JP6606974B2 - Separating material and method for producing the same - Google Patents

Description

  The present invention relates to a separation material that can be used as a column packing material for liquid chromatography, and a method for producing the same.

  In recent years, research for creating functional polymer materials having molecular discrimination ability has been actively conducted. As one of the development methods, attention has been focused on the “molecular imprint method”. The molecular imprint method is a method in which a monomer is polymerized in the presence of a target molecule (template molecule) for molecular identification, and then the template molecule is removed to store information on the shape and properties of the crosslinked polymer network. This is a very simple method. Intelligent gels containing cross-linked polymers obtained by molecular imprinting can selectively adsorb or capture template molecules, and can be applied to highly selective and sensitive separations, sensors, and catalysts. Expected.

  For example, Derek et al. Produced polyacrylamide gel in the presence of bobbin hemoglobin (BHb) as a target molecule (Non-patent Document 1). Here, polyacrylamide (PAAm), which is an acrylate-based nitrogen-containing polymer, is water-soluble, inexpensive, and can be easily produced. Polyacrylamide has been reported to be suitable as a substrate for imprinting biomolecules because it is designed to have attractive structural parameters. By removing BHb from the obtained gel, a gel having an imprint void of BHb in the gel is produced, and this shows high selectivity for BHb compared to other structural analogs.

  However, in the bulk gel obtained by the conventional molecular imprinting method, it has been difficult to control the form of the field where the imprint voids are formed. In order to capture the template molecule in the imprint void formed inside the gel having a three-dimensional network structure, it is necessary that the template molecule not only approaches the gel but also enters the inside thereof. When the template molecule is a polymer having a certain large molecular weight such as protein, it is not easy to allow the protein to enter the gel.

  Patent Document 1 discloses a material in which a polymer having a protein molecular shape template is provided on the surface of porous inorganic fine particles.

  Wang et al. Immobilized BHb on the pore wall (inner wall) of nanoporous alumina, and then filled the pores with a mixture of acrylamide (AAm) and methylenebisacrylamide (MBAAm) to polymerize them. Thereafter, by removing the alumina film, a thin tubular polymer having imprint voids only in the vicinity of the outermost surface was formed (Non-Patent Document 2).

  Patent Document 2 synthesizes particles having a molecular imprint shape on the surface through living radical polymerization after the protein is immobilized on the surface of the monodisperse polymer fine particles.

Japanese Patent No. 30627745 Japanese Patent No. 4558097

Derek et al. , Anal. Chim. Acta, 542, 61-65 (2005) Wang et al. , Anal. Chem, 78, 317-320 (2006)

  Conventional separation materials using molecular imprint technology tend to lack the amount of protein target molecule adsorption or adsorption selectivity.

  Therefore, an object of the present invention is to further improve the adsorption amount and adsorption selectivity of the target molecule with respect to the separation material used for separating the target molecule.

  One aspect of the present invention is a porous polymer particle containing a cross-linked polymer, a molecular template film that is provided on the surface of the porous polymer and that contains a polymer for imprinting and forms a template by a molecular imprint method, A separating material is provided.

  This separation material can exhibit sufficient performance in terms of the adsorption amount and adsorption selectivity of the target molecule.

  The molecular template film has an underlayer that covers at least a part of the surface of the porous polymer particles, and a surface layer provided on the underlayer, and the template may be formed by the surface layer.

  The imprinting polymer may contain polydopamine. The polydopamine may contain a modified polydopamine having a structural unit derived from dopamine and a structural unit derived from dihydroxyphenylalanine.

  The separation material may have an average pore diameter of 0.1 to 0.5 μm. C. of the particle size of the separation material. V. The value may be 5-15%.

The porous polymer particles may have a 5% compression deformation elastic modulus of 100 to 500 MPa. The specific surface area of the porous polymer particles may be 30 m ² / g or more.

  The molecular template membrane may form a template that interacts with the target protein, and the separation material may adsorb the target protein with a selectivity of 2 to 10 times with respect to bovine serum albumin. The adsorption amount of the target protein may be 5 mg or more per 1 g of the separation material.

  Another aspect of the present invention is a step of forming an underlayer containing a polymer for an underlayer on the surface of a porous polymer particle containing a crosslinked polymer, a step of adsorbing a template molecule to the underlayer, and an adsorbed template molecule Forming a surface layer containing a surface layer polymer on the underlying layer, and removing a template molecule to form a molecular template film having a surface layer and an underlying layer to form a template derived from the template molecule And a step of forming the separation material.

  According to this method, it is possible to produce a separation material that can exhibit sufficient performance in terms of the amount of target molecule adsorption and adsorption selectivity.

  According to the present invention, it is possible to further improve the adsorption amount and adsorption selectivity of the target molecule with respect to the separation material. The separating material of the present invention can also have high durability.

  The separation material according to the present invention is useful, for example, as a column packing material for liquid chromatography. When used as a column packing material, the separation material of the present invention can exhibit excellent properties in terms of liquid flow rate, durability (handleability), reduction of non-specific protein adsorption, and the like. By using the separation material of the present invention as a column packing material, proteins can be efficiently purified using conventional purification process equipment.

  In the case of the separating material according to the present invention, it is possible to easily form a mold on the surface of the particle or in the vicinity thereof, while easily controlling the form of the particle. This point is advantageous for industrial use.

Hereinafter, preferred embodiments of the present invention will be described in detail. However, the present invention is not limited to the following embodiments.
In this specification, the term “process” includes a process that is independent of other processes and includes the process if the purpose of the process is achieved even if it cannot be clearly distinguished from the other processes. It is.
In the present specification, the numerical ranges indicated by using “to” include numerical values described before and after “to” as the minimum value and the maximum value, respectively.
In this specification, the term “layer” refers to the case where the layer is formed only in a part of the region in addition to the case where the layer is formed over the entire region. Is also included.

  According to one embodiment, a separating material includes a porous polymer particle containing a cross-linked polymer, and a molecular template membrane that is provided on the surface of the porous polymer and includes a polymer for imprinting and forms a template by a molecular imprint method. And having.

(Porous polymer particles)
The porous polymer particles may include a monomer-based crosslinked polymer including a polyfunctional monomer having two or more double bonds. 90% by mass or more of the monomer raw material may be a polyfunctional monomer. The porous polymer particles can be synthesized, for example, by suspension polymerization in an emulsion containing a monomer raw material, a porous agent and an aqueous medium.

  Although the monomer used as a monomer raw material is not specifically limited, Vinyl monomers, such as styrene and a styrene derivative, may be sufficient. The monomer raw material may contain a monofunctional monomer.

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

  Examples of the monofunctional monomer include styrene, o-methylstyrene, m-methylstyrene, p-methylstyrene, α-methylstyrene, o-ethylstyrene, m-ethylstyrene, p-ethylstyrene, 2,4- Dimethyl styrene, pn-butyl styrene, pt-butyl styrene, pn-hexyl styrene, pn-octyl styrene, pn-nonyl styrene, pn-decyl styrene, pn-dodecyl Examples thereof include styrene such as styrene, p-methoxystyrene, p-phenylstyrene, p-chlorostyrene, and 3,4-dichlorostyrene and derivatives thereof. These can be used alone or in combination of two or more. Among these, it is preferable to use styrene having acid resistance and alkali resistance. Moreover, the styrene derivative which has functional groups, such as a carboxyl group, an amino group, a hydroxyl group, and an aldehyde group, can also be used.

  The polyfunctional monomer may be a polyfunctional acrylic monomer. As polyfunctional acrylic monomers, (poly) alkylene glycol di (meth) acrylates such as (poly) ethylene glycol di (meth) acrylate, (poly) propylene glycol di (meth) acrylate, and (poly) tetramethylene glycol di (meth) acrylate ) Acrylate; neopentyl glycol di (meth) acrylate, trimethylolpropane tri (meth) acrylate, tetramethylolmethane tri (meth) acrylate, tetramethylolpropane tetra (meth) acrylate, pentaerythritol tri (meth) acrylate, ethoxylated cyclohexane Dimethanol di (meth) acrylate, ethoxylated bisphenol A di (meth) acrylate, tricyclodecane dimethanol di (meth) acrylate, propoxylated ethoxy Bisphenol A di (meth) acrylate, 1,1,1-trishydroxymethylethane di (meth) acrylate, 1,1,1-trishydroxymethylethanetri (meth) acrylate, 1,1,1-trishydroxymethylpropane Examples include triacrylate, diallyl phthalate and its isomer, triallyl isocyanurate and its derivatives. Commercially available polyfunctional acrylic monomers include NK esters (A-TMPT-6P0, A-TMPT-3E0, A-TMM-3LMN, A-GLY series, A-manufactured by Shin-Nakamura Chemical Co., Ltd.) 9300, AD-TMP, AD-TMP-4CL, ATM-4E, A-DPH) and the like. These monomers may be used alone or in combination of two or more.

  Monofunctional monomers that can be copolymerized with the above monomers include methyl acrylate, ethyl acrylate, propyl acrylate, n-butyl acrylate, isobutyl acrylate, hexyl acrylate, 2-ethylhexyl acrylate, and n-octyl acrylate. , Dodecyl acrylate, lauryl acrylate, stearyl acrylate, 2-chloroethyl acrylate, phenyl acrylate, methyl α-chloroacrylate, glycidyl methacrylate, methyl methacrylate, ethyl methacrylate, propyl methacrylate, n-methacrylate (Meth) acrylic acid esters such as butyl, isobutyl methacrylate, hexyl methacrylate, 2-ethylhexyl methacrylate, n-octyl methacrylate, dodecyl methacrylate, lauryl methacrylate, stearyl methacrylate (Iii) vinyl esters such as vinyl acetate, vinyl propionate, vinyl benzoate and vinyl butyrate; (iv) N-vinyl such as N-vinylpyrrole, N-vinylcarbazole, N-vinylindole and N-vinylpyrrolidone. Compound, (v) vinyl fluoride, vinylidene fluoride, tetrafluoroethylene, hexafluoropropylene, trifluoroethyl acrylate, fluorinated alkyl group-containing (meth) acrylic acid ester such as tetrafluoropropyl acrylate, (vi) butadiene And conjugated dienes such as isoprene. These are used individually by 1 type or in combination of 2 or more types.

  The porosifying agent is a component that promotes phase separation at the time of polymerization and promotes the porous formation of particles. Porous agents include aliphatic or aromatic hydrocarbons, esters, ketones, ethers, and alcohols. Specifically, the porosifying agent is selected from toluene, xylene, cyclohexane, octane, butyl acetate, dibutyl phthalate, methyl ethyl ketone, dibutyl ether, 1-hexanol, 2-octanol, decanol, lauryl alcohol, and cyclohexanol. There may be at least one.

  The amount of the porosifying agent may be 0 to 200% by mass with respect to the monomer raw material. The porosity of the particles can be controlled by the amount of the porous agent. Depending on the type of the porous agent, the size and shape of the pores can be controlled.

  Water or an aqueous medium used as a solvent may function as a porous agent. In this case, by dissolving the oil-soluble surfactant in the monomer raw material, the monomer raw material can absorb water and make the particles porous.

  The oil-soluble surfactant used for the pore formation is 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 monooleate, sorbitan mono Sorbitan monoesters derived from myristate and coconut fatty acids); diglycerol monoesters of branched C16-C24 fatty acids, chain unsaturated C16-C22 fatty acids or chain saturated C12-C14 fatty acids (eg, diglycerol monooleate ( (E.g., C18: 1 fatty acid diglycerol monoester), diglycerol monomyristate, diglycerol monoisostearate and coconut fatty acid diglycerol monoester); branched C16-C24 alcohols (e.g. Guerbet alcohol), unchained Saturated C16 C22 alcohol and chain saturated C12~C14 alcohols (e.g., coconut fatty alcohols) diglycerol mono-fatty ethers of; include and combinations thereof. Preferred oil-soluble surfactants include sorbitan monolaurate (eg, SPAN® 20, preferably greater than about 40%, more preferably greater than about 50%, most preferably greater than about 70%. Laurate), sorbitan monooleate (eg, SPAN®, preferably greater than about 40%, more preferably greater than about 50%, most preferably greater than about 70% sorbitan monooleate), diglycerol monooleate ( For example, greater than about 40%, more preferably greater than about 50%, most preferably greater than about 70% diglycerol monooleate), diglycerol monoisostearate (eg, preferably greater than about 40%, more Preferably greater than about 50%, most preferably greater than about 70% diglycero Monoisostearate), diglycerol monomyristate (preferably more than about 40%, more preferably more than about 50%, most preferably more than about 70% sorbitan monomyristate), cocoyl of diglycerol (e.g. Lauryl and myristoyl) ethers, and mixtures thereof.

  The amount of the oil-soluble surfactant is preferably in the range of 5 to 80% by mass with respect to the monomer raw material. When this amount is 5% by mass or more, the stability of the water droplets becomes good and the formation of large single holes tends to be suppressed. When this amount is 80% by mass or less, the particle shape tends to be easily retained after polymerization.

  Examples of the aqueous medium include water or a mixed medium of water and a water-soluble solvent (for example, lower alcohol). 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 alkyl phenyl ether sulfates Salt, polyoxyethylene alkyl ether sulfate such as 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.

  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-based nonionic surfactants such as polyethylene oxide adducts of silicon and polypropylene oxide adducts, and fluorine-based nonionic surfactants such as perfluoroalkyl glycols.

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

  One surfactant may be used alone, or two or more surfactants may be used in combination. Among the above surfactants, an anionic surfactant is preferable from the viewpoint of dispersion stability during polymerization.

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

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

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

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

  Water-soluble polymerization inhibitors such as nitrites, sulfites, hydroquinones, ascorbic acids, water-soluble vitamin Bs, citric acid, polyphenols, etc., in order to suppress the generation of particles in which monomers are polymerized alone in an aqueous medium May be used.

  The average particle diameter of the porous polymer particles is preferably 500 μm or less, more preferably 300 μm or less, and still more preferably 100 μm or less. The average particle diameter of the porous polymer particles is preferably 10 μm or more, more preferably 30 μm or more, and further preferably 50 μm or more. When the average particle size is reduced, the column pressure after column filling may increase.

  C. of the particle size of the porous polymer particles and / or the separating material. V. The value is preferably 5 to 15% and more preferably 5 to 10% in order to improve liquid permeability. Particle size C.I. V. In order to reduce the value, it is possible to monodisperse the particles with an emulsifier such as a microprocess server (Hitachi).

  The pore volume of the porous polymer particles and / or the separating material may be 30% or more and 70% or less of the true volume of the particles. The diameter of most of the pores (or the average pore diameter) of the porous polymer particles and / or the separating material may be 0.1 μm to 0.5 μm. More preferably, the pore volume is 40% or more and 70% or less, and the pore diameter is 0.2 μm or more and less than 0.5 μm. If the pores are smaller than this, there is a tendency that the number of substances that cannot enter the pores increases. When the pore is larger than this, the surface area tends to be small. These can be adjusted by the above-mentioned pore regulator.

The porous polymer particles usually have a specific surface area of about 30 m ² / g or more. In view of practicality, the specific surface area of the porous polymer particles is preferably 35 m ² / g or more, and more preferably 40 m ² / g or more. If the surface area is small, the amount of adsorbed substances tends to decrease. The upper limit of the specific surface area of the porous polymer particles is not particularly limited, but may be 500 m ² / g or less.

  The 5% compression deformation elastic modulus of the porous polymer particles may be 100 to 500 MPa. Thereby, since it does not deform | transform when it fills with a column, the effect which makes a column pressure lower is acquired.

  The average pore diameter, specific surface area, and pore volume (or porosity) of the particles are values measured by a mercury intrusion measuring device (Autopore: Shimadzu Corporation). For example, about 0.05 g of a particle sample is put in a standard 5 cc powder cell (stem volume 0.4 cc), and the average pore diameter and the like are determined under the conditions of an initial pressure of 21 kPa (about 3 psia, about 60 μm pore diameter). Can be measured. The mercury parameters are set to an initial mercury contact angle of 130 degrees 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.

(Molecular template film)
The molecular template film includes a polymer for imprinting provided on the surface of porous polymer particles, and forms a template by a molecular imprinting method.
The molecular template film according to one embodiment has an underlayer containing a polymer for an underlayer (simply referred to as an underlayer) and a surface layer containing a polymer for a surface layer (sometimes simply referred to as a surface layer). The surface layer is formed on the base layer. That is, the molecular template film according to one embodiment has an underlayer that covers at least a part of the surface of the porous polymer particles, and a surface layer provided on the underlayer, and the template is formed by the surface layer. It may be formed.
The polymer for the underlayer and the polymer for the surface layer may be the same or different. In the present specification, these polymers may be collectively referred to as “imprinting polymer”. By combining the underlayer and the surface layer, a template that interacts with the target molecule can be formed on or near the outermost surface of the separating material. Thereby, efficient capture of the template molecule can be realized.
The molecular template film having an underlayer and a surface layer includes, for example, a step of forming an underlayer containing a polymer for an underlayer on the surface of porous polymer particles containing a crosslinked polymer, a step of adsorbing template molecules on the underlayer, A step of forming a surface layer containing a surface layer polymer on the underlayer on which the template molecule is adsorbed, and removing the template molecule to form a template derived from the template molecule having the surface layer and the underlayer. And forming a molecular template film.

The imprinting polymer may contain polydopamine. The polydopamine may contain a modified polydopamine having a structural unit derived from dopamine and a structural unit derived from dihydroxyphenylalanine.
As an underlayer according to an embodiment, a polydopamine film can be formed on the surface of the porous polymer particles by polymerization of dopamine. The underlayer (here, polydopamine film) is formed so as to cover at least a part of the porous polymer particles including the surface in the pores. The underlayer may penetrate some or all of the pores of the porous polymer particles. By covering the porous polymer particles with an underlayer, the diffusibility of the protein into the particles can be improved. Moreover, the protein adsorption selectivity can be improved by immobilizing a substance that interacts with the protein to be adsorbed on the polydopamine membrane.

  The polydopamine film can be formed, for example, by a method of oxidative polymerization of dopamine in the presence of porous polymer particles. Dopamine's excellent adhesion and spontaneous thin film-forming ability make it possible to form a polydopamine film on the surface of the porous polymer particles. Specifically, when porous polymer particles are immersed in an aqueous solution of dopamine (pH = about 8), dopamine self-oxidation polymerization starts quickly, and polymerized dopamine is deposited on the surface of the porous polymer particles to form a thin film. Form. This thin film (polydopamine film) adheres stably to the surface of the porous polymer particles.

  The concentration of the aqueous dopamine solution is not particularly limited, but is generally about 0.01 to 0.1 mol / liter. This concentration is preferably from 0.01 to 0.05 mol / liter. The pH of the dopamine solution is generally about 6.4 to 10.8, and most preferably 8.0 to 8.5. The temperature of the aqueous dopamine solution when the porous polymer particles are immersed is preferably about 10 to 50 ° C. The soaking time varies depending on the thickness of the target polydopamine film, and is generally about 6 to 48 hours. The thickness of the polydopamine film is preferably about 10 to 30 nm.

  The aqueous dopamine solution may contain dihydroxyphenylalanine. Thereby, the polydopamine film | membrane (underlayer) containing the modified polydopamine containing the structural unit derived from dopamine and the structural unit derived from dihydroxyphenylalanine can be formed. When the underlayer contains this modified polydopamine, even higher adsorption selectivity can be achieved.

  By forming a surface layer on the underlayer as follows, a template (molecular template) that interacts with a target molecule such as a protein can be created. A template molecule (for example, protein) is adsorbed on a dopamine film formed on the surface of the porous polymer particle. Thereafter, by further polymerizing dopamine, a dopamine film as a surface layer is formed while forming a template derived from the template molecule. The protein as a template molecule may be an enzyme, an antibody, or the like.

  In order to improve the selectivity of the target molecule, a substance having a functional group that interacts with the target molecule can also be immobilized. The term “interaction” as used herein is a generic term for actions that exert a strong influence on the target molecule, such as electrostatic interactions, enzyme-substrate similar interactions, enzyme-inhibitor interactions, and enzyme-coenzyme interactions. Say it.

  The porous polymer particles and the particles having the molecular template membrane are separation materials suitable for capturing target molecules such as proteins. For example, if the separation material of this embodiment is added to a mixed solution containing proteins, the protein is adsorbed on the template, and then the separation material is added to an aqueous solution having a high salt concentration, the adsorption material is adsorbed on the particles of the separation material. It can be easily detached and recovered.

  The target molecule that can be separated using the separation material of the present embodiment may be a biopolymer such as a protein, and is preferably a water-soluble substance. Specifically, target molecules include proteins such as blood proteins including serum albumin and immunoglobulin, enzymes existing in the living body, protein bioactive substances produced by biotechnology, DNA, peptides having bioactivity, etc. It can be a biopolymer. The molecular weight of the target molecule may be 2 million or less, more preferably 500,000 or less.

  It is preferable that the molecular template membrane forms a template that adsorbs a protein (target protein) as a target molecule, and the separation material adsorbs the target protein with 2 to 10 times selectivity to bovine serum albumin. . The separation material of this embodiment can easily achieve such high selectivity.

  The amount of target protein adsorbed on the separation material can be 5 mg or more per 1 g of the separation material. The separating material of this embodiment can easily achieve such a high adsorption amount. This adsorption amount is the maximum value of the mass of the target protein adsorbed on the separation material per 1 g of the separation material. The upper limit of the adsorption amount is not particularly limited, but is usually about 200 mg or less per 1 g of the separating material.

  The particle size of the separating material is usually preferably 10 to 300 μm. When used as a column packing material in preparative or industrial chromatography, the particle size of the separation material is preferably 50 to 100 μm in order to avoid an extreme increase in the internal pressure of the column. The particles of the separation material of this embodiment, when used as a column packing material for column chromatography, have an excellent operability effect that there is almost no volume change in the column, regardless of the properties of the eluate used. It can be demonstrated.

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

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

Example 1
<Synthesis of porous polymer particles>
In a 500 mL three-necked flask, 14 g of divinylbenzene having a purity of 96% (purity 96%), 5 g of octanol, 3 g of span 80 (surfactant), and 0.64 g of benzoyl peroxide were added to an aqueous polyvinyl alcohol solution (concentration 0.5 mass). %) And emulsification using a microprocess server. The obtained emulsified liquid was transferred to another flask and stirred for about 8 hours using a stirrer while heating in an 80 ° C. water bath to produce polymer particles. The polymer particles were collected by filtration and washed with acetone to obtain porous polymer particles. Further, the 5% compression deformation modulus of the porous polymer particles was measured using a micro compression tester.

<Formation of molecular template film>
Coating with polydopamine (formation of underlayer)
1 g of the obtained porous polymer particles were immersed in 50 g (concentration 2 mg / g) of an aqueous dopamine hydrochloride adjusted to pH 8.5 with a phosphate buffer, and the aqueous solution was stirred at room temperature for 24 hours. Thereafter, the surfaces of the particles recovered from the aqueous solution were analyzed by XPS measurement. Since nitrogen atoms could be confirmed on the particle surface, it was confirmed that a polydopamine film was formed on the particle surface.

<Formation of protein template (formation of surface layer)>
1 g of particles having a polydopamine membrane was put into a 5 mL glycylglycine aqueous solution (0.1 mol / L, pH 8.5) containing 10 mg / mL glucose-6-phosphate dehydrogenase (G-6-PDH). , G-6-PDH was adsorbed on the polydopamine membrane over 12 hours. Then, a template corresponding to G-6-PDH was formed again by forming a polydopamine film under the above conditions. Particles were immersed in NaCl 0.1M aqueous solution to desorb the template protein (G-6-PDH). Thereafter, the particles recovered from the aqueous solution were washed with water to obtain separation material particles having a molecular template membrane (polydopamine membrane) containing a template corresponding to G-6-PDH.

  The particle size of the obtained separation material was measured with a flow-type particle size measuring device, and the average particle size and particle size C.I. V. The value was calculated. Further, the average pore diameter of the separating material was measured by a mercury intrusion method.

(Example 2)
A separation material having a molecular template membrane (polydopamine membrane) containing a template corresponding to G-6-PDH in the same manner as in Example 1 except that the amount of octanol was changed to 2 g and the amount of span 80 was changed to 5 g. Obtained particles. The amount of divinylbenzene was not changed from 14 g. The average particle size and C.I. V. Values and average pore diameters were measured.

(Example 3)
Separation material particles having a molecular template membrane (polydopamine membrane) containing a template corresponding to G-6-PDH were obtained in the same manner as in Example 1 except that the amount of span 80 was changed to 7 g. The amount of divinylbenzene, 14 g, and the amount of octanol, 5 g, were unchanged from Example 1. The average particle size and C.I. V. Values and average pore diameters were measured.

Example 4
A solution for forming a polydopamine film was prepared by mixing 25 g of a dopamine hydrochloride aqueous solution (concentration 1 mg / g) adjusted to pH 8.5 with a phosphate buffer and 25 g of a dihydroxyphenylalanine aqueous solution (concentration 1 mg / g). Got ready. Porous polymer particles obtained in the same manner as in Example 1 were immersed in this solution, and the aqueous solution was stirred at room temperature for 24 hours. Thereafter, polymer particles having a polydopamine film containing dihydroxyphenylalanine as a copolymer component were recovered from the aqueous solution. On the surface of the obtained polymer particles, separation material particles having a molecular template membrane (polydopamine membrane) containing a template corresponding to G-6-PDH were obtained in the same manner as in Example 1. The average particle size and C.I. V. Values and average pore diameters were measured.

(Example 5)
In the same manner as in Example 1 except that the monomer was changed from divinylbenzene to 14 g of ethylene glycol dimethacrylate and the amount of span 80 was changed to 5 g, a molecular template film containing a template corresponding to G-6-PDH (poly Separation material particles having a dopamine membrane) were obtained. The amount of octanol was not changed from 5 g. The average particle size and C.I. V. Values and average pore diameters were measured.

(Comparative Example 1)
Porous polymer particles were obtained in the same manner as in Example 1 except that the monomer was changed from divinylbenzene to 5 g of ethylene glycol dimethacrylate and 9 g of glycidyl methacrylate, and the amount of span 80 was changed to 5 g. The amount of octanol was not changed from 5 g. 10 g of the obtained porous polymer particles, 80 g of water, and 20 g of diethylamine were placed in a flask and reacted at 50 ° C. for 3 hours to introduce amino groups on the particle surfaces. The amount of amino groups in the polymer particles obtained was 3.2 mmol / g based on the mass of the particles. The average particle size and C.I. V. Values and average pore diameters were measured.

(Comparative Example 2)
9 g of acrylamide as a monomer and 1 g of methylenebisacrylamide and 150 mg of enzyme (G-6-PDH) were added to 20 mL of phosphate buffer (pH 6.3). The obtained solution was added to a toluene solution of ethyl cellulose (concentration 0.2% by mass). After performing nitrogen substitution for 15 minutes, 10 mL of an aqueous solution containing 0.1 g of potassium persulfate and 0.05 g of sodium hydrogen sulfite was dropped, and the monomer was polymerized at 25 ° C. over 3 hours.

  After polymerization, the produced particles were washed with a 10% by volume aqueous acetic acid solution (containing 10 wt% sodium dodecyl sulfate) to remove the enzyme as a template molecule. The particles were washed with water and then used as a separation material in Comparative Example 2. The average particle size and C.I. V. Values and average pore diameters were measured.

(Comparative Example 3)
Double bonds were imparted to Liclosolve (registered trademark) RP-18 (particle diameter: 10 μm, manufactured by Merck), which is a particle for a column filler, with 3-methacryloxypropyltrimethoxysilane. 10 g of this particle is 12 mM containing 180 mg of enzyme (G-6-PDH), 2.5 mL of acrylic acid as a monomer, 2.3 g of acrylamide, 1.2 g of dihydroxyethylenebisacrylamide, and 1.2 g of methylenebisacrylamide. The solution was stirred by adding to 300 mL of an aqueous phosphate buffer solution. Thereto were added 1.5 g of an aqueous solution containing 0.6 g of ammonium persulfate and 1.2 g of tetramethylenediamine, and the monomer was polymerized over 6 hours. After polymerization, the particles were washed as in Comparative Example 2 to remove the template molecules. Thereafter, the particles were washed with water and used as a separation material of Comparative Example 3. The average particle size and C.I. V. Values and average pore diameters were measured.

(Comparative Example 4)
To 15 mM phosphate buffer (50 mM) in which 15 mg of enzyme (G-6-PDH) is dissolved, 250 mg of macroporous silica (Lichlorosphere Si1000) is added, and the solution is stirred for 3 hours to adsorb the enzyme to microporous silica. I let you. Thereafter, the microporous silica was removed by filtration and washed to separate the unadsorbed enzyme. 50 mg of the enzyme was adsorbed on the microporous. The resulting particles were added to 10 mL Tris buffer (10 mM, pH 8). Thereto 20 mg of dopamine was added and the solution was stirred for 24 hours. Thereafter, an enzyme used as a template was desorbed using a 0.5 M NaCl aqueous solution, and the particles were washed with water, and this was used as a separation material of Comparative Example 4. The average particle size and C.I. V. Values and average pore diameters were measured.

  Table 1 shows the average particle size and particle size of each of the examples and comparative examples. V. Indicates the value.

Evaluation of protein adsorption capacity 5 mL (pH 8.5) of an enzyme solution containing BSA and enzyme (G-6-PDH) at a concentration of 10 mg / mL and glycylglycine at a concentration of 0.1 mol / L was prepared. A standard solution was prepared by mixing 10 ml of 0.1 M glycylglycine buffer solution, 228 mg of MgCl ₂ , 18 mg of glucose-6-phosphate disodium, and 40 mg of NADP ⁺ . The enzyme solution was put into this standard solution, and a peak at 340 nm was traced every minute with a spectrophotometer to measure the reduction reaction rate of NADP ⁺ .

The particles of Example or Comparative Example (0.5 g) were added to the enzyme solution and stirred for 30 minutes to adsorb BSA and the enzyme to the particles. Thereafter, the enzyme solution was put into a standard solution, and a peak at 340 nm was traced every minute with a spectrophotometer to measure the reduction reaction rate of NADP ⁺ . The amount of enzyme adsorbed onto the particles was determined by comparing the measured reduction reaction rate with the reduction reaction rate in the standard solution. Further, the amount of BSA adsorbed on the particles was measured based on the absorbance at 280 nm. The adsorption selectivity (enzyme adsorption amount / BSA adsorption amount) was calculated from the enzyme and BSA adsorption amounts. Regarding the adsorption capacity of each particle, the case where the total amount of adsorbed enzyme was 15 mg / particle g or more was judged as ◯, and the case where it was 15 mg / particle g or less was judged as x.

Shaking test 1 g of the particles of the examples or comparative examples were dispersed in 10 g of water. The obtained dispersion was put into a 20 mL sample tube and shaken with a shaker for 6 hours. After shaking, the sample tube was allowed to stand for 10 minutes, and then the turbidity of the supernatant was measured. Regarding the resistance of the particles to shaking, the case where the water permeability was 3% or more was judged as ◯ when the water permeability was within 3%.

Column pressure evaluation The particles of the examples or comparative examples were mixed with methanol to prepare a slurry (particle concentration 30% by mass). This slurry was packed in a stainless steel column of φ7.8 × 300 mm over 15 minutes. The initial value of the column pressure was measured while flowing water through the column at a flow rate of 800 cm / h. Next, the flow rate was increased to 3000 cm / h, and water was allowed to flow for 1 hour. Thereafter, the flow rate was lowered to 800 cm / h, and the column pressure was measured. The case where the column pressure was improved by 10% or more from the initial value (before increasing the flow rate to 3000 cm / h) was evaluated as x when the column pressure was within 10%.

  As can be seen from the results in Table 2, it was found that the target protein can be adsorbed with high selectivity by forming a molecular template membrane with polydopamine on the surface of the porous polymer particles. It was also found that the column pressure did not fluctuate even at high flow rates. Furthermore, by forming a molecular template film on the surface of the porous polymer particles, particles that did not break in the shaking test were obtained. From the above results, it was found that the particles of the present invention are very useful as a separating material such as a column filler.

Claims (11)

Porous polymer particles comprising a crosslinked polymer;
A molecular template film that is provided on the surface of the porous polymer particles and includes a polymer for imprinting and forms a template by a molecular imprinting method;
Have a,
The molecular template film has an underlayer that covers at least a part of the surface of the porous polymer particle, and a surface layer provided on the underlayer, and the template is formed by the surface layer. , Separation material. The separation material according to claim 1, wherein the imprinting polymer contains polydopamine. The separation material according to claim 2 , wherein the polydopamine includes a modified polydopamine having a structural unit derived from dopamine and a structural unit derived from dihydroxyphenylalanine. Porous polymer particles comprising a crosslinked polymer;
A molecular template film that is provided on the surface of the porous polymer particles and includes a polymer for imprinting and forms a template by a molecular imprinting method;
Have
  The separation material, wherein the imprinting polymer includes polydopamine, and the polydopamine includes a modified polydopamine having a structural unit derived from dopamine and a structural unit derived from dihydroxyphenylalanine.   The separation material according to any one of claims 1 to 4, wherein the average pore diameter of the separation material is 0.1 to 0.5 µm.   The separation material according to any one of claims 1 to 5, wherein the porous polymer particles have a 5% compressive deformation elastic modulus of 100 to 500 MPa. The separation material according to any one of claims 1 to 6, wherein the specific surface area of the porous polymer particles is 30 m ² / g or more.   C. of the particle size of the separation material. V. The separating material according to any one of claims 1 to 7, wherein the value is 5 to 15%. The molecular template membrane forms a template that interacts with the target protein,
The separation material according to any one of claims 1 to 8, wherein the separation material adsorbs the target protein with a selectivity of 2 to 10 times with respect to bovine serum albumin.   The separation material according to claim 9, wherein an adsorption amount of the target protein is 5 mg or more per 1 g of the separation material. Forming a base layer containing the polymer for the base layer on the surface of the porous polymer particles containing the crosslinked polymer;
Adsorbing template molecules to the underlayer;
Forming a surface layer containing a surface layer polymer on the underlayer on which the template molecule is adsorbed;
Removing the template molecule to form a molecular template film having the surface layer and the base layer to form a template derived from the template molecule;
A method for producing a separating material.