WO2024029394A1

WO2024029394A1 - Method for producing carrier for chromatography, and carrier for chromatography

Info

Publication number: WO2024029394A1
Application number: PCT/JP2023/027099
Authority: WO
Inventors: 透矢井上; 健宮島; 倫敬上出; 浩志野口; 聡中村
Original assignee: Jsr株式会社
Priority date: 2022-08-01
Filing date: 2023-07-25
Publication date: 2024-02-08

Abstract

The present invention provides a carrier which is for chromatography, which has a large dynamic binding capacity with respect to an antibody or a fragment thereof, and through which a protein ligand is unlikely to leak even when the carrier is repeatedly used for isolation of an antibody.　Provided is a method for producing a carrier for chromatography, said method comprising the following steps A-1 and B.　(Step A-1) A step for immobilizing, to porous particles, one or more ligands selected from protein A, protein G, protein L, and analogues thereof. (Step B) A step for reacting the porous particles to which the one or more ligands have been immobilized in the step A-1 and a compound which has at least one ligand reactive group selected from groups represented by -C(=O)-O-C(=O)-, carbodiimide groups, and cyclic ether groups.

Description

Method for producing chromatography carrier and chromatography carrier

The present invention relates to a method for producing a chromatography carrier, a chromatography carrier, a chromatography column, and a method for isolating an antibody or a fragment thereof.

In recent years, antibodies have been widely used as research reagents, antibody drugs, etc. These reagents and antibodies for pharmaceutical use are generally produced through isolation by chromatography. The carrier used for such chromatography is required to have a dynamic binding capacity for antibodies and fragments thereof, to be difficult to leak out of the ligand during isolation, and to be difficult to non-specifically adsorb impurities. For example, a carrier is known in which the dynamic binding capacity for antibodies is increased by adjusting the median particle size to a specific range (Patent Document 1).

Special Publication No. 2008-523140 WO2020/040307 publication JP2022-62096A

In addition, it has been proposed to use immunoglobulin-binding proteins with mutated polypeptide chains as ligands in order to improve alkaline tolerance and prevent the ligand from leaking even when used repeatedly for antibody isolation. Patent Document 2, Patent Document 3), there is a need for further improvement in low leakage of protein ligands. In addition, mutations in the polypeptide chain to improve alkaline tolerance as described above can reduce the dynamic binding capacity of the original immunoglobulin binding protein, and thus increase the dynamic binding capacity for antibodies or fragments thereof. It was difficult to achieve both this and suppressing the leakage of protein ligands.
The problem to be solved by the present invention is to provide a chromatography carrier that has a large dynamic binding capacity for antibodies or fragments thereof, and that protein ligands do not easily leak out even when used repeatedly for antibody isolation.

The above problem was solved by the following means <1> to <19>.
<1> A method for producing a chromatography carrier (hereinafter also referred to as a method for producing a chromatography carrier of the present invention), comprising the following Step A-1 and Step B.
(Step A-1) Step of immobilizing one or more kinds of ligands selected from protein A, protein G, protein L, and related substances to porous particles. (Step B) Ligand after step A-1. porous particles on which is fixed, and at least one ligand-reactive group selected from a group represented by -C(=O)-OC(=O)-, a carbodiimide group, and a cyclic ether group. Process of reacting with a compound

<2> The compound having the ligand-reactive group is a compound represented by the following formula (1) and its salt, a compound represented by the following formula (2), and a compound represented by the following formula (3). The method for producing a chromatography carrier according to <1>, which is one or more selected types.

[In formula (1), R ¹ and R ² each independently represent a hydrogen atom or a substituted or unsubstituted hydrocarbon group. ]

[In formula (2), R ³ and R ⁴ independently represent a substituted or unsubstituted hydrocarbon group, and R ³ and R ⁴ may be bonded to each other to form a cyclic structure. ]

[In formula (3), R ⁵ represents a substituted or unsubstituted hydrocarbon group, and X represents a cyclic ether group. ]

<3> The compound having the ligand-reactive group is 1-[3-(dimethylamino)propyl]-3-ethylcarbodiimide, a salt of 1-[3-(dimethylamino)propyl]-3-ethylcarbodiimide, N , N'-dicyclohexylcarbodiimide, N,N'-diisopropylcarbodiimide, maleic anhydride, propionic anhydride, acetic anhydride, pivalic anhydride, succinic anhydride, glutaric anhydride, propylene oxide, butylene oxide, glycidyl methyl ether, The method for producing a chromatography carrier according to <1> or <2>, which is one or more compounds selected from ethyl glycidyl ether, glycidol, epichlorohydrin, and epibromohydrin.

<4> The compound having a ligand-reactive group is at least one compound selected from the compound represented by the formula (1) and a salt thereof, according to any one of <1> to <3>. A method for producing a carrier for chromatography.
<5> Any one of <1> to <4>, wherein the amount of the compound having a ligand-reactive group used is 0.01 to 15 mmol per 1 g of dry weight of the porous particles on which the ligand is immobilized. A method for producing a chromatography carrier as described in .

<6> At least one or two or more substitutions selected from the following (a) to (i) to the amino acid sequence in which the ligand has 85% or more homology with the amino acid sequence shown in SEQ ID NO: 2. The method for producing a chromatography carrier according to any one of <1> to <5>, which is a protein ligand having an amino acid sequence in which the amino acid sequence has been changed.
(a) Substitution of the amino acid residue at the position corresponding to position 1 of the amino acid sequence of SEQ ID NO: 2 with a valine residue (b) Substitution of the amino acid residue at the position corresponding to position 3 of the amino acid sequence of SEQ ID NO: 2 with an alanine residue (c) Substitution of the amino acid residue at the position corresponding to position 6 of the amino acid sequence of SEQ ID NO: 2 with an alanine residue or aspartic acid residue (d) Corresponding to position 9 of the amino acid sequence of SEQ ID NO: 2 (e) Substitution of the amino acid residue at the position corresponding to position 11 of the amino acid sequence of SEQ ID NO: 2 with an alanine residue, glutamine residue, or glutamic acid residue (f ) Substitution of the amino acid residue at the position corresponding to position 23 of the amino acid sequence of SEQ ID NO: 2 with a leucine residue (g) Substitution of the amino acid residue at the position corresponding to position 29 of the amino acid sequence of SEQ ID NO: 2 with an alanine residue (h) Substitution of the amino acid residue at the position corresponding to position 43 of the amino acid sequence of SEQ ID NO: 2 with an alanine residue (i) Substitution of the amino acid residue at the position corresponding to position 49 of the amino acid sequence of SEQ ID NO: 2 Substitution to arginine residue

<7> The ligand has at least three or more substitutions selected from the following (a) to (i) to the amino acid sequence having 85% or more homology with the amino acid sequence shown in SEQ ID NO: 2. The method for producing a chromatography carrier according to any one of <1> to <5>, which is a protein ligand having an amino acid sequence.
(a) Substitution of the amino acid residue at the position corresponding to position 1 of the amino acid sequence of SEQ ID NO: 2 with a valine residue (b) Substitution of the amino acid residue at the position corresponding to position 3 of the amino acid sequence of SEQ ID NO: 2 with an alanine residue (c) Substitution of the amino acid residue at the position corresponding to position 6 of the amino acid sequence of SEQ ID NO: 2 with an alanine residue or aspartic acid residue (d) Corresponding to position 9 of the amino acid sequence of SEQ ID NO: 2 (e) Substitution of the amino acid residue at the position corresponding to position 11 of the amino acid sequence of SEQ ID NO: 2 with an alanine residue, glutamine residue, or glutamic acid residue (f ) Substitution of the amino acid residue at the position corresponding to position 23 of the amino acid sequence of SEQ ID NO: 2 with a leucine residue (g) Substitution of the amino acid residue at the position corresponding to position 29 of the amino acid sequence of SEQ ID NO: 2 with an alanine residue (h) Substitution of the amino acid residue at the position corresponding to position 43 of the amino acid sequence of SEQ ID NO: 2 with an alanine residue (i) Substitution of the amino acid residue at the position corresponding to position 49 of the amino acid sequence of SEQ ID NO: 2 Substitution to arginine residue

<8> The method for producing a chromatography carrier according to any one of <1> to <7>, wherein the reaction in step B is carried out in an aqueous medium at a pH of 8 to 14.

<9> The following step A-2 is further provided between step A-1 and step B, and the hydrophilic group-containing ligand-immobilized porous particles obtained in step A-2 are treated with the ligand after step A-1. The method for producing a chromatography carrier according to any one of <1> to <8>, which is used in Step B as a fixed porous particle.
(Step A-2) The porous particles on which the ligand was fixed in Step A-1 are reacted with a compound having a total of two or more hydrophilic groups of at least one type selected from hydroxy groups and mercapto groups in the molecule. process of letting

<10> Step A further comprising the following steps A-P1 and A-P2, using porous particles reacted with at least one selected from a crosslinking agent and a hydrophilizing agent in step A-P2 as the porous particles. -1, the method for producing a chromatography carrier according to any one of <1> to <9>.
(Step A-P1) A step of dispersing the monomer composition in an aqueous medium and carrying out suspension polymerization. (Step A-P2) The porous particles obtained in step A-P1 and at least one selected from a crosslinking agent and a hydrophilizing agent. Step of reacting with one species

<11> A porous particle, a ligand fixed to the porous particle, and a group selected from -C(=O)-OC(=O)-, a carbodiimide group, and a cyclic ether group and a partial structure derived from a compound having at least one type of ligand-reactive group,
The ligand is one or more ligands selected from protein A, protein G, protein L and related substances,
At least one functional group selected from an amino group and a carboxy group of the ligand is chemically modified with the partial structure,
The following formula:
Chemical modification rate (mol%) = (number of moles of chemically modified functional groups) / (sum of number of moles of chemically modified functional groups and number of moles of non-chemically modified functional groups) × 100
A chromatography carrier (hereinafter also referred to as the chromatography carrier of the present invention) having a chemical modification rate calculated from 1 to 70 mol%.

<12> The chromatography carrier according to <11>, wherein the amino group of the ligand is chemically modified with the partial structure.
<13> The partial structure is -C(=O)-, -NR ¹ -C(=O)-, -C(=NR ¹ )-, or -CH ₂ -CH(-OH)- (R ¹ is , a hydrogen atom, an alkyl group, a cycloalkyl group, an aminoalkyl group, a monoalkylaminoalkyl group, or a dialkylaminoalkyl group. chromatography carrier.

<14> The partial structure is -NR ¹ -C(=O)-NR ² - or -C(=NR ¹ )-NR ² - (R ¹ and R ² are each independently a hydrogen atom, an alkyl The chromatograph according to any one of <11> to <13>, which has a group represented by the following in its molecule: cycloalkyl group, aminoalkyl group, monoalkylaminoalkyl group, or dialkylaminoalkyl group. Carrier for graphics.
<15> The chromatography carrier according to <14>, wherein at least one of R ¹ and R ² is an aminoalkyl group, a monoalkylaminoalkyl group, or a dialkylaminoalkyl group.

<16> At least one or two or more substitutions selected from the following (a) to (i) to the amino acid sequence in which the ligand has 85% or more homology with the amino acid sequence shown in SEQ ID NO: 2. The chromatography carrier according to any one of <11> to <15>, which is a protein ligand having an amino acid sequence in which the amino acid sequence has been changed.
(a) Substitution of the amino acid residue at the position corresponding to position 1 of the amino acid sequence of SEQ ID NO: 2 with a valine residue (b) Substitution of the amino acid residue at the position corresponding to position 3 of the amino acid sequence of SEQ ID NO: 2 with an alanine residue (c) Substitution of the amino acid residue at the position corresponding to position 6 of the amino acid sequence of SEQ ID NO: 2 with an alanine residue or aspartic acid residue (d) Corresponding to position 9 of the amino acid sequence of SEQ ID NO: 2 (e) Substitution of the amino acid residue at the position corresponding to position 11 of the amino acid sequence of SEQ ID NO: 2 with an alanine residue, glutamine residue, or glutamic acid residue (f ) Substitution of the amino acid residue at the position corresponding to position 23 of the amino acid sequence of SEQ ID NO: 2 with a leucine residue (g) Substitution of the amino acid residue at the position corresponding to position 29 of the amino acid sequence of SEQ ID NO: 2 with an alanine residue (h) Substitution of the amino acid residue at the position corresponding to position 43 of the amino acid sequence of SEQ ID NO: 2 with an alanine residue (i) Substitution of the amino acid residue at the position corresponding to position 49 of the amino acid sequence of SEQ ID NO: 2 Substitution to arginine residue

<17> The ligand has at least three or more substitutions selected from the following (a) to (i) to the amino acid sequence having 85% or more homology with the amino acid sequence shown in SEQ ID NO: 2. The chromatography carrier according to any one of <11> to <15>, which is a protein ligand having an amino acid sequence.
(a) Substitution of the amino acid residue at the position corresponding to position 1 of the amino acid sequence of SEQ ID NO: 2 with a valine residue (b) Substitution of the amino acid residue at the position corresponding to position 3 of the amino acid sequence of SEQ ID NO: 2 with an alanine residue (c) Substitution of the amino acid residue at the position corresponding to position 6 of the amino acid sequence of SEQ ID NO: 2 with an alanine residue or aspartic acid residue (d) Corresponding to position 9 of the amino acid sequence of SEQ ID NO: 2 (e) Substitution of the amino acid residue at the position corresponding to position 11 of the amino acid sequence of SEQ ID NO: 2 with an alanine residue, glutamine residue, or glutamic acid residue (f ) Substitution of the amino acid residue at the position corresponding to position 23 of the amino acid sequence of SEQ ID NO: 2 with a leucine residue (g) Substitution of the amino acid residue at the position corresponding to position 29 of the amino acid sequence of SEQ ID NO: 2 with an alanine residue (h) Substitution of the amino acid residue at the position corresponding to position 43 of the amino acid sequence of SEQ ID NO: 2 with an alanine residue (i) Substitution of the amino acid residue at the position corresponding to position 49 of the amino acid sequence of SEQ ID NO: 2 Substitution to arginine residue

<18> A chromatography column comprising the chromatography carrier according to any one of <11> to <17>.
<19> A method for isolating an antibody or a fragment thereof using the chromatography carrier according to any one of <11> to <17> or the chromatography column according to <18> (hereinafter, a method for isolating an antibody or a fragment thereof of the present invention) (also referred to as fragment isolation method).

According to the method for producing a chromatography carrier of the present invention, it is possible to easily produce a chromatography carrier that has a large dynamic binding capacity for antibodies or fragments thereof and that is resistant to leakage of protein ligands even when used repeatedly for antibody isolation. Can be manufactured.
The chromatography carrier of the present invention has a large dynamic binding capacity for antibodies or fragments thereof, and protein ligands do not easily leak out even when used repeatedly for antibody isolation.
Therefore, according to the present invention, it is possible to provide a chromatography column that has a large dynamic binding capacity for antibodies or fragments thereof and that does not leak protein ligands even when used repeatedly for antibody isolation.

[Method for manufacturing carrier for chromatography]
The method for producing a chromatography carrier of the present invention includes (Step A-1) immobilizing one or more ligands selected from protein A, protein G, protein L, and their related substances onto porous particles. It has a process.
-Process A-1-
Step A-1 is a step of immobilizing one or more ligands selected from protein A, protein G, protein L, and related substances to porous particles.
As the porous particles, porous particles containing a polymer are preferred. Such porous particles may be natural polymer porous particles or synthetic polymer porous particles composed of polysaccharides such as agarose, dextran, cellulose, etc., but in order to increase the dynamic binding capacity or In order to improve the uniformity of particle diameter, synthetic polymer porous particles are preferred. Also, the porous particles are preferably water-insoluble.

As the porous particles, commercially available products or those manufactured according to conventional methods may be used. Here, a method for manufacturing porous particles will be explained.
Porous particles can be produced by a method including a step of dispersing a monomer composition in an aqueous medium and carrying out suspension polymerization (hereinafter also referred to as steps A-P1).

-Process A-P1-
The monomer composition used in step A-P1 preferably contains a functional group-containing monomer. The functional group contained in this monomer is preferably one that can be used for additional chemical reactions (such as reaction with a crosslinking agent), and may be one that can immobilize a ligand. Examples of the functional group include a cyclic ether group, a carboxy group, -C(=O)-OC(=O)-, a succinimidoxycarbonyl group, a formyl group, a hydroxyl group, and an isocyanate group. Examples include functional groups such as Among these, cyclic ether groups are preferred.
Here, the "cyclic ether group" is preferably a cyclic ether group having 3 to 7 ring atoms. The cyclic ether group may have an alkyl group as a substituent. Specific examples of the cyclic ether group include cyclic ether groups represented by the following formulas (4) to (9); is preferred, and a cyclic ether group represented by formula (4) is more preferred.

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

The number of carbon atoms in the alkyl group represented by R ¹¹ to R ¹⁴ is preferably 1 to 4, more preferably 1 or 2. The alkyl group may be linear or branched, and includes, for example, a methyl group, ethyl group, n-propyl group, isopropyl group, n-butyl group, sec-butyl group, tert-butyl group, and the like. Furthermore, R ¹¹ to R ¹⁴ are preferably hydrogen atoms.

As the functional group-containing monomer, a monomer having a functional group capable of immobilizing a ligand and a polymerizable unsaturated group is preferable. Examples of such monomers include glycidyl (meth)acrylate, 3-oxiranylpropyl (meth)acrylate, 4-oxiranylbutyl (meth)acrylate, 5-oxiranylpentyl (meth)acrylate, and 6-oxiranylpentyl (meth)acrylate. Oxiranylhexyl (meth)acrylate, 7-oxiranylheptyl (meth)acrylate, 8-oxiranyl octyl (meth)acrylate, (3-methyloxiranyl)methyl (meth)acrylate, 4-hydroxybutyl (meth)acrylate ) Acrylate glycidyl ether, glycerin mono(meth)acrylate glycidyl ether, 3,4-epoxycyclohexylmethyl (meth)acrylate, 3,4-epoxycyclohexyl ethyl (meth)acrylate, 3,4-epoxycyclohexylpropyl (meth)acrylate, (Meth)acrylate monomers having a cyclic ether group such as α-(meth)acrylic-ω-glycidyl polyethylene glycol, tetrahydrofurfuryl (meth)acrylate; (vinylbenzyl)glycidyl ether, (isopropenylbenzyl)glycidyl ether, ( Vinylphenethyl)glycidyl ether, (vinylphenylbutyl)glycidyl ether, (vinylbenzyloxyethyl)glycidyl ether, (vinylphenyl)glycidyl ether, (isopropenylphenyl)glycidyl ether, 1,2-epoxy-3-(4-vinyl) Aromatic vinyl monomers having a cyclic ether group such as benzyl) propane; Allyl ether monomers having a cyclic ether group such as allyl glycidyl ether; (meth)acrylate monomers having an isocyanate group such as isocyanatoethyl (meth)acrylate ; In addition to unsaturated dicarboxylic anhydride monomers such as maleic anhydride, methylmaleic anhydride, and glutaconic anhydride, (meth)acrylic acid, 3,4-epoxy-1-butene, 3,4-epoxy -3-methyl-1-butene and the like. These monomers can be used alone or in combination of two or more.
Among these monomers, (meth)acrylate monomers having a cyclic ether group are preferred, and glycidyl (meth)acrylate is particularly preferred.

The total amount of functional group-containing monomers used is preferably 35 parts by mass or more, more preferably 45 parts by mass or more, particularly preferably 55 parts by mass or more, based on 100 parts by mass of the total amount of monomers used in Step A-P1. Also, it is preferably 99 parts by mass or less, more preferably 90 parts by mass or less, particularly preferably 85 parts by mass or less, based on 100 parts by mass of the total amount of monomers used in Step A-P1.

In addition to the functional group-containing monomer, the monomer composition used in Step A-P1 may further contain a monomer other than the functional group-containing monomer (hereinafter also referred to as other monomer).
Other monomers include polymerizable unsaturated group-containing monomers that do not have a functional group capable of immobilizing a ligand. Other monomers are broadly classified into non-crosslinkable monomers and crosslinkable monomers, and one or both of these monomers may be used. Furthermore, according to the present invention, even when a monomer that does not contain a hydrophilic group such as a hydroxy group is used as another monomer, it is possible to satisfy the stain resistance, and it is applicable to a wide range of monomer compositions. be.

Examples of the non-crosslinking monomer include (meth)acrylate non-crosslinking monomer, (meth)acrylamide non-crosslinking monomer, aromatic vinyl non-crosslinking monomer, vinyl ketone non-crosslinking monomer, (meth)acrylonitrile. Examples include non-crosslinking monomers, N-vinylamide non-crosslinking monomers, and the like. These can be used alone or in combination of two or more. Among the non-crosslinking monomers, (meth)acrylate non-crosslinking monomers and aromatic vinyl non-crosslinking monomers are preferred.

Examples of the (meth)acrylate non-crosslinking monomer include methyl (meth)acrylate, ethyl (meth)acrylate, n-butyl (meth)acrylate, 4-tert-butyl (meth)acrylate, and isobutyl (meth)acrylate. , n-octyl (meth)acrylate, 2-ethylhexyl (meth)acrylate, cyclohexyl (meth)acrylate, methoxyethyl (meth)acrylate, hydroxyethyl (meth)acrylate, hydroxypropyl (meth)acrylate, glycerol mono(meth)acrylate , trimethylolethane mono(meth)acrylate, trimethylolpropane mono(meth)acrylate, butanetriol mono(meth)acrylate, polyethylene glycol mono(meth)acrylate, methoxypolyethylene glycol(meth)acrylate, pentaerythritol mono(meth)acrylate , dipentaerythritol mono(meth)acrylate, inositol mono(meth)acrylate, and the like. These can be used alone or in combination of two or more.

Examples of the (meth)acrylamide non-crosslinkable monomer include (meth)acrylamide, dimethyl (meth)acrylamide, hydroxyethyl (meth)acrylamide, (meth)acryloylmorpholine, diacetone (meth)acrylamide, etc. It will be done. These can be used alone or in combination of two or more.

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

Examples of the vinyl ketone non-crosslinkable monomer include ethyl vinyl ketone, propyl vinyl ketone, isopropyl vinyl ketone, and the like. These can be used alone or in combination of two or more.
Further, examples of the (meth)acrylonitrile non-crosslinking monomer include acrylonitrile, methacrylonitrile, and the like. These can be used alone or in combination of two or more.
Further, examples of the N-vinylamide non-crosslinking monomer include N-vinylacetamide, N-vinylpropionamide, and the like. These can be used alone or in combination of two or more.

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, particularly preferably 0. .1 parts by mass or more, and preferably 30 parts by mass or less, more preferably 15 parts by mass or less, particularly preferably 5 parts by mass or less, based on 100 parts by mass of the total amount of monomers used in Step A-P1. .

Examples of the crosslinkable monomer include (meth)acrylate crosslinkable monomers, aromatic vinyl crosslinkable monomers, allyl crosslinkable monomers, and the like. These can be used alone or in combination of two or more. Further, as the crosslinking monomer, a di- to penta-functional cross-linking monomer is preferable, and a di- or tri-functional cross-linking monomer is more preferable. Among the crosslinking monomers, (meth)acrylate crosslinking monomers and aromatic vinyl crosslinking monomers are preferred.

Examples of the (meth)acrylate crosslinking monomer include ethylene glycol di(meth)acrylate, diethylene glycol di(meth)acrylate, triethylene glycol di(meth)acrylate, tetraethylene glycol di(meth)acrylate, and polyethylene glycol di(meth)acrylate. (meth)acrylate, propylene glycol di(meth)acrylate, dipropylene glycol di(meth)acrylate, tripropylene glycol di(meth)acrylate, tetrapropylene glycol di(meth)acrylate, polypropylene glycol di(meth)acrylate, 1, 4-butanediol di(meth)acrylate, 1,6-hexanediol di(meth)acrylate, glycerin di(meth)acrylate, trimethylolethane di(meth)acrylate, trimethylolpropane di(meth)acrylate, trimethylolpropane Tri(meth)acrylate, butanetriol di(meth)acrylate, pentaerythritol di(meth)acrylate, pentaerythritol tri(meth)acrylate, pentaerythritol tetra(meth)acrylate, glucose di(meth)acrylate, glucose tri(meth)acrylate Acrylate, glucose tetra(meth)acrylate, dipentaerythritol di(meth)acrylate, dipentaerythritol tri(meth)acrylate, dipentaerythritol tetra(meth)acrylate, dipentaerythritol penta(meth)acrylate, inositol di(meth)acrylate Acrylate, inositol tri(meth)acrylate, inositol tetra(meth)acrylate, mannitol di(meth)acrylate, mannitol tri(meth)acrylate, mannitol tetra(meth)acrylate, mannitol penta(meth)acrylate, and the like. These can be used alone or in combination of two or more.

Examples of the aromatic vinyl crosslinking monomers include divinylbenzene, trivinylbenzene, divinyltoluene, divinylxylene, divinylethylbenzene, and divinylnaphthalene. These can be used alone or in combination of two or more.

Examples of the allyl crosslinking monomer include diallyl phthalate, diallyl isophthalate, diallyl terephthalate, diallyl maleate, diallyl fumarate, diallyl itaconate, diallyl trimellitate, triallyl trimellitate, and triallyl cyanurate. , diallyl isocyanurate, triallyl isocyanurate, and the like. These can be used alone or in combination of two or more.
Furthermore, in addition to the above-mentioned examples, examples of crosslinking monomers include dehydration condensation products of amino alcohols such as diaminopropanol, trishydroxymethylaminomethane, and glucosamine with (meth)acrylic acid, and conjugated monomers such as butadiene and isoprene. Examples include olefins.

The total amount of crosslinking monomers used is preferably 1 part by mass or more, more preferably 5 parts by mass or more, particularly preferably 10 parts by mass or more, based on 100 parts by mass of the total amount of monomers used in Steps A-P1. Also, it is preferably 50 parts by mass or less, more preferably 40 parts by mass or less, particularly preferably 30 parts by mass or less, based on 100 parts by mass of the total amount of monomers used in Step A-P1.

Examples of the aqueous medium used in Step A-P1 include an aqueous solution of a water-soluble polymer, and examples of the water-soluble polymer include hydroxyethyl cellulose, polyvinyl alcohol, carboxymethyl cellulose, polyvinylpyrrolidone, starch, and gelatin.
The total amount of the aqueous medium used is usually about 200 parts by mass or more and 7000 parts by mass or less based on 100 parts by mass of the total amount of monomers.
Furthermore, when water is used as a dispersion medium in an aqueous medium, a dispersion stabilizer such as sodium carbonate, calcium carbonate, sodium sulfate, calcium phosphate, or sodium chloride may be used.

Further, as a specific method for Step A-P1, for example, a polymerization initiator is dissolved in a mixed solution (monomer solution) containing a monomer composition and, if necessary, a porosity-forming agent, and suspended in an aqueous medium. Alternatively, a polymerization initiator may be dissolved in a mixed solution (monomer solution) containing a monomer composition and, if necessary, a porosity agent, and then added to an aqueous medium heated to a predetermined temperature. A method in which a mixed solution (monomer solution) containing a monomer composition and, if necessary, a porosity-forming agent is suspended in an aqueous medium, heated to a predetermined temperature, and a polymerization initiator is added and polymerized. etc.

As the polymerization initiator, a radical polymerization initiator is preferable. Examples of the radical polymerization initiator include azo initiators, peroxide initiators, redox initiators, etc. Specifically, azobisisobutyronitrile, methyl azobisisobutyrate, azobis-2, Examples include 4-dimethylvaleronitrile, benzoyl peroxide, di-tert-butyl peroxide, benzoyl peroxide-dimethylaniline, and the like. The total amount of the polymerization initiator used is usually about 0.01 parts by mass or more and 10 parts by mass or less based on 100 parts by mass of the total amount of monomers.

The above-mentioned porosity agent is used to produce porous particles, exists together with the monomer during polymerization within the oil droplets, and has the role of forming pores as a non-polymerized component. The pore-forming agent is not particularly limited as long as it can be easily removed from the porous surface, and examples include linear polymers that are soluble in various organic solvents and mixed monomers. May be used together.

Examples of the porosity-forming agent include aliphatic hydrocarbons such as hexane, heptane, octane, nonane, decane, and undecane; alicyclic hydrocarbons such as cyclopentane and cyclohexane; benzene, toluene, xylene, naphthalene, and ethylbenzene. Aromatic hydrocarbons such as carbon tetrachloride, 1,2-dichloroethane, tetrachloroethane, chlorobenzene, etc.; halogenated hydrocarbons such as butanol, pentanol, hexanol, heptanol, 4-methyl-2-pentanol, 2- Aliphatic alcohols such as ethyl-1-hexanol; alicyclic alcohols such as cyclohexanol; aromatic alcohols such as 2-phenylethyl alcohol and benzyl alcohol; diethyl ketone, methyl isobutyl ketone, diisobutyl ketone, acetophenone, 2 -Ketones such as octanone and cyclohexanone; ethers such as dibutyl ether, diisobutyl ether, anisole, and ethoxybenzene; esters such as isopentyl acetate, butyl acetate, 3-methoxybutyl acetate, diethyl malonate, and non-crosslinkable Examples include linear polymers such as homopolymers of vinyl monomers. Porosifying agents can be used alone or in combination of two or more.
The total amount of the porosity-forming agent used is usually about 40 parts by mass or more and 600 parts by mass or less based on 100 parts by mass of the total amount of monomers.

Furthermore, various surfactants including anionic surfactants such as alkyl sulfate salts, alkylaryl sulfate salts, alkyl phosphate salts, and fatty acid salts may be used in Step A-P1. Further, polymerization inhibitors such as nitrite salts such as sodium nitrite, iodide salts such as potassium iodide, tert-butylpyrocatechol, benzoquinone, picric acid, hydroquinone, copper chloride, and ferric chloride can also be used. Additionally, a polymerization regulator such as dodecyl mercaptan may be used.

Further, the polymerization temperature in Steps A-P1 may be determined depending on the polymerization initiator, but is usually about 2 to 100°C, preferably 50 to 100°C. The polymerization time is usually 5 minutes to 48 hours, preferably 10 minutes to 24 hours.

-Process A-P2-
Further, prior to step A-1, a step (hereinafter also referred to as step A-P2) of reacting the porous particles obtained in step A-P1 with at least one selected from a crosslinking agent and a hydrophilic agent is performed. You may go. When both a crosslinking agent and a hydrophilic agent are used, the hydrophilic reaction may be performed after the crosslinking reaction, or the crosslinking reaction may be performed after the hydrophilic reaction. Further, the crosslinking reaction and the hydrophilization reaction may be performed simultaneously.
When a monomer composition containing a functional group-containing monomer is used in Step A-P1, the crosslinking reaction causes an addition reaction of the crosslinking agent to a part of the functional groups that the porous particles have in the polymer molecules. A partial structure derived from the crosslinking agent is introduced. As a result, the residues of the functional groups are crosslinked with each other via the partial structure derived from the crosslinking agent.
In addition, when a monomer composition containing a functional group-containing monomer is used in Step A-P1, the hydrophilic agent is attached to a part of the functional groups that the porous particles have in the polymer molecule due to the above hydrophilic reaction. An addition reaction is carried out, and a partial structure derived from the hydrophilizing agent is introduced.

The crosslinking agent used in Steps A-P2 may be one that can introduce a crosslinked structure by reacting with a functional group that can immobilize a ligand, but it may also be one that can introduce a crosslinked structure by reacting with a functional group that can immobilize a ligand. A crosslinking agent containing at least two groups represented by , -C(=O)-NH- in the molecule is preferred.

When the porous particles obtained in step A-P1 have a cyclic ether group, specifically, at least a group represented by -C(=O)-NH-NH ₂ is included in the molecule as a crosslinkable group. Use a crosslinking agent containing at least two groups represented by -C(=O)-NH- in the molecule and at least two carboxy groups as crosslinkable groups in the molecule. I can do it.
When the porous particles obtained in step A-P1 have a carboxy group, -C(=O)-OC(=O)-, succinimidoxycarbonyl group, formyl group or isocyanate group, specific Specifically, a crosslinking agent containing at least two groups represented by -C(=O)-NH-NH ₂ as crosslinkable groups in the molecule can be used.

Examples of crosslinking agents containing at least two groups represented by -C(=O)-NH- in the molecule include oxalyl dihydrazide, malonic acid dihydrazide, succinic acid dihydrazide, and 2,3-dihydroxysuccinic acid. Dihydrazide, glutaric acid dihydrazide, adipic acid dihydrazide, pimelic acid dihydrazide, octanedioic acid dihydrazide, nonanedioic acid dihydrazide, sebacic acid dihydrazide, dodecanedioic acid dihydrazide, phthalic acid dihydrazide, isophthalic acid dihydrazide, terephthalic acid dihydrazide, quinolic acid dihydrazide, etc. Dicarboxylic acid dihydrazides; Tricarboxylic acid trihydrazides such as cyclohexanetricarboxylic acid trihydrazide; (alkylenebisimino)bis(oxoalkanoic acids) such as N1,N1-(ethane-1,2-diyl)bis(succinic acid monoamide); etc. One type of crosslinking agent can be used alone or two or more types can be used in combination. Among these crosslinking agents, dicarboxylic acid dihydrazides and (alkylenebisimino)bis(oxoalkanoic acids) are preferable in order to improve liquid permeability, pressure resistance characteristics during liquid passage, and stain resistance. Dihydrazides are more preferred.

Furthermore, in step A-P2, a crosslinking agent other than the crosslinking agent containing at least two groups represented by -C(=O)-NH- in the molecule can also be used. Examples of such crosslinking agents include polyfunctional isocyanate crosslinking agents, polyfunctional epoxy crosslinking agents, polyfunctional aldehyde crosslinking agents, polyfunctional thiol crosslinking agents, polyfunctional oxazoline crosslinking agents, and polyfunctional aziridine crosslinking agents. agent, metal chelate type crosslinking agent, etc.

The total amount of the crosslinking agent used is preferably 0.01 molar equivalent or more and 0.8 molar equivalent or less, more preferably 0.05 molar equivalent or more and 0.8 molar equivalent or less, per mole of the functional group derived from the functional group-containing monomer. .7 molar equivalent or less, particularly preferably 0.1 molar equivalent or more and 0.6 molar equivalent or less.

The hydrophilic agent used in Steps A-P2 contains a total of two hydrophilic groups of at least one kind selected from hydroxy groups and mercapto groups in the molecule in order to improve antifouling properties and low leakage of protein ligands. Compounds having the above are preferred, and compounds having a total of 2 to 4 at least one kind of hydrophilic group selected from hydroxy groups and mercapto groups in the molecule are more preferred. Examples include alcohols having a mercapto group in the molecule such as mercaptoethanol and thioglycerol; polyhydric alcohols such as glycerol and diglycerol. The hydrophilic agents can be used alone or in combination of two or more.
Among these, alcohols having a mercapto group in the molecule are preferred, and thioglycerol is particularly preferred, in order to improve stain resistance and low leakage of protein ligands.

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

Steps A-P2 may be performed in the presence of a basic catalyst. Examples of the basic catalyst include triethylamine, N,N-dimethyl-4-aminopyridine, sodium hydroxide, diisopropylethylamine, etc., and one type can be used alone or two or more types can be used in combination.

Further, the reaction time of Steps A-P2 is not particularly limited, but is usually about 0.5 to 72 hours, preferably 0.5 to 48 hours. Further, the reaction temperature may be appropriately selected below the boiling point of the solvent, but is usually about 2 to 100°C.

The ligand used in step A-1 is one or more ligands selected from protein A, protein G, protein L, and related substances thereof. As the ligand, protein A or a substance analogous to protein A is preferable, and a substance analogous to protein A is more preferable, in order to enhance the binding property with an antibody or a fragment thereof.
Furthermore, protein A contains five domains, E, D, A, B, and C, which have the ability to bind to immunoglobulins. Protein A analogs are preferred, and protein A analogs having a modified C domain are more preferred.
In addition, other amino acids of the amino acid residue at the position corresponding to position 23 of the amino acid sequence, such as modified protein A which is a hexamer of the amino acid sequence domain of SEQ ID NO: 1 (SEQ ID NO: 29 of WO2020/040307). In the case of a modified protein A having a modified domain in which at least one residue has been substituted, it may tend to leak when used repeatedly for antibody isolation. Even when protein ligands are used as ligands, leakage of protein ligands can be suppressed when used repeatedly. Also, modified domains in which one or more amino acid residues in the C domain are replaced with other amino acid residues, such as modified protein A, which is a hexamer of amino acid sequence domains of SEQ ID NOs: 3 to 14. The modified protein A having the above structure also has excellent low leakage of protein ligands when used repeatedly.

In addition, among the ligands, in order to increase the dynamic binding capacity and improve the low leakage of protein ligands, it has more than 85% homology with the amino acid sequence shown in SEQ ID NO: 2 (C domain of protein A). Preferably, the protein ligand has an amino acid sequence in which at least one or two or more substitutions selected from (a) to (i) below have been made to the amino acid sequence. Among such protein ligands, those having an amino acid sequence with three or more substitutions selected from (a) to (i) below are preferred, and three to nine substitutions selected from (a) to (i) below are preferable. It is more preferable to have an amino acid sequence with the following substitutions, and particularly preferable is an amino acid sequence with 3 to 5 substitutions selected from (a) to (i) below. Furthermore, a ligand having two or more such amino acid sequences is preferred, a ligand having 2 to 12 such amino acid sequences is more preferred, and a ligand having 4 to 7 is particularly preferred. When it contains two or more amino acid sequences, those amino acid sequences may be the same or different.

(a) Substitution of the amino acid residue at the position corresponding to position 1 of the amino acid sequence of SEQ ID NO: 2 with a valine residue (b) Substitution of the amino acid residue at the position corresponding to position 3 of the amino acid sequence of SEQ ID NO: 2 with an alanine residue (c) Substitution of the amino acid residue at the position corresponding to position 6 of the amino acid sequence of SEQ ID NO: 2 with an alanine residue or aspartic acid residue (d) Corresponding to position 9 of the amino acid sequence of SEQ ID NO: 2 (e) Substitution of the amino acid residue at the position corresponding to position 11 of the amino acid sequence of SEQ ID NO: 2 with an alanine residue, glutamine residue, or glutamic acid residue (f ) Substitution of the amino acid residue at the position corresponding to position 23 of the amino acid sequence of SEQ ID NO: 2 with a leucine residue (g) Substitution of the amino acid residue at the position corresponding to position 29 of the amino acid sequence of SEQ ID NO: 2 with an alanine residue (h) Substitution of the amino acid residue at the position corresponding to position 43 of the amino acid sequence of SEQ ID NO: 2 with an alanine residue (i) Substitution of the amino acid residue at the position corresponding to position 49 of the amino acid sequence of SEQ ID NO: 2 Substitution to arginine residue

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

As used herein, a "corresponding position" on an amino acid sequence refers to a position where the target sequence and a reference sequence (e.g., the amino acid sequence of SEQ ID NO: 2) have the greatest homology to the conserved amino acid residues present in each amino acid sequence. It can be determined by alignment to give . Alignment can be performed using known algorithms and procedures are known to those skilled in the art. For example, alignment can be performed using the Clustal W multiple alignment program (Thompson, J. D. et al, 1994, Nucleic Acids Res., 22:4673-4680) with default settings. Clustal W is used, for example, by the European Bioinformatics Institute (EBI [www.ebi.ac.uk/index.html]) and the DNA Data Bank of Japan (DDBJ [www. It can be used on the ddbj.nig.ac.jp/index.html) website.

Amino acid residues are also written herein 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 a peptide is described in accordance with a conventional method such that the amino terminus (hereinafter referred to as N-terminus) is located on the left side and the carboxyl terminus (hereinafter referred to as C-terminus) is located on the right side.

As used herein, the positions "before" and "after" a specific position in an amino acid sequence refer to positions adjacent to the N-terminus and C-terminus, respectively, of the specific position. For example, when inserting an amino acid residue into positions "before" and "after" a specific position, the amino acid residues after insertion are placed at positions adjacent to the N-terminus and C-terminus of the specific position. be done.

In a preferred embodiment, the ligand used in step A-1 is a protein ligand (parent domain) having an amino acid sequence having 85% or more homology with the amino acid sequence shown in SEQ ID NO: 2, and the above (a). Produced by performing one or more substitutions selected from ~(i).

The above substitution may be one or more of the above substitutions (a) to (i), but
Two or more substitutions selected from substitution (a), substitution (b), substitution (c), substitution (d), substitution (g), substitution (h), and substitution (i), and substitution (e) and substitution ( Preferably, two or more substitutions include at least a combination with one or more substitutions selected from f),
Three or more substitutions selected from substitution (a), substitution (b), substitution (c), substitution (d), substitution (g), substitution (h), and substitution (i), and substitution (e) and substitution ( More preferably, two or more substitutions include at least a combination with one or more substitutions selected from f).

Generally, when substitution (f) is made, protein ligands tend to leak out when used repeatedly; however, according to the present invention, even when substitution (f) is made, even when used repeatedly, It is possible to suppress leakage of protein ligands when the protein is present.

In addition, as the ligand used in step A-1,
For the amino acid sequence having 85% or more homology with the amino acid sequence shown in SEQ ID NO: 2, at least substitution (a), substitution (b), substitution (c), substitution (d), substitution (g), An amino acid sequence in which two or more substitutions have been made, including at least a combination of two or more substitutions selected from substitution (h) and substitution (i), and one or more substitutions selected from substitution (e) and substitution (f). , preferably has 2 to 12 ligands,
For the amino acid sequence having 85% or more homology with the amino acid sequence shown in SEQ ID NO: 2, at least substitution (a), substitution (b), substitution (c), substitution (d), substitution (g), An amino acid sequence in which two or more substitutions have been made, including at least a combination of two or more substitutions selected from substitution (h) and substitution (i), and one or more substitutions selected from substitution (e) and substitution (f). , 4 to 7 ligands are more preferred.

In addition, two or more substitutions selected from substitution (a), substitution (b), substitution (c), substitution (d), substitution (g), substitution (h), and substitution (i), and substitution (e) and Two or more substitutions including at least a combination with one or more substitutions selected from substitution (f) are:
Preferably, two or more substitutions include at least a combination of one or more substitutions selected from substitution (a) and substitution (d) and one or more substitutions selected from substitution (e) and substitution (f),
One or more substitutions selected from substitution (a) and substitution (d), one or more substitutions selected from substitution (e) and substitution (f), substitution (b), substitution (c), substitution (g) More preferably, three or more substitutions include at least a combination with one or more substitutions selected from , substitution (h), and substitution (i).
Specifically, such a replacement is
A combination of substitution (a), substitution (e), substitution (f), and substitution (g);
Combination of substitution (d), substitution (e) and substitution (h);
Combination of substitution (a), substitution (e) and substitution (g);
A combination of substitution (a), substitution (e), substitution (g), and substitution (i);
A combination of substitution (a), substitution (b), substitution (c), substitution (e), and substitution (g);
A combination of substitution (a), substitution (b), substitution (c), substitution (e), substitution (g), and substitution (i);
can be mentioned.

In addition, two or more substitutions selected from substitution (a), substitution (b), substitution (c), substitution (d), substitution (g), substitution (h), and substitution (i), and substitution (e) and Specifically, substitutions other than two or more substitutions that include at least a combination with one or more substitutions selected from substitution (f) include:
A combination of substitution (a), substitution (b) and substitution (g);
Combination of substitution (a), substitution (c) and substitution (g);
A combination of substitution (a), substitution (b), substitution (c) and substitution (g);
Combination of substitution (a), substitution (d) and substitution (g);
can be mentioned.

As a means of substituting a protein ligand (parent domain) having an amino acid sequence having 85% or more homology with the amino acid sequence shown in SEQ ID NO: 2, desired amino acid residues are substituted for the polynucleotide encoding the parent domain. Examples include a method of introducing a mutation so that a substitution occurs. Specific methods for introducing mutations into polynucleotides include site-specific mutation, homologous recombination, SOE (splicing by overlap extension)-PCR method (Gene, 1989, 77:61-68), etc. These detailed procedures are well known to those skilled in the art.
The produced ligand has immunoglobulin binding activity and functions as an immunoglobulin binding domain.

Preferred examples of the ligand used in step A-1 include at least the following (a) for the amino acid sequence having 85% or more homology with the amino acid sequence shown in SEQ ID NO: 2 (C domain of protein A). Examples include those in which two or more amino acid sequences with one or more substitutions selected from (i) are connected in a linear chain. Note that the term "linearly connected" amino acid sequences refers to a structure in which two or more amino acid sequences are connected in series with or without a linker. For example, in the case of using a linker, "linearly connected" means a structure in which the C-terminus of one amino acid sequence and the N-terminus of another amino acid sequence are connected in series via a linker; When not mediated by a linker, "linearly connected" means a structure in which the C-terminus of one amino acid sequence and the N-terminus of another amino acid sequence are connected in series by a peptide bond.

In order to increase the dynamic binding capacity, the immobilized amount of the ligand is preferably 10 mg or more and 300 mg or less, more preferably 25 mg or more and 150 mg or less per gram of dry weight of the porous particles.

The immobilization of the ligand onto the porous particles in step A-1 may be carried out in the same manner as a conventional method. As the ligand immobilization method, a chemical bonding method is preferable. For example, there is a method in which a ligand is bonded to a functional group capable of immobilizing the ligand. This method may be performed with reference to the descriptions in International Publication No. 2015/119255 pamphlet, International Publication No. 2015/041218 pamphlet, and the like. Specifically, a method of bonding a cyclic ether group, carboxy group, -C(=O)-OC(=O)-, formyl group, etc. of a porous particle to an amino group, etc. of a ligand may be mentioned. The ligand immobilization reaction is preferably carried out in a buffer having a pH of 7 to 14 in order to increase reaction efficiency. Further, the reaction time of the ligand immobilization reaction is not particularly limited, but is usually about 0.1 to 72 hours. Further, the reaction temperature may be appropriately selected below the boiling point of the solvent, but is usually about 2 to 100°C.
In addition, in step A-1, -C(=O)-O-C(=O)-, which is used in step B described later, is used to increase dynamic binding capacity and improve low leakage of protein ligands. It is preferable to carry out the reaction in the absence of a compound having at least one ligand-reactive group selected from the group shown above, a carbodiimide group, and a cyclic ether group.

In addition, methods for controlling the orientation of ligands (US Pat. No. 6,399,750, Ljungquist C. et al., "Eur. J. Biochem.", 1989, vol. 186, pp. 557-561) and linker (spacer) (U.S. Patent No. 5,260,373, JP 2010-133733, JP 2010-133734). It may be fixed using a method such as stacking on top (Japanese Patent Laid-Open No. 2011-256176).

Examples of the compound providing the linker include ethylene glycol diglycidyl ether, diethylene glycol diglycidyl ether, polyethylene glycol diglycidyl ether, 1,2-propylene glycol diglycidyl ether, dipropylene glycol diglycidyl ether, polypropylene glycol diglycidyl ether, 1 Diglycidyl ethers of aliphatic polyhydroxy compounds such as , 4-butanediol diglycidyl ether, neopentyl glycol diglycidyl ether, 1,6-hexanediol diglycidyl ether, glycerol diglycidyl ether; sorbitol polyglycidyl ether, glycerol poly Examples include polyglycidyl ethers of aliphatic polyhydroxy compounds such as glycidyl ether, trimethylolpropane polyglycidyl ether, diglycerol polyglycidyl ether, and polyglycerol polyglycidyl ether. Among these, diglycidyl ethers of aliphatic polyhydroxy compounds are preferred when the hydrophilization reaction is carried out in steps A-P2.
The linker introduction reaction is preferably carried out in a buffer having a pH of 7 to 14 in order to increase reaction efficiency. Further, the reaction time of the linker introduction reaction is not particularly limited, but is usually about 0.5 to 72 hours. Further, the reaction temperature may be appropriately selected below the boiling point of the solvent, but is usually about 2 to 100°C.

-Process A-2-
The method for producing a chromatography carrier of the present invention further includes the following step A-2 between step A-1 and step B in order to improve the antifouling property and the low protein ligand leakage property. It is preferable to use the hydrophilic group-containing ligand-immobilized porous particles obtained in Step-2 as the ligand-immobilized porous particles obtained in Step A-1 in Step B.
(Step A-2) The porous particles on which the ligand was fixed in Step A-1 are reacted with a compound having a total of two or more hydrophilic groups of at least one kind selected from hydroxy groups and mercapto groups in the molecule. process of letting

The compound having a total of two or more hydrophilic groups in the molecule used in step A-2 is at least one type selected from hydroxy groups and mercapto groups in order to improve antifouling properties and low leakage of protein ligands. Compounds having a total of 2 to 4 hydrophilic groups in the molecule are preferred. Examples include alcohols having a mercapto group in the molecule such as mercaptoethanol and thioglycerol; polyhydric alcohols such as glycerol and diglycerol. Compounds having a total of two or more hydrophilic groups in the molecule can be used singly or in combination of two or more.
Among these, alcohols having a mercapto group in the molecule are preferred, and thioglycerol is particularly preferred, in order to improve stain resistance and low leakage of protein ligands.

The total amount of the compound having two or more hydrophilic groups in the molecule in step A-2 is preferably 1 part by mass or more based on 100 parts by mass of the porous particles (dry weight) on which the ligand is immobilized. The content is 1000 parts by mass or less, more preferably 10 parts by mass or more and 800 parts by mass or less, particularly preferably 100 parts by mass or more and 600 parts by mass or less.

Step A-2 may be performed in the presence of a basic catalyst. Examples of the basic catalyst include triethylamine, N,N-dimethyl-4-aminopyridine, sodium hydroxide, diisopropylethylamine, etc., and one type can be used alone or two or more types can be used in combination.

Further, the reaction time in step A-2 is not particularly limited, but is usually about 0.5 to 72 hours, preferably 0.5 to 48 hours. Further, the reaction temperature may be appropriately selected below the boiling point of the solvent, but is usually about 2 to 100°C.

-Process B-
The method for producing a chromatography carrier of the present invention includes (Step B) porous particles on which the ligand after Step A-1 is immobilized, and -C(=O)-OC(=O)-. The method comprises a step of reacting the compound with a compound having at least one type of ligand-reactive group selected from a group consisting of a group consisting of a group containing a ligand, a carbodiimide group, and a cyclic ether group.
When a compound having at least one selected from a group represented by -C(=O)-OC(=O)- and a cyclic ether group is used as the compound having the above-mentioned ligand-reactive group, the compound has Reacts primarily with the amino group of the ligand. Further, when a compound having a carbodiimide group is used as the compound having a ligand-reactive group, the compound mainly reacts with the amino group of the ligand and also reacts with the carboxy group. The present inventors conjecture that such a reaction in step B increases the dynamic binding capacity for antibodies or fragments thereof, and also makes it difficult for protein ligands to leak out even when used repeatedly to isolate antibodies.

Step B uses porous particles on which the ligands obtained in Step A-1 are immobilized. The porous particles may be those to which the ligand after the reaction in step A-1 is immobilized. The porous particles with immobilized ligands obtained in step A-1 may be used, or the porous particles containing a hydrophilic group and immobilized with ligands obtained by further performing step A-2 may be used.

As the compound having a ligand-reactive group used in step B, in order to increase the dynamic binding capacity and improve the low leakage of protein ligand, the compound represented by the following formula (1) and its salt, the following formula One or more compounds selected from the compound represented by (2) and the compound represented by the following formula (3) are preferred, and the compound represented by the formula (1) and its salt and the compound represented by the formula (2) are preferred. One or more types selected from the compounds represented by the formula (1) are more preferred, and one or more types selected from the compounds represented by the formula (1) and salts thereof are particularly preferred. The salts of the compound represented by formula (1) include inorganic acid salts such as hydrochloride, sulfate, nitrate, hydrofluoride, and hydrobromide; acetate, tartrate, and citrate. , organic acid salts such as fumarate, and the like.
When one or more selected from the compound represented by formula (1) and its salt and the compound represented by formula (2) are used, especially from the compound represented by formula (1) and its salt When one or more selected types are used, it becomes easier to achieve both dynamic binding capacity and low protein ligand leakage.

Here, each symbol in formulas (1) to (3) will be explained.
In formulas (1) to (3), the number of carbon atoms in the hydrocarbon groups represented by R ¹ to R ⁵ is preferably 1 to 1 in order to increase dynamic binding capacity and improve low leakage of protein ligands. 30, more preferably 1-14, even more preferably 1-8, particularly preferably 1-4.
Examples of the "hydrocarbon group" for R ¹ to R ⁵ include an alkyl group, an alkenyl group, a cycloalkyl group, a bridged ring hydrocarbon group, an aryl group, and an aralkyl group.

The number of carbon atoms in the alkyl group is preferably 1 to 30, more preferably 1 to 14, even more preferably 1 to 8, particularly preferably 1 to 4. The alkyl group may be linear or branched. Specific examples of the alkyl group include methyl group, ethyl group, n-propyl group, isopropyl group, n-butyl group, isobutyl group, sec-butyl group, tert-butyl group, pentyl group, hexyl group, and heptyl group. , octyl group, 2-ethylhexyl group, nonyl group, decyl group, undecyl group, dodecyl group and the like.
The alkenyl group preferably has 2 to 30 carbon atoms, more preferably 2 to 14 carbon atoms, still more preferably 2 to 8 carbon atoms, and particularly preferably 2 to 4 carbon atoms. The alkenyl group may be linear or branched. Specific examples of the alkenyl group include a vinyl group, a propenyl group, a butenyl group, and the like.

The number of carbon atoms in the cycloalkyl group and the bridged ring hydrocarbon group is preferably 3 to 30, more preferably 3 to 12, particularly preferably 3 to 8. Specific examples of the cycloalkyl group include a cyclopropyl group and a cyclohexyl group. Examples of the bridged ring hydrocarbon group include an isobornyl group and the like.

The number of carbon atoms in the aryl group is preferably 6 to 30, more preferably 6 to 12. For example, phenyl group etc. can be mentioned. The aralkyl group preferably has 7 to 30 carbon atoms, more preferably 7 to 13 carbon atoms. Examples include benzyl group and phenethyl group.

Furthermore, the "hydrocarbon group" in R ¹ to R ⁵ may have a substituent. The substituent is preferably a substituent containing a heteroatom, such as a halogen atom such as a fluorine atom, a chlorine atom, a bromine atom, or an iodine atom; a hydroxy group; an alkoxy group such as a methoxy group or an ethoxy group (preferably a carbon number of 1 -4 alkoxy group); Amino group; Monoalkylamino group (preferably mono C _1-4 alkylamino group) such as monomethylamino group, monoethylamino group, mono n-propylamino group; dimethylamino group, diethylamino group , a dialkylamino group (preferably a di-C _1-4 alkylamino group) such as methylethylamino group; a carboxy group; a cyano group; a sulfo group; a nitro group. In addition, a mono-C _1-4 alkylamino group means a mono-alkylamino group in which the alkyl group has 1 to 4 carbon atoms, and a di-C _1-4 alkylamino group refers to a mono-C 1-4 alkylamino group in which the two alkyl groups have 1 to 4 carbon atoms. 4 dialkylamino group. The number of substituents is preferably 0 to 8, more preferably 0 to 2.

R ¹ and R ² in formula (1) are hydrogen atoms, substituted or unsubstituted alkyl groups, or cycloalkyl groups in order to increase dynamic binding capacity and improve low leakage of protein ligands. Preferably, a hydrogen atom, an alkyl group, a cycloalkyl group, an aminoalkyl group, a monoalkylaminoalkyl group, or a dialkylaminoalkyl group, and a hydrogen atom, an alkyl group having 1 to 14 carbon atoms, and a cycloalkyl group having 3 to 12 carbon atoms. Alkyl group, aminoalkyl group having 1 to 14 carbon atoms, alkyl group having 1 to 14 carbon atoms having a mono-C _1-4 alkylamino group as a substituent, or carbon having a di-C _1-4 alkylamino group as a substituent More preferred are alkyl groups having 1 to 14 carbon atoms, such as a hydrogen atom, an alkyl group having 1 to 8 carbon atoms, a cycloalkyl group having 3 to 8 carbon atoms, an aminoalkyl group having 1 to 8 carbon atoms, and a mono-C _1-4 alkylamino group. An alkyl group having 1 to 8 carbon atoms having a group as a substituent, or an alkyl group having 1 to 8 carbon atoms having a diC _1-4 alkylamino group as a substituent is more preferable, and an alkyl group, a cycloalkyl group having 3 to 8 carbon atoms, an aminoalkyl group having 1 to 4 carbon atoms, an alkyl group having 1 to 4 carbon atoms having a mono-C _1-4 alkylamino group as a substituent, or a di-C _1- Particularly preferred is an alkyl group having ₁ to 4 carbon atoms having a 4-alkylamino group as a substituent.
Further, R ¹ and R ² in formula (1) are such that at least one of R ¹ and R ² is an aminoalkyl group or A monoalkylaminoalkyl group or a dialkylaminoalkyl group is preferred.

The hydrocarbon groups represented by R ³ and R ⁴ in formula (2) are substituted or unsubstituted alkyl groups, or substituted or an unsubstituted alkenyl group, more preferably an alkyl group or an alkenyl group, even more preferably an alkyl group having 1 to 14 carbon atoms or an alkenyl group having 1 to 14 carbon atoms, an alkyl group having 1 to 8 carbon atoms or an alkenyl group having 1 to 8 carbon atoms. An alkenyl group having 1 to 8 carbon atoms is more preferred, an alkyl group having 1 to 4 carbon atoms or an alkenyl group having 1 to 4 carbon atoms is even more preferred, an alkyl group having 1 to 4 carbon atoms is even more preferred, an alkyl group having 2 to 4 carbon atoms Particularly preferred are groups.
Furthermore, the number of carbon atoms in the cyclic structure formed by R ³ and R ⁴ bonding to each other is preferably 4 to 8, more preferably 4 to 6. In the case where R ³ and R ⁴ are bonded to each other to form a cyclic structure, examples of the compound having a ligand-reactive group include succinic anhydride, maleic anhydride, citraconic anhydride, dimethylmaleic anhydride, and glutaric acid. Examples include anhydrides.

In formula (3), R ⁵ is preferably a substituted or unsubstituted alkyl group, and a substituted or unsubstituted alkyl group having 1 to 1 carbon atoms, in order to increase dynamic binding capacity and improve low leakage of protein ligands. 14 alkyl groups are more preferred, substituted or unsubstituted alkyl groups having 1 to 8 carbon atoms are even more preferred, substituted or unsubstituted alkyl groups having 1 to 4 carbon atoms are even more preferred, and alkyl groups having 1 to 4 carbon atoms are more preferred. , or an alkyl group having 1 to 4 carbon atoms having a substituent selected from a halogen atom, a hydroxy group, and an alkoxy group, more preferably an alkyl group having 1 to 4 carbon atoms, or a halogen atom, a hydroxy group, and a 1 to 4 carbon atoms. An alkyl group having 1 to 4 carbon atoms having a substituent selected from alkoxy groups is more preferred, and an alkyl group having 1 to 4 carbon atoms is particularly preferred.
X in formula (3) represents a cyclic ether group. This cyclic ether group is the same as that mentioned as the functional group in Step A-P1, and a cyclic ether group having 3 to 7 atoms constituting the ring is preferable. The cyclic ether group may have an alkyl group as a substituent. Specific examples of the cyclic ether group include cyclic ether groups represented by formulas (4) to (9) listed as functional groups in step A-P1; ) is preferred, and a cyclic ether group represented by formula (4) is more preferred.

The compound having a ligand-reactive group used in Step B is 1-[3-(dimethylamino)propyl]-3-ethylcarbodiimide in order to increase dynamic binding capacity and improve low leakage of protein ligands. , 1-[3-(dimethylamino)propyl]-3-ethylcarbodiimide salt, N,N'-dicyclohexylcarbodiimide, N,N'-diisopropylcarbodiimide, maleic anhydride, citraconic anhydride, dimethylmaleic anhydride , propionic anhydride, acetic anhydride, pivalic anhydride, succinic anhydride, glutaric anhydride, propylene oxide, butylene oxide, glycidyl methyl ether, ethyl glycidyl ether, glycidol, epichlorohydrin, and epibromohydrin. 1-[3-(dimethylamino)propyl]-3-ethylcarbodiimide, a salt of 1-[3-(dimethylamino)propyl]-3-ethylcarbodiimide, N, N'-dicyclohexylcarbodiimide, N,N'-diisopropylcarbodiimide, maleic anhydride, propionic anhydride, acetic anhydride, pivalic anhydride, succinic anhydride, glutaric anhydride, propylene oxide, butylene oxide, glycidyl methyl ether, ethyl One or more compounds selected from glycidyl ether, glycidol, epichlorohydrin, and epibromohydrin are more preferred, and 1-[3-(dimethylamino)propyl]-3-ethylcarbodiimide, 1-[ 3-(dimethylamino)propyl]-3-ethylcarbodiimide salt, N,N'-dicyclohexylcarbodiimide, N,N'-diisopropylcarbodiimide, maleic anhydride, propionic anhydride, acetic anhydride, pivalic anhydride, succinic anhydride One or more compounds selected from acids and glutaric anhydride are more preferred, such as 1-[3-(dimethylamino)propyl]-3-ethylcarbodiimide, 1-[3-(dimethylamino)propyl] More preferably, one or more compounds selected from a salt of -3-ethylcarbodiimide, N,N'-dicyclohexylcarbodiimide, and N,N'-diisopropylcarbodiimide, and 1-[3-(dimethylamino)propyl] More preferred are compounds selected from -3-ethylcarbodiimide and its salts, and 1-[3-(dimethylamino)propyl]-3-ethylcarbodiimide hydrochloride is particularly preferred.
1-[3-(dimethylamino)propyl]-3-ethylcarbodiimide, 1-[3-(dimethylamino)propyl]-3-ethylcarbodiimide salt, N,N'-dicyclohexylcarbodiimide, N,N'-diisopropyl When one or more compounds selected from carbodiimide, maleic anhydride, propionic anhydride, acetic anhydride, pivalic anhydride, succinic anhydride, and glutaric anhydride are used, especially 1-[3-( 1 selected from dimethylamino)propyl]-3-ethylcarbodiimide, a salt of 1-[3-(dimethylamino)propyl]-3-ethylcarbodiimide, N,N'-dicyclohexylcarbodiimide, and N,N'-diisopropylcarbodiimide When a species or two or more kinds of compounds are used, it becomes easier to achieve both dynamic binding capacity and low protein ligand leakage.

In order to maintain high dynamic binding capacity while sufficiently modifying the ligand, the total amount of the compound having a ligand-reactive group used in Step B is determined per 1 g of dry weight of the porous particles on which the ligand is immobilized. The amount is preferably 0.01 mmol or more and 15 mmol or less, more preferably 0.02 mmol or more and 10 mmol or less, still more preferably 0.05 mmol or more and 5 mmol or less, and even more preferably 0.1 mmol. The amount is 3 mmol or less, particularly preferably 0.3 mmol or more and 3 mmol or less.
Both dynamic binding capacity and low leakage of protein ligands can be achieved when the total amount of compounds having ligand-reactive groups used is 0.3 mmol or more per 1 g of dry weight of porous particles on which ligands are immobilized. It becomes easier.

Step B is preferably carried out in an aqueous medium in order to increase reaction efficiency. Examples of the aqueous medium include water and various buffers such as carbonate buffer, CHES buffer, CAPS buffer, TAPS buffer, sodium borate buffer, and phosphate buffer. Among these, carbonate buffers and CAPS buffers are preferred, and carbonate buffers are more preferred, in order to improve low leakage of protein ligands.
When a carbonate buffer or a CAPS buffer is used as the aqueous medium, especially when a carbonate buffer is used, it becomes easier to achieve both dynamic binding capacity and low protein ligand leakage.
The reaction pH in Step B is preferably 7 or higher, more preferably 8 to 14, even more preferably 9 to 12, particularly preferably 9, in order to increase the dynamic binding capacity and improve low leakage of protein ligands. ~11.
When the reaction pH in Step B is set to 12 or less, particularly when it is set to 11 or less, it becomes easier to achieve both dynamic binding capacity and low protein ligand leakage.

The reaction time in step B is not particularly limited, but is usually about 0.1 to 72 hours, preferably 0.3 to 48 hours. Further, the reaction temperature may be appropriately selected below the boiling point of the solvent, but is usually about 2 to 100°C.

Note that the reaction products obtained in each of the above steps may be purified by separation means such as filtration and washing. Furthermore, it may be classified.

According to the method for producing a chromatography carrier of the present invention, a chromatography carrier that has a large dynamic binding capacity for antibodies or fragments thereof and that does not easily leak protein ligands even when used repeatedly for antibody isolation can be obtained. Easy to manufacture.
Furthermore, the chromatography carrier thus obtained has a large dynamic binding capacity for antibodies or fragments thereof, and protein ligands are unlikely to leak out even when used repeatedly for antibody isolation. Furthermore, when isolating antibodies, non-specific adsorption of contaminants and the like to protein ligands can be suppressed. It is also suitable for use in affinity chromatography. Next, the chromatography carrier of the present invention obtained in this way will be explained.

One or more compounds selected from the compound represented by formula (1) and its salts were used as the compound having a ligand-reactive group used in step B, and a carbonate buffer or CAPS buffer was used as the aqueous medium. In case,
Furthermore, one or more compounds selected from the compound represented by formula (1) and its salts are used as the compound having a ligand-reactive group used in step B, and a carbonate buffer or CAPS buffer is used as the aqueous medium. and when the reaction pH in step B is 12 or less or 11 or less,
In particular, one or more compounds selected from the compound represented by formula (1) and its salts are used as the compound having a ligand-reactive group used in step B, a carbonate buffer is used as the aqueous medium, and the ligand is When the total amount of compounds having reactive groups used is 0.3 mmol or more per 1 g of dry weight of porous particles with immobilized ligands, both dynamic binding capacity and low leakage of protein ligands can be achieved. It becomes easier.

[Carrier for chromatography]
The chromatography carrier of the present invention comprises porous particles, a ligand fixed to the porous particles, a group represented by -C(=O)-OC(=O)-, a carbodiimide group, and a partial structure derived from a compound having at least one ligand-reactive group selected from a cyclic ether group, and the ligand is one selected from Protein A, Protein G, Protein L, and related substances thereof; Two or more types of ligands, at least one type of functional group selected from an amino group and a carboxy group of the ligands are chemically modified with the above partial structure, and have the following formula:
Chemical modification rate (mol%) = (number of moles of chemically modified functional groups) / (sum of number of moles of chemically modified functional groups and number of moles of non-chemically modified functional groups) × 100
It is characterized in that the chemical modification rate calculated by is 1 to 70 mol%. The porous particles, the ligand, and the compound having a ligand-reactive group have the same meanings as those explained in the method for producing a chromatography carrier of the present invention.

Examples of the compound having a ligand-reactive group include those similar to those used in Step B. As partial structures derived from compounds having a ligand-reactive group, -C(=O)-, -NR ¹ -C(= Those having a group represented by O)-, -C(=NR ¹ )- or -CH ₂ -CH(-OH)- in the molecule are preferable, and -C(=O)-, -C(=NR ¹ )- or -NR ¹ -C(=O)- in the molecule, and -C(=NR ¹ )- or -NR ¹ -C(=O)-. Those having the group represented by the formula in the molecule are more preferable, and those having the group represented by -C(=NR ¹ )- in the molecule are particularly preferable.
In addition, as a partial structure having a group represented by -C(=O)- in the molecule, -C(=O)- A group represented by -R ³ or a group represented by -C(=O)-R ²¹ -C(=O)OH is preferred.
In addition, as a partial structure having a group represented by -C(=NR ¹ )- in the molecule, -C(=NR ¹ ) Those having a group represented by --NR ² -- in the molecule are preferred, and groups represented by --C(=NR ¹ )-NR ² --H are more preferred.
In addition, as a partial structure having a group represented by -NR ¹ -C(=O)- in the molecule, -NR ¹ Those having a group represented by --C(=O)-NR ² -- in the molecule are preferred, and groups represented by --NR ¹ --C(=O)-NR ² --H are more preferred.
In addition, as a partial structure having a group represented by -CH ₂ -CH(-OH)- in the molecule, -CH ₂ A group represented by -CH(-OH)-R ⁵ is preferred.
R ¹ , R ² , R ³ and R ⁵ in the above partial structure have the same meanings as R ¹ , R ² , R ³ and R ⁵ in formulas (1) to (3), and R ¹ and R ² are Each independently represents a hydrogen atom or a substituted or unsubstituted hydrocarbon group. Moreover, R ³ and R ⁵ each independently represent a substituted or unsubstituted hydrocarbon group.
-C(=O)-R ²¹ -C(=O)OH is represented by formula (2), and R ³ and R ⁴ in formula (2) combine with each other to form a cyclic structure. Any group formed by ring opening of a compound may be used. R ²¹ is preferably a divalent hydrocarbon group having 2 to 6 carbon atoms. For example, alkanediyl groups such as ethane-1,2-diyl group, propane-1,3-diyl group, butane-1,4-diyl group, pentane-1,5-diyl group; ethene-1,2-diyl group; Examples include alkenediyl groups such as.

In the chromatography carrier of the present invention, the compound having the ligand-reactive group is at least one selected from a group represented by -C(=O)-OC(=O)-, a carbodiimide group, and a cyclic ether group. If one type is present, it is preferable that the amino group of the ligand is chemically modified with the partial structure. In addition, when it has a carbodiimide group, the amino group of the said ligand may be chemically modified with the said partial structure, and the carboxy group of the said ligand may also be chemically modified with the said partial structure.

The chemical modification rate is preferably 1 mol% or more, more preferably 3 mol% or more, even more preferably 10 mol% or more, in order to increase the dynamic binding capacity and improve the low leakage of protein ligands. More preferably 20 mol% or more, particularly preferably 30 mol% or more, and in order to maintain the function that the protein ligand should have, preferably 70 mol% or less, more preferably 60 mol% or less, More preferably, it is 55 mol% or less, particularly preferably 50 mol% or less. The specific range is preferably 1 mol% or more and 70 mol% or less, more preferably 3 mol% or more and 70 mol% or less, even more preferably 10 mol% or more and 60 mol% or less, and 20 mol% or more and 55 mol% or less. is more preferable, and particularly preferably 30 mol% or more and 50 mol% or less.

The chemical modification rate can be calculated according to the following formula using, for example, amino acid analysis, neutralization titration, or potentiometric titration. Specifically, when the compound having the ligand-reactive group has at least one selected from a carbodiimide group and a cyclic ether group, and the amino group of the ligand is chemically modified with the partial structure, for example, , based on the amino acid analysis method: Chemical modification rate (mol%) = {(1-Number of moles of protein ligand lysine after step B) / (Number of moles of protein ligand in porous particles 5 × number of moles in porous particles 5) Number of lysines present per protein ligand) × 100}
It can be calculated by
Further, the compound having the ligand-reactive group has a group represented by -C(=O)-OC(=O)-, and the amino group of the ligand is chemically modified with the partial structure. For example, based on the neutralization titration method, the following formula: Chemical modification rate (mol%) = (1-Amino group content before step B / amino group content in chromatography carrier) × 100
It can be calculated by In addition, when the compound having a ligand-reactive group has a group represented by -C(=O)-OC(=O)-, the amino group of the ligand and the compound having the ligand-reactive group react. By doing so, an amide group can be generated. The occurrence of such amide groups may impede accurate measurement in the above amino acid analysis method. Therefore, when a compound having a ligand-reactive group has a group represented by -C(=O)-OC(=O)-, it should be calculated based on the neutralization titration method as described above. is preferred.

The volume average particle diameter of the carrier for chromatography of the present invention is preferably 40 to 150 μm, more preferably 50 to 100 μm. Further, the coefficient of variation of the volume average particle diameter is preferably 40% or less, more preferably 30% or less.
Further, the specific surface area of the carrier for chromatography of the present invention is preferably 1 to 500 m ² /g, more preferably 10 to 300 m ² /g.
Further, the volume average pore diameter of the chromatography carrier of the present invention is preferably 10 to 300 nm.
The volume average particle diameter, coefficient of variation, specific surface area, and volume average pore diameter can be measured by laser diffraction, scattering particle size distribution measurement, or the like.

[Chromatography column]
The chromatography column of the present invention is characterized by containing the chromatography carrier of the present invention. The chromatography column of the present invention is similar to a conventional chromatography column except that it contains the chromatography carrier of the present invention. Specifically, a chromatography column including a column container and the chromatography carrier of the present invention filled in the column container can be mentioned.
The chromatography column of the invention is suitable for use in affinity chromatography.

[Method for isolating antibodies or fragments thereof]
The method for isolating antibodies or fragments thereof of the present invention is characterized by using the chromatography carrier of the present invention or the chromatography column of the present invention.
As used herein, the term "antibody" is a concept that includes any class of immunoglobulin, such as IgG, IgA, IgD, IgE, IgM, and subclasses thereof, as well as variants thereof. Furthermore, in this specification, the term "antibody" may be a chimeric antibody such as a humanized antibody, an antibody complex, or another modified immunoglobulin containing an antigen recognition site.
Furthermore, as used herein, the term "antibody fragment" may be an antibody fragment that includes an antigen recognition site or an antibody fragment that does not include an antigen recognition site. Examples of antibody fragments that do not contain an antigen recognition site include proteins consisting only of the Fc region of immunoglobulin, Fc fusion proteins, and variants and modifications thereof.

The method for isolating the antibody or fragment thereof of the present invention can be carried out in the same manner as the general method for isolating antibodies or fragments thereof, except for using the chromatography carrier of the present invention or the chromatography column of the present invention. good. Specifically, a method including a step of bringing the chromatography carrier of the present invention into contact with a sample containing an antibody or a fragment thereof can be mentioned. In addition, in this step, the antibody or its fragment is captured on a chromatography carrier, and after separating impurities (e.g., proteins other than the antibody or its fragment) from the antibody or its fragment, the antibody or its fragment is captured on the chromatography carrier. Preferably, an elution step is performed to elute the antibody or its fragment. By collecting this eluate, antibodies or fragments thereof can be isolated from the sample. In the elution step, a dissociation solution that dissociates the immunoglobulin binding protein and the antibody or its fragment is usually used.
Isolation may also be performed using a chromatography column of the invention. Such a method includes a method that includes a step of passing a sample containing an antibody or a fragment thereof through the chromatography column of the present invention, and by this step, the antibody or a fragment thereof is captured on a chromatography carrier. After separating contaminants from the antibody or fragment thereof, it is preferable to perform an elution step in the same manner as above.

Samples containing antibodies or fragments thereof are not particularly limited, but include, for example, whole blood, serum, plasma, various blood cells, blood components such as blood clots and platelets, urine, semen, breast milk, sweat, interstitial fluid, and interstitial fluid. Various liquid samples such as qualitative lymph fluid, bone marrow fluid, tissue fluid, saliva, gastric fluid, joint fluid, pleural effusion, bile, ascites fluid, amniotic fluid, etc., bacterial fluid, cell culture medium, cell culture supernatant, tissue cell disruption fluid, etc. can be mentioned.

Hereinafter, the present invention will be explained in detail with reference to Examples, but the present invention is not limited to these Examples.

(Preparation example: Preparation of ligand (immunoglobulin binding protein))
Immunoglobulin binding proteins PrA-0 to PrA-13 were obtained. PrA-0 is an immunoglobulin binding protein that contains a homohexamer in which the C domain of Protein A (SEQ ID NO: 2) is linked in tandem. PrA-1 to PrA-13 are mutants in which the mutations listed in Table 1 were introduced into each immunoglobulin binding domain of PrA-0.

Expression and purification of PrA-0 to PrA-13 were performed as follows. Escherichia coli BL21 (DE3) was transformed using the plasmids encoding PrA-0 to PrA-13, and the resulting transformants were cultured in a rich medium at 37°C until the logarithmic growth phase. Thereafter, the target protein was expressed by adding isopropyl-β-thiogalactopyranoside (manufactured by Wako Pure Chemical Industries, Ltd.) to the medium at a final concentration of 1 mM and culturing at 37° C. for 4 hours. Next, the culture solution was centrifuged to remove the supernatant, and the resulting bacterial cells were treated with egg white-derived lysozyme (manufactured by Wako Pure Chemical Industries, Ltd.) and polyoxyethylene (10) octylphenyl ether (manufactured by Wako Pure Chemical Industries, Ltd.). The bacterial cells were disrupted by adding 30 mM Tris buffer containing pH 9.5. The obtained cell lysate was subjected to cation exchange chromatography (SP-Sepharose FF, manufactured by GE Healthcare Biosciences) and anion exchange chromatography (Q-Sepharose FF, manufactured by GE Healthcare Biosciences). Globulin binding proteins were purified. The purified immunoglobulin binding protein was dialyzed against 10 mM citrate buffer pH 6.0. The purity of the recombinant immunoglobulin binding protein confirmed by SDS-PAGE was greater than 95%.

(Example 1)
(Step A-1) Immobilization of Ligand 2.69 g of polyvinyl alcohol (PVA-217 manufactured by Kuraray Co., Ltd.) was added to 448 g of pure water, and the mixture was heated and stirred to dissolve the polyvinyl alcohol to obtain an aqueous solution. On the other hand, a monomer 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.) The composition was dissolved in 29.38 g of 2-octanone (manufactured by Toyo Gosei Co., Ltd.) to prepare a monomer solution. Next, the entire amount of the aqueous solution was poured into a separable flask, which was equipped with a thermometer, a stirring blade, and a cooling tube, set in a hot water bath, and stirred under a nitrogen atmosphere. Pour the entire amount of the monomer solution into a separable flask, warm it with a hot water bath, and when the internal temperature reaches 85°C, 2,2'-azobis(methyl isobutyrate) (manufactured by Wako Pure Chemical Industries, Ltd.) 1.34 g was added, and the internal temperature was brought to 86°C. Thereafter, stirring was performed for 3 hours while maintaining the temperature at 86°C. Next, after cooling the reaction solution, the reaction solution was filtered and washed with pure water and ethanol. The washed particles were dispersed in pure water and decanted three times to remove small particles. Next, the particles were dispersed in pure water so that the particle concentration was 10% by mass to obtain a porous particle dispersion. The porous particles contained in this dispersion are referred to as "porous particles 1."
Then, 0.956 g of adipic acid dihydrazide (manufactured by Tokyo Kasei Kogyo Co., Ltd.), 8 g of thioglycerol (manufactured by Tokyo Kasei Kogyo Co., Ltd.) and 1.418 g of diisopropylethylamine (manufactured by Tokyo Kasei Kogyo Co., Ltd.) were then added to 100 g of Porous Particle 1 Dispersion. The mixture was heated to 70°C and stirred for 8 hours while maintaining the temperature at 70°C. Next, after cooling the reaction solution, the reaction solution was filtered and washed with pure water and ethanol. Next, the particles were dispersed in pure water so that the particle concentration was 10% by mass to obtain a porous particle dispersion. The porous particles contained in this dispersion are referred to as "porous particles 2."
Next, the thioglycerol-derived hydroxy groups contained in the porous particles 2 were reacted with ethylene glycol diglycidyl ether. That is, 8.7 g of pure water, 1.2 g of sodium sulfate (manufactured by Wako Pure Chemical Industries, Ltd.), and 0.10 g of sodium carbonate (manufactured by Wako Pure Chemical Industries, Ltd.) were mixed to obtain a carbonate buffer (pH 11.2). To this carbonate buffer, 0.5 g of ethylene glycol diglycidyl ether (Denacol EX810 manufactured by Nagase ChemteX) and 8 mL of porous particle 2 dispersion were added and stirred with shaking at 23° C. for 16 hours. Next, the particles were dispersed in pure water so that the particle concentration was 50% by volume to obtain a porous particle dispersion. The porous particles contained in this dispersion are referred to as "porous particles 3."
Next, a ligand was immobilized on the porous particles 3. That is, 28.8 g of pure water, 5.4 g of sodium sulfate (manufactured by Wako Pure Chemical Industries, Ltd.), 0.2 g of sodium hydrogen carbonate (manufactured by Wako Pure Chemical Industries, Ltd.), and 0.2 g of sodium carbonate (manufactured by Wako Pure Chemical Industries, Ltd.). 16 g were mixed to obtain a carbonate buffer (pH 9.4). To 34 mL of this carbonate buffer was added 0.0% of the immunoglobulin binding protein PrA-1 (modified protein A, which is a hexamer of the amino acid sequence domain of SEQ ID NO: 1 (SEQ ID NO: 29 of WO2020/040307)) prepared in the preparation example. 21 g and 8 mL of porous particle 3 dispersion were added, and the mixture was shaken and stirred at 23° C. for 1.5 hours. Next, the particles were dispersed in pure water so that the particle concentration was 50% by volume to obtain a porous particle dispersion. The porous particles contained in this dispersion are referred to as "porous particles 4."

(Step A-2) Hydrophilization reaction Mix 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.) to create a buffer. After that, 4.5 g of thioglycerol (manufactured by Tokyo Kasei Kogyo Co., Ltd.) was added to obtain a hydrophilic reaction solution. 8 mL of the porous particle 4 dispersion obtained in step A-1 was added to this hydrophilization reaction solution, and the mixture was shaken and stirred at 23° C. for 16 hours to perform a hydrophilization reaction. The porous particles after this hydrophilization reaction were filtered and washed in sequence with a 0.1M aqueous sodium hydroxide solution and a 0.1M sodium citrate buffer (pH 3.2). Next, the particles were dispersed in pure water so that the particle concentration was 50% by volume to obtain a porous particle dispersion. The porous particles contained in this dispersion are referred to as "porous particles 5."

(Step B) Post-modification reaction 1M sodium hydroxide (manufactured by Wako Pure Chemical Industries, Ltd.) and pure water were mixed into a solution in which 67.2 mg of sodium hydrogen carbonate (manufactured by Wako Pure Chemical Industries, Ltd.) was dissolved in 6 mL of pure water. 8 mL of carbonate buffer 1 (pH 10 at 23°C) was obtained. To this carbonate buffer 1, 8 mL of the porous particle 5 dispersion obtained in step A-2 and 0.16 g of 1-[3-(dimethylamino)propyl]-3-ethylcarbodiimide (hereinafter also referred to as EDC) were added. The carrier 1 was obtained by adding the mixture and shaking and stirring at 23° C. for 1.5 hours. This carrier 1 was sequentially filtered and washed with 0.1M aqueous sodium hydroxide solution, 0.1M sodium citrate buffer (pH 3.2), and 16wt% ethanol/50mM phosphate buffer (pH 7.3), and then the carrier 1 was prepared as a carrier for affinity chromatography. A containing liquid (carrier content: 50% by volume) was obtained.

Calculation of modification rate (amino acid analysis method)
90 μL of the carrier-containing solution for affinity chromatography obtained in the post-modification reaction of Step B was placed in a 2 mL tube (Eppendorf, product number 0030120094), and using 100 μL of a 12N hydrochloric acid aqueous solution, the mixture was stirred at 110° C. for 24 hours to undergo a hydrolysis reaction. Ta. After the hydrolysis reaction was completed, the aqueous hydrochloric acid solution was completely removed by removing the supernatant and drying under reduced pressure. Furthermore, 600 μL of 0.1N hydrochloric acid aqueous solution was added to the tube to dissolve it, and the entire amount of the resulting carrier-containing solution was transferred to a 0.22 μm filter tube (Merck Millipore, product number UFC30GV00), and solid components were removed by centrifugation using a centrifuge. The removed filtrate was collected. UHPLC measurement (using Thermo Fisher scientific, Vanquish HPLC, based on the following measurement conditions) was performed on the obtained filtrate.
Column: ACQUITY UPLC HSS T3 Column (C18), 100Å, 1.8μm, 2.1mm x 150nm
Mobile phase A: 0.4% heptafluorobutyric acid + 0.02% formic acid in H ₂ O
Mobile phase B: 0.02% formic acid in MeCN
Gradient conditions: 0-0.75min Mobile phase B 1%, 0.75-1.5min Mobile phase B 1→5%, 1.5-5min Mobile phase B 5→7.5%, 5-17.5min movement Phase B 7.5→27.5%, 17.5-18 min Mobile phase B 27.5→80%, 18-20 min Mobile phase B 80%, 20-20.1 min Mobile phase B 80→1%, 20. 1-25min Mobile phase B 1%
Flow rate: 0.2ml/min
Column temperature: 25℃
Detection: Thermo Scientific Corona Veo RS (Charged Aerosol Detector: CAD)
Injection volume: 2μl
The number of moles of leucine was determined from the peak area value of the obtained chromatogram, and the number of moles of protein A ligand used in this example was calculated. Next, the number of moles of lysine was quantified from the peak area value of the chromatogram, and the modification rate (mol%) was calculated from the reduction rate of lysine in the protein A ligand used in this example using the following formula.
Modification rate (mol%) = {(1-number of moles of lysine in the filtrate from which solid components were removed by the above operation) / (number of moles of protein A ligand in porous particles 5 × protein A ligand in porous particles 5 Number of lysine present per lysine) × 100}

(Example 2)
Step A-1 and Step A- of Example 1 except that 0.16 g of 1-[3-(dimethylamino)propyl]-3-ethylcarbodiimide used in Step B was changed to 0.14 g of propionic anhydride. The same operations as in Step 2 and Step B were performed to obtain a carrier-containing liquid for affinity chromatography (carrier content: 50% by volume). The carrier contained in this liquid is referred to as carrier 2.
Buffer replacement was performed by suction filtering 10 mL of the carrier 2-containing solution 10 times with 5 mL of 0.01 M NaOH aqueous solution using JIS P3801 standard 5 type A filter paper. The obtained dispersion and pure water were mixed in a bottle so that 0.6 g of carrier, 8 g of 0.01M NaOH aqueous solution, and 30 g of pure water were prepared. The obtained carrier-containing liquid was titrated with a 0.01M aqueous hydrochloric acid solution while stirring with a stirrer. In this titration, the above hydrochloric acid aqueous solution is added dropwise in 0.1 mL increments, pH 7 is set as the neutralization point, and from the amount of hydrochloric acid aqueous solution required to reach the neutralization point, the amino group content per 1 g of carrier solid content is determined as follows. Calculated using the formula.
Amino group content (μmol/g-bz) = {Amount of hydrochloric acid aqueous solution (mL) required to reach neutralization point x 0.01-8 x 0.01}/0.6
Similarly, the same operation was performed on the porous particles 5 obtained in Step A-2, and the amino group content per 1 g of porous particle solid content was calculated. Next, the modification rate (mol %) was calculated from the ratio of the amino group content per gram of solid content before and after post-modification using the following formula.
Modification rate (mol%) = (1-amino group content in carrier 2/amino group content in porous particles 5) × 100

(Example 3)
Step A-1 and Step A-2 of Example 1 except that 0.16 g of 1-[3-(dimethylamino)propyl]-3-ethylcarbodiimide used in Step B was changed to 0.10 g of acetic anhydride. Then, the same operation as in step B was performed to obtain carrier 3. The modification rate (mol %) was measured in the same manner as in Example 2.

(Example 4)
Step A-1 and Step A- of Example 1 except that 0.16 g of 1-[3-(dimethylamino)propyl]-3-ethylcarbodiimide used in Step B was changed to 0.10 g of succinic anhydride. Carrier 4 was obtained by carrying out the same operations as in Step 2 and Step B. The modification rate (mol %) was measured in the same manner as in Example 2.

(Example 5)
Step A-1 and Step A of Example 1 except that 0.16 g of 1-[3-(dimethylamino)propyl]-3-ethylcarbodiimide used in Step B was changed to 0.19 g of pivalic anhydride. -2 and Step B were performed to obtain carrier 5. The modification rate (mol %) was measured in the same manner as in Example 2.

(Example 6)
Step A-1, Step A-2, and Step A-2 of Example 1 except that 0.16 g of 1-[3-(dimethylamino)propyl]-3-ethylcarbodiimide used in Step B was changed to 0.08 g of glycidol. The same operation as in step B was performed to obtain carrier 6. The modification rate (mol %) was measured in the same manner as in Example 1.

(Example 7)
Step A-1 and Step A- of Example 1 except that the amount of 1-[3-(dimethylamino)propyl]-3-ethylcarbodiimide used in Step B was changed from 0.16 g to 0.03 g. Carrier 7 was obtained by carrying out the same operations as in Step 2 and Step B. The modification rate (mol %) was measured in the same manner as in Example 1.

(Example 8)
Step A-1 and Step A- of Example 1 except that the amount of 1-[3-(dimethylamino)propyl]-3-ethylcarbodiimide used in Step B was changed from 0.16 g to 0.32 g. Carrier 8 was obtained by carrying out the same operations as in Step 2 and Step B. The modification rate (mol %) was measured in the same manner as in Example 1.

(Example 9)
After dissolving 67.2 mg of sodium hydrogen carbonate (manufactured by Wako Pure Chemical Industries, Ltd.) in 5 mL of pure water, 5M sodium hydroxide (manufactured by Wako Pure Chemical Industries, Ltd.) and pure water were mixed with this to create carbonate buffer 2 (23 pH 13) was obtained.
The same steps as Step A-1, Step A-2 and Step B of Example 1 were carried out, except that carbonate buffer 1 (pH 10 at 23°C) used in Step B was changed to carbonate buffer 2 (pH 13 at 23°C). The operation was performed to obtain carrier 9. The modification rate (mol %) was measured in the same manner as in Example 1.

(Example 10)
After dissolving 151.0 mg of N-Cyclohexyl-3-aminopropanesulfonic acid (manufactured by Wako Pure Chemical Industries, Ltd., hereinafter also referred to as CAPS) in 5 mL of pure water, 1M sodium hydroxide (manufactured by Wako Pure Chemical Industries, Ltd.) and pure Water was mixed to obtain CAPS buffer (pH 10 at 23°C).
The same operations as Step A-1, Step A-2, and Step B of Example 1 were performed, except that carbonate buffer 1 (pH 10 at 23° C.) used in Step B was changed to CAPS buffer (pH 10 at 23° C.). A carrier 10 was obtained. The modification rate (mol %) was measured in the same manner as in Example 1.

(Example 11)
After dissolving 177.0 mg of N-Cyclohexyl-2-aminoethanesulfonic acid (manufactured by Wako Pure Chemical Industries, Ltd., hereinafter also referred to as CHES) in 5 mL of pure water, 1M sodium hydroxide (manufactured by Wako Pure Chemical Industries, Ltd.) and pure Water was mixed to obtain CHES buffer (pH 10 at 23°C).
The same operation as in Step A-1, Step A-2, and Step B of Example 1, except that carbonate buffer 1 (pH 10 at 23 °C) used in Step B was changed to CHES buffer (pH 10 at 23 °C). A carrier 11 was obtained. The modification rate (mol %) was measured in the same manner as in Example 1.

(Examples 12-23)
The carrier was prepared and the modification rate (mol%) was calculated in the same manner as in Example 1, except that the immunoglobulin binding protein PrA-1 prepared in Preparation Example was changed to PrA-2 to PrA-13.

(Comparative example 1)
Porous particles 5 were obtained by performing the same operations as in Step A-1 and Step A-2 of Example 1. This porous particle 5 is used as a carrier in Comparative Example 1.

(Comparative example 2)
The carrier was prepared and the modification rate (mol %) was calculated in the same manner as in Comparative Example 1, except that the immunoglobulin binding protein PrA-1 prepared in Preparation Example was changed to PrA-2.

(Test Example 1) Dynamic binding capacity (DBC) measurement test Using AKTA avant25 manufactured by Cytiva, each carrier of Examples and Comparative Examples was measured against protein (human IgG antibody, 1875-0007 manufactured by LGC) at a retention time of 4 minutes. DBC was measured. The column container had a capacity of 4 mL (5 mm φ x 200 mm length), the protein was 5 mg/mL dissolved in 20 mM sodium phosphate/150 mM sodium chloride aqueous solution (pH 7.5), and the elution tip had a 10% break. DBC was determined from the amount of protein captured during through-through and the column packing volume, and evaluated based on the following criteria. The results are shown in Tables 2 and 3.

(DBC evaluation criteria)
AAA (excellent): 60 mg/mL or more AA (excellent): 55 mg/mL or more and less than 60 mg/mL A (good): 50 mg/mL or more and less than 55 mg/mL B (poor): less than 50 mg/mL

(Test Example 2) Protein A Leakage Amount Measurement Test Using AKTA avant 25 manufactured by Cytiva, 7.5 mL of cell culture solution (Herceptin, titer: 4.38 mg/ml) was added to the cells of Examples and Comparative Examples with a holding time of 4 minutes. The antibodies were loaded onto each carrier, and then the antibodies were collected in the eluate. Considering actual use, a carrier soaked in 0.5 M NaOH for 100 cycles (25 hours) was used. In addition, a column container with a capacity of 0.8 mL (5 mm φ x 40 mm length) was used, and the eluent was a 100 mM aqueous sodium acetate solution (pH 3.3). Washing according to .5) was carried out.
Next, use the Protein A ELISA kit (F740) to determine the amount of protein A in the eluate and the antibody concentration in the eluate from the absorbance, and calculate the amount of antibody per amount of antibody in the eluate collected from these values. The amount of protein A leakage (leach) was calculated. It can be said that the smaller the value of protein A leakage (leach), the less likely the protein ligand will leak out even when it is repeatedly used for antibody isolation. The results are shown in Tables 2 and 3.

Claims

A method for producing a chromatography carrier, comprising the following steps A-1 and B.
(Step A-1) Step of immobilizing one or more kinds of ligands selected from protein A, protein G, protein L, and related substances to porous particles. (Step B) Ligand after step A-1. porous particles on which is fixed, and at least one ligand-reactive group selected from a group represented by -C(=O)-OC(=O)-, a carbodiimide group, and a cyclic ether group. Process of reacting with a compound
The compound having the ligand-reactive group is selected from a compound represented by the following formula (1) and its salt, a compound represented by the following formula (2), and a compound represented by the following formula (3). The method for producing a chromatography carrier according to claim 1, wherein the carrier is one or more kinds.

[In formula (1), R 1 and R 2 each independently represent a hydrogen atom or a substituted or unsubstituted hydrocarbon group. ]

[In formula (2), R 3 and R 4 each independently represent a substituted or unsubstituted hydrocarbon group, and R 3 and R 4 may be bonded to each other to form a cyclic structure. ]

[In formula (3), R 5 represents a substituted or unsubstituted hydrocarbon group, and X represents a cyclic ether group. ]
The compound having the ligand-reactive group is 1-[3-(dimethylamino)propyl]-3-ethylcarbodiimide, a salt of 1-[3-(dimethylamino)propyl]-3-ethylcarbodiimide, N,N' -Dicyclohexylcarbodiimide, N,N'-diisopropylcarbodiimide, maleic anhydride, propionic anhydride, acetic anhydride, pivalic anhydride, succinic anhydride, glutaric anhydride, propylene oxide, butylene oxide, glycidyl methyl ether, ethyl glycidyl ether The method for producing a chromatography carrier according to claim 1 or 2, wherein the carrier is one or more compounds selected from , glycidol, epichlorohydrin, and epibromohydrin.
The compound for chromatography according to any one of claims 1 to 3, wherein the compound having a ligand-reactive group is at least one compound selected from a compound represented by the following formula (1) and a salt thereof. Method for manufacturing carrier.

[In formula (1), R 1 and R 2 each independently represent a hydrogen atom, an alkyl group, a cycloalkyl group, an aminoalkyl group, a monoalkylaminoalkyl group, or a dialkylaminoalkyl group. ]
According to any one of claims 1 to 4, the amount of the compound having a ligand-reactive group used is 0.01 to 15 mmol per 1 g of dry weight of the porous particles on which the ligand is immobilized. A method for producing a carrier for chromatography.
The ligand has at least one or two or more substitutions selected from the following (a) to (i) to the amino acid sequence that has 85% or more homology to the amino acid sequence shown in SEQ ID NO: 2. The method for producing a chromatography carrier according to any one of claims 1 to 5, which is a protein ligand having an amino acid sequence.
(a) Substitution of the amino acid residue at the position corresponding to position 1 of the amino acid sequence of SEQ ID NO: 2 with a valine residue (b) Substitution of the amino acid residue at the position corresponding to position 3 of the amino acid sequence of SEQ ID NO: 2 with an alanine residue (c) Substitution of the amino acid residue at the position corresponding to position 6 of the amino acid sequence of SEQ ID NO: 2 with an alanine residue or aspartic acid residue (d) Corresponding to position 9 of the amino acid sequence of SEQ ID NO: 2 (e) Substitution of the amino acid residue at the position corresponding to position 11 of the amino acid sequence of SEQ ID NO: 2 with an alanine residue, glutamine residue, or glutamic acid residue (f ) Substitution of the amino acid residue at the position corresponding to position 23 of the amino acid sequence of SEQ ID NO: 2 with a leucine residue (g) Substitution of the amino acid residue at the position corresponding to position 29 of the amino acid sequence of SEQ ID NO: 2 with an alanine residue (h) Substitution of the amino acid residue at the position corresponding to position 43 of the amino acid sequence of SEQ ID NO: 2 with an alanine residue (i) Substitution of the amino acid residue at the position corresponding to position 49 of the amino acid sequence of SEQ ID NO: 2 Substitution to arginine residue
An amino acid sequence in which the ligand has at least three or more substitutions selected from the following (a) to (i) to an amino acid sequence that has 85% or more homology with the amino acid sequence shown in SEQ ID NO: 2. The method for producing a chromatography carrier according to any one of claims 1 to 5, which is a protein ligand having the following.
(a) Substitution of the amino acid residue at the position corresponding to position 1 of the amino acid sequence of SEQ ID NO: 2 with a valine residue (b) Substitution of the amino acid residue at the position corresponding to position 3 of the amino acid sequence of SEQ ID NO: 2 with an alanine residue (c) Substitution of the amino acid residue at the position corresponding to position 6 of the amino acid sequence of SEQ ID NO: 2 with an alanine residue or aspartic acid residue (d) Corresponding to position 9 of the amino acid sequence of SEQ ID NO: 2 (e) Substitution of the amino acid residue at the position corresponding to position 11 of the amino acid sequence of SEQ ID NO: 2 with an alanine residue, glutamine residue, or glutamic acid residue (f ) Substitution of the amino acid residue at the position corresponding to position 23 of the amino acid sequence of SEQ ID NO: 2 with a leucine residue (g) Substitution of the amino acid residue at the position corresponding to position 29 of the amino acid sequence of SEQ ID NO: 2 with an alanine residue (h) Substitution of the amino acid residue at the position corresponding to position 43 of the amino acid sequence of SEQ ID NO: 2 with an alanine residue (i) Substitution of the amino acid residue at the position corresponding to position 49 of the amino acid sequence of SEQ ID NO: 2 Substitution to arginine residue
The method for producing a chromatography carrier according to any one of claims 1 to 7, wherein the reaction in step B is carried out in an aqueous medium at a pH of 8 to 14.
The following step A-2 is further provided between step A-1 and step B, and the ligand after step A-1 is immobilized on the hydrophilic group-containing ligand-immobilized porous particles obtained in step A-2. The method for producing a chromatography carrier according to any one of claims 1 to 8, which is used in step B as porous particles.
(Step A-2) The porous particles on which the ligand was fixed in Step A-1 are reacted with a compound having a total of two or more hydrophilic groups of at least one kind selected from hydroxy groups and mercapto groups in the molecule. process of letting
The process further comprises the following steps A-P1 and A-P2, and in step A-1, porous particles reacted with at least one selected from a crosslinking agent and a hydrophilic agent are used as the porous particles. A method for producing a chromatography carrier according to any one of claims 1 to 9.
(Step A-P1) A step of dispersing the monomer composition in an aqueous medium and carrying out suspension polymerization. (Step A-P2) The porous particles obtained in step A-P1 and at least one selected from a crosslinking agent and a hydrophilizing agent. Step of reacting with one species
Porous particles, a ligand fixed to the porous particles, and at least one type selected from a group represented by -C(=O)-OC(=O)-, a carbodiimide group, and a cyclic ether group. and a partial structure derived from a compound having a ligand-reactive group,
The ligand is one or more ligands selected from protein A, protein G, protein L and related substances,
At least one functional group selected from an amino group and a carboxy group of the ligand is chemically modified with the partial structure,
The following formula:
Chemical modification rate (mol%) = (number of moles of chemically modified functional groups) / (sum of number of moles of chemically modified functional groups and number of moles of non-chemically modified functional groups) × 100
A carrier for chromatography, which has a chemical modification rate calculated from 1 to 70 mol%.
The chromatography carrier according to claim 11, wherein the amino group of the ligand is chemically modified with the partial structure.
The partial structure is -C(=O)-, -NR 1 -C(=O)-, -C(=NR 1 )- or -CH 2 -CH(-OH)- (R 1 is a hydrogen atom , an alkyl group, a cycloalkyl group, an aminoalkyl group, a monoalkylaminoalkyl group, or a dialkylaminoalkyl group) in its molecule. .
The partial structure is -NR 1 -C(=O)-NR 2 - or -C(=NR 1 )-NR 2 - (R 1 and R 2 are each independently a hydrogen atom, an alkyl group, a cyclo The chromatography carrier according to any one of claims 11 to 13, which has a group represented by an alkyl group, an aminoalkyl group, a monoalkylaminoalkyl group, or a dialkylaminoalkyl group in its molecule. .
The chromatography carrier according to claim 14, wherein at least one of R 1 and R 2 is an aminoalkyl group, a monoalkylaminoalkyl group, or a dialkylaminoalkyl group.
The ligand has at least one or two or more substitutions selected from the following (a) to (i) to the amino acid sequence that has 85% or more homology to the amino acid sequence shown in SEQ ID NO: 2. The chromatography carrier according to any one of claims 11 to 15, which is a protein ligand having an amino acid sequence.
(a) Substitution of the amino acid residue at the position corresponding to position 1 of the amino acid sequence of SEQ ID NO: 2 with a valine residue (b) Substitution of the amino acid residue at the position corresponding to position 3 of the amino acid sequence of SEQ ID NO: 2 with an alanine residue (c) Substitution of the amino acid residue at the position corresponding to position 6 of the amino acid sequence of SEQ ID NO: 2 with an alanine residue or aspartic acid residue (d) Corresponding to position 9 of the amino acid sequence of SEQ ID NO: 2 (e) Substitution of the amino acid residue at the position corresponding to position 11 of the amino acid sequence of SEQ ID NO: 2 with an alanine residue, glutamine residue, or glutamic acid residue (f ) Substitution of the amino acid residue at the position corresponding to position 23 of the amino acid sequence of SEQ ID NO: 2 with a leucine residue (g) Substitution of the amino acid residue at the position corresponding to position 29 of the amino acid sequence of SEQ ID NO: 2 with an alanine residue (h) Substitution of the amino acid residue at the position corresponding to position 43 of the amino acid sequence of SEQ ID NO: 2 with an alanine residue (i) Substitution of the amino acid residue at the position corresponding to position 49 of the amino acid sequence of SEQ ID NO: 2 Substitution to arginine residue
An amino acid sequence in which the ligand has at least three or more substitutions selected from the following (a) to (i) to an amino acid sequence that has 85% or more homology with the amino acid sequence shown in SEQ ID NO: 2. The chromatography carrier according to any one of claims 11 to 15, which is a protein ligand having the following.
(a) Substitution of the amino acid residue at the position corresponding to position 1 of the amino acid sequence of SEQ ID NO: 2 with a valine residue (b) Substitution of the amino acid residue at the position corresponding to position 3 of the amino acid sequence of SEQ ID NO: 2 with an alanine residue (c) Substitution of the amino acid residue at the position corresponding to position 6 of the amino acid sequence of SEQ ID NO: 2 with an alanine residue or aspartic acid residue (d) Corresponding to position 9 of the amino acid sequence of SEQ ID NO: 2 (e) Substitution of the amino acid residue at the position corresponding to position 11 of the amino acid sequence of SEQ ID NO: 2 with an alanine residue, glutamine residue, or glutamic acid residue (f ) Substitution of the amino acid residue at the position corresponding to position 23 of the amino acid sequence of SEQ ID NO: 2 with a leucine residue (g) Substitution of the amino acid residue at the position corresponding to position 29 of the amino acid sequence of SEQ ID NO: 2 with an alanine residue (h) Substitution of the amino acid residue at the position corresponding to position 43 of the amino acid sequence of SEQ ID NO: 2 with an alanine residue (i) Substitution of the amino acid residue at the position corresponding to position 49 of the amino acid sequence of SEQ ID NO: 2 Substitution to arginine residue
A chromatography column comprising the chromatography carrier according to any one of claims 11 to 17.
A method for isolating an antibody or a fragment thereof using the chromatography carrier according to any one of claims 11 to 17 or the chromatography column according to claim 18.