Substrate for filler, method for producing substrate for filler, filler, and method for producing protein

Abstract

This substrate for a filler has a hydroxyl group, which is derived from a hydrophilic monomer that is solidified by means of graft polymerization, at the surface of a porous organic polymer carrier. The porous organic polymer carrier contains 40 mol% or more of a constituent unit derived from a polyfunctional monomer relative to the total amount of all monomer components that constitute the porous organic polymer carrier. The hydrophilic monomer is a hydroxyl group-containing (meth)acrylate.

Description

Base material for filler, method for producing base material for filler, method for producing filler and protein

[0001] The present invention relates to a base material for a filler, a method for producing a base material for a filler, and a method for producing a filler and a protein.

[0002] Conventionally, chromatography has been used for the adsorption, separation, and purification of biopolymers such as proteins.

[0003] In recent years, there has been a demand for the development of columns that can accommodate applications requiring more precise and rapid separation and purification. For example, in gel filtration chromatography (GFC), the diameter of the packing material packed in the column must be reduced to achieve precise and rapid separation and purification. Furthermore, in applications such as separating high-molecular-weight proteins, the pore size of the packing material must be increased. However, reducing the diameter of the packing material and increasing the pore size poses a problem: a decrease in mechanical strength, such as pressure resistance.

[0004] Patent Document 1 discloses a technique for improving the mechanical strength of a filler substrate by adjusting the degree of crosslinking to 50 to 85 mol%. Specifically, the raw material monomers used to produce the filler substrate contain a monofunctional monomer having an epoxy group and a crosslinkable monomer, and when polymerizing the raw material monomers in the presence of a diluent and a polymerization initiator to obtain a polymer α, the amount of crosslinkable monomer in the raw material monomers is adjusted to 50 to 85 mol%, thereby obtaining a polymer with a crosslinking degree of 50 to 85 mol%. However, increasing the amount of crosslinkable monomer in the raw material monomers decreases the amount of monofunctional monomer having an epoxy group, resulting in a decrease in the hydrophilicity of the surface of the filler substrate.

[0005] Because GFC separates molecules based on molecular size, it is necessary to prevent interactions between the solute and the gel. In other words, it is necessary to prevent adsorption on the gel surface (hereinafter referred to as nonspecific adsorption). When the packing material is a synthetic organic polymer, such nonspecific adsorption is caused by hydrophobic interactions. It is known that nonspecific adsorption can lead to phenomena such as reduced resolution and peak tailing. Various measures have been proposed to reduce nonspecific adsorption. Among these various measures, the most commonly used method is to modify the packing material surface with hydrophilic functional groups to alleviate the hydrophobic interactions and suppress nonspecific adsorption (e.g., Patent Documents 2 and 3).

[0006] Patent Document 2 discloses a technique for grafting glycidol onto 4-8 μm polymer particles composed of a polyfunctional monomer. Patent Document 3 relates to a separation material composed of large particles (100 μm) suitable for protein adsorption, and discloses a technique for grafting a crosslinked polymer having hydroxyl groups and a linear hydrophilic polymer having hydroxyl groups or polyethylene glycol chains onto porous polymer particles.

[0007] International Publication No. 2019 / 187377 Patent No. 6731402 JP 2020-22940 A

[0008] However, neither Patent Document 2 nor Patent Document 3 considers the suppression of nonspecific adsorption when the particle size of the packing material is reduced to 10 μm or less. An object of the present invention is to provide a packing material that is suitable for use in high-precision, rapid separation and purification, has sufficient mechanical strength, and is capable of reducing nonspecific adsorption, and to provide a packing material base material that realizes such a packing material, and a method for producing the same.

[0009] The present inventors have found that the above-mentioned problems can be solved by fixing a predetermined hydrophilic monomer by graft polymerization to the surface of a porous organic polymer support containing a predetermined amount or more of structural units derived from a polyfunctional monomer.

[0010] That is, the present invention is as follows. <Filler substrate> [1] A filler substrate having hydroxyl groups derived from hydrophilic monomers fixed on the surface of a porous organic polymer carrier by graft polymerization, the porous organic polymer carrier containing 40 mol % or more of structural units derived from polyfunctional monomers relative to all monomer components constituting the porous organic polymer carrier, and the hydrophilic monomer being a (meth)acrylate having a hydroxyl group. [2] The filler substrate according to [1], which is made of particles having a number-average particle size of 3 to 10 μm and a CV value of the particle size distribution of 8% or less. [3] The filler substrate according to [1] or [2], wherein the porous organic polymer carrier has a pore size distribution determined by mercury intrusion porosimetry that shows a pore size peak in the range of 30 nm to 500 nm. [4] A polymer having atom transfer radical polymerization (hereinafter referred to as ATRP) initiating groups remaining after the graft polymerization, wherein the amount of bromine atoms derived from the ATRP initiating groups per specific surface area is 0.3 to 2.5 mg / m ２[5] The packing substrate according to any one of [1] to [3], wherein the pore ratio, which is the ratio of the total pore volume of the packing to the internal volume of a column packed with a packing using the packing substrate, is 12 to 30%. [6] The packing substrate according to [2], wherein the graft amount of the (meth)acrylate having a hydroxyl group to the particles is 50 to 200 mg / g of particles. [7] The packing substrate according to any one of [1] to [6], wherein the (meth)acrylate having a hydroxyl group is at least either glycerin monomethacrylate or glycerin monoacrylate. [8] The packing substrate according to any one of [1] to [7], wherein the 10% thermal weight loss in nitrogen is 290 to 330°C. <Method for producing a packing substrate> [9] A method for producing the packing substrate according to any one of [1] to [8], comprising the steps of: polymerizing raw material monomers containing 40 mol % or more of a polyfunctional monomer to obtain a porous organic polymer support; introducing ATRP initiator groups into the porous organic polymer support; and grafting a hydrophilic monomer to the initiator groups. <Packing>

[10] A packing for size exclusion chromatography using the packing substrate according to any one of [1] to [8]. <Method for producing a protein>

[11] A method for producing a protein, comprising the steps of passing a solution containing a protein through a column packed with the size exclusion chromatography packing according to

[10] , and purifying the protein loaded into the size exclusion chromatography packing.

[0011] According to the present invention, there are provided a packing material that is suitable for use in high-precision and rapid separation and purification, has sufficient mechanical strength, and is capable of reducing nonspecific adsorption, as well as a packing material base material that realizes said packing material and a method for producing the same.

[0012] FIG. 1 is a diagram illustrating a method for determining a specific surface area from a pore size distribution measured by mercury intrusion porosimetry.

[0013] Hereinafter, embodiments of the present invention will be described in detail. In this specification, the expression "to" is used to express a range that includes the upper and lower limits of a numerical value or physical quantity. The upper and lower limits of a numerical range can be combined in any combination.

[0014] <Filler substrate> In one embodiment, the filler substrate has a hydroxyl group derived from a hydrophilic monomer fixed on the surface of a porous organic polymer carrier by graft polymerization, and the porous organic polymer carrier contains 40 mol% or more of a structural unit derived from a polyfunctional monomer relative to the total monomer components constituting the porous organic polymer carrier, and the hydrophilic monomer is a (meth)acrylate having a hydroxyl group.In this specification, the "surface of the porous organic polymer carrier" includes not only the outer surface of the porous organic polymer carrier, but also the surface of the pores inside the porous organic polymer carrier.

[0015] [Porous Organic Polymer Carrier] The porous organic polymer carrier contains 40 mol% or more of structural units derived from polyfunctional monomers relative to the total monomer components constituting the porous organic polymer carrier. The porous organic polymer carrier may contain 50 mol% or more, or even 60 mol% or more, of structural units derived from polyfunctional monomers relative to the total monomer components constituting the porous organic polymer carrier. The porous organic polymer carrier may contain structural units derived from monofunctional monomers. It is preferable that the porous organic polymer carrier contains structural units derived from monofunctional monomers to the extent that its properties are not significantly changed. For example, from the viewpoint of obtaining large pores, it is preferable to increase the content of structural units derived from monofunctional monomers, but from the viewpoint of avoiding a decrease in elasticity and an increase in column pressure, it is preferable that the content of structural units derived from monofunctional monomers is not excessively high. The monofunctional monomer is a crosslinkable compound having one ethylenic double bond in the molecule. The monofunctional monomer may have one vinyl group or one (meth)acryloyl group in the molecule.The monofunctional monomer is not particularly limited, and may be, for example, epoxy compounds such as glycidyl (meth)acrylate, 4,5-epoxypentyl (meth)acrylate, 4-(2,3-epoxypropyl)-n-butyl (meth)acrylate, 9,10-epoxystearyl acrylate, 4-(2,3-epoxypropyl)cyclohexylmethyl acrylate, and allyl glycidyl ether; alicyclic epoxy compounds such as 3,4-epoxycyclohexylmethyl (meth)acrylate, 3,4-epoxycyclohexylethyl (meth)acrylate, 3,4-epoxycyclohexylpropyl (meth)acrylate, and vinylbenzyl glycidyl ether; or styrene, methylstyrene, ethylstyrene, hydroxystyrene, chlorostyrene, etc. Any aromatic monomers, (meth)acrylates such as methyl (meth)acrylate, ethyl (meth)acrylate, hydroxyethyl (meth)acrylate, hydroxypropyl (meth)acrylate, hydroxybutyl (meth)acrylate, hydroxypentyl (meth)acrylate, 2-chloroethyl (meth)acrylate, and polyethylene glycol (meth)acrylate, (meth)acrylamides such as dimethyl (meth)acrylamide, diethyl (meth)acrylamide, hydroxyethyl (meth)acrylamide, hydroxypropyl (meth)acrylamide, and hydroxybutyl (meth)acrylamide, haloalkyl (C1-C4) vinyl ethers, hydroxyalkyl (C1-C4) vinyl ethers, and vinyl acetates may be used. Among these, compounds having a glycidyl group are preferred because they can introduce a hydroxyl group for introducing an ATRP initiating group. Specifically, glycidyl methacrylate is preferably used.

[0016] (Polyfunctional Monomer) The polyfunctional monomer is a crosslinkable compound having two or more ethylenic double bonds in the molecule. The polyfunctional monomer may have two or more (meth)acryloyl groups in the molecule. Specific examples include alkanediol di(meth)acrylate (the alkane has 1 to 12 carbon atoms), trimethylolpropane tri(meth)acrylate, ditrimethylolpropane tetra(meth)acrylate, pentaerythritol tetra(meth)acrylate, pentaerythritol penta(meth)acrylate, dipentaerythritol penta(meth)acrylate, dipentaerythritol hexa(meth)acrylate, and the like. Other examples include polyfunctional urethane (meth)acrylate. These compounds may be used alone or in combination of two or more.

[0017] The polyfunctional monomer may include at least one of ethylene glycol dimethacrylate or glycerol dimethacrylate. The glycerol dimethacrylate may be glycerol 1,3-dimethacrylate, glycerol 1,2-dimethacrylate, or an isomer mixture. At least one of ethylene glycol dimethacrylate or glycerol dimethacrylate may be contained in an amount of 50 mol% or more, or 80 mol% or more, based on the total amount of polyfunctional monomers. In particular, in the case of polyfunctional monomers having hydroxyl groups, such as glycerol dimethacrylate, it may be contained in an amount of 100 mol%. The use of glycerol dimethacrylate results in a particularly high degree of crosslinking and high strength.

[0018] (Proportion of polyfunctional monomer) The proportion of polyfunctional monomer, i.e., the degree of crosslinking of the porous organic polymer carrier, is preferably 40 to 100 mol%, more preferably 50 to 10 mol%, and most preferably 60 to 100 mol%. When the proportion of polyfunctional monomer is 40 mol% or more, sufficient mechanical strength can be obtained.

[0019] The proportion of the polyfunctional monomer can be calculated by the following formula: Proportion of polyfunctional monomer (mol %)=(total number of moles of polyfunctional monomer / total number of moles of raw material monomers)×100 The total number of moles of raw material monomers is the total number of moles of monomers used in the synthesis of the porous organic polymer support.

[0020] [Hydrophilic Monomer] As will be described later, the hydrophilic monomer is immobilized by graft polymerization to the ATRP initiation group introduced onto the surface of the porous organic polymer support.

[0021] The method for introducing an ATRP initiator group in the ATRP method is not particularly limited. However, when a double bond remains in the porous organic polymer support, the ATRP initiator group can be introduced by reacting hydrobromic acid, hydrochloric acid, or the like with the double bond. Furthermore, when a hydroxyl group exists on the surface of the porous organic polymer support, the initiator group can be easily introduced by reacting 2-bromopropionyl bromide with the porous organic polymer support. From the viewpoint of suppressing nonspecific adsorption, the amount of the ATRP initiator group introduced is 0.3 to 2.5 mg / m per specific surface area of ​​the porous organic polymer support. ２ is preferred, and 0.35 to 2.5 mg / m ２ More preferably, 0.4 to 2.5 mg / m ２ is most preferred.

[0022] The catalyst for the ATRP method is not particularly limited and can be selected from a wide range of catalysts commonly used in the ATRP method. For example, a transition metal complex can be used as the catalyst. The transition metal complex is not particularly limited and can be selected from a wide range of catalysts. The transition metal complex can be used by appropriately selecting a ligand and a transition metal from the ligand group and transition metal group exemplified below and combining them. Examples of the ligand include 2,2'-bipyridyl, 4,4'-dimethyl-2,2'-dipyridyl, 4,4'-di-tert-butyl-2,2'-dipyridyl, 4,4'-dinonyl-2,2'-dipyridyl, N-butyl-2-pyridylmethanimine, N-octyl-2-pyridylmethanimine, N-dodecyl-N-(2-pyridyl-methylene)amine, N-octadecyl-N-(2-pyridylmethylene)amine, and N,N,N',N',N'-pentane. Examples of the transition metal include trimethyl-diethylenetriamine, tris(2-pyridylmethyl)amine, 1,1,4,7,10,10-hexamethyltriethylenetetramine, tris[2-(dimethylamino)ethylamine], 1,4,8,11-tetraazacyclotetradecane, 1,4,8,11-tetramethyl-1-4-8-11-tetraazacyclotetradecane, and N,N,N',N'-tetrakis(2-pyridylmethyl)-ethylenediamine. ２ , CuBr, CuBr ２ , TiCl ２ , TiCl ３ , TiCl ４ , TiBr ４ , FeCl ２ , FeCl ３ , FeBr ２ , FeBr ３ , CoCl ２ , COBr ２ , NiCl ２ , NiBr ２ , MoCl ３ , MoCl ５ and RuCl ３ As the transition metal complex, it is preferable to use a monovalent or divalent copper complex. The divalent copper complex is not particularly limited, but may be CuBr ２It is preferable to use a pentamethyldiethylenetriamine complex or the like in terms of cost.

[0023] When a solvent is used in the ATRP method, it is not particularly limited, but a solvent used in free radical polymerization that can dissolve the catalyst relatively uniformly is preferred. For example, at least one solvent selected from the group consisting of water, ethers, amides, nitriles, and alcohols can be used. Examples of ethers include, but are not limited to, diethyl ether, tetrahydrofuran, diphenyl ether, anisole, and dimethoxybenzene. Examples of amides include, but are not limited to, N,N-dimethylformamide (DMF) and N,N-dimethylacetamide. Examples of nitriles include, but are not limited to, acetonitrile, propionitrile, and benzonitrile. Examples of alcohols include, but are not limited to, methanol, ethanol, propanol, isopropanol, n-butyl alcohol, t-butyl alcohol, and isoamyl alcohol. The solvent is preferably at least one selected from the group consisting of water, ethers, amides, and alcohols. From the viewpoint of polymerization rate, it is preferable to use water, dimethyl sulfoxide, DMF, or a mixture thereof.

[0024] The hydrophilic monomer is preferably water-soluble from the viewpoint of suppressing nonspecific adsorption. The hydrophilic monomer may be a (meth)acrylate having a hydroxyl group. Use of a (meth)acrylate having a hydroxyl group particularly improves hydrophilicity.

[0025] Examples of the (meth)acrylate having a hydroxyl group include methacrylates such as hydroxyethyl methacrylate, glycerin monomethacrylate, and polyethylene glycol monomethacrylate; and acrylates such as hydroxyethyl acrylate, glycerin monoacrylate, and polyethylene glycol monoacrylate.

[0026] The graft amount of hydrophilic monomer is preferably 50 to 200 mg / g of particle, more preferably 75 to 200 mg / g of particle, and most preferably 75 to 180 mg / g of particle, relative to the particle weight (hereinafter referred to as "particle g") of the porous organic polymer carrier. When the graft amount of hydrophilic monomer is 50 mg / g of particle or more, sufficient hydrophilicity is imparted to the surface of the porous organic polymer carrier, reducing nonspecific adsorption. When the graft amount of hydrophilic monomer is 200 mg / g of particle or less, the pores of the porous organic polymer carrier are not buried by the graft chains, ensuring pore volume. Specifically, the graft amount can be calculated from the dry particle weight before and after grafting using the method described in the Examples.

[0027] [Bromine Atom Mass of Filler Base Material] When an ATRP initiating group is introduced and the ATRP initiating group and a hydrophilic monomer are graft polymerized as described above, the ATRP initiating group remains in the filler base material after the hydrophilic monomer has been graft polymerized. By measuring the amount of bromine atoms derived from the ATRP initiating group in the filler base material, it can be confirmed that the filler base material has been produced through the above process. The bromine atom mass is 0.3 to 2.5 mg / m from the viewpoint of suppressing nonspecific adsorption. ２ is preferred, and 0.35 to 2.5 mg / m ２ More preferably, 0.4 to 2.5 mg / m ２ is most preferred.

[0028] [Number average particle size and CV value of filler substrate] The number average particle size and the coefficient of variation of the particle size distribution (hereinafter referred to as CV value) of the filler substrate can be determined by the following measurement method. 1) Particles that are the filler substrate are dispersed in water (containing a dispersant such as a surfactant) to prepare a dispersion containing 1 mass % of particles. 2) Using a particle size distribution analyzer (Sysmex Flow, manufactured by Sysmex Corporation), the number average particle size and the CV value of the particle size are measured using images of approximately 10,000 particles in the dispersion.

[0029] The number average particle size of the filler base material is preferably 2 to 10 μm, more preferably 2.5 to 10 μm, and most preferably 3 to 10 μm.

[0030] The CV value of the main peak of the particle size distribution of the filler base material is preferably 8% or less, more preferably 7 to 1%, and most preferably 6 to 1%. A method for reducing the CV value includes monodispersion by seed polymerization.

[0031] [Pore Size Peak of Filler Substrate] The pore size peak of the filler substrate can be measured using a mercury intrusion measurement device (Autopore: manufactured by Shimadzu Corporation). Specifically, approximately 0.05 g of sample is added to a standard 5 mL powder cell (stem volume 0.4 mL) and measured under an initial pressure of 21 kPa (approximately 3 psia, equivalent to a pore diameter of approximately 60 μm). The mercury parameters are set to the default mercury contact angle of 130° and mercury surface tension of 485 dynes / cm. Furthermore, measurements are performed in the pore size range of 0.005 to 90 μm, and the pore size peak can be calculated from the maximum value between 0.005 and 1 μm.

[0032] The pore size distribution determined by the mercury intrusion method preferably exhibits a pore size peak in the range of 30 nm to 500 nm, more preferably in the range of 50 nm to 450 nm, and most preferably in the range of 30 nm to 400 nm.

[0033] A packing base material having an average particle size, CV value and pore size peak within the above ranges is suitable for use in high-precision and rapid separation and purification.

[0034] [Pore ratio of packing substrate] The pore ratio, which is the ratio of the total pore volume of the packing to the internal volume of a column packed with a packing made from the packing substrate, is preferably 12 to 30%, more preferably 15 to 30%, and most preferably 18 to 30%. A higher pore ratio can improve separation performance during size exclusion chromatography.

[0035] [10% Thermal Weight Loss Temperature of Filler Base Material] The filler base material preferably has a "10% thermal weight loss temperature in nitrogen" measured with a simultaneous differential scanning calorimeter and thermogravimetry analyzer of 290 to 330° C., more preferably 295 to 330° C., and most preferably 300 to 330° C. The higher the lower limit of the thermal weight loss temperature, the more progressed the grafting on the surface, and the greater the effect of suppressing nonspecific adsorption.

[0036] <Method for producing a filler substrate> In one embodiment, a method for producing a filler substrate includes the steps of: polymerizing raw material monomers containing 40 mol % or more of a polyfunctional monomer to obtain a porous organic polymer support; introducing ATRP initiation groups into the porous organic polymer support molecules; and grafting hydrophilic monomers onto the ATRP initiation groups.

[0037] [Step of Obtaining Porous Organic Polymer Carrier] The porous organic polymer carrier may be obtained by polymerizing raw material monomers containing 40 mol% or more of the above-mentioned polyfunctional monomer in the presence of a porosifying agent. The porous organic polymer carrier may be obtained by impregnating seed particles (such as polymethyl methacrylate or polystyrene having a weight average molecular weight (Mw) of 50,000 or less) with the raw material monomers and then polymerizing them (seed polymerization). The seed particles may generally be synthesized by emulsion polymerization, soap-free emulsion polymerization, or dispersion polymerization.

[0038] Examples of the porosity-inducing agent include aliphatic or aromatic hydrocarbons, esters, ketones, ethers, alcohols, and the like, which are organic solvents that promote phase separation during polymerization and facilitate the formation of porous particles. Specific examples include toluene, xylene, diethylbenzene, cyclohexane, octane, butyl acetate, diethyl succinate, dibutyl phthalate, methyl ethyl ketone, dibutyl ether, 1-hexanol, 1-octanol, decanol, lauryl alcohol, and cyclohexanol. These porosity-inducing agents can be used alone or in combination of two or more.

[0039] The porosifying agent can be used in an amount of, for example, 0 to 250% by mass based on the total mass of the monomers.

[0040] The pore size, exclusion limit molecular weight, etc. of the porous organic polymer carrier particles can be controlled by the type and amount of the porosity-inducing agent, so the type and amount may be selected according to the purpose.

[0041] Examples of the aqueous medium used in the polymerization reaction include water and a mixture of water and a water-soluble solvent (e.g., a lower alcohol). The aqueous medium may contain a surfactant. The surfactant may be any of anionic, cationic, nonionic, and amphoteric surfactants.

[0042] Examples of anionic surfactants include fatty acid oils such as sodium oleate and castor oil potassium, alkyl sulfates such as sodium lauryl sulfate and ammonium lauryl sulfate, alkylbenzenesulfonates such as sodium dodecylbenzenesulfonate, alkylnaphthalenesulfonates, alkanesulfonates, dialkylsulfosuccinates such as sodium dioctyl sulfosuccinate, alkenylsuccinates (dipotassium salts), alkyl phosphates, naphthalenesulfonate-formalin condensates, polyoxyethylene alkylphenyl ether sulfates, polyoxyethylene alkyl ether sulfates such as sodium polyoxyethylene lauryl ether sulfate, and polyoxyethylene alkyl sulfates. Examples of cationic surfactants include alkylamine salts such as laurylamine acetate and stearylamine acetate, and quaternary ammonium salts such as lauryltrimethylammonium chloride. Examples of nonionic surfactants include hydrocarbon-based nonionic surfactants such as polyethylene glycol alkyl ethers, polyethylene glycol alkylaryl ethers, polyethylene glycol esters, polyethylene glycol sorbitan esters, and polyalkylene glycol alkylamines or amides; polyether-modified silicon-based nonionic surfactants such as silicon polyethylene oxide adducts and polypropylene oxide adducts; and fluorine-based nonionic surfactants such as perfluoroalkyl glycols. Examples of amphoteric surfactants include hydrocarbon surfactants such as lauryl dimethylamine oxide, phosphate ester surfactants, and phosphite ester surfactants. The surfactants may be used alone or in combination of two or more. Anionic surfactants may be selected from the viewpoint of dispersion stability during polymerization.

[0043] A polymerization initiator may be used. Examples of the polymerization initiator include organic peroxides such as benzoyl peroxide, lauroyl peroxide, orthochlorobenzoyl peroxide, orthomethoxybenzoyl peroxide, 3,5,5-trimethylhexanoyl peroxide, tert-butylperoxy-2-ethylhexanoate, and di-tert-butyl peroxide; and azo compounds such as 2,2'-azobisisobutyronitrile, 1,1'-azobiscyclohexanecarbonitrile, and 2,2'-azobis(2,4-dimethylvaleronitrile). The polymerization initiator may be used in an amount of, for example, 0.1 to 7.0 parts by mass per 100 parts by mass of the monomer.

[0044] The polymerization temperature can be appropriately selected depending on the types of monomer and polymerization initiator, and is preferably 25 to 110°C, more preferably 50 to 100°C.

[0045] In the synthesis of a porous organic polymer carrier, a polymer dispersion stabilizer may be added to improve the dispersion stability of the particles. Examples of polymer dispersion stabilizers include polyvinyl alcohol, polycarboxylic acid, celluloses (hydroxyethyl cellulose, carboxymethyl cellulose, methyl cellulose, etc.), and polyvinylpyrrolidone. Inorganic water-soluble polymer compounds such as sodium tripolyphosphate can also be used in combination. Of these, polyvinyl alcohol or polyvinylpyrrolidone is preferred. The amount of polymer dispersion stabilizer added may be, for example, 0.01 to 10 parts by mass per 100 parts by mass of monomer. From the viewpoint of handleability and dispersion stability, 0.01 to 5 parts by mass is preferred, 0.05 to 5 parts by mass is more preferred, and 0.1 to 3 parts by mass is most preferred.

[0046] In order to suppress polymerization of the raw material monomers in the aqueous phase, a water-soluble polymerization inhibitor such as nitrites, sulfites, hydroquinones, ascorbic acids, water-soluble vitamin B, citric acid, polyphenols, etc. may be used.

[0047] [Step of Introducing ATRP Initiating Groups] In this step, ATRP initiating groups are introduced to hydroxyl groups on the surface of the porous organic polymer support. Introduction of the ATRP initiating groups makes it possible to graft hydrophilic monomers onto the hydroxyl groups. Examples of methods for introducing ATRP initiating groups include a method using an alkyl halide compound.

[0048] The alkyl halide compound is preferably a bromide or chloride, and examples of the alkyl halide compound include compounds having an alkyl halide such as 2-bromobutyryl bromide.

[0049] Since the ATRP initiation group is introduced into the hydroxyl group on the surface of the particle, which is a porous organic polymer support, the particles are filtered to remove water, then immersed in an organic solvent, and an alkyl halide is added in the presence of a predetermined concentration of amine to cause the reaction. The reaction temperature for this reaction is preferably 10°C or lower when adding the alkyl halide, and then the reaction is continued at room temperature for 12 to 24 hours.

[0050] When the particle surface contains epoxy groups, a step of ring-opening the epoxy groups may be carried out prior to the introduction of the ATRP initiation group. Ring-opening the epoxy groups means adding water to the epoxy groups on the particle surface to convert the epoxy groups into 1,2-diol groups (-CH(OH)-CH ２ The method for ring-opening the epoxy group is not particularly limited, and any method may be used. Examples include a method using an acidic aqueous solution containing hydrochloric acid, sulfuric acid, phosphoric acid, etc., and a method using a basic aqueous solution containing sodium hydroxide, potassium hydroxide, etc.

[0051] [Step of Grafting Hydrophilic Monomer] In this step, the above-mentioned hydrophilic monomer is graft-polymerized onto the particles into which the ATRP initiation group has been introduced.

[0052] Since the reaction of the hydrophilic monomer is carried out at the ATRP initiator group, the particles to which the ATRP initiator group has been introduced may be filtered or otherwise removed, then immersed in an organic solvent, to which copper halide, tris(dimethylaminoethylamine), ascorbic acid, and the hydrophilic monomer are added to give predetermined concentrations, and the reaction may be carried out under an inert gas atmosphere. The reaction temperature is preferably 20 to 40°C, and more preferably 12 to 24 hours.

[0053] <Filler> In one embodiment, the filler may be a filler for size exclusion chromatography. The above-mentioned filler base material is a porous organic polymer carrier containing 40 mol% or more of a constitutional unit derived from a crosslinkable polyfunctional monomer relative to the total monomer components constituting the porous organic polymer carrier, and a (meth)acrylate having a hydroxyl group is fixed to the surface of the porous organic polymer carrier by graft polymerization. Therefore, the filler using the above-mentioned filler base material can have both suitable mechanical strength and hydrophilic properties suitable for suppressing nonspecific adsorption.

[0054] The average particle size of the packing material is preferably 10 μm or less, more preferably 3.0 to 8.0 μm. When it is 10 μm or less, a high number of theoretical plates is easily obtained. When it is 3.0 μm or more, the column pressure is less likely to become high.

[0055] The pore size of the filler is preferably 50 to 500 nm, more preferably 100 to 500 nm, and particularly preferably 100 to 400 nm.

[0056] The packing material preferably has an exclusion limit molecular weight of 10,000 to 20,000,000, more preferably 100,000 to 20,000,000, and particularly preferably 100,000 to 10,000,000. Specifically, the exclusion limit molecular weight can be determined by evaluating the elution positions of standard substances with low to different molecular weights: pullulan standard (trade name: Shodex (registered trademark) STANDARD P-82, manufactured by Resonac Corporation), using the method described in the Examples, and calculating the molecular weight of the pullulan standard whose elution position no longer changes as the exclusion molecular weight.

[0057] Packing materials having an average particle size, pore size and exclusion limit molecular weight within the above ranges are suitable for use in high-precision and rapid separation and purification.

[0058] The average particle size, pore size and exclusion limit molecular weight can be adjusted by appropriately selecting raw material monomers, porosifying agents, hydrophilic monomers for grafting and the like during the production of the filler base material.

[0059] <Protein Production Method> The protein production method of this embodiment is carried out by GFC using the above-mentioned packing material, and includes a step of passing a protein-containing solution through a column packed with the above-mentioned packing material, and a purification step of eluting the protein loaded into the packing material. The protein contained in the solution may be extracted from nature or may be artificially synthesized. The mass (kDa) of the protein contained in the solution is, for example, preferably 10 to 10,000 kDa, more preferably 100 to 10,000 kDa, and most preferably 1,000 to 5,000 kDa.

[0060] In the protein production method of this embodiment, any known method can be used except for using the above-mentioned packing material. The protein production method of this embodiment, which uses a column packed with the above-mentioned packing material, is suitable for use in highly accurate and rapid separation and purification.

[0061] The present invention will be described below with reference to examples, but the present invention is not limited to these examples.

[0062] Example 1 Synthesis of Porous Organic Polymer Carrier (1) 3.8 g of glycerol dimethacrylate (GDMA), 9.0 g of ethylene glycol dimethacrylate (EGDM) (100 mol% polyfunctional monomer), 0.17 g of benzoyl peroxide as a polymerization initiator, 24.5 g of octanol, and 2.7 g of dichloroethane were added to prepare an oil phase. 5.2 g of an aqueous solution of sodium dodecylbenzenesulfonate (Kao, G-15) and 107 g of water were prepared as an aqueous phase. The oil and aqueous phases were mixed and emulsified using a homomixer (18,000 rpm, 10 min), and the resulting emulsion was transferred to a 200 mL flask. Subsequently, 1.5 g of polymethyl methacrylate (PMMA) seed particle dispersion (particle size 0.63 μm, 10 wt %, Mw 9700) was added, and stirring was continued at 30°C for 24 hours to swell the seed particles. While heating in an 80°C water bath, stirring was continued for 6 hours using a stirrer. The temperature was then raised to 95°C, and polymerization was continued for 2 hours. The resulting particles were centrifuged, the supernatant was discarded, and 300 mL of water was added and redispersed three times. The resulting particles were redispersed in 300 mL of acetone and filtered three times to obtain porous organic polymer carrier (1). (Introduction of ATRP Initiating Group) 5 g of porous organic polymer carrier (1), 0.8 g of triethylamine, and 180 g of acetonitrile were added to a 500 mL flask and stirred while cooling on ice. Then, a solution of 2.3 mL of 2-bromoisobutyryl bromide in 20 g of acetonitrile was added dropwise over 10 minutes. After stirring for 1 hour, the temperature was raised to 30°C, and the reaction was continued for 18 hours. The resulting particle dispersion was filtered and washed with an additional 600 mL of acetone to obtain initiator group-introduced particles (1). (Graft Polymerization) 5 g of initiator group-introduced particles (1), 28.5 g of Blemmer GLM (glycerin methacrylate, NOF Corporation, abbreviated name: GMMA), 5.4 mg of copper (II) bromide, 4.2 mg of pentamethyldiethylenetriamine, 4.2 mg of ascorbic acid, and 117 g of a methanol / water mixed solution (5 / 5 vol% ratio) were added to a 500 mL flask and stirred at 30°C. Nitrogen bubbling was performed to initiate polymerization, and the reaction was continued for 6 hours. The resulting particles were filtered and washed by pouring 200 mL of water three times.Further, 200 mL of methanol was poured three times, and then 200 mL of acetone was poured further to obtain a GMMA-grafted base material for fillers (1).

[0063] (Calculation of Graft Amount) The obtained filler substrate (1) was dried at 60° C. for 12 hours, and then its weight was measured. The graft amount was calculated from the weight difference between the filler substrate (1) and the initiator group-introduced particles (1). The calculation results are shown in Table 1.

[0064] (Calculation of number average particle diameter and CV value of particle diameter) The particle diameter of the obtained filler base material (1) was measured with a flow type particle diameter measuring device (FPIA-3000, manufactured by Sysmex Corporation), and the number average particle diameter and CV value of particle diameter were calculated. The calculation results are shown in Table 1.

[0065] (Pore diameter peak) The pore diameter of the filler substrate (1) was evaluated using an Autopore IV9505 (Shimadzu Corporation). The sample amount was 50 mg, added to a standard 5 mL powder cell (stem volume 0.4 mL), and measured in the range of pore diameters of 0.005 to 90 μm under conditions of an initial pressure of 21 kPa (approximately 3 psia, equivalent to a pore diameter of approximately 60 μm). The mercury parameters were set to the default mercury contact angle of 130 ° and mercury surface tension of 485 dynes / cm. The evaluation results are shown in Table 1.

[0066] (Specific Surface Area Determined by Mercury Intrusion Porosimetry) The sum of the measured values ​​obtained by converting the amount of mercury intrusion into specific surface area for the pore diameter (c) with the smallest amount of mercury intrusion between the interparticle pore peak (a) and the filler peak (b) in FIG. 1 up to 10 nm was taken as the "specific surface area determined by mercury intrusion porosimetry" and was used to calculate the bromine atomic weight described below.

[0067] (Bromine atom weight) The bromine atom weight of the filler substrate (1) was measured as follows. Approximately 0.6 to 8 mg of sample was weighed and subjected to thermal decomposition in a sample combustion device (AQF-100) under oxygen flow while passing water vapor through it. The combustion tube decomposition conditions were as follows. The generated gas was absorbed in a weak alkaline solution (containing hydrogen peroxide solution). Then, the bromine atom weight was measured by anion chromatography. －was analyzed and converted into the Br concentration in the sample. The anion chromatography measurement conditions were as follows. The amount of bromine contained in the particle weight obtained from the bromine concentration was calculated. The "bromine atomic weight per specific surface area determined by mercury porosimetry" was calculated from the amount of bromine and the "specific surface area determined by mercury porosimetry." The results are shown in Table 1. <Combustion tube decomposition conditions> Apparatus: AQF-100 (Nitto Seiko Analytech) Combustion tube temperature: 1000°C (heat increase due to movement of the combustion boat) Absorption liquid: carbonate-based weak alkaline liquid (H ２ O ２ (including water) [correction standard: tartaric acid; 5 ppm by mass] Initial absorption solution volume: 10 ml <Conditions for anion chromatography measurement> Measurement device: DIONEX ICS-1600 Eluent: 2.7 mM Na ２ CO ３ +0.3mM NaHCO ３ Column (temperature): DIONEX AG12A / AS12A (30°C) Flow rate: 1.5 mL / min Injection volume: 100 μL Detector: Electrical conductivity detector (with suppressor)

[0068] (10% Thermal Weight Loss Temperature) The 10% thermal weight loss temperature of the filler base material (1) was measured using a simultaneous thermogravimetry and differential thermal analyzer (NEXTA STA200RV, manufactured by Hitachi) under the following conditions. The measurement conditions were as follows. The measurement results are shown in Table 1. <Measurement conditions> - Sample amount: 5 to 10 mg - Heating program: 40°C, hold for 30 minutes → heat to 500°C at 10°C / min → hold at 500°C for 15 minutes → air cool - Measurement atmosphere: 100 mL / min, nitrogen - The temperature at which a further 10% weight loss occurred from 150°C by weight was taken as the 10% weight loss temperature.

[0069] (Column Packing) The obtained packing base material (1) was packed into a stainless steel column having a diameter of 4.6 mm and a length of 150 mm under the conditions of pure water as a packing solvent, 0.1 M aqueous potassium chloride solution as a packing dispersion solvent, a packing pressure of 4 MPa, and a packing time of 25 minutes.

[0070] (Evaluation of Protein Recovery Rate) Protein was measured under the following recovery rate evaluation conditions using the stainless steel column obtained by column packing, and the protein recovery rate was evaluated. The protein recovery rate was calculated using the area value of the chromatogram obtained when the sample was measured without connecting the stainless steel column (area value 1) and the area value of the chromatogram obtained when the sample was measured with connecting the stainless steel column (area value 2) using the following formula. The calculation results are shown in Table 2. Recovery rate (%) = 100 × area value (2) / area value (1) Recovery rate evaluation conditions: - Solution delivery pump LC-20AD (Shimadzu Corporation) - Manual injector 8125 (Rheodyne) - Detector SPD-20A (Shimadzu Corporation) - Column oven CTO-20A (Shimadzu Corporation) - Eluent: 50 mmol / phosphate buffer (pH 6.7) containing 0.3 M sodium chloride - Measurement time: 20 minutes - Flow rate: 0.35 mL / min - Column temperature: 30°C - Detection wavelength: 280 nm - Sample: Proteins (bovine serum albumin (BSA), human γ-globulin G (IgG)) diluted with the eluent were used as samples - Sample injection volume: 1 μl

[0071] (Evaluation of pore volume) Using the stainless steel column obtained by column packing, the exclusion limit molecular weight was measured under the following conditions, and the pore volume was evaluated. The pore volume was calculated using the following formula, measuring pullulan standards of Mp 6300 and Mp 739000, and using the retention time and measurement flow rate of the obtained pullulan standards. The calculation results are shown in Table 2. Pore ​​volume (mL) = {retention time of pullulan standard (molecular weight 6300) - retention time of pullulan standard (molecular weight 73900)} x 0.35 Pore volume evaluation conditions: - Solution delivery pump: Agilent 1200 binary pump (manufactured by Agilent) - Manual injector 4908 (manufactured by Rheodyne) - Detector: RI-201H (manufactured by Shoko Science) - Column oven: CTO-20A (manufactured by Shimadzu Corporation) - Eluent: pure water - Measurement time: 8 minutes - Flow rate: 0.35 mL / min - Column temperature: 30°C - Sample: pullulan standard (trade name: Shodex (registered trademark) STANDARD P-82, manufactured by Resonac) diluted with pure water was used as the sample - Sample injection volume: 10 μl

[0072] (Calculation of Pore Ratio) The pore ratio was calculated from the internal volume of the column (diameter 4.6 mm, length 150 mm) and the pore volume. The calculation results are shown in Table 2.

[0073] (Evaluation of the number of theoretical plates) The number of theoretical plates (N) is an index representing the efficiency of separation. It was calculated from the shape of the elution peak in the above chromatogram using the following formula. Specifically, ethylene glycol was measured under the following conditions using the stainless steel column obtained by column packing, and the number of theoretical plates was evaluated. The evaluation results are shown in Table 2. Theoretical number of plates (N) = 5.54 × (elution time of peak / half-width) ２<Conditions for evaluating the number of theoretical plates> - Solution delivery pump: Agilent 1200 binary pump (manufactured by Agilent) - Manual injector 4908 (manufactured by Rheodyne) - Detector: RI-201H (manufactured by Shoko Science) - Column oven: CTO-20A (manufactured by Shimadzu Corporation) - Eluent: pure water - Measurement time: 8 minutes - Flow rate: 0.35 mL / min - Column temperature: 30°C - Sample: Ethylene glycol diluted with pure water was used as the sample - Sample injection amount: 1 μl

[0074] Example 2 (Synthesis of Porous Organic Polymer Carrier (2)) Particles were synthesized and washed in the same manner as in Example 1, except that 11.8 g of EGDM, 5 g of glycidyl methacrylate (63 mol % polyfunctional monomer), 4.6 g of chlorobenzene, 2.3 g of dodecanol, 16 g of butyl acetate, and 0.17 g of benzoyl peroxide were used as the oil phase. 14 g of the obtained particles, 131 g of water, and 2 g of sulfuric acid were placed in a 300 mL flask. The temperature was raised to 85°C with stirring, and the reaction was continued for 4 hours. The obtained particles were filtered, and an operation of pouring 200 mL of water was carried out three times, an operation of pouring 200 mL of acetone was carried out twice, and then 200 mL of acetonitrile was poured to obtain a porous organic polymer carrier (2). (Introduction of ATRP Initiating Group) The same procedure as in Example 1 was carried out, except that 2.7 g of triethylamine and 3.2 mL of 2-bromoisobutyryl bromide were used. (Graft Polymerization) The same procedure as in Example 1 was carried out except that 1.55 g of GMMA was used, to obtain a filler substrate (2).

[0075] Calculation of the graft amount, calculation of the number average particle diameter and the CV value of the particle diameter, measurement of the pore diameter peak, measurement of the bromine atom weight, measurement of the 10% thermal weight loss temperature, column packing, evaluation of the protein recovery rate, evaluation of the pore volume, calculation of the pore ratio, and evaluation of the theoretical plate number were carried out in the same manner as in Example 1.

[0076] Example 3 (Synthesis of porous organic polymer carrier (3)) A porous organic polymer carrier (3) was synthesized in the same manner as in Example 2, except that 9.2 g of EGDM, 9.2 g of glycidyl methacrylate (42 mol % polyfunctional monomer), 16.2 g of octanol, 5.4 g of chlorobenzene, and 0.17 g of benzoyl peroxide were used as the oil phase. (Introduction of ATRP initiating group) This was carried out in the same manner as in Example 2. (Graft polymerization) A filler substrate (3) was obtained in the same manner as in Example 2.

[0077] Calculation of the graft amount, calculation of the number average particle diameter and the CV value of the particle diameter, measurement of the pore diameter peak, measurement of the bromine atom weight, measurement of the 10% thermal weight loss temperature, column packing, evaluation of the protein recovery rate, evaluation of the pore volume, calculation of the pore ratio, and evaluation of the theoretical plate number were carried out in the same manner as in Example 1.

[0078] Example 4 (Synthesis of porous organic polymer carrier (4)) A porous organic polymer carrier (4) was synthesized in the same manner as in Example 2, except that 9.2 g of EGDM, 9.2 g of glycidyl methacrylate (42 mol % polyfunctional monomer), 16.2 g of octanol, 5.4 g of diethyl succinate, and 0.17 g of VE-073 (manufactured by Wako Pure Chemical Industries, Ltd.) were used as the oil phase. (Introduction of ATRP initiation group) This was carried out in the same manner as in Example 2. (Graft polymerization) A filler substrate (4) was obtained in the same manner as in Example 2.

[0079] Calculation of the graft amount, calculation of the number average particle diameter and the CV value of the particle diameter, measurement of the pore diameter peak, measurement of the bromine atom weight, measurement of the 10% thermal weight loss temperature, column packing, evaluation of the protein recovery rate, evaluation of the pore volume, calculation of the pore ratio, and evaluation of the theoretical plate number were carried out in the same manner as in Example 1.

[0080] Comparative Example 1 (Synthesis of Porous Organic Polymer Carrier (4)) In the synthesis of porous organic polymer carrier (3), 5 g of particles that had been polymerized and washed, 50 g of ethylene glycol, 14.5 g of diethylene glycol dimethyl ether (42 mol% polyfunctional monomer), and 0.2 g of boron trifluoride diethyl ether complex were placed in a 200 mL flask. The temperature was raised to 80°C with stirring, and the reaction was continued for 3 hours. After filtering the dispersion after the reaction, an operation of pouring 200 mL of acetone was carried out three times to obtain porous organic polymer carrier (4). The introduction of an ATRP initiating group and graft polymerization were not carried out, and the porous organic polymer carrier (4) was used as a filler substrate (4).

[0081] Calculation of the number average particle size and the CV value of the particle size, measurement of the pore size peak, measurement of the bromine atomic weight, measurement of the 10% thermal weight loss temperature, column packing, evaluation of the protein recovery rate, evaluation of the pore volume, calculation of the pore ratio, and evaluation of the theoretical plate number were carried out in the same manner as in Example 1.

[0082] Comparative Example 2 (Synthesis of porous organic polymer carrier (5)) 48 g of EGDM, 84 g of glycidyl methacrylate (29 mol % polyfunctional monomer), 60.2 g of chlorobenzene, 60 g of lauryl alcohol, and 4.4 g of V-65 (Wako Pure Chemical Industries) were used as the oil phase. Furthermore, a solution of 26 g of polyvinyl alcohol (GH-20) dissolved in 800 g of water was used as the aqueous phase. The oil phase and the aqueous phase were mixed in a 2 L flask and stirred to obtain porous organic polymer carrier (5). Introduction of an ATRP initiator group and graft polymerization were not carried out, and the porous organic polymer carrier (5) was used as the filler substrate (5).

[0083] Calculation of the graft amount, calculation of the number average particle diameter and the CV value of the particle diameter, measurement of the pore diameter peak, measurement of the bromine atom weight, measurement of the 10% thermal weight loss temperature, column packing, evaluation of the protein recovery rate, evaluation of the pore volume, calculation of the pore ratio, and evaluation of the theoretical plate number were carried out in the same manner as in Example 1.

[0084]

[0085]

Claims

1. A base material for fillers, comprising a porous organic polymer support having hydroxyl groups derived from hydrophilic monomers immobilized on the surface thereof by graft polymerization, the porous organic polymer support containing 40 mol% or more of structural units derived from polyfunctional monomers relative to the total monomer components constituting the porous organic polymer support, and the hydrophilic monomers being (meth)acrylates having hydroxyl groups.

2. The filler base material according to claim 1, wherein the particles have a number-average particle size of 3 to 10 μm and a CV value of the particle size distribution of 8% or less.

3. The filler substrate according to claim 1 or 2, wherein the pore size distribution of the porous organic polymer carrier determined by mercury intrusion porosimetry shows a pore size peak in the range of 30 nm to 500 nm.

4. The ATRP initiation group remains after the graft polymerization, and the amount of bromine atoms derived from the ATRP initiation group per specific surface area is 0.3 to 2.5 mg / m ２ The filler base material according to claim 1 or 2, 5. The packing base material according to claim 1 or 2, wherein the pore ratio, which is the ratio of the total pore volume of the packing material to the internal volume of a column packed with a packing material made from said packing base material, is 12 to 30%.

6. The filler base material according to claim 2, wherein the graft amount of the (meth)acrylate having a hydroxyl group to the particles is 50 to 200 mg / g of particles.

7. The filler base material according to claim 1 or 2, wherein the (meth)acrylate having a hydroxyl group is at least one of glycerin monomethacrylate and glycerin monoacrylate.

8. The base material for fillers according to claim 1 or 2, which has a 10% thermal weight loss in nitrogen of 290 to 330°C.

9. A method for producing a filler substrate according to claim 1 or 2, comprising the steps of: polymerizing raw material monomers containing 40 mol % or more of a polyfunctional monomer to obtain a porous organic polymer support; introducing an initiating group for ATRP into the porous organic polymer support; and grafting a hydrophilic monomer onto the initiating group.

10. A packing material for size exclusion chromatography, which uses the packing material base material according to claim 1 or 2.

11. A method for producing a protein, comprising the steps of: passing a solution containing the protein through a column packed with the size exclusion chromatography packing material described in claim 10; and purifying the protein loaded onto the size exclusion chromatography packing material by elution.

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