Separation material and method for producing separation material

Abstract

To provide an ion exchange separation material in which a copolymer is covalently bonded on a carrier that is a water-insoluble porous particle satisfying recently required high separation performance even in separation purification of a large molecule such as protein and an application technology thereof.SOLUTION: A separation material formed by binding a copolymer to a carrier, where the copolymer is a structural unit derived from a monomer represented by a specific formula (1) and a structural unit derived from a monomer represented by a particular formula (2), and a number average molecular weight of the copolymer is 10,000 or more and 1,000,000,000 or less.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to a separation material in which a copolymer is covalently bonded to a carrier, which is a water-insoluble porous particle; and a method for producing the same, a method for separating biopolymers such as proteins using the separation material, and This relates to chromatography columns. [Background technology]

[0002] In research and development of biopolymers such as proteins, chlo- rophores are used for their adsorption, separation, and purification. Math is widely used. The carriers used for liquid chromatography separation materials include silica gel, hydroxy Inorganic carriers such as apatite, natural carriers such as agarose, dextran, cellulose, and chitosan Polymer-based carriers and synthetic polymers such as polystyrene and poly(meth)acrylic esters Supports are known. These supports can be used as is or in a variety of separation modes. In order to obtain the above structure, various functional groups may be added as necessary. In recent years, there has been a demand for further improvement in the separation and purification capabilities of proteins. do.

[0003] For example, Patent Document 1 describes a separation material in which a polymer is immobilized on a porous cross-linked particle by covalent bonding. According to this technology, by having a specific specific surface area, the amount of adsorption is large and the strength is high. Thus, an ion exchange separation material having excellent adsorption properties and improved dynamic adsorption capacity can be obtained. [Prior art documents] [Patent documents]

[0004] [Patent Document 1] JP 2018-155745 A Summary of the Invention [Problem to be solved by the invention]

[0005] The separation material disclosed in Patent Document 1 had sufficient adsorption capacity and strength. The separation and purification capabilities required in recent years were insufficient.

[0006] The present invention has been made in view of the above problems, and is directed to a method for producing a large molecule such as a protein. The present invention relates to an ion exchange separation material with improved separation and purification capabilities even in children, and its application technology. do. [Means for solving the problem]

[0007] As a result of intensive research, the present inventors have found that the molecular weight of the copolymer to be immobilized on the carrier can be adjusted. It was found that the separation and purification ability of the separation material is improved by this, and the present invention was completed.

[0008] That is, the gist of the present invention is as follows. [1] A separation material comprising a copolymer bound to a carrier, The copolymer comprises a structural unit derived from a monomer represented by the following formula (1) and a structural unit derived from a monomer represented by the following formula (2 ) a structural unit derived from a monomer represented by The number average molecular weight of the copolymer is 10,000 or more and 1,000,000 or less. Material. [ka] [ka] (In formula (1), R1 represents -NR3-R4-R5 or -O-R4-R5. R2 represents a hydrogen atom. R3 represents a hydrogen atom or an alkyl group having 1 to 4 carbon atoms. R4 represents an alkylene group having 1 to 6 carbon atoms and containing an aliphatic ring, or It represents a linear or branched alkylene group having 1 to 6 carbon atoms. R5 represents -SO3M. M represents a hydrogen atom, Na or K. In formula (2), R6 represents -NR8-R9 or -O-R9. R7 represents a hydrogen atom or a carbon number R8 represents an alkyl group having 1 to 4 carbon atoms. R8 is a hydrogen atom or an alkyl group having 1 to 4 carbon atoms. R9 represents a linear or branched alkyl group having 1 to 4 carbon atoms, or an alkoxymethyl group. represents a group or a hydroxyalkyl group. [2] The copolymer contains a structural unit derived from the monomer represented by the above formula (1), The separation material according to [1], wherein the content is in the range of 35 mol % or more and 65 mol % or less of the total. [3] The monomer represented by the above formula (1) is 2-acrylamide-2-methylpropanediol. Sulfonic acid, acrylic acid-3-sulfopropyl acid, methacrylic acid-3-sulfopropyl acid, The separation material according to [1] or [2], which is one or more selected from the group consisting of tautomers, tautomers, and salts thereof. [4] The monomer represented by the above formula (2) is N-hydroxyethylacrylamide. The separation material according to any one of [1] to [3]. [5] The exchange capacity of the ion exchange group bound to the carrier is 2.5 × 10 -2 mmol / mL -R or above 1.2×10 -1 mmol / mL-R or less, [1] to [4] The separation material described. [6] The carrier comprises a structural unit derived from a monomer represented by the following formula (3), and a structural unit derived from a monomer represented by the following formula ( The molecule according to any one of [1] to [5], which contains a structural unit derived from a monomer represented by the following formula (4): Separation. [ka] [ka] (In formula (3), R 11 R represents a hydrogen atom or an alkyl group having 1 to 4 carbon atoms. 10 teeth -OR 12 -R 13 Represents R 12 is a straight-chain or is a branched alkylene group, or a linear or branched alkylene group having 1 to 6 carbon atoms. Represents. R 13 is an alkyl group, a halogen atom, a hydroxyl group, an amino group, a glycidyl group, or Represents an epoxy group. In formula (4), R 14 represents a linear or branched alkylene group having 1 to 6 carbon atoms. R 15 represents a hydrogen atom or an alkyl group having 1 to 4 carbon atoms.

[0009] [7] A method for producing a separation material in which a copolymer is bound to a carrier, comprising the steps of: In the presence of the carrier, a monomer represented by the formula (1) and a monomer represented by the formula (2) are reacted with each other. and a chain transfer agent, The monomer represented by the above formula (1) is 35 mol% based on the total monomer (100 mol%). or more and 65 mol % or less of a monomer composition is polymerized. [8] The content of the chain transfer agent in the monomer composition is a monomer represented by the above formula (1), 0.4 mol% or more relative to the total amount (100 mol%) of the monomers represented by the above formula (2) The method for producing a separation material according to [7], wherein the content is 2.0 mol % or less. [9] The monomer represented by the above formula (1) is 2-acrylamide-2-methylpropanediol. Sulfonic acid, acrylic acid-3-sulfopropyl acid, methacrylic acid-3-sulfopropyl acid, and a salt thereof. .

[10] The monomer represented by the above formula (2) is N-hydroxyethylacrylamide. The method for producing a separation material according to any one of [7] to [9].

[11] The chain transfer agent according to any one of [7] to

[10] , wherein the chain transfer agent has a thiol group. Manufacturing method of separation material.

[0010]

[12] A method for producing an antibody, comprising a purification step using the separation material described in [1]1.

[13] A method for isolating a target molecule, comprising the following steps (a) and (b): Step (a): A solution containing a target molecule is contacted with the separation material described in [1], and the target molecule is transferred to the separation material. adsorbing the Step (b): Eluting the target molecule from the separation material treated in step (a).

[14] The target molecule is a monoclonal or polyclonal antibody, or A method for isolating a target molecule described in

[13] , which is a chemically denatured product.

[15] A chromatograph comprising at least one container comprising the separation material according to [1]. Column for HPLC. Effect of the Invention

[0011] According to the present invention, a suitable chromatographic packing material based on a synthetic polymer is The water-soluble monomer is sufficiently dispersed in the pores of the support and polymerized to fix the copolymer to the support. The molecular chain length of this copolymer can be adjusted by using a chain transfer agent. By adjusting the size of the gel, the separation performance of the target molecule is improved, making it useful as a packing material for chromatography. do. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0012] The present invention will be described in detail below. The following examples are merely examples of the embodiments of the present invention. The present invention is not limited to these examples without departing from the spirit of the invention. There is none.

[0013] In this specification, "(meth)acrylic" means "acrylic" and "methacrylic". The same applies to "(meth)acrylate".

[0014] [Separation material] The separation material of the present invention is a separation material in which a copolymer is bound to a carrier, A structural unit derived from a monomer represented by formula (1) and a monomer represented by formula (2) The copolymer contains structural units derived from 1,000 to 1,000,000, and the number average molecular weight of the copolymer is 10,000 to 1,000,000. 000 or less. The copolymer contains structural units derived from the monomer represented by the above formula (1) in the total structural units. The content is preferably in the range of 35 mol % or more and 65 mol % or less.

[0015] [Copolymer] The copolymer of the present invention comprises a structural unit derived from the monomer represented by the above formula (1) and It contains a structural unit derived from a monomer represented by formula (2). The monomer represented by the above formula (1) has good polymerization reactivity, good copolymerizability, and a high degree of polymerization completion. It has excellent properties, a wide variety of post-treatment and post-reaction options, and is easily available industrially. From this viewpoint, the copolymer of the present invention contains a structural unit derived from the monomer represented by the above formula (1): Includes.

[0016] In the above formula (1), R1 represents -NR3-R4-R5 or -O-R4-R5. R2 represents water. R3 represents a hydrogen atom or an alkyl group having 1 to 4 carbon atoms. R4 represents an alkyl group having 3 to 6 carbon atoms and containing an aliphatic ring, represents a linear or branched alkylene group having 1 to 6 carbon atoms. R5 represents -SO3M. M represents a hydrogen atom, Na or K.

[0017] The monomer represented by the above formula (1) is, for example, 2-acrylamide-2-methylpropanediol. propanesulfonic acid, acrylic acid-3-sulfopropyl acid, methacrylic acid-3-sulfopropanesulfonic acid Pyric acid and its salts. Of these, 2-acrylamido-2-methylpropanesulfonic acid is preferred. The structural unit derived from the monomer represented by the above formula (1) may contain one type alone, or two It may contain more than one species.

[0018] The monomer represented by the above formula (2) has good polymerization reactivity, good copolymerizability, and a high degree of polymerization completion. It has excellent properties, a wide variety of post-treatment and post-reaction options, and is easily available industrially. From this viewpoint, the copolymer of the present invention contains a structural unit derived from the monomer represented by the above formula (2): Includes. In the above formula (2), R6 represents -NR8-R9 or -O-R9. R7 represents a hydrogen atom or a carbon atom. R8 is a hydrogen atom or an alkyl group having 1 to 4 carbon atoms. R9 represents a linear or branched alkyl group having 1 to 4 carbon atoms, an alkoxy group, or represents a methyl group or a hydroxyalkyl group.

[0019] The monomer represented by the above formula (2) is, for example, N-methyl(meth)acrylamide, N-ethyl Alkyl (meth)acrylamides such as N-hydroxymethyl (meth)acrylamide; Hydroxyalkyl (methacrylamide, N-hydroxyethyl acrylamide, etc.) )acrylamides; (meth)acrylates such as methyl (meth)acrylate and ethyl (meth)acrylate; ) Alkyl acrylates; (meth) hydroxymethyl acrylate, (meth) hydroxymethyl acrylate and hydroxyalkyl (meth)acrylates such as hydroxyethyl. Among these, N-hydroxymethyl (meth)acrylamide, N-hydroxyethyl (meth)acrylamide, (meth)acrylic acid hydroxymethyl, (meth)acrylic acid Hydroxyethyl is preferred, N-hydroxymethyl(meth)acrylamide, N-hydroxymethyl(meth)acrylamide, More preferred is N-hydroxyethyl (meth)acrylamide. Mido is more preferred. The structural unit derived from the monomer represented by the above formula (2) may contain one type alone, or two It may contain more than one species.

[0020] The copolymer of the present invention comprises a structural unit derived from the monomer represented by the above formula (1) and In addition to the structural unit derived from the monomer represented by formula (2), structural units derived from other monomers are Other monomers may include, for example, aromatic vinyl monomers such as styrene, (meth) ) acrylamide, and (meth)acrylic acid.

[0021] The copolymer of the present invention contains a structural unit derived from the monomer represented by the above formula (1), It is preferable that the content of the fluorine-containing compound is in the range of 35 mol % or more and 65 mol % or less of the total, and more preferably 42 mol % or less. It is more preferable that the content be in the range of 58 mol % or less.

[0022] Structural units derived from other monomers may be contained in an amount of 5 mol % or less of the total structural units. .

[0023] The number average molecular weight of the copolymer of the present invention is 10,000 or more and 1,000,000 or less, A value between 50,000 and 120,000 is more preferable. If the number average molecular weight is within the above range, the degree of separation is high when used for separating antibodies.

[0024] [Carrier] The carrier of the present invention comprises a structural unit derived from a monomer represented by the above formula (3) and a structural unit derived from a monomer represented by the above formula ( 4) is a structural unit derived from a monomer represented by the formula (I).

[0025] In the above formula (3), R 11 R1 represents a hydrogen atom or an alkyl group having 1 to 4 carbon atoms. 0 is -OR 12 -R 13 Represents R 12 Also includes straight chains with 1 to 6 carbon atoms including aliphatic rings. or a linear or branched alkylene group having 1 to 6 carbon atoms; R 13 is an alkyl group, a halogen atom, a hydroxyl group, an amino group, or a glycidyl group. or an epoxy group.

[0026] Examples of the monomer represented by the above formula (3) include methyl (meth)acrylate, ) Ethyl acrylate, 2-hydroxymethyl (meth)acrylate, 2-hydroxyethyl (meth)acrylate, 2-aminoethyl (meth)acrylate, 2-(dimethylamino)ethyl (meth)acrylate (meth) acrylate, 2-(dimethylamino)ethyl (meth) acrylate , glycidyl (meth)acrylate, 4,5-epoxybutyl (meth)acrylate, 9 ,10-epoxystearyl(meth)acrylate. The structural unit derived from the monomer represented by the above formula (3) may contain one type alone, or two It may contain more than one species.

[0027] In the above formula (4), R 14represents a linear or branched alkylene group having 1 to 6 carbon atoms. R 15 represents a hydrogen atom or an alkyl group having 1 to 4 carbon atoms.

[0028] Examples of the monomer represented by (4) above include ethylene glycol di(meth)acrylate. acrylate, 1,3-propanediol di(meth)acrylate, tetramethylene glycol Di(meth)acrylates are examples. The structural unit derived from the monomer represented by the above formula (4) may contain one type alone, or two It may contain more than one species.

[0029] [Separation material] The separation material of the present invention is formed by bonding a copolymer to a carrier. The method involves combining a preformed copolymer with a carrier, or reacting a carrier with a monomer composition in a solvent. The monomer composition may be polymerized in the presence of a carrier in addition to a solvent to effect the bonding.

[0030] The separation material of the present invention has an exchange capacity of 2.5 × 10 -2 MMO l / mL-R or more 1.2×10 -1 mmol / mL-R or less is preferable, and 3.0×10 - 2 mmol / mL-R or more 5.0×10 -2 More preferably, it is less than mmol / mL-R. If the exchange capacity of the ion exchange group is equal to or higher than the lower limit, the peak shape of the antibody is not easily distorted. Below the upper limit, the selectivity of the antibody and its impurities is increased.

[0031] [Method of manufacturing separation material] The method for producing a separation material of the present invention is a method for producing a separation material in which a copolymer is bonded to a carrier. In the presence of the carrier, the monomer represented by the formula (1) and the monomer represented by the formula (2) are reacted with each other. and a chain transfer agent, and a monomer represented by the above formula (1) and a monomer represented by the above formula (2) The monomer represented by the above formula (1) is 35 mol per 100 mol of the total monomer. % or more and 65 mol % or less of a monomer composition is polymerized.

[0032] The carrier is the same as that explained in the section on separation materials. The carrier may be used alone or in combination of two or more kinds.

[0033] The monomer represented by the above formula (1) and the monomer represented by the above formula (2) are This is the same as that explained in the section on separation materials. The monomer represented by the above formula (1) may be used alone or in combination of two or more. good. The monomer represented by the above formula (2) may be used alone or in combination of two or more. good.

[0034] The chain transfer agent used in the present invention may be any commonly used one, and is not particularly limited. It is not something that can be achieved. The chain transfer agent preferably has a thiol group, for example, 1-butanethiol, Cyclohexanethiol, 1-decanethiol, 2-ethylhexyl mercaptoacetate, mercaptoacetate ethyl mercaptoacetate, 1-hexanedecanethiol, 2-mercaptoethanol, 3-mercaptoethanol Mercapto-1,2-propanediol, mercaptoacetic acid, 2-mercaptopropanesulfone Sodium mercaptoacetate, 3-mercaptopropionic acid, methyl mercaptoacetate, mercaptosuccinic acid, α -thioglycerol, 1-octanethiol, and thiophenol.

[0035] Among these, 2-mercaptoethanol and 3-mercaptoethanol are preferred because they use water as a reaction solvent. Mercapto-1,2-propanediol, mercaptoacetic acid, 2-mercaptopropanesulfonyl Sodium phosphate, 3-mercaptopropionic acid, mercaptosuccinic acid, α-thioglycerol preferable. The chain transfer agent may be used alone or in combination of two or more kinds.

[0036] The content of the monomer represented by the above formula (1) in the monomer composition is The total amount of the monomers represented by the above formula (2) and the monomers represented by the above formula (100 mol%) is 35m % or more and 65 mol % or less, and preferably 42 mol % or more and 58 mol % or less. The content of the chain transfer agent in the monomer composition is determined by the ratio of the monomer represented by the formula (1) to the monomer represented by the formula (2) 0.4 mol% or more and 2.0 mol% or more based on the total amount (100 mol%) of the monomers represented by mol % or less, and more preferably 0.4 mol % or more and 1.0 mol % or less. Within this range, the degree of separation is high when used to separate antibodies.

[0037] [Antibody production method] The antibody production method of the present invention may be any method as long as it includes a purification step using the separation material of the present invention, Generally, the method is similar to that used for antibody purification.

[0038] [Method for isolating target molecules] The method for isolating a target molecule of the present invention is characterized in that it comprises the following steps (a) and (b): . Step (a): A solution containing a target molecule is brought into contact with the separation material of the present invention, and the target molecule is adsorbed onto the separation material. A process of Step (b): Eluting the target molecule from the separation material treated in step (a).

[0039] The target molecule may be a monoclonal or polyclonal antibody, or a chemical modification thereof. It is preferable that the

[0040] [Chromatography columns] The chromatography column of the present invention contains the separation material of the present invention as a packing material and has at least There are no particular limitations as long as each device has one container. EXAMPLES

[0041] The present invention will be described in more detail below with reference to examples, but the present invention will not deviate from the gist of the invention. Unless otherwise stated, the present invention is not limited to the following examples. In the following description, "parts" and "%" mean "quantity" unless otherwise specified. "Parts by weight" and "% by weight" are indicated.

[0042] [Evaluation and Evaluation Method]

[0043] <Measurement of number average molecular weight (Mn) of copolymer> (1) Equipment and reagents used Dextran: Fujifilm Wako Pure Chemical LC system: Shimadzu CBM-20A Mobile phase: Acetate buffer pH 5.0 Column: TSKgel G4000PW XL

[0044] (2)Measurement method The column was connected to an LC system and equilibrated with the mobile phase. The polymerization reaction solution of the example was filtered. After removing the separation material, the reaction solution was diluted to 2.5 mg / mL. This sample was applied to a column and subjected to gel filtration chromatography. Using a calibration curve made from dextrans with different molecular weights, the number average molecular weight of the copolymer was calculated. The amount (Mn) was calculated.

[0045] <Measurement of exchange capacity> Put the slurry solution containing the separating agent into a 10 mL measuring cylinder and tap the cylinder to remove the separating agent. The mixture was allowed to settle, and 5.0 mL of separation material was weighed out. This separation material was filtered off and placed in a column, and the resulting mixture was diluted to 1M. Regenerate by pouring 100mL of hydrochloric acid, rinse thoroughly with water, and confirm that the pH of the effluent water is neutral. I acknowledged. The resulting separation material was filtered off, mixed with 50 mL of a 5% aqueous sodium chloride solution, and the resulting solution was The acid was titrated, and the titrated value was divided by the volume of the separation material to obtain the exchange capacity per mL of separation material.

[0046] <Method for preparing monoclonal antibodies containing impurities> (1) Equipment and reagents used LC system: ACTA avant 150 A buffer: Acetate buffer pH 3.0 B buffer: 0.1M NaOH C buffer: PBS buffer Column: Mabspeed rP202

[0047] (2)Adjustment method The column was connected to the LC system and equilibrated with C buffer. The cell supernatant PrA affinity purified antibody culture solution was applied. Buffer A was then passed through the tube in an increasing volumetric percentage from 0 to 100%. Finally, buffer B was passed through the tube. The monoclonal antibody solution was obtained by mixing the eluent with 100 mL of ethanol. carried out. This monoclonal antibody solution was diluted 5-fold, adjusted to pH 2.5 with 1M hydrochloric acid, and incubated for 90 minutes. The mixture was then allowed to stand. Then, the pH was adjusted to 5.0 with 1M Tris solution, and the mixture was diluted with 10% impurities. A monoclonal antibody solution was prepared.

[0048] <Isolation of monoclonal antibodies containing impurities> (1) Equipment and reagents used LC system: ACTA avant 150 A buffer: acetate buffer pH 5.0 B buffer: acetate buffer pH 5.0, 0.5 mol / L NaCl Column: diameter 7mm, length 26mm

[0049] (2) Preparative separation method The column packed with the separation material was connected to the LC system and equilibrated with A buffer. 13.5 mL of the monoclonal antibody solution containing impurities was applied to the column. After the completion of the above, buffer A was passed through the column. The proportion of buffer B was changed from 0% to 10% by volume. The volume of the solution was increased to 0%, and the adsorbed monoclonal antibody was eluted and collected at 1 mL intervals. All flows were carried out at 1.0 mL / min.

[0050] <How to calculate the degree of separation> (1) Equipment and reagents used LC system: Shimadzu CBM-20A Buffer: PBS buffer pH 6.7 Column: TSKgel SuperSW mAb HR (7.8mm I.D. x 30cm m)

[0051] (2) Analysis of monoclonal antibody solutions The column was connected to the LC system and equilibrated with PBS buffer. 10 μL of the antibody solution was applied, and the analysis was carried out at a flow rate of 0.8 mL / min. This was repeated for each sample taken.

[0052] (3) Calculation method for resolution (Rs) Analysis of (2) yielded peaks for monoclonal antibodies and impurities. The area values ​​were plotted against the fractionation time to produce a chromatogram. The time when the monoclonal antibody peak tops is t1 (min), and the half-width of the peak is W 0.5h1 , the peak top of the impurity is t2(min), and the half-width of the peak is W 0.5h2 and Then, Rs was calculated using the following formula. Rs = 1.18 × (t2-t1) / (W 0.5h1 +W 0.5h2 )

[0053] [Manufacture of carrier] <Production Example 1> Polyvinyl alcohol (manufactured by Nippon Synthetic Chemical Industry) was dissolved in 1000 parts of water to make a 2% solution. 40 parts of methacrylic acid (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.), ethylene glycol dimethacrylate Relate (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) 60 parts, 2,2'-azobis(2,4-dimethylba 1.3 parts of 4-methyl-2-pentaerythronitrile (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) and A mixture of 225 parts of non (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) was added at room temperature and stirred in a nitrogen atmosphere. The mixture was stirred to form a suspension. At this time, the stirring speed was adjusted so that the average diameter of the droplets became 30 μm. The suspension was heated to 70° C. and reacted for 3 hours in a nitrogen atmosphere. The porous particles made of cross-linked polymer are washed with water, then washed with methanol, dried, and classified to obtain the desired product. A carrier was obtained.

[0054] [Example 1] <Production of separation materials> 100 parts of the carrier obtained in the above Production Example 1, as the monomer represented by formula (1), Amido-2-methylpropanesulfonic acid (Shinryo Co., Ltd.) 166 parts (40 mol%), As a monomer represented by the formula, N-(2-hydroxyethyl)acrylamide (manufactured by Tokyo Chemical Industry Co., Ltd.) ) 138 parts (60 mol%), as a chain transfer agent, 3-mercapto-1-propanesulfonyl 2 parts (0.5 mol%) of sodium phosphate (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.), 883 parts of water, and As an initiator, 2,2'-azobis[N-(2-carboxyethyl)-2-methylpropionate Three parts of 1,2-dimethylaminopropane hydrate (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) were mixed and dissolved in this reaction. The reaction mixture was reacted at 50° C. for 5 hours. After that, 180 parts of 47% sulfuric acid (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) was added and reacted at 50°C for 6 hours. Thereafter, the temperature was lowered, and the particles were filtered and washed with water to obtain a separation material. Here, 2-acrylamido-2-methylpropanesulfonic acid, N-(2-hydroxyethyl) The molar ratio of ethyl acrylamide to ethyl acrylamide was 40:60. For the separation material of Example 1, a sample containing monoclonal antibodies and their aggregates was used. The separation performance was evaluated. The evaluation results are shown in Table 1.

[0055] [Examples 2 to 4] 2-Acrylamido-2-methylpropanesulfonic acid, N-(2-hydroxyethyl) Acrylamide, 3-mercapto-1-propanesulfonic acid Na, and 2,2'-azo Contains bis[N-(2-carboxyethyl)-2-methylpropionamidine] hydrate A separation material was obtained in the same manner as in Example 1, except that the amount was changed as shown in Table 2, and the separation properties were similarly evaluated. Performance evaluation was carried out.

[0056] [Comparative Example 1] <Synthesis of copolymer> 2-acrylamido-2-methylpropanesulfonic acid 70 parts, N-(2-hydroxy 50 parts of ethyl acrylamide, 460 parts of water, and 2,2-azobisulfite as a polymerization initiator. Mix 1 part of 2-methylpropionamidine dihydrochloride and polymerize at 70°C. This resulted in an aqueous solution of the copolymer. Here, 2-acrylamido-2-methylpropanesulfonic acid and N-(2-hydroxy The molar ratio of ethyl acrylamide to ethyl acrylamide was 44:56.

[0057] <Immobilization of copolymer> 5 parts of carrier, 20 parts of water, 25 parts of the aqueous solution of the copolymer, and 10 parts of 47% sulfuric acid were added and mixed. The mixture was reacted at 50° C. for 6 hours. After that, the temperature was lowered, and the particles were filtered and washed with water to obtain a separation material.

[0058] [Table 1]

[0059] [Table 2]

[0060] As can be seen from Table 1, the separation materials of Examples 1 to 4 within the scope of the present invention contain monoclonal antibodies and It was found that the separation of various proteins and impurities was excellent. It is expected that this will provide excellent separation of carbon and its impurities. In addition, the separation material of Comparative Example 1, which is outside the scope of the present invention, has a number average molecular weight of the copolymer bonded to the carrier. Since the amount exceeds 1,000,000, the separation of monoclonal antibodies and their impurities is poor. It was found that... [Industrial Applicability]

[0061] The separation material of the present invention exhibits high selectivity for antibodies and exhibits high adsorption and separation performance. It has great practical value in the diagnostic field.

Claims

1. A separation material in which a copolymer is bound to a carrier, The copolymer comprises a structural unit derived from a monomer represented by the following formula (1) and a structural unit derived from a monomer represented by the following formula (2): ) a structural unit derived from a monomer represented by the formula The number average molecular weight of the copolymer is 10,000 or more and 1,000,000 or less. Material. 【Chemistry 1】 【Chemistry 2】 (In formula (1), R １ Ha-NR ３ -R ４ -R ５ Or -O-R ４ -R ５ Represents R ２ Hydrogen R represents an alkyl group having 1 to 4 carbon atoms. ３ is a hydrogen atom or a carbon atom having 1 to 4 carbon atoms R represents an alkyl group. ４ represents an alkylene group having 1 to 6 carbon atoms and containing an aliphatic ring, or R represents a linear or branched alkylene group having 1 to 6 carbon atoms. ５ Ha-SO ３ Represents M M represents a hydrogen atom, Na or K. In formula (2), R ６ Ha-NR ８ -R ９ Or -O-R ９ Represents R ７ is a hydrogen atom or carbon number R represents an alkyl group having 1 to 4 alkyl groups. ８ is a hydrogen atom or an alkyl group having 1 to 4 carbon atoms Represents R ９ is a straight-chain or branched alkyl group having 1 to 4 carbon atoms, or an alkoxymethyl group. represents a group or a hydroxyalkyl group.

2. The copolymer contains structural units derived from the monomer represented by the formula (1) as a whole structural units. The separation material according to claim 1 , wherein the content is in the range of 35 mol % to 65 mol %.

3. The monomer represented by the above formula (1) is 2-acrylamide-2-methylpropanesulfone Acid, acrylic acid-3-sulfopropyl acid, methacrylic acid-3-sulfopropyl acid, and The separation material according to claim 1 , which is one or more selected from the group consisting of fluorine-containing compounds and salts thereof.

4. The monomer represented by the above formula (2) is N-hydroxyethylacrylamide. Item 2. The separation material according to item 1.

5. The exchange capacity of the ion exchange group bound to the carrier is 2.5×10 －２ mmol / mL-R or more Top 1.2 x 10 －１ The separation material according to claim 1, wherein the R is mmol / mL-R or less.

6. The carrier has a structural unit derived from a monomer represented by the following formula (3), and a structural unit derived from a monomer represented by the following formula (4): The separation material according to claim 1 , comprising structural units derived from the monomers represented by the formula: 【Chemistry 3】 【Chemistry 4】 (In formula (3), R １１ represents a hydrogen atom or an alkyl group having 1 to 4 carbon atoms. １０ teeth -O-R １２ -R １３ Represents R １２ is a straight-chain or represents a branched alkylene group, or a linear or branched alkylene group having 1 to 6 carbon atoms; Represents: R １３ is an alkyl group, a halogen atom, a hydroxyl group, an amino group, a glycidyl group, or Represents an epoxy group. In formula (4), R １４ represents a linear or branched alkylene group having 1 to 6 carbon atoms. R １５ represents a hydrogen atom or an alkyl group having 1 to 4 carbon atoms.

7. A method for producing a separation material in which a copolymer is bound to a carrier, comprising the steps of: In the presence of the carrier, a monomer represented by the formula (1) and a monomer represented by the formula (2) are reacted with each other. and a chain transfer agent, The monomer represented by the above formula (1) is 35 mol % relative to the total monomer (100 mol %). % or more and 65 mol % or less of a monomer composition is polymerized.

8. The content of the chain transfer agent in the monomer composition is the ratio of the monomer represented by the formula (1) to the monomer represented by the formula (2) (2) 0.4 mol % or more and 2.0 mol % or more relative to the total amount (100 mol %) of the monomers represented by The method for producing a separation material according to claim 7 , wherein the content of the cations in the separation material is 100% by mass or less.

9. The monomer represented by the above formula (1) is 2-acrylamide-2-methylpropanesulfone Acid, acrylic acid-3-sulfopropyl acid, methacrylic acid-3-sulfopropyl acid, and The method for producing a separation material according to claim 7, wherein the organic solvent is at least one selected from the group consisting of fluororesin, fluoroisotope, fluorosulfate, fluoroisotope and fluorosulfur salts.

10. The monomer represented by the above formula (2) is N-hydroxyethylacrylamide. Item 8. A method for producing the separation material according to Item 7.

11. The method for producing a separation material according to claim 7 , wherein the chain transfer agent has a thiol group.

12. A method for producing an antibody, comprising a purification step using the separation material according to claim 1.

13. A method for isolating a target molecule, comprising the steps of: Step (a): Contacting a solution containing a target molecule with the separation material according to claim 1 to separate the target molecule. A step of adsorbing the Step (b): Eluting the target molecule from the separation material treated in step (a).

14. The target molecule is a monoclonal or polyclonal antibody, or a chemical modification thereof. The method for isolating a target molecule according to claim 13 .

15. A method for chromatography comprising at least one container comprising the separation material according to claim 1. column.

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