JP2021007915A - Production method for coated particle and column production method - Google Patents

Abstract

To provide a production method for particles usable as a separation material excellent in dynamic absorption amount, and to provide a column including the particle.SOLUTION: There is provided a production method for a coated particle comprising a hydrophobic porous polymer particle and a coating layer coating at least a part of the surface of the hydrophobic porous polymer particle. The method includes: preparing the hydrophobic porous polymer particle; obtaining an adsorption particle by dispersing the above porous polymer particle in a solution containing a hydroxy group-containing polymer and a solvent to adsorb the hydroxy group-containing polymer onto the above porous polymer particle; drying the adsorption particle; and cross-linking the hydroxy group-containing polymer using a cross-linking agent after dispersing the adsorption particle in a liquid medium whose solubility of the above hydroxy group-containing polymer is lower than that of the solvent.SELECTED DRAWING: None

Description

The present invention relates to a method for producing coated particles and a method for producing a column.

Conventionally, when biopolymers typified by proteins are separated and purified, generally, ion exchanges based on porous synthetic polymers and ion exchanges based on crosslinked gels of hydrophilic natural polymers are used. The body etc. are used. In the case of an ion exchanger based on a porous synthetic polymer, since the volume change due to salt concentration is small, when it is packed in a column and used for chromatography, it tends to have excellent pressure resistance during liquid passage. However, when this ion exchanger is used for separating proteins and the like, non-specific adsorption such as irreversible adsorption based on hydrophobic interaction occurs, so peak asymmetry occurs or ion exchange occurs due to hydrophobic interaction. There is a problem that the protein adsorbed on the body cannot be recovered while being adsorbed.

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

In order to overcome the drawbacks of crosslinked gels of hydrophilic natural polymers, complexes in which gels such as natural polymer gels are held in the pores of porous polymers are known in the field of peptide synthesis (for example, See Patent Document 1). By using such a complex, the loading coefficient of the reactive substance is increased, and high-yield synthesis becomes possible. Further, since the gel is surrounded by a hard synthetic polymer substance, there is an advantage that the volume does not change and the pressure of the flow-through passing through the column does not change even when used in the form of a column bed.

An ion exchanger of a hybrid copolymer in which the pores of a copolymer having a macronetwork structure are filled with a gel of a crosslinked copolymer synthesized from a monoma is known (see, for example, Patent Document 2). When the degree of cross-linking is low, the cross-linked copolymer gel has problems such as pressure loss and volume change, but by using a hybrid copolymer, the liquid passage characteristics are improved, the pressure loss is small, and the ion exchange capacity is improved. Leak behavior is improved.

Further, a composite filler in which a crosslinked gel of a hydrophilic natural polymer having a huge network structure is filled in the pores of an organic synthetic polymer substrate has been proposed.

U.S. Pat. No. 4,965,289 U.S. Pat. No. 4,335,017

The conventional separating material provided with a coating layer has room for improvement in terms of the amount of dynamic adsorption. Therefore, an object of the present invention is to provide particles that can be used as a separating material having an excellent amount of dynamic adsorption, and a column provided with the particles.

The present invention provides the method for producing coated particles and the method for producing a column according to the following [1] to [13].
[1] To prepare hydrophobic porous polymer particles, the hydrophobic porous polymer particles are dispersed in a solution containing a hydroxyl group-containing polymer and a solvent, and the hydroxyl group-containing polymer is used as the hydrophobic porous polymer. After adsorbing the adsorbed particles on the particles, drying the adsorbed particles, and dispersing the adsorbed particles in a liquid medium having a solubility of the hydroxyl group-containing polymer lower than that of the solvent, a cross-linking agent. A method for producing coated particles, comprising: cross-linking the hydroxyl group-containing polymer with the above-mentioned, and a coating layer covering at least a part of the surface of the hydrophobic porous polymer particles.
[2] The method according to [1], wherein the hydrophobic porous polymer particles contain a polymer having a structural unit derived from a styrene-based monoma.
[3] The method according to [1] or [2], wherein the solvent is water.
[4] The method according to any one of [1] to [3], wherein the liquid medium is methanol.
[5] The method according to any one of [1] to [4], wherein the hydroxyl group-containing polymer is a polysaccharide or a modified product thereof.
[6] The method according to any one of [1] to [5], wherein the cross-linking agent is at least one selected from the group consisting of epihalohydrin, a diglycidyl compound and a triglycidyl compound.
[7] The method according to any one of [1] to [6], wherein the average particle size of the coated particles is 10 to 500 μm.
[8] The method according to any one of [1] to [7], wherein the mode diameter in the pore size distribution of the coated particles is 0.05 to 0.5 μm.
[9] The method according to any one of [1] to [8], wherein the coefficient of variation of the particle size of the coated particles is 5 to 15%.
[10] The method according to any one of [1] to "9], wherein the specific surface area of the coated particles is 30 m ² / g or more.
[11] The method according to any one of [1] to [10], wherein the amount of the coating layer of the coating particles is 30 to 500 mg with respect to 1 g of the hydrophobic porous polymer particles.
[12] The method according to any one of [1] to [11], wherein the coating particles are a separating material.
[13] A method for producing a column, which comprises obtaining coated particles by the method according to any one of [1] to [12] and filling the column tube with the coated particles.

According to the present invention, it is possible to provide particles that can be used as a separating material having an excellent amount of dynamic adsorption, and a column having the particles.

Hereinafter, some embodiments of the present invention will be described in detail. However, the present invention is not limited to the following embodiments. In the present specification, the "surface of the hydrophobic porous polymer particles" includes not only the outer surface of the hydrophobic porous polymer particles but also the surface of the pores inside the hydrophobic porous polymer particles. .. Further, in the present specification, (meth) acrylic acid means acrylic acid or methacrylic acid, and the same applies to similar expressions such as (meth) acrylate.

The method for producing coated particles according to the present embodiment relates to a method for producing coated particles including hydrophobic porous polymer particles and a coating layer that covers at least a part of the surface of the hydrophobic porous polymer particles. The method for producing the coated particles according to the present embodiment is to prepare hydrophobic porous polymer particles and to disperse the hydrophobic porous polymer particles in a solution containing a hydroxyl group-containing polymer and a solvent to increase the hydroxyl group content. Adsorbed particles are obtained by adsorbing molecules on the hydrophobic porous polymer particles, the adsorbed particles are dried, and the adsorbed particles are dispersed in a liquid medium having a lower solubility of a hydroxyl group-containing polymer than water. This includes cross-linking the hydroxyl group polymer with a cross-linking agent.

(Hydrophobic porous polymer particles)
The porous polymer particles used in the production method according to the present embodiment are hydrophobic. Hydrophobic porous polymer particles are particles containing a hydrophobic porous polymer. Porous polymer particles can be obtained, for example, by polymerizing a monoma capable of forming a hydrophobic porous polymer. The porous polymer particles may be particles containing crosslinked polymers. The crosslinked polymer has, for example, a structural unit derived from a crosslinkable monoma having an aromatic group and two or more vinyl groups bonded to the aromatic group. Porous polymer particles formed by the crosslinked polymer can have a high elastic modulus, and therefore tend to be less likely to be broken when filled in a column tube.

Hydrophobic porous polymer particles containing crosslinked polymers can be obtained, for example, by suspension polymerization in a reaction solution containing a crosslinked monoma and, if necessary, other monoma components, a porosifying agent, and an aqueous medium. It can be synthesized by a method including polymerizing a monoma component. The crosslinkable monoma may be, for example, a styrene-based monoma. The styrene-based monoma refers to a monoma having a styrene skeleton.

The crosslinkable monoma may be, for example, a divinyl compound such as divinylbenzene, divinylbiphenyl, divinylnaphthalene, and divinylphenanthrene. These crosslinkable monomas may be used alone or in combination of two or more. From the viewpoint of durability, acid resistance, and alkalinity, divinylbenzene is preferable as the crosslinkable monoma.

The monofunctional monoma may be polymerized with the crosslinkable monoma. Examples of the monofunctional monoma include styrene, o-methylstyrene, m-methylstyrene, p-methylstyrene, α-methylstyrene, o-ethylstyrene, m-ethylstyrene, p-ethylstyrene, 2,4-. Dimethylstyrene, pn-butylstyrene, pt-butylstyrene, pn-hexylstyrene, pn-octylstyrene, pn-nonylstyrene, pn-decylstyrene, pn-dodecyl Examples thereof include styrenes such as styrene, p-methoxystyrene, p-phenylstyrene, p-chlorostyrene, and 3,4-dichlorostyrene, and derivatives thereof. These may be used alone or in combination of two or more. From the viewpoint of acid resistance and alkali resistance, the monofunctional monoma may be styrene. Styrene derivatives having functional groups such as carboxy group, amino group, hydroxyl group and aldehyde group can also be used.

The porosifying agent is a component that promotes phase separation of particles during polymerization, thereby forming hydrophobic porous polymer particles. An example of a porosifying agent is an organic solvent. Examples of organic solvents that can be used as the porosifying agent include aliphatic or aromatic hydrocarbons, esters, ketones, ethers, and alcohols. The porosifying agent is at least selected from the group consisting of, for example, toluene, xylene, cyclohexane, octane, butyl acetate, dibutyl phthalate, methyl ethyl ketone, dibutyl ether, 1-hexanol, 2-octanol, decanol, lauryl alcohol, and cyclohexanol. One type can be included.

The amount of the porosifying agent may be 0 to 300% by mass based on the total amount of the crosslinkable monoma and other monomas. The porosity of the hydrophobic porous polymer particles can be controlled by the amount of the porosifying agent. The size and shape of the pores of the hydrophobic porous polymer particles can be controlled by the type of the porosifying agent.

The aqueous medium may contain water. This water may function as a porosifying agent. For example, when an oil-soluble surfactant is added to the reaction solution, particles containing monoma and the oil-soluble surfactant are formed, and the particles can absorb water to promote phase separation in the particles. By removing one phase from the phase-separated particles, the particles are made porous.

Examples of oil-soluble surfactants include branched C16 to C24 fatty acids, chain unsaturated C16 to C22 fatty acids or chain saturated C12 to C14 fatty acids and sorbitan monoester formed from sorbitan, branched C16 to C24 fatty acids, and chains. Examples thereof include diglycerol monoaliphatic ether formed from state unsaturated C16 to C22 fatty acid or chain saturated C12 to C14 fatty acid and diglycerol, and diglycerol monoaliphatic ether. Examples of sorbitan monoesters include sorbitan monooleate, sorbitan monomillistate, and sorbitan monoesters of coconut fatty acids. Examples of diglycerol monoesters include diglycerol monooleates, diglycerol monomillistates, diglycerol monoisostearates, and diglycerol monoesters of coconut fatty acids. Examples of diglycerol monoaliphatic ethers include branched C16-C24 alcohols (eg, Gerve alcohol), chain unsaturated C16-C22 alcohols or chain saturated C12-C14 alcohols (eg palm fatty alcohols) and diglycerol. Examples include monoaliphatic ethers formed from. These can be used alone or in combination of two or more.

The amount of the oil-soluble surfactant may be 5 to 80% by mass based on the total mass of the monoma. As a result, droplets containing the monoma and the oil-soluble surfactant are likely to be stably formed.

The aqueous medium contains, for example, water or a mixed solvent of water and a water-soluble solvent (eg, lower alcohol). The aqueous medium may contain a surfactant. The surfactant may be an anionic, cationic, nonionic or zwitterionic surfactant.

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

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

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

Examples of the amphoteric ion-based surfactant include hydrocarbon surfactants such as lauryldimethylamine oxide, phosphate ester-based surfactants, and phosphite ester-based surfactants.

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

The reaction solution for suspension polymerization may contain a polymerization initiator. Examples of the polymerization initiator include benzoyl peroxide, lauroyl peroxide, benzoyl orthochloro peroxide, benzoyl orthomethoxy peroxide, 3,5,5-trimethylhexanoyl peroxide, and tert-butylperoxy-2-ethylhexano. Organic peroxides such as ate, di-tert-butyl peroxide; 2,2'-azobisisobutyronitrile, 1,1'-azobiscyclohexanecarbonitrile, 2,2'-azobis (2,4-azobis) Examples thereof include azo compounds such as dimethylvaleronitrile). The amount of the polymerization initiator may be 0.1 to 7.0 parts by mass with respect to 100 parts by mass of the monoma.

In order to improve the dispersion stability of the particles containing the monoma, the reaction solution may contain a dispersion stabilizer. Examples of the dispersion stabilizer include polyvinyl alcohol, polycarboxylic acid, celluloses (hydroxyethyl cellulose, carboxymethyl cellulose, methyl cellulose, etc.), and polyvinylpyrrolidone. These may be used in combination with an inorganic water-soluble polymer compound such as sodium tripolyphosphate. The dispersion stabilizer may be polyvinyl alcohol or polyvinylpyrrolidone. The amount of the dispersion stabilizer may be 1 to 10 parts by mass with respect to 100 parts by mass of the monoma.

The reaction solution for suspension polymerization may contain a water-soluble polymerization inhibitor such as nitrites, sulfites, hydroquinones, ascorbic acids, water-soluble B vitamins, citric acid, and polyphenols.

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

(Coating layer)
The coating layer covers at least a part of the surface of the porous polymer particles. By providing a coating layer that covers the surface of the porous polymer particles, it is possible to suppress an increase in column pressure. The coating layer contains a crosslinked body of a hydroxyl group-containing polymer. Since the coating layer contains a crosslinked product of a hydroxyl group-containing polymer, non-specific adsorption of the protein can be suppressed.

The hydroxyl group-containing polymer may have two or more hydroxyl groups. The hydroxyl group-containing polymer is preferably hydrophilic. The hydroxyl group-containing polymer may be, for example, a polysaccharide or a modified product thereof, or may be polyvinyl alcohol. Polysaccharides include agarose, dextran, cellulose, and chitosan. A modified product of a polysaccharide refers to a polysaccharide in which a hydrophobic group is introduced. By introducing a hydrophobic group, the interfacial adsorption ability of the coating layer can 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 can be introduced by reacting a functional group (for example, an epoxy group) that reacts with a hydroxyl group and a compound having a hydrophobic group (for example, glycidyl phenyl ether) with a hydroxyl group-containing polymer.

The weight average molecular weight of the hydroxyl group-containing polymer may be about 10,000 to 2 million. The content of the hydrophobic group in the modified product of the hydroxyl group-containing polymer is 5 to 30% by mass from the balance between the retention of the hydrophobic interaction force for adsorbing on the particle surface and the suppression of non-specific adsorption of the protein. It is more preferable, it is more preferably 10 to 20% by mass, and further preferably 12 to 17% by mass.

The coating layer containing the hydroxyl group contains the adsorbed particles by dispersing the hydrophobic porous polymer particles in the solution containing the hydroxyl group and the solvent and adsorbing the hydroxyl group to the hydrophobic porous polymer particles. To obtain, to dry the adsorbed particles, to disperse the adsorbed particles in a liquid medium having a solubility of the hydroxyl group-containing polymer lower than that of the above solvent, and then to crosslink the hydroxyl group-containing polymer with a cross-linking agent. It can be formed by a method including.

By forming the coating layer by the above method, it is possible to obtain coating particles capable of exhibiting a sufficiently high dynamic adsorption amount without applying special chemical modification. Although the shape of the coating layer in the coating particles according to the present embodiment is not clear, the present inventors infer as follows. It is considered that the hydroxyl group-containing polymer on the adsorbed particles is in a swollen and spread state in a solvent such as water having a high solubility of the hydroxyl group-containing polymer. On the other hand, since the liquid medium in which the adsorbed particles are once dried and then dispersed again has a low solubility of the hydroxyl group-containing polymer, the hydroxyl group-containing polymer adsorbed on the particles does not easily spread in the medium, and the hydroxyl groups on the particles are difficult to spread. It is considered that the layer of the containing polymer shrinks to become a thin state, and then the coating layer formed by cross-linking the hydroxyl group-containing polymer becomes thinner. Further, when a solvent having a high solubility of the hydroxyl group-containing polymer is used and when a solvent having a low solubility as in the present embodiment is used, the swelling / contracting state of the hydroxyl group-containing polymer at the time of cross-linking is different. It is considered that the method of cross-linking the chains is also different.

The solvent used in the hydroxyl group-containing polymer solution is one capable of dissolving the hydroxyl group-containing polymer. The solvent may be, for example, water or saline solution. The solvent is preferably water. The concentration of the hydroxyl group-containing polymer in the solution containing the hydroxyl group-containing polymer and the solvent may be 5 to 50 mg / ml, preferably 10 to 30 mg / ml.

The adsorption of the hydroxyl group-containing polymer may be chemical adsorption or physical adsorption. The adsorption of the hydroxyl group-containing polymer can be performed, for example, by the interaction between the hydrophobic porous polymer particles and the hydroxyl group-containing polymer into which a hydrophobic group has been introduced. Specifically, the porous polymer particles are dispersed in a solution containing a hydroxyl group-containing polymer and a solvent, and the porous polymer particles are left in that state for a certain period of time, so that the hydroxyl group-containing polymer solution is placed in the pores of the porous polymer particles. Can be impregnated with, and a hydroxyl group-containing polymer can be adsorbed on the porous polymer particles. The leaving time for impregnation varies depending on the surface condition of the porous polymer particles, but if left for one day, the concentration of the hydroxyl group-containing polymer inside the pores of the porous polymer particles is in equilibrium with the external concentration. It becomes. Then, the adsorbed particles are washed with a solvent such as water or alcohol to remove the unadsorbed hydroxyl group-containing polymer. At this stage, a solution of the hydroxyl group-containing polymer is usually held in the pores of the particles.

The method for drying the adsorbed particles is not particularly limited, but for example, the adsorbed particles can be dried in a dryer. The drying temperature may be, for example, 25 ° C. to 90 ° C., preferably 40 to 85 ° C., more preferably 50 to 80 ° C. The time for drying is not particularly limited, but may be, for example, 4 to 24 hours. By drying, the water content of the adsorbed particles is preferably 10% by mass or less, and more preferably 5% by mass or less.

The liquid medium has a lower solubility of the hydroxyl group-containing polymer than the above solvent. The liquid medium may be, for example, an alcohol such as methanol, ethanol or 2-propanol.

The liquid medium can continue to be used as a medium for the reaction of the subsequent cross-linking step. As the medium for the cross-linking reaction, it is preferable that the hydroxyl group-containing polymer does not dissolve from the adsorbed particles, it is difficult to extract the cross-linking agent or the like, and it is inactive in the cross-linking reaction. By adding a cross-linking agent to the medium containing the adsorbed particles after drying and cross-linking the hydroxyl group-containing polymer on the adsorbed particles, a cross-linked gel of the hydroxyl group-containing polymer can be formed. The reaction solution may be heated if necessary in the crosslinking step.

Examples of the cross-linking agent include epihalohydrins such as divinyl sulfone and epichlorohydrin, dialdehyde compounds such as glutaraldehyde, methylene diisocyanate, diisocyanate compounds, glycidyl compounds, and other compounds having two or more functional groups active on hydroxyl groups. Examples of the glycidyl compound include diglycidyl compounds such as ethylene glycol diglycidyl ether and triglycidyl compounds. When the hydroxyl group-containing polymer has an amino group such as chitosan, dihalide such as dichlorooctane can be used as a cross-linking agent. A catalyst may be used for the cross-linking reaction. The catalyst differs depending on the type of the cross-linking agent. For example, when the cross-linking agent is epichlorohydrin or the like, an alkali such as sodium hydroxide is effective, and when the cross-linking agent is a dialdehyde compound, a mineral acid such as hydrochloric acid is effective.

When the hydroxyl group-containing polymer is a polysaccharide or a modified product thereof, the amount of the cross-linking agent may be in the range of 0.001 to 100 times, assuming that one unit of the monosaccharide is 1 mol. When the amount of the cross-linking agent is 0.001 times or more, the coating layer tends to be difficult to peel off from the porous polymer particles. When the amount of the cross-linking agent is 100 times or less, the characteristics of the hydroxyl group-containing polymer are likely to be exhibited. When the hydroxyl group-containing polymer is a polysaccharide or a modified product thereof, the amount of the catalyst at the time of the cross-linking reaction is 0.01 to 10 times that of 1 mol of the monosaccharide forming the polysaccharide. , Or 0.1 to 5 times the ratio.

The cross-linking reaction can be carried out, for example, at a temperature of 5 to 90 ° C. over 1 to 10 hours. The temperature of the cross-linking reaction may be 30 to 90 ° C. After the cross-linking reaction is completed, the particles taken out by filtration are washed with a hydrophilic solvent such as water, methanol, ethanol, etc. to remove the unreacted hydroxyl group-containing polymer, medium, etc., and the particles have porous polymer particles and a coating layer. Coated particles are obtained. The coating layer is also usually formed in the pores.

The amount of the coating layer may be, for example, 30 to 500 mg per 1 g of the porous polymer particles. The amount of the coating layer is preferably 100 mg or more, more preferably 150 mg or more, further preferably 180 mg or more, still more preferably 200 mg or more, based on 1 g of the porous polymer particles. .. When the amount of the coating layer is 100 mg or more, non-specific adsorption of the protein can be further reduced, which is preferable. The amount of the coating layer may be 400 mg or less, or 300 mg or less, with respect to 1 g of the porous polymer particles. The amount of the coating layer can be measured by the weight loss of thermal decomposition, the Anthrone method or the like.

(Introduction of ion exchange group)
The coating particles provided with the coating layer can be used as a separating material. The coated particles can be used as a separating material in ion exchange purification, affinity purification, etc. by introducing, for example, an ion exchange group, a ligand (protein A), or the like via a hydroxyl group on the surface. Examples of the method for introducing an ion exchange group include a method using an alkyl halide compound.

Examples of the alkyl halide compound include monohalogenocarboxylic acids such as monohalogenoacetic acid and monohalogenopropionic acid, sodium salts thereof, and primary, secondary or tertiary amines and halogens having at least one alkyl halide group such as diethylaminoethyl chloride. Examples thereof include quaternary ammonium hydrochloride having an alkylated group. These alkyl halide compounds are preferably bromides or chlorides. The amount of the alkyl halide compound used is preferably 0.2% by mass or more with respect to the total mass of the separating material to which the ion exchange group is imparted.

For the introduction of 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.

The introduction of the ion exchange group can be performed on the hydroxyl group of the coating layer on the surface of the coating particles. To introduce the ion exchange group, the wet particles are drained by filtration or the like, immersed in an alkaline aqueous solution having a predetermined concentration, left to stand for a certain period of time, and then the above alkyl halide compound is added in a water-organic solvent mixed system. And react. This reaction is preferably carried out at a temperature of 40 to 90 ° C. for 0.5 to 12 hours. The type of alkyl halide used in the above reaction determines the ion exchange group to be added.

As a method for introducing an amino group which is a weakly basic group as an ion exchange group, among the above alkyl halide compounds, a mono-alkazole compound having at least one alkyl group in which a part of hydrogen atoms is replaced with chlorine atoms. , Di- or tri-alkylamine, mono-, di- or tri-alkanolamine, mono-alkyl-mono-alkanolamine, di-alkyl-mono-alkanolamine, mono-alkyl-di-alkanolamine, etc. The method can be mentioned. The amount of these alkyl halide compounds used is preferably 0.2% by mass or more with respect to 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 quaternary ammonium group of a strongly basic group as an ion exchange group, first, a tertiary amino group is introduced, and the tertiary amino group is reacted with an alkyl halide such as epichlorohydrin to be quaternary. A method of converting to an ammonium group can be mentioned. Further, quaternary ammonium hydrochloride or the like may be reacted with the coated particles.

Examples of the method for introducing a carboxy group, which is a weakly acidic group, as an ion exchange group include a method in which a monohalogenocarboxylic acid such as monohalogenacetic acid or monohalogenopropionic acid or a sodium salt thereof is reacted as the alkyl halide compound. .. The amount of these alkyl halide compounds used is preferably 0.2% by mass or more with respect to 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 the coated particles, and a sulfite such as sodium sulfite or sodium bisulfite or a sulfite is obtained. A method of adding a separating material to the saturated aqueous solution of the above can be mentioned. The reaction conditions are preferably 30 to 90 ° C. for 1 to 10 hours.

On the other hand, as a method for introducing an ion exchange group, a method of reacting 1,3-propane sultone with coated particles in an alkaline atmosphere can also be mentioned. It is preferable to use 1,3-propane sultone in an amount of 0.4% by mass or more based on the total mass of the coated particles. The reaction conditions are preferably 0 to 90 ° C. for 0.5 to 12 hours.

The average particle size of the porous polymer particles and the coated particles is preferably 500 μm or less, more preferably 300 μm or less, still more preferably 100 μm or less. The average particle size of the porous polymer particles and the coated particles is preferably 10 μm or more, more preferably 30 μm or more, still more preferably 50 μm or more. When the average particle size is 10 μm or more, the pressure of the packed column packed with particles tends to be difficult to increase. The average particle size of the porous polymer particles and the coated particles may be 10 to 500 μm. When used as a packing material for preparative or industrial chromatography, the particle size of the coated particles is preferably 50 to 300 μm, preferably 50 to 100 μm, in order to avoid an extreme increase in column internal pressure. Is more preferable.

The coefficient of variation (CV) of the particle size of the porous polymer particles and the coated particles is preferably 5 to 15%, more preferably 5 to 10% from the viewpoint of liquid permeability. As a method of reducing the coefficient of variation of the particle size, there is a method of monodispersing droplets containing monoma by an emulsifying device such as a microprocess server (manufactured by Hitachi, Ltd.).

The average particle size and the coefficient of variation of the particle size of the hydrophobic porous polymer particles and the coated particles can be obtained by the following measurement methods.
1) The particles are dispersed in water (including a dispersant such as a surfactant) to prepare a dispersion liquid containing 1% by mass of particles.
2) Using a particle size distribution meter (Cysmex Flow: manufactured by Sysmex), measure the average particle size and the coefficient of variation of the particle size from images of about 10,000 particles in the dispersion.

The ratio (porosity) of the pore volume to the total volume (including the pore volume) of the hydrophobic porous polymer particles or the coating particles is preferably 30% or more and 70% or less, preferably 40% or more and 70% or less. More preferably.

Most of the pores of the hydrophobic porous polymer and the coated particles may be macropores having a diameter of 0.1 μm or more and 0.5 μm or less. The mode diameter in the pore size distribution of the hydrophobic porous polymer particles and the coated particles may be 0.05 to 0.5 μm, 0.05 to 0.3 μm, or 0.1 to 0.5 μm. When the mode diameter in the pore diameter distribution is 0.1 μm or more, the substance tends to easily enter the pores. When the mode diameter in the pore diameter distribution is 0.5 μm or less, a sufficient specific surface area tends to be obtained. The porosity and the mode diameter in the pore size distribution can be adjusted by the porosity agent.

The specific surface area of the hydrophobic porous polymer particles and coated particles is preferably ^{30 m} 2 / g or more, more preferably at least ^35m 2 / g, more ^{40 m} 2 / g is more preferable. The larger the specific surface area, the more the adsorbed amount of the separated substance tends to increase relatively. The specific surface area of the hydrophobic porous polymer particles and the coated particles may be, for example, 150 m ² / g or less or 100 m ² / g or less.

The mode diameter (or average pore diameter), specific surface area, and porosity of the hydrophobic porous polymer particles and the coated particles in the pore diameter distribution can be measured by a mercury intrusion measuring device (Autopore: manufactured by Shimadzu Corporation). These can be measured as follows. A sample of about 0.05 g is collected in a standard 5 mL powder cell (stem volume 0.4 cc), and the pore distribution is measured under the condition of an initial pressure of 21 kPa (about 3 psia, equivalent to a pore diameter of about 60 μm). The mercury parameters are set to the device default mercury contact angle of 130 degrees and mercury surface tension of 485 days / cm. Each value is calculated by limiting the pore diameter to the range of 0.05 to 5 μm.

The coated particles according to this embodiment can be used as a separating material. The separating material can be used, for example, for separation by electrostatic interaction of proteins or for affinity purification. For example, a separating material is added to a mixed solution containing a protein, only the protein is adsorbed on the separating material by electrostatic interaction, and then the separating material is filtered off from the solution and added to an aqueous solution having a high salt concentration. Then, the protein adsorbed on the separating material can be easily desorbed and recovered. The coated particles according to the present embodiment have a high dynamic adsorption amount, a high static adsorption amount, and a reduced nonspecific adsorption amount, and can also have excellent liquid permeability when used as a column. .. Further, the separating material according to the present embodiment can exhibit an excellent effect in operability that when the column tube is filled, there is almost no volume change in the column tube regardless of the nature of the eluate.

The separating material is also useful as a column packing material in column chromatography. The packed column used for column chromatography can include a column tube and a separating material (coating particles, column packing material) filled in the column tube. The packed column can be produced by a method including filling the column tube with the coated particles obtained by the above method. A separating material into which a cation exchange group or an anion exchange group has been introduced can also be used as an ion exchange carrier.

As the biopolymer that can be separated using the separating material, a water-soluble substance is preferable. The biopolymer may be a protein such as blood protein such as serum albumin or immunoglobulin, an enzyme present in the living body, a protein bioactive substance produced by biotechnology, or DNA. The molecular weight of the biopolymer may be 2 million or less, or 500,000 or less. The properties and conditions of the ion exchange group can be selected according to the isoelectric point of the protein, the ionization state, and the like. In this regard, for example, Japanese Patent Application Laid-Open No. 60-169427 can be referred to.

When the protein is separated on the column, the flow rate of the protein solution or the like passed through the column is generally in the range of 400 cm / h or less. On the other hand, the separating material according to the present embodiment can be used with a high adsorption capacity even at a liquid passing rate of 800 cm / h or more. The liquid passing speed here means the liquid passing speed when a stainless column tube having a diameter of 7.8 × 300 mm is filled with a filler and the liquid is allowed to flow.

By coating the surface of the porous polymer particles with a crosslinked hydroxyl group-containing polymer, non-specific adsorption is less likely to occur. By introducing a cation exchange group or an anion exchange group, in the separation of biopolymers such as proteins, the advantages of hydroxyl group-containing polymers, porous polymer particles, and graft chains, if any, are combined. The characteristics it has can be shown. In particular, the coated particles according to the present embodiment have a preferable property as compared with the conventional ion exchange resin in that the protein adsorption capacity (dynamic adsorption capacity) under the same flow velocity is large.

Hereinafter, the present invention will be described in more detail with reference to examples. However, the present invention is not limited to these examples.

1. 1. In a three-necked flask of 500 mL of porous polymer particles, 16 g of divinylbenzene (manufactured by Nippon Steel & Sumikin Chemical Co., Ltd.) with a purity of 96%, 6 g of sorbitan monooleate (span 80), and 0.64 g of benzoyl peroxide were added to an aqueous polyvinyl alcohol solution (polyvinyl alcohol solution). The concentration was 0.5% by mass). The obtained mixed solution was emulsified by a microprocess server to form an emulsified solution containing a polyvinyl alcohol aqueous solution as a continuous phase and a monoma phase dispersed in the continuous phase. The obtained emulsion was stirred at 80 ° C. for 8 hours to allow the polymerization of divinylbenzene to proceed. The polymer was filtered and washed with acetone to give hydrophobic porous polymer particles. The particle size of the obtained porous polymer particles was measured with a flow type particle size measuring device. The average particle size was 99 μm, and the coefficient of variation (CV value) of the particle size was 8%.

2. 2. Separator (Example 1)
(Formation of coating layer with hydroxyl group-containing polymer)
A mixed solution was prepared by adding 1.00 g of sodium hydroxide and 9.20 g of glycidyl phenyl ether to 490 mL of an aqueous dextran solution (concentration: 2% by mass). The mixture was reacted for 12 hours to introduce a phenyl group into dextran. Dextran (modified dextran) into which a phenyl group was introduced was washed with methanol. The hydrophobic group content of the modified dextran was calculated by the following method and found to be 12.0%.

A dry powdered dextran (unmodified dextran) and a modified dextran 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. did. The hydrophobic group content was calculated from the following formula by measuring the absorbance of each aqueous solution at 269 nm with a spectrophotometer to determine the concentration.
Hydrophobic group content (%) = (C _AG / (C _HAG + C _AG )) × 100
_CAG : Concentration of modified dextran constituent unit (mmol / L) = A / ε _GPE × 1000
C _HAG : Concentration of unmodified dextran constituent unit (mmol / L) = [Concentration of unmodified dextran constituent unit (g / L) / dextran constituent unit (306 g / mol)] × 1000
-A: True absorbance of denatured dextran = Absorbance of denatured dextran-Absorption of _unmodified dextran-ε _GPE : Absorbance coefficient of glycidyl phenyl ether = 1372 (L / (mol · cm))
Concentration of unmodified dextran constituent unit (g / L) = sample concentration of modified dextran (% by mass) x 10-concentration of modified dextran constituent unit (g / L)
-Absorption of undenatured dextran = Absorbance of undenatured dextran x [Sample concentration of denatured dextran (mmol / L) / Sample concentration of undenatured dextran (mmol / L)]
Concentration of modified dextran building unit (g / L) = ( _CAG x modified dextran building block (456 g / mol)) / 1000

450 mL of a modified dextran aqueous solution having a concentration of 14.1 mg / mL was prepared, and 10 g of porous polymer particles were dispersed therein. By stirring the dispersion for 24 hours, the modified dextran was adsorbed on the surface of the porous polymer particles to form adsorbed particles. The adsorbed particles were filtered and washed with hot water.

The adsorbed particles after washing were air-dried in a blower dryer at 60 ° C. for 16 hours. Then, 10 g of the adsorbed particles were dispersed in 350 g of a 0.4 M sodium hydroxide methanol solution, and 13 g of epichlorohydrin was added to the obtained dispersion. Then, the dispersion was stirred for 8 hours to crosslink the modified dextran on the adsorbed particles with epichlorohydrin. The adsorbed particles taken out by filtration were washed with water and a 2% by mass aqueous sodium dodecyl sulfate solution to obtain coated particles having a coating layer containing crosslinked modified dextran. When the amount of the coating layer with respect to the porous polymer particles was calculated from the thermogravimetric analysis, it was 184 mg per 1 g of the porous polymer particles.

(Non-specific adsorption of proteins)
0.2 g of the coated particles were added to 20 mL of Tris buffer (pH 8.0) containing bovine serum albumin (BSA) at a concentration of 24 mg / mL. The dispersion was stirred for 24 hours at room temperature. Then, the supernatant obtained by centrifugation was filtered, and the BSA concentration of the filtrate was measured with a spectrophotometer. From the obtained BSA concentration, the amount of BSA adsorbed on the coated particles was calculated. The concentration of BSA was confirmed by a spectrophotometer from the absorbance at 280 nm.

(Introduction of ion exchange group)
The coated particles (dry weight 20 g) obtained by filtering the aqueous suspension containing the coated particles were added to 200 mL of a 5 M aqueous sodium hydroxide solution. Further, 200 mL of a predetermined amount (120.5 g) of diethylaminoethyl chloride hydrochloride was added, and the mixture was reacted at 70 ° C. for 8 hours with stirring. After completion of the reaction, the mixture was filtered and washed with water / ethanol (volume ratio 5/1) to obtain a separating material (DEAE-modified separating material) having a diethylaminoethyl (DEAE) group as an ion exchange group. A DEAE-modified separator was used for the subsequent evaluation of the specific surface area, ion exchange capacity, and column characteristics.

The mode diameter, specific surface area and porosity of the obtained DEAE-modified separator in the pore size distribution were measured by mercury intrusion measurement. The particle size of the DEAE-modified separator was measured with a flow-type particle size measuring device, and the average particle size and the coefficient of variation (CV) of the particle size were calculated from the measurement results.

The ion exchange capacity of the DEAE modified separator was measured as follows. The particles having a volume of 1 mL were immersed in 20 mL of 0.1 N sodium hydroxide for 1 hour and stirred at room temperature. Then, it was washed with water so that the pH of the solution became 7 or less. The obtained particles were immersed in 20 mL of 0.1N hydrochloric acid and stirred for 1 hour. After filtering the particles, the ion exchange capacity of the particles was measured by neutralizing and titrating the aqueous hydrochloric acid solution.

(Example 2)
A DEAE-modified separator was obtained in the same manner as in Example 1 except that the modified dextran concentration at the time of adsorption was changed to 18.6 g / L.

(Example 3)
A DEAE-modified separator was obtained in the same manner as in Example 1 except that the modified dextran concentration at the time of adsorption was changed to 22.3 g / L.

(Comparative Example 1)
The modified dextran concentration during adsorption was changed to 13.9 g / L, the adsorption process was completed in a wet state in water without drying after the modified dextran was adsorbed, and water was used instead of methanol during cross-linking. A DEAE-modified adsorbent was obtained in the same manner as in Example 1 except that the cross-linking reaction was carried out using.

(Comparative Example 2)
The concentration of modified dextran during adsorption was changed to 17.8 g / L, the adsorption process was completed in a wet state in water without drying after adsorption of modified dextran, and water was used instead of methanol during cross-linking. A DEAE-modified adsorbent was obtained in the same manner as in Example 1 except that the cross-linking reaction was carried out using.

(Comparative Example 3)
The modified dextran concentration during adsorption was changed to 22.5 g / L, the adsorption process was completed in a wet state in water without drying after the modified dextran was adsorbed, and water was used instead of methanol during cross-linking. A DEAE-modified adsorbent was obtained in the same manner as in Example 1 except that the cross-linking reaction was carried out using.

(Comparative Example 4)
DEAE was carried out in the same manner as in Example 1 except that the modified dextran concentration at the time of adsorption was changed to 13.7 g / L and the cross-linking reaction was carried out using water instead of methanol as a medium for cross-linking. A modified separator was obtained.

(Comparative Example 5)
The concentration of modified dextran during adsorption was changed to 13.7 g / L, the adsorption process was completed in a wet state in water without drying the particles after adsorption of modified dextran, and the water-dispersed slurry was changed to a methanol-dispersed slurry. After the medium exchange, a DEAE-modified adsorbent was obtained in the same manner as in Example 1 except that the cross-linking reaction was carried out using methanol as the medium.

3. 3. Packing Column The DEAE-modified separator was dispersed in ethanol to prepare a slurry having a particle concentration of 30% by mass. This slurry was filled in a stainless column tube having a diameter of 7.8 × 300 mm over 15 minutes. As a result, a packed column having a separating material filled in the column tube was obtained.

(Liquid permeability)
Water was passed through the packed column while changing the flow velocity, and the relationship between the linear flow velocity and the column pressure at that time was recorded. From the measurement results, the linear flow velocity at the time when the column pressure was 0.3 MPa was determined. Based on the linear flow velocity, the liquid permeability of the separating material was evaluated according to the following criteria.
A: Linear flow velocity 1500 cm / h or more B: Linear flow velocity 1000 cm / h or more 1500 cm / h or less C: Linear flow velocity less than 1000 cm / h

(Dynamic adsorption amount)
A 10-column volume buffer solution was passed through the packed column. Then, the protein concentration at the outlet of the column was measured based on the absorption of ultraviolet rays while flowing a buffer solution containing protein at a concentration of 2 mg / mL through the packed column. A buffer containing the protein was passed through the packed column until the protein concentrations matched at the inlet and outlet of the column. Subsequently, a solution prepared by adding 1M NaCl to the buffer solution was passed through a packed column for 10 column volumes, whereby the protein adsorbed on the separating material was recovered. The following proteins and buffers were used in the evaluation of each packed column.
Buffer: 40 mmol / L Tris-hydrochloric acid buffer (pH 8.0)
Protein: BSA (bovine serum albumin)

The amount of dynamic adsorption at 10% breakthrough was calculated by the following formula.
q ₁₀ = c _f F (t ₁₀ −t ₀ ) / V _B
q ₁₀ : Dynamic adsorption amount at 10% breakthrough (mg / mL wet resin)
cf: Injecting protein concentration F: Flow velocity (mL / min)
V _B : Bed volume (mL)
time at t ₁₀ : 10% breakthrough (min)
t ₀ : Protein injection start time (min)

(Static adsorption amount)
0.2 g of DEAE-modified separator was added to 20 mL of Tris buffer (pH 8.0) containing bovine serum albumin (BSA) at a concentration of 24 mg / mL. The dispersion was stirred for 24 hours at room temperature. Then, the supernatant obtained by centrifugation was filtered, and the BSA concentration of the filtrate was measured with a spectrophotometer. From the obtained BSA concentration, the amount of BSA adsorbed on the coated particles was calculated. The concentration of BSA was confirmed by a spectrophotometer from the absorbance at 280 nm.

It was confirmed that the separating material obtained in the examples suppressed non-specific adsorption of proteins and exhibited good liquid permeability. The adsorbents of the examples were compared with those subjected to the crosslinking reaction in water, those subjected to the crosslinking reaction in methanol without drying, or those subjected to the crosslinking reaction in water without drying. The dynamic adsorption amount was 10% or more higher. Further, in the examples, it was confirmed that the dynamic adsorption amount tends to increase as the coating amount increases.

Claims (13)

Preparing hydrophobic porous polymer particles and
The hydrophobic porous polymer particles are dispersed in a solution containing a hydroxyl group-containing polymer and a solvent, and the hydroxyl group-containing polymer is adsorbed on the hydrophobic porous polymer particles to obtain adsorbed particles.
Drying the adsorbed particles and
This includes dispersing the adsorbed particles in a liquid medium having a solubility of the hydroxyl group-containing polymer lower than that of the solvent, and then cross-linking the hydroxyl group-containing polymer with a cross-linking agent.
A method for producing coated particles, comprising: a hydrophobic porous polymer particle and a coating layer that covers at least a part of the surface of the hydrophobic porous polymer particle. The method according to claim 1, wherein the hydrophobic porous polymer particles contain a polymer having a structural unit derived from a styrene-based monoma. The method according to claim 1 or 2, wherein the solvent is water. The method according to any one of claims 1 to 3, wherein the liquid medium is methanol. The method according to any one of claims 1 to 4, wherein the hydroxyl group-containing polymer is a polysaccharide or a modified product thereof. The method according to any one of claims 1 to 5, wherein the cross-linking agent is at least one selected from the group consisting of epihalohydrin, a diglycidyl compound and a triglycidyl compound. The method according to any one of claims 1 to 6, wherein the average particle size of the coated particles is 10 to 500 μm. The method according to any one of claims 1 to 7, wherein the mode diameter in the pore size distribution of the coated particles is 0.05 to 0.5 μm. The method according to any one of claims 1 to 8, wherein the coefficient of variation of the particle size of the coated particles is 5 to 15%. The method according to any one of claims 1 to 9, wherein the specific surface area of the coated particles is 30 m ² / g or more. The method according to any one of claims 1 to 10, wherein the amount of the coating layer of the coating particles is 30 to 500 mg with respect to 1 g of the hydrophobic porous polymer particles. The method according to any one of claims 1 to 11, wherein the coating particles are a separating material. A method for producing a column, which comprises obtaining coated particles by the method according to any one of claims 1 to 12 and filling the column tube with the coated particles.