Method for producing chromatography carrier and chromatography carrier

Abstract

To provide a chromatography carrier that has a large dynamic binding capacity for antibodies or fragments thereof and is resistant to leakage of protein ligands even when repeatedly used for antibody isolation.SOLUTION: A method for manufacturing a chromatography carrier comprises the following steps A-1 and B: (Step A-1) a step of immobilizing one or more ligands selected from Protein A, Protein G, Protein L, and their related substances onto porous particles; and (Step B) a step of reacting the porous particles with the ligands immobilized after the step A-1 with a compound having at least one ligand-reactive group selected from a group represented by -C(=O)-O-C(=O)-, a carbodiimide group, and a cyclic ether group.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to a method for producing a chromatographic support, a chromatographic support, a chromatographic column, and a method for isolating an antibody or a fragment thereof. [Background technology]

[0002] In recent years, antibodies have been widely used as research reagents, antibody drugs, and the like. Antibodies for these reagents and drugs are generally produced through isolation by chromatography. Carriers used in such chromatography are required to have a dynamic binding capacity for antibodies or their fragments, to be less susceptible to ligand leakage during isolation, and to be less susceptible to nonspecific adsorption of impurities. For example, carriers with increased dynamic binding capacity for antibodies are known, for example, by adjusting the median particle size to a specific range (Patent Document 1). [Prior art documents] [Patent documents]

[0003] [Patent Document 1] Special Publication No. 2008-523140 [Patent Document 2] WO2020 / 040307 publication Summary of the Invention [Problem to be solved by the invention]

[0004] Furthermore, it has been proposed to use immunoglobulin-binding proteins with mutated polypeptide chains as ligands to improve alkali resistance and reduce ligand leakage even when repeatedly used for antibody isolation (Patent Document 2), but further improvements in low protein ligand leakage are needed. Furthermore, because the above-mentioned polypeptide chain mutations for improving alkali resistance can reduce the dynamic binding capacity of the original immunoglobulin-binding protein, it has been difficult to simultaneously increase the dynamic binding capacity for antibodies or fragments thereof and reduce protein ligand leakage. The problem to be solved by the present invention is to provide a chromatography support that has a large dynamic binding capacity for antibodies or fragments thereof and that is less likely to leak protein ligands even when used repeatedly for antibody isolation. [Means for solving the problem]

[0005] The above issues are addressed as follows: <1> ~ <15> was resolved by the following means. <1> A method for producing a chromatography carrier (hereinafter also referred to as the method for producing a chromatography carrier of the present invention) comprises the following steps A-1 and B. (Step A-1) A step of immobilizing one or more ligands selected from protein A, protein G, protein L, and their analogs onto porous particles. (Step B) A step of reacting the porous particles to which the ligands have been immobilized after Step A-1 with a compound having at least one ligand-reactive group selected from a group represented by -C(=O)-OC(=O)-, a carbodiimide group, and a cyclic ether group.

[0006] <2> The compound having a ligand reactive group is one or more selected from the group consisting of a compound represented by the following formula (1) and a salt thereof, a compound represented by the following formula (2), and a compound represented by the following formula (3): <1> A method for producing the chromatography carrier according to claim 1.

[0007] [ka]

[0008] [In formula (1), R 1 and R 2 are each independently a hydrogen atom or a substituted or unsubstituted hydrocarbon group.

[0009] [ka]

[0010] [In formula (2), R 3 and R 4 each independently represents a substituted or unsubstituted hydrocarbon group, R 3 and R 4 may be bonded to each other to form a cyclic structure.

[0011] [ka]

[0012] [In formula (3), R 5 represents a substituted or unsubstituted hydrocarbon group, and X represents a cyclic ether group.

[0013] <3> the compound having a ligand reactive group is one or more compounds selected from 1-[3-(dimethylamino)propyl]-3-ethylcarbodiimide, a salt of 1-[3-(dimethylamino)propyl]-3-ethylcarbodiimide, N,N'-dicyclohexylcarbodiimide, N,N'-diisopropylcarbodiimide, maleic anhydride, propionic anhydride, acetic anhydride, pivalic anhydride, succinic anhydride, glutaric anhydride, propylene oxide, butylene oxide, glycidyl methyl ether, ethyl glycidyl ether, glycidol, epichlorohydrin, and epibromohydrin; <1> or <2> A method for producing the chromatography carrier according to claim 1.

[0014] <4> the compound having a ligand reactive group is at least one compound selected from the compound represented by formula (1) and a salt thereof; <1> ~ <3> 1. A method for producing a chromatography support according to any one of the preceding claims.

[0015] <5> The amount of the compound having a ligand reactive group used is 0.01 to 15 millimoles per 1 g of the dry weight of the porous particles to which the ligand is immobilized. <1> ~ <4> 1. A method for producing a chromatography support according to any one of the preceding claims. <6> The reaction in step B is carried out in an aqueous medium at a pH of 8 to 14. <1> ~ <5> 1. A method for producing a chromatography support according to any one of the preceding claims.

[0016] <7> The method further includes the following step A-2 between step A-1 and step B, and the hydrophilic group-containing, ligand-immobilized porous particles obtained in step A-2 are used in step B as the ligand-immobilized porous particles after step A-1. <1> ~ <6> 1. A method for producing a chromatography support according to any one of the preceding claims. (Step A-2) A step of reacting the porous particles to which the ligands have been immobilized in Step A-1 with a compound having a total of two or more hydrophilic groups of at least one type selected from hydroxyl groups and mercapto groups in the molecule.

[0017] <8> The method further includes the following steps A-P1 and A-P2, and the porous particles reacted with at least one agent selected from a crosslinking agent and a hydrophilizing agent in step A-P2 are used as the porous particles in step A-1. <1> ~ <7> 1. A method for producing a chromatography support according to any one of the preceding claims. (Step A-P1) A step of dispersing a monomer composition in an aqueous medium and carrying out suspension polymerization (Step A-P2) A step of reacting the porous particles obtained in Step A-P1 with at least one agent selected from a crosslinking agent and a hydrophilizing agent.

[0018] <9> A polymerizable composition comprising porous particles, a ligand immobilized on the porous particles, and a partial structure derived from a compound having at least one ligand-reactive group selected from a group represented by -C(=O)-OC(=O)-, a carbodiimide group, and a cyclic ether group, the ligand is one or more ligands selected from protein A, protein G, protein L, and analogs thereof; at least one functional group selected from an amino group and a carboxy group of the ligand is chemically modified with the partial structure; The following formula: Chemical modification rate (mol%) = (number of moles of chemically modified functional groups) / (sum of number of moles of chemically modified functional groups and number of moles of unmodified functional groups) × 100 The degree of chemical modification calculated by the above formula is 1 to 70 mol % (hereinafter also referred to as the chromatography carrier of the present invention).

[0019] <10> an amino group of the ligand is chemically modified with the partial structure; <9> The chromatography carrier according to claim 1. <11> The partial structure is -C(=O)-, -NR 1 -C(=O)-, -C(=NR 1 )- or -CH2-CH(-OH)-(R 1 represents a hydrogen atom, an alkyl group, a cycloalkyl group, an aminoalkyl group, a monoalkylaminoalkyl group, or a dialkylaminoalkyl group. <9> or <10> The chromatography carrier according to claim 1.

[0020] <12> The partial structure is -NR 1 -C(=O)-NR 2 - or -C(=NR 1 )-NR 2 -(R 1 and R 2 are each independently a hydrogen atom, an alkyl group, a cycloalkyl group, an aminoalkyl group, a monoalkylaminoalkyl group, or a dialkylaminoalkyl group. <9> ~ <11> 2. The chromatography carrier according to claim 1, wherein <13> R 1 and R 2 at least one of the groups is an aminoalkyl group, a monoalkylaminoalkyl group, or a dialkylaminoalkyl group; <12> The chromatography carrier according to claim 1.

[0021] <14> <9> ~ <13> A chromatography column comprising the chromatography support according to any one of the preceding items. <15> <9> ~ <13> or a chromatography carrier according to any one of <14> (hereinafter also referred to as the method for isolating the antibody or fragment thereof of the present invention) [Effects of the Invention]

[0022] According to the method for producing a chromatography carrier of the present invention, it is possible to simply produce a chromatography carrier that has a large dynamic binding capacity for an antibody or a fragment thereof and is less likely to leak protein ligands even when used repeatedly for antibody isolation. The chromatographic carrier of the present invention has a large dynamic binding capacity for antibodies or fragments thereof, and is less likely to leak protein ligands even when used repeatedly for isolating antibodies. Therefore, the present invention can provide a chromatography column that has a large dynamic binding capacity for antibodies or fragments thereof and is less susceptible to leakage of protein ligands even when used repeatedly for isolating antibodies. DETAILED DESCRIPTION OF THE INVENTION

[0023] [Method for producing a chromatography carrier] The method for producing a chromatography carrier of the present invention comprises (Step A-1) a step of immobilizing one or more ligands selected from protein A, protein G, protein L, and their analogs onto porous particles. -Process A-1- Step A-1 is a step of immobilizing one or more ligands selected from protein A, protein G, protein L, and their analogs onto porous particles. The porous particles are preferably porous particles containing a polymer. Such porous particles may be natural polymer-based porous particles or synthetic polymer-based porous particles composed of polysaccharides such as agarose, dextran, or cellulose, but synthetic polymer-based porous particles are preferred in order to increase the dynamic binding capacity and improve the uniformity of particle size. Furthermore, the porous particles are preferably water-insoluble.

[0024] The porous particles may be commercially available products or may be produced by a conventional method. Here, a method for producing the porous particles will be described. The porous particles can be produced by a method including a step of dispersing a monomer composition in an aqueous medium and carrying out suspension polymerization (hereinafter also referred to as step A-P1).

[0025] -Process A-P1- The monomer composition used in step A-P1 preferably contains a functional group-containing monomer. The functional group contained in this monomer is preferably one that can be used for 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 cyclic ether groups, carboxy groups, -C(=O)-OC(=O)-, succinimideoxycarbonyl groups, formyl groups, hydroxyl groups, and isocyanate groups. Among these, cyclic ether groups are 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. Specific examples of the cyclic ether group include cyclic ether groups represented by the following formulas (4) to (9), with the cyclic ether group represented by formula (4), (6) or (9) being preferred, and the cyclic ether group represented by formula (4) being more preferred.

[0026] [ka]

[0027] [In the formula, R 11 ~R 14 each independently represents a hydrogen atom or an alkyl group, and * represents a bond.

[0028] R 11 ~R 14 The number of carbon atoms in the alkyl group represented by the formula (I) is preferably 1 to 4, 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. 11 ~R 14 is preferably a hydrogen atom.

[0029] 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 can 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 particularly preferred.

[0030] The total amount of functional group-containing monomers used is preferably 35 parts by mass or more, more preferably 45 parts by mass or more, and particularly preferably 55 parts by mass or more, relative to 100 parts by mass of the total amount of monomers used in step A-P1, and is preferably 99 parts by mass or less, more preferably 90 parts by mass or less, and particularly preferably 85 parts by mass or less, relative to 100 parts by mass of the total amount of monomers used in step A-P1.

[0031] Furthermore, the monomer composition used in step A-P1 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). The other monomer may be a polymerizable unsaturated group-containing monomer that does not have a functional group capable of fixing a ligand. The other monomer is roughly classified into a non-crosslinkable monomer and a crosslinkable monomer, and either one of these may be used alone or in combination. Note that, according to the present invention, even when a monomer that does not contain a hydrophilic group such as a hydroxyl group is used as the other monomer, satisfactory antifouling properties can be achieved, and the present invention is applicable to a wide range of monomer compositions.

[0032] Examples of the non-crosslinkable 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 may 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.

[0033] 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.

[0034] 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.

[0035] 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 may be used alone or in combination of two or more.

[0036] 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. Furthermore, examples of the (meth)acrylonitrile-based non-crosslinkable monomer include acrylonitrile, methacrylonitrile, etc. These can be used alone or in combination of two or more. Examples of the N-vinylamide non-crosslinkable monomer include N-vinylacetamide, N-vinylpropionamide, etc. These can be used alone or in combination of two or more.

[0037] The total amount of non-crosslinkable monomers used is preferably 0.01 parts by mass or more, more preferably 0.05 parts by mass or more, and particularly preferably 0.1 parts by mass or more, relative to 100 parts by mass of the total amount of monomers used in step A-P1, and is preferably 30 parts by mass or less, more preferably 15 parts by mass or less, and particularly preferably 5 parts by mass or less, relative to 100 parts by mass of the total amount of monomers used in step A-P1.

[0038] Examples of the crosslinkable monomer include (meth)acrylate crosslinkable monomers, aromatic vinyl crosslinkable monomers, and allyl crosslinkable monomers. These may 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 the crosslinkable monomers, (meth)acrylate crosslinkable monomers and aromatic vinyl crosslinkable monomers are preferred.

[0039] 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.

[0040] 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.

[0041] 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. Further, in addition to the above-mentioned crosslinkable monomers, examples thereof include 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.

[0042] The total amount of crosslinkable monomers used is preferably 1 part by mass or more, more preferably 5 parts by mass or more, and particularly preferably 10 parts by mass or more, relative to 100 parts by mass of the total amount of monomers used in step A-P1, and is preferably 50 parts by mass or less, more preferably 40 parts by mass or less, and particularly preferably 30 parts by mass or less, relative to 100 parts by mass of the total amount of monomers used in step A-P1.

[0043] The aqueous medium used in step A-P1 may be, for example, an aqueous solution of a water-soluble polymer, and examples of the water-soluble polymer include hydroxyethyl cellulose, polyvinyl alcohol, carboxymethyl cellulose, polyvinylpyrrolidone, starch, and gelatin. The total amount of the aqueous medium used is usually about 200 parts by mass or more and 7000 parts by mass or less per 100 parts by mass of the total amount of the monomers. When water is used as the dispersion medium of the aqueous medium, a dispersion stabilizer such as sodium carbonate, calcium carbonate, sodium sulfate, calcium phosphate, or sodium chloride may be used.

[0044] Specific examples of the method for step A-P1 include a method in which a polymerization initiator is dissolved in a mixed solution (monomer solution) containing a monomer composition and, if necessary, a porosifying agent, and the resulting solution is suspended in an aqueous medium and heated to a predetermined temperature to polymerize; a method in which a polymerization initiator is dissolved in a mixed solution (monomer solution) containing a monomer composition and, if necessary, a porosifying agent, and the resulting solution is added to an aqueous medium heated to a predetermined temperature to polymerize; and a method in which a mixed solution (monomer solution) containing a monomer composition and, if necessary, a porosifying agent is suspended in an aqueous medium and heated to a predetermined temperature, and a polymerization initiator is added to polymerize.

[0045] 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 is usually about 0.01 to 10 parts by mass per 100 parts by mass of the total amount of monomers.

[0046] The porosifying agent is used to produce porous particles, and is present together with the monomer in the polymerization of the oil droplets, and plays a role in forming pores as a non-polymerized component. The porosifying agent is not particularly limited as long as it can be easily removed from the porous surface, and examples thereof include linear polymers soluble in various organic solvents and mixed monomers, and these may be used in combination.

[0047] 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, and ethylbenzene; 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; Examples of the porosifying agent include alicyclic alcohols such as cyclohexanol; aromatic alcohols such as 2-phenylethyl alcohol and benzyl alcohol; ketones such as diethyl ketone, methyl isobutyl ketone, diisobutyl ketone, acetophenone, 2-octanone and cyclohexanone; ethers such as dibutyl ether, diisobutyl ether, anisole and ethoxybenzene; esters such as isopentyl acetate, butyl acetate, 3-methoxybutyl acetate and diethyl malonate, as well as linear polymers such as homopolymers of non-crosslinkable vinyl monomers. The porosifying agent can be used alone or in combination of two or more. The total amount of the porosifying agent used is usually about 40 parts by mass or more and 600 parts by mass or less per 100 parts by mass of the total amount of the monomers.

[0048] In addition, various surfactants, including anionic surfactants such as alkyl sulfate ester salts, alkylaryl sulfate ester salts, alkyl phosphate ester salts, and fatty acid salts, may be used in Step A-P1. 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.

[0049] The polymerization temperature in step A-P1 may be determined depending on the polymerization initiator, but is usually about 2 to 100° C., preferably 50 to 100° C. The polymerization time is usually 5 minutes to 48 hours, preferably 10 minutes to 24 hours.

[0050] -Process A-P2- Furthermore, prior to step A-1, a step (hereinafter also referred to as step A-P2) of reacting the porous particles obtained in step A-P1 with at least one selected from a crosslinking agent and a hydrophilizing agent may be carried out. When both a crosslinking agent and a hydrophilizing agent are used, the crosslinking reaction may be carried out after the hydrophilizing reaction, or the hydrophilizing reaction may be carried out after the crosslinking reaction. Alternatively, the crosslinking reaction and the hydrophilizing reaction may be carried out simultaneously. When a monomer composition containing a functional group-containing monomer is used in step A-P1, the crosslinking reaction causes an addition reaction of the crosslinking agent with some of the functional groups in the polymer molecules of the porous particles, introducing a partial structure derived from the crosslinking agent, thereby crosslinking the residues of the functional groups via the partial structure derived from the crosslinking agent. Furthermore, when a monomer composition containing a functional group-containing monomer is used in step A-P1, the hydrophilization reaction causes an addition reaction of a 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.

[0051] The crosslinking agent used in step A-P2 may be any agent capable of reacting with a functional group capable of immobilizing a ligand to introduce a crosslinked structure, but 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.

[0052] When the porous particles obtained in step A-P1 have cyclic ether groups, specifically, 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 can be used. When the porous particles obtained in step A-P1 have a carboxy group, -C(=O)-OC(=O)-, a succinimideoxycarbonyl group, a formyl group, or an isocyanate group, specifically, a crosslinking agent containing at least two groups represented by -C(=O)-NH-NH2 as crosslinkable groups in the molecule can be used.

[0053] Examples of crosslinking agents containing at least two groups represented by -C(=O)-NH- in the molecule include 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, and dodeca Examples of suitable crosslinking agents include dicarboxylic acid dihydrazides such as quinolinic acid dihydrazide, phthalic acid dihydrazide, isophthalic acid dihydrazide, terephthalic acid dihydrazide, and quinolinic acid dihydrazide; tricarboxylic acid trihydrazides such as cyclohexanetricarboxylic acid trihydrazide; and (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 liquid permeability, pressure resistance during liquid passage, and antifouling properties.

[0054] In step A-P2, a crosslinking agent other than the crosslinking agent containing at least two -C(=O)-NH- groups in the molecule can also be used. Examples of such crosslinking agents include polyfunctional isocyanate-based crosslinking agents, polyfunctional epoxy-based crosslinking agents, polyfunctional aldehyde-based crosslinking agents, polyfunctional thiol-based crosslinking agents, polyfunctional oxazoline-based crosslinking agents, polyfunctional aziridine-based crosslinking agents, and metal chelate-based crosslinking agents.

[0055] The total amount of crosslinking agent used is preferably 0.01 molar equivalents or more and 0.8 molar equivalents or less, more preferably 0.05 molar equivalents or more and 0.7 molar equivalents or less, and particularly preferably 0.1 molar equivalents or more and 0.6 molar equivalents or less, relative to 1 mole of functional group derived from the functional group-containing monomer.

[0056] The hydrophilizing agent used in step A-P2 is preferably a compound having a total of two or more hydrophilic groups of at least one type selected from hydroxyl groups and mercapto groups in the molecule, in order to improve antifouling properties and low protein ligand leakage, and more preferably a compound having a total of two to four hydrophilic groups of at least one type selected from hydroxyl groups and mercapto groups in the molecule. Examples include alcohols having a mercapto group in the molecule, such as mercaptoethanol and thioglycerol, and polyhydric alcohols, such as glycerol and diglycerol. The hydrophilizing agent can be used alone or in combination of two or more. Among these, in order to improve antifouling properties and low protein ligand leakage, alcohols having a mercapto group in the molecule are preferred, and thioglycerol is particularly preferred.

[0057] The total amount of the hydrophilizing agent used is preferably 0.5 to 10 molar equivalents, more preferably 1 to 8 molar equivalents, and particularly preferably 2 to 6 molar equivalents, relative to 1 mole of the functional group derived from the functional group-containing monomer.

[0058] Step A-P2 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.

[0059] The reaction time for step A-P2 is not particularly limited, but is usually about 0.5 to 72 hours, preferably 0.5 to 48 hours. The reaction temperature may be selected appropriately as long as it is equal to or lower than the boiling point of the solvent, but is usually about 2 to 100°C.

[0060] The ligand used in step A-1 is preferably protein A or a protein A analogue, more preferably a protein A analogue, in order to enhance the binding affinity to the antibody or a fragment thereof. Protein A contains five domains, E, D, A, B, and C, which have the ability to bind to immunoglobulins. Among the above-mentioned ligands, Protein A analogs having modified B and C domains are preferred, and Protein A analogs having modified C domains are more preferred. Furthermore, in the case of modified protein A having a modified domain in which one or more amino acid residues in the C domain have been replaced with other amino acid residues, such as modified protein A that is a hexamer of the amino acid sequence domain of SEQ ID NO: 1 (SEQ ID NO: 29 in WO2020 / 040307), leakage may be more likely when the protein is repeatedly used to isolate antibodies. However, according to the present invention, even when such a protein is used as a ligand, leakage of the protein ligand can be suppressed when the protein is repeatedly used. Among such protein ligand leakage, the use of protein ligands having the following amino acid sequences can suppress leakage: an amino acid sequence in which the glycine at position 29 of the amino acid sequence of SEQ ID NO: 2 (C domain of protein A) is substituted with alanine; an amino acid sequence in which the glycine at position 29 of the amino acid sequence of SEQ ID NO: 2 is substituted with alanine and the asparagine at position 11 or the threonine at position 23 is substituted with another amino acid residue; an amino acid sequence in which the glycine at position 29 of the amino acid sequence of SEQ ID NO: 2 is substituted with alanine and the asparagine at position 11 is substituted with glutamine or the threonine at position 23 is substituted with leucine; an amino acid sequence in which the glycine at position 29 of the amino acid sequence of SEQ ID NO: 2 is substituted with alanine, the asparagine at position 11 is substituted with glutamine and the threonine at position 23 is substituted with leucine; an amino acid sequence having 95% or more homology to these amino acid sequences; an amino acid sequence having 98% or more homology to these amino acid sequences; or an amino acid sequence having 99% or more homology to these amino acid sequences.

[0061] Furthermore, when using a protein ligand in which multiple domains capable of binding to immunoglobulins of Protein A or modified domains thereof are linked, in order to increase the dynamic binding capacity and suppress leakage of the protein ligand, the following amino acid sequences are available: an amino acid sequence in which the glycine at position 29 of the amino acid sequence of SEQ ID NO: 2 is substituted with alanine; an amino acid sequence in which the glycine at position 29 of the amino acid sequence of SEQ ID NO: 2 is substituted with alanine and the asparagine at position 11 or the threonine at position 23 is substituted with another amino acid residue; an amino acid sequence in which the glycine at position 29 of the amino acid sequence of SEQ ID NO: 2 is substituted with alanine and the asparagine at position 11 is substituted with glutamine; It is preferable to use a ligand having 2 to 12 amino acid sequences selected from the amino acid sequence of SEQ ID NO: 2 in which glycine at position 29 is substituted with alanine, asparagine at position 11 is substituted with glutamine, and threonine at position 23 is substituted with leucine, an amino acid sequence in which glycine at position 29 of the amino acid sequence of SEQ ID NO: 2 is substituted with alanine, asparagine at position 11 is substituted with glutamine, and threonine at position 23 is substituted with leucine, as well as amino acid sequences having 95% or more homology to these amino acid sequences, amino acid sequences having 98% or more homology to these amino acid sequences, and amino acid sequences having 99% or more homology to these amino acid sequences.

[0062] To increase the dynamic binding capacity, the amount of immobilized ligand is preferably 10 mg or more and 300 mg or less, more preferably 25 mg or more and 150 mg or less, per gram of dry weight of the porous particles.

[0063] The immobilization of the ligand to the porous particles in step A-1 may be carried out in a conventional manner. Chemical bonding is preferred as the ligand immobilization method. For example, a method of binding the ligand to a functional group capable of immobilizing the ligand may be employed. This method may be carried out with reference to the descriptions in WO 2015 / 119255, WO 2015 / 041218, etc. Specific examples include a method of binding a cyclic ether group, carboxy group, -C(=O)-OC(=O)-, or formyl group of the porous particles to an amino group or the like of the ligand. To increase the reaction efficiency, the ligand immobilization reaction is preferably carried out in a buffer solution with a pH of 7 to 14. The reaction time for the ligand immobilization reaction is not particularly limited, but is typically about 0.1 to 72 hours. The reaction temperature may be selected as appropriate below the boiling point of the solvent, but is typically about 2 to 100°C. Furthermore, in order to increase the dynamic binding capacity and improve the low protein ligand leakage, step A-1 is preferably carried out in the absence of a compound having at least one ligand-reactive group selected from a group represented by -C(=O)-OC(=O)-, a carbodiimide group, and a cyclic ether group, which is used in step B described below.

[0064] Alternatively, the ligand may be immobilized using a method of controlling the orientation of the ligand (U.S. Pat. No. 6,399,750; Ljungquist C. et al., rEur. J. Biochem., 1989, Vol. 186, pp. 557-561), a method of immobilizing the ligand on the porous particle via a linker (spacer) (U.S. Pat. No. 5,260,373; JP-A Nos. 2010-133733 and 2010-133734), or a method of accumulating the ligand on the porous particle using an associative group (JP-A No. 2011-256176).

[0065] Examples of compounds that provide 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 a hydrophilization reaction is carried out in step A-P2. To increase the reaction efficiency, the linker introduction reaction is preferably carried out in a buffer having a pH of 7 to 14. The reaction time for the linker introduction reaction is not particularly limited, but is usually about 0.5 to 72 hours. The reaction temperature may be selected appropriately as long as it is equal to or lower than the boiling point of the solvent, but is usually about 2 to 100°C.

[0066] -Process A-2- In order to improve antifouling properties and low protein ligand leakage, the method for producing the chromatography carrier of the present invention preferably further includes the following step A-2 between step A-1 and step B, and uses the hydrophilic group-containing ligand-immobilized porous particles obtained in step A-2 in step B as the ligand-immobilized porous particles after step A-1. (Step A-2) A step of reacting the porous particles to which the ligands have been immobilized in Step A-1 with a compound having a total of two or more hydrophilic groups of at least one type selected from hydroxyl groups and mercapto groups in the molecule.

[0067] The compound having two or more hydrophilic groups in total in the molecule used in step A-2 is preferably a compound having two to four hydrophilic groups in total in the molecule, each of which is at least one type selected from hydroxyl groups and mercapto groups, in order to improve antifouling properties and low protein ligand leakage. Examples include alcohols having a mercapto group in the molecule, such as mercaptoethanol and thioglycerol, and polyhydric alcohols, such as glycerol and diglycerol. Compounds having two or more hydrophilic groups in total in the molecule can be used alone or in combination. Among these, in order to improve antifouling properties and low protein ligand leakage, alcohols having a mercapto group in the molecule are preferred, and thioglycerol is particularly preferred.

[0068] The total amount of compounds having a total of two or more hydrophilic groups in the molecule used in step A-2 is preferably 1 part by mass or more and 1,000 parts by mass or less, more preferably 10 parts by mass or more and 800 parts by mass or less, and particularly preferably 100 parts by mass or more and 600 parts by mass or less, relative to 100 parts by mass of the porous particles (dry weight) to which the ligands are fixed.

[0069] Step A-2 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.

[0070] The reaction time in step A-2 is not particularly limited, but is usually about 0.5 to 72 hours, and 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.

[0071] -Process B- The method for producing a chromatography carrier of the present invention includes (Step B) a step of reacting the porous particles to which the ligands have been immobilized after Step A-1 with a compound having at least one ligand-reactive group selected from a group represented by -C(=O)-OC(=O)-, a carbodiimide group, and a cyclic ether group. When the compound having the ligand-reactive group is one having at least one selected from a group represented by -C(=O)-OC(=O)- and a cyclic ether group, the compound reacts primarily with the amino group of the ligand. Furthermore, when the compound having a carbodiimide group is used as the compound having the ligand-reactive group, the compound reacts primarily with the amino group of the ligand and also with the carboxy group. The inventors speculate that the reaction in step B increases the dynamic binding capacity for the antibody or a fragment thereof and reduces the risk of leakage of the protein ligand even when the compound is repeatedly used to isolate the antibody.

[0072] Step B uses porous particles on which a ligand has been immobilized after step A-1. The porous particles may be those on which a ligand has been immobilized after the reaction in step A-1 has been completed. The porous particles may be those on which a ligand has been immobilized obtained in step A-1, or may be those on which a hydrophilic group-containing ligand has been immobilized obtained by further performing step A-2.

[0073] The compound having a ligand-reactive group used in step B is preferably one or more selected from the group consisting of compounds represented by the following formula (1) and salts thereof, compounds represented by the following formula (2), and compounds represented by the following formula (3), in order to increase the dynamic binding capacity and improve low protein ligand leakage. More preferably, it is one or more selected from the group consisting of compounds represented by formula (1) and salts thereof and compounds represented by formula (2), and particularly preferably, it is one or more selected from the group consisting of compounds represented by formula (1) and salts thereof. Examples of salts of the compound represented by formula (1) include inorganic acid salts such as hydrochloride, sulfate, nitrate, hydrofluoride, and hydrobromide; and organic acid salts such as acetate, tartrate, citrate, and fumarate.

[0074] [ka]

[0075] [In formula (1), R 1 and R 2 are each independently a hydrogen atom or a substituted or unsubstituted hydrocarbon group.

[0076] [ka]

[0077] [In formula (2), R 3 and R 4 each independently represents a substituted or unsubstituted hydrocarbon group, R 3 and R 4 may be bonded to each other to form a cyclic structure.

[0078] [ka]

[0079] [In formula (3), R 5 represents a substituted or unsubstituted hydrocarbon group, and X represents a cyclic ether group.

[0080] Here, each symbol in formulas (1) to (3) will be explained. In formulas (1) to (3), R 1 ~R 5 The number of carbon atoms in the hydrocarbon group represented by is preferably 1 to 30, more preferably 1 to 14, even more preferably 1 to 8, and particularly preferably 1 to 4, in order to increase the dynamic binding capacity and improve low protein ligand leakage. R 1 ~R 5 The "hydrocarbon group" in the above formula includes an alkyl group, an alkenyl group, a cycloalkyl group, a bridged ring hydrocarbon group, an aryl group, and an aralkyl group.

[0081] The number of carbon atoms in the alkyl group is preferably 1 to 30, more preferably 1 to 14, even more preferably 1 to 8, and particularly preferably 1 to 4, in order to increase the dynamic binding capacity and improve low protein ligand leakage. The alkyl group may be linear or branched. Specific examples of the alkyl group include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, pentyl, hexyl, heptyl, octyl, 2-ethylhexyl, nonyl, decyl, undecyl, and dodecyl groups. The number of carbon atoms in the alkenyl group is preferably 2 to 30, more preferably 2 to 14, still more preferably 2 to 8, and particularly preferably 2 to 4. The alkenyl group may be linear or branched. Specific examples of the alkenyl group include a vinyl group, a propenyl group, and a butenyl group.

[0082] The number of carbon atoms in the cycloalkyl group or bridged ring hydrocarbon group is preferably 3 to 30, more preferably 3 to 12, and particularly preferably 3 to 8. Specific examples of the cycloalkyl group include a cyclopropyl group and a cyclohexyl group. Examples of the bridged ring hydrocarbon group include an isobornyl group.

[0083] The number of carbon atoms in the aryl group is preferably 6 to 30, more preferably 6 to 12. For example, a phenyl group can be mentioned. The number of carbon atoms in the aralkyl group is preferably 7 to 30, more preferably 7 to 13. For example, a benzyl group, a phenethyl group can be mentioned.

[0084] Also, R 1 ~R 5 The "hydrocarbon group" in the formula (I) may have a substituent. The substituent is preferably a substituent containing a hetero atom, and examples thereof include: a halogen atom such as a fluorine atom, a chlorine atom, a bromine atom, or an iodine atom; a hydroxy group; an alkoxy group such as a methoxy group or an ethoxy group (preferably an alkoxy group having 1 to 4 carbon atoms); an amino group; a monoalkylamino group such as a monomethylamino group, a monoethylamino group, or a mono-n-propylamino group (preferably a mono-C 1-4alkylamino group; dialkylamino group such as dimethylamino group, diethylamino group, methylethylamino group (preferably di-C 1-4 alkylamino group; carboxy group; cyano group; sulfo group; nitro group, etc. 1-4 The alkylamino group means a monoalkylamino group having 1 to 4 carbon atoms in the alkyl group, and a diC 1-4 The alkylamino group refers to a dialkylamino group in which the carbon numbers of the two alkyl groups are 1 to 4. The number of substituents is preferably 0 to 8, and more preferably 0 to 2.

[0085] R in formula (1) 1 and R 2 In order to increase the dynamic binding capacity or improve the low leakage of protein ligands, the alkyl group is preferably a hydrogen atom, a substituted or unsubstituted alkyl group, or a cycloalkyl group, and more preferably a hydrogen atom, an alkyl group, a cycloalkyl group, an aminoalkyl group, a monoalkylaminoalkyl group, or a dialkylaminoalkyl group, and more preferably a hydrogen atom, an alkyl group having 1 to 14 carbon atoms, a cycloalkyl group having 3 to 12 carbon atoms, an aminoalkyl group having 1 to 14 carbon atoms, a monoC 1-4 an alkyl group having 1 to 14 carbon atoms and an alkylamino group as a substituent, or a diC 1-4 An alkyl group having 1 to 14 carbon atoms and having an alkylamino group as a substituent is more preferred, and examples thereof include a hydrogen atom, an alkyl group having 1 to 8 carbon atoms, a cycloalkyl group having 3 to 8 carbon atoms, an aminoalkyl group having 1 to 8 carbon atoms, and a mono-C 1-4 an alkyl group having 1 to 8 carbon atoms and an alkylamino group as a substituent, or a diC 1-4 An alkyl group having 1 to 8 carbon atoms and having an alkylamino group as a substituent is more preferred, and examples thereof include a hydrogen atom, an alkyl group having 1 to 4 carbon atoms, a cycloalkyl group having 3 to 8 carbon atoms, an aminoalkyl group having 1 to 4 carbon atoms, and a mono-C 1-4 an alkyl group having 1 to 4 carbon atoms and an alkylamino group as a substituent, or a diC 1-4 An alkyl group having 1 to 4 carbon atoms and having an alkylamino group as a substituent is particularly preferred. Furthermore, R in formula (1) 1 and R 2R 1 and R 2 At least one of the groups is preferably an aminoalkyl group, a monoalkylaminoalkyl group, or a dialkylaminoalkyl group.

[0086] R in equation (2) 3 and R 4 In order to increase the dynamic binding capacity or improve low protein ligand leakage, the hydrocarbon group represented by the formula (I) is preferably a substituted or unsubstituted alkyl group or a substituted or unsubstituted alkenyl group, more preferably an alkyl group or an alkenyl group, even more preferably an alkyl group having 1 to 14 carbon atoms or an alkenyl group having 1 to 14 carbon atoms, even more preferably an alkyl group having 1 to 8 carbon atoms or an alkenyl group having 1 to 8 carbon atoms, even more preferably an alkyl group having 1 to 4 carbon atoms or an alkenyl group having 1 to 4 carbon atoms, even more preferably an alkyl group having 1 to 4 carbon atoms, and particularly preferably an alkyl group having 2 to 4 carbon atoms. Also, R 3 and R 4 The number of carbon atoms in the cyclic structure formed by bonding R to each other is preferably 4 to 8, and more preferably 4 to 6. 3 and R 4 In the case where the groups are bonded to each other to form a cyclic structure, examples of the compound having a ligand reactive group include succinic anhydride, maleic anhydride, citraconic anhydride, dimethylmaleic anhydride, and glutaric anhydride.

[0087] R in equation (3) 5As the alkyl group, in order to increase the dynamic binding capacity or improve low protein ligand leakage, a substituted or unsubstituted alkyl group is preferable, a substituted or unsubstituted alkyl group having 1 to 14 carbon atoms is more preferable, a substituted or unsubstituted alkyl group having 1 to 8 carbon atoms is even more preferable, a substituted or unsubstituted alkyl group having 1 to 4 carbon atoms is even more preferable, an alkyl group having 1 to 4 carbon atoms or an alkyl group having 1 to 4 carbon atoms with a substituent selected from a halogen atom, a hydroxy group, and an alkoxy group is even more preferable, an alkyl group having 1 to 4 carbon atoms or an alkyl group having 1 to 4 carbon atoms with a substituent selected from a halogen atom, a hydroxy group, and an alkoxy group having 1 to 4 carbon atoms is even more preferable, and an alkyl group having 1 to 4 carbon atoms is particularly preferable. X in formula (3) represents a cyclic ether group. This cyclic ether group is the same as those exemplified as functional groups in step A-P1, and 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. Specific examples of the cyclic ether group include the cyclic ether groups represented by formulas (4) to (9) exemplified as functional groups in step A-P1, with the cyclic ether groups represented by formula (4), (6) or (9) being preferred, and the cyclic ether group represented by formula (4) being more preferred.

[0088] Compounds having a ligand-reactive group used in step B include 1-[3-(dimethylamino)propyl]-3-ethylcarbodiimide, salts of 1-[3-(dimethylamino)propyl]-3-ethylcarbodiimide, N,N'-dicyclohexylcarbodiimide, N,N'-diisopropylcarbodiimide, maleic anhydride, citraconic anhydride, dimethylmaleic anhydride, propionic anhydride, and acetic anhydride, in order to increase the dynamic binding capacity and improve the low leakage of protein ligands. Preferred are one or more compounds selected from the group consisting of 1-[3-(dimethylamino)propyl]-3-ethylcarbodiimide, a salt of 1-[3-(dimethylamino)propyl]-3-ethylcarbodiimide, N,N'-dicyclohexyl ... More preferred are one or more compounds selected from diisopropylcarbodiimide, maleic anhydride, propionic anhydride, acetic anhydride, pivalic anhydride, succinic anhydride, glutaric anhydride, propylene oxide, butylene oxide, glycidyl methyl ether, ethyl glycidyl ether, glycidol, epichlorohydrin, and epibromohydrin, and 1-[3-(dimethylamino)propyl]-3-ethylcarbodiimide, 1-[3-(dimethylamino)propyl]-3-ethyl More preferred are one or more compounds selected from the group consisting of 1-[3-(dimethylamino)propyl]-3-ethylcarbodiimide, salts of 1-[3-(dimethylamino)propyl]-3-ethylcarbodiimide, N,N'-dicyclohexylcarbodiimide, N,N'-diisopropylcarbodiimide, maleic anhydride, propionic anhydride, acetic anhydride, pivalic anhydride, succinic anhydride, and glutaric anhydride, and more preferred are 1-[3-(dimethylamino)propyl]-3-ethylcarbodiimide, salts of 1-[3-(dimethylamino)propyl]-3-ethylcarbodiimide, N,N'-dicyclohexylcarbodiimide, and N,More preferred are one or more compounds selected from N'-diisopropylcarbodiimide, even more preferred are compounds selected from 1-[3-(dimethylamino)propyl]-3-ethylcarbodiimide and salts thereof, and particularly preferred is 1-[3-(dimethylamino)propyl]-3-ethylcarbodiimide hydrochloride.

[0089] The total amount of the compound having a ligand-reactive group used in step B is preferably 0.01 mmol to 15 mmol, more preferably 0.02 mmol to 10 mmol, even more preferably 0.05 mmol to 5 mmol, still more preferably 0.1 mmol to 3 mmol, and particularly preferably 0.3 mmol to 3 mmol, per gram of dry weight of the porous particles to which the ligand is immobilized, in order to maintain a high dynamic binding capacity while sufficiently modifying the ligand.

[0090] To increase the reaction efficiency, step B is preferably carried out in an aqueous medium. Examples of aqueous media include water and various buffers such as carbonate buffer, CHES buffer, CAPS buffer, TAPS buffer, sodium borate buffer, and phosphate buffer. Among these, carbonate buffer and CAPS buffer are preferred, with carbonate buffer being more preferred, in order to improve low protein ligand leakage. The reaction pH in step B is preferably 7 or higher, more preferably 8 to 14, even more preferably 9 to 12, and particularly preferably 9 to 11, in order to increase the dynamic binding capacity and improve low protein ligand leakage.

[0091] The reaction time in step B is not particularly limited, but is usually about 0.1 to 72 hours, and preferably 0.3 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.

[0092] The reaction products obtained in each of the above steps may be purified by separation means such as filtration, washing, etc. Also, they may be classified.

[0093] Furthermore, the method for producing a chromatography carrier of the present invention makes it possible to easily produce a chromatography carrier that has a large dynamic binding capacity for an antibody or a fragment thereof and is less likely to leak protein ligands even when used repeatedly for antibody isolation. Furthermore, the chromatography carrier thus obtained has a large dynamic binding capacity for antibodies or their fragments, and is less likely to leak protein ligands even when repeatedly used for antibody isolation. Furthermore, nonspecific adsorption of contaminants to the protein ligand can be suppressed during antibody isolation. Furthermore, the chromatography carrier of the present invention thus obtained is suitable for use in affinity chromatography. Next, the chromatography carrier of the present invention thus obtained will be described.

[0094] [Chromatography Support] The chromatography carrier of the present invention comprises porous particles, a ligand immobilized on the porous particles, and a partial structure derived from a compound having at least one ligand-reactive group selected from a group represented by -C(=O)-OC(=O)-, a carbodiimide group, and a cyclic ether group, wherein the ligand is one or more ligands selected from Protein A, Protein G, Protein L, and their analogs, and at least one functional group selected from an amino group and a carboxy group of the ligand is chemically modified with the partial structure, and has a structure represented by the following formula: Chemical modification rate (mol%) = (number of moles of chemically modified functional groups) / (sum of number of moles of chemically modified functional groups and number of moles of unmodified functional groups) × 100 The porous particles, the ligand, and the compound having a ligand-reactive group are the same as those explained in the method for producing a chromatography support of the present invention.

[0095] Examples of compounds having a ligand-reactive group include those used in step B. Examples of partial structures derived from compounds having a ligand-reactive group include -C(=O)-, -NR 1-C(=O)-, -C(=NR 1 )- or -CH2-CH(-OH)- in the molecule, and -C(=O)-, -C(=NR 1 )- or -NR 1 More preferred are those having a group represented by —C(═O)— in the molecule, and —C(═NR 1 )- or -NR 1 More preferred are those having a group represented by -C(=O)- in the molecule, and -C(=NR 1 Particularly preferred are those having a group represented by the formula: In addition, as a partial structure having a group represented by -C(=O)- in a molecule, -C(=O)-R is preferred in order to increase the dynamic binding capacity and improve the low leakage of protein ligands. 3 or a group represented by -C(=O)-R 21 A group represented by —C(═O)OH is preferred. Also, -C(=NR 1 As a partial structure having a group represented by -C(=NR)- in a molecule, in order to increase the dynamic binding capacity or improve the low leakage of protein ligands, 1 )-NR 2 Preferably, the compound has a group represented by - in the molecule, and 1 )-NR 2 A group represented by -H is more preferred. Also, -NR 1 As a partial structure having a group represented by -C(=O)- in the molecule, -NR 1 -C(=O)-NR 2 Preferably, the compound has a group represented by -NR 1 -C(=O)-NR 2 A group represented by -H is more preferred. In addition, as a partial structure having a group represented by -CH2-CH(-OH)- in a molecule, -CH2-CH(-OH)-R is used in order to increase the dynamic binding capacity and improve the low leakage of protein ligands. 5 A group represented by the following formula is preferred. R in the above partial structure1 , R 2 , R 3 and R 5 is R in formulas (1) to (3). 1 , R 2 , R 3 and R 5 is synonymous with R 1 and R 2 are each independently a hydrogen atom or a substituted or unsubstituted hydrocarbon group. 3 and R 5 are each independently a substituted or unsubstituted hydrocarbon group. Also, -C(=O)-R 21 -C(=O)OH is represented by formula (2) and R 3 and R 4 Any group may be formed by ring-opening a compound in which R 21 is preferably a divalent hydrocarbon group having 2 to 6 carbon atoms. Examples include alkanediyl groups such as ethane-1,2-diyl, propane-1,3-diyl, butane-1,4-diyl, and pentane-1,5-diyl; and alkenediyl groups such as ethene-1,2-diyl.

[0096] When the compound having a ligand-reactive group has at least one selected from a group represented by -C(=O)-OC(=O)-, a carbodiimide group, and a cyclic ether group, the chromatography support of the present invention is preferably one in which the amino group of the ligand is chemically modified with the partial structure. When the compound has a carbodiimide group, the amino group of the ligand may be chemically modified with the partial structure, and the carboxyl group of the ligand may also be chemically modified with the partial structure.

[0097] The chemical modification rate is preferably 1 mol% or more, more preferably 3 mol% or more, even more preferably 10 mol% or more, even more preferably 20 mol% or more, and particularly preferably 30 mol% or more in order to increase the dynamic binding capacity and improve low leakage of the protein ligand, and is preferably 70 mol% or less, more preferably 60 mol% or less, even more preferably 55 mol% or less, and particularly preferably 50 mol% or less in order to maintain the functions that the protein ligand should have. Specific ranges are preferably 1 mol% to 70 mol%, more preferably 3 mol% to 70 mol%, more preferably 10 mol% to 60 mol%, even more preferably 20 mol% to 55 mol%, and particularly preferably 30 mol% to 50 mol%.

[0098] The chemical modification rate can be calculated, for example, by amino acid analysis, neutralization titration, or potentiometric titration according to the following formula: Specifically, when the compound having a ligand-reactive group has at least one selected from a carbodiimide group and a cyclic ether group, and the amino group of the ligand is chemically modified with the partial structure, the chemical modification rate can be calculated, for example, by amino acid analysis according to the following formula: Chemical modification rate (mol %)={(1−number of moles of lysine in protein ligand after step B) / (number of moles of protein ligand in porous particle 5×number of lysines present per protein ligand in porous particle 5)×100} It can be calculated as follows. Furthermore, when the compound having the ligand reactive group has a group represented by -C(=O)-OC(=O)- and the amino group of the ligand is chemically modified with the partial structure, for example, the compound can be reacted with the compound represented by the following formula based on the neutralization titration method: Chemical modification rate (mol%) = (1 - amino group content before step B / amino group content in chromatography support) × 100 It can be calculated by the formula: When the compound having a ligand-reactive group has a group represented by -C(=O)-OC(=O)-, an amide group may be generated by reaction between the amino group of the ligand and the compound having the ligand-reactive group. The generation of such an amide group may interfere with accurate measurement in the above-mentioned amino acid analysis method. Therefore, when the compound having a ligand-reactive group has a group represented by -C(=O)-OC(=O)-, it is preferable to perform the calculation based on the neutralization titration method as described above.

[0099] The volume average particle diameter of the chromatography carrier of the present invention is preferably 40 to 150 μm, more preferably 50 to 100 μm, and the coefficient of variation of the volume average particle diameter is preferably 40% or less, more preferably 30% or less. The specific surface area of ​​the chromatography carrier of the present invention is preferably 1 to 500 m 2 / g, more preferably 10 to 300m 2 / g. The volume average pore diameter of the chromatography carrier of the present invention is preferably 10 to 300 nm. The volume average particle size, coefficient of variation, specific surface area, and volume average pore size can be measured by laser diffraction / scattering particle size distribution measurement or the like.

[0100] [Chromatography column] The chromatography column of the present invention is characterized by containing the chromatography support of the present invention. The chromatography column of the present invention is similar to a conventional chromatography column except that it contains 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 columns of the present invention are suitable for use in affinity chromatography.

[0101] [Method for isolating antibodies or fragments thereof] The method for isolating an antibody or a fragment thereof of the present invention is characterized by using the chromatographic support of the present invention or the chromatographic column of the present invention. As used herein, the term "antibody" is a concept that encompasses any class of immunoglobulin, such as IgG, IgA, IgD, IgE, IgM, and subclasses thereof, as well as variants thereof. Furthermore, as used herein, the term "antibody" may also include chimeric antibodies such as humanized antibodies, antibody complexes, and other modified immunoglobulins that contain an antigen-recognition site. Furthermore, as used herein, the term "antibody fragment" may refer to either an antibody fragment containing an antigen-recognition site or an antibody fragment not containing an antigen-recognition site. Examples of antibody fragments not containing an antigen-recognition site include proteins consisting of only the Fc region of immunoglobulin, Fc fusion proteins, and mutants and modified forms thereof.

[0102] The method for isolating the antibody or fragment thereof of the present invention may be carried out in the same manner as a general method for isolating an antibody or fragment thereof, except that the chromatography support of the present invention or the chromatography column of the present invention is used. Specifically, the method includes a step of contacting the chromatography support of the present invention with a sample containing the antibody or fragment thereof. Furthermore, after capturing the antibody or fragment thereof on the chromatography support through this step and separating contaminants (e.g., proteins other than the antibody or fragment thereof) from the antibody or fragment thereof, it is preferable to carry out an elution step in which the antibody or fragment thereof captured on the chromatography support is eluted. The antibody or fragment thereof can be isolated from the sample by collecting this eluate. A dissociation solution that dissociates the immunoglobulin-binding protein and the antibody or fragment thereof is usually used in the elution step. Alternatively, the isolation may be carried out using the chromatography column of the present invention. Such a method includes a step of passing a sample containing an antibody or a fragment thereof through the chromatography column of the present invention, and preferably, this step captures the antibody or a fragment thereof on the chromatography carrier, thereby separating contaminants from the antibody or the fragment thereof, followed by an elution step similar to that described above.

[0103] The sample containing an antibody or a fragment thereof is not particularly limited, and examples thereof include blood components such as whole blood, serum, plasma, various blood cells, blood clots, and platelets; body fluids such as urine, semen, breast milk, sweat, interstitial fluid, interstitial lymph fluid, bone marrow fluid, tissue fluid, saliva, gastric juice, synovial fluid, pleural effusion, bile, ascites, and amniotic fluid; and various liquid samples such as bacterial fluid, cell culture medium, cell culture supernatant, and tissue cell homogenate. [Example]

[0104] The present invention will be described in detail below with reference to examples, but the present invention is not limited to these examples.

[0105] Example 1 (Step A-1) Immobilization of Ligand 2.69 g of polyvinyl alcohol (PVA-217, manufactured by Kuraray Co., Ltd.) was added to 448 g of pure water and heated and stirred to dissolve the polyvinyl alcohol, yielding an aqueous solution. Separately, 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 29.38 g of 2-octanone (manufactured by Toyo Gosei Co., Ltd.) to prepare a monomer solution. The entire amount of the aqueous solution was then poured into a separable flask, which was then fitted with a thermometer, stirring blade, and condenser, placed in a hot water bath, and stirring was initiated under a nitrogen atmosphere. The entire amount of the monomer solution was placed in a separable flask and heated in a 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 raised to 86°C. Stirring was then carried out for 3 hours while maintaining the temperature at 86°C. The reaction solution was then cooled, filtered, and washed with pure water and ethanol. The washed particles were dispersed in pure water and decanted three times to remove small particles. The particles were then dispersed in pure water to a particle concentration of 10% by mass, yielding a porous particle dispersion. The porous particles contained in this dispersion are referred to as "porous particles 1." Next, 0.956 g of adipic acid dihydrazide (Tokyo Chemical Industry Co., Ltd.), 8 g of thioglycerol (Tokyo Chemical Industry Co., Ltd.), and 1.418 g of diisopropylethylamine (Tokyo Chemical Industry Co., Ltd.) were added to 100 g of the porous particle 1 dispersion, and the mixture was heated to 70°C and stirred for 8 hours while maintaining the temperature at 70°C. The reaction solution was then cooled, filtered, and washed with pure water and ethanol. The particles were then dispersed in pure water to a particle concentration of 10% by mass, yielding a porous particle dispersion. The porous particles contained in this dispersion are referred to as "porous particles 2." Next, ethylene glycol diglycidyl ether was reacted with the thioglycerol-derived hydroxyl groups contained in porous particles 2. Specifically, 8.7 g of pure water, 1.2 g of sodium sulfate (manufactured by Wako Pure Chemical Industries, Ltd.), and 0.10 g of sodium carbonate (manufactured by Wako Pure Chemical Industries, Ltd.) were mixed to obtain a carbonate buffer (pH 11.2). 0.5 g of ethylene glycol diglycidyl ether (Denacol EX810, manufactured by Nagase ChemteX Corporation) and 8 mL of the porous particle 2 dispersion were added to this carbonate buffer and shaken and stirred at 23°C for 16 hours. Next, the particles were 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 3." Next, a ligand was immobilized on porous particle 3. 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 9.4). 0.21 g of modified protein A, a hexamer of the amino acid sequence domain of SEQ ID NO: 1 (SEQ ID NO: 29 in WO 2020 / 040307 ), and 8 mL of porous particle 3 dispersion were added to 34 mL of this carbonate buffer, and the mixture was shaken and stirred at 23°C for 1.5 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 4."

[0106] (Step A-2) Hydrophilization reaction A buffer was prepared by mixing 8.8 g of pure water, 0.1 g of sodium sulfate (manufactured by Wako Pure Chemical Industries, Ltd.), and 0.03 g of sodium hydroxide (manufactured by Wako Pure Chemical Industries, Ltd.), and then 4.5 g of thioglycerol (manufactured by Tokyo Chemical Industry Co., Ltd.) was added to obtain a hydrophilization reaction solution. 8 mL of the porous particle 4 dispersion obtained in step A-1 was added to this hydrophilization reaction solution, and the mixture was shaken and stirred at 23°C for 16 hours to carry out a hydrophilization reaction. The porous particles after this hydrophilization reaction were filtered and washed sequentially with a 0.1 M aqueous sodium hydroxide solution and a 0.1 M sodium citrate buffer (pH 3.2). Next, the particles were 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 5."

[0107] (Step B) Post-modification reaction A solution of 67.2 mg of sodium bicarbonate (Wako Pure Chemical Industries, Ltd.) dissolved in 6 mL of purified water was mixed with 1 M sodium hydroxide (Wako Pure Chemical Industries, Ltd.) and purified water to obtain 8 mL of carbonate buffer 1 (pH 10 at 23°C). 8 mL of the porous particle 5 dispersion obtained in step A-2 and 0.16 g of 1-[3-(dimethylamino)propyl]-3-ethylcarbodiimide (hereinafter also referred to as EDC) were added to this carbonate buffer 1, and the mixture was shaken and stirred at 23°C for 1.5 hours to obtain carrier 1. This carrier 1 was filtered and washed sequentially with 0.1 M aqueous sodium hydroxide, 0.1 M sodium citrate buffer (pH 3.2), and 16 wt% ethanol / 50 mM phosphate buffer (pH 7.3) to obtain a carrier-containing solution for affinity chromatography (carrier content 50% by volume).

[0108] Calculation of modification rate (amino acid analysis method) 90 μL of the affinity chromatography carrier-containing solution obtained in the post-modification reaction in step B was placed in a 2 mL tube (Eppendolf, product number 0030120094) and hydrolyzed with 100 μL of 12 N aqueous hydrochloric acid at 110°C for 24 hours with stirring. After completion of the hydrolysis reaction, the supernatant was removed and the aqueous hydrochloric acid solution was completely removed by vacuum drying. 600 μL of 0.1 N aqueous hydrochloric acid was added to the tube to dissolve the carrier. The entire volume of the resulting carrier-containing solution was transferred to a 0.22 μm filter tube (Merck Millipore, product number UFC30GV00). The solid components were removed by centrifugation, and the filtrate was collected. The resulting filtrate was subjected to UHPLC analysis (using a Thermo Fisher Scientific Vanquish HPLC under the following measurement conditions). Column: ACQUITY UPLC HSS T3 Column (C18), 100Å, 1.8μm, 2.1mm x 150nm Mobile phase A: 0.4% heptafluorobutyric acid + 0.02% formic acid in H2O Mobile phase B: 0.02% formic acid in MeCN Gradient conditions: 0-0.75min Mobile phase B 1%, 0.75-1.5min Mobile phase B 1→5%, 1.5-5min Mobile phase B 5→7.5%, 5-17.5min Mobile phase B 7.5→27.5%, 17.5-18min Mobile phase B 27.5→80%, 18-20min Mobile phase B 80%, 20-20.1min Mobile phase B 80→1%, 20.1-25min Mobile phase B 1% Flow rate: 0.2ml / min Column temperature: 25℃ Detection: Thermo Scientific Corona Veo RS (Charged Aerosol Detector; CAD) Injection volume: 2μl The number of moles of leucine was determined from the peak area of ​​the obtained chromatogram, and the number of moles of the Protein A ligand used in this example was calculated. Next, the number of moles of lysine was determined from the peak area of ​​the chromatogram, and the modification rate (mol %) was calculated from the reduction rate of lysine in the Protein A ligand used in this example using the following formula. Modification rate (mol %)={(1−number of moles of lysine in the filtrate obtained by removing solid components by the above procedure) / (number of moles of protein A ligand in porous particle 5×number of lysines present per protein A ligand in porous particle 5)×100}

[0109] Example 2 A carrier-containing solution for affinity chromatography (carrier content 50% by volume) was obtained by the same procedures as in Steps A-1, A-2, and B of Example 1, except that 0.16 g of 1-[3-(dimethylamino)propyl]-3-ethylcarbodiimide used in Step B was replaced with 0.14 g of propionic anhydride. The carrier contained in this solution is referred to as Carrier 2. 10 mL of the carrier 2-containing solution was subjected to suction filtration 10 times with 5 mL of 0.01 M NaOH aqueous solution using JIS P3801 Type 5A filter paper to perform buffer replacement. The resulting dispersion and pure water were mixed in a bottle to make 0.6 g of carrier, 8 g of 0.01 M NaOH aqueous solution, and 30 g of pure water. The resulting carrier-containing solution was titrated with 0.01 M hydrochloric acid aqueous solution while stirring with a stirrer. The titration was performed by adding the hydrochloric acid aqueous solution dropwise in 0.1 mL increments, and the neutralization point was set at pH 7. The amino group content per 1 g of carrier solids was calculated using the following formula from the amount of hydrochloric acid aqueous solution required to reach the neutralization point. Amino group content (μmol / g-bz) = {amount of hydrochloric acid solution required to reach neutralization point (mL) × 0.01 - 8 × 0.01} / 0.6 Similarly, the same procedure was carried out for the porous particles 5 obtained in step A-2, and the amino group content per 1 g of the porous particle solid content was calculated. Next, the modification rate (mol %) was calculated using the following formula from the ratio of the amino group content per 1 g of the solid content before and after post-modification. Modification rate (mol%) = (1 - amino group content in support 2 / amino group content in porous particle 5) × 100

[0110] Example 3 Carrier 3 was obtained by the same procedures as in Steps A-1, A-2, and B of Example 1, except that 0.16 g of 1-[3-(dimethylamino)propyl]-3-ethylcarbodiimide used in Step B was changed to 0.10 g of acetic anhydride. The modification rate (mol %) was measured in the same manner as in Example 2.

[0111] Example 4 Carrier 4 was obtained by the same procedures as in steps A-1, A-2, and B of Example 1, except that 0.16 g of 1-[3-(dimethylamino)propyl]-3-ethylcarbodiimide used in step B was changed to 0.10 g of succinic anhydride. The modification rate (mol %) was measured in the same manner as in Example 2.

[0112] Example 5 Carrier 5 was obtained by the same procedures as in Steps A-1, A-2, and B of Example 1, except that 0.16 g of 1-[3-(dimethylamino)propyl]-3-ethylcarbodiimide used in Step B was changed to 0.19 g of pivalic anhydride. The modification rate (mol %) was measured in the same manner as in Example 2.

[0113] Example 6 Carrier 6 was obtained by the same procedures as in steps A-1, A-2, and B of Example 1, except that 0.16 g of 1-[3-(dimethylamino)propyl]-3-ethylcarbodiimide used in step B was changed to 0.08 g of glycidol. The modification rate (mol %) was measured in the same manner as in Example 1.

[0114] Example 7 Carrier 7 was obtained by the same procedures as in steps A-1, A-2, and B of Example 1, except that the amount of 1-[3-(dimethylamino)propyl]-3-ethylcarbodiimide used in step B was changed from 0.16 g to 0.03 g. The modification rate (mol %) was measured in the same manner as in Example 1.

[0115] Example 8 Carrier 8 was obtained by the same procedures as in steps A-1, A-2, and B of Example 1, except that the amount of 1-[3-(dimethylamino)propyl]-3-ethylcarbodiimide used in step B was changed from 0.16 g to 0.32 g. The modification rate (mol %) was measured in the same manner as in Example 1.

[0116] Example 9 67.2 mg of sodium bicarbonate (Wako Pure Chemical Industries, Ltd.) was dissolved in 5 mL of pure water, and then this was mixed with 5 M sodium hydroxide (Wako Pure Chemical Industries, Ltd.) and pure water to obtain carbonate buffer 2 (pH 13 at 23° C.). Carrier 9 was obtained by the same procedures as in steps A-1, A-2, and B of Example 1, except that carbonate buffer 1 (pH 10 at 23°C) used in step B was changed to carbonate buffer 2 (pH 13 at 23°C). The modification rate (mol%) was measured in the same manner as in Example 1.

[0117] Example 10 151.0 mg of N-cyclohexyl-3-aminopropanesulfonic acid (manufactured by Wako Pure Chemical Industries, Ltd., hereinafter referred to as CAPS) was dissolved in 5 mL of pure water, and then this was mixed with 1 M sodium hydroxide (manufactured by Wako Pure Chemical Industries, Ltd.) and pure water to obtain CAPS buffer (pH 10 at 23°C). Carrier 10 was obtained by the same procedures as in steps A-1, A-2, and B of Example 1, except that the carbonate buffer 1 (pH 10 at 23°C) used in step B was changed to CAPS buffer (pH 10 at 23°C). The modification rate (mol%) was measured in the same manner as in Example 1.

[0118] Example 11 177.0 mg of N-cyclohexyl-2-aminoethanesulfonic acid (manufactured by Wako Pure Chemical Industries, Ltd., hereinafter referred to as CHES) was dissolved in 5 mL of pure water, and then this was mixed with 1 M sodium hydroxide (manufactured by Wako Pure Chemical Industries, Ltd.) and pure water to obtain CHES buffer (pH 10 at 23°C). Carrier 11 was obtained by the same procedures as in steps A-1, A-2, and B of Example 1, except that the carbonate buffer 1 (pH 10 at 23°C) used in step B was changed to CHES buffer (pH 10 at 23°C). The modification rate (mol%) was measured in the same manner as in Example 1.

[0119] (Comparative Example 1) The same operations as in steps A-1 and A-2 of Example 1 were carried out to obtain porous particles 5. These porous particles 5 were used as the carrier of Comparative Example 1.

[0120] (Test Example 1) Dynamic Binding Capacity (DBC) Measurement Test Using a Cytiva AKTA avant25, the DBC of each carrier in the Examples and Comparative Examples was measured for a protein (human IgG antibody, LGC 1875-0007) at a retention time of 4 minutes. A 4 mL column (5 mm diameter x 200 mm length) was used, and the protein was dissolved at 5 mg / mL in a 20 mM sodium phosphate / 150 mM sodium chloride aqueous solution (pH 7.5). The DBC was calculated from the amount of protein captured at 10% breakthrough at the elution tip and the column volume, and evaluated according to the following criteria. The results are shown in Table 1.

[0121] (DBC evaluation criteria) AAA (excellent): 60mg / mL or more AA (Excellent): 55 mg / mL or more and less than 60 mg / mL A (Good): 50 mg / mL or more and less than 55 mg / mL B (poor): Less than 50 mg / mL

[0122] (Test Example 2) Protein A leakage measurement test Using a Cytiva AKTA avant25, 7.5 mL of cell culture medium (Herceptin, titer: 4.38 mg / mL) was loaded onto each carrier in the Examples and Comparative Examples with a retention time of 4 minutes, and the antibody was then recovered with the eluate. Considering practical use, the carrier was soaked in 0.5 M NaOH for 100 cycles (25 hours). The column container had a capacity of 0.8 mL (5 mm diameter x 40 mm length), and the eluate was 100 mM sodium acetate aqueous solution (pH 3.3). Prior to elution, the column was washed with 20 mM sodium phosphate / 500 mM sodium chloride aqueous solution (pH 7.5). Next, the amount of Protein A in the eluate was determined using a Protein A ELISA kit (F740), and the antibody concentration in the eluate was determined from the absorbance. These values ​​were then used to calculate the amount of Protein A leaked per amount of antibody in the recovered eluate. The smaller the value of Protein A leaked, the less likely the protein ligand will leak, even when the sample is repeatedly used to isolate antibodies. The results are shown in Table 1.

[0123] [Table 1]

Claims

1. A method for producing a chromatography carrier, comprising the following steps A-1 and B: (Step A-1) A step of immobilizing one or more ligands selected from protein A, protein G, protein L, and their analogs onto porous particles. (Step B) A step of reacting the porous particles to which the ligands have been immobilized after Step A-1 with a compound having at least one ligand-reactive group selected from a group represented by —C(═O)—O—C(═O)—, a carbodiimide group, and a cyclic ether group.

2. 2. The method for producing a chromatography carrier according to claim 1, wherein the compound having a ligand reactive group is one or more selected from the group consisting of a compound represented by the following formula (1) and a salt thereof, a compound represented by the following formula (2), and a compound represented by the following formula (3): 【Chemical 1】 [In formula (1), R 1 and R 2 are each independently a hydrogen atom or a substituted or unsubstituted hydrocarbon group. 【Chemistry 2】 [In formula (2), R 3 and R 4 each independently represents a substituted or unsubstituted hydrocarbon group, R 3 and R 4 may be bonded to each other to form a cyclic structure. 【Chemistry 3】 [In formula (3), R 5 represents a substituted or unsubstituted hydrocarbon group, and X represents a cyclic ether group.

3. 2. The method for producing a chromatography carrier according to claim 1, wherein the compound having a ligand reactive group is one or more compounds selected from 1-[3-(dimethylamino)propyl]-3-ethylcarbodiimide, a salt of 1-[3-(dimethylamino)propyl]-3-ethylcarbodiimide, N,N'-dicyclohexylcarbodiimide, N,N'-diisopropylcarbodiimide, maleic anhydride, propionic anhydride, acetic anhydride, pivalic anhydride, succinic anhydride, glutaric anhydride, propylene oxide, butylene oxide, glycidyl methyl ether, ethyl glycidyl ether, glycidol, epichlorohydrin, and epibromohydrin.

4. 2. The method for producing a chromatography carrier according to claim 1, wherein the compound having a ligand reactive group is at least one compound selected from the group consisting of compounds represented by the following formula (1) and salts thereof: 【Chemistry 4】 [In formula (1), R 1 and R 2 are each independently a hydrogen atom, an alkyl group, a cycloalkyl group, an aminoalkyl group, a monoalkylaminoalkyl group, or a dialkylaminoalkyl group.

5. 2. The method for producing a chromatography carrier according to claim 1, wherein the amount of the compound having a ligand reactive group used is 0.01 to 15 mmol per gram of dry weight of the porous particles to which the ligand is immobilized.

6. 2. The method for producing a chromatography carrier according to claim 1, wherein the reaction in step B is carried out in an aqueous medium at a pH of 8 to 14.

7. The method for producing a chromatography carrier according to claim 1, further comprising the following step A-2 between step A-1 and step B, wherein the hydrophilic group-containing, ligand-immobilized porous particles obtained in step A-2 are used in step B as the ligand-immobilized porous particles after step A-1. (Step A-2) A step of reacting the porous particles to which the ligands have been immobilized in Step A-1 with a compound having a total of two or more hydrophilic groups of at least one type selected from hydroxyl groups and mercapto groups in the molecule.

8. The method for producing a chromatography carrier according to claim 7, further comprising the following steps A-P1 and A-P2, in which porous particles reacted with at least one agent selected from a crosslinking agent and a hydrophilizing agent in step A-P2 are used as the porous particles in step A-1. (Step A-P1) A step of dispersing a monomer composition in an aqueous medium and carrying out suspension polymerization (Step A-P2) A step of reacting the porous particles obtained in step A-P1 with at least one agent selected from a crosslinking agent and a hydrophilizing agent.

9. The present invention relates to a method for producing a porous particle, a ligand fixed to the porous particle, and a partial structure derived from a compound having at least one ligand-reactive group selected from a group represented by -C(=O)-O-C(=O)-, a carbodiimide group, and a cyclic ether group, the ligand is one or more ligands selected from protein A, protein G, protein L, and analogs thereof; at least one functional group selected from an amino group and a carboxy group of the ligand is chemically modified with the partial structure; The following formula: Chemical modification rate (mol %)=(number of moles of chemically modified functional groups) / (sum of number of moles of chemically modified functional groups and number of moles of unmodified functional groups)×100 A chromatography carrier having a chemical modification rate calculated by the following formula: 1 to 70 mol %.

10. The chromatography carrier according to claim 9, wherein the amino group of the ligand is chemically modified with the partial structure.

11. The partial structure is -C(=O)-, -NR 1 -C(=O)-, -C(=NR 1 )- or -CH 2 -CH(-OH)-(R 1 The chromatography carrier according to claim 9 , which has in its molecule a group represented by the following formula:

12. The partial structure is -NR 1 —C(═O)—NR 2 - or -C(=NR 1 )-NR 2 - (R 1 and R 2 and each independently represent a hydrogen atom, an alkyl group, a cycloalkyl group, an aminoalkyl group, a monoalkylaminoalkyl group, or a dialkylaminoalkyl group.

13. R 1 and R 2 13. The chromatography support according to claim 12, wherein at least one of the groups is an aminoalkyl group, a monoalkylaminoalkyl group, or a dialkylaminoalkyl group.

14. A chromatography column comprising the chromatographic support according to claim 9.

15. A method for isolating an antibody or a fragment thereof, which uses the chromatographic support according to claim 9 or the chromatographic column according to claim 14.

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