JP2017196544A - Separation material and column - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a separation material that reduces nonspecific absorption of protein, and has excellent liquid permeability when charged into a column.SOLUTION: A separation material has a hydrophobic polymer particle, and a coating layer that coats at least part of the surface of the hydrophobic polymer particle, the coating layer containing a functional group-containing polymer having a hydroxy group and another functional group.SELECTED DRAWING: None

Description

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

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

  On the other hand, an ion exchanger based on a cross-linked gel of a hydrophilic natural polymer typified by polysaccharides such as dextran and agarose has an advantage that there is almost no non-specific adsorption of protein. However, this ion exchanger swells remarkably in an aqueous solution, and has the disadvantage that the volume change due to the ionic strength of the solution, the volume change between the free acid form and the loaded form is large, and the mechanical strength is not sufficient. In particular, when a cross-linked gel is used in chromatography, there is a disadvantage that the pressure loss during liquid passage is large and the gel is consolidated by liquid passage.

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

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

  However, the conventional separation material has many problems such as non-specific adsorption of proteins and poor liquid permeability when packed in a column.

  Therefore, an object of the present invention is to provide a separation material that reduces nonspecific adsorption of proteins and has excellent liquid permeability when packed in a column.

Specific embodiments of the present invention are shown below.
[1] A hydrophobic polymer particle and a coating layer that covers at least a part of the surface of the hydrophobic polymer particle, wherein the coating layer has a functional group-containing polymer having a hydroxyl group and a functional group other than a hydroxyl group. Separation material containing molecules.
[2] The separating material according to [1], wherein the functional group other than the hydroxyl group is a carboxy group, an epoxy group, or an amino group.
[3] The separation material according to [1] or [2], wherein a 5% compressive deformation elastic modulus in water is 70 MPa or more.
[4] The separation material according to any one of [1] to [3], wherein when the column is packed, the liquid flow rate is 500 cm / h or more when the column pressure is 0.3 MPa.
[5] The separating material according to any one of [1] to [4], wherein the hydrophobic polymer particles include a hydrophobic polymer having a structural unit derived from a styrene monomer.
[6] The separating material according to any one of [1] to [5], wherein the functional group-containing polymer includes a structure derived from a polysaccharide or a modified product thereof.
[7] The separating material according to any one of [1] to [6], wherein the functional group-containing polymer includes a structure derived from agarose or a modified product thereof.
[8] The separating material according to any one of [1] to [7], wherein the functional group-containing polymer is crosslinked.
[9] The separation material according to any one of [1] to [8], wherein the hydrophobic polymer particles have an average particle size of 10 to 500 μm.
[10] The separation material according to any one of [1] to [9], wherein the hydrophobic polymer particles have a porous structure.
[11] A column comprising the separation material according to any one of [1] to [10].

  ADVANTAGE OF THE INVENTION According to this invention, the nonspecific adsorption | suction of protein is reduced and a column provided with the separation material which is excellent in liquid permeability when it fills a column, and the said separation material can be provided.

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

<Separation material>
The separation material of this embodiment includes hydrophobic polymer particles and a coating layer that covers at least a part of the surface of the hydrophobic polymer particles, and the coating layer has hydroxyl groups and functional groups other than hydroxyl groups. A functional group-containing polymer. For example, in the case where the hydrophobic polymer particles have a porous structure, the “surface of the hydrophobic polymer particles” means not only the outer surface of the hydrophobic polymer particles but also the hydrophobic polymer particles. It shall include the surface of the pores inside the molecular particle. According to the separation material of the present embodiment, non-specific adsorption of proteins is reduced, and excellent liquid permeability is obtained when packed in a column. In addition, the separation material of this embodiment is excellent in durability, alkali resistance, and pressure resistance, and the adsorption amount (dynamic adsorption amount) when packed in a column is considered to be sufficiently high in practice.

  The present inventors presume the reason why the separating material of the present embodiment exhibits the above-described effects as follows.

  The separation material of this embodiment is considered to have the advantages of both the hydrophobic polymer particles and the functional group-containing polymer by including the hydrophobic polymer particles and the coating layer. For example, it is considered that the separation material of this embodiment is excellent in durability, alkali resistance and pressure resistance by providing the hydrophobic polymer particles, and exhibits excellent liquid permeability when packed in a column. In addition, since the separation material of the present embodiment includes the coating layer, nonspecific adsorption of proteins is reduced, and adsorption and desorption of proteins and the like are likely to occur. It is considered that the amount of adsorption is large.

(Hydrophobic polymer particles)
The hydrophobic polymer particles according to the present embodiment are particles containing a polymer having hydrophobicity (hereinafter sometimes referred to as “hydrophobic polymer”). Although there is no restriction | limiting in particular in the manufacturing method of hydrophobic polymer particle, For example, the method of polymerizing the monomer which can form the polymer which has hydrophobicity is mentioned. The monomer is not particularly limited as long as it can form a hydrophobic polymer, and examples thereof include a styrene monomer. That is, the hydrophobic polymer particles may include, for example, a hydrophobic polymer having a structural unit derived from a styrene monomer.

  The hydrophobic polymer particles according to the present embodiment may have a porous structure. That is, the hydrophobic polymer particles according to the present embodiment may be, for example, hydrophobic porous polymer particles. The porous polymer particle is, for example, a particle containing a polymer obtained by polymerizing a monomer in the presence of a porosifying agent, and can be synthesized by conventional suspension polymerization, emulsion polymerization, or the like.

  Examples of the styrenic monomer include the following polyfunctional monomers and monofunctional monomers.

  Examples of the polyfunctional monomer include divinyl compounds such as divinylbenzene, divinylbiphenyl, divinylnaphthalene, and divinylphenanthrene. These polyfunctional monomers can be used alone or in combination of two or more. Among these, divinylbenzene is preferable from the viewpoint of 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 alone or in combination of two or more. Among these, styrene is preferable from the viewpoint of excellent acid resistance and alkali resistance. The monomer may be a styrene derivative having a functional group such as a carboxy group, an amino group, a hydroxyl group, and an aldehyde group.

  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, diethylbenzene and the like. These porous materials can be used alone or in combination of two or more.

  The amount of the porosifying agent may be, for example, 0 to 200% by mass with respect to the total mass of the monomer. The porosity of the hydrophobic polymer particles can be controlled by the amount of the porous agent. Furthermore, the size and shape of the pores of the hydrophobic polymer particles can be controlled by the type 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 greater than about 40% purity, more preferably greater than about 50% purity, and even more preferably greater than about 70% purity. Sorbitan monooleate (eg, SPAN 80, preferably about 40% purity, more preferably about 50% purity, more preferably more than about 70% purity sorbitan monooleate); Diglycerol monooleate (eg, greater than about 40% purity, more preferably greater than about 50% purity, more preferably greater than about 70% purity); diglycerol monoisostearate (eg, Preferably greater than about 40% purity, more preferably greater than about 50% purity More preferably a diglycerol monoisostearate of greater than about 70% purity); diglycerol monomyristate (preferably greater than about 40% purity, more preferably greater than about 50% purity, even more preferably about 70% purity). More preferred sorbitan monomyristate); cocoyl (eg, lauryl, myristoyl, etc.) ethers of diglycerol; and mixtures thereof.

  These oil-soluble surfactants are preferably used in the range of 5 to 80% by mass with respect to the total mass of the monomer. When the content of the oil-soluble surfactant is 5% by mass or more, the stability of the water droplets becomes sufficient, so that a large single hole is easily formed. Further, when the content of the oil-soluble surfactant is 80% by mass or less, it becomes easier for the hydrophobic polymer particles to retain the shape after polymerization.

  An aqueous medium can also be used for the polymerization reaction. Examples of the aqueous medium 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 nonionic surfactants such as silicon polyethylene oxide adducts and polypropylene oxide adducts, and fluorine 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.

  In the said polymerization reaction, a polymerization initiator can also be added as needed. Examples of the polymerization initiator include benzoyl peroxide, lauroyl peroxide, orthochlorobenzoyl peroxide, orthomethoxybenzoyl peroxide, 3,5,5-trimethylhexanoyl peroxide, tert-butylperoxy-2-ethylhexa Organic peroxides such as noate and di-tert-butyl peroxide; and 2,2′-azobisisobutyronitrile, 1,1′-azobiscyclohexanecarbonitrile, 2,2′-azobis (2, Azo compounds such as 4-dimethylvaleronitrile). A polymerization initiator can be used in 0.1-7.0 mass parts with respect to 100 mass parts of monomers, for example.

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

  Examples of the polymer dispersion stabilizer include polyvinyl alcohol, polycarboxylic acid, celluloses (hydroxyethyl cellulose, carboxymethyl cellulose, methyl cellulose, etc.), and polyvinyl pyrrolidone, and inorganic water-soluble polymer compounds such as sodium tripolyphosphate are also included. Can be used together. 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 hydrophobic polymer particles is preferably 10 μm or more from the viewpoint of further improving liquid permeability. The average particle diameter of the hydrophobic polymer particles is preferably 500 μm or less from the viewpoint of column separability. From these viewpoints, the hydrophobic polymer particles may have an average particle size of, for example, 10 to 500 μm.

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

C. of average particle diameter and particle diameter of hydrophobic polymer particles or separation material V. (Coefficient of variation) can be determined by the following measurement method.
1) Disperse particles or a separating material in water (including a dispersing agent such as a surfactant) using an ultrasonic dispersion device to prepare a dispersion liquid containing 1% by mass of the particles or the separating material.
2) Using a particle size distribution meter (Sysmex Flow, manufactured by Sysmex Corporation), the average particle size and particle size of C.I. V. (Coefficient of variation) is measured.

  When the hydrophobic polymer particles have a porous structure, the porosity (pore volume) of the particles is preferably 30% by volume or more and 70% by volume or less based on the total volume of the hydrophobic polymer particles. It is more preferable that the volume be not less than 70% by volume. In addition, the hydrophobic polymer particles have pores having a mode diameter (mode of pore diameter distribution, maximum frequency pore diameter) of 0.1 μm or more and less than 0.5 μm, that is, macropores. It is preferable to have. The mode diameter in the pore size distribution of the hydrophobic polymer particles is more preferably 0.2 μm or more and less than 0.5 μm. If the mode diameter in the pore size distribution is 0.1 μm or more, the substance tends to easily enter the pores. If the mode diameter in the pore diameter distribution is less than 0.5 μm, the specific surface area is sufficient. Become. These can be adjusted by the above-mentioned porous agent.

The specific surface area of the hydrophobic 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.

  The mode diameter, specific surface area and porosity in the pore size distribution of the hydrophobic polymer particles or separation material according to the present embodiment are values measured with a mercury intrusion measuring apparatus (Autopore: manufactured by Shimadzu Corporation), and the following Measure 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. Moreover, it limits to the range of 0.1-3 micrometers of pore diameters, and calculates each value.

(Coating layer)
The coating layer according to the present embodiment includes a functional group-containing polymer having a hydroxyl group and a functional group other than the hydroxyl group. In the functional group-containing polymer, the functional group is bonded to the polymer by a chemical bond. When the coating layer is such, ion exchange property or binding property with a ligand is imparted.

  Examples of the functional group include a carboxy group, an epoxy group, an amino group, a quaternary ammonium group, and a sulfonic acid group. The functional group is preferably a carboxy group, an epoxy group, or an amino group. Specific examples of amino groups include diethylaminoethyl (DEAE) groups.

  The functional group-containing polymer may contain, for example, a group represented by the following formula (a) or (b).

  In formula (a), X represents a group containing a carboxy group. Specific examples of X include an alkyl group substituted with a carboxy group.

  In formula (b), Y represents a group containing an amino group or a group containing an epoxy group. Specific examples of Y include a glycidyl group and a diethylaminoethyl group.

  The functional group-containing polymer may be a hydrophilic polymer.

  From the viewpoint of improving hydrophilicity, the functional group-containing polymer may include a structure derived from a polymer having a hydroxyl group or a modified product thereof. For example, the functional group-containing polymer may have a structure in which one or more hydroxyl groups are removed from a polymer having a hydroxyl group or a modified product thereof. 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 and polyvinyl alcohol. Examples of polysaccharides include agarose, dextran, cellulose, and chitosan. That is, the functional group-containing polymer may include a structure derived from a polysaccharide or a modified product thereof, or may include a structure derived from agarose or a modified product thereof.

  The weight average molecular weight (Mw) of the polymer having a hydroxyl group may be, for example, about 10,000 to 200,000.

  Examples of the modified polymer having a hydroxyl group include those obtained by modifying a polymer having a hydroxyl group with a hydrophobic group (hereinafter also referred to as “hydrophobic group-modified product”). When the functional group-containing polymer contains a structure derived from such a modified product, the interfacial adsorption ability tends to be improved. Examples of the hydrophobic group include an alkyl group having 1 to 6 carbon atoms and an aryl group having 6 to 10 carbon atoms. Examples of the alkyl group having 1 to 6 carbon atoms include a methyl group, an ethyl group, and a propyl group. Examples of the aryl group having 6 to 10 carbon atoms include a phenyl group and a naphthyl group. The hydrophobic group is introduced, for example, 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.

  In the hydrophobic group-modified product, a structure containing a hydrophobic group relative to the total molar amount of all the structural units constituting the hydrophobic group-modified product (sum of the molar amounts of the structural unit containing the hydrophobic group and the structural unit not containing the hydrophobic group). The content ratio of the unit is 5 to 30 from the viewpoint of balance between retention of the hydrophobic interaction force for adsorbing the functional group-containing polymer to the surface of the hydrophobic polymer particle and suppression of nonspecific adsorption of the protein. %, More preferably 10 to 20%, and still more preferably 12 to 17%.

  From the viewpoint of further suppressing the increase in column pressure, the functional group-containing polymer may be crosslinked.

(Formation method of coating layer)
The coating layer according to the present embodiment includes, for example, a step of adsorbing a polymer having a hydroxyl group or a modified product thereof on the surface of the hydrophobic polymer particles (adsorption treatment step), and a polymer having the hydroxyl group or a modified product thereof. It can be formed by a method comprising a step of crosslinking (crosslinking treatment step) and a step of introducing a functional group into the polymer having a crosslinked hydroxyl group or a modified product thereof (functional group introduction step). Hereinafter, these steps will be specifically described.

(Adsorption treatment process)
In the adsorption treatment step, for example, a polymer having a hydroxyl group or a modified product thereof is formed on the surface of the hydrophobic polymer particle by impregnating the polymer solution having a hydroxyl group or a modified product thereof with the hydrophobic polymer particle. Adsorb. Thereby, the polymer which has a hydroxyl group, or its modified body is fix | immobilized on the hydrophobic polymer particle surface. The solvent for the polymer having a hydroxyl group or a modified product thereof is not particularly limited as long as it can dissolve the polymer having a hydroxyl group or a modified product thereof, but water is most commonly used. The concentration of the polymer dissolved in the solvent is preferably 5 to 20 (mg / mL).

  Examples of the impregnation method include a method in which hydrophobic polymer particles are added to a solution of a polymer having a hydroxyl group or a modified product thereof and left for a predetermined time. Although the impregnation time varies depending on the surface state of the hydrophobic polymer particles, for example, even when the hydrophobic polymer particles have a porous structure, the polymer concentration is usually equal to the external concentration inside the porous body if it is impregnated all day and night. It becomes an equilibrium state.

  Next, the polymer is washed with a solvent such as water or alcohol to remove the polymer having an unadsorbed hydroxyl group or a modified product thereof.

(Crosslinking process)
In the crosslinking treatment step, for example, using a crosslinking agent, a polymer having a hydroxyl group adsorbed on the surface of the hydrophobic polymer particles or a modified product thereof is subjected to a crosslinking reaction to form these crosslinked products. The crosslinked body has, for example, a three-dimensional crosslinked network structure having a hydroxyl group.

  Examples of the crosslinking agent include two functional groups active on hydroxyl such as epihalohydrins such as epichlorohydrin, dialdehyde compounds such as glutaraldehyde, diisocyanate compounds such as methylene diisocyanate, and glycidyl compounds such as ethylene glycol diglycidyl ether. The compound which has the above is mentioned. Further, when the polymer having a hydroxyl group or a modified product thereof is, for example, a compound having an amino group (such as chitosan or a modified product thereof), a dihalide compound such as dichlorooctane can also be used as a crosslinking agent.

  A catalyst is usually used for this crosslinking reaction. As the catalyst, a conventionally known catalyst can be appropriately used according to the type of the crosslinking agent. For example, when the crosslinking agent is epichlorohydrin or the like, an alkali such as sodium hydroxide is effective, and in the case of a dialdehyde compound. Mineral acids such as hydrochloric acid are effective.

  The crosslinking reaction with a crosslinking agent is usually performed by adding a crosslinking agent to a system in which hydrophobic polymer particles adsorbed with a polymer having a hydroxyl group or a modified product thereof are dispersed and suspended in an appropriate medium. Is called. When the polysaccharide is used as the polymer having a hydroxyl group, the addition amount of the cross-linking agent is, for example, within a range of 0.1 to 100 mol times with respect to one unit of the monosaccharide as 1 mol. It can be selected according to the performance of the material. When the addition amount of the crosslinking agent is not less than the above lower limit value, the coating layer tends to be favorably retained on the hydrophobic polymer particles. Moreover, if the addition amount of a crosslinking agent is below the said upper limit, even if the reaction rate of the polymer which has a hydroxyl group or its modified body is high, the characteristic of the polymer which has a hydroxyl group or its modified body is hard to be impaired.

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

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

  A medium in which hydrophobic polymer particles to which a polymer having a hydroxyl group or a modified product thereof has been adsorbed is dispersed or suspended does not extract a polymer component, a crosslinking agent, etc. It can be active. Specific examples of the medium include water, toluene, dichlorobenzene, nitromethane and the like.

  The crosslinking reaction may be performed at a temperature in the range of 1 to 90 ° C, for example. The reaction time may be, for example, 1 to 36 hours, 1 to 24 hours, or 1 to 10 hours.

  When adjusting the degree of crosslinking, the crosslinking reaction can be carried out stepwise. For example, the degree of progress of crosslinking can be adjusted by performing the crosslinking reaction once and then performing the crosslinking reaction again.

  The degree of crosslinking can be evaluated from a value obtained by measuring a 5% weight loss temperature due to thermal decomposition. When the degree of crosslinking is high, the weight reduction start temperature is high, and when the degree of crosslinking is low, the weight reduction start temperature is also low. The 5% weight loss temperature due to thermal decomposition can be measured using, for example, a differential thermothermal gravimetric apparatus.

  After completion of the crosslinking reaction, the obtained particles are filtered off and then washed with a hydrophilic organic solvent such as water, methanol, ethanol, etc. to remove unreacted polymer, suspending medium and the like. Thereby, the crosslinked body covering particle | grains by which the coating layer containing the bridge | crosslinking body of the polymer which has a hydroxyl group, or its modified body was formed in at least one part of the surface of hydrophobic polymer particle can be produced.

(Functional group introduction process)
The functional group introduction step is a step of introducing a functional group into the crosslinked product. There is no restriction | limiting in particular in the method of introduce | transducing a functional group into the said crosslinked body, Arbitrary methods can be selected. Examples of the method for introducing a functional group into the crosslinked body include a method for directly introducing a functional group into the crosslinked body, and a method for introducing a functional group via a hydroxyl group of the crosslinked body. There is no restriction | limiting in particular in the introduction method of a functional group, According to the kind etc. of functional group, it can select suitably. Examples of the method for introducing a functional group through a hydroxyl group include a method using a halogenated alkyl compound. In addition, when introduce | transducing a functional group through a hydroxyl group, the functional group should just be introduce | transduced into at least one part of the hydroxyl group which a crosslinked body has.

  Examples of the halogenated alkyl compounds 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. Can be mentioned. The alkyl halide compound may be a sulfur compound, bromide or chloride.

  Examples of the method for introducing a carboxy group, which is a weakly acidic group, as the functional 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. It is done. The amount of these halogenated alkyl compounds used is preferably 0.2% by mass or more based on the total mass of the crosslinked-coated particles.

  The method for introducing a carboxy group as a functional group is, for example, by reacting a halogen group-containing glycidyl compound such as epichlorohydrin with the hydroxyl group of the crosslinked product, introducing an epoxy group, and then introducing the carboxylic acid or a salt thereof into the epoxy group. May be a method of reacting. Examples of the carboxylic acid or a salt thereof include mercaptopropionic acid and sodium mercaptoacetate.

  As a method for introducing an epoxy group as a functional group, for example, a method of reacting an epoxy group-containing compound such as epihalohydrin, ethylene glycol diglycidyl ether, 1,2,3,4-diepoxybutane, or the like with the above crosslinked product. Can be mentioned. Preferable examples of the epihalohydrin include epichlorohydrin, epibromohydrin, epiiodohydrin, β-methylepichlorohydrin, α-methylepichlorohydrin, γ-methylepichlorohydrin, and the like. It is preferable that the usage-amount of these epoxy group containing compounds is 0.2 mass% or more with respect to the total mass of bridge | crosslinking body covering particle | grains. The reaction conditions are preferably 40 to 90 ° C. and 0.5 to 12 hours.

  The method for introducing an epoxy group as a functional group may be, for example, a method in which a halogen group-containing glycidyl compound such as epichlorohydrin is reacted with the hydroxyl group of the crosslinked product.

  As a method for introducing an amino group as a functional group, for example, among the above-mentioned halogenated alkyl compounds, mono-, di- or tri-containing compounds having at least one alkyl group in which a part of hydrogen atoms are substituted with chlorine atoms. -Alkylamine, mono-, di- or tri-alkanolamine, mono-alkyl-mono-alkanolamine, di-alkyl-mono-alkanolamine, mono-alkyl-di-alkanolamine and the like can be mentioned. The amount of these halogenated alkyl compounds used is preferably 0.2% by mass or more based on the total mass of the crosslinked product-coated particles. The reaction conditions are preferably 40 to 90 ° C. and 0.5 to 12 hours. Specific examples of the alkyl halide compound used in the method include diethylaminoethyl chloride hydrochloride.

  As a method for introducing a sulfonic acid group that is a strongly acidic group as a functional group, a glycidyl compound such as epichlorohydrin is reacted with the cross-linked product, and a sulfite or bisulfite such as sodium sulfite or sodium bisulfite. And the like, and the like. The reaction conditions are preferably 30 to 90 ° C. and 1 to 10 hours.

  Also, a functional group can be introduced into the crosslinked product by a method of reacting the crosslinked product with 1,3-propane sultone in an alkaline atmosphere. 1,3-propane sultone is preferably used in an amount of 0.4% by mass or more based on the total mass of the crosslinked product-coated particles. The reaction conditions are preferably 0 to 90 ° C. and 0.5 to 12 hours.

  In the separation material of this embodiment, the molar amount (mmol) of the functional group per gram of the separation material in a dry state is preferably adjusted as appropriate according to the size of the protein to be adsorbed, the surface area of the separation material, the specific gravity and the like.

  The separation material of this embodiment can be suitably used as an ion exchange carrier. Moreover, the separation material of this embodiment can be suitably used for affinity purification, separation by electrostatic interaction of proteins, and the like. 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 (for example, liquid chromatography). More specifically, the separation material of the present embodiment can be suitably used, for example, in the affinity chromatography field (particularly in the field of antibody drug purification).

  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, etc. of the separation material depending on the isoelectric point, ionization state, etc. of the protein. Examples of known methods include the methods described in JP-A-60-169427.

  The separating material of the present embodiment preferably includes a coating layer of 30 to 600 mg per 1 g of the hydrophobic polymer particles. The amount of the coating layer can be measured by reducing the weight of pyrolysis.

  The 5% compressive deformation modulus in water (5% compressive deformation modulus in a wet state) of the separation material of the present embodiment may be, for example, 70 MPa or more, 75 MPa or more, or 80 MPa or more. It may be.

In this specification, the 5% compressive deformation modulus of the separating material in water (also referred to as compressive modulus and 5% K value when the separating material undergoes 5% compressive deformation) is a value calculated by the following method. Say.
Separation using a micro compression tester (manufactured by Fisher) at room temperature (20 to 25 ° C.) at a load rate of 1 mN / sec. The load and compression displacement when the material is compressed to 50 mN are measured. From the obtained measured value, the compression elastic modulus (5% K value) when the separating material is 5% compressively deformed is 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 separating material is 5% compressively deformed
S: Compression displacement (mm) when the separating material is 5% compressively deformed
R: radius of separating material (mm)

  When the separation material of this embodiment is packed in a column, it is preferable that the liquid passing speed is 500 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 500 cm / h or more faster than the separating material. In addition, the liquid flow rate in this specification represents the liquid flow rate when the separation material of this embodiment is filled in a stainless steel column of φ7.8 × 300 mm and the liquid is passed.

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

  For example, a ligand (for example, protein A) or an ion exchange group can be introduced into the separation material of the present embodiment via the functional group or separately from the functional group. A separation material into which a ligand or ion exchange group is further introduced can be suitably used for applications such as affinity purification and ion exchange purification.

  The column of the present embodiment includes the separation material of the present embodiment. The column of this embodiment can be manufactured, for example, by filling the column with the separation material of this embodiment. The method for filling the column with the separation material of the present embodiment is not particularly limited, and for example, a known method can be employed.

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

Example 1
(Synthesis of hydrophobic porous polymer particles)
In a 500 mL three-necked flask, 16 g of divinylbenzene having a purity of 96% as a monomer (manufactured by Nippon Steel & Sumikin Co., Ltd., trade name: DVB960), 16 g of hexanol and 16 g of diethylbenzene as a porous agent, and benzoyl peroxide 0 as an initiator .64 g was mixed with an aqueous polyvinyl alcohol (0.5% by mass) dispersant solution to obtain a mixed solution. The resulting mixture is emulsified using a microprocess server, and then the resulting emulsion is transferred to a flask and stirred with a stirrer for about 8 hours while heating in a water bath at 80 ° C. Got. The obtained particles were collected by filtration and further washed with acetone to obtain hydrophobic porous polymer particles 1. The particle size of the obtained porous polymer particles 1 was measured with a flow type particle size measuring device, and the average particle size was calculated. The results are shown in Table 1.

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

(Evaluation of hydrophobic group content of modified agarose)
Dry powder agarose (unmodified agarose) and denatured agarose dried to a volatile content of less than 0.1% by mass are each dissolved in pure water at 70 ° C. to prepare a 0.05% by mass aqueous solution sample. did.

The absorbance at 269 nm of each aqueous solution was measured with a spectrophotometer to determine the concentration, and the hydrophobic group content was calculated from the following formula.
Hydrophobic group content (%) = (C _AG / (C _HAG + C _AG )) × 100
C _AG : concentration of denatured agarose constituent unit (mmol / L)
= A / ε _GPE × 1000
C _HAG : Concentration of unmodified agarose constituent unit (mmol / L)
= (Concentration of unmodified agarose constituent unit (g / L) / Agarose constituent unit (306 g / mol)) × 1000
A: True absorbance of hydrophobic group-introduced agarose = Absorbance of agarose with hydrophobic group introduced-Absorption of _unmodified agarose • ε _GPE : Absorption coefficient of glycidyl phenyl ether = 1372 (L / (mol · cm))
Absorption of undenatured agarose = absorbance of undenatured agarose × (sample concentration of denatured agarose (mmol / L) / sample concentration of unmodified agarose (mmol / L))
Non-denatured agarose constituent unit concentration (g / L) = denatured agarose sample concentration (mass%) × 10—denatured agarose constituent unit concentration (g / L)
Denatured agarose constituent unit concentration (g / L) = (C _AG × modified agarose constituent unit (456 g / mol)) / 1000

(Coating on particles of modified polymer having a hydroxyl group)
Porous polymer particles 1 were put into a 20 mg / mL modified agarose aqueous solution at a concentration of 70 mL / particle g and stirred at 55 ° C. for 24 hours to adsorb the modified agarose to the porous polymer particles 1. Subsequently, the porous polymer particles 1 on which the modified agarose was adsorbed were separated by filtration and further washed with hot water.

(Crosslinking of a modified polymer having a coated hydroxyl group)
The modified agarose adsorbed on the surface of the porous polymer particles 1 and inside the pores was crosslinked as follows. 10 g of particles adsorbed with modified agarose were dispersed in a 0.4 M aqueous sodium hydroxide solution, 0.02 M epichlorohydrin was added, and the mixture was stirred at room temperature for 24 hours. Then, after washing with a 2% by mass of hot sodium dodecyl sulfate aqueous solution, further washing with pure water and drying were performed to obtain crosslinked-coated particles having a crosslinked product of modified agarose as a coating layer.

(Evaluation of non-specific adsorption ability of protein)
0.2 g of the obtained crosslinked product-coated particles was added to 20 mL of Tris-hydrochloric acid buffer (pH 8.0) having a BSA (Bovine Serum Albumin) concentration of 24 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 supernatant obtained by measuring the absorbance at 280 nm of the supernatant with a spectrophotometer. The case where the amount of adsorbed BSA per mL of particles was 1 mg or less was evaluated as “A”, the case where it was more than 1 mg and less than 5 mg as “B”, and the case where it was 5 mg or more as “C”. The results are shown in Table 1.

(Introduction of carboxy group)
The obtained crosslinked product-coated particles (dry mass 1 g) were added to 10 mL of a 0.4 M aqueous sodium hydroxide solution, 0.6 g of epichlorohydrin was further added, and the mixture was reacted at room temperature for 3 hours with stirring. After completion of the reaction, the particles were filtered off and washed with 500 mL of water to introduce epoxy groups into the crosslinked product. Next, 10 mL of water and 10 mL of 1M aqueous solution of mercaptoacetate were added to the particles, and the mixture was stirred at room temperature for 24 hours to be reacted. After completion of the reaction, the particles were filtered off and washed with 1000 mL of water to introduce a carboxy group into the crosslinked product. Thereby, the separation material of this embodiment was obtained. In addition, the functional group introducing agent in this example is sodium mercaptoacetate.

(Measurement of carboxy group amount)
The functional group amount of the obtained separating material was measured as follows. 20 g of 0.1N aqueous hydrochloric acid solution was added to 0.5 g of the wet separating material, and the mixture was stirred at room temperature for 30 minutes. The separation material after stirring was filtered by suction, and further washed with 500 mL of water, and it was confirmed that the pH of the cleaning solution was 5 or more. 10 g of a 0.5 M aqueous sodium chloride solution was added to the separation material after washing, and the mixture was stirred at room temperature for 30 minutes. By titrating the suspension of the separated material after stirring with a 0.01N sodium hydroxide aqueous solution at an end point of pH 7, the molar amount (mmol) of carboxy groups per 1 g of the separated material in a dry state was measured. This result is shown in Table 1 as the functional group density (mmol / particle g).

(Immobilization of ligand)
To 0.09 g of the obtained separating material, 0.02 g of protein A, 0.02 g of WSC (1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride), and 50 mM adjusted to pH 5.8 After adding 2 ml of PBS, the mixture was stirred at room temperature for 24 hours. Thereafter, the particles were filtered and washed to obtain ligand-immobilized particles.

(Calculation of ligand immobilization amount)
After filtering the particles from the reaction solution after stirring for 24 hours, a sample containing particles having a predetermined concentration was prepared. The absorbance (280 nm) of the sample was measured, and the amount of immobilized ligand was calculated using a calibration curve prepared in advance. The results are shown in Table 1.

(Calculation of 5% compressive deformation modulus in water)
The 5% compressive deformation elastic modulus of the obtained separating material in water was calculated as follows. The results are shown in Table 1.

  Separation using a micro compression tester (manufactured by Fisher) at room temperature (20 to 25 ° C.) at a load load rate of 1 mN / sec. And a smooth end face (50 μm × 50 μm) of a quadrangular column in advance. The load and compression displacement when the material is compressed to 50 mN are measured. From the measured value obtained, the compression modulus (5% K value) when the separating material is 5% compressively deformed can be obtained by the following equation. Further, the load at the point at which the displacement during the measurement changes most greatly is defined as the breaking strength (mN).

5% K value (MPa) = (3/2 ^1/2 ) · F · S ⁻³ / ² · R ^−1/2
F: Load (mN) when the separating material is 5% compressively deformed
S: Compression displacement (mm) when the separating material is 5% compressively deformed
R: radius of separating material (mm)

(Column characteristic evaluation)
The obtained separating material was packed in a φ7.8 × 300 mm stainless steel column as a slurry (solvent: methanol) with a concentration of 30% by mass for 15 minutes. Thereafter, water was passed through the column while changing the flow rate, the relationship between the flow rate and the column pressure was measured, and the liquid flow rate (linear flow rate) at 0.3 MPa was measured. The results are shown in Table 1.

(Example 2)
In the same manner as in Example 1, crosslinked product-coated particles were produced.

(Introduction of carboxy group)
The obtained crosslinked product-coated particles (dry mass 1 g) were added to 10 mL of a 0.4 M aqueous sodium hydroxide solution, 0.6 g of epichlorohydrin was further added, and the mixture was reacted at room temperature for 3 hours with stirring. After completion of the reaction, the particles were filtered off and washed with 500 mL of water to introduce epoxy groups into the crosslinked product. Next, 15 mL of an aqueous sodium hydroxide solution in which 5 g of sodium hydroxide was dissolved in 15 mL of water and 5 g of mercaptopropionic acid were added to the particles, and the mixture was stirred at room temperature for 24 hours to be reacted. After completion of the reaction, the particles were filtered off and washed with 1000 mL of water to introduce a carboxy group into the crosslinked product. Thereby, the separation material of this embodiment was obtained. The functional group introducing agent in this example is mercaptopropionic acid.

  Further, in the same manner as in Example 1, the average particle size, the nonspecific adsorption ability of the protein, the amount of carboxy groups (functional group density), the 5% compressive deformation elastic modulus in water, and the column characteristics (liquid flow rate) were evaluated. did. Further, in the same manner as in Example 1, the ligand was immobilized, and the amount of immobilized ligand was calculated. The results are shown in Table 1.

(Example 3)
In the same manner as in Example 1, crosslinked product-coated particles were produced.

(Introduction of epoxy group)
The obtained crosslinked product-coated particles (dry mass 1 g) were added to 10 mL of a 0.4 M aqueous sodium hydroxide solution, 0.6 g of epichlorohydrin was further added, and the mixture was reacted at room temperature for 3 hours with stirring. After completion of the reaction, the particles were filtered off and washed with 500 mL of water to introduce epoxy groups into the crosslinked product. Thereby, the separation material of this embodiment was obtained.

(Measurement of epoxy group content)
The functional group amount of the obtained separating material was measured as follows. 10 g of water and 1 g of a 1N aqueous hydrochloric acid solution were added to 0.5 g of a wet separating material, and the mixture was stirred at 70 ° C. for 1 hour to be reacted. After the reaction, neutralization titration with 0.1N sodium hydroxide was performed to measure the molar amount (mmol) of the epoxy group per 1 g of the separating material in a dry state. This result is shown in Table 1 as the functional group density (mmol / particle g).

(Immobilization of ligand)
To 0.09 g of the obtained separation material, 0.02 g of protein A, and 2 mL of 0.5 M carbonate buffer (prepared with sodium hydrogen carbonate and sodium carbonate manufactured by Wako Pure Chemical Industries, Ltd. and RO water) at pH 10 are added. , And stirred at 30 ° C. for 24 hours. Thereafter, the particles were filtered and washed to obtain ligand-immobilized particles.

(Calculation of ligand immobilization amount)
After filtering the particles from the reaction solution after stirring for 24 hours, a sample containing particles having a predetermined concentration was prepared. The absorbance (280 nm) of the sample was measured, and the amount of immobilized ligand was calculated using a calibration curve prepared in advance. The results are shown in Table 1.

  Further, in the same manner as in Example 1, the average particle size, the nonspecific adsorption ability of the protein, the 5% compressive deformation modulus in water, and the column characteristics (liquid passing speed) were evaluated. The results are shown in Table 1.

Example 4
In the same manner as in Example 1, crosslinked product-coated particles were produced.

(Introduction of amino group)
The obtained crosslinked product-coated particles (dry mass 20 g) were added to 200 mL of a 5 M aqueous sodium hydroxide solution and left at room temperature for 1 hour. Separately, 200 mL of a predetermined amount (10 g) of diethylaminoethyl chloride hydrochloride was added, the temperature of the aqueous solution was raised to 70 ° C., and the mixture was reacted for 2 hours with stirring. After completion of the reaction, the mixture was filtered and washed three times with water / ethanol (volume ratio 5/1) to introduce a diethylaminoethyl (DEAE) group into the crosslinked product. Thereby, the separation material of this embodiment was obtained.

(Measurement of amino group content)
The functional group amount of the obtained separating material was measured as follows. 0.5 g of the wet separating material was immersed in 20 mL of 0.1N sodium hydroxide for 1 hour and stirred at room temperature. Thereafter, the solution was washed with water so that the pH of the solution was 7 or less. The separation material after washing was immersed in 20 mL of 0.1N hydrochloric acid and stirred for 1 hour. The separation material after stirring was filtered off, and the hydrochloric acid aqueous solution was neutralized and titrated to measure the molar amount (mmol) of amino groups per 1 g of the separation material in a dry state. This result is shown in Table 1 as the functional group density (mmol / particle g).

  Further, in the same manner as in Example 1, the average particle size, the nonspecific adsorption ability of the protein, the 5% compressive deformation modulus in water, and the column characteristics (liquid passing speed) were evaluated. The results are shown in Table 1.

(Comparative Example 1)
Commercially available agarose particles (Capto DEAE (manufactured by GE Healthcare, trade name)) were used as they were as separation materials, and in the same manner as in Example 1, average particle size, nonspecific adsorption ability of proteins, 5% compression deformation elasticity in water The rate and column characteristics (liquid flow rate) were evaluated. The results are shown in Table 1.

(Comparative Example 2)
Using the porous polymer particles 1 as a separating material as they were, the same as in Example 1, the average particle size, the nonspecific adsorption ability of the protein, the 5% compressive deformation elastic modulus in water, and the column characteristics (liquid flow rate) evaluated. The results are shown in Table 1.

  From the results in Table 1, it can be seen that the separation materials of Examples 1 to 4 have reduced nonspecific adsorption of proteins and are excellent in liquid permeability when packed in a column. Moreover, it turns out that the separating material of Examples 1-4 has a high 5% compressive deformation elastic modulus in water.

Claims (11)

Hydrophobic polymer particles,
A coating layer covering at least a part of the surface of the hydrophobic polymer particles,
The separating material, wherein the coating layer includes a functional group-containing polymer having a hydroxyl group and a functional group other than a hydroxyl group.   The separating material according to claim 1, wherein the functional group other than the hydroxyl group is a carboxy group, an epoxy group, or an amino group.   The separation material according to claim 1 or 2, wherein a 5% compression deformation elastic modulus in water is 70 MPa or more.   The separation material according to any one of claims 1 to 3, wherein when the column is packed, the liquid flow rate is 500 cm / h or more when the column pressure is 0.3 MPa.   The separation material according to any one of claims 1 to 4, wherein the hydrophobic polymer particles include a hydrophobic polymer having a structural unit derived from a styrene monomer.   The separating material according to any one of claims 1 to 5, wherein the functional group-containing polymer includes a structure derived from a polysaccharide or a modified product thereof.   The separating material according to any one of claims 1 to 6, wherein the functional group-containing polymer includes a structure derived from agarose or a modified product thereof.   The separating material according to any one of claims 1 to 7, wherein the functional group-containing polymer is crosslinked.   The separation material according to any one of claims 1 to 8, wherein the hydrophobic polymer particles have an average particle size of 10 to 500 µm.   The separation material according to any one of claims 1 to 9, wherein the hydrophobic polymer particles have a porous structure.   A column comprising the separation material according to claim 1.