JP6705189B2 - Separation material and column - Google Patents

Description

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

Conventionally, when separating and purifying a biopolymer represented by a protein, generally, an ion exchanger having a porous synthetic polymer as a matrix and an ion exchange having a crosslinked gel of a hydrophilic natural polymer as a matrix. The body is used.

An ion exchanger having a porous synthetic polymer as a matrix has a small volume change due to salt concentration, and when packed in a column and used for chromatography, tends to have excellent pressure resistance during liquid passage. However, when this ion exchanger is used for separation of proteins, etc., nonspecific adsorption such as irreversible adsorption based on hydrophobic interaction occurs, asymmetry of peaks occurs, or nonspecifically adsorbed proteins are adsorbed. There is a problem that it cannot be collected as it is.

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

In order to overcome the drawbacks of the crosslinked gel of hydrophilic natural polymer, attempts have been made so far to combine it with a rigid substance that is, so to speak, a “skeleton” (for example, Patent Documents 1 to 7).

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

However, the separation material as described above does not sufficiently suppress non-specific adsorption of proteins and the like, and also has excellent liquid permeability and durability even when packed in a column and used in chromatography. It did not have sufficient properties as a separating material.

Therefore, the present invention provides a separation material which can sufficiently suppress non-specific adsorption of proteins and the like, and which can obtain excellent liquid permeability and durability even when it is packed in a column and used in chromatography, and the separation material. It is an object to provide a column provided with wood.

The separating material according to the present invention is a coating containing a porous polymer particle containing a cross-linked polymer, a polymer having a hydroxyl group that covers at least a part of the surface of the porous polymer particle, and a crosslinking agent that cross-links the polymer. A separating material comprising a layer and a linker group bonded to the crosslinked polymer and the crosslinking agent, wherein the linker group has a thio group bonded to the crosslinked polymer.

Non-specific adsorption of proteins and the like is sufficiently suppressed in the separating material, and excellent liquid permeability and durability are obtained even when packed in a column and used for chromatography. The above separating material also improves the dynamic adsorption amount.

The crosslinked polymer may contain monomer units derived from divinylbenzene. When the crosslinked polymer contains such a monomer unit, the durability and swelling property of the separating material are improved.

The linker group can be derived from a linker compound having a molecular weight of 500 or less. When the molecular weight of the linker compound is not more than the above upper limit, it is preferable from the viewpoint of reactivity with the particles or the crosslinking agent.

The specific surface area of the separating material may be 20 m ² /g or more. When the specific surface area of the separating material is equal to or more than the above lower limit value, the adsorption amount of the substance to be separated can be improved.

The maximum value of the pore volume in the pore volume distribution of the separation material, which is measured by the mercury intrusion method, can be in the range of the pore diameter of 0.05 to 0.7 μm. When the maximum value of the pore volume is in the above range, the specific surface area of the particles tends to be large, and the amount of adsorbed protein or the like tends to increase.

The average particle size of the porous polymer particles can be 10 to 300 μm. When the average particle diameter of the porous polymer particles is within the above range, it is possible to suppress an increase in column pressure and to purify the protein at a high flow rate.

The polymer having a hydroxyl group can be a polysaccharide or a modified product thereof, and can be agarose or a modified product thereof. When the polymer having a hydroxyl group is such a compound, nonspecific adsorption of proteins can be reduced.

The amount of the coating layer per 1 g of the porous polymer particles can be 30 to 400 mg. When the amount of the coating layer is within the above range, the coating layer can be formed into a thin film, and not only the liquid permeability when used as a column is further improved, but also the adsorption amount and dynamic adsorption amount of protein etc. Will tend to increase.

The flow rate of water when water is passed through the column filled with the separating material so that the pressure in the column is 0.3 MPa can be 800 cm/h or more.

A column according to the present invention comprises the above separating material.

According to the present invention, a non-specific adsorption of proteins and the like is sufficiently suppressed, and even when packed in a column and used in chromatography, excellent separation properties and durability can be obtained, and a separation material A column provided with a separating material can be provided.

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

The separating material according to the present invention includes porous polymer particles, a coating layer, and a linker group. The porous polymer particles include a crosslinked polymer. The coating layer contains a polymer having a hydroxyl group and a crosslinking agent that crosslinks the polymer, and coats at least a part of the surface of the porous polymer particles. The linker group has a thio group bonded to the crosslinked polymer and is bonded to the crosslinked polymer and the crosslinking agent. 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 pores inside the porous polymer particle.

The crosslinked polymer in the porous polymer particles can be a polymer of monomers including crosslinkable monomers having two or more double bonds. The cross-linked polymer can have double bonds.

Porous polymer particles are obtained, for example, by polymerizing droplets containing a monomer and a porosifying agent dispersed in an aqueous medium. Examples of the polymerization method include conventional suspension polymerization and emulsion polymerization. The crosslinkable monomer having two or more double bonds is not particularly limited, and examples thereof include acrylic and styrene vinyl monomers. Specific examples of the crosslinkable monomer having two or more double bonds include the following polyfunctional monomers and monofunctional monomers.

Examples of polyfunctional monomers include divinyl compounds such as divinylbenzene, divinylbiphenyl and divinylnaphthalene; (poly)ethylene glycol di(meth)acrylate, (poly)propylene glycol di(meth)acrylate and (poly)tetramethylene glycol. (Poly)alkylene glycol-based di(meth)acrylate such as di(meth)acrylate; trimethylolpropane tri(meth)acrylate, tetramethylolmethane tri(meth)acrylate, tetramethylolpropane tetra(meth)acrylate, pentaerythritol tri( (Meth)acrylates, tri- or more-functional (meth)acrylates such as 1,1,1-trishydroxymethylethane tri(meth)acrylate and 1,1,1-trishydroxymethylpropane triacrylate; ethoxylated bisphenol A-based di( (Meth)acrylate, propoxylated ethoxylated bisphenol A-based di(meth)acrylate, tricyclodecane dimethanol di(meth)acrylate, 1,1,1-trishydroxymethylethane di(meth)acrylate and ethoxylated cyclohexanedimethanol di Di(meth)acrylates such as (meth)acrylate; diallyl phthalate and its isomers; triallyl isocyanurate and its derivatives. Among these monomers, NK ester (A-TMPT-6P0, A-TMPT-3E0, A-TMM-3LMN, A-GLY series, A-9300, AD-TMP, AD-TMP manufactured by Shin-Nakamura Chemical Co., Ltd. -4CL, ATM-4E, A-DPH) and the like are commercially available. These polyfunctional monomers can be used alone or in combination of two or more. Among these, divinylbenzene is preferable from the viewpoint of durability and swelling property. That is, the crosslinked polymer preferably contains a monomer unit derived from divinylbenzene. The proportion of divinylbenzene in the total amount of the monomers is preferably 60% by mass or more, more preferably 70% by mass or more, and further preferably 80% by mass or more. Note that (meth)acrylate means acrylate or methacrylate, and the same applies to similar compounds.

Examples of monofunctional monomers include styrene, o-methylstyrene, m-methylstyrene, p-methylstyrene, α-methylstyrene, o-ethylstyrene, m-ethylstyrene, p-ethylstyrene, 2,4-. Dimethylstyrene, pn-butylstyrene, pt-butylstyrene, pn-hexylstyrene, pn-octylstyrene, pn-nonylstyrene, pn-decylstyrene, pn-dodecyl Styrene and its derivatives such as styrene, p-methoxystyrene, p-phenylstyrene, p-chlorostyrene, and 3,4-dichlorostyrene; methyl acrylate, ethyl acrylate, propyl acrylate, n-butyl acrylate, acrylic Isobutyl acrylate, hexyl acrylate, 2-ethylhexyl acrylate, n-octyl acrylate, dodecyl acrylate, lauryl acrylate, stearyl acrylate, 2-chloroethyl acrylate, phenyl acrylate, methyl α-chloroacrylate, methacrylic acid Such as methyl, ethyl methacrylate, propyl methacrylate, n-butyl methacrylate, isobutyl methacrylate, hexyl methacrylate, 2-ethylhexyl methacrylate, n-octyl methacrylate, dodecyl methacrylate, lauryl methacrylate and stearyl methacrylate ( (Meth)acrylic acid ester; vinyl ester such as vinyl acetate, vinyl propionate, vinyl benzoate and vinyl butyrate; N-vinyl compound such as N-vinylpyrrole, N-vinylcarbazole, N-vinylindole and N-vinylpyrrolidone; Fluorine-containing monomers such as vinyl fluoride, vinylidene fluoride, tetrafluoroethylene, hexafluoropropylene, trifluoroethyl acrylate and tetrafluoropropyl acrylate; conjugated dienes such as butadiene and isoprene. These monofunctional monomers may be used alone or in combination of two or more. Among these, styrene is preferable from the viewpoint of excellent acid resistance and alkali resistance.

Examples of the aqueous medium include water and a mixed solvent of water and a water-soluble solvent (eg, lower alcohol). The aqueous medium can include a surfactant. Examples of the surfactant include anionic, cationic, nonionic and zwitterionic surfactants.

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

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

Examples of nonionic surfactants include hydrocarbon nonionic surfactants such as polyethylene glycol alkyl ether, polyethylene glycol alkylaryl ether, polyethylene glycol ester, polyethylene glycol sorbitan ester, polyalkylene glycol alkylamine and amide, and polyethylene of silicon. Examples thereof include polyether-modified silicone-based nonionic surfactants such as oxide adducts and polypropylene oxide adducts, and fluorine-based nonionic surfactants such as perfluoroalkyl glycols.

Examples of the zwitterionic surfactants include hydrocarbon surfactants such as lauryldimethylamine oxide, phosphoric acid ester surfactants and phosphorous acid ester surfactants.

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

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

The porosifying agent may be 0 to 200% by mass based on the total mass of the monomers. The porosity of the porous polymer particles and the particles of the separating material can be controlled by the amount of the porosifying agent. The pore size and shape of the porous polymer particles and the separating material can be controlled by the kind of the porosifying agent.

Water, which is an aqueous medium, can be used as the porosifying agent. When water is used as the porosifying agent, an oil-soluble surfactant (emulsifier) can be added to water.

Examples of the oil-soluble surfactant include branched fatty acids having 16 to 24 carbon atoms, chain unsaturated fatty acids having 16 to 22 carbon atoms, and sorbitan monoesters of chain saturated fatty acids having 12 to 14 carbon atoms (for example, sorbitan mono Laurate, sorbitan monooleate, sorbitan monomyristate or sorbitan monoester derived from coconut fatty acid); branched fatty acid having 16 to 24 carbon atoms, chain unsaturated fatty acid having 16 to 22 carbon atoms or 12 to 14 carbon atoms Monoesters of chain saturated fatty acids (for example, diglycerol monooleate such as diglycerol monoesters of fatty acids having 18 carbon atoms and 1 double bond); diglycerol monomyristate, diglycerol monoisostea Diglycerol monoester of coconut or coconut fatty acid; branched alcohol having 16 to 24 carbon atoms, chain unsaturated alcohol having 16 to 22 carbon atoms, or chain saturated alcohol having 12 to 14 carbon atoms (for example, coconut fatty alcohol) Glycerol monoaliphatic ethers; as well as mixtures thereof.

Among these, sorbitan monolaurate (for example, SPAN (registered trademark) 20, preferably 40% or more in purity, more preferably 50% or more in purity, further preferably 70% or more in purity); sorbitan monooleate Ate (eg, SPAN® 80, preferably sorbitan monooleate having a purity of 40% or more, more preferably 50% or more, further preferably 70% or more); diglycerol monooleate (eg, preferably 40% or more of purity, more preferably 50% or more, further preferably 70% or more of purity of diglycerol monoisoate; diglycerol monoisostearate (eg, preferably 40% or more of purity, more preferably 50% of purity) Or more, more preferably diglycerol monoisostearate having a purity of 70% or more; diglycerol monomyristate (eg, sorbitan having a purity of preferably 40% or more, more preferably 50% or more, still more preferably 70% or more) Monomyristate); cocoyl (eg lauryl and myristoyl) ethers of diglycerol; or mixtures thereof are preferred.

From the viewpoint of making the particles porous, it is preferable to use these oil-soluble surfactants in the range of 5 to 80 mass% with respect to the total mass of the monomers. When the content of the oil-soluble surfactant is 5% by mass or more, the particles are preferably made porous, which is preferable. When the content of the oil-soluble surfactant is 80% by mass or less, it becomes easier for the porous polymer particles to retain their shape after polymerization.

The droplets containing the monomer and porosifying agent can further include soluble particles. Examples of the soluble particles include particles that can be dissolved with an acid, an alkali, a solvent, or the like. Specific examples of the soluble particles include calcium carbonate, tricalcium phosphate, silica, polymers and metal colloids. Among them, calcium carbonate and tricalcium phosphate are preferable from the viewpoint of easy removal of soluble particles. The average particle diameter of the soluble particles is preferably 0.6 to 5 μm, and from the viewpoint of improving the liquid permeability of the soluble particles, the average particle diameter of the soluble particles is more preferably 1 to 5 μm.

If necessary, a polymerization initiator may be further added to the aqueous medium. Examples of the polymerization initiator include benzoyl peroxide, lauroyl peroxide, benzoyl orthochloroperoxide, benzoyl orthomethoxy peroxide, 3,5,5-trimethylhexanoyl peroxide, and tert-butylperoxy-2-ethylhexano. And organic peroxides such as di-tert-butyl peroxide; and 2,2′-azobisisobutyronitrile, 1,1′-azobiscyclohexanecarbonitrile and 2,2′-azobis(2,4) -Dimethylvaleronitrile) and other azo compounds. The amount of the polymerization initiator may be 0.1 to 7.0 parts by mass with respect to 100 parts by mass of the monomer.

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

In order to improve the dispersion stability of the particles, a polymer dispersion stabilizer can be added to the aqueous medium. Examples of the polymer dispersion stabilizer include polyvinyl alcohol, polycarboxylic acid, cellulose (eg, hydroxyethyl cellulose, carboxymethyl cellulose and methyl cellulose) and polyvinyl pyrrolidone. Of these, polyvinyl alcohol or polyvinylpyrrolidone is preferable as the polymer dispersion stabilizer. An inorganic water-soluble polymer compound such as sodium tripolyphosphate can also be used in combination. 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 emulsion polymerization of the monomer alone, a water-soluble polymerization inhibitor such as nitrite, sulfite, hydroquinone, ascorbic acid, water-soluble vitamin B, citric acid and polyphenol can be used.

The average particle size of the porous polymer particles and the separating material is preferably 300 μm or less, more preferably 150 μm or less, and further preferably 100 μm or less, from the viewpoint of the amount of adsorbed protein and the separation performance. The average particle size of the porous polymer particles and the separating material is preferably 10 μm or more, more preferably 30 μm or more, and further preferably 50 μm or more from the viewpoint of suppressing an increase in the column pressure of the column packed with the porous polymer particles and the separating material. preferable.

The average particle size of the porous polymer particles and the separating material can be determined by the following measuring method.
1) The particles are dispersed in water (including a dispersant such as a surfactant) by an ultrasonic dispersion device to prepare a dispersion liquid containing 1% by mass of the porous polymer particles.
2) Using a particle size distribution meter (Sysmex Flow, manufactured by Sysmex Co., Ltd.), the average particle size is measured by an image of about 10,000 particles in the dispersion.

The pore volume of the porous polymer particles and the particles of the separating material is preferably 30 to 70% by volume, and more preferably 50 to 70% by volume, based on the total volume of the porous polymer particles and the separating material, respectively. ..

The porous polymer particles and the separating material preferably have macropores (macropores). The mode diameter (mode of pore size distribution, maximum frequency pore size) in the pore size distribution of the porous polymer particles and the separating material is preferably 0.01 to 0.6 μm, and 0.1 to 0.6 μm. Is more preferable. If the mode diameter is 0.01 μm or more, the substance tends to easily enter the pores, and if the mode diameter is 0.6 μm or less, the specific surface area of the particles becomes sufficient. The pore volume and the mode diameter in the pore size distribution of the porous polymer particles and the separating material can be adjusted by the porosifying agent.

In the pore volume distribution of the porous polymer particles and the separating material, the maximum value of the pore volume is preferably within the range of 0.05 to 0.7 μm, and within the range of 0.05 to 0.6 μm. More preferably. When the maximum value of the pore volume is within the above range, the specific surface area of the particles tends to be large and the protein adsorption amount tends to increase.

The specific surface area of the porous polymer particles and the particles of the separating material is preferably 20 m ² /g or more from the viewpoint of improving the adsorption amount of the substance to be separated, and 35 m ² /g or more from the viewpoint of improving practicality. Is more preferable, and 40 m ² /g or more is further preferable.

The mode diameter, the pore volume distribution, and the specific surface area of the pore size distribution of the porous polymer particles and the particles of the separation material should be measured as follows using a mercury intrusion measuring device (Autopore: Shimadzu Corporation). You can 0.05 g of the sample is added to a standard 5 mL powder cell (stem volume 0.4 mL), and the initial pressure is set to 21 kPa (about 3 psia, pore diameter of about 60 μm). The mercury parameters are a mercury contact angle of 130 degrees and a mercury surface tension of 485 dynes/cm. The mode diameter, the pore volume distribution, and the specific surface area in the pore diameter distribution of the porous polymer particles and the particles of the separating material are calculated within the range of the average pore diameter of 0.05 to 5 μm.

The coating layer of the present embodiment contains a polymer having a hydroxyl group and a crosslinking agent that crosslinks the polymer, and coats at least a part of the surface of the porous polymer particles.

The polymer having a hydroxyl group is preferably a hydrophilic polymer. Examples of the polymer having a hydroxyl group include polysaccharides, polyvinyl alcohol and modified products thereof. Examples of polysaccharides include agarose, dextran, cellulose and chitosan. Here, the modified product refers to a product in which a hydrophobic group is introduced into the molecule.

The polymer having a hydroxyl group is preferably a polysaccharide from the viewpoint of reducing non-specific adsorption of proteins, and a modified polysaccharide is preferred from the viewpoint of improving the interfacial adsorption capacity of the separating material. Among them, agarose or its modified product is more preferable. The weight average molecular weight of the polymer having a hydroxyl group may be 10,000 to 200,000.

The polymer having a hydroxyl group is crosslinked with a crosslinking agent. The crosslinker can be a compound having two or more crosslinkable groups (eg, epoxy groups). In the present specification, for convenience, the cross-linking agent after reacting with at least one of the polymer having a hydroxyl group and the linker compound is also referred to as a cross-linking agent.

As the cross-linking agent, for example, an epihalohydrin such as epichlorohydrin, a dialdehyde compound such as glutaraldehyde, a diisocyanate compound such as methylene diisocyanate, and a diglycidyl compound such as ethylene glycol diglycidyl ether, a functional group reactive with a hydroxyl group. And a compound having two or more of When a compound having an amino group such as chitosan is used as the polymer having a hydroxyl group, a dihalide compound such as dichlorooctane can be used as the crosslinking agent.

The amount of the coating layer is preferably 30 to 400 mg, more preferably 50 to 400 mg, and further preferably 100 to 400 mg with respect to 1 g of the porous polymer particles. When the amount of the coating layer is less than or equal to the above upper limit, the coating layer can be made into a thin film, and the liquid permeability when used as a column tends to be further improved. When the amount of the coating layer is equal to or more than the above lower limit, the adsorption amount of proteins and the like and the dynamic adsorption amount tend to be further increased. The amount of coating layer can be measured, for example, by the weight loss of the thermal decomposition of the separating material. That is, first, a predetermined amount of porous polymer particles is heated from 30° C. to 600° C. (10° C./min) by the thermogravimetric analysis method, and the 5% thermogravimetric reduction temperature T of the porous polymer particles is measured. Next, a predetermined amount of the separating material is heated from 30° C. at a heating rate of 10° C./min, and the thermogravimetric reduction amount per unit mass of the separating material at the temperature T is measured. Separately, the thermogravimetric weight loss amount of the coating layer at a predetermined temperature T is measured, and the thermogravimetric weight loss amount of the coating layer per unit mass at the temperature T is calculated. Here, the rate of weight loss of the porous polymer particles at the temperature T is set to 5%, and the coating amount of the separating material can be calculated from these values.

By coating the porous polymer particles with the coating layer containing the polymer having cross-linked hydroxyl groups, the rise of the pressure inside the column (column pressure) when the liquid is passed through the column packed with the separation material is suppressed. be able to. In addition, non-specific adsorption of proteins to the separation material is suppressed, and excellent protein adsorption tends to be obtained.

The linker group of the present embodiment has a thio group (-S-) bonded to the crosslinked polymer and is bonded to the crosslinked polymer and the crosslinking agent. Since the cross-linking polymer and the cross-linking agent are bound by the linker group, the coating layer is favorably held on the porous polymer particles, and thus the durability of the separating material is improved. The linker group can be formed by reacting a linker compound with a crosslinking polymer and a crosslinking agent. For convenience, the linker compound that has reacted with the cross-linking polymer but not with the cross-linking agent will also be referred to as a linker group.

The linker compound can be a compound having a thiol group and a reactive functional group that reacts with the crosslinkable group of the crosslinking agent. Examples of the reactive functional group that reacts with the crosslinkability of the crosslinking agent include a carboxy group, an amino group, a hydroxyl group and a thiol group. The linker compound may not have a reactive functional group other than a thiol group.

Examples of the linker compound having a carboxy group as a reactive functional group include mercaptoacetic acid, mercaptopropionic acid, 2-mercapto-5-benzimidazolecarboxylic acid, mercaptobenzoic acid, 3-mercaptohexyl acetate, 4-mercaptohydrosilicic acid. Examples include cinnamic acid, 3-mercaptoisobutyric acid, 2-mercaptonicotinic acid and thiomalic acid.

Examples of the linker compound having an amino group as a reactive functional group include aminobenzenethiol, aminoethanethiol, 5-amino-2-mercaptobenzimidazole, 2-amino-5-mercapto-1,3,4-thiadiazole, 3-amino-5-mercapto-1,2,4-triazole, 2,5-diamino-1,4-benzenedithiol dihydrochloride, 4,6-diaminopyrimidine-2-thiol, 7-amino-1-heptane Examples include thiol, 2-propylaminoethanethiol, 2-[[3-(ethylamino)propyl]amino]ethanethiol and 8-amino-3-azaoctane-1-thiol.

Examples of the linker compound having a hydroxyl group as a reactive functional group include dithioerythritol, hydroxybenzenethiol, 1-(4-hydroxyphenyl)-5-mercapto-1H-tetrazole and α-thioglycerol.

Examples of the linker compound having a thiol group as a reactive functional group include toluene-3,4-dithiol, benzenedithiol, 4,4-biphenyldithiol, 1,2-butanedithiol, 1,10-decanedithiol, 1, 5-dimercaptonaphthalene, 3,6-dioxa-1,8-octanedithiol, 3,7-dithia-1,9-nonanedithiol, dithioerythritol, 1,2-ethanedithiol, 1,6-hexanedithiol, 1 , 8-octanedithiol, 1,5-pentanedithiol, 1,3-propanedithiol, bis(2-mercaptoethyl)ether, and Karens MT (registered trademark) BD1 (manufactured by Showa Denko KK). Be done.

From the viewpoint of reactivity with the particles or the crosslinking agent, the molecular weight of the linker compound is preferably 500 to 100, more preferably 400 to 100, and even more preferably 300 to 100.

Separation material according to the present embodiment, the step of introducing a linker group to the cross-linked polymer, on the surface of at least a portion of the porous polymer particles containing a cross-linked polymer introduced linker group, a polymer having a hydroxyl group. It can be produced by a method including a step of adsorbing, and a step of crosslinking at least a part of the polymer having a hydroxyl group with a crosslinking agent to form a coating layer and binding the crosslinking agent and a linker group. ..

The step of introducing a linker group into the crosslinked polymer is a step of binding the crosslinked molecule and the linker compound via a thio group by a thiolene reaction between a thiol group in the linker compound and a double bond in the crosslinked polymer. It is preferable to include. The thiolene reaction can be carried out by a known method. For example, it can be carried out by dispersing porous polymer particles in a solution containing a linker compound and a radical polymerization initiator and then heating the solution.

The step of adsorbing the polymer having a hydroxyl group on the surface of at least a part of the porous polymer particles containing the crosslinked polymer having the linker group introduced therein may include the following operations. First, a solution containing a polymer having a hydroxyl group is impregnated into porous polymer particles containing a cross-linked polymer having a linker group introduced therein. Then, the particles are filtered and washed with a solvent such as water and alcohol to remove unadsorbed polymer.

The solvent of the solution of the polymer having a hydroxyl group is not particularly limited as long as it can dissolve the polymer having a hydroxyl group, and can be, for example, water. The concentration of the polymer having a hydroxyl group in the solution is preferably 5 to 20 mg/mL. The impregnation method is not particularly limited, but a method of adding porous polymer particles to a solution of a polymer having a hydroxyl group and allowing it to stand for a certain period of time can be mentioned. Although the impregnation time depends on the surface state of the porous body, normally, when impregnating for one day, the polymer concentration is in equilibrium between the inside and outside of the porous body.

The step of forming a coating layer by cross-linking at least a part of the polymer having a hydroxyl group with a cross-linking agent and binding the cross-linking agent and the linker group can include the following operations, for example. That is, porous polymer particles impregnated with a solution of a polymer having a hydroxyl group are dispersed and suspended in an appropriate medium, and a crosslinking agent is added thereto. The cross-linking agent cross-links the polymer having a hydroxyl group, which is held in solution on the surface of the porous polymer particles, and coats the polymer having a cross-linked hydroxyl group and the cross-linking agent on the porous polymer particles. Is formed. The coating layer has a three-dimensional crosslinked network structure having a hydroxyl group.

When a cross-linking agent is added, along with the cross-linking reaction of the polymer having a hydroxyl group as described above, the reaction between the linker group and the cross-linking agent introduced on the porous polymer particles also proceeds, and the linker group in the porous polymer particles The cross-linked polymer and the cross-linking agent are bound (immobilized). As a result, the coating layer is favorably held on the porous polymer particles, and the durability of the separating material is improved. It is also preferable from the viewpoint of improving productivity that the coating layer can be formed and the coating layer can be immobilized on the porous polymer at the same time.

After completion of the crosslinking reaction, the generated porous polymer particles having a coating layer are separated by filtration, and then washed with a hydrophilic organic solvent such as methanol and ethanol, water or an aqueous solution to obtain unreacted polymer and suspension medium. By removing at least a part of the surface of the porous polymer particle is covered with the coating layer, a separation material in which the porous polymer particle and the coating layer are bonded via a thio group can be obtained.

A medium for dispersing and suspending the porous polymer particles impregnated with a solution of a polymer having a hydroxyl group does not extract the polymer having a hydroxyl group and a crosslinking agent impregnated in the porous polymer particles, and , Can be inert to the crosslinking reaction. Examples of the medium include water, an aqueous solution, and alcohol.

When a polysaccharide is used as the polymer having a hydroxyl group, the addition amount of the cross-linking agent may be selected within the range of 0.1 to 100 equivalents per equivalent of monosaccharide, depending on the performance of the separating material. it can. When the addition amount of the cross-linking agent is not more than the above lower limit value, the coating layer tends to be well retained on the porous polymer particles. When the addition amount of the cross-linking agent is not more than the above upper limit, the characteristics of the polymer having a hydroxyl group are not easily impaired even when the reaction rate between the cross-linking agent and the polymer having a hydroxyl group is high.

A catalyst can be used for the crosslinking reaction. As the catalyst, a conventionally known catalyst can be appropriately used according to the kind of the crosslinking agent. For example, when the crosslinking agent is epichlorohydrin, an alkali such as sodium hydroxide is effective as a catalyst, and when it is a dialdehyde compound, a mineral acid such as hydrochloric acid is effective.

The amount of the catalyst used depends on the kind of the cross-linking agent, but when a polysaccharide is used as the polymer having a hydroxyl group, it is preferably 0.01 to 10 equivalents relative to 1 equivalent of the polysaccharide. , 0.1 to 5 equivalents are more preferable.

When the cross-linking reaction between the cross-linking agent and the polymer having a hydroxyl group can be controlled by the addition of a catalyst or the like, the porous polymer particles are impregnated with a solution of the polymer having a hydroxyl group mixed with a cross-linking agent in advance. By dispersing and suspending in a suitable medium and adding a catalyst and the like thereto, the crosslinking agent and the polymer having a hydroxyl group can be crosslinked. When a catalyst or the like is not used, the crosslinking reaction can be caused by changing the crosslinking reaction conditions such as temperature. For example, porous polymer particles impregnated with a solution in which a polymer having a hydroxyl group and a cross-linking agent are dissolved are dispersed and suspended in an appropriate medium, and then the cross-linking reaction conditions (for example, temperature) are the conditions under which the reaction proceeds. By adjusting the ratio to 1, the crosslinking reaction between the crosslinking agent and the polymer having a hydroxyl group can be carried out. When the cross-linking reaction conditions are temperature conditions, the temperature of the reaction system is raised, and when the temperature reaches the reaction temperature, the cross-linking reaction occurs.

The crosslinking reaction can be performed at 5 to 90° C. for 1 to 10 hours. The temperature of the crosslinking reaction is preferably 30 to 90°C.

The separating material according to this embodiment may have an ion exchange group or a ligand (for example, protein A) in addition to the porous polymer particles, the linker group and the coating layer. The ion exchange group or ligand can be introduced, for example, via a hydroxyl group or the like on the surface of the separation material. Since the separation material has an ion exchange group or a ligand, the separation material can be used for ion exchange purification, affinity purification and the like. Examples of the method of introducing the ion exchange group include a method using a halogenated alkyl group-containing compound.

Examples of the ion exchanger include an amino group that is a weakly basic group, a quaternary ammonium group that is a strongly basic group, a carboxy group that is a weakly acidic group, and a sulfonic acid group that is a strongly acidic group.

As a method of using a halogenated alkyl group-containing compound, for example, after the particles of the separating material in a wet state are drained by filtration or the like, immersed in an alkaline aqueous solution of a predetermined concentration for a certain period of time, and then halogenated in a water-organic solvent mixture system. Examples include adding an alkyl group-containing compound and reacting. This reaction is preferably carried out under heating under reflux at 40 to 90°C for 0.5 to 12 hours. The type of the halogenated alkyl group-containing compound determines the ion exchange group to be provided.

As the halogenated alkyl group-containing compound, a monohalogen carboxylic acid such as monohalogen acetic acid or monohalogen propionic acid, a sodium salt thereof or a primary halogen, secondary or tertiary compound having at least one halogenated alkyl group such as diethylaminoethyl chloride is used. Included are primary amines as well as quaternary ammonium hydrochlorides having alkyl halide groups. These halogenated alkyl group-containing compounds are preferably bromides or chlorides. The addition amount of the halogenated alkyl group-containing compound is preferably 0.2% by mass or more based on the total mass of the separation material to which the ion exchange group is added.

When an amino group that is a weakly basic group is introduced as the ion-exchange group, examples of the halogenated alkyl group-containing compound include, for example, mono-, in which at least one of the alkyl groups is substituted with a halogenated alkyl group. Di- or tri-alkylamines, mono-, di- or tri-alkanolamines, mono-alkyl-mono-alkanolamines, di-alkyl-mono-alkanolamines and mono-alkyl-di-alkanolamines can be used.

As a method for introducing a quaternary ammonium group which is a strongly basic group as an ion exchange group, for example, first, a tertiary amino group is introduced, and a compound containing a halogenated alkyl group such as epichlorohydrin is reacted with the tertiary amino group. Then, a method of converting to a quaternary ammonium group and a method of reacting a quaternary ammonium hydrochloride or the like with the separating material can be mentioned.

When a carboxy group, which is a weakly acidic group, is introduced as the ion-exchange group, examples of the halogenated alkyl group-containing compound include monohalogen carboxylic acids such as monohalogen acetic acid and monohalogen propionic acid, and sodium salts thereof. ..

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

Examples of the method of introducing the ion exchange group also include a method of reacting 1,3-propane sultone with the separation material in an alkaline atmosphere. The amount of 1,3-propane sultone is preferably 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.

Other methods for introducing an ion exchanger include, for example, a method of reacting sulfopropyl with a separation material, and after adding an epihalohydrin diglycidyl compound to the separation material, an ion exchange group is added to an epoxy group in the added compound. There is also a method of introduction.

To introduce the ion exchange group, an organic solvent can be used in order to accelerate the reaction. Examples of the organic solvent include alcohols such as ethanol, 1-propanol, 2-propanol, 1-butanol, isobutanol, 1-pentanol and isopentanol.

The separation material of the present embodiment is suitable for separation by electrostatic interaction of proteins or affinity purification. For example, the separation material of the present embodiment is added to a solution containing a protein, and the protein is adsorbed to the separation material by electrostatic interaction, and then the separation material is filtered from the solution to obtain a solution in a high salt concentration solution. The protein adsorbed on the separation material can be easily desorbed and collected by adding to. The separation material of this embodiment can be used in column chromatography. That is, the column of this embodiment includes the separating material of this embodiment.

When the column is filled with the separating material of the present embodiment, it is preferable that the liquid passing rate when the column pressure is 0.3 MPa is 800 cm/h or more. The liquid passing rate in this specification is such that water is passed through a stainless steel column having a size of φ7.8×300 mm filled with the separating material of the present embodiment so that the pressure in the column becomes a predetermined value. Represents the flow velocity of water when it is struck. When proteins and the like are separated by column chromatography using a separation material that has been commonly used in the past, the flow rate of the protein solution and the like is generally in the range of 400 cm/h or less. On the other hand, when the separation material of the present embodiment is used, a high dynamic adsorption amount can be maintained even when the liquid passing speed is 800 cm/h or more, which is faster than the conventional separation material for protein separation.

The biopolymer that is separated using the separation material of this embodiment is preferably a water-soluble substance. Specific examples thereof include blood proteins such as serum albumin and immunoglobulins, enzymes existing in the living body, physiologically active substances of proteins produced by biotechnology, DNA and biopolymers such as peptides having physiological activity. . The molecular weight of the biopolymer is preferably 2,000,000 or less, more preferably 500,000 or less. The properties and conditions of the separating material can be selected according to a known method depending on the isoelectric point and ionization state of the protein. Known methods include, for example, the method described in JP-A-60-169427.

The separating material of the present embodiment has the advantages of the natural polymer particles and the synthetic polymer particles in the separation of biopolymers such as proteins, and introduces ion-exchange groups and ligands on the surface of the separating material. By doing so, a more remarkable effect can be obtained. The separation material of the present embodiment has the advantages that it has durability and alkali resistance, reduces nonspecific adsorption of proteins, easily adsorbs and desorbs proteins, and has a large dynamic adsorption amount. Furthermore, the separation material of the present embodiment is excellent in operability when the column is filled with the separation material and the volume of the separation material hardly changes in the column regardless of the property of the eluent. Be effective.

The preferred embodiments of the present invention have been described above in detail, but the present invention is not limited to the above embodiments, and various modifications are possible. For example, a separation material having no ion exchange group can be used for gel filtration chromatography.

Hereinafter, the present invention will be specifically described based on Examples, but the present invention is not limited to the following Examples.
(Example 1)
1-1. Synthesis of Porous Polymer Particles In a 500 mL three-necked flask, 16 g of 96% pure divinylbenzene (manufactured by Nippon Steel & Sumikin Co., Ltd., trade name: DVB960) as a monomer, 16 g of dodecanol and 16 g of toluene as a porosifying agent, and a peroxide as a polymerization initiator. 0.64 g of benzoyl oxide was added to a polyvinyl alcohol (0.5% by mass) aqueous solution containing a dispersant to prepare a mixed solution. This mixed solution was emulsified using a micro process server, and then the emulsified solution was transferred to a flask and stirred with a stirrer for about 8 hours while being heated in a water bath at 80°C. The particles produced by polymerizing the monomer were filtered and washed with acetone to obtain porous polymer particles 1. The particle size of the porous polymer particles 1 was measured by a flow-type particle size measuring device (FPIA-3000, manufactured by Sysmex Co., Ltd.), and the average particle size (μm) and the coefficient of variation C.I. V. (%) was calculated. The results are shown in Table 1.

1-2. Introduction of Linker Group on Porous Polymer Particle Surface 10 g of porous polymer particle 1, 0.1 mmol of 4-aminobenzenethiol and 1 mmol of azobisisobutyronitrile were added to 100 mL of N,N-dimethylformamide. The resulting dispersion was stirred at a temperature of 70° C. for 12 hours to introduce a linker group derived from 4-aminobenzenethiol on the surface of the porous polymer particles. The porous polymer particles having the linker group introduced therein were taken out from the dispersion and washed with acetone.

1-3. Introduction of Hydrophobic Group to Polymer Having Hydroxyl Group To 100 mL of agarose aqueous solution (concentration 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 phenyl group into agarose. did. The obtained denatured agarose was reprecipitated with isopropyl alcohol and washed.

2-1. Adsorption of Polymer Having Hydroxyl Group on Surface of Porous Polymer Particles Addition of 1 g of porous polymer particles 1 having a linker group introduced to 70 mL of 20 mg/mL modified agarose aqueous solution, and stirring at 55° C. for 24 hours, The modified agarose was adsorbed on the porous polymer particles 1. After the adsorption, the porous polymer particles 1 were separated by filtration and washed with hot water.

2-2. Formation of coating layer The modified agarose adsorbed on the surface of the porous polymer particles 1 was crosslinked as follows. Particles having denatured agarose adsorbed thereto were added at a ratio of 1 g to 35 mL of an aqueous solution having an ethylene glycol diglycidyl ether concentration of 0.64 M and a sodium hydroxide concentration of 0.4 M, and the mixture was stirred at room temperature for 24 hours. As a result, the modified agarose was crosslinked with ethylene glycol diglycidyl ether and the ethylene glycol diglycidyl ether and the linker group were bonded. Then, the particles were washed with a 2% by mass aqueous solution of sodium dodecyl sulfate and then with pure water to obtain a separating material.

2-3. Evaluation of Non-Specific Adsorption Ability of Protein 0.5 g of a separation material was added to 50 ml of a phosphate buffer (pH 7.4) having a BSA (Bovine Serum Albumin) concentration of 20 mg/mL, and the mixture was stirred at room temperature for 24 hours. Then, the supernatant was taken by centrifugation, and the amount of BSA adsorbed on the separation material was calculated from the BSA concentration of the supernatant obtained by measuring the absorbance of the supernatant at 280 nm with a spectrophotometer. The non-specific adsorption amount was evaluated as “◯” when the BSA adsorption amount per 1 mL of the separation material was less than 1 mg, “Δ” when it was 1 to 10 mg, and “x” when it was more than 10 mg. The results are shown in Table 3.

3-1. Introduction of ion-exchange group The separating material after cross-linking (dry weight: 20 g) was added to 200 mL of a 5 M aqueous sodium hydroxide solution, and the mixture was allowed to stand at room temperature for 1 hour. A solution obtained by dissolving 60 g of diethylaminoethyl chloride hydrochloride in 200 mL of water was added to this aqueous sodium hydroxide solution, and the mixture was stirred at 70° C. for 2 hours. After completion of the reaction, the reaction solution was filtered and the separation material was washed with water to obtain a DEAE-modified separation material having a diethylaminoethyl (DEAE) group as an ion exchange group. The DEAE-modified separation material was used for the subsequent evaluation. After the obtained DEAE-modified separation material was dried, the amount of the coating layer was measured by thermogravimetric analysis. That is, first, a predetermined amount of porous polymer particles was heated (30° C./min) from 30° C. to 600° C. by thermogravimetric analysis, and the 5% thermogravimetric reduction temperature of the porous polymer particles was measured. Next, a predetermined amount of the separating material was heated from 30° C. at a heating rate of 10° C./min, and the thermogravimetric reduction amount per 1 g of the separating material at the above temperature was measured. Separately, the thermogravimetric reduction amount of a predetermined amount of the coating layer at the above temperature was measured, and the thermogravimetric reduction amount per 1 g of the coating layer at the above temperature was calculated. Here, the rate of weight loss of the porous polymer particles at the above temperature was set to 5%, and the coating amount of the separating material was calculated from these values.

3-2. Measurement of Mode Pore Diameter and Specific Surface Area After drying the DEAE-modified separation material, the mode diameter and specific surface area in the pore size distribution were measured by the mercury porosimetry method. The results are shown in Table 2.

3-3-1. Packing in column The DEAE-modified separation material was mixed with water to obtain a slurry having a concentration of 30% by mass. This slurry was filled in a φ7.8 mm×300 mm stainless steel column under a pressure of 4 MPa for 15 minutes in an amount of 65 mL, and used for the following evaluation of liquid permeability and dynamic adsorption amount.

3-3-2. Evaluation of Liquid Permeability Water was passed through the column packed with the DEAE-modified separation material while changing the liquid passage speed, and the relationship between the liquid passage speed and the column pressure was measured. The liquid passage rate (flow rate) when the column pressure was 0.3 MPa was determined. When the column pressure is 0.3 MPa, the liquid passage rate is 1500 cm/h or more, it is “◯”, when it is 800 cm/h or more and less than 1500 cm/h, it is “Δ”, and it is less than 800 cm/h. The case was designated as "x". The results are shown in Table 3.

3-3-3. Evaluation of Dynamic Adsorption Amount of 20 mmol/L of Tris-hydrochloric acid buffer solution (pH 8.0) was applied to the column packed with the DEAE-modified separation material in 10 column volumes. Then, the BSA concentration in the eluate at the column outlet was measured by UV absorption measurement while flowing a 20 mmol/L Tris-hydrochloric acid buffer solution having a BSA concentration of 2 mg/mL. The liquid passing speed was the same as the speed when the column pressure was 0.3 MPa in the liquid passing evaluation. The buffer solution was flowed until the BSA concentrations at the column inlet and outlet coincided with each other, and then 5 column volumes of 1 M NaCl Tris-hydrochloric acid buffer solution were caused to flow. The amount of dynamic adsorption at 10% breakthrough was calculated using the following formula. When the dynamic adsorption amount is 50 mg/particle mL or more, it is indicated by "○", when it is 20-50 mg/particle mL, it is indicated by "△", and when it is 20 mg/particle mL or less, it is indicated by "x", and the dynamic adsorption amount is indicated by evaluated. The results are shown in Table 3. The 10% breakthrough refers to a state in which 10% by mass of BSA is adsorbed on the separation material with respect to 100% by mass of the BSA adsorption amount when the adsorption of the separation material is saturated.
_{· Q 10 = c f F (} t 10 -t 0) / V B
· _Q 10: dynamic adsorption amount at 10% breakthrough (mg / separation member mL)
_-Cf : BSA concentration (mg/mL) of the injectate
・F: Flow rate (mL/min)
・V _B : Bed volume (mL)
· _T 10: 10% break time in the through (min)
・T ₀ : BSA injection start time (min)

3-4. Durability Evaluation A DEAE-modified separation material was packed in a φ4.6×150 mm column in the same manner as in the evaluation of column characteristics above. A 20 mmol/L Tris-hydrochloric acid buffer solution (pH 8.0) having a BSA concentration of 2 mg/mL was flowed until the BSA was saturated and adsorbed (until the column eluate was about the same as the sample solution concentration), and the DEAE denaturing separation material was used. BSA was adsorbed on the column packed with and the adsorption amount of BSA was measured. The amount of BSA adsorbed was measured by measuring the absorbance of the eluate. 6 column volumes of 0.5 M NaCl/0.05 M Tris buffer (pH 8.0) was applied to desorb the adsorbed BSA, and 0.5 column of 0.5 M NaOH aqueous solution was applied to 3 column volumes to remove the column. Was washed. This cycle was repeated 100 times, and the reduction rate of the adsorption amount of BSA was calculated. The case where the decrease rate of the BSA adsorption amount was 15% or less was represented by “◯”, the case of 15-40% was represented by “Δ”, and the case of 40% or more was represented by “x”. The results are shown in Table 3.

(Example 2)
Porous polymer particles 2 were synthesized in the same manner as porous polymer particles 1 except that 18 g of dodecanol and 14 g of toluene were used as the porosifying agent. The obtained porous polymer particles 2 were treated in the same manner as in Example 1 to obtain a separating material. The same evaluation as in Example 1 was performed on the porous polymer particles 2 and the separating material.
(Example 3)
Porous polymer particles 3 were synthesized in the same manner as porous polymer particles 1 except that 22 g of dodecanol and 10 g of toluene were used as the porosifying agent. The obtained porous polymer particles 3 were treated in the same manner as in Example 1 to obtain a separating material. The same evaluation as in Example 1 was performed on the porous polymer particles 3 and the separating material.
(Example 4)
The porous polymer particles 1 and the separating material were evaluated in the same manner as in Example 1 except that bis(2-mercaptoethyl)ether was used as the linker compound.
(Example 5)
The porous polymer particles 1 and the separating material were evaluated in the same manner as in Example 1 except that thioglycerol was used as the linker compound.
(Example 6)
The porous polymer particles 1 and the separating material were evaluated in the same manner as in Example 1 except that 4-hydroxybenzenethiol was used as the linker compound.
(Example 7)
The porous polymer particles 1 and the separating material were evaluated in the same manner as in Example 1 in the same manner as in Example 1 except that decandithiol was used as the linker compound.
(Example 8)
In the same manner as in Example 1 except that trimethylolethanetris(3-mercaptobutyrate) was used as the linker compound, the same evaluation as in Example 1 was performed on the porous polymer particles 1 and the separating material.
(Comparative Example 1)
Various evaluations were performed in the same manner as in Example 1 except that a coating layer was not formed on the surface of the porous polymer particle 1 and the porous polymer particle 1 having a functional group and a thioether bond introduced therein was used as a separating material. It was
(Comparative example 2)
The porous polymer particles 1 and the separation material were evaluated in the same manner as in Example 1 in the same manner as in Example 1 except that the linker group was not introduced into the porous polymer particles 1.
(Comparative example 3)
The porous polymer particles 1 and the separating material were evaluated in the same manner as in Example 1 except that benzenethiol was used instead of the linker compound.
(Comparative example 4)
Various evaluations were performed in the same manner as in Example 1 except that CaptoDEAE (manufactured by GE Healthcare Co., Ltd.) was used instead of the porous polymer particles 1 and the separating material.
(Comparative example 5)
Similar to the porous polymer particle 1 except that 2,3-dihydroxypropyl methacrylate (11.2 g), ethylene glycol dimethacrylate (4.8 g) as a monomer and SPAN80 (5 g) as an oil-soluble surfactant (emulsifier) are used. Then, the porous polymer particles 4 were synthesized. Various evaluations were performed in the same manner as in Example 1 except that the linker groups were not introduced into the porous polymer particles 4 and the coating layer was not formed, and the porous polymer particles 4 themselves were used as the separating material.

As shown in Table 3, it was found that the separation materials of Examples 1 to 6 had good column flow rate, non-specific adsorption amount, dynamic adsorption amount and durability.

Claims (11)

Porous polymer particles containing a cross-linked polymer,
A coating layer containing at least a part of the surface of the porous polymer particles, a polymer having a hydroxyl group and a crosslinking agent that crosslinks the same,
A linker group bonded to the cross-linked polymer and the cross-linking agent in the porous polymer particles ,
Equipped with
The linker group has a thio group bonded to the cross-linked polymer,
Separation material. The separation material according to claim 1, wherein the crosslinked polymer contains a monomer unit derived from divinylbenzene. The separation material according to claim 1 or 2, wherein the linker group is derived from a linker compound having a molecular weight of 500 or less. The separation material according to any one of claims 1 to 3, which is a particle having a specific surface area of 20 m ² /g or more. The maximum value of the pore volume in the pore volume distribution of the separation material measured by the mercury porosimetry is in the range of 0.05 to 0.7 μm of the pore diameter. Separation material. The separating material according to any one of claims 1 to 5, wherein the average particle diameter of the porous polymer particles is 10 to 300 µm. The separation 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 separation 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. When water is passed through a stainless steel column having a size of φ7.8×300 mm filled with the separation material so that the pressure in the column is 0.3 MPa, the flow rate of water is 800 cm/h or more. The separation material according to any one of claims 1 to 9, which is A column comprising the separation material according to claim 1.