JP2018169354A - Separation material and column - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a separating material in which non-specific adsorption of a protein is reduced, which is excellent in alkali resistance and durability, and which is excellent in column characteristics such as liquid permeability when used as a column. The present invention comprises a porous polymer particle and a coating layer that covers at least a part of the surface of the porous polymer particle, the coating layer containing a crosslinked polymer having a hydroxyl group, and the water. The present invention relates to a separating material in which a hydrophilic monomer is grafted on a crosslinked polymer. [Selection diagram] None

Description

  The present invention relates to a separation material and a column.

  Conventionally, when separating and purifying biopolymers typified by proteins, generally, ion exchangers based on porous synthetic polymers and ion exchanges based on cross-linked gels of hydrophilic natural polymers are generally used. The body is used. In the case of an ion exchanger based on a porous synthetic polymer, the volume change due to the salt concentration is small. Therefore, when packed in a column and used for chromatography, the pressure resistance during liquid passage tends to be excellent. However, when this ion exchanger is used for separation of proteins, etc., nonspecific adsorption such as irreversible adsorption based on hydrophobic interaction occurs, resulting in peak asymmetry, or ion exchange by hydrophobic interaction. There is a problem that the protein adsorbed on the body cannot be recovered while adsorbed.

  On the other hand, an ion exchanger based on a cross-linked gel of a hydrophilic natural polymer typified by polysaccharides such as dextran and agarose has an advantage that there is almost no non-specific adsorption of protein. However, this ion exchanger swells remarkably in an aqueous solution, and has the disadvantage that 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 cross-linked gel is used in chromatography, there is a disadvantage that the pressure loss during liquid passage is large and the gel is consolidated by liquid passage.

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

  Separation materials are known in which xerogels such as polysaccharides such as dextran and cellulose are held in an inorganic porous material such as celite (see, for example, Patent Documents 2 and 3). In order to add sorption performance to this gel, a diethylaminoethyl (DEAE) group or the like is added, which is used for removing hemoglobin. Such a separation material has good liquid permeability in the column.

  A hybrid copolymer ion exchanger in which pores of a macro-network structure copolymer are filled with a gel of a crosslinked copolymer synthesized from a monomer is known (for example, see Patent Document 4). When the cross-linked copolymer gel has a low degree of cross-linking, there are problems such as pressure loss and volume change, but by making it a hybrid copolymer, the liquid flow characteristics are improved, the pressure loss is small, the ion exchange capacity is improved, Leakage behavior is improved.

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

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

  However, the conventional separation material has a sufficient level of all properties such as reduced non-specific adsorption of protein, excellent alkali resistance and durability, and excellent column characteristics such as liquid permeability when used as a column. It's not something you can combine.

  Accordingly, the present invention provides a separation material having reduced non-specific adsorption of protein, excellent alkali resistance and durability, and excellent column characteristics such as liquid permeability when used as a column, a method for producing the separation material, and the separation An object is to provide a column comprising a material.

The present invention provides a separation material according to the following [1] to [13], a method for producing the separation material, and a column including 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, the coating layer includes a crosslinked polymer having a hydroxyl group, and the crosslinked polymer has a hydrophilic monomer Separation material to which is grafted.
[2] The separating material according to [1], wherein the hydrophilic monomer is a (meth) acrylate compound having a hydroxyl group.
[3] The separating material according to [1] or [2], wherein the porous polymer particles include a polymer having a structural unit derived from a styrene monomer.
[4] The separating material according to any one of [1] to [3], wherein the crosslinked polymer having a hydroxyl group is a crosslinked polymer derived from a polysaccharide or a modified product thereof.
[5] The separating material according to any one of [1] to [4], wherein the crosslinked polymer having a hydroxyl group is a crosslinked polymer derived from agarose, dextran, or pullulan.
[6] The separation material according to any one of [1] to [5], wherein the average particle diameter is 10 to 300 μm and the mode pore diameter is 0.05 to 0.6 μm.
[7] The separating material according to any one of [1] to [6], wherein the porosity is 40 to 70% by volume.
[8] The separating material according to any one of [1] to [7], wherein the specific surface area is 20 m ² / g or more.
[9] The separating material according to any one of [1] to [8], wherein the coefficient of variation is 5 to 15%.
[10] The separating material according to any one of [1] to [9], comprising a coating layer containing a crosslinked polymer having 30 to 500 mg of hydroxyl groups per gram of porous polymer particles.
[11] When water is passed through the column filled with the separation material so that the pressure of the column is 0.3 MPa, the water flow rate is 800 cm / h or more. [10] The separation material according to any one of [10].
[12] A column comprising the separation material according to any one of [1] to [11].
[13] A method for producing a separating material according to any one of [1] to [11], wherein a polymer having a hydroxyl group is adsorbed on the surface of porous polymer particles, and the polymer is crosslinked. A method comprising a step of introducing an initiation group for atom transfer radical polymerization into a crosslinked polymer, and a step of grafting a hydrophilic monomer onto the initiation group.

  According to the present invention, non-specific adsorption of proteins is reduced, the separation material is excellent in alkali resistance and durability, and has excellent column characteristics such as liquid permeability when used as a column, the method for producing the separation material, and the separation It is possible to provide a column including a material.

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

<Separation material>
The separating 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. The coating layer includes a crosslinked polymer having a hydroxyl group, The molecule is grafted with a hydrophilic monomer. 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 pores inside the porous polymer particle. In the present specification, (meth) acrylic acid means acrylic acid or methacrylic acid, and the same applies to similar expressions such as (meth) acrylate.

  In the separation material of this embodiment, a polymer having a hydroxyl group is adsorbed on the surface of the porous polymer particles, and the polymer is crosslinked, and an initiation group for atom transfer radical polymerization is introduced into the crosslinked polymer. It can be produced by a production method comprising a step and a step of grafting a hydrophilic monomer to an initiating group.

[Porous polymer particles]
The porous polymer particles according to the present embodiment are particles obtained by curing a monomer containing a porosifying agent, and can be synthesized by, for example, conventional suspension polymerization, emulsion polymerization, or the like. Although it does not specifically limit as a monomer, For example, a styrene-type monomer can be used. That is, the porous polymer particles can include a polymer having a structural unit derived from a styrene monomer. Specific examples of the monomer include the following polyfunctional monomers and monofunctional monomers.

  Examples of the polyfunctional monomer include divinyl compounds such as divinylbenzene, divinylbiphenyl, divinylnaphthalene, and divinylphenanthrene. These polyfunctional monomers can be used singly or in combination of two or more. Among these, it is preferable that the monomer contains divinylbenzene from the viewpoint of durability, acid resistance, and alkali resistance.

  Examples of the monofunctional monomer include styrene, o-methylstyrene, m-methylstyrene, p-methylstyrene, α-methylstyrene, o-ethylstyrene, m-ethylstyrene, p-ethylstyrene, 2,4- Dimethyl styrene, pn-butyl styrene, pt-butyl styrene, pn-hexyl styrene, pn-octyl styrene, pn-nonyl styrene, pn-decyl styrene, pn-dodecyl Examples thereof include styrene such as styrene, p-methoxystyrene, p-phenylstyrene, p-chlorostyrene, and 3,4-dichlorostyrene and derivatives thereof. These monofunctional monomers can be used alone or in combination of two or more. Among these, it is preferable to use styrene from the viewpoint of excellent acid resistance and alkali resistance. Moreover, the styrene derivative which has functional groups, such as a carboxyl group, an amino group, a hydroxyl group, and an aldehyde group, can also be used.

  Examples of the porosifying agent include aliphatic or aromatic hydrocarbons, esters, ketones, ethers, alcohols, and the like, which are organic solvents that promote phase separation at the time of polymerization and promote pore formation of particles. It is done. 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, and cyclohexanol. These porosifying agents can be used alone or in combination of two or more.

  The porosifying agent can be used in an amount of 0 to 200% by mass based on the total monomer mass. The porosity of the porous polymer particles can be controlled by the amount of the porous agent. Furthermore, the size and shape of the pores of the porous polymer particles can be controlled by the kind of the porous agent.

  Water used as a solvent can also be used as a porosifying agent. When water is used as a porosifying agent, the oil-soluble surfactant is dissolved in the monomer and the water can be absorbed to make the particles porous.

  Examples of the oil-soluble surfactant used for the porous formation 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, such as sorbitan monolaurate, sorbitan Sorbitan monoesters derived from monooleate, sorbitan monomyristate or coconut fatty acid; diglycerol monoesters of branched C16-C24 fatty acids, chain unsaturated C16-C22 fatty acids or chain saturated C12-C14 fatty acids, for example di- Glycerol monooleate (for example, diglycerol monoester of C18: 1 (18 carbon atoms, 1 double bond) fatty acid), diglycerol monomyristate, diglycerol monoisostearate or diglycerol monoester of coconut fatty acid Ester; Branch 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 (eg, SPAN 20), preferably having a purity of greater than about 40%, more preferably greater than about 50%, and even more preferably greater than about 70%. Rate), sorbitan monooleate (eg, SPAN 80, sorbitan monooleate, preferably greater than about 40%, more preferably greater than about 50%, and even more preferably greater than about 70%. ), Diglycerol monooleate (eg, diglycerol monooleate having a purity preferably greater than about 40%, more preferably greater than about 50%, even more preferably greater than about 70%), diglycerol monoisostearate (For example, the purity is preferably greater than about 40%, more preferably about 50%. More preferably, greater than about 70% diglycerol monoisostearate), diglycerol monomyristate (purity preferably greater than about 40%, more preferably greater than about 50%, even more preferably about 70%. Sorbitan monomyristate), diglycerol cocoyl (eg, lauryl, myristoyl, etc.) ethers, or mixtures thereof.

  These oil-soluble surfactants are preferably used in the range of 5 to 80% by mass relative to the total monomer mass. When the content of the oil-soluble surfactant is 5% by mass or more, the stability of the water droplets becomes sufficient, so that a large single hole is easily formed. In addition, when the content of the oil-soluble surfactant is 80% by mass or less, the porous polymer particles are more easily retained in shape after 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, 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 potassium, alkyl sulfate salts such as sodium lauryl sulfate and ammonium lauryl sulfate, alkylbenzene sulfonates such as sodium dodecylbenzenesulfonate, and alkylnaphthalene sulfone. Acid salts, alkane sulfonates, dialkyl sulfosuccinates such as sodium dioctyl sulfosuccinate, alkenyl succinates (dipotassium salts), alkyl phosphate esters, naphthalene sulfonate formalin condensates, polyoxyethylene alkylphenyl ether sulfates Salts, polyoxyethylene alkyl ether sulfates such as sodium polyoxyethylene lauryl ether sulfate, and polyoxyethylene alkyl sulfate salts.

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

  Nonionic surfactants 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, or amides. Agents, polyether-modified silicon-based nonionic surfactants such as polyethylene oxide adducts of silicon and polypropylene oxide adducts, and fluorine-based nonionic surfactants such as perfluoroalkyl glycols.

  Examples of zwitterionic surfactants include hydrocarbon surfactants such as lauryl dimethylamine oxide, phosphate ester surfactants, and phosphite ester surfactants.

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

  Examples of the polymerization initiator added as necessary include benzoyl peroxide, lauroyl peroxide, orthochlorobenzoyl peroxide, 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 ′ -Azo compounds such as azobis (2,4-dimethylvaleronitrile). A polymerization initiator can be used in 0.1-7.0 mass parts with respect to 100 mass parts of monomers.

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

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

  Examples of the polymer dispersion stabilizer include polyvinyl alcohol, polycarboxylic acid, celluloses (hydroxyethyl cellulose, carboxymethyl cellulose, methyl cellulose, etc.), polyvinyl pyrrolidone, and inorganic water-soluble polymer compounds such as sodium tripolyphosphate. can do. Of these, polyvinyl alcohol or polyvinyl pyrrolidone is 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 vitamin Bs, citric acid, and polyphenols may be used.

  The average particle diameter of the porous polymer particles is preferably 300 μm or less, more preferably 150 μm or less, and still more preferably 100 μm or less. Further, the average particle diameter of the porous polymer particles is preferably 10 μm or more, more preferably 30 μm or more, and further preferably 50 μm or more, from the viewpoint of improving liquid permeability.

  The coefficient of variation (CV) of the particle size of the porous polymer particles is preferably 3 to 15%, more preferably 5 to 15%, from the viewpoint of improving liquid permeability. More preferably, it is 10%. C. V. As a method for reducing the above, monodispersion can be performed by an emulsification apparatus such as a microprocess server (Hitachi).

C. of average particle size and particle size of porous polymer particles or separator V. Can be determined by the following measurement method.
1) Disperse the particles in water (including a dispersant such as a surfactant) using an ultrasonic dispersion device to prepare a dispersion containing 1% by mass of porous polymer particles.
2) Using a particle size distribution meter (Sysmex Flow, manufactured by Sysmex Corporation), an average particle size and particle size of C.I. V. Measure.

  The pore volume of the porous polymer particles is preferably 30% to 70% by volume, more preferably 40% to 70% by volume, based on the total volume of the porous polymer particles. The porous polymer particles preferably have pores having a pore diameter of 0.1 μm or more and less than 0.5 μm, that is, macropores (macropores). The pore diameter of the porous polymer particles is more preferably 0.2 μm or more and less than 0.5 μm. If the pore diameter is 0.1 μm or more, the substance tends to easily enter the pores. If the pore diameter is less than 0.5 μm, the specific surface area is sufficient. These can be adjusted by the above-mentioned porous agent.

The specific surface area of the porous polymer particles is preferably 30 m ² / g or more. From the viewpoint of higher practicality, the specific surface area is more preferably 35 m ² / g or more, and further preferably 40 m ² / g or more. When the specific surface area is 30 m ² / g or more, the adsorption amount of the substance to be separated tends to increase.

[Coating layer]
The coating layer according to this embodiment includes a crosslinked polymer having a hydroxyl group. A hydrophilic monomer is grafted to the crosslinked polymer having a hydroxyl group. That is, the coating layer has a graft chain based on a hydrophilic monomer bonded to a crosslinked polymer. By covering the porous polymer particles with a cross-linked polymer having a hydroxyl group, it is possible to suppress an increase in column pressure and to suppress non-specific adsorption of protein, and to reduce the amount of protein adsorbed on the separation material. It is possible to make it equal to or higher than when natural polymers are used. Further, by having a graft chain based on a hydrophilic monomer, it is possible to impart high mobility to the coating material while maintaining the hydrophilicity of the particles.

(Crosslinked polymer having a hydroxyl group)
The crosslinked polymer having a hydroxyl group preferably has two or more hydroxyl groups in one molecule, and more preferably a hydrophilic polymer. The crosslinked polymer having a hydroxyl group is a crosslinked polymer having a hydroxyl group. Examples of the polymer having a hydroxyl group include polysaccharides or modified products thereof, and polyvinyl alcohol or modified polymers thereof. Examples of the polysaccharide include agarose, dextran, pullulan, cellulose, and chitosan. As the polymer having a hydroxyl group, for example, a polymer having an average molecular weight of 10,000 or more can be used. The crosslinked polymer having a hydroxyl group is preferably a crosslinked polymer derived from a polysaccharide or a modified product thereof. The crosslinked polymer having a hydroxyl group may be a crosslinked polymer derived from agarose, dextran or pullulan.

  The polymer having a hydroxyl group is preferably a modified body having a hydrophobic group introduced 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. As a C1-C6 alkyl group, a methyl group, an ethyl group, and a propyl group are mentioned, for example. 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. be able to.

  The content of the hydrophobic group in the modified polymer having a hydroxyl group into which a hydrophobic group has been introduced depends on the balance between retention of hydrophobic interaction force to adsorb on the particle surface and suppression of nonspecific adsorption of protein. 5 to 30% by mass, more preferably 10 to 20% by mass, and further preferably 12 to 17% by mass.

(Formation method of coating layer)
The coating layer according to this embodiment, for example, adsorbs a polymer having a hydroxyl group on the surface of the porous polymer particle, and then crosslinks the polymer to form a crosslinked polymer having a hydroxyl group on the surface of the porous polymer particle. Then, after introducing an atom transfer radical polymerization initiating group into the crosslinked polymer, a hydrophilic monomer is grafted onto the initiating group. Hereinafter, specific examples of the method for forming the coating layer will be described.

  First, a polymer solution having a hydroxyl group is adsorbed on the surface of the porous polymer particles. The solvent for the polymer solution 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).

  This solution is impregnated into porous polymer particles. In the impregnation method, porous polymer particles are added to a polymer solution having a hydroxyl group and allowed to stand for a predetermined time. Although the impregnation time varies depending on the surface state of the porous body, the polymer concentration is in an equilibrium state with the external concentration inside the porous body if it is usually impregnated day and night. Then, it wash | cleans with solvents, such as water and alcohol, and remove | eliminates the polymer which has a hydroxyl group which is not adsorbed.

(Crosslinking treatment)
Next, a crosslinking agent is added to cause the polymer having a hydroxyl group adsorbed on the surface of the porous polymer particles to undergo a crosslinking reaction to form a crosslinked body. At this time, the crosslinked body has a three-dimensional crosslinked network structure having a hydroxyl group.

  As the crosslinking agent, for example, an epihalohydrin such as epichlorohydrin, a dialdehyde compound such as glutaraldehyde, a diisocyanate compound such as methylene diisocyanate, a glycidyl compound such as ethylene glycol diglycidyl ether, and two or more functional groups active on a hydroxyl group. The compound which has is mentioned. Further, when a compound having an amino group such as chitosan is used as the polymer having a hydroxyl group, a dihalide compound 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 epichlorohydrin or the like, an alkali such as sodium hydroxide is effective, and in the case of a dialdehyde compound. Mineral acids such as hydrochloric acid are effective.

  The crosslinking reaction with the crosslinking agent is usually performed by adding the crosslinking agent to a system in which the separating material is dispersed and suspended in an appropriate medium. When the polysaccharide is used as the polymer having a hydroxyl group, the addition amount of the crosslinking agent is within a range of 0.1 to 100 mol times per unit of the monosaccharide, and the performance of the separating material. It can be selected according to. Generally, when the addition amount of the crosslinking agent is reduced, the coating layer tends to be easily peeled off from the porous polymer particles. Moreover, when the addition amount of a crosslinking agent is excessive and the reaction rate with the polymer which has a hydroxyl group is high, the characteristic of the polymer which has a hydroxyl group of a raw material tends to be impaired.

  The amount of the catalyst used varies depending on the type of the crosslinking agent. Usually, when a polysaccharide is used as the polymer having a hydroxyl group, if one unit of the monosaccharide forming the polysaccharide is 1 mole, In a range of 0.01 to 10 mol times, preferably 0.1 to 5 mol times.

  For example, when the crosslinking reaction condition is a temperature condition, a crosslinking reaction occurs when the temperature of the reaction system is raised and the temperature reaches the reaction temperature.

  As a medium for dispersing and suspending porous polymer particles impregnated with a polymer solution having a hydroxyl group, etc., without extracting a polymer, a crosslinking agent or the like from the impregnated polymer solution, and without crosslinking It must be inert to the reaction. Specific examples of the medium include water and alcohol.

  The crosslinking reaction is usually performed at a temperature in the range of 5 to 90 ° C. over 1 to 10 hours. Preferably, the temperature is in the range of 30 to 90 ° C.

  After completion of the crosslinking reaction, the produced particles are filtered off, washed with a hydrophilic organic solvent such as methanol and ethanol, and unreacted polymer, suspending medium, etc. are removed to remove the surface of the porous polymer particles. A separation material that is at least partially coated with a coating layer containing a polymer having a hydroxyl group is obtained.

  The separating material of this embodiment preferably includes a coating layer containing a crosslinked polymer having 30 to 500 mg of hydroxyl group per gram of porous polymer particles. The coating amount of the crosslinked polymer is more preferably 100 to 450 mg, and further preferably 200 to 400 mg, per 1 g of the porous polymer particles. When the coating amount of the crosslinked polymer is 500 mg or less with respect to 1 g of the porous polymer particles, the coating layer can be made into a thin film, and the liquid permeability when used as a column tends to be further improved. Moreover, when the coating amount of the crosslinked polymer is 30 mg or more with respect to 1 g of the porous polymer particles, the protein adsorption amount tends to be further increased. The coating amount of the crosslinked polymer having a hydroxyl group can be measured by, for example, reducing the weight of thermal decomposition.

(Introduction of ATRP initiation group)
Initiating groups of ATRP (Atom Transfer Radical Polymerization) can be introduced into the porous polymer particles coated with a crosslinked polymer having a hydroxyl group by converting the hydroxyl group on the surface. By introducing an ATRP initiating group, a hydrophilic monomer can be grafted to the hydroxyl group. Examples of the method for introducing the ATRP initiating group include a method using an alkyl halide compound.

  The halogenated alkyl compound is preferably bromide or chloride. Examples of the halogenated alkyl compound include compounds having an alkyl halide such as 2-bromobutyryl bromide.

  Since the introduction of the ATRP initiating group is performed on the hydroxyl group on the particle surface, the particle is drained by filtration or the like, then immersed in an organic solvent, and an alkyl halide is added in the presence of a predetermined concentration of amine to react. The reaction temperature of this reaction is preferably 0 ° C. when the alkyl halide is added, and then preferably 12 to 24 hours at room temperature.

(Graft of hydrophilic monomer)
Next, a hydrophilic monomer is reacted with the particles into which the ATRP initiating group has been introduced. Usually, since the monomer reaction is carried out on the ATRP initiation group, the particles are drained by filtration or the like and then immersed in an organic solvent to obtain a predetermined concentration of copper halide, trisdimethylaminoethylamine, ascorbic acid, and hydrophilic. A reactive monomer is added and reacted under an inert gas. This reaction temperature is preferably 20 to 40 ° C. and preferably 12 to 24 hours.

  As the hydrophilic monomer, a hydrophilic (meth) acrylate compound can be used. From the viewpoint of improving hydrophilicity, a (meth) acrylate compound having a hydroxyl group is preferable. Examples of the (meth) acrylate compound 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.

  The graft amount of the hydrophilic monomer is preferably 10 to 500 mg per 1 g of the porous polymer particles, more preferably 20 to 450 mg, and further preferably 30 to 430 mg. When the graft amount is 500 mg or less with respect to 1 g of the porous polymer particles, the pores tend not to be buried by the graft chains. Further, when the graft amount is 10 mg or more with respect to 1 g of the porous polymer particles, the compression elastic modulus is improved and the protein adsorption amount tends to be improved. The amount of grafting can be measured by reducing the weight of thermal decomposition.

(Introduction of ion exchange groups)
The separation material provided with the coating layer can be used for ion exchange purification, affinity purification, etc. by introducing ion exchange groups, ligands (protein A), etc. via hydroxyl groups on the surface. Examples of the method for introducing an ion exchange group include a method using an alkyl halide compound.

  Examples of the halogenated alkyl compound include monohalogenocarboxylic acids such as monohalogenoacetic acid and monohalogenopropionic acid and sodium salts thereof, primary, secondary or tertiary amines having at least one halogenated alkyl group such as diethylaminoethyl chloride, halogen And quaternary ammonium hydrochloride having an alkyl group. These halogenated alkyl compounds are preferably bromides or chlorides. The amount of the halogenated alkyl compound used is preferably 0.2% by mass or more based on the total mass of the separating material imparting ion exchange groups.

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

  In general, since the ion exchange group is introduced into the hydroxyl group on the surface of the separation material, the wet particles are drained by filtration or the like, immersed in an alkaline aqueous solution of a predetermined concentration, left for a certain period of time, and then water-organic. The halogenated alkyl compound is added and reacted in a solvent mixture system. This reaction is preferably carried out at a temperature of 40 to 90 ° C. for 0.5 to 12 hours. The ion exchange group to be provided is determined depending on the kind of the halogenated alkyl compound used in the above reaction.

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

  As a method for introducing a strongly basic quaternary ammonium group as an ion exchange group, first, a tertiary amino group is introduced, and then the tertiary amino group is reacted with a halogenated alkyl compound such as epichlorohydrin. The method of converting into an ammonium group is mentioned. Further, quaternary ammonium hydrochloride or the like may be reacted with the separation material.

  Examples of the method for introducing a carboxy group that is a weakly acidic group as an ion exchange group include a method in which a monohalogenocarboxylic acid such as monohalogenoacetic acid or monohalogenopropionic acid or a sodium salt thereof is reacted as the halogenated alkyl compound. . The amount of the halogenated alkyl compound used is preferably 0.2% by mass or more based on the total mass of the separating 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 a bisulfite such as sodium sulfite or sodium bisulfite. And a method of adding a separating material to the saturated aqueous solution. The reaction conditions are preferably 30 to 90 ° C. and 1 to 10 hours.

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

  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. It is more preferable that it is 50-100 micrometers.

  The separating material preferably has pores having a mode pore diameter of 0.05 to 0.6 μm, that is, macropores (macropores). The mode pore diameter is preferably 0.1 to 0.5 μm. When the pore diameter is 0.05 μm or more, a substance tends to easily enter the pores. When the pore diameter is 0.6 μm or less, the specific surface area is sufficient.

The specific surface area of the separating material is preferably 20 m ² / g or more. From the viewpoint of higher practicality, the specific surface area is more preferably 25 m ² / g or more. When the specific surface area is 20 m ² / g or more, the adsorption amount of the substance to be separated tends to increase. The upper limit value of the specific surface area of the separating material can be 1000 m ² / g or less.

  The porosity of the separating material is preferably 40 to 70% by volume. When the porosity is in this range, the amount of protein adsorption can be increased.

  The mode pore diameter, specific surface area, and porosity of the separating material or porous polymer particle according to the present embodiment are values measured with a mercury intrusion measuring device (Autopore: manufactured by Shimadzu Corporation), and are as follows. taking measurement. 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 an initial pressure of 21 kPa (about 3 psia, corresponding to a pore diameter of about 60 μm). Mercury parameters are set to a device default mercury contact angle of 130 ° and a mercury surface tension of 485 dynes / cm. Moreover, it limits to the range of 0.1-3 micrometers of pore diameters, and calculates each value.

  The mode pore diameter, specific surface area, etc. of the separating material can be adjusted by appropriately selecting the raw material of the porous polymer particles, the porosifying agent, the polymer having a hydroxyl group, and the like.

The 5% compressive deformation elastic modulus of the separating material of the present embodiment can be calculated as follows.
Using a micro compression tester (manufactured by Fisher), the load when the separation material is compressed to 50 mN with a smooth end face (50 μm × 50 μm) of a square column at a load load rate of 1 mN / sec at room temperature (25 ° C.). And measure the compression displacement. From the measured value obtained, the compression modulus (5% K value) when the separating material is 5% compressively deformed can be obtained by the following equation. Further, the load at the point at which the displacement during the measurement changes most greatly is defined as the breaking strength (mN).
5% K value (MPa) = (3/2 ^1/2 ) · F · S ⁻³ / ² · R ^−1/2
5% K value (MPa) = (3/2 ^1/2 ) · F · S ⁻³ / ² · R ^−1/2
F: Load (mN) when the separating material is 5% compressively deformed
S: Compression displacement (mm) when the separating material is 5% compressively deformed
R: radius of separating material (mm)

  The compression elastic modulus (5% K value) when the separating material is 5% compressively deformed is preferably 100 to 1000 MPa, more preferably 150 to 1000 MPa, and still more preferably 170 to 1000 MPa. When the compression elastic modulus is 100 MPa or more, the rigidity of the separating material increases, and it becomes difficult to deform when a liquid is flowed in the column, and the column pressure is difficult to increase.

  The separation material of this embodiment is suitable for use in separation of proteins by electrostatic interaction and affinity purification. For example, after adding the separation material of the present embodiment to a mixed solution containing protein, adsorbing only the protein to the separation material by electrostatic interaction, the separation material is filtered from the solution, and the salt concentration If added to a high aqueous solution, the protein adsorbed on the separation material can be easily desorbed and recovered. Moreover, the separation material of this embodiment can also be used in column chromatography.

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

  In the separation material of the present embodiment, after the coating layer on the porous polymer particles is cross-linked, an ion exchange group and protein A are introduced into the surface of the separation material, thereby separating the natural polymer in the separation of biopolymers such as proteins. Each has the advantage of particles made of molecules or particles made of synthetic polymer. 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 tends to reduce non-specific adsorption of proteins and easily cause protein adsorption / desorption. Furthermore, the separation material of the present embodiment tends to have a large adsorption amount (dynamic adsorption amount) of protein or the like under the same flow rate.

  The liquid passing speed in this specification represents the liquid passing speed when the separation material of this embodiment is packed in a stainless steel column of φ7.8 × 300 mm and the liquid is passed. When the separation material of this embodiment is packed in a column, it is preferable that the flow rate of water is 800 cm / h or more when water is passed so that the column pressure becomes 0.3 MPa. When separating proteins by column chromatography, the flow rate of protein solution or the like is generally in the range of 400 cm / h or less. However, when the separation material of the present embodiment is used, the separation rate for normal protein separation is as follows. It can be used at a liquid passing speed of 800 cm / h or more faster than the separating material.

  In addition, although this embodiment demonstrated the separation material of the form which introduce | transduces an ion exchange group, even if it does not introduce | transduce an ion exchange group, it can be used as a separation material. Such a separating material can be used for, for example, gel filtration chromatography.

  EXAMPLES Hereinafter, although an Example demonstrates this invention, this invention is not limited to these Examples.

(Porous polymer particles)
In a 500 mL three-necked flask, 16 g of divinylbenzene having a purity of 96% (trade name “DVB960” manufactured by Nippon Steel & Sumikin Chemical Co., Ltd.), 16 g of hexanol, 16 g of diethylbenzene and 0.64 g of benzoyl peroxide were added to 0.5% by mass of polyvinyl alcohol. In addition to the aqueous solution, after emulsification using a microprocess server, the obtained emulsion was transferred to a flask and stirred for about 8 hours using a stirrer while heating in a water bath at 80 ° C. The obtained particles were filtered and then washed with acetone to obtain porous polymer particles. The particle size of the obtained porous polymer particles was measured with a flow type particle size measuring device, and the average particle size and particle size C.I. V. The value (coefficient of variation) was calculated. The results are shown in Table 1.

(Denatured agarose)
0.98 g of sodium hydroxide and 4.90 g of glycidyl phenyl ether were added to 480 mL of a 2% by mass agarose aqueous solution and reacted at 60 ° C. for 6 hours to introduce a phenyl group into the agarose. The obtained modified agarose was reprecipitated with isopropyl alcohol and washed. The hydrophobic group content of the modified agarose was calculated by the following method and found to be 14.2%.

Dry powdered agarose (unmodified agarose) and hydrophobic group-introduced agarose dried to a volatile content of less than 0.1% by mass were each dissolved in pure water at 70 ° C., and 0.05% by mass An aqueous solution was prepared. The hydrophobic group content was calculated from the following formula by measuring the absorbance at 269 nm of each aqueous solution with a spectrophotometer to determine the concentration.
Hydrophobic group content (%) = (C _AG / (C _HAG + C _AG )) × 100
C _AG : Density of denatured agarose structural unit (mmol / L) = A / ε _GPE × 1000
C _HAG : concentration of unmodified agarose constituent unit (mmol / L) = [concentration of unmodified agarose constituent unit (g / L) / agarose constituent unit (306 g / mol)] × 1000
A: True absorbance of denatured agarose = Absorbance of denatured agarose-Absorption of _unmodified agarose-ε _GPE : Absorption coefficient of glycidyl phenyl ether = 1372 (L / (mol · cm))
Non-denatured agarose constituent unit concentration (g / L) = denatured agarose sample concentration (mass%) × 10—denatured agarose constituent unit concentration (g / L)
Absorption of undenatured agarose = absorbance of undenatured agarose × [sample concentration of denatured agarose (mmol / L) / sample concentration of unmodified agarose (mmol / L)]
Concentration of modified agarose unit (g / L) = (C _AG × modified agarose constituent unit (456 g / mol)) / 1000

[Example 1]
<Formation of coating layer>
Into a 20 mg / mL modified agarose aqueous solution, the porous polymer particles are added at a concentration of 70 mL / particle g, and stirred at 55 ° C. for 24 hours to adsorb the modified agarose to the porous polymer particles, followed by filtration. Washed with hot water.

(Crosslinking treatment)
The modified agarose adsorbed on the porous polymer particles was crosslinked as follows. 10 g of particles adsorbed with modified agarose were dispersed in a 0.4 M aqueous sodium hydroxide solution, 0.04 M epichlorohydrin was added, and the mixture was stirred at room temperature for 8 hours. Then, it was washed with hot water of 2% by mass sodium dodecyl sulfate aqueous solution and then with pure water. After the obtained particles were dried, the coating amount of the crosslinked polymer having a hydroxyl group was measured by thermogravimetric analysis.

(Introduction of ATRP initiation group)
Introduction of the ATRP initiating group into the particles coated with the crosslinked polymer having a hydroxyl group was performed as follows. 2 g of the particles washed with an aqueous solution of sodium dodecyl sulfate are dispersed in 250 mL of N, N-dimethylformamide, the dispersion added with 1.1 g of triethylamine is immersed in an ice bath, and 2.5 g of 2-bromobutyryl bromide is added to 50 mL of DMF. The solution dissolved in was added. After the addition, the mixture was stirred for 24 hours at room temperature, washed with pure water, and then washed with acetone to obtain particles introduced with ATRP initiating groups.

(Graft of hydrophilic monomer)
2 g of particles introduced with ATRP initiating groups were dispersed in 50 mL of methanol, and 3.1 g of ascorbic acid was added to prepare solution a. Further, 60 g of glycerin monomethacrylate (GLM), 8 mg of copper dibromide and 16 mg of trisdimethylaminoethylamine were added to 150 mL of methanol to prepare a solution b. Nitrogen gas was bubbled into solutions a and b for 15 minutes, and then solutions a and b were mixed and stirred for 24 hours. Thereafter, washing with pure water was performed to obtain an aqueous suspension of a separating material grafted with a hydrophilic monomer. After drying the obtained particles, the graft amount of the hydrophilic monomer was measured by thermogravimetric analysis.

(Non-specific adsorption evaluation of protein)
After 0.2 g of particles grafted with a hydrophilic monomer was added to 20 mL of Tris-hydrochloric acid buffer (pH 8.0) having a BSA (Bovine Serum Albumin, Mw 66000) concentration of 24 mg / mL, the mixture was stirred at room temperature for 24 hours. The supernatant was collected by centrifugation, and the amount of BSA adsorbed on the particles was calculated from the BSA concentration of the filtrate with a spectrophotometer. The concentration of BSA was confirmed from the absorbance at 280 nm using a spectrophotometer. Moreover, the same evaluation was performed using insulin (Mw5800) instead of BSA. The nonspecific adsorption amount was 10 mg / mL or less as “◯”, and more than 10 mg / mL as “x”. The results are shown in Table 3.

<Introduction of ion exchange groups>
The separating material (dry mass 20 g) recovered by filtering the aqueous suspension was put into 200 mL of 5M aqueous sodium hydroxide solution and allowed to stand at room temperature for 1 hour. Separately, 200 mL of a predetermined amount (60 g) of diethylaminoethyl chloride hydrochloride was added, the temperature of the aqueous solution was raised to 70 ° C., and the reaction was allowed to proceed for 8 hours with stirring. After the completion of the reaction, filtration and washing with water / ethanol (volume ratio 5/1) three times gave a DEAE-modified separation material having diethylaminoethyl (DEAE) groups as ion exchange groups. The mode pore diameter and specific surface area of the obtained DEAE-modified separation material were measured by a mercury intrusion method. The results are shown in Table 2.

<Measurement of ion exchange group capacity>
The ion exchange group capacity of the obtained particles was measured as follows. 5 mL capacity | capacitance particle | grains were immersed in 20 mL of 0.1N sodium hydroxide for 1 hour, and were stirred at room temperature. Thereafter, the solution was washed with water so that the pH of the solution was 7 or less. The obtained particles were immersed in 10 mL of 0.1N hydrochloric acid and stirred for 1 hour. After filtering the particles, the ion exchange group capacity of the particles was measured by neutralizing titration with an aqueous hydrochloric acid solution. The results are shown in Table 3.

(5% compressive deformation modulus)
The 5% compressive deformation modulus of the obtained DEAE-modified separating material was measured by the method described above. The results are shown in Table 3.

(Column characteristics)
The obtained DEAE-modified separation material was packed as a slurry (solvent: methanol) with a concentration of 30% by mass into a stainless steel column of φ7.8 × 300 mm over 15 minutes. Thereafter, water was allowed to flow through the column while changing the flow rate, the relationship between the flow rate and the column pressure was measured, and the liquid flow rate (linear flow rate) at 0.3 MPa was measured. The results are shown in Table 2.
The dynamic adsorption amount was measured as follows. 10 column volumes of 20 mmol / L Tris-HCl buffer (pH 8.0) were passed through the column. Thereafter, a 20 mmol / L Tris-hydrochloric acid buffer solution having a BSA concentration of 2 mg / mL was poured, and the BSA concentration at the column outlet was measured by UV measurement. The solution was allowed to flow until the BSA concentrations at the column inlet and outlet coincided with each other, and diluted with 1 M NaCl Tris-HCl buffer solution for 5 column volumes. The amount of dynamic adsorption at 10% breakthrough was calculated using the following formula. The results are shown in Table 3.
_{q 10 = c f F (t} 10 -t 0) / V B
q ₁₀ : dynamic adsorption amount in 10% breakthrough (mg / mL wet resin)
cf: Injected BSA concentration F: Flow rate (mL / min)
V _B : Bed volume (mL)
t ₁₀ : time (min) at 10% breakthrough
t ₀ : BSA injection start time (min)

(Alkali resistance evaluation)
The obtained DEAE-modified separation material was stirred in a 0.5 M aqueous sodium hydroxide solution for 24 hours, washed with a Tris-hydrochloric acid buffer (pH 8.0), and then packed in a column under the above conditions. The 10% breakthrough dynamic adsorption amount of BSA was measured and compared with the dynamic adsorption amount before alkali treatment. The case where the decrease in the amount of dynamic adsorption was less than 3% was designated as “◯”, 3-20% as “Δ”, and over 20% as “x”. The results are shown in Table 3.

(Durability evaluation)
Water was allowed to flow through the column at a flow rate of 800 cm / h, and after measuring the column pressure, the flow rate was increased to 3000 cm / h and allowed to flow for 1 h. When the column pressure is lowered to 800 cm / h again, “×” indicates that the column pressure has increased by 10% or more from the initial value (before increasing the flow rate to 3000 cm / h), and “○” indicates that the column pressure is less than 10%. It was. The results are shown in Table 3.

[Example 2]
In the step of introducing the ATRP initiating group, except that triethylamine 820 mg and 2-bromobutyryl bromide 1.9 g were used, the same treatment as in Example 1 was carried out to produce a DEAE-modified separation material. Evaluation was performed in the same manner.

[Example 3])
In the step of introducing an ATRP initiating group, except that triethylamine 270 mg and 2-bromobutyryl bromide 0.63 g were used, the same treatment as in Example 1 was performed to prepare a DEAE-modified separation material. Evaluation was performed in the same manner.

[Example 4]
In the step of introducing ATRP initiating group, 270 mg of triethylamine and 0.63 g of 2-bromobutyryl bromide were used, and in the step of grafting hydrophilic monomer, except that hydroxyethyl methacrylate (HEMA) was used instead of GLM The same treatment as in Example 1 was performed to prepare a DEAE-modified separation material, and evaluation was performed in the same manner as in Example 1.

[Comparative Example 1]
Except for performing the step of introducing an ATRP initiating group and the step of introducing an ion exchange group without performing the grafting step of the hydrophilic monomer, the same treatment as in Example 1 was performed to produce a DEAE-modified separation material, Evaluation was performed in the same manner as in Example 1.

[Comparative Example 2]
Except that phenyl methacrylate (PMA) was used instead of GLM in the hydrophilic monomer grafting process, the same treatment as in Example 1 was performed to prepare a DEAE-modified separation material, and evaluation was performed in the same manner as in Example 1. went.

[Comparative Example 3]
Evaluation was performed in the same manner as in Example 1 using commercially available agarose particles (manufactured by GE Healthcare, trade name “Capto DEAE”) as a separating material.

  It was found that the separation material of the example had little nonspecific adsorption, a high liquid passing speed at 0.3 MPa, and a high dynamic adsorption amount. Moreover, it has confirmed that the amount of amino groups and the amount of dynamic adsorption improved by the separation material of an Example having the graft chain based on a hydrophilic monomer. Furthermore, it was found that the separators of the examples were sufficiently excellent in alkali resistance and durability.

Claims (13)

A porous polymer particle, and a coating layer covering at least a part of the surface of the porous polymer particle,
The separation material, wherein the coating layer includes a crosslinked polymer having a hydroxyl group, and a hydrophilic monomer is grafted to the crosslinked polymer.   The separating material according to claim 1, wherein the hydrophilic monomer is a (meth) acrylate compound having a hydroxyl group.   The separation material according to claim 1 or 2, wherein the porous polymer particles include a polymer having a structural unit derived from a styrene-based monomer.   The separation material according to any one of claims 1 to 3, wherein the crosslinked polymer having a hydroxyl group is a crosslinked polymer derived from a polysaccharide or a modified product thereof.   The separating material according to any one of claims 1 to 4, wherein the crosslinked polymer having a hydroxyl group is a crosslinked polymer derived from agarose, dextran, or pullulan.   The separation material according to any one of claims 1 to 5, having an average particle diameter of 10 to 300 µm and a mode pore diameter of 0.05 to 0.6 µm.   The separation material according to any one of claims 1 to 6, wherein the porosity is 40 to 70% by volume. The separation material according to claim 1, wherein the specific surface area is 20 m ² / g or more.   The separation material according to any one of claims 1 to 8, wherein a coefficient of variation in particle diameter is 5 to 15%.   The separation material according to any one of claims 1 to 9, further comprising a coating layer containing a crosslinked polymer having 30 to 500 mg of the hydroxyl group per 1 g of the porous polymer particles.   The water flow rate is 800 cm / h or more when water is passed through the column filled with the separation material so that the pressure of the column is 0.3 MPa. The separation material according to claim 1.   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 11,
Adsorbing a polymer having a hydroxyl group on the surface of the porous polymer particles, and crosslinking the polymer;
Introducing an initiation group for atom transfer radical polymerization into the crosslinked polymer;
Grafting a hydrophilic monomer to the initiating group.