JP2018163004A - Separation material and column - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a separation material which is excellent in desorption rate of protein and also excellent in alkali resistance.SOLUTION: A separation material includes: a porous polymer particle; and a coating layer which covers at least a part of a surface of the porous polymer particle and contains a polymer having a hydroxyl group. A fluorescence peak wavelength after protein adsorption is not in a range of 430 to 440 nm.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 a porous synthetic polymer, the volume change due to the salt concentration is small. Therefore, when packed in a column and used for chromatography, there is a tendency that pressure resistance during liquid passage is excellent. However, when this ion exchanger is used for separation of proteins, etc., nonspecific adsorption such as irreversible adsorption based on hydrophobic interaction occurs, resulting in peak asymmetry or ion exchange due to the hydrophobic interaction. There is a problem that the protein adsorbed on the body cannot be recovered while adsorbed.

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

  In order to overcome the drawbacks of the hydrophilic natural polymer cross-linked gel, a complex in which a gel such as a natural polymer gel is held in the pores of the porous polymer is known in the field of peptide synthesis (for example, Patent Document 1). In Patent Document 1, the use of such a complex increases the load factor of the reactive substance, enables high yield synthesis, and surrounds the gel with a hard synthetic polymer substance. It is described that there is no change in volume even when used in, and the flow-through pressure passing through the column does not change.

  Separation materials in which xerogels such as polysaccharides such as dextran and cellulose are held in an inorganic porous material such as celite are known (see, for example, Patent Document 2 and Patent Document 3). In order to add adsorption performance to this gel, a diethylaminomethyl (DEAE) group or the like is added, and the gel is used for removal of hemoglobin. As its effect, the above literature mentions good liquid permeability in a column.

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

  There has been proposed a composite filler in which a hydrophilic natural polymer cross-linked gel having a giant network structure is filled in the pores of an organic synthetic polymer substrate (see, for example, Patent Document 5 and Patent Document 6).

  Porous particles formed by copolymerization of glycidyl methacrylate and an acrylic cross-linked monomer have been synthesized (see, for example, Patent Document 7).

US Pat. No. 4,965,289 US Pat. No. 4,335,017 US Pat. No. 4,336,161 US Pat. No. 3,966,489 JP-A-1-254247 US Pat. No. 5,114,577 JP 2009-244067 A JP-A-60-169427

  When a conventional separation material is used for protein purification or the like, there is a problem that a part of the protein adsorbed on the separation material cannot be desorbed and recovered.

  Further, conventional separators do not have a sufficient level of alkali resistance.

  Therefore, an object of the present invention is to provide a separation material that is excellent in protein desorption rate and excellent in alkali resistance.

The present invention provides the separation material or column described in [1] to [11] below.
[1] A porous polymer particle and a coating layer containing a polymer having a hydroxyl group that covers at least a part of the surface of the porous polymer particle, and has a fluorescence peak in a wavelength range of 430 to 440 nm after protein adsorption. Separation material that does not have.
[2] The separating material according to [1], wherein the polymer having a hydroxyl group is reduced.
[3] The separation material according to [1] or [2], wherein the polymer having a hydroxyl group is crosslinked.
[4] The separation material according to any one of [1] to [3], wherein the polymer having a hydroxyl group includes a reduced polysaccharide or a modified product thereof.
[5] The separation material according to any one of [1] to [4], wherein the porous polymer particles include a polymer containing a monomer unit derived from a styrene monomer.
[6] The average particle size of the separating material is 10 to 500 μm, and the mode diameter in the pore size distribution of the separating material is 0.05 to 0.6 μm, according to any one of [1] to [5] Separation material.
[7] The separation material according to any one of [1] to [6], wherein the porosity of the separation material is 40 to 70% by volume.
[8] The separation material according to any one of [1] to [7], wherein the separation material has a specific surface area of 30 m ² / g or more.
[9] The separation material according to any one of [1] to [8], wherein the coefficient of variation of the particle size of the separation material is 5 to 15%.
[10] When water is passed through the column packed with the separation material so that the pressure in the column is 0.3 MPa, the water flow rate is 500 cm / h or more. [1 ] To [9].
[11] A column comprising the separation material according to any one of [1] to [10].

  According to the present invention, it is possible to provide a separation material that is excellent in protein detachment rate and excellent in alkali resistance.

4 is a graph showing a fluorescence spectrum of a separating material in Example 1. 6 is a graph showing a fluorescence spectrum of a separating material in Comparative Example 1.

  Hereinafter, although embodiment of this invention is described in detail, this invention is not limited to these embodiment at all.

<Separation material>
The separating material of the present embodiment includes porous polymer particles and a coating layer that covers at least a part of the surface of the porous polymer particles. In the present specification, the “surface of the porous polymer particle” includes not only the outer surface of the porous polymer particle but also the surface of the pores inside the porous polymer particle. In the present specification, (meth) acrylic acid means acrylic acid or methacrylic acid, and the same applies to similar expressions such as (meth) acrylate.

(Porous polymer particles)
The porous polymer particle of this embodiment is a porous particle containing a polymer containing a monomer unit derived from one or more kinds of monomers. The porous polymer particle is, for example, a particle obtained by polymerizing a monomer containing a porosifying agent. The porous polymer particles can be synthesized, for example, by conventional suspension polymerization or emulsion polymerization. Although it does not specifically limit as a monomer, For example, a styrene-type monomer can be used. That is, the porous polymer particles may contain a monomer unit derived from a styrene monomer. Specific examples of the monomer include the following polyfunctional monomers and monofunctional monomers.

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

  Examples of the styrenic monofunctional monomer include styrene, o-methyl styrene, m-methyl styrene, p-methyl styrene, α-methyl styrene, o-ethyl styrene, m-ethyl styrene, p-ethyl styrene, 2 , 4-dimethylstyrene, pn-butylstyrene, pt-butylstyrene, pn-hexylstyrene, pn-octylstyrene, pn-nonylstyrene, pn-decylstyrene, p- Examples thereof include styrene and derivatives thereof such as n-dodecylstyrene, p-methoxystyrene, p-phenylstyrene, p-chlorostyrene, and 3,4-dichlorostyrene. These monofunctional monomers can be used singly or in combination of two or more. Of these, styrene is preferably used from the viewpoint of excellent acid resistance and alkali resistance. Moreover, the styrene derivative which has functional groups, such as a carboxyl group, an amino group, a hydroxyl group, and an aldehyde group, can also be used.

  Examples of the porosifying agent include aliphatic or aromatic hydrocarbons, esters, ketones, ethers, and alcohols, which are organic solvents that promote phase separation during polymerization and promote pore formation of particles. . Specific examples include toluene, xylene, diethylbenzene, cyclohexane, octane, butyl acetate, dibutyl phthalate, methyl ethyl ketone, dibutyl ether, 1-hexanol, 2-octanol, decanol, lauryl alcohol, and cyclohexanol. These porosifying agents can be used singly or in combination of two or more.

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

  Water used as a solvent for the polymerization reaction can also be used as a porous agent. When water is used as the porosifying agent, the oil-soluble surfactant is dissolved in the monomer, and the monomer droplets absorb the water, thereby making the particles porous.

  Examples of the oil-soluble surfactant used for the porous formation include sorbitan monoesters of branched C16 to C24 fatty acids, chain unsaturated C16 to C22 fatty acids or chain saturated C12 to C14 fatty acids (for example, sorbitan monolaurate). Sorbitan monoesters derived from sorbitan 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 (eg C18: 1 (18 carbon atoms, 1 double bond) diglycerol monooleate such as diglycerol monoester of fatty acid), diglycerol monomyristate, diglycerol monoisostearate or diglycerol of coconut fatty acid Monoester; Branch C16 ~ 24 alcohol (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 oil-soluble surfactants, sorbitan monolaurate (eg, SPAN® 20, preferably greater than about 40% purity, more preferably greater than about 50% purity, most preferably about purity Greater than 70% sorbitan monolaurate; sorbitan monooleate (eg, SPAN 80, preferably about 40% purity, more preferably about 50% purity, most preferably more than about 70% purity) Sorbitan monooleate); diglycerol monooleate (eg, diglycerol monooleate having a purity of greater than about 40%, more preferably greater than about 50%, most preferably greater than about 70%); Isostearate (eg, preferably greater than about 40% purity, more preferably pure Diglycerol monoisostearate greater than about 50%, most preferably greater than about 70% purity; diglycerol monomyristate (preferably greater than about 40% purity, more preferably greater than about 50% purity, most preferably Sorbitan monomyristate having a purity of greater than about 70%); 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. If the content of the oil-soluble surfactant is 5% by mass or more, the stability of the water droplet is sufficient, and it is difficult to form a large single hole. Further, when the content of the oil-soluble surfactant is 80% by mass or less, it becomes easier for the porous polymer particles to retain the shape after polymerization.

  Examples of the aqueous medium used for the polymerization reaction include water, a mixed medium of water and a water-soluble solvent (for example, lower alcohol), and the like. The aqueous medium may contain a surfactant. As the surfactant, any of anionic, cationic, nonionic and zwitterionic surfactants can be used.

  Examples of the anionic surfactant include fatty acid oils such as sodium oleate and castor oil potassium, alkyl sulfate salts such as sodium lauryl sulfate and ammonium lauryl sulfate, alkylbenzene sulfonates such as sodium dodecylbenzenesulfonate, and alkylnaphthalene sulfone. Acid salts, alkane sulfonates, dialkyl sulfosuccinates such as sodium dioctyl sulfosuccinate, alkenyl succinates (dipotassium salts), alkyl phosphate esters, naphthalene sulfonate formalin condensates, polyoxyethylene alkylphenyl ether sulfates Examples thereof include 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 silicone 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, phosphite ester surfactants, and the like.

  Surfactant can be used individually by 1 type or in combination of 2 or more types. Among the surfactants, anionic surfactants are preferable from the viewpoint of dispersion stability during monomer polymerization.

  Examples of the polymerization initiator added as necessary include benzoyl peroxide, lauroyl peroxide, orthochlorobenzoyl peroxide, orthomethoxybenzoyl peroxide, 3,5,5-trimethylhexanoyl peroxide, tert-butyl peroxide. Organic peroxides such as oxy-2-ethylhexanoate and di-tert-butyl peroxide; 2,2′-azobisisobutyronitrile, 1,1′-azobiscyclohexanecarbonitrile, 2,2 ′ -Azo compounds such as azobis (2,4-dimethylvaleronitrile). A polymerization initiator can be used in 0.1-7.0 mass parts with respect to 100 mass parts of monomers.

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

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

  Examples of the polymer dispersion stabilizer include polyvinyl alcohol, polycarboxylic acid, celluloses (hydroxyethyl cellulose, carboxymethyl cellulose, methyl cellulose, etc.), polyvinyl pyrrolidone, and inorganic water-soluble polymer compounds such as sodium tripolyphosphate. can do. Of these, polyvinyl alcohol or polyvinyl pyrrolidone is preferred. The addition amount of the polymer dispersion stabilizer is preferably 1 to 10 parts by mass with respect to 100 parts by mass of the monomer.

  In order to 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 size of the porous polymer particles and the separating material is preferably 500 μm or less, more preferably 300 μm or less, still more preferably 150 μm or less, and still more preferably 100 μm or less. Further, the average particle diameter 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 improving liquid permeability.

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

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

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

The specific surface areas of the porous polymer particles and the separating material are 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 (mode value of pore diameter distribution, maximum frequency pore diameter) in the pore diameter distribution of the separation material of the present embodiment is preferably 0.05 to 0.6 μm. If the mode diameter in the pore size distribution is 0.01 μm or more, the liquid tends to flow in the particles and the dynamic adsorption capacity tends to increase. If the mode diameter is 0.6 μm or less, the specific surface area is more It tends to grow.

  The average pore diameter, the mode diameter in the pore diameter distribution, the specific surface area, and the porosity of the separation material or porous polymer particle of the present embodiment are values measured with a mercury intrusion measuring apparatus (Autopore: manufactured by Shimadzu Corporation). Measured as follows. About 0.05 g of a sample is added to a standard 5 mL powder cell (stem volume 0.4 mL), and measurement is performed under an initial pressure of 21 kPa (about 3 psia, corresponding to a pore diameter of about 60 μm). Mercury parameters are set to a device default mercury contact angle of 130 degrees and a mercury surface tension of 485 dynes / cm. Moreover, it limits to the range of 0.1-3 micrometers of pore diameters, and calculates each value.

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

  The separation material of this embodiment does not have a fluorescence peak in the wavelength range of 430 to 440 nm after the separation material adsorbs protein. In the separation material of the present embodiment, the fluorescence peak wavelength after protein adsorption is preferably 420 nm or more and less than 430 nm. The fluorescence peak wavelength is measured with a fluorometer. Specifically, a separation material adsorbed with protein such as BSA (Bovine Serum Albumin) is dried and then wetted with water again as a measurement sample, and measurement is performed using a fluorometer. The excitation wavelength is preferably 370 nm.

(Coating layer)
The coating layer of the present embodiment includes a polymer having a hydroxyl group. The coating layer covers at least a part of the surface of the porous polymer particle. By coating porous polymer particles with a polymer having a hydroxyl group, it is possible to suppress an increase in column pressure and to suppress nonspecific adsorption of biopolymers such as proteins. The amount can be high enough. Furthermore, when the polymer having a hydroxyl group is crosslinked, it is possible to further suppress an increase in column pressure.

(Polymer having a hydroxyl group)
The polymer having a hydroxyl group preferably has two or more hydroxyl groups in one molecule, and more preferably a hydrophilic polymer. Examples of the polymer having a hydroxyl group include polysaccharides such as agarose, dextran, cellulose, chitosan, glycogen, pectin, chondroitin, hyaluronic acid, starch, alginic acid, fructan, heparin, carrageenan, curdlan, xanthan gum, and polyvinyl alcohol. These compounds may be those obtained by further treatment such as reduction or modification. As the polymer having a hydroxyl group, for example, a polymer having an average molecular weight of 10,000 or more can be used.

  The polymer having a hydroxyl group is preferably a modified body having a hydrophobic group introduced from the viewpoint of improving the interfacial adsorption ability. Examples of the hydrophobic group include an alkyl group having 1 to 6 carbon atoms and an aryl group having 6 to 10 carbon atoms. 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. Hydrophobic groups are introduced by reacting a functional group that reacts with a hydroxyl group (for example, an epoxy group) and a compound having a hydrophobic group (for example, glycidyl phenyl ether) with a polymer having a hydroxyl group by a conventionally known method. can do.

  The content of the hydrophobic group in the modified polymer having a hydroxyl group introduced with a hydrophobic group is based on the balance between retention of the hydrophobic interaction force for adsorption to the particle surface and suppression of nonspecific adsorption of the protein. The number of structural units in the polymer constituting the hydroxyl group is preferably 0.05 to 0.3, more preferably 0.1 to 0.2, and 0.12 to 0.17. More preferably, it is individual.

  The structural unit of the polymer having a hydroxyl group in the present specification may be the smallest unit among units constituting the polymer having a hydroxyl group by substantially repeating it, and may be any unit such as two smallest units. Also good. When the polymer having a hydroxyl group is a polysaccharide, the structural unit can be, for example, a monosaccharide or disaccharide constituting the polymer, and the polymer having a hydroxyl group is a synthetic polymer. The structure may be derived from a monomer that is the minimum unit constituting the polymer.

  The content ratio of the hydrophobic group in the polymer having a hydroxyl group is, for example, the amount of the hydrophobic group introduction agent (a compound having a functional group that reacts with a hydroxyl group and a hydrophobic group), the amount of catalyst used when the hydrophobic group is introduced. The temperature can be adjusted by the temperature at the time of introducing the hydrophobic group.

  The present invention is characterized in that when a protein is adsorbed on the particles and irradiated with fluorescence at 370 nm, a fluorescence peak is not newly developed at 430 to 440 nm. It is known that a carbonyl group and a protein-derived amino group react irreversibly to generate saccharification. When this saccharification product is irradiated with fluorescence of 350 nm to 370 nm, it is known that a fluorescence peak appears in the range of 430 to 440 nm. Therefore, it is preferable that the polymer contained in the coating layer does not have a carbonyl group. When the carbonyl group content in the polymer is small, it is possible to suppress a decrease in the desorption rate after protein adsorption.

  The polymer having a hydroxyl group of the present embodiment is preferably subjected to a reduction treatment. The polysaccharide usually has a carbonyl group such as an aldehyde group at the terminal. In particular, when the polymer having a hydroxyl group is made from a polymer having a carbonyl group, such as a polysaccharide, it is preferably subjected to a reduction treatment. In the separation material of the present embodiment, at least a part of a carbonyl group such as an aldehyde group in the polymer having a hydroxyl group is reduced, whereby the carbonyl group is converted to a hydroxyl group, and the carbonyl group and the amino group in the protein It is considered that the protein can be prevented from reacting and denatured and the protein can be prevented from sticking to the porous polymer particles.

(Method for manufacturing separation material)
The separating material of the present embodiment can be manufactured, for example, by a method including a step of preparing porous polymer particles and a step of forming a coating layer on at least a part of the surface of the porous polymer particles. The step of forming the coating layer may include, for example, a step of adsorbing the polysaccharide or a modified product thereof on the surface of the porous polymer particles and a step of reducing the polysaccharide or the modified product thereof. The polysaccharide adsorbed on the porous polymer particles or a modified product thereof may be crosslinked. That is, the step of forming the coating layer may further include a step of crosslinking the adsorbed polysaccharide or a modified product thereof.

  The step of reducing the polysaccharide or a modified product thereof (reduction treatment step) may be performed in advance before the polysaccharide or the modified product thereof is adsorbed on the surface of the porous polymer particles. When cross-linking a polysaccharide or a modified product thereof, the reduction treatment step may be performed before cross-linking or may be performed at the time of cross-linking or after cross-linking. The reduction treatment step is preferably performed during or after crosslinking. Hereinafter, specific examples of the coating layer forming method will be described.

(adsorption)
First, a polymer solution having a hydroxyl group is adsorbed on the surface of the porous polymer particles. The solvent for the polymer solution having a hydroxyl group is not particularly limited as long as it can dissolve the polymer having a hydroxyl group, but water is the most common. The concentration of the polymer dissolved in the solvent is preferably 5 to 20 (mg / mL).
The solution is impregnated into porous polymer particles. Examples of the impregnation method include a method in which porous polymer particles are added to a polymer solution having a hydroxyl group and left for a predetermined time. Although the impregnation time varies depending on the surface state of the porous body, the polymer concentration is usually in equilibrium with the external concentration inside the porous polymer particles if it is impregnated overnight. Then, it wash | cleans with solvents, such as water and alcohol, and remove | eliminates the polymer which has a hydroxyl group which is not adsorb | sucked.

(Crosslinking treatment)
The polymer having a hydroxyl group adsorbed on the surface of the porous polymer particles is preferably immobilized. Immobilization can be performed by, for example, crosslinking a polymer having a hydroxyl group. Crosslinking can be performed, for example, by adding a crosslinking agent to a polymer having a hydroxyl group such as a polysaccharide adsorbed on the porous polymer particles to cause a crosslinking reaction. That is, in the separation material of this embodiment, the polymer having a hydroxyl group may be crosslinked. At this time, the crosslinked polymer having a hydroxyl group obtained by crosslinking has a three-dimensional crosslinked network structure having a hydroxyl group.

  As a crosslinking agent, for example, an epihalohydrin such as epichlorohydrin, a dialdehyde compound such as glutaraldehyde, a diisocyanate compound such as methylene diisocyanate, a glycidyl compound such as ethylene glycol diglycidyl ether, and two or more functional groups active on a hydroxyl group. The compound which has is mentioned. Further, when a compound having an amino group such as chitosan is used as the polymer having a hydroxyl group, a dihalide compound such as dichlorooctane can also be used as a crosslinking agent.

  A catalyst is usually used for the 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 porous polymer particles impregnated in a pore with a polymer solution having a hydroxyl group are dispersed and suspended in an appropriate medium. Done. When the polysaccharide is used as the polymer having a hydroxyl group, the addition amount of the crosslinking agent is within a range of 0.1 to 100 mol times per unit of the monosaccharide, and the performance of the separating material. It can be selected according to. When the addition amount of the crosslinking agent is 0.1 mol times or more, the coating layer tends to be difficult to peel off from the porous polymer particles. Moreover, when the addition amount of the crosslinking agent is 100 mol times or less, even when the reaction rate with the polymer having a hydroxyl group is high, the properties of the polymer having a hydroxyl group as a raw material tend not 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, The catalyst is used in the range of 0.01 to 10 mol times, preferably 0.1 to 5 mol times.

  The cross-linking reaction may be caused by changing the cross-linking reaction conditions such as temperature. For example, when the crosslinking reaction condition is a temperature condition, a crosslinking reaction occurs when the temperature of the reaction system is raised and the temperature reaches the reaction temperature.

  As a medium for dispersing and suspending porous polymer particles impregnated with a polymer solution having a hydroxyl group, the polymer or crosslinking agent is not extracted from the impregnated polymer solution, and a crosslinking reaction is performed. Must be inert. Specific examples thereof include water and alcohol.

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

  After completion of the crosslinking reaction, the produced particles are filtered, washed with a hydrophilic organic solvent such as water, methanol, ethanol, etc. to remove unreacted polymer, suspending medium, etc. A separation material in which at least a part of the surface is coated with a coating layer containing a polymer having a hydroxyl group is obtained. The separating material of the present embodiment preferably includes a coating layer of 30 to 500 mg per 1 g of porous polymer particles. The amount of the coating layer can be measured by reducing the weight of pyrolysis.

(Reduction treatment)

  When the polymer has a carbonyl group, a reduction treatment is performed in the separation material manufacturing process. The reduction treatment can be performed using a reducing agent, for example. The reducing agent is preferably one that can reduce a carbonyl group, and more preferably one that can reduce an aldehyde group and / or a keto group. Examples of the reducing agent include sodium borohydride, lithium borohydride, lithium aluminum hydride, diisobutylaluminum hydride, sodium cyanoborohydride, lithium triethylborohydride, sodium bis (2-methoxyethoxy) aluminum hydride, Examples thereof include borohydride, oxalic acid, formic acid, hydrazine, sulfur dioxide, hydrogen peroxide, hydrogen sulfide, sodium sulfite, and metal catalysts (palladium, nickel, etc.). By the reduction treatment, a part or all of the carbonyl group in the polymer, for example, the terminal aldehyde group in the polysaccharide or a modified product thereof can be reduced.

  The reduction reaction is usually performed by adding a reducing agent to a system in which a separating material is dispersed and suspended in an appropriate medium. When the polysaccharide is used as the polymer having a hydroxyl group, the amount of the reducing agent added is within the range of 0.01 to 10 moles per unit of the monosaccharide, and the performance of the separating material. It can be selected according to.

  The reduction reaction is usually performed in an organic solvent, but may be performed in water. Moreover, it is preferable to carry out for 0.5 to 6 hours on 0 degreeC-60 degreeC temperature conditions according to the kind of reducing agent. When the reduction treatment is performed at the time of crosslinking or after crosslinking, the particles are separated by filtration after the reduction reaction, and the unreacted reducing agent is washed with water. At this time, it may be further washed with salt.

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

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

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

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

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

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

  Examples of the method for introducing a carboxy group that is a weakly acidic group as an ion exchange group include a method in which a monohalogenocarboxylic acid such as monohalogenoacetic acid or monohalogenopropionic acid or a sodium salt thereof is reacted as the halogenated alkyl compound. . The amount of the halogenated alkyl compound used is preferably 0.2% by mass or more based on the total mass of the separating material into which the ion exchange group is introduced.

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

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

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

  The biopolymer that can be separated using the separation material of the present embodiment is preferably a water-soluble substance. Specifically, for example, blood proteins such as serum albumin and immunoglobulin, proteins such as enzymes present in the living body, bioactive substances produced by biotechnology, DNA, biopolymers such as bioactive peptides Preferably, the molecular weight is 2 million or less, more preferably 500,000 or less. In addition, according to a known method, it is necessary to select properties, conditions, and the like of the separation material depending on the isoelectric point, ionization state, and the like of the protein. As a known method, for example, the method described in Patent Document 8 and the like can be mentioned.

  The separation material of this embodiment has respective advantages of particles made of natural polymers and particles made of synthetic polymers in the separation of biopolymers such as proteins. Moreover, when the particle | grains after protein adsorption | suction are irradiated with the fluorescence of 370 nm as excitation light, for example, by limiting to the thing in which a fluorescence peak does not appear in wavelength 430-440 nm, the quantity of the protein which cannot be desorbed but cannot collect | recover Can be reduced. Thereby, the fall of the adsorption | suction capacity | capacitance of protein etc. and the quantity which can be collect | recovered can also be suppressed. Further, the porous polymer particles in the separation material of the present embodiment have small non-specific adsorption, and are excellent in durability and alkali resistance.

  The liquid passing speed in this specification represents the liquid passing speed when a separating material is filled in a stainless steel column of φ7.8 × 300 mm and the liquid is passed. When the separation material of the present embodiment is packed in a column, the flow rate is preferably 500 cm / h or more when water is passed so that the pressure in the column becomes 0.3 MPa. More preferably, it is h or more. 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, although this embodiment demonstrated the separation material of the form which introduce | transduces an ion exchange group, even if it does not introduce | transduce an ion exchange group, it can be used as a separation material. Such a separating material can be used for, for example, gel filtration chromatography.

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

(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 (2% by mass) having a molecular weight (Mw) of 150,000 and reacted at 60 ° C. for 6 hours. Introduced. The obtained modified agarose was precipitated with isopropyl alcohol and washed. When the hydrophobic group content of the modified agarose was calculated by the following method, it was 0.135.

(Evaluation of introduction ratio of hydrophobic group in modified polymer having hydroxyl group)
Dry powdered agarose (unmodified agarose) and hydrophobic group-introduced agarose dried to a volatile content of less than 0.1% by weight are each dissolved in pure water at 70 ° C. to prepare a 0.05% by weight aqueous solution sample. Prepared. By measuring the absorbance at 269 nm of each aqueous solution with a spectrophotometer to obtain the concentration, the hydrophobic group content per one structural unit of the polymer having a hydroxyl group was calculated from the following formula.
-Hydrophobic group content (pieces) = (C _AG / (C _HAG + C _AG ))
C _AG : Denatured dextran or agarose constituent unit concentration (mmol / l) C _AG = A / ε _GPE × 1000
A: True absorbance of dextran or agarose having a hydrophobic group introduced A = Absorbance of dextran or agarose having a hydrophobic group introduced—Absorption of unmodified dextran or agarose / Absorption of unmodified dextran or agarose = modified Absorbance of unextracted dextran or agarose × (sample concentration of dextran or agarose introduced with a hydrophobic group (mmol / l) / sample concentration of unmodified dextran or agarose (mmol / l))
.Epsilon. _GPE : extinction coefficient of glycidyl phenyl _{ether.epsilon.GPE} = 1372 (l / (mol.cm))
C _HAG : concentration of unmodified dextran or agarose constituent unit (mmol / l)
C _HAG = (Concentration of unmodified dextran or agarose building blocks (g / l) / dextran building blocks (324 g / mol) or agarose building blocks (306 g / mol)) × 1000
Non-denatured dextran or agarose constituent unit concentration (g / l) = Dextran or agarose sample concentration (mass%) introduced with a hydrophobic group × 10-denatured dextran or agarose constituent unit concentration (g / L)
Denatured dextran or agarose constituent unit concentration (g / l) = (C _AG × modified dextran or agarose constituent unit (456 g / mol)) / 1000

  The hydrophobic group content of the modified agarose or dextran adsorbed on the particles was obtained by treating 0.2 g of the particles in 10 ml of 1 M sulfuric acid at 70 ° C. for 5 hours, and treating the treatment liquid with an absorbance of 269 nm using a spectrophotometer. It can calculate similarly by measuring and calculating | requiring a process liquid density | concentration.

Example 1
(Synthesis of porous polymer particles)
In a 500 mL three-necked flask, 16 g of divinylbenzene having a purity of 96% as a monomer (trade name: DVB960, manufactured by Nippon Steel & Sumikin Co., Ltd.), 16 g of hexanol and 16 g of diethylbenzene as a porous material, and 0.64 g of benzoyl peroxide as an initiator are added to polyvinyl alcohol ( In addition to the 0.5 wt% dispersant aqueous solution, a mixed solution was prepared. The mixture was emulsified using a microprocess server, and the resulting emulsion was transferred to a flask and stirred for about 8 hours using a stirrer while heating in a water bath at 80 ° C. The obtained particles were filtered and then washed with acetone to obtain porous polymer particles having an average particle size of 100 μm. The average particle size of the porous polymer particles was measured with a flow type particle size measuring device (FPIA-3000, manufactured by Sysmex Corporation).

(Coating of particles of modified polymer having a hydroxyl group)
Porous polymer particles were added at a rate of 1 g to 70 mL of a 20 mg / mL modified agarose aqueous solution and stirred at 55 ° C. for 24 hours to adsorb the modified agarose to the porous polymer particles. After adsorption, the porous polymer particles were filtered and washed with hot water.

(Crosslinking of polymer with coated hydroxyl group)
The agarose adsorbed on the surface of the porous polymer particles was crosslinked as follows. 10 g of particles adsorbed with modified agarose were dispersed in a 0.4 M aqueous sodium hydroxide solution, 0.4 M epichlorohydrin was added, and the mixture was stirred at room temperature for 8 hours to crosslink the agarose. Thereafter, the particles were washed with hot water of 2 wt% sodium dodecyl sulfate aqueous solution and further washed with pure water.

(Reduction treatment)
The particles after crosslinking were subjected to a reduction treatment. The reaction was performed at room temperature (23 ° C.) for 1 hour in an aqueous solution prepared by adding 0.2% by weight of sodium borohydride as a reducing agent to water. After completion of the reaction, the particles obtained by filtration were washed with stirring with 1M NaCl Tris-HCl buffer for 3 hours. Thereafter, it was filtered and washed with pure water.

(Evaluation of nonspecific adsorption ability of protein)
0.2 g of the obtained separating material was put into 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. Then, after removing the supernatant by centrifugation, the amount of BSA adsorbed on the separating material was calculated from the BSA concentration in the supernatant obtained by measuring the absorbance at 280 nm of the supernatant with a spectrophotometer. . The BSA adsorption amount (non-specific adsorption amount) per 1 mL of the separation material was “◯” when 1 mg or less, “Δ” when 1 mg or more and less than 10 mg, and “x” when 10 mg or more. The results are shown in Table 1.

(Introduction of ion exchange groups)
The separating material (dry weight 20 g) was put into 200 mL of 5M aqueous sodium hydroxide solution and left at room temperature for 1 hour. Separately, 200 mL of an aqueous solution in which a predetermined amount (60 g) of diethylaminoethyl chloride hydrochloride was dissolved was added, the temperature of the aqueous solution was raised to 70 ° C., and the mixture was reacted for 8 hours with stirring. After completion of the reaction, the mixture was filtered and washed three times with water / ethanol (volume ratio 5/1) to obtain a separating material (DEAE-modified separating material) having diethylaminoethyl (DEAE) groups as ion exchange groups.

(Evaluation of ion exchange capacity)
The ion exchange capacity of the DEAE-modified separation material was measured as follows. A 5 mL capacity separating material was immersed in 20 mL of 0.1 N sodium hydroxide aqueous solution for 1 hour and stirred at room temperature. Thereafter, washing was performed until the pH of water used as the washing liquid became 7 or less. The washed separation material was immersed in 20 mL of 0.1N hydrochloric acid and stirred for 1 hour. After removing the separating material by filtration, the ion exchange capacity of the DEAE separating material was measured by neutralizing titrating the hydrochloric acid aqueous solution of the filtrate. The results are shown in Table 1.

(Static adsorption of protein)
The amount of static protein adsorption was measured as follows. 0.2 g of DEAE-modified separation material was put into 10 g of Tris-hydrochloric acid buffer (pH 8.0) and sufficiently wetted. Thereafter, a Tris-hydrochloric acid buffer solution (pH 8.0) having a BSA (Bovine Serum Albumin) concentration of 24 mg / mL was added so that the total volume became 20 ml, followed by stirring at room temperature for 24 hours. Thereafter, the supernatant was collected by centrifugation, the BSA concentration of the filtrate was measured with a spectrophotometer, and the amount of BSA adsorbed on the particles (static adsorption amount) was calculated based on the concentration. The concentration of BSA was confirmed by measuring the absorbance at 280 nm with a spectrophotometer.

(Protein detachment)
The protein adsorbed by the above method was desorbed by the following method. After the supernatant was collected, the residual solution containing the DEAE-denatured separation material was added with NaCl adjusted to 0.1 M, and stirred for 24 hours at room temperature to desorb the protein from the separation material. Then, after removing the supernatant by centrifugation, the BSA concentration of the supernatant was measured with a spectrophotometer, and the amount of BSA desorbed from the particles was calculated based on the concentration. The concentration of BSA was confirmed from the absorbance at 280 nm using a spectrophotometer. The desorption rate was calculated assuming that the total amount of adsorbed protein was desorbed as 100%. The results are shown in Table 1.

(Fluorescence observation)
The fluorescence measurement of the DEAE separating material adsorbed with protein was performed by the following method. The separator obtained by statically adsorbing BSA obtained by the above method was dried at 80 ° C. for 5 hours, then wet with pure water for 1 hour, and put into a quartz cell. After the separating material was sufficiently settled, it was measured with a fluorometer. At this time, light having a wavelength of 370 nm was irradiated as excitation light, and fluorescence observation was performed in a wavelength range of 300 to 600 nm. The slit width was 5 nm on both the excitation side and the fluorescence side, and the measurement was performed at a scanning speed of 240 nm / min and a photomultiplier voltage of 400V. Table 1 shows the fluorescence peak wavelengths. The fluorescence spectra of Example 1 and Comparative Example 1 are shown in FIGS. In the fluorescence measurement, extremely strong fluorescence having a peak in the vicinity of a wavelength of 370 nm was detected. Since this was considered to be derived from excitation light, the peak wavelength other than the peak was taken as the fluorescence peak wavelength.

(Alkali resistance evaluation)
First, the dynamic adsorption capacity (dynamic binding capacity) of the separation material before alkali treatment was measured as follows. The DEAE-modified separation material was mixed with methanol to prepare a slurry having a concentration of 30% by mass. This slurry was packed in a φ7.8 × 300 mm stainless steel column over 15 minutes. 10 column volumes of 20 mmol / L Tris-hydrochloric acid buffer (pH 8.0) were passed through the column. Thereafter, a 20 mmol / L Tris-hydrochloric acid buffer solution having a BSA concentration of 2 mg / mL was poured, and the BSA concentration at the column outlet was measured by UV absorbance measurement. The buffer was allowed to flow until the BSA concentrations at the column inlet and outlet coincided, and then diluted with 5 column volumes of 1 M NaCl Tris-HCl buffer. The dynamic adsorption capacity at 10% breakthrough was calculated using the following formula.
_{q 10 = c f F (t} 10 -t 0) / V B
q ₁₀ : dynamic adsorption capacity at 10% breakthrough (mg / mL wet resin)
cf: Injected BSA concentration F: Flow rate (mL / min)
V _B : Bed volume (mL)
t ₁₀ : time (min) at 10% breakthrough
t ₀ : BSA injection start time (min)

  The DEAE separation material was stirred in a 0.5 M aqueous sodium hydroxide solution for 24 hours and washed with a Tris-hydrochloric acid buffer (pH 8.0). The washed separation material was packed in a column under the same conditions as described above. The 10% breakthrough dynamic adsorption capacity of BSA was measured by the same method as described above, and compared with the dynamic adsorption capacity before alkali treatment. The case where the decrease in the dynamic adsorption capacity was 3% or less was “◯”, the case where more than 3% was less than 20% was “Δ”, and the case where 20% or more was “X”. The results are shown in the table.

(Example 2)
The same treatment as in Example 1 was carried out except that a modified dextran having a hydrophobic group introduced as described below was used instead of the modified agarose as the polymer modified product having a hydroxyl group to be coated on the porous polymer particles. Evaluation was performed as 2.
Sodium hydroxide 0.98 g and glycidyl phenyl ether 9.80 g were added to 480 mL of a dextran aqueous solution (2 wt%) having a molecular weight (Mw) of 300,000 and reacted at 60 ° C. for 6 hours to introduce a phenyl group into dextran. did. The resulting modified dextran was precipitated with methanol and washed. When the hydrophobic group content of the modified dextran was calculated by the following method, it was 0.103.

(Comparative Example 1)
The same treatment as in Example 1 was performed except that the reduction treatment was not performed, and evaluation was performed as Comparative Example 1.

(Comparative Example 2)
The same treatment as in Example 2 was performed except that the reduction treatment was not performed, and evaluation was performed as Comparative Example 2.

(Comparative Example 3)
Evaluation was performed as Comparative Example 3 using commercially available agarose particles (Capto DEAE, manufactured by GE Healthcare).

  In the separation material obtained in the examples, the fluorescence peak wavelength after BSA adsorption was not in the range of 430 to 440 nm, and the desorption rate after adsorption of BSA was higher than that of the separation material obtained in the comparative example. The separation material obtained in the comparative example exhibited a fluorescence peak wavelength at 432 to 436 nm when BSA was adsorbed. It was shown that by preparing particles that do not have a fluorescence peak wavelength in the range of 430 to 440 nm after protein adsorption, the desorption rate after protein adsorption is improved and the repeatability when using the column is improved.

Claims (11)

  Separation comprising porous polymer particles and a coating layer containing a polymer having a hydroxyl group covering at least a part of the surface of the porous polymer particles, and having no fluorescence peak in the wavelength range of 430 to 440 nm after protein adsorption Wood.   The separation material according to claim 1, wherein the polymer having a hydroxyl group is subjected to a reduction treatment.   The separation material according to claim 1 or 2, wherein the polymer having a hydroxyl group is crosslinked.   The separation material according to any one of claims 1 to 3, wherein the polymer having a hydroxyl group includes a reduced polysaccharide or a modified product thereof.   The separation material according to any one of claims 1 to 4, wherein the porous polymer particles include a polymer containing a monomer unit derived from a styrenic monomer.   The separation material according to any one of claims 1 to 5, wherein the average particle size of the separation material is 10 to 500 µm, and the mode diameter in the pore size distribution of the separation material is 0.05 to 0.6 µm.   The separation material according to any one of claims 1 to 6, wherein the porosity of the separation material is 40 to 70% by volume. The separation material according to claim 1, wherein the separation material has a specific surface area of 30 m ² / g or more.   The separation material according to any one of claims 1 to 8, wherein a coefficient of variation of the particle size of the separation material is 5 to 15%.   The water flow rate is 500 cm / h or more when water is passed through the column filled with the separation material so that the pressure in the column is 0.3 MPa. The separation material according to any one of the above.   A column comprising the separation material according to claim 1.

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