Method for producing a chromatography carrier, method for producing a chromatography column, and chromatography carrier

Abstract

The objective of the present invention is to provide a chromatographic carrier having excellent liquid permeability and excellent pressure resistance characteristics during liquid passage.　This method for producing a chromatographic carrier includes the following step 1 and step 2, wherein a solid phase support prepared in step 1 comprises porous particles to which ligands are not fixed, or porous particles to which ligands are fixed.　(Step 1) A step of preparing a solid phase support (Step 2) A step of performing an adjustment by sieving and classifying the solid phase support such that the coefficient of variation of a volume-based particle size distribution of the porous particles when ligands are fixed thereto is at least equal to 1% and at most equal to 22%, and the degree of skew of the volume-based particle size distribution of the porous particles when ligands are fixed thereto is at least equal to -0.1 and at most equal to 5

Description

[Technical field]

[0001] The present invention relates to a method for producing a chromatography support, a method for producing a chromatography column, and a chromatography support. [Background technology]

[0002] In recent years, in the field of biopharmaceuticals, such as antibody drugs, the expression technology of target substances such as proteins has progressed significantly, and there is a demand for improving the productivity in purification processes using chromatography and the like. One method for improving productivity is to reduce the concentration of impurities present in pharmaceutical raw materials, such as host cell-derived proteins and deoxyribonucleic acid, as much as possible in one purification step, thereby reducing the number of purification steps and steps, and there is an increasing demand for chromatography carriers that can achieve this. As such chromatography carriers, for example, a carrier in which a ligand is bound to porous cross-linked particles obtained by polymerizing divinylbenzene, ethylvinylbenzene, and glycidyl methacrylate and further cross-linking with adipic acid dihydrazide (Patent Document 1) and a carrier in which a ligand is bound to cross-linked agarose particles (Patent Document 2) have been reported. [Prior art documents] [Patent documents]

[0003] [Patent Document 1] International Publication No. 2019 / 039545 Brochure [Patent Document 2] Special Publication No. 2016-507729 Summary of the Invention [Problem to be solved by the invention]

[0004] However, the chromatography supports described in Patent Documents 1 and 2 have insufficient liquid permeability and pressure resistance characteristics during liquid passage, and have problems such as a tendency for compression to occur when the flow rate is increased or the scale is increased, and a tendency for the load to be imposed on later steps in the purification process, resulting in a decrease in productivity.

[0005] Therefore, an object of the present invention is to provide a chromatography carrier which is excellent in liquid permeability and pressure resistance characteristics during liquid passage. [Means for solving the problem]

[0006] The above issues are as follows: <1> ~ <12> This was resolved by the following means. <1> The method for producing a chromatography carrier comprises the following steps 1 and 2, in which the solid phase support prepared in step 1 is a porous particle having no ligand immobilized thereon or a porous particle having a ligand immobilized thereon (hereinafter also referred to as the method for producing a chromatography carrier of the present invention). (Step 1) preparing a solid support (Step 2) A step of adjusting the coefficient of variation of the volume-based particle size distribution of the porous particles when the ligand is immobilized to be 1% or more and 22% or less by sieving the solid phase support, and adjusting the coefficient of variation of the volume-based particle size distribution of the porous particles when the ligand is immobilized to be -0.1 or more and 5 or less.

[0007] <2> A method for producing a chromatography support, comprising the following steps 1 and 2, in which the solid support prepared in step 1 is a porous particle having a ligand immobilized thereon. (Step 1) preparing a solid support (Step 2) A step of adjusting the coefficient of variation of the volume-based particle size distribution of the porous particles to which the ligand is fixed to be 1% or more and 22% or less, and the skewness of the volume-based particle size distribution of the porous particles to which the ligand is fixed to be -0.1 or more and 5 or less, by sieving the solid phase support. <3> A method for producing a chromatography support, comprising the following steps 1 and 2, in which the solid support prepared in step 1 is a porous particle having no ligand immobilized thereon. (Step 1) preparing a solid support (Step 2) A step of adjusting the coefficient of variation of the volume-based particle size distribution of the porous particles when the ligand is immobilized to be 1% or more and 22% or less by sieving the solid phase support, and adjusting the coefficient of variation of the volume-based particle size distribution of the porous particles when the ligand is immobilized to be -0.1 or more and 5 or less. <4> (Step 3) further comprising a step of immobilizing a ligand on the porous particles classified by sieving in step 2; <3> A method for producing the chromatography carrier according to claim 1.

[0008] <5> The sieve classification in step 2 includes, in this order or in reverse order, (step 2a) a step of passing the solid support through a sieve having a mesh size of 10% or more and less than 200% of (2a-d), when the volume average particle size (2a-d) of the solid support before the sieve classification is taken as 100%, and recovering the sieve residue, and (step 2b) a step of passing the solid support through a sieve having a mesh size larger than that of the sieve in step 2a and less than 600% of (2a-d), and recovering the sieve-passing part. <1> ~ <4> 2. A method for producing a chromatography support according to any one of the preceding claims. <6> The sieve classification in step 2 is a wet method. <1> ~ <5> 2. A method for producing a chromatography support according to any one of the preceding claims. <7> The coefficient of variation is 5% or more and 20% or less, and the skewness is 0.01 or more and 3 or less. <1> ~ <6> 2. A method for producing a chromatography support according to any one of the preceding claims. <8> Step 1 includes a step of dispersing a monomer composition in an aqueous medium and carrying out suspension polymerization. <1> ~ <7> 2. A method for producing a chromatography support according to any one of the preceding claims.

[0009] <9> A method for producing a chromatography column, comprising the step of packing a carrier into a column container, the carrier being porous particles having ligands fixed thereto, the carrier having a coefficient of variation of a volumetric particle size distribution of 1% or more and 22% or less and a skewness of the volumetric particle size distribution of -0.1 or more and 5 or less (hereinafter also referred to as a method for producing a chromatography column of the present invention).

[0010] <10> A chromatography carrier dispersed in a dispersion medium, the carrier being composed of porous particles having ligands fixed thereto, the carrier having a coefficient of variation of a volume-based particle size distribution of 1% or more and 22% or less, and a skewness of the volume-based particle size distribution of -0.1 or more and 5 or less (hereinafter also referred to as the chromatography carrier of the present invention). <11> The carrier has a volume-based cumulative 50% particle diameter d50 of 55 μm or more and 100 μm or less. <10> The chromatography carrier according to claim 1, <12> The carrier has a volume-based cumulative 1% particle size d1 of 30 μm or more and 75 μm or less. <10> or <11> The chromatography carrier according to claim 1, Effect of the Invention

[0011] According to the present invention, it is possible to provide a chromatography carrier which is excellent in liquid permeability and pressure resistance characteristics during liquid passage. [Brief description of the drawings]

[0012] [Figure 1] FIG. 2 is a graph showing the volume-based particle size distribution of carrier W1 (Example 1). [Diagram 2] FIG. 13 shows the volume-based particle size distribution of carrier W3 (Example 3). [Diagram 3] FIG. 1 shows the volume-based particle size distribution of carrier W5 (Example 5). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0013] <Method of manufacturing chromatography carrier> The method for producing a chromatography support of the present invention comprises the following steps 1 and 2, and is characterized in that the solid phase support prepared in step 1 is a porous particle having no ligand immobilized thereon or a porous particle having a ligand immobilized thereon. (Step 1) preparing a solid support (Step 2) A step of adjusting the coefficient of variation of the volume-based particle size distribution of the porous particles when the ligand is immobilized to be 1% or more and 22% or less by sieving the solid phase support, and adjusting the coefficient of variation of the volume-based particle size distribution of the porous particles when the ligand is immobilized to be -0.1 or more and 5 or less.

[0014] (Process 1) Step 1 is a step of preparing a solid support. The solid support prepared in step 1 is porous particles to which no ligand is immobilized or porous particles to which a ligand is immobilized. The porous particles are preferably those containing a polymer. Such porous particles may be natural polymer-based porous particles composed of polysaccharides such as agarose, dextran, cellulose, etc., or synthetic polymer-based porous particles, but are preferably synthetic polymer-based porous particles in order to increase the dynamic binding capacity, improve the uniformity of the particle size, and simplify the sieve classification process. In addition, the porous particles are preferably water-insoluble. The solid support may be produced by a conventional method, but step 1 preferably includes a step of preparing porous particles to which no ligand is immobilized (hereinafter also referred to as step 1a). -Process 1a- In step 1a, a monomer composition is preferably dispersed in an aqueous medium and suspended in the aqueous medium to facilitate preparation of synthetic polymer-based porous particles having a desired particle size. The monomer composition used in step 1a preferably contains a functional group-containing monomer. The functional group contained in this monomer is preferably one that can be used in additional chemical reactions (such as a reaction with a crosslinking agent), and may be one that can fix 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 (1) to (6), with the cyclic ether group represented by formula (1), (3) or (6) being preferred, and the cyclic ether group represented by formula (1) being more preferred.

[0015] [ka]

[0016] [In the formula, R 1 ~R 4 each independently represents a hydrogen atom or an alkyl group, and * represents a bond.

[0017] 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.

[0018] The functional group-containing monomer is preferably a monomer having a functional group capable of fixing a ligand and a polymerizable unsaturated group. Examples of such monomers include cyclic ether group-containing monomers such as 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-glycidylpolyethylene glycol, and tetrahydrofurfuryl (meth)acrylate. Meth)acrylate monomers; 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, 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 anhydride monomers such as maleic anhydride, methylmaleic anhydride, glutaconic anhydride, etc., as well as (meth)acrylic acid, 3,4-epoxy-1-butene, 3,4-epoxy-3-methyl-1-butene, etc. 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.

[0019] 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 1a, 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 1a.

[0020] Furthermore, the monomer composition used in step 1a may further 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 monomers include polymerizable unsaturated group-containing monomers that do not have a functional group capable of fixing a ligand. The other monomers are broadly classified into non-crosslinkable monomers and crosslinkable monomers, and either one of them may be used or they may be used in combination. According to the present invention, even if a monomer that does not contain a hydrophilic group such as a hydroxyl group is used as the other monomer, the antifouling properties can be satisfied, and the present invention is applicable to a wide range of monomer compositions.

[0021] 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-vinyl amide-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.

[0022] Examples of the (meth)acrylate-based 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 can be used alone or in combination of two or more.

[0023] 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.

[0024] 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.

[0025] 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. 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. 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.

[0026] 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 1a, 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 1a.

[0027] 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.

[0028] 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, 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.

[0029] 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.

[0030] Examples of the allyl-based 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 examples, examples of the crosslinkable monomer include dehydration condensation products of aminoalcohols such as diaminopropanol, trishydroxymethylaminomethane, and glucosamine with (meth)acrylic acid, and conjugated diolefins such as butadiene and isoprene.

[0031] 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 1a, 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 1a.

[0032] The aqueous medium may, for example, be 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 7,000 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.

[0033] In addition, specific methods of step 1a include, for example, a method of dissolving a polymerization initiator in a mixed solution (monomer solution) containing a monomer composition and, if necessary, a porogen, suspending the mixture in an aqueous medium and polymerizing the mixture to a predetermined temperature, a method of dissolving a polymerization initiator in a mixed solution (monomer solution) containing a monomer composition and, if necessary, a porogen, adding the mixture to an aqueous medium heated to a predetermined temperature and polymerizing the mixture, a method of suspending a mixed solution (monomer solution) containing a monomer composition and, if necessary, a porogen, in an aqueous medium and heating the mixture to a predetermined temperature, adding a polymerization initiator and polymerizing the mixture, etc. By providing step 1a in any of these methods, the uniformity of the particle size is improved, and porous particles with excellent liquid permeability can be easily prepared.

[0034] The polymerization initiator is preferably a radical polymerization initiator. Examples of the radical polymerization initiator include azo initiators, peroxide initiators, and redox initiators, and specifically 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 parts by mass or more and 10 parts by mass or less with respect to 100 parts by mass of the total amount of monomers.

[0035] The porosity-inducing agent is used to produce porous particles, and is present together with the monomer in the polymerization in the oil droplets, and plays a role of forming pores as a non-polymerized component. The porosity-inducing agent is not particularly limited as long as it is easily removable, and examples thereof include linear polymers soluble in various organic solvents and mixed monomers, and these may be used in combination.

[0036] 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 porosifier 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, and linear polymers such as homopolymers of non-crosslinkable vinyl monomers. The porosifier can be used alone or in combination of two or more kinds. The total amount of the porosifying agents used is usually about 20 parts by mass or more and 1000 parts by mass or less per 100 parts by mass of the total amount of monomers.

[0037] In addition, in step 1a, 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 addition, nitrites 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 be used. In addition, a polymerization regulator such as dodecyl mercaptan may be used.

[0038] The polymerization temperature in step 1a may be determined depending on the polymerization initiator, but is usually about 2 to 100° C., and when azobisisobutyronitrile is used as the polymerization initiator, the temperature is preferably 50 to 100° C., more preferably 60 to 95° C. The polymerization time is usually 5 minutes to 48 hours, and preferably 10 minutes to 24 hours.

[0039] -Process 1b- The method for producing a chromatography carrier of the present invention preferably further includes a step of contacting the porous particles to which no ligand is fixed obtained in step 1a with a crosslinking agent (hereinafter also referred to as step 1b). When a monomer composition containing a functional group-containing monomer is used in step 1a, step 1b causes an addition reaction of the crosslinking agent with some of the functional groups in the polymer molecule of the porous particles, introducing a partial structure derived from the crosslinking agent. As a result, the residues of the functional groups are crosslinked with each other via the partial structure derived from the crosslinking agent. In addition, a step of contacting the solid support after the sieve classification in step 2 with a crosslinking agent may be provided after step 1a without performing step 1b. The manufacturing method including such a step includes, for example, a mode in which the solid support is contacted with a crosslinking agent between step 2a and step 2b described below (specifically, a mode in which the steps are performed in the order of step 2a, crosslinking step, and step 2b, or a mode in which the steps are performed in the order of step 2b, crosslinking step, and step 2a), and a mode in which the solid support is contacted with a crosslinking agent after steps 2a and 2b (specifically, a mode in which the steps are performed in the order of step 2a, step 2b, and crosslinking step, or a mode in which the steps are performed in the order of step 2b, step 2a, and crosslinking step). Among these, a mode in which the solid support is contacted with a crosslinking agent between step 2a and step 2b is preferred.

[0040] The crosslinking agent used in step 1b 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.

[0041] When the porous particles obtained in step 1a have a cyclic ether group, specifically, a crosslinking agent containing at least two groups represented by -C(=O)-NH-NH2 as crosslinkable groups in the molecule, a crosslinking agent containing at least two groups represented by -C(=O)-NH- in the molecule, etc. can be used. In the case where the porous particles obtained in step 1a 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.

[0042] 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 dodecadihydrochloride. Examples of the crosslinking agent 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 acid)s such as N1,N1-(ethane-1,2-diyl)bis(succinic acid monoamide). The crosslinking agent may be used alone or in combination of two or more. Among these crosslinking agents, dicarboxylic acid dihydrazides and (alkylenebisimino)bis(oxoalkanoic acid)s are preferred, and dicarboxylic acid dihydrazides are more preferred, in order to improve the liquid permeability, pressure resistance characteristics during liquid passage, and antifouling properties.

[0043] In step 1b, a crosslinking agent other than the crosslinking agent containing at least two groups represented by -C(=O)-NH- 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.

[0044] The total amount of the 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 the functional group derived from the functional group-containing monomer.

[0045] Step 1b may be carried out in the presence of a basic catalyst. Examples of the basic catalyst include triethylamine, N,N-dimethyl-4-aminopyridine, sodium hydroxide, and diisopropylethylamine, and one type may be used alone or two or more types may be used in combination.

[0046] The reaction time in step 1b 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 below the boiling point of the solvent, but is usually about 2 to 100° C.

[0047] -Process 1c- When preparing porous particles having a ligand immobilized thereon as the solid phase support, the method for producing a chromatography support of the present invention preferably further includes a step of immobilizing a ligand on the porous particles obtained in step 1a or step 1b (hereinafter also referred to as step 1c).When a monomer composition containing a functional group-containing monomer is used in step 1a, the ligand is bound to the functional group that the porous particles have in the polymer molecule in step 1c. The ligand may be a molecule that binds to a target substance or an ion exchange group that can be introduced via a functional group on the surface of a porous particle, and examples of the ligand include cation exchange groups such as sulfo groups and phosphonic groups; anion exchange groups such as amino groups and quaternary ammonium groups; proteins such as protein A, protein G, and avidin; peptides such as insulin; nucleic acids such as DNA and RNA; enzymes; chelating compounds such as iminodiacetic acid; antibodies; antigens; hormones; carbohydrates such as heparin, Lewis X, and gangliosides; receptors; aptamers; vitamins such as biotin and its derivatives; metal ions; synthetic dyes; and low molecular weight compounds such as 2-aminophenylboronic acid, 4-aminobenzamidine, and glutathione. The ligands exemplified above may be used in their entirety, or fragments obtained by recombinant or enzyme treatment may be used. Artificially synthesized peptides and peptide derivatives may also be used. Among the above ligands, proteins, peptides, nucleic acids, enzymes, and chelating compounds are preferred, proteins and peptides are more preferred, and proteins are particularly preferred. In particular, among antibody affinity ligands targeting antibodies, immunoglobulin-binding proteins are preferred.

[0048] Examples of the antibody affinity ligand include a peptide ligand, a protein ligand, and a chemically synthesized ligand (synthetic compound), and are preferably a peptide or protein ligand. Among them, Protein A, Protein G, Protein L, Protein H, Protein D, Protein Arp, Protein FcγR, an antibody-binding synthetic peptide ligand, and their analogues are preferred, Protein A, Protein G, Protein L, and their analogues are more preferred, and Protein A and its analogues are particularly preferred.

[0049] In order to increase the dynamic binding capacity, the amount of the immobilized ligand is preferably 10 mg to 300 mg, more preferably 25 mg to 150 mg, per gram of dry weight of the porous particles obtained in step 1a or step 1b.

[0050] The ligand may be fixed to the porous particles in the same manner as in the conventional method. A chemical bonding method is preferable as the ligand fixing method. For example, a method of bonding a ligand to a functional group capable of fixing the ligand may be mentioned. This method may be carried out with reference to the descriptions in WO 2015 / 119255, WO 2015 / 041218, etc. Specifically, a method of bonding a cyclic ether group, carboxy group, -C(=O)-OC(=O)-, formyl group, etc. of the porous particles to an amino group, etc. of the ligand may be mentioned. 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). The porous particles to which the ligands are fixed may be contacted with a thiol compound such as methanethiol or thioglycerol to open the ring of the unreacted functional groups, etc. This treatment may be performed with reference to the description in the pamphlet of International Publication No. WO 2015 / 119255, etc.

[0051] The volume average particle size of the solid support prepared in step 1 is preferably 30 μm or more, more preferably 40 μm or more, particularly preferably 50 μm or more, and is preferably 120 μm or less, more preferably 100 μm or less, particularly preferably 95 μm or less. The specific range of the volume average particle size of the solid support prepared in step 1 is preferably 30 μm or more and 120 μm or less, more preferably 40 μm or more and 100 μm or less, and particularly preferably 50 μm or more and 95 μm or less. The volume average particle size of the solid support prepared in step 1 can be measured in accordance with ISO13320:2009.

[0052] (Process 2) Step 2 is a step of adjusting the coefficient of variation of the volume-based particle size distribution of the porous particles when the ligand is fixed to be 1% or more and 22% or less by sieving the solid phase support prepared in step 1, and adjusting the coefficient of variation of the volume-based particle size distribution of the porous particles when the ligand is fixed to be -0.1 or more and 5 or less. The sieve classification in step 2 may be performed so that the coefficient of variation of the volumetric particle size distribution measured after the ligand is fixed to the porous particles is 1% to 22%, and the skewness of the volumetric particle size distribution measured after the ligand is fixed to the porous particles is -0.1 to 5. In addition, as long as the coefficient of variation and skewness measured after the ligand is fixed to the porous particles are within the above ranges, sieve classification may be performed either before or after the ligand is fixed, or may be performed both. In addition, a classification method such as a centrifugation method or a sedimentation method may be used in combination so that the coefficient of variation and skewness measured after the ligand is fixed to the porous particles are within the above ranges.

[0053] By adjusting the coefficient of variation of the volumetric particle size distribution to 1% or more, the productivity of the carrier is improved, and by adjusting the coefficient of variation to 22% or less, the liquid permeability of the chromatography carrier and the pressure resistance characteristics during liquid passage are improved. The coefficient of variation of the volume-based particle size distribution is preferably 5% or more, more preferably 6% or more, even more preferably 8% or more, and particularly preferably 10% or more in order to improve the productivity of the carrier, and is preferably 20% or less, more preferably 18% or less, even more preferably 17% or less, and particularly preferably 16% or less in order to improve the liquid permeability and pressure resistance characteristics during liquid passage. As a specific range, 5% or more and 20% or less are preferable, 6% or more and 20% or less are more preferable, 8% or more and 18% or less are more preferable, 8% or more and 17% or less are even more preferable, and 10% or more and 16% or less are particularly preferable. When the coefficient of variation of the volume-based particle size distribution is set to 18% or less or 17% or less, the liquid permeability and pressure resistance characteristics during liquid passage of the chromatography carrier are particularly improved. The coefficient of variation of the volume-based particle size distribution is a coefficient of variation of the volume-based particle size distribution measured for ligand-immobilized porous particles in accordance with ISO13319:2007, and specifically, can be measured by the method described in the examples described later.

[0054] By adjusting the skewness of the volumetric particle size distribution to -0.1 or more, the liquid permeability and pressure resistance characteristics during liquid passage of the chromatography carrier are improved. In addition, by adjusting the skewness to 5 or less, the dynamic binding capacity is increased. The skewness of the volume-based particle size distribution is preferably -0.01 or more, more preferably 0.01 or more, even more preferably 0.1 or more, even more preferably 0.3 or more, and particularly preferably 0.4 or more in order to improve the liquid permeability and the pressure resistance characteristics during liquid passage, and is preferably 4 or less, more preferably 3 or less, and particularly preferably 2 or less in order to increase the dynamic binding capacity. As a specific range, -0.01 or more and 4 or less are preferable, 0.01 or more and 3 or less are more preferable, 0.1 or more and 3 or less are even more preferable, 0.3 or more and 2 or less are even more preferable, and 0.4 or more and 2 or less are particularly preferable. When the skewness of the volume-based particle size distribution is 0.1 or more or 0.3 or more, the liquid permeability and pressure resistance characteristics during liquid passage of the chromatography carrier are particularly improved. The skewness of the volume-based particle size distribution is the skewness of the volume-based particle size distribution measured for porous particles having ligands fixed thereto in accordance with ISO13319:2007, and specifically, can be measured by the method described in the examples described below.

[0055] In step 2, the sieve is classified so that the volume average particle diameter is preferably 40 μm or more, more preferably 50 μm or more, and particularly preferably 55 μm or more, and the volume average particle diameter is preferably 150 μm or less, more preferably 100 μm or less, even more preferably 90 μm or less, and particularly preferably 80 μm or less. The specific range of the volume average particle diameter is preferably 40 μm or more and 150 μm or less, more preferably 50 μm or more and 100 μm or less, even more preferably 55 μm or more and 90 μm or less, and particularly preferably 55 μm or more and 80 μm or less. The volume average particle diameter refers to a volume average particle diameter measured by a laser diffraction scattering method in accordance with ISO13320:2009.

[0056] In step 2, in order to improve the liquid permeability and pressure resistance characteristics during liquid passage, the 1% volumetric cumulative particle diameter d1 is preferably 20 μm or more, more preferably 30 μm or more, even more preferably 35 μm or more, and particularly preferably 40 μm or more. In order to increase the dynamic binding capacity, the 1% volumetric cumulative particle diameter d1 is preferably 85 μm or less, more preferably 75 μm or less, even more preferably 65 μm or less, even more preferably 58 μm or less, and particularly preferably 55 μm or less. The specific range of the 1% volumetric cumulative particle diameter d1 is preferably 20 μm or more and 85 μm or less, more preferably 30 μm or more and 75 μm or less, even more preferably 35 μm or more and 65 μm or less, even more preferably 40 μm or more and 58 μm or less, and particularly preferably 40 μm or more and 55 μm or less. When the volume-based cumulative 1% particle diameter d1 is set to 35 μm or more or 40 μm or more, the liquid permeability of the chromatography carrier and the pressure resistance characteristics during liquid passage are particularly improved.

[0057] In step 2, in order to improve the liquid permeability and pressure resistance characteristics during liquid passage, the 5% volumetric cumulative particle diameter d5 is preferably 20 μm or more, more preferably 30 μm or more, even more preferably 35 μm or more, even more preferably 40 μm or more, and particularly preferably 45 μm or more. In order to increase the dynamic binding capacity, the 5% volumetric cumulative particle diameter d5 is preferably 100 μm or less, more preferably 80 μm or less, even more preferably 70 μm or less, even more preferably 65 μm or less, and particularly preferably 60 μm or less. The specific range of the 5% volumetric cumulative particle diameter d5 is preferably 20 μm or more and 100 μm or less, more preferably 30 μm or more and 80 μm or less, more preferably 35 μm or more and 70 μm or less, even more preferably 40 μm or more and 65 μm or less, and particularly preferably 45 μm or more and 60 μm or less. When the volume-based cumulative 5% particle diameter d5 is 40 μm or more or 45 μm or more, the liquid permeability of the chromatography carrier and the pressure resistance characteristics during liquid passage are particularly improved.

[0058] In step 2, in order to improve the dynamic binding capacity and pressure resistance characteristics, the particle is sieved so that the volume-based cumulative 50% particle diameter d50 is preferably 20 μm or more, more preferably 30 μm or more, even more preferably 40 μm or more, and particularly preferably 55 μm or more. In addition, in order to improve the dynamic binding capacity and pressure resistance characteristics, the particle is sieved so that the volume-based cumulative 50% particle diameter d50 is preferably 100 μm or less, more preferably 90 μm or less, even more preferably 85 μm or less, even more preferably 75 μm or less, and particularly preferably 65 μm or less. The specific range of the volume-based cumulative 50% particle diameter d50 is preferably 20 μm or more and 100 μm or less, more preferably 30 μm or more and 90 μm or less, even more preferably 30 μm or more and 85 μm or less, even more preferably 40 μm or more and 75 μm or less, and particularly preferably 55 μm or more and 65 μm or less.

[0059] In step 2, in order to improve the liquid permeability and pressure resistance characteristics during liquid passage, the particles are sieved and classified so that the value [d1 / d50] obtained by dividing the volume-based cumulative 1% particle diameter d1 by the volume-based cumulative 50% particle diameter d50 is preferably 0.55 or more, more preferably 0.60 or more, even more preferably 0.63 or more, even more preferably 0.65 or more, and particularly preferably 0.70 or more. In addition, in order to facilitate an appropriate packing state when packed into a column, the particles are sieved and classified so that the value [d1 / d50] obtained by dividing the volume-based cumulative 1% particle diameter d1 by the volume-based cumulative 50% particle diameter d50 is preferably 1.0 or less, more preferably 0.95 or less, even more preferably 0.90 or less, and particularly preferably 0.85 or less. The specific range of the value [d1 / d50] obtained by dividing the volume-based cumulative 1% particle diameter d1 by the volume-based cumulative 50% particle diameter d50 is preferably 0.55 or more and 1.0 or less, more preferably 0.60 or more and 0.95 or less, even more preferably 0.63 or more and 0.90 or less, still more preferably 0.65 or more and 0.90 or less, and particularly preferably 0.70 or more and 0.85 or less. When the value [d1 / d50] obtained by dividing the volume-based cumulative 1% particle diameter d1 by the volume-based cumulative 50% particle diameter d50 is 0.63 or more or 0.70 or more, the liquid permeability and pressure resistance characteristics during liquid passage of the chromatography carrier are particularly improved.

[0060] In step 2, in order to improve the liquid permeability and pressure resistance characteristics during liquid passage, the particles are sieved and classified so that the value [d5 / d50] obtained by dividing the volume-based cumulative 5% particle diameter d5 by the volume-based cumulative 50% particle diameter d50 is preferably 0.50 or more, more preferably 0.60 or more, even more preferably 0.70 or more, and particularly preferably 0.80 or more. In addition, in order to facilitate an appropriate packing state when packed into a column, the particles are sieved and classified so that the value [d5 / d50] obtained by dividing the volume-based cumulative 5% particle diameter d5 by the volume-based cumulative 50% particle diameter d50 is preferably 1.0 or less, more preferably 0.97 or less, even more preferably 0.95 or less, and particularly preferably 0.90 or less. The specific range of the value [d5 / d50] obtained by dividing the volume-based cumulative 5% particle diameter d5 by the volume-based cumulative 50% particle diameter d50 is preferably 0.50 or more and 1.0 or less, more preferably 0.60 or more and 0.97 or less, even more preferably 0.70 or more and 0.95 or less, and particularly preferably 0.80 or more and 0.90 or less. When the value [d5 / d50] obtained by dividing the volume-based cumulative 5% particle diameter d5 by the volume-based cumulative 50% particle diameter d50 is 0.70 or more or 0.80 or more, the liquid permeability and pressure resistance characteristics during liquid passage of the chromatography carrier are particularly improved.

[0061] The volume-based cumulative 1% particle diameter d1, the volume-based cumulative 5% particle diameter d5, and the volume-based cumulative 50% particle diameter d50 refer to values ​​of d1, d5, and d50 measured in accordance with ISO13319:2007 corrected using a shape factor that makes the value of the volume average particle diameter measured in accordance with ISO13319:2007 the value of the volume average particle diameter measured in accordance with ISO13320:2009, and specifically, they can be measured by the method described in the examples described later.

[0062] The sieve classification in step 2 may be a dry method or a wet method, but is preferably a wet method in order to facilitate adjustment to porous particles having a desired particle size and to prevent the porous particles from being damaged. Sieve classification can be performed using a normal classifier such as a Sato type vibrating sieve machine, a direct discharge type vibrating sieve machine, a vibrating sieve machine with a crushing mechanism, a high vibration type vibrating sieve machine, or a vibrating sieve machine with an ultrasonic oscillator, all manufactured by Koei Sangyo Co., Ltd. In addition, the sieve classification in step 2 is preferably a classification operation in which (step 2a) when the volume average particle size (2a-d) of the solid phase support before sieve classification is taken as 100%, the solid phase support is sieved through a sieve with a mesh size of 10% or more and less than 200% of (2a-d) and the residue on the sieve is recovered, and (step 2b) the solid phase support is sieved through a sieve with a mesh size larger than that of the sieve in step 2a and less than 600% of (2a-d) and the part that passed through the sieve is recovered, in this order or in the reverse order.

[0063] The opening of the sieve used in step 2a is preferably 30% or more, more preferably 50% or more, even more preferably 60% or more, even more preferably 62% or more, and particularly preferably 68% or more, when (2a-d) is taken as 100%, in order to improve the liquid permeability and the pressure resistance characteristics during liquid passage, and is preferably 150% or less, more preferably 120% or less, even more preferably 105% or less, and particularly preferably 90% or less, in order to increase the dynamic binding capacity, when (2a-d) is taken as 100%. The mesh size of the sieve used in step 2a, when (2a-d) is taken as 100%, is preferably 30% or more and 150% or less, more preferably 50% or more and 120% or less, even more preferably 60% or more and 105% or less, still more preferably 62% or more and 90% or less, and particularly preferably 68% or more and 90% or less. When the mesh size of the sieve used in step 2a is 62% or more or 68% or more when (2a-d) is taken as 100%, the liquid permeability and pressure resistance characteristics during liquid passage of the chromatography carrier are particularly improved.

[0064] The mesh size of the sieve used in step 2a is preferably 16 μm or more, more preferably 20 μm or more, more preferably 25 μm or more, more preferably 30 μm or more, more preferably 35 μm or more, and particularly preferably 40 μm or more in order to improve the liquid permeability and pressure resistance characteristics during liquid passage, and is preferably less than 100 μm, more preferably 80 μm or less, more preferably 70 μm or less, more preferably 60 μm or less, and particularly preferably 50 μm or less in order to increase the dynamic binding capacity. As a specific range, 16 μm or more and less than 100 μm is preferable, 20 μm or more and 80 μm or less is more preferable, 25 μm or more and 70 μm or less is more preferable, 30 μm or more and 60 μm or less is more preferable, 35 μm or more and 60 μm or less is more preferable, and 40 μm or more and 50 μm or less is particularly preferable.

[0065] When the mesh size of the sieve used in step 2a is greater than 70 μm and not greater than 80 μm, the mesh size of the sieve used in step 2b is preferably 90 μm or more; when the mesh size of the sieve used in step 2a is greater than 60 μm and not greater than 70 μm, the mesh size of the sieve used in step 2b is preferably 75 μm or more; when the mesh size of the sieve used in step 2a is greater than 50 μm and not greater than 60 μm, the mesh size of the sieve used in step 2b is preferably 65 μm or more; and when the mesh size of the sieve used in step 2a is not greater than 50 μm, the mesh size of the sieve used in step 2b is preferably 55 μm or more. In addition, in order to improve the liquid permeability and the pressure resistance characteristics during liquid passage, the mesh size of the sieve used in step 2b is preferably less than 300 μm, more preferably 250 μm or less, even more preferably 200 μm or less, even more preferably 150 μm or less, and particularly preferably 100 μm or less.

[0066] Regardless of the order of steps 2a and 2b, the sieve classification in step 2 may be either sieve classification A or sieve classification B described below, but is preferably sieve classification B. When the sieve classification in step 2 is sieve classification B, damage to the sieve classifier can be reduced and adjustment to a desired particle size becomes easier. Sieve classification A (step 2a) The solid support prepared in step 1 is sieved through a sieve having an opening size of 10% or more and less than 200% of (2a-d), where the volume average particle size (2a-d) of the solid support before sieve classification is taken as 100%, and the residue on the sieve is recovered; (step 2b) The residue on the sieve recovered in step 2a is sieved through a sieve having an opening size larger than that of the sieve used in step 2a and less than 600% of (2a-d), and the part passing through the sieve is recovered. Sieve classification B (Step 2b) The solid support prepared in step 1 is sieved through a sieve having a mesh size larger than that of the sieve used in step 2a and less than 600% of (2a-d), and the portion that passes through the sieve is collected. (Step 2a) The portion that passes through the sieve collected in step 2b is sieved through a sieve having a mesh size of 10% or more and less than 200% of (2a-d), where the volume average particle size (2a-d) of the solid support before sieve classification is taken as 100%, and the portion remaining on the sieve is collected.

[0067] (Step 3) In the case where porous particles to which no ligand is immobilized are prepared as the solid phase support in step 1, the method for producing a chromatography support of the present invention preferably further includes a step of immobilizing a ligand on the porous particles sieved in step 2 (hereinafter also referred to as step 3). By providing step 3 after step 2 in this manner, the dynamic binding capacity is increased and the productivity of the support can be improved. In the case where a monomer composition containing a functional group-containing monomer is used as the monomer composition in step 1a, the ligand is bound to the functional group that the porous particles have in the polymer molecule in step 3. Step 3 can be carried out in the same manner as step 1c.

[0068] The reaction products obtained in each of the above steps may be purified by separation means such as filtration and washing.

[0069] According to the method for producing a chromatography carrier of the present invention, a chromatography carrier having excellent liquid permeability and pressure resistance characteristics during liquid passage can be produced simply and efficiently. The thus obtained chromatography support is highly hydrophilic, has excellent antifouling properties, and has a large dynamic binding capacity for a target substance when a ligand is immobilized thereon, and is suitable for use in affinity chromatography.

[0070] <Method of manufacturing a chromatography column and a carrier for chromatography> The method for producing a chromatography column of the present invention includes a step of packing a carrier into a column container, the carrier being a porous particle having a ligand fixed thereto, and the carrier has a coefficient of variation of a volumetric particle size distribution of 1% or more and 22% or less and a skewness of the volumetric particle size distribution of -0.1 or more and 5 or less. The chromatography carrier of the present invention is a chromatography carrier dispersed in a dispersion medium, the carrier being characterized in that the carrier has ligands fixed to porous particles, and the carrier has a coefficient of variation of a volume-based particle size distribution of 1% or more and 22% or less, and a skewness of the volume-based particle size distribution of -0.1 or more and 5 or less. The method for producing a chromatography column of the present invention may be carried out in the same manner as a conventional method, except that a carrier having a ligand fixed to a porous particle and a coefficient of variation of a volume-based particle size distribution of 1% or more and 22% or less and a skewness of the volume-based particle size distribution of -0.1 or more and 5 or less is used. The carrier used in the method for producing a chromatography column of the present invention and the chromatography carrier of the present invention are the same as those obtained by the method for producing a chromatography carrier of the present invention, in which a ligand is fixed. The coefficient of variation of the volumetric particle size distribution, the skewness of the volumetric particle size distribution, the volume average particle size, the volumetric cumulative 1% particle size d1, the volumetric cumulative 5% particle size d5, the volumetric cumulative 50% particle size d50, [d1 / d50], and [d5 / d50] of the above carrier and the chromatography carrier of the present invention are preferably within the ranges adjusted in step 2.

[0071] The specific surface area of ​​the above carrier, the chromatography carrier of the present invention, is preferably 1 to 500 m 2 / g, more preferably 10 to 300m2 / g. The volume average pore diameter of the above carrier, that is, the carrier for chromatography of the present invention, is preferably 10 to 300 nm. The specific surface area and the volume average pore diameter can be measured by a gas adsorption method, a mercury porosimeter method, or the like.

[0072] When the porous particle is a porous particle containing a polymer, the polymer has a residue of a functional group capable of fixing a ligand, which is represented by Z. 1 The structural unit (A) represented by the following formula (7) is preferably represented by the following formula (7), and the structural unit (A) represented by the following formula (7) and the residue of a functional group capable of fixing a ligand are preferably represented by Z 2 More preferred is one having a structural unit (B) represented by the following formula (8): The ligand can be immobilized on the polymer-containing porous particles by a conventional method.

[0073] [ka]

[0074] [In formula (7), R 5 represents a hydrogen atom or a methyl group, R 6 represents a single bond or a divalent linking group, Z 1 represents a residue of a functional group capable of immobilizing a ligand, X is -R 15 -R 16 (R 15 represents a single bond or a divalent linking group, R 16 indicates the ligand.

[0075] [ka]

[0076] [In formula (8), R 7 represents a hydrogen atom or a methyl group, R 8represents a single bond or a divalent linking group, Z 2 represents a residue of a functional group capable of immobilizing a ligand, ** indicates a bond that bonds to the partial structure derived from the crosslinking agent (specifically, the crosslinking agent listed in step 1b).

[0077] Z in formula (7) 1 and Z in formula (8) 2 Specific examples of the functional group residue capable of immobilizing a ligand represented by the formula (I) include divalent groups formed by ring-opening of a cyclic ether group, -C(=O)- and -NH-C(=O)-, and divalent groups formed by ring-opening of a cyclic ether group are preferred. The "cyclic ether group" in the divalent group formed by ring-opening of the cyclic ether group is preferably the same as one of the examples of the functional group possessed by the functional group-containing monomer. When the cyclic ether group is an epoxy group, the divalent group formed by ring-opening of the cyclic ether group is a ring-opened epoxy group (-CHOH-CH2-). In addition, ** in formula (8) indicates a bond that bonds to the partial structure derived from the crosslinking agent, and means that the compound is crosslinked by the partial structure derived from the crosslinking agent.

[0078] R in Equation (7) 6 , R in Equation (8) 8 Examples of the divalent linking group represented by the formula (I) include an alkanediyl group, a group having an ether bond between the carbon atoms of an alkanediyl group having two or more carbon atoms, an arylene group, -C(=O)OR 9 -*, -C(=O)NH-R 10 -*, -Ar-R 11 -* or -Ar-OR 12 In this case, Ar represents an arylene group, and R 6 , R 8 The * in the above Z 1 or Z 2 R 6 , R 8 Examples of the arylene group represented by Ar include a phenylene group, a naphthylene group, and a phenanthrenylene group. Also, R 9 ~R 12each independently represents an alkanediyl group or a group having an ether bond between carbon atoms of an alkanediyl group having 2 or more carbon atoms.

[0079] R 6 , R 8 , R 9 ~R 12 The number of carbon atoms of the alkanediyl group represented by the formula (I) is preferably 1 to 12, more preferably 1 to 6, and particularly preferably 1 to 3. The alkanediyl group may be linear or branched. Specific examples of the alkanediyl group include a methane-1,1-diyl group, an ethane-1,1-diyl group, an ethane-1,2-diyl group, a propane-1,1-diyl group, a propane-1,2-diyl group, a propane-1,3-diyl group, a propane-2,2-diyl group, a butane-1,1-diyl group, a butane-1,2-diyl group, a butane-1,3-diyl group, a butane-1,4-diyl group, and a pentane-1,1-diyl group. group, pentane-1,2-diyl group, pentane-1,3-diyl group, pentane-1,4-diyl group, pentane-1,5-diyl group, hexane-1,1-diyl group, hexane-1,2-diyl group, hexane-1,3-diyl group, hexane-1,4-diyl group, hexane-1,5-diyl group, hexane-1,6-diyl group, heptane-1,7-diyl group, and octane-1,8-diyl group.

[0080] R 6 , R 8 , R 9 ~R 12 The "group having an ether bond between the carbon atoms of an alkanediyl group having two or more carbon atoms" represented by the formula: -R a (OR b ) p OR c A group represented by the formula - (R a , R b and R c each independently represents an alkanediyl group having 1 to 4 carbon atoms, and p represents an integer of 0 to 30. R a , R b and R cThe alkanediyl group represented by the formula may be linear or branched. Specific examples include methane-1,1-diyl, ethane-1,1-diyl, ethane-1,2-diyl, propane-1,1-diyl, propane-1,2-diyl, propane-1,3-diyl, propane-2,2-diyl, butane-1,1-diyl, butane-1,2-diyl, butane-1,3-diyl, and butane-1,4-diyl. p is preferably an integer of 0 to 20, more preferably an integer of 0 to 10, even more preferably an integer of 0 to 5, and particularly preferably 0. When p is an integer of 2 to 30, p R b may be the same or different. Preferable specific examples of the alkanediyl group having 2 or more carbon atoms and having an ether bond between the carbon atoms include C 1-4 Alkanediyloxy C 1-4 An example is an alkanediyl group. In addition, R 6 , R 8 , R 9 ~R 12 and an alkanediyl group having 2 or more carbon atoms and an ether bond between the carbon atoms of the alkanediyl group may have a substituent. Examples of the substituent include a hydroxyl group.

[0081] R as above 6 , R 8 In order to make it easier to produce polymers, -C(=O)OR 9 -* is especially preferred.

[0082] R in Equation (7) 15 represents a single bond or a divalent linking group, R 16 R represents the ligand. 16 Examples of the ligand represented by the formula (I) include those listed in step 1c. R 15 The divalent linking group represented by the formula (I) may be any group capable of linking a ligand to a residue of a functional group capable of immobilizing the ligand. R 15Examples of the divalent linking group represented by the formula (I) include a partial structure derived from a crosslinking agent (specifically, the crosslinking agent listed in step 1b), a partial structure derived from a diglycidyl ether, and a partial structure containing a hetero atom other than an oxygen atom. Examples of diglycidyl ethers include 1,4-butanediol diglycidyl ether, 1,6-hexanediol diglycidyl ether, bisphenol A diglycidyl ether, ethylene glycol diglycidyl ether, diethylene glycol diglycidyl ether, polyethylene glycol diglycidyl ether, propylene glycol diglycidyl ether, tripropylene glycol diglycidyl ether, polypropylene glycol diglycidyl ether, neopentyl glycol diglycidyl ether, and glycerin diglycidyl ether. Examples of the partial structure containing a heteroatom other than an oxygen atom include a partial structure derived from thioglycerol. R 15 The divalent linking group represented by the formula (I) is preferably a divalent linking group derived from a diglycidyl ether, a divalent linking group composed of a partial structure derived from the crosslinking agent (preferably adipic acid dihydrazide) listed in step 1b and a partial structure derived from a diglycidyl ether, or a divalent linking group composed of a partial structure derived from thioglycerol and a partial structure derived from a diglycidyl ether.

[0083] As the monomer from which the structural unit (A) or the structural unit (B) is derived, the above-mentioned functional group-containing monomer is preferred.

[0084] The polymer may have a structural unit other than the structural units (A) and (B). Examples of the monomer from which such a structural unit is derived include the other monomers listed in step 1a. The polymer content in the porous particles (excluding the ligand) is preferably 90% by mass or more and 100% by mass or less, more preferably 95% by mass or more and 100% by mass or less, and particularly preferably 99% by mass or more and 100% by mass or less. The content of the partial structure derived from the crosslinking agent in the polymer is preferably from 0.5 to 50% by mass, more preferably from 1 to 40% by mass, and particularly preferably from 10 to 35% by mass.

[0085] Examples of the dispersion medium for dispersing the chromatographic carrier of the present invention include various buffers, water, alcohol, etc. In addition, a preservative or the like may be added to the dispersion medium. The content of the chromatography carrier is preferably 0.5% by mass or more and 20% by mass or less, and more preferably 1% by mass or more and 15% by mass or less, when the total of the chromatography carrier and the dispersion medium is 100% by mass.

[0086] The column obtained by the method for producing a chromatography column of the present invention and the chromatography carrier of the present invention are useful for affinity chromatography. The chromatography support and chromatography column obtained by the present invention, as well as the chromatography support of the present invention, can be used for purifying a target substance. Purification of the target substance may be carried out according to a conventional method. Examples of target substances include antigens; antibodies such as monoclonal antibodies and polyclonal antibodies; cells (normal cells; cancer cells such as colon cancer cells and circulating cancer cells); nucleic acids such as DNA and RNA; and biological substances such as proteins, peptides, amino acids, sugars, polysaccharides, lipids, and vitamins, and may also be low molecular weight compounds such as drugs that are targets for drug discovery, and biotin. EXAMPLES

[0087] The present invention will be described in detail below with reference to examples, but the present invention is not limited to these examples.

[0088] Example 1 (1) 2.69 g of polyvinyl alcohol (PVA-217 manufactured by Kuraray Co., Ltd.) was added to 448 g of pure water, and the polyvinyl alcohol was dissolved by heating and stirring to prepare an aqueous solution S. Meanwhile, 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., Ltd.) and 14.15 g of glycidyl methacrylate (manufactured by Mitsubishi Gas Chemical Co., Ltd.) was dissolved in 29.38 g of 2-octanone (manufactured by Toyo Gosei Co., Ltd.) to prepare a monomer solution. Next, the entire amount of the aqueous solution S was poured into a separable flask, and the flask was fitted with a thermometer, a stirring blade and a cooling tube, set in a hot water bath, and stirring was started 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. (2) After that, the mixture was stirred 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, to obtain a porous particle dispersion. The porous particles contained in this dispersion are referred to as "porous particles 1". (3) After that, 0.956 g of adipic acid dihydrazide (manufactured by Tokyo Chemical Industry Co., Ltd.) and 1.418 g of diisopropylethylamine (manufactured by 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. Next, the reaction liquid was cooled, filtered, and washed with pure water and ethanol. Next, the particles were dispersed in pure water so that the particle concentration was 10 mass %, to obtain a porous particle dispersion. The porous particles contained in this dispersion are referred to as “porous particles 2”. The volume average particle diameter of the porous particles 2 was measured in the same manner as in Test Example 1, and the value was 53 μm. (4) After that, the porous particles 2 were put into a Sato-type vibrating sieve machine (400DB-2S manufactured by Koei Sangyo Co., Ltd.) equipped with a wire mesh with an opening of 77 μm (manufactured by Taiyo Wire Net Co., Ltd.), and the sieve-passing portion (liquid that passed through the sieve after wet classification) was collected. Next, the sieve-passing portion was put into the Sato-type vibrating sieve machine in which the wire mesh was changed to a wire mesh with an opening of 32 μm (manufactured by Taiyo Wire Net Co., Ltd.), and the sieve-passing portion (porous particles remaining on the sieve after wet classification) was collected with pure water, to obtain a porous particle dispersion. The porous particles contained in this dispersion are referred to as "porous particles 3". (5) Next, 0.069 g of ethylene glycol diglycidyl ether (manufactured by Tokyo Chemical Industry Co., Ltd.) was reacted with the porous particles 3 (equivalent to 1 g in terms of particle dry mass) for 16 hours. Next, 0.15 g of modified protein A (rSPA manufactured by Repligen) was dissolved in 40 mL of 1.2 M sodium sulfate / 0.1 M sodium phosphate buffer (pH 6.6) to obtain a protein A solution, and the porous particles 3 (equivalent to 1 g in terms of particle dry mass) were added to this protein A solution. This dispersion was shaken and stirred at 25° C. for 10 hours to immobilize the protein A on the particles. The resulting protein A-immobilized particles were then dispersed in 40 mL of 1.0 M α-thioglycerol (Tokyo Chemical Industry Co., Ltd.) / 0.1 M sodium sulfate (pH 8.3) and stirred at 25° C. for 17 hours to open the unreacted epoxy groups. The resulting protein A-immobilized particles with the unreacted epoxy groups opened were washed with 0.1 M sodium phosphate buffer (pH 6.6), 0.1 M aqueous sodium hydroxide solution, and 0.1 M sodium citrate buffer (pH 3.2) to obtain a filler-containing solution for affinity chromatography. The filler contained in this solution is referred to as "ligand-immobilized carrier W1".

[0089] Example 2 A porous particle dispersion was obtained by carrying out the same operations as in steps (1) to (4) of Example 1, except that in step (4) of Example 1, the wire mesh having an opening of 32 μm was changed to a wire mesh having an opening of 34 μm (manufactured by Taiyo Wire Mesh Co., Ltd.). Next, a liquid containing a packing material for affinity chromatography was obtained by carrying out the same step as step (5) in Example 1. The packing material contained in this liquid is referred to as "ligand-immobilized carrier W2."

[0090] Example 3 A porous particle dispersion was obtained by carrying out the same operations as in steps (1) to (4) of Example 1, except that in step (4) of Example 1, the wire mesh having an opening of 32 μm was changed to a wire mesh having an opening of 38 μm (manufactured by Asada Mesh Co., Ltd.). Next, a liquid containing a packing material for affinity chromatography was obtained by carrying out the same step as step (5) in Example 1. The packing material contained in this liquid is referred to as "ligand-immobilized carrier W3."

[0091] Example 4 A porous particle dispersion was obtained by carrying out the same operations as in steps (1) to (4) of Example 1, except that in step (4) of Example 1, the wire mesh having an opening of 32 μm was changed to a wire mesh having an opening of 43 μm (manufactured by Taiyo Wire Mesh Co., Ltd.). Next, a liquid containing a packing material for affinity chromatography was obtained by carrying out the same step as step (5) in Example 1. The packing material contained in this liquid is referred to as "ligand-immobilized carrier W4."

[0092] Example 5 A porous particle dispersion was obtained by carrying out the same operations as in steps (1) to (4) of Example 1, except that in step (4) of Example 1, the wire mesh having an opening of 32 μm was changed to a wire mesh having an opening of 45 μm (manufactured by Taiyo Wire Mesh Co., Ltd.). Next, a liquid containing a packing material for affinity chromatography was obtained by carrying out the same step as step (5) in Example 1. The packing material contained in this liquid is referred to as "ligand-immobilized carrier W5."

[0093] Example 6 A porous particle dispersion was obtained by carrying out the same operations as in steps (1) to (4) of Example 1, except that in step (4) of Example 1, the wire mesh having an opening of 32 μm was changed to a wire mesh having an opening of 54 μm (manufactured by Taiyo Wire Mesh Co., Ltd.). Next, a liquid containing a packing material for affinity chromatography was obtained by carrying out the same step as step (5) in Example 1. The packing material contained in this liquid is referred to as "ligand-immobilized carrier W6."

[0094] Example 7 Mabselect SuRe (Cytiva, volume average particle size: 92 μm), an agarose-based carrier, was placed in the Sato-type vibrating sieve equipped with a 104 μm mesh (Taiyo Wire Mesh), and the portion that passed through the sieve was collected. Next, it was placed in the Sato-type vibrating sieve equipped with a 62 μm mesh (Taiyo Wire Mesh), and the residue on the sieve (particles remaining on the sieve after wet classification) was collected with pure water, to obtain a liquid containing a filler for affinity chromatography. The filler contained in this liquid is referred to as "ligand-immobilized carrier W7".

[0095] Example 8 A liquid containing a packing material for affinity chromatography was obtained by the same procedure as in Example 7, except that the wire mesh with an opening of 104 μm was replaced with a wire mesh with an opening of 115 μm (manufactured by Taiyo Kanamo Co., Ltd.) The packing material contained in this liquid is referred to as "ligand immobilized carrier W8".

[0096] Example 9 An agarose-based carrier, Mabselect SuRe (manufactured by Cytiva, volume average particle size: 92 μm), was placed in the Sato-type vibrating sieve equipped with a 54 μm mesh (manufactured by Taiyo Kanamo Co., Ltd.), and the residue on the sieve (particles remaining on the sieve after wet classification) was collected with pure water to obtain a liquid containing a packing material for affinity chromatography. The packing material contained in this liquid is referred to as "ligand-immobilized carrier W9".

[0097] Example 10 A liquid containing a packing material for affinity chromatography was obtained by the same procedure as in Example 9, except that the 54 μm mesh was replaced with a 62 μm mesh (manufactured by Taiyo Kanamo Co., Ltd.) The packing material contained in this liquid is referred to as "ligand immobilized carrier W10".

[0098] Comparative Example 1 The same operations as in steps (1) to (3) of Example 1 were carried out to obtain "porous particles 2." Next, a liquid containing a packing material for affinity chromatography was obtained by carrying out the same step as step (5) in Example 1. The packing material contained in this liquid is referred to as "ligand-immobilized carrier Y1."

[0099] Comparative Example 2 The same operations as in steps (1) to (3) of Example 1 were carried out to obtain "porous particles 2." The dispersion of porous particles 2 was filled into a 1 L plastic bottle and allowed to stand for 1 hour, after which the supernatant liquid was removed. Next, a liquid containing a packing material for affinity chromatography was obtained by carrying out the same step as step (5) in Example 1. The packing material contained in this liquid is referred to as "ligand-immobilized carrier Y2."

[0100] Comparative Example 3 A porous particle dispersion was obtained by carrying out the same operations as in steps (1) to (4) of Example 1, except that in step (4) of Example 1, the wire mesh having an opening of 77 μm was changed to a wire mesh having an opening of 62 μm (manufactured by Taiyo Wire Mesh Co., Ltd.) and the subsequent classification operation using a Sato type vibrating sieve equipped with a wire mesh having an opening of 32 μm (manufactured by Taiyo Wire Mesh Co., Ltd.) was omitted. Next, a liquid containing a packing material for affinity chromatography was obtained by carrying out the same step as step (5) in Example 1. The packing material contained in this liquid is referred to as "ligand-immobilized carrier Y3."

[0101] (Test Example 1: Measurement of particle size and particle size distribution) The volume average particle diameter of the carriers W1 to W10 and Y1 to Y3 obtained in each Example and Comparative Example was measured by a laser diffraction scattering type particle size distribution analyzer (MT3000II manufactured by MicrotracBEL) in accordance with ISO13320:2009. The concentration of the carrier in the measurement sample was adjusted so that the transmittance was 0.1%, and the refractive index of the solvent (water) was set to 1.333, and the refractive index of the particles (carrier) was set to 1.50. The measured values ​​of the volume average particle diameter measured by the laser diffraction scattering type particle size distribution analyzer are shown in Table 1.

[0102] Next, for the carriers W1 to W10 and Y1 to Y3 obtained in each Example and Comparative Example, the values ​​of d1, d5, and d50 based on the volumetric particle size distribution were measured using a Beckman Coulter Multisizer 4e in accordance with ISO13319:2007. Since the particle size is estimated to be small when measuring porous particles using this device, d1, d5, and d50 were calculated by correcting using a shape factor such that the volume average particle size measured by this device is the value of the volume average particle size measured according to ISO13320:2009. The measured values ​​of d1, d5, and d50 are shown in Table 1. The volumetric particle size distribution of carrier W1 (Example 1) is shown in FIG. 1, the volumetric particle size distribution of carrier W3 (Example 3) is shown in FIG. 2, and the volumetric particle size distribution of carrier W5 (Example 5) is shown in FIG. 3. For particle size measurement in accordance with ISO13319, a 200 μm aperture was used, and an ISOTON II manufactured by Beckman Coulter was used as the electrolyte. The measurement conditions were: total number of measurements 30,000, number of particle size bins 400, current value 1600 μA, and gain 2.

[0103] d1, d5 and d50 respectively mean the particle sizes below. d1 = Particle size equivalent to 1% cumulative volume from the small particle size side based on the volume-based particle size distribution d5 = Particle size equivalent to cumulative volume 5% from the small particle size side based on the volume-based particle size distribution d50 = Particle size equivalent to 50% cumulative volume from the smallest particle size side based on the volume-based particle size distribution

[0104] In addition, the coefficient of variation (CV value) and skewness were calculated from each measured value measured in accordance with ISO 13319. The calculation method is described below.

[0105] CV value (%) = {(Standard deviation of volume-based particle size distribution) / (Volume average particle size)} × 100

[0106]

number

[0107]

number

[0108] n = total number of measurements x i = Particle size of each measured particle s = standard deviation of particle size

[0109] (Test Example 2: Evaluation of Liquid Permeability) Carriers W1 to W10 and Y1 to Y3 obtained in each Example and Comparative Example were packed into two connected Hiscale 26 / 20 columns (manufactured by Cytiva) so that the inner diameter was 26 mm and the packed height was 200 mm, and this column was connected to AKTA AVANT 150 manufactured by Cytiva. Next, pure water was passed through the column at a linear flow rate of 100 cm / hr for 30 minutes, and then the upper column was removed, and the carrier was packed in the column by adhering an adapter to the carrier layer. Then, the linear flow rate was adjusted until a back pressure of 0.25 MPa was generated, and the linear flow rate at that time was recorded. The results are shown in Table 1. It can be said that the higher the linear flow rate, the better the liquid permeability.

[0110] (Test Example 3: Evaluation of pressure resistance characteristics during liquid passage) The carriers W1 to W10 and Y1 to Y3 obtained in each of the Examples and Comparative Examples were packed in the same manner as in Test Example 2, and then pure water was passed through them at a linear flow rate of 800 cm / hr, and the back pressure and compression ratio (rate of change in packed height) were recorded. The compression ratio was calculated using the following formula. The results are shown in Table 1. It can be said that the smaller the back pressure and compression ratio values, the better the pressure resistance characteristics.

[0111] (Compressibility) = 1-{(Filling height after passing liquid at 800 cm / h) / (Filling height before passing liquid at 800 cm / h)}

[0112] (Test Example 4: Evaluation of dynamic binding capacity) Using AKTAprime plus manufactured by GE Healthcare, the DBC of carriers W1 to W10 and Y2 to Y3 of the examples and comparative examples for a protein (human IgG antibody, HGG-1000 manufactured by Equitech Bio) at a linear flow rate of 300 cm / hr was measured. A column container with a capacity of 4 mL (5 mm diameter x 200 mm length) and a protein dissolved at 5 mg / mL in a 20 mM sodium phosphate / 150 mM sodium chloride aqueous solution (pH 7.5) were used, and the DBC was calculated from the protein capture amount at 10% breakthrough at the elution tip and the column packing volume. The results are shown in Table 1.

[0113] [Table 1]

Claims

1. A method for producing a support for antibody affinity chromatography, comprising the following steps 1 and 2, in which the solid phase support prepared in step 1 is a porous particle having no antibody affinity ligand fixed thereto, or a porous particle having an antibody affinity ligand fixed thereto. (Step 1) Preparing a solid support (Step 2) A step of adjusting the coefficient of variation of the volume-based particle size distribution of the porous particles having the antibody affinity ligand immobilized thereon to be 1% or more and 22% or less, and the skewness of the volume-based particle size distribution of the porous particles having the antibody affinity ligand immobilized thereon to be -0.1 or more and 5 or less, by sieving the solid phase support using a wet method.

2. A method for producing a support for antibody affinity chromatography, comprising the following steps 1 and 2, in which the solid phase support prepared in step 1 is a porous particle having an antibody affinity ligand fixed thereto. (Step 1) Preparing a solid support (Step 2) A step of adjusting the coefficient of variation of the volume-based particle size distribution of the porous particles having an antibody affinity ligand immobilized thereon to be 1% or more and 22% or less, and the skewness of the volume-based particle size distribution of the porous particles having an antibody affinity ligand immobilized thereon to be -0.1 or more and 5 or less, by sieving the solid-phase support using a wet method.

3. A method for producing a support for antibody affinity chromatography, comprising the following steps 1 and 2, in which the solid phase support prepared in step 1 is a porous particle having no antibody affinity ligand immobilized thereon. (Step 1) Preparing a solid support (Step 2) A step of adjusting the coefficient of variation of the volume-based particle size distribution of the porous particles having the antibody affinity ligand immobilized thereon to be 1% or more and 22% or less, and the skewness of the volume-based particle size distribution of the porous particles having the antibody affinity ligand immobilized thereon to be -0.1 or more and 5 or less, by sieving the solid phase support using a wet method.

4. (Step 3) further comprising a step of immobilizing an antibody affinity ligand on the porous particles classified by sieve in step 2; A method for producing the chromatography carrier according to claim 3.

5. The sieve classification in step 2 includes, in this order or in reverse order, (step 2a) a step of passing the solid support through a sieve having a mesh size of 10% or more and less than 200% of (2a-d), when the volume average particle size (2a-d) of the solid support before the sieve classification is taken as 100%, and recovering the sieve residue, and (step 2b) a step of passing the solid support through a sieve having a mesh size larger than that of the sieve in step 2a and less than 600% of (2a-d), and recovering the sieve-passing portion. A method for producing the chromatography carrier according to any one of claims 1 to 4.

6. A method for producing a chromatography carrier as described in claim 5, wherein the mesh size of the sieve used in step 2a is 35 μm or more and 60 μm or less, and in step 2, the sieve classification is performed so that the volume-based cumulative 50% particle diameter d50 is 55 μm or more and 100 μm or less.

7. The coefficient of variation is 5% or more and 20% or less, and the skewness is 0.01 or more and 3 or less. A method for producing the chromatography carrier according to any one of claims 1 to 6.

8. Step 1 includes a step of dispersing a monomer composition in an aqueous medium and carrying out suspension polymerization. A method for producing the chromatography carrier according to any one of claims 1 to 7.

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