Chromatographic support for purifying fc fusion protein and fc fusion protein purification method using same

Abstract

The present invention provides a chromatographic support for purifying an Fc fusion protein and an Fc fusion protein purification method using said chromatographic support. The chromatographic support comprises porous particles to which a ligand is immobilized, wherein: the porous particles are synthetic polymer-based or natural polymer-based porous particles; the ligand is at least one substance selected from the group consisting of protein A, protein G, protein L, and related substances of these; and the average pore diameter of the porous particles to which the ligand is immobilized is 75-120 nm. The Fc fusion protein purification method comprises: (step 1) a step for passing a solution containing an Fc fusion protein through a column that is provided with said chromatographic support and causing the Fc fusion protein to bind with the support; (step 2) a step for washing the support; and (step 3) a step for collecting the Fc fusion protein from the support.

Description

Chromatography support for purifying Fc fusion proteins and method for purifying Fc fusion proteins using the same

[0001] The present invention relates to a chromatographic support for purifying an Fc fusion protein, and a method for purifying an Fc fusion protein using the same.

[0002] In the manufacturing process of biopharmaceuticals, including antibody drugs, the target pharmaceutical protein, such as an antibody, must be separated from the expression medium and purified to a purity acceptable for use as a therapeutic or diagnostic agent. Pharmaceutical proteins are generally produced through purification by affinity chromatography. The affinity support used in such chromatographic purification is required to have an improved binding capacity for the target protein and reduced nonspecific adsorption of impurities such as culture medium components.

[0003] Patent Document 1 discloses that a carrier for affinity chromatography is produced by polymerizing divinylbenzene, ethyldivinylbenzene, and glycidyl methacrylate, then crosslinking the polymer to obtain porous crosslinked particles, which are then sieved to have a predetermined particle size distribution, and then bound to a protein ligand, and that the dynamic binding capacity of the carrier for antibodies is improved.

[0004] International Publication No. 2022 / 202466

[0005] Fc fusion proteins are proteins in which an Fc fragment of an immunoglobulin is fused with a protein (such as the extracellular domain of a receptor) that has the ability to specifically bind to a target molecule, and are clinically used as molecularly targeted therapeutics. Fc fusion proteins are purified by affinity chromatography, similar to antibody drugs. However, because conventional affinity supports are primarily produced for the purpose of purifying antibodies, their use in purifying Fc fusion proteins has not achieved performance equivalent to that of antibody purification in terms of dynamic binding capacity (DBC) or impurity contamination. It is desirable to separate Fc fusion proteins with higher efficiency and purity. The present invention provides a chromatography support that enables the separation of highly pure Fc fusion proteins with a large DBC for Fc fusion proteins and fewer impurities, and a method for purifying Fc fusion proteins using the same.

[0006] The present invention provides the following as representative embodiments: [1] A chromatography support for purifying an Fc fusion protein, comprising porous particles having immobilized ligands, the porous particles being synthetic or natural polymer-based porous particles, the ligand being at least one selected from the group consisting of protein A, protein G, protein L, and related substances thereof, and the porous particles having immobilized ligands have an average pore diameter of 75 to 120 nm. [2] The chromatography support according to [1], wherein the porous particles having immobilized ligands have an average pore diameter of 101 to 110 nm. [3] The chromatography support according to [1] or [2], wherein the Fc fusion protein has a molecular weight of 160 to 300 kDa. [4] The chromatography support according to any one of [1] to [3], wherein the porous particles having immobilized ligands have a volume average particle diameter of 45 to 100 μm and a porosity of 70 to 95% (v / v). [5] The chromatography support according to any one of [1] to [4], wherein the Fc fusion protein is at least one selected from the group consisting of etanercept, rilonacept, and efmoroctocog alfa. [6] The chromatography support according to any one of [1] to [5], wherein the Fc fusion protein is an Fc fusion protein consisting of the amino acid sequence of SEQ ID NO: 4 or SEQ ID NO: 5, or an amino acid sequence having 85% or more identity to either of these sequences. [7] The chromatography support according to any one of [1] to [6], wherein the porous particles are a polymer of a monomer having a functional group capable of immobilizing a ligand and a polymerizable unsaturated group, and a polymerizable unsaturated group-containing monomer not having the functional group. [8] The chromatography support according to [7], wherein the polymerizable unsaturated group-containing monomer not having a functional group includes a non-crosslinkable monomer and a crosslinkable monomer.[9] A method for purifying an Fc fusion protein, comprising the following steps: (Step 1) passing a solution containing the Fc fusion protein through a column containing the chromatography support according to any one of [1] to [8] to bind the Fc fusion protein to the support; (Step 2) washing the support; and (Step 3) recovering the Fc fusion protein from the support.

[10] The method of [9], further comprising the following step: (Step 4) passing an alkaline solution through the column after step 3.

[11] The method of

[10] , comprising repeating steps 1 to 4 multiple times.

[0007] The chromatography support of the present invention has a large DBC for Fc fusion proteins and is excellent in the ability to separate the target Fc fusion proteins from contaminants. Therefore, the method for purifying Fc fusion proteins using the chromatography support of the present invention enables the separation of Fc fusion proteins with higher efficiency and higher purity.

[0008] All patents, non-patent documents, and other publications cited herein are hereby incorporated by reference in their entirety.

[0009] In this specification, the expression "A to B" or the like expressing a range of numerical values ​​is synonymous with "greater than or equal to A and less than or equal to B," and A and B are included in the range of numerical values.

[0010] As used herein, the term "ligand" in relation to affinity chromatography refers to a molecule that binds to a target substance in affinity chromatography. A "protein ligand" refers to a ligand in which the portion that binds to the target substance is composed of a protein.

[0011] As used herein, "Fc" and "Fc region" refer to the Fc region or Fc fragment of an immunoglobulin. As used herein, an "Fc-binding domain" refers to a functional unit of a polypeptide that has Fc-binding activity by itself. As used herein, an "Fc-binding protein" refers to a protein that contains the "Fc-binding domain" and has specific affinity for the Fc of an immunoglobulin.

[0012] As used herein, the term "Fc fusion protein" refers to a protein in which a heterologous protein is fused to an Fc fragment of immunoglobulin. The heterologous protein may be of human or non-human origin and is a protein other than immunoglobulin Fab. In this respect, Fc fusion proteins are distinguished from immunoglobulins such as IgG, IgA, IgD, IgE, and IgM, and antibodies and fragments thereof, including chimeric antibodies. Typically, the heterologous protein is a protein capable of specifically binding to a target molecule, such as an extracellular domain of a receptor, a receptor antagonist, or an enzyme. In clinical settings, Fc fusion proteins are used as molecularly targeted therapeutic agents for the treatment of inflammatory diseases such as rheumatoid arthritis and bleeding disorders such as hemophilia. Examples of molecularly targeted therapeutic agents using Fc fusion proteins include etanercept, rilonacept, and efmoroctocog alfa.

[0013] As used herein, "85% or greater identity" with respect to amino acid sequences refers to preferably 90% or greater identity, more preferably 94% or greater, even more preferably 96% or greater, even more preferably 98% or greater, and even more preferably 99% or greater identity. Amino acid sequence identity can be determined using the BLAST algorithm (Pro. Natl. Acad. Sci. USA., 1993, 90:5873-5877). Based on this BLAST algorithm, programs called BLASTN, BLASTX, BLASTP, TBLASTN, and TBLASTX have been developed (J. Mol. Biol., 1990, 215:403-410). When using these programs, the default parameters of each program can be used. Specific techniques for these analysis methods are known (see [www.ncbi.nlm.nih.gov]).

[0014] As used herein, a "corresponding position" in an amino acid sequence can be determined by aligning a target sequence with a reference sequence (e.g., the amino acid sequence of SEQ ID NO: 1) to maximize homology between conserved amino acid residues present in each amino acid sequence. Alignment can be performed using known algorithms, and the procedures are well known to those skilled in the art. For example, alignment can be performed using the Clustal W multiple alignment program (Thompson J.D. et al., Nucleic Acids Res., 1994, 22:4673-4680) with default settings. Clustal W is available, for example, on the website of the DNA Data Bank of Japan (DDBJ [www.ddbj.nig.ac.jp / index.html]), operated by the National Institute of Genetics. A position in a target sequence aligned to a given position in a reference sequence by the above-described alignment is considered to be a "position corresponding to" that given position.

[0015] As used herein, amino acid residues are also abbreviated as follows: alanine (Ala or A), arginine (Arg or R), asparagine (Asn or N), aspartic acid (Asp or D), cysteine ​​(Cys or C), glutamine (Gln or Q), glutamic acid (Glu or E), glycine (Gly or G), histidine (His or H), isoleucine (Ile or I), leucine (Leu or L), lysine (Lys or K), methionine (Met or M), phenylalanine (Phe or F), proline (Pro or P), serine (Ser or S), threonine (Thr or T), tryptophan (Trp or W), tyrosine (Tyr or Y), valine (Val or V), and any amino acid residue (Xaa or X). In this specification, the amino acid sequence of a peptide is written in accordance with conventional practice, with the amino terminus (hereinafter referred to as the N-terminus) on the left and the carboxyl terminus (hereinafter referred to as the C-terminus) on the right.

[0016] [Chromatography Support for Purifying Fc-Fusion Proteins] In one embodiment, the present invention provides a chromatography support for purifying Fc-fusion proteins, which comprises porous particles to which ligands capable of binding to Fc-fusion proteins are immobilized, and the support has a controlled pore size.

[0017] (Porous particles) The porous particles used in the chromatography carrier of the present invention may be natural polymer-based porous particles or synthetic polymer-based porous particles, but synthetic polymer-based porous particles are preferred in order to adjust the pore size of the carrier. In addition, the porous particles are preferably water-insoluble. The porous particles can be produced by suspending a monomer composition together with a porogen in an aqueous medium and polymerizing the monomer. The method for producing the porous particles used in the chromatography carrier of the present invention will be described below.

[0018] The monomer composition preferably contains a functional group-containing monomer. The functional group contained in the monomer is preferably one that can be used in additional chemical reactions (such as reactions with a crosslinking agent) and may be one that can immobilize a ligand. Examples of the functional group include functional groups selected from the group consisting of a cyclic ether group, a carboxy group, -C(=O)-O-C(=O)-, a succinimideoxycarbonyl group, a formyl group, a hydroxyl group, and an isocyanate group. Among these, a cyclic ether group is preferred. Here, the "cyclic ether group" is preferably a cyclic ether group having 3 to 7 atoms constituting the ring. The cyclic ether group may have an alkyl group as a substituent. Examples of the cyclic ether group include cyclic ether groups represented by the following formulas (1) to (6). Of these, cyclic ether groups represented by formulas (1), (3), or (6) are preferred, and a cyclic ether group represented by formula (1) is more preferred.

[0019]

[0020] [In the formula, R 1 ~R 4 each independently represents a hydrogen atom or an alkyl group, and * represents a bond.

[0021] R 1 ~R 4 The number of carbon atoms in the alkyl group represented by the formula (I) is preferably 1 to 4, and more preferably 1 or 2. The alkyl group may be linear or branched, and examples thereof include a methyl group, an ethyl group, an n-propyl group, an isopropyl group, an n-butyl group, a sec-butyl group, and a tert-butyl group. 1 ~R 4 is preferably a hydrogen atom.

[0022] The functional group-containing monomer is preferably a monomer having a functional group capable of immobilizing a ligand and a polymerizable unsaturated group. Examples of such monomers include glycidyl (meth)acrylate, 3-oxiranylpropyl (meth)acrylate, 4-oxiranylbutyl (meth)acrylate, 5-oxiranylpentyl (meth)acrylate, 6-oxiranylhexyl (meth)acrylate, 7-oxiranylheptyl (meth)acrylate, 8-oxiranyloctyl (meth)acrylate, (3-methyloxiranyl)methyl (meth)acrylate, 4-hydroxybutyl (meth)acrylate glycidyl ether, glycerin mono(meth)acrylate glycidyl ether, 3,4-epoxycyclohexylmethyl (meth)acrylate, 3,4-epoxycyclohexylethyl (meth)acrylate, 3,4-epoxycyclohexylpropyl (meth)acrylate, α-(meth)acryl-ω-glycidyl polyethylene glycol, tetrahydrofurfuryl (meth)acrylate, and other monomers having a cyclic ether group ( Examples of suitable methacrylate monomers include aromatic vinyl monomers having a cyclic ether group, such as (vinylbenzyl)glycidyl ether, (isopropenylbenzyl)glycidyl ether, (vinylphenethyl)glycidyl ether, (vinylphenylbutyl)glycidyl ether, (vinylbenzyloxyethyl)glycidyl ether, (vinylphenyl)glycidyl ether, (isopropenylphenyl)glycidyl ether, and 1,2-epoxy-3-(4-vinylbenzyl)propane; allyl ether monomers having a cyclic ether group, such as allyl glycidyl ether; (meth)acrylate monomers having an isocyanate group, such as isocyanatoethyl (meth)acrylate; unsaturated dicarboxylic acid anhydride monomers, such as maleic anhydride, methylmaleic anhydride, and glutaconic anhydride; (meth)acrylic acid, 3,4-epoxy-1-butene, and 3,4-epoxy-3-methyl-1-butene. These monomers may be used alone or in combination of two or more. Among these monomers, (meth)acrylate monomers having a cyclic ether group are preferred, and glycidyl (meth)acrylate is more preferred.

[0023] The total content of the functional group-containing monomers in the monomer composition is preferably 35 parts by mass or more, more preferably 45 parts by mass or more, and even more preferably 55 parts by mass or more, relative to 100 parts by mass of the total amount of monomers in the monomer composition, and on the other hand, is preferably 99 parts by mass or less, more preferably 90 parts by mass or less, and even more preferably 85 parts by mass or less, relative to 100 parts by mass of the total amount of monomers in the monomer composition.

[0024] The monomer composition may contain, in addition to the functional group-containing monomer, a monomer other than the functional group-containing monomer (hereinafter also referred to as "other monomer"). Examples of the other monomer include a polymerizable unsaturated group-containing monomer that does not have a functional group capable of immobilizing a ligand. The other monomer is roughly classified into a non-crosslinkable monomer and a crosslinkable monomer, and either one of them may be used alone or in combination.

[0025] Examples of non-crosslinkable monomers that can be used as the other monomer include (meth)acrylate-based non-crosslinkable monomers, (meth)acrylamide-based non-crosslinkable monomers, aromatic vinyl-based non-crosslinkable monomers, vinyl ketone-based non-crosslinkable monomers, (meth)acrylonitrile-based non-crosslinkable monomers, and N-vinylamide-based non-crosslinkable monomers. These can be used alone or in combination of two or more. Among the non-crosslinkable monomers, (meth)acrylate-based non-crosslinkable monomers and aromatic vinyl-based non-crosslinkable monomers are preferred.

[0026] Examples of the (meth)acrylate non-crosslinkable monomer include methyl (meth)acrylate, ethyl (meth)acrylate, n-butyl (meth)acrylate, 4-tert-butyl (meth)acrylate, isobutyl (meth)acrylate, n-octyl (meth)acrylate, 2-ethylhexyl (meth)acrylate, cyclohexyl (meth)acrylate, methoxyethyl (meth)acrylate, hydroxyethyl (meth)acrylate, hydroxypropyl (meth)acrylate, glycerol mono(meth)acrylate, trimethylolethane mono(meth)acrylate, trimethylolpropane mono(meth)acrylate, butanetriol mono(meth)acrylate, polyethylene glycol mono(meth)acrylate, methoxypolyethylene glycol (meth)acrylate, pentaerythritol mono(meth)acrylate, dipentaerythritol mono(meth)acrylate, and inositol mono(meth)acrylate. These may be used alone or in combination of two or more.

[0027] Examples of the (meth)acrylamide-based non-crosslinkable monomer include (meth)acrylamide, dimethyl(meth)acrylamide, hydroxyethyl(meth)acrylamide, (meth)acryloylmorpholine, diacetone(meth)acrylamide, etc. These may be used alone or in combination of two or more.

[0028] Examples of the aromatic vinyl non-crosslinkable monomer include styrenes such as styrene, α-methylstyrene, halogenated styrene, 4-methylstyrene, 2,4-dimethylstyrene, 2,4,6-trimethylstyrene, ethylvinylbenzene, 4-isopropylstyrene, 4-n-butylstyrene, 4-isobutylstyrene, and 4-tert-butylstyrene; vinylnaphthalenes such as 1-vinylnaphthalene and 2-vinylnaphthalene; and the like. These can be used alone or in combination of two or more.

[0029] Examples of the vinyl ketone-based non-crosslinkable monomer include ethyl vinyl ketone, propyl vinyl ketone, isopropyl vinyl ketone, etc. These may be used alone or in combination of two or more.

[0030] Examples of the (meth)acrylonitrile-based non-crosslinkable monomer include acrylonitrile, methacrylonitrile, etc. These may be used alone or in combination of two or more.

[0031] Examples of the N-vinylamide non-crosslinkable monomer include N-vinylacetamide, N-vinylpropionamide, etc. These may be used alone or in combination of two or more.

[0032] The total content of the non-crosslinkable monomers in the monomer composition is preferably 0.01 parts by mass or more, more preferably 0.05 parts by mass or more, and even more preferably 0.1 parts by mass or more, relative to 100 parts by mass of the total amount of monomers in the monomer composition, and on the other hand, is preferably 30 parts by mass or less, more preferably 15 parts by mass or less, and even more preferably 5 parts by mass or less, relative to 100 parts by mass of the total amount of monomers in the monomer composition.

[0033] Examples of crosslinkable monomers that can be used as the other monomer include (meth)acrylate crosslinkable monomers, aromatic vinyl crosslinkable monomers, and allyl crosslinkable monomers. These can be used alone or in combination of two or more. As the crosslinkable monomer, di- to penta-functional crosslinkable monomers are preferred, and di- or tri-functional crosslinkable monomers are more preferred. Among these crosslinkable monomers, (meth)acrylate crosslinkable monomers and aromatic vinyl crosslinkable monomers are preferred.

[0034] Examples of the (meth)acrylate crosslinkable monomer include ethylene glycol di(meth)acrylate, diethylene glycol di(meth)acrylate, triethylene glycol di(meth)acrylate, tetraethylene glycol di(meth)acrylate, polyethylene glycol di(meth)acrylate, propylene glycol di(meth)acrylate, dipropylene glycol di(meth)acrylate, tripropylene glycol di(meth)acrylate, tetrapropylene glycol di(meth)acrylate, polypropylene glycol di(meth)acrylate, 1,4-butanediol di(meth)acrylate, 1,6-hexanediol di(meth)acrylate, glycerin di(meth)acrylate, trimethylolethane di(meth)acrylate, trimethylolpropane di(meth)acrylate, and trimethylolpropane tri(meth)acrylate. acrylate, butanetriol di(meth)acrylate, pentaerythritol di(meth)acrylate, pentaerythritol tri(meth)acrylate, pentaerythritol tetra(meth)acrylate, glucose di(meth)acrylate, glucose tri(meth)acrylate, glucose tetra(meth)acrylate, dipentaerythritol di(meth)acrylate, dipentaerythritol tri(meth)acrylate, dipentaerythritol tetra(meth)acrylate, dipentaerythritol penta(meth)acrylate, inositol di(meth)acrylate, inositol tri(meth)acrylate, inositol tetra(meth)acrylate, mannitol di(meth)acrylate, mannitol tri(meth)acrylate, mannitol tetra(meth)acrylate, mannitol penta(meth)acrylate, etc. These can be used alone or in combination of two or more.

[0035] Examples of the aromatic vinyl crosslinkable monomer include divinylbenzene, trivinylbenzene, divinyltoluene, divinylxylene, divinylethylbenzene, divinylnaphthalene, etc. These may be used alone or in combination of two or more.

[0036] Examples of the allyl crosslinkable monomer include diallyl phthalate, diallyl isophthalate, diallyl terephthalate, diallyl maleate, diallyl fumarate, diallyl itaconate, diallyl trimellitate, triallyl trimellitate, triallyl cyanurate, diallyl isocyanurate, triallyl isocyanurate, etc. These may be used alone or in combination of two or more.

[0037] Furthermore, examples of the crosslinkable monomer include, in addition to the above-mentioned examples, dehydration condensation reaction products of amino alcohols such as diaminopropanol, trishydroxymethylaminomethane, and glucosamine with (meth)acrylic acid, and conjugated diolefins such as butadiene and isoprene.

[0038] The total content of the crosslinkable monomers in the monomer composition is preferably 1 part by mass or more, more preferably 5 parts by mass or more, and even more preferably 10 parts by mass or more, relative to 100 parts by mass of the total amount of monomers in the monomer composition, and on the other hand, is preferably 50 parts by mass or less, more preferably 40 parts by mass or less, and even more preferably 30 parts by mass or less, relative to 100 parts by mass of the total amount of monomers in the monomer composition.

[0039] Examples of the aqueous medium used in producing the porous particles include aqueous solutions of water-soluble polymers. Examples of the water-soluble polymers include hydroxyethyl cellulose, polyvinyl alcohol, carboxymethyl cellulose, polyvinylpyrrolidone, starch, and gelatin. The total amount of the aqueous medium used in producing the porous particles is usually about 200 to 7,000 parts by mass per 100 parts by mass of the total amount of monomers. When water is used as the dispersion medium for the aqueous medium, a dispersion stabilizer such as sodium carbonate, calcium carbonate, sodium sulfate, calcium phosphate, or sodium chloride may be used.

[0040] In the production of the porous particles, the porosifying agent exists as a non-polymerized component during the polymerization of the monomer and serves to form pores in the particles obtained by the polymerization. The porosifying agent is not particularly limited as long as it can be easily removed from the surface of the produced porous particles. Examples of the porosifying agent include linear polymers soluble in various organic solvents and mixed monomers, and these may be used in combination.

[0041] Examples of the porosifying agent include aliphatic hydrocarbons such as hexane, heptane, octane, nonane, decane, and undecane; alicyclic hydrocarbons such as cyclopentane and cyclohexane; aromatic hydrocarbons such as benzene, toluene, xylene, naphthalene, ethylbenzene, and polystyrene; halogenated hydrocarbons such as carbon tetrachloride, 1,2-dichloroethane, tetrachloroethane, and chlorobenzene; aliphatic alcohols such as butanol, pentanol, hexanol, heptanol, 4-methyl-2-pentanol, and 2-ethyl-1-hexanol; alicyclic alcohols such as cyclohexanol; 2-phenylethyl alcohol, benzyl alcohol, and the like. Examples of suitable porosifying agents include aromatic alcohols such as ethanol; ketones such as diethyl ketone, methyl isobutyl ketone, diisobutyl ketone, acetophenone, 2-octanone, and cyclohexanone; ethers such as dibutyl ether, diisobutyl ether, anisole, ethoxybenzene, polyethylene glycol, and polypropylene glycol; esters such as isopentyl acetate, butyl acetate, 3-methoxybutyl acetate, and diethyl malonate; and linear polymers such as polysaccharides such as methyl cellulose, ethyl cellulose, acetyl cellulose, cellulose acetate, cellulose triacetate, and alkyl cellulose, and homopolymers of non-crosslinkable vinyl monomers. These porosifying agents can be used alone or in combination of two or more.

[0042] The weight-average molecular weight of the porogen is preferably 40,000 or more, more preferably 50,000 or more, and preferably 180,000 or less, more preferably 100,000 or less. The total amount of the porogen used in producing the porous particles is preferably 40 parts by mass or more, more preferably 70 parts by mass or more, and preferably 600 parts by mass or less, more preferably 400 parts by mass or less, per 100 parts by mass of the total amount of monomers. The weight-average molecular weight and amount of the porogen can affect the pore size of the ligand-immobilized porous particles described below. For example, as the weight-average molecular weight of the porogen decreases or the amount used decreases, the pore size of the ligand-immobilized porous particles tends to decrease. The pore size of the porous particles can be adjusted by adjusting the molecular weight and amount of the porogen used.

[0043] Specific methods for producing porous particles used in the present invention include, for example, dissolving a polymerization initiator in a mixed solution (monomer solution) containing the monomer composition and a porosifying agent, suspending the mixture in the aqueous medium, and polymerizing the mixture to a predetermined temperature; dissolving a polymerization initiator in a mixed solution (monomer solution) containing the monomer composition and a porosifying agent, and adding the mixture to the aqueous medium heated to a predetermined temperature to polymerize the mixture; suspending the mixed solution (monomer solution) containing the monomer composition and a porosifying agent in the aqueous medium, heating the mixture to a predetermined temperature, and adding a polymerization initiator to polymerize the mixture. When using some polysaccharides such as ethyl cellulose as the porosifying agent, it is preferable to heat and stir the porosifying agent in advance, then mix it with the monomer composition to prepare a monomer solution, and then add a polymerization initiator to the mixture.

[0044] The polymerization initiator is preferably a radical polymerization initiator. Examples of the radical polymerization initiator include azo initiators, peroxide initiators, and redox initiators. Specific examples include azobisisobutyronitrile, methyl azobisisobutyrate, azobis-2,4-dimethylvaleronitrile, benzoyl peroxide, di-tert-butyl peroxide, and benzoyl peroxide-dimethylaniline. The total amount of the polymerization initiator used in producing the porous particles is usually about 0.01 to 10 parts by mass per 100 parts by mass of the total amount of monomers.

[0045] Various surfactants, including anionic surfactants such as alkyl sulfates, alkylaryl sulfates, alkyl phosphates, and fatty acid salts, may be used to produce the porous particles. Nitrite salts such as sodium nitrite, iodide salts such as potassium iodide, and polymerization inhibitors such as tert-butylpyrocatechol, benzoquinone, picric acid, hydroquinone, copper chloride, and ferric chloride may also be used. Polymerization regulators such as dodecyl mercaptan may also be used.

[0046] In the production of the porous particles, the polymerization temperature of the monomer may be determined depending on the polymerization initiator, but is usually about 2 to 100° C., preferably 50 to 100° C. The polymerization time of the monomer is usually 5 minutes to 48 hours, preferably 10 minutes to 24 hours.

[0047] The resulting porous particles may be reacted with at least one selected from the group consisting of a crosslinking agent and a hydrophilizing agent. When both a crosslinking agent and a hydrophilizing agent are used, the crosslinking reaction may be performed after the hydrophilizing reaction, or the hydrophilizing reaction may be performed after the crosslinking reaction. The crosslinking reaction and the hydrophilizing reaction may also be performed simultaneously. Alternatively, the crosslinking reaction and the hydrophilizing reaction of the porous particles may each be performed in parallel with the polymerization reaction of the monomers using the polymerization initiator. When a monomer composition containing a functional group-containing monomer is used, the crosslinking reaction involves an addition reaction of the crosslinking agent with some of the functional groups present in the polymer molecules of the porous particles, thereby introducing a partial structure derived from the crosslinking agent. This crosslinks the residues of the functional groups via the partial structure derived from the crosslinking agent. When a monomer composition containing a functional group-containing monomer is used, the hydrophilizing reaction involves an addition reaction of the hydrophilizing agent with some of the functional groups present in the polymer molecules of the porous particles, thereby introducing a partial structure derived from the hydrophilizing agent.

[0048] The crosslinking agent used in the crosslinking reaction may be any agent capable of reacting with a functional group capable of immobilizing a ligand to introduce a crosslinked structure. A crosslinking agent capable of reacting with a functional group capable of immobilizing a ligand to introduce a crosslinked structure and containing at least two groups represented by —C(═O)—NH— in the molecule is preferred.

[0049] When the porous particles have a cyclic ether group, it is preferable to use a crosslinking agent containing at least two groups represented by -C(=O)-NH-NH2 as crosslinkable groups in the molecule, or a crosslinking agent containing at least two groups represented by -C(=O)-NH- in the molecule and at least two carboxy groups as crosslinkable groups in the molecule. Furthermore, when the porous particles have a carboxy group, -C(=O)-O-C(=O)-, succinimidooxycarbonyl group, formyl group, or isocyanate group, it is preferable to use a crosslinking agent containing at least two groups represented by -C(=O)-NH-NH2 as crosslinkable groups in the molecule.

[0050] Examples of crosslinking agents containing at least two groups represented by -C(=O)-NH-NH in the molecule include dicarboxylic acid dihydrazides such as oxalyl dihydrazide, malonic acid dihydrazide, succinic acid dihydrazide, 2,3-dihydroxysuccinic acid dihydrazide, glutaric acid dihydrazide, adipic acid dihydrazide, pimelic acid dihydrazide, octanedioic acid dihydrazide, nonanedioic acid dihydrazide, sebacic acid dihydrazide, dodecanedioic acid dihydrazide, phthalic acid dihydrazide, isophthalic acid dihydrazide, terephthalic acid dihydrazide, and quinolinic acid dihydrazide; and tricarboxylic acid trihydrazides such as cyclohexanetricarboxylic acid trihydrazide. Examples of crosslinking agents containing at least two groups represented by -C(=O)-NH- in the molecule and at least two carboxy groups as crosslinkable groups in the molecule include (alkylenebisimino)bis(oxoalkanoic acids) such as N1,N1-(ethane-1,2-diyl)bis(succinic acid monoamide). These crosslinking agents can be used alone or in combination of two or more. Among these crosslinking agents, dicarboxylic acid dihydrazides and (alkylenebisimino)bis(oxoalkanoic acids) are preferred, with dicarboxylic acid dihydrazides being more preferred, in order to improve the liquid permeability of the chromatography support, or the pressure resistance characteristics and antifouling properties during liquid passage.

[0051] In the crosslinking reaction, a crosslinking agent other than the above-mentioned crosslinking agents can also be used, such as a polyfunctional isocyanate-based crosslinking agent, a polyfunctional epoxy-based crosslinking agent, a polyfunctional aldehyde-based crosslinking agent, a polyfunctional thiol-based crosslinking agent, a polyfunctional oxazoline-based crosslinking agent, a polyfunctional aziridine-based crosslinking agent, a metal chelate-based crosslinking agent, etc.

[0052] The total amount of the crosslinking agent used in the crosslinking reaction is preferably 0.01 molar equivalents or more, more preferably 0.05 molar equivalents or more, and even more preferably 0.1 molar equivalents or more, relative to 1 mole of the functional group derived from the functional group-containing monomer used in producing the porous particles, and is preferably up to 0.8 molar equivalents or less, more preferably 0.7 molar equivalents or less, and even more preferably 0.6 molar equivalents or less.

[0053] As the hydrophilizing agent used in the hydrophilization reaction, in order to improve the antifouling properties or low ligand leakage properties of the chromatography support, a compound having a total of two or more hydrophilic groups selected from hydroxyl groups and mercapto groups in the molecule is preferred, and a compound having a total of 2 to 4 hydrophilic groups selected from hydroxyl groups and mercapto groups in the molecule is more preferred. Examples of the hydrophilizing agent include alcohols having a mercapto group in the molecule, such as mercaptoethanol and thioglycerol; and polyhydric alcohols, such as glycerol and diglycerol. These hydrophilizing agents can be used alone or in combination of two or more. Among these, in order to improve the antifouling properties or low ligand leakage properties of the support, alcohols having a mercapto group in the molecule are preferred, and thioglycerol is more preferred.

[0054] The total amount of the hydrophilizing agent used in the hydrophilization reaction is preferably 0.5 molar equivalents or more, more preferably 1 molar equivalent or more, and even more preferably 2 molar equivalents or more, relative to 1 mole of the functional group derived from the functional group-containing monomer used in producing the porous particles, and is preferably 10 molar equivalents or less, more preferably 8 molar equivalents or less, and even more preferably 6 molar equivalents or less.

[0055] The crosslinking reaction or hydrophilization reaction may be carried out in the presence of a basic catalyst, such as triethylamine, N,N-dimethyl-4-aminopyridine, sodium hydroxide, or diisopropylethylamine, which may be used singly or in combination of two or more.

[0056] The reaction time for the crosslinking reaction or the hydrophilization reaction is not particularly limited, but is usually about 0.5 to 72 hours, preferably 0.5 to 48 hours. The reaction temperature may be appropriately selected as long as it is equal to or lower than the boiling point of the solvent, but is usually about 2 to 100°C.

[0057] (Ligand) The ligand immobilized on the porous particles is a protein ligand capable of binding to an Fc fusion protein, and is preferably one or more protein ligands selected from the group consisting of Protein A, Protein G, Protein L, and their analogs. The ligand is preferably Protein A or a Protein A analog, and more preferably a Protein A analog.

[0058] Protein A contains five domains, E, D, A, B, and C, which have the ability to bind to the Fc of immunoglobulin and are therefore Fc-binding domains. Preferably, the Protein A analog used as a ligand in the present invention is an Fc-binding protein comprising the Fc-binding domain of Protein A or a modified Fc-binding domain thereof. Preferably, the ligand of the present invention comprises one or more Fc-binding domains selected from the group consisting of the B domain, C domain, or modified domains thereof of Protein A. More preferably, the ligand comprises one or more Fc-binding domains selected from the group consisting of the C domain of Protein A consisting of the amino acid sequence of SEQ ID NO: 1 or modified domains thereof. Even more preferably, the Fc-binding domain contained in the ligand is a modified domain of the C domain of Protein A consisting of the amino acid sequence of SEQ ID NO: 1.

[0059] In order to improve DBC for an Fc fusion protein or to suppress leakage of the ligand in a chromatography support, it is preferable that the ligand comprises an Fc-binding domain consisting of an amino acid sequence in which one or more of the following substitutions selected from the group consisting of (a) to (i) have been made with respect to the amino acid sequence of SEQ ID NO: 1 or an amino acid sequence having 85% or more identity thereto (amino acid sequence of a parent Fc-binding domain): (a) substitution of the amino acid residue at position 1 of the amino acid sequence of SEQ ID NO: 1 with a valine residue; (b) substitution of the amino acid residue at position 3 of the amino acid sequence of SEQ ID NO: 1 with an alanine residue; (c) substitution of the amino acid residue at position 6 of the amino acid sequence of SEQ ID NO: 1 with an alanine residue or an aspartic acid residue; (d) substitution of the amino acid residue at position 9 of the amino acid sequence of SEQ ID NO: 1 with an alanine residue; (e) substitution of the amino acid residue at position 11 of the amino acid sequence of SEQ ID NO: 1 with an alanine residue, glutamine residue, or glutamic acid residue; (f) substitution of the amino acid residue at the position corresponding to position 23 in the amino acid sequence of SEQ ID NO: 1 with a leucine residue; (g) substitution of the amino acid residue at the position corresponding to position 29 in the amino acid sequence of SEQ ID NO: 1 with an alanine residue; (h) substitution of the amino acid residue at the position corresponding to position 43 in the amino acid sequence of SEQ ID NO: 1 with an alanine residue; (i) substitution of the amino acid residue at the position corresponding to position 49 in the amino acid sequence of SEQ ID NO: 1 with an arginine residue.

[0060] Preferably, the Fc-binding domain contained in the ligand consists of an amino acid sequence in which three or more substitutions, more preferably 3 to 9 substitutions, even more preferably 3 to 6 substitutions, and even more preferably 3 to 5 substitutions selected from the group consisting of (a) to (i) above have been made relative to the amino acid sequence of the parent Fc-binding domain.

[0061] Preferred examples of substitutions in the Fc-binding domain contained in the ligand include the following: three or more substitutions selected from the group consisting of (a), (b), (c), (d), (g), (h), and (i); three or more substitutions including a combination of two or more substitutions selected from the group consisting of (a), (b), (c), (d), (g), (h), and (i) with one or more substitutions selected from the group consisting of (e) and (f); four or more substitutions including a combination of three or more substitutions selected from the group consisting of (a), (b), (c), (d), (g), (h), and (i) with one or more substitutions selected from the group consisting of (e) and (f); three or more substitutions including a combination of one or more substitutions selected from the group consisting of (a) and (d), one or more substitutions selected from the group consisting of (e) and (f), and one or more substitutions selected from the group consisting of (b), (c), (g), (h), and (i). Specific examples of such substitutions include: a combination of (a), (b), and (g); a combination of (a), (c), and (g); a combination of (a), (b), (c), and (g); a combination of (a), (d), and (g); a combination of (a), (e), and (g); a combination of (d), (e), and (h); a combination of (a), (e), (f), and (g); a combination of (a), (e), (g), and (i); a combination of (a), (b), (c), (e), and (g); a combination of (a), (b), (c), (e), (g), and (i).

[0062] The Fc-binding domain contained in the ligand used in the present invention can be prepared according to known techniques. For example, the Fc-binding domain can be prepared by expressing a polynucleotide encoding it. The polynucleotide encoding the Fc-binding domain can be isolated from cells or chemically synthesized. For example, a polynucleotide encoding an Fc-binding domain consisting of the amino acid sequence of SEQ ID NO: 1 can be isolated from a microorganism that expresses protein A (e.g., Staphylococcus aureus). Desired mutations can also be introduced into the isolated polynucleotide using known methods. For example, an Fc-binding domain containing the above-mentioned amino acid residue substitutions can be prepared by introducing mutations into a polynucleotide encoding a parent domain (e.g., an Fc-binding domain consisting of SEQ ID NO: 1 or an amino acid sequence having 85% or more identity thereto) so that the desired amino acid residue substitution occurs. Specific techniques for introducing mutations into polynucleotides include site-specific mutagenesis, homologous recombination, and SOE (splicing by overlap extension)-PCR (Gene, 1989, 77:61-68), and detailed procedures for these are well known to those skilled in the art.

[0063] The ligand used in the present invention may contain one or more Fc-binding domains, preferably two or more, more preferably 2 to 12, and even more preferably 4 to 7. When the ligand contains two or more Fc-binding domains, the domains may be the same or different. Preferably, the ligand is an Fc-binding protein in which the amino acid sequences of two or more Fc-binding domains are linked in a linear chain. Here, "linearly linked" amino acid sequences refers to a structure in which two or more amino acid sequences are linked in series with or without a linker. For example, when a linker is used, "linearly linked" refers to a structure in which the C-terminus of one amino acid sequence and the N-terminus of another amino acid sequence are linked in series with a linker. On the other hand, when a linker is not used, "linearly linked" refers to a structure in which the C-terminus of one amino acid sequence and the N-terminus of another amino acid sequence are linked in series by a peptide bond.

[0064] The ligand can be produced using known genetic recombination techniques such as those described in "Current Protocols in Molecular Biology" by Frederick M. Ausbel et al., or "Molecular Cloning" edited by Sambrook et al. (Cold Spring Harbor Laboratory Press, 3rd edition, 2001). For example, an expression vector containing a polynucleotide encoding the Fc-binding protein can be transformed into host cells, and the resulting recombinant can be cultured in an appropriate liquid medium, allowing the target protein ligand to be isolated from the cultured cells. Preferred expression vectors include any known vectors that can replicate in host cells, such as those described in U.S. Pat. No. 5,151,350 and those described in Molecular Cloning (edited by Sambrook et al.) (Cold Spring Harbor Laboratory Press, 3rd edition, 2001). The host for transformation is not particularly limited, and known hosts used to express recombinant proteins, such as bacteria such as Escherichia coli, fungal cells, insect cells, and mammalian cells, can be used. To introduce a polynucleotide into a host to transform it, any method known in the art can be used depending on the host. For example, known methods described in Molecular Cloning (edited by Sambrook et al.) (Cold Spring Harbor Laboratory Press, 3rd edition, 2001) can be used. Methods for culturing the resulting transformants (preferably cells such as bacteria) and recovering the expressed protein are well known to those skilled in the art. Alternatively, the Fc-binding proteins of the present invention may be expressed using a cell-free protein synthesis system.

[0065] (Immobilization of Ligand to Porous Particles) The immobilization of the ligand to the porous particles may be carried out according to a conventional method. A preferred method for immobilizing the ligand to the particles is a chemical bonding method, for example, a method in which the ligand is bonded to a functional group contained in the porous particles that can immobilize the ligand. Specific examples include a method in which a cyclic ether group, a carboxy group, -C(=O)-O-C(=O)-, a formyl group, or the like contained in the porous particles is bonded to an amino group or the like of the ligand. This method may be carried out with reference to the descriptions in WO 2015 / 119255, WO 2015 / 041218, and the like.

[0066] The ligand can be immobilized on the porous particles via a linker (spacer). Introduction of a linker to the porous particles and immobilization of the ligand using the linker can be performed with reference to U.S. Pat. No. 5,260,373, JP-A-2010-133733, and JP-A-2010-133734. Examples of compounds that provide such linkers include diglycidyl ethers of aliphatic polyhydroxy compounds such as ethylene glycol diglycidyl ether, diethylene glycol diglycidyl ether, polyethylene glycol diglycidyl ether, 1,2-propylene glycol diglycidyl ether, dipropylene glycol diglycidyl ether, polypropylene glycol diglycidyl ether, 1,4-butanediol diglycidyl ether, neopentyl glycol diglycidyl ether, 1,6-hexanediol diglycidyl ether, and glycerol diglycidyl ether; and polyglycidyl ethers of aliphatic polyhydroxy compounds such as sorbitol polyglycidyl ether, glycerol polyglycidyl ether, trimethylolpropane polyglycidyl ether, diglycerol polyglycidyl ether, and polyglycerol polyglycidyl ether. Among these, diglycidyl ethers of aliphatic polyhydroxy compounds are preferred when the aforementioned hydrophilization reaction of porous particles is carried out.

[0067] In order to increase the reaction efficiency, the linker introduction reaction and the ligand immobilization reaction onto the porous particles are preferably carried out in a buffer having a pH of 7 to 14. In the linker introduction reaction, the reaction time is not particularly limited, but is usually about 0.5 to 72 hours, and the reaction temperature may be appropriately selected below the boiling point of the solvent, but is usually about 2 to 100°C. In the ligand immobilization reaction, the reaction time is not particularly limited, but is usually about 0.1 to 72 hours, and the reaction temperature may be appropriately selected below the boiling point of the solvent, but is usually about 2 to 100°C.

[0068] Alternatively, the ligand may be immobilized on the porous particle by using a method of controlling the orientation of the ligand (U.S. Pat. No. 6,399,750; Ljungquist C et al., Eur. J. Biochem., 1989, 186: 557-561), a method of accumulating the ligand on the porous particle by an associative group (JP 2011-256176 A), or the like.

[0069] In order to increase the DBC, the amount of ligand immobilized on the porous particles is preferably 10 mg or more, more preferably 25 mg or more, per gram of dry weight of the porous particles, and is preferably 300 mg or less, more preferably 150 mg or less.

[0070] The resulting ligand-immobilized porous particles are preferably hydrophilized to improve the antifouling properties or low ligand leakage of the chromatography support. In the hydrophilization reaction, the ligand-immobilized porous particles are reacted with a compound having a hydrophilic group. The compound having a hydrophilic group is preferably a compound having a total of 2 to 4 hydrophilic groups in the molecule, each of which is at least one type of hydrophilic group selected from the group consisting of a hydroxy group and a mercapto group. Examples of such compounds include alcohols having a mercapto group in the molecule, such as mercaptoethanol and thioglycerol; and polyhydric alcohols, such as glycerol and diglycerol. These compounds can be used alone or in combination of two or more. Among these compounds, alcohols having a mercapto group in the molecule are preferred, and thioglycerol is more preferred, to improve the antifouling properties or low ligand leakage of the support.

[0071] The total amount of the compounds having hydrophilic groups used in the hydrophilization reaction is preferably 1 part by mass or more, more preferably 10 parts by mass or more, and even more preferably 100 parts by mass or more, relative to 100 parts by mass of the ligand-immobilized porous particles (dry weight), and is preferably 1,000 parts by mass or less, more preferably 800 parts by mass or less, and even more preferably 600 parts by mass or less.

[0072] The hydrophilization reaction may be carried out in the presence of a basic catalyst, such as triethylamine, N,N-dimethyl-4-aminopyridine, sodium hydroxide, or diisopropylethylamine, which may be used alone or in combination of two or more.

[0073] In the hydrophilization reaction, the reaction time is not particularly limited, but is usually about 0.5 to 72 hours, preferably 0.5 to 48 hours. The reaction temperature may be appropriately selected so long as it is equal to or lower than the boiling point of the solvent, but is usually about 2 to 100°C.

[0074] The resulting ligand-immobilized porous particles can be purified by separation means such as filtration, washing, etc. The ligand-immobilized porous particles may also be classified.

[0075] The ligand-immobilized porous particles have a volume-average particle diameter of preferably 40 to 150 μm, more preferably 45 to 100 μm, even more preferably 50 to 100 μm, and even more preferably 50 to 80 μm. When the ligand-immobilized porous particles have a volume-average particle diameter within the above range, the DBC for Fc fusion proteins and the pressure resistance characteristics during liquid passage are improved. The coefficient of variation of the volume-average particle diameter is preferably 40% or less, more preferably 30% or less. Furthermore, the chromatography carrier of the present invention has a specific surface area of ​​preferably 1 to 500 m 2 / g, more preferably 10 to 300 m 2 / g. In this specification, the volume average particle size of the ligand-immobilized porous particles refers to the volume average particle size measured by laser diffraction scattering in accordance with JIS Z 8825 (2013). Specifically, the volume average particle size of the particles can be determined by measuring the volume-based particle size distribution of the particles using a laser diffraction scattering particle size distribution analyzer (e.g., LS13320 manufactured by Beckman Coulter, Inc.) in accordance with JIS Z 8825 (2013), as described in the Examples below. Furthermore, in this specification, the specific surface area of ​​the chromatography support refers to a value measured by laser diffraction / scattering particle size distribution measurement or the like.

[0076] (Application to Fc fusion protein purification) The ligand-immobilized porous particles used in the present invention have an average pore size adjusted to fall within a specific range, thereby improving the ability to purify Fc fusion proteins. The average pore size of the ligand-immobilized porous particles is preferably 75 nm or more, more preferably 101 nm or more, and is preferably 120 nm or less, more preferably 110 nm or less. Having an average pore size within the above range, the ligand-immobilized porous particles improve DBC for Fc fusion proteins and pressure resistance characteristics during liquid flow.

[0077] In this specification, the average pore diameter is calculated according to the method described by Hagel et al. (Journal of Chromatography A, 1996, Vol. 743: 32-42). Specifically, the average pore diameter may be calculated according to the method described in the Examples below. That is, a plot of the molecular size of pullulan versus the partition coefficient Kd is prepared, an approximation line is drawn, and since Kd = ε (1 - molecular size / average pore size)^(1 / 2) holds, the average pore diameter is calculated from the intercept and slope. The partition coefficient Kd of pullulan is expressed as Kd = (elution volume of pullulan - elution volume of dextran) / (elution volume of sodium chloride - elution volume of dextran). The elution volumes of pullulan, dextran, and sodium chloride can be measured by passing a 20 mM sodium phosphate / 150 mM sodium chloride aqueous solution containing pullulan, a 20 mM sodium phosphate / 150 mM sodium chloride aqueous solution containing dextran, and a 500 mM sodium chloride aqueous solution through a column packed with the ligand-immobilized porous particles, respectively.

[0078] The ligand-immobilized porous particles preferably have a porosity in a wet state of 70 to 95% (v / v), more preferably 75 to 85% (v / v). When the porosity is within the above range, the ligand-immobilized porous particles exhibit improved DBC for Fc fusion proteins and improved pressure resistance during liquid passage. Herein, the porosity of the ligand-immobilized porous particles in a wet state refers to the ratio of the pore volume (volume of the pores) to the particle volume (the total volume of the non-pores, pores, and ligand portions) of the ligand-immobilized porous particles measured in a wet state. For example, the porosity can be determined based on the elution volume of a 500 mM sodium chloride aqueous solution from a column packed with the ligand-immobilized porous particles and the elution volume of a 20 mM sodium phosphate / 150 mM sodium chloride aqueous solution containing dextran from the column.

[0079] Specifically, the average pore size and porosity in a wet state of the ligand-immobilized porous particles may be measured according to the method described in the Examples below.

[0080] When used to purify Fc fusion proteins, the ligand-immobilized porous particles can exhibit high performance in terms of high DBC and low nonspecific binding (contamination by impurities). Considering that conventional supports did not achieve high performance when used to purify Fc fusion proteins, this indicates that the chromatography support of the present invention has a high ability to purify Fc fusion proteins. Therefore, the ligand-immobilized porous particles can be used as a chromatography support for purifying Fc fusion proteins.

[0081] [Chromatography column] A chromatography column comprising the chromatography support of the present invention is provided. The chromatography column of the present invention is similar to a conventional chromatography column except that it comprises the chromatography support of the present invention. Specifically, the chromatography column includes a column container and the chromatography support of the present invention packed in the column container. The chromatography column of the present invention is suitable for use in affinity chromatography.

[0082] [Method for purifying Fc fusion proteins] In one embodiment, the present invention provides a method for purifying Fc fusion proteins using the aforementioned chromatography support of the present invention. The method for purifying Fc fusion proteins according to the present invention can be carried out according to a general procedure for purifying antibodies by affinity chromatography, except that the chromatography support of the present invention is used.

[0083] In a preferred embodiment, the method for purifying an Fc fusion protein according to the present invention comprises the steps of passing a solution containing the Fc fusion protein through a column containing the chromatography support of the present invention to allow the Fc fusion protein to bind to the support (Step 1); washing the support (Step 2); and recovering the Fc fusion protein from the support (Step 3).

[0084] In step 1, a solution (sample solution) containing an Fc fusion protein is passed through a column containing the chromatography carrier under conditions in which the Fc fusion protein binds to the protein ligand of the carrier. Examples of the sample solution include an extract from a culture of cells expressing the Fc fusion protein, or a buffer solution containing the extract. Conditions in which the Fc fusion protein binds include, for example, a protein concentration of 0.1 to 10 g / L, a solution pH of 5 to 9, a column residence time of 0.5 to 50 minutes, and a temperature of 0 to 40°C.

[0085] In step 1, most of the substances in the sample solution other than the Fc fusion protein pass through the column. In step 2, impurities remaining in the column (e.g., proteins other than the Fc fusion protein in the sample solution) are removed by washing. For this washing, the packing material can be washed with a neutral buffer containing a salt such as NaCl, such as a sodium phosphate / sodium chloride solution, sodium dihydrogen phosphate / disodium hydrogen phosphate solution, citric acid / disodium hydrogen phosphate solution, hydrochloric acid / tris(hydroxymethyl)aminomethane solution, or HEPES / sodium hydroxide solution.

[0086] In step 3, the purified Fc fusion protein is recovered by passing an eluent through the column to elute the Fc fusion protein from the column. The eluent may be a buffer solution of pH 2 to 5, such as a citric acid / sodium citrate solution, an acetic acid / sodium acetate solution, or a hydrochloric acid / glycine solution.

[0087] The Fc fusion protein contained in the eluate eluted from the column in step 3 may be further purified. The Fc fusion protein can be purified using, for example, cation exchange chromatography, anion exchange chromatography, mixed-mode chromatography, hydrophilic interaction chromatography, hydrophobic interaction chromatography, size exclusion chromatography, etc., either alone or in appropriate combination.

[0088] The type of Fc fusion protein targeted in the Fc fusion protein purification method of the present invention is not particularly limited, but one with a molecular weight of 160 to 300 kDa is preferred. Preferred examples of target Fc fusion proteins include etanercept, rilonacept, and efmoroctocog alfa, with etanercept being more preferred. Other preferred examples of target Fc fusion proteins include biosimilars of etanercept, rilonacept, or efmoroctocog alfa, such as Fc fusion proteins that share 85% or more amino acid sequence identity with etanercept, rilonacept, or efmoroctocog alfa and whose region corresponding to the Fc fragment has binding affinity to the ligand contained in the chromatography support of the present invention. The amino acid sequence of etanercept can be represented by SEQ ID NO: 4. The amino acid sequence of rilonacept can be represented by SEQ ID NO: 5. Therefore, an Fc fusion protein consisting of the amino acid sequence of SEQ ID NO: 4 or SEQ ID NO: 5, or an amino acid sequence having 85% or more identity to either of these sequences, is a preferred example of the target Fc fusion protein.

[0089] The method for purifying an Fc fusion protein according to the present invention may further comprise, after step 3, a step (step 4) of passing an alkaline solution through the chromatography support. Examples of alkaline solutions used in step 4 include aqueous sodium hydroxide, aqueous potassium hydroxide, triethylamine, and tetrabutylammonium hydroxide. The molar concentration of the alkali salt in the alkaline solution used in step 4 is preferably 0.01 to 4.0 M, more preferably 0.1 to 2.0 M or more. The pH of the alkaline solution used is preferably 11.0 to 15.0, more preferably 12.0 to 14.0.

[0090] After step 4, the chromatography support can be reused to purify an Fc fusion protein. Thus, in one embodiment, the method for purifying an Fc fusion protein according to the present invention comprises repeating steps 1 to 4 multiple times, preferably 50 or more times, and more preferably 100 or more times. The chromatography support of the present invention can maintain a high DBC for the Fc fusion protein and low nonspecific binding (contamination by impurities) even after repeated use. Furthermore, the chromatography support of the present invention has excellent pressure resistance characteristics when packed into a column and passed through, which is also an advantage for repeated use.

[0091] The present invention will be described in detail below with reference to examples, but the present invention is not limited to these examples.

[0092] (Preparation Example 1) Preparation of Ligand Fc-binding proteins PrA-1 and PrA-2 were prepared. PrA-1 and PrA-2 are Fc-binding proteins comprising a homopentamer in which Fc-binding domains are linked in tandem. The Fc-binding domains contained in PrA-1 and PrA-2 are modified domains of the C domain of Protein A (SEQ ID NO: 1), and each consists of an amino acid sequence in which the mutations listed in Table 1 have been introduced into the parent domain of SEQ ID NO: 1.

[0093]

[0094] Expression and purification of PrA-1 and PrA-2 were carried out as follows. Escherichia coli BL21(DE3) was transformed with the plasmids encoding PrA-1 and PrA-2, respectively, and the resulting transformants were cultured in a rich medium at 37°C until the logarithmic growth phase. Subsequently, isopropyl-β-thiogalactopyranoside (Wako Pure Chemical Industries, Ltd.) was added to the medium to a final concentration of 1 mM, and the transformants were further cultured at 37°C for 4 hours to express the target proteins. The culture medium was then centrifuged to remove the supernatant, and the resulting cells were disrupted by adding 30 mM Tris buffer (pH 9.5) containing egg white-derived lysozyme (Wako Pure Chemical Industries, Ltd.) and polyoxyethylene (10) octylphenyl ether (Wako Pure Chemical Industries, Ltd.). The recombinant Fc-binding protein was purified from the resulting cell lysate by cation exchange chromatography (SP-Sepharose FF, GE Healthcare Biosciences) and anion exchange chromatography (Q-Sepharose FF, GE Healthcare Biosciences). The purified Fc-binding protein was dialyzed against 10 mM citrate buffer, pH 6.0. The purity of the recombinant Fc-binding protein confirmed by SDS-PAGE was 95% or higher.

[0095] Example 1: Production of a chromatography carrier and purification of an Fc fusion protein (1) Preparation of porous particles Step (a): 2.69 g of polyvinyl alcohol (PVA-217 manufactured by Kuraray Co., Ltd.) was added to 448 g of pure water, and the mixture was heated and stirred to dissolve the polyvinyl alcohol, thereby preparing an aqueous solution S. 26.44 g of 2-octanone (manufactured by Toyo Gosei Co., Ltd.) was mixed with ethyl cellulose (ETHOCEL manufactured by The Dow Chemical Company) to prepare an aqueous solution S. TM 14) 2.94 g of ethyl cellulose was added, and the mixture was heated and stirred to dissolve the ethyl cellulose, thereby preparing an organic solution P. Next, a monomer composition consisting of 3.63 g of divinylbenzene (manufactured by Wako Pure Chemical Industries, Ltd.), 0.36 g of 1-ethyl-4-vinylbenzene (manufactured by ChemSampCo., Inc.), and 14.15 g of glycidyl methacrylate (manufactured by Mitsubishi Gas Chemical Company, Inc.) was dissolved in the organic solution P to prepare a monomer solution.

[0096] Step (b): The entire amount of the aqueous solution S was poured into a separable flask, which was then fitted with a thermometer, stirring blades, and a condenser. The flask was then placed in a hot water bath, and stirring was initiated at 470 rpm under a nitrogen atmosphere. The entire amount of the monomer solution was poured into the separable flask, which was then heated in the hot water bath. When the internal temperature reached 85°C, 1.34 g of 2,2'-azobis(methyl isobutyrate) (manufactured by Wako Pure Chemical Industries, Ltd.) was added, and the internal temperature was adjusted to 86°C. Subsequently, while maintaining the solution at 86°C, 86.38 g of thioglycerol (manufactured by Tokyo Chemical Industry Co., Ltd.) and 10.15 g of diisopropylethylamine (manufactured by Tokyo Chemical Industry Co., Ltd.) were added, and the mixture was stirred for a total of 3 hours. The resulting reaction solution was cooled, filtered, and the particles were recovered and washed with pure water and ethanol. The washed particles were dispersed in pure water and decanted three times to remove small particles. Next, the particles were dispersed in pure water so that the particle concentration was 10% by mass, to obtain a dispersion of porous particles.

[0097] Step (c): Ethylene glycol diglycidyl ether was reacted with the thioglycerol-derived hydroxyl groups contained in the porous particles in the obtained dispersion. Specifically, 8.8 g of pure water, 0.91 g of sodium sulfate (manufactured by Wako Pure Chemical Industries, Ltd.), and 0.08 g of sodium carbonate (manufactured by Wako Pure Chemical Industries, Ltd.) were mixed to obtain a carbonate buffer (pH 9.5). 0.27 g of ethylene glycol diglycidyl ether (Denacol EX810, manufactured by Nagase ChemteX Corporation) and 8 mL of porous particles were added to the carbonate buffer, and the mixture was shaken and stirred at 23°C for 16 hours. The particles were then dispersed in pure water to a particle concentration of 50% by volume, obtaining a porous particle dispersion. The porous particles contained in this dispersion are referred to as "porous particles 1."

[0098] (2) Preparation of Ligand-Immobilized Porous Particles A ligand was immobilized on porous particle 1. Specifically, 28.8 g of pure water, 5.4 g of sodium sulfate (Wako Pure Chemical Industries, Ltd.), 0.2 g of sodium bicarbonate (Wako Pure Chemical Industries, Ltd.), and 0.16 g of sodium carbonate (Wako Pure Chemical Industries, Ltd.) were mixed to obtain a carbonate buffer (pH 9.3). 0.17 g of the Fc fusion protein PrA-1 prepared in Preparation Example 1 and 8 mL of porous particle 1 were added to 25 mL of this carbonate buffer, and the mixture was shaken and stirred at 23°C for 1.5 hours. The resulting reaction solution was filtered to recover the particles. A buffer was obtained by mixing 8.8 g of pure water, 0.1 g of sodium sulfate (Wako Pure Chemical Industries, Ltd.), and 0.03 g of sodium hydroxide (Wako Pure Chemical Industries, Ltd.), and 4.5 g of thioglycerol (Tokyo Chemical Industry Co., Ltd.) was added to prepare a hydrophilization reaction solution. The hydrophilization reaction solution was added to the particles, and the particles were shaken and stirred at 23°C for 16 hours to carry out a hydrophilization reaction. The particles were then dispersed in pure water to a particle concentration of 50% by volume, yielding a ligand-immobilized porous particle dispersion. The average pore diameter of the ligand-immobilized porous particles contained in this dispersion was calculated using the following procedure. This ligand-immobilized porous particle is referred to as "carrier 1."

[0099] <Calculation of Volume Average Particle Diameter> The volume average particle diameter of the carrier 1 was measured using a laser diffraction / scattering particle size distribution analyzer (LS13320 manufactured by Beckman Coulter, Inc.) in accordance with JIS Z 8825 (2013), where the refractive index of the solvent (water) was set to 1.333, and the refractive index of the ligand-immobilized porous particles was set to 1.50.

[0100] <Calculation of Porosity> The porosity of the carrier 1 in a wet state was measured by the following method. The carrier 1 was packed into a 4 mL volume (1) column (5 mm diameter x 200 mm length) using a 20 mM sodium phosphate / 150 mM sodium chloride aqueous solution. 50 μL of a 500 mM sodium chloride aqueous solution was loaded into the column, and the elution volume (2) was measured. Also, 50 μL of a 20 mM sodium phosphate / 150 mM sodium chloride aqueous solution containing dextran (molecular weight: 5,000,000 to 40,000,000, manufactured by Wako Pure Chemical Industries, Ltd.) was loaded into the column, and the elution volume (3) was measured. The value obtained by subtracting the elution volume (3) of the dextran-containing aqueous solution (particle void volume) from the column volume was determined as the "particle volume," and the value obtained by subtracting the elution volume (3) of the dextran from the elution volume of the sodium chloride aqueous solution (2) was determined as the "pore volume." The ratio (%) of pore volume to particle volume was calculated as "porosity in a wet state" according to the following formula: Porosity (%) = pore volume / particle volume × 100 = {(2) - (3)} / {(1) - (3)} × 100 (1): column volume (2): elution volume of 500 mM sodium chloride aqueous solution (3): elution volume of dextran-containing aqueous solution

[0101] <Calculation of average pore size> The average pore size of carrier 1 in a wet state was calculated according to the method described by Hagel et al. (Journal of Chromatography A, 1996, Vol. 743: 32-42). Using a 20 mM sodium phosphate / 150 mM sodium chloride aqueous solution, carrier 1 was packed into a 4 mL column (5 mmφ x 200 mm length). 50 μL of a 500 mM sodium chloride aqueous solution was loaded into the column, and the elution volume (1) of the sodium chloride aqueous solution was measured. Furthermore, 50 μL of a 20 mM sodium phosphate / 150 mM sodium chloride aqueous solution containing dextran (molecular weight: 5,000,000 to 40,000,000, manufactured by Wako Pure Chemical Industries, Ltd.) was loaded into the column, and the elution volume (2) of the dextran-containing aqueous solution was measured. Furthermore, 50 μL of a 20 mM sodium phosphate / 150 mM sodium chloride aqueous solution containing a pullulan standard sample (P-82, manufactured by Showa Denko K.K.) was loaded onto the column, and the elution volume (3) of the pullulan-containing aqueous solution was measured. The partition coefficient Kd of pullulan was calculated according to the following formula: Kd of pullulan = {(3) - (2)} / {(1) - (2)} (1): Elution volume of 500 mM sodium chloride aqueous solution (2): Elution volume of dextran-containing aqueous solution (3): Elution volume of pullulan-containing aqueous solution A plot of the molecular size of pullulan versus the partition coefficient Kd was made, and an approximate straight line was drawn. Since Kd = ε(1 - molecular size / average pore size)^(1 / 2) holds, the average pore size of carrier 1 was calculated from the intercept and slope.

[0102] The volume average particle size, porosity, and average pore size of Carriers 2 to 8 and Carrier 01 described below were also calculated using the same procedures as above.

[0103] (3) Purification of Fc Fusion Protein by Chromatography The Fc fusion protein was purified by chromatography using the carrier 1. A Cytiva Tricorn 50 / 200 column was packed with 4 mL of the carrier 1, and this column was connected to a Cytiva AKTA avant 25. A culture medium (etanercept concentration: 1.1 mg / mL) of cells expressing the Fc fusion protein (etanercept) was prepared as a sample solution. The following steps (i) to (iv) constituted one cycle, and this cycle was repeated 100 times. (i) The column was equilibrated by passing 3 CV (column volume) of a 20 mM sodium phosphate / 150 mM sodium chloride aqueous solution (pH 7.5) through the column at a downstream flow rate of 1 mL / min at 23°C. (ii) 36.36 mL of the sample solution was loaded onto the column with a retention time of 4 minutes, followed by column washing with a wash solution (20 mM sodium phosphate / 500 mM sodium chloride aqueous solution, pH 7.5). (iii) An eluent (100 mM sodium acetate aqueous solution, pH 3.3) was passed through the column, and the eluate was collected. The column was then equilibrated with a 20 mM sodium phosphate / 150 mM sodium chloride aqueous solution (pH 7.5). (iv) 3 CV of an alkaline solution (0.1 M sodium hydroxide aqueous solution) was passed through the column at a flow rate of 1 mL / min, followed by equilibration with a 20 mM sodium phosphate / 150 mM sodium chloride aqueous solution (pH 7.5).

[0104] Example 2 Ethyl cellulose (ETHOCEL manufactured by The Dow Chemical Company) contained in the organic solution P TM 14) was mixed with ethyl cellulose (ETHOCEL manufactured by The Dow Chemical Company) TM Porous particles (referred to as "porous particles 2") were prepared in the same manner as in Example 1(1), except that the procedure for preparing the carrier 2 was changed to 10). Ligand-immobilized porous particles (referred to as "carrier 2") were prepared using the porous particles 2 in the same manner as in Example 1(2). Fc fusion protein was purified using the carrier 2 in the same manner as in Example 1(3).

[0105] (Example 3) Ethyl cellulose (ETHOCEL manufactured by The Dow Chemical Company) contained in the organic solution P TM14) was mixed with ethyl cellulose (ETHOCEL manufactured by The Dow Chemical Company) TM Porous particles (referred to as "porous particles 3") were prepared in the same manner as in Example 1(1), except that the carrier 20 was changed to the carrier 20. Ligand-immobilized porous particles (referred to as "carrier 3") were prepared using porous particles 3 in the same manner as in Example 1(2). Fc fusion protein was purified using carrier 3 in the same manner as in Example 1(3).

[0106] (Example 4) Porous particles (referred to as "porous particles 4") were prepared in the same manner as in Example 1(1), except that the stirring speed in step (b) was changed to 490 rpm. Ligand-immobilized porous particles (referred to as "carrier 4") were prepared using porous particles 4 in the same manner as in Example 1(2). Fc fusion protein was purified using carrier 4 in the same manner as in Example 1(3).

[0107] (Example 5) Porous particles (referred to as "porous particles 5") were prepared in the same manner as in Example 1(1), except that the stirring speed in step (b) was changed to 230 rpm. Ligand-immobilized porous particles (referred to as "carrier 5") were prepared using porous particles 5 in the same manner as in Example 1(2). Fc fusion protein was purified using carrier 5 in the same manner as in Example 1(3).

[0108] Example 6 In step (a), the amounts of the reagents were changed to 23.70 g of 2-octanone (manufactured by Toyo Gosei Co., Ltd.) and 1.0 g of ethyl cellulose (ETHOCEL® manufactured by The Dow Chemical Company). TMPorous particles (referred to as "porous particles 6") were prepared in the same manner as in Example 1(1), except that the amounts of 14) and 16.94 g of 2,64 g of divinylbenzene (manufactured by Wako Pure Chemical Industries, Ltd.), 4.35 g of 1-ethyl-4-vinylbenzene (manufactured by ChemSampCo., Inc.), 0.43 g of glycidyl methacrylate (manufactured by Mitsubishi Gas Chemical Company, Inc.), and in step (b), the amounts of 2,2'-azobis(methyl isobutyrate) (manufactured by Wako Pure Chemical Industries, Ltd.) and 1.60 g of thioglycerol (manufactured by Tokyo Chemical Industry Co., Ltd.), 103.39 g of diisopropylethylamine (manufactured by Tokyo Chemical Industry Co., Ltd.) were changed. Using porous particles 6, ligand-immobilized porous particles (referred to as "carrier 6") were prepared in the same manner as in Example 1(2). Using the carrier 6, the Fc fusion protein was purified in the same manner as in Example 1(3).

[0109] Example 7 In step (a), the amounts of the reagents were changed to 28.37 g of 2-octanone (manufactured by Toyo Gosei Co., Ltd.) and 10.0 g of ethyl cellulose (ETHOCEL® manufactured by The Dow Chemical Company). TM Porous particles (referred to as "porous particles 7") were prepared in the same manner as in Example 1(1), except that the amounts of 14) and 14.15 g were changed, the amount of divinylbenzene (manufactured by Wako Pure Chemical Industries, Ltd.) was changed to 3.12 g, the amount of 1-ethyl-4-vinylbenzene (manufactured by ChemSampCo., Inc.) was changed to 0.31 g, the amount of glycidyl methacrylate (manufactured by Mitsubishi Gas Chemical Company, Inc.) was changed to 12.17 g, and in step (b), the amount of 2,2'-azobis(methyl isobutyrate) (manufactured by Wako Pure Chemical Industries, Ltd.) was changed to 1.15 g, the amount of thioglycerol (manufactured by Tokyo Chemical Industry Co., Ltd.) was changed to 74.29 g, and the amount of diisopropylethylamine (manufactured by Tokyo Chemical Industry Co., Ltd.) was changed to 8.73 g. Using porous particles 7, ligand-immobilized porous particles (referred to as "carrier 7") were prepared in the same manner as in Example 1(2). Using carrier 7, purification of Fc fusion proteins was carried out in the same manner as in Example 1(3).

[0110] (Example 8) Ligand-immobilized porous particles (referred to as "carrier 8") were prepared from the porous particles 1 in the same manner as in Examples 1(1) and 1(2), except that the ligand was changed to PrA-2. Using carrier 8, Fc fusion protein was purified in the same manner as in Example 1(3).

[0111] (Example 9) Purification of an Fc fusion protein (rilonacept) was carried out using the carrier 1 in the same manner as in Example 1(3), except that the sample solution was changed to a culture medium of rilonacept-expressing cells (rilonacept concentration: 1.1 mg / mL) and 29.09 mL of the sample solution was loaded onto the column with a retention time of 4 minutes in step (ii).

[0112] Example 10 The Fc fusion protein (efmoroctocog alfa) was purified using the carrier 1 in the same manner as in Example 1(3), except that the sample solution was changed to a culture medium of efmoroctocog alfa-expressing cells (efmoroctocog alfa concentration: 1.1 mg / mL) and that in step (ii), 22.59 mL of the sample solution was loaded onto the column with a retention time of 4 minutes.

[0113] (Example 11) Purification of an Fc fusion protein was carried out using the carrier 1 in the same manner as in Example 1(3), except that the washing solution used in the step (ii) was changed to a 20 mM sodium phosphate / 150 mM sodium chloride aqueous solution (pH 7.5).

[0114] (Example 12) Purification of an Fc fusion protein was carried out using the carrier 1 in the same manner as in Example 1(3), except that the washing solution used in the step (ii) was changed to a 20 mM sodium phosphate / 500 mM sodium chloride aqueous solution (pH 6.5).

[0115] Example 13 The Fc fusion protein was purified using the carrier 1 in the same manner as in Example 1(3), except that the alkaline solution used in the step (iv) was changed to a 0.5 M NaOH aqueous solution.

[0116] Comparative Example 1 Porous particles (referred to as "porous particles 01") were prepared in the same manner as in Example 1(1), except that 29.44 g of 2-octanone (manufactured by Toyo Gosei Co., Ltd.) was used as the organic solution P. Using the porous particles 01, ligand-immobilized porous particles (referred to as "carrier 01") were prepared in the same manner as in Example 1(2). Using the carrier 01, Fc fusion protein purification was carried out in the same manner as in Example 1(3).

[0117] Test Example 1: Measurement of dynamic binding capacity (DBC) The DBC for the target protein was measured at the first and 100th cycles of chromatography in Examples 1 to 13 and Comparative Example 1. That is, the DBC was calculated from the amount of target protein captured at 10% breakthrough at the elution front and the column packed volume. The DBC (mg / mL) at the first cycle and the percentage of DBC at the 100th cycle relative to the first cycle (% DBC) were calculated. The results are shown in Tables 2 and 3.

[0118] Test Example 2: Measurement of host cell-derived protein (HCP) amount The residual HCP content per target protein content (HCP residual rate: HCP / target) was calculated for the eluates obtained at the first and 100th cycles of chromatography in Examples 1 to 13 and Comparative Example 1. The residual HCP content in the eluates was measured using an HCP ELISA kit (F550-1). The target protein content in the eluates was measured based on absorbance. The residual HCP content (ppm) at the first cycle and the percentage of the HCP residual rate at the 100th cycle relative to the first cycle (% [HCP / target]) were calculated. The results are shown in Tables 2 and 3.

[0119] (Test Example 3) Evaluation of Pressure Resistance Characteristics During Liquid Flow Two Hiscale 26 / 20 columns (manufactured by Cytiva) were connected together to prepare a column with an inner diameter of 26 mm and a packed height of 200 mm. The column was packed with one of Carriers 1, 4, or 7. Each column was connected to an AKTA AVANT 150 (manufactured by Cytiva), and pure water was passed through the column at a linear flow rate of 100 cm / hr for 30 minutes. The upper column was then removed, and an adapter was attached to the carrier layer to pack the carrier into the column. Pure water was then passed through the column at a linear flow rate of 800 cm / hr, and the back pressure during this period was measured. The results are shown in Table 2. The smaller the back pressure, the better the pressure resistance characteristics.

[0120]

[0121]

Claims

1. A chromatography carrier for purifying an Fc fusion protein, comprising porous particles having immobilized ligands, the porous particles being synthetic or natural polymer-based porous particles, the ligand being at least one selected from the group consisting of protein A, protein G, protein L, and related substances thereof, and the porous particles having immobilized ligands have an average pore diameter of 75 to 120 nm.

2. The chromatography carrier according to claim 1, wherein the porous particles to which the ligands are immobilized have an average pore size of 101 to 110 nm.

3. The chromatography carrier according to claim 1, wherein the Fc fusion protein has a molecular weight of 160 to 300 kDa.

4. The chromatography carrier according to claim 1, wherein the porous particles to which the ligands are immobilized have a volume average particle size of 45 to 100 μm and a porosity of 70 to 95% (v / v).

5. The chromatography support according to claim 1, wherein the Fc fusion protein is at least one selected from the group consisting of etanercept, rilonacept, and efmoroctocog alfa.

6. The chromatography carrier according to claim 1, wherein the Fc fusion protein is an Fc fusion protein consisting of the amino acid sequence of SEQ ID NO: 4 or SEQ ID NO: 5, or an amino acid sequence having 85% or more identity to either of these sequences.

7. The chromatography carrier according to claim 1, wherein the porous particles are polymers of a monomer having a functional group capable of immobilizing a ligand and a polymerizable unsaturated group, and a polymerizable unsaturated group-containing monomer that does not have the functional group.

8. The chromatography support according to claim 7, wherein the polymerizable unsaturated group-containing monomers having no functional group include non-crosslinkable monomers and crosslinkable monomers.

9. A method for purifying an Fc fusion protein, comprising the following steps: (Step 1) passing a solution containing the Fc fusion protein through a column containing the chromatography carrier according to any one of claims 1 to 8, thereby allowing the Fc fusion protein to bind to the carrier; (Step 2) washing the carrier; and (Step 3) recovering the Fc fusion protein from the carrier.

10. The method according to claim 9, further comprising the following step: (Step 4) after Step 3, passing an alkaline liquid through the column.

11. The method of claim 10, comprising repeating steps 1 through 4 multiple times.

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