Method of manufacturing column filled with separation agent

Abstract

To provide a method of manufacturing a column filled with a separation agent, which comprises a new step for introducing a functional group into an insoluble carrier.SOLUTION: The above problem is solved by providing a method of manufacturing a column filled with a separation agent, the method comprising a step of introducing a functional group into an insoluble carrier by feeding a reagent capable of introducing a functional group into the insoluble carrier to the column loaded with the insoluble carrier having pores.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] The present invention relates to a method for producing a separating agent, and more particularly to a method for producing a separating agent by introducing functional groups into an insoluble carrier having fine pores. [Background technology]

[0002] The separation material is packed in a column and used for the analysis and separation of amino acids, peptides, proteins, nucleic acids, sugars, etc. In particular, in recent years, it has been widely used for the analysis and separation of bioparticles used in the medical field, such as viruses, virus-like particles, and viral vectors.

[0003] Patent Document 1 discloses that an anion exchange column is prepared by stirring an insoluble carrier and a reagent capable of introducing a functional group in a flask, introducing the functional group into the insoluble carrier, and then filling the insoluble carrier into a column. [Prior art documents] [Patent documents]

[0004] [Patent Document 1] Japanese Patent Application Publication No. 2018-197294 Summary of the Invention [Problem to be solved by the invention]

[0005] The present invention provides a method for producing a column packed with a separating material, which includes a novel step for introducing functional groups into an insoluble carrier. [Means for solving the problem]

[0006] In order to solve the above problems, the inventors conducted extensive research and discovered that functional groups can be introduced into an insoluble carrier by pumping a reagent capable of introducing functional groups into a column filled with an insoluble carrier having fine pores.

[0007] That is, the present invention includes the following aspects [1] to [5]. [1] A method for producing a column packed with a separating agent, comprising the step of introducing functional groups into an insoluble carrier by feeding a reagent capable of introducing functional groups into the insoluble carrier into a column packed with an insoluble carrier having fine pores. [2] The insoluble carrier has hydroxy groups on the surfaces of the pores, and the step of introducing functional groups into the insoluble carrier is a step of introducing a reagent capable of introducing a highly nucleophilic functional group into the insoluble carrier into a column packed with the insoluble carrier, thereby introducing the highly nucleophilic functional group into the hydroxy group; and a method for producing a column according to [1], further comprising at least a step of feeding a reagent capable of introducing an activated functional group into the insoluble carrier into the column packed with the insoluble carrier, thereby introducing the activated functional group into the highly nucleophilic functional group. [3] The reagent capable of introducing an activated functional group into the insoluble support has an electrophilic reactive group, and the step of introducing the activating functional group into the highly nucleophilic functional group is carried out by reacting the highly nucleophilic functional group with the electrophilic reactive group. [4] The method for producing a column according to [2] or [3], wherein the highly nucleophilic functional group is an amino group. [5] The method for producing a column according to [3] or [4], wherein the electrophilic reactive group is an epoxy group. [Effects of the Invention]

[0008] According to the present invention, a reagent capable of introducing a functional group is delivered to a column packed with an insoluble carrier having fine pores, thereby introducing the functional group into the insoluble carrier. By including a step of delivering a reagent capable of introducing a functional group to a column packed with an insoluble carrier having fine pores, a column packed with a separating agent can be produced.

[0009] Furthermore, according to one aspect of the present invention, when a highly nucleophilic functional group is introduced into a hydroxy group having a pore surface on which a reagent capable of introducing a functional group is used, and then a separation material-packed column is prepared by introducing a reagent capable of introducing a functional group, the performance of the separation material is significantly improved. [Brief explanation of the drawings]

[0010] [Figure 1] 1 shows the results of measuring the dynamic adsorption capacity for bovine serum albumin using a column packed with a separating agent prepared by the method of the present disclosure. DETAILED DESCRIPTION OF THE INVENTION

[0011] The present invention will be described in detail below.

[0012] The insoluble carrier may be any substance that can be introduced with a functional group and is insoluble in the solution or solvent used for adsorption onto the separating agent to be produced and elution from the separating agent. For example, the insoluble carrier may be a carrier derived from an inorganic substance such as zirconia, zeolite, silica, or coated silica, a carrier derived from a natural organic polymer such as cellulose, agarose, or dextran, or a carrier derived from a synthetic organic polymer such as polyacrylic acid, polystyrene, polyacrylamide, polymethacrylamide, polymethacrylate, vinyl polymer, or other copolymer.

[0013] The insoluble porous carrier, which is the raw material for the separating agent, may be prepared by crushing a material with interconnected pores such as a monolith into particles, or may be produced directly by emulsion polymerization, etc. Commercially available chromatography carriers such as POROS (manufactured by Thermo Fisher Scientific) and Dulocore (manufactured by Tantti) may also be used as is.

[0014] The pores of the insoluble carrier may be closed pores or interconnected pores, and the pores may include both closed pores and interconnected pores.

[0015] Unlike closed pores, which transport the substance to be analyzed / separated by diffusion, open pores transport the substance by convection, allowing for high flow rate processing. Therefore, it is more preferable that the pores of the insoluble carrier are open pores.

[0016] The pore diameter is preferably 10 nm or more and 8 μm or less, and more preferably 50 nm or more and 2 μm or less.

[0017] In the present invention, it has been found that functional groups can be introduced into an insoluble carrier by feeding a reagent capable of introducing functional groups into the insoluble carrier into a column packed with the insoluble carrier having fine pores.

[0018] We found that by pumping a reagent capable of introducing functional groups into a column packed with this insoluble carrier, the reagent is pumped from a location in the column where liquid can easily pass through, and therefore the reaction to introduce functional groups proceeds efficiently from the site that acts on column adsorption.

[0019] Alternatively, a reagent capable of introducing functional groups into an insoluble carrier may be introduced into a column packed with an insoluble carrier having fine pores, thereby introducing functional groups onto the surface of the pores. It is believed that introducing functional groups onto the surface of the pores of the insoluble carrier can increase the surface area of ​​the sites that act on column adsorption. It is believed that the increased surface area improves column performance.

[0020] The liquid delivery rate is calculated as column volume per hour (CV) and is 0.1 CV / min to 3.0 CV / min, preferably 0.2 CV / min to 1.0 CV / min, and more preferably 0.3 CV / min to 0.7 CV / min.

[0021] Examples of functional groups that can be introduced into the column include N-hydroxysuccinimide (NHS) activated ester groups, hydroxy groups, epoxy groups, amino groups, maleimide groups, haloacetyl groups, tresyl groups, formyl groups, and haloacetamide groups (iodoacetamide groups, bromoacetamide groups, etc.).

[0022] The introduction of the functional group into the insoluble carrier can be carried out by the same method as in the production of conventional particulate fillers, for example, by introducing the functional group into the pores of the insoluble carrier under appropriate reaction conditions.

[0023] When the surface of the pores has hydroxy groups, epoxy groups can be introduced into the pores by reacting with an epoxidizing agent such as epihalohydrins (e.g., epichlorohydrin, epibromohydrin), diglycidyl ethers (e.g., ethanediol diglycidyl ether, butanediol diglycidyl ether, hexanediol diglycidyl ether, neopentyl glycol diglycidyl ether, polyethylene glycol diglycidyl ether, bisphenol A diglycidyl ether), or alkyl diene diepoxides (e.g., 1,7-octadiene diepoxide). Among these, polyethylene glycol diglycidyl ethers with a repeating unit of -CHO- of 1 to 10 are preferred. Epoxy groups can be introduced by reaction in an aqueous solution of an inorganic base (e.g., sodium hydroxide or potassium carbonate) or in an aqueous solution of an inorganic base (e.g., sodium hydroxide or potassium carbonate) containing sodium borohydride.

[0024] When introducing amino groups as functional groups into the pores, they may be introduced using an aminating reagent such as ethylenediamine, 1,3-propanediamine, putrescine, cadaverine, hexamethylenediamine, 1,8-octanediamine, 1,10-decanediamine, 1,12-dodecanediamine, o-phenylenediamine, m-phenylenediamine, p-phenylenediamine, trans-1,4-cyclohexanediamine, trans-1,2-cyclohexanediamine, 4,4'-diaminodiphenyl ether, 4,4'-ethylenedianiline, spermidine, spermine, bis(hexamethylene)triamine, N,N'-bis(2-aminoethyl)-1,3-propanediamine, N,N'-bis(3-aminopropyl)ethylenediamine, triethylene glycol bis(2-aminoethyl)ether, tris(2-aminoethyl)amine, tris(3-aminopropyl)amine, or tris(4-aminophenyl)amine.

[0025] When the surface of the pores has an epoxy group, a hydroxy group, or an amino group, for example, N-(ε-maleimidocaproic acid) hydrazide, N-(ε-maleimidopropionic acid) hydrazide, 4-[4-N-maleimidophenyl]acetic acid hydrazide, 2-aminomaleimide, 3-aminomaleimide, 4-aminomaleimide, 6-aminomaleimide, 1-(4-aminophenyl)maleimide, 1-(3-aminophenyl)maleimide, 4-(maleimido)phenyl isocyanate, 2-maleimidoacetic acid, 3-maleimidopropionic acid, 4-maleimidobutyric acid, 6-maleimidohexa The maleimide group, an activated functional group, can be introduced using maleimidation reagents such as succinimidyl-4-(maleimidomethyl)cyclohexane-1-carbonyl-(6-aminohexanoic acid), N-(α-maleimidoacetoxy)succinimide ester, (m-maleimidobenzoyl)N-hydroxysuccinimide ester, succinimidyl-4-(maleimidomethyl)cyclohexane-1-carboxylic acid, (p-maleimidobenzoyl)N-hydroxysuccinimide ester, and (m-maleimidobenzoyl)N-hydroxysuccinimide ester.

[0026] When introducing a haloacetyl group as a functional group into an epoxy group, a hydroxy group, or an amino group on the surface of a pore, the introduction can be carried out using a haloacetylating reagent such as chloroacetic acid, bromoacetic acid, iodoacetic acid, chloroacetic acid chloride, bromoacetic acid chloride, bromoacetic acid bromide, chloroacetic acid anhydride, bromoacetic acid anhydride, iodoacetic acid anhydride, 2-(iodoacetamido)acetic acid-N-hydroxysuccinimide ester, 3-(bromoacetamido)propionic acid-N-hydroxysuccinimide ester, or 4-(iodoacetyl)aminobenzoic acid-N-hydroxysuccinimide ester. Another method for introducing a haloacetyl group involves reacting an ω-alkenylalkane glycidyl ether with an epoxy group, hydroxy group, or amino group on the surface of a pore, and then halogenating the ω-alkenyl moiety with a halogenating agent. Examples of the ω-alkenylalkane glycidyl ether include allyl glycidyl ether, 3-butenyl glycidyl ether, and 4-pentenyl glycidyl ether, and examples of the halogenating agent include N-chlorosuccinimide, N-bromosuccinimide, and N-iodosuccinimide.

[0027] When carboxyl groups exist on the surface of the pores, functional groups can be introduced to the carboxyl groups using a condensation agent and an additive. Examples of the condensation agent include 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC), dicyclohexylcarbodiamide, and carbonyldiimidazole. Examples of the additive include N-hydroxysuccinimide (NHS), 4-nitrophenol, and 1-hydroxybenzotriazole.

[0028] Water is typically used as the reaction solvent for introducing functional groups into an insoluble support. However, other solvents may also be used: hydrocarbons such as n-hexane, benzene, and xylene; ethers such as tetrahydrofuran and dioxane; halogenated hydrocarbons such as chloroform and chlorobenzene; alcohols such as ethanol and methyl cellosolve; ketones such as acetone and methyl isobutyl ketone; or 1,4-dioxane, dimethylformamide, and dimethyl sulfoxide. These solvents may be used alone or in a mixed solvent system. Although no particular catalyst is required for introducing functional groups onto the surface of an insoluble support, hydroxides or carbonates of alkali or alkaline earth metals, such as sodium hydroxide and potassium carbonate, may also be used as catalysts.

[0029] In one aspect of the present invention, the insoluble carrier has hydroxy groups on the surfaces of the pores, and the step of introducing functional groups into the insoluble carrier comprises the steps of: feeding a reagent capable of introducing highly nucleophilic functional groups into the insoluble carrier into a column packed with the insoluble carrier; and introducing the highly nucleophilic functional groups into the hydroxy groups; The present invention also discloses a method for producing a column packed with a separation material, which includes at least a step of feeding a reagent capable of introducing an activated functional group into the insoluble carrier into the column packed with the insoluble carrier, thereby introducing the activated functional group into the highly nucleophilic functional group.

[0030] The highly nucleophilic functional group may be, for example, a thiol group or an amino group (amines). The highly nucleophilic functional group may preferably be an amino group.

[0031] Reagents capable of introducing highly nucleophilic functional groups into an insoluble support may be, for example, ethylenediamine, 1,4-butanediamine, 1,6-hexanediamine, 1,8-octanediamine, 1,10-decanediamine, tris(2-aminoethyl)amine, tris(3-aminopropyl)amine, 1,2-ethanedithiol, 1,4-butanedithiol, 1,6-hexanedithiol, 1,8-octanedithiol, or 1,10-decanedithiol.

[0032] The activated functional group may be, for example, a cyano group, a halogenated silane group, a halogenated phosphorus (pentavalent) group, a halogenated phosphorus (trivalent) group, a halogenated boron group, a group having a boron-oxygen bond, a halogenated metal group, a heterosilyl group, an imino group, a halogenated acyl group, a disulfide group, a carboxylic acid anhydride, an imidoyl halide group, an aziridine group, a thiol group, an amino group, a secondary amine, a tertiary amine, a phosphate group, a sulfo group, a carboxyl group, a hydroxy group, an epoxy group, a haloacetyl group, a carbonyl group (a ketone group, an aldehyde group, a carboxylic acid amide group, a carboxylic acid ester group), or a halogenated alkyl group.

[0033] The step of introducing an activating functional group into the highly nucleophilic functional group may be carried out by reacting it with an electrophilic reactive group.

[0034] The reagent capable of introducing an activated functional group into the insoluble support may contain an electrophilic reactive group.

[0035] Examples of reagents capable of introducing activated functional groups into an insoluble support include carboxylation reagents such as 2-mercaptoacetic acid, 3-mercaptopropionic acid, 4-mercaptobutyric acid, 6-mercaptobutyric acid, glycine, 3-aminopropionic acid, and 4-aminobutyric acid, secondary amines such as dimethylaminoethanol and diethylaminoethanol, tertiary amines such as choline chloride, glycidyltrimethylammonium chloride, iodomethyltrimethylammonium iodide, (3-bromopropyl)trimethylammonium bromide, (2-chloroethyl)trimethylammonium chloride, 1,3-propane sultone, 1,4-butane sultone, sodium iodoacetate, sodium bromoacetate, sodium chloroacetate, and sodium fluoroacetate.

[0036] The electrophilic reactive group may be, for example, an epoxy group, a haloacetyl group, a carbonyl group (a ketone group, an aldehyde group, a carboxylic acid amide group, a carboxylic acid ester group), or a halogenated alkyl group. The electrophilic reactive group may preferably be an epoxy group. Examples of reagents having an electrophilic reactive group and capable of introducing an activated functional group include glycidyltrimethylammonium chloride, iodomethyltrimethylammonium iodide, (3-bromopropyl)trimethylammonium bromide, (2-chloroethyl)trimethylammonium chloride, 1,3-propane sultone, 1,4-butane sultone, sodium iodoacetate, sodium bromoacetate, sodium chloroacetate, and sodium fluoroacetate.

[0037] The activated functional group may be an ion exchange group. The ion exchange group can be introduced by reacting it with a reagent capable of reacting with the activated functional group. For example, cation exchange groups can be introduced by introducing sulfo groups using sultones such as 1,4-butane sultone, or by introducing carboxy groups using carboxylation reagents such as sodium chloroacetate, 2-mercaptoacetic acid, 3-mercaptopropionic acid, 4-mercaptobutyric acid, 6-mercaptobutyric acid, glycine, 3-aminopropionic acid, or 4-aminobutyric acid. Anion exchange groups can be introduced by introducing secondary amines using dimethylaminoethanol, diethylaminoethanol, or the like, or by introducing tertiary amines using choline chloride, glycidyl trimethylammonium chloride, or the like.

[0038] After introducing a functional group into the insoluble carrier, for example, an affinity ligand may be covalently bonded to the insoluble carrier via the functional group to produce a separating agent.

[0039] The affinity ligand to be covalently bonded to the aforementioned functional group may be any ligand that corresponds to the substance to be analyzed / separated. Examples of such ligands include Protein A derived from Staphylococcus aureus, Protein G derived from group G hemolytic streptococci, Protein L derived from Finegoldia bacteria, and Fc receptors (see, for example, WO2015 / 041303) when the substance to be analyzed / separated is immunoglobulin (antibody), and AAV receptors (see, for example, WO2021 / 106882) when the substance to be analyzed / separated is adeno-associated virus (AAV).

[0040] Examples of buffer solutions used for covalently bonding the affinity ligand to the functional group introduced into the insoluble carrier include acetate buffer, phosphate buffer, MES (2-morpholinoethanesulfonic acid) buffer, HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) buffer, Tris buffer, and borate buffer. The reaction temperature during immobilization may be appropriately set within the range of 5°C to 50°C, taking into consideration the reactivity of the functional group and the stability of the ligand, and is preferably within the range of 10°C to 40°C.

[0041] The adsorption performance of the prepared separation material can be evaluated by adsorbing and desorbing a biological sample onto a column packed with the separation material. One example is a method in which a bovine serum albumin (BSA) solution is added to a separation material column containing quaternary ammonium groups, allowing the BSA to adsorb to the separation material, and then the BSA is eluted by switching to an elution buffer. The amount of BSA adsorbed to the column can be calculated by monitoring the absorbance at 280 nm, which is a convenient and advantageous feature. [Example]

[0042] Each embodiment of the present invention will be described in more detail using examples, but the present invention is not limited to these examples in any way.

[0043] Example 1 Preparation of a column packed with a separating agent (Part 1) (1) Column filling with insoluble carrier (1-1) An insoluble support having pores and hydroxyl groups on the surfaces of the pores (a porous hydrophilic vinyl polymer for liquid chromatography manufactured by Tosoh Corporation) was subjected to suction filtration on a glass filter to obtain a suction dry gel (weight: 0.8 g). The insoluble support was then washed four times with pure water to prepare a slurry.

[0044] (1-2) The slurry prepared in (1-1) was poured into a measuring cylinder and left for 6 hours to allow the gel to settle. The supernatant was carefully removed without disturbing the gel at the bottom, and then pure water was added. The gel was resuspended by repeatedly inverting the measuring cylinder while holding the top of the cylinder.

[0045] (1-3) The resuspension procedure in (1-2) was repeated three more times, and then the amount of pure water was adjusted so that the resin volume accounted for 50% of the total volume, and the resin was resuspended (hereinafter referred to as "50% slurry").

[0046] (1-4) The 50% slurry prepared in (1-3) was loaded into a column container with a diameter of 4.6 mm and a length of 75 mm (column volume: 1.25 mL). Using a diaphragm pump, purified water was pumped through the column while gradually increasing the flow rate: 1.5 mL / min for 5 minutes, 3.0 mL / min for 5 minutes, and 5.0 mL / min for 15 minutes, to fill the column with the insoluble carrier.

[0047] (2) Introduction of trimethylammonium groups (hereinafter also referred to as "Q groups") into the hydroxyl groups of the insoluble carrier (2-1) A diaphragm pump was connected to a column packed with the insoluble carrier prepared in (1) so that a mixed solution of 32 mmol of 80% (w / v) aqueous glycidyltrimethylammonium chloride solution, which is a reagent having an epoxy group as an electrophilic reactive group and a Q group as an activated functional group, and 32 mmol of 5 mol / L sodium hydroxide could be circulated. The column was then placed in a column oven set at 45°C, and the mixed solution was pumped at a flow rate of 0.5 mL / min for 4 hours, thereby introducing the Q group into the hydroxyl groups on the surface of the pores of the insoluble carrier.

[0048] (2-2) After the introduction was completed, the column oven was set to 35°C, and 32 mmol of 6 mol / L hydrochloric acid was pumped at a flow rate of 0.5 mL / min for 1 hour.

[0049] (2-3) After hydrochloric acid was pumped, the glycidyltrimethylammonium chloride aqueous solution and hydrochloric acid were discharged, and the column was washed by pumping pure water at room temperature at a flow rate of 0.5 mL / min for 16 hours, to prepare a column packed with a separation agent (an insoluble carrier with Q groups introduced).

[0050] Example 2: Preparation of a column packed with a separating agent (part 2) (1) Introduction of amino groups into the hydroxyl groups of the insoluble carrier (1-1) A diaphragm pump was connected to a column packed with the insoluble carrier prepared in Example 1(1) so that a mixed solution of 0.4 mL of pure water, 0.4 mL of dimethyl sulfoxide, 24 mmol of epichlorohydrin, and 24 mmol of NaOH could be circulated. The column was then placed in a column oven set at 30°C, and the reagent was pumped at a flow rate of 0.5 mL / min for 3 hours. (1-2) After the reaction was completed, the mixed solution delivered in (1-1) was discharged, and the column was washed by delivering pure water at room temperature for 16 hours at a flow rate of 0.5 mL / min. (1-3) After washing the column, a diaphragm pump was connected to the column packed with the insoluble carrier prepared in (1-2) so that a mixture of 2.4 mL of pure water and 24 mmol of ethylenediamine could be circulated. The column was then placed in a column oven set at 45°C, and the reagent was pumped at a flow rate of 0.5 mL / min for 4 hours, thereby introducing amino groups, which are highly nucleophilic functional groups, into the hydroxyl groups on the surface of the pores of the insoluble carrier. (1-4) After the amino group introduction reaction was completed, the mixed solution delivered in (1-3) was discharged, and the column was washed by delivering pure water at room temperature at a flow rate of 0.5 mL / min for 16 hours, thereby preparing a column packed with an insoluble carrier to which an amino group had been introduced.

[0051] (2) Introduction of Q groups into the amino groups of the insoluble carrier The column used was the one prepared in (1), but the Q group was introduced to the amino group on the surface of the pores of the insoluble carrier by the method described in Example 1(2), and a column packed with a separation agent (an insoluble carrier with a Q group introduced therein) was prepared.

[0052] Example 3 Evaluation of separation material packed column The dynamic binding capacity (DBC) and ion exchange capacity of columns packed with the separating agent (insoluble carrier with Q groups introduced therein) prepared by the methods described in Examples 1 and 2 were evaluated.

[0053] (1) Evaluation of dynamic adsorption capacity (DBC) (1-1) A bovine serum albumin (BSA) solution diluted to 4.0 mg / mL with equilibration buffer (50 mmol / L Tris (hydroxymethyl) aminomethane) buffer (pH 8.5)) was directly delivered to Cytiva's AKTA avant 25, and the absorbance (280 nm) was measured.

[0054] (1-2) A column packed with the separating agent prepared by the method described in Example 1 or Example 2 was connected to an AKTA avant 25, and the column was equilibrated by feeding an equilibration buffer solution. Then, the BSA solution prepared in (1-1) was fed into the column, and BSA was adsorbed onto the separating agent. The flow rate of the BSA solution was 0.2 CV (column volume) / min.

[0055] (1-3) The absorbance (280 nm) of the eluate from the column was monitored, and when the absorbance reached 10% of the value measured in (1-1), the flow of the BSA solution was stopped. The time at which the flow was stopped was recorded (10% breakthrough time).

[0056] (1-4) A 50 mmol / L Tris buffer solution (pH 8.5) containing 1 mol / L sodium chloride was pumped through the column to elute the BSA adsorbed to the separating agent.

[0057] (1-5) The dynamic adsorption amount of BSA onto each column was calculated using the following formula 1.

[0058] [Number 1] Dynamic adsorption amount of BSA onto the column (mg / mL) = 10% breakthrough time (min) × flow rate (mL / min) × antibody concentration (mg / mL) / column volume (mL) The antibody concentration in Equation 1 is the concentration of the BSA solution before it is sent to the column, and is therefore 4.0 mg / mL.

[0059] The results are shown in Figure 1. In Figure 1, the vertical axis represents the dynamic adsorption capacity (DBC) of BSA onto a column packed with a separating agent. The separating agent-packed columns prepared in both Examples 1 and 2 showed adsorption of BSA, indicating that functional groups can be introduced into an insoluble carrier by feeding a reagent capable of introducing functional groups into a column packed with an insoluble carrier having pores. It can also be seen that DBC is significantly improved when a separating agent-packed column is prepared by introducing a highly nucleophilic functional group into the hydroxy group and then introducing a reagent capable of introducing a functional group (Example 2), compared to when a separating agent-packed column is prepared by directly introducing a reagent capable of introducing a functional group into the hydroxy group on the surface of the pores (Example 1).

[0060] (2) Evaluation of ion exchange capacity (2-1) A column packed with the separating agent prepared in Example 1 or Example 2 was filled with 0.1 mol / L sodium hydroxide at a volume 30 to 50 times the volume of the column at a flow rate of 0.5 mL / min, and then thoroughly washed with pure water until the pH of the washing solution reached 7.0.

[0061] (2-2) Next, a 0.5 mol / L aqueous sodium chloride solution in an amount 25 times the volume of the separating agent was pumped at a flow rate of 0.5 mL / min, and the eluate discharged from the column was collected.

[0062] (2-3) The eluate collected in (2-2) was titrated with 0.01 mol / L hydrochloric acid until the pH reached 7.0, and the amount of hydrochloric acid used in the titration was recorded.

[0063] (2-4) The ion exchange capacity per 1 g of the suction-dried gel was calculated using the following formula 2.

[0064] [Number 2] Ion exchange capacity per 1 g of suction dry gel (mol / g) = hydrochloric acid titration (mL) × hydrochloric acid concentration (1.0 × 10 -5mol / mL) / amount of suction-dried gel (g) The results are shown in Table 1. Table 1 shows relative values ​​when the ion exchange capacity per 1 g of the suction-dried gel in Example 1 is set to 1. Because the separating agent-packed columns prepared in Examples 1 and 2 both have anion exchange capacity, it is clear that by feeding a reagent capable of introducing a functional group into a column packed with an insoluble carrier having pores, the functional group (Q group, an anion exchange group in this example) can be introduced into the insoluble carrier. It is also clear that the performance of the separating agent (anion exchange capacity in this example) is significantly improved when a separating agent-packed column is prepared by first introducing a highly nucleophilic functional group into the hydroxy group and then introducing a reagent capable of introducing a functional group (Example 2), rather than when a separating agent-packed column is prepared by directly introducing a reagent capable of introducing a functional group into the hydroxy group on the surface of the pores (Example 1).

[0065] [Table 1]

Claims

1. A method for manufacturing a column filled with a separation material, comprising a step of introducing functional groups into an insoluble carrier by feeding a reagent capable of introducing functional groups into the insoluble carrier into a column filled with an insoluble carrier having fine pores.

2. The insoluble carrier has hydroxy groups on the surfaces of the pores, and the step of introducing functional groups into the insoluble carrier comprises: a step of introducing a reagent capable of introducing a highly nucleophilic functional group into the insoluble carrier into a column packed with the insoluble carrier, thereby introducing the highly nucleophilic functional group into the hydroxy group; and a step of feeding a reagent capable of introducing an activated functional group into the insoluble carrier into the column packed with the insoluble carrier, thereby introducing the activated functional group into the highly nucleophilic functional group.

3. the reagent capable of introducing an activated functional group into the insoluble support has an electrophilic reactive group; The method for producing a column according to claim 2, wherein the step of introducing the activating functional group into the highly nucleophilic functional group is carried out by reacting the highly nucleophilic functional group with the electrophilic reactive group.

4. The method for producing a column according to claim 2 or 3, wherein the highly nucleophilic functional group is an amino group.

5. The method for producing a column according to claim 3, wherein the electrophilic reactive group is an epoxy group.

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