Production method for chromatographic carrier

Abstract

The present invention provides a method for easily producing a chromatographic carrier that has a high dynamic binding capacity to antibodies or fragments thereof, inhibits the leakage of protein ligands during isolation, and inhibits the aggregation of carriers.　Provided is a production method for a chromatographic carrier comprising the ligand binding step, ligand-bound carrier bed forming step, ligand-bound carrier flushing and cleaning step, and ligand-bound carrier stirring and cleaning step described hereafter. Ligand binding step: a step in which a protein ligand is bound to a solid phase carrier. Ligand-bound carrier bed forming step: a step in which a container is filled with the ligand-bound carrier obtained in the ligand binding step to form a ligand-bound carrier bed. Ligand-bound carrier flushing and cleaning step: a step in which the ligand-bound carrier bed formed in the ligand-bound carrier bed forming step is cleaned one or more times by flushing the bed with a cleaning liquid. Ligand-bound carrier stirring and cleaning step: a step in which the ligand-bound carrier after the ligand-bound carrier flushing and cleaning step is cleaned one or more times by stirring in a cleaning liquid.

Description

Method for producing chromatography support

[0001] The present invention relates to a method for producing a chromatography support.

[0002] In recent years, in the field of biopharmaceuticals, such as antibody drugs, remarkable progress has been made in expression technologies for target substances such as proteins, which has led to a demand for improved productivity in purification processes using chromatography, etc. One method for improving productivity is to reduce the concentrations of impurities present in pharmaceutical raw materials, such as host cell-derived proteins and deoxyribonucleic acid, as much as possible in a single purification run, thereby reducing the number of purification runs and the number of steps. There is a growing demand for chromatography supports that can achieve this, and the demand for dynamic binding capacity for antibodies or fragments thereof is particularly high. Known methods for producing such chromatography supports include, for example, a method in which a synthetic polymer solid phase support or a natural polymer solid phase support is obtained by a polymerization reaction, a crosslinking reaction, a functional group introduction reaction, etc., and then a proteinaceous ligand is bound to the support (Patent Document 1).

[0003] Japanese Patent Application Laid-Open No. 2017-37069 WO2008 / 146906 Pamphlet WO2019 / 121296 Pamphlet

[0004] Furthermore, the carrier to which the proteinaceous ligand is bound obtained as described above is contaminated with impurities generated in each production step, including unreacted proteinaceous ligand. Therefore, in order to prevent unreacted proteinaceous ligand from leaking and contaminating the antibody during antibody purification, agitation washing is sometimes performed after the ligand is bound (Patent Documents 2 and 3). However, this washing does not sufficiently remove the unreacted proteinaceous ligand, and the proteinaceous ligand sometimes leaks during isolation. Therefore, the present inventors performed a liquid-flow washing instead of agitation washing on the ligand-bound carrier and investigated the washing performance, and found that carriers are prone to aggregation. Carriers prone to aggregation can cause performance variations between lots.

[0005] The problem to be solved by the present invention is to provide a method for easily producing a chromatography carrier that has a large dynamic binding capacity for an antibody or a fragment thereof, is less likely to leak proteinaceous ligands during isolation, and is less likely to aggregate with other carriers.

[0006] The present inventors have found that, after binding a proteinaceous ligand to a solid phase carrier, performing a combination of flow-through washing and agitation washing in this order at least once each not only increases the dynamic binding capacity of the chromatography carrier for an antibody or a fragment thereof, but also reduces the likelihood of aggregation between carriers and leakage of the proteinaceous ligand during isolation.

[0007] That is, the present invention provides the following items <1> to <7>. <1> A method for producing a chromatography carrier (hereinafter also referred to as a method for producing a chromatography carrier of the present invention), comprising the following steps: a ligand binding step, a ligand-bound carrier bed formation step, a ligand-bound carrier liquid passing washing step, and a ligand-bound carrier agitation washing step. (Ligand binding step) A step of binding a proteinaceous ligand to a solid phase carrier. (Ligand-bound carrier bed formation step) A step of filling a container with the ligand-bound carrier obtained in the ligand binding step to form a ligand-bound carrier bed. (Ligand-bound carrier liquid passing washing step) A step of passing a washing solution through the ligand-bound carrier bed formed in the ligand-bound carrier bed formation step and washing it one or more times. (Ligand-bound carrier agitation washing step) A step of agitating and washing the ligand-bound carrier after the ligand-bound carrier liquid passing washing step in a washing solution one or more times.

[0008] <2> The method for producing a chromatography carrier according to <1>, wherein the number of times of washing in the ligand-bound carrier washing step is 2 to 5.

[0009] <3> The method for producing a chromatography carrier according to <1> or <2>, further comprising a solid phase carrier washing step, the solid phase carrier washing step comprising the following solid phase carrier bed formation step and solid phase carrier liquid passing washing step, and the solid phase carrier washed in the solid phase carrier washing step is used as the solid phase carrier in the ligand binding step: (Solid phase carrier bed formation step) A step of filling a vessel with the solid phase carrier to form a solid phase carrier bed, and (Solid phase carrier liquid passing washing step) A step of passing a washing solution through the solid phase carrier bed formed in the solid phase carrier bed formation step to wash the solid phase carrier bed formed in the solid phase carrier bed formation step one or more times.

[0010] <4> The method for producing a chromatography carrier according to any one of <1> to <3>, further comprising a solid phase carrier washing step, the solid phase carrier washing step comprising the following steps: a solid phase carrier bed formation step, a solid phase carrier liquid passing washing step, and a solid phase carrier agitation washing step, and the solid phase carrier washed in the solid phase carrier washing step is used as the solid phase carrier in the ligand binding step. (Solid phase carrier bed formation step) A step of filling a vessel with the solid phase carrier to form a solid phase carrier bed (Solid phase carrier liquid passing washing step) A step of passing a liquid through the solid phase carrier bed formed in the solid phase carrier bed formation step and washing it one or more times with a washing solution (Solid phase carrier agitation washing step) A step of agitating and washing the solid phase carrier after the solid phase carrier liquid passing washing step in a washing solution one or more times.

[0011] <5> The method for producing a chromatography carrier according to <3> or <4>, wherein the number of times of liquid-passage washing in the solid-phase carrier liquid-passage washing step is 2 to 5. <6> The method for producing a chromatography carrier according to any of <3> to <5>, wherein the total number of times of liquid-passage washing in the solid-phase carrier liquid-passage washing step and the ligand-bound carrier liquid-passage washing step is 2 to 8. <7> The method for producing a chromatography carrier according to any of <1> to <6>, wherein the proteinaceous ligand is one or more ligands selected from protein A, protein G, protein L, and analogs thereof.

[0012] According to the method for producing a chromatography carrier of the present invention, it is possible to easily produce a chromatography carrier that has a large dynamic binding capacity for an antibody or a fragment thereof, is less likely to leak protein ligands during isolation, and is less likely to aggregate with other carriers.

[0013] [Method for manufacturing a chromatography carrier] The method for manufacturing a chromatography carrier of the present invention comprises the following ligand binding step, ligand-bound carrier bed formation step, ligand-bound carrier liquid passing washing step, and ligand-bound carrier stirring washing step: (Ligand binding step) A step of binding a proteinaceous ligand to a solid phase carrier; (Ligand-bound carrier bed formation step) A step of filling a container with the ligand-bound carrier obtained in the ligand binding step to form a ligand-bound carrier bed; (Ligand-bound carrier liquid passing washing step) A step of passing a washing solution through the ligand-bound carrier bed formed in the ligand-bound carrier bed formation step and washing it one or more times; (Ligand-bound carrier stirring washing step) A step of stirring and washing the ligand-bound carrier after the ligand-bound carrier liquid passing washing step in the washing solution one or more times.

[0014] Here, the solid-phase carrier used in the ligand binding step will be described. A solid-phase carrier preferably has a functional group capable of binding a ligand (e.g., a functional group selected from the group consisting of a cyclic ether group, a carboxy group, -C(=O)-O-C(=O)-, a succinimidooxycarbonyl group, a formyl group, a hydroxyl group, and an isocyanate group) within the molecule. Use of such a solid-phase carrier makes it easier to obtain a carrier with particularly low proteinaceous ligand leakage. Examples of solid-phase carriers include particulate solid-phase carriers, monolithic solid-phase carriers, plate-like solid-phase carriers, membrane-like solid-phase carriers, fibrous solid-phase carriers, and chip-like solid-phase carriers. Particulate solid-phase carriers are preferred, and porous particulate solid-phase carriers (hereinafter simply referred to as "porous particles") are more preferred. Porous particles containing a polymer are preferred. 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. The solid phase carrier may be a commercially available product or one manufactured according to a conventional method. Here, a method for manufacturing the solid phase carrier will be described.

[0015] When porous particles are obtained as the solid phase support, 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 P1).

[0016] -Step P1- The monomer composition used in step P1 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 bond to a ligand. Examples of the functional group include functional groups selected from the group consisting of a cyclic ether group, a carboxy group, -C(=O)-O-C(=O)-, a succinimideoxycarbonyl group, a formyl group, a hydroxyl group, and an isocyanate group. Among these, a cyclic ether group is preferred. Here, the "cyclic ether group" is preferably a cyclic ether group having 3 to 7 atoms constituting the ring. The cyclic ether group may have an alkyl group as a substituent. Specific examples of the cyclic ether group include cyclic ether groups represented by the following formulas (4) to (9). Cyclic ether groups represented by formulas (4), (6), or (9) are preferred, with the cyclic ether group represented by formula (4) being more preferred.

[0017]

[0018] [In the formula, R 11 ~R 14 each independently represents a hydrogen atom or an alkyl group, and * represents a bond.

[0019] R 11 ~R 14 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. 11 ~R 14 is preferably a hydrogen atom.

[0020] The functional group-containing monomer is preferably a monomer having a functional group capable of binding to a ligand and a polymerizable unsaturated group. Examples of such monomers include glycidyl (meth)acrylate, 3-oxiranylpropyl (meth)acrylate, 4-oxiranylbutyl (meth)acrylate, 5-oxiranylpentyl (meth)acrylate, 6-oxiranylhexyl (meth)acrylate, 7-oxiranylheptyl (meth)acrylate, 8-oxiranyloctyl (meth)acrylate, (3-methyloxiranyl)methyl (meth)acrylate, 4-hydroxybutyl (meth)acrylate glycidyl ether, glycerin mono(meth)acrylate glycidyl ether, 3,4-epoxycyclohexylmethyl (meth)acrylate, 3,4-epoxycyclohexylethyl (meth)acrylate, 3,4-epoxycyclohexylpropyl (meth)acrylate, α-(meth)acryl-ω-glycidyl polyethylene glycol, tetrahydrofurfuryl (meth)acrylate, and other monomers having a cyclic ether group ( Examples of suitable methacrylate monomers include aromatic vinyl monomers having a cyclic ether group, such as (vinylbenzyl)glycidyl ether, (isopropenylbenzyl)glycidyl ether, (vinylphenethyl)glycidyl ether, (vinylphenylbutyl)glycidyl ether, (vinylbenzyloxyethyl)glycidyl ether, (vinylphenyl)glycidyl ether, (isopropenylphenyl)glycidyl ether, and 1,2-epoxy-3-(4-vinylbenzyl)propane; allyl ether monomers having a cyclic ether group, such as allyl glycidyl ether; (meth)acrylate monomers having an isocyanate group, such as isocyanatoethyl (meth)acrylate; unsaturated dicarboxylic acid anhydride monomers, such as maleic anhydride, methylmaleic anhydride, and glutaconic anhydride; (meth)acrylic acid, 3,4-epoxy-1-butene, and 3,4-epoxy-3-methyl-1-butene. These monomers may be used alone or in combination of two or more. Among these monomers, (meth)acrylate monomers having a cyclic ether group are preferred, and glycidyl (meth)acrylate is particularly preferred.

[0021] 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 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 P1.

[0022] The monomer composition used in step 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"). Examples of the other monomer include a polymerizable unsaturated group-containing monomer that does not have a functional group capable of binding to 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 or a combination of these may be used.

[0023] 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.

[0024] 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.

[0025] 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.

[0026] Examples of the aromatic vinyl non-crosslinkable monomer include styrenes such as styrene, α-methylstyrene, halogenated styrene, 4-methylstyrene, 2,4-dimethylstyrene, 2,4,6-trimethylstyrene, ethylvinylbenzene, 4-isopropylstyrene, 4-n-butylstyrene, 4-isobutylstyrene, and 4-tert-butylstyrene; vinylnaphthalenes such as 1-vinylnaphthalene and 2-vinylnaphthalene; and the like. These can be used alone or in combination of two or more.

[0027] Examples of the vinyl ketone-based non-crosslinkable monomer include ethyl vinyl ketone, propyl vinyl ketone, and isopropyl vinyl ketone. These may be used alone or in combination of two or more. Examples of the (meth)acrylonitrile-based non-crosslinkable monomer include acrylonitrile and methacrylonitrile. These may be used alone or in combination of two or more. Examples of the N-vinyl amide-based non-crosslinkable monomer include N-vinyl acetamide and N-vinyl propionamide. These may be used alone or in combination of two or more.

[0028] 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 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 P1.

[0029] 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. The crosslinkable monomer is preferably a di- to penta-functional crosslinkable monomer, more preferably a di- or tri-functional crosslinkable monomer. Among the crosslinkable monomers, (meth)acrylate crosslinkable monomers and aromatic vinyl crosslinkable monomers are preferred.

[0030] 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.

[0031] 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.

[0032] 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, and triallyl isocyanurate. These may be used alone or in combination of two or more. Furthermore, in addition to the above-mentioned examples, examples of the crosslinkable monomer include dehydration condensation products of amino alcohols such as diaminopropanol, trishydroxymethylaminomethane, and glucosamine with (meth)acrylic acid, and conjugated diolefins such as butadiene and isoprene.

[0033] 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 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 P1.

[0034] The aqueous medium used in step 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 to 7,000 parts by mass per 100 parts by mass of the total amount of monomers. When water is used as the dispersion medium for the aqueous medium, a dispersion stabilizer such as sodium carbonate, calcium carbonate, sodium sulfate, calcium phosphate, or sodium chloride may be used.

[0035] Specific examples of the method for step 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.

[0036] The polymerization initiator is preferably a radical polymerization initiator. Examples of the radical polymerization initiator include azo initiators, peroxide initiators, and redox initiators, and 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 parts by mass or more and 10 parts by mass or less per 100 parts by mass of the total amount of monomers.

[0037] 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.

[0038] 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 suitable porogens 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. Porous agents can be used alone or in combination of two or more. The total amount of the porous agents used is typically about 40 to 600 parts by weight per 100 parts by weight of the total amount of monomers.

[0039] In step P1, various surfactants may be used, including anionic surfactants such as alkyl sulfates, alkylaryl sulfates, alkyl phosphates, and fatty acid salts. Also, polymerization inhibitors such as nitrites such as sodium nitrite, iodides such as potassium iodide, tert-butylpyrocatechol, benzoquinone, picric acid, hydroquinone, copper chloride, and ferric chloride may also be used. Furthermore, polymerization regulators such as dodecyl mercaptan may also be used.

[0040] The polymerization temperature in step 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.

[0041] -Step P2- Furthermore, prior to the solid phase carrier washing step, a step (hereinafter also referred to as step P2) of reacting the porous particles obtained in step P1 with at least one selected from a crosslinking agent and a hydrophilizing agent may be performed. When both a crosslinking agent and a hydrophilizing agent are used, the crosslinking reaction may be performed after the hydrophilizing reaction, or the hydrophilizing reaction may be performed after the crosslinking reaction. Alternatively, the crosslinking reaction and the hydrophilizing reaction may be performed simultaneously. When a monomer composition containing a functional group-containing monomer is used in step P1, the crosslinking reaction involves an addition reaction of the crosslinking agent with some of the functional groups present in the polymer molecules of the porous particles, thereby introducing a partial structure derived from the crosslinking agent. As a result, residues of the functional groups are crosslinked via the partial structure derived from the crosslinking agent. When a monomer composition containing a functional group-containing monomer is used in step P1, the hydrophilizing reaction involves an addition reaction of the hydrophilizing agent with some of the functional groups present in the polymer molecules of the porous particles, thereby introducing a partial structure derived from the hydrophilizing agent.

[0042] The crosslinking agent used in step P2 may be any agent capable of reacting with a functional group capable of binding a ligand to introduce a crosslinked structure, but a crosslinking agent capable of reacting with a functional group capable of binding a ligand to introduce a crosslinked structure and containing at least two groups represented by -C(=O)-NH- in the molecule is preferred.

[0043] When the porous particles obtained in step 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, 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, etc. can be used. When the porous particles obtained in step P1 have carboxy groups, -C(=O)-O-C(=O)-, succinimidooxycarbonyl groups, formyl groups, or isocyanate groups, specifically, a crosslinking agent containing at least two groups represented by -C(=O)-NH-NH2 as crosslinkable groups in the molecule can be used.

[0044] 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.

[0045] In step P2, 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 a crosslinking agent include a polyfunctional isocyanate-based crosslinking agent, a polyfunctional epoxy-based crosslinking agent, a polyfunctional aldehyde-based crosslinking agent, a polyfunctional thiol-based crosslinking agent, a polyfunctional oxazoline-based crosslinking agent, a polyfunctional aziridine-based crosslinking agent, and a metal chelate-based crosslinking agent.

[0046] 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.

[0047] The hydrophilizing agent used in step 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 proteinaceous ligand leakage, and more preferably a compound having a total of 2 to 4 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 types. Among these, alcohols having a mercapto group in the molecule are preferred, in order to improve antifouling properties and low proteinaceous ligand leakage, and thioglycerol is particularly preferred.

[0048] The total amount of the hydrophilizing agent used is preferably 0.5 molar equivalents or more and 10 molar equivalents or less, more preferably 1 molar equivalents or more and 8 molar equivalents or less, and particularly preferably 2 molar equivalents or more and 6 molar equivalents or less, relative to 1 mole of the functional group derived from the functional group-containing monomer.

[0049] Step 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.

[0050] The reaction time in step 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.

[0051] -Step P3- Furthermore, when a ligand is bound to the porous particles via a linker (spacer) in the ligand binding step, a step of reacting the porous particles obtained in step P1 or step P2 with a compound that provides a linker (hereinafter also referred to as step P3) may be carried out prior to the solid phase carrier washing step.

[0052] 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 performed in step P2. The linker introduction reaction is preferably carried out in a buffer having a pH of 7 to 14 to increase the reaction efficiency. 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 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.

[0053] (Solid phase carrier washing step) The method for producing a chromatography carrier of the present invention preferably further comprises a solid phase carrier washing step in addition to the ligand binding step, ligand-bound carrier bed formation step, ligand-bound carrier liquid passing washing step, and ligand-bound carrier stirring washing step, in order to increase the hydrophilicity of the solid phase carrier surface and to make it less likely for proteinaceous ligands to leak out during isolation, and the solid phase carrier washed in the solid phase carrier washing step is preferably used as the solid phase carrier in the ligand binding step.

[0054] The solid phase carrier washing step is a step of washing the solid phase carrier. In the solid phase carrier washing step, washing is preferably performed with a washing liquid (hereinafter also referred to as "solid phase carrier washing liquid"). The solid phase carrier washing liquid is preferably an aqueous washing liquid. "Aqueous washing liquid" means a washing liquid containing at least water. Examples of aqueous washing liquids include those containing water or a mixture of water and a lower alcohol. Examples of lower alcohols include one or more selected from ethanol and isopropanol.

[0055] Furthermore, as the solid phase carrier washing solution, at least one washing solution selected from washing solutions containing hydrogen peroxide, washing solutions containing peracetic acid, and washing solutions having a pH of 0 to 3 or more and a pH of 12.5 to 14 or less (excluding washing solutions containing hydrogen peroxide and washing solutions containing peracetic acid) is preferred in order to increase the hydrophilicity of the solid phase carrier surface, reduce leakage of proteinaceous ligands during isolation, increase the dynamic binding capacity, and suppress aggregation of carriers themselves. When such a washing solution is used, the solid phase carrier surface is hydrophilized, and the dynamic binding capacity for antibodies or fragments thereof, low proteinaceous ligand leakage, and low aggregation of the resulting chromatography carrier are improved. Furthermore, washing solutions with various pH values ​​can be used in the ligand-binding carrier flow-through washing step and the ligand-binding carrier agitation washing step. For example, even when a washing solution having a pH of more than 3 and less than 12.5 is used in the ligand-binding carrier flow-through washing step or the ligand-binding carrier agitation washing step, a chromatography carrier that is less susceptible to leakage of proteinaceous ligands during isolation can be obtained. Although the reason why ligand leakage during isolation is thus reduced is not entirely clear, the inventors speculate that this is because the washing solution hydrophilizes the solid phase carrier surface, providing excellent antifouling properties, making it easier to wash away unreacted proteinaceous ligands that typically adhere in the subsequent ligand binding step, even when the pH of the washing solution is greater than 3 and less than 12.5. For example, the inventors speculate that if the solid phase carrier contains, in its molecule, functional groups capable of binding ligands, such as cyclic ether groups or hydroxyl groups, or functional groups such as carbonyl bonds, hydrolysis of these functional groups or the presence of a large number of hydroxyl groups or carboxyl groups increases hydrophilicity and provides excellent antifouling properties. Furthermore, even when a solid phase carrier washing step is performed prior to ligand binding, the method for producing a chromatography carrier of the present invention allows sufficient ligand binding to the solid phase carrier in the ligand binding step, thereby producing a chromatography carrier with a high dynamic binding capacity for antibodies or fragments thereof. The reason why such dynamic binding capacity is achieved is also not entirely clear, but the inventors speculate that one reason is that multiple washings performed in an appropriate manner significantly reduce damage to the chromatography carrier.In the present invention, "hydrophilization" refers to increasing affinity for water.

[0056] As the solid phase carrier washing solution, a washing solution having a pH of 0 to 3 or more or a pH of more than 12.5 to 14 is preferred, and a washing solution having a pH of more than 12.5 to 14 is more preferred, in order to increase the hydrophilicity of the solid phase carrier surface and to make it less likely for the proteinaceous ligand to leak during isolation.

[0057] To prevent leakage of the proteinaceous ligand during isolation, the pH of the washing solution containing hydrogen peroxide is preferably 4.5 or more and less than 7, more preferably 5 or more and 6.8 or less, particularly preferably 5.5 or more and 6.5 or less, and most preferably 6 or more and 6.5 or less. Furthermore, to prevent leakage of the proteinaceous ligand during isolation, the concentration of hydrogen peroxide in the washing solution is preferably 0.001 M or more and 10 M or less, more preferably 0.005 M or more and 5 M or less, and particularly preferably 0.01 M or more and 1 M or less.

[0058] To prevent leakage of the proteinaceous ligand during isolation, the pH of the washing solution containing peracetic acid is preferably 1 or more and less than 7, more preferably 2 or more and 6 or less, particularly preferably 3 or more and 5 or less, and most preferably 4 or more and 5 or less. Furthermore, to prevent leakage of the proteinaceous ligand during isolation, the concentration of peracetic acid in the washing solution is preferably 0.001 M or more and 5 M or less, more preferably 0.005 M or more and 3 M or less, and particularly preferably 0.01 M or more and 1 M or less.

[0059] The solid phase carrier washing solution includes washing solutions containing hydrogen peroxide and peracetic acid, as well as washing solutions having a pH of 0 to 3 and a pH of greater than 12.5 to 14. The pH of a washing solution having a pH of 0 to 3 is preferably 0 to 2.5, more preferably 0 to 2, in order to increase the hydrophilicity of the solid phase carrier surface and to reduce the leakage of proteinaceous ligands during isolation. The pH of a washing solution having a pH of greater than 12.5 to 14 is preferably 12.7 to 14, more preferably 13 to 14, and particularly preferably 13.2 to 14, in order to increase the hydrophilicity of the solid phase carrier surface and to reduce the leakage of proteinaceous ligands during isolation. When the pH of a washing solution having a pH of greater than 12.5 to 14 is set to 13.2 to 14, the hydrophilicity of the solid phase carrier surface of the resulting chromatography carrier is increased, and the proteinaceous ligand is particularly reduced from leaking during isolation.

[0060] The solid support washing solution preferably contains a strongly acidic or strongly basic pH adjuster, more preferably a strongly basic pH adjuster, in order to increase the hydrophilicity of the solid support surface and to prevent leakage of proteinaceous ligands during isolation. As the strongly acidic pH adjuster, inorganic strong acid pH adjusters such as sulfuric acid, hydrochloric acid, and nitric acid are preferred, with hydrochloric acid being more preferred. Furthermore, as the strongly basic pH adjuster, alkali metal hydroxides such as lithium hydroxide, potassium hydroxide, and sodium hydroxide are preferred, with potassium hydroxide and sodium hydroxide being more preferred. When a strongly basic pH adjuster is used, it becomes easier to adjust the pH of the washing solution to a range of more than 12.5 and not more than 14, and when a strongly acidic pH adjuster is used, it becomes easier to adjust the pH of the washing solution to a range of 0 to 5. Furthermore, in order to reduce leakage of proteinaceous ligands during isolation, the concentration of the strongly acidic pH adjuster or strongly basic pH adjuster in the solid phase carrier washing solution is preferably 0.001 M to 25 M, more preferably 0.005 M to 10 M, even more preferably 0.01 M to 5 M, even more preferably 0.05 M to 2.5 M, even more preferably 0.25 M to 1.5 M, and particularly preferably 0.5 M to 1 M. When the concentration of the strongly acidic pH adjuster or strongly basic pH adjuster is 0.25 M or more or 0.5 M or more, the solution exhibits particularly excellent low leakage of proteinaceous ligands. The solid phase carrier washing solution may contain a buffer or two or more pH adjusters to adjust the pH to a desired level or to maintain the target pH.

[0061] Washing with a solid phase carrier washing solution may be performed once or twice or more times in total. However, in order to increase the hydrophilicity of the solid phase carrier surface, to prevent leakage of the proteinaceous ligand during isolation, and to suppress aggregation between carriers, washing is preferably performed twice or more times in total, more preferably 2 to 20 times in total, even more preferably 2 to 15 times in total, even more preferably 2 to 10 times in total, even more preferably 2 to 8 times in total, and particularly preferably 3 to 4 times in total. When washing is performed twice or more times in total, the types of solid phase carrier washing solutions used for each washing may be the same or different, but the same type is preferred. When washing is performed three or more times in total with a solid phase carrier washing solution, particularly excellent low leakage of proteinaceous ligands is achieved. Furthermore, when washing is performed four or fewer times in total with a solid phase carrier washing solution, particularly excellent low aggregation is achieved.

[0062] The amount of solid phase carrier washing solution used per washing is preferably 40 parts by volume or more, more preferably 60 parts by volume or more, and particularly preferably 80 parts by volume or more, relative to 100 parts by volume of the dry solid content of the solid phase carrier, and is preferably 1000 parts by volume or less, more preferably 500 parts by volume or less, and particularly preferably 300 parts by volume or less, relative to 100 parts by volume of the dry solid content of the solid phase carrier. Specific ranges are preferably 40 parts by volume or more and 1000 parts by volume or less, more preferably 60 parts by volume or more and 500 parts by volume or less, and particularly preferably 80 parts by volume or more and 300 parts by volume or less, relative to 100 parts by volume of the dry solid content of the solid phase carrier.

[0063] The washing temperature is not particularly limited, but is usually in the range of 10 to 50° C., preferably 15 to 45° C. Furthermore, either batch washing or continuous washing may be used.

[0064] The washing method using the solid phase carrier washing solution is not particularly limited. Examples include flow-through washing, stirring washing, and static washing. Among these, flow-through washing and stirring washing are preferred for increasing the hydrophilicity of the solid phase carrier surface, preventing leakage of proteinaceous ligands during isolation, increasing the dynamic binding capacity, and suppressing aggregation between carriers. Flow-through washing is more preferred for further improving the replacement efficiency, and stirring washing is more preferred for suppressing aggregation between carriers. A combination of flow-through washing and stirring washing is particularly preferred. When flow-through washing and stirring washing are performed, the order and number of times are not particularly limited. However, it is preferred that stirring washing be performed after flow-through washing, and that these be performed at least once each. When flow-through washing and stirring washing are performed in combination as the solid phase carrier washing step, particularly excellent low leakage of proteinaceous ligands, low aggregation, and replacement efficiency are achieved. Note that when washing is performed two or more times in total, the types of washing methods used for each step may be the same or different.

[0065] The liquid passing washing may be performed by passing a solid carrier washing solution through the solid carrier bed one or more times, but when liquid passing washing is performed, the solid carrier washing step preferably includes the following solid carrier bed formation step and solid carrier liquid passing washing step: (Solid carrier bed formation step) A step of filling a vessel with solid carriers to form a solid carrier bed, (Solid carrier liquid passing washing step) A step of passing the solid carrier bed formed in the solid carrier bed formation step one or more times with a solid carrier washing solution (preferably at least one cleaning solution selected from a cleaning solution containing hydrogen peroxide, a cleaning solution containing peracetic acid, and a cleaning solution having a pH of 0 to 3 or a pH of more than 12.5 and 14 (excluding cleaning solutions containing hydrogen peroxide and cleaning solutions containing peracetic acid)).

[0066] (Solid-phase carrier bed formation step) In the present invention, the term "solid-phase carrier bed" refers to a layer of solid carrier solids, and may be dry or wet. Specific methods for forming a solid-phase carrier bed include pouring a solid-phase carrier slurry (for example, one using water, alcohol (ethanol, isopropanol, etc.), a mixture of water and alcohol, or a buffer solution (carbonate buffer, etc.) as a dispersion medium) into a container, allowing the solid-phase carrier solids to settle to the bottom of the container by gravitational sedimentation or centrifugal sedimentation, and removing the liquid phase of the slurry by a solid-liquid separation operation (for example, decantation, centrifugation, filtration, etc.). Furthermore, a solid-liquid separator is preferred as the container, since it is simple and convenient to pass the washing solution directly through the solid-phase carrier bed formed in the solid-phase carrier bed formation step in the solid-phase carrier liquid-washing step. Specific examples include a filter-equipped column container, a filter-equipped funnel, a filter-equipped centrifuge, a filter reactor, etc.

[0067] The content of the solid phase carrier in the slurry is preferably 5% by volume or more, more preferably 15% by volume or more, particularly preferably 30% by volume or more, and is preferably 80% by volume or less, more preferably 75% by volume or less, particularly preferably 70% by volume or less. Specific ranges are preferably 5% by volume or more and 80% by volume or less, more preferably 15% by volume or more and 75% by volume or less, and particularly preferably 30% by volume or more and 70% by volume or less. The volume percentage of the carrier in the slurry can be calculated, for example, by filling a 250 mL glass graduated cylinder (JIS R3505 Class A compliant) manufactured by Corning Incorporated with 200 mL of the slurry, and dividing the sedimentation volume after 3 hours by the volume of the slurry filled in the graduated cylinder.

[0068] (Solid Phase Carrier Liquid-Passing Washing Step) In the present invention, "liquid-passing washing" of a solid phase carrier refers to passing a wash solution through the solid phase carrier bed. For example, washing is performed by passing the wash solution through the solid phase carrier bed from one direction to the other. Specifically, washing can be performed using a solid-liquid separator such as a filter-equipped column container, a filter plate, a filter-equipped funnel, a Buchner funnel, a Nutsch filter, a filter-equipped centrifuge, or a filter reactor. The wash solution flowed into the solid phase carrier bed may be incubated preferably for 30 seconds to 3 hours, more preferably 2 minutes to 60 minutes, and then drained. The number of liquid-passing washes in the solid phase carrier liquid-passing washing step is preferably one or more times, more preferably 1 to 20 times, even more preferably 1 to 10 times, even more preferably 1 to 5 times, even more preferably 2 to 5 times, and particularly preferably 2 to 3 times, in order to increase the hydrophilicity of the solid phase carrier surface, to reduce leakage of proteinaceous ligands during isolation, and to suppress aggregation between carriers. When the number of liquid-passing washes is two or more times, particularly low leakage of proteinaceous ligands is achieved. Furthermore, when the number of times of liquid-passage washing is set to three or less, the low flocculation property is particularly excellent. The type of solid phase carrier washing solution and the amount of solid phase carrier washing solution used per washing are as described above. Note that liquid-passage washing may be liquid-passage washing using gravity, washing by pressurized liquid passage, or liquid-passage washing by reduced pressure.

[0069] (Solid-Phase Carrier Agitation Washing Step) The agitation washing step may involve agitating the solid-phase carrier in a solid-phase carrier washing solution. However, it is preferable to agitate and wash the solid-phase carrier after the solid-phase carrier solution-passing washing step in the solid-phase carrier washing solution at least once in order to increase the hydrophilicity of the solid-phase carrier surface, to reduce leakage of the proteinaceous ligand during isolation, to increase the dynamic binding capacity, and to suppress aggregation between carriers. The number of agitation washes in the solid-phase carrier agitation washing step is preferably at least once, more preferably 1 to 20 times, even more preferably 1 to 10 times, even more preferably 1 to 5 times, even more preferably 1 to 2 times, and particularly preferably 1 time, in order to increase the hydrophilicity of the solid-phase carrier surface, to reduce leakage of the proteinaceous ligand during isolation, and to suppress aggregation between carriers. When the number of agitation washes is at least once, particularly excellent low aggregation properties are achieved. Furthermore, when the number of agitation washes is 2 or less, particularly excellent low proteinaceous ligand leakage properties are achieved. The agitation speed in the solid carrier agitation washing step is preferably 10 to 150 rpm, more preferably 20 to 100 rpm, and particularly preferably 30 to 80 rpm. The agitation time per solid carrier agitation washing step is preferably 30 seconds to 300 minutes, more preferably 1 minute to 180 minutes, even more preferably 2 minutes to 120 minutes, and particularly preferably 5 minutes to 90 minutes. The type of solid carrier washing solution and the amount of solid carrier washing solution used per step are as described above. When the solid carrier liquid-passing washing step is performed using a solid-liquid separator, continuous agitation washing can be performed by carrying out the solid carrier agitation washing step while leaving the filtrate outlet of the solid-liquid separator used in the solid carrier liquid-passing washing step open. Alternatively, batchwise agitation washing can be performed by using a closed container in the solid carrier agitation washing step.

[0070] After washing the solid phase carrier, the washing solution may be removed or the solid phase carrier may be dispersed in water or a mixture of water and a lower alcohol, if necessary, prior to the ligand binding step.

[0071] Here, the ligand binding step will be described. (Ligand Binding Step) The ligand binding step is a step of binding a proteinaceous ligand to a solid-phase support. The proteinaceous ligand is preferably one or more ligands selected from Protein A, Protein G, Protein L, and their analogs. Protein A and modified Protein A are preferred, with modified Protein A being more preferred, in order to increase the dynamic binding capacity and to reduce leakage of the proteinaceous ligand during isolation. Protein A contains five domains, E, D, A, B, and C, which have the ability to bind to immunoglobulins. Among the above ligands, a Protein A analog having modified B and C domains is preferred, and a Protein A analog having a modified C domain is more preferred.

[0072] Furthermore, among proteinaceous ligands, in order to increase the dynamic binding capacity and to reduce leakage of the proteinaceous ligand during isolation, proteinaceous ligands having an amino acid sequence with at least one or two or more substitutions selected from the following (a) to (i) relative to an amino acid sequence having 85% or more homology to the amino acid sequence shown in SEQ ID NO: 1 (C domain of protein A) are preferred. Among such proteinaceous ligands, those having an amino acid sequence with two or more substitutions selected from the following (a) to (i) are preferred, those having an amino acid sequence with 2 to 9 substitutions selected from the following (a) to (i) are more preferred, and those having an amino acid sequence with 2 to 6 substitutions selected from the following (a) to (i) are particularly preferred. Furthermore, ligands having two or more of such amino acid sequences are preferred, those having 2 to 12 substitutions are more preferred, and those having 4 to 7 substitutions are particularly preferred. When the proteinaceous ligand contains two or more amino acid sequences, the amino acid sequences may be of the same or different types.

[0073] (a) substitution of the amino acid residue at position 1 in the amino acid sequence of SEQ ID NO: 1 with a valine residue; (b) substitution of the amino acid residue at position 3 in the amino acid sequence of SEQ ID NO: 1 with an alanine residue; (c) substitution of the amino acid residue at position 6 in the amino acid sequence of SEQ ID NO: 1 with an alanine residue or an aspartic acid residue; (d) substitution of the amino acid residue at position 9 in the amino acid sequence of SEQ ID NO: 1 with an alanine residue; (e) substitution of the amino acid residue at position 11 in the amino acid sequence of SEQ ID NO: 1 with an alanine residue, a glutamine residue or a glutamic acid residue; (f) substitution of the amino acid residue at position 23 in the amino acid sequence of SEQ ID NO: 1 with a leucine residue; (g) substitution of the amino acid residue at position 29 in the amino acid sequence of SEQ ID NO: 1 with an alanine residue; (h) substitution of the amino acid residue at position 43 in the amino acid sequence of SEQ ID NO: 1 with an alanine residue; (i) substitution of the amino acid residue at position 49 in the amino acid sequence of SEQ ID NO: 1 with an arginine residue.

[0074] Methods for substituting amino acid residues include known methods such as site-specific mutation of a polynucleotide encoding a domain. Here, "85% or more homology" with respect to amino acid sequence preferably means 90% or more homology, more preferably 95% or more homology, even more preferably 97% or more homology, even more preferably 98% or more homology, and particularly preferably 99% or more homology.

[0075] As used herein, a "corresponding position" in an amino acid sequence can be determined by aligning a target sequence with a reference sequence (e.g., the amino acid sequence of SEQ ID NO: 1) to maximize homology between conserved amino acid residues present in each amino acid sequence. Alignment can be performed using known algorithms, and the procedures are well known to those skilled in the art. For example, alignment can be performed using the Clustal W multiple alignment program (Thompson, JD et al., 1994, Nucleic Acids Res., 22:4673-4680) with default settings. Clustal W is available, for example, on the websites of the European Bioinformatics Institute (EBI) [www.ebi.ac.uk / index.html] and the DNA Data Bank of Japan (DDBJ) [www.ddbj.nig.ac.jp / index.html], operated by the National Institute of Genetics.

[0076] As used herein, amino acid residues are also abbreviated as follows: alanine (Ala or A), arginine (Arg or R), asparagine (Asn or N), aspartic acid (Asp or D), cysteine ​​(Cys or C), glutamine (Gln or Q), glutamic acid (Glu or E), glycine (Gly or G), histidine (His or H), isoleucine (Ile or I), leucine (Leu or L), lysine (Lys or K), methionine (Met or M), phenylalanine (Phe or F), proline (Pro or P), serine (Ser or S), threonine (Thr or T), tryptophan (Trp or W), tyrosine (Tyr or Y), valine (Val or V), and any amino acid residue (Xaa or X). In this specification, the amino acid sequence of a peptide is written in accordance with the conventional method, with the amino terminus (hereinafter referred to as the N-terminus) on the left and the carboxyl terminus (hereinafter referred to as the C-terminus) on the right.

[0077] As used herein, the terms "before" and "after" a specific position in an amino acid sequence refer to the positions adjacent to the N-terminus and C-terminus of the specific position, respectively. For example, when an amino acid residue is inserted "before" or "after" a specific position, the inserted amino acid residue will be located at the positions adjacent to the N-terminus and C-terminus of the specific position.

[0078] In a preferred embodiment, the proteinaceous ligand is produced by making one or more substitutions selected from the above (a) to (i) to a proteinaceous ligand (parent domain) having an amino acid sequence that is 85% or more homologous to the amino acid sequence shown in SEQ ID NO: 1.

[0079] A method for substituting a proteinaceous ligand (parent domain) having an amino acid sequence with 85% or more homology to the amino acid sequence shown in SEQ ID NO: 1 includes a method of introducing a mutation into a polynucleotide encoding the parent domain so that a desired amino acid residue is substituted. Specific techniques for introducing a mutation into a polynucleotide include site-specific mutagenesis, homologous recombination, and SOE (splicing by overlap extension)-PCR (Gene, 1989, 77:61-68), and detailed procedures for these are well known to those skilled in the art. The produced ligand has immunoglobulin-binding activity and functions as an immunoglobulin-binding domain.

[0080] A preferred example of a proteinaceous ligand is an amino acid sequence having 85% or more homology to the amino acid sequence shown in SEQ ID NO: 1 (C domain of protein A), and in which two or more amino acid sequences are linearly linked, with at least one or more substitutions selected from the following (a) to (i): (a) a) a) b) c) d ... Specifically, examples include modified protein A that is a trimer to pentamer of amino acid sequence domains set forth in SEQ ID NOS: 2 to 4 and that have 85% or more homology thereto (but have 85% or more homology with the amino acid sequence shown in SEQ ID NO: 1). However, to prevent leakage of the proteinaceous ligand during isolation, modified protein A that is a trimer to pentamer of amino acid sequence domains set forth in SEQ ID NOS: 2 to 3 and that have 85% or more homology thereto (but have 85% or more homology with the amino acid sequence shown in SEQ ID NO: 1) is preferred.

[0081] To increase the dynamic binding capacity, the binding amount of the proteinaceous ligand is preferably 10 mg to 300 mg, more preferably 25 mg to 150 mg, per gram of dry weight of the solid phase carrier.

[0082] The binding of the proteinaceous ligand to the solid-phase support in the ligand binding step may be carried out in the same manner as in the conventional method. Chemical binding methods are preferred as ligand binding methods. For example, a method of binding the ligand to a functional group capable of binding the ligand may be used. 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)-O-C(=O)-, formyl group, etc. of the solid-phase support to an amino group, etc. of the ligand. The ligand binding reaction is preferably carried out in a buffer with a pH of 7 to 14 to increase the reaction efficiency. The reaction time for the ligand binding 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.

[0083] Alternatively, the ligand may be bound 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 binding the ligand to the solid phase support 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 solid phase support using an associative group (JP-A No. 2011-256176).

[0084] In order to enhance the antifouling properties and to prevent the protein ligand from leaking during isolation, the method for producing the chromatography carrier of the present invention preferably further comprises a ligand-binding carrier hydrophilization step between the ligand binding step and the ligand-binding carrier bed formation step, and the hydrophilic group-containing ligand-binding carrier obtained in the ligand-binding carrier hydrophilization step is used in the ligand-binding carrier bed formation step as the ligand-binding carrier after the ligand binding step. (Ligand-binding carrier hydrophilization step) A step of reacting the carrier to which the ligand has been bound in the ligand binding step 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.

[0085] The compound having a total of two or more hydrophilic groups in the molecule used in the ligand-binding support hydrophilization step is 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, in order to enhance antifouling properties and reduce leakage of proteinaceous ligands during isolation. 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 a total of two or more hydrophilic groups in the molecule can be used alone or in combination of two or more. Among these, alcohols having a mercapto group in the molecule are preferred in order to enhance antifouling properties and reduce leakage of proteinaceous ligands during isolation, and thioglycerol is particularly preferred.

[0086] The total amount of compounds having two or more hydrophilic groups in the molecule used in the ligand-binding carrier hydrophilization step 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 ligand-binding carrier (dry solid content).

[0087] The ligand-bound support hydrophilization step 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.

[0088] The reaction time for the ligand-binding carrier hydrophilization step 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.

[0089] (Ligand-binding carrier bed formation step, ligand-binding carrier liquid passing washing step, ligand-binding carrier stirring washing step) The ligand-binding carrier bed formation step is a step in which the ligand-binding carrier obtained in the ligand binding step is filled into a container to form a ligand-binding carrier bed. The ligand-binding carrier liquid passing washing step is a step in which the ligand-binding carrier bed formed in the ligand-binding carrier bed formation step is washed with a washing liquid (hereinafter also referred to as "ligand-binding carrier washing liquid") at least once. The ligand-binding carrier stirring washing step is a step in which the ligand-binding carrier after the ligand-binding carrier liquid passing washing step is stirred and washed at least once in the ligand-binding carrier washing liquid. In the method for producing a chromatography carrier of the present invention, by combining the ligand-binding carrier liquid passing washing step and the ligand-binding carrier stirring washing step in this order, not only is the dynamic binding capacity of the chromatography carrier for antibodies or fragments thereof increased, but aggregation between carriers is less likely to occur, and proteinaceous ligands are less likely to leak during isolation. Furthermore, the substitution efficiency is also improved.

[0090] In the present invention, the term "ligand-bonded carrier bed" refers to a solid layer of the ligand-bonded carrier, and may be dry or wet. Specific methods for forming the ligand-bonded carrier bed include pouring a slurry of the ligand-bonded carrier (e.g., one using water, alcohol (ethanol, isopropanol, etc.), a mixture of water and alcohol, or a buffer solution (carbonate buffer, etc.) as a dispersion medium) into a container, allowing the ligand-bonded carrier solids to settle to the bottom of the container by gravitational sedimentation or centrifugal sedimentation, and then removing the liquid phase of the slurry by a solid-liquid separation procedure (e.g., decantation, centrifugation, filtration, etc.). Furthermore, a solid-liquid separator is preferred as the container, since it allows the washing solution to be passed directly through the ligand-bonded carrier bed formed in the ligand-bonded carrier bed formation step in the ligand-bonded carrier liquid-passage washing step, which is convenient. Specific examples include a filter-equipped column container, a filter-equipped funnel, a filter-equipped centrifuge, and a filter reactor.

[0091] The content of the ligand-binding carrier in the slurry is preferably 5% by volume or more, more preferably 15% by volume or more, particularly preferably 30% by volume or more, and preferably 80% by volume or less, more preferably 75% by volume or less, particularly preferably 70% by volume or less. Specific ranges are preferably 5% by volume or more and 80% by volume or less, more preferably 15% by volume or more and 75% by volume or less, and particularly preferably 30% by volume or more and 70% by volume or less. The volume percentage of the carrier in the slurry can be calculated, for example, by filling a 250 mL glass graduated cylinder (manufactured by Corning Incorporated) with 200 mL of the slurry, leaving it to stand for 3 hours, and dividing the sedimentation volume by the volume of the slurry filled in the graduated cylinder.

[0092] The ligand-binding carrier washing solution used in the ligand-binding carrier flow washing step and the ligand-binding carrier agitation washing step is preferably an aqueous washing solution. "Aqueous washing solution" means a washing solution containing at least water. Examples of aqueous washing solutions include those containing water or a mixture of water and a lower alcohol. Examples of lower alcohols include one or more selected from ethanol and isopropanol.

[0093] The pH of the ligand-binding carrier washing solution used in the ligand-binding carrier flow washing step and the ligand-binding carrier stirring washing step is preferably greater than 3, more preferably greater than 6, even more preferably greater than 7, even more preferably greater than 8.5, even more preferably greater than 9.5, and particularly preferably greater than 10.5, in order to increase the hydrophilicity of the solid phase carrier surface, to make it less likely for the protein ligand to leak during isolation, and to increase the dynamic binding capacity. Also, in order to increase the dynamic binding capacity, the pH is preferably 12.5 or less, more preferably 12 or less, and particularly preferably 11.8 or less. Specific ranges include a pH greater than 3 and less than 12.5, more preferably a pH of 6 to 12.5, even more preferably a pH of 7 to 12.5, even more preferably a pH of 8.5 to 12, even more preferably a pH of 9.5 to 12, even more preferably a pH of 10.5 to 12, and particularly preferably a pH of 10.5 to 11.8.

[0094] According to the method for producing a chromatography carrier of the present invention, a chromatography carrier can be obtained in which proteinaceous ligands are less likely to leak during isolation, even when the pH of the washing solution used in the ligand-binding carrier flow-through washing step or the ligand-binding carrier stirring-washing step is in the mild pH range as described above. Furthermore, when the pH of the washing solution used in the ligand-binding carrier flow-through washing step or the ligand-binding carrier stirring-washing step is in the mild pH range as described above, the dynamic binding capacity is increased, thereby achieving both excellent dynamic binding capacity and excellent low leakage. When the pH of the ligand-binding carrier washing solution is 8.5 or higher, 9.5 or higher, or 10.5 or higher, the dynamic binding capacity and low proteinaceous ligand leakage are particularly excellent. Furthermore, when the pH of the ligand-binding carrier washing solution is 12 or lower or 11.8 or lower, the dynamic binding capacity is particularly excellent.

[0095] The ligand-binding carrier washing solution used in the ligand-binding carrier flow-through washing step and the ligand-binding carrier stirring and washing step preferably contains a pH adjuster to achieve the above pH range. pH adjusters can be broadly classified into acidic pH adjusters and basic pH adjusters. Specific examples include strongly acidic inorganic acid pH adjusters such as sulfuric acid, hydrochloric acid, and nitric acid; weakly acidic inorganic acid pH adjusters such as sodium dihydrogen phosphate, carbonic acid, phosphoric acid, hydrogen fluoride, and hydrogen sulfide; weakly acidic organic acid pH adjusters such as acetic acid and oxalic acid; strongly basic inorganic base pH adjusters such as alkali metal hydroxides, sodium carbonate, and trisodium phosphate; strongly basic organic base pH adjusters such as triethylamine; weakly basic inorganic base pH adjusters such as sodium bicarbonate, disodium hydrogen phosphate, ammonia, copper hydroxide, magnesium hydroxide, zinc hydroxide, iron hydroxide, and aluminum hydroxide; and weakly basic organic base pH adjusters such as diethanolamine. Examples of alkali metal hydroxides include lithium hydroxide, potassium hydroxide, and sodium hydroxide.

[0096] Furthermore, in order to reduce leakage of the proteinaceous ligand during isolation, the concentration of the pH adjuster in the ligand-binding carrier washing solution is preferably 0.001 M to 10 M, more preferably 0.005 M to 2.5 M, even more preferably 0.01 M to 0.5 M, and particularly preferably 0.05 M to 0.3 M. When the concentration of the pH adjuster is 0.05 M or higher, the solution is particularly excellent in terms of low proteinaceous ligand leakage. Furthermore, when the concentration of the pH adjuster is 0.3 M or lower, the solution is particularly excellent in terms of dynamic binding capacity. The ligand-binding carrier washing solution may contain a buffer or two or more pH adjusters to adjust the pH to a desired level or to maintain the target pH.

[0097] The number of washes using the ligand-binding carrier washing solution in the ligand-binding carrier flow washing step and the ligand-binding carrier stirring washing step may be two or more in total. However, in order to increase the hydrophilicity of the solid phase carrier surface, to make it less likely for the protein ligand to leak during isolation, to further improve the substitution efficiency, and to suppress aggregation between carriers, the number of washes is preferably 2 to 20 in total, more preferably 2 to 15 in total, even more preferably 2 to 10 in total, even more preferably 2 to 8 in total, and particularly preferably 3 to 4 in total. The types of ligand-binding carrier washing solutions used in each wash may be the same or different, but the same type is preferred. Furthermore, the types of washing techniques used in each wash may be the same or different. When the number of washes using the ligand-binding carrier washing solution is three or more in total, the substitution efficiency is improved, and the resulting chromatography carrier exhibits particularly excellent low protein ligand leakage properties. Furthermore, when the number of washes using the ligand-binding carrier washing solution is four or less in total, the carrier exhibits particularly excellent low aggregation properties.

[0098] The amount of the ligand-binding carrier washing solution used per washing in the ligand-binding carrier liquid passing washing step and the ligand-binding carrier stirring washing step is preferably 40 parts by volume or more, more preferably 60 parts by volume or more, and particularly preferably 80 parts by volume or more, relative to 100 parts by volume of the dry solid content of the ligand-binding carrier, and is preferably 1000 parts by volume or less, more preferably 500 parts by volume or less, and particularly preferably 300 parts by volume or less, relative to 100 parts by volume of the dry solid content of the ligand-binding carrier. Specific ranges are preferably 40 parts by volume or more and 1000 parts by volume or less, more preferably 60 parts by volume or more and 500 parts by volume or less, and particularly preferably 80 parts by volume or more and 300 parts by volume or less, relative to 100 parts by volume of the dry solid content of the ligand-binding carrier.

[0099] The washing temperature in the ligand-bound carrier solution-passing washing step and the ligand-bound carrier agitation washing step is not particularly limited, but is usually in the range of 10 to 50° C., preferably in the range of 15 to 45° C. Furthermore, either batch washing or continuous washing may be used.

[0100] Here, the ligand-binding carrier liquid-flow washing step will be described in more detail. In the present invention, "liquid-flow washing" of a ligand-binding carrier refers to passing a wash solution through the ligand-binding carrier bed. For example, washing is performed by passing the wash solution through the ligand-binding carrier bed from one direction to the other. Specifically, washing can be performed using a solid-liquid separator such as a filter-equipped column container, a filter plate, a filter-equipped funnel, a Buchner funnel, a Nutsch filter, a filter-equipped centrifuge, or a filter reactor. The wash solution flowed into the ligand-binding carrier bed may be incubated for preferably 30 seconds to 3 hours, more preferably 2 minutes to 60 minutes, and then drained. The number of washes in the ligand-binding carrier liquid-flow washing step is preferably one or more, more preferably 1 to 20 times, even more preferably 1 to 10 times, even more preferably 1 to 5 times, even more preferably 2 to 5 times, and particularly preferably 2 to 3 times, in order to increase the hydrophilicity of the solid phase carrier surface, to reduce leakage of proteinaceous ligands during isolation, to further improve substitution efficiency, and to suppress aggregation between carriers. When the number of times of washing by passing the liquid is two or more, the replacement efficiency is improved and the resulting chromatography carrier has particularly excellent low protein ligand leakage properties, whereas when the number of times of washing by passing the liquid is three or less, the carrier has particularly excellent low aggregation properties.

[0101] The total number of washes in the solid phase carrier washing step and the ligand-binding carrier washing step is preferably 2 or more, more preferably 2 to 30, even more preferably 2 to 16, even more preferably 2 to 8, even more preferably 3 to 6, and particularly preferably 4 to 5, in order to increase the hydrophilicity of the solid phase carrier surface, to make it less likely for the proteinaceous ligand to leak during isolation, to further improve the substitution efficiency, and to suppress aggregation between carriers. When the total number of washes in the solid phase carrier washing step and the ligand-binding carrier washing step is 4 or more, the substitution efficiency is improved, and the resulting chromatography carrier has particularly excellent low proteinaceous ligand leakage properties. Furthermore, when the total number of washes in the solid phase carrier washing step and the ligand-binding carrier washing step is 5 or less, the chromatography carrier has particularly excellent low aggregation properties.

[0102] The type of ligand-binding carrier washing solution and the amount of the ligand-binding carrier washing solution used per wash are as described above. The liquid-flow washing may be a liquid-flow washing using gravity, a liquid-flow washing under pressure, or a liquid-flow washing under reduced pressure.

[0103] Here, the ligand-bound carrier agitation and washing step will be described in more detail. The number of agitation washes in the ligand-bound carrier agitation and washing step is preferably at least once, more preferably 1 to 20 times, even more preferably 1 to 10 times, even more preferably 1 to 5 times, even more preferably 1 to 2 times, and particularly preferably 1 time, in order to increase the hydrophilicity of the solid phase carrier surface, to make it difficult for the proteinaceous ligand to leak during isolation, and to suppress aggregation between carriers. When the number of agitation and washings is 1 or more, particularly low aggregation properties are achieved. Furthermore, when the number of agitation and washings is 2 or less, particularly low leakage of the proteinaceous ligand is achieved.

[0104] The total number of times of stirring and washing in the solid-phase carrier stirring and washing step and the ligand-bound carrier stirring and washing step is preferably 2 or more, more preferably 2 to 30 times, even more preferably 2 to 16 times, even more preferably 2 to 8 times, still more preferably 2 to 6 times, and particularly preferably 2 to 3 times, in order to increase the hydrophilicity of the solid-phase carrier surface, to make it less likely that the proteinaceous ligand will leak during isolation, to further improve the substitution efficiency, and to suppress aggregation of the carriers themselves.

[0105] The stirring speed in the ligand-binding carrier stirring and washing step is preferably 10 to 150 rpm, more preferably 20 to 100 rpm, and particularly preferably 30 to 80 rpm. The stirring time per ligand-binding carrier stirring and washing step is preferably 30 seconds to 300 minutes, more preferably 1 minute to 180 minutes, even more preferably 2 minutes to 60 minutes, and particularly preferably 5 minutes to 30 minutes. The type of ligand-binding carrier washing solution and the amount of ligand-binding carrier washing solution used per step are as described above. When the ligand-binding carrier liquid-passing washing step is performed using a solid-liquid separator, continuous stirring and washing can be performed by carrying out the ligand-binding carrier stirring and washing step while leaving the filtrate outlet of the solid-liquid separator used in the ligand-binding carrier liquid-passing washing step open. Alternatively, batchwise stirring and washing can be performed by using a closed container in the ligand-binding carrier stirring and washing step.

[0106] 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.

[0107] Furthermore, the method for producing a chromatography carrier of the present invention makes it possible to produce a chromatography carrier that has a large dynamic binding capacity for antibodies or fragments thereof, is less likely to leak proteinaceous ligands during isolation, and is less likely to aggregate between carriers. Furthermore, the method has good substitution efficiency and is easy to produce. Furthermore, washing solutions with various pH values ​​can be used in the ligand-binding carrier flow-through washing step and the ligand-binding carrier stirring washing step. For example, even when a washing solution with a pH greater than 3 and equal to or less than 12.5 is used, a chromatography carrier that is less likely to leak proteinaceous ligands during isolation can be obtained.

[0108] As used herein, the term "antibody" encompasses any class of immunoglobulin, such as IgG, IgA, IgD, IgE, IgM, and their subclasses, as well as variants thereof. Furthermore, as used herein, the term "antibody" may also refer to chimeric antibodies such as humanized antibodies, antibody complexes, and other modified immunoglobulins containing 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 variants and modifications thereof.

[0109] The volume-average particle size of the chromatography carrier obtained as described above is preferably 40 to 150 μm, more preferably 50 to 100 μm. The coefficient of variation of the volume-average particle size is preferably 40% or less, more preferably 30% or less. The specific surface area of ​​the chromatography carrier is preferably 1 to 500 m. 2 / g, more preferably 10 to 300m 2 / g. The volume average pore diameter of the chromatography carrier is preferably 10 to 300 nm. The volume average particle diameter, coefficient of variation, specific surface area, and volume average pore diameter can be measured by laser diffraction / scattering particle size distribution measurement or the like.

[0110] Furthermore, the chromatography carrier obtained as described above is useful for separating an antibody or a fragment thereof from a sample containing the antibody or a fragment thereof. Examples of the sample 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.

[0111] The present invention will be described in detail below with reference to examples, but the present invention is not limited to these examples.

[0112] (Preparation Example: Preparation of Ligand (Immunoglobulin-Binding Protein)) Immunoglobulin-binding proteins PrA-0 to PrA-3 were obtained. PrA-0 is an immunoglobulin-binding protein containing a homopentamer in which the C domains of Protein A (SEQ ID NO: 1) are linked in tandem. PrA-1 to PrA-3 are mutants in which the mutations listed in Table 1 have been introduced into each immunoglobulin-binding domain of PrA-0.

[0113]

[0114] Expression and purification of PrA-0 to PrA-3 were carried out as follows. Escherichia coli BL21(DE3) was transformed with the plasmids encoding PrA-0 to PrA-3, and the resulting transformants were cultured in a rich medium at 37°C until the logarithmic growth phase. Subsequently, isopropyl-β-thiogalactopyranoside (manufactured by Wako Pure Chemical Industries, Ltd.) was added to the medium to a final concentration of 1 mM, and the transformants were further cultured at 37°C for 4 hours to express the target proteins. The culture medium was then centrifuged to remove the supernatant, and the resulting cells were disrupted by adding 30 mM Tris buffer (pH 9.5) containing egg white-derived lysozyme (manufactured by Wako Pure Chemical Industries, Ltd.) and polyoxyethylene (10) octylphenyl ether (manufactured by Wako Pure Chemical Industries, Ltd.). The recombinant immunoglobulin-binding protein was purified from the resulting cell lysate by cation exchange chromatography (SP-Sepharose FF, GE Healthcare Biosciences) and anion exchange chromatography (Q-Sepharose FF, GE Healthcare Biosciences). The purified immunoglobulin-binding protein was dialyzed against 10 mM citrate buffer, pH 6.0. The purity of the recombinant immunoglobulin-binding protein confirmed by SDS-PAGE was 95% or higher.

[0115] Example 1 (Step 1: Polymerization of Porous Particles) 2.69 g of polyvinyl alcohol (PVA-217 manufactured by Kuraray Co., Ltd.) was added to 448 g of pure water, and the mixture was heated and stirred to dissolve the polyvinyl alcohol, yielding an aqueous solution. 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., 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, and placed in a hot water bath. Stirring was commenced 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 (manufactured by Tokyo Chemical Industry Co., Ltd.), 8 g of thioglycerol (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. 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 to obtain 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 hydroxy 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).To this carbonate buffer, 0.5 g of ethylene glycol diglycidyl ether (Denacol EX810, manufactured by Nagase ChemteX Corporation) and 8 mL of porous particles 2 were added, and the mixture was shaken and stirred at 23°C for 16 hours. The reaction solution was then filtered and washed with pure water, after which the particles were dispersed in pure water to a particle concentration of 50% by volume, yielding a porous particle dispersion. The porous particles contained in this dispersion are referred to as "porous particles 3."

[0116] (Step 2: Porous Particle Washing Step) Next, porous particles 3 were washed. That is, a funnel (Kiriyama Funnel manufactured by Kiriyama Seisakusho) equipped with a filter (Kiriyama Funnel filter paper manufactured by Kiriyama Seisakusho) was prepared, and 16 mL of the dispersion of porous particles 3 was poured into the funnel under reduced pressure. In this way, pure water was filtered from the dispersion by suction filtration, and the solid content of the dispersion was allowed to settle to the bottom of the funnel (filter bed) to form a bed. Thereafter, "cut-out washing (hereinafter also simply referred to as "S" or "washing S")" was performed twice, in which 8 mL of a 0.5 M aqueous sodium hydroxide solution (pH 13.7) was poured in while the vacuum suction was being continued, and the liquid was allowed to flow from the top to the bottom of the bed, and filtered as it was without stirring. Next, with the tip of the funnel closed, 8 mL of a 0.5 M aqueous sodium hydroxide solution (pH 13.7) was added, and the entire slurry in the funnel was stirred (10 minutes, 60 rpm) and then suction filtered. This "reslurry washing" (hereinafter also referred to simply as "R" or "washing R") was carried out once. Next, the particles were dispersed in pure water to a particle concentration of 50% by volume, yielding a porous particle dispersion. The porous particles contained in this dispersion are referred to as "porous particles 4."

[0117] (Step 3: Ligand Binding Step) Next, a ligand was bound to porous particles 4. That is, 28.8 g of pure water, 5.4 g of sodium sulfate (manufactured by Wako Pure Chemical Industries, Ltd.), 0.2 g of sodium bicarbonate (manufactured by Wako Pure Chemical Industries, Ltd.), and 0.16 g of sodium carbonate (manufactured by Wako Pure Chemical Industries, Ltd.) were mixed to obtain a carbonate buffer (pH 9.3). 0.17 g of the immunoglobulin-binding protein PrA-1 (modified protein A which is a pentamer of the amino acid sequence domain of SEQ ID NO: 2) prepared in Preparation Example and 8 mL of porous particles 4 were added to 25 mL of this carbonate buffer, and the mixture was shaken and stirred at 23°C for 1.5 hours, and the reaction solution was filtered. Next, 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.) were mixed to obtain a buffer, and 4.5 g of thioglycerol (manufactured by Tokyo Chemical Industry Co., Ltd.) was added to prepare a hydrophilization reaction solution. This solution was then added to the ligand-bound porous particles, and the mixture was shaken and stirred at 23°C for 16 hours to carry out a hydrophilization reaction. Next, the particles were dispersed in pure water to a particle concentration of 50% by volume, to obtain a porous particle dispersion. The porous particles contained in this dispersion are referred to as "porous particles 5."

[0118] (Step 4: Washing Step After Bonding Step) Next, the porous particles 5 were washed. That is, a funnel (Kiriyama Funnel manufactured by Kiriyama Manufacturing Co., Ltd.) equipped with a filter (Kiriyama Funnel filter paper manufactured by Kiriyama Manufacturing Co., Ltd.) was prepared, and 16 mL of the dispersion of porous particles 5 was poured into the funnel under reduced pressure. In this way, pure water was filtered from the dispersion by suction filtration, and the solid content of the dispersion was allowed to settle to the bottom of the funnel (filter bed) to form a bed. Thereafter, "Washing S" was performed twice by pouring 8 mL of a 0.1 M aqueous sodium carbonate solution (pH 11.4) and allowing the solution to pass through while the vacuum suction was maintained. Next, with the tip of the funnel closed, 8 mL of a 0.1 M aqueous sodium carbonate solution (pH 11.4) was poured in, and the entire slurry in the funnel was stirred (10 minutes, 60 rpm) and then suction filtered, followed by one "Washing R" process. The mixture was then neutralized with a sodium citrate buffer and dispersed in pure water to a particle concentration of 50% by volume to obtain a porous particle dispersion, which is referred to as "carrier 1."

[0119] [Example 2] Carrier 2 was obtained by the same operation as in Example 1, except that the 0.5 M aqueous sodium hydroxide solution used in "Step 2: Porous particle washing step" was changed to 0.1 M hydrochloric acid. [Example 3] Carrier 3 was obtained by the same operation as in Example 1, except that the 0.5 M aqueous sodium hydroxide solution used in "Step 2: Porous particle washing step" was changed to 1.0 M hydrochloric acid. [Example 4] Carrier 4 was obtained by the same operation as in Example 1, except that the 0.5 M aqueous sodium hydroxide solution used in "Step 2: Porous particle washing step" was changed to 0.01 M hydrochloric acid.

[0120] [Example 5] Carrier 5 was obtained by the same operation as in Example 1, except that the 0.5 M aqueous sodium hydroxide solution used in "Step 2: Porous particle washing step" was changed to a 0.1 M aqueous hydrogen peroxide solution. [Example 6] Carrier 6 was obtained by the same operation as in Example 1, except that the 0.5 M aqueous sodium hydroxide solution used in "Step 2: Porous particle washing step" was changed to a 0.1 M aqueous peracetic acid solution. [Example 7] Carrier 7 was obtained by the same operation as in Example 1, except that the 0.5 M aqueous sodium hydroxide solution used in "Step 2: Porous particle washing step" was changed to a 1.0 M aqueous sodium hydroxide solution.

[0121] [Example 8] Carrier 8 was obtained by the same operation as in Example 1, except that the 0.5 M aqueous sodium hydroxide solution used in "Step 2: Porous particle washing step" was changed to a 0.1 M aqueous sodium hydroxide solution. [Example 9] Carrier 9 was obtained by the same operation as in Example 1, except that the number of times of "Washing S" performed in "Step 2: Porous particle washing step" was changed from two to four. [Example 10] Carrier 10 was obtained by the same operation as in Example 1, except that the number of times of "Washing S" performed in "Step 2: Porous particle washing step" was changed from two to one.

[0122] [Example 11] The same operation as in Example 1 was carried out, except that the 0.1 M aqueous sodium carbonate solution (pH 11.4) used in "Step 4: Washing step after binding step" was changed to a 0.1 M aqueous sodium phosphate solution (prepared by mixing a 0.1 M aqueous sodium dihydrogen phosphate solution and a 0.1 M aqueous sodium disodium hydrogen phosphate solution to a pH of 7.5), to obtain carrier 11. [Example 12] The same operation as in Example 1 was carried out, except that the 0.1 M aqueous sodium carbonate solution (pH 11.4) used in "Step 4: Washing step after binding step" was changed to a 0.5 M aqueous sodium phosphate solution (prepared by mixing a 0.5 M aqueous sodium dihydrogen phosphate solution and a 0.5 M aqueous sodium disodium hydrogen phosphate solution to a pH of 7.5), to obtain carrier 12. Example 13 Carrier 13 was obtained by the same procedure as in Example 1, except that the 0.1 M aqueous sodium carbonate solution (pH 11.4) used in "Step 4: Washing step after binding step" was changed to a 0.01 M aqueous sodium phosphate solution (prepared by mixing a 0.01 M aqueous sodium dihydrogen phosphate solution with a 0.01 M aqueous sodium dihydrogen phosphate solution to a pH of 7.5).

[0123] [Example 14] Carrier 14 was obtained by the same procedure as in Example 1, except that the 0.1 M sodium carbonate aqueous solution (pH 11.4) used in "Step 4: Washing step after binding step" was changed to 0.1 M sodium carbonate aqueous solution (prepared by adding sodium hydroxide aqueous solution to pH 12.5). [Example 15] Carrier 15 was obtained by the same procedure as in Example 1, except that the 0.1 M sodium carbonate aqueous solution (pH 11.4) used in "Step 4: Washing step after binding step" was changed to 0.1 M sodium carbonate aqueous solution (prepared by mixing 0.1 M sodium bicarbonate aqueous solution and 0.1 M sodium carbonate aqueous solution to pH 10). [Example 16] Carrier 16 was obtained by the same procedure as in Example 1, except that the "Washing S" performed in "Step 4: Washing step after binding step" was changed from two to four times.

[0124] [Example 17] Carrier 17 was obtained by the same procedure as in Example 1, except that "Washing S" performed in "Step 4: Washing step after binding step" was changed from two times to one time. [Example 18] Carrier 18 was obtained by the same procedure as in Example 1, except that the porous particles 3 obtained in "Step 1: Porous particle polymerization step" were changed to agarose-based particles (WorkBeads 40 ACT (manufactured by Bio-works)). [Example 19] Carrier 19 was obtained by the same procedure as in Example 1, except that the immunoglobulin-binding protein PrA-1 used in "Step 3: Ligand binding step" was changed to immunoglobulin-binding protein PrA-0.

[0125] [Example 20] A carrier 20 was obtained by the same procedure as in Example 1, except that the immunoglobulin-binding protein PrA-1 used in "Step 3: Ligand binding step" was changed to immunoglobulin-binding protein PrA-3. [Example 21] A carrier 21 was obtained by the same procedure as in Example 1, except that the immunoglobulin-binding protein PrA-1 used in "Step 3: Ligand binding step" was changed to immunoglobulin-binding protein PrA-2.

[0126] Example 22: Carrier 22 was obtained by the same procedure as in Example 1, except that the immunoglobulin-binding protein PrA-1 used in "Step 3: Ligand binding step" was changed to Pierce (registered trademark) Recombinant Protein L (21189, manufactured by Thermo Fisher Scientific). Example 23: Carrier 23 was obtained by the same procedure as in Example 1, except that the immunoglobulin-binding protein PrA-1 used in "Step 3: Ligand binding step" was changed to Pierce (registered trademark) Recombinant Protein G (21193, manufactured by Thermo Fisher Scientific).

[0127] Comparative Example 1: The same operation as in Example 1 was carried out, except that the number of "Washing S" performed in "Step 4, Washing Step After Bonding Step" was changed from two to three times, and the number of "Washing R" was changed from one to zero, to obtain a carrier of Comparative Example 1. Comparative Example 2: The same operation as in Example 1 was carried out, except that the number of "Washing S" performed in "Step 4, Washing Step After Bonding Step" was changed from two to zero, and the number of "Washing R" was changed from one to three, to obtain a carrier of Comparative Example 2. Comparative Example 3: The same operation as in Example 1 was carried out, except that the "Step 2, Washing Step of Porous Particles" was not carried out, and the 0.1 M aqueous sodium carbonate solution (pH 11.4) used in "Step 4, Washing Step After Bonding Step" was changed to a 0.5 M aqueous sodium hydroxide solution (pH 13.7), and the number of "Washing S" performed in "Step 4, Washing Step After Bonding Step" was changed from two to six times, and the number of "Washing R" was changed from one to zero, to obtain a carrier of Comparative Example 3. Comparative Example 4 The same operations as in Example 1 were carried out to obtain a carrier of Comparative Example 4, except that "Step 2: Porous particle washing step" was not carried out, the 0.1 M sodium carbonate aqueous solution (pH 11.4) used in "Step 4: Washing step after bonding step" was changed to a 0.5 M sodium hydroxide aqueous solution (pH 13.7), the number of "Washing S" performed in "Step 4: Washing step after bonding step" was changed from two to zero, and the number of "Washing R" performed in "Step 4: Washing step after bonding step" was changed from one to three.

[0128] Test Example 1: Dynamic Binding Capacity (DBC) Measurement Test Using an AKTA avant25 manufactured by Cytiva, the DBC of each carrier in Examples 1 to 21 and the Comparative Example for a protein (human IgG antibody, 1875-0007 manufactured by LGC) at a retention time of 4 minutes was measured. A column container with a capacity of 4 mL (5 mm diameter x 200 mm length) was used, and the protein was dissolved in a 20 mM sodium phosphate / 150 mM sodium chloride aqueous solution (pH 7.5) at 5 mg / mL. The DBC was calculated from the amount of protein captured at 10% breakthrough at the elution tip and the column packing volume, and evaluated according to the following criteria. The results are shown in Tables 2 to 4 and 6.

[0129] (DBC evaluation criteria) AA (Excellent): 62 mg / mL or more A (Good): 61 mg / mL or more and less than 62 mg / mL B (Poor): Less than 61 mg / mL

[0130] Test Example 2: Protein Leakage Measurement Test Using an AKTA avant 25 manufactured by Cytiva, 7.5 mL of cell culture medium (Herceptin, titer: 4.38 mg / mL) was loaded onto each carrier of the Examples and Comparative Examples with a retention time of 4 minutes, and the antibody was then recovered with the eluate. The column container had a capacity of 0.8 mL (5 mm diameter x 40 mm length), and the eluate was 100 mM aqueous sodium acetate solution (pH 3.3). Prior to elution, the column was washed with 20 mM sodium phosphate / 500 mM aqueous sodium chloride solution (pH 7.5). Next, the amount of protein in the eluate was determined using a Protein L-Ligand Leakage ELISA Kit (manufactured by Genaxxon Bioscience) for the carrier of Example 22, a Protein G ELISA Kit (manufactured by Alpha Diagnostic International) for the carrier of Example 23, and a Protein A ELISA kit (F740) for the other carriers, and the antibody concentration in the eluate was determined from the absorbance. From these values, the amount of protein leakage (leach) per amount of antibody in the recovered eluate was calculated and evaluated according to the following criteria. The results are shown in Tables 2 to 6.

[0131] (Evaluation criteria for protein leakage) AAA (Excellent): 8 ppm / IgG or less AA (Excellent): More than 8 ppm / IgG and less than 10 ppm / IgG A (Good): 10 ppm / IgG or more and less than 13 ppm / IgG B (Poor): 13 ppm / IgG or more

[0132] (Test Example 3) Evaluation of the amount of aggregation 16 mL of the dispersion of each carrier in the Examples and Comparative Examples was passed through a wire mesh (replaceable mesh funnel, 30 mesh, manufactured by Taiyo Co., Ltd.), and the particles remaining on the mesh were collected using pure water. The collected particle dispersion was transferred to an aluminum dish and heated on a hot plate at 200°C for 10 minutes to obtain dried particles. The weight of these dried particles was measured and used as the amount of aggregation (g). The smaller the value of the amount of aggregation obtained, the less aggregation there was. The results are shown in Tables 2 to 6.

[0133] (Test Example 4) Replacement efficiency of cleaning solution The electrical conductivity of the cleaning solution used first in "Step 4: Cleaning step after binding step" was measured using a HORIBA electrical conductivity meter. Next, the final filtrate immediately before washing with sodium citrate buffer in "Step 4: Cleaning step after binding step" was collected, and the electrical conductivity was measured in the same manner as above. The replacement efficiency (%) of the cleaning solution was calculated using the following formula. The closer this value is to 100%, the better the replacement efficiency. The results are shown in Tables 2 to 6. Replacement efficiency (%) of cleaning solution = (electrical conductivity of filtrate / electrical conductivity of cleaning solution) x 100

[0134] Test Example 5: Evaluation of relative hydrophilicity 30 parts by mass of water was added to 10 parts by mass of dried porous particles 4 (particles before ligand binding) obtained in steps 1 and 2 of each Example and Comparative Example, and the state of dispersion was visually confirmed. Particles that were not agglomerated and were well dispersed were rated "A," particles that were dispersed but small agglomerates were visible were rated "B," and particles that still had larger agglomerates remaining were rated "C." The results are shown in Tables 2 to 6.

[0135]

[0136]

[0137]

[0138]

[0139]

Claims

1. A method for producing a chromatography carrier, comprising the following ligand binding step, ligand-bound carrier bed formation step, ligand-bound carrier flow-through washing step, and ligand-bound carrier agitation washing step: (ligand binding step) a step of binding a protein ligand to a solid-phase carrier, (ligand-bound carrier bed formation step) a step of filling a container with the ligand-bound carrier obtained in the ligand binding step to form a ligand-bound carrier bed, (ligand-bound carrier flow-through washing step) a step of washing the ligand-bound carrier bed formed in the ligand-bound carrier bed formation step with a washing solution one or more times, and (ligand-bound carrier agitation washing step) a step of agitating and washing the ligand-bound carrier after the ligand-bound carrier flow-through washing step in a washing solution one or more times.

2. The method for producing a chromatography carrier according to claim 1, wherein the number of washing cycles in the ligand-bound carrier washing step is 2 to 5.

3. The method for producing a chromatography carrier according to claim 1 or 2, further comprising a solid phase carrier washing step, the solid phase carrier washing step comprising the following solid phase carrier bed formation step and solid phase carrier liquid passing washing step, and the solid phase carrier washed in the solid phase carrier washing step is used as the solid phase carrier in the ligand binding step: (Solid phase carrier bed formation step) A step of filling a vessel with the solid phase carrier to form a solid phase carrier bed (Solid phase carrier liquid passing washing step) A step of passing a washing liquid through the solid phase carrier bed formed in the solid phase carrier bed formation step and washing it at least once.

4. The method for producing a chromatography carrier according to any one of claims 1 to 3, further comprising a solid phase carrier washing step, the solid phase carrier washing step including the following steps of forming a solid phase carrier bed, passing a solid phase carrier through a washing solution, and stirring and washing the solid phase carrier, and the solid phase carrier washed in the solid phase carrier washing step is used as the solid phase carrier in the ligand binding step. (Solid phase carrier bed forming step) A step of filling a vessel with a solid phase carrier to form a solid phase carrier bed (Solid phase carrier passing a liquid washing step) A step of passing a liquid through the solid phase carrier bed formed in the solid phase carrier bed forming step and washing it one or more times with a washing solution (Solid phase carrier stirring and washing step) A step of stirring and washing the solid phase carrier after the solid phase carrier passing a liquid washing step in the washing solution one or more times.

5. The method for producing a chromatography carrier according to claim 3 or 4, wherein the number of washing cycles in the solid phase carrier washing step is 2 to 5.

6. A method for producing a chromatography carrier described in any one of claims 3 to 5, wherein the total number of times of washing in the solid phase carrier washing step and the ligand-bound carrier washing step is 2 to 8 times.

7. A method for producing a chromatography carrier according to any one of claims 1 to 6, wherein the proteinaceous ligand is one or more ligands selected from protein A, protein G, protein L and related substances thereof.

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