JP2017125811A - Separation material and column - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a separation material with satisfactory liquid permeability capable of improving protein absorption while reducing non specific adsorption.SOLUTION: The separation material includes: porous polymer particle; and a coating layer covering at least a part of the surface of the porous polymer particle. The coating layer contains a high polymer which has a hydroxyl group. The separation material contains at least one component A of an organic solvent which has an HLB value of 1 to 9 or an oil-soluble surfactant which has an HLB value of 1 to 9. Before and after immersing the separating material in a washing solution containing acetonitrile for 24 hours, the amount of change in the content of the component A in the cleaning solution is 5000 ppm or less per 1 g of the separation material.SELECTED DRAWING: None

Description

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

  Conventionally, when separating and purifying biopolymers typified by proteins, generally, porous particles based on synthetic polymers and particles based on cross-linked gels of hydrophilic natural polymers are used. . The ion exchanger based on the above-mentioned porous type synthetic polymer has the advantage that the volume change due to the salt concentration is small, and when packed in a column and used for chromatography, the pressure resistance during passage is good. Yes. However, when this ion exchanger is used for separation of proteins and the like, non-specific adsorption such as irreversible adsorption based on hydrophobic interaction occurs, resulting in the problem of peak asymmetry, or the hydrophobic interaction However, there is a problem that the protein adsorbed on the ion exchanger may not be recovered while adsorbed.

  On the other hand, the above-mentioned 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 nonspecific 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 disadvantages of the hydrophilic natural polymer cross-linked gel, attempts have been made so far to combine it with a rigid substance that becomes a “skeleton”.

  For example, in Patent Document 1, there is a description that a complex in which a gel such as a natural polymer gel is held in pores of a porous polymer is used in the field of peptide synthesis. The effect of the invention is that it can be synthesized at a high yield.

  Moreover, in this Patent Document 1, since the gel is surrounded by a hard synthetic polymer substance, there is no effect that even if it is used in the form of a column bed, there is no volume change and the flow-through pressure passing through the column does not change. Are listed.

  In Patent Documents 2 and 3, an inorganic porous material such as celite is held with a xerogel such as polysaccharides such as dextran and cellulose, and a diethylaminomethyl (DEAE) group or the like is added to the gel to add sorption performance. Is used to remove hemoglobin. As its effect, liquid permeability in the column is mentioned.

  Patent Document 4 discloses an ion exchanger of a hybrid copolymer in which pores of a so-called macro network-structured copolymer are filled with a crosslinked copolymer gel synthesized from a monomer. In Patent Document 4, when the degree of cross-linking of the gel of the cross-linked copolymer is low, there are problems such as pressure loss, volume change, etc., but by using a hybrid copolymer, the liquid flow characteristics are improved and the pressure loss is reduced. It is also described that the ion exchange capacity is improved and the leakage behavior is improved.

  Patent Documents 5 and 6 propose a composite filler in which pores of an organic synthetic polymer substrate are filled with a crosslinked gel of a hydrophilic natural polymer having a huge network structure.

  In Patent Document 7, porous particles composed of glycidyl methacrylate and an acrylic crosslinked monomer are synthesized.

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

  With conventional column packing materials, it is difficult to solve liquid permeability, which is a problem with natural polymers, and protein adsorption amount and nonspecific adsorption, which are problems with polymer particles, at a sufficient level.

  Therefore, an object of the present invention is to provide a separation material capable of ensuring liquid permeability, improving the amount of protein adsorption and reducing nonspecific adsorption, and a column using the separation material.

The present invention provides the separation material and column described in the following [1] to [10].
[1] A separation material comprising porous polymer particles and a coating layer covering at least a part of the surface of the porous polymer particles, wherein the coating layer contains a polymer having a hydroxyl group, and the separation The material contains at least one component A of an organic solvent having an HLB value of 1 to 9 and an oil-soluble surfactant having an HLB value of 1 to 9, and before and after immersing the separating material in a cleaning liquid containing acetonitrile for 24 hours, The separating material, wherein the amount of change in the content of the component A in the cleaning liquid is 5000 ppm or less per 1 g of the separating material.
[2] The separation material according to [1], wherein the porous polymer particles include a polymer obtained by polymerizing a monomer containing 50% by mass or more of divinylbenzene.
[3] The separation material according to [1] or [2], wherein the specific surface area is 30 m ² / g or more.
[4] The separation material according to any one of [1] to [3], wherein the mode diameter of the pore size distribution is 0.05 to 0.6 μm.
[5] The separation material according to any one of [1] to [4], wherein the porous polymer particles have an average particle size of 10 to 300 μm.
[6] The separating material according to any one of [1] to [5], wherein the polymer having a hydroxyl group is a polysaccharide or a modified product thereof.
[7] The separation material according to any one of [1] to [6], wherein the polymer having a hydroxyl group is agarose or a modified product thereof.
[8] The separating material according to any one of [1] to [7], comprising 30 to 400 mg of the coating layer per gram of the porous polymer particles.
[9] The separation material according to any one of [1] to [8], wherein when the column is packed, the liquid flow rate is 800 cm / h or more when the column pressure is 0.3 MPa.
[10] A column comprising the separation material according to any one of [1] to [9].

  ADVANTAGE OF THE INVENTION According to this invention, liquid permeability can be ensured, and the separation material which can improve a protein adsorption amount and can reduce nonspecific adsorption, and the column using this separation material can be provided.

  Hereinafter, although embodiment of this invention is described, this invention is not limited to the following embodiment.

  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, and the coating layer includes a polymer having a hydroxyl group. 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. For example, a polymer having a hydroxyl group may be coated on the outer surface of the porous polymer particles and / or the surface of the pores inside the porous polymer particles.

  According to this embodiment, while ensuring liquid permeability, the amount of protein adsorption can be improved and nonspecific adsorption can be reduced. Further, according to the present embodiment, non-specific adsorption due to hydrophobic interaction is achieved while maintaining excellent separation ability for separation of biopolymers such as proteins, which the filler based on a hydrophilic natural polymer has. In addition, a column packing material that separates and purifies biopolymers by electrostatic interaction or affinity purification can be provided.

(Porous polymer particles)
The porous polymer particles of the present embodiment include, for example, a polymer obtained by polymerizing a monomer and can have a structural unit derived from the monomer. The porous polymer particles may contain 50% by mass or more of the polymer based on the total mass of the particles, or may be particles made of a polymer. The porous polymer particles can be obtained by polymerizing a composition containing a monomer and a porosifying agent and then removing the porosifying agent. The porous polymer particles can be synthesized, for example, by conventional suspension polymerization or emulsion polymerization. Although it does not specifically limit as a monomer, For example, vinyl monomers, such as a (meth) acrylic-type monomer and a styrene-type monomer, can be used. Specific examples of the monomer include the following polyfunctional monomers and monofunctional monomers.

  Examples of the multifunctional monomer include divinyl compounds such as divinylbenzene, divinylbiphenyl, divinylnaphthalene, divinylphenanthrene; (poly) ethylene glycol di (meth) acrylate, (poly) propylene glycol di (meth) acrylate, (poly) (Poly) alkylene glycol di (meth) acrylates such as tetramethylene glycol di (meth) acrylate; trimethylolpropane tri (meth) acrylate, tetramethylolmethane tri (meth) acrylate, tetramethylolpropane tetra (meth) acrylate, penta Erythritol tri (meth) acrylate, 1,1,1-trishydroxymethylethane tri (meth) acrylate, 1,1,1-trishydroxymethylpropane triacrylate, etc. Tri- or higher functional (meth) acrylate; ethoxylated bisphenol A di (meth) acrylate, propoxylated ethoxylated bisphenol A di (meth) acrylate, tricyclodecane dimethanol di (meth) acrylate, 1,1,1- Examples include di (meth) acrylates such as trishydroxymethylethane di (meth) acrylate and ethoxylated cyclohexanedimethanol di (meth) acrylate; diallyl phthalate and isomers thereof; triallyl isocyanurate and derivatives thereof. Examples of the multifunctional monomer include NK ester (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, etc.), Nippon Steel & Sumikin Chemical Co., Ltd. divinylbenzene (DVB960) and the like are commercially available. A polyfunctional monomer can be used individually by 1 type or in combination of 2 or more types.

  Among these, divinylbenzene is preferably used from the viewpoint of excellent durability and swelling properties. When the monomer contains divinylbenzene, the content of divinylbenzene is preferably 50% by mass or more, more preferably 60% by mass or more, and more preferably 70% by mass or more based on the total mass of the monomer, from the viewpoint of excellent alkali resistance and pressure resistance. Is more preferable.

  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 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) acrylic acid esters such as isobutyl acid, 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, tetrafluoroethylene Emissions, hexafluoropropylene, trifluoroethyl acrylate, containing fluorinated monomers such as acrylic acid tetrafluoropropyl; butadiene include conjugated dienes such as isoprene. A monofunctional monomer can be used individually by 1 type or in combination of 2 or more types. Among these, styrene is preferably used from the viewpoint of excellent acid resistance and alkali resistance. Moreover, the styrene derivative which has functional groups, such as a carboxy group, an amino group, a hydroxyl group, and an aldehyde group, can also be used.

  The porosifying agent is an additive that promotes phase separation at the time of polymerization and promotes the porosity of particles. Examples of the porosifying agent include a solvent (such as an organic solvent) and an oil-soluble surfactant (emulsifier).

  Examples of the solvent include alcohol and fatty acid. Examples of the alcohol include 2-butanol, tert-butyl alcohol, hexanol (for example, 1-hexanol), ethyl hexanol, heptanol, octanol (for example, 2-octanol), decanol, dodecanol, isoamyl alcohol, lauryl alcohol, cyclohexanol, dimethyl phthalate, Examples include diethylene glycol monobutyl alcohol, heptylene glycol, dipropylene glycol, and triethylene glycol. Examples of fatty acids include butanoic acid, hexanoic acid, heptanoic acid, octanoic acid, decanoic acid, dodecanoic acid, lauric acid and the like. As the solvent, an organic solvent having an HLB value of 1 to 9 can be used.

  Examples of the oil-soluble surfactant include polyoxyethylene alkyl ethers; branched C16 to C24 (carbon number 16 to 24, the same applies to the following “C”) fatty acids, chain unsaturated C16 to C22 fatty acids, or chain saturated C12 to C18. Sorbitan monoesters of fatty acids (eg sorbitan monolaurate, sorbitan monooleate, sorbitan monomyristate, sorbitan monopalmitate, sorbitan monostearate or sorbitan monoesters derived from coconut fatty acid); branched C16-C24 Sorbitan diesters of fatty acids, chain unsaturated C16-C22 fatty acids or chain saturated C12-C18 fatty acids (eg sorbitan distearate); branched C16-C24 fatty acids, chain unsaturated C16-C22 fatty acids or chain saturated C12- C18 fatty acid propylene glycol Cole monoesters (eg propylene glycol monolaurate); glycerol monoesters of branched C16-C24 fatty acids, chain unsaturated C16-C22 fatty acids or chain saturated C12-C14 fatty acids (eg glycerol monooleate or glycerol Monostearate); diglycerol monoesters of branched C16-C24 fatty acids, chain unsaturated C16-C22 fatty acids or chain saturated C12-C14 fatty acids (eg diglycerol monooleate (eg C18: 1 (carbon number 18) , Diglycerol monoester of fatty acid), diglycerol monomyristate, diglycerol monoisostearate, or diglycerol monoester of coconut fatty acid); branched C16-C24 alcohol (eg, gel bear) Lumpur), chain unsaturated C16~C22 alcohol or chain saturated C12~C14 alcohols (e.g., diglycerol mono-fatty ethers of coconut fatty alcohols); and, mixtures thereof. As the oil-soluble surfactant, an oil-soluble surfactant having an HLB value of 1 to 9 can be used.

  Preferred oil-soluble surfactants include sorbitan monolaurate (eg, SPAN® 20, preferably greater than about 40% purity, more preferably greater than about 50% purity, most preferably about 70% purity). Sorbitan monooleate (eg, SPAN® 80. preferably greater than about 40% purity, more preferably greater than about 50% purity, most preferably greater than about 70% purity) Diglycerol monooleate (e.g., preferably diglycerol monooleate, preferably greater than about 40%, more preferably greater than about 50%, and most preferably greater than about 70%); diglycerol Monoisostearate (eg, preferably greater than about 40% purity, more Preferably greater than about 50% purity, most preferably greater than about 70% purity diglycerol monoisostearate); diglycerol monomyristate (preferably greater than about 40% purity, more preferably about 50% purity). Sorbitan monomyristate with greater than about 70% purity); cocoyl (eg, lauryl, myristoyl, etc.) ethers of diglycerol; and mixtures thereof.

  The solvent which is a porous agent can be used in the range of 0 to 200% by mass based on the total mass of the monomer. The oil-soluble surfactant that is a porosifying agent can be used in the range of 5 to 80% by mass based on the total mass of the monomer. When the content of the oil-soluble surfactant is 5% by mass or more, the stability of the water droplets is sufficient, so that a large single hole is easily formed. 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.

  A porous agent can be used individually by 1 type or in combination of 2 or more types. 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 depending on the kind of the porous agent. When a substance that easily absorbs water is used as a porosifying agent, large pores are easily formed inside the particles. Moreover, the water used as a solvent can also be used as a porous agent. When water is used as a porosifying agent, the oil-soluble surfactant is dissolved in the monomer, so that water can be absorbed and the particles can be easily made porous.

  The porous agent as described above needs to be removed by washing with a solvent capable of dissolving the porous agent after the synthesis of the porous polymer particles. In particular, non-specific adsorption occurs when an organic solvent having an HLB value (Hydrophile-Lipophile Banlance value) of 1 to 9 or an oil-soluble surfactant having an HLB value of 1 to 9 remains on the particles (for example, the particle surface). Therefore, it is preferable to remove these porosifying agents by washing with Soxhlet or the like.

  From such a viewpoint, the separation material of the present embodiment contains at least one component A of an organic solvent having an HLB value of 1 to 9 and an oil-soluble surfactant having an HLB value of 1 to 9, and contains acetonitrile (for example, The amount of change in the content of component A in the cleaning liquid is 5000 ppm or less per 1 g of the separating material before and after immersing the separating material in acetonitrile for 24 hours. In this case, the occurrence of non-specific adsorption can be suppressed, the amount of protein adsorption can be improved and non-specific adsorption can be reduced while ensuring liquid permeability. When the amount of change exceeds 5000 ppm, non-specific adsorption is likely to occur because an excessive amount of the porous agent remains on the particles (for example, the particle surface). The amount of change in the content of the porous agent is, for example, the amount of elution of the porous agent that is eluted from the separation material into the cleaning liquid containing acetonitrile. In the separation material of the present embodiment, it is sufficient that at least one kind of the component A satisfies the amount of change. The remaining amount of the porosifying agent can be determined by immersing the separating material in a cleaning solution containing acetonitrile for 24 hours and measuring it with liquid chromatography or the like.

  The amount of the cleaning liquid containing acetonitrile is, for example, 10 g with respect to 1 g of the separation material. The temperature at which the separation material is immersed is, for example, not more than the boiling point (82 ° C.) of acetonitrile (for example, 25 ° C.). You may stir the liquid containing a separating material during immersion. Examples of the stirring means include a three-one motor, a stirrer, a mix rotor, and the like. The stirring speed is, for example, 500 rpm or less.

  The upper limit of the amount of change in the content of Component A is preferably 4500 ppm or less, more preferably 4000 ppm or less, further preferably 3000 ppm or less, and particularly preferably less than 3000 ppm from the viewpoint of further suppressing the occurrence of nonspecific adsorption. 10 ppm may be sufficient as the minimum of the variation | change_quantity of content of the said component A, for example.

  Examples of the organic solvent having an HLB value of 1 to 9 include C16 or lower alkyl alcohol and C16 or lower fatty acid. Examples of oil-soluble surfactants having an HLB value of 1 to 9 include polyoxyethylene alkyl ether, sorbitan monolaurate, sorbitan monooleate, sorbitan monopalmitate, sorbitan monostearate, propylene glycol monolaurate, sorbitan distea Rate, glycerol monostearate, glycerol monooleate and the like.

  The composition for obtaining porous polymer particles may contain soluble particles as a porosifying agent. The soluble particles are particles that can be dissolved in an acid, an alkali, a solvent, or the like, for example. The soluble particles are not dissolved during the polymerization, and can be removed by a method of dissolving the powder porosifying agent by immersing the polymer particles in an acid solution after the particles are formed. Specific examples of the constituent material of the soluble particles include calcium carbonate, tricalcium phosphate, silica, polymer, metal colloid, and the like. As a constituent material of the soluble particles, calcium carbonate, tricalcium phosphate, or the like is preferably used from the viewpoint of easy removal. The particle size of the soluble particles is preferably 0.6 to 5 μm. The particle diameter of the soluble particles is more preferably 1 to 5 μm from the viewpoint of further improving the liquid permeability in the separation material. In addition, the average particle diameter of a soluble particle can be measured by the method similar to the measuring method of the average particle diameter of the below-mentioned porous polymer particle.

  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 anionic surfactants 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; alkylnaphthalenesulfone 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 alkylphenyl ether sulfate Salts; polyoxyethylene alkyl ether sulfates such as sodium polyoxyethylene lauryl ether sulfate; polyoxyethylene alkyl sulfate esters.

  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 silicon polyethylene oxide adducts 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 excellent 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 type of monomer and polymerization initiator. The polymerization temperature is preferably 25 to 110 ° C, more preferably 50 to 100 ° C.

  In the synthesis (polymerization step) of the porous polymer particles, a polymer dispersion stabilizer may be used in order to improve the dispersion stability of the particles, and a polymer dispersion stabilizer may be added to the emulsion.

  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 polymerization of the monomer alone (for example, to suppress the generation of particles obtained by emulsion polymerization of the monomer alone in water), nitrites, sulfites, hydroquinones, ascorbic acids, water-soluble vitamin Bs, Water-soluble polymerization inhibitors such as 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, from the viewpoint of further improving the liquid permeability and suppressing an increase in the column pressure after column filling. More preferably, it is 50 μm or more.

  The pore volume (porosity) of the porous polymer particles is preferably 30 to 70% by volume, more preferably 40 to 70% by volume, and more preferably 50 to 70% by volume based on the total volume of the porous polymer particles. % Is more preferable. The porous polymer particles preferably have pores having a pore diameter (mode diameter) of 0.01 μm or more and less than 0.6 μm, that is, macropores (macropores). The pore diameter is more preferably 0.02 μm or more and less than 0.6 μm. If the pore diameter is 0.01 μm or more, the substance tends to easily enter the pores. If the pore diameter is less than 0.6 μm, the specific surface area is sufficient. These can be adjusted by the above-mentioned porous agent.

  The coefficient of variation (C.V.) of the particle size of the porous polymer particles is preferably 3 to 15%, more preferably 3 to 12%, further preferably 3 to 10%, from the viewpoint of further improving liquid permeability. preferable. C. V. As a method for reducing the above, monodispersion may be performed by an emulsification apparatus such as a microprocess server (for example, manufactured by Hitachi, Ltd.).

The specific surface area of the porous polymer particles is preferably 30 m ² / g or more. There exists a tendency for the adsorption amount of the substance to isolate | separate that the specific surface area is 30 m < ² > / g or more to become large. The specific surface area of the porous polymer particles, from the viewpoint of more highly practical, more preferably at least ^35m 2 / g, more ^{40 m} 2 / g is more preferred.

(Coating layer)
The coating layer of the present embodiment includes a polymer having a hydroxyl group (for example, a water-soluble polymer). By covering the porous polymer particles with a polymer having a hydroxyl group, an increase in the column pressure can be suppressed, and non-specific adsorption of the protein can be suppressed, and the protein adsorption amount of the separation material can be reduced. It can be equal to or more than when natural polymers are used. Furthermore, when the polymer having a hydroxyl group is cross-linked, it is possible to further suppress an increase in column pressure.

  The polymer having a hydroxyl group preferably has two or more hydroxyl groups in one molecule. The polymer having a hydroxyl group is preferably a hydrophilic polymer. Examples of the polymer having a hydroxyl group include polysaccharides (agarose, dextran, cellulose, polyvinyl alcohol, chitosan, etc.), and those having a weight average molecular weight of about 10,000 to 200,000 can be used.

  As the polymer having a hydroxyl group, a modified product modified with a hydrophobic group (such as a modified product having a hydrophobic group introduced) can be used 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 can be introduced by reacting a functional group that reacts with a hydroxyl group (such as an epoxy group) and a compound having a hydrophobic group (such as glycidyl phenyl ether) with a polymer having a hydroxyl group by a conventionally known method. Examples of such a modified body include a modified body of a polysaccharide. Specifically, a modified body of agarose (modified agarose), a modified body of dextran, a modified body of cellulose, a modified body of polyvinyl alcohol, chitosan. And the like.

[Method for forming coating layer]
The coating layer containing a polymer having a hydroxyl group can be formed by, for example, the following method. The coating layer can be formed by coating porous polymer particles with a polymer having a hydroxyl group. As a method for forming the coating layer, for example, there is a method in which a polymer solution having a hydroxyl group is adsorbed on the surface of the porous polymer particles, the unadsorbed portion is removed, and then a crosslinking reaction is performed with a crosslinking agent to be supported in the pores. Can be mentioned. 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. Examples of the impregnation method include a method in which porous polymer particles are added to a polymer solution having a hydroxyl group and left for a predetermined time. Although the impregnation time varies depending on the surface state of the porous polymer particles, the polymer concentration is in equilibrium with the external concentration inside the porous polymer particles if it is usually impregnated overnight. Then, it wash | cleans with solvents, such as water and alcohol, and removes the unadsorbed part of the polymer which has a hydroxyl group.

[Crosslinking treatment]
Subsequently, a crosslinking agent is added to cause a polymer having a hydroxyl group adsorbed on the surface of the porous polymer particles to undergo a crosslinking reaction to form a crosslinked body. For example, a crosslinking agent is added to cause a polymer having a hydroxyl group adsorbed on the surface of the porous polymer particles to undergo a crosslinking reaction to form a polymer crosslinked gel. At this time, in the crosslinked body, for example, a polymer having a hydroxyl group has a three-dimensional crosslinked network structure.

  Examples of the crosslinking agent include hydroxyl groups (OH groups) such as epihalohydrins such as epichlorohydrin, dialdehyde compounds such as glutaraldehyde, diisocyanate compounds such as methylene diisocyanate, and glycidyl compounds such as ethylene glycol diglycidyl ether. Examples thereof include compounds having two or more active functional groups. 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.

  For this crosslinking reaction, a catalyst is usually used. The catalyst varies depending on 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. In the case of a dialdehyde compound, a mineral acid such as hydrochloric acid is effective.

  The crosslinking reaction with the crosslinking agent can be usually performed by adding the crosslinking agent to a system in which the separating material before crosslinking is dispersed and suspended in an appropriate medium. When a polysaccharide or a modified product thereof is used as a polymer having a hydroxyl group, the addition amount of the crosslinking agent is within a range of 0.1 to 100 mol times with respect to 1 mol of a monosaccharide. It can be selected according to the performance of the target separation material. Generally, when the addition amount of the crosslinking agent is less than 0.1 mol times, the coating layer tends to be easily peeled off from the porous polymer particles. Moreover, when the addition amount of a crosslinking agent exceeds 100 mol times 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 in the crosslinking reaction depends on the type of the crosslinking agent. Usually, when a polysaccharide is used as the polymer having a hydroxyl group, 1 mol of one unit of the monosaccharide forming the polysaccharide is used. Then, it is preferably used in the range of 0.01 to 10 mole times, more preferably in the range of 0.1 to 5 mole 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.

  Specific examples of the medium for dispersing and suspending the porous polymer particles adsorbed with a polymer solution having a hydroxyl group include a cross-linking reaction without extracting the adsorbed polymer, a cross-linking agent and the like. There is no limitation as long as it is inactive. Specific examples of such a medium include water and alcohol.

  The crosslinking reaction is usually carried out at a temperature in the range of 5 to 90 ° C. for 1 to 24 hours. Preferably, the temperature is in the range of 30 to 90 ° C.

  After completion of the crosslinking reaction, the produced composite of porous polymer particles (porous body) and the crosslinked gel is filtered off, then washed with water, a hydrophilic organic solvent (methanol, ethanol, etc.), etc., and unreacted. By removing the polymer, the suspending medium, etc., a separating material in which at least a part of the surface of the porous polymer particles is coated with a coating layer containing a polymer having a hydroxyl group can be obtained. The separating material of the present embodiment preferably includes a coating layer of 30 to 400 mg per 1 g of porous polymer particles. The amount of the coating layer can be measured by reducing the weight of pyrolysis or the like.

[Introduction of ion exchange groups]
The separation material having a coating layer can be used for ion exchange purification, affinity purification, and the like by introducing ion exchange groups, ligands (protein A), and the like through hydroxyl groups on the particle surface. Examples of the method for introducing an ion exchange group include a method using a halogenated alkyl compound (halogenated alkyl group-containing 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; Examples thereof include quaternary ammonium hydrochloride having a halogenated alkyl group. As the halogenated alkyl compound, bromide or chloride is preferred. The amount of the halogenated alkyl compound used is preferably 0.2% by mass or more based on the total mass of the separation material for introducing the ion exchange group (separation material before introducing the ion exchange group).

  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.

  Usually, since the introduction of the ion exchange group is performed on the hydroxyl group on the surface of the separation material, the wet particles are drained by filtration or the like, then immersed in an alkaline aqueous solution of a predetermined concentration, left for a certain period of time, water, or In the water-organic solvent mixed system, the halogenated alkyl compound is added and reacted. As a method for introducing an ion exchange group, generally, there is a method in which a hydrophilic natural polymer is dissolved in an aqueous sodium hydroxide solution and reacted with a halogenated alkyl compound in water or a water-organic solvent mixed system. It is done. The amount of the halogenated alkyl compound used is, for example, 0.2% by mass or more based on the total mass of the hydrophilic natural polymer. This reaction is preferably performed at a temperature of 40 to 90 ° C. under reflux for 0.5 to 12 hours. Depending on the type of the halogenated alkyl compound used in the above reaction, the ion exchange group provided is determined.

  As a method for introducing an amino group which is a weakly basic group as an ion exchange group, among the above halogenated alkyl compounds, mono-, di- or tri-alkylamino chloride, mono-, di- or tri-alkanol amino chloride And a method of reacting a secondary or tertiary amino halogenide such as mono (or di-) alkyl-mono (or di-) alkanolamino chloride. The amount of these amines used is, for example, 0.2% by mass or more based on the total mass of the separating material into which the ion exchange group is introduced. The reaction conditions are, for example, 40 to 90 ° C. and 0.5 to 12 hours.

  As a method for introducing a quaternary ammonium group which is a strongly basic group as an ion exchange group, first, a tertiary amino group is introduced, and a halogenated alkyl compound such as epichlorohydrin is reacted with the tertiary amino group. And a method of converting it to a quaternary ammonium group. Alternatively, a quaternary amino halide such as quaternary ammonium chloride may be reacted with a separating material (composite) in the same manner as the above-described primary to tertiary amino chlorides.

  Examples of the method for introducing a weakly acidic carboxy 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 these halogenated alkyl compounds used is, for example, 0.2% by mass or more based on the total mass of the separating material into which ion exchange groups are 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. And a method of adding a separating material to the saturated aqueous solution. The reaction conditions are, for example, 30 to 90 ° C. and 1 to 10 hours.

  As another method for introducing an ion exchange group, there may be mentioned a method in which 1,3-propane sultone is reacted with a separating material in an alkaline atmosphere. For example, 1,3-propane sultone is used in an amount of 0.4% by mass or more based on the total mass of the separation material into which the ion exchange group is introduced. The reaction conditions are, for example, 0 to 90 ° C. and 0.5 to 12 hours.

  Examples of methods other than these methods for introducing ion-exchange groups include a method of reacting sulfopropyl and a method of introducing an ion-exchange group into a glycidyl group after adding an epihalohydrin diglycidyl compound or the like. In general, there is a method in which a hydrophilic natural polymer is dissolved in an aqueous sodium hydroxide solution and reacted with a halogenated alkyl group-containing compound in water or a water-organic solvent mixed system. The amount of the halogenated alkyl group-containing compound used is, for example, 0.2% by mass or more based on the total mass of the hydrophilic natural polymer, and this reaction is carried out at a temperature of 40 to 90 ° C. under reflux, 0.5 to It is preferably performed for 12 hours.

  1-30 mass% is preferable, as for the moisture absorption of the separating material of this embodiment, 1-20 mass% is more preferable, and 1-10 mass% is further more preferable. It can further suppress that the liquid permeability of a separating material falls by the thickness of a coating layer as moisture absorption is 30 mass% or less.

  The mode diameter (mode of pore diameter distribution, maximum frequency diameter) of the pore diameter distribution of the separation material of the present embodiment is preferably 0.05 to 0.6 μm, and preferably 0.1 to 0.5 μm. It is more preferable. When the mode diameter of the pore size distribution is within this range, the liquid easily flows in the particles, and the amount of dynamic adsorption is easily increased.

The specific surface area of the separating material of the present embodiment 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, there is a tendency that the adsorption amount of the substance to be separated tends to increase. Although the upper limit of the specific surface area of a separating material is not specifically limited, For example, it can be 300 m < ² > / g or less.

  The pore volume, pore diameter (mode diameter), and specific surface area of the separation material or porous polymer particle of the present embodiment are measured as follows, for example, using a mercury intrusion measuring apparatus (Autopore: manufactured by Shimadzu Corporation). Can be measured. 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 degrees and a mercury surface tension of 485 dynes / cm. Further, each value is calculated by limiting to a pore diameter range of 0.05 to 5 μm.

C. of the average particle diameter and particle diameter of the separating material or porous polymer particle of this embodiment. V. (Coefficient of variation) can be determined by the following measurement method.
1) Using an ultrasonic dispersion device, particles are dispersed in water (including a dispersant such as a surfactant) to prepare a dispersion containing 1% by mass of particles.
2) Using a particle size distribution meter (Sysmex Flow FPIA-3000, manufactured by Sysmex Corporation), an average particle size and a particle size of C.I. V. (Coefficient of variation) is measured.

  The average particle size of the separation material of the present embodiment is 10 from the viewpoint of avoiding an extreme increase in the internal pressure of the column (for example, from the viewpoint of avoiding an extreme increase in the internal pressure of the column in use in preparative or industrial chromatography). ˜300 μm is preferred.

  The 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 separation material of this embodiment is suitable for use in separation of proteins by electrostatic interaction, affinity purification, and the like. For example, protein can be recovered by the following method using a separation material (ion exchanger) into which an ion exchange group has been introduced. First, the separation material of this embodiment is added to the mixed solution containing protein, and after adsorbing only the protein to the separation material by electrostatic interaction, the separation material is filtered from the solution, and the salt concentration is high. If added to an aqueous solution, the protein adsorbed on the separating material can be easily desorbed and recovered. Moreover, the separation material of this embodiment can also be used in column chromatography as a separation material for liquid chromatography, for example. The column of this embodiment includes the separation material of this embodiment, and is formed by, for example, filling the separation material of this embodiment.

  The biopolymer that can be separated using the separation material of the present embodiment is preferably a water-soluble substance. Specifically, proteins such as serum albumin (BSA: Bovine Serum Albumin) and blood proteins such as immunoglobulins; enzymes present in living bodies; protein physiologically active substances produced by biotechnology; DNA; peptides having physiological activity And other biopolymers. The molecular weight of the biopolymer is preferably 2 million or less, and more preferably 500,000 or less. Further, according to a known method, the properties, conditions, etc. of the separation material can be selected according to the isoelectric point, ionization state, etc. of the protein. Examples of known methods include the methods described in JP-A-60-169427.

  Separation of biopolymers such as proteins by coating porous polymer particles with a crosslinked polymer of a hydroxyl group and then introducing ion exchange groups, protein A, etc. into the particle surface and / or pores However, it is easy to show the characteristics having the advantages of natural polymer and polymer particles. This performance has not been demonstrated by conventional techniques. In particular, since the porous polymer particles that serve as the skeleton of the separation material of the present embodiment are particles obtained by the above method, they are excellent in durability and alkali resistance. Further, by coating a crosslinked polymer having a hydroxyl group, non-specific adsorption is less likely to occur, and protein desorption tends to occur. Furthermore, the separation material of the present embodiment has a preferable property compared to the conventional ion exchange resin in that the adsorption capacity (dynamic adsorption capacity) of protein or the like under the same flow rate is large.

  In the present specification, the “liquid passing speed” represents a liquid passing speed when the separation material of this embodiment is filled in a stainless steel column of φ7.8 × 300 mm and the liquid is allowed to flow. When the separation material of this embodiment is packed in a column, the liquid flow rate (flow rate) when the column pressure is 0.3 MPa is preferably 800 cm / h or more. When separating proteins by column chromatography, the flow rate of a protein solution or the like that is passed through the column is generally in the range of 400 cm / h or less, but when the separation material of this embodiment is used Can be used with a high adsorption capacity even at a liquid flow rate of 800 cm / h or higher, which is faster than a normal separation material for protein separation.

  When used as a column packing material in column chromatography, the separation material of this embodiment is excellent in operability because there is little volume change in the column regardless of the properties of the eluate used.

  In addition, although this embodiment mainly 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.

<Example 1>
(Synthesis of porous polymer particle 1)
In a 500 mL three-necked flask, 10.7 g of divinylbenzene (DVB960, Nippon Steel & Sumikin Chemical Co., Ltd.) with a purity of 96% by mass and 5.3 g of styrene, 14.4 g of toluene and 21.6 g of dodecanol as a porosifying agent Then, 0.64 g of benzoyl peroxide as an initiator was added to an aqueous polyvinyl alcohol solution (0.5% by mass) as a dispersion stabilizer, and emulsified 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. to obtain particles. The obtained particles were filtered and washed with water. Furthermore, acetone washing was performed with a Soxhlet extraction apparatus for 8 hours to obtain porous polymer particles 1. The particle size of the obtained particles was measured with a flow-type particle size measuring device, and the average particle size and the C. V. The value was calculated. The results are shown in Table 1.

(Formation of coating layer: Coating with water-soluble polymer having hydroxyl group)
To 100 mL of an agarose aqueous solution (2% by mass), 4 g of sodium hydroxide and 0.14 g of glycidyl phenyl ether were added and reacted at 70 ° C. for 12 hours to introduce a phenyl group into the agarose. The obtained modified agarose was precipitated with isopropyl alcohol and washed.

  The porous polymer particle 1 is adsorbed to the porous polymer particle 1 by charging the porous polymer particle 1 with a content of 10 g in 400 mL of a 20 mg / mL modified agarose aqueous solution and stirring at 55 ° C. for 24 hours. A coating layer was formed on the surface. Then, it filtered and wash | cleaned with hot water.

  The modified agarose adsorbed on the particles was crosslinked as follows. Particles were added to an aqueous solution containing ethylene glycol diglycidyl ether 0.64M and sodium hydroxide 0.4M at a ratio of 1 g of particles to 35 mL of the aqueous solution, and stirred at room temperature for 24 hours. Then, it wash | cleaned with the pure water after wash | cleaning with the heated 2 mass% sodium dodecyl sulfate aqueous solution.

  Particles (dry mass 20 g) obtained by filtering the aqueous suspension containing the particles obtained above were put into 200 mL of 5M aqueous sodium hydroxide solution and allowed to stand at room temperature for 1 hour. Water was added to a predetermined amount (60 g) of diethylaminoethyl chloride hydrochloride to obtain 200 g of an aqueous solution. The temperature of the aqueous solution was raised to 70 ° C. and reacted for 2 hours with stirring. After completion of the reaction, the product was filtered off and washed with water to obtain a separating material (DEAE-modified separating material) having diethylaminoethyl (DEAE) groups as ion exchange groups. The pore diameter (mode diameter) and specific surface area of the obtained DEAE-modified separation material were measured by a mercury intrusion method. Further, after the DEAE-modified separation material was dried, the coating amount of the coating layer (the coating amount of the water-soluble polymer) was quantified by thermogravimetric analysis. The results are shown in Table 2.

(Measurement of elution amount of porous agent)
After putting 5 g of DEAE-modified separating material into 50 g of acetonitrile at 25 ° C., the mixture was stirred for 24 hours at 100 rpm using a mix rotor. Next, by using liquid chromatography, the eluate (porosifying agent) eluted in acetonitrile is detected, and the content of eluate in acetonitrile (elution amount. Amount per 1 g of separation material (particles)). Was measured. In addition, before immersing the separating material, it was confirmed that acetonitrile did not contain the porous agent.

(Evaluation of nonspecific adsorption ability of protein)
0.5 g of the obtained particles (separating material) was put into 50 mL of a phosphate buffer solution (pH 7.4) having a BSA concentration of 20 mg / mL and stirred at room temperature for 24 hours. Thereafter, the supernatant was collected by centrifugation, and then the BSA concentration of the filtrate was calculated with a spectrophotometer to calculate the amount of BSA adsorbed on the particles. The BSA concentration was calculated from the absorbance at 280 nm using a spectrophotometer. The case where the amount of non-specific adsorption was 5 mg or less was evaluated as “◯”, and the case where it exceeded 5 mg was evaluated as “x”. The results are shown in Table 3.

(Column characteristic evaluation)
The DEAE-modified separation material was packed in a stainless steel column of φ7.8 × 300 mm at a slurry (solvent: methanol) concentration of 30% by mass over 15 minutes. Then, the flow rate was changed, water was passed through the column, the relationship between the flow rate and the column pressure was measured, and the linear flow rate (column flow rate) at a column pressure of 0.3 MPa was calculated. The results are shown in Table 3.

The protein recovery rate was measured as follows. First, 10 column volumes of 20 mmol / L Tris-hydrochloric acid buffer (pH 8.0) were passed through the column. Thereafter, a BSA solution having a BSA concentration of 2 mg / mL prepared using 20 mmol / L Tris-HCl buffer was flowed at 800 cm / h, and the BSA concentration at the column outlet was measured by UV. The dynamic adsorption amount (dynamic binding capacity, (BSA adsorption amount)) at 10% breakthrough was calculated by the following formula. Next, a BSA solution with a BSA concentration of 2 mg / mL is allowed to flow until the BSA concentrations at the column inlet and outlet match, and a 1 M NaCl Tris-hydrochloric acid buffer solution for 5 column volumes is allowed to flow. ) The protein recovery rate was calculated based on “BSA recovery amount / BSA adsorption amount × 100 (%)”. 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)
c _f : Injected BSA concentration (mg / mL)
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>
Porous polymer particles 2 were synthesized in the same manner as the porous polymer particles 1. A separation material was obtained by the same treatment as in Example 1 except that the washing time in Soxhlet extraction after the synthesis of the porous polymer particles was changed to 5 hours. The separation material was evaluated in the same manner as in Example 1.

<Example 3>
Porous polymer particles 3 were synthesized in the same manner as the porous polymer particles 1. A separation material was obtained by the same treatment as in Example 1 except that the washing time in Soxhlet extraction after the synthesis of the porous polymer particles was changed to 3 hours. The separation material was evaluated in the same manner as in Example 1.

<Example 4>
Porous polymer particles 4 were synthesized in the same manner as the porous polymer particles 1. A separation material was obtained by the same treatment as in Example 1 except that the washing time in Soxhlet extraction after the synthesis of the porous polymer particles was changed to 2 hours. The separation material was evaluated in the same manner as in Example 1.

<Example 5>
The same procedure as in Example 1 was conducted except that 14.4 g of divinylbenzene and 5.3 g of styrene were used as monomers, and 14.4 g of divinylbenzene and 3.6 g of styrene were used to synthesize porous polymer particles 5. Thus, a separating material was obtained. The separation material was evaluated in the same manner as in Example 1.

<Example 6>
As a monomer, a separator was obtained in the same manner as in Example 1 except that porous polymer particles 6 were synthesized using 18 g of divinylbenzene instead of 10.7 g of divinylbenzene and 5.3 g of styrene. . The separation material was evaluated in the same manner as in Example 1.

<Example 7>
Separation was carried out in the same manner as in Example 1 except that porous polymer particles 7 were synthesized using 8 g of sorbitan monooleate instead of 14.4 g of toluene and 21.6 g of dodecanol as the porosifying agent. The material was obtained. The separation material was evaluated in the same manner as in Example 1.

<Example 8>
Separation was carried out in the same manner as in Example 1 except that porous polymer particles 8 were synthesized using 8 g of sorbitan monolaurate instead of 14.4 g of toluene and 21.6 g of dodecanol as the porosifying agent. The material was obtained. The separation material was evaluated in the same manner as in Example 1.

<Example 9>
Separation was carried out in the same manner as in Example 1 except that porous polymer particles 9 were synthesized using 8 g of glycerol monooleate instead of 14.4 g of toluene and 21.6 g of dodecanol as the porosifying agent. The material was obtained. The separation material was evaluated in the same manner as in Example 1.

<Comparative Example 1>
A separation material was obtained by the same treatment as in Example 1 except that the porous polymer particles 10 were synthesized by performing the washing with stirring for 10 minutes twice in 500 mL of acetone instead of Soxhlet extraction as a particle washing method. The separation material was evaluated in the same manner as in Example 1.

<Comparative example 2>
A separation material was obtained by the same treatment as in Example 1 except that the porous polymer particles 11 were synthesized by performing washing once with stirring for 10 minutes in 500 mL of acetone instead of Soxhlet extraction as a particle washing method. The separation material was evaluated in the same manner as in Example 1.

<Comparative Example 3>
After synthesizing particles in the same manner as in Example 1 except that 14.4 g of toluene and 8 g of sorbitan monooleate were used instead of 14.4 g of toluene and 21.6 g of dodecanol as a porosifying agent, instead of Soxhlet extraction as a particle washing method A separation material was obtained by treating in the same manner as in Example 1 except that the porous polymer particles 12 were synthesized by stirring and washing twice in 500 mL of acetone twice. The separation material was evaluated in the same manner as in Example 1.

<Comparative Example 4>
Commercially available agarose particles (Capto DEAE, manufactured by GE Healthcare) were used as the porous polymer particles 13, and the porous polymer particles 13 were evaluated in the same manner as in Example 1.

  As can be seen from the results in Table 3, Examples 1 to 9 of the present application have a high protein recovery rate while reducing non-specific adsorption of the protein compared to Comparative Examples 1 to 4, and when used as a column. It was found that the column characteristics such as liquidity were excellent.

Claims (10)

Porous polymer particles;
A separating layer comprising a coating layer covering at least a part of the surface of the porous polymer particle,
The coating layer includes a polymer having a hydroxyl group,
The separating material contains at least one component A of an organic solvent having an HLB value of 1 to 9 and an oil-soluble surfactant having an HLB value of 1 to 9,
The separation material, wherein the amount of change in the content of the component A in the cleaning liquid is 5000 ppm or less per 1 g of the separation material before and after the separation material is immersed in a cleaning liquid containing acetonitrile for 24 hours.   The separation material according to claim 1, wherein the porous polymer particles include a polymer obtained by polymerizing a monomer containing 50% by mass or more of divinylbenzene. The separation material according to claim 1 or 2, wherein the specific surface area is 30 m ² / g or more.   The separation material according to any one of claims 1 to 3, wherein the mode diameter of the pore size distribution is 0.05 to 0.6 µm.   The separation material according to any one of claims 1 to 4, wherein an average particle diameter of the porous polymer particles is 10 to 300 µm.   The separation material according to any one of claims 1 to 5, wherein the polymer having a hydroxyl group is a polysaccharide or a modified product thereof.   The separating material according to any one of claims 1 to 6, wherein the polymer having a hydroxyl group is agarose or a modified product thereof.   The separation material according to any one of claims 1 to 7, comprising 30 to 400 mg of the coating layer per 1 g of the porous polymer particles.   The separation material according to any one of claims 1 to 8, wherein when the column is packed, the liquid flow rate is 800 cm / h or more when the column pressure is 0.3 MPa.   A column comprising the separation material according to claim 1.