JP2024178581A - Separation material and method for producing same - Google Patents

Abstract

[Problem] The object of the present invention is to provide an ion exchange separation material in which a copolymer is covalently bonded to a carrier, which is a water-insoluble porous particle, and which satisfies the high level of separation and purification capability that has been demanded in recent years, even in the separation and purification of large molecules such as proteins, and an application technology thereof. [Solution] A separation material in which a copolymer is bonded to a carrier, the copolymer containing a structural unit derived from a monomer represented by a specific formula (1) and a structural unit derived from a monomer represented by a specific formula (2), and the number average molecular weight of the copolymer is 10,000 or more and 1,000,000 or less. [Selected Figure] None

Description

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, a method for producing the same, a method for separating biopolymers such as proteins using the separation material, and a chromatography column.

In research and development of biopolymers such as proteins, chromatography is often used for their adsorption, separation, and purification.
Known carriers used as separation materials for liquid chromatography include inorganic carriers such as silica gel and hydroxyapatite, natural polymer carriers such as agarose, dextran, cellulose, chitosan, and synthetic polymer carriers such as polystyrene and poly(meth)acrylic acid esters. These carriers are used as they are, or are used after various functional groups are added as necessary to enable use in various separation modes.
Moreover, in recent years, there has been a demand for further improvement in the separation and purification capabilities of proteins.

For example, Patent Document 1 discloses a separation material in which a polymer is covalently fixed to porous cross-linked particles, and this technology provides an ion-exchange separation material that has a specific specific surface area, resulting in a large adsorption capacity, excellent strength, and improved dynamic adsorption capacity.

JP 2018-155745 A

The separation material disclosed in Patent Document 1 had sufficient adsorption capacity and strength, but was insufficient in terms of the separation and purification capabilities required in recent years.

The present invention has been made in view of the above problems, and relates to an ion exchange separation material with improved separation and purification capabilities even for large molecules such as proteins, and an application technology thereof.

As a result of extensive research, the present inventors have found that the separation and purification ability of a separation material can be improved by adjusting the molecular weight of a copolymer immobilized on a carrier, and have arrived at the present invention.

（式（１）中、Ｒ_１は－ＮＲ_３－Ｒ_４－Ｒ_５又は－Ｏ－Ｒ_４－Ｒ_５を表す。Ｒ_２は水素原
子又は炭素数１以上４以下のアルキル基を表す。Ｒ_３は水素原子又は炭素数１以上４以下
のアルキル基を表す。Ｒ_４は脂肪族環を含む炭素数１以上６以下のアルキレン基、又は、
炭素数１以上６以下の直鎖もしくは分岐のアルキレン基を表す。Ｒ_５は－ＳＯ_３Ｍを表す
。Ｍは水素原子、Ｎａ又はＫを表す。
式（２）中、Ｒ_６は－ＮＲ_８－Ｒ_９又は－Ｏ－Ｒ_９を表す。Ｒ_７は水素原子又は炭素数
１以上４以下のアルキル基を表す。Ｒ_８は水素原子又は炭素数１以上４以下のアルキル基
を表す。Ｒ_９は炭素数１以上４以下の直鎖もしくは分岐のアルキル基、アルコキシメチル
基又はヒドロキシアルキル基を表す。）
［２］ 前記共重合体が上記式（１）で表される単量体に由来する構造単位を、構造単位
全体の３５ｍｏｌ％以上６５ｍｏｌ％以下の範囲で含む、［１］に記載の分離材。
［３］ 上記式（１）で表される単量体が、２－アクリルアミド－２－メチルプロパンス
ルホン酸、アクリル酸－３－スルホプロピル酸、メタクリル酸－３－スルホプロピル酸、
及び、その塩から選ばれる１種以上である、［１］又は［２］に記載の分離材。
［４］ 上記式（２）で表される単量体が、Ｎ－ヒドロキシエチルアクリルアミドである
、［１］～［３］のいずれかに記載の分離材。
［５］ 前記担体に結合したイオン交換基の交換容量が２．５×１０^－２ｍｍｏｌ／ｍＬ
－Ｒ以上１．２×１０^－１ｍｍｏｌ／ｍＬ－Ｒ以下である、［１］～［４］のいずれかに
記載の分離材。
［６］ 前記担体が下記式（３）で表される単量体に由来する構造単位、及び、下記式（
４）で表される単量体に由来する構造単位を含む、［１］～［５］のいずれかに記載の分
離材。

（式（３）中、Ｒ_１１は水素原子又は炭素数１以上４以下のアルキル基を表す。Ｒ_１０は
－Ｏ－Ｒ_１２－Ｒ_１３を表す。Ｒ_１２は脂肪族環を含む炭素数１以上６以下の直鎖もしく
は分岐のアルキレン基、又は、炭素数１以上６以下の直鎖もくしは分岐のアルキレン基を
表す。Ｒ_１３はアルキル基、ハロゲン原子、ヒドロキシ基、アミノ基、グリシジル基又は
エポキシ基を表す。
式（４）中、Ｒ_１４は炭素数１以上６以下の直鎖もしくは分岐のアルキレン基を表す。
Ｒ_１５は水素原子又は炭素数１以上４以下のアルキル基を表す。） 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 formula
The number average molecular weight of the copolymer is 10,000 or more and 1,000,000 or less.

(In formula (1), R ₁ represents -NR ₃ -R ₄ -R ₅ or -O-R ₄ -R _5. R ₂ represents a hydrogen atom or an alkyl group having 1 to 4 carbon atoms. R ₃ represents a hydrogen atom or an alkyl group having 1 to 4 carbon atoms. R ₄ represents an alkylene group having 1 to 6 carbon atoms and containing an aliphatic ring, or
_R5 represents a linear or branched alkylene group having from 1 to 6 carbon atoms. R5 represents -SO ₃ M. M represents a hydrogen atom, Na or K.
In formula (2), _R6 represents _-NR8 - _R9 or -O- _R9 . _R7 represents a hydrogen atom or an alkyl group having 1 to 4 carbon atoms. _R8 represents a hydrogen atom or an alkyl group having 1 to 4 carbon atoms. _R9 represents a linear or branched alkyl group, an alkoxymethyl group, or a hydroxyalkyl group having 1 to 4 carbon atoms.
[2] The separation material according to [1], wherein the copolymer contains structural units derived from the monomer represented by the above formula (1) in an amount of 35 mol % to 65 mol % of all structural units.
[3] The monomer represented by the above formula (1) is 2-acrylamido-2-methylpropanesulfonic acid, acrylic acid-3-sulfopropyl acid, methacrylic acid-3-sulfopropyl acid,
and salts thereof. The separation material according to [1] or [2],
[4] The separation material according to any one of [1] to [3], wherein the monomer represented by the formula (2) is N-hydroxyethylacrylamide.
[5] The exchange capacity of the ion exchange group bound to the carrier is 2.5×10 ⁻² mmol/mL.
The separation material according to any one of [1] to [4], wherein −R is equal to or greater than 1.2×10 ⁻¹ mmol/mL-R.
[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 separation material according to any one of [1] to [5], which contains a structural unit derived from a monomer represented by the formula (4).

In formula (3), R ₁₁ represents a hydrogen atom or an alkyl group having 1 to 4 carbon atoms. R ₁₀ represents --O--R ₁₂ -R _13. R ₁₂ represents a linear or branched alkylene group containing an aliphatic ring and having 1 to 6 carbon atoms, or a linear or branched alkylene group having 1 to 6 carbon atoms. R ₁₃ represents an alkyl group, a halogen atom, a hydroxy group, an amino group, a glycidyl group, or 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, the monomer represented by the formula (1), the monomer represented by the formula (2), and a chain transfer agent are contained, and the monomer represented by the formula (1) is 35 mol % relative to the total (100 mol %) of the monomer represented by the formula (1) and the monomer represented by the formula (2),
% or more and 65 mol % or less of a monomer composition is polymerized.
[8] The method for producing a separation material according to [7], wherein the content of the chain transfer agent in the monomer composition is 0.4 mol % or more and 2.0 mol % or less with respect to the total (100 mol %) of the monomer represented by the above formula (1) and the monomer represented by the above formula (2).
[9] The monomer represented by the above formula (1) is 2-acrylamido-2-methylpropanesulfonic acid, acrylic acid-3-sulfopropyl acid, methacrylic acid-3-sulfopropyl acid,
and salts thereof.
[10] The method for producing a separation material according to any one of [7] to [9], wherein the monomer represented by the formula (2) is N-hydroxyethylacrylamide.
[11] The method for producing a separation material according to any one of [7] to [10], 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 [1] 1.
[13] A method for isolating a target molecule, comprising the following steps (a) and (b):
Step (a): contacting a solution containing a target molecule with the separation material according to [1] to adsorb the target molecule to the separation material;
Step (b): Eluting the target molecule from the separation material treated in step (a).
[14] The method for isolating a target molecule according to [13], wherein the target molecule is a monoclonal antibody, a polyclonal antibody, or a chemically modified version of these.
[15] A chromatography column comprising the separation material according to [1] and at least one container.

According to the present invention, a separation material can be produced in which a copolymer is immobilized on a support by sufficiently diffusing and polymerizing a water-soluble monomer into the pores of a support suitable as a packing material for chromatography based on a synthetic polymer. The molecular chain length of this copolymer can be adjusted with a chain transfer agent to improve the separation performance of target molecules, making the material useful as a packing material for chromatography.

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 gist of the present invention.

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

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

[Copolymer]
The copolymer of the present invention contains a structural unit derived from a monomer represented by the above formula (1) and a structural unit derived from a monomer represented by the above formula (2).
The monomer represented by the above formula (1) has good polymerization reactivity, good copolymerizability, good polymerization completion, a wide variety of post-treatments and post-reactions, and is easily available industrially. From these viewpoints, the copolymer of the present invention contains a structural unit derived from the monomer represented by the above formula (1).

In the above formula (1), R ₁ represents -NR ₃ -R ₄ -R ₅ or -O-R ₄ -R _5. R ₂ represents a hydrogen atom or an alkyl group having 1 to 4 carbon atoms. R ₃ represents a hydrogen atom or an alkyl group having 1 to 4 carbon atoms.
R ₄ represents an alkyl group having 3 to 6 carbon atoms containing an aliphatic ring, or a linear or branched alkylene group having 1 to 6 carbon atoms. R ₅ represents -SO ₃ M. M represents a hydrogen atom, Na or K.

Examples of the monomer represented by the above formula (1) include 2-acrylamido-2-methylpropanesulfonic acid, acrylic acid-3-sulfopropyl acid, methacrylic acid-3-sulfopropyl acid, and salts thereof.
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,
It may contain more than one species.

The monomer represented by the above formula (2) has good polymerization reactivity, good copolymerizability, good polymerization completion, a wide variety of post-treatments and post-reactions, and is easily available industrially. From these viewpoints, the copolymer of the present invention contains a structural unit derived from the monomer represented by the above formula (2).
In the above formula (2), _R6 represents _-NR8 - _R9 or -O- _R9 . _R7 represents a hydrogen atom or an alkyl group having 1 to 4 carbon atoms. _R8 represents a hydrogen atom or an alkyl group having 1 to 4 carbon atoms. _R9 represents a linear or branched alkyl group, an alkoxymethyl group, or a hydroxyalkyl group having 1 to 4 carbon atoms.

Examples of the monomer represented by the above formula (2) include alkyl (meth)acrylamides such as N-methyl(meth)acrylamide and N-ethyl(meth)acrylamide; hydroxyalkyl (meth)acrylamides such as N-hydroxymethylacrylamide and N-hydroxyethylacrylamide; alkyl (meth)acrylates such as methyl (meth)acrylate and ethyl (meth)acrylate; and hydroxyalkyl (meth)acrylates such as hydroxymethyl (meth)acrylate and hydroxyethyl (meth)acrylate.
Among these, N-hydroxymethyl(meth)acrylamide, N-hydroxyethyl(meth)acrylamide, hydroxymethyl(meth)acrylate, and hydroxyethyl(meth)acrylate are preferred, N-hydroxymethyl(meth)acrylamide and N-hydroxyethyl(meth)acrylamide are more preferred, and N-hydroxyethylacrylamide is even more preferred.
The structural unit derived from the monomer represented by the above formula (2) may contain one type alone,
It may contain more than one species.

The copolymer of the present invention may contain structural units derived from other monomers in addition to the structural units derived from the monomer represented by the above formula (1) and the structural units derived from the monomer represented by the above formula (2). Examples of the other monomers include aromatic vinyl monomers such as styrene, (meth)acrylamide, and (meth)acrylic acid.

The copolymer of the present invention preferably contains structural units derived from the monomer represented by the above formula (1) in a range of 35 mol % or more and 65 mol % or less, and more preferably in a range of 42 mol % or more and 58 mol % or less, of the total structural units.

The structural units derived from other monomers may account for 5 mol % or less of the total structural units.

The number average molecular weight of the copolymer of the present invention is 10,000 or more and 1,000,000 or less,
More preferably, it is 50,000 or more and 120,000 or less.
If the number average molecular weight is within the above range, the degree of separation is high when used for separating antibodies.

[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).

In the above formula (3), R ₁₁ represents a hydrogen atom or an alkyl group having 1 to 4 carbon atoms _.
0 represents -O-R ₁₂ -R _13. R ₁₂ represents a linear or branched alkylene group containing an aliphatic ring and having from 1 to 6 carbon atoms, or a linear or branched alkylene group having from 1 to 6 carbon atoms. R ₁₃ represents an alkyl group, a halogen atom, a hydroxy group, an amino group, a glycidyl group, or an epoxy group.

Examples of the monomer represented by the above formula (3) include methyl (meth)acrylate, ethyl (meth)acrylate, 2-hydroxymethyl (meth)acrylate, 2-hydroxyethyl (meth)acrylate, 2-aminoethyl (meth)acrylate, 2-(dimethylamino)methyl (meth)acrylate, 2-(dimethylamino)ethyl (meth)acrylate, glycidyl (meth)acrylate, 4,5-epoxybutyl (meth)acrylate, 9,10-tetramethylphenyl (meth)acrylate, 1,1 ...
,10-epoxystearyl(meth)acrylate.
The structural unit derived from the monomer represented by the above formula (3) may contain one type alone,
It may contain more than one species.

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

Examples of the monomer represented by (4) above include ethylene glycol di(meth)acrylate, 1,3-propanediol di(meth)acrylate, and tetramethylene glycol di(meth)acrylate.
The structural unit derived from the monomer represented by the above formula (4) may contain one type alone,
It may contain more than one species.

[Separation material]
The separation material of the present invention comprises a copolymer bound to a carrier, and the method for binding the copolymer to the carrier may involve binding a pre-prepared copolymer to the carrier, or may involve adding the carrier and a monomer composition to a reaction solvent and polymerizing the monomer composition in the presence of the carrier.

The separation material of the present invention has an exchange capacity of 2.5×10 ⁻² mmo of the ion exchange group bound to the carrier.
Preferably, the concentration is 1.2×10 ⁻¹ mmol/mL-R or more, and 3.0×10 −1 mmol/mL-R or less ^.
It is more preferable that the concentration is ² mmol/mL-R or more and 5.0×10 ⁻² mmol/mL-R or less.
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 less likely to be distorted, whereas if it is equal to or lower than the upper limit, the selectivity between the antibody and impurities increases.

[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 bound to a carrier, the method comprising the steps of: in the presence of the carrier, producing a separation material which contains a monomer represented by the above formula (1), a monomer represented by the above formula (2), and a chain transfer agent; and, based on the total amount (100 mol%) of the monomer represented by the above formula (1) and the monomer represented by the above formula (2), the amount of the monomer represented by the above formula (1) is 35 mol%.
% or more and 65 mol % or less of a monomer composition is polymerized.

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.

The monomer represented by the above formula (1) and the monomer represented by the above formula (2) are the same as those explained in the section on the separation material.
The monomer represented by the above formula (1) may be used alone or in combination of two or more kinds.
The monomer represented by the above formula (2) may be used alone or in combination of two or more kinds.

The chain transfer agent used in the present invention is not particularly limited as long as it is a commonly used one.
The chain transfer agent preferably has a thiol group, and examples thereof include 1-butanethiol, cyclohexanethiol, 1-decanethiol, 2-ethylhexyl mercaptoacetate, ethyl mercaptoacetate, 1-hexanedecanethiol, 2-mercaptoethanol, 3-mercapto-1,2-propanediol, mercaptoacetic acid, sodium 2-mercaptopropanesulfonate, 3-mercaptopropionic acid, methyl mercaptoacetate, mercaptosuccinic acid, α
-thioglycerol, 1-octanethiol, and thiophenol.

Among these, since water is used as the reaction solvent, 2-mercaptoethanol, 3-mercapto-1,2-propanediol, mercaptoacetic acid, sodium 2-mercaptopropanesulfonate, 3-mercaptopropionic acid, mercaptosuccinic acid, and α-thioglycerol are preferred.
The chain transfer agent may be used alone or in combination of two or more kinds.

The content of the monomer represented by the formula (1) in the monomer composition is 35 mol % or less relative to the total (100 mol %) of the monomer represented by the formula (1) and the monomer represented by the formula (2).
% 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 0.4 mol % or more and 2.0 mol % or less with respect to the total (100 mol %) of the monomer represented by the above formula (1) and the monomer represented by the above formula (2).
% or less, and more preferably 0.4 mol % to 1.0 mol %. Within the above range, the degree of separation is high when used for separating antibodies.

[Method of producing antibodies]
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.

[Method for isolating target molecules]
The method for isolating a target molecule of the present invention is characterized by comprising the following steps (a) and (b):
Step (a): contacting a solution containing a target molecule with the separation material of the present invention to adsorb the target molecule to the separation material;
Step (b): Eluting the target molecule from the separation material treated in step (a).

The target molecule is preferably a monoclonal or polyclonal antibody, or a chemical modification thereof.

[Chromatography columns]
The chromatography column of the present invention is not particularly limited as long as it contains the separation material of the present invention as a packing material and has at least one container.

The present invention will be described in more detail below using examples. However, the present invention is not limited to the description of the following examples as long as it does not deviate from the gist of the present invention.
In the following description, "parts" and "%" indicate "parts by mass" and "% by mass", respectively, unless otherwise specified.

[Evaluation and evaluation method]

<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

(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 to remove the separation material, and then the reaction solution was diluted to 2.5 mg/mL.
The sample was applied to a column and subjected to gel filtration chromatography, after which the number average molecular weight (Mn) of the copolymer was calculated using a calibration curve prepared from dextrans having different molecular weights.

<Measurement of exchange capacity>
The aqueous slurry containing the separation material was placed in a 10 mL graduated cylinder, and the separation material was allowed to settle while tapping, and 5.0 mL of the separation material was measured out. This separation material was filtered and placed in a column to obtain a 1M
The filter was regenerated by passing 100 mL of hydrochloric acid through it, and then thoroughly washed with water, after which it was confirmed that the pH of the effluent water had become neutral.
The resulting separation material was filtered and mixed with 50 mL of a 5% aqueous sodium chloride solution, and the liberated acid was titrated. The titration value was divided by the volume of the separation material to obtain the exchange capacity per mL of the separation material.

<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

(2) Preparation method The column was connected to an LC system and equilibrated with C buffer.
The cell supernatant PrA affinity purified antibody culture solution was applied. After application, the A buffer was increased from 0% to 100% by volume and passed through, and finally the B buffer was passed through to obtain a monoclonal antibody solution. The flow rate was 4.0 mL/min in all cases.
This monoclonal antibody solution was diluted 5-fold, adjusted to pH 2.5 with 1 M hydrochloric acid, and allowed to stand for 90 minutes, and then adjusted to pH 5.0 with 1 M Tris aqueous solution to prepare a monoclonal antibody solution containing 10% impurities.

<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 7 mm, length 26 mm

(2) Separation Method A column packed with the separation material was connected to an LC system and equilibrated with buffer A.
13.5 mL of the monoclonal antibody solution containing impurities was applied to the column. After application, buffer A was passed through the column. The proportion of buffer B was changed from 0% to 10% by volume.
The volumetric flow rate was increased to 0% and the adsorbed monoclonal antibody was eluted and collected at 1 mL intervals.

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

(2) Analysis of monoclonal antibody solution The column was connected to an LC system and equilibrated with PBS buffer. 10 μL of the collected monoclonal antibody solution was applied and analyzed at a flow rate of 0.8 mL/min. This was repeated for the number of collected samples.

(3) Method for calculating resolution (Rs) Peaks of the monoclonal antibody and impurities were obtained by the analysis in (2). The area value of each peak was plotted against the fractionation time to draw a chromatogram.
The time when the monoclonal antibody peak tops is t ₁ (min), and the half-width of the peak is W
_0.5h1 , the peak top of the impurities is _t2 (min), and the half-width of the peak is _W0.5h2 , and Rs was calculated from the following formula.
Rs=1.18×(t ₂ - _{t 1} )/(W _0.5h1 +W _0.5h2 )

[Preparation of Carrier]
<Production Example 1>
A mixture of 40 parts of glycidyl methacrylate (manufactured by FUJIFILM Wako Pure Chemical Industries, Ltd.), 60 parts of ethylene glycol dimethacrylate (manufactured by FUJIFILM Wako Pure Chemical Industries, Ltd.), 1.3 parts of 2,2'-azobis(2,4-dimethylvaleronitrile) (manufactured by FUJIFILM Wako Pure Chemical Industries, Ltd.), and 225 parts of 4-methyl-2-pentanone (manufactured by FUJIFILM Wako Pure Chemical Industries, Ltd.) was added to 1,000 parts of water in which polyvinyl alcohol (manufactured by Nippon Synthetic Chemical Industries, Ltd.) was dissolved to a concentration of 2% at room temperature, and the mixture was stirred in a nitrogen flow atmosphere 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. After cooling, the resulting porous particles made of crosslinked polymer were washed with water, washed with methanol, dried and classified to obtain the desired carrier.

[Example 1]
<Production of separation material>
100 parts of the carrier obtained in Production Example 1 above, 166 parts (40 mol%) of 2-acrylamide-2-methylpropanesulfonic acid (manufactured by Shinryo Co., Ltd.) as a monomer represented by formula (1),
138 parts (60 mol%) of N-(2-hydroxyethyl)acrylamide (manufactured by Tokyo Chemical Industry Co., Ltd.) as a monomer represented by the formula (I), 2 parts (0.5 mol%) of 3-mercapto-1-propanesulfonic acid Na (manufactured by Fuji Film Wako Pure Chemical Industries, Ltd.) as a chain transfer agent, 883 parts of water, and 3 parts of 2,2'-azobis[N-(2-carboxyethyl)-2-methylpropionamidine]n-hydrate (manufactured by Fuji Film Wako Pure Chemical Industries, Ltd.) as a polymerization initiator were mixed and dissolved. This reaction solution was reacted at 50°C for 5 hours.
Thereafter, 180 parts of 47% sulfuric acid (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) was added, and the mixture was reacted for 6 hours at 50° C. After that, the temperature was lowered, and the particles were filtered and washed with water to obtain a separating material.
The molar ratio of 2-acrylamido-2-methylpropanesulfonic acid to N-(2-hydroxyethyl)acrylamide was 40:60.
The separation performance of the separation material of Example 1 was evaluated using a sample containing a monoclonal antibody and its aggregates.
The evaluation results are shown in Table 1.

[Examples 2 to 4]
2-Acrylamido-2-methylpropanesulfonic acid, N-(2-hydroxyethyl)
A separation material was obtained in the same manner as in Example 1, except that the amounts of acrylamide, 3-mercapto-1-propanesulfonic acid Na, and 2,2'-azobis[N-(2-carboxyethyl)-2-methylpropionamidine] n-hydrate were changed as shown in Table 2, and the separation performance was evaluated in the same manner.

[Comparative Example 1]
<Synthesis of copolymer>
70 parts of 2-acrylamido-2-methylpropanesulfonic acid, 50 parts of N-(2-hydroxyethyl)acrylamide, 460 parts of water, and 1 part of 2,2-azobis(2-methylpropionamidine)dihydrochloride as a polymerization initiator were mixed and polymerized at 70°C to obtain an aqueous solution of a copolymer.
Here, the molar ratio of 2-acrylamido-2-methylpropanesulfonic acid to N-(2-hydroxyethyl)acrylamide was 44:56.

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

From Table 1, it was found that the separation materials of Examples 1 to 4, which are within the scope of the present invention, have excellent separation properties for monoclonal antibodies and their impurities. Therefore, it is expected that the separation material of the present invention has excellent separation properties for various proteins and their impurities.
Moreover, the separation material of Comparative Example 1, which is outside the scope of the present invention, was found to have poor separation properties for monoclonal antibodies and their impurities because the number average molecular weight of the copolymer bound to the carrier exceeded 1,000,000.

The separation material of the present invention exhibits separation and adsorption performance with high selectivity, particularly for antibodies, and is useful for the treatment of pharmaceuticals and
It has great practical value in the diagnostic field.

Claims (15)

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.

(In formula (1), R ₁ represents -NR ₃ -R ₄ -R ₅ or -O-R ₄ -R _5. R ₂ represents a hydrogen atom or an alkyl group having 1 to 4 carbon atoms. R ₃ represents a hydrogen atom or an alkyl group having 1 to 4 carbon atoms. R ₄ represents an alkylene group having 1 to 6 carbon atoms and containing an aliphatic ring, or
_R5 represents a linear or branched alkylene group having from 1 to 6 carbon atoms. R5 represents -SO ₃ M. M represents a hydrogen atom, Na or K.
In formula (2), _R6 represents _-NR8 - _R9 or -O- _R9 . _R7 represents a hydrogen atom or an alkyl group having 1 to 4 carbon atoms. _R8 represents a hydrogen atom or an alkyl group having 1 to 4 carbon atoms. _R9 represents a linear or branched alkyl group, an alkoxymethyl group, or a hydroxyalkyl group having 1 to 4 carbon atoms. 2. The separation material according to claim 1, wherein the copolymer contains structural units derived from the monomer represented by formula (1) in an amount of 35 mol % to 65 mol % of all structural units. The monomer represented by the above formula (1) is 2-acrylamido-2-methylpropanesulfonic 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. 2. The separation material according to claim 1, wherein the monomer represented by the formula (2) is N-hydroxyethylacrylamide. 2. The separation material according to claim 1, wherein the exchange capacity of the ion exchange group bound to the carrier is 2.5×10 ⁻² mmol/mL-R or more and 1.2×10 ⁻¹ mmol/mL-R or less. The separation material according to claim 1 , wherein the carrier contains 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):

In formula (3), R ₁₁ represents a hydrogen atom or an alkyl group having 1 to 4 carbon atoms. R ₁₀ represents --O--R ₁₂ -R _13. R ₁₂ represents a linear or branched alkylene group containing an aliphatic ring and having 1 to 6 carbon atoms, or a linear or branched alkylene group having 1 to 6 carbon atoms. R ₁₃ represents an alkyl group, a halogen atom, a hydroxy group, an amino group, a glycidyl group, or 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. 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, the monomer represented by the formula (1), the monomer represented by the formula (2), and a chain transfer agent are contained, and the monomer represented by the formula (1) is 35 mol % relative to the total (100 mol %) of the monomer represented by the formula (1) and the monomer represented by the formula (2),
% or more and 65 mol % or less of a monomer composition is polymerized. The content of the chain transfer agent in the monomer composition is 0.4 mol % or more and 2.0 mol % or less with respect to the total (100 mol %) of the monomer represented by the formula (1) and the monomer represented by the formula (2).
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. The monomer represented by the above formula (1) is 2-acrylamido-2-methylpropanesulfonic 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, fluoroalkylsulfuric acid, fluorophenylsulfuric acid, fluoro ... The method for producing a separation material according to claim 7, wherein the monomer represented by the formula (2) is N-hydroxyethylacrylamide. The method for producing a separation material according to claim 7, wherein the chain transfer agent has a thiol group. An antibody production method comprising a purification step using the separation material according to claim 1. 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 adsorb the target molecule to the separation material;
Step (b): Eluting the target molecule from the separation material treated in step (a). The method for isolating a target molecule according to claim 13, wherein the target molecule is a monoclonal or polyclonal antibody, or a chemically modified version thereof. A chromatography column comprising at least one container comprising the separation material according to claim 1.