JP6676975B2 - Separation materials and columns - Google Patents

Description

  The present invention relates to a separation material and a column.

  Conventionally, when separating and purifying a biopolymer represented by a protein, generally, a porous type particle having a synthetic polymer as a base and a particle having a crosslinked gel of a hydrophilic natural polymer as a base have been generally used. . Ion exchangers based on the above-mentioned porous synthetic polymers have the advantage that the volume change due to salt concentration is small, and when packed in a column and used for chromatography, the pressure resistance during passage is good. I have. However, when this ion exchanger is used for separation of proteins and the like, nonspecific adsorption such as irreversible adsorption based on hydrophobic interaction occurs, and asymmetry of peaks occurs or the hydrophobic interaction causes There is a problem that the protein adsorbed on the ion exchanger cannot be recovered while being adsorbed.

  On the other hand, the above-mentioned ion exchanger having a crosslinked gel of a hydrophilic natural polymer represented by a polysaccharide such as dextran or agarose as a base has the advantage that there is almost no non-specific adsorption of proteins. However, this ion exchanger has the disadvantage that it swells significantly in an aqueous solution, changes in volume due to the ionic strength of the solution, changes in volume between the free acid form and the loaded form are large, and has insufficient mechanical strength. In particular, when a crosslinked gel is used for chromatography, there is a disadvantage that the pressure loss at the time of passing the liquid is large, and the gel is compacted by the passing of the liquid.

  In order to overcome the drawbacks of the crosslinked gel of a hydrophilic natural polymer, attempts have been made to combine it with a so-called "skeleton" rigid substance.

  For example, Patent Document 1 describes that a complex in which a gel such as a natural polymer gel is held in the pores of a porous polymer is used in the field of peptide synthesis, thereby increasing the load coefficient of a reactive substance. It can be synthesized in high yield as an effect of the invention. Moreover, in Patent Document 1, since the gel is surrounded by a hard synthetic polymer substance, even when used in the form of a column bed, there is no change in volume and the pressure of the flow-through passing through the column does not change. Have been.

  Patent Documents 2 and 3 disclose a method in which a xerogel such as a polysaccharide such as dextran or cellulose is held on an inorganic porous material such as celite, and a diethylaminomethyl (DEAE) group or the like is added to the gel to add sorption performance. Used to remove hemoglobin. As an effect thereof, good liquid permeability in the column is mentioned.

  Patent Literature 4 discloses an ion exchanger of a hybrid copolymer in which pores of a copolymer having a so-called macro network structure are filled with a gel of a crosslinked copolymer synthesized from a monomer. In Patent Document 4, when the degree of cross-linking of the gel of the cross-linked copolymer is low, there are problems such as pressure loss and volume change, but by using a hybrid copolymer, the liquid-permeability is improved and the pressure loss is reduced. It is also mentioned that the ion exchange capacity is improved and the leak behavior is improved.

  Further, a composite filler in which a crosslinked gel of a hydrophilic natural polymer having a huge network structure is filled in pores of an organic synthetic polymer substrate has been proposed (see Patent Documents 5 and 6).

  In Patent Document 7, porous particles composed of glycidyl methacrylate and an acrylic cross-linked monomer are synthesized.

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

  With conventional column packing materials, sufficient consideration was given to the problems of natural polymers such as liquid permeability and durability, and problems of polymer particles such as alkali resistance, pressure resistance, reduction of non-specific adsorption, and low protein adsorption. It is difficult to solve at the level.

  Therefore, an object of the present invention is to provide a separation material and a column capable of securing liquid permeability, improving the amount of protein adsorption, and reducing non-specific adsorption.

The present invention provides a separation material described in the following [1] to [10] and a column described in the following [11].
[1] A porous polymer particle obtained by polymerizing a monomer containing a styrene-based monomer, and a coating layer containing at least a part of the surface of the porous polymer particle and containing a hydrophilic polymer having an amino group. And a mode diameter of 0.10 to 0.40 μm.
[2] The separation material according to [1], wherein the porosity is 30 to 70% by volume.
[3] The separation material according to [1] or [2], wherein the specific surface area of the porous polymer particles is 30 m ² / g or more.
[4] The separation material according to any one of [1] to [3], wherein the monomer containing a styrene-based monomer contains 50% by mass or more of divinylbenzene based on the total mass of the monomer.
[5] The separation material according to any one of [1] to [4], wherein the coefficient of variation of the particle diameter of the porous polymer particles is 3 to 15%.
[6] The separation material according to any one of [1] to [5], wherein the hydrophilic polymer having an amino group is a polymer having an amino group introduced into a polysaccharide or a modified product thereof.
[7] The separation material according to any one of [1] to [5], wherein the hydrophilic polymer having an amino group is a polymer having an amino group introduced into agarose or a modified product thereof.
[8] The separation material according to any one of [1] to [7], comprising a coating layer of 30 to 500 mg per 1 g of the porous polymer particles.
[9] The separation material according to any one of [1] to [8], wherein, when packed in a column, the liquid passing speed is 800 cm / h or more when the column pressure is 0.3 MPa.
[10] The separating material according to any one of [1] to [9], wherein the breaking strength of the porous polymer particles is 10 mN or more.
[11] A column comprising the separation material according to any one of [1] to [10].

  Advantageous Effects of Invention According to the present invention, it is possible to provide a separation material and a column capable of securing liquid permeability, improving the amount of adsorbed protein and reducing non-specific adsorption. Further, the separation material and the column according to the present invention are also excellent in alkali resistance.

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

<Separation material>
The separation material of the present embodiment includes porous polymer particles and a coating layer that covers at least a part of the surface of the porous polymer particles. In this specification, the “surface of the porous polymer particle” includes not only the outer surface of the porous polymer particle but also the surface of the pore inside the porous polymer particle.

(Porous polymer particles)
The porous polymer particles of the present embodiment are obtained by polymerizing a monomer including a styrene monomer, and have a structural unit derived from a styrene monomer. Further, the porous polymer particles of the present embodiment can be obtained by reacting a composition containing a styrene-based monomer and a porogen. The porous polymer particles can be synthesized by, for example, conventional suspension polymerization, emulsion polymerization, or the like. As the monomer, a styrene monomer can be used. Specific examples of the styrene monomer include the following polyfunctional styrene monomers and monofunctional styrene monomers.

  Examples of the polyfunctional styrene monomer include divinyl compounds such as divinylbenzene, divinylbiphenyl, divinylnaphthalene, and divinylphenanthrene. These polyfunctional styrene-based monomers can be used alone or in combination of two or more. Among them, divinylbenzene is preferably used because it is more excellent in durability, acid resistance and alkali resistance.

  When the monomer contains divinylbenzene as a styrene-based monomer, it preferably contains divinylbenzene in an amount of 50% by mass or more, more preferably 60% by mass or more, even more preferably 70% by mass or more based on the total mass of the monomer. . By containing 50% by mass or more of divinylbenzene based on the total mass of the monomer, alkali resistance and pressure resistance tend to be further improved.

  Examples of the monofunctional styrene-based monomer include, for example, styrene, o-methylstyrene, m-methylstyrene, p-methylstyrene, α-methylstyrene, o-ethylstyrene, m-ethylstyrene, p-ethylstyrene, 2, 4-dimethylstyrene, pn-butylstyrene, pt-butylstyrene, pn-hexylstyrene, pn-octylstyrene, pn-nonylstyrene, pn-decylstyrene, pn Styrene such as -dodecylstyrene, p-methoxystyrene, p-phenylstyrene, p-chlorostyrene, and 3,4-dichlorostyrene, and derivatives thereof. These monofunctional styrene monomers can be used alone or in combination of two or more. Of these, it is preferable to use styrene from the viewpoint of more excellent acid resistance and alkali resistance. Further, a styrene derivative having a functional group such as a carboxy group, an amino group, a hydroxyl group, and an aldehyde group can also be used.

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

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

  Further, water used as a solvent can also be used as a porosifying agent. When water is used as the porosifying agent, the oil-soluble surfactant can be dissolved in the monomer, and water can be absorbed to make the monomer porous.

  Examples of the oil-soluble surfactant used for making the porous material include a sorbitan monoester of a branched C16-C24 fatty acid, a chain unsaturated C16-C22 fatty acid or a chain saturated C12-C14 fatty acid, for example, sorbitan monolaurate, sorbitan Sorbitan monoesters derived from monooleate, sorbitan monomyristate or coconut fatty acids; diglycerol monoesters of branched C16-C24 fatty acids, chain unsaturated C16-C22 fatty acids or chain saturated C12-C14 fatty acids, e.g. Glycerol monooleate (for example, diglycerol monoester of C18: 1 (18 carbon atoms, 1 double bond) fatty acid), diglycerol monomyristate, diglycerol monoisostearate, or diglycerol mono of coconut fatty acid Ester; branched C16-C 4 alcohols (e.g., Guerbet alcohols), linear unsaturated C16~C22 alcohol or chain saturated C12~C14 alcohols (e.g., coconut fatty alcohols) diglycerol mono-fatty ethers; and mixtures thereof.

  Of these, sorbitan monolaurate (eg, SPAN® 20, preferably having a purity of greater than about 40%, more preferably greater than about 50%, and most preferably having a purity of greater than about 70%) Laurate); sorbitan monooleate (eg, SPAN® 80, preferably more than about 40% pure, more preferably more than about 50% pure, most preferably more than about 70% pure Diglycerol monooleate (eg, preferably greater than about 40% pure, more preferably greater than about 50% pure, most preferably greater than about 70% pure); diglycerol monooleate; Isostearate (e.g., preferably greater than about 40% pure, more preferably Or more than about 50% pure, most preferably more than about 70% pure diglycerol monoisostearate); diglycerol monomyristate (preferably more than about 40% pure, more preferably more than about 50% pure) Sorbitan monomyristate, most preferably greater than about 70% pure); cocoyl (eg, lauryl, myristoyl, etc.) ethers of diglycerol; and mixtures thereof.

  These oil-soluble surfactants are preferably used in a range of 5 to 80% by mass based on the total mass of the monomers. When the content of the oil-soluble surfactant is 5% by mass or more, the stability of water droplets becomes sufficient, so that large single pores are easily formed. When the content of the oil-soluble surfactant is 80% by mass or less, the shape of the porous polymer particles can be more easily maintained 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 amphoteric surfactants can be used.

  Examples of anionic surfactants include fatty acid oils such as sodium oleate and castor oil, alkyl sulfate esters such as sodium lauryl sulfate and ammonium lauryl sulfate, alkyl benzene sulfonates such as sodium dodecyl benzene sulfonate, and alkyl naphthalene sulfones. Acid, alkane sulfonate, dialkyl sulfosuccinate such as sodium dioctyl sulfosuccinate, alkenyl succinate (dipotassium salt), alkyl phosphate ester salt, naphthalene sulfonic acid formalin condensate, polyoxyethylene alkyl phenyl ether sulfate 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.

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

  Examples of amphoteric 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, anionic surfactants are preferable from the viewpoint of dispersion stability during polymerization of monomers.

  Examples of the polymerization initiator added as needed include, for example, benzoyl peroxide, lauroyl peroxide, orthochlorobenzoyl peroxide, orthomethoxybenzoyl peroxide, 3,5,5-trimethylhexanoyl peroxide, tert-butyl peroxide Organic peroxides such as oxy-2-ethylhexanoate and di-tert-butyl peroxide; 2,2′-azobisisobutyronitrile, 1,1′-azobiscyclohexanecarbonitrile, 2,2 ′ Azo compounds such as -azobis (2,4-dimethylvaleronitrile). The polymerization initiator can be used in a 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 depending on the type of the monomer and the polymerization initiator. The polymerization temperature is preferably from 25 to 110 ° C, more preferably from 50 to 100 ° C.

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

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

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

  The average particle size of the porous polymer particles is preferably 300 μm or less, more preferably 150 μm or less, and further preferably 100 μm or less. The average particle size of the porous polymer particles is preferably 10 μm or more, more preferably 30 μm or more, and further preferably 50 μm or more, from the viewpoint of further improving liquid permeability.

  The coefficient of variation (CV) of the particle size of the porous polymer particles is preferably 3 to 15%, more preferably 5 to 15%, from the viewpoint of further improving liquid permeability. More preferably, it is 5 to 10%. C. V. As a method for reducing the dispersion, monodispersion may be performed using an emulsifying apparatus such as a microprocess server (for example, manufactured by Hitachi, Ltd.).

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

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

  Further, the mode diameter (mode of pore diameter distribution, maximum frequency diameter) of the pore diameter distribution of the porous polymer particles of the present embodiment is preferably 0.01 to 0.5 μm, and 0.05 to 0 μm. More preferably, it is 0.5 μm. When the mode diameter of the pore diameter distribution is in this range, the liquid easily flows into the particles, and the dynamic adsorption amount is easily increased.

The specific surface area of the porous polymer particles is preferably 30 m ² / g or more. In light of higher practicality, the specific surface area is more preferably equal to or greater than 35 m ² / g, and still more preferably equal to or greater than 40 m ² / g. 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 breaking strength of the porous polymer particles is preferably 10 mN or more, more preferably 15 mN or more. When the breaking strength of the porous polymer particles is within the above range, the durability of the porous polymer particles is excellent, and the particles are crushed when filling the column, or the particles are crushed when the flow rate of the column is increased. Column pressure does not easily increase.

The breaking strength of the porous polymer particles of the present embodiment can be calculated as follows.
Using a micro-compression tester (manufactured by Fisher), the load when the particles were compressed to 50 mN by the smooth end face (50 μm × 50 μm) of a square pillar at a load application speed of 1 mN / sec at room temperature (25 ° C.). Measure the compression displacement. The load at the point where the displacement amount changes the most during the measurement is defined as the breaking strength (mN).

(Coating layer)
The coating layer of the present embodiment includes a hydrophilic polymer having an amino group. By coating the porous polymer particles with a hydrophilic polymer having an amino group, it is possible to suppress an increase in column pressure, to suppress nonspecific adsorption of proteins, and to adsorb proteins on the separation material. The amount can be equal to or greater than that when using a natural polymer. Further, when the hydrophilic polymer having an amino group is crosslinked, it is possible to further suppress the increase in column pressure.

(Hydrophilic polymer having amino group)
The hydrophilic polymer having an amino group is, for example, a polymer having an amino group introduced into a polymer having a hydroxyl group. Amino group, a group represented by ^-NR 1 ^{R 2, R 1} and ^{R 2} are, for example, each independently represent a hydrogen atom or an alkyl group having 1 to 3 carbon atoms.

  The polymer having a hydroxyl group preferably has two or more hydroxyl groups in one molecule, and is more preferably a hydrophilic polymer. Examples of the polymer having a hydroxyl group include a polysaccharide or a modified product thereof. More preferred are agarose, dextran, cellulose, polyvinyl alcohol, and chitosan, and modified products thereof. The weight average molecular weight of the polymer having a hydroxyl group may be 10,000 to 200,000.

  Here, the denatured product refers to a product in which a hydrophobic group is introduced into a molecule. The polymer having a hydroxyl group is preferably a modified form from the viewpoint of improving interfacial adsorption ability. Examples of the hydrophobic group include an alkyl group having 1 to 6 carbon atoms and an aryl group having 6 to 10 carbon atoms. Examples of the alkyl group having 1 to 6 carbon atoms include a methyl group, an ethyl group, and a propyl group. Examples of the aryl group having 6 to 10 carbon atoms include a phenyl group and a naphthyl group. The hydrophobic group is introduced by reacting a functional group that reacts with a hydroxyl group (for example, an epoxy group) and a compound having a hydrophobic group (for example, glycidyl phenyl ether) with a polymer having a hydroxyl group by a conventionally known method. Can be. Examples of such a modified product include a modified product of a polysaccharide, preferably a modified product of agarose, a modified product of dextran, a modified product of cellulose, a modified product of polyvinyl alcohol, a modified product of chitosan, and the like.

  That is, the hydrophilic polymer having an amino group may be a polymer in which an amino group is introduced into a polysaccharide or a modified product thereof, preferably, agarose or a modified product thereof, dextran or a modified product thereof, cellulose or a modified product thereof. It is a polymer in which an amino group is introduced into a modified product, polyvinyl alcohol or a modified product thereof, or chitosan or a modified product thereof.

(Method of forming coating layer)
The coating layer containing the hydrophilic polymer having an amino group can be formed, for example, by the following method.
First, a solution of a polymer having a hydroxyl group is adsorbed on the surface of the porous polymer particles. The solvent for the solution of the polymer having a hydroxyl group is not particularly limited as long as it can dissolve the polymer having a hydroxyl group, but water is the most common. The concentration of the polymer dissolved in the solvent is preferably 5 to 20 (g / L).
The solution is impregnated with the porous polymer particles at, for example, 40 to 95 ° C. In the impregnation method, porous polymer particles are added to a solution of a polymer having a hydroxyl group, and the mixture is allowed to stand for a certain time. Although the impregnation time varies depending on the surface state of the porous body, the polymer concentration is usually in equilibrium with the external concentration inside the porous body when the impregnation is carried out day and night. Thereafter, the polymer is washed with water, alcohol, or the like at a temperature at which the polymer is dissolved at room temperature to wash the polymer excessively adsorbed on the particle surface. Thereby, a separation material precursor is obtained.

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

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

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

  The crosslinking reaction by the crosslinking agent is usually performed by adding the crosslinking agent to a system in which the precursor of the separating material is dispersed and suspended in an appropriate medium. When the polysaccharide is used as the polymer having a hydroxyl group, the amount of the crosslinking agent is within the range of 0.1 to 100 moles per 1 unit of the monosaccharide. Can be selected according to the performance of In general, when the amount of the cross-linking agent is reduced, the coating layer tends to be easily separated from the porous polymer particles. When the amount of the crosslinking agent added is excessive and the reaction rate with the polymer having a hydroxyl group is high, the properties of the polymer having a hydroxyl group as a raw material tend to be impaired.

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

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

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

  The crosslinking reaction is usually performed at a temperature in the range of 5 to 90 ° C. for 1 to 10 hours. Preferably, the temperature is in the range of 20 to 90C.

  After the cross-linking reaction is completed, the generated particles are separated by filtration, and then washed with a hydrophilic organic solvent such as water, methanol, and ethanol to remove unreacted polymer and a suspending medium. A separation material precursor having at least a part of its surface covered with a coating layer containing a polymer having a hydroxyl group is obtained.

(Introduction of amino group)
As a method for introducing an amino group into a polymer having a hydroxyl group, a polymer having a hydroxyl group, a mono-, di- or tri-alkylamine, a mono-, di- or tri-alkanolamine, a mono-alkyl-mono- A method of reacting with an amine compound such as alkanolamine, di-alkyl-mono-alkanolamine and mono-alkyl-di-alkanolamine is exemplified. These amine compounds have at least one alkyl group such as diethylaminoethyl chloride in which some of the hydrogen atoms have been replaced with chlorine atoms. The use amount of these amine compounds is preferably 0.2% by mass or more based on the total mass of the separation material. The reaction conditions are preferably at 40 to 90 ° C. for 0.5 to 12 hours. Thereby, the separation material according to the present embodiment is obtained.

  The separating material has a coating layer of preferably 30 to 500 mg, more preferably 30 to 400 mg, per 1 g of the porous polymer particles. The amount of the coating layer can be measured by a weight loss due to thermal decomposition or the like.

  An ion exchange group other than an amino group, a ligand (protein A), or the like may be introduced into the polymer having a hydroxyl group. Examples of a 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, and quaternary ammonium hydrochlorides having a halogenated alkyl group. These alkyl halide compounds are preferably bromide or chloride. The amount of the halogenated alkyl compound to be used is preferably 0.2% by mass or more based on the total mass of the separation material to which the ion exchange groups are provided.

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

  Usually, the ion-exchange group is introduced into the hydroxyl group on the surface of the separation material. Therefore, after the wet particles are drained by filtration or the like, the particles are immersed in an aqueous alkaline solution having a predetermined concentration, and left for a certain period of time. The above-mentioned alkyl halide compound is added and reacted in a solvent mixture system. This reaction is preferably performed at a temperature of 40 to 90 ° C. for 0.5 to 12 hours. The ion exchange group to be provided is determined by the type of the alkyl halide compound used in the above reaction.

  As a method of introducing a quaternary ammonium group as a strongly basic group as an ion exchange group, first, a tertiary amino group is introduced, and a halogenated alkyl compound such as epichlorohydrin is reacted with the tertiary amino group. A method of converting to an ammonium group may be mentioned. Alternatively, a quaternary ammonium hydrochloride or the like may be reacted with the separating material.

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

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

  On the other hand, as a method for introducing an ion exchange group, a method of reacting 1,3-propane sultone with a separating material in an alkaline atmosphere can also be mentioned. 1,3-Propane sultone is preferably used in an amount of 0.4% by mass or more based on the total mass of the separation material. The reaction conditions are preferably 0 to 90 ° C. for 0.5 to 12 hours. The step of introducing an amino group into a polymer having a hydroxyl group may be performed after or before the above-described crosslinking treatment.

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

  The mode diameter (mode of pore diameter distribution, maximum frequency diameter) of the separation material of the present embodiment is 0.10 to 0.40 μm from the viewpoint of excellent liquid permeability and protein adsorption amount and reduction of nonspecific adsorption. It is. The mode diameter of the separation material is preferably 0.10 to 0.35 μm, more preferably 0.10 to 0.30 μm, from the viewpoint of further improving the liquid permeability.

The specific surface area of the separation material of the present embodiment is preferably 30 m ² / g or more. In light of higher practicality, the specific surface area is more preferably equal to or greater than 35 m ² / g, and still more preferably equal to or greater than 40 m ² / g. 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 upper limit of the specific surface area of the separation material is not particularly limited, but may be, for example, 300 m ² / g or less.

  The porosity of the separation material of the present embodiment is preferably 30 to 70% by volume, and more preferably 40 to 70% by volume. When the porosity is in this range, the protein adsorption amount can be increased.

  The separation material of the present embodiment is suitable for use in separation of proteins by electrostatic interaction, ion exchange purification, and affinity purification. For example, the separating material of the present embodiment is added to a mixed solution containing a protein, and only the protein is adsorbed on the separating material by electrostatic interaction. When added to a high aqueous solution, the protein adsorbed on the separation material can be easily desorbed and recovered. Further, the separation material of the present embodiment can also be used in column chromatography. That is, the column of the present embodiment includes the separating material of the present embodiment, and is, for example, packed with the separating material of the present embodiment.

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

  The separation material of the present embodiment is obtained by introducing a ion-exchange group and protein A to the surface of the separation material after cross-linking the coating layer on the porous polymer particles, and thereby separating natural macromolecules such as proteins. It has the advantages of particles of molecules or particles of synthetic polymers, respectively. In particular, since the porous polymer particles in the separation material of the present embodiment are obtained by the above-described method, they have durability and alkali resistance. In addition, the separation material of the present embodiment tends to reduce non-specific adsorption of proteins and easily cause adsorption and desorption of proteins. Furthermore, the separation material of the present embodiment tends to have a large amount of adsorption of protein and the like (dynamic adsorption amount) under the same flow rate.

  The liquid passing rate in the present specification indicates a liquid passing rate when a stainless steel column of φ7.8 × 300 mm is filled with the separating material of the present embodiment and liquid is passed. When the separation material of the present embodiment is packed in a column, it is preferable that the liquid passing speed is 800 cm / h or more when the column pressure is 0.3 MPa. When separating proteins by column chromatography, the flow rate of a protein solution or the like is generally in the range of 400 cm / h or less. However, when the separation material of the present embodiment is used, a normal protein separation speed is used. It can be used at a flow rate of 800 cm / h or more, which is faster than the separation material.

  The average particle size of the separation material of the present embodiment is preferably from 10 to 300 μm. For use in preparative or industrial chromatography, it is preferably from 10 to 100 μm in order to avoid an extreme increase in column internal pressure.

  When the separation material of this embodiment is used as a column packing material in column chromatography, there is almost no change in volume in the column regardless of the properties of the eluent used, so that the operability is excellent.

  The mode diameter, specific surface area and the like of the separation material are appropriately selected from the raw materials of the porous polymer particles, the porosifying agent, the polymer having a hydroxyl group, the crosslinking agent for crosslinking the polymer having a hydroxyl group, the method for forming the coating layer, and the like. This can be adjusted.

  Note that, in the present embodiment, the separation material in which an ion exchange group is introduced has been described, but the separation material can be used as a separation material without introducing an ion exchange group. Such a separating material can be used, for example, for gel filtration chromatography.

  Hereinafter, the present invention will be described with reference to 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, 16 g of 96% pure divinylbenzene (DVB960, Nippon Steel & Sumikin Chemical Co., Ltd.), 6 g of sorbitan monooleate (Span 80), and 0.64 g of benzoyl peroxide were added to a polyvinyl alcohol aqueous solution (0.5 mass %) And emulsified 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. to obtain particles. After filtering the obtained particles, the particles were washed with acetone to obtain porous polymer particles. The particle size of the obtained porous polymer particles was measured with a flow type particle size measuring device, and the average particle size and the C.I. V. Values were calculated. The mode diameter, specific surface area, and porosity of the porous polymer particles were measured by a mercury intrusion method. Table 1 shows the results.

(Evaluation of breaking strength)
The breaking strength of the porous polymer particles was evaluated as follows. Using a micro compression tester (manufactured by Fisher) at room temperature (25 ° C.) at a load application speed of 1 mN / sec, the load when the separation material is compressed to 50 mN by the smooth end face (50 μm × 50 μm) of a square prism. And the compression displacement were measured. The load at the point where the displacement amount changed the most during the measurement was defined as the breaking strength (mN). Table 1 shows the results.

(Evaluation of linear velocity)
The porous polymer particles were packed into a stainless steel column of φ7.8 × 300 mm at a slurry (solvent: methanol) concentration of 30% by mass over 15 minutes. Thereafter, water was flowed through the column at different flow rates, the relationship between the flow rate and the column pressure was measured, and the linear flow rate at a column pressure of 0.3 MPa was calculated. Table 1 shows the results.

(Formation of coating layer)
A phenyl group was introduced into agarose by adding 4 g of sodium hydroxide and 0.4 g of glycidyl phenyl ether to 100 mL of an agarose aqueous solution (2% by mass) and reacting at 70 ° C. for 12 hours.

  The resulting denatured agarose was reprecipitated three times with isopropyl alcohol and washed. 10 g of the porous polymer particles was added to 700 mL of a 4.5 g / L modified agarose aqueous solution, and the mixture was stirred at 55 ° C. for 24 hours to form a coating layer on the surface of the porous polymer particles. Thereafter, the surface of the porous polymer particles was washed with 55 ° C. pure water at room temperature. The amount of the modified agarose adsorbed on 1 g of the porous polymer particles (the amount of the coating layer) was calculated by measuring the thermal weight loss of the dried particles. Table 2 shows the results.

  The modified agarose adsorbed on the particles was crosslinked as follows. 39 g of ethylene glycol diglycidyl ether was added to a 0.4 M aqueous sodium hydroxide solution in which 10 g of particles were dispersed, and the mixture was stirred at room temperature for 8 hours. Then, after washing with a heated 2% by mass aqueous solution of sodium dodecyl sulfate, the particles were washed with pure water, and the particles (separating material) were stored in water.

(Introduction of amino group)
After water was removed from the dispersion of the separation material by centrifugation, 20 g of the separation material was dispersed in 100 mL of an aqueous solution in which a predetermined amount of diethylaminoethyl chloride hydrochloride was dissolved, and the mixture was stirred at 70 ° C. for 10 minutes. Thereafter, 100 mL of a 5M NaOH aqueous solution heated to 70 ° C. was added, and the mixture was reacted for 1 hour. After completion of the reaction, the mixture was filtered and washed twice with water / ethanol (volume ratio: 8/2) to obtain a DEAE-modified separation material into which a diethylaminoethyl (DEAE) group was introduced. The mode diameter, specific surface area, and porosity of the obtained DEAE-modified separation material were measured by a mercury intrusion method. Table 2 shows the results.

(Evaluation of ion exchange capacity)
The ion exchange capacity of the separation material was measured as follows. A 5 mL DEAE-modified separation material was immersed in 20 mL of a 0.1 N aqueous sodium hydroxide solution for 1 hour and stirred at room temperature. Thereafter, washing was performed until the pH of water used as the washing liquid became 7 or less. The washed separating material was immersed in 20 mL of 0.1N hydrochloric acid and stirred for 1 hour. After removing the DEAE-modified separation material by filtration, the ion exchange capacity of the DEAE-modified separation material was measured by neutralizing and titrating the hydrochloric acid aqueous solution of the filtrate. Table 3 shows the results.

(Column characteristics evaluation)
The linear flow velocity of the obtained DEAE-modified separation material was evaluated in the same manner as in the evaluation of the linear flow velocity of the porous polymer particles. Table 3 shows the results.
Further, the amount of dynamic adsorption was measured as follows. 20 column volumes of 20 mmol / L Tris-HCl buffer (pH 8.0) were applied to the column. Thereafter, a 20 mmol / L Tris-HCl buffer having a BSA concentration of 2 mg / mL was passed, and the BSA concentration at the column outlet was measured by UV. The solution was allowed to flow until the BSA concentration at the column inlet and the BSA concentration at the outlet matched, and the column was diluted with 5 column volumes of 1 M NaCl Tris-HCl buffer. The dynamic adsorption amount at 10% breakthrough was calculated using the following equation. Table 3 shows the results.
_{q 10 = c f F (t} 10 -t 0) / V B
q ₁₀ : Dynamic adsorption amount at 10% breakthrough (mg / mL wet resin)
c _f : BSA concentration injected F: flow rate (mL / min)
V _B : bed volume (mL)
t ₁₀ : Time at 10% breakthrough t ₀ : BSA injection start time

(Evaluation of nonspecific adsorption ability of protein)
0.5 g of the DEAE-modified separation material was added to 50 mL of a phosphate buffer (pH 7.4) having a BSA concentration of 20 mg / mL, and the mixture was stirred at room temperature for 24 hours. Then, after taking the supernatant by centrifugation, the amount of BSA adsorbed on the particles was calculated by calculating the BSA concentration of the filtrate using a spectrophotometer. The concentration of BSA was calculated from the absorbance at 280 nm using a spectrophotometer. Table 3 shows the results.

(Alkali resistance evaluation)
The DEAE-modified separating material was stirred in a 0.5 M aqueous sodium hydroxide solution for 24 hours, washed with a phosphate buffer, and then packed under the same conditions as in the column characteristic evaluation. The 10% breakthrough dynamic adsorption of BSA was measured and compared with the dynamic adsorption before alkali treatment. The case where the reduction rate of the dynamic adsorption amount was 3% or less was evaluated as ○, the case where it exceeded 3% and 20% or less was evaluated as Δ, and the case where it exceeded 20% was evaluated as ×. Table 3 shows the results.

<Example 2>
The production and evaluation of the separation material were performed in the same manner as in Example 1 except that the amount of ethylene glycol diglycidyl ether was changed to 10 g in forming the coating layer.

<Example 3>
The production and evaluation of the separation material were performed in the same manner as in Example 1 except that the amount of ethylene glycol diglycidyl ether added was changed to 40 g in forming the coating layer.

<Comparative Example 1>
In forming the coating layer, separation was performed in the same manner as in Example 1 except that the porous polymer particles were sufficiently filtered and washed with pure water at 80 ° C. instead of washing the surface of the porous polymer particles with pure water at 55 ° C. The material was produced and evaluated.

<Comparative Example 2>
Preparation and evaluation of a separation material in the same manner as in Example 1 except that the amount of ethylene glycol diglycidyl ether added was changed to 40 g in forming the coating layer, and the surfaces of the porous polymer particles were washed with pure water at 15 ° C. Was done.

  From the results in Tables 2 and 3, when the mode diameter of the separation material is 0.10 to 0.40 μm, the dynamic adsorption amount is improved, and further, the liquid permeability is improved and the non-specific adsorption is reduced. You can see that.

Claims (11)

Porous polymer particles obtained by polymerizing monomers including styrene-based monomers,
Covering at least a portion of the surface of the porous polymer particles, and a coating layer containing a hydrophilic polymer having an amino group, a separation material which Ru provided with,
The separation material, wherein the maximum frequency diameter of the pore size distribution of the separation material is 0.10 to 0.40 μm .   The separation material according to claim 1, wherein the porosity is 30 to 70% by volume. The separation material according to claim 1, wherein the specific surface area of the porous polymer particles is 30 m ² / g or more.   The separation material according to any one of claims 1 to 3, wherein the monomer containing the styrene-based monomer contains divinylbenzene in an amount of 50% by mass or more based on the total mass of the monomer.   The separation material according to any one of claims 1 to 4, wherein the coefficient of variation of the particle size of the porous polymer particles is 3 to 15%.   The separation material according to any one of claims 1 to 5, wherein the hydrophilic polymer having an amino group is a polymer having an amino group introduced into a polysaccharide or a modified product thereof.   The separation material according to any one of claims 1 to 5, wherein the hydrophilic polymer having an amino group is a polymer having an amino group introduced into agarose or a modified product thereof.   The separation material according to any one of claims 1 to 7, comprising 30 to 500 mg of the coating layer per 1 g of the porous polymer particles.   The separation material according to any one of claims 1 to 8, wherein, when packed in a column, the liquid passing speed 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 9, wherein the breaking strength of the porous polymer particles is 10 mN or more.   A column comprising the separation material according to claim 1.