WO2018174022A1

WO2018174022A1 - Separation material

Info

Publication number: WO2018174022A1
Application number: PCT/JP2018/010880
Authority: WO
Inventors: 優渡邊; 史彦河内; 健安江; 後藤　泰史; 道男佛願
Original assignee: 日立化成株式会社
Priority date: 2017-03-21
Filing date: 2018-03-19
Publication date: 2018-09-27
Also published as: JPWO2018174022A1

Abstract

The present invention relates to a separation material which is provided with: porous polymer particles that contain a polymer which contains a styrene monomer as a monomer unit; and a coating layer that covers at least a part of the surface of each porous polymer particle, while containing a polymer that has a hydroxyl group. The change ratio of the specific surface area of the separation material before and after coating is from 0.50 to 0.85.

Description

Separation material

The present invention relates to a separating material.

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

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

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

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

However, conventional separation materials do not satisfy a sufficient level of low non-specific adsorption of protein, excellent protein adsorption, and excellent liquid permeability when used as a column.

Therefore, an object of the present invention is to provide a separation material that has a small amount of nonspecific adsorption of protein, is excellent in protein adsorption, and has excellent liquid permeability when used as a column.

The present invention provides the separating material described in [1] to [10] below.
[1] A porous polymer particle containing a polymer containing a styrene monomer as a monomer unit, and a coating layer containing a polymer having a hydroxyl group and covering at least a part of the surface of the porous polymer particle A separation material having a change ratio of a specific surface area before and after that of 0.50 to 0.85.
[2] The separation material according to [1], wherein the specific surface area is 30 m ² / g or more.
[3] The separation material according to [1] or [2], wherein the coefficient of variation of the particle size of the porous polymer particles is 5 to 15%.
[4] The separation material according to any one of [1] to [3], wherein the polymer having a hydroxyl group is a polysaccharide or a modified product thereof.
[5] The separating material according to any one of [1] to [4], wherein the polymer having a hydroxyl group is at least one selected from the group consisting of dextran, agarose, pullulan, and modified products thereof.
[6] The separating material according to any one of [1] to [5], comprising 30 to 450 mg of the coating layer per gram of the porous polymer particles, wherein the polymer having a hydroxyl group is crosslinked.
[7] The separation material according to any one of [1] to [6], wherein the mode diameter in the pore size distribution of the porous polymer particles is 0.05 to 0.50 μm.
[8] The separation material according to any one of [1] to [7], wherein when the column is packed, the liquid flow rate is 800 cm / h or more when the column pressure is 0.3 MPa.
[9] The separating material according to any one of [1] to [8], wherein the polymer having a hydroxyl group has a weight average molecular weight of 10,000 to 5,000,000.
[10] The separation material according to any one of [1] to [9], which has a cation exchange group or an anion exchange group and is used for ion exchange.

According to the present invention, it is possible to provide a separation material with less non-specific protein adsorption, excellent protein adsorption, and excellent liquid permeability when used as a column.

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

<Separation material>
The separating material of the present embodiment includes a porous polymer particle containing a polymer containing a styrene monomer as a monomer unit, and a coating layer containing a polymer having a hydroxyl group that covers at least a part of the surface of the porous polymer particle. The change ratio of the specific surface area before and after coating is 0.50 to 0.85. Such a separation material has less non-specific protein adsorption, and is excellent in protein adsorption and liquid permeability when used as a column. The reason why the non-specific adsorption is small in the separation material of the present embodiment is considered to be that the hydrophobic interaction is small. The separating material of the present embodiment is also excellent in alkali resistance, pressure resistance and durability. For example, the separation material of the present embodiment is excellent in dynamic adsorptivity when the liquid is passed at a high flow rate. The separation material of the present embodiment is excellent in strength and can have a shape close to a true sphere. Spherical separators are considered hydrodynamically advantageous when used in chromatography. Therefore, it is considered that such a separation material is easy to suppress pressure loss and perform a chromatography operation, for example.

In this specification, “the surface of the porous polymer particle” includes not only the surface of the porous polymer particle but also the surface of the pores inside the porous polymer particle.

[Porous polymer particles]
The porous polymer particle according to the present embodiment includes a polymer containing a styrene monomer as a monomer unit. Such porous polymer particles are considered to be excellent in durability and alkali resistance. The porous polymer particles according to this embodiment are, for example, particles obtained by polymerizing a monomer containing a styrenic monomer in the presence of a porosifying agent. The porous polymer particles can be synthesized by, for example, conventional suspension polymerization. Here, the styrene monomer means a monomer having a styrene skeleton. Examples of the styrenic monomer include the following polyfunctional monomers and monofunctional monomers.

Examples of the styrenic polyfunctional monomer include divinyl compounds having a styrene skeleton such as divinylbenzene, divinylbiphenyl, divinylnaphthalene, and divinylphenanthrene. These polyfunctional monomers can be used alone or in combination of two or more. Among these, divinylbenzene is preferably used from the viewpoint of further improving durability, acid resistance and alkali resistance. That is, the porous polymer particles may contain a polymer containing divinylbenzene as a monomer unit.

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

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

The above porous agent can be used, for example, in an amount of 0 to 200% by mass with respect to the total mass of the monomer. The porosity of the porous polymer particles can be controlled by the amount of the porous agent. Further, the size and shape of the pores of the porous polymer particles can be controlled by the kind of the porous agent.

Water used as a solvent can be used as a porous agent. When water is used as the porosifying agent, the particles can be made porous by, for example, dissolving an oil-soluble surfactant in the monomer and absorbing water.

Examples of the oil-soluble surfactant used for the porosification include sorbitan monoesters of branched C16 to C24 fatty acids, chain unsaturated C16 to C22 fatty acids or chain saturated C12 to C14 fatty acids, such as sorbitan monolaurate, sorbitan Sorbitan monoesters derived from monooleate, sorbitan monomyristate or coconut fatty acid; diglycerol monoesters of branched C16-C24 fatty acids, chain unsaturated C16-C22 fatty acids or chain saturated C12-C14 fatty acids, for example di- Glycerol monooleate (for example, diglycerol monoester of C18: 1 (18 carbon atoms, 1 double bond) fatty acid), diglycerol monomyristate, diglycerol monoisostearate or diglycerol monoester of coconut fatty acid Ester; Branch C16 ~ 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 (eg, SPAN 20), preferably having a purity of greater than about 40%, more preferably greater than about 50%, and even more preferably greater than about 70%. Sorbitan monooleate (e.g., SPAN 80), preferably greater than about 40%, more preferably greater than about 50%, and even more preferably greater than about 70% sorbitan monooleate ); Diglycerol monooleate (eg, diglycerol monooleate having a purity of preferably greater than about 40%, more preferably greater than about 50%, even more preferably greater than about 70%); diglycerol monoisostearate (For example, the purity is preferably greater than about 40%, more preferably about 50%. Greater than, more preferably greater than about 70% diglycerol monoisostearate); diglycerol monomyristate (eg, preferably greater than about 40%, more preferably greater than about 50%, even more preferably about 70% purity). % Of sorbitan monomyristate); a cocoyl (eg, lauryl and myristoyl group) ether of diglycerol; or a mixture thereof.

These oil-soluble surfactants are preferably used in the range of 5 to 80% by mass relative to the total mass of the monomer. When the content of the oil-soluble surfactant is 5% by mass or more, the stability of the water droplets is easily improved, so that a large single hole is easily formed. When the content of the oil-soluble surfactant is 80% by mass or less, the porous polymer particles are more easily retained in shape after polymerization.

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

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

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

Nonionic surfactants include, for example, hydrocarbon nonionic surfactants such as polyethylene glycol alkyl ethers, polyethylene glycol alkyl aryl ethers, polyethylene glycol esters, polyethylene glycol sorbitan esters, polyalkylene glycol alkylamines, or amides. Agents, polyether-modified silicon nonionic surfactants such as silicon polyethylene oxide adducts and polypropylene oxide adducts, and fluorine nonionic surfactants such as perfluoroalkyl glycols.

Examples of zwitterionic surfactants include hydrocarbon surfactants such as lauryl dimethylamine oxide, phosphate ester surfactants, and phosphite ester surfactants.

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

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

The polymerization temperature can be appropriately selected according to the type of monomer and polymerization initiator. The polymerization temperature may be, for example, 25 to 110 ° C. or 50 to 100 ° C.

In the above polymerization step, a polymer dispersion stabilizer may be added in order to improve the dispersion stability of the particles.

Examples of the polymer dispersion stabilizer include polyvinyl alcohol, polycarboxylic acid, celluloses (hydroxyethyl cellulose, carboxymethyl cellulose, methyl cellulose, etc.), and polyvinyl pyrrolidone, and inorganic water-soluble polymer compounds such as sodium tripolyphosphate are also included. Can be used together. Of these, polyvinyl alcohol or polyvinyl pyrrolidone is preferred. The amount of the polymer dispersion stabilizer added may be, for example, 1 to 10 parts by mass with respect to 100 parts by mass of the monomer.

In order to suppress the polymerization of the monomer alone, a water-soluble polymerization inhibitor such as nitrites, sulfites, hydroquinones, ascorbic acids, water-soluble vitamin Bs, citric acid, polyphenols and the like may be used.

The average particle diameter of the porous polymer particles may be, for example, 500 μm or less, 300 μm or less, or 200 μm or less. In addition, the average particle diameter of the porous polymer particles may be, for example, 10 μm or more, 30 μm or more, or 50 μm or more from the viewpoint of easily suppressing an increase in column pressure after column filling. Good.

The coefficient of variation (CV) of the particle size of the porous polymer particles may be, for example, 5 to 15% or 5 to 13% from the viewpoint of improving liquid permeability. It may be up to 10%. C. V. As a method for reducing the above, monodispersion by an emulsification apparatus such as a microprocess server (Hitachi Ltd.) can be mentioned.

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

The porosity (pore volume) of the porous polymer particles may be, for example, 30% to 70% by volume, and 40% to 70% by volume based on the total volume of the porous polymer particles. May be. The porous polymer particles preferably have macropores (macropores).

The mode diameter in the pore size distribution of the porous polymer particles is preferably from 0.05 to 0.50 μm, more preferably from 0.10 to 0.50 μm, and from 0.10 μm to less than 0.50 μm. More preferably. When the mode diameter in the pore size distribution is 0.05 μm or more, substances tend to easily enter the pores, and when the mode diameter in the pore diameter distribution is 0.50 μm or less, the specific surface area is sufficient. It tends to be.

The porosity and pore diameter of the porous polymer particles can be adjusted by, for example, the above-described porosifying agent.

The specific surface area of the porous polymer particles is preferably 30 m ² / g or more. From the viewpoint of higher practicality, the specific surface area is more preferably 35 m ² / g or more, and further preferably 40 m ² / g or more. When the specific surface area is 30 m ² / g or more, the adsorption amount of the substance to be separated tends to increase. The specific surface area of the porous polymer particles may be, for example, 500 m ² / g or less, 200 m ² / g or less, or 100 m ² / g or less.

The mode diameter (mode value of pore diameter distribution, maximum frequency pore diameter), specific surface area and porosity in the pore diameter distribution of porous polymer particles or separation materials are measured with a mercury intrusion measuring device (Autopore: manufactured by Shimadzu Corporation). Measured in the following manner. About 0.05 g of a sample is added to a standard 5 mL powder cell (stem volume 0.4 mL), and measurement is performed under an initial pressure of 21 kPa (about 3 psia, corresponding to a pore diameter of about 60 μm). Mercury parameters are set to a device default mercury contact angle of 130 ° and a mercury surface tension of 485 dynes / cm. Each value is calculated by limiting the pore diameter to a range of 0 to 3 μm.

[Coating layer]
The coating layer according to the present embodiment includes a polymer having a hydroxyl group. When the coating layer contains a polymer having a hydroxyl group, it is easy to suppress nonspecific adsorption of protein and to easily improve the protein adsorption amount. The polymer having a hydroxyl group may be cross-linked, for example, from the viewpoint of further suppressing the increase in the column pressure and from the viewpoint that the porous polymer particles and the coating layer are difficult to peel off.

(Polymer having a hydroxyl group)
The polymer having a hydroxyl group preferably has two or more hydroxyl groups in one molecule, and more preferably a hydrophilic polymer. Examples of the polymer having a hydroxyl group include polysaccharides and polyvinyl alcohol. Examples of the polysaccharide include agarose, dextran, cellulose, pullulan, and chitosan. The weight average molecular weight of the polymer having a hydroxyl group may be, for example, 10,000 or more, 50000 or more, or 100,000 or more. The weight average molecular weight of the polymer having a hydroxyl group may be, for example, 5000000 or less, 4500000 or less, or 4000000 or less. That is, the weight average molecular weight of the polymer having a hydroxyl group may be, for example, 10,000 to 5000000, 50000 to 450,000, or 100000 to 4000000. The polymer having a hydroxyl group may be used singly or in combination of two or more.

In this specification, the weight average molecular weight (Mw) refers to a value measured by the following method. A solution obtained by dissolving 0.5% by mass of a sample in ultrapure water is measured using a gel permeation chromatography apparatus with 0.2M NaCl as an eluent. A calibration curve is prepared by measuring pullulan and ethylene glycol as standard samples.

The polymer having a hydroxyl group may be a modified body (hydrophobic group-modified body) modified with a hydrophobic group from the viewpoint of improving the interfacial adsorption ability with the particles. 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 polymer having a hydroxyl group may be, for example, a polysaccharide or a modified product thereof from the viewpoint of alkali resistance. Examples of modified polysaccharides include hydrophobic group-modified products. From the viewpoint of molecular mobility, the polymer having a hydroxyl group may be at least one selected from the group consisting of dextran, agarose, pullulan, and modified products thereof.

(Method for forming a coating layer containing a polymer having a hydroxyl group)
The coating layer according to the present embodiment can be formed by, for example, the following method.

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

Specifically, for example, the polymer solution is impregnated with the above solution. In the impregnation method, porous polymer particles are added to a polymer solution having a hydroxyl group and left for a predetermined time. At this time, for example, for the purpose of dispersing the particles in the solution, stirring may be performed. The impregnation time varies depending on the surface state of the porous polymer particles. However, when the impregnation is performed for 6 to 50 hours, the polymer concentration is in equilibrium with the external concentration inside the porous polymer particles. Then, it wash | cleans with solvents, such as water and alcohol, and remove | eliminates the polymer which has a hydroxyl group which is not adsorb | sucked.

(Crosslinking treatment)
Next, a crosslinking agent is added to cause the polymer having a hydroxyl group adsorbed on the surface of the porous polymer particles to undergo a crosslinking reaction to form a crosslinked body.

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

A catalyst is usually used for this crosslinking reaction. The catalyst varies depending on the type of crosslinking agent. For example, when the crosslinking agent is epichlorohydrin or the like, an alkali such as sodium hydroxide is effective, and when the crosslinking agent is a dialdehyde compound, a mineral acid such as hydrochloric acid is effective. It is.

The crosslinking reaction with a crosslinking agent is usually performed by adding a crosslinking agent to a system in which porous polymer particles adsorbing a polymer having a hydroxyl group are dispersed and suspended in an appropriate medium. When the polysaccharide or a modified product thereof is used as the polymer having a hydroxyl group, the amount of the crosslinking agent added is within a range of 0.1 to 100 mol times with respect to 1 mol of one unit of the monosaccharide. It can be selected according to the performance of the target separation material. When the addition amount of the crosslinking agent is 0.1 mol times or more, the coating layer tends to hardly peel from the porous polymer particles. When the addition amount of the crosslinking agent is 100 mol times or less, even when the reaction rate with the polymer having a hydroxyl group is high, the characteristics of the polymer having a hydroxyl group tend to be easily maintained.

The amount of the catalyst used in the crosslinking reaction depends on the type of crosslinking agent, but when a polysaccharide is used as the polymer having a hydroxyl group, usually 1 mol of one unit of monosaccharide forming the polysaccharide is used. Then, it is preferably used in the range of 0.01 to 10 mole times, more preferably 0.1 to 5 mole times.

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

As a medium for dispersing and suspending the porous polymer particles on which the polymer having a hydroxyl group is adsorbed, the polymer, the cross-linking agent, etc. are not extracted from the adsorbed polymer solution and are not effective in the crosslinking reaction. It needs to be active. Specific examples thereof include water and alcohol.

The crosslinking reaction can be usually performed at a temperature in the range of 5 to 90 ° C. for 1 to 30 hours. The crosslinking reaction may be performed, for example, at a temperature in the range of 5 to 90 ° C. for 1 to 10 hours. The temperature of the crosslinking reaction is preferably 25 to 90 ° C.

After completion of the crosslinking reaction, the particles are filtered off and then washed with a hydrophilic organic solvent such as methanol or ethanol or water to remove unreacted polymer, suspending medium and the like. As a result, a separating material is obtained in which at least a part of the surface of the porous polymer particles is covered with a coating layer containing a polymer having a hydroxyl group, and the polymer having a hydroxyl group is crosslinked. If necessary, the above crosslinking treatment may be omitted.

The separating material of the present embodiment may include, for example, a coating layer of 30 mg or more per 1 g of porous polymer particles, may include a coating layer of 50 mg or more, or may include a coating layer of 100 mg or more. Good. The separating material of the present embodiment may include, for example, a coating layer of 450 mg or less per 1 g of porous polymer particles, may include a coating layer of 400 mg or less, or may include a coating layer of 400 mg or less. Good. When the ratio of the coating layer is 450 mg or less with respect to 1 g of the porous polymer particles, the coating layer tends to be a thin film, and the column pressure tends to be suppressed when used as a column. The separating material of the present embodiment preferably has a coating layer of 30 to 450 mg per 1 g of porous polymer particles, preferably has a coating layer of 50 to 400 mg, and preferably has a coating layer of 100 to 400 mg. The amount of the coating layer can be measured by reducing the weight of pyrolysis.

The separation material of the present embodiment includes a coating layer of 30 to 450 mg per 1 g of the porous polymer particles from the viewpoint of further suppressing the increase in the column pressure and from the viewpoint of difficult separation of the porous polymer particles and the coating layer. The polymer having a hydroxyl group is preferably crosslinked.

(Introduction of ion exchange groups)
The separation material provided with the coating layer may have an ion exchange group, a ligand (protein A), and the like. The separation material can be used for ion exchange purification, affinity purification, and the like by introducing these via a hydroxyl group on the surface.

Examples of the method for introducing an ion exchange group include a method using a halogenated alkyl compound.

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

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

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

The separation material of this embodiment may have a cation exchange group or an anion exchange group as an ion exchange group. Such a separating material can be used for ion exchange. That is, the separation material of the present embodiment may have a cation exchange group or an anion exchange group and be used for ion exchange. Examples of the cation exchange group include a carboxy group and a sulfonic acid group. Examples of the anion exchange group include an amino group and a quaternary ammonium group.

As a method for introducing an amino group which is a weakly basic group as an ion exchange group, among the above halogenated alkyl compounds, a mono- having at least one alkyl group in which a part of hydrogen atoms is substituted with a chlorine atom. , Di- or tri-alkylamine, mono-, di- or tri-alkanolamine, monoalkyl-monoalkanolamine, dialkyl-monoalkanolamine, monoalkyl-dialkanolamine and the like. The amount of the alkyl halide compound used is preferably 0.2% by mass or more based on the total mass of the separating material into which the ion exchange group is introduced. The reaction conditions are preferably 40 to 90 ° C. and 0.5 to 12 hours.

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

Examples of the method for introducing a carboxy group that is a weakly acidic group as an ion exchange group include a method in which a monohalogenocarboxylic acid such as monohalogenoacetic acid or monohalogenopropionic acid or a sodium salt thereof is reacted as the halogenated alkyl compound. . The amount of these halogenated alkyl compounds used is preferably 0.2% by mass or more based on the total mass of the separating material into which ion exchange groups are 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 ebichlorohydrin is reacted with a separating material, and a sulfite or bisulfite such as sodium sulfite or sodium bisulfite. And a method of adding a separating material to the saturated aqueous solution. The reaction conditions are preferably 30 to 90 ° C. and 1 to 10 hours.

On the other hand, as a method for introducing a sulfonic acid group, there may be mentioned a method in which 1,3-propane sultone is reacted with a separating material in an alkaline cloud atmosphere. 1,3-propane sultone is preferably used in an amount of 0.4% by mass or more based on the total mass of the separating material into which the ion exchange group is introduced. The reaction conditions are preferably 0 to 90 ° C. and 0.5 to 12 hours.

In the present specification, the change ratio of the specific surface area before and after coating refers to a value calculated from the following formula using the specific surface area before and after coating the porous polymer particles with a polymer having a hydroxyl group. The specific surface area before and after coating is measured by the mercury intrusion method described above.
(Change ratio of specific surface area) = (specific surface area after coating) / (specific surface area before coating)

That is, in this embodiment, the change ratio of the specific surface area is the ratio of the specific surface area of the separation material to the specific surface area of the porous polymer particles, and is expressed by (specific surface area of the separation material) / (specific surface area of the porous polymer particles). expressed.

In the separation material of the present embodiment, the change ratio of the specific surface area before and after coating is 0.50 to 0.85. The change ratio may be 0.55 or more, for example. The change ratio may be, for example, 0.80 or less, 0.75 or less, or 0.70 or less. From the viewpoint of further improving the dynamic adsorption amount, the change ratio of the specific surface area before and after coating may be, for example, 0.50 to 0.80, 0.55 to 0.80, .55 to 0.75 or 0.55 to 0.70.

The change ratio of the specific surface area before and after coating is determined by appropriately selecting the type and weight average molecular weight of the polymer having a hydroxyl group, the raw material of the porous polymer particles, the mode diameter in the porosity and pore size distribution, the amount of the coating layer, and the like. Can be adjusted.

When the separation material of this embodiment is packed in a column, the liquid flow rate is preferably 800 cm / h or more when the column pressure is 0.3 MPa.

When separating proteins by column chromatography, the flow rate of protein solution or the like is generally in the range of 400 cm / h or less. However, when the separation material of the present embodiment is used, the separation rate for normal protein separation is as follows. Even when used at a liquid flow rate of 800 cm / h or higher, which is faster than that of the ion exchanger, it is considered that a high adsorption amount can be maintained. The liquid passing speed may be, for example, 4000 cm / h or less.

In the present specification, the liquid passing speed represents a liquid passing speed when the separation material of the present embodiment is filled in a glass column of φ5.0 × 200 mm and the liquid is passed.

The average particle diameter of the separation material of the present embodiment may be, for example, 500 μm or less, 300 μm or less, or 200 μm or less. Further, the average particle diameter of the separation material may be, for example, 10 μm or more, 30 μm or more, or 50 μm or more from the viewpoint of easily suppressing an increase in the column pressure after column filling. . From the above viewpoint, the average particle diameter of the separating material may be, for example, 10 to 500 μm, 10 to 300 μm, 30 to 300 μm, or 50 to 200 μm. When used in preparative or industrial chromatography, the average particle size of the separating material is preferably, for example, 50 to 200 μm from the viewpoint of easily avoiding an extreme increase in the internal pressure of the column.

The mode diameter in the pore size distribution of the separating material may be, for example, 0.05 μm or more, 0.075 μm or more, or 0.10 μm or more. The mode diameter may be, for example, 0.50 μm or less, or less than 0.50 μm. The mode diameter in the pore size distribution of the separating material is preferably 0.05 to 0.50 μm, more preferably 0.075 to 0.50, and further preferably 0.10 to 0.50 μm. It is particularly preferably 0.10 μm or more and less than 0.50 μm. When the mode diameter in the pore size distribution is 0.05 μm or more, substances tend to easily enter the pores, and when the mode diameter in the pore diameter distribution is 0.50 μm or less, the specific surface area is sufficient. It tends to be. The mode diameter may be, for example, 0.50 to 0.75.

The specific surface area of the separating material is preferably 30 m ² / g or more from the viewpoint of easy adsorption of the substance to be separated and higher practicality. The specific surface area of the separating material may be, for example, 500 m ² / g or less, 200 m ² / g or less, or 100 m ² / g or less.

The separation material of this embodiment has the respective advantages of particles made of natural polymers or particles made of synthetic polymers in the separation of biopolymers such as proteins. Moreover, the separation material of the present embodiment tends to reduce non-specific adsorption and easily cause protein adsorption / desorption. Furthermore, the separation material according to the present embodiment tends to have a large adsorption amount (dynamic adsorption amount) of protein or the like under the same flow rate.

When the separation material of this embodiment is used as a column packing material in column chromatography, it has excellent operability because there is almost no volume change in the column regardless of the properties of the eluate used. The separation material of the present embodiment may be used for liquid chromatography, for example. That is, the separation material of this embodiment may be, for example, a liquid chromatography column packing material.

The separation material of this embodiment is suitable for use in separation of proteins by electrostatic interaction and affinity purification. For example, after a separation material having an ion exchange group (hereinafter also referred to as “ion exchanger”) is added to a mixed solution containing protein and only the protein is adsorbed to the ion exchanger by electrostatic interaction. If the ion exchanger is filtered from the solution and added to an aqueous solution having a high salt concentration, the protein adsorbed on the ion exchanger can be easily desorbed and recovered. The ion exchanger can also be used in column chromatography.

As a biopolymer that can be separated using the ion exchanger according to the present embodiment, a water-soluble substance is preferable. Specific examples include proteins such as serum albumin and blood proteins such as immunoglobulins, enzymes present in the living body, protein bioactive substances produced by biotechnology, DNA, and peptides having bioactivity. The molecular weight of these substances may be, for example, 2 million or less, or 500,000 or less. Further, according to a known method, the nature and conditions of the ion exchanger may be selected according to the isoelectric point, ionization state, etc. of the protein. Examples of known methods include the method described in JP-A-60-169427.

Hereinafter, the present invention will be described by way of examples, but the present invention is not limited to these examples.

[Example 1]
(Synthesis of porous polymer particles)
In a 500 mL three-necked flask, 12 g of 96% pure divinylbenzene (manufactured by NS Was added to obtain a dispersed phase. Moreover, 0.5 mass% polyvinyl alcohol aqueous solution was used as a continuous phase. After emulsifying the continuous phase and the dispersed phase using a microprocess server, the obtained emulsion was transferred to a flask and stirred for about 8 hours using a stirrer while heating in a water bath at 80 ° C. The obtained particles were filtered off and then washed with acetone to obtain porous polymer particles. The particle size of the porous polymer particles was measured with a flow-type particle size measuring device, and the average particle size and particle size C.I. V. The value (coefficient of variation) was calculated. The results are shown in Table 1.

(Formation of coating layer)
To 100 mL of dextran (weight average molecular weight (Mw): 3 million) aqueous solution (2% by mass), 4 g of sodium hydroxide and 0.4 g of glycidyl phenyl ether were added and reacted at 70 ° C. for 12 hours to introduce a phenyl group into dextran. .

The obtained modified dextran was reprecipitated three times with isopropyl alcohol and washed.

10 g of porous polymer particles were put into 700 mL of a 20 mg / mL modified dextran aqueous solution and stirred at 55 ° C. for 24 hours to adsorb the modified dextran to the porous polymer particles. The particles adsorbed with the modified dextran were separated by filtration and further washed with hot water. The coating amount (adsorption amount) of the modified dextran per 1 g of the porous polymer particles was calculated from the concentration of the modified dextran in the filtrate. The results are shown in Table 1.

The modified dextran was crosslinked as follows. 10 g of particles adsorbed with modified dextran were dispersed in a 0.4 M aqueous sodium hydroxide solution, 39 g of ethylene glycol diglycidyl ether was added, and the mixture was stirred at room temperature for 24 hours. Then, after washing with 2% by mass of hot sodium dodecyl sulfate aqueous solution, it was washed with pure water. The obtained particles were stored as a dispersion in water.

(Evaluation of non-specific adsorption amount of protein)
0.5 g of the particles filtered off from the dispersion was put into 50 mL of Tris-hydrochloric acid buffer (pH 8.0) having a BSA (Bovine Serum Albumin) concentration of 12 mg / mL and stirred at room temperature for 24 hours. Then, it centrifuged and took the supernatant liquid. The amount of BSA adsorbed on the particles was calculated from the BSA concentration in the supernatant obtained by measuring the absorbance at 280 nm of the supernatant with a spectrophotometer. The amount of BSA adsorbed per mL of particles was evaluated as the amount of nonspecific adsorption. The results are shown in Table 2.

(Introduction of ion exchange groups)
20 g of the particles filtered off from the dispersion were dispersed in 100 mL of an aqueous solution in which a predetermined amount of diethylaminoethyl chloride hydrochloride was dissolved, and stirred at 70 ° C. for 10 minutes. Thereafter, 100 mL of 5 M NaOH aqueous solution was added by heating to 70 ° C., and the mixture was reacted for 1 hour. After completion of the reaction, the particles were filtered off and washed twice with water / ethanol (volume ratio 8/2). Thereby, a separation material (ion exchanger) having a diethylaminoethyl (DEAE) group as an ion exchange group was obtained.

(Evaluation of change ratio of specific surface area before and after coating and pore diameter)
The specific surface areas of the porous polymer particles and the separating material were measured by the mercury intrusion method, and the change ratio of the specific surface area before and after coating was calculated by the method described above. Further, the pore diameter (mode diameter in the pore diameter distribution) of the porous polymer particles was measured by mercury porosimetry. The results are shown in Table 1.

(Evaluation of static adsorption capacity of protein)
0.5 g of the obtained separating material was put into 50 mL of Tris-hydrochloric acid buffer (pH 8.0) having a BSA (Bovine Serum Albumin) concentration of 12 mg / mL and stirred at room temperature for 24 hours. Then, it centrifuged and took the supernatant liquid. The amount of BSA adsorbed on the particles was calculated from the BSA concentration in the supernatant obtained by measuring the absorbance at 280 nm of the supernatant with a spectrophotometer. The amount of BSA adsorbed per 1 mL of the separation material was evaluated as the amount of static adsorption. The results are shown in Table 2.

(Column characteristic evaluation)
The obtained separation material was charged as a slurry (solvent: 1 M NaCl) with a concentration of 30% by mass over a glass column of φ5.0 × 200 mm over 15 minutes, and then the solvent in the column was replaced with water.

Thereafter, water was passed through the column at different flow rates, the relationship between the flow rate and the column pressure was measured, and the liquid flow rate (linear flow rate) at 0.3 MPa was measured. The results are shown in Table 2.

The dynamic adsorption amount (dynamic binding capacity) was measured as follows. 10 column volumes of 40 mmol / L Tris-HCl buffer (pH 8.0) were passed through the column. Thereafter, a 40 mmol / L Tris-HCl buffer solution having a BSA concentration of 0.5 mg / mL was passed at 800 cm / h, and the BSA concentration at the column outlet was measured by UV measurement. The solution was passed through until the BSA concentration at the column inlet and outlet coincided, and diluted with 1 M NaCl Tris-HCl buffer solution for 5 column volumes. The dynamic binding capacity at 10% breakthrough was calculated using the following equation. The results are shown in Table 2.
q ₁₀ = c _f F (t ₁₀ −t ₀ ) / V _B
q ₁₀ : dynamic binding capacity at 10% breakthrough (mg / mL wet resin)
cf: Injected BSA concentration F: Flow rate (mL / min)
V _B : Bed volume (mL)
t ₁₀ : Time at 10% breakthrough t ₀ : BSA injection start time

(Dynamic adsorption amount relative to static adsorption amount)
The dynamic adsorption amount (dynamic adsorption amount / static adsorption amount) relative to the static adsorption amount was calculated. The results are shown in Table 2. The larger the dynamic adsorption amount relative to the static adsorption amount, the better.

[Example 2]
In the synthesis of the porous polymer particles, a separating material was produced and evaluated in the same manner as in Example 1 except that the amount of the porosifying agent was changed to 8 g of diethylbenzene and 16 g of hexanol.

[Example 3]
In the synthesis of the porous polymer particles, a separation material was prepared and evaluated in the same manner as in Example 1 except that the amount of the porosifying agent was changed to 4 g of diethylbenzene and 20 g of hexanol.

[Example 4]
A separation material was prepared and evaluated in the same manner as in Example 1 except that the Mw of dextran used was changed to 1 million.

[Example 5]
A separation material was produced and evaluated in the same manner as in Example 1 except that Mw of dextran used was changed to 500,000.

[Comparative Example 1]
A separation material was prepared and evaluated in the same manner as in Example 1 except that Mw of dextran used was changed to 40,000.

[Comparative Example 2]
The porous polymer particles synthesized in Example 1 were used as they were as a separating material and evaluated in the same manner as in Example 1.

[Comparative Example 3]
Commercially available agarose particles (Capto DEAE: GE Healthcare) were used as they were as a separating material and evaluated in the same manner as in Example 1.

As shown in Table 2, it can be seen that the separation materials of the examples have less protein non-specific adsorption, excellent protein adsorption, and excellent liquid permeability when used as a column.

Claims

Porous polymer particles containing a polymer containing a styrenic monomer as a monomer unit;
A coating layer containing a polymer having a hydroxyl group, covering at least a part of the surface of the porous polymer particles;
A separating material having a change ratio of a specific surface area before and after coating of 0.50 to 0.85.
The separation material according to claim 1, wherein the specific surface area is 30 m 2 / g or more.
The separation material according to claim 1 or 2, wherein the porous polymer particles have a coefficient of variation in particle diameter of 5 to 15%.
The separation material according to any one of claims 1 to 3, wherein the polymer having a hydroxyl group is a polysaccharide or a modified product thereof.
The separation material according to any one of claims 1 to 4, wherein the polymer having a hydroxyl group is at least one selected from the group consisting of dextran, agarose, pullulan, and modified products thereof.
The separation material according to any one of claims 1 to 5, comprising 30 to 450 mg of the coating layer per gram of the porous polymer particles, wherein the polymer having a hydroxyl group is crosslinked.
The separation material according to any one of claims 1 to 6, wherein a mode diameter in a pore size distribution of the porous polymer particles is 0.05 to 0.50 µm.
The separation material according to any one of claims 1 to 7, wherein when the column is packed, the liquid flow rate is 800 cm / h or more when the column pressure is 0.3 MPa.
The separation material according to any one of claims 1 to 8, wherein the polymer having a hydroxyl group has a weight average molecular weight of 10,000 to 5,000,000.
The separation material according to any one of claims 1 to 9, which has a cation exchange group or an anion exchange group and is used for ion exchange.