JP6627514B2 - Separation material, column, and method for producing separation material - Google Patents

Description

  The present invention relates to a separation material, a column, and a method for producing the separation material.

  Conventionally, when a biopolymer represented by a protein is separated and purified, generally, an ion exchanger based on a porous synthetic polymer or an ion exchanger based on a crosslinked gel of a hydrophilic natural polymer is generally used. Are used. In the case of an ion exchanger having a porous synthetic polymer as a base, since the volume change due to salt concentration is small, when the ion exchanger is packed in a column and used for chromatography, it tends to be excellent in pressure resistance during passage. However, when this ion exchanger is used for separation of proteins and the like, nonspecific adsorption such as irreversible adsorption based on hydrophobic interaction occurs, resulting in peak asymmetry or ion exchange due to the hydrophobic interaction. There is a problem that the protein adsorbed on the body cannot be recovered while being adsorbed.

  On the other hand, an ion exchanger having a crosslinked gel of a hydrophilic natural polymer represented by a polysaccharide such as dextran or agarose as a base has the advantage that there is almost no nonspecific adsorption of proteins. However, this ion exchanger has the disadvantage that it swells remarkably in an aqueous solution, the volume change due to the ionic strength of the solution, the volume change between the free acid form and the loaded form is large, and the mechanical strength is not sufficient. In particular, when a crosslinked gel is used in chromatography, there is a disadvantage that the pressure loss at the time of passing the liquid is large and the gel is compacted by the passing of the liquid.

  In order to overcome the drawbacks of the crosslinked gel of a hydrophilic natural polymer, for example, a complex in which a gel such as a natural polymer gel is held in the pores of a porous polymer is known in the field of peptide synthesis ( For example, see Patent Document 1). By using such a complex, the load coefficient of the reactive substance can be increased, and high-yield synthesis can be achieved. In addition, since the gel is surrounded by a hard synthetic polymer material, there is an advantage that even when used in the form of a column bed, there is no change in volume and the pressure of the flow-through passing through the column does not change.

  BACKGROUND ART Separating materials in which xerogels such as polysaccharides such as dextran and cellulose are held on an inorganic porous material such as celite (for example, see Patent Documents 2 and 3). This gel is provided with a diethylaminoethyl (DEAE) group or the like in order to add adsorption performance, and is used for removing hemoglobin. Such a separation material has good liquid permeability in a column.

  There is known an ion exchanger of a hybrid copolymer in which pores of a copolymer having a macro network structure are filled with a crosslinked copolymer gel synthesized from a monomer (for example, see Patent Document 4). Crosslinked copolymer gel has problems such as pressure loss and volume change when the degree of crosslinking is low.However, by using a hybrid copolymer, the liquid permeation characteristics are improved, the pressure loss is reduced, and the ion exchange capacity is improved. The leak behavior is improved.

  Further, a composite filler in which a crosslinked gel of a hydrophilic natural polymer having a huge network structure is filled in pores of an organic synthetic polymer substrate has been proposed (for example, see Patent Documents 5 and 6). Furthermore, a technique for synthesizing porous particles composed of glycidyl methacrylate and an acrylic cross-linked monomer is known (for example, see Patent Document 7).

U.S. Pat. No. 4,965,289 U.S. Pat. No. 4,335,017 U.S. Pat. No. 4,336,161 U.S. Pat. No. 3,966,489 JP-A-1-254247 U.S. Pat. No. 5,114,577 JP 2009-244067 A

  However, the conventional separation material has a problem that the amount of nonspecific adsorption of protein is large, the amount of adsorption is not sufficient, and the liquid permeability when used as a column is poor.

  Therefore, the present invention provides a separation material having reduced non-specific adsorption of proteins, a high adsorption amount, and excellent liquid permeability when used as a column, a column including the separation material, and a method for producing the separation material. The purpose is to:

The present invention provides a separation material described in the following [1] to [12], a column including the separation material, and a method for producing the separation material.
[1] A porous polymer particle, and a coating layer that covers at least a part of the surface of the porous polymer particle, wherein the coating layer has an active hydrogen group and a hydroxyl group in a crosslinked polymer having a hydroxyl group. A separating material containing a graft polymer on which a polymer is grafted.
[2] The separation material according to [1], wherein the porous polymer particles include a polymer having a structural unit derived from a monomer containing divinylbenzene.
[3] The separation material according to [1] or [2], wherein the active hydrogen group is an amino group or a thiol group.
[4] The separation material according to any one of [1] to [3], wherein the pore diameter (mode diameter) is 0.1 to 0.5 μm.
[5] The separation material according to any one of [1] to [4], wherein the coefficient of variation of the particle diameter of the porous polymer particles is 5 to 15%.
[6] The separation material according to any one of [1] to [5], wherein the crosslinked polymer having a hydroxyl group is a crosslinked polymer derived from a polysaccharide or a modified product thereof.
[7] The separation material according to any one of [1] to [6], wherein the crosslinked polymer having a hydroxyl group is a crosslinked polymer derived from agarose or a modified product thereof.
[8] The separation material according to any one of [1] to [7], wherein the polymer having an active hydrogen group and a hydroxyl group is dextran, polyglycerin monomethacrylate or polyhydroxyethyl methacrylate having an active hydrogen group. .
[9] The separation material according to any one of [1] to [8], wherein the polymer having an active hydrogen group and a hydroxyl group has a weight average molecular weight of 5,000 to 100,000.
[10] The separating material according to any one of [1] to [9], comprising a coating layer of 30 to 400 mg per 1 g of the porous polymer particles.
[11] A column comprising the separation material according to any one of [1] to [10].
[12] The method for producing a separation material according to any one of [1] to [10],
A step of adsorbing a polymer having a hydroxyl group on the surface of the porous polymer particles and crosslinking the polymer,
A step of introducing a group capable of reacting with an active hydrogen group into the crosslinked polymer;
Reacting a polymer having an active hydrogen group and a hydroxyl group with a group capable of reacting with the active hydrogen group.

  ADVANTAGE OF THE INVENTION According to this invention, the nonspecific adsorption | suction of a protein is reduced, the adsorption amount is high, and the separation material which is excellent in liquid permeability when used as a column, a column provided with the separation material, and the manufacturing method of a separation material are provided. be able to.

  Hereinafter, preferred embodiments of the present invention will be described, but the present invention is not limited to these embodiments.

<Separation material>
The separation material of the present embodiment includes porous polymer particles and a coating layer that covers at least a part of the surface of the porous polymer particles. In the present specification, the “surface of the porous polymer particle” includes not only the outer surface of the porous polymer particle but also the surface of the pore inside the porous polymer particle. In this specification, (meth) acrylic acid means acrylic acid or methacrylic acid, and the same applies to other similar expressions such as (meth) acrylate.

(Porous polymer particles)
The porous polymer particles according to the present embodiment are particles containing a polymer obtained by polymerizing a monomer containing a porogen, and can be synthesized by, for example, conventional suspension polymerization, seed polymerization, or the like. That is, the porous polymer particles include, for example, a polymer having a structural unit derived from the above monomer. The monomer is not particularly limited, but for example, an acrylic monomer or a styrene monomer can be used. Specific monomers include the following polyfunctional monomers and monofunctional monomers.

  Examples of the polyfunctional monomer include divinyl compounds such as divinylbenzene, divinylbiphenyl, divinylnaphthalene, and divinylphenanthrene; (poly) ethylene glycol di (meth) acrylate, (poly) propylene glycol di (meth) acrylate, and (poly) (Poly) alkylene glycol-based di (meth) acrylates such as tetramethylene glycol di (meth) acrylate; trimethylolpropane tri (meth) acrylate, tetramethylol methane tri (meth) acrylate, tetramethylol propane tetra (meth) acrylate, penta Erythritol tri (meth) acrylate, 1,1,1-trishydroxymethylethanetri (meth) acrylate, 1,1,1-trishydroxymethylpropane triacrylate, etc. Trifunctional or higher (meth) acrylate; ethoxylated bisphenol A di (meth) acrylate, propoxylated ethoxylated bisphenol A di (meth) acrylate, tricyclodecane dimethanol di (meth) acrylate, 1,1,1- Di (meth) acrylates such as trishydroxymethylethanedi (meth) acrylate and ethoxylated cyclohexanedimethanol di (meth) acrylate; diallyl phthalate and its isomers; triallyl isocyanurate and its derivatives. Among these monomers, for example, NK esters (A-TMPT-6P0, A-TMPT-3E0, A-TMM-3LMN, A-GLY series, A-9300, AD-TMP, AD manufactured by Shin-Nakamura Chemical Co., Ltd.) -TMP-4CL, ATM-4E, A-DPH) and the like are commercially available. These polyfunctional monomers can be used alone or in combination of two or more. It is preferable that the monomer contains divinylbenzene from the viewpoints of improving durability, acid resistance, alkali resistance and elastic modulus, and suppressing the destruction of particles during column filling and particle handling. That is, the porous polymer particles preferably include a polymer having a structural unit derived from a monomer containing divinylbenzene.

  When the porous polymer particle contains divinylbenzene as a monomer unit, it preferably contains divinylbenzene in an amount of 60% by mass or more, more preferably 70% by mass or more, and more preferably 80% by mass or more based on the total mass of the monomer. More preferred. It is preferable to include divinylbenzene in an amount of 60% by mass or more because alkali resistance is further improved.

  As the monofunctional monomer, for example, styrene, o-methylstyrene, m-methylstyrene, p-methylstyrene, α-methylstyrene, o-ethylstyrene, m-ethylstyrene, p-ethylstyrene, 2,4- Dimethylstyrene, pn-butylstyrene, pt-butylstyrene, pn-hexylstyrene, pn-octylstyrene, pn-nonylstyrene, pn-decylstyrene, pn-dodecyl Styrene such as styrene, p-methoxystyrene, p-phenylstyrene, p-chlorostyrene, 3,4-dichlorostyrene and derivatives thereof; methyl acrylate, ethyl acrylate, propyl acrylate, n-butyl acrylate, acrylic acid Isobutyl, hexyl acrylate, 2-ethylhexyl acrylate, n-octyl acrylate , Dodecyl acrylate, lauryl acrylate, stearyl acrylate, 2-chloroethyl acrylate, phenyl acrylate, methyl α-chloroacrylate, methyl methacrylate, ethyl methacrylate, propyl methacrylate, n-butyl methacrylate, methacryl (Meth) acrylates such as isobutyl acrylate, hexyl methacrylate, 2-ethylhexyl methacrylate, n-octyl methacrylate, dodecyl methacrylate, lauryl methacrylate, stearyl methacrylate; vinyl acetate, vinyl propionate, vinyl benzoate, Vinyl esters such as vinyl butyrate; N-vinyl compounds such as N-vinyl pyrrole, N-vinyl carbazole, N-vinyl indole, N-vinyl pyrrolidone; vinyl fluoride, vinylidene fluoride, tetrafluoroethyl Emissions, hexafluoropropylene, trifluoroethyl acrylate, containing fluorinated monomers such as acrylic acid tetrafluoropropyl; butadiene include conjugated dienes such as isoprene. These monofunctional monomers can be used alone or in combination of two or more. Among them, styrene is preferably used from the viewpoint of excellent acid resistance and alkali resistance. Further, a styrene derivative having a functional group such as a carboxy group, an amino group, a hydroxyl group, or an aldehyde group can also be used.

  Examples of the porosifying agent include aliphatic or aromatic hydrocarbons, esters, ketones, ethers, and alcohols that are organic solvents that promote phase separation during polymerization and promote the porosity of particles. Can be Specific examples of the porosifying agent include toluene, xylene, cyclohexane, octane, butyl acetate, dibutyl phthalate, methyl ethyl ketone, dibutyl ether, 1-hexanol, 2-octanol, decanol, lauryl alcohol, cyclohexanol, and the like. . These porosifying agents can be used alone or in combination of two or more.

  The above-mentioned porous agent can be used, for example, in a range of more than 0 to 300% by mass based on the total mass of the monomers. The porosity of the porous polymer particles can be controlled by the amount of the porogen. Further, the size and shape of the pores of the porous polymer particles can be controlled by the type of the porosifying agent.

  Water used as a solvent can also be used as a porosifying agent. When water is used as the porosifying agent, the particles can be made porous by dissolving the oil-soluble surfactant in the monomer and absorbing the water.

  Examples of the oil-soluble surfactant used for making porous include sorbitan monoesters of branched C16 to C24 fatty acids, chain unsaturated C16 to C22 fatty acids or chain saturated C12 to C14 fatty acids, for example, sorbitan monolaurate, sorbitan Sorbitan monoester derived from monooleate, sorbitan monomyristate or coconut fatty acid; diglycerol monoester of branched C16-C24 fatty acid, chain unsaturated C16-C22 fatty acid or chain saturated C12-C14 fatty acid, such as diester Glycerol monooleate (for example, diglycerol monoester of C18: 1 (18 carbon atoms, 1 double bond) fatty acid), diglycerol monomyristate, diglycerol monoisostearate, or diglycerol mono of coconut fatty acid Ester; branched C16-C 4 alcohols (e.g., Guerbet alcohols), linear unsaturated C16~C22 alcohol or chain saturated C12~C14 alcohols (e.g., coconut fatty alcohols) diglycerol mono-fatty ethers, and mixtures thereof.

  Of these, sorbitan monolaurate (e.g., SPAN® 20, having a purity preferably greater than about 40%, more preferably greater than about 50%, and even more preferably greater than about 70%) Rate), sorbitan monooleate (eg, SPAN® 80, preferably having a purity of greater than about 40%, more preferably greater than about 50%, even more preferably greater than about 70%) ), Diglycerol monooleate (eg, having a purity preferably greater than about 40%, more preferably greater than about 50%, even more preferably greater than about 70%), diglycerol monoisostearate (E.g., the purity is preferably greater than about 40%, more preferably about 50% Diglycerol monoisostearate), diglycerol monomyristate (purity preferably greater than about 40%, more preferably greater than about 50%, even more preferably about 70%). Sorbitan monomyristate), cocoyl (e.g., lauryl, myristoyl, etc.) ethers of diglycerol, or mixtures thereof.

  It is preferable to use these oil-soluble surfactants in the range of 5 to 80% by mass based on the total mass of the monomers. When the content of the oil-soluble surfactant is 5% by mass or more, the stability of water droplets becomes sufficient, so that it is difficult to form large single pores. When the content of the soluble surfactant is 80% by mass or less, the shape of the porous polymer particles can be more easily maintained after the polymerization.

  Examples of the aqueous medium used for the polymerization reaction include water, a mixed medium of water and a water-soluble solvent (for example, a lower alcohol), and the like. The aqueous medium may contain a surfactant. As the surfactant, any of anionic, cationic, nonionic and zwitterionic surfactants can be used.

  Examples of the anionic surfactant include fatty acid oils such as sodium oleate and castor oil, alkyl sulfates such as sodium lauryl sulfate and ammonium lauryl sulfate, alkylbenzene sulfonates such as sodium dodecylbenzenesulfonate, and alkylnaphthalene sulfone. Acid salt, alkane sulfonate, dialkyl sulfosuccinate such as sodium dioctyl sulfosuccinate, alkenyl succinate (dipotassium salt), alkyl phosphate ester salt, naphthalene sulfonic acid formalin condensate, polyoxyethylene alkyl phenyl ether sulfate Salts, polyoxyethylene alkyl ether sulfates such as sodium polyoxyethylene lauryl ether sulfate, and polyoxyethylene alkyl sulfates.

  Examples of the cationic surfactant include alkylamine salts such as laurylamine acetate and stearylamine acetate, and quaternary ammonium salts such as lauryltrimethylammonium chloride.

  Examples of the nonionic surfactant include, for example, hydrocarbon nonionic surfactants such as polyethylene glycol alkyl ethers, polyethylene glycol alkyl aryl ethers, polyethylene glycol esters, polyethylene glycol sorbitan esters, polyalkylene glycol alkylamines and amides. And polyether modified silicone nonionic surfactants such as polyethylene oxide adducts and polypropylene oxide adducts of silicon, and fluorine nonionic surfactants such as perfluoroalkyl glycols.

  Examples of the amphoteric surfactant include a hydrocarbon surfactant such as lauryl dimethylamine oxide, a phosphate ester surfactant, and a phosphite ester surfactant.

  One type of surfactant may be used alone, or two or more types may be used in combination. Among the above surfactants, anionic surfactants are preferred from the viewpoint of dispersion stability during polymerization of the monomer.

  Examples of the polymerization initiator added as needed include, for example, benzoyl peroxide, lauroyl peroxide, orthochloroperoxybenzoyl, orthomethoxybenzoyl peroxide, 3,5,5-trimethylhexanoyl peroxide, tert-butyl peroxide Organic peroxides such as oxy-2-ethylhexanoate and di-tert-butyl peroxide; 2,2′-azobisisobutyronitrile, 1,1′-azobiscyclohexanecarbonitrile, 2,2 ′ And azo compounds such as -azobis (2,4-dimethylvaleronitrile). The polymerization initiator can be used, for example, in a range of 0.1 to 7.0 parts by mass with respect to 100 parts by mass of the monomer.

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

  In the synthesis of the porous polymer particles, a polymer dispersion stabilizer may be added in order to improve the dispersion stability of the particles.

  Examples of the polymer dispersion stabilizer include polyvinyl alcohol, polycarboxylic acid, celluloses (hydroxyethylcellulose, carboxymethylcellulose, methylcellulose, and the like), and polyvinylpyrrolidone, and an inorganic water-soluble polymer compound such as sodium tripolyphosphate is also used. can do. Of these, polyvinyl alcohol and polyvinylpyrrolidone are preferred. The addition amount of the polymer dispersion stabilizer is preferably 1 to 10 parts by mass with respect to 100 parts by mass of the monomer.

  In order to suppress the monomer from being polymerized alone, a water-soluble polymerization inhibitor such as nitrites, sulfites, hydroquinones, ascorbic acids, water-soluble vitamins B, citric acid, and polyphenols may be used.

  The average particle size of the porous polymer particles is preferably 500 μm or less, more preferably 300 μm or less, and still more preferably 100 μm or less. The average particle size of the porous polymer particles is preferably 10 μm or more, more preferably 30 μm or more, and further preferably 50 μm or more. When the average particle size of the porous polymer particles is 10 μm or more, there is a tendency that the column pressure after filling the column can be suppressed.

  The coefficient of variation (CV) of the particle diameter of the porous polymer particles is preferably from 5 to 15%, more preferably from 5 to 14%, from the viewpoint of improving liquid permeability. More preferably, it is 13%. C. V. As a method for reducing the dispersion, monodispersion is carried out using an emulsifying apparatus such as a microprocess server (manufactured by Hitachi, Ltd.).

C. average particle size and particle size of the porous polymer particles or the separating material. V. Can be determined by the following measurement method.
1) A dispersion liquid containing 1% by mass of the porous polymer particles or the separating material by dispersing the porous polymer particles or the separating material in water (including a dispersant such as a surfactant) using an ultrasonic dispersion device. Is prepared.
2) Using a particle size distribution meter (Sysmex Flow, manufactured by Sysmex Corporation), the average particle diameter and the C.I. V. Is measured.

  The porosity (pore volume) of the porous polymer particles is preferably 30% by volume or more and 70% by volume or less, more preferably 40% by volume or more and 70% by volume or less based on the total volume of the porous polymer particles. preferable. The porous polymer particles preferably have macropores (macropores), and the pore diameter (mode diameter) of the porous polymer particles is preferably 0.05 μm or more and less than 0.5 μm. The pore diameter of the porous polymer particles is more preferably 0.05 μm or more and less than 0.3 μm. When the pore diameter is 0.05 μm or more, the substance tends to easily enter the pores, and when the pore diameter is less than 0.5 μm, the specific surface area becomes sufficient. These can be adjusted by the above-mentioned porosifying agent.

The specific surface area of the porous polymer particles is preferably 30 m ² / g or more. In light of higher practicality, the specific surface area is more preferably equal to or greater than 35 m ² / g, and still more preferably equal to or greater than 40 m ² / g. When the specific surface area is 30 m ² / g or more, the adsorption amount of the substance to be separated tends to increase.

  The pore diameter (mode diameter), specific surface area and porosity of the porous polymer particles or the separating material can be measured using a mercury intrusion measuring device (Autopore: manufactured by Shimadzu Corporation), for example, as follows. About 0.05 g of a sample is added to a standard 5 mL powder cell (stem volume: 0.4 mL), and measurement is performed under the conditions of an initial pressure of 21 kPa (about 3 psia, a pore diameter of about 60 μm). The mercury parameters are set to a device default mercury contact angle of 130 degrees and a mercury surface tension of 485 dynes / cm. Further, each value is calculated limited to the range of the pore diameter of 0.05 to 5 μm.

(Coating layer)
The coating layer according to this embodiment includes a graft polymer in which a polymer having an active hydrogen group and a hydroxyl group is grafted to a crosslinked polymer having a hydroxyl group. By coating the porous polymer particles with such a coating layer, it is possible to suppress an increase in column pressure, to suppress non-specific adsorption of proteins, and to prevent protein adsorption of the separation material. The amount can be made larger than that when a natural polymer is used.

  Examples of the graft polymer include a graft obtained by introducing a group capable of reacting with an active hydrogen group into a crosslinked polymer having a hydroxyl group, and reacting the group with a polymer having an active hydrogen group and a hydroxyl group. Polymers. The graft polymer preferably contains a structure in which a group capable of reacting with an active hydrogen group and an active hydrogen group are bonded. When the graft polymer contains such a structure, the amount of dynamic adsorption tends to increase.

  The crosslinked polymer having a hydroxyl group preferably has two or more hydroxyl groups in one molecule, and is more preferably a hydrophilic polymer. Examples of the crosslinked polymer having a hydroxyl group include a crosslinked polymer derived from a polysaccharide or a modified product thereof, and a crosslinked polymer derived from polyvinyl alcohol or a modified product thereof. Examples of the polysaccharide include agarose, dextran, cellulose, chitosan and the like. From the viewpoint of the number of hydroxyl groups in the molecule, the crosslinked polymer having a hydroxyl group is preferably a polysaccharide, and more preferably agarose or dextran.

  Further, the crosslinked polymer having a hydroxyl group may be a modified product modified with a hydrophobic group from the viewpoint of improving the interfacial adsorption ability. Examples of the hydrophobic group include an alkyl group having 1 to 6 carbon atoms and an aryl group having 6 to 10 carbon atoms. Examples of the alkyl group having 1 to 6 carbon atoms include a methyl group, an ethyl group, and a propyl group. Examples of the aryl group having 6 to 10 carbon atoms include a phenyl group and a naphthyl group. The hydrophobic group is introduced by reacting a functional group that reacts with a hydroxyl group (for example, an epoxy group) and a compound having a hydrophobic group (for example, glycidyl phenyl ether) with a polymer having a hydroxyl group by a conventionally known method. Can be.

  The polymer having an active hydrogen group and a hydroxyl group preferably has two or more hydroxyl groups in one molecule, and is more preferably a hydrophilic polymer.

  Examples of the polymer having an active hydrogen group and a hydroxyl group include, for example, a polysaccharide having an active hydrogen group and a modified product thereof, polyvinyl alcohol and a modified product thereof, polyglycerin monomethacrylate and a modified product thereof, and polyhydroxyethyl methacrylate. And denatured products thereof. Examples of the polysaccharide include agarose, dextran, cellulose, chitosan and the like. When the polysaccharide has a reducing end, the polymer having an active hydrogen group and a hydroxyl group is preferably a polymer obtained by reacting a compound having two or more active hydrogen groups with the reducing end. Examples of the polysaccharide having a reducing end include agarose and dextran.

  From the viewpoint of molecular mobility, the polymer having an active hydrogen group and a hydroxyl group is preferably dextran having an active hydrogen group, polyglycerin monomethacrylate having an active hydrogen group or polyhydroxyethyl methacrylate having an active hydrogen group. .

  The polymer having an active hydrogen group and a hydroxyl group preferably has 1 to 5 active hydrogen groups per one polymer molecule.

  The polymer having an active hydrogen group and a hydroxyl group can be synthesized, for example, by a method of reacting a polymer having a hydroxyl group with a compound having an active hydrogen group. When a polymer having an active hydrogen group and a hydroxyl group is synthesized from a monomer, for example, a method using an initiator having an active hydrogen group at the time of polymerization may be mentioned. The polymerization method is not particularly limited, and examples thereof include raft polymerization and living polymerization by ATRP.

  Specific examples of the active hydrogen group include a functional group known as a functional group having active hydrogen. The active hydrogen group is preferably an amino group or a thiol group because it can be easily introduced into a polymer having a hydroxyl group.

  The weight average molecular weight (Mw) of the polymer having an active hydrogen group and a hydroxyl group is preferably 5,000 to 100,000, more preferably 10,000 to 100,000, and still more preferably 30,000 to 100,000. When Mw is 100,000 or less, the possibility of closing the pores of the porous polymer particles is reduced. When Mw is 5000 or more, the ability to adsorb proteins is further improved.

  In this specification, Mw indicates a numerical value measured by GPC using pullulan having a different molecular weight as a standard substance and using water as a solvent.

  The group that can react with the active hydrogen group is not particularly limited as long as it is a group that can react with the corresponding active hydrogen group, and examples thereof include an epoxy group, a carboxyl group, an isocyanate group, and an active ester group.

  Examples of the active ester group include those obtained by esterifying a carboxyl group with a condensing agent such as N-hydroxysuccinimide (NHS) and a triazine-based condensing agent (DMT-MM). Among them, the active ester group is preferably one obtained by esterifying a carboxyl group with N-hydroxysuccinimide (NHS) as a condensing agent. Such an active ester group has high reactivity with an active hydrogen group such as an amino group.

  The graft amount of the polymer having an active hydrogen group and a hydroxyl group can be calculated by, for example, XPS before and after grafting, weight change, thermogravimetric analysis and the like. From the viewpoint of reducing that the pores are closed, and from the viewpoint of further improving the protein adsorption amount, the graft amount of the polymer per 1 g of the porous polymer particles is preferably 5 to 100 mg, and more preferably 10 to 50 mg. Is more preferable, and 15 to 30 mg is still more preferable.

  The amount of the coating layer is preferably 30 to 400 mg, more preferably 50 to 400 mg, even more preferably 100 to 400 mg per 1 g of the porous polymer particles. When the ratio of the coating layer is 400 mg or less per 1 g of the porous polymer particles, the coating layer can be formed into a thin film, and the liquid permeability when used as a column tends to be further improved. Further, when the ratio of the coating layer is 30 mg or more per 1 g of the porous polymer particles, the protein adsorption amount tends to be further increased. The amount of the coating layer can be measured by a weight loss due to thermal decomposition, anthrone method or the like.

<Production method of separation material>
The separation material of the present embodiment includes, for example, 1) a step of adsorbing a polymer having a hydroxyl group on the surface of porous polymer particles and crosslinking the polymer (adsorption crosslinking step); Introducing a group capable of reacting with an active hydrogen group (introducing a group capable of reacting with an active hydrogen group), and 3) a polymer having an active hydrogen group and a hydroxyl group as a group capable of reacting with an active hydrogen group (Grafting step).

  According to such a method, a group capable of reacting with the active hydrogen group and the active hydrogen group can be selectively reacted in the grafting step. Hereinafter, a specific example of the method will be described.

(Adsorption crosslinking process)
First, a solution of a polymer having a hydroxyl group is adsorbed on the surface of the porous polymer particles.

  Examples of the polymer having a hydroxyl group include a polysaccharide or a modified product thereof, and polyvinyl alcohol or a modified product thereof. Examples of the polysaccharide include agarose, dextran, cellulose, chitosan and the like. In light of ease of coating the pores of the particles, the Mw of the polymer having a hydroxyl group is preferably 10,000 to 200,000, more preferably 20,000 to 180,000, and more preferably 20,000 to 150,000. Is more preferred.

  The solvent for the solution of the polymer having a hydroxyl group is not particularly limited as long as it can dissolve the polymer having a hydroxyl group, but water is the most common. The concentration of the polymer dissolved in the solvent is preferably 5 to 20 (mg / mL). In order to adsorb the polymer having a hydroxyl group to the surface of the porous body (including the inside of the pores), the porous body is impregnated with this solution. In the impregnation method, porous polymer particles are added to a solution of a polymer having a hydroxyl group, and the solution is stirred for a certain time. Although the impregnation time varies depending on the surface state of the porous body, usually, when the mixture is stirred for 6 to 12 hours, the polymer concentration becomes equilibrium with the external concentration inside the porous body. Thereafter, the polymer having a hydroxyl group that has not been adsorbed is removed by washing with a solvent such as water or alcohol.

  Next, a crosslinking agent is added, and a polymer having a hydroxyl group adsorbed on the surface of the porous polymer particles undergoes a crosslinking reaction to form a crosslinked product. At this time, the crosslinked body has a three-dimensional crosslinked network structure having a hydroxyl group.

  Examples of the crosslinking agent include functional groups active on hydroxyl groups such as divinyl sulfone, epihalohydrin such as epichlorohydrin, dialdehyde compounds such as glutaraldehyde, diisocyanate compounds such as methylene diisocyanate, and glycidyl compounds such as ethylene glycol diglycidyl ether. And compounds having two or more of the following. When a compound having an amino group such as chitosan is used as the polymer having a hydroxyl group, a dihalide such as dichlorooctane can also be used as a crosslinking agent.

  A catalyst is usually used for this crosslinking reaction. As the catalyst, a conventionally known catalyst can be appropriately used according to the type of the crosslinking agent.For example, when the crosslinking agent is a compound having an epoxy group such as epichlorohydrin or ethylene glycol diglycidyl ether, sodium hydroxide or the like is used. An alkali is effective, and in the case of a dialdehyde compound, a mineral acid such as hydrochloric acid is effective.

  Cross-linking reaction by a cross-linking agent is usually performed by adding a cross-linking agent to a system in which porous polymer particles impregnated with a solution of a polymer having a hydroxyl group or the like in pores are dispersed and suspended in an appropriate medium. Done. When a polysaccharide is used as the polymer having a hydroxyl group, the amount of the cross-linking agent added is within the range of 0.1 to 100 moles per mole of one unit of the monosaccharide. The selection may be made according to the performance of the material. In general, when the amount of the cross-linking agent is reduced, the coating layer tends to be easily separated from the porous polymer particles. When the amount of the crosslinking agent added is excessive and the reaction rate with the polymer having a hydroxyl group is high, the properties of the polymer having a hydroxyl group as a raw material tend to be impaired.

  The amount of the catalyst used varies depending on the type of the cross-linking agent. Usually, when a polysaccharide is used as the polymer having a hydroxyl group, when one unit of the monosaccharide forming the polysaccharide is 1 mol, Is preferably used in the range of 0.01 to 10 times by mole, more preferably in the range of 0.1 to 5 times by mole.

  For example, when the cross-linking reaction conditions are temperature conditions, the temperature of the reaction system is increased, and when the temperature reaches the reaction temperature, a cross-linking reaction occurs.

  As a medium for dispersing and suspending the porous polymer particles impregnated with a solution of a polymer having a hydroxyl group, the polymer, the crosslinking agent, etc. are not extracted from the impregnated polymer solution, and the cross-linking is performed. It must be inert to the reaction. Specific examples thereof include water, alcohol, and the like.

  The crosslinking reaction is usually performed at a temperature in the range of 5 to 90 ° C for 1 to 15 hours. Preferably, the temperature is in the range of 30 to 90C.

  After the cross-linking reaction is completed, the generated particles are separated by filtration, and then washed with a hydrophilic organic solvent such as water, methanol, and ethanol to remove the unreacted polymer and the suspending medium. Particles at least partially coated with a coating layer containing a crosslinked polymer having a hydroxyl group are obtained.

(Step of introducing a group capable of reacting with an active hydrogen group)
Next, a group capable of reacting with an active hydrogen group is introduced into the crosslinked polymer having a hydroxyl group. The method for introducing a group capable of reacting with an active hydrogen group is not particularly limited, and a known method or the like can be used. For example, the epoxy group can be introduced by immersing particles in an aqueous alkaline solution having a predetermined concentration to prepare a dispersion, then adding an alkyl halide compound having an epoxy group to the dispersion, and reacting the dispersion. Examples of the alkaline aqueous solution include a sodium hydroxide aqueous solution. Examples of the halogenated alkyl compound having an epoxy group include epichlorohydrin.

(Graft process)
Particles obtained by the step of introducing a group capable of reacting with an active hydrogen group are dispersed in an aqueous solution of a polymer having an active hydrogen group and a hydroxyl group. Is reacted with a polymer having an active hydrogen group and a hydroxyl group. Examples of the catalyst include an aqueous solution of sodium hydroxide.

  Then, the obtained particles are separated by filtration, washed, and unreacted polymer and the like are removed, so that at least a part of the surface of the porous polymer particles becomes a crosslinked polymer having a hydroxyl group, an active hydrogen group and a hydroxyl group. Thus, a separating material covered with a coating layer containing a graft polymer to which a polymer having the following is grafted is obtained.

(Introduction of ion exchange group)
The separation material provided with the coating layer can be used for ion exchange purification, affinity purification, and the like by introducing an ion exchange group, a ligand (protein A), or the like via a hydroxyl group on the surface. As a method for introducing an ion exchange group, for example, a method using a halogenated alkyl compound can be mentioned.

  Examples of the halogenated alkyl compound include a monohalogenocarboxylic acid and its sodium salt, a primary, secondary or tertiary amine having at least one halogenated alkyl group and its hydrochloride, and a quaternary having at least one halogenated alkyl group. Ammonium salts and the like. Examples of the monohalogenocarboxylic acid include monohalogenoacetic acid, monohalogenopropionic acid and the like. Examples of the tertiary amine having at least one halogenated alkyl group include diethylaminoethyl chloride. These alkyl halide compounds are preferably bromide or chloride. The amount of the halogenated alkyl compound to be used is preferably 0.2% by mass or more based on the total mass of the separation material to which the ion exchange group is provided.

  It is effective to use an organic solvent to promote the reaction when introducing an ion exchange group. Examples of the organic solvent include alcohols such as ethanol, 1-propanol, 2-propanol, 1-butanol, isobutanol, 1-pentanol, and isopentanol.

  Usually, the ion-exchange group is introduced into the hydroxyl group on the surface of the separation material. Therefore, after the wet particles are drained by filtration or the like, the particles are immersed in an alkaline aqueous solution having a predetermined concentration, left for a certain period of time, and then mixed with water-organic solution. The above-mentioned alkyl halide compound is added and reacted in a solvent mixture system. Examples of the alkaline aqueous solution include a sodium hydroxide aqueous solution. This reaction is preferably performed at a temperature of 40 to 90 ° C. under reflux for 0.5 to 12 hours. The type of the ion exchange group is determined by the type of the alkyl halide compound used in the above reaction.

  As a method for introducing an amino group, which is a weakly basic group, as an ion exchange group, a mono-, di- or di-alkyl compound in which at least one of the alkyl groups is substituted with a halogenated alkyl group among the above-mentioned halogenated alkyl compounds. -Or tri-alkylamine, mono-alkyl-mono-alkanolamine, di-alkyl-mono-alkanolamine, mono-alkyl-di-alkanolamine, etc., or at least one of the alkanol groups is halogenated A method of reacting a mono-, di- or tri-alkanolamine, mono-alkyl-mono-alkanolamine, di-alkyl-mono-alkanolamine, mono-alkyl-di-alkanolamine substituted with an alkanol group, etc. Is mentioned. The use amount of these alkyl halide compounds is preferably 0.2% by mass or more based on the total mass of the separation material. The reaction conditions are preferably 40 to 90 ° C. and 0.5 to 12 hours.

  As a method for introducing a quaternary ammonium group as a strongly basic group as an ion exchange group, a tertiary amino group is first introduced, and a compound containing a halogenated alkyl group such as epichlorohydrin is reacted with the tertiary amino group. And a method of converting to a quaternary ammonium group. In addition, a quaternary ammonium halide such as quaternary ammonium chloride may be reacted with the separating material.

  Examples of a method for introducing a carboxy group that is a weakly acidic group as the ion exchange group include a method of reacting a monohalogenocarboxylic acid such as monohalogenoacetic acid or monohalogenopropionic acid or a sodium salt thereof as the halogenated alkyl compound. . The amount of the halogenated alkyl compound to be used is preferably 0.2% by mass or more based on the total mass of the separation material into which the ion exchange group is introduced.

  As a method for introducing a sulfonic acid group, which is a strongly acidic group, as an ion exchange group, a glycidyl compound such as epichlorohydrin is reacted with a separating material, and a sulfite or bisulfite such as sodium sulfite or sodium bisulfite is used. A method of adding a separating material to a saturated aqueous solution of The reaction conditions are preferably 30 to 90 ° C. for 1 to 10 hours.

  On the other hand, as a method for introducing an ion exchange group, a method of reacting 1,3-propane sultone with a separating material in an alkaline atmosphere can also be mentioned. 1,3-Propane sultone is preferably used in an amount of 0.4% by mass or more based on the total mass of the separation material. The reaction conditions are preferably 0 to 90 ° C. for 0.5 to 12 hours.

  The separation material of the present embodiment is suitable for use in separation by protein electrostatic interaction and affinity purification. For example, the separating material of the present embodiment is added to a mixed solution containing a protein, and only the protein is adsorbed to the separating material by electrostatic interaction. When added to a high aqueous solution, the protein adsorbed on the separation material can be easily desorbed and recovered. Further, the separation material of the present embodiment can be used in column chromatography.

  As the biopolymer that can be separated using the separation material of the present embodiment, a water-soluble substance is preferable. Specifically, proteins such as serum albumin, blood proteins such as immunoglobulin, enzymes present in the living body, protein bioactive substances produced by biotechnology, DNA, biopolymers such as bioactive peptides, etc. The molecular weight is preferably 2,000,000 or less, more preferably 500,000 or less. In addition, it is necessary to select the properties, conditions, and the like of the separation material according to the isoelectric point, ionization state, and the like of the protein according to a known method. Known methods include, for example, the method described in JP-A-60-169427.

  The separation material of the present embodiment, after forming the coating layer, by introducing an ion-exchange group, protein A on the surface of the separation material, in the separation of biopolymers such as proteins, particles composed of natural polymers or synthetic It has the advantages of each of the polymer particles. In particular, since the porous polymer particles in the separation material of the present embodiment are obtained by the above-described method, they have durability and alkali resistance. In addition, the separation material of the present embodiment includes a coating layer containing the above graft polymer (a graft polymer in which a polymer having an active hydrogen group and a hydroxyl group is grafted on a crosslinked polymer having a hydroxyl group). Specific adsorption is reduced, and protein adsorption and desorption tend to occur. Furthermore, the separation material of the present embodiment tends to have a large amount of adsorption of protein and the like (dynamic adsorption amount) under the same flow rate.

  The liquid passing speed in the present specification indicates a liquid passing speed when a separating column of the present embodiment is packed in a stainless steel column of φ7.8 × 300 mm and liquid is passed. When the separation material of the present embodiment is packed in a column, it is preferable that the liquid passing speed is 800 cm / h or more at a column pressure of 0.3 MPa. When proteins and the like are separated by column chromatography, the flow rate of the protein solution or the like passed through the column is generally 400 cm / h or less. On the other hand, when the separation material of the present embodiment is used, a high adsorption amount can be maintained even when the separation material is used at a flow rate of 800 cm / h or more, which is faster than a conventional separation material for protein separation.

  The average particle size of the separation material of the present embodiment is preferably 10 to 300 μm, and for use in preparative or industrial chromatography, in order to avoid an extreme increase in column internal pressure, 10 to 100 μm And more preferably 50 to 100 μm.

  When the separation material of the present embodiment is used as a column packing material in column chromatography, there is almost no change in volume in the column irrespective of the properties of the eluate used, so that the operability is excellent.

  The porosity of the separating material is preferably 30% by volume or more and 70% by volume or less, more preferably 40% by volume or more and 70% by volume or less based on the total volume of the separating material. The separating material preferably has macropores (macropores), and the pore size (mode diameter) of the separating material is preferably 0.1 to 0.5 μm. The pore diameter of the separation material is more preferably 0.2 to 0.5 μm. When the pore diameter is 0.1 μm or more, the substance tends to easily enter the pores, and when the pore diameter is 0.5 μm or less, the specific surface area becomes sufficient.

The specific surface area of the separating material is preferably 30 m ² / g or more. In light of higher practicality, the specific surface area is more preferably equal to or greater than 35 m ² / g, and still more preferably equal to or greater than 40 m ² / g. When the specific surface area is 30 m ² / g or more, the adsorption amount of the substance to be separated tends to increase.

Note that, in the present embodiment, the separation material in which an ion exchange group is introduced has been described. However, the separation material can be used without introducing an ion exchange group. Such a separating material can be used, for example, for gel filtration chromatography.
Further, the column of the present embodiment includes the separation material of the present embodiment. The column of the present embodiment can be manufactured by filling the column with the separation material of the present embodiment. The method for filling the column with the separating material is not particularly limited, and for example, a known method can be adopted.

  Hereinafter, the present invention will be described with reference to examples, but the present invention is not limited to these examples.

(Example 1)
<Synthesis of Porous Polymer Particle 1>
In a 500 mL three-necked flask, 16 g of 96% pure divinylbenzene, 6 g of Span80, and 0.64 g of benzoyl peroxide were mixed to prepare a monomer solution. A 0.5% by mass aqueous solution of polyvinyl alcohol was used as a solvent. After emulsifying the monomer solution and the solvent using a microprocess server, the obtained emulsion (monomer concentration: 25% by volume) is transferred to a flask, and heated to about 80 ° C. with a stirrer while heating in a water bath at 80 ° C. Stirred for hours. The obtained particles were filtered and washed with acetone to obtain porous polymer particles 1. The particle size of the porous polymer particles 1 was measured with a flow type particle size measuring device, and the average particle size and the C.I. V. Values were calculated. Table 1 shows the results.

<Formation of coating layer>
To 100 mL of an aqueous solution (2% by mass) of agarose (Mw: 100,000), 4 g of sodium hydroxide and 0.4 g of glycidyl phenyl ether were added, and reacted at 70 ° C. for 12 hours to introduce a phenyl group into the agarose. The resulting denatured agarose was precipitated with isopropyl alcohol. The precipitated denatured agarose was separated by filtration, dissolved again in water, and the operation of precipitating the denatured agarose with isopropyl alcohol was further repeated twice, followed by drying to obtain denatured agarose. The resulting modified agarose was dissolved again in water to prepare a 20 mg / mL modified agarose aqueous solution. The modified agarose was adsorbed on the porous polymer particles 1 by adding 10 g of the porous polymer particles 1 to 700 mL of this aqueous solution and stirring the mixture at 55 ° C. for 24 hours. The porous polymer particles 1 on which the modified agarose was adsorbed were filtered and washed with hot water.

(Cross-linking treatment)
The modified agarose adsorbed on the surface of the porous polymer particles 1 was crosslinked as follows. After preparing a dispersion in which 10 g of the porous polymer particles 1 are dispersed in 350 g of a 0.4 M sodium hydroxide aqueous solution, 10 g of ethylene glycol diglycidyl ether is added to the dispersion, and the mixture is stirred at room temperature for 12 hours. The modified agarose was crosslinked. The obtained particles were separated by filtration and washed with pure water.

(Introduction of epoxy group into cross-linked modified agarose)
10 g of the obtained particles were dispersed in 350 g of a 0.4 M aqueous sodium hydroxide solution. 20 g of epichlorohydrin was successively added to the dispersion and stirred for 3 hours to introduce an epoxy group into the crosslinked product of the modified agarose. The obtained particles were washed with a 2% by mass aqueous solution of hot sodium dodecyl sulfate, and then washed with pure water to obtain particles modified with an epoxy.

(Preparation of polymer having active hydrogen group and hydroxyl group)
5 g of dextran (Mw: 6000) was dissolved in 50 mL of dimethyl sulfoxide (DMSO) to obtain a dextran solution. 0.1 g of ethylenediamine and 20 mg of sodium cyanoborohydride were added to the obtained dextran solution, followed by stirring at 60 ° C. for 7 days. During this time, every other day (ie, on days 3, 5, and 7), 20 mg of sodium cyanoborohydride was added.

Next, dextran having an amino group terminal (hereinafter, sometimes referred to as “Dex-NH ₂ ”) was obtained by reprecipitation using methanol. The weight average molecular weight (Mw) of Dex-NH ₂ was 6,100.

(Graft)
10 g of the epoxy-modified body-coated particles were dispersed in an aqueous Dex-NH ₂ solution in which 5 g of Dex-NH ₂ was dissolved in 50 g of water. The obtained dispersion was stirred at 80 ° C. for 12 hours, and Dex-NH ₂ was grafted onto the particles coated with the epoxy-modified product. The particles after grafting were filtered off and washed with hot water. The obtained particles were evaluated by the following method.

(Coating layer amount evaluation)
The amount (mg) of the coating layer per 1 g of the porous polymer particles was calculated by thermogravimetric analysis.
Table 3 shows the results.

(Evaluation of graft amount)
By performing thermogravimetric analysis on the particles before and after the grafting, the amount of grafting (mg) per 1 g of the porous polymer particles was calculated. Table 3 shows the results.

(Evaluation of nonspecific adsorption of protein)
0.5 g of the obtained separation material was added to 50 mL of a phosphate buffer (pH 7.4) having a concentration of 20 mg / mL of BSA (Bovine Serum Alubumin), and the mixture was stirred at room temperature for 24 hours. Thereafter, centrifugation was performed and a supernatant was obtained. The amount of BSA adsorbed on the separating material was calculated from the BSA concentration in the supernatant determined by measuring the absorbance of the supernatant at 280 nm with a spectrophotometer. Table 3 shows the results.

<Introduction of ion exchange group>
Water was removed from the obtained particle dispersion by centrifugation. 10 g of the obtained particles were dispersed in an aqueous solution of diethylaminoethyl chloride hydrochloride (an aqueous solution obtained by dissolving 60 g of diethylaminoethyl chloride hydrochloride in water to make 100 g), stirred at 70 ° C for 10 minutes, and then heated to 70 ° C. 100 mL of a 5M aqueous sodium hydroxide solution was added and reacted for 1 hour. After the completion of the reaction, the product was collected by filtration and washed twice with water / ethanol (volume ratio: 8/2) to obtain a (DEAE-modified) separation material having a diethylaminoethyl (DEAE) group as an ion exchange group. The pore size (modal size), specific surface area and porosity (porosity) of the obtained separation material were measured by a mercury intrusion method. Table 2 shows the results.

<Evaluation of column characteristics>
(Evaluation of liquid passing and evaluation of dynamic adsorption amount)
The obtained separation material was packed as a slurry (solvent: methanol) with a concentration of 30% by mass into a φ7.8 × 300 mm stainless steel column for 15 minutes. Thereafter, water was passed through the column while changing the flow rate, the relationship between the flow rate and the column pressure was measured, and the liquid passing rate (linear flow rate) at 0.3 MPa was measured. Table 3 shows the results.

The amount of dynamic adsorption was measured as follows. A 20 mmol / L Tris-HCl buffer (pH 8.0) was passed through the column in 10 column volumes. Thereafter, the solution was passed through a 20 mmol / L Tris-HCl buffer having a BSA concentration of 2 mg / mL, and the BSA concentration at the column outlet was measured by UV measurement. The buffer solution was passed until the BSA concentration at the column inlet and the BSA concentration at the outlet matched, and the column was diluted with 5 column volumes of 1 M NaCl Tris-HCl buffer. The dynamic adsorption amount at 10% breakthrough was calculated using the following equation. Table 3 shows the results.
_{q 10 = c f F (t} 10 -t 0) / V B
q ₁₀ : Dynamic adsorption amount (mg / mL wet resin) at 10% breakthrough
cf: BSA concentration injected F: Flow rate (mL / min)
V _B : bed volume (mL)
t ₁₀ : time (min) at 10% breakthrough
t ₀ : BSA injection start time (min)

(Example 2)
A separation material was produced in the same manner as in Example 1 except that the Mw of dextran used for producing a polymer having an active hydrogen group and a hydroxyl group was changed to 23,000, and the separation material was evaluated in the same manner as in Example 1. There was no change in the polymer molecular weight before and after the introduction of the active hydrogen groups, and the Mw of the obtained Dex-NH ₂ was 23,000.

(Example 3)
A separation material was prepared in the same manner as in Example 1 except that the Mw of dextran used for the preparation of the polymer having an active hydrogen group and a hydroxyl group was changed to 45,000, and evaluated in the same manner as in Example 1. There was no change in the polymer molecular weight before and after the introduction of the active hydrogen group, and the Mw of the obtained Dex-NH ₂ was 45,000.

(Example 4)
A separation material was prepared in the same manner as in Example 1 except that Dex-NH ₂ was changed to polyglycerin monomethacrylate (PGMA-SH, Mw: 75000) having a thiol terminal synthesized as described below. Evaluation was performed in the same manner as in Example 1.

(Synthesis of thiol-terminated polyglycerol monomethacrylate)
0.4 g of 2-cyano-2-propyldithiobenzoate, 78.1 g of glycerin monomethacrylate, 0.38 g of 4,4′-azobis (4-cyanovaleric acid) and 140 mL of ethanol were mixed, and the mixture was polymerized at 70 ° C. for 5 hours. After that, a solution obtained by dissolving sodium borohydride (0.2 g) in 5 g of water was added, and the mixture was stirred for 3 hours. The resulting polymer was reprecipitated using dichloromethane. When Mw of the obtained polymer was measured by GPC, it was 75,000.

(Example 5)
A separation material was prepared in the same manner as in Example 1 except that Dex-NH ₂ was changed to polyhydroxyethyl methacrylate having a thiol terminal (PHEMA-SH, Mw: 92000) synthesized as described below. Evaluation was performed in the same manner as in Example 1.

(Synthesis of thiol-terminated polyhydroxyethyl methacrylate)
0.4 g of 2-cyano-2-propyldithiobenzoate, 78.1 g of hydroxyethyl methacrylate, 0.38 g of 4,4′-azobis (4-cyanovaleric acid) and 140 mL of ethanol were mixed, and the mixture was polymerized at 70 ° C. for 5 hours. After that, a solution obtained by dissolving sodium borohydride (0.2 g) in 5 g of water was added, and the mixture was stirred for 3 hours. The resulting polymer was reprecipitated using dichloromethane. The Mw of the obtained polymer measured by GPC was 92,000.

(Comparative Example 1)
The evaluation was performed in the same manner as in Example 1 except that the porous polymer particles 1 were used as a separating material as they were.

(Comparative Example 2)
Except that Dex-NH ₂ was changed to dextran (Mw: 45000), a separation material was prepared in the same manner as in Example 1, and evaluated in the same manner as in Example 1.

(Comparative Example 3)
Epoxy-modified body-coated particles were produced in the same manner as in Example 1. Next, 10 g of the epoxy-modified body-coated particles were put into 100 mL of a 10% by mass aqueous solution of polyvinyl alcohol (PVA, Mw: 12000), and stirred for 1 hour to obtain a dispersion. To the obtained dispersion, 100 mL of a 1 M aqueous sodium hydroxide solution and 0.36 g of sodium borohydride were further added and stirred for 18 hours, so that polyvinyl alcohol was grafted on the particles coated with the epoxy-modified product. The particles after grafting were filtered off and washed with hot water. The obtained particles were used as a separating material and evaluated in the same manner as in Example 1.

(Comparative Example 4)
Except that polyvinyl alcohol was changed to agarose (Mw: 21000), a separation material was prepared in the same manner as in Comparative Example 2, and evaluated in the same manner as in Comparative Example 3.

(Comparative Example 5)
Commercially available agarose particles (Capto DEAE: GE Healthcare) (hereinafter referred to as “porous polymer particles 2”) were used as a separating material as they were, and evaluated in the same manner as in Example 1.

(Comparative Example 6)
<Synthesis of porous polymer particles 3>
Same as the porous polymer particle 1 except that 11.2 g of 2,3-dihydroxypropyl methacrylate and 4.8 g of ethylene glycol dimethacrylate were used instead of 16 g of divinylbenzene, and that the amount of Span80 was changed from 6 g to 5 g. Thus, porous polymer particles 3 were obtained. The particle size of the porous polymer particles 3 was measured with a flow type particle size measuring device, and the average particle size and the C.I. V. Values were calculated. Table 1 shows the results.

<Formation of coating layer>
1 g of dextran (Mw: 150,000), 0.6 g of sodium hydroxide and 0.15 g of sodium borohydride were dissolved in 6 g of distilled water. 4 g of the porous polymer particles 3 were mixed with 6 g of the obtained solution, and dextran was adsorbed on the porous polymer particles 3. The dextran-adsorbed porous polymer particles 3 were added to 1 L of a mixed solution of ethyl cellulose and toluene (ethyl cellulose concentration: 1% by mass) and stirred to disperse the particles to obtain a suspension. To the obtained suspension, 5 mL of epichlorohydrin was added, the temperature was raised to 50 ° C., and the mixture was stirred at this temperature for 6 hours to crosslink the dextran adsorbed on the surface of the porous polymer particles 3. After completion of the reaction, the particles were separated from the suspension by filtration and washed sequentially with toluene, ethanol, and distilled water to prepare a separating material. The obtained separation material was evaluated in the same manner as in Example 1.

  From the results in Table 3, it can be seen that the separation materials of Examples 1 to 5 have reduced nonspecific adsorption of proteins, have a high adsorption amount, and have excellent liquid permeability when used as a column.

Claims (12)

Porous polymer particles, comprising a coating layer covering at least a part of the surface of the porous polymer particles,
A separating material, wherein the coating layer includes a graft polymer in which a polymer having an active hydrogen group and a hydroxyl group is grafted on a crosslinked polymer having a hydroxyl group.   The separation material according to claim 1, wherein the porous polymer particles include a polymer having a structural unit derived from a monomer containing divinylbenzene.   The separation material according to claim 1, wherein the active hydrogen group is an amino group or a thiol group.   The separation material according to any one of claims 1 to 3, wherein the pore diameter (mode diameter) is 0.1 to 0.5 µm.   The separation material according to any one of claims 1 to 4, wherein the coefficient of variation of the particle size of the porous polymer particles is 5 to 15%.   The separation material according to any one of claims 1 to 5, wherein the crosslinked polymer having a hydroxyl group is a crosslinked polymer derived from a polysaccharide or a modified product thereof.   The separation material according to any one of claims 1 to 6, wherein the crosslinked polymer having a hydroxyl group is a crosslinked polymer derived from agarose or a modified product thereof.   The separation material according to any one of claims 1 to 7, wherein the polymer having an active hydrogen group and a hydroxyl group is dextran, polyglycerin monomethacrylate, or polyhydroxyethyl methacrylate having an active hydrogen group.   The separation material according to any one of claims 1 to 8, wherein the polymer having an active hydrogen group and a hydroxyl group has a weight average molecular weight of 5,000 to 100,000.   The separation material according to any one of claims 1 to 9, comprising 30 to 400 mg of the coating layer per 1 g of the porous polymer particles.   A column comprising the separation material according to claim 1. It is a manufacturing method of the separation material according to any one of claims 1 to 10,
A step of adsorbing a polymer having a hydroxyl group on the surface of the porous polymer particles and crosslinking the polymer,
Introducing a group capable of reacting with an active hydrogen group into the crosslinked polymer;
Reacting a polymer having an active hydrogen group and a hydroxyl group with a group capable of reacting with the active hydrogen group.