Method for producing ion exchange resin, method for separating compound, and cation exchange resin

Abstract

The present invention provides a low-cost method for producing an ion exchange resin capable of efficiently separating a compound which is used for medium- and large-molecule pharmaceuticals and is typified by lactoferrin. The method for producing an ion exchange resin according to the present invention includes: mixing, in an aqueous medium, a vinyl monomer mixture containing 0-90 mass% of a hydrophilic monofunctional vinyl monomer and / or a monofunctional vinyl monomer having a functional group capable of forming a hydrophilic group and 10-100 mass% of a polyfunctional vinyl monomer, a radical polymerization initiator, and a porogenic solvent to prepare a suspension for polymerization; heating the suspension for polymerization up to a polymerization temperature; performing a polymerization reaction, during which a hydrophilic monofunctional vinyl monomer and / or a monofunctional vinyl monomer having a functional group capable of forming a hydrophilic group is added to the suspension for polymerization to form porous polymer particles; and introducing an ion exchange group into the porous polymer particles.

Description

Method for producing ion exchange resin, method for separating compounds, and cation exchange resin

[0001] The present invention relates to a method for producing an ion exchange resin, a method for separating compounds, and a cation exchange resin. This application claims priority based on Japanese Patent Application No. 2024-025166, filed on February 22, 2024, the contents of which are incorporated herein by reference.

[0002] In recent years, there has been a significant shift in pharmaceutical development from small molecules with molecular weights of several hundred to so-called medium-sized molecules such as peptides and oligonucleotides, as well as antibody drugs and mRNA vaccines, with molecular weights of approximately 1,000 to 10,000. The application of chromatography is essential for the separation of compounds used in these medium-sized and high-molecular-weight drugs, and ion exchange chromatography using ion exchange resins is the most commonly used method. Furthermore, in the food industry, there is a growing demand for the separation of proteins, which are high-molecular-weight compounds, not only from naturally occurring raw materials, but also from materials derived from precision fermentation in response to recent food and decarbonization issues. For these purposes, adsorption / desorption methods using ion exchange resins and ion exchange chromatography are widely used.

[0003] As an example, lactoferrin derived from cow's milk is in increasing demand as an additive to infant formula and for use in health foods, pharmaceuticals, etc. due to its high physiological activity. However, since its content in cow's milk is low, it is separated from raw materials such as cheese whey and skim milk by an adsorption / desorption method using a cation exchange resin (Patent Document 1).

[0004] When separating drugs used in low-molecular-weight pharmaceuticals from biological samples, styrene-based ion exchange resins with highly hydrophobic substrates are used (Patent Document 2).

[0005] When separating lipoproteins such as cholesterol in plasma or serum, a resin made of an anion exchanger in which anion exchange groups are introduced into a water-insoluble, hydrophilic matrix is ​​used (Patent Document 3).

[0006] Japanese Unexamined Patent Publication No. 63-152400 Japanese Unexamined Patent Publication No. 2002-55093 Unexamined Japanese Patent Application No. 9-12629

[0007] GRAS Notice No. 423, U. S. Food & Drug Administration.

[0008] When separating compounds used in medium- and high-molecular-weight drugs, such as lactoferrin derived from milk, using ion exchange resins, for example, styrene-based ion exchange resins with a highly hydrophobic substrate require a high amount of ion exchange groups to suppress irreversible adsorption of the compounds used in medium- and high-molecular-weight drugs. However, a drawback of this approach is that ionic compounds in the raw material may exchange with the ion exchange groups in excess of what is necessary after the liquid is passed through the resin, causing compositional fluctuations. This can result in pH fluctuations and other alterations in the raw material after the liquid is passed through the resin, potentially impairing the quality of the raw material for food applications. To avoid these problems, a method for adsorption / desorption purification using an ion exchange resin has been disclosed, which uses cross-linked polysaccharides, such as cross-linked dextran or cross-linked agarose, as the ion exchange resin substrate and does not introduce excessive ion exchange groups, thereby avoiding excessive ion exchange of ionic compounds other than lactoferrin and suppressing irreversible adsorption (Non-Patent Document 1). However, ion exchange resins derived from these cross-linked polysaccharides have low mechanical strength and are expensive, so there is an industrial demand for inexpensive ion exchange resins with excellent mechanical strength.

[0009] The present invention has been made in view of these problems, and the object of the present invention is to provide a method for producing an ion exchange resin at low production cost, which can efficiently separate compounds used in medium-sized molecular weight drugs and high molecular weight drugs, such as lactoferrin.Another object of the present invention is to provide a method for efficiently separating compounds used in medium-sized molecular weight drugs and high molecular weight drugs using the ion exchange resin obtained by the production method of the present invention, particularly a method for efficiently separating compounds used in high molecular weight drugs, such as lactoferrin.Furthermore, another object of the present invention is to provide a (meth)acrylate cation exchange resin, which can efficiently separate compounds used in high molecular weight drugs, such as lactoferrin.

[0010] As a result of extensive research, the inventors discovered that the ion exchange resin obtained by the manufacturing method of the present invention can efficiently separate compounds used in medium-sized and high-molecular-weight drugs, such as lactoferrin, and thus completed the present invention.

[0011] That is, the gist of the present invention is as follows: [1] A method for producing an ion exchange resin, comprising: mixing an aqueous medium with a vinyl monomer mixture containing 0 to 90% by mass of a hydrophilic monofunctional vinyl monomer and / or a monofunctional vinyl monomer having a functional group capable of generating a hydrophilic group, and 10 to 100% by mass of a polyfunctional vinyl monomer, a radical polymerization initiator, and a porosity-generating solvent to obtain a polymerization suspension; heating the polymerization suspension to a polymerization temperature; and adding the hydrophilic monofunctional vinyl monomer and / or the monofunctional vinyl monomer having a functional group capable of generating a hydrophilic group to the polymerization suspension during the polymerization reaction, thereby introducing ion exchange groups into the resulting porous polymer particles. [2] The production method of [1], wherein the hydrophilic monofunctional vinyl monomer, the monofunctional vinyl monomer having a functional group capable of generating a hydrophilic group, and the polyfunctional vinyl monomer are (meth)acrylate monomers. [3] The manufacturing method of [1] or [2], wherein the hydrophilic monofunctional vinyl monomer and / or the monofunctional vinyl monomer having a functional group capable of generating a hydrophilic group added during the polymerization reaction is glycidyl (meth)acrylate. [4] The manufacturing method of any of [1] to [3], wherein the ion exchange group is a cation exchange group. [5] The manufacturing method of any of [1] to [4], wherein the amount of the ion exchange group introduced is 0.01 milliequivalent / mL or more and 2 milliequivalent / mL or less. [6] The manufacturing method of any of [1] to [5], which is a manufacturing method for an ion exchange resin for separating compounds used in medium-sized molecular weight drugs and polymeric molecular weight drugs. [7] A method for separating compounds used in medium-sized molecular weight drugs and polymeric molecular weight drugs, using the ion exchange resin obtained by the manufacturing method of any of [1] to [6]. [8] A method for separating lactoferrin, using the ion exchange resin obtained by the manufacturing method of any of [1] to [6]. [9] A (meth)acrylate cation exchange resin, wherein the lactoferrin adsorption capacity is 20 mg / mL or more.

[10] The cation exchange resin according to [9], wherein the amount of cation exchange groups introduced is 0.01 meq / mL or more and 2 meq / mL or less.

[0012] According to the manufacturing method of the present invention, an ion exchange resin capable of efficiently separating compounds used in medium-sized molecular weight drugs and polymeric drugs, such as lactoferrin, can be provided at low manufacturing cost. Furthermore, according to the separation method of the present invention, compounds used in medium-sized molecular weight drugs and polymeric drugs, such as lactoferrin, can be efficiently separated. Furthermore, a cation exchange resin capable of efficiently separating compounds used in polymeric drugs, such as lactoferrin, can be provided.

[0013] The present invention will be described in detail below, but the present invention is not limited to the following embodiments and can be practiced with various modifications within the scope of the gist. When the expression "to" is used in this specification, it is intended to be an expression including the numerical values ​​or physical property values ​​before and after it. In this specification, "(meth)acrylic" refers to "acrylic", "methacrylic", or both, and "(meth)acrylate" refers to "acrylate", "methacrylate", or both.

[0014] [Porous Polymer Particles] The porous polymer particles into which ion exchange groups have been introduced by the production method of the present invention are polymer particles obtained by heating a polymerization suspension (hereinafter also simply referred to as "suspension") obtained by mixing a vinyl monomer mixture containing 0 to 90% by mass of a hydrophilic monofunctional vinyl monomer and / or a monofunctional vinyl monomer having a functional group capable of generating a hydrophilic group, and 10 to 100% by mass of a polyfunctional vinyl monomer, a radical polymerization initiator, and a porosifying solvent into an aqueous medium, to a polymerization temperature, and adding the hydrophilic monofunctional vinyl monomer and / or the monofunctional vinyl monomer having a functional group capable of generating a hydrophilic group to the suspension during the ongoing polymerization reaction.

[0015] [Vinyl Monomer Mixture] The vinyl monomer mixture used as the raw material for the porous polymer particles contains 0 to 90% by mass of a hydrophilic monofunctional vinyl monomer and / or a monofunctional vinyl monomer having a functional group capable of generating a hydrophilic group, in order to suppress hydrophobic adsorption of the resulting ion exchange resin. Furthermore, in order to maintain the mechanical strength and solvent solubility resistance of the porous polymer particles, it contains 10 to 100% by mass of a multifunctional vinyl monomer. As these monofunctional vinyl monomers, (meth)acrylate-based monomers and styrene-based monomers are preferred due to their excellent mechanical strength. As vinyl monomer mixtures, (meth)acrylate-based monomer mixtures and styrene-based monomer mixtures are preferred due to their excellent mechanical strength. When separating compounds used in medium-molecule and polymer-molecule drugs, such as lactoferrin, (meth)acrylate-based monomer mixtures are more preferred due to their excellent hydrophilicity.

[0016] Here, the (meth)acrylate monomer mixture refers to a mixture containing 50% by mass or more of (meth)acrylate monomers in 100% by mass of the monomer mixture. This proportion is preferably 80% by mass or more. Furthermore, the styrene monomer mixture refers to a mixture containing 50% by mass or more of styrene monomers in 100% by mass of the monomer mixture. This proportion is preferably 80% by mass or more.

[0017] Examples of hydrophilic monofunctional vinyl monomers include unsaturated carboxylic acids such as (meth)acrylic acid, itaconic acid, and maleic acid; (meth)acrylic acid esters having hydrophilic groups such as hydroxyl groups and amino groups, such as hydroxyethyl (meth)acrylate, hydroxypropyl (meth)acrylate, diethylene glycol (meth)acrylate, triethylene glycol (meth)acrylate, tetraethylene glycol (meth)acrylate, 2,3-dihydroxypropyl (meth)acrylate, dimethylaminoethyl (meth)acrylate, diethylaminoethyl (meth)acrylate, and tetrahydrofurfuryl (meth)acrylate; (meth)acrylamide derivatives such as (meth)acrylamide, N,N-dimethyl (meth)acrylamide, and N-isopropylacrylamide; vinyl ethers such as methyl vinyl ether and ethyl vinyl ether; allyl alcohol, and vinylpyrrolidone. One type of hydrophilic monofunctional vinyl monomer may be used alone, or two or more types may be used in combination.

[0018] Examples of monofunctional vinyl monomers having a functional group capable of generating a hydrophilic group include alkyl acrylates such as methyl acrylate and ethyl acrylate; (meth)acrylic acid esters such as 3-chloro-2-hydroxypropyl(meth)acrylate, 2-chloroethyl(meth)acrylate and glycidyl(meth)acrylate; (meth)acrylamide derivatives such as N-butyl(meth)acrylamide; vinyl esters such as vinyl acetate and vinyl propionate; esters or ethers of allyl alcohol; and (meth)acrylonitrile. As monofunctional vinyl monomers having a functional group capable of generating a hydrophilic group, 3-chloro-2-hydroxypropyl(meth)acrylate, 2-chloroethyl(meth)acrylate, glycidyl(meth)acrylate, vinyl acetate and vinyl propionate are preferred because they can generate a hydroxyl group by hydrolysis, and glycidyl(meth)acrylate is more preferred because of its ease of hydrolysis. The monofunctional vinyl monomer having a functional group capable of generating a hydrophilic group may be used alone or in combination of two or more kinds.

[0019] Suitable polyfunctional vinyl monomers include aromatic polyvinyl monomers and aliphatic polyvinyl monomers. Examples of aromatic polyvinyl monomers include divinylbenzene. Examples of aliphatic polyvinyl monomers include poly(meth)acrylates of polyhydric alcohols and alkylene poly(meth)acrylamides. Specific examples include glycerol tri(meth)acrylate, allyl (meth)acrylate, trimethylolpropane tri(meth)acrylate, tetrahydroxybutane di(meth)acrylate, 1,4-cyclohexanediol di(meth)acrylate, methylene bisacrylamide, glycerol di(meth)acrylate, pentaerythritol di(meth)acrylate, ethylene glycol di(meth)acrylate, and polyethylene glycol di(meth)acrylate. One type of polyfunctional vinyl monomer may be used alone, or two or more types may be used in combination.

[0020] The hydrophilic monofunctional vinyl monomer to be added to the suspension during the polymerization reaction can be the same as the "hydrophilic monofunctional vinyl monomer" described above. Also preferred are vinyl monomers having an ion exchange group such as an amino group, a quaternary ammonium group, a carboxyl group, a phosphonic acid group, or a sulfonic acid group. The monofunctional vinyl monomer having a functional group capable of generating a hydrophilic group to be added to the suspension during the polymerization reaction can be the same as the "monofunctional vinyl monomer having a functional group capable of generating a hydrophilic group" described above. The hydrophilic monofunctional vinyl monomer and / or the monofunctional vinyl monomer having a functional group capable of generating a hydrophilic group to be added to the suspension during the polymerization reaction can be used alone or in combination of two or more.

[0021] The monofunctional vinyl monomer having a functional group capable of generating a hydrophilic group does not need to be completely miscible with the aqueous medium of the suspension. In particular, when the monofunctional vinyl monomer having a functional group capable of generating a hydrophilic group is more likely to be distributed in the porosity-forming solvent than in the aqueous medium, the added monofunctional vinyl monomer having a functional group capable of generating a hydrophilic group is concentrated in the pore-forming portion, which is preferable because the pore surface is efficiently modified.

[0022] In addition to the hydrophilic monofunctional vinyl monomer and / or the monofunctional vinyl monomer having a functional group capable of generating a hydrophilic group, a hydrophilic and crosslinkable polyfunctional vinyl monomer such as glycerol di(meth)acrylate, pentaerythritol di(meth)acrylate, ethylene glycol di(meth)acrylate, or polyethylene glycol di(meth)acrylate may be added to the suspension during the polymerization reaction. The amount of the hydrophilic and crosslinkable polyfunctional vinyl monomer used is preferably 10% by mass or less, based on 100% by mass of the total amount of vinyl monomers used in the polymerization reaction.

[0023] [Porosity-forming solvent] Examples of the porosity-forming solvent include aliphatic or aromatic hydrocarbons, esters, ketones, alcohols, and ethers, which are organic solvents that act as phase separation agents during polymerization and promote the porosity of polymer particles. Examples of the porosity-forming solvent include toluene, xylene, cyclohexane, octane, isooctane, butyl acetate, dimethyl phthalate, methyl ethyl ketone, methyl isobutyl ketone, dibutyl ether, 1-hexanol, 2-octanol, decanol, lauryl alcohol, and cyclohexanol. One type of porosity-forming solvent may be used alone, or two or more types may be used in combination.

[0024] [Aqueous Suspension Polymerization] Porous polymer particles are produced by aqueous suspension polymerization of a polymerization suspension obtained by mixing a vinyl monomer mixture, a radical polymerization initiator, and a porosifying solvent in an aqueous medium.

[0025] Aqueous suspension polymerization is a method in which an organic phase containing a vinyl monomer mixture, a radical polymerization initiator, and a porosifying agent is dispersed in an aqueous phase containing, for example, a dispersion stabilizer described below, and a polymerization reaction is carried out by, for example, heating.

[0026] [Radical Polymerization Initiator] Examples of the radical polymerization initiator include peroxide-based polymerization initiators such as di-t-hexyl peroxide, dicumyl peroxide, di-t-butyl peroxide, t-hexyl perbenzoate, t-hexylperoxyisopropyl carbonate, t-butylcumyl peroxide, diisopropylbenzene hydroperoxide, benzoyl peroxide, di-(3-methylbenzoyl) peroxide, benzoyl(3-methylbenzoyl) peroxide, di-n-propyl peroxydicarbonate, diisopropyl peroxydicarbonate, di-2-ethoxyethyl peroxydicarbonate, di-sec-butyl peroxydicarbonate, t-hexyl peroxypivalate, t-butyl peroxybenzoate, hydrogen peroxide, and persulfates; and azo-based polymerization initiators such as azobisisobutyronitrile and 2,2′-azobis(2,4-dimethylvaleronitrile). The radical polymerization initiator may be used alone or in combination of two or more kinds.

[0027] The amount of the radical polymerization initiator used may be appropriately determined depending on the type of vinyl monomer and polymerization initiator used, but is preferably 0.05 to 10 parts by mass, and more preferably 0.1 to 5 parts by mass, relative to 100 parts by mass of the total amount of vinyl monomers. When the amount of the polymerization initiator used is equal to or greater than the above lower limit, polymerization easily proceeds.

[0028] [Dispersion stabilizer] Examples of the dispersion stabilizer include gelatin, starch, polyvinyl alcohol, partial hydrolyzed polyvinyl acetate, polyacrylamide, poly(dimethyldiallyl)ammonium chloride, carboxymethyl-methylcellulose, ethyl cellulose, methyl cellulose, hydroxypropyl methylcellulose, hydroxyethyl methylcellulose, and magnesium silicate. One type of dispersion stabilizer may be used alone, or two or more types may be used in combination.

[0029] The content of the dispersion stabilizer in the aqueous phase may be appropriately set depending on the type of aqueous phase components and the desired particle size, but is preferably 0.01 to 5 mass% and more preferably 0.05 to 3 mass% relative to the total amount (100 mass%) of the aqueous phase. When the content of the dispersion stabilizer is equal to or greater than the lower limit, coalescence and crushing of droplets in the organic phase are suppressed, resulting in excellent particle size uniformity of the droplets in the organic phase. Furthermore, when the content of the dispersion stabilizer is equal to or less than the upper limit, it is preferable from the viewpoint of production costs.

[0030] In aqueous suspension polymerization, an organic phase and an aqueous phase are fed into a reactor, and vinyl monomers are polymerized while the organic phase and the aqueous phase are kept in a suspended state by stirring or other means. The bath ratio of the organic phase to the aqueous phase, in terms of the volume of the organic phase:the volume of the aqueous phase, is preferably 1:0.5 to 15, and more preferably 1:1.5 to 10, in order to achieve excellent production reproducibility.

[0031] The polymerization temperature is preferably 20 to 150° C., more preferably 40 to 100° C. When the polymerization temperature is equal to or higher than the lower limit, the polymerization easily proceeds. When the polymerization temperature is equal to or lower than the upper limit, depolymerization can be suppressed.

[0032] The polymerization time is preferably 1 to 24 hours, more preferably 2 to 12 hours. When the polymerization time is equal to or greater than the lower limit, the polymerization proceeds easily. When the polymerization time is equal to or less than the upper limit, the productivity of the resin is excellent.

[0033] The polymerization atmosphere may be air or an inert gas, but an inert gas is preferred due to its safety and reproducibility, such as nitrogen, carbon dioxide, and argon.

[0034] [Method of Adding a Hydrophilic Monofunctional Vinyl Monomer and / or a Monofunctional Vinyl Monomer Having a Functional Group Capable of Forming a Hydrophilic Group to a Suspension During a Polymerization Reaction] The porous polymer particles according to the present invention are produced by heating a polymerization suspension obtained by mixing a vinyl monomer mixture, a radical polymerization initiator, and a porosifying solvent in an aqueous medium to a polymerization temperature, and adding a hydrophilic monofunctional vinyl monomer and / or a monofunctional vinyl monomer having a functional group capable of forming a hydrophilic group to the polymerization suspension during the polymerization reaction. The amount of vinyl monomer added during the polymerization reaction (including the amount of hydrophilic and crosslinkable multifunctional vinyl monomer when a hydrophilic and crosslinkable multifunctional vinyl monomer is also added) is preferably 1 to 30% by mass, more preferably 2 to 20% by mass, based on 100% by mass of the total amount of vinyl monomer used in the polymerization reaction. The vinyl monomer to be added during the polymerization reaction is added to the reaction system all at once or in portions over a period of 0.5 to 12 hours, preferably 1 to 6 hours, after the temperature has been raised to a predetermined polymerization temperature and polymerization has commenced. When the vinyl monomer is added in portions, it is preferably added, for example, every 10 to 60 minutes. The vinyl monomer to be added during the polymerization reaction may be added as is, or may be diluted or dissolved in a solvent before being added.

[0035] [Ion Exchange Groups] The ion exchange resin of the present invention is produced by heating a polymerization suspension obtained by mixing a vinyl monomer mixture, a radical polymerization initiator, and a porous-forming solvent in an aqueous medium to a polymerization temperature, and adding a hydrophilic monofunctional vinyl monomer and / or a monofunctional vinyl monomer having a functional group capable of generating a hydrophilic group to the polymerization suspension during the polymerization reaction, thereby introducing ion exchange groups into the resulting porous polymer particles. Examples of methods for introducing ion exchange groups include a method in which a vinyl monomer having an ion exchange group such as an amino group, a quaternary ammonium group, a carboxyl group, a phosphonic acid group, or a sulfonic acid group is used as the vinyl monomer added during the polymerization reaction, or a method in which ion exchange groups are introduced into the resulting porous polymer particles by a post-reaction using a known method.

[0036] The ion exchange groups are preferably cation exchange groups, particularly from the viewpoint of use in separating cationic polymer compounds such as lactoferrin. The amount of ion exchange groups introduced is 0.01 to 2 milliequivalents / mL, preferably 0.05 to 1 milliequivalent / mL. When the amount is equal to or greater than the lower limit, the adsorption ability for compounds used in medium-sized and high-molecular-weight drugs is excellent. Furthermore, when the amount is equal to or less than the upper limit, unnecessary ion exchange of ionic compounds in the raw material after passing the liquid is suppressed, which is preferable.

[0037] [Physical properties of porous polymer particles] The shape of the porous polymer particles according to the present invention may be spherical or amorphous. However, spherical shape is preferred because it can suppress pressure loss and increase the liquid passage rate when an ion exchange resin obtained from the polymer particles is packed into a column and a liquid is passed through it, and it is excellent in productivity when separating compounds used in medium-molecule drugs and polymer drugs.

[0038] The volume average particle diameter of the porous polymer particles according to the present invention is preferably 1 to 1,000 μm, more preferably 4 to 700 μm, and even more preferably 10 to 500 μm. When the volume average particle diameter of the polymer particles is equal to or greater than the lower limit, pressure loss can be suppressed when an ion exchange resin obtained from the polymer particles is packed into a column and a liquid is passed through the column, increasing the liquid passage rate and improving productivity in separating compounds used in medium-sized molecule drugs and polymeric drug drugs. Furthermore, when the volume average particle diameter of the polymer particles is equal to or less than the upper limit, excellent column efficiency, adsorption capacity, and separation performance can be achieved.

[0039] The volume average particle diameter of the porous polymer particles can be adjusted by setting the polymerization conditions of the aqueous suspension polymerization, specifically, the type and amount of the monomer, the type and amount of the dispersion stabilizer, the stirring rotation speed, etc. After the polymerization is completed, the polymer particles may be classified using a sieve, a water sieve, an air sieve, or other method to make the volume average particle diameter of the polymer particles uniform.

[0040] The uniformity coefficient of the porous polymer particles is preferably 2.0 or less, more preferably 1.0 to 2.0, and even more preferably 1.0 to 1.6, because this can suppress pressure loss when an ion exchange resin obtained from the polymer particles is packed into a column and a liquid is passed through it. In this specification, the uniformity coefficient of the polymer particles is an index of the particle size distribution width, and is defined as the value obtained by dividing the particle size of the largest 40% of the particle sizes in the volume distribution of the polymer particles by the particle size of the largest 90% of the particle sizes.

[0041] The porous polymer particles have porosity. The specific surface area of ​​the porous polymer particles is 1 to 1000 m. ２ / g is preferred, and 10 to 800m ２ / g is more preferable. When the specific surface area of ​​the polymer particles is equal to or greater than the above lower limit, the ion exchange resin obtained from the polymer particles has excellent separation processing ability for compounds used in medium-molecular-weight drugs and polymeric drugs. Furthermore, when the specific surface area of ​​the polymer particles is equal to or less than the above upper limit, the compounds used in medium-molecular-weight drugs are less likely to be hindered from diffusing and penetrating into the pores of the polymer particles, resulting in excellent separation processing ability. In this specification, the specific surface area of ​​the polymer particles is measured by nitrogen gas adsorption method (BET method) after drying under reduced pressure. Specifically, the monolayer adsorption amount is calculated using the BET formula from the pressure change before and after nitrogen gas adsorption, and the specific surface area of ​​the polymer particles is calculated from the cross-sectional area of ​​one nitrogen gas molecule, according to ISO 9277.

[0042] The specific surface area of ​​the polymer particles can be adjusted, for example, by setting the reaction conditions for aqueous suspension polymerization.

[0043] The pore diameter of the polymer particles is preferably 1 to 1,000 nm, more preferably 2 to 500 nm, and even more preferably 3 to 200 nm. When the pore diameter of the polymer particles is equal to or greater than the above-mentioned lower limit, the frequency of contact of compounds used in medium-molecule drugs and polymeric drugs with the inner surface of the pores of the polymer particles is excellent. When the pore diameter of the polymer particles is equal to or less than the above-mentioned upper limit, the mechanical strength of the polymer particles is excellent. In this specification, the pore diameter of the polymer particles is the most frequent diameter measured by mercury intrusion porosimetry when the most frequent diameter after reduced-pressure drying is 100 nm or more, and by nitrogen gas adsorption when the most frequent diameter is less than 100 nm. Specifically, in the case of mercury intrusion porosimetry, pressure is applied to the polymer particles to cause mercury to penetrate into the pores, and the pressure value and the corresponding volume of penetrated mercury are used to calculate the pore size using the Washburn equation, assuming that the pores are cylindrical in shape, and ISO 15901-1 is applied mutatis mutandis. In the case of the nitrogen gas adsorption method, ISO 15901-2 is applied.

[0044] The pore diameter of the polymer particles can be adjusted by setting the aqueous suspension polymerization conditions, specifically, for example, the type and amount of the monomer, the type and amount of the porosifying agent, or the type and amount of the polymerization initiator.

[0045] The pore volume of the polymer particles is preferably 0.01 to 3.0 mL / g, more preferably 0.1 to 2.5 mL / g, and even more preferably 0.2 to 2.0 mL / g. When the pore volume of the polymer particles is equal to or greater than the above-mentioned lower limit, the ion exchange resin obtained from the polymer particles has excellent adsorption ability for compounds used in medium-molecular-weight drugs and polymeric drugs. When the pore volume of the polymer particles is equal to or less than the above-mentioned upper limit, the polymer particles have excellent mechanical strength. In this specification, the pore volume of the polymer particles is the modal volume measured by mercury porosimetry when the modal diameter after reduced-pressure drying is 100 nm or more, or by nitrogen gas adsorption when the modal diameter is less than 100 nm.

[0046] The pore volume of the polymer particles can be adjusted, for example, by setting the reaction conditions for aqueous suspension polymerization.

[0047] [Method for separating compounds used in medium-sized molecule drugs and polymeric drug] The method for separating compounds used in the medium-sized molecule drugs and polymeric drug of the present invention is a method for separating compounds by adsorption / desorption and / or ion exchange chromatography using an ion exchange resin obtained by the production method of the present invention.

[0048] Examples of methods for separating compounds used in the medium-sized molecule drugs and polymeric drug of the present invention include a batch processing method in which an aqueous solution containing the compound used in the medium-sized molecule drug and polymeric drug is mixed and contacted with polymer particles in a container, and a column processing method in which polymer particles are packed in a column and a liquid containing the compound used in the medium-sized molecule drug and polymeric drug is passed through. As a method for separating compounds used in medium-sized molecule drugs and polymeric drug, the column processing method is preferred because it can efficiently separate the compounds used in medium-sized molecule drugs and polymeric drug.

[0049] [Ion exchange resin] The ion exchange resin of the present invention is highly practically valuable in the industrial field because it can efficiently separate compounds used in medium-sized molecular weight drugs and high-molecular weight drugs from aqueous solutions, particularly compounds used in medium-sized molecular weight drugs and high-molecular weight drugs such as lactoferrin.

[0050] The ion exchange resin of the present invention is a (meth)acrylate-based cation exchange resin. When a (meth)acrylate-based cation exchange resin with a highly hydrophilic substrate is used, there is no need to suppress irreversible adsorption due to the hydrophobicity of compounds used in, for example, medium- and high-molecular-weight drugs, and there is no need to increase the amount of ion exchange groups introduced. As a result, ionic compounds in the raw material do not undergo excessive ion exchange with the ion exchange groups after passing the liquid, causing compositional changes. As a result, there is no pH change or other deterioration in the raw material after passing the liquid, and the quality of the raw material for food use after passing the liquid is not impaired. Furthermore, (meth)acrylate-based cation exchange resins can overcome the drawbacks of ion exchange resins derived from cross-linked polysaccharides, such as low mechanical strength and high cost, which make them difficult to use industrially, and can provide an ion exchange resin that is inexpensive and has excellent mechanical strength.

[0051] In order to efficiently separate lactoferrin, the ion exchange resin of the present invention has a lactoferrin adsorption capacity of 20 mg / mL or more, preferably 22 mg / mL or more, and even more preferably 24 mg / mL or more, as measured by the method described below.

[0052] The amount of ion exchange groups introduced into the ion exchange resin of the present invention is preferably 0.01 to 2 milliequivalents / mL, more preferably 0.05 to 1 milliequivalent / mL. When the amount is equal to or greater than the lower limit, the adsorption of compounds used in medium-sized and large molecule drugs is excellent. On the other hand, when the amount is equal to or less than the upper limit, unnecessary ion exchange of ionic compounds in the raw material after passing the liquid is suppressed, which is preferable.

[0053] The present invention will be explained in more detail below using examples, but the present invention is not limited to the description of the following examples as long as it does not deviate from the gist of the invention.

[0054] (1) Specific Surface Area The obtained polymer particles were dried under reduced pressure, and then the specific surface area was measured by the nitrogen gas adsorption method (BET method) using a specific surface area measuring device (model name "Flowsorb", manufactured by Micromeritics Co., Ltd.).

[0055] (2) Pore diameter and pore volume After drying the obtained polymer particles under reduced pressure, the pore diameter and pore volume were measured by a mercury intrusion method using an automatic porosimeter (model name "Autopore 9520", manufactured by Micromeritics Co., Ltd.), or by a nitrogen gas adsorption method using a pore distribution measuring device (model name "ASAP2400", manufactured by Micromeritics Co., Ltd.).

[0056] (3) Total Exchange Capacity The total exchange capacity of the ion exchange resin was calculated from the results of precisely weighing an amount of dried ion exchange resin equivalent to 0.5 to 1.5 mL, adding it to 250 mL of a 0.2 mol / L aqueous sodium hydroxide solution, and shaking it at 30°C for 8 hours, and then measuring the sodium hydroxide concentration in the supernatant by titration.

[0057] Comparative Example 1 Production of Porous Polymer Particles Without Adding a Hydrophilic Monofunctional Vinyl Monomer and / or a Monofunctional Vinyl Monomer Having a Functional Group Capable of Forming a Hydrophilic Group to a Suspension During Polymerization Reaction A reactor equipped with a stirrer, a condenser, a thermometer, and a nitrogen gas inlet tube was charged with 1,950 parts by mass of a 0.39% by mass aqueous solution of partially saponified polyvinyl alcohol, followed by addition of a mixture of 100 parts by mass of glycidyl methacrylate, 100 parts by mass of ethylene glycol dimethacrylate, 300 parts by mass of toluene, and 0.8 parts by mass of 2,2'-azobis(2,4-dimethylvaleronitrile), followed by stirring to form a suspension. The suspension was heated to 70°C under a nitrogen atmosphere and polymerized for 6 hours.

[0058] The resulting porous polymer particles were isolated, washed with water, extracted with methanol to remove toluene, and then dried under reduced pressure. The porous polymer particles had a specific surface area of ​​81 m ２ The physical properties were 50.6 nm / g, pore diameter 50.6 nm, and pore volume 0.32 mL / g.

[0059] Example 1 Production of Porous Polymer Particles by the Production Method of the Present Invention A reactor equipped with a stirrer, a condenser, a thermometer, and a nitrogen gas inlet tube was charged with 1,950 parts by mass of a 0.39% by mass aqueous solution of partially saponified polyvinyl alcohol, followed by the addition of a mixture of 100 parts by mass of glycidyl methacrylate, 100 parts by mass of ethylene glycol dimethacrylate, 300 parts by mass of toluene, and 0.8 parts by mass of 2,2'-azobis(2,4-dimethylvaleronitrile), followed by stirring to form a suspension. The suspension was heated to 70°C under a nitrogen atmosphere.

[0060] After raising the temperature to 70° C., 40 parts by mass of glycidyl methacrylate was added dropwise over 30 minutes after 1 hour had passed, and polymerization was continued for a further 4.5 hours (total 6 hours) to obtain porous polymer particles.

[0061] The resulting porous polymer particles were isolated, washed with water, extracted with methanol to remove toluene, and then dried under reduced pressure. The porous polymer particles had a specific surface area of ​​45 m ２ The physical properties were 45.4 nm / g, 45.4 μm, and 0.23 mL / g.

[0062] [Formation of Hydrophilic Groups] 1,440 parts by mass of a 10% by mass aqueous sulfuric acid solution was added to 240 parts by mass of the porous polymer particles obtained in Example 1 and Comparative Example 1, and the mixture was stirred to form a suspension. The temperature of this suspension was raised to 50°C and maintained for 6 hours, thereby carrying out a reaction to form diol groups, which are hydrophilic groups, through hydrolysis of the epoxy groups derived from glycidyl methacrylate.

[0063] [Introduction of Ion Exchange Groups] 886 parts by mass of a 13% by mass aqueous sodium hydroxide solution was added to the total amount of porous polymer particles obtained in Example 1 and Comparative Example 1, in which a diol group-forming reaction was performed, and the mixture was stirred to form a suspension. After heating the suspension to 50°C, 838 parts by mass of a 40% by mass aqueous sodium monochloroacetate solution was added dropwise over 1 hour and maintained for 4 hours, thereby carrying out a carboxymethyl etherification reaction to form diol groups. The resulting cation exchange resin was sieved using a 74 μm pore size sieve to remove fine particles smaller than this size, and using a 212 μm pore size sieve to remove larger particles. The total exchange capacity of the resulting cation exchange resin was 0.18 meq / mL in Example 1 and 0.15 meq / mL in Comparative Example 1.

[0064] <Measurement of Equilibrium Adsorption Amount of Lactoferrin> 2 mL of cation exchange resin obtained from the porous polymer particles from Example 1 and Comparative Example 1 was placed in an Erlenmeyer flask, and 200 mL of a solution of 1 mg / mL lactoferrin (a reagent manufactured by Tokyo Chemical Industry Co., Ltd.) dissolved in 50 mM sodium phosphate buffer (pH: 6.5) was added. This was subjected to rotational shaking at 130 rpm for 5 hours at 25°C in a thermostatic shaker. The lactoferrin concentration in the solution was determined by measuring the ultraviolet absorbance at a wavelength of 280 nm of the solution before and after shaking, and the lactoferrin equilibrium adsorption amount was calculated. The lactoferrin equilibrium adsorption amount of the obtained cation exchange resin was 52 mg / mL in Example 1 and 51 mg / mL in Comparative Example 1. The specific surface area of ​​the porous polymer particles obtained in Example 1 was 45 m ２ / g and 81m in Comparative Example 1 ２ / g, the ion exchange resins obtained showed equivalent equilibrium lactoferrin adsorption amounts. This suggests that the ion exchange resins obtained according to the present invention not only adsorb lactoferrin to the surface of the pores of porous polymer particles during ion exchange adsorption of lactoferrin, but also that by adding hydrophilic monofunctional vinyl monomers and / or monofunctional vinyl monomers having functional groups capable of generating hydrophilic groups to the suspension during the polymerization reaction of porous polymer particles, a polymer layer is formed on the surface of the pores of the porous polymer particles, and lactoferrin is three-dimensionally ion-exchanged onto the ion exchange groups introduced into this polymer layer.

[0065] Example 2 Separation of Lactoferrin from Skim Milk 200 mL of the cation exchange resin obtained in Example 1 was packed into a column with an inner diameter of 25 mm (layer height: 41 cm). After equilibration with 25 mM phosphate buffer (pH: 8.0), 25 L of bovine skim milk containing 0.21 mg / mL lactoferrin was passed through the column at a flow rate of 9 resin volumes / hour to adsorb lactoferrin onto the ion exchange resin. There was no change in pH or color of the skim milk after the adsorption procedure. After the adsorption procedure, 1.0 L of 25 mM phosphate buffer (pH: 8.0) was passed through the column to wash the column. Next, 0.8 L of 25 mM phosphate buffer (pH: 8.0) containing 1.8% by weight of sodium chloride was passed through the column at a flow rate of 1 resin volume / hour to elute impurities from the column. Lactoferrin was eluted from the ion exchange resin by passing 0.4 L of 25 mM phosphate buffer (pH 8.0) containing 5.8% by mass of sodium chloride through the resin at a flow rate of 1 resin volume / hour. HPLC analysis of each fraction determined the lactoferrin adsorption amount to be 25 mg / mL, the purity of the lactoferrin eluted fraction to be 96%, and the recovery rate to be 94%.

[0066] Comparative Example 2: Separation of lactoferrin from skim milk using a commercially available styrene-based cation exchange resin. 500 mL of commercially available styrene-based cation exchange resin WK10 (Mitsubishi Chemical Corporation, functional group: carboxyl group, total exchange capacity: >2.5 meq / mL) was packed into a 25 mm inner diameter column (layer height: 100 cm). After equilibration with 20 mM phosphate buffer (pH: 8.0), 40 L of bovine skim milk containing 0.21 mg / mL lactoferrin was passed through the column at a flow rate of 10 resin volumes / hour to adsorb lactoferrin to the ion exchange resin. The pH of the skim milk flowing out of the column at the beginning of the flow increased to approximately 10, and its color changed from the pale yellow color before flow to a pale yellow-green color. After the adsorption procedure, the column was washed with 1.5 L of 20 mM phosphate buffer (pH: 8.0) containing 0.5% by weight of sodium chloride. Next, 1.25 L of 20 mM phosphate buffer (pH: 8.0) containing 1.8% by weight of sodium chloride was passed through the column at a flow rate of 1 resin volume / hour to elute impurities.Furthermore, 1.25 L of 20 mM phosphate buffer (pH: 8.0) containing 5.8% by weight of sodium chloride was passed through the column at a flow rate of 1 resin volume / hour to elute lactoferrin from the ion exchange resin.The lactoferrin adsorption amount determined by HPLC analysis of each fraction was 15 mg / mL, the purity of the lactoferrin eluted fraction was 95%, and the recovery rate was 92%.

[0067] Example 3 Production of Porous Polymer Particles by the Production Method of the Present Invention A reactor equipped with a stirrer, a condenser, a thermometer, and a nitrogen gas inlet tube was charged with 1,942 parts by mass of a 0.39% by mass aqueous solution of partially saponified polyvinyl alcohol, followed by the addition of a mixture of 100 parts by mass of glycidyl methacrylate, 100 parts by mass of ethylene glycol dimethacrylate, 300 parts by mass of toluene, and 0.8 parts by mass of 2,2'-azobis(2,4-dimethylvaleronitrile), followed by stirring to form a suspension. The suspension was heated to 70°C under a nitrogen atmosphere.

[0068] After the temperature was raised to 70°C, 24 parts by mass of glycidyl methacrylate and 16 parts by mass of 3-chloro-2-hydroxypropyl methacrylate were added dropwise over 30 minutes after 1 hour had passed, and polymerization was continued for a further 4.5 hours (total of 6 hours) to obtain porous polymer particles.

[0069] The resulting porous polymer particles were isolated, washed with water and extracted with methanol to remove toluene, and then dried under reduced pressure.

[0070] [Introduction of ion exchange groups] 240 parts by mass of the obtained porous polymer particles were added with 240 parts by mass of sodium sulfite and 770 parts by mass of deionized water, and the mixture was stirred to form a suspension. The suspension was heated to 90°C and maintained for 6 hours to carry out a sulfonic acid group introduction reaction.

[0071] [Hydrophilic Group Formation] 1,440 parts by weight of a 10% by weight aqueous sulfuric acid solution was added to the total amount of porous polymer particles subjected to the sulfonic acid group introduction reaction, and the mixture was stirred to form a suspension. The suspension was heated to 50°C and held for 6 hours to hydrolyze the epoxy groups derived from glycidyl methacrylate, thereby forming diol groups, which are hydrophilic groups. The resulting cation exchange resin was sieved using a 74 μm pore size sieve to remove fine particles smaller than this size, and a 212 μm pore size sieve to remove larger particles. The total exchange capacity of the resulting cation exchange resin was 0.19 meq / mL.

[0072] <Measurement of lactoferrin equilibrium adsorption amount> 2 mL of the obtained cation exchange resin was placed in an Erlenmeyer flask, and 200 mL of a solution of lactoferrin (a reagent manufactured by Tokyo Chemical Industry Co., Ltd.) dissolved at a concentration of 1 mg / mL in 50 mM sodium phosphate buffer (pH: 6.5) was added. This was subjected to rotational shaking at 130 rpm for 5 hours at a temperature of 25 ° C in a thermostatic shaker. The lactoferrin concentration in the solution was determined by measuring the ultraviolet absorbance at a wavelength of 280 nm of the solution before and after shaking, and the lactoferrin equilibrium adsorption amount was calculated. The lactoferrin equilibrium adsorption amount of the obtained cation exchange resin was 45 mg / mL.

[0073] Example 4 Separation of Lactoferrin from Whey The cation exchange resin obtained in Example 3 was packed into a column with an inner diameter of 9 mm and a bed height of 20 cm. After equilibration with 20 mM phosphate buffer (pH: 7.0), 1,019 mL of bovine colostrum-derived whey containing 0.49 mg / mL lactoferrin was passed through the column at a flow rate of 10 resin volumes / hour to adsorb lactoferrin onto the ion exchange resin. After the adsorption procedure, 32 mL of 20 mM phosphate buffer (pH: 7.0) was passed through the column at a flow rate of 5 resin volumes / hour to wash the column. Next, 24.5 mL of a 1.5% by mass sodium chloride aqueous solution was passed through the column at a flow rate of 2 resin volumes / hour to elute impurities from the column. Furthermore, 24.5 mL of a 5.8% by mass sodium chloride aqueous solution was passed through the column at a flow rate of 2 resin volumes / hour to elute lactoferrin from the ion exchange resin. The amount of lactoferrin adsorbed to each fraction determined by HPLC analysis was 39 mg / mL, the purity of the lactoferrin elution fraction was 97%, and the recovery rate was 99%.

[0074] The ion exchange resin produced by the production method of the present invention can efficiently separate compounds used in medium- and high-molecular-weight drugs, such as lactoferrin, especially on an industrial scale. Furthermore, the separation method of the present invention can efficiently separate compounds used in medium- and high-molecular-weight drugs, such as lactoferrin, at low cost, and is therefore of great practical value in the industrial field.

Claims

1. A method for producing an ion exchange resin, comprising: mixing a vinyl monomer mixture containing 0 to 90% by mass of a hydrophilic monofunctional vinyl monomer and / or a monofunctional vinyl monomer having a functional group capable of generating a hydrophilic group, and 10 to 100% by mass of a polyfunctional vinyl monomer; a radical polymerization initiator; and a porosity-generating solvent into an aqueous medium to obtain a polymerization suspension; heating the polymerization suspension to a polymerization temperature; and introducing ion exchange groups into the resulting porous polymer particles by adding the hydrophilic monofunctional vinyl monomer and / or the monofunctional vinyl monomer having a functional group capable of generating a hydrophilic group to the polymerization suspension during the polymerization reaction.

2. The manufacturing method according to claim 1, wherein the hydrophilic monofunctional vinyl monomer, the monofunctional vinyl monomer having a functional group capable of generating a hydrophilic group, and the polyfunctional vinyl monomer are (meth)acrylate monomers.

3. The method according to claim 1 or 2, wherein the hydrophilic monofunctional vinyl monomer and / or the monofunctional vinyl monomer having a functional group capable of generating a hydrophilic group, which is added during the polymerization reaction, is glycidyl (meth)acrylate.

4. The method according to claim 1 or 2, wherein the ion exchange group is a cation exchange group.

5. The method of claim 1 or 2, wherein the amount of the ion exchange group introduced is 0.01 milliequivalent / mL or more and 2 milliequivalent / mL or less.

6. The method according to claim 1 or 2, which is a method for producing an ion exchange resin for separating compounds used in medium-molecular-weight and high-molecular-weight drugs.

7. A method for separating compounds used in medium- and high-molecular-weight drugs, using an ion exchange resin obtained by the manufacturing method described in claim 1 or 2.

8. A method for separating lactoferrin using an ion exchange resin obtained by the manufacturing method described in claim 1 or 2.

9. A (meth)acrylate-based cation exchange resin having a lactoferrin adsorption capacity of 20 mg / mL or more.

10. The cation exchange resin according to claim 9, wherein the amount of cation exchange groups introduced is 0.01 meq / mL or more and 2 meq / mL or less.