Separation agent

Abstract

Provided is a separation agent having a carrier and a ligand that is supported on the surface of the carrier by means of physical adsorption or chemical bonding, wherein the ratio of the particle diameter d90 to the particle diameter d10 of the separation agent is 1.00-1.38, the carrier is core-shell particles having an inorganic non-porous core and a porous shell, the porous shell contains silica gel, and the ligand is one or more selected from the group consisting of optically active polymers, optically inactive polyesters, proteins, and nucleic acids. The separation agent has good separation performance.

Description

Separating agent

[0001] The present disclosure relates to a separating agent.

[0002] BACKGROUND ART Conventionally, as a separating agent used for separation such as chromatography, a carrier having a ligand supported on its surface is known.

[0003] For example, Patent Documents 1 and 2 disclose separating agents in which a ligand selected from an optically active polymer, an optically inactive polyester, a protein, a nucleic acid, etc. is supported on core-shell type particles, and the shell of the core-shell type particles is made of a hydrolyzate of polyalkoxysiloxane.

[0004] International Publication No. WO 2013 / 176215 International Publication No. WO 2014 / 087937

[0005] In the field of separation chemistry, research and development is being conducted to improve the separation performance of separation agents. An object of the present disclosure is to provide a separation agent having good separation performance.

[0006] In order to solve the above problems, the present inventors have conducted extensive research. As a result, they have found that in a separating agent in which a ligand is supported on a carrier, the separation performance can be improved by using core-shell particles having a specific particle size distribution as the carrier. That is, the gist of the present disclosure is as follows.

[0007] [1] A separating agent comprising a carrier and a ligand supported on the surface of the carrier by physical adsorption or chemical bonding, wherein the ratio of the particle diameter d90 to the particle diameter d10 of the separating agent is 1.00 or more and 1.38 or less, the carrier is a core-shell particle having an inorganic nonporous core and a porous shell, the porous shell comprises silica gel, and the ligand is one or more selected from the group consisting of an optically active polymer, an optically inactive polyester, a protein, and a nucleic acid. [2] The separating agent according to [1], wherein the optically active polymer is one or more selected from the group consisting of a polysaccharide, a polysaccharide derivative, an optically active poly(meth)acrylic acid amide, an optically active polyamino acid, and an optically active polyamide. [3] The separating agent according to [1], wherein the optically active polymer is one or more selected from the group consisting of a polysaccharide, a polysaccharide derivative, an optically active polyamino acid, and an optically active polyamide. [4] The separating agent according to any one of [1] to [3], wherein the protein is one or more selected from the group consisting of glycoprotein, protein A, protein G, protein L, and functional variants thereof. [5] The separating agent according to any one of [1] to [4], wherein the optically inactive polyester is one or more selected from the group consisting of polybutylene terephthalate, polyethylene terephthalate, and polylactic acid. [6] The separating agent according to any one of [1] to [5], wherein the nucleic acid is one or more selected from the group consisting of DNA, DNA derivatives, RNA, and RNA derivatives, and the number of bases of the nucleic acid is 5 to 10,000. [7] The separating agent according to any one of [1] to [6], wherein the ligand is supported on the surface of the carrier by physical adsorption. [8] The separating agent according to any one of [1] to [7], wherein the separating agent is a chromatographic separating agent.

[0008] According to the present disclosure, it is possible to provide a separating agent having good separation performance. The problems and advantages of the present disclosure are not limited to those specifically described above, but also include those that will become apparent to those skilled in the art from the entire specification.

[0009] FIG. 10 is a schematic diagram of peaks for explaining elements related to calculation of resolution.

[0010] The present disclosure will be described below with reference to specific embodiments. However, each configuration and combination thereof in each embodiment is merely an example, and addition, omission, substitution, and other modifications of the configuration are possible as appropriate within the scope of the gist of the present disclosure. The present disclosure is not limited by the embodiments. Furthermore, each aspect disclosed in this specification can be combined with any other feature disclosed in this specification.

[0011] In the present disclosure, the range "X to Y" means "X or more and Y or less." Furthermore, when a numerical range expressed as "X to Y" or "X or more and Y or less" is described in stages (for example, in order of preference), the upper and lower limits of each numerical range can be combined in any way.

[0012] In the present disclosure, a description such as "one or more selected from the group consisting of X, Y, and Z" means any of X, Y, Z, a combination of X and Y, a combination of X and Z, a combination of Y and Z, or a combination of X, Y, and Z.

[0013] In the present disclosure, a description such as "X such as x1, x2, and x3" lists x1, x2, and x3 as examples of X, and does not mean that X is limited to x1, x2, x3, and the like.

[0014] <Separating Agent> One embodiment of the present disclosure is a separating agent having a carrier and a ligand supported on the surface of the carrier by physical adsorption or chemical bonding, wherein the ratio of the particle diameter d90 to the particle diameter d10 of the separating agent (d90 / d10) is 1.00 or more and 1.38 or less, the carrier is a core-shell type particle having an inorganic non-porous core and a porous shell, the porous shell contains silica gel, and the ligand is one or more selected from the group consisting of an optically active polymer, an optically inactive polyester, a protein, and a nucleic acid.

[0015] In this embodiment, from the viewpoint of obtaining better separation performance, it is preferable that the ligand is supported on the surface of the carrier by physical adsorption.

[0016] The separating agent according to this embodiment exhibits good separation performance because a specific ligand is supported on the surface of core-shell particles having a shell containing silica gel and a specific particle size distribution.

[0017] From the viewpoint of obtaining good separation performance, the d90 / d10 of the separating agent is usually 1.00 or more and 1.38 or less, preferably 1.05 or more and 1.36 or less, more preferably 1.10 or more and 1.17 or less, even more preferably 1.15 or more and 1.17 or less, and still more preferably 1.17.

[0018] The d90 / d10 of a separating agent is correlated with the variation in particle size, and the larger the particle distribution, the larger the d90 / d10 of the separating agent. Therefore, by selecting core-shell particles based on the particle size distribution, a separating agent with a desired d90 / d10 can be obtained. Since the d90 / d10 of a separating agent and the d90 / d10 of core-shell particles are almost equivalent, the d90 / d10 of the core-shell particles may be treated as the d90 / d10 of the separating agent, or the d90 / d10 of a separating agent may be treated as the d90 / d10 of the core-shell particles.

[0019] In the present disclosure, "d10" and "d90" refer to particle sizes at which the cumulative distribution is 10% and 90%, respectively, in a number-based particle size distribution determined by observing the core-shell particles with a scanning electron microscope (SEM) (for example, "SU-5000" manufactured by Hitachi High-Technologies Corporation), randomly selecting approximately 300 particles from the obtained image, and analyzing the circular particles using image analysis software (for example, "Image J" manufactured by Wyne Rasband). The d10 and d90 of core-shell particles can be determined not only by SEM observation of the core-shell particles but also by SEM observation of the separating agent.

[0020] In this disclosure, the indices used to evaluate the separation performance of a separating agent are the separation factor (α), the number of theoretical plates (N1), and the resolution (Rs) when the separating agent is used as a chromatographic separating agent. In this disclosure, a high value for one or more, preferably two, and more preferably all of the separation factor (α), the number of theoretical plates (N1), and the resolution (Rs) is considered to indicate good separation performance. Each indices is defined as follows:

[0021] Separation factor (α) α = k2 / k1 Where, k1: retention factor of the component that is more weakly retained, and is calculated by the following formula: k1 = (t1 - t0) / t0 k2: retention factor of the component that is more strongly retained, and is calculated by the following formula: k2 = (t2 - t0) / t0 t0: dead time (the time from when a substance that does not interact with the separating agent is introduced into the column until it is eluted. For convenience, the elution time of tri-tert-butylbenzene is taken as the dead time.) t1: elution time of the component that is more weakly retained t2: elution time of the component that is more strongly retained

[0022] Number of theoretical plates (N1) N1 = 16 × (tr / W) ２ In the formula, tr: retention time W: peak width

[0023] ・Separation degree (Rs)

[0024]

[0025] In the formula, t Ｒ１ , t Ｒ２ : Retention time (t Ｒ１ ≦t Ｒ２ ) (See Figure 1) W １ , W ２ : Peak width (see Figure 1) W ０．５ｈ１ , W ０．５ｈ２ : Peak width at half the peak height (half width; see Figure 1)

[0026] A resolution Rs of 1.5 or more generally indicates complete separation (baseline separation). According to a standard resolution curve for two peaks with a peak height ratio of 1 / 1, an Rs of 1.25 or more indicates that the valley depth between the two peaks is 99.4%, indicating nearly complete baseline separation. Therefore, an Rs of 1.25 or more is preferred, and an Rs of 1.5 or more is more preferred.

[0027] 1. Carrier The carrier of the separating agent according to this embodiment is a core-shell particle having an inorganic non-porous core (hereinafter sometimes simply referred to as the "core") and a porous shell (hereinafter sometimes simply referred to as the "shell").

[0028] In the present disclosure, "non-porous" refers to a material having a specific surface area (m ２ / g) is defined as A, and the surface area calculated from the core particle diameter (calculated from the particle radius r, 4πr ２ ) can be calculated from the surface area per unit mass (m ２ / g) is defined as B, this means that (A−B) / B×100 is less than 20.

[0029] In the present disclosure, "porous" means a porous material having a specific surface area of ​​10 mm2 or less as measured by the BET method. ２ / g or more.

[0030] The average particle size of the core-shell particles is not particularly limited, but is preferably 0.5 μm or more and 1000 μm or less, more preferably 1.0 μm or more and 200 μm or less, even more preferably 1.5 μm or more and 50 μm or less, still more preferably 2.0 μm or more and 10 μm or less, and particularly preferably 2.5 μm or more and 5.0 μm or less.

[0031] In the present disclosure, the term "average particle size" refers to the number-average particle size. The number-average particle size is determined as the arithmetic mean value of the particle sizes of particles measured by observing particles with a scanning electron microscope (SEM) (for example, "SU-5000" manufactured by Hitachi High-Technologies Corporation), randomly selecting about 300 particles from the obtained image, and analyzing the circular particle size using image analysis software (for example, "Image J" manufactured by Wyne Rasband).

[0032] The core material of the core-shell particles is an inorganic substance. Examples of inorganic substances include glass; metals such as titanium and zirconium; metal oxides such as titanium oxide, zirconium oxide, and silicon oxide; and clay minerals such as bentonite and mica; with glass being preferred. The shape of the core may be, for example, spherical, granular, powdery, or any irregular shape. Among these, a uniform particle shape makes it easier to ensure packing density, so the core is preferably spherical. The term "spherical" encompasses not only a perfect sphere but also an approximately spherical shape that can generally be recognized as spherical.

[0033] The average particle size of the core is not particularly limited, but is preferably 0.3 μm or more and 900 μm or less, more preferably 0.5 μm or more and 200 μm or less, even more preferably 1.0 μm or more and 50 μm or less, still more preferably 1.5 μm or more and 10 μm or less, and particularly preferably 1.7 μm or more and 3.0 μm or less.

[0034] The average particle size of the core is measured by the same method as that for the average particle size of the core-shell type particles.

[0035] The shell material constituting the core-shell particles includes, and is preferably, made of, silica gel. The silica gel may be synthesized by any synthesis method, but from the viewpoint of facilitating the production of core-shell particles, it is preferably synthesized by further hydrolyzing polyalkoxysiloxane obtained by partial hydrolysis of alkoxysilane.

[0036] The alkoxysilane is preferably a tetraalkoxysilane, such as tetramethoxysilane, tetraethoxysilane, tetrapropoxysilane, and tetrabutoxysilane, and is preferably tetraethoxysilane.

[0037] The maximum pore diameter of the shell of the core-shell particle is preferably 13 nm or more and 500 nm or less, more preferably 20 nm or more and 300 nm or less, even more preferably 30 nm or more and 200 nm or less, and even more preferably 40 nm or more and 100 nm or less. In the present disclosure, the maximum pore diameter of the shell is synonymous with the maximum pore diameter of the core-shell particle.

[0038] By setting the maximum pore size of the shell of the core-shell particle within the above range, the ligand substance can penetrate into the interior of the shell of the core-shell particle, and a sufficient amount of ligand can be supported on the core-shell particle, which is expected to ensure the interaction between the separating agent and the object to be separated and contribute to good separation of the object to be separated.

[0039] The maximum pore size of the shell of a core-shell particle is measured by mercury intrusion porosimetry. Mercury intrusion porosimetry is a method in which mercury is forced into the pores by applying pressure, and the diameter of the pores, assumed to be cylindrical, is calculated using the pressure value and the corresponding volume of invaded mercury according to the Washburn equation. The measurement of the maximum pore size of the shell by mercury intrusion porosimetry is performed in accordance with JIS R 1655:2003 (Method for testing pore size distribution of fine ceramics molded bodies by mercury intrusion porosimetry, established May 20, 2003), which is specified for ceramic molded bodies.

[0040] The average thickness of the shell of the core-shell particle is not particularly limited, but is preferably from 0.1 μm to 100 μm, more preferably from 0.2 μm to 50 μm, even more preferably from 0.3 μm to 10 μm, still more preferably from 0.5 μm to 5.0 μm, and particularly preferably from 0.5 μm to 1.0 μm.

[0041] By setting the average shell thickness to the above lower limit or more, the ligand can be sufficiently supported in the porous portion of the shell, and the substance to be separated can be sufficiently retained. On the other hand, by setting the average shell thickness to the above upper limit or less, the ratio of the shell to the entire core-shell particle is reduced, and the advantage of the core-shell particle can be more easily exhibited in that the diffusion of the solute in the porous portion of the shell is suppressed.

[0042] The average shell thickness can be determined by observing the core-shell particles with a transmission electron microscope (TEM). Specifically, a thin sample of an arbitrarily selected core-shell particle is irradiated with an electron beam accelerated at a high voltage, and the transmitted electron beam is analyzed to obtain a TEM image. Next, the maximum length of the entire particle in this TEM image minus the maximum length of the non-porous portion is divided by 2, and the resulting value is used as the average shell thickness.

[0043] The ratio of the average particle diameter of the core to the average thickness of the shell of the core-shell particle is not particularly limited, but from the viewpoint of obtaining good separation properties, it is preferably from 0.11 to 9.00, more preferably from 0.50 to 7.00, even more preferably from 1.00 to 6.00, still more preferably from 2.00 to 5.00, and particularly preferably from 3.00 to 4.00.

[0044] The specific surface area of ​​the shell is not particularly limited, but is preferably 10 m ２ / g or more 100m ２ / g or less, more preferably 15m ２ / g or more 50m ２ / g or less, more preferably 19m ２ / g or more 25m ２ / g or less. In the present disclosure, the specific surface area of ​​the shell is synonymous with the specific surface area of ​​the core-shell particles.

[0045] When the specific surface area of ​​the shell is equal to or greater than the lower limit, the pore volume is ensured, and a sufficient amount of ligand can be supported on the core-shell particles. As a result, the interaction between the separating agent and the object to be separated is ensured, and separation performance can be improved. Furthermore, when the specific surface area of ​​the shell is equal to or less than the upper limit, the shell has a large pore diameter, so the ligand can sufficiently penetrate into the pores of the shell, and a sufficient amount of ligand can be supported on the core-shell particles. As a result, the interaction between the separating agent and the object to be separated is ensured, and separation performance can be improved.

[0046] The specific surface area of ​​the shell is measured by the same mercury intrusion method as used to measure the maximum pore diameter of the shell.

[0047] The pore volume of the shell is not particularly limited, but is preferably 0.05 cm ３ / g or more 0.50cm ３ / g or less, more preferably 0.10 cm ３ / g or more 0.30cm ３ / g or less, more preferably 0.12 cm ３ / g or more 0.25cm ３ / g or less. In the present disclosure, the pore volume of the shell is synonymous with the pore volume of the core-shell particle.

[0048] When the pore volume of the shell is equal to or greater than the lower limit, the pore volume is ensured, and a sufficient amount of ligand can be supported on the core-shell particles. As a result, the interaction between the separating agent and the separation target is ensured, and separation performance can be improved. Furthermore, when the pore volume of the shell is equal to or less than the upper limit, the shell has a large pore diameter, so the ligand can sufficiently penetrate into the pores of the shell, and a sufficient amount of ligand can be supported on the core-shell particles. As a result, the interaction between the separating agent and the separation target is ensured, and separation performance can be improved.

[0049] The pore volume of the shell is measured by the same mercury intrusion method as in the measurement of the maximum pore diameter of the shell.

[0050] The method for producing the core-shell type particles is not particularly limited, but for example, the method described in JP-A-49-36396 or a method based thereon can be employed.

[0051] Specifically, first, polyalkoxysiloxane is prepared by partial hydrolysis of alkoxysilane. Next, the obtained polyalkoxysiloxane is dissolved in a solvent (e.g., ether, acetone, dichloromethane, etc.) to prepare a polyalkoxysiloxane solution. Subsequently, the obtained polyalkoxysiloxane solution is applied to a core or the core is immersed in the polyalkoxysiloxane solution, and then the solvent is removed, thereby depositing the polyalkoxysiloxane on the surface of the core. Then, polycondensation (hydrolysis) of the polyalkoxysiloxane on the core is carried out to form a porous shell. This results in core-shell particles.

[0052] The specific surface area, pore volume, and maximum pore diameter of the shell can be adjusted by the pH of the reaction solution (aqueous solution) used in polycondensation of the polyalkoxysiloxane deposited on the core. For example, the maximum pore diameter of the shell can be increased by increasing the pH of the reaction solution. In addition, the specific surface area and pore volume of the shell can be increased by decreasing the pH of the reaction solution.

[0053] The average thickness of the shell can be adjusted by the viscosity of the alkoxysilane. For example, the average thickness of the shell can be increased by using an alkoxysilane with a low viscosity.

[0054] The ratio of the average particle size of the core to the average thickness of the shell is adjusted by adjusting the average particle size of the core and the average thickness of the shell. The average thickness of the shell can be adjusted by the method described above.

[0055] As the core-shell type particles, commercially available products may be used.

[0056] The core-shell particles serving as carriers in the separating agent according to this embodiment may be surface-treated. Examples of surface treatment methods include a surface treatment method using a silane coupling agent having an amino group, such as 3-aminopropyltriethoxysilane. The amino group introduced by such surface treatment can also be used as a reactive group when chemically bonding with a ligand, as described below.

[0057] 2. Ligand In the present disclosure, the term "ligand" refers to a substance supported on the surface of a core-shell particle serving as a carrier, which exhibits physical affinity for the separation target or is capable of chiral recognition. The ligand is one or more selected from the group consisting of optically active polymers, optically inactive polyesters, proteins, and nucleic acids, and is preferably an optically active polymer, in terms of high affinity for the separation target, stability, and analytical reproducibility.

[0058] From the viewpoint of obtaining better separation performance, the ligand loading rate in the separating agent (average loading rate SR; the ratio of the total amount of ligands loaded on the carrier when the total amount of the separating agent is taken as 100% by mass) is preferably 1.0% by mass or more and 25.0% by mass or less, more preferably 2.0% by mass or more and 20.0% by mass or less, even more preferably 3.0% by mass or more and 15.0% by mass or less, still more preferably 4.0% by mass or more and 10.0% by mass or less, particularly preferably 5.0% by mass or more and 8.0% by mass or less, and especially preferably 6.0% by mass or more and 7.0% by mass or less.

[0059] By setting the ligand loading rate to the above lower limit or higher, sufficient interaction occurs between the separating agent and the object to be separated, thereby improving the separation factor. On the other hand, by setting the ligand loading rate to the above upper limit or lower, the interaction between the ligand and the object to be separated does not become excessively strong, and the migration speed of the object to be separated can be ensured, thereby improving the number of theoretical plates.

[0060] The ligand loading rate in the separating agent is expressed by the following formula (1) and is calculated from the carbon content measured by elemental analysis of the separating agent.

[0061]

[0062] In formula (1), %C ＣＳＰ is the carbon content of the entire separating agent (mass%), %C Ｓｕｐｐｏｒｔ is the carbon content (mass%) of the entire support, %C Ｌｉｇａｎｄ represents the carbon content (% by mass) of the ligand. The carbon content of each of the above-mentioned substances (separating agent, carrier, ligand) can be measured using an elemental analysis (CHN analysis) device (for example, "Flash Smart CHNS MAS Plus" manufactured by Yamato Scientific Co., Ltd.).

[0063] 2-1. Optically Active Polymer In the present disclosure, "optically active polymer" refers to a polymer having chirality, and therefore, when plane polarized light is transmitted through a solution of the optically active polymer, it exhibits optical rotation, rotating the plane of polarization. Optically active polymers are generally polymers of optically active monomers, or polymers obtained by polymerizing optically inactive monomers using an optically active polymerization catalyst. The weight-average molecular weight (Mw) of the optically active polymer is preferably 1,000 or more and 1,000,000 or less.

[0064] The weight average molecular weight (Mw) of the polymer is calculated in terms of polystyrene by size exclusion chromatography (SEC).

[0065] Examples of optically active polymers include polysaccharides, polysaccharide derivatives, optically active poly(meth)acrylic acid amides, optically active polyamino acids, and optically active polyamides, and are preferably at least one selected from the group consisting of polysaccharides, polysaccharide derivatives, optically active polyamino acids, and optically active polyamides, more preferably at least one selected from the group consisting of polysaccharides and polysaccharide derivatives, and even more preferably polysaccharides. These polymers will be described in more detail below.

[0066] In the present disclosure, "(meth)acrylic acid" is a general term for acrylic acid and methacrylic acid, i.e., acrylic acid and / or methacrylic acid. Similarly, "(meth)acryloyl" is a general term for acryloyl and methacryloyl, i.e., acryloyl and / or methacryloyl.

[0067] 2-1-1. Polysaccharides and Polysaccharide Derivatives Examples of polysaccharides include β-1,4-glucan (cellulose), α-1,4-glucan (amylose, amylopectin), α-1,6-glucan (dextran), β-1,6-glucan (pustulan), β-1,3-glucan (curdlan, schizophyllan), α-1,3-glucan, and β-1,2-glucan (Crown Examples of suitable polysaccharides include β-1,4-galactan, β-1,4-mannan, α-1,6-mannan, β-1,2-fructan (inulin), β-2,6-fructan (levan), β-1,4-xylan, β-1,3-xylan, β-1,4-chitosan, β-1,4-N-acetylchitosan (chitin), pullulan, agarose, alginic acid, α-cyclodextrin, β-cyclodextrin, γ-cyclodextrin, nigeran, and starch containing amylose. These polysaccharides are preferred because they have high optical isomer separation performance and can be easily supported on core-shell particles.

[0068] Of these, the polysaccharide is preferably one or more selected from the group consisting of cellulose, amylose, β-1,4-chitosan, chitin, β-1,4-mannan, β-1,4-xylan, inulin, curdlan, pullulan, α-cyclodextrin, β-cyclodextrin, γ-cyclodextrin, and nigeran, more preferably one or more selected from the group consisting of cellulose, amylose, pullulan, and nigeran, even more preferably one or more selected from the group consisting of cellulose and amylose, and particularly preferably cellulose, in terms of ease of purification or easy availability of highly purified products.

[0069] The number-average degree of polymerization of the polysaccharide (the average number of pyranose rings or furanose rings contained in one molecule) is preferably from 5 to 1,000, more preferably from 10 to 500. Polysaccharides having a number-average degree of polymerization equal to or less than the above upper limit are preferred in terms of ease of handling.

[0070] Polysaccharide derivatives are chemically modified polysaccharides. The polysaccharide in the polysaccharide derivative has the same meaning as above. The polysaccharide derivative is not particularly limited, but preferred examples include polysaccharide ester derivatives and polysaccharide carbamate derivatives. These polysaccharide derivatives are known to have high optical resolution as chiral separating agents. Furthermore, these polysaccharide derivatives are preferred in that they have high optical isomer separation performance and are easily supported on core-shell particles.

[0071] Any derivative can be used as the polysaccharide ester derivative and polysaccharide carbamate derivative. Examples of such derivatives include polysaccharide derivatives in which the hydroxyl groups of cellulose are modified with phenylcarbamoyl groups, aralkylcarbamoyl groups, or benzoate groups, in which some or all of the hydrogen atoms on the aromatic ring are substituted with halogeno groups (fluoro or chloro groups), alkyl groups, or alkoxy groups; and polysaccharide derivatives in which the hydroxyl groups of cellulose are modified with phenylcarbamoyl groups, aralkylcarbamoyl groups, or benzoate groups, in which some or all of the hydrogen atoms on the aromatic ring are substituted with halogeno groups (fluoro or chloro groups), alkyl groups, or alkoxy groups. In the polysaccharide derivatives, the substituents substituting the hydrogen atoms on the aromatic ring are preferably halogeno groups only, halogeno groups and alkyl groups, or alkyl groups only. The halogeno group is preferably a chloro group. The alkyl group is preferably an alkyl group having 1 to 3 carbon atoms, more preferably a methyl group.

[0072] From the viewpoint of obtaining better separation performance, the loading rate of polysaccharides or polysaccharide derivatives in the separating agent (the ratio of the total amount of polysaccharides or polysaccharide derivatives loaded on the carrier when the total amount of the separating agent is taken as 100% by mass) is preferably 1.0% by mass or more and 25.0% by mass or less, more preferably 2.0% by mass or more and 20.0% by mass or less, even more preferably 3.0% by mass or more and 15.0% by mass or less, still more preferably 4.0% by mass or more and 10.0% by mass or less, and particularly preferably 5.0% by mass or more and 8.0% by mass or less.

[0073] Examples of a method for supporting a polysaccharide or a polysaccharide derivative on core-shell particles by physical adsorption include a method in which the core-shell particles are immersed in a solution containing the polysaccharide or the polysaccharide derivative, and then the solvent is removed by evaporation, for example, by drying under reduced pressure.

[0074] Methods for chemically bonding a polysaccharide or polysaccharide derivative to a core-shell particle include a method of forming a chemical bond between the core-shell particle and the polysaccharide or polysaccharide derivative; a method of chemically bonding the core-shell particle and the polysaccharide or polysaccharide derivative via a third component (spacer); and a method of physically adsorbing the polysaccharide or polysaccharide derivative onto a carrier and crosslinking the polysaccharide or polysaccharide derivative on the carrier.

[0075] A method for forming a chemical bond between a core-shell particle and a polysaccharide or a polysaccharide derivative (hereinafter abbreviated as "reductive amination method") can be, for example, the method described in JP-A-07-138301. Specifically, this method involves forming a Schiff base between the reducing end of the polysaccharide and a surface-treated support, for example by reacting an aldehyde group with an amino group, and then reducing this to a secondary amine in the presence of a reducing agent (see ELISABETH KALLIN et al., Glycoconjugate J (1986) 3, 311-319), thereby supporting the polysaccharide on the support through a chemical bond, and then derivatizing the polysaccharide as needed.

[0076] A method for chemically bonding a core-shell particle and a polysaccharide or polysaccharide derivative via a third component (spacer) can be, for example, the method described in the examples of JP-A No. 2002-148247. Specifically, this method involves copolymerizing a polymerizable polysaccharide derivative having a polymerizable group such as a vinyl group introduced therein with a core-shell particle having a polymerizable group such as a vinyl group introduced therein in the presence of a third component (polymerizable monomer) having a polymerizable group such as a vinyl group.

[0077] A method for physically adsorbing a polysaccharide or polysaccharide derivative onto a support and crosslinking the polysaccharide or polysaccharide derivative on the support can be, for example, the method described in the Examples of JP-A-11-510193. Specifically, this method involves supporting a polysaccharide derivative on core-shell particles by coating, and then irradiating the polysaccharide derivative on the core-shell particles with light from an immersion mercury lamp to photochemically crosslink the polysaccharide derivative. The method for crosslinking the polysaccharide or polysaccharide derivative is not limited to the above method, but may also be used, for example, a method for crosslinking by a reaction induced by irradiation with radiation such as gamma rays or electromagnetic waves such as microwaves; or a method for crosslinking by a radical reaction using a radical initiator, etc.

[0078] As a method for crosslinking a polysaccharide or polysaccharide derivative coated on core-shell particles by a reaction induced by irradiation with radiation such as gamma rays or electromagnetic waves such as microwaves, for example, the method described in JP-A-2004-167343 can be used. This method involves coating a polysaccharide or polysaccharide derivative on surface-treated core-shell particles, and then irradiating the polysaccharide or polysaccharide derivative on the core-shell particles with gamma rays, thereby crosslinking the polysaccharide or polysaccharide derivative.

[0079] Other methods for chemically bonding core-shell particles to polysaccharides or polysaccharide derivatives include, for example, coating the core-shell particles with a polysaccharide derivative obtained by introducing alkoxysilyl groups into some of the hydroxyl groups or amino groups of the polysaccharide or polysaccharide derivative, and then carrying out a reaction such as polycondensation of the alkoxysilyl groups in an arbitrary solvent.

[0080] 2-1-2. Optically active poly(meth)acrylic acid amides Optically active poly(meth)acrylic acid amides (hereinafter, may be simply referred to as "poly(meth)acrylic acid amides") include those obtained by polymerizing optically active (meth)acrylic acid amides represented by the following formula (I):

[0081] The polymerization method for poly(meth)acrylic acid amide is not particularly limited, but a preferred example is radical polymerization using a radical polymerization initiator such as AIBN (azobisisobutyronitrile) in the presence of a Lewis acid catalyst.

[0082] The Lewis acid used in the radical polymerization is preferably a metal Lewis acid, which is a metal salt, such as scandium triflate, yttrium triflate, magnesium bromide, hafnium chloride, ytterbium triflate, and lutetium triflate.

[0083] In the polymerization, if the (meth)acrylic acid amide is liquid under the reaction conditions, the polymerization reaction can be carried out even under solvent-free conditions. If the (meth)acrylic acid amide is solid under the reaction conditions, the polymerization is carried out in a reaction solvent. The reaction solvent is not particularly limited as long as it is an organic solvent that can dissolve the (meth)acrylic acid amide and does not have a radical scavenging effect, and examples thereof include tetrahydrofuran, chloroform, and methanol.

[0084] Other polymerization conditions can be selected as appropriate by referring to WO 02 / 088204.

[0085]

[0086] In the formula, R １ , R ２ , and R ３ are each a hydrogen atom, a monovalent hydrocarbon group having 1 to 30 carbon atoms, or a monovalent atomic group containing a heteroatom; R ４ is a hydrogen atom or a monovalent hydrocarbon group having 1 to 30 carbon atoms; R ５ is a hydrogen atom or a methyl group.

[0087] R １ , R ２ , and R ３ are preferably each a hydrogen atom, an alkyl group having 1 to 6 carbon atoms, an aryl group, an aralkyl group, a carboalkoxy group, a carbamoyl group, an amino-substituted alkyl group, an amino group, an alkoxy-substituted alkyl group, an alkoxy group, or a silyl group.

[0088] R ４ is preferably a hydrogen atom, an alkyl group having 1 to 6 carbon atoms, an aryl group, or an aralkyl group, and particularly preferably a hydrogen atom.

[0089] From the viewpoint of obtaining better separation performance, the loading rate of poly(meth)acrylic acid amide in the separating agent (the ratio of the amount of poly(meth)acrylic acid amide supported on the carrier when the total amount of the separating agent is taken as 100% by mass) is preferably 1.0% by mass or more and 25.0% by mass or less, more preferably 2.0% by mass or more and 20.0% by mass or less, even more preferably 3.0% by mass or more and 15.0% by mass or less, still more preferably 4.0% by mass or more and 10.0% by mass or less, and particularly preferably 5.0% by mass or more and 8.0% by mass or less.

[0090] Examples of a method for supporting poly(meth)acrylic acid amide on core-shell particles by physical adsorption include a method in which the core-shell particles are immersed in a solution containing poly(meth)acrylic acid amide, and then the solvent is removed by evaporation by drying under reduced pressure, etc. Examples of the solvent for the solution include chloroform and dichloromethane, from the viewpoint of the solubility of poly(meth)acrylic acid amide.

[0091] The method of making poly(meth)acrylic acid amide supported on core-shell type particles by chemical bonding can be, for example, the method of introducing reactive functional groups into core-shell type particles, and making this reactive functional group react with the amide group that poly(meth)acrylic acid amide has; and the method of introducing reactive functional groups into core-shell type particles, and making the functional group that can react with this reactive functional group react with poly(meth)acrylic acid amide, and making both reactive functional groups react.The former method is carried out by, for example, carrying out surface treatment using a silane coupling agent or the like that has epoxy group, introducing epoxy group into core-shell type particles, and making this epoxy group react with the amide group that poly(meth)acrylic acid amide has.

[0092] Other methods for supporting poly(meth)acrylic acid amide on core-shell type particles by chemical bonding include, for example, a method in which a polymerizable functional group is introduced into the core-shell type particles by surface treatment using a silane coupling agent or the like having a polymerizable functional group such as a vinyl group and a (meth)acryloyl group, and a polymerizable functional group is also introduced into the poly(meth)acrylic acid amide by reaction with an isocyanate ester or the like (see JP 2006-177795 A), and the polymerizable functional groups of both are copolymerized.

[0093] 2-1-3. Optically active polyamino acids Optically active polyamino acids (hereinafter sometimes simply referred to as "polyamino acids") do not include proteins, which will be described later. Examples of polyamino acids include polymers represented by the following formula (II). Such polyamino acids can be synthesized, for example, by the method described in JP-A-60-193538.

[0094]

[0095] In formula (II), n １ is 5 or more; R ６ are each independently an alkyl group having 1 to 5 carbon atoms, a phenyl group, an aralkyl group having 7 to 12 carbon atoms, or a heterocyclic group having 3 to 12 carbon atoms, and the alkyl group, the phenyl group, the aralkyl group, and the heterocyclic group may have a substituent; R ７ are each independently an alkyl group having 1 to 5 carbon atoms.

[0096] n １ is preferably 100 or less, more preferably 10 or more and 40 or less.

[0097] R ６ When the alkyl group, phenyl group, aralkyl group, and heterocyclic group represented by the formula (I) have a substituent, examples of the substituent include a hydroxy group, a carboxy group, a mercapto group, an amino group, and a methylthio group. Note that the number of carbon atoms does not include the number of carbon atoms contained in the substituent.

[0098] R ６Examples of the heterocyclic group represented by the formula (I) include groups in which one hydrogen atom has been removed from a heterocycle such as 5-pyrazolone, pyrazole, triazole, oxazolone, isoxazolone, barbituric acid, pyridone, pyridine, rhodanine, pyrazolidinedione, pyrazolopyridone, and Meldrum's acid; and groups in which a ring such as an aromatic ring or a heterocycle is further condensed with the heterocyclic group.

[0099] R ７ The alkyl group represented by the formula (I) is preferably a methyl group or an ethyl group.

[0100] Polyamino acids are polymers of amino acids and amino acid derivatives. Examples of amino acids include alanine, valine, leucine, phenylalanine, proline, glutamic acid, and aspartic acid. Examples of amino acid derivatives include benzyl aspartate, methyl glutamate, benzyl glutamate, carbobenzoxylysine, carbobenzoxyornithine, acetyltyrosine, and benzylserine.

[0101] From the viewpoint of obtaining better separation performance, the loading rate of polyamino acid in the separating agent (the ratio of the amount of polyamino acid loaded on the carrier when the total amount of the separating agent is taken as 100% by mass) is preferably 1.0% by mass or more and 25.0% by mass or less, more preferably 2.0% by mass or more and 20.0% by mass or less, even more preferably 3.0% by mass or more and 15.0% by mass or less, still more preferably 4.0% by mass or more and 10.0% by mass or less, and particularly preferably 5.0% by mass or more and 8.0% by mass or less.

[0102] A method for supporting a polyamino acid on core-shell particles by physical adsorption includes immersing the core-shell particles in a solution containing the polyamino acid, and then distilling off the solvent by drying under reduced pressure, etc. Examples of the solvent for the solution include dimethylformamide and dioxane, from the viewpoint of the solubility of the polyamino acid.

[0103] The method of supporting polyamino acid on core-shell type particles by chemical bonding includes the method of introducing reactive functional group into core-shell type particles, and reacting this reactive functional group with the amino group of polyamino acid; and the method of introducing reactive functional group into core-shell type particles, and introducing functional group that can react with this reactive functional group into polyamino acid, and reacting both reactive functional groups;.The reactive functional group that is bonded to core-shell type particles can be, for example, epoxy group that reacts with the amino group of polyamino acid.The method of introducing reactive functional group into core-shell type particles can be, for example, surface-treating core-shell type particles with silane coupling agent having epoxy group.

[0104] Other methods for supporting polyamino acids on core-shell particles by chemical bonding include, for example, a method in which a surface treatment is performed using a silane coupling agent having a polymerizable functional group such as a vinyl group and a (meth)acryloyl group to introduce a polymerizable functional group into the core-shell particles, and a method in which a compound having a group reactive with the amino group of the polyamino acid and a polymerizable functional group, such as acrylic acid chloride, glycidyl methacrylate, and chloromethylstyrene, is reacted with the polyamino acid to introduce a polymerizable functional group into the polyamino acid, and the polymerizable functional groups of both are copolymerized.

[0105] 2-1-4. Optically Active Polyamides Optically active polyamides (hereinafter sometimes simply referred to as "polyamides") include polymers derived from optically active amino acids, each of which has one optically active amino acid residue in the main chain of the repeating unit.

[0106] Monomers used in the synthesis of polymers derived from optically active amino acids include N-substituted amino acids, which are optically active dicarboxylic acids, and diamines. Examples of N-substituted amino acids include N-substituted glutamic acid and N-substituted aspartic acid. Preferred diamines include aromatic diamines such as 4,4'-diaminodiphenylmethane and 1,3-phenylenediamine.

[0107] An example of a method for synthesizing a polymer derived from an optically active amino acid will be described. As described above, a polymer derived from an optically active amino acid is synthesized by polymerizing an N-substituted amino acid, which is an optically active dicarboxylic acid, with a diamine. A more specific example of the synthesis method will be described below, but the synthesis method for a polymer derived from an optically active amino acid is not limited to this example, and various reaction conditions such as the monomer, condensing agent, reaction scale, reaction temperature, and reaction time, as well as the purification method, can be changed as appropriate.

[0108] 7.5 cm of a solution containing N-methylpyrrolidone and pyridine in a volume ratio of 4:1 and containing lithium chloride at a concentration of 4% by mass ３ To this, 3 mmol of benzoyl-L-glutamic acid, 3 mmol of 4,4'-diaminodiphenylmethane, and 6 mmol of triphenyl phosphite are added, and the reaction is carried out for 3 hours at 80°C while stirring. Thereafter, the reaction mixture is added dropwise to methanol, and the precipitated polymer is recovered by filtration and dried under reduced pressure to obtain a polymer derived from an optically active amino acid.

[0109] The method for synthesizing the polymer derived from an optically active amino acid is not limited to the above-mentioned method, and any method other than the above-mentioned method may be used.

[0110] The polymer derived from an optically active amino acid has a residue of an N-substituted amino acid, which is an optically active dicarboxylic acid, and therefore has a D- or L-optical recognition site in the main chain. Therefore, the separating agent according to this embodiment exhibits optical resolution ability in an embodiment having a polymer derived from an optically active amino acid as a ligand.

[0111] Examples of optically active polyamides include polymers derived from optically active amino acids, as well as polymers represented by the following formula (III) and polymers represented by the following formula (IV).

[0112]

[0113]

[0114] In formula (III) and formula (IV), R ８ and R ９are each independently an alkylene group having 2 to 20 carbon atoms which may have a branched structure, a group having one or more aromatic groups having 6 to 10 carbon atoms, or a group having one or more alicyclic groups having 3 to 10 carbon atoms; ２ and n ３ are each an integer between 50 and 100,000.

[0115] R ８ and R ９ Examples of the alkylene group represented by the formula (I) include alkylene groups having 4, 6, 8, or 10 carbon atoms, and preferred examples include a tetramethylene group, a hexamethylene group, an octamethylene group, and an octamethylene group.

[0116] R ８ and R ９ Examples of the group having one or more aromatic groups represented by the formula (I) include a phenylene group and an oxydiphenylene group.

[0117] R ８ and R ９ Examples of the group having one or more alicyclic groups represented by the formula (I) include groups containing a cycloalkylene group such as a cyclohexanediyl group and a cyclobutanediyl group.

[0118] The polymer represented by formula (III) and the polymer represented by formula (IV) can be synthesized by the method described in JP-B-4-77737. As raw materials for these polymers, (+)- or (-)-trans-stilbenediamine and dicarboxylic acid, or derivatives thereof can be used.

[0119] As the dicarboxylic acid, a compound represented by the following formula (V) can be used: HOOC-R １０ -COOH (V) In formula (V), R １０ is R in formula (III). ８ and R in formula (IV) ９ is synonymous with.

[0120] The polymer represented by formula (III) or the polymer represented by formula (IV) can be synthesized by any method such as a known polyamide synthesis method or a method similar thereto.

[0121] From the viewpoint of obtaining better separation performance, the polyamide loading rate in the separating agent (the ratio of the amount of polyamide supported on the carrier when the total amount of the separating agent is taken as 100% by mass) is preferably 1.0% by mass or more and 25.0% by mass or less, more preferably 2.0% by mass or more and 20.0% by mass or less, even more preferably 3.0% by mass or more and 15.0% by mass or less, still more preferably 4.0% by mass or more and 10.0% by mass or less, and particularly preferably 5.0% by mass or more and 8.0% by mass or less.

[0122] A method for supporting an optically active polyamide on core-shell particles by physical adsorption includes immersing the core-shell particles in a solution containing the optically active polyamide, and then distilling off the solvent by drying under reduced pressure, etc. From the viewpoint of the solubility of the optically active polyamide, examples of the solvent for the solution include hexafluoroisopropanol, dimethylformamide, and dichloromethane.

[0123] The method of making polyamide supported on core-shell particle by chemical bonding can be, for example, the method of introducing reactive functional group into core-shell particle, and making this reactive functional group react with the amide group that polyamide has; and the method of introducing reactive functional group into core-shell particle, and making the functional group that can react with this reactive functional group react with polyamide, and making both reactive functional groups react.The former method is carried out by, for example, carrying out surface treatment using a silane coupling agent etc. that has epoxy group, introducing epoxy group into core-shell particle, and making this epoxy group react with the amide group that polyamide has.

[0124] Other methods for supporting polyamide on core-shell particles by chemical bonding include, for example, a method in which a surface treatment is performed using a silane coupling agent or the like having a polymerizable functional group such as a vinyl group or a (meth)acryloyl group to introduce a polymerizable functional group into the core-shell particles, and a method in which a polymerizable functional group is also introduced into the polyamide by a reaction with an isocyanate ester or the like (see JP 2006-177795 A), and the polymerizable functional groups of both are copolymerized.

[0125] 2-2. Optically Inactive Polyesters Examples of optically inactive polyesters include polyethylene terephthalate, polybutylene terephthalate, polyethylene-polybutylene terephthalate, polytrimethylene terephthalate, polyethylene naphthalate, polybutylene naphthalate, polylactic acid, polyglycolic acid, poly(ε-caprolactone), and poly(oxycarbonyloxy-1,4-phenylene-2,2-isopropylidene-1,4-phenylene) (polycarbonate of bisphenol A).

[0126] Of these, the optically inactive polyester is preferably one or more selected from the group consisting of polyethylene terephthalate, polybutylene terephthalate, polylactic acid, and polyglycolic acid, in terms of higher affinity for the separation target, and higher stability and analytical reproducibility, and more preferably one or more selected from the group consisting of polybutylene terephthalate, polyethylene terephthalate, and polylactic acid.

[0127] The weight average molecular weight (Mw) of the optically inactive polyester is preferably 10,000 or more and 1,000,000 or less, more preferably 20,000 or more and 200,000 or less, from the viewpoints of strength of physical adsorption to the carrier and ease of handling due to increased viscosity of the solvent in which the polymer is dissolved.

[0128] From the viewpoint of obtaining better separation performance, the polyester loading rate in the separating agent (the ratio of the amount of polyester loaded on the carrier when the total amount of the separating agent is taken as 100% by mass) is preferably 1.0% by mass or more and 25.0% by mass or less, more preferably 2.0% by mass or more and 20.0% by mass or less, even more preferably 3.0% by mass or more and 15.0% by mass or less, still more preferably 4.0% by mass or more and 10.0% by mass or less, and particularly preferably 5.0% by mass or more and 8.0% by mass or less.

[0129] The separation material containing the optically inactive polyester as a ligand is expected to be useful for separating high molecular weight molecules such as proteins.

[0130] An example of a method for supporting an optically inactive polyester on core-shell particles by physical adsorption is a method in which the core-shell particles are immersed in a solution containing an optically inactive polyester, and then the solvent is removed by drying under reduced pressure or the like.

[0131] Examples of methods for supporting polyester on core-shell particles by chemical bonding include a method in which reactive functional groups are introduced into the core-shell particles, functional groups reactive with the reactive functional groups are introduced into the polyester, and the two reactive functional groups are reacted. For example, such a method is carried out by surface treatment using a silane coupling agent or the like having an epoxy group to introduce epoxy groups into the core-shell particles, and chemical treatment using a polyamine to introduce amino groups into the polyester, and then reacting the epoxy groups introduced into the core-shell particles with the amino groups introduced into the polyester in vacuum or air.

[0132] Examples of polyamines for introducing amino groups into polyesters include (poly)alkylene polyamines having 2 to 8 carbon atoms. Specific examples of (poly)alkylene polyamines having 2 to 8 carbon atoms include ethylene diamine, propylene diamine, butylene diamine, diethylene triamine, triethylene tetramine, and tetraethylene pentamine. Preferably, the polyamine is one or more selected from the group consisting of ethylene diamine, diethylene triamine, triethylene tetramine, and tetraethylene pentamine, with diethylene triamine being particularly preferred. In particular, the use of a polyamine containing at least three amino groups improves the introduction of amino groups into the polyester. Furthermore, low molecular weight amines are preferred because they are easily removed after chemical treatment.

[0133] An example of a chemical treatment method using a polyamine is a method in which polyester is heat-treated in a solution in which a polyamine is dissolved in an organic solvent or a mixed solvent of water and an organic solvent.

[0134] 2-3. Proteins Proteins are preferably used as ligands for separation agents for separating proteins such as antibodies. As proteins, substances that have affinity for the target to be separated can be used. The molecular mass of the protein is preferably 3 kDa or more and 300 kDa or less, more preferably 30 kDa or more and 150 kDa or less.

[0135] Specific examples of proteins used as ligands include glycoproteins, protein A, protein G, protein L, albumin, and functional variants thereof, and preferably one or more selected from the group consisting of glycoproteins, protein A, protein G, protein L, and functional variants thereof. These have high selectivity when used to separate antibodies, and are therefore particularly preferred as ligands for separation agents for separating proteins such as antibodies. When antibody separation is the main purpose, ligands that can specifically bind to a portion of immunoglobulin are also preferred.

[0136] The glycoprotein that can be used is α1-acid glycoprotein. Origins of α1-acid glycoprotein include mammals such as humans, cows, and rabbits, and birds such as chickens, swans, and turkeys. Of these, specific examples of preferred α1-acid glycoproteins include human α1-acid glycoprotein (hereinafter also referred to as h-AGP) and chicken AGP (hereinafter also referred to as c-AGP).

[0137] Human α1-acid glycoprotein (h-AGP) can be a commercially available product (for example, manufactured by Merck Co., Ltd.) If necessary, the commercially available product may be purified by high performance liquid chromatography before immobilization.

[0138] Chicken α1-acid glycoprotein (c-AGP) can be obtained by separating ovomucoid and chicken α1-acid glycoprotein (c-AGP) from crude chicken ovomucoid by liquid chromatography using a cation exchange carrier (e.g., "SP Sepharose" manufactured by Merck) using a stepwise elution method with ammonium acetate buffer (pH 4.6). At this time, a fraction containing c-AGP may be collected and further purified using an ion exchange chromatography carrier such as SP Sepharose.

[0139] Human α1-acid glycoprotein is a glycoprotein consisting of 183 amino acid residues and five sugar chains with a molecular weight of approximately 41,000 to 43,000. Chicken α1-acid glycoprotein has the same amino acid residues and sugar chains as human α1-acid glycoprotein, but its molecular weight is approximately 30,000.

[0140] Examples of albumin include ovalbumin and human serum albumin (molecular weight approximately 66 kDa).

[0141] The functional variant refers to a protein that has at least one alteration in its native amino acid sequence and has at least one function associated with the native sequence. The native sequence includes an amino acid sequence that originally occurs in nature. Alterations of the native amino acid sequence can include, for example, substitution of one or more amino acids with other amino acids; deletion of one or more amino acids; addition of one or more amino acids; and combinations thereof (e.g., a combination of substitution of one or more amino acids with other amino acids, addition of one or more amino acids, and deletion of one or more amino acids from the native sequence).

[0142] Functional variants can also include fragments or domains of a protein. The amino acid sequence of a functional variant can be 70% or more identical, 75% or more identical, 80% or more identical, 85% or more identical, 90% or more identical, 95% or more identical, or 98% or more identical to the native amino acid sequence.

[0143] Alternatively, cellobiohydrolases (CBHs) known as cellobiohydrolases I and II shown below can also be used as proteins. Cellobiohydrolase I (CBH I) and cellobiohydrolase II (CBH II) are known to be major enzymes that account for the majority of cellulases (80% or more together, with small amounts of endoglucanases, β-glucosidases, and the like also included). Methods for producing these proteins include expressing large amounts of the desired cellobiohydrolase in host cells using genetic recombination techniques.

[0144] From the viewpoint of obtaining better separation performance, the protein loading rate in the separating agent (the ratio of the amount of protein loaded on the carrier when the total amount of the separating agent is taken as 100% by mass) is preferably 1.0% by mass or more and 25.0% by mass or less, more preferably 2.0% by mass or more and 20.0% by mass or less, even more preferably 3.0% by mass or more and 15.0% by mass or less, still more preferably 4.0% by mass or more and 10.0% by mass or less, and particularly preferably 5.0% by mass or more and 8.0% by mass or less.

[0145] A method for supporting a protein on core-shell particles by physical adsorption includes immersing the core-shell particles in a solution containing the protein, and then evaporating the solvent by drying under reduced pressure, etc. Examples of the solvent for the solution include hexane and chloroform, from the viewpoint of the solubility of the protein.

[0146] Methods for chemically bonding a protein to a core-shell particle typically include, but are not limited to, a method in which reactive functional groups are introduced into the core-shell particle and then this reactive functional group is reacted directly with a functional group possessed by the protein; or a method in which reactive functional groups are introduced into the core-shell particle and then the core-shell particle and the protein are chemically bonded via a compound having one or more functional groups capable of reacting with the reactive functional groups bonded to the core-shell particle and one or more functional groups capable of reacting with a functional group possessed by the protein (hereinafter, such compounds are collectively referred to as a "spacer").

[0147] An example of the former method is a method in which, when a ligand having an amino group (e.g., Protein A) is carried out, the core-shell particles are surface-treated with a silane coupling agent or the like having a functional group such as an epoxy group that can react with an amino group, thereby introducing a functional group that reacts with an amino group into the core-shell particles, and then this functional group is allowed to react directly with Protein A.

[0148] Examples of the latter method include a method in which an amino acid (amine carboxylic acid) is used as a spacer, and the amino group of the amino acid is reacted with an epoxy group introduced into the core-shell particle, and then the carboxyl group of the amino acid is reacted with an amino group of Protein A; and a method in which a diamine or diol and a diglycidyl compound such as (poly)ethylene glycol diglycidyl ether are successively used as spacers, and the epoxy group introduced into the core-shell particle is reacted with one end group of the diamine or diol, and then the other end group of the diamine or diol is reacted with one epoxy group of the diglycidyl compound, thereby introducing an epoxy group into the core-shell particle and reacting this epoxy group with Protein A.

[0149] Examples of the diamine spacer include aliphatic diamines such as tetramethylenediamine and hexamethylenediamine. Examples of the diol spacer include aliphatic diols such as propylene glycol, butanediol, diethylene glycol, and triethylene glycol, and polyalkylene glycols such as polyethylene glycol.

[0150] The spacer preferably has a linear structure, taking into consideration the reactivity with the ligand and the steric hindrance with the core-shell particles when supporting by chemical bonding. A separating agent in which the core-shell particles and the ligand are bonded via a spacer with a linear structure is less likely to suffer from problems caused by steric hindrance, such as the prevention of the formation of affinity bonds between the protein ligand and the target to be separated (e.g., antibody), and therefore is more likely to ensure good separation performance.

[0151] Alternatively, the protein can be reacted with carbonic acid disuccinimide (N,N'-disuccinimidyl carbonate: DSC) and then reacted with core-shell particles to which amino groups have been introduced using 3-aminopropyltriethoxysilane or the like, thereby allowing the protein to be supported on the core-shell particles by chemical bonding.

[0152] 2-4. Nucleic Acids Nucleic acids include, but are not limited to, DNA, DNA derivatives, RNA, RNA derivatives, oligonucleotides, and oligonucleotide derivatives. Of these, the nucleic acid is preferably one or more selected from the group consisting of DNA, DNA derivatives, RNA, and RNA derivatives, in terms of higher affinity for the target to be separated, as well as higher stability and analytical reproducibility. The number of bases in the nucleic acid is not particularly limited, but is preferably 5 to 10,000.

[0153] The DNA and RNA may be of natural type or artificial type, but in consideration of the stability as a separation agent, it is preferable that they are of structurally stable artificial type.

[0154] Artificial DNA and RNA can have base sequences that do not exist in natural forms. In order to prevent thymine dimerization, it is preferable that the artificial nucleic acid does not have adjacent thymines.

[0155] The number of bases in the artificial nucleic acid is preferably 50 or more and 200 or less, and more preferably 80 or more and 120 or less, in terms of enabling efficient synthesis.

[0156] The nucleic acid may be modified with a protecting group to improve durability as a separating agent. Specifically, the nucleic acid may be modified with a protecting group in one or both of the 5'-position hydroxy group and the 3'-position hydroxy group. Examples of the protecting group include a phosphate ester group, an acyl group, an alkoxycarbonyl group, a benzyl group, a substituted benzyl group, and an allyl group. The DNA derivative, the RNA derivative, and the oligonucleotide derivative are DNA, RNA, and nucleotides, respectively, in which one or both of the 5'-position hydroxy group and the 3'-position hydroxy group are modified with a protecting group.

[0157] From the viewpoint of obtaining better separation performance, the nucleic acid loading rate in the separation agent (the ratio of the amount of nucleic acid loaded on the carrier when the total amount of the separation agent is taken as 100% by mass) is preferably 1.0% by mass or more and 25.0% by mass or less, more preferably 2.0% by mass or more and 20.0% by mass or less, even more preferably 3.0% by mass or more and 15.0% by mass or less, still more preferably 4.0% by mass or more and 10.0% by mass or less, and particularly preferably 5.0% by mass or more and 8.0% by mass or less.

[0158] By setting the nucleic acid loading rate to the above lower limit or higher, the nucleic acid can be stably present in the separating agent, resulting in sufficient separation performance.By setting the nucleic acid loading rate to the above upper limit or lower, it is possible to prevent free nucleic acid that was not fully loaded on the core-shell particles when preparing the separating agent from remaining in the separating agent.Since free nucleic acid may have a negative effect on separation performance, reducing the amount of remaining free nucleic acid can ensure good separation performance.

[0159] Examples of methods for supporting nucleic acids on core-shell particles by physical adsorption include adding nucleic acids to a suspension prepared by dispersing core-shell particles in distilled water, followed by drying. When adding nucleic acids to the suspension, the nucleic acids may be added directly to the suspension, or an aqueous solution prepared by dissolving the nucleic acids in distilled water may be added to the suspension, or a portion of the nucleic acids may be added directly to the suspension, and the remainder of the nucleic acids may be added to the suspension in distilled water.

[0160] To make the core-shell particles carry the nucleic acid by chemical bonding, for example, there is mentioned a method of immobilizing the nucleic acid on the carrier via chitosan and the amino group of the chitosan, as described in JP-A-2010-259405.

[0161] In this method, core-shell particles are first subjected to a vapor deposition reaction with an aminosilane such as 3-aminopropyltriethoxysilane, followed by a heat treatment. The aminosilane-treated core-shell particles are then immersed in a glutaraldehyde solution, washed, and air-dried. The glutaraldehyde-treated core-shell particles are then immersed in a chitosan solution and washed with ultrapure water. This allows the aldehyde groups of the glutaraldehyde to react with the amino groups of the chitosan, introducing numerous amino groups onto the surface of the core-shell particles and increasing the surface area for binding nucleic acids. The chitosan-treated core-shell particles are then immersed in a glutaraldehyde solution, washed, and air-dried. This introduces aldehyde groups into the core-shell particles. An avidin solution is dropped onto the core-shell particles and allowed to stand, whereby the aldehyde groups introduced into the core-shell particles react with the amino groups of the avidin, immobilizing the avidin to the core-shell particles via chitosan. A biotin-labeled nucleic acid solution is then dropped onto the avidin-immobilized core-shell particles for reaction, whereby the nucleic acid is immobilized to the core-shell particles via chemical bonds.

[0162] 3. Applications The separating material according to this embodiment can be suitably used as a packing material for columns used in chromatography, such as liquid chromatography, supercritical fluid chromatography, gas chromatography, capillary electrochromatography (CEC), and micellar electrokinetic chromatography (MEKC). The separating material according to this embodiment can also be used as a packing material for columns used in electrophoresis, such as capillary zone electrophoresis (CZE).

[0163] The separating agent according to this embodiment can be used as a separating agent for optical isomers by selecting an optically active ligand as the ligand. The separating agent according to this embodiment can be used as a separating agent for affinity chromatography by selecting an optically inactive ligand as the ligand.

[0164] The present disclosure will be explained in more detail below using examples, but the present disclosure is not limited to the following examples as long as it does not deviate from the gist of the disclosure.

[0165] Synthesis Example 1 Synthesis of Cellulose Tris(3-chloro-4-methylphenylcarbamate) Cellulose tris(3-chloro-4-methylphenylcarbamate) was obtained as a white solid (1) by reacting 3-chloro-4-methylphenylisocyanate with cellulose in pyridine solvent under the conditions described in B. Chankvetadze, E. Yashima, Y. Okamoto, J. Chromatogr. A 670 (1994) 39.

[0166] Synthesis Example 2: Synthesis of amylose tris(3-chloro-4-methylphenylcarbamate) Amylose tris(3-chloro-4-methylphenylcarbamate) was obtained as a white solid (2) by reacting 3-chloro-4-methylphenyl isocyanate with amylose in pyridine solvent under the conditions described in B. Chankvetadze, E. Yashima, Y. Okamoto, J. Chromatogr. A 670 (1994) 39.

[0167] Example 1 0.6 g of the white solid (1) obtained in Synthesis Example 1 was dissolved in 4.8 mL of acetone. The obtained solution was uniformly applied to 5.4 g of core-shell particles A shown in Table 1, and the acetone was removed by distillation under reduced pressure to obtain a separating agent in which cellulose tris(3-chloro-4-methylphenylcarbamate) was supported on the core-shell particles A by physical adsorption.

[0168] Example 2 3.3 g of the separating agent obtained in Example 1 was suspended in 500 mL of acetonitrile / water = 60 / 40 (vol / vol) to obtain a suspension. This suspension was irradiated with light from a high-pressure mercury lamp (Philips HPK-125W, quartz-encapsulated) for 10 minutes. The resulting precipitate was collected by filtration, washed with tetrahydrofuran, and then dried to obtain 3.1 g of a separating agent in which cellulose tris(3-chloro-4-methylphenylcarbamate) was supported by a core-shell particle A via chemical bonding.

[0169] Example 3 A separating agent in which cellulose tris(3-chloro-4-methylphenylcarbamate) was supported by core-shell particles B by physical adsorption was obtained in the same manner as in Example 1, except that the carrier was changed to core-shell particles B shown in Table 1.

[0170] Example 4 A separating agent in which cellulose tris(3-chloro-4-methylphenylcarbamate) was supported by a core-shell particle B via a chemical bond was obtained in the same manner as in Example 2, except that the separating agent was changed to 3.1 g of the separating agent obtained in Example 3.

[0171] Example 5 0.6 g of the white solid (2) obtained in Synthesis Example 2 was dissolved in 4.8 mL of tetrahydrofuran. The resulting solution was uniformly applied to 5.4 g of core-shell particles A shown in Table 1, and the tetrahydrofuran was distilled off under reduced pressure to obtain a separating agent in which amylose tris(3-chloro-4-methylphenylcarbamate) was supported on the core-shell particles A by physical adsorption.

[0172] Example 6 3.0 g of the separating agent obtained in Example 5 was suspended in 500 mL of acetonitrile / water = 60 / 40 (vol / vol) to obtain a suspension. This suspension was irradiated with light from a high-pressure mercury lamp (Philips HPK-125W, quartz-encapsulated) for 18 minutes. The resulting precipitate was collected by filtration, washed with tetrahydrofuran, and then dried to obtain 2.6 g of a separating agent in which amylose tris(3-chloro-4-methylphenylcarbamate) was supported by a chemical bond on core-shell particles A.

[0173] [d10 and d90 of separating agent] The separating agent was observed with a scanning electron microscope (SEM), and approximately 300 particles were randomly selected from the obtained image and subjected to circular particle analysis using image analysis software. The d10 and d90 of the separating agent were calculated from the obtained particle size distribution.

[0174] [Average particle size of core-shell type particles and separating agent] The separating agent or core-shell type particles were observed with a scanning electron microscope (SEM), and approximately 300 particles were randomly selected from the obtained image and analyzed for circular particle size using image analysis software. The arithmetic mean of the particle sizes measured for each particle was taken as the average particle size of the separating agent or core-shell type particles. The results are shown in Tables 1 and 2.

[0175] [Maximum pore diameter, specific surface area, and pore volume of the shell of core-shell particles, and maximum pore diameter of the separating agent] The maximum pore diameter, specific surface area, and pore volume of the shell of the core-shell particles, and the maximum pore diameter of the separating agent were measured by mercury intrusion porosimetry in accordance with JIS R 1655:2003 (Method for testing pore size distribution of molded bodies of fine ceramics by mercury intrusion porosimetry, established on May 20, 2003). The results are shown in Tables 1 and 2.

[0176] [Ligand Loading Rate] The ligand loading rate was calculated by the following formula (1): % C ＣＳＰ is the carbon content of the entire separating agent (mass%), %C Ｓｕｐｐｏｒｔ is the carbon content (mass%) of the entire support, %C Ｌｉｇａｎｄ represents the carbon content (mass%) of the ligand. The carbon content of each object (separating agent, carrier, and ligand) was measured using an elemental analysis (CHN analysis) device ("Flash Smart CHNS MAS Plus" manufactured by Yamato Scientific Co., Ltd.). The results are shown in Table 2.

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[0178] [Evaluation of Separation Performance] The separating agent obtained in the examples was pressurized and packed into a stainless steel column of φ0.21 cm×L25 cm by a slurry packing method to prepare a chromatography column.

[0179] The prepared chromatography column was connected to a liquid chromatograph ("X-LC" manufactured by JASCO Corporation). Using this liquid chromatograph under the analytical conditions described above, optical resolution of the racemate of trans-stilbene oxide (TSO) was performed to evaluate the separation performance of the separating agent. The retention factors (k1, k2), separation factor (α), number of theoretical plates (N1), and resolution (Rs) are shown in Table 3.

[0180] (Analysis conditions) Mobile phase: n-Hexane / 2-Propanol = 9 / 1 (v / v) Flow rate: 0.21 mL / min Temperature: 25°C Detection: 254 nm Conditioning time: 10 minutes

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Claims

1. A separating agent having a carrier and a ligand supported on the surface of the carrier by physical adsorption or chemical bonding, wherein the ratio of particle diameter d90 to particle diameter d10 of the separating agent is 1.00 or more and 1.38 or less, the carrier is a core-shell type particle having an inorganic non-porous core and a porous shell, the porous shell comprises silica gel, and the ligand is one or more selected from the group consisting of optically active polymers, optically inactive polyesters, proteins, and nucleic acids.

2. The separating agent according to claim 1, wherein the optically active polymer is one or more selected from the group consisting of polysaccharides, polysaccharide derivatives, optically active poly(meth)acrylic acid amides, optically active polyamino acids, and optically active polyamides.

3. The separating agent according to claim 1, wherein the optically active polymer is at least one selected from the group consisting of polysaccharides, polysaccharide derivatives, optically active polyamino acids, and optically active polyamides.

4. The separation agent according to any one of claims 1 to 3, wherein the protein is one or more selected from the group consisting of glycoprotein, protein A, protein G, protein L, and functional mutants thereof.

5. The separating agent according to any one of claims 1 to 4, wherein the optically inactive polyester is at least one selected from the group consisting of polybutylene terephthalate, polyethylene terephthalate, and polylactic acid.

6. The separating agent according to any one of claims 1 to 5, wherein the nucleic acid is one or more types selected from the group consisting of DNA, DNA derivatives, RNA, and RNA derivatives, and the number of bases of the nucleic acid is 5 to 10,000.

7. The separating agent according to any one of claims 1 to 6, wherein the ligand is supported on the surface of the carrier by physical adsorption.

8. The separation material according to any one of claims 1 to 7, which is a chromatographic separation material.

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