WO2020036151A1

WO2020036151A1 - Separating material, method of manufacturing separating material, and column

Info

Publication number: WO2020036151A1
Application number: PCT/JP2019/031740
Authority: WO
Inventors: 史彦河内; 真裕青嶌; 健安江; 後藤　泰史
Original assignee: 日立化成株式会社
Priority date: 2018-08-14
Filing date: 2019-08-09
Publication date: 2020-02-20
Also published as: JP2021181037A

Abstract

The purpose of the present invention is to provide a separating material which has excellent liquid permeability when used as a column. This separating material comprises a hydrophilic polymer that includes hydrophobic polymer particles and a coating layer that coats at least a portion of the surface of the hydrophobic polymer particles. The coating layer comprises a hydroxyl group and a group represented by -O-R-L (O is an oxygen atom derived from a hydroxyl group, R is an alkylene group having 1 to 5 carbon atoms, and L is a carboxyl group or alkali metal salt of a carboxyl group).

Description

Separation material, method for producing separation material, and column

The present invention relates to a separation material, a method for producing the separation material, and a column.

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

On the other hand, an ion exchanger whose base is a crosslinked gel of a hydrophilic natural polymer represented by a polysaccharide such as dextran or agarose has an advantage that there is almost no nonspecific adsorption of proteins. However, this ion exchanger has the disadvantage that it swells remarkably in an aqueous solution, 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 crosslinked gel is used for chromatography, there is a disadvantage that the pressure loss at the time of passing the liquid is large and the gel is compacted by the passing of the liquid.

In order to overcome the drawbacks of the crosslinked gel of a hydrophilic natural polymer, a complex in which a gel such as a natural polymer gel is retained in the pores of a porous polymer is known in the field of peptide synthesis (for example, Patent Document 1). By using such a complex, the load coefficient of the reactive substance can be increased, and high-yield synthesis can be achieved. Further, since the gel is surrounded by the hard synthetic polymer material, there is an advantage that even when used in the form of a column bed, there is no change in volume and the pressure of the flow-through passing through the column does not change.

(2) There is known an ion exchanger of a hybrid copolymer in which pores of a copolymer having a macro network structure are filled with a gel of a crosslinked copolymer synthesized from a monomer (for example, see Patent Document 2). The crosslinked copolymer gel has problems such as pressure loss and volume change when the degree of crosslinking is low.However, by using a hybrid copolymer, the liquid permeation characteristics are improved, the pressure loss is reduced, and the ion exchange capacity is improved. The leak behavior is improved.

複合 Further, composite fillers in which a crosslinked gel of a hydrophilic natural polymer having a giant network structure is filled in pores of an organic synthetic polymer substrate have been proposed (for example, see Patent Documents 3 and 4).

U.S. Pat. No. 4,965,289 U.S. Pat. No. 3,966,489 JP-A-1-254247 U.S. Pat. No. 5,114,577

The conventional separation material has room for improvement in column characteristics such as liquid permeability when used as a column.

Therefore, an object of the present invention is to provide a separation material having excellent liquid permeability when used as a column, a column including the separation material, and a method for producing the separation material.

The present invention provides a separation material, a method for producing a separation material, and a column according to the following [1] to [8].
[1] Hydrophobic polymer particles and a coating layer that covers at least a part of the surface of the hydrophobic polymer particles, wherein the coating layer is formed of a hydroxyl group and —ORL (O is derived from a hydroxyl group). R represents an alkylene group having 1 to 5 carbon atoms, L represents a carboxy group or a group represented by an alkali metal salt of a carboxy group.) , Separation material.
[2] The separation material according to [1], wherein the content of the group represented by —ORL is 100 μmol or more per 1 mL of the separation material.
[3] The separation material according to [1] or [2], wherein the hydrophobic polymer particles are particles containing a polymer having a structural unit derived from a styrene-based monomer.
[4] The separation material according to any one of [1] to [3], wherein the hydrophilic polymer contains a hydrophilic polymer derived from at least one selected from the group consisting of polysaccharides and modified products thereof.
[5] The method of any of [1] to [4], wherein the hydrophilic polymer includes a hydrophilic polymer derived from at least one selected from the group consisting of agarose, dextran, cellulose, pullulan, chitosan, and modified products thereof. The separating material according to any one of the above.
[6] The separation material according to any one of [1] to [5], wherein the hydrophilic polymer is crosslinked.
[7] A column comprising the separation material according to any one of [1] to [6].
[8] The method for producing a separation material according to any one of [1] to [6], wherein at least a part of the surface of the hydrophobic polymer particles is coated with a hydrophilic polymer having a hydroxyl group. A part of the hydrogen atom of the hydroxyl group of the coated hydrophilic polymer is -RL (R represents an alkylene group having 1 to 5 carbon atoms, and L represents a carboxy group or an alkali metal salt of a carboxy group. ), A method of producing a separating material.
[9] The method for producing a separation material according to [8], wherein the hydrophilic polymer having a hydroxyl group contains at least one selected from the group consisting of polysaccharides and modified products thereof.
[10] The separation material according to [8] or [9], wherein the hydrophilic polymer having a hydroxyl group contains at least one selected from the group consisting of agarose, dextran, cellulose, pullulan, chitosan, and modified products thereof. Manufacturing method.

According to the present invention, it is possible to provide a separating material having excellent liquid permeability when used as a column, a column including the separating material, and a method for producing the separating material. Further, the present invention can also provide a separation material which is excellent in column characteristics such as protein dynamic adsorption and recovery while reducing non-specific adsorption of proteins.

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

<Separation material>
The separation material according to the present embodiment includes hydrophobic polymer particles and a coating layer that covers at least a part of the surface of the hydrophobic polymer particles. The coating layer includes a hydrophilic polymer, and has a high hydrophilicity. When the molecule is a hydroxyl group, -ORL (O represents an oxygen atom derived from the hydroxyl group, R represents an alkylene group having 1 to 5 carbon atoms, and L represents a carboxy group or an alkali metal salt of a carboxy group. And a group represented by: The separation material of the present embodiment has excellent liquid permeability when packed in a column. In addition, the separation material of the present embodiment has excellent durability and alkali resistance, can reduce non-specific adsorption of protein, and has a sufficiently high dynamic adsorption amount and recovery rate of protein when packed in a column. It is considered high. The separating material of the present embodiment has excellent strength and can have a shape close to a true sphere. A truly spherical separating material is considered hydrodynamically advantageous when used in chromatography. Therefore, such a separation material is considered to be easy to suppress the pressure loss and to perform the chromatography operation, for example. In the present specification, the “surface of the hydrophobic polymer particles” includes not only the outer surface of the hydrophobic polymer particles but also the surface of the pores inside the hydrophobic polymer particles.

[Hydrophobic polymer particles]
The hydrophobic polymer particles according to the present embodiment are particles containing a polymer having hydrophobicity. The method for producing the hydrophobic polymer particles is not particularly limited, and examples thereof include a method of polymerizing a monomer capable of forming a polymer having hydrophobicity. The monomer is not particularly limited as long as it can form a polymer having hydrophobicity, and examples thereof include a styrene-based monomer. That is, the hydrophobic polymer particles may include, for example, a polymer having a structural unit derived from a styrene-based monomer. Such hydrophobic polymer particles are considered to be excellent in durability and alkali resistance.

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

(4) Examples of the polyfunctional monomer include divinyl compounds such as divinylbenzene, divinylbiphenyl, divinylnaphthalene, and divinylphenanthrene. These polyfunctional monomers can be used alone or in combination of two or more. Among them, the monomer preferably contains divinylbenzene from the viewpoint of durability, acid resistance and alkali resistance. That is, the hydrophobic polymer particles preferably include a polymer having a structural unit derived from divinylbenzene.

Examples of the monofunctional monomer include styrene, o-methylstyrene, m-methylstyrene, p-methylstyrene, α-methylstyrene, o-ethylstyrene, m-ethylstyrene, p-ethylstyrene, 2,4- Dimethylstyrene, pn-butylstyrene, pt-butylstyrene, pn-hexylstyrene, pn-octylstyrene, pn-nonylstyrene, pn-decylstyrene, pn-dodecyl Styrene such as styrene, p-methoxystyrene, p-phenylstyrene, p-chlorostyrene, and 3,4-dichlorostyrene, and derivatives thereof. These monofunctional monomers can be used alone or in combination of two or more. Among them, the monomer preferably contains styrene from the viewpoint of excellent acid resistance and alkali resistance. Further, a styrene derivative having a functional group such as a carboxy group, an amino group, a hydroxyl group, and an aldehyde group can also be used.

Examples of the porosifying agent include an organic solvent that promotes phase separation during polymerization and promotes porosity of particles, such as aliphatic or aromatic hydrocarbons, esters, ketones, ethers, and alcohols. Can be used. Examples of the porosifying agent include diethylbenzene, toluene, xylene, 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 alone or in combination of two or more.

The above-mentioned porous agent can be used in an amount of 0 to 200% by mass based on the total mass of the monomers. The porosity of the hydrophobic polymer particles can be controlled by the amount of the porosifying agent. Furthermore, the size and shape of the pores of the hydrophobic polymer particles can be controlled by the type of the porosifying agent.

水 Water used as a solvent can be used as a porosifying agent. When water is used as the porosifying agent, the oil-soluble surfactant is dissolved in the monomer, and the droplets of the monomer absorb the water, whereby the particles can be made porous.

Examples of the oil-soluble surfactant used for making the porous material include a sorbitan monoester of a branched C16-C24 fatty acid, a chain unsaturated C16-C22 fatty acid or a chain saturated C12-C14 fatty acid, for example, sorbitan monolaurate, sorbitan Sorbitan monoesters derived from monooleate, sorbitan monomyristate or coconut fatty acids; diglycerol monoesters of branched C16-C24 fatty acids, chain unsaturated C16-C22 fatty acids or chain saturated C12-C14 fatty acids, such as Glycerol monooleate (for example, diglycerol monoester of C18: 1 fatty acid (18 carbon atoms, 1 double bond) fatty acid), diglycerol monomyristate, diglycerol monoisostearate or coconut fatty acid diglycerol monoester Ester; Branch C16 ~ 24 alcohol (e.g., Guerbet alcohols), linear unsaturated C16 ~ C22 alcohols or linear saturated C12 ~ C14 alcohols (e.g., coconut fatty alcohols) diglycerol mono-fatty ethers; and mixtures thereof.

Of these, sorbitan monolaurate (e.g., SPAN® 20, having a purity preferably greater than about 40%, more preferably greater than about 50%, and even more preferably greater than about 70%) Rate), sorbitan monooleate (eg, SPAN® 80, preferably having a purity of greater than about 40%, more preferably greater than about 50%, even more preferably greater than about 70%) ), Diglycerol monooleate (eg, diglycerol monooleate having a purity preferably greater than about 40%, more preferably greater than about 50%, even more preferably greater than about 70%), diglycerol monoisostearate (Eg, having a purity preferably greater than about 40%, more preferably about 50% More preferably more than about 70% diglycerol monoisostearate), diglycerol monomyristate (purity preferably more than about 40%, more preferably more than about 50%, even more preferably more than about 70%). Diglycerol monomyristate), cocoyl (e.g., lauryl, myristoyl, etc.) ethers of diglycerol, or mixtures thereof.

These oil-soluble surfactants are preferably used in the range of 5 to 80% by mass based on the total mass of the monomers. When the use amount of the oil-soluble surfactant is 5% by mass or more, the stability of water droplets becomes sufficient, so that it is difficult to form large single holes. When the amount of the oil-soluble surfactant used is 80% by mass or less, the shape of the hydrophobic polymer particles can be more easily maintained after the polymerization.

水性 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 anionic surfactants include fatty acid oils such as sodium oleate and castor oil, alkyl sulfates such as sodium lauryl sulfate and ammonium lauryl sulfate, alkylbenzene sulfonates such as sodium dodecylbenzenesulfonate, and alkylnaphthalene sulfones. Acid salts, alkane sulfonates, dialkyl sulfosuccinates such as sodium dioctyl sulfosuccinate, etc., alkenyl succinates (eg, dipotassium alkenyl succinate), alkyl phosphate salts, naphthalene sulfonic acid formalin condensates, polyoxyethylene Polyoxyethylene alkyl ether sulfates such as alkyl phenyl ether sulfate, sodium polyoxyethylene lauryl ether sulfate, and polyoxyethylene alkyl sulfate; Ester salts.

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

Examples of the nonionic surfactant 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, and amides. Agents, polyether-modified silicon-based nonionic surfactants such as polyethylene oxide adducts of silicon and polypropylene oxide adducts of silicon, and fluorine-based nonionic surfactants such as perfluoroalkyl glycols.

Examples of the amphoteric surfactant include a hydrocarbon surfactant such as lauryl dimethylamine oxide, a phosphate ester surfactant, and a phosphite ester surfactant.

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

Examples of the polymerization initiator that is added as needed 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); The polymerization initiator can be used in the range of 0.1 to 7.0 parts by mass based on 100 parts by mass of the monomer.

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

において In the synthesis of the hydrophobic polymer particles, a polymer dispersion stabilizer may be used to improve the dispersion stability of the particles.

Examples of the polymer dispersion stabilizer include polyvinyl alcohol, polycarboxylic acid, celluloses (such as hydroxyethylcellulose, carboxymethylcellulose, and methylcellulose) and polyvinylpyrrolidone, and an inorganic water-soluble polymer compound such as sodium tripolyphosphate is also used in combination. can do. Of these, polyvinyl alcohol or polyvinylpyrrolidone is preferred. The addition amount of the polymer dispersion stabilizer is preferably 1 to 10 parts by mass based on 100 parts by mass of the monomer.

In order to suppress the monomer from being polymerized alone, a water-soluble polymerization inhibitor such as nitrites, sulfites, hydroquinones, ascorbic acids, water-soluble vitamins B, citric acid, and polyphenols may be used.

平均 The average particle size of the hydrophobic polymer particles is preferably 500 µm or less, more preferably 150 µm or less, and further preferably 110 µm or less. The average particle size of the hydrophobic polymer particles is preferably 10 μm or more, more preferably 30 μm or more, and further preferably 50 μm or more, from the viewpoint of improving liquid permeability.

The coefficient of variation (CV) of the particle diameter of the hydrophobic polymer particles is preferably from 3 to 15%, more preferably from 5 to 15%, from the viewpoint of improving liquid permeability. More preferably, it is 10%. C. V. For example, a method of monodispersing with an emulsifying apparatus such as a microprocess server (manufactured by Hitachi, Ltd.) can be mentioned.

The average particle diameter and the coefficient of variation of the particle diameter of the hydrophobic polymer particles or the separating material can be determined by the following measurement methods.
1) Disperse the hydrophobic polymer particles or the separating material in water (including a dispersant such as a surfactant) using an ultrasonic dispersing device and include 1% by mass of the hydrophobic polymer particles or the separating material. Prepare a dispersion.
2) Using a particle size distribution meter (Sysmex Flow, manufactured by Sysmex Corporation), the average particle size and the coefficient of variation of the particle size are measured from the image of the hydrophobic polymer particles or about 10,000 separating materials in the dispersion. .

The pore volume (porosity) of the hydrophobic polymer particles is preferably 30% by volume to 70% by volume, and more preferably 40% by volume to 70% by volume based on the total volume of the hydrophobic polymer particles. Is more preferred. The hydrophobic polymer particles preferably have pores having a pore diameter (mode diameter) of 0.05 μm or more and less than 0.6 μm, that is, macropores (macropores). The pore diameter of the hydrophobic polymer particles is more preferably 0.2 μm or more and less than 0.5 μm. When the pore diameter is 0.05 μm or more, the substance tends to easily enter the pores, and when the pore diameter is less than 0.6 μm, the specific surface area tends to be sufficient. The pore volume and the pore diameter can be adjusted by the above-mentioned porosifying agent.

The specific surface area of the hydrophobic polymer particles is preferably 20 m ² / g or more. In light of higher practicality, the specific surface area is more preferably equal to or greater than 35 m ² / g, and still more preferably equal to or greater than 40 m ² / g. If the specific surface area is 20 m ² / g or more, the amount of adsorption of the substance to be separated tends to increase.

The mode diameter (or average pore diameter), specific surface area, and porosity in the pore diameter distribution of the hydrophobic polymer particles and the separating material are values 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 taken in a standard 5 mL powder cell (stem volume: 0.4 mL) and measured under conditions of an initial pressure of 21 kPa (about 3 psia, pore diameter of about 60 μm). The mercury parameters are set to a device default mercury contact angle of 130 degrees and a mercury surface tension of 485 dynes / cm. Each value is calculated limited to the range of the pore diameter of 0.05 to 5 μm.

[Coating layer]
The coating layer according to this embodiment includes a hydroxyl group and —ORL (O represents an oxygen atom derived from the hydroxyl group, R represents an alkylene group having 1 to 5 carbon atoms, and L represents a carboxy group or a carboxy group. And a group represented by an alkali metal salt of a group). The separation material according to the present embodiment is excellent in liquid permeability when used as a column, because the coating layer contains a hydrophilic polymer having a hydroxyl group and a group represented by —ORL. In addition, the separation material according to the present embodiment tends to be excellent in characteristics such as dynamic adsorption property and recovery rate of protein while reducing non-specific adsorption of protein by providing the above-mentioned coating layer.

The alkylene group constituting R may be linear or branched, but is preferably linear from the viewpoint of better liquid permeability. R is preferably an alkylene group having 1 to 4 carbon atoms, and more preferably an alkylene group having 1 to 3 carbon atoms, from the viewpoint of more excellent liquid permeability. L preferably contains at least one selected from the group consisting of a carboxy group, a sodium salt of a carboxy group, and a potassium salt of a carboxy group, from the viewpoint of more excellent liquid permeability, and is composed of a sodium salt of a carboxy group and a carboxy group. More preferably, it contains at least one member selected from the group.

The hydrophilic polymer having a hydroxyl group and a group represented by —ORL may include a hydrophilic polymer derived from at least one selected from the group consisting of polysaccharides and modified products thereof. The hydrophilic polymer may be a hydrophilic polymer derived from at least one selected from the group consisting of agarose, dextran, cellulose, pullulan, chitosan, and modified products thereof. The hydrophilic polymer may be cross-linked.

非 By coating the hydrophobic polymer particles with a hydrophilic polymer having a hydroxyl group, non-specific adsorption of proteins tends to be easily suppressed. Further, the hydrophilic polymer having a hydroxyl group is preferably crosslinked. That is, the coating layer preferably contains a crosslinked body of a hydrophilic polymer having a hydroxyl group. Since the hydrophilic polymer having a hydroxyl group is crosslinked, an increase in column pressure tends to be suppressed.

(Hydrophilic polymer having hydroxyl group)
The hydrophilic polymer having a hydroxyl group preferably has two or more hydroxyl groups in one molecule. Examples of the hydrophilic polymer having a hydroxyl group include a polysaccharide or a modified product thereof, and a polyvinyl alcohol or a modified product thereof. The hydrophilic polymer having a hydroxyl group is preferably a polysaccharide or a modified product thereof. The hydrophilic polymer having a hydroxyl group may include at least one selected from the group consisting of polysaccharides and modified products thereof. Polysaccharides include, for example, agarose, dextran, cellulose, pullulan and chitosan. As the hydrophilic polymer having a hydroxyl group, for example, a polymer having an average molecular weight of about 10,000 to 200,000 can be used. The hydrophilic polymer having a hydroxyl group may include at least one selected from the group consisting of agarose, dextran, cellulose, pullulan, chitosan, and modified products thereof.

The hydrophilic polymer having a hydroxyl group is preferably a modified polymer 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. The hydrophobic group is obtained by reacting a functional group (for example, an epoxy group) that reacts with a hydroxyl group and a compound (for example, glycidyl phenyl ether) having a hydrophobic group with a hydrophilic polymer having a hydroxyl group by a conventionally known method. , Can be introduced.

The content of the hydrophobic group in the modified hydrophilic polymer in which the hydrophobic group is introduced is determined from the balance between the retention of the hydrophobic interaction force for adsorption on the particle surface and the suppression of non-specific adsorption of the protein. It is preferably from 5 to 30% by mass, more preferably from 10 to 20% by mass, even more preferably from 12 to 17% by mass.

<Production method of separation material>
The separating material of the present embodiment includes a step (1) of coating at least a part of the surface of the hydrophobic polymer particles with a hydrophilic polymer having a hydroxyl group, and a step of coating a hydrogen atom of the hydroxyl group of the coated hydrophilic polymer. Step (2) of partially substituting a part of the group represented by -RL (R represents an alkylene group having 1 to 5 carbon atoms, and L represents a carboxy group or an alkali metal salt of a carboxy group). And can be manufactured by a method comprising: The coated hydrophilic polymer may be cross-linked before step (2). The method for producing the separation material of the present embodiment includes, for example, a step of adsorbing a hydrophilic polymer having a hydroxyl group on the surface of the hydrophobic polymer particles, a step of cross-linking the adsorbed hydrophilic polymer, And introducing a group represented by —RL into the hydrophilic polymer via a hydroxyl group.

[Method of forming coating layer]
The coating layer according to the present embodiment can be formed, for example, by the following method.

(Adsorption of hydrophilic polymer having hydroxyl group)
First, a solution of a hydrophilic polymer having a hydroxyl group is adsorbed on the surface of the hydrophobic polymer particles. The solvent for the solution of the hydrophilic polymer having a hydroxyl group is not particularly limited as long as it can dissolve the hydrophilic polymer having a hydroxyl group, but water is the most common. The concentration of the hydrophilic polymer dissolved in the solvent is preferably 5 to 20 (mg / mL).

This solution is impregnated with hydrophobic polymer particles. In the impregnation method, hydrophobic polymer particles are added to a solution of a hydrophilic polymer having a hydroxyl group, and the solution is left for a predetermined time. The impregnation time varies depending on the surface state of the hydrophobic polymer particles. However, if the impregnation is carried out for 24 hours, the concentration of the hydrophilic polymer is in an equilibrium state between the inside and the outside of the hydrophobic polymer particles. Thereafter, washing with a solvent such as water or alcohol is performed to remove the hydrophilic polymer having a hydroxyl group that has not been adsorbed.

(Cross-linking treatment)
Next, a crosslinking agent is added to cause a crosslinking reaction of the hydrophilic polymer having a hydroxyl group adsorbed on the surface of the hydrophobic polymer particles, thereby forming a crosslinked body. At this time, the crosslinked body has a three-dimensional crosslinked network structure having a hydroxyl group.

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

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

架橋 The crosslinking reaction with a crosslinking agent is usually performed by adding a crosslinking agent to a system in which a separating material is dispersed and suspended in an appropriate medium. When a polysaccharide is used as the hydrophilic polymer having a hydroxyl group, the amount of the crosslinking agent is in the range of 0.1 to 100 moles per mole of one unit of the monosaccharide forming the polysaccharide. Within, it can be selected according to the performance of the separating material. In general, when the amount of the cross-linking agent is reduced, the coating layer tends to be easily separated from the hydrophobic polymer particles. If the amount of the crosslinking agent added is excessive and the reaction rate with the hydroxyl group-containing hydrophilic polymer is high, the properties of the raw material hydroxyl group-containing hydrophilic polymer tend to be impaired.

The amount of the catalyst used depends on the type of the cross-linking agent. Usually, when a polysaccharide is used as the hydrophilic polymer having a hydroxyl group, when one unit of the monosaccharide forming the polysaccharide is 1 mol, Is used in a range of 0.01 to 10 mole times, preferably 0.1 to 5 mole times.

For example, when the cross-linking reaction conditions are temperature conditions, the temperature of the reaction system is increased, and when the temperature reaches the reaction temperature, a cross-linking reaction occurs.

A medium for dispersing and suspending hydrophobic polymer particles impregnated with a solution of a hydrophilic polymer having a hydroxyl group, without extracting polymers, cross-linking agents, etc. from the impregnated polymer solution, and Must be inert to the crosslinking reaction. Specific examples of the medium include water, alcohol, and the like.

The crosslinking reaction is usually performed at a temperature in the range of 5 to 90 ° C. for 1 to 24 hours. Preferably, the temperature is in the range of 30 to 90 ° C.

調整 When adjusting the degree of progress of crosslinking, the crosslinking reaction may be performed stepwise. For example, the degree of crosslinking can also be adjusted by subjecting the particles that have undergone a crosslinking reaction to a crosslinking reaction again. The degree of cross-linking is evaluated at the temperature at which 5% weight loss due to thermal decomposition occurs. When the degree of progress of crosslinking is high, the temperature at which weight loss starts is high, and when the degree of progress of crosslinking is low, the temperature at which weight loss starts is also low.

The temperature at the time of 5% thermal weight loss is preferably from 200 to 350 ° C, more preferably from 220 to 330 ° C, and more preferably from 230 to 320 ° C, from the viewpoint of maintaining the characteristics of the hydrophilic polymer having a hydroxyl group. Is more preferable. If the degree of cross-linking is too low, the hydrophilic polymer having a hydroxyl group tends to fall off the particles.If the degree of cross-linking is too high, the hydrophilic polymer having a hydroxyl group can be prevented from falling off the particles, but the hydrophilic polymer has Swelling or a decrease in the amount of functional groups tends to decrease the amount of dynamic adsorption.

After the cross-linking reaction is completed, the generated particles are separated by filtration, and then washed with a hydrophilic organic solvent such as methanol and ethanol to remove unreacted polymer and suspending medium. Particles at least part of the surface of which are coated with a coating layer containing a hydrophilic polymer having a hydroxyl group (hereinafter sometimes referred to as “coated particles”) are obtained.

(Introduction of a group represented by —ORL)
A carboxy group can be introduced into the coated particles via a hydroxyl group of the hydrophilic polymer. That is, in the coating layer according to the present embodiment, a group represented by —ORL, in which an alkylene group having a carboxy group is directly bonded to an oxygen atom derived from a hydroxyl group of the hydrophilic polymer, is introduced. ing.

As a method for introducing a group represented by —ORL, for example, a method using a halogenated alkyl compound can be mentioned. Examples of the alkyl halide compound include monohalogenoacetic acid, monohalogenopropionic acid, monohalogenobutanoic acid, monohalogenopentanoic acid, monohalogenohexanoic acid, and alkali metal salts thereof. Preferably, the alkyl halide compound is a bromide or chloride. By reacting the hydroxyl group of the hydrophilic polymer with an alkyl halide compound under alkaline conditions, the hydrogen atom of the hydroxyl group can be replaced with a group represented by -RL.

Here, the hydroxyl group of the hydrophilic polymer is reacted with a diglycidyl compound such as epichlorohydrin or ethylene glycol diglycidyl ether such as epichlorohydrin to introduce an epoxy group, and then the epoxy group is converted into a carboxylic acid thiol such as thioglycolic acid. By reacting, a carboxy group can be introduced. However, the introduction of the epoxy group also causes the cross-linking reaction of the coating layer to the hydrophilic polymer to proceed in parallel, so that the swellability of the hydrophilic polymer such as a sugar chain is impaired, and the amount of dynamic adsorption of the protein decreases. There is a tendency.

On the other hand, the method for introducing a group represented by —ORL according to the present embodiment does not involve a step of introducing an epoxy group, and thus avoids the progress of the crosslinking reaction as described above. It is possible to improve the protein dynamic adsorption amount. By introducing a carboxy group through the hydroxyl group of the hydrophilic polymer, a separation material having excellent liquid permeability can be produced.

The content of the group represented by -ORL in the separation material (the amount of the introduced carboxy group) is preferably 100 µmol or more, more preferably 110 µmol or more, and more preferably 120 µmol per 1 mL of the separation material. More preferably, it is more preferably at least 125 μmol. When the amount of the carboxy group is 100 μmol or more per 1 mL of the separation material, the liquid permeability of the separation material can be further improved, and the electrostatic force of the separation material increases. Can be increased. If the content of the group represented by —ORL in the separation material (the amount of the introduced carboxy group) is too large, the protein is hardly diffused into the separation material from the viewpoint of difficulty in diffusing the protein into the separation material. The amount is preferably 1000 μmol or less, more preferably 950 μmol or less per 1 mL of the separation material.

分離 It is preferable that the separation material of the present embodiment has a coating layer of 30 to 500 mg per 1 g of the hydrophobic polymer particles. The amount of the coating layer can be measured by a weight loss due to thermal decomposition or the like.

通 The liquid passing speed in the present specification indicates a liquid passing speed when a separating material of the present embodiment is packed 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 of water is preferably 1300 cm / h or more when water is passed so that the pressure of the column becomes 0.3 MPa. It is more preferably at least 1500 cm / h, even more preferably at least 2000 cm / h. When separating proteins by column chromatography, the flow rate of a 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, a normal protein separation speed is used. It can be used at a flow rate of 1300 cm / h or more, which is faster than the separation material.

The average particle size of the separating material is preferably from 10 to 500 μm, more preferably from 30 to 300 μm, further preferably from 50 to 150 μm, and more preferably from 50 to 110 μm, from the viewpoint of improving liquid permeability. Is particularly preferred. The coefficient of variation (CV) of the particle size of the separating material is preferably 3 to 15%, more preferably 5 to 15%, and more preferably 5 to 10% from the viewpoint of improving liquid permeability. It is even more preferred.

The pore volume of the separating material is preferably 30% by volume or more and 70% by volume or less, more preferably 40% by volume or more and 70% by volume or less, and more preferably 50% by volume or more and 70% by volume based on the total volume of the separating material. % Is more preferable. The separation material preferably has pores having a pore diameter of 0.05 μm or more and less than 0.6 μm, that is, macropores (macropores). The pore size of the separation material is more preferably 0.1 μm or more and less than 0.5 μm. When the pore diameter is 0.05 μm or more, the substance tends to easily enter the pores, and when the pore diameter is less than 0.6 μm, the specific surface area tends to be sufficient.

The specific surface area of the separation material is preferably 20 m ² / g or more. In light of higher practicality, the specific surface area is more preferably equal to or greater than 35 m ² / g, and still more preferably equal to or greater than 40 m ² / g. If the specific surface area is 20 m ² / g or more, the amount of adsorption of the substance to be separated tends to increase.

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

The 5% compressive deformation elastic modulus in water of the separating material (5% compressive deformation elastic modulus in a wet state) may be, for example, 70 MPa or more, 100 MPa or more, 150 MPa or more, or 200 MPa or more. The upper limit of the 5% compressive deformation elastic modulus is not particularly limited.

The 5% compressive deformation elastic modulus (5% K value) of the separating material in water can be calculated as follows. Using a micro-compression tester (manufactured by Fisher), the separation material previously immersed in water was applied at a load application speed of 1 mN / sec at room temperature (25 ° C.) and a smooth end surface (50 μm × 50 μm) of a square pillar. The load and the compression displacement when compressed to 50 mN are measured. From the obtained measured values, the compression elastic modulus (5% K value) when the separation material undergoes 5% compression deformation can be obtained by the following equation. The load at the point where the amount of displacement changes the most during the measurement is defined as the breaking strength (mN).
5% K value (MPa) = (3/2 ^1/2 ) ・^FS -3 ^{/ 2}・ R- ^／
F: Load (mN) when the separation material is compressed and deformed by 5%
S: Compressive displacement (mm) when the separating material is compressed and deformed by 5%
R: radius of separation material (mm)

リガンド For example, a ligand (eg, protein A) or an ion exchange group can be introduced into the separation material of the present embodiment via the carboxy group in the group represented by —ORL. In addition, for example, a ligand (for example, protein A) or an ion exchange group can be introduced into the separation material of the present embodiment in addition to the group represented by —ORL.

分離 The separation material of the present embodiment can be suitably used for ion exchange purification, affinity purification, separation of biological macromolecules such as proteins by electrostatic interaction, and the like. For example, the separating material of the present embodiment is added to a mixed solution containing a protein, and only the protein is adsorbed to the separating material by electrostatic interaction. When added to an aqueous solution having a high concentration, the protein adsorbed on the separation material can be easily desorbed and recovered. Further, the separation material of the present embodiment can be used in column chromatography.

水溶 As the biopolymer that can be separated using the separation material of the present embodiment, a water-soluble substance is preferable. Specific examples include proteins such as blood proteins such as serum albumin and immunoglobulin, enzymes present in living bodies, protein bioactive substances produced by biotechnology, DNA, and biopolymers such as bioactive peptides. Can be The molecular weight of the biopolymer is preferably 2,000,000 or less, more preferably 500,000 or less. In addition, according to a known method, the properties of the separation material, separation conditions, and the like can be selected depending on the isoelectric point, ionization state, and the like of the protein. Known methods include, for example, the method described in JP-A-60-169427.

In the separation material of the present embodiment, by introducing a carboxy group or an alkali metal salt of a carboxy group which serves as an ion exchange group into the coating layer, particles of natural polymer or synthetic polymer can be used for separating biopolymers such as proteins. Each particle has the advantages of molecules. By providing the above-described coating layer, the separation material of the present embodiment has excellent liquid permeability, reduces nonspecific adsorption of proteins, and tends to easily cause protein adsorption and desorption. Further, the separation material of the present embodiment tends to have a large adsorption amount (dynamic adsorption amount) of proteins and the like under the same flow rate.

<Column>
The column of the present embodiment includes the above-described separation material. The column can be manufactured by packing the above-mentioned separating material in the column. The method for filling the column with the separating material is not particularly limited, and for example, a known method can be employed.

Hereinafter, the present invention will be described with reference to examples, but the present invention is not limited to these examples.

(Hydrophobic polymer particles)
In a 500 mL three-necked flask, 16 g of 96% pure divinylbenzene (trade name “DVB960” manufactured by Nippon Steel & Sumikin Chemical Co., Ltd.), 16 g of hexanol, 16 g of diethylbenzene, and 0.64 g of benzoyl peroxide were added to 0.5% by mass of polyvinyl alcohol. In addition to the aqueous solution, the mixture was emulsified using a micro process server (manufactured by Hitachi, Ltd.), and the obtained emulsion was transferred to a flask and heated with a water bath at 80 ° C. for about 8 hours using a stirrer. Stirred. After the obtained particles were taken out by filtration, they were washed with acetone to obtain hydrophobic polymer particles.

粒径 The particle size of the hydrophobic polymer particles was measured by a flow type particle size measuring device, and the average particle size and the coefficient of variation (CV) of the particle size were calculated. The average particle size of the hydrophobic polymer particles is 102 μm, and the C.I. V. Was 8%.

(Denatured agarose)
0.98 g of sodium hydroxide and 4.90 g of glycidyl phenyl ether were added to 480 mL of a 2% by mass aqueous agarose solution, and reacted at 60 ° C. for 6 hours to introduce a phenyl group into the agarose as a hydrophobic group. The resulting denatured agarose was reprecipitated with isopropyl alcohol and washed. When the hydrophobic group content of the modified agarose was calculated by the following method, it was 14.2%.

Dry agarose (unmodified agarose) and hydrophobic group-introduced agarose (modified agarose) dried to a volatile content of less than 0.1% by mass were each dissolved in pure water at 70 ° C. A 05% by mass aqueous solution sample was prepared. The hydrophobic group content was calculated from the following formula by measuring the absorbance at 269 nm of each aqueous solution using a spectrophotometer to determine the concentration. In the following, an agarose constituent unit means a disaccharide unit of agarose, and a modified agarose constituent unit means one in which a phenyl group is introduced into at least one of the hydroxyl groups in the disaccharide unit of agarose. I do.
Hydrophobic group content _{_{(%) = (C AG /}} (C HAG + C AG)) × 100
_CAG : Concentration of denatured agarose constituent unit (mmol / L) = A / _εGPE × 1000
C _HAG : concentration of unmodified agarose constituent unit (mmol / L) = (concentration of unmodified agarose constituent unit (g / L) / agarose constituent unit (306 g / mol)
) × 1000
A: true absorbance of denatured agarose = absorbance of denatured agarose-absorbance of undenatured agarose ε _GPE : extinction coefficient of glycidyl phenyl ether = 1372 (L / (mol · cm))
-Concentration of undenatured agarose constituent unit (g / L) = sample concentration of denatured agarose (% by mass) x 10-concentration of denatured agarose constituent unit (g / L)
Absorption of undenatured agarose = absorbance of undenatured agarose × (sample concentration of denatured agarose (mmol / L) / sample concentration of undenatured agarose (mmol / L))
-Concentration of denatured agarose constituent unit (g / L) = ( _CAG x denatured agarose constituent unit (456 g / mol)) / 1000

[Example 1]
<Formation of coating layer>
Hydrophobic polymer particles are added to a 20 mg / mL modified agarose aqueous solution at a concentration of 70 mL / g of particles, and the mixture is stirred at 55 ° C. for 24 hours to adsorb the modified agarose to the hydrophobic polymer particles. And washed with hot water.

(Cross-linking treatment)
The modified agarose adsorbed on the hydrophobic polymer particles was crosslinked as follows. 10 g of the particles onto which the modified agarose had been adsorbed were dispersed in a 0.4 M aqueous sodium hydroxide solution, and 0.02 M epichlorohydrin was added thereto, followed by stirring at room temperature for 24 hours. Thereafter, the resultant was washed with a 2% by mass aqueous solution of hot sodium dodecyl sulfate, and then washed with pure water to obtain coated particles having a crosslinked product of modified agarose as a coating layer. The amount of the coating layer was measured by weight loss due to thermal decomposition, and the coating amount (mg / g) per 1 g of the hydrophobic polymer particles was calculated. Table 1 shows the results.

(Non-specific adsorption amount of protein)
0.2 g of the obtained coated particles was added to 20 mL of a Tris-hydrochloride buffer (pH 8.0) having a BSA (Bovine Serum Albumin) concentration of 24 mg / mL, and the mixture was stirred at room temperature for 24 hours. Thereafter, the supernatant was collected by centrifugation. Based on the BSA concentration of the supernatant, the amount of BSA adsorbed on the particles was calculated. The BSA concentration was confirmed from the absorbance at 280 nm of the supernatant with a spectrophotometer. Non-specific adsorption was evaluated as “A” when the amount of BSA adsorbed per 1 mL of the separating material was less than 1 mg, “B” when it was 1 to 10 mg, and “C” when it exceeded 10 mg. Table 1 shows the results. In Comparative Example 1, nonspecific adsorption could not be evaluated.

(Introduction of carboxy group)
40 g of monochloroacetic acid was added to a dispersion obtained by dispersing 10 g of the obtained coated particles in 80 g of a 10 M aqueous NaOH solution, and reacted at 70 ° C. for 3 hours. After the reaction, the resultant was washed with water to remove excess monochloroacetic acid to obtain a separation material having a group in which —CH ₂ COOH or —CH ₂ COONa was bonded to an oxygen atom derived from a hydroxyl group of modified agarose.

(Measurement of carboxy group content)
The carboxy group content of the obtained separation material was measured as follows. 20 g of a 0.1 mol / L aqueous HCl solution was added to 1.0 mL of the wet separating material, and the mixture was stirred at room temperature for 30 minutes. After stirring, the separation material was washed with pure water by suction filtration, and the washing was continued until the pH of the washing solution exceeded 5. Thereafter, the separating material was dispersed in 20 g of a 0.5 M NaCl solution and stirred for 30 minutes. An aqueous solution of 0.01 M NaOH was added dropwise to the particle dispersion. The carboxy group content was calculated from the following equation. Table 1 shows the results.
Carboxy group introduction amount (μmol / mL) = 0.01 (mol / L) × Drop amount of 0.01M NaOH aqueous solution (mL) / Volume of separation material used for measurement (mL) × 1000

(Measurement of average particle size of separation material)
The particle size of the separation material 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. The mode pore diameter, specific surface area, and porosity of the separation material were measured with a mercury intrusion measuring device. Table 1 shows the results.

(5% compression deformation elastic modulus)
The 5% compressive deformation elastic modulus in water of the separating material was measured by the method described above. Table 2 shows the results.

(Liquid permeability)
The obtained separation material was dispersed in 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 to obtain a packed column. Thereafter, water was flowed through the column while changing the flow rate, the relationship between the flow rate and the column pressure was measured, and the liquid passing rate (linear flow rate) at a column pressure of 0.3 MPa was measured. Table 2 shows the results.

(Measurement of dynamic adsorption amount and recovery rate)
The buffer was flowed through the packed column for 10 column volumes. Thereafter, a buffer containing a protein at a concentration of 2 mg / mL was passed through the packed column with a residence time of 2 minutes, and the protein concentration at the column outlet was measured by UV measurement. The solution was allowed to flow until the protein concentration at the inlet and the column of the column coincided with each other. Thereafter, a solution obtained by adding a 1 M NaCl solution to the buffer was passed through 10 column volumes as a desorption solution, and the adsorbed protein was recovered. The dynamic adsorption amount (10% Dynamic Binding Capacity: hereinafter, 10% DBC) at 10% breakthrough was calculated using the following equation. Further, the amount of protein in the recovered solution was calculated, and the recovery rate R (%) of the protein relative to the amount of dynamic adsorption was calculated from the following equation. As a buffer, a 20 mmol / L sodium acetate buffer (pH 5.0) was used. The protein used was IgG (immunoglobulin G). Table 2 shows the results.
q ₁₀ = c _ff (t ₁₀ −t ₀ ) / V _B
_{_{_{r 10 = c r × V r}}} / V B
R = r ₁₀ / q ₁₀ × 100
q ₁₀ : Dynamic adsorption amount (mg / mL wet resin) at 10% breakthrough
cf: IgG concentration injected F: flow rate (mL / min)
V _B : bed volume (mL)
t ₁₀ : time (min) at 10% breakthrough
t ₀ : IgG injection start time (min)
r ₁₀ : Recovered amount of protein (mg / mL wet resin)
_cr = IgG concentration (mg / mL) in the collected desorbed solution
_Vr = volume of recovered desorbed liquid (mL)
R: Recovery rate (%)

[Example 2]
A separation material having a group in which —CH ₂ COOH or —CH ₂ COONa is bonded to an oxygen atom derived from a hydroxyl group of modified agarose was prepared in the same manner as in Example 1 except that monochloroacetic acid 40 g was changed to monochloroacetic acid 20 g. Evaluation was performed in the same manner as in Example 1.

[Example 3]
Performed in the same manner as in Example 1 except that 40 g of monochloroacetic acid was changed to 40 g of 3-chloropropionic acid, and then 40 g of DMF (N, N-dimethylformamide) was added after adding 3-chloropropionic acid. A separation material having a group in which — (CH ₂ ) ₂ COOH or — (CH ₂ ) ₂ COONa was bonded to the oxygen atom of was prepared, and evaluated in the same manner as in Example 1.

[Example 4]
Monochloroacetic acid 40g 4-chlorobutane was changed to acid 40g, 4-chlorobutane except that after the acid addition further DMF was added 40g is as in Example 1 and the oxygen atom derived from the hydroxyl group of the denaturing agarose - _(CH _{2) 3} A separating material having a group to which COOH or — (CH ₂ ) ₃ COONa was bonded was prepared and evaluated in the same manner as in Example 1.

[Example 5]
The same procedure as in Example 1 was carried out except that 40 g of monochloroacetic acid was changed to 40 g of 5-chloropentanoic acid, and 40 g of DMF was further added after the addition of 5-chloropentanoic acid, and the oxygen atom derived from the hydroxyl group of the modified agarose was replaced with-(CH ₂ ) ₄ COOH, or _- _(CH 2) 4 COONa to form a separation member having the bonded groups, was evaluated in the same manner as in example 1.

[Example 6]
The same procedure as in Example 1 was carried out except that 40 g of monochloroacetic acid was changed to 40 g of 6-chlorohexanoic acid, and 40 g of DMF was further added after the addition of 6-chlorohexanoic acid. The oxygen atom derived from the hydroxyl group of the modified agarose was replaced with-(CH ₂ ) ₅ COOH or _- _(CH 2) 5 COONa is prepared separation material having attached groups, was evaluated in the same manner as in example 1.

[Comparative Example 1]
Evaluation was performed in the same manner as in Example 1 using a commercially available ion exchange chromatography carrier “CM Sepharose Fast Flow” (manufactured by GE Healthcare Japan) as a separating material.

[Comparative Example 2]
Change the monochloroacetic acid 40g in 11-bromo undecanoic acid 40g, 11 bromo undecanoic acid added after addition DMF except for adding 40g the same manner as in Example 1, the oxygen atom derived from the hydroxyl group of the denaturing agarose - (CH ₂ A separation material having a group to which ₁₀ COOH or — (CH ₂ ) ₁₀ COONa was bonded was prepared and evaluated in the same manner as in Example 1.

分離 It was confirmed that the separation materials of Examples 1 to 6 were excellent in liquid permeability when packed in a column, and high in the amount of dynamic adsorption of protein and the recovery rate. In addition, it was also confirmed that the separation materials of Examples 1 to 6 can suppress nonspecific adsorption of proteins and have a high 5% compressive deformation elastic modulus.

As described above, according to the present invention, it is possible to provide a separation material having excellent liquid permeability when used as a column, a column including the separation material, and a method for producing the separation material.

Claims

Hydrophobic polymer particles, comprising a coating layer covering at least a part of the surface of the hydrophobic polymer particles,
The coating layer comprises a hydroxyl group and —ORL (O represents an oxygen atom derived from the hydroxyl group, R represents an alkylene group having 1 to 5 carbon atoms, and L represents a carboxy group or an alkali carboxy group. And a hydrophilic polymer having a group represented by the following formula:
分離 The separation material according to claim 1, wherein the content of the group represented by -ORL is 100 µmol or more per 1 mL of the separation material.
The separation material according to claim 1 or 2, wherein the hydrophobic polymer particles are particles containing a polymer having a structural unit derived from a styrene-based monomer.
4. The separation material according to claim 1, wherein the hydrophilic polymer includes a hydrophilic polymer derived from at least one selected from the group consisting of polysaccharides and modified products thereof.
5. The hydrophilic polymer according to claim 1, wherein the hydrophilic polymer includes a hydrophilic polymer derived from at least one selected from the group consisting of agarose, dextran, cellulose, pullulan, chitosan, and modified products thereof. The separating material according to the above.
分離 The separation material according to any one of claims 1 to 5, wherein the hydrophilic polymer is cross-linked.
カラム A column comprising the separation material according to any one of claims 1 to 6.
A method for producing a separation material according to any one of claims 1 to 6, wherein
A step of coating at least a part of the surface of the hydrophobic polymer particles with a hydrophilic polymer having a hydroxyl group,
A part of the hydrogen atom of the hydroxyl group of the coated hydrophilic polymer may be represented by -RL (R represents an alkylene group having 1 to 5 carbon atoms, and L represents a carboxy group or an alkali metal salt of a carboxy group. A) a group represented by the following formula:
A method for producing a separation material, comprising:
The method for producing a separation material according to claim 8, wherein the hydrophilic polymer having a hydroxyl group contains at least one selected from the group consisting of polysaccharides and modified products thereof.
The method for producing a separation material according to claim 8, wherein the hydrophilic polymer having a hydroxyl group includes at least one selected from the group consisting of agarose, dextran, cellulose, pullulan, chitosan, and modified products thereof.