Process for the manufacture of chromatographic supports

Abstract

This invention provides a method for easily manufacturing a chromatographic carrier with a large dynamic binding capacity to antibodies or their fragments, minimal leakage of protein ligands during separation, and minimal aggregation of carriers. The method for manufacturing this chromatographic carrier includes the following steps: ligand binding step, ligand binding carrier bed formation step, ligand binding carrier liquid flushing step, and ligand binding carrier stirring and cleaning step. The ligand binding step involves binding a protein ligand to a solid-phase carrier; the ligand binding carrier bed formation step involves filling the ligand binding carrier obtained in the ligand binding step into a container to form a ligand binding carrier bed; the ligand binding carrier liquid flushing step involves flushing the ligand binding carrier bed formed in the ligand binding carrier bed formation step with a cleaning solution at least once; and the ligand binding carrier stirring and cleaning step involves stirring the ligand binding carrier after liquid flushing at least once in a cleaning solution.

Description

Technical Field

[0001] This invention relates to a method for manufacturing a chromatographic carrier. Background Technology

[0002] In recent years, significant progress has been made in the expression technology of target substances such as proteins in the biopharmaceutical field, represented by antibody drugs. This has led to a demand for increased productivity in purification processes based on chromatography and other methods. Methods to improve productivity include minimizing the concentration of impurities mixed in with pharmaceutical raw materials, such as host cell-derived proteins and deoxyribonucleic acid, during a single purification step, and reducing the number of purification steps. The demand for chromatographic carriers capable of achieving this goal is constantly increasing, particularly regarding the increasing requirements for the dynamic binding capacity of antibodies or their fragments. Methods for manufacturing such chromatographic carriers include, for example, obtaining synthetic polymer-based solid-phase carriers or natural polymer-based solid-phase carriers through polymerization reactions, cross-linking reactions, and functional group introduction reactions, and then binding protein ligands to them (Patent Document 1).

[0003] Existing technical documents

[0004] Patent documents

[0005] Patent Document 1: Japanese Patent Application Publication No. 2017-37069

[0006] Patent Document 2: Booklet No. WO2008 / 146906

[0007] Patent Document 3: Booklet No. WO2019 / 121296 Summary of the Invention

[0008] Furthermore, the carriers containing the protein ligands obtained above are covered with impurities generated during the various manufacturing processes, such as unreacted protein ligands. Therefore, in order to prevent unreacted protein ligands from leaking out and mixing into the antibody during antibody purification, stirring and washing are sometimes performed after ligand binding (Patent Documents 2 and 3). However, sometimes this washing is not sufficient to remove the unreacted protein ligands, and the protein ligands leak out during separation.

[0009] Therefore, the inventors conducted a liquid-flow cleaning process on the ligand-binding carriers instead of agitation cleaning and studied its cleaning performance. The results showed that the carriers were prone to aggregation. Carriers prone to aggregation may exhibit batch-to-batch performance variations.

[0010] The problem to be solved by the present invention is to provide a method for easily manufacturing a chromatographic carrier with a large dynamic binding capacity to antibodies or their fragments, which is not prone to leakage of protein ligands during separation and is not prone to aggregation between carriers.

[0011] The inventors have discovered that by sequentially performing liquid-pass washing and stirring washing at least once each after binding protein ligands to a solid-phase support, not only does the dynamic binding capacity of the chromatographic support to antibodies or their fragments increase, but it also makes it less likely for the supports to aggregate with each other, and the protein ligands are less likely to leak out during separation.

[0012] That is, the present invention provides the following <1> to <7>.

[0013] <1> A method for manufacturing a chromatographic carrier (hereinafter also referred to as the method for manufacturing a chromatographic carrier of the present invention) comprises the following steps: a ligand binding step, a ligand binding carrier bed formation step, a ligand binding carrier liquid flushing step, and a ligand binding carrier stirring and cleaning step.

[0014] (Ligand binding process) The process of binding protein ligands to a solid support.

[0015] (Ligand binding carrier bed formation process) The process of filling the ligand binding carrier obtained in the ligand binding process into a container to form a ligand binding carrier bed.

[0016] (Ligand binding carrier liquid flushing process) A process in which the ligand binding carrier bed formed in the ligand binding carrier bed formation process is flushed with a cleaning solution at least once.

[0017] (Ligand binding carrier stirring and cleaning process) The ligand binding carrier after the liquid flushing cleaning process is stirred and cleaned in the cleaning solution more than once.

[0018] <2> According to the manufacturing method of the chromatographic carrier described in <1>, the liquid flushing process of the ligand binding carrier is performed 2 to 5 times.

[0019] <3> The method for manufacturing the chromatographic carrier according to <1> or <2> further includes a solid-phase carrier cleaning step, which includes a solid-phase carrier bed formation step and a solid-phase carrier liquid-pass cleaning step. The solid-phase carrier cleaned in the solid-phase carrier cleaning step is used as the solid-phase carrier in the ligand binding step.

[0020] (Solid support bed formation process) The process of filling a container with a solid support to form a solid support bed.

[0021] (Solid carrier liquid cleaning process) The solid carrier bed formed in the solid carrier bed formation process is cleaned by liquid cleaning solution at least once.

[0022] <4> The method for manufacturing a chromatographic carrier according to any one of <1> to <3>, further comprising a solid-phase carrier cleaning step, wherein the solid-phase carrier cleaning step includes the following solid-phase carrier bed formation step, solid-phase carrier liquid-pass cleaning step, and solid-phase carrier stirring cleaning step, and the solid-phase carrier cleaned in the solid-phase carrier cleaning step is used as the solid-phase carrier in the ligand binding step.

[0023] (Solid support bed formation process) The process of filling a container with a solid support to form a solid support bed.

[0024] (Solid support liquid cleaning process) A process in which the solid support bed formed in the solid support bed formation process is cleaned with a cleaning solution at least once.

[0025] (Solid carrier stirring and cleaning process) The solid carrier after the liquid flushing and cleaning process is stirred and cleaned in the cleaning solution more than once.

[0026] <5> The method for manufacturing the chromatographic carrier according to <3> or <4>, wherein the liquid-flushing cleaning process of the solid-phase carrier is performed 2 to 5 times.

[0027] <6> The method for manufacturing a chromatographic carrier according to any one of <3> to <5>, wherein the total number of liquid-purging cleaning steps of the solid-phase carrier and the ligand-binding carrier is 2 to 8 times.

[0028] <7> A method for manufacturing a chromatographic carrier according to any one of <1> to <6>, wherein the protein ligand is one or more ligands selected from protein A, protein G, protein L and their analogues.

[0029] According to the method for manufacturing chromatographic carriers of the present invention, it is possible to easily manufacture chromatographic carriers with large dynamic binding capacity to antibodies or their fragments, which are not prone to leakage of protein ligands during separation and are not prone to aggregation of carriers. Detailed Implementation

[0030] [Method for manufacturing chromatographic carriers]

[0031] The method for manufacturing the chromatographic carrier of the present invention includes the following steps: ligand binding step, ligand binding carrier bed formation step, ligand binding carrier liquid flushing step, and ligand binding carrier stirring and cleaning step.

[0032] (Ligand binding process) The process of binding protein ligands to a solid support;

[0033] (Ligand binding carrier bed formation process) The process of filling the ligand binding carrier obtained in the ligand binding process into a container to form a ligand binding carrier bed;

[0034] (Ligand binding carrier liquid flushing process) A process in which the ligand binding carrier bed formed in the ligand binding carrier bed formation process is flushed with a cleaning solution at least once.

[0035] (Ligand binding carrier stirring and cleaning process) The ligand binding carrier after the liquid flushing cleaning process is stirred and cleaned in the cleaning solution more than once.

[0036] Here, the solid-phase carrier used in the ligand binding process is described.

[0037] As a solid-phase support, a solid-phase support having a functional group within the molecule capable of binding ligands (e.g., a functional group selected from cyclic ether groups, carboxyl groups, -C(=O)-OC(=O)-, succinimideoxycarbonyl groups, formyl groups, hydroxyl groups, and isocyanate groups) is preferred. When using such a solid-phase support, it is easy to obtain a support with particularly excellent low leakage of protein ligands.

[0038] Examples of solid phase supports include granular solid phase supports, monolithic solid phase supports, plate-shaped solid phase supports, film-shaped solid phase supports, fibrous solid phase supports, and chip-shaped solid phase supports. Granular solid phase supports are preferred, and porous granular solid phase supports (hereinafter also referred to as "porous particles") are more preferred.

[0039] As porous particles, porous particles containing polymers are preferred. Such porous particles can be natural polymer-based porous particles composed of polysaccharides such as agarose, dextran, and cellulose, or they can be synthetic polymer-based porous particles. Synthetic polymer-based porous particles are preferred to increase dynamic binding capacity and improve particle size uniformity. Furthermore, the porous particles are preferably water-insoluble.

[0040] The solid support can be a commercially available product or a product manufactured using conventional methods. The manufacturing method of the solid support is described here.

[0041] When porous particles are obtained as solid-phase carriers, the porous particles can be manufactured by a method including a step (hereinafter also referred to as step P1) in which the monomer composition is dispersed in an aqueous medium for suspension polymerization.

[0042] -Process P1-

[0043] The monomer composition used in step P1 preferably contains a functionalized monomer. The functional group contained in this monomer is preferably capable of being used in an additional chemical reaction (such as a reaction with a crosslinking agent) or is a ligand-binding functional group. Examples of functional groups selected from cyclic ether groups, carboxyl groups, -C(=O)-OC(=O)-, succinimideoxycarbonyl groups, formyl groups, hydroxyl groups, and isocyanate groups are cited. Cyclic ether groups are preferred.

[0044] Here, as a "cyclic ether group", a cyclic ether group having 3 to 7 atoms constituting the ring is preferred. The cyclic ether group may have an alkyl group as a substituent. Specific examples of cyclic ether groups include cyclic ether groups represented by the following formulas (4) to (9), preferably cyclic ether groups represented by formulas (4), (6) or (9), and more preferably cyclic ether groups represented by formula (4).

[0045]

[0046] [In the formula, R] 11 ~R 14 Each atom independently represents a hydrogen atom or an alkyl group; * indicates a binding site.

[0047] R 11 ~R 14 The alkyl group represented preferably has 1 to 4 carbon atoms, more preferably 1 or 2. The alkyl group can be straight-chain or branched, and examples include methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, tert-butyl, etc. Furthermore, as R... 11 ~R 14 Hydrogen atoms are preferred.

[0048] As a functionalized monomer, monomers having ligand-binding functional groups and polymerizable unsaturated groups are preferred. Examples of such monomers include glycidyl (meth)acrylate, 3-oxetyl propanediol (meth)acrylate, 4-oxetyl propanediol (meth)acrylate, 5-oxetyl propanediol (meth)acrylate, 6-oxetyl propanediol (meth)acrylate, 7-oxetyl propanediol (meth)acrylate, 8-oxetyl propanediol (meth)acrylate, methyl (3-methyloxetyl)acrylate, 4-hydroxybutyl (meth)acrylate glycidyl ether, glyceryl mono(meth)acrylate glycidyl ether, 3,4-epoxycyclohexyl methyl (meth)acrylate, 3,4-epoxycyclohexyl ethyl (meth)acrylate, 3,4-epoxycyclohexyl propanediol (meth)acrylate, α-(meth)acrylate-ω-glycidyl polyethylene glycol, and tetrahydrofurfural (meth)acrylate. The monomers include (meth)acrylate monomers with cyclic ether groups, such as esters; aromatic vinyl monomers with cyclic ether groups, such as (vinylbenzyl)glycidyl ether, (isopropenylbenzyl)glycidyl ether, (vinylphenylethyl)glycidyl ether, (vinylphenylbutyl)glycidyl ether, (vinylphenyl)glycidyl ether, (isopropenylphenyl)glycidyl ether, and 1,2-epoxy-3-(4-vinylbenzyl)propane; allyl ether monomers with cyclic ether groups, such as allyl glycidyl ether; (meth)acrylate monomers with isocyanate groups, such as ethyl isocyanate of (meth)acrylate; unsaturated dicarboxylic anhydride monomers, such as maleic anhydride, methylmaleic anhydride, and pentene anhydride; and (meth)acrylic acid, 3,4-epoxy-1-butene, and 3,4-epoxy-3-methyl-1-butene. These monomers can be used alone or in combination of two or more.

[0049] Among these monomers, (meth)acrylate monomers having cyclic ether groups are preferred, and glycidyl (meth)acrylates are particularly preferred.

[0050] As for the total amount of monomers containing functional groups used, it 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 the total amount of monomers used in step P1 of 100 parts by mass. In addition, relative to the total amount of monomers used in step P1 of 100 parts by mass, it 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.

[0051] In addition, the monomer composition used in process P1 may contain monomers other than the functional group monomers mentioned above (hereinafter also referred to as other monomers).

[0052] Other monomers include monomers containing polymerizable unsaturated groups that do not have functional groups capable of binding ligands. These other monomers are broadly classified into non-crosslinked monomers and crosslinked monomers; one type can be used, or a combination thereof.

[0053] Examples of non-crosslinking monomers include (meth)acrylate-based non-crosslinking monomers, (meth)acrylamide-based non-crosslinking monomers, aromatic vinyl-based non-crosslinking monomers, vinyl ketone-based non-crosslinking monomers, (meth)acrylonitrile-based non-crosslinking monomers, and N-vinylamide-based non-crosslinking monomers. These can be used individually or in combination of two or more. Among non-crosslinking monomers, (meth)acrylate-based non-crosslinking monomers and aromatic vinyl-based non-crosslinking monomers are preferred.

[0054] Examples of non-crosslinking monomers of the aforementioned (meth)acrylates include methyl methacrylate, ethyl methacrylate, n-butyl methacrylate, 4-tert-butyl methacrylate, isobutyl methacrylate, n-octyl methacrylate, 2-ethylhexyl methacrylate, cyclohexyl methacrylate, methoxyethyl methacrylate, hydroxyethyl methacrylate, hydroxypropyl methacrylate, glyceryl mono(meth)acrylate, trimethylolethane mono(meth)acrylate, trimethylolpropane mono(meth)acrylate, glycerol mono(meth)acrylate, polyethylene glycol mono(meth)acrylate, methoxy polyethylene glycol (meth)acrylate, pentaerythritol mono(meth)acrylate, dipentaerythritol mono(meth)acrylate, and inositol mono(meth)acrylate. These can be used alone or in combination of two or more.

[0055] In addition, examples of non-crosslinking monomers of the aforementioned (meth)acrylamide system include (meth)acrylamide, dimethacrylamide, hydroxyethyl (meth)acrylamide, (meth)acryloylmorpholine, and diacetone (meth)acrylamide. These can be used alone or in combination of two or more.

[0056] In addition, examples of the aforementioned aromatic vinyl non-crosslinking monomers include styrene, α-methylstyrene, halostyrene, 4-methylstyrene, 2,4-dimethylstyrene, 2,4,6-trimethylstyrene, ethylvinylbenzene, 4-isopropylstyrene, 4-n-butylstyrene, 4-isobutylstyrene, 4-tert-butylstyrene, and other styrene derivatives; and 1-vinylnaphthalene, 2-vinylnaphthalene, and other vinylnaphthalene derivatives. These can be used alone or in combination of two or more.

[0057] In addition, examples of non-crosslinking monomers in the vinyl ketone system mentioned above include ethyl vinyl ketone, propyl vinyl ketone, and isopropyl vinyl ketone. These can be used alone or in combination of two or more.

[0058] In addition, examples of non-crosslinking monomers of the aforementioned (meth)acrylonitrile system include acrylonitrile and methacrylonitrile. These can be used alone or in combination of two or more.

[0059] In addition, examples of non-crosslinking monomers of the aforementioned N-vinylamide system include N-vinylacetamide and N-vinylpropionamide. These can be used alone or in combination of two or more.

[0060] The total amount of non-crosslinking 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 the total amount of monomers used in step P1 of 100 parts by mass. In addition, it 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 the total amount of monomers used in step P1 of 100 parts by mass.

[0061] Furthermore, examples of crosslinking monomers include (meth)acrylate-based crosslinking monomers, aromatic vinyl-based crosslinking monomers, and allyl-based crosslinking monomers. These can be used individually or in combination of two or more. Moreover, as crosslinking monomers, 2- to 5-functional crosslinking monomers are preferred, and 2- or 3-functional crosslinking monomers are more preferred. Among the crosslinking monomers, (meth)acrylate-based crosslinking monomers and aromatic vinyl-based crosslinking monomers are preferred.

[0062] Examples of crosslinking monomers for the aforementioned (meth)acrylate systems 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, glycerol di(meth)acrylate, trimethylolpropane di(meth)acrylate, trimethylolpropane tri(meth)acrylate, and trimethylolpropane tri(meth)acrylate. The following are examples of methacrylates: butylene glycerol dimethacrylate, pentaerythritol dimethacrylate, pentaerythritol trimethacrylate, pentaerythritol tetramethacrylate, glucose dimethacrylate, glucose trimethacrylate, glucose tetramethacrylate, dipentaerythritol dimethacrylate, dipentaerythritol trimethacrylate, dipentaerythritol tetramethacrylate, dipentaerythritol pentamethacrylate, inositol dimethacrylate, inositol trimethacrylate, inositol tetramethacrylate, mannitol dimethacrylate, mannitol trimethacrylate, mannitol tetramethacrylate, mannitol pentamethacrylate, etc. These can be used alone or in combination of two or more.

[0063] In addition, examples of the aforementioned aromatic vinyl crosslinking monomers include divinylbenzene, trivinylbenzene, divinyltoluene, divinylxylbenzene, divinylethylbenzene, and divinylnaphthalene. These can be used alone or in combination of two or more.

[0064] In addition, examples of allyl crosslinking monomers mentioned above include diallyl phthalate, diallyl isophthalate, diallyl terephthalate, diallyl maleate, diallyl fumarate, diallyl itaconic acid, diallyl trimellitate, triallyl trimellitate, triallyl cyanurate, diallyl isocyanurate, and triallyl isocyanurate. These can be used alone or in combination of two or more.

[0065] In addition to the monomers exemplified above, other crosslinking monomers include dehydration condensates of amino alcohols such as diaminopropanol, tris(hydroxymethyl)aminomethane, and glucosamine with (meth)acrylic acid, as well as conjugated dienes such as butadiene and isoprene.

[0066] The total amount of crosslinking monomers used is preferably 1 part or more, more preferably 5 parts or more, and particularly preferably 10 parts or more, relative to the total amount of monomers used in step P1 of 100 parts by mass. In addition, relative to the total amount of monomers used in step P1 of 100 parts by mass, it is preferably 50 parts or less, more preferably 40 parts or less, and particularly preferably 30 parts or less.

[0067] Aqueous media used in process P1 can include, for example, aqueous solutions of water-soluble polymers. Examples of water-soluble polymers include, for example, hydroxyethyl cellulose, polyvinyl alcohol, carboxymethyl cellulose, polyvinylpyrrolidone, starch, and gelatin.

[0068] Compared to a total of 100 parts by mass of a single component, the total amount of water-based media used is typically around 200 to 7000 parts by mass.

[0069] In addition, when water is used as the dispersion medium in an aqueous system, dispersion stabilizers such as sodium carbonate, calcium carbonate, sodium sulfate, calcium phosphate, and sodium chloride can also be used.

[0070] Furthermore, specific methods for process P1 may include: dissolving a polymerization initiator in a mixed solution (monomer solution) containing a monomer composition and a porogen to be used as needed, suspending it in an aqueous medium, and heating it to a specified temperature for polymerization; dissolving a polymerization initiator in a mixed solution (monomer solution) containing a monomer composition and a porogen to be used as needed, adding it to an aqueous medium heated to a specified temperature for polymerization; suspending a mixed solution (monomer solution) containing a monomer composition and a porogen to be used as needed in an aqueous medium, heating it to a specified temperature, and then adding a polymerization initiator for polymerization, etc.

[0071] As a polymerization initiator, a free radical polymerization initiator is preferred. Examples of free radical polymerization initiators include azo-based initiators, peroxide-based initiators, and redox initiators. Specifically, examples include azobisisobutyronitrile, methyl azobisisobutyrate, azobis-2,4-dimethylpentanonitrile, benzoyl peroxide, di-tert-butyl peroxide, and benzoyl peroxide-dimethylaniline. The total amount of polymerization initiator used is typically about 0.01 to 10 parts by mass relative to 100 parts by mass of the monomer.

[0072] The aforementioned porogen is used to manufacture porous particles. During polymerization within oil droplets, it coexists with the monomers and acts as a non-polymerizing component, forming pores. There are no particular limitations on the porogen as long as it can be easily removed from the porous surface; examples include various organic solvents and linear polymers soluble in the mixed monomers, and these can also be used in combination.

[0073] Examples of porogens mentioned above 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; alicyclic alcohols such as cyclohexanol; aromatic alcohols such as 2-phenylethanol 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 isoamyl acetate, butyl acetate, 3-methoxybutyl acetate, and diethyl malonate; and linear polymers such as homopolymers of non-crosslinked vinyl monomers. Pore-forming agents can be used alone or in combination of two or more.

[0074] Relative to a total monomer content of 100 parts by mass, the total amount of the above-mentioned porogen used is usually around 40 to 600 parts by mass.

[0075] In addition, in process P1, various surfactants, such as anionic surfactants including alkyl sulfates, alkyl aryl sulfates, alkyl phosphates, and fatty acid salts, can be used. Furthermore, polymerization inhibitors such as sodium nitrite and other nitrites, potassium iodide and other iodide salts, tert-butylcatechol, benzoquinone, picric acid, hydroquinone, copper chloride, and ferric chloride can also be used. Additionally, polymerization regulators such as dodecyl mercaptan can also be used.

[0076] Furthermore, the polymerization temperature of step P1 can be determined based on the polymerization initiator, and is typically around 2 to 100°C, preferably 50 to 100°C. Additionally, the polymerization time is typically 5 minutes to 48 hours, preferably 10 minutes to 24 hours.

[0077] -Process P2-

[0078] Additionally, prior to the solid support cleaning process, a step (hereinafter also referred to as step P2) can be performed in which the porous particles obtained in step P1 react with at least one selected from a crosslinking agent and a hydrophilizing agent. When using both a crosslinking agent and a hydrophilizing agent, the hydrophilizing reaction can be performed after the crosslinking reaction, or after the hydrophilizing reaction. Alternatively, the crosslinking reaction and the hydrophilizing reaction can be performed simultaneously.

[0079] When a composition containing a functional group monomer is used as the monomer composition in process P1, the crosslinking agent undergoes an addition reaction with a portion of the functional groups present in the polymer molecule by the aforementioned crosslinking reaction, introducing a partial structure from the crosslinking agent. Thus, the residues of the aforementioned functional groups are crosslinked to each other through the partial structure from the crosslinking agent.

[0080] In addition, when a composition containing a functional group monomer is used as a monomer composition in process P1, the hydrophilic agent undergoes an addition reaction with a portion of the functional groups present in the polymer molecule by the above-mentioned hydrophilication reaction, thereby introducing a portion of the structure from the hydrophilic agent.

[0081] The crosslinking agent used in step P2 only needs to be able to react with the functional group of the ligand-binding ligand to introduce a crosslinked structure. Preferably, it is a crosslinking agent that can react with the functional group of the ligand-binding ligand to introduce a crosslinked structure and contains at least two groups represented by -C(=O)-NH- in the molecule.

[0082] When the porous particles obtained in process P1 have cyclic ether groups, specifically, crosslinking agents that contain at least two groups represented by -C(=O)-NH-NH2 in the molecule as crosslinking groups, or crosslinking agents that contain at least two groups represented by -C(=O)-NH- in the molecule and at least two carboxyl groups in the molecule as crosslinking groups, can be used.

[0083] When the porous particles obtained in process P1 have carboxyl groups, -C(=O)-OC(=O)-, succinimideoxycarbonyl groups, formyl groups or isocyanate groups, specifically, crosslinking agents that contain at least two groups represented by -C(=O)-NH-NH2 in the molecule as crosslinking groups can be used.

[0084] Examples of crosslinking agents containing at least two -C(=O)-NH- groups within the molecule, as described above, include dicarboxylic acid dihydrazides such as oxaloyl dihydrazide, malonyl dihydrazide, succinic acid dihydrazide, 2,3-dihydroxysuccinic acid dihydrazide, glutaric acid dihydrazide, adipic acid dihydrazide, pimelic acid dihydrazide, octanoic acid dihydrazide, azelaic acid dihydrazide, sebacic acid dihydrazide, dodecanoic acid dihydrazide, phthalic acid dihydrazide, isophthalic acid dihydrazide, terephthalic acid dihydrazide, and quinoline acid dihydrazide; tricarboxylic acid trihydrazides such as cyclohexanetricarboxylic acid trihydrazide; and (alkylene diimino) bis(oxoalkyl acid) compounds such as N1,N1-(ethane-1,2-diyl)bis(succinic acid monoamide). One type of crosslinking agent can be used alone or in combination of two or more. Among these crosslinking agents, dicarboxylic acid dihydrazides and (alkylene diimino) bis(oxoalkyl) acids are preferred to improve liquid permeability, pressure resistance and antifouling properties during liquid permeation, and dicarboxylic acid dihydrazides are even more preferred.

[0085] Additionally, in step P2, crosslinking agents other than those containing at least two groups represented by -C(=O)-NH- can be used. Examples of such crosslinking agents include polyfunctional isocyanate-based crosslinking agents, polyfunctional epoxy-based crosslinking agents, polyfunctional aldehyde-based crosslinking agents, polyfunctional thiol-based crosslinking agents, and polyfunctional... Crosslinking agents such as zoline-based crosslinking agents, polyfunctional aziridine-based crosslinking agents, and metal chelate-based crosslinking agents.

[0086] The total amount of crosslinking agent used relative to 1 mole of functional group from a functional group-containing monomer is preferably 0.01 molar equivalent to 0.8 molar equivalent, more preferably 0.05 molar equivalent to 0.7 molar equivalent, and particularly preferably 0.1 molar equivalent to 0.6 molar equivalent.

[0087] As a hydrophilizing agent used in step P2, to improve antifouling properties and low leakage of protein ligands, it is preferable to be a compound having at least two hydrophilic groups selected from hydroxyl and thiol groups in total within the molecule; more preferably, it is a compound having at least two to four hydrophilic groups selected from hydroxyl and thiol groups in total within the molecule. Examples include alcohols containing thiol groups such as mercaptoethanol and thioglycerol; and polyols such as glycerol and diglycerol. One hydrophilizing agent can be used alone or in combination of two or more.

[0088] In order to improve the antifouling properties and the low leakage of protein ligands, alcohols with intramolecular thiol groups are preferred, and thioglycerol is particularly preferred.

[0089] The total amount of hydrophilizing agent used relative to 1 mole of functional group from a functional group-containing monomer is preferably 0.5 molar equivalent to 10 molar equivalents, more preferably 1 molar equivalent to 8 molar equivalents, and particularly preferably 2 molar equivalents to 6 molar equivalents.

[0090] Step P2 can be carried out in the presence of an alkaline catalyst. Examples of alkaline catalysts include triethylamine, N,N-dimethyl-4-aminopyridine, sodium hydroxide, and diisopropylethylamine, and one or more catalysts can be used alone or in combination.

[0091] Furthermore, the reaction time for step P2 is not particularly limited, typically ranging from 0.5 to 72 hours, preferably from 0.5 to 48 hours. Additionally, the reaction temperature can be appropriately selected below the boiling point of the solvent, typically ranging from 2 to 100°C.

[0092] -Process P3-

[0093] In addition, when binding ligands to porous particles via a connector (spacer arm) in the ligand binding process, a process (hereinafter also referred to as process P3) can be performed before the solid support cleaning process to react the porous particles obtained in process P1 or process P2 with the compound that provides the connector.

[0094] Examples of compounds providing the connector include diglycidyl ethers of aliphatic polyhydroxy compounds such as ethylene glycol diglycidyl ether, diethylene glycol diglycidyl ether, polyethylene glycol diglycidyl ether, 1,2-propanediol 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 performing the hydrophilication reaction in step P2.

[0095] To improve reaction efficiency, the linker introduction reaction is preferably carried out in a buffer solution with a pH of 7–14. Furthermore, the reaction time is not particularly limited, typically ranging from 0.5 to 72 hours. Additionally, the reaction temperature can be appropriately selected below the boiling point of the solvent, typically between 2 and 100°C.

[0096] (Solid carrier cleaning process)

[0097] As a method for manufacturing the chromatographic carrier of the present invention, in order to improve the hydrophilicity of the solid carrier surface or to prevent protein ligands from leaking out during separation, it is preferable to further include a solid carrier cleaning step in addition to the ligand binding step, the ligand binding carrier bed formation step, the ligand binding carrier liquid flushing step, and the ligand binding carrier stirring cleaning step, and the solid carrier cleaned by the solid carrier cleaning step is used as the solid carrier in the ligand binding step.

[0098] The solid support cleaning process is the process of cleaning the solid support.

[0099] The cleaning process of the solid carrier is preferably carried out using a cleaning solution (hereinafter also referred to as "solid carrier cleaning solution").

[0100] As a solid-phase carrier cleaning solution, an aqueous cleaning solution is preferred. "Aqueous cleaning solution" means a cleaning solution containing at least water. Examples of aqueous cleaning 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.

[0101] Furthermore, as a solid-phase carrier washing solution, in order to improve the hydrophilicity of the solid-phase carrier surface, prevent protein ligands from leaking out during separation, increase the dynamic binding capacity, and inhibit carrier aggregation, at least one washing solution selected from the following is preferred: a washing solution containing hydrogen peroxide, a washing solution containing peracetic acid, and a washing solution with a pH of 0 to 3 or a pH greater than 12.5 and less than 14 (excluding washing solutions containing hydrogen peroxide and peracetic acid). When such a washing solution is used, the solid-phase carrier surface is hydrophilized, and the dynamic binding capacity of the resulting chromatographic carrier against antibodies or their fragments, the low leakage rate of protein ligands, and the low aggregation rate are improved. In addition, washing solutions of various pH values ​​can be used as washing solutions in the ligand-binding carrier liquid-pass washing process or the ligand-binding carrier stirring washing process. For example, even when a washing solution with a pH greater than 3 and less than 12.5 is used in the ligand-binding carrier liquid-pass washing process or the ligand-binding carrier stirring washing process, a chromatographic carrier in which protein ligands are not easily leaked out during separation can be obtained. While the reason why ligands are less likely to leak out during separation is not yet clear, the inventors speculate that the aforementioned cleaning solution hydrophilizes the surface of the solid support, providing excellent antifouling properties. This makes it easier to clean unreacted protein ligands that typically adhere to the ligand binding process, even when the pH of the cleaning solution is, for example, greater than 3 and less than 12.5. For example, when the solid support contains ligand-binding functional groups such as cyclic ether groups or hydroxyl groups, or functional groups such as carbonyl bonds, the inventors speculate that the hydrophilicity is increased due to the hydrolysis of these functional groups, or due to the presence of a large number of hydroxyl and carboxyl groups, thus providing excellent antifouling properties.

[0102] Furthermore, the method for manufacturing the chromatographic carrier of the present invention ensures that the ligand can be sufficiently bound to the solid-phase carrier during the ligand binding step, even during the solid-phase carrier cleaning step before ligand binding. This enables the manufacture of chromatographic carriers with a large dynamic binding capacity for antibodies or their fragments. The reason for achieving such a dynamic binding capacity is not yet clear, but the inventors speculate that one reason is that multiple washing operations using appropriate methods can significantly reduce damage to the chromatographic carrier.

[0103] It should be noted that in this invention, "hydrophilization" refers to an increased affinity for water.

[0104] As a cleaning solution for solid-phase carriers, in order to improve the hydrophilicity of the solid-phase carrier surface and prevent protein ligands from leaking out during separation, a cleaning solution with a pH of 0 to 3 or a pH greater than 12.5 and less than 14 is preferred, and a cleaning solution with a pH greater than 12.5 and less than 14 is even more preferred.

[0105] To prevent the protein ligands from leaking out during separation, the pH of the washing solution containing hydrogen peroxide is preferably 4.5 or higher and less than 7, more preferably 5 to 6.8, particularly preferably 5.5 to 6.5, and most preferably 6 to 6.5.

[0106] In addition, in order to prevent the protein ligands from leaking out during separation, the concentration of hydrogen peroxide in the above-mentioned cleaning solution is preferably 0.001M to 10M, more preferably 0.005M to 5M, and particularly preferably 0.01M to 1M.

[0107] To prevent the protein ligands from leaking out during separation, the pH of the washing solution containing peracetic acid is preferably 1 or higher and less than 7, more preferably 2 to 6, particularly preferably 3 to 5, and most preferably 4 to 5.

[0108] In addition, in order to prevent the protein ligands from leaking out during separation, the concentration of peracetic acid in the above-mentioned washing solution is preferably 0.001M to 5M, more preferably 0.005M to 3M, and particularly preferably 0.01M to 1M.

[0109] In addition to cleaning solutions containing hydrogen peroxide and peracetic acid, solid-phase carrier cleaning solutions also include cleaning solutions with a pH of 0 to 3 and cleaning solutions with a pH greater than 12.5 and less than 14.

[0110] In order to improve the hydrophilicity of the solid support surface and prevent protein ligands from leaking out during separation, the pH of the washing solution with a pH of 0 to 3 is preferably 0 to 2.5, and more preferably 0 to 2.

[0111] In order to improve the hydrophilicity of the solid support surface and prevent protein ligands from leaking out during separation, the pH of the washing solution with a pH greater than 12.5 and less than 14 is preferably 12.7 to 14, more preferably 13 to 14, and particularly preferably 13.2 to 14.

[0112] When the pH of the cleaning solution, which has a pH greater than 12.5 and less than 14, is between 13.2 and 14, the hydrophilicity of the solid-phase carrier surface of the resulting chromatographic carrier increases, and protein ligands are particularly less likely to leak out during separation.

[0113] As a solid-phase carrier cleaning solution, in order to improve the hydrophilicity of the solid-phase carrier surface and prevent protein ligands from leaking out during separation, it is preferable to contain a strong acidic pH adjuster or a strong alkaline pH adjuster, and more preferably, a strong alkaline pH adjuster.

[0114] As a strong acid pH adjuster, inorganic acid-based strong acid pH adjusters such as sulfuric acid, hydrochloric acid, and nitric acid are preferred, with hydrochloric acid being more preferred. Furthermore, as a strong alkaline 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.

[0115] When using a strong alkaline pH adjuster, it is easy to adjust the pH of the cleaning solution to a range greater than 12.5 and less than 14. In addition, when using a strong acidic pH adjuster, it is easy to adjust the pH of the cleaning solution to a range of 0 to 5.

[0116] In addition, to prevent the protein ligands from leaking out during separation, the concentration of the strong acidic pH adjuster or the strong alkaline pH adjuster in the solid-phase carrier washing solution is preferably 0.001M to 25M, more preferably 0.005M to 10M, even more preferably 0.01M to 5M, even more preferably 0.05M to 2.5M, even more preferably 0.25M to 1.5M, and particularly preferably 0.5M to 1M.

[0117] When the concentration of a strong acidic pH adjuster or a strong alkaline pH adjuster is above 0.25M or above 0.5M, the low leakage of protein ligands is particularly excellent.

[0118] To adjust to the desired pH and maintain the target pH, the solid-phase carrier cleaning solution may contain buffers and two or more pH adjusters.

[0119] The cleaning process using the solid-phase carrier cleaning solution can be performed once or more than twice. To improve the hydrophilicity of the solid-phase carrier surface, prevent the leakage of protein ligands during separation, and inhibit carrier aggregation, it is preferable to perform the cleaning process twice or more, more preferably 2 to 20 times, even more preferably 2 to 15 times, even more preferably 2 to 10 times, even more preferably 2 to 8 times, and particularly preferably 3 to 4 times. It should be noted that when cleaning is performed twice or more, the type of solid-phase carrier cleaning solution used each time can be the same or different, but is preferably the same.

[0120] When the total number of washes using the solid-phase carrier cleaning solution is 3 or more, the low leakage of protein ligands is particularly excellent. Furthermore, when the total number of washes using the solid-phase carrier cleaning solution is 4 or fewer, the low aggregation is particularly excellent.

[0121] The amount of cleaning solution used for each cleaning of the solid support 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 solids component of the solid support. Furthermore, it 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 solids component of the solid support. Specifically, the range relative to 100 parts by volume of the dry solids component of the solid support is preferably 40 parts by volume to 1000 parts by volume, more preferably 60 parts by volume to 500 parts by volume, and particularly preferably 80 parts by volume to 300 parts by volume.

[0122] There is no particular limitation on the cleaning temperature, which is usually in the range of 10 to 50°C, and preferably in the range of 15 to 45°C.

[0123] Alternatively, it can be batch cleaning or continuous cleaning.

[0124] There are no particular limitations on the cleaning method using the solid-phase support cleaning solution. Examples include liquid-pass cleaning, stirring cleaning, and static cleaning. Among these, liquid-pass cleaning and stirring cleaning are preferred in order to improve the hydrophilicity of the solid-phase support surface, prevent protein ligands from leaking out during separation, increase dynamic binding capacity, and inhibit the aggregation of the supports. Liquid-pass cleaning is more preferred in order to further improve substitution efficiency, and stirring cleaning is more preferred in order to inhibit the aggregation of the supports. A combination of liquid-pass cleaning and stirring cleaning is particularly preferred. There are no particular limitations on the order or number of liquid-pass cleaning and stirring cleaning, but it is preferred that stirring cleaning be performed after liquid-pass cleaning, and that each be performed at least once.

[0125] When protein ligands are used in combination with liquid-pass cleaning and agitation cleaning as a solid-phase carrier cleaning process, they exhibit excellent low leakage, low aggregation, and substitution efficiency.

[0126] It should be noted that when cleaning is performed more than twice, the cleaning methods used each time can be the same or different.

[0127] Liquid flushing only requires flushing the solid carrier bed with the solid carrier cleaning solution once or more. When performing liquid flushing, the solid carrier cleaning process preferably includes the following solid carrier bed formation process and solid carrier liquid flushing process.

[0128] (Solid support bed formation process) The process of filling a container with a solid support to form a solid support bed.

[0129] (Solid-phase carrier liquid cleaning process) A process in which the solid-phase carrier bed formed in the solid-phase carrier bed formation process is cleaned by liquid flow at least once with a solid-phase carrier cleaning solution (preferably selected from a cleaning solution containing hydrogen peroxide, a cleaning solution containing peracetic acid, and a cleaning solution with a pH of 0 to 3 or a pH greater than 12.5 and less than 14 (excluding cleaning solutions containing hydrogen peroxide and cleaning solutions containing peracetic acid)).

[0130] (Solid support bed formation process)

[0131] In this invention, a "solid support bed" refers to a solid component layer of a solid support, which can be dry or wet. A specific method for forming a solid support bed can be described as follows: a slurry of the solid support (e.g., a slurry using water, alcohol (ethanol, isopropanol, etc.), a mixture of water and alcohol, or a buffer solution (carbonate buffer, etc.) as a dispersion medium) is placed into a container, and the solid components of the solid support are allowed to settle to the bottom of the container by gravity sedimentation or centrifugal sedimentation. The liquid phase of the slurry is removed using solid-liquid separation operations (e.g., decantation, centrifugation, filtration, etc.).

[0132] Furthermore, as a container, a solid-liquid separator is preferred because it is relatively simple to directly pass the cleaning solution through the solid support bed formed in the solid support bed formation process during the solid support liquid cleaning process. Specifically, examples include column containers with filters, funnels with filters, centrifuges with filters, and reaction filters.

[0133] The content of the solid 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. Specifically, the range is preferably 5% to 80% by volume, more preferably 15% to 75% by volume, and particularly preferably 30% to 70% by volume.

[0134] It should be noted that the volume percentage of the carrier in the slurry can be calculated, for example, by filling 200 mL of slurry into a 250 mL glass graduated cylinder (compliant with JIS R3505 Class A) manufactured by Corning and allowing it to stand for 3 hours, and then dividing the settling volume by the volume of slurry filled into the graduated cylinder.

[0135] (Solid-phase carrier liquid cleaning process)

[0136] In this invention, "liquid flushing" of the solid support refers to passing a cleaning solution into the solid support bed, for example, by passing the cleaning solution from one direction to the opposite direction. Specifically, solid-liquid separators such as column containers with filters, filter plates, funnels with filters, Buchner funnels, Nutsch filters, centrifuges with filters, and reaction filters can be used for flushing. It should be noted that the cleaning solution flowing into the solid support bed can preferably be incubated for 30 seconds to 3 hours, more preferably for 2 minutes to 60 minutes, before flowing out.

[0137] In order to improve the hydrophilicity of the solid support surface, prevent protein ligands from leaking out during separation, and inhibit the aggregation of the supports, the liquid flushing process of the solid support is preferably performed once 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.

[0138] When the number of liquid flushing cycles is 2 or more, the low leakage of protein ligands is particularly excellent. Conversely, when the number of liquid flushing cycles is 3 or fewer, the low aggregation is particularly excellent.

[0139] The types of solid carrier cleaning solutions and the amount of solid carrier cleaning solution used per cleaning cycle are as described above.

[0140] It should be noted that liquid flushing can be gravity-flow liquid flushing, pressurized liquid flushing, or depressurized liquid flushing.

[0141] (Solid carrier stirring and cleaning process)

[0142] Stirring and cleaning simply involves stirring the solid carrier in the solid carrier cleaning solution. In order to improve the hydrophilicity of the solid carrier surface, prevent protein ligands from leaking out during separation, increase the dynamic binding capacity, and inhibit the aggregation of carriers, it is preferable to stir and clean the solid carrier at least once in the solid carrier cleaning solution after the liquid flushing process.

[0143] In order to improve the hydrophilicity of the solid support surface, prevent protein ligands from leaking out during separation, and inhibit the aggregation of the supports, the stirring and washing process of the solid support is preferably stirred and washed more than 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 once.

[0144] When the number of stirring and washing cycles is more than 1, the low aggregation properties are particularly excellent. In addition, when the number of stirring and washing cycles is less than 2, the low leakage properties of protein ligands are particularly excellent.

[0145] The stirring speed for the solid carrier stirring and cleaning process is preferably 10 to 150 rpm, more preferably 20 to 100 rpm, and particularly preferably 30 to 80 rpm.

[0146] The stirring time for each solid-phase carrier stirring and cleaning process is preferably 30 seconds to 300 minutes, more preferably 1 minute to 180 minutes, further preferably 2 minutes to 120 minutes, and particularly preferably 5 minutes to 90 minutes.

[0147] The types of solid carrier cleaning solutions and the amount of solid carrier cleaning solution used per cleaning cycle are as described above.

[0148] It should be noted that when using a solid-liquid separator for the solid carrier liquid-pass cleaning process, continuous stirring cleaning can be carried out by performing the solid carrier stirring cleaning process while keeping the filtrate outlet of the solid-liquid separator used in the solid carrier liquid-pass cleaning process open. Alternatively, batch stirring cleaning can be carried out by using the solid-liquid separator with the filtrate outlet closed as a container for the solid carrier stirring cleaning process.

[0149] It should be noted that after the solid support is cleaned, the cleaning solution can be removed as needed before the ligand binding process, or the solid support can be dispersed in water or a mixture of water and lower alcohol.

[0150] The ligand binding process will be explained here.

[0151] (Ligand binding process)

[0152] The ligand binding process is the process of binding protein ligands to a solid support.

[0153] As a protein ligand, it is preferred to select one or more ligands from protein A, protein G, protein L and their analogues. In order to increase the dynamic binding capacity and make the protein ligand less prone to leakage during separation, protein A and modified protein A are preferred, and modified protein A is more preferred.

[0154] In addition, protein A contains five domains, namely E, D, A, B and C, which have the ability to bind to immunoglobulins. Among the above ligands, protein A analogs with modified domains having B and C domains are preferred, and protein A analogs with modified domains having C domains are more preferred.

[0155] Furthermore, in the protein ligand, to increase the dynamic binding capacity and prevent leakage during separation, it is preferable to have a protein ligand in which an amino acid sequence having at least 85% homology with the amino acid sequence shown in sequence number 1 (the C domain of protein A) has been substituted with at least one or two amino acid sequences selected from (a) to (i) below. In such a protein ligand, it is preferable to have a ligand with an amino acid sequence substituted with two or more amino acid sequences selected from (a) to (i) below, more preferably a ligand with an amino acid sequence substituted with two to nine amino acid sequences selected from (a) to (i) below, and particularly preferably a ligand with an amino acid sequence substituted with two to six amino acid sequences selected from (a) to (i) below. Additionally, it is preferable to have a ligand with two or more such amino acid sequences, more preferably a ligand with two to twelve such sequences, and particularly preferably a ligand with four to seven such sequences. When containing two or more amino acid sequences, these amino acid sequences can be of the same or different types.

[0156] (a) Replace the amino acid residue at position 1 of the amino acid sequence corresponding to sequence number 1 with a valine residue.

[0157] (b) Replace the amino acid residue at position 3 corresponding to the amino acid sequence of sequence number 1 with an alanine residue.

[0158] (c) Replace the amino acid residue at position 6 of the amino acid sequence corresponding to sequence number 1 with an alanine residue or an aspartic acid residue.

[0159] (d) Replace the amino acid residue at position 9 of the amino acid sequence of sequence number 1 with an alanine residue.

[0160] (e) Replace the amino acid residue at position 11 of the amino acid sequence of sequence number 1 with an alanine residue, a glutamine residue, or a glutamic acid residue.

[0161] (f) Replace the amino acid residue at position 23 of the amino acid sequence corresponding to sequence number 1 with a leucine residue.

[0162] (g) Replace the amino acid residue at position 29 of the amino acid sequence of sequence number 1 with an alanine residue.

[0163] (h) Replace the amino acid residue at position 43 of the amino acid sequence corresponding to sequence number 1 with an alanine residue.

[0164] (i) Replace the amino acid residue at position 49 of the amino acid sequence of sequence number 1 with an arginine residue.

[0165] As a method for replacing amino acid residues, well-known methods include site-specific mutations of polynucleotides encoding structural domains.

[0166] Here, "homology of 85% or more" for amino acid sequences means preferably 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 especially preferably 99% or more homology.

[0167] In this specification, the “corresponding position” on an amino acid sequence can be determined by aligning the target sequence with a reference sequence (e.g., the amino acid sequence of sequence number 1) in a manner that imparts maximum homology to conserved amino acid residues present in each amino acid sequence. Alignment can be performed using well-known algorithms, the steps of which are well known to those skilled in the art. For example, alignment can be performed using the Clustal W multiple sequence alignment program (Thompson, JD et al, 1994, Nucleic Acids Res., 22:4673-4680) with default settings. ClustalW can be used on websites such as the European Bioinformatics Institute (EBI [www.ebi.ac.uk / index.html]) and the Japanese DNA database (DDBJ [www.ddbj.nig.ac.jp / index.html]) operated by the National Institute of Genetics.

[0168] In this specification, amino acid residues are also referred to by the following abbreviations: 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). Furthermore, in this specification, the amino acid sequence of the peptide is described in a conventional manner 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.

[0169] In this specification, the "before" and "after" positions for a specific location in an amino acid sequence refer to the positions adjacent to the N-terminus and C-terminus of that specific location, respectively. For example, when an amino acid residue is inserted into the "before" and "after" positions of a specific location, the inserted amino acid residue is positioned adjacent to the N-terminus and C-terminus of that specific location.

[0170] In a preferred embodiment, the protein ligand is manufactured by substituting one or more of the amino acid sequences (parental domain) having an amino acid sequence that is more than 85% homologous to the amino acid sequence shown in Serial No. 1 into one or more of the sequences (a) to (i) above.

[0171] As a method for replacing a protein ligand (parental domain) having an amino acid sequence with more than 85% homology to the amino acid sequence shown in Serial No. 1, one example is the introduction of mutations into a polynucleotide encoding the parental domain to produce the desired amino acid residue substitution. Specific methods for introducing mutations into polynucleotides include site-directed mutagenesis, homologous recombination, and SOE (splicing by overlap extension)-PCR (Gene, 1989, 77:61-68), the detailed steps of which are well known to those skilled in the art.

[0172] The manufactured ligands have immunoglobulin binding activity and function as immunoglobulin binding domains.

[0173] As a preferred example of a protein ligand, a protein ligand is formed by linking two or more amino acid sequences in a straight chain that have undergone substitution of at least one or two of the amino acid sequences selected from (a) to (i) below, to form an amino acid sequence having at least 85% homology with the amino acid sequence shown in sequence number 1 (the C domain of protein A). It should be noted that "linked in a straight chain" means a structure in which two or more amino acid sequences are linked in tandem with or without a linker. For example, in the case of a linker, "linked in a straight chain" means a structure in which the C-terminus of one amino acid sequence is linked in tandem with the N-terminus of another amino acid sequence via a linker; on the other hand, in the case of no linker, "linked in a straight chain" means a structure in which the C-terminus of one amino acid sequence is linked in tandem with the N-terminus of another amino acid sequence via a peptide bond.

[0174] Specifically, a modified protein A can be described as a trimer to pentamer of sequence numbers 2 to 4 and amino acid sequence domains having more than 85% homology with those sequence numbers (wherein, it has more than 85% homology with the amino acid sequence shown in sequence number 1). In order to prevent the protein ligand from leaking out during separation, a modified protein A is preferably a trimer to pentamer of sequence numbers 2 to 3 and amino acid sequence domains having more than 85% homology with those sequence numbers (wherein, it has more than 85% homology with the amino acid sequence shown in sequence number 1).

[0175] To increase the dynamic binding capacity, the amount of protein ligands bound is preferably 10 mg to 300 mg per 1 g of dry weight of the solid support, more preferably 25 mg to 150 mg.

[0176] The binding of protein ligands to the solid support in the ligand binding process can be carried out using conventional methods. Chemical binding is preferred as the ligand binding method. For example, methods involving binding the ligand to a functional group capable of binding the ligand can be cited. This method can be performed by referring to the descriptions in International Publication No. 2015 / 119255, International Publication No. 2015 / 041218, etc. Specifically, methods can be used to bind cyclic ether or carboxyl groups, -C(=O)-OC(=O)-, formyl groups, etc., of the solid support to the amino groups of the ligand. To improve reaction efficiency, the ligand binding reaction is preferably carried out in a buffer solution with a pH of 7–14. Furthermore, the reaction time is not particularly limited, typically ranging from 0.1 to 72 hours. Additionally, the reaction temperature can be appropriately selected below the boiling point of the solvent, typically ranging from 2 to 100°C.

[0177] Alternatively, ligands can be bound using methods such as controlling ligand orientation (US Patent No. 6,399,750, Ljungquist C. et al., *Eur. J. Biochem.*, 1989, Vol. 186, pp. 557-561), binding ligands to a solid support via a linker (spacer arm) (US Patent No. 5,260,373, Japanese Patent Application Publication No. 2010-133,733, and 2010-133,734), or accumulating ligands on a solid support via associative groups (Japanese Patent Application Publication No. 2011-256,176).

[0178] In order to improve the antifouling properties and prevent the protein ligands from leaking out during separation, the following method is preferred in the method of manufacturing the chromatographic carrier of the present invention: a ligand-binding carrier hydrophilization step is further provided between the ligand binding step and the ligand-binding carrier bed formation step, and the ligand-binding carrier containing hydrophilic groups obtained in the ligand-binding carrier hydrophilization step is used as the ligand-binding carrier after the ligand binding step in the ligand-binding carrier bed formation step.

[0179] (Ligand binding carrier hydrophilization process) is a process in which a carrier that has bound ligands in the ligand binding process reacts with a compound having a total of two or more hydrophilic groups selected from hydroxyl and thiol groups.

[0180] For compounds used in the hydrophilization process of ligand-binding carriers that have a total of two or more intramolecular hydrophilic groups, in order to improve antifouling properties and prevent leakage of protein ligands during separation, it is preferable that the compound has a total of 2 to 4 intramolecular hydrophilic groups selected from hydroxyl and thiol groups. Examples include alcohols with intramolecular thiol groups such as mercaptoethanol and thioglycerol; and polyols such as glycerol and diglycerol. Compounds with a total of two or more intramolecular hydrophilic groups can be used alone or in combination of two or more.

[0181] In order to improve antifouling properties and prevent protein ligands from leaking out during separation, alcohols with thiol groups in the molecule are preferred, and thioglycerol is particularly preferred.

[0182] The total amount of compounds having two or more hydrophilic groups in the ligand-binding carrier hydrophilization process used in the process relative to 100 parts by mass of the ligand-binding carrier (dry solid component) is preferably 1 to 1000 parts by mass, more preferably 10 to 800 parts by mass, and particularly preferably 100 to 600 parts by mass.

[0183] The ligand-binding support hydrophilization process can be carried out in the presence of a basic catalyst. Examples of basic catalysts include triethylamine, N,N-dimethyl-4-aminopyridine, sodium hydroxide, and diisopropylethylamine, and one or more of them can be used alone or in combination.

[0184] Furthermore, the reaction time for the ligand-carrier hydrophilization process is not particularly limited, typically ranging from 0.5 to 72 hours, preferably from 0.5 to 48 hours. Additionally, the reaction temperature can be appropriately selected as long as it is below the boiling point of the solvent, typically ranging from 2 to 100°C.

[0185] (Ligand binding carrier bed formation process, ligand binding carrier liquid flushing and cleaning process, ligand binding carrier stirring and cleaning process)

[0186] The ligand-binding carrier bed formation process is the process of filling the ligand-binding carrier obtained in the ligand-binding process into a container to form a ligand-binding carrier bed.

[0187] The ligand binding carrier liquid cleaning process is a process of cleaning the ligand binding carrier bed formed in the ligand binding carrier bed formation process with a cleaning solution (hereinafter also referred to as "ligand binding carrier cleaning solution") at least once.

[0188] The ligand binding carrier stirring and cleaning process is a process in which the ligand binding carrier after the liquid flushing and cleaning process is stirred and cleaned more than once in the ligand binding carrier cleaning solution.

[0189] The method for manufacturing the chromatographic carrier of the present invention, by sequentially combining a ligand-binding carrier liquid-flushing and a ligand-binding carrier stirring and washing step, not only increases the dynamic binding capacity of the chromatographic carrier to antibodies or their fragments, but also reduces the likelihood of carrier aggregation and leakage of protein ligands during separation. Furthermore, the substitution efficiency is also good.

[0190] In this invention, a "ligand-binding carrier bed" refers to a solid component layer of a ligand-binding carrier, which can be dry or wet. A specific method for forming a ligand-binding carrier bed can be described as follows: a slurry of the ligand-binding carrier (e.g., a slurry using water, alcohol (ethanol, isopropanol, etc.), a mixture of water and alcohol, or a buffer solution (buffer carbonate, etc.) as a dispersion medium) is added to a container; the solid component of the ligand-binding carrier settles to the bottom of the container by gravity sedimentation or centrifugal sedimentation; and the liquid phase of the slurry is removed by solid-liquid separation operations (e.g., decantation, centrifugation, filtration, etc.).

[0191] Furthermore, as a container, a solid-liquid separator is preferred because it is relatively simple to directly pass the cleaning solution through the ligand-binding carrier bed formed in the ligand-binding carrier bed formation process during the ligand-binding carrier liquid cleaning process. Specifically, examples include column containers with filters, funnels with filters, centrifuges with filters, and reaction filters.

[0192] 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. Specifically, the range in the slurry is preferably 5% to 80% by volume, more preferably 15% to 75% by volume, and particularly preferably 30% to 70% by volume.

[0193] It should be noted that the volume percentage of the carrier in the slurry can be calculated, for example, by filling 200 mL of slurry into a 250 mL glass graduated cylinder (compliant with JIS R3505 Class A) manufactured by Corning and allowing it to stand, and then dividing the sedimentation volume after 3 hours by the volume of slurry filled into the graduated cylinder.

[0194] As the cleaning solution used in the ligand-binding carrier flushing and stirring cleaning processes, an aqueous cleaning solution is preferred. "Aqueous cleaning solution" means a cleaning solution containing at least water. Examples of aqueous cleaning 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.

[0195] To improve the hydrophilicity of the solid-phase support surface, prevent protein ligands from leaking out during separation, and increase the dynamic binding capacity, the pH of the ligand-binding carrier cleaning solution used in the ligand-binding carrier flushing and stirring cleaning processes is preferably greater than 3, more preferably 6 or higher, even more preferably 7 or higher, even more preferably 8.5 or higher, even more preferably 9.5 or higher, and particularly preferably 10.5 or higher. Furthermore, to increase the dynamic binding capacity, it is preferably 12.5 or lower, more preferably 12 or lower, and particularly preferably 11.8 or lower. Specifically, a pH greater than 3 and lower than 12.5 is preferred, a pH of 6 to 12.5 is more preferred, a pH of 7 to 12.5 is more preferred, a pH of 8.5 to 12 is more preferred, a pH of 9.5 to 12 is more preferred, a pH of 10.5 to 12 is more preferred, and a pH of 10.5 to 11.8 is particularly preferred.

[0196] According to the method for manufacturing the chromatographic carrier of the present invention, even when the pH of the cleaning solution used in the ligand-binding carrier flushing and stirring cleaning steps is within the mild pH range described above, a chromatographic carrier in which protein ligands are not easily leaked during separation can be obtained. Furthermore, when the pH of the cleaning solution used in the ligand-binding carrier flushing and stirring cleaning steps is within the mild pH range described above, the dynamic binding capacity increases, thus achieving both excellent dynamic binding capacity and excellent low leakage.

[0197] 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 leakage of protein ligands are particularly excellent. Furthermore, when the pH of the ligand-binding carrier washing solution is below 12 or below 11.8, the dynamic binding capacity is particularly excellent.

[0198] To achieve the aforementioned pH range, the ligand-binding carrier cleaning solution used in the ligand-binding carrier flushing and stirring cleaning processes preferably contains a pH adjuster.

[0199] pH adjusters are broadly classified into acidic pH adjusters and alkaline pH adjusters. Specifically, examples include: strongly acidic pH adjusters based on inorganic acids such as sulfuric acid, hydrochloric acid, and nitric acid; weakly acidic pH adjusters based on inorganic acids such as sodium dihydrogen phosphate, carbonic acid, phosphoric acid, hydrogen fluoride, and hydrogen sulfide; weakly acidic pH adjusters based on organic acids such as acetic acid and oxalic acid; strongly alkaline pH adjusters based on inorganic bases such as alkali metal hydroxides, sodium carbonate, and trisodium phosphate; strongly alkaline pH adjusters based on organic bases such as triethylamine; weakly alkaline pH adjusters based on inorganic bases such as sodium bicarbonate, disodium hydrogen phosphate, ammonia, copper hydroxide, magnesium hydroxide, zinc hydroxide, iron hydroxide, and aluminum hydroxide; and weakly alkaline pH adjusters based on organic bases such as diethanolamine.

[0200] Examples of alkali metal hydroxides include lithium hydroxide, potassium hydroxide, and sodium hydroxide.

[0201] In addition, in order to prevent the protein ligands from leaking out during separation, the concentration of the pH adjuster in the ligand-binding carrier washing solution is preferably 0.001M to 10M, more preferably 0.005M to 2.5M, even more preferably 0.01M to 0.5M, and particularly preferably 0.05M to 0.3M.

[0202] When the concentration of the above-mentioned pH adjuster is 0.05 M or higher, the low leakage of protein ligands is particularly excellent. When the concentration of the above-mentioned pH adjuster is 0.3 M or lower, the dynamic binding capacity is particularly excellent.

[0203] To adjust to the desired pH and maintain the target pH, the ligand-binding carrier cleaning solution may contain a buffer and two or more pH adjusters.

[0204] The ligand-binding carrier cleaning process, including both the liquid-passing cleaning step and the stirring cleaning step, requires a total of two or more cleaning cycles using the ligand-binding carrier cleaning solution. To improve the hydrophilicity of the solid-phase carrier surface, prevent leakage of protein ligands during separation, enhance substitution efficiency, and inhibit carrier aggregation, a total of 2 to 20 cycles is preferred, more preferably 2 to 15 cycles, further preferably 2 to 10 cycles, even more preferably 2 to 8 cycles, and particularly preferably 3 to 4 cycles. It should be noted that the type of ligand-binding carrier cleaning solution used each time can be the same or different, but the same type is preferred. Furthermore, the cleaning methods used each time can be the same or different.

[0205] When the total number of washes using the ligand-binding carrier washing solution is 3 or more, the substitution efficiency is good, and the resulting chromatographic carrier exhibits particularly excellent low leakage of protein ligands. Furthermore, when the total number of washes using the ligand-binding carrier washing solution is 4 or fewer, the low aggregation properties are particularly excellent.

[0206] The amount of cleaning solution used for each cleaning step in the ligand-binding carrier liquid-passing cleaning process and the ligand-binding carrier stirring cleaning process 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 solids component of the ligand-binding carrier. Furthermore, it 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 solids component of the ligand-binding carrier. Specifically, the range is preferably 40 to 1000 parts by volume, more preferably 60 to 500 parts by volume, and particularly preferably 80 to 300 parts by volume, relative to 100 parts by volume of the dry solids component of the ligand-binding carrier.

[0207] The cleaning temperature for the liquid flushing and stirring cleaning processes of ligand-binding carriers is not particularly limited, but is usually in the range of 10 to 50°C, preferably in the range of 15 to 45°C.

[0208] Alternatively, it can be batch cleaning or continuous cleaning.

[0209] Here, the liquid flushing process for ligand-binding carriers is explained in more detail.

[0210] In this invention, "liquid flushing" of the ligand-binding carrier refers to flushing the ligand-binding carrier bed with a cleaning solution, for example, by flushing the bed in one direction from the other. Specifically, a solid-liquid separator such as a column container with a filter, a filter plate, a funnel with a filter, a Buchner funnel, a Nutsch filter, a centrifuge with a filter, or a reaction filter can be used for flushing. It should be noted that the cleaning solution flowing into the ligand-binding carrier bed can preferably be incubated for 30 seconds to 3 hours, more preferably for 2 minutes to 60 minutes, before flowing out.

[0211] In order to improve the hydrophilicity of the solid support surface, prevent protein ligands from leaking out during separation, improve substitution efficiency, and inhibit the aggregation of the supports, the number of liquid washing cycles in the ligand-binding support liquid washing process is preferably more than 1 time, 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.

[0212] When the number of liquid flushing cycles is 2 or more, the substitution efficiency is good, and the protein ligands of the resulting chromatographic carrier exhibit particularly excellent low leakage. Furthermore, when the number of liquid flushing cycles is 3 or fewer, the low aggregation properties are particularly excellent.

[0213] To improve the hydrophilicity of the solid support surface, prevent protein ligands from leaking out during separation, improve substitution efficiency, and inhibit the aggregation of supports, the total number of liquid-washing cycles for the solid support and the ligand-binding support 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, even more preferably 3 to 6 times, and particularly preferably 4 to 5 times.

[0214] When the total number of liquid-pass cleaning steps for the solid-phase support and the ligand-binding support is 4 or more, the substitution efficiency is good, and the resulting chromatographic support exhibits particularly excellent low leakage of protein ligands. When the total number of liquid-pass cleaning steps for the solid-phase support and the ligand-binding support is 5 or less, the low aggregation properties are particularly excellent.

[0215] The types of ligand-binding carrier cleaning solutions and the amount of ligand-binding carrier cleaning solution used per session are as described above.

[0216] It should be noted that liquid flushing can be gravity-flow liquid flushing, pressurized liquid flushing, or depressurized liquid flushing.

[0217] Here, the stirring and cleaning process of the ligand binding carrier is explained in more detail.

[0218] In order to improve the hydrophilicity of the solid support surface, prevent protein ligands from leaking out during separation, and inhibit the aggregation of the supports, the stirring and washing process of the ligand-binding support is preferably stirred and washed more than 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 once.

[0219] When the number of stirring and washing cycles is more than 1, the low coagulation properties are particularly excellent. When the number of stirring and washing cycles is less than 2, the low leakage properties of protein ligands are particularly excellent.

[0220] In order to improve the hydrophilicity of the solid support surface, prevent protein ligands from leaking out during separation, improve substitution efficiency, and inhibit the aggregation of the supports, the total number of stirring and cleaning steps of the solid support stirring and cleaning process and the stirring and cleaning steps of the ligand-binding support stirring and cleaning process 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, even more preferably 2 to 6 times, and particularly preferably 2 to 3 times.

[0221] The stirring speed for the ligand binding carrier stirring and cleaning process is preferably 10 to 150 rpm, more preferably 20 to 100 rpm, and particularly preferably 30 to 80 rpm.

[0222] The stirring time for each stirring and cleaning process of the ligand binding carrier is preferably 30 seconds to 300 minutes, more preferably 1 minute to 180 minutes, further preferably 2 minutes to 60 minutes, and particularly preferably 5 minutes to 30 minutes.

[0223] The types of ligand-binding carrier cleaning solutions and the amount of ligand-binding carrier cleaning solution used per session are as described above.

[0224] It should be noted that when using a solid-liquid separator for the liquid-pass cleaning process of ligand-binding carriers, continuous stirring cleaning can be carried out by keeping the filtrate outlet of the solid-liquid separator open during the liquid-pass cleaning process of ligand-binding carriers. Alternatively, batch stirring cleaning can be carried out by using a solid-liquid separator with the filtrate outlet closed as a container in the stirring cleaning process of ligand-binding carriers.

[0225] It should be noted that the reaction products obtained in each step can be purified by separation methods such as filtration and washing. Alternatively, fractionation can also be performed.

[0226] Furthermore, the method for manufacturing chromatographic carriers according to the present invention can produce chromatographic carriers with large dynamic binding capacity to antibodies or their fragments, low leakage of protein ligands during separation, and low tendency for carrier aggregation. In addition, it exhibits good substitution efficiency and is simple to manufacture.

[0227] Furthermore, the cleaning solution used in the ligand-binding carrier liquid-passing cleaning process and the ligand-binding carrier stirring cleaning process can be liquids of various pH values. For example, even when using a cleaning solution with a pH greater than 3 and less than 12.5, a chromatographic carrier in which protein ligands are not easily leaked during separation can be obtained.

[0228] It should be noted that, in this specification, "antibody" refers to any class of immunoglobulins, including, for example, IgG, IgA, IgD, IgE, IgM, and their subclasses, as well as their mutants. Additionally, in this specification, "antibody" can also refer to chimeric antibodies such as humanized antibodies, antibody complexes, and other immunoglobulin modifications containing antigen recognition sites.

[0229] Furthermore, in this specification, "antibody fragment" can refer to 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 only of the Fc region of immunoglobulins, Fc fusion proteins, and their mutants and modified forms.

[0230] The volume average particle size of the chromatographic carrier obtained as described above is preferably 40–150 μm, more preferably 50–100 μm. Furthermore, the coefficient of variation of the volume average particle size is preferably 40% or less, more preferably 30% or less.

[0231] In addition, the specific surface area of ​​the chromatographic carrier is preferably 1 to 500 m² / g, more preferably 10 to 300 m² / g.

[0232] In addition, the volume average pore size of the chromatographic carrier is preferably 10 to 300 nm.

[0233] It should be noted that the above-mentioned volume average particle size, coefficient of variation, specific surface area, and volume average pore size can be determined by laser diffraction / scattering particle size distribution measurement, etc.

[0234] In addition, the chromatographic carrier obtained as described above is useful for separating antibodies or their fragments from samples containing antibodies or their fragments. Examples of such samples 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, bone marrow fluid, tissue fluid, saliva, gastric juice, synovial fluid, pleural effusion, bile, ascites, and amniotic fluid; bacterial fluids; and various liquid samples such as cell culture medium, cell culture supernatant, and lysate of tissue cells.

[0235] Example

[0236] The present invention will be described in detail below with reference to specific embodiments, but the present invention is not limited to these embodiments.

[0237] (Preparation of ligand (immunoglobulin-binding protein))

[0238] Immunoglobulin-binding proteins PrA-0 to PrA-3 were obtained. PrA-0 is an immunoglobulin-binding protein containing a homopentamer of the C domain (sequence number 1) of protein A linked together. PrA-1 to PrA-3 are mutants of PrA-0 in which the mutations listed in Table 1 were introduced into each immunoglobulin-binding domain.

[0239] [Table 1]

[0240]

[0241] The expression and purification of PrA-0 to PrA-3 were performed as follows. E. coli BL21(DE3) were transformed using plasmids encoding PrA-0 to PrA-3, and the resulting transformants were cultured in nutrient-rich medium at 37°C to the logarithmic growth phase. Then, isopropyl-β-thiogalactopyranoside (manufactured by Wako Pure Chemical Industries, Ltd.) was added to the medium to a final concentration of 1 mM, and the culture was further incubated at 37°C for 4 hours to express the target proteins. Next, the culture medium was centrifuged to remove the supernatant, and the resulting bacterial cells were lysed by adding 30 mM Tris buffer at 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 obtained cell lysate using cation exchange chromatography (SP-Sepharose FF, GE Healthcare Bio-Sciences) and anion exchange chromatography (Q-Sepharose FF, GE Healthcare Bio-Sciences). The purified immunoglobulin-binding protein was dialyzed against 10 mM citrate buffer at pH 6.0. The purity of the recombinant immunoglobulin-binding protein, confirmed by SDS-PAGE, was above 95%.

[0242] [Example 1]

[0243] (Process 1: Polymerization of porous particles)

[0244] 2.69 g of polyvinyl alcohol (PVA-217, manufactured by Kuraray) was added to 448 g of pure water, and the mixture was heated and stirred until the polyvinyl alcohol dissolved, 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.), and 14.15 g of glycidyl methacrylate (manufactured by Mitsubishi Gas Chemical Co., Ltd.) was dissolved in 29.38 g of 2-octanone (manufactured by Toyo Synthetic Co., Ltd.) to prepare a monomer solution. Next, all of the above aqueous solution was added to a separable flask, a thermometer, a stirrer, and a condenser were installed, and the flask was placed in a warm water bath under a nitrogen atmosphere and stirred. All of the above monomer solution was added to the separable flask, and the mixture was heated in a warm water bath until the internal temperature reached 85°C. Then, 1.34 g of 2,2'-azobis(methyl isobutyrate) (manufactured by Wako Pure Chemical Industries, Ltd.) was added to bring the internal temperature to 86°C. Then, the temperature was maintained at 86°C, and the mixture was stirred for 3 hours. Afterward, the reaction solution was cooled, filtered, and washed with pure water and ethanol. The washed particles were dispersed in pure water and subjected to three decantations to remove small particles. Then, the particles were dispersed in pure water at a concentration of 10% by mass to obtain a porous particle dispersion. The porous particles contained in this dispersion are referred to as "porous particles 1".

[0245] Then, 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 porous particle dispersion 1. The mixture was heated to 70°C and stirred at 70°C for 8 hours. After cooling the reaction solution, it was filtered and washed with pure water and ethanol. The particles were then dispersed in pure water at 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".

[0246] Next, the hydroxyl groups from thioglycerol contained in porous particles 2 were reacted with ethylene glycol diglycidyl ether. Specifically, 8.7 g of pure water, 1.2 g of sodium sulfate (manufactured by Wako Pure Chemical Industries, Ltd.), and 0.10 g of sodium carbonate (manufactured by Wako Pure Chemical Industries, Ltd.) were mixed to obtain a carbonate buffer (pH 11.2). 0.5 g of ethylene glycol diglycidyl ether (Denacol EX810, manufactured by Nagase ChemteX) and 8 mL of porous particles 2 were added to this carbonate buffer, and the mixture was stirred and shaken at 23°C for 16 hours. The reaction solution was then filtered, washed with pure water, and the particles were dispersed in pure water at a concentration of 50% by volume to obtain a porous particle dispersion. The porous particles contained in this dispersion were designated as "porous particles 3".

[0247] (Step 2: Cleaning of porous particles)

[0248] Next, the porous particles 3 are cleaned.

[0249] That is, a funnel (Kiriyama funnel made by Kiriyama Works) lined with filter paper (Kiriyama funnel filter paper made by Kiriyama Works) is prepared, and 16 mL of a dispersion of porous particles 3 is added into the funnel under reduced pressure. Pure water is then filtered out from the dispersion by suction filtration, causing the solid components of the dispersion to settle at the bottom of the funnel (filter bed) to form a bed.

[0250] Then, two "retention cleaning" (hereinafter referred to as "S" or "cleaning S") was carried out by directly filtering the fluid from the top to the bottom of the bed without stirring by adding 8 mL of 0.5M sodium hydroxide aqueous solution (pH 13.7) under reduced pressure suction.

[0251] Next, a "re-slurry cleaning" (hereinafter referred to as "R" or "cleaning R") was carried out by closing the tip of the funnel and adding 8 mL of 0.5M sodium hydroxide aqueous solution (pH 13.7) to stir the slurry in the funnel (10 minutes, 60 rpm) and then filtering it.

[0252] Then, the particles were dispersed in pure water at 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 4".

[0253] (Step 3: Ligand binding process)

[0254] Next, the ligand was attached to porous particles 4. Specifically, 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 (a modified protein A, the pentamer of the amino acid sequence domain at sequence number 2) prepared in the preparation example and 8 mL of porous particles 4 were added to this carbonate buffer. The mixture was stirred and vortexed at 23°C for 1.5 hours, and the reaction solution was filtered. Next, a buffer solution was prepared by mixing 8.8 g of pure water, 0.1 g of sodium sulfate (manufactured by Wako Pure Chemical Industries, Ltd.), and 0.03 g of sodium hydroxide (manufactured by Wako Pure Chemical Industries, Ltd.). Then, 4.5 g of thioglycerol (manufactured by Tokyo Chemical Industries, Ltd.) was added to prepare a hydrophilization reaction solution. This hydrophilization reaction solution was added to porous particles bound with ligands, and the mixture was stirred and vibrated at 23°C for 16 hours to carry out the hydrophilization reaction. Next, the particles were dispersed in pure water at a particle concentration of 50% by volume to obtain a porous particle dispersion. The porous particles contained in this dispersion were designated as "porous particle 5".

[0255] (Step 4: Cleaning step following the previous step)

[0256] Next, clean the porous particles 5.

[0257] That is, a funnel (Kiriyama funnel made by Kiriyama Works) lined with filter paper (Kiriyama funnel filter paper made by Kiriyama Works) is prepared, and 16 mL of a dispersion of porous particles 5 is added into the funnel under reduced pressure. Pure water is then filtered out from the dispersion by suction filtration, causing the solid components of the dispersion to settle at the bottom of the funnel (filter bed) to form a bed.

[0258] Then, while maintaining reduced pressure suction, 8 mL of 0.1 M sodium carbonate aqueous solution (pH 11.4) was added for liquid flow, and "cleaning S" was performed twice.

[0259] Next, a "washing R" process was performed, in which the funnel tip was closed and 8 mL of 0.1 M sodium carbonate aqueous solution (pH 11.4) was added. The slurry in the funnel was stirred for 10 minutes at 60 rpm and then filtered.

[0260] Next, after neutralization with sodium citrate buffer, the particles were dispersed in pure water at a particle concentration of 50% by volume to obtain a porous particle dispersion. The porous particles contained in this dispersion are referred to as "carrier 1".

[0261] [Example 2]

[0262] The 0.5M sodium hydroxide aqueous solution used in "Step 2: Cleaning of Porous Particles" was changed to 0.1M hydrochloric acid. Otherwise, the same operation as in Example 1 was performed to obtain carrier 2.

[0263] [Example 3]

[0264] The 0.5M sodium hydroxide aqueous solution used in "Step 2: Cleaning of Porous Particles" was changed to 1.0M hydrochloric acid. Otherwise, the same operation as in Example 1 was performed to obtain carrier 3.

[0265] [Example 4]

[0266] The 0.5M sodium hydroxide aqueous solution used in "Step 2: Cleaning of Porous Particles" was changed to 0.01M hydrochloric acid. Otherwise, the same operation as in Example 1 was performed to obtain carrier 4.

[0267] [Example 5]

[0268] The 0.5M sodium hydroxide aqueous solution used in "Step 2: Cleaning of Porous Particles" was changed to a 0.1M hydrogen peroxide aqueous solution. Otherwise, the same operation as in Example 1 was performed to obtain carrier 5.

[0269] [Example 6]

[0270] The 0.5M sodium hydroxide aqueous solution used in "Step 2: Cleaning of Porous Particles" was changed to a 0.1M peracetic acid aqueous solution. Otherwise, the same operation as in Example 1 was performed to obtain carrier 6.

[0271] [Example 7]

[0272] The 0.5M sodium hydroxide aqueous solution used in "Step 2: Cleaning of Porous Particles" was changed to a 1.0M sodium hydroxide aqueous solution. Otherwise, the same operation as in Example 1 was performed to obtain carrier 7.

[0273] [Example 8]

[0274] The 0.5M sodium hydroxide aqueous solution used in "Step 2: Cleaning of Porous Particles" was changed to a 0.1M sodium hydroxide aqueous solution. Otherwise, the same operation as in Example 1 was performed to obtain carrier 8.

[0275] [Example 9]

[0276] Except for changing the "cleaning S" in "step 2, cleaning of porous particles" from 2 times to 4 times, the same operation as in Example 1 was performed to obtain carrier 9.

[0277] [Example 10]

[0278] The "cleaning S" in "Step 2 Cleaning of porous particles" was changed from 2 times to 1 time. Otherwise, the same operation as in Example 1 was performed to obtain carrier 10.

[0279] [Example 11]

[0280] The 0.1M sodium carbonate aqueous solution (pH 11.4) used in “Step 4 Cleaning Step after Step” was changed to a 0.1M sodium phosphate aqueous solution (prepared by mixing 0.1M sodium dihydrogen phosphate aqueous solution and 0.1M disodium hydrogen phosphate aqueous solution to pH 7.5). Otherwise, the same operation as in Example 1 was performed to obtain carrier 11.

[0281] [Example 12]

[0282] The 0.1M sodium carbonate aqueous solution (pH 11.4) used in “Step 4 Cleaning Step after Step” was changed to a 0.5M sodium phosphate aqueous solution (prepared by mixing 0.5M sodium dihydrogen phosphate aqueous solution with 0.5M disodium hydrogen phosphate aqueous solution to pH 7.5). Otherwise, the same operation as in Example 1 was performed to obtain carrier 12.

[0283] [Example 13]

[0284] The 0.1M sodium carbonate aqueous solution (pH 11.4) used in “Step 4 Cleaning Step after Step” was changed to a 0.01M sodium phosphate aqueous solution (prepared by mixing 0.01M sodium dihydrogen phosphate aqueous solution with 0.01M disodium hydrogen phosphate aqueous solution to pH 7.5). Otherwise, the same operation as in Example 1 was performed to obtain carrier 13.

[0285] [Example 14]

[0286] The 0.1M sodium carbonate aqueous solution (pH 11.4) used in "Step 4 Cleaning Step after Step" was changed to a 0.1M sodium carbonate aqueous solution (prepared to pH 12.5 by adding sodium hydroxide aqueous solution). Otherwise, the same operation as in Example 1 was performed to obtain carrier 14.

[0287] [Example 15]

[0288] The 0.1M sodium carbonate aqueous solution (pH 11.4) used in “Step 4 Cleaning Step after Step” was changed to a 0.1M sodium carbonate aqueous solution (prepared by mixing 0.1M sodium bicarbonate aqueous solution with 0.1M sodium carbonate aqueous solution to pH 10). Otherwise, the same operation as in Example 1 was performed to obtain carrier 15.

[0289] [Example 16]

[0290] Except for changing the "cleaning S" in "step 4 combined with the cleaning step" from 2 times to 4 times, the same operation as in Example 1 was performed to obtain carrier 16.

[0291] [Example 17]

[0292] Except for changing the "cleaning S" in "step 4 combined with the cleaning step" from 2 times to 1 time, the same operation as in Example 1 is performed to obtain carrier 17.

[0293] [Example 18]

[0294] The porous particles 3 obtained in "Process 1: Polymerization of Porous Particles" were replaced with agarose-based particles (WorkBeads 40 ACT (Bio-works)). Otherwise, the same operation as in Example 1 was performed to obtain carrier 18.

[0295] [Example 19]

[0296] The immunoglobulin-binding protein PrA-1 used in "Step 3: Ligand Binding Step" was changed to immunoglobulin-binding protein PrA-0. Otherwise, the same operation as in Example 1 was performed to obtain vector 19.

[0297] [Example 20]

[0298] The immunoglobulin-binding protein PrA-1 used in "Step 3: Ligand Binding Step" was changed to immunoglobulin-binding protein PrA-3. Otherwise, the same operation as in Example 1 was performed to obtain vector 20.

[0299] [Example 21]

[0300] The immunoglobulin-binding protein PrA-1 used in "Step 3: Ligand Binding Step" was changed to immunoglobulin-binding protein PrA-2. Otherwise, the same operation as in Example 1 was performed to obtain vector 21.

[0301] [Example 22]

[0302] The immunoglobulin-binding protein PrA-1 used in "Step 3: Ligand Binding Step" was replaced with Pierce (registered trademark) recombinant protein L (Thermo Fisher Scientific, 21189). Otherwise, the same operation as in Example 1 was performed to obtain vector 22.

[0303] [Example 23]

[0304] The immunoglobulin-binding protein PrA-1 used in "Step 3: Ligand Binding Step" was replaced with Pierce (registered trademark) recombinant protein G (Thermo Fisher Scientific, 21193). Otherwise, the same operation as in Example 1 was performed to obtain vector 23.

[0305] [Comparative Example 1]

[0306] The "cleaning S" performed in "step 4, cleaning step after the combined step" was changed from 2 times to 3 times, and the "cleaning R" was changed from 1 time to 0 times. Otherwise, the same operation as in Example 1 was performed to obtain the carrier of Comparative Example 1.

[0307] [Comparative Example 2]

[0308] In the "cleaning process after the combined process" of "process 4", the "cleaning S" was changed from 2 times to 0 times, and the "cleaning R" was changed from 1 time to 3 times. Otherwise, the same operation as in Example 1 was performed to obtain the carrier of Comparative Example 2.

[0309] [Comparative Example 3]

[0310] The "Step 2 Cleaning of Porous Particles" step was omitted. In addition, the 0.1M sodium carbonate aqueous solution (pH 11.4) used in "Step 4 Cleaning after the Combining Step" was changed to a 0.5M sodium hydroxide aqueous solution (pH 13.7). The "cleaning S" in "Step 4 Cleaning after the Combining Step" was changed from 2 times to 6 times, and the "cleaning R" was changed from 1 time to 0 times. Otherwise, the same operation as in Example 1 was performed to obtain the carrier of Comparative Example 3.

[0311] [Comparative Example 4]

[0312] The "Step 2 Cleaning of Porous Particles" step was omitted. In addition, the 0.1M sodium carbonate aqueous solution (pH 11.4) used in "Step 4 Cleaning after the Combining Step" was changed to a 0.5M sodium hydroxide aqueous solution (pH 13.7). The "cleaning S" in "Step 4 Cleaning after the Combining Step" was changed from 2 times to 0 times, and the "cleaning R" was changed from 1 time to 3 times. Otherwise, the same operation as in Example 1 was performed to obtain the carrier of Comparative Example 4.

[0313] (Experimental Example 1) Dynamic Binding Capacity (DBC) Determination Test

[0314] Using Cytiva AKTA avant 25, the DBC of each vector in Examples 1–21 and the Comparative Examples against the protein (human IgG antibody, LGC 1875-0007) at a retention time of 4 minutes was determined. 4 mL columns (5 mm φ × 200 mm length) were used. The protein was dissolved in a 5 mg / mL solution of 20 mM sodium phosphate / 150 mM sodium chloride aqueous solution (pH 7.5). The DBC was calculated based on the protein capture at the 10% breakthrough point and the column packing volume, and evaluated according to the following criteria. The results are shown in Tables 2–4 and 6.

[0315] (DBC Evaluation Benchmark)

[0316] AA (Excellent): 62 mg / mL or higher

[0317] A (Good): 61 mg / mL or higher but less than 62 mg / mL

[0318] B (Adverse): Less than 61 mg / mL

[0319] (Experimental Example 2) Protein Leakage Measurement Test

[0320] Using Cytiva AKTA avant 25, 7.5 mL of cell culture medium (Herceptin, titer: 4.38 mg / mL) was loaded onto each vector of the examples and comparative examples at a retention time of 4 minutes, and the antibody was then recovered with elution buffer. 0.8 mL (5 mm φ × 40 mm length) columns were used. Elution buffer was 100 mM sodium acetate aqueous solution (pH 3.3), and the column was washed with 20 mM sodium phosphate / 500 mM sodium chloride aqueous solution (pH 7.5) before elution.

[0321] Next, the protein L-Ligand Leakage ELISA Kit (manufactured by Genaxxon Bioscience) was used for the vector of Example 22, the protein G ELISA Kit (manufactured by Alpha Diagnostic International) was used for the vector of Example 23, and the protein A ELISA kit (F740) was used for the other vectors. The amount of protein in the elution buffer was determined, and the antibody concentration in the elution buffer was determined based on the absorbance. Based on these values, the amount of protein leakage per unit amount of antibody in the recovered elution buffer was calculated, and the results were evaluated according to the following criteria. The results are shown in Tables 2-6.

[0322] (Bachelor's degree of protein leakage assessment)

[0323] AAA (Excellent): Below 8 ppm / IgG

[0324] AA (Excellent): Greater than 8 ppm / IgG and less than 10 ppm / IgG

[0325] A (Good): 10 ppm / IgG or higher and less than 13 ppm / IgG

[0326] B (Poor): Above 13 ppm / IgG

[0327] (Experimental Example 3) Evaluation of Coagulation Amount

[0328] 16 mL of the dispersions of each carrier from the examples and comparative examples were passed through a metal mesh sieve (manufactured by Taiyo Corporation, a replaceable mesh funnel with a 30-mesh screen), and the particles remaining on the sieve were recovered with pure water. The recovered particle dispersions were transferred to an aluminum dish and heated on a hot plate at 200°C for 10 minutes to obtain dried particles. The weight of the dried particles was measured as the agglomeration amount (g). The smaller the agglomeration amount value, the less agglomeration. The results are shown in Tables 2-6.

[0329] (Experimental Example 4) Replacement efficiency of cleaning solution

[0330] The conductivity of the cleaning solution initially used in "Step 4: Cleaning after the Combination Step" was measured using a conductivity meter manufactured by HORIBA. Next, the final filtrate from the "Cleaning after the Combination Step" before rinsing with sodium citrate buffer was recovered, and its conductivity was measured in the same manner. The substitution efficiency (%) of the cleaning solution was calculated using the following formula. The closer this value is to 100%, the better the substitution efficiency. The results are shown in Tables 2–6.

[0331] Cleaning solution replacement efficiency (%) = (filtrate conductivity / cleaning solution conductivity) × 100

[0332] (Experimental Example 5) Evaluation of Relative Hydrophilicity

[0333] Add 30 parts by mass of water to 10 parts by mass of dried porous particles 4 (particles before ligand binding) obtained in steps 1 to 2 of each embodiment and comparative example, and visually confirm the dispersion.

[0334] The case where the particles are completely dispersed without clumping is classified as "A", the case where they are dispersed but small pieces are visible is classified as "B", and the case where large pieces remain is classified as "C". The results are shown in Tables 2-6.

[0335] [Table 2]

[0336]

[0337] [Table 3]

[0338]

[0339] [Table 4]

[0340]

[0341] [Table 5]

[0342]

[0343] [Table 6]

[0344]

Claims

1. A method for manufacturing a chromatographic carrier, comprising the following steps: ligand binding step, ligand binding carrier bed formation step, ligand binding carrier liquid flushing and cleaning step, and ligand binding carrier stirring and cleaning step. Ligand binding process: The process of binding protein ligands to a solid support; Ligand binding carrier bed formation process: The process of filling the ligand binding carrier obtained in the ligand binding process into a container to form a ligand binding carrier bed; Ligand binding carrier liquid flushing process: The process of flushing the ligand binding carrier bed formed in the ligand binding carrier bed formation process with a cleaning solution at least once; The ligand-binding carrier stirring and cleaning process involves stirring and cleaning the ligand-binding carrier, which has undergone the liquid flushing and cleaning process, in the cleaning solution at least once.

2. The method for manufacturing a chromatographic carrier according to claim 1, wherein, The liquid cleaning process for the ligand binding carrier involves 2 to 5 liquid cleaning cycles.

3. The method for manufacturing the chromatographic carrier according to claim 1 or 2, wherein, The system further includes a solid-phase carrier cleaning process, which comprises a solid-phase carrier bed formation process and a solid-phase carrier liquid-flushing cleaning process. The solid-phase carrier cleaned in the solid-phase carrier cleaning process is then used as the solid-phase carrier in the ligand binding process. Solid-phase carrier bed formation process: The process of filling a container with a solid-phase carrier to form a solid-phase carrier bed; Solid-phase carrier liquid cleaning process: a process of cleaning the solid-phase carrier bed formed in the solid-phase carrier bed formation process with a cleaning solution at least once.

4. The method for manufacturing the chromatographic carrier according to any one of claims 1 to 3, wherein, The system further includes a solid-phase carrier cleaning process, which comprises a solid-phase carrier bed formation process, a solid-phase carrier liquid-passing cleaning process, and a solid-phase carrier stirring cleaning process. The solid-phase carrier cleaned in the solid-phase carrier cleaning process is then used as the solid-phase carrier in the ligand binding process. Solid-phase carrier bed formation process: The process of filling a container with a solid-phase carrier to form a solid-phase carrier bed; Solid support liquid cleaning process: a process of cleaning the solid support bed formed in the solid support bed formation process with a cleaning solution at least once; Solid carrier stirring and cleaning process: The solid carrier after the liquid flushing and cleaning process is stirred and cleaned in the cleaning solution more than once.

5. The method for manufacturing the chromatographic carrier according to claim 3 or 4, wherein, The liquid-flushing cleaning process of the solid carrier involves 2 to 5 flushes.

6. The method for manufacturing a chromatographic carrier according to any one of claims 3 to 5, wherein, The total number of liquid-flushing cleaning cycles for the solid-phase carrier and the ligand-binding carrier is 2 to 8.

7. The method for manufacturing a chromatographic carrier according to any one of claims 1 to 6, wherein, The protein ligand is one or more ligands selected from protein A, protein G, protein L and their analogues.

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