "seventh pipe." One end of the dissociation liquid supply pipe 62 is connected to the third tank 61, and the other end is connected to the membrane module 41. The dissociation liquid is supplied to the concentrated liquid containing the complex (C) after membrane separation in the membrane module 41 via the dissociation liquid supply pipe 62. In addition, a seventh pump 63 is installed midway along the dissociation liquid supply pipe 62.

[0068] (Recovery device) The polypeptide and ligand dissociated in the dissociation liquid supply device can be recovered from the primary side or secondary side of the membrane module. When recovering from the primary side of the membrane module, the fifth pump 45 is operated and a solution containing the polypeptide and ligand in a dissociated state is discharged from the fifth pipe 44. When recovering from the secondary side of the membrane module, the fourth pump 43 is operated and a solution containing the polypeptide and ligand in a dissociated state is discharged from the fourth pipe 42. From the viewpoint of being able to remove impurities larger than the pore size of the micropores formed in the separation membrane, recovery from the secondary side of the membrane module is preferred.

[0069] (Polypeptide purification) When purifying a polypeptide using the polypeptide purification apparatus 1 shown in FIG. 2 , first, the first pump 12 is operated to supply a solution containing the polypeptide from the storage tank 10 to the mixing vessel 31 of the complex formation apparatus 30 via the first pipe 11. Separately, the second pump 22 is operated to supply a ligand or a solution containing the ligand from the first tank 20 to the mixing vessel 31 of the complex formation apparatus 30 via the second pipe 21. The polypeptide-containing solution and the ligand or the solution containing the ligand supplied to the complex formation apparatus 30 are mixed in the mixing vessel 31, forming a complex (C) between the polypeptide and the ligand, and obtaining a liquid containing the complex (C). The conditions for mixing the polypeptide and the ligand, such as the ratio of the two, are the same as those in the complex formation step of the polypeptide purification method of the present invention described above.

[0070] Next, the first pump 12 and the second pump 22 are stopped. Separately, the third pump 34 and the fourth pump 43 are operated to supply the liquid containing the complex (C) from the mixing vessel 31 to the membrane module 41 of the membrane separation device 40 via the third pipe 33, and membrane separation is performed. The liquid containing the complex (C) supplied to the membrane module 41 is separated by the separation membrane provided in the membrane module 41 and separated into a concentrated liquid in which the complex (C) is concentrated and does not permeate through the separation membrane, and a permeate that permeates through the separation membrane. The permeate that permeates through the separation membrane contains impurities and is discharged from the membrane module 41 via the fourth pipe 42 and discarded.

[0071] Separately, the sixth pump 53 is operated to supply the buffer solution from the second tank 51 to the concentrated liquid (i.e., the liquid containing the complex (C) after membrane separation) in the membrane module 41 via the buffer solution supply pipe 52. At this time, the buffer solution may be supplied while continuing membrane separation, or the membrane separation and the supply of the buffer solution may be performed separately. That is, the sixth pump 53 may be operated while the fourth pump 43 is operating, or the fourth pump 43 may be stopped and then the sixth pump 53 may be operated. The amount of buffer solution supplied is preferably approximately the same as the amount of permeate discharged from the membrane module 41. Furthermore, when the buffer solution is supplied while continuing membrane separation, the supply rate of the buffer solution is preferably approximately the same as the membrane permeation rate (filtration flow rate) of the liquid containing the complex (C). That is, the fourth pump 43 and the sixth pump 53 preferably have the same output. If the fourth pump 43 has been stopped, the fourth pump 43 is operated again to perform membrane separation of the concentrated liquid containing the complex (C) to which the buffer solution has been added. By repeating this supply of buffer solution and membrane separation, a highly pure polypeptide from which impurities have been removed is obtained in the form of a complex (C) with a ligand. When membrane separation and supply of buffer solution are performed separately, the fourth pump 43 and the sixth pump 53 are alternately operated and stopped. Note that when all of the liquid containing the complex (C) has been supplied from the mixing vessel 31 to the membrane module 41, the third pump 34 is stopped.

[0072] Next, the fourth pump 43 and the sixth pump 53 are stopped. Separately, the seventh pump 63 is operated to supply the dissociation liquid from the third tank 61 to the concentrated liquid (i.e., the liquid containing the complex (C) after membrane separation) in the membrane module 41 via the dissociation liquid supply pipe 62, thereby dissociating the complex (C) into the polypeptide and the ligand, and obtaining a solution containing the dissociated polypeptide and the ligand. The dissociated polypeptide and the ligand are dissolved in the solution. The amount of dissociation liquid added is not particularly limited, as long as it is an amount that does not adversely affect the pH of the concentrated liquid containing the complex (C) and is an amount at which the polypeptide and the ligand are dissociated, i.e., the bond between the polypeptide and the ligand is cleaved. For example, the amount is preferably less than 7, more preferably 3 to 7, even more preferably 3 to 5, and particularly preferably 4 to 5.

[0073] Next, the seventh pump 63 is stopped. When recovering from the primary side of the membrane module, the fifth pump 45 is operated, and a solution containing the polypeptide and the ligand in a dissociated state is discharged from the membrane module 41 via the fifth pipe 44 and recovered. When recovering from the secondary side of the membrane module, the fourth pump 43 is operated, and a solution containing the polypeptide and the ligand in a dissociated state is discharged from the membrane module 41 via the fourth pipe 42 and recovered. Next, the polypeptide and the ligand are separated, allowing the purified polypeptide to be recovered alone at a high recovery rate.

[0074] (Other Aspects) The polypeptide purification apparatus of this embodiment is not limited to the one described above. For example, the membrane module 41 provided in the membrane separation apparatus 40 shown in Figure 2 is a casing-type membrane module in which a separation membrane (not shown) is housed in a casing, but it may also be a non-casing-type membrane module in which the separation membrane is not housed in a casing. When a non-casing-type membrane module is used, membrane separation can be performed by immersing the membrane module in a membrane separation tank that stores a liquid containing the complex (C) supplied from the complex formation apparatus.

[0075] Alternatively, a solution containing the polypeptide may be directly supplied from the storage tank 10 to the membrane module 41 of the membrane separation apparatus 40 via the first pipe 11, and a ligand or a solution containing the ligand may be directly supplied from the first tank 20 to the membrane module 41 of the membrane separation apparatus 40 via the second pipe 21. In this case, a complex between the polypeptide and the ligand is formed in the membrane module 41, and a liquid containing complex (C) is obtained, so that the membrane separation apparatus 40 also serves as the complex formation apparatus 30. When the membrane separation apparatus 40 also serves as the complex formation apparatus 30, after the liquid containing complex (C) is obtained, the fourth pump 43 is operated to perform membrane separation.

[0076] <Second Aspect> Figure 3 shows an example of a polypeptide purification apparatus according to the second aspect of the present invention. The polypeptide purification apparatus 2 shown in Figure 3 comprises a storage tank 10 for storing a solution containing the polypeptide, a first tank 20 for storing a ligand, a complex formation apparatus 30, a membrane separation apparatus 40, a buffer solution supplying apparatus 50, and a dissociation solution supplying apparatus 60. The polypeptide purification apparatus 2 of the second aspect is similar to the polypeptide purification apparatus 1 of the first aspect shown in Figure 2, except that the membrane separation method using the membrane module 46 provided in the membrane separation apparatus 40 is a cross-flow filtration method, and the buffer solution supplying apparatus 50 is a mechanism for supplying a buffer solution to the concentrated solution containing the complex (C) after membrane separation outside the membrane module 46. In Figure 3, the same components as those in Figure 2 are designated by the same reference numerals, and their description will be omitted.

[0077] (Membrane Separation Apparatus) The membrane separation apparatus 40 shown in FIG. 3 comprises a casing-type membrane module 46 equipped with a separation membrane (not shown), a fourth pipe 42 for discharging the permeate from the membrane module 46, a fifth pipe 44 for discharging the solution containing the polypeptide and the ligand in a dissociated state from the membrane module 46, and an eighth pipe 47 for discharging the concentrated solution from the membrane module 46 and returning it to the complex formation apparatus 30.

[0078] The other end of the third pipe 33 is connected to the membrane module 46. A liquid containing the complex (C) is supplied to the membrane module 46. The membrane separation method using the membrane module 46 shown in FIG. 3 is a cross-flow filtration method. The membrane module 46 performs filtration by pressure. The pressure method is not particularly limited, and may be an external pressure type or an internal pressure type. In addition, one end of the fourth pipe 42, one end of the fifth pipe 44, and the other end of the dissociation liquid supply pipe 62 are each connected to the membrane module 46.

[0079] The eighth pipe 47 is a pipe that discharges a concentrated liquid (liquid containing the complex (C) after membrane separation) that does not permeate the separation membrane and in which the complex (C) is concentrated, from the membrane module 46 and returns it to the complex formation apparatus 30. One end of the eighth pipe 47 is connected to the membrane module 46, and the other end is connected to the mixing vessel 31 of the complex formation apparatus 30. The concentrated liquid is discharged from the membrane module 46 via the eighth pipe 47 and returned to the mixing vessel 31 of the complex formation apparatus 30. An eighth pump 48 is installed midway along the eighth pipe 47.

[0080] (Buffer Solution Supply Device) The buffer solution supply device 50 shown in Fig. 3 includes a second tank 51 that stores a buffer solution, and a buffer solution supply pipe 52 that supplies the buffer solution to a concentrate containing the complex (C). One end of the buffer solution supply pipe 52 shown in Fig. 3 is connected to the second tank 51, and the other end is connected to the mixing vessel 31 of the complex formation apparatus 30. The buffer solution is supplied through the buffer solution supply pipe 52 in the mixing vessel 31 to the concentrate containing the complex (C) after membrane separation, which has been discharged from the membrane module 46 and returned to the mixing vessel 31.

[0081] (Recovery Device) The recovery device is the same as that described in the first embodiment.

[0082] (Polypeptide purification) When purifying a polypeptide using the polypeptide purification apparatus 2 shown in FIG. 3 , first, the first pump 12 is operated to supply a solution containing the polypeptide from the storage tank 10 via the first pipe 11 to the mixing vessel 31 of the complex formation apparatus 30. Separately, the second pump 22 is operated to supply a ligand or a solution containing the ligand from the first tank 20 via the second pipe 21 to the mixing vessel 31 of the complex formation apparatus 30. The polypeptide-containing solution and the ligand or the solution containing the ligand supplied to the complex formation apparatus 30 are mixed in the mixing vessel 31, forming a complex (C) between the polypeptide and the ligand, and obtaining a liquid containing the complex (C). The conditions for mixing the polypeptide and the ligand, such as the ratio of the two, are the same as those in the complex formation step of the polypeptide purification method of the present invention described above.

[0083] Next, the first pump 12 and the second pump 22 are stopped. Separately, the third pump 34 and the eighth pump 48 are operated to circulate the liquid containing the complex (C) between the complex formation apparatus 30 and the membrane separation apparatus 40. While circulating the liquid containing the complex (C), the fourth pump 43 is operated to membrane separate the liquid containing the complex (C). The liquid containing the complex (C) is separated by a separation membrane provided in the membrane module 46 and separated into a concentrate in which the complex (C) is concentrated and which does not permeate the separation membrane, and a permeate containing impurities which permeated the separation membrane. The permeate which permeated the separation membrane is discharged from the membrane module 46 via the fourth pipe 42 and discarded. The concentrate (liquid containing the complex (C) after membrane separation) is discharged from the membrane module 46 via the eighth pipe 47 and returned to the mixing vessel 31 of the complex formation apparatus 30.

[0084] Separately, the sixth pump 53 is operated to supply the buffer solution from the second tank 51 to the mixing vessel 31 of the complex formation device 30 via the buffer solution supply pipe 52. At this time, the buffer solution may be supplied while membrane separation is continuing, or membrane separation and buffer solution supply may be performed separately. That is, the sixth pump 53 may be operated while the fourth pump 43 is operating, or the fourth pump 43 may be stopped and then operated. The amount of buffer solution supplied is preferably approximately the same as the amount of permeate discharged from the membrane module 46. Furthermore, when the buffer solution is supplied while membrane separation is continuing, the supply rate of the buffer solution is preferably approximately the same as the membrane permeation rate (filtration flow rate) of the liquid containing the complex (C). That is, the fourth pump 43 and the sixth pump 53 preferably have the same output. If the fourth pump 43 was stopped, the fourth pump 43 is operated again. The concentrated liquid (liquid containing the complex (C) after membrane separation) returned to the mixing vessel 31 is mixed with the buffer solution and circulated between the complex formation device 30 and the membrane separation device 40 for membrane separation. By repeating the supply of the buffer solution and membrane separation in this manner, a highly pure polypeptide from which impurities have been removed is obtained in the form of a complex (C) with the ligand. When membrane separation and the supply of the buffer solution are carried out separately, the fourth pump 43 and the sixth pump 53 are alternately operated and stopped.

[0085] Next, the fourth pump 43 and the sixth pump 53 are stopped. While the third pump 34 and the eighth pump 48 are operating, i.e., while circulating the concentrated solution containing the complex (C) after membrane separation between the membrane separation device 40 and the complex formation device 30, the seventh pump 63 is operated separately to supply the dissociation liquid from the third tank 61 to the concentrated solution containing the complex (C) after membrane separation in the membrane module 46 via the dissociation liquid supply pipe 62, thereby dissociating the complex (C) into the polypeptide and the ligand, and obtaining a solution containing the dissociated polypeptide and the ligand. The dissociated polypeptide and the ligand are dissolved in the solution. The supply of the dissociation liquid is continued until the pH of the concentrated solution containing the complex (C) in the membrane module 46 reaches a pH that is unlikely to adversely affect the structural stability of the polypeptide and the ligand and at which the polypeptide and the ligand are dissociated, i.e., the bond between the polypeptide and the ligand is broken. The pH of the concentrate containing the complex (C) in the membrane module 46 is, for example, preferably less than 7, more preferably 3 or more but less than 7, even more preferably 3 to 5, and particularly preferably 4 to 5.

[0086] Next, the eighth pump 48 and the seventh pump 63 are stopped. When recovering from the primary side of the membrane module, the fifth pump 45 is operated, and a solution containing the polypeptide and the ligand in a dissociated state is discharged from the membrane module 46 via the fifth pipe 44 and recovered. When recovering from the secondary side of the membrane module, the fourth pump 43 is operated, and a solution containing the polypeptide and the ligand in a dissociated state is discharged from the membrane module 46 via the fourth pipe 42 and recovered. Next, the polypeptide and the ligand are separated, allowing the purified polypeptide to be recovered alone at a high recovery rate.

[0087] (Other Aspects) The polypeptide purification device of this embodiment is not limited to the one described above. For example, although the other end of the buffer solution supply pipe 52 shown in Fig. 3 is connected to the mixing vessel 31, the other end of the buffer solution supply pipe 52 may be joined to the middle of the eighth pipe 47. In this case, in the eighth pipe 47, the concentrated solution containing the complex (C) after membrane separation is mixed with the buffer solution and returned to the mixing vessel 31.

[0088] 3 is connected to the membrane module 46, the other end of the dissociation liquid supply pipe 62 may be connected to the mixing vessel 31. In this case, the complex (C) contained in the concentrate in the mixing vessel 31 is dissociated into the polypeptide and the ligand. Furthermore, the dissociation liquid supplied to the mixing vessel 31 is circulated between the membrane separation device 40 and the complex formation device 30, so that the complex (C) attached to the separation membrane of the membrane module 46 is dissociated into the polypeptide and the ligand.

[0089] <Action and Effect> According to the polypeptide purification devices of the first and second aspects described above, after a liquid containing a complex (C) of a polypeptide and a ligand is subjected to membrane separation, the pH of the concentrated liquid containing the complex (C) after membrane separation is subsequently adjusted in the membrane module used for membrane separation to dissociate the complex (C) into the polypeptide and the ligand.

[0090] Because the complex (C) before pH adjustment is in a solid state, the solid-state complex (C) attached to the separation membrane is difficult to recover. This tends to reduce the recovery rate of the polypeptide. However, in the polypeptide purification device of this embodiment, the complex (C) after membrane separation is dissociated into the polypeptide and the ligand, and the polypeptide and the ligand are dissolved in solution. Therefore, even if the complex (C) is attached to the separation membrane, the complex (C) can be dissociated into the polypeptide and the ligand and recovered in a dissolved state in solution, allowing the polypeptide to be recovered with a high recovery rate.

[0091] In particular, when a polypeptide is bound to a ligand in a complex formation device to form a complex (C), the complex (C) has a larger particle size than the polypeptide before binding. Furthermore, since the complex (C) is dispersed or precipitated in a liquid, it can be easily separated from impurities and purified in a membrane separation device. Furthermore, since separation by membrane separation can be performed sufficiently using a microfiltration membrane, there is no need to use an ultrafiltration membrane for separation, making it suitable for industrialization. Therefore, polypeptides can be easily separated and purified to a high degree of purity.

[0092] <Applications> The polypeptide purification apparatus of this embodiment enables highly efficient purification and production of polypeptides that can be applied even on an industrial scale. The polypeptides obtained by the polypeptide purification apparatus of this embodiment can be suitably used, for example, in prescription drugs, functional foods, and intermediates for the synthesis of high-value-added compounds, and are particularly suitable for use in prescription drugs because of their excellent health-improving effects.

[0093] The present invention will be described in more detail below with reference to examples, but the present invention is not limited to the following examples as long as the gist of the invention is not exceeded.

[0094] [Materials Used] <Polypeptides> The following antibodies were used as polypeptides: Monoclonal antibody A (concentration in culture medium: 1.2 mg / ml).

[0095] <Ligand> A modified protein A having the amino acid sequence of SEQ ID NO: 7 shown below was used as the ligand: DHHKEQQNAFYEILHLPNLTEEQRNGFIQSLKHDPSVSKEILAEAKKLNDAQAPDHHKEQQNAFYEILHLPNLTEEQRNGFIQSLKHDPSVSKEILAEAKKLNDAQAPDHHKEQQNAFYEILHLPNLTEEQRNGFIQSLKHDPSVSKEILAEAKKLNDAQAPDHH KEQQNAFYEILHLPNLTEEQRNGFIQSLKHDPSVSKEILAEAKKLNDAQAPDHHKEQQNAFYEILHLPNLTEEQRNGFIQSLK HDPSVSKEILAEAKKLNDAQAPDHHKEQQNAFYEILHLPNLTEEQRNGFIQSLKHDPSVSKEILAEAKKLNDAQAPHHHHHHC

[0096] <Separation membrane> A high-density polyethylene (HDPE) hollow fiber membrane having a porous structure was used as the separation membrane. The hollow fiber membrane had an outer diameter of 0.38 mm, an inner diameter of 0.27 mm, and a pore size of 0.1 μm. A casing-type membrane module was used in which the separation membrane was housed in a cylindrical casing (container).

[0097] Example 1 A polypeptide was purified as follows using the polypeptide purification apparatus 1 shown in Figure 2. In Example 1, the solution containing the polypeptide and the ligand were directly supplied to the membrane module 41 of the membrane separation apparatus 40.

[0098] First, the first pump 12 was operated to supply 50 mL of a solution containing the polypeptide from the storage tank 10 via the first pipe 11 to the membrane module 41 of the membrane separation device 40. Separately, the second pump 22 was operated to supply 131 μL of a ligand from the first tank 20 via the second pipe 21 to the membrane module 41 of the membrane separation device 40. The polypeptide-containing solution and the ligand supplied to the membrane module 41 were mixed by stirring, and a liquid containing complex (C) was obtained as a white solid precipitate. Next, the first pump 12 and the second pump 22 were stopped. Separately, the fourth pump 43 was operated to perform membrane separation on 25 mL of a liquid containing complex (C), and the permeate that permeated the separation membrane was discharged from the membrane module 41 via the fourth pipe 42.

[0099] Next, the fourth pump 43 was stopped. Separately, the sixth pump 53 was operated, and 25 mL of phosphate buffer solution (PBS) as a buffer solution was supplied from the second tank 51 via the buffer solution supply pipe 52 to the concentrated solution (i.e., the liquid containing the complex (C) after membrane separation) in the membrane module 41. Next, the sixth pump 53 was stopped, and the fourth pump 43 was operated again, and 25 mL of the concentrated solution containing the complex (C) to which the buffer solution had been added was subjected to membrane separation. This operation (supply of the buffer solution and membrane separation) was repeated five times.

[0100] Next, the fourth pump 43 was stopped. Separately, the seventh pump 63 was operated to supply 0.5 mL of a 1 M aqueous acetic acid solution as a dissociation solution from the third tank 61 through the dissociation solution supply pipe 62 to the concentrated solution (i.e., the liquid containing complex (C) after membrane separation) in the membrane module 41, thereby dissociating the complex (C) into the polypeptide and the ligand, and obtaining a solution containing the dissociated polypeptide and the ligand in a dissolved state. The pH of the solution in the membrane module 41 after the addition of the dissociation solution was 4.5.

[0101] Next, after confirming that all of the complex (C) in the membrane module 41 had dissociated into the polypeptide and the ligand and dissolved, the seventh pump 63 was stopped. Separately, the fifth pump 45 was operated to discharge and recover a solution containing the polypeptide and the ligand in a dissociated state from the membrane module 41 via the fifth pipe 44 (the primary side of the membrane module), and this was used as a solution containing the purified polypeptide.

[0102] The solution containing the polypeptide before purification and the solution containing the polypeptide after purification were each subjected to measurement by high performance liquid chromatography (HPLC) under the conditions shown below. A calibration curve was prepared in advance using a purified polypeptide of known concentration as a standard sample, and the concentration of the polypeptide (antibody concentration) in each solution was calculated from the peak area detected at the same time as the standard sample and the calibration curve. The antibody weight was calculated from the calculated antibody concentration and the flow rate used for HPLC measurement, and the polypeptide recovery rate was determined using the following formula (1). The results are shown in Table 1. Polypeptide recovery rate [%] = (antibody weight in solution containing purified polypeptide / antibody weight in solution containing polypeptide before purification) × 100 (1)

[0103] <HPLC Measurement Conditions> Column: YMC Triart Bio C4 (300 Å, 3 μm). Column size: 75 × 4.6 mm. Eluent A: A solution obtained by adding trifluoroacetic acid to a mixed solution of water and acetonitrile in a mass ratio of water:acetonitrile = 9:1 to a concentration of 0.1 mass%. Eluent B: A solution obtained by adding trifluoroacetic acid to a mixed solution of water and acetonitrile in a mass ratio of water:acetonitrile = 1:4 to a concentration of 0.1 mass%. Gradient: The concentration of eluent B was increased from 25 mass% to 60 mass% over 10 minutes, and then the concentration of eluent B was held at 75 mass% for 1 minute. Flow rate: 0.8 ml / min. Column temperature: 80°C. Detection: UV (280 nm). Sample injection volume: 5 μl.

[0104] Example 2 A polypeptide was purified as follows using the polypeptide purification apparatus 1 shown in Figure 2. In Example 2, the polypeptide-containing solution and the ligand were directly supplied to the membrane module 41 of the membrane separation device 40.

[0105] First, the first pump 12 was operated to supply 100 mL of a solution containing the polypeptide from the storage tank 10 via the first pipe 11 to the membrane module 41 of the membrane separation device 40. Separately, the second pump 22 was operated to supply 500 μL of a ligand from the first tank 20 via the second pipe 21 to the membrane module 41 of the membrane separation device 40. The polypeptide-containing solution and the ligand supplied to the membrane module 41 were mixed by stirring, and a liquid containing complex (C) was obtained as a white solid precipitate. Next, the first pump 12 and the second pump 22 were stopped. Separately, the fourth pump 43 was operated to perform membrane separation on 50 mL of a liquid containing complex (C), and the permeate that permeated the separation membrane was discharged from the membrane module 41 via the fourth pipe 42.

[0106] Next, the fourth pump 43 was stopped. Separately, the sixth pump 53 was operated to supply 50 mL of 20 mM sodium phosphate buffer as a buffer solution from the second tank 51 to the concentrated solution (i.e., the liquid containing the complex (C) after membrane separation) in the membrane module 41 via the buffer solution supply pipe 52. Next, the sixth pump 53 was stopped, and the fourth pump 43 was operated again to perform membrane separation on 50 mL of the concentrated solution containing the complex (C) to which the buffer solution had been added. This operation (supply of the buffer solution and membrane separation) was repeated 10 times.

[0107] Next, the fourth pump 43 was stopped. Separately, the seventh pump 63 was operated, and 1 mL of 1 M acetic acid aqueous solution and 10 mL of 20 mM sodium phosphate buffer containing 200 μL of 1 M acetic acid aqueous solution were sequentially supplied from the third tank 61 via the dissociation solution supply pipe 62 to the concentrated solution (i.e., the liquid containing complex (C) after membrane separation) in the membrane module 41, dissociating the complex (C) into polypeptide and ligand, and obtaining a solution containing the dissociated polypeptide and ligand in a dissolved state. The pH of the solution in the membrane module 41 after the addition of the dissociation solution was 4.5. The third tank 61 had previously stored therein 1 M acetic acid aqueous solution and 20 mM sodium phosphate buffer containing 200 μL of 1 M acetic acid aqueous solution, separately.

[0108] Next, after confirming that all of the complex (C) in the membrane module 41 had dissociated into the polypeptide and the ligand and dissolved, the seventh pump 63 was stopped. Separately, the fifth pump 45 was operated, and the solution containing the polypeptide and the ligand in a dissociated state was discharged and recovered from the membrane module 41 through the fifth pipe 44 (the primary side of the membrane module), and this was used as a solution containing the purified polypeptide. HPLC measurements were performed on the solutions containing the polypeptide before and after purification in the same manner as in Example 1, and the recovery rate of the polypeptide was determined. The results are shown in Table 1.

[0109] Example 3 Using the polypeptide purification apparatus 2 shown in FIG. 3, a polypeptide was purified as follows.

[0110] First, the first pump 12 was operated to supply 100 mL of a solution containing the polypeptide from the storage tank 10 via the first pipe 11 to the mixing vessel 31 of the complex formation device 30. Separately, the second pump 22 was operated to supply 261 μL of a ligand from the first tank 20 via the second pipe 21 to the mixing vessel 31 of the complex formation device 30. The polypeptide-containing solution and the ligand supplied to the mixing vessel 31 were mixed by stirring, and a liquid containing complex (C) was obtained as a white solid precipitate. Next, the first pump 12 and the second pump 22 were stopped. Separately, the third pump 34 and the eighth pump 48 were operated to circulate the liquid containing complex (C) between the complex formation device 30 and the membrane separation device 40 at a flow rate of 100 mL / min. While circulating the liquid containing the complex (C), the fourth pump 43 was operated to perform membrane separation on 50 mL of the liquid containing the complex (C). The permeate that permeated the separation membrane was discharged from the membrane module 46 via the fourth pipe 42, and the concentrated liquid (liquid containing the complex (C) after membrane separation) was returned from the membrane module 46 via the eighth pipe 47 to the mixing vessel 31 of the complex formation apparatus 30. The filtration flow rate at this time was 2 mL / min. Separately, the sixth pump 53 was operated to supply phosphate buffer solution (PBS) as a buffer solution at a rate of 2 mL / min from the second tank 51 via the buffer supply pipe 52 to the mixing vessel 31 of the complex formation apparatus 30. During this time, the circulation of the concentrated liquid containing the complex (C) continued.

[0111] After 90 minutes had elapsed since the sixth pump 53 was started, the fourth pump 43 and the sixth pump 53 were stopped. While the third pump 34 and the eighth pump 48 were operating, i.e., while the concentrated solution containing the complex (C) after membrane separation was circulated between the membrane separation apparatus 40 and the complex formation apparatus 30, the seventh pump 63 was separately operated to supply 1 mL of a 1 M aqueous acetic acid solution as a dissociation solution from the third tank 61 via the dissociation solution supply pipe 62 to the concentrated solution containing the complex (C) after membrane separation in the membrane module 46, thereby dissociating the complex (C) into the polypeptide and the ligand, and obtaining a solution containing the dissociated polypeptide and the ligand in a dissolved state. The pH of the solution in the membrane module 46 after the addition of the dissociation solution was 4.5.

[0112] Next, after confirming that all of the complex (C) circulating between the membrane separation device 40 and the complex formation device 30 had dissociated into the polypeptide and the ligand and dissolved, the eighth pump 48 and the seventh pump 63 were stopped. Separately, the fifth pump 45 was operated, and the solution containing the polypeptide and the ligand in a dissociated state was discharged and recovered from the membrane module 46 through the fifth pipe 44 (the primary side of the membrane module), and this was used as a solution containing the purified polypeptide. The solutions containing the polypeptide before and after purification were subjected to HPLC measurement in the same manner as in Example 1, and the recovery rate of the polypeptide was determined. The results are shown in Table 1.

[0113] Comparative Example 1 The polypeptide was purified in the same manner as in Example 1, except that the concentrate containing the purified complex (C) was transferred to another container, and then the dissociation solution was added to the concentrate containing complex (C) in the container, and the recovery rate of the polypeptide was determined. The results are shown in Table 1. In Comparative Example 1 and Comparative Examples 2 and 3 described below, a 0.2-fold volume of 1M aqueous acetic acid solution was added to the recovered concentrate containing complex (C) after membrane separation to dissociate complex (C) into the polypeptide and the ligand, followed by HPLC measurement.

[0114] The polypeptide was purified and the recovery rate of the polypeptide was determined in the same manner as in Comparative Example 1, except that the amount of solution containing the polypeptide was 100 mL, the amount of ligand added was 261 μL, and the amount of buffer solution supplied and the amount of membrane permeation per each cycle of buffer solution supply and membrane separation was 50 mL. The results are shown in Table 1.

[0115] The polypeptide was purified and the recovery rate of the polypeptide was determined in the same manner as in Example 3, except that the concentrated solution containing the purified complex (C) was transferred to another container and then the dissociation solution was added to the concentrated solution containing complex (C) in the container. The results are shown in Table 1.

[0116]

[0117] As is clear from the results in Table 1, in Examples 1 to 3 in which the dissociation liquid was supplied to the membrane module, the polypeptide was recovered at a higher recovery rate than in Comparative Examples 1 to 3.

[0118] Example 4 The same procedure as in Example 3 was repeated, except that the antibody concentration in the culture medium was 2.1 mg / mL, the volume of the solution containing the polypeptide was 82 mL, the amount of ligand added was 782 μL, the volume filtered in the first membrane separation step was 76 mL, the volume of buffer solution supplied and the volume permeated through the membrane per each subsequent cycle of buffer supply and membrane separation were 6 mL, and the dissociation solution was 0.1 mL of 2M aqueous acetic acid. As a result, the recovery rate of the polypeptide reached 100%.

[0119] Example 5 was carried out in the same manner as in Example 1, except that recovery was performed on the secondary side of the membrane. As a result, the recovery rate of the polypeptide reached 100%. Specifically, the fourth pump 43 was operated, and a solution containing the polypeptide and the ligand in a dissociated state was discharged from the membrane module 41 through the fourth pipe 42 (the secondary side of the membrane module) and recovered, and this was used as a solution containing the purified polypeptide.

[0120] According to the present invention, a method for purifying a polypeptide and an apparatus for purifying a polypeptide, which are capable of recovering a polypeptide at a high recovery rate, can be provided.

[0121] 1 Polypeptide purification apparatus, 2 Polypeptide purification apparatus, 10 Storage tank, 11 First pipe, 12 First pump, 20 First tank, 21 Second pipe, 22 Second pump, 30 Complex formation apparatus, 31 Mixing vessel, 32 Agitator, 33 Third pipe, 34 Third pump, 40 Membrane separation apparatus, 41 Membrane module, 42 Fourth pipe, 43 Fourth pump, 44 Fifth pipe, 45 Fifth pump, 46 Membrane module, 47 Eighth pipe, 48 Eighth pump, 49 Pressure gauge, 50 Buffer solution supply apparatus, 51 Second tank, 52 Buffer solution supply pipe (sixth pipe), 53 Sixth pump, 60 Dissociation liquid supply apparatus, 61 Third tank, 62 Dissociation liquid supply pipe (seventh pipe), 63 Seventh pump

Claims

1. A method for purifying a polypeptide, comprising: a membrane separation step of separating a liquid containing a complex of a polypeptide and a ligand into a concentrate containing the complex and a permeate using a separation membrane provided in a membrane module; and a dissociation step of supplying a dissociation liquid that dissociates the complex to the membrane module after the membrane separation step, thereby dissociating the complex into the polypeptide and the ligand.

2. The method for purifying a polypeptide according to claim 1, wherein the polypeptide is one or more selected from the group consisting of an antibody, an antibody fragment, an antibody derivative, and an antibody fragment derivative.

3. A method for purifying a polypeptide according to claim 1 or 2, further comprising, prior to the membrane separation step, a complex formation step of mixing the polypeptide and the ligand in a liquid to form a complex between the polypeptide and the ligand, thereby obtaining a liquid containing the complex.

4. A method for purifying a polypeptide according to claim 1 or 2, wherein the polypeptide and the ligand dissociated in the dissociation step are recovered from the secondary side of the membrane module.

5. A method for purifying a polypeptide according to claim 1 or 2, wherein in the dissociation step, the dissociation solution is supplied to a concentrated solution containing the complex to adjust the pH of the concentrated solution containing the complex.

6. A method for purifying a polypeptide according to claim 5, wherein the pH of the concentrated solution containing the complex is adjusted to less than 7 in the dissociation step.

7. The method for purifying a polypeptide according to claim 1 or 2, wherein the pore size of the micropores formed in the separation membrane is 0.05 to 3 μm.

8. The method for purifying a polypeptide according to claim 1 or 2, wherein the membrane separation step is a cross-flow filtration method or a dead-end filtration method.

9. A method for purifying a polypeptide according to claim 1 or 2, wherein the separation membrane comprises a polyolefin resin.

10. The method for purifying a polypeptide according to claim 1 or 2, wherein the separation membrane comprises polyethylene.

11. The method for purifying a polypeptide according to claim 1 or 2, wherein the separation membrane comprises high-density polyethylene.

12. A method for purifying a polypeptide according to claim 1 or 2, further comprising a buffer supply step of supplying a buffer to a concentrated solution containing the complex.

13. A polypeptide purification device comprising: a membrane separation device including a membrane module equipped with a separation membrane, which separates a liquid containing a polypeptide-ligand complex into a concentrate containing the complex and a permeate using the separation membrane; and a dissociation liquid supply device which supplies a dissociation liquid to the membrane module to dissociate the complex.

14. The apparatus for purifying a polypeptide according to claim 13, wherein the polypeptide is one or more selected from the group consisting of an antibody, an antibody fragment, an antibody derivative, and an antibody fragment derivative.

15. A polypeptide purification apparatus as described in claim 13 or 14, further comprising a complex formation device, located upstream of the membrane separation device, for mixing the polypeptide and the ligand in a liquid to form a complex between the polypeptide and the ligand and obtaining a liquid containing the complex.

16. The polypeptide purification apparatus according to claim 13 or 14, further comprising a recovery device for recovering the polypeptide and the ligand dissociated in the dissociation liquid supply device from the secondary side of the membrane module.

17. The polypeptide purification apparatus according to claim 13 or 14, wherein the dissociation liquid supply device comprises a dissociation liquid supply pipe for supplying the dissociation liquid to the concentrated solution containing the complex.

18. The polypeptide purification apparatus according to claim 13 or 14, wherein the dissociation liquid supplying device adjusts the pH of the concentrated liquid containing the complex to less than 7.

19. The polypeptide purification device according to claim 13 or 14, wherein the pore diameter of the micropores formed in the separation membrane is 0.05 to 3 μm.

20. The polypeptide purification apparatus according to claim 13 or 14, wherein the membrane separation in the membrane separation apparatus is a cross-flow filtration method or a dead-end filtration method.

21. The polypeptide purification device according to claim 13 or 14, wherein the separation membrane comprises a polyolefin resin.

22. The polypeptide purification device according to claim 13 or 14, wherein the separation membrane comprises polyethylene.

23. The polypeptide purification device according to claim 13 or 14, wherein the separation membrane comprises high-density polyethylene.

24. The polypeptide purification apparatus according to claim 13 or 14, further comprising a buffer supply device for supplying a buffer to the concentrated solution containing the complex.

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