JP2017122654A - Separation material and column - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a separating material which reduces non-specific adsorption of a protein and has excellent column characteristics such as liquid permeability when used as a column, and a column provided with the separating material. According to the present invention, a porous polymer particle containing a copolymer containing a structural unit derived from a polymerizable monomer having an adamantan skeleton and a structural unit derived from a styrene-based monomer, and a surface of the porous polymer particle. Provided is a separating material comprising a coating layer containing a polymer having a hydroxyl group, which covers at least a part of the above. [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 exchangers based on cross-linked gels of hydrophilic natural polymers Etc. are 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 disadvantages that the volume change due to the ionic strength of the solution, the volume change between the free acid form and the loaded form are 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.

  An ion exchanger of a hybrid copolymer 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). Cross-linked copolymer gel has problems such as pressure loss and volume change when the degree of cross-linking is low, but by making it a hybrid copolymer, liquid flow characteristics are improved, pressure loss is low, ion exchange capacity is improved, Leakage behavior is improved.

  In addition, composite fillers in which pores of an organic synthetic polymer substrate are filled with a crosslinked gel of a hydrophilic natural polymer having a giant network structure have been proposed (see, for example, Patent Documents 5 and 6). Furthermore, synthesis of porous particles formed by copolymerization of glycidyl methacrylate and an acrylic cross-linked 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, conventional separation materials do not have sufficient characteristics such as reduced nonspecific adsorption of proteins and excellent column characteristics such as liquid permeability when used as a column.

  Then, this invention aims at providing the separation material which reduces nonspecific adsorption | suction of protein, and is excellent in column characteristics, such as liquid permeability when used as a column, and a column provided with this separation material. .

The present invention provides the separating material described in [1] to [11] below.
[1] A porous polymer particle containing a structural unit derived from a polymerizable monomer having an adamantane skeleton and a copolymer containing a structural unit derived from a styrene monomer, and at least a part of the surface of the porous polymer particle And a covering layer containing a polymer having a hydroxyl group.
[2] The separating material according to [1], wherein the 5% compression deformation elastic modulus is 300 to 1500 MPa.
[3] The separating material according to [1] or [2], wherein the copolymer contains 15 to 90% by mass of a structural unit derived from a polymerizable monomer having an adamantane skeleton based on the total mass of the monomer.
[4] The separating material according to any one of [1] to [3], wherein the specific surface area is 30 m ² / g or more.
[5] The separation material according to any one of [1] to [4], wherein the average pore diameter is 0.05 to 0.6 μm.
[6] The separation material according to any one of [1] to [5], wherein the average particle diameter of the porous polymer particles is 10 to 300 μm.
[7] The separating material according to any one of [1] to [6], wherein the polymer having a hydroxyl group is a polysaccharide or a modified product thereof.
[8] 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.
[9] The separating material according to any one of [1] to [8], comprising a coating layer of 30 to 400 mg per 1 g of porous polymer particles.
[10] The separation material according to any one of [1] to [9], wherein when the column is packed, the liquid flow rate is 800 cm / h or more when the column pressure is 0.3 MPa.
[11] A column comprising the separation material according to any one of [1] to [10].

  According to the present invention, it is possible to provide a separation material that reduces nonspecific adsorption of proteins and has excellent column characteristics such as liquid permeability when used as a column, and a column including the separation material. .

  Hereinafter, preferred embodiments of the present invention will be described, 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. 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 other similar expressions such as (meth) acrylate.

(Porous polymer particles)
The porous polymer particles according to the present embodiment are particles obtained by curing a monomer including a porosifying agent, soluble particles, and the like, and can be synthesized by copolymerization by conventional suspension polymerization, seed polymerization, or the like. it can. The porous polymer particles can be obtained by copolymerizing a polymerizable monomer having an adamantane skeleton and a monomer containing a styrenic monomer. That is, the porous polymer particles contain a copolymer containing a structural unit derived from a polymerizable monomer having an adamantane skeleton and a structural unit derived from a styrenic monomer. By including a polymerizable monomer having an adamantane skeleton as a monomer, it becomes possible to make the porous polymer particles highly elastic, and when packed in a column, the particles do not become compact and the liquid flow rate is increased. Can do.

  Examples of the polymerizable monomer having an adamantane skeleton include adamantyl (meth) acrylate, N- (1-adamantyl) (meth) acrylamide, adamantane divinyl, adamantane vinyl, adamantane di (meth) acrylate, adamantane tri (meth) acrylate, 2 -Isopropenyl-2-phenyladamantane, 1- (1-phenylvinyloxy) adamantane, 1-adamantyl vinyl ether, 1,3-bis (vinyloxy) adamantane, 4- (1-adamantyl) styrene, 4-vinylbenzoic acid 1 -Adamantyl and the like. Among these, it is preferable to use a bifunctional or higher polymerizable monomer. These polymerizable monomers having an adamantane skeleton can be used singly or in combination of two or more.

  The copolymer preferably contains 15 to 90% by mass, more preferably 30 to 90% by mass, and more preferably 40 to 85% by mass of structural units derived from a polymerizable monomer having an adamantane skeleton, based on the total mass of the monomer. % Is more preferable. When the copolymer contains a structural unit derived from a polymerizable monomer having an adamantane skeleton in an amount of 15% by mass or more based on the total mass of the monomer, swelling of the separation material can be suppressed, and an increase in column pressure can be easily suppressed.

  Examples of the styrenic monomer include the following polyfunctional monomers and monofunctional monomers. In the present specification, the styrenic monomer represents one having no adamantane skeleton.

  Examples of the polyfunctional monomer include divinyl compounds such as divinylbenzene, divinylbiphenyl, divinylnaphthalene, and divinylphenanthrene. These polyfunctional monomers can be used alone or in combination of two or more. Of these, divinylbenzene is preferably used because of its excellent 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 singly 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 carboxy group, an amino group, a hydroxyl group, and an aldehyde group, can also be used.

  The copolymer preferably contains 85 mass% or less, more preferably 70 mass% or less, and still more preferably 60 mass% or less of a structural unit derived from a styrene monomer based on the total mass of the monomer. The lower limit of the content of the structural unit derived from the styrenic monomer is preferably 5% by mass or more, preferably 10% by mass or more, and more preferably 15% by mass or more, based on the total mass of the monomer. preferable.

  The copolymer may contain a monomer other than a polymerizable monomer having an adamantane skeleton and a styrene monomer as a monomer. Examples of such monomers include (poly) alkylene glycols such as (poly) ethylene glycol di (meth) acrylate, (poly) propylene glycol di (meth) acrylate, and (poly) tetramethylene glycol di (meth) acrylate. Di (meth) acrylate; trimethylolpropane tri (meth) acrylate, tetramethylolmethane tri (meth) acrylate, tetramethylolpropane tetra (meth) acrylate, pentaerythritol tri (meth) acrylate, 1,1,1-trishydroxymethyl Tri- or higher functional (meth) acrylates such as ethane tri (meth) acrylate, 1,1,1-trishydroxymethylpropane triacrylate; ethoxylated bisphenol A di (meth) acrylate, propoxy Ethoxylated bisphenol A-based di (meth) acrylate, tricyclodecane dimethanol di (meth) acrylate, 1,1,1-trishydroxymethylethane di (meth) acrylate, ethoxylated cyclohexanedimethanol di (meth) acrylate, etc. Di (meth) acrylate; diallyl phthalate and isomers thereof; triallyl isocyanurate and derivatives thereof; methyl acrylate, ethyl acrylate, propyl acrylate, n-butyl acrylate, isobutyl acrylate, hexyl acrylate, acrylic acid 2 -Ethylhexyl, n-octyl acrylate, dodecyl acrylate, lauryl acrylate, stearyl acrylate, 2-chloroethyl acrylate, phenyl acrylate, methyl α-chloroacrylate, methyl methacrylate, meta (Meta) such as ethyl crylate, propyl methacrylate, n-butyl methacrylate, isobutyl methacrylate, hexyl methacrylate, 2-ethylhexyl methacrylate, n-octyl methacrylate, dodecyl methacrylate, lauryl methacrylate, stearyl methacrylate Acrylic acid esters; vinyl esters such as vinyl acetate, vinyl propionate, vinyl benzoate and vinyl butyrate; N-vinyl compounds such as N-vinylpyrrole, N-vinylcarbazole, N-vinylindole and N-vinylpyrrolidone; Fluorinated monomers such as vinyl, vinylidene fluoride, tetrafluoroethylene, hexafluoropropylene, trifluoroethyl acrylate, tetrafluoropropyl acrylate and the like can be mentioned. These can be used alone or in combination of two or more.

  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. Specific examples of the porosifying agent include toluene, xylene, cyclohexane, octane, butyl acetate, dibutyl phthalate, methyl ethyl ketone, dibutyl ether, 1-hexanol, 2-octanol, decanol, lauryl alcohol, cyclohexanol and the like. . These porosifying agents can be used singly or in combination of two or more.

  The porosifying agent can be used in an amount of 0 to 200% by mass relative to the total mass of the monomer. 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 the porosifying agent, it is possible to make the particles porous by dissolving the oil-soluble surfactant in the monomer and absorbing water.

  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 mass of the monomer. When the content of the oil-soluble surfactant is 5% by mass or more, the stability of water droplets is sufficient, and it is difficult to form a large single hole. Further, 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.

  The soluble particles are particles that are soluble in acids, alkalis, solvents, and the like, for example. As the soluble particles, for example, calcium carbonate, tricalcium phosphate, silica, polymer, metal colloid, and the like can be used. From the viewpoint of easy removal, it is preferable to use calcium carbonate or tricalcium phosphate. The particle diameter of the soluble particles is preferably 0.6 to 5 μm and more preferably 1 to 5 μm from the viewpoint of improving liquid permeability in the particles.

  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 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 type of monomer and 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 added 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 prevent the monomer from polymerizing alone, water-soluble polymerization inhibitors 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. 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 average particle diameter of the porous polymer particles or the separating material can be determined by the following measurement method.
(1) The particles are dispersed in water (including a dispersant such as a surfactant) using an ultrasonic dispersion device to prepare a dispersion liquid containing 1% by mass of porous polymer particles.
(2) Using a particle size distribution meter (Sysmex Flow, manufactured by Sysmex Corporation), the average particle size is measured from an image of about 10,000 particles in the dispersion.

  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 0.01 μm or more and less than 0.6 μm, that is, macropores (macropores). The pore diameter of the porous polymer particles is more preferably 0.1 μm or more and less than 0.6 μm. If the pore diameter is 0.01 μm or more, a 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 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 the present embodiment includes a polymer having a hydroxyl group. 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 becomes possible to make it equal to or higher than when natural polymers are used. Furthermore, when the polymer having a hydroxyl group is crosslinked, it is possible to further suppress an increase in column pressure.

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

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

(Formation method of coating layer)
The coating layer according to the present embodiment can be formed by, for example, the following method.

  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 left 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 adsorb | sucked.

(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.

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

  A catalyst is usually used for this crosslinking reaction. As the catalyst, a conventionally known catalyst can be appropriately used according to the type of the crosslinking agent. For example, when the crosslinking agent is 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 mol, this is used. It is used in the range of 0.01 to 10 mol times, more preferably 0.1 to 5 mol times with respect to.

  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, the polymer or crosslinking agent is not extracted from the impregnated polymer solution, and a crosslinking reaction is performed. Must be inert. Specific examples thereof 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, washed with a hydrophilic organic solvent such as water, methanol, ethanol, etc. to remove unreacted polymer, suspending medium, etc. A separation material in which at least a part of the surface is coated with a coating layer containing a polymer having a hydroxyl group is 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, more preferably includes a coating layer of 50 to 400 mg, and further includes a coating layer of 100 to 350 mg. preferable. When the ratio of the coating layer is 400 mg or less with respect to 1 g of the porous polymer particles, the coating layer can be a thin film, and the liquid permeability when used as a column tends to be further improved. Moreover, it exists in the tendency for protein adsorption amount to rise more that the ratio of a coating layer is 30 mg or more with respect to 1 g of porous polymer particles. The amount of the coating layer can be measured by reducing the weight of pyrolysis.

(Introduction of ion exchange groups)
The separation material having a 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 halogenated alkyl compounds, a mono- having at least one alkyl group in which a part of hydrogen atoms is substituted with a chlorine atom. , Di- or tri-alkylamines, mono-, di- or tri-alkanolamines, mono-alkyl-mono-alkanolamines, di-alkyl-mono-alkanolamines, mono-alkyl-di-alkanolamines, etc. A method is 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 pore diameter and specific surface area of the separating material or porous polymer particle of the present embodiment are values measured with a mercury intrusion measuring apparatus (Autopore: manufactured by Shimadzu Corporation), and are measured as follows. About 0.05 g of a sample is added to a standard 5 mL powder cell (stem volume 0.4 mL), and measurement is performed under 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. Further, each value is calculated by limiting to a pore diameter range of 0.05 to 5 μm.

  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 polymers. 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 liquid passing speed is 800 cm / h or more when the column pressure is 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.

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

  When used as a column packing material in column chromatography, the separation material of this embodiment is excellent in operability because there is almost no volume change in the column regardless of the properties of the eluent used.

  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 particles are 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.) Measure the compression displacement. From the measured values obtained, the compression modulus (5% K value) when the particles are compressively deformed by 5% can be obtained by the following formula. 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
F: Load (mN) when the crosslinked polymer particles are 5% compressively deformed
S: Compression displacement (mm) when the crosslinked polymer particles are 5% compressively deformed.
R: radius of cross-linked polymer particles (mm)

  The compression elastic modulus (5% K value) when the separating material is subjected to compression deformation by 5% is preferably 300 to 1500 MPa, and more preferably 400 to 1500 MPa. When the compression elastic modulus is 300 MPa or more, the flexibility of the porous polymer particles becomes low, so that it is difficult to be deformed when a liquid is flowed in the column, and the column pressure can be suppressed.

  The average pore diameter of the separating material is preferably 0.05 to 0.6 μm, more preferably 0.05 to 0.5 μm, and still more preferably 0.1 to 0.5 μm. When the average pore diameter is 0.05 μm or more, a substance tends to easily enter the pores. When the average pore diameter is 0.6 μm or less, the specific surface area is sufficient.

The specific surface area of the separating material 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. The upper limit value of the specific surface area of the separating material can be 300 m ² / g or less.

  The 5% deformation elastic modulus, average pore diameter, specific surface area, etc. of the separating material can be adjusted by appropriately selecting the raw material for the porous polymer particles, the porosifying agent, the polymer having a hydroxyl group, and the like.

  The separation material of this embodiment can be used for a column. That is, the column of this embodiment includes the separation 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 1)
In a 500 mL three-necked flask, 9.6 g of adamantane trimethacrylate (ADTMA) as a monomer, 2.4 g of divinylbenzene having a purity of 96% (DVB, manufactured by Nippon Steel & Sumikin Chemical Co., Ltd., trade name “DVB960”), as a porous agent As a polymerization initiator, 12 g of diethylbenzene, 12 g of hexanol and 0.5 g of benzoyl peroxide were added to prepare a monomer solution. An aqueous solution containing 0.5% by mass of polyvinyl alcohol was used as a solvent, and the monomer solution was emulsified using a microprocess server to obtain a 25% by volume emulsion. The obtained emulsified liquid was transferred to a flask and stirred for about 8 hours using a stirrer while heating in an 80 ° C. water bath. The obtained particles were filtered and then washed with acetone to obtain porous polymer particles 1. The particle size of the porous polymer particle 1 was measured with a flow type particle size measuring device (FPIA-3000, manufactured by Sysmex Corporation), and the average particle size was calculated. The results are shown in Table 1.

(Porous polymer particles 2)
Porous polymer particles 2 were synthesized in the same manner as the porous polymer particles 1 except that the amounts of adamantane trimethacrylate, divinylbenzene, diethylbenzene, and hexanol were changed to 6 g, 6 g, 10 g, and 14 g, respectively.

(Porous polymer particles 3)
Porous polymer particles 3 were synthesized in the same manner as the porous polymer particles 1 except that the amounts of adamantane trimethacrylate, divinylbenzene, diethylbenzene and hexanol were changed to 3 g, 9 g, 8 g and 16 g, respectively.

(Porous polymer particles 4)
Synthesis of porous polymer particles 1 except that 9.6 g of adamantane trimethacrylate, 2.4 g of divinylbenzene, 12 g of diethylbenzene and 12 g of hexanol were changed to 6 g of adamantane diacrylate (ADDA), 6 g of divinylbenzene, 10 g of diethylbenzene and 14 g of hexanol. In the same manner, porous polymer particles 4 were synthesized.

(Porous polymer particles 5)
The synthesis of porous polymer particles 1 except that 9.6 g of adamantane trimethacrylate, 2.4 g of divinylbenzene, 12 g of diethylbenzene and 12 g of hexanol were changed to 6 g of adamantane methacrylate (ADMA), 6 g of divinylbenzene, 16 g of diethylbenzene and 8 g of hexanol. Similarly, porous polymer particles 5 were synthesized.

(Porous polymer particle 6)
Porous polymer particles 6 were synthesized in the same manner as the synthesis of the porous polymer particles 2 except that 14 g of hexanol was changed to 14 g of octanol.

(Porous polymer particle 7)
Porous polymer particles 7 were synthesized in the same manner as the synthesis of the porous polymer particles 2 except that 14 g of hexanol was changed to 14 g of isoamyl alcohol.

(Porous polymer particles 8)
A porous polymer except that 9.6 g of adamantane trimethacrylate, 2.4 g of divinylbenzene, 12 g of diethylbenzene and 12 g of hexanol were changed to 4 g of divinylbenzene, 8 g of 2,3-dihydroxypropyl methacrylate (GMA) and span 80 (4 g). In the same manner as the synthesis of the particles 1, porous polymer particles 8 were synthesized.

(Porous polymer particle 9)
Except for changing 9.6 g of adamantane trimethacrylate, 2.4 g of divinylbenzene, 12 g of diethylbenzene and 12 g of hexanol to 11.2 g of 2,3-dihydroxypropyl methacrylate, 4.8 g of ethylene glycol dimethacrylate (EGDMA) and 805 g of span. The porous polymer particles 9 were synthesized in the same manner as the synthesis of the porous polymer particles 1.

Example 1
<Formation and cross-linking of coating layer>
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 reprecipitated with isopropyl alcohol and washed. Next, the porous polymer particles 1 are added to a 20 mg / mL modified agarose aqueous solution at a ratio of 1 g of the porous polymer particles 1 to 70 mL of the aqueous solution, and stirred at 55 ° C. for 24 hours. The denatured agarose was adsorbed on the surface. After adsorption, it was filtered and washed with hot water.

  The modified agarose was crosslinked as follows. 1 g of porous polymer particles 1 on which modified agarose is adsorbed with respect to 35 mL of the aqueous solution are added to an aqueous solution having ethylene glycol diglycidyl ether and sodium hydroxide concentrations of 0.64 M and 0.4 M, respectively, for 24 hours. Stir at room temperature. Then, after washing with 2% by mass of hot sodium dodecyl sulfate aqueous solution, it was washed with pure water.

(Evaluation of nonspecific adsorption ability of protein)
0.5 g of the obtained particles were put into 50 mL of a phosphate buffer solution (pH 7.4) having a BSA (Bovine Serum Albumin) concentration of 20 mg / mL and stirred at room temperature for 24 hours. Thereafter, 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. The results are shown in Table 3.

<Introduction of ion exchange groups>
Particles were filtered from an aqueous suspension containing particles cross-linked with modified agarose. The obtained particles (dry mass 20 g) were put into 200 mL of 5M aqueous sodium hydroxide solution and allowed to stand at room temperature for 1 hour. Separately, a predetermined amount (60 g) of diethylaminoethyl chloride hydrochloride was added to 200 mL of water to prepare an aqueous solution of diethylaminoethyl chloride hydrochloride. This aqueous solution was added to the aqueous sodium hydroxide solution, the temperature was raised to 70 ° C., and the mixture was reacted for 2 hours with stirring. After completion of the reaction, the product was filtered and washed with water to obtain a separating material having a diethylaminoethyl (DEAE) group as an ion exchange group (DEAE-modified). The average pore diameter and specific surface area of the obtained separating material were measured by a mercury intrusion method. Moreover, after drying the obtained separating material, the mass (coating amount) of the coating layer was measured by thermogravimetric analysis. The results are shown in Table 2.

(5% compressive deformation modulus)
The 5% compressive deformation modulus of the separating material was measured by the method described above. The results are shown in Table 2.

(Column characteristic evaluation)
The obtained separation material was packed in a φ7.8 × 300 mm stainless steel column as a slurry (solvent: methanol) having a concentration of 30% by mass over 15 minutes. Thereafter, water was passed through the column while changing the flow rate, the relationship between the flow rate and the column pressure was measured, and the liquid flow rate at 0.3 MPa was measured. The results are shown in Table 3.

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 BSA concentration at a column outlet was measured by UV measurement through a 20 mmol / L Tris-HCl buffer solution having a BSA concentration of 2 mg / mL. The buffer was passed through until the BSA concentration at the column inlet and outlet coincided, and diluted with 1 M NaCl Tris-HCl buffer 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 (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)
A separator was prepared in the same manner as in Example 1 except that the porous polymer particle 1 was changed to the porous polymer particle 2 and evaluated in the same manner as in Example 1.

(Example 3)
A separator was prepared in the same manner as in Example 1 except that the porous polymer particle 1 was changed to the porous polymer particle 3 and evaluated in the same manner as in Example 1.

Example 4
A separator was prepared in the same manner as in Example 1 except that the porous polymer particle 1 was changed to the porous polymer particle 4 and evaluated in the same manner as in Example 1.

(Example 5)
A separator was prepared in the same manner as in Example 1 except that the porous polymer particle 1 was changed to the porous polymer particle 5 and evaluated in the same manner as in Example 1.

(Example 6)
A separator was prepared in the same manner as in Example 1 except that the porous polymer particle 1 was changed to the porous polymer particle 6 and evaluated in the same manner as in Example 1.

(Example 7)
A separator was prepared in the same manner as in Example 1 except that the porous polymer particle 1 was changed to the porous polymer particle 7 and evaluated in the same manner as in Example 1.

(Comparative Example 1)
The porous polymer particle 1 was changed to the porous polymer particle 8, and a separation material was obtained in the same manner as in Example 1 except that an ion exchange group was introduced without forming a coating layer of a polymer having a hydroxyl group (agarose). This was prepared and evaluated in the same manner as in Example 1.

(Comparative Example 2)
4 g of porous polymer particles 9 were added to 6 g of a solution in which 1 g of dextran (molecular weight 150,000), 0.6 g of sodium hydroxide and 0.15 g of sodium borohydride were dissolved in distilled water. The pores were impregnated with dextran. The obtained dextran-impregnated polymer was added to 1 L of a 1% by weight ethylcellulose toluene solution and stirred, dispersed, and suspended. 5 mL of epichlorohydrin was added to the resulting suspension, the temperature was raised to 50 ° C., and the mixture was stirred at this temperature for 6 hours to crosslink dextran impregnated in the pores of the porous polymer particles 9. After completion of the reaction, the suspension was filtered to separate the gel substance, and washed with toluene, ethanol and distilled water in order to obtain a separating material. This separator was evaluated in the same manner as in Example 1.

(Comparative Example 3)
The porous polymer particles 7 were used as a separating material as they were and evaluated in the same manner as in Example 1.

  As shown in Table 3, Examples 1 to 7 of the present application reduce non-specific adsorption of proteins compared to Comparative Examples 1 to 3, have excellent durability, and have a liquid permeability when used as a column. It was found that the characteristics were excellent.

Claims (11)

Porous polymer particles containing a copolymer comprising a structural unit derived from a polymerizable monomer having an adamantane skeleton and a structural unit derived from a styrenic monomer;
A coating layer containing a polymer having a hydroxyl group, covering at least a part of the surface of the porous polymer particles;
Separation material comprising.   The separation material according to claim 1, wherein the 5% compressive deformation elastic modulus is 300 to 1500 MPa.   The separation material according to claim 1 or 2, wherein the copolymer contains 15 to 90% by mass of a structural unit derived from a polymerizable monomer having the adamantane skeleton based on the total mass of the monomer. The separation material according to claim 1, wherein the specific surface area is 30 m ² / g or more.   The separation material according to any one of claims 1 to 4, wherein the average pore diameter is 0.05 to 0.6 µm.   The separation material according to any one of claims 1 to 5, wherein an average particle diameter of the porous polymer particles is 10 to 300 µm.   The separating material according to any one of claims 1 to 6, 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 8, 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 9, 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.