Filler and method for producing the same, and column for size exclusion chromatography

Abstract

Provided are a packing for size exclusion chromatography having high alkali resistance and suppressed non-specific adsorption, and a method for producing the same. A packing is a block copolymer represented by the following Formula 1, one end of which is bound to a porous organic high-molecular carrier via an ether bond derived from a terminal hydroxyl group, the porous organic high-molecular carrier containing 60 to 95 mol% of a repeating unit derived from glycidyl methacrylate and 5 to 40 mol% of a repeating unit derived from a multifunctional monomer. (Formula 1) HO-(C2H4O) l -(C3H6O) m -(C2H4O) n -H (in Formula 1, l is an integer of 5 to 140, m is an integer of 15 to 75, and n is an integer of 5 to 140. The proportion of m in the total of l, m, and n is 5 to 85%).

Description

Technical Field

[0001] This invention relates to packing materials for size exclusion chromatography used in protein purification. More specifically, it relates to packing materials with high alkali resistance. Background Technology

[0002] Size exclusion chromatography, which separates molecules according to their size, is widely used in the separation of water-soluble macromolecules such as polysaccharides, peptides, proteins, DNA, and RNA. Particularly in the separation and sorting of biological macromolecules such as proteins, size exclusion chromatography allows for the treatment of these macromolecules under mild conditions, thus offering the advantage of separation and sorting with minimal denaturation. The separated and sorted biological macromolecules are used in applications in biochemical reactions, food, pharmaceuticals, and chemical industries, and the market has experienced significant growth in recent years.

[0003] To address this demand, there is a need for fillers capable of large-scale, industrial-scale separation and purification. In particular, in the gene therapy field, where market expansion is anticipated, further functional improvements in fillers are desired, with the aim of significantly improving the productivity of purification processes for viral vectors, virus-like particles, etc. Specifically, there is a need for fillers that can be used for the precise separation and purification of large-molecule viral vectors and virus-like particles, simplifying precise size separation through a series of steps from culture medium to purification.

[0004] Therefore, for packing materials, it is desirable to have good sorting efficiency that can recover the separated substances, which is a different subject / requirement from analytical columns. For example, it is desirable to be able to suppress the non-specific adsorption of various components such as impurities and unknown components contained in biopharmaceuticals derived from fermentation products, and to be able to be regenerated by washing with strong alkali.

[0005] However, no such alkali-resistant filler has yet been proposed.

[0006] Furthermore, in International Publication No. 2018 / 155241 (Patent Document 1), the applicant proposed a size exclusion chromatography packing material using methacrylate monomers. While Patent Document 1 discloses a packing material that involves hydrophilizing porous particles composed of copolymers obtained by polymerizing monomers having glycidyl groups and crosslinking agents in the presence of a polymerization initiator with sugar alcohols, and then ring-opening the remaining glycidyl groups using an inorganic acid, a higher level of alkali resistance is required.

[0007] Furthermore, Japanese Patent No. 5315691 (Patent Document 2) discloses a filler that improves resistance to alkaline aqueous solutions by hydrophilically treating the surface of crosslinked polymer particles using a monomer with a special structure among methacryloyl monomers. The monomer used in Patent Document 2 is not a universal monomer, thus presenting the problem of being difficult to obtain easily and simply, leading to higher costs.

[0008] Existing technical documents

[0009] Patent documents

[0010] Patent Document 1: International Publication No. 2018 / 155241

[0011] Patent Document 2: Japanese Patent No. 5315691 Summary of the Invention

[0012] The problem that the invention aims to solve

[0013] Thus, despite various reports, there is still a strong expectation for fillers with high alkali resistance and suitability for sorting.

[0014] The present invention addresses the problems identified above. More specifically, the subject of the present invention is to provide a packing material for size exclusion chromatography with high alkali resistance and a method for manufacturing the same.

[0015] Methods for solving problems

[0016] The inventors conducted repeated and in-depth research to solve the above-mentioned problems, and as a result, discovered that introducing a filler consisting of a specified block copolymer into porous particles composed of a monomer having a glycidyl group and a crosslinking agent exhibits high alkali resistance, thus completing the present invention. Specifically, the present invention relates to the following matters.

[0017] [1] A filler is formed by linking one end of a block copolymer of Formula 1 to a porous organic polymer carrier via an ether bond derived from a terminal hydroxyl group, wherein the porous organic polymer carrier contains 60-95 mol% repeating units derived from glycidyl methacrylate and 5-40 mol% repeating units derived from a multifunctional monomer.

[0018] (Equation 1)

[0019] HO-(C2H4O) l -(C3H6O) m -(C2H4O) n -H

[0020] (In Equation 1, l is an integer from 5 to 140, m is an integer from 15 to 75, and n is an integer from 5 to 140. The proportion of m in the total of l, m, and n is 5% to 85%.)

[0021] [2] According to the filler described in [1], the total of l, m and n of the above block copolymer is 30 to 330.

[0022] [3] According to the filler described in [1] or [2], the above-mentioned multifunctional monomer is a (meth)acryloyl monomer containing two or more (meth)acryloyl groups.

[0023] [4] The filler according to any one of [1] to [3], wherein the multifunctional monomer is any one of ethylene glycol dimethacrylate and glycerol-1,3-dimethacrylate.

[0024] [5] The filler according to any one of [1] to [4] has an exclusion limit molecular weight of 1 million or more.

[0025] [6] A method for manufacturing a filler, which is a method for manufacturing the filler described in any one of [1] to [5], comprising the following steps:

[0026] Step (A) is a process of polymerizing a raw material monomer containing glycidyl methacrylate and a multifunctional monomer in the presence of a diluent and a polymerization initiator to obtain a porous organic polymer carrier α.

[0027] The concentration of the multifunctional monomer in the above-mentioned raw material monomer is 5 mol% to 40 mol%, and the above-mentioned diluent is used at a volume of 0.8 to 4.0 times the total volume of the above-mentioned raw material monomer; and

[0028] Step (B) involves ring-opening the glycidyl group of the porous organic polymer carrier α derived from glycidyl methacrylate with the block copolymer shown in Formula 1 to obtain a filler β in which one end of the block copolymer shown in Formula 1 is bonded to the porous organic polymer carrier α via an ether bond derived from the terminal hydroxyl group.

[0029] (Equation 1)

[0030] HO-(C2H4O) l -(C3H6O) m -(C2H4O) n -H

[0031] (In Equation 1, l is an integer from 5 to 140, m is an integer from 15 to 75, and n is an integer from 5 to 140. The proportion of m in the total of l, m, and n is 5% to 85%.)

[0032] [7] A size exclusion chromatography column, wherein the packing agent described in any one of [1] to [5] is filled in a liquid chromatography shell.

[0033] The effects of the invention

[0034] According to the present invention, a filler with high alkali resistance and excellent sorting efficiency can be obtained. This enables high-speed separation processing. Furthermore, the filler can be manufactured using inexpensive raw materials and simple processes, and is easily applied on an industrial scale using large quantities of filler. Detailed Implementation

[0035] Hereinafter, embodiments of the present invention will be described in detail. However, the materials, dimensions, etc., illustrated in the following description are examples only, and the embodiments are not limited to them. They can be implemented by appropriate modifications without changing their spirit.

[0036] In addition, the term "filler" refers to a substance that can be used alone as a filler, and also includes substances that can be used to modify surfaces for a purpose.

[0037] Furthermore, in this specification, "(meth)acrylic acid" refers to acrylic acid and methacrylic acid, and "(meth)acryloyl" is the same.

[0038] [Filler]

[0039] The filler in this embodiment has a specific framework that is combined with a porous organic polymer carrier α as shown below.

[0040] (Porous organic polymer carrier α)

[0041] The porous organic polymer carrier α is composed of a copolymer containing 60-95 mol% repeating units derived from glycidyl methacrylate and 5-40 mol% repeating units derived from multifunctional monomers. Preferably, it contains 65-95 mol% repeating units derived from glycidyl methacrylate and 5-35 mol% repeating units derived from multifunctional monomers; more preferably, it contains 75-92 mol% repeating units derived from glycidyl methacrylate and 8-25 mol% repeating units derived from multifunctional monomers. When the ratio of repeating units derived from multifunctional monomers is low, the final filler sometimes results in high back pressure, making it unsuitable for use; when the ratio of repeating units derived from multifunctional monomers is high, non-specific adsorption sometimes occurs, preventing target sorting. Furthermore, the porous organic polymer carrier of this embodiment can also be obtained using glycidyl acrylate.

[0042] A multifunctional monomer is a compound having two or more olefinic double bonds within its molecule. Preferably, a multifunctional monomer has two or more (meth)acryloyl groups within its molecule. Specifically, examples include alkyldiol di(meth)acrylate (alkane with 1 to 12 carbon atoms), trimethylolpropane tri(meth)acrylate, bis(trimethylolpropane)tetra(meth)acrylate, pentaerythritol tetra(meth)acrylate, pentaerythritol penta(meth)acrylate, dipentaerythritol penta(meth)acrylate, dipentaerythritol hexa(meth)acrylate, etc. Additionally, multifunctional carbamate (meth)acrylates are also possible. Only one of these compounds may be used, or two or more may be used in combination.

[0043] Preferably, it contains at least one of ethylene glycol dimethacrylate and glycerol-1,3-dimethacrylate. Ethylene glycol dimethacrylate and glycerol-1,3-dimethacrylate may be present in at least 50 mol% relative to the total amount of the multifunctional monomer, preferably at least 80 mol%. For the formation of fine pores suitable for sorting, a total amount of ethylene glycol dimethacrylate and / or glycerol-1,3-dimethacrylate is most preferred.

[0044] If the copolymer contains a total of 95 mol% or more of the above-mentioned glycidyl methacrylate and multifunctional monomers as monomer units, other monomer units may be included within a range that does not significantly alter the properties of the porous particles. Other monomers, including those with glycidyl groups, such as 3,4-epoxycyclohexyl methacrylate, 4-hydroxybutyl acrylate glycidyl ether, etc., may also include methyl (meth)acrylate, ethyl (meth)acrylate, etc.

[0045] The degree of crosslinking of the copolymer is 5 mol% to 40 mol%, preferably 5 mol% to 35 mol%, and more preferably 8 to 25 mol%.

[0046] The above degree of crosslinking is determined by

[0047] (Total moles of multifunctional monomers / Total moles of all monomers) × 100

[0048] = Degree of crosslinking (mol%)

[0049] express.

[0050] When the degree of cross-linking is low, the back pressure of the final packing material sometimes becomes too high for size exclusion chromatography columns, making them unsuitable for use. If the degree of cross-linking is high, non-specific adsorption sometimes occurs, making it impossible to sort the target.

[0051] (Surface structure)

[0052] In this embodiment, the block copolymer shown in Formula 1 is bonded to the porous organic polymer carrier α via ether bonds derived from the terminal hydroxyl groups. That is, the bonding occurs via ether bonds formed by the reaction of the glycidyl groups derived from glycidyl methacrylate in the copolymer constituting the carrier with the terminal hydroxyl groups of the block copolymer.

[0053] (Equation 1)

[0054] HO-(C2H4O) l -(C3H6O) m -(C2H4O) n -H

[0055] In Equation 1, l is an integer from 5 to 140, m is an integer from 15 to 75, and n is an integer from 5 to 140. The proportion of m in the total of l, m, and n is 5% to 85%. l and n can be the same integer or different integers, and l can be greater than n or less than n. l is preferably 6 to 135, m is preferably 16 to 70, and n is preferably an integer from 6 to 135.

[0056] The C3 alkyl group constituting (C3H6O) can be either n-propyl or isopropyl, but isopropyl is preferred to improve hydrophobicity.

[0057] That is, (C2H4O) exists on the surface of the carrier. l Or (C2H4O) n The highly hydrophilic block fraction is derived from (C3H8O). m The highly hydrophobic block fraction is derived from (C2H4O). l Or (C2H4O) n The highly hydrophilic block portion (of the component not bound to the carrier) allows for the suppression of non-specific adsorption, resulting in a filler with high alkali resistance.

[0058] The proportion of m in the total of l, m, and n of the above-mentioned block copolymer is 5-85%, preferably 10-75%, and more preferably 15-65%. Within this range, a filler with particularly high alkali resistance and suppressed non-specific adsorption can be obtained.

[0059] The total of l, m, and n in the above-mentioned block copolymer is preferably 30 to 330, more preferably 35 to 200. Within this range, a filler with particularly high alkali resistance and suppressed non-specific adsorption can be obtained.

[0060] In addition, l, m, and n can be calculated from the weights of the (C2H4O) and (C3H8O) portions based on Fig. 2 and Table 1 of the Journal of the Japanese Society of Oil Chemistry (JOS) vol. 49, No. 10, 1071, for example, by referring to the values ​​in the catalogs of various companies.

[0061] As such block copolymers, the Pullonnic (registered trademark) L, P, and F series manufactured by ADEKA Co., Ltd. and BASF can be used, including L35, F38, L42, L43, L44, L62, L63, L64, L72, P65, F68, P75, F77, P84, P85, F87, F88, L92, P94, F98, P103, P104, P105, F108, L122, P123, F127, etc.

[0062] (Properties of fillers)

[0063] For example, the following shows a schematic structure of the filler in this embodiment.

[0064]

[0065] There are no particular restrictions on the average particle size of the filler as long as it is 10 μm or more, but from the viewpoint of column filling performance, a range of 15 to 100 μm is preferred. Here, the average particle size is expressed as the volume-resolved average particle size. The volume-resolved average particle size is a value obtained by an image resolution particle size distribution measuring device. When using an image resolution particle size distribution measuring device for volume-resolved average particle size, the equivalent circle diameter (the diameter of a circle with an area equal to the projected area of ​​the particle image) of each particle is obtained from a two-dimensional particle image (preferably a still image) obtained by taking pictures of 2000 or more crosslinked polymer particles using the image resolution particle size distribution measuring device. The volume of each particle is calculated from the equivalent circle diameter, and the particle size is averaged based on the volume. At this time, each particle is considered to be a sphere with the same diameter as the equivalent circle diameter. As an image resolution particle size distribution measuring device, for example, a flow cytometry particle image analyzer (trade name: FPIA-3000, manufactured by Sismex Co., Ltd.) can be used. In addition, the average particle size of the filler can be adjusted according to the polymerization conditions during carrier manufacturing.

[0066] Preferably, the size exclusion molecular weight of the packing material in this embodiment is 1 million or more. The size exclusion molecular weight is adjusted according to the size of the pores of the packing material. The size exclusion molecular weight can be determined by connecting a column packed with the packing material to a high-performance liquid chromatography (HPLC) system, injecting standards of various molecular weights into the column at a flow rate of 1.0 mL per minute using ion-exchanged water as the mobile phase, and using the elution capacity by a generally known method. In this embodiment, a differential refractive index detector (trade name: RI-201H, manufactured by Showa Denko) is used for detection, and pullulan polysaccharide standard (trade name: Shodex (registered trademark) STANDARD P-82, manufactured by Showa Denko) is used as the standard.

[0067] To separate large proteins using size exclusion chromatography, a size exclusion limit molecular weight of 1 million or higher is preferred. For example, when separating large proteins such as IgM with a molecular weight of approximately 900,000 and IgG with a molecular weight of approximately 150,000, if the size exclusion limit is less than 1 million, IgM will fall into the size exclusion limit region and cannot be separated.

[0068] The exclusion limit molecular weight can be adjusted by the type and amount of diluent used in the manufacturing process, or the ratio of glycidyl methacrylate to the multifunctional monomer, as described later. Furthermore, the average particle size and pore size of the filler do not vary depending on the surface structure.

[0069] (Method for manufacturing fillers)

[0070] The method for manufacturing the filler in this embodiment includes the following steps:

[0071] Step (A) is a process of polymerizing a raw material monomer containing glycidyl methacrylate and a multifunctional monomer in the presence of a diluent and a polymerization initiator to obtain a porous organic polymer carrier α.

[0072] Furthermore, the concentration of the multifunctional monomer in the above-mentioned raw material monomer is made to be 5 mol% to 40 mol%, and the above-mentioned diluent is used at a volume of 0.8 to 4.0 times that of the raw material monomer; and

[0073] Step (B) involves ring-opening the glycidyl group of the porous organic polymer carrier α derived from glycidyl methacrylate with the block copolymer shown in Formula 1 to obtain a filler β in which one end of the block copolymer shown in Formula 1 is bonded to the porous organic polymer carrier α via an ether bond derived from the terminal hydroxyl group.

[0074] [Process (A)]

[0075] A porous organic polymer carrier α is prepared, consisting of copolymers with glycidyl methacrylate and multifunctional monomer units. The porous organic polymer carrier α is obtained by copolymerizing these monomers in the presence of a diluent and a polymerization initiator. These porous particles can be manufactured using methods described in Japanese Patent Application Laid-Open No. 2007-170907 and WO2006 / 132333, etc.

[0076] The concentration of glycidyl methacrylate in the above-mentioned raw material monomers is expected to be 60-95 mol%, preferably 70-95 mol%. The concentration of the above-mentioned multifunctional monomers in the above-mentioned raw material monomers is preferably 5 mol%-40 mol%, preferably 5 mol%-30 mol%.

[0077] In addition, as mentioned above, it may contain other monomeric components.

[0078] To introduce micropores into the porous organic polymer carrier α, a diluent is added to the monomer mixture during polymerization in step (A). The diluent is an organic solvent that, while soluble in the monomer mixture, is inactive in the polymerization reaction and does not dissolve the resulting copolymer. After polymerization, the diluent is removed by washing or similar means, thereby partially transforming the diluent into voids and forming porous micropores within the porous organic polymer carrier α.

[0079] The amount of this diluent added affects the exclusion limit molecular weight of the filler and the volume percentage of the pore volume (representing the ratio of pore volume to the total volume of filler particles). Therefore, its amount is adjusted appropriately before addition. The amount of these diluents added is 0.8 to 4.0 times, preferably 1.0 to 3.0 times, the total volume of the raw material monomers at the temperature at which they are added.

[0080] As diluents, aromatic hydrocarbons such as toluene, xylene, diethylbenzene, dodecylbenzene, and chlorobenzene can be used; saturated hydrocarbons such as hexane, heptane, pentane, octane, nonane, and decane; alcohols such as isoamyl alcohol, hexanol, heptanol, octanol, and nonanol; aliphatic halogenated hydrocarbons such as dichloromethane, dichloroethane, and trichloroethane; and aliphatic or aromatic esters such as ethyl acetate, propyl acetate, butyl acetate, amyl acetate, diethyl succinate, methyl benzoate, ethyl benzoate, and propyl benzoate. These diluents can be used alone or in mixtures of two or more.

[0081] The polymerization initiator used during polymerization is not particularly limited as long as it is a known free radical polymerization initiator that generates free radicals. Examples include azo initiators such as 2,2'-azobisisobutyronitrile, 2,2'-azobis(methyl isobutyrate), and 2,2'-azobis(2,4-dimethylpentanonitrile). Among these, 2,2'-azobis(2,4-dimethylpentanonitrile) is preferred due to its chemical affinity. The concentration of the polymerization initiator is not particularly limited, but it is preferably 0.1 to 5 parts by mass relative to 100 parts by mass of the total monomer.

[0082] An oil phase containing the raw monomers is prepared by mixing a raw monomer mixture, a diluent, and a polymerization initiator. The oil phase is then stirred and suspended in an aqueous medium containing a suitable dispersant and stabilizer, forming oil droplets. Polymerization (suspension polymerization) is then performed in this state, resulting in a copolymer that is a porous particle with a suitable particle size. In addition to the stirring method described above, another method for preparing oil droplets is to dropwise add a monomer solvent containing a diluent into the aqueous medium through a microflow path formed on a porous membrane or a quartz substrate.

[0083] Known substances can be used as dispersants and stabilizers in aqueous media. Water-soluble polymers such as gelatin, sodium polyacrylate, and polyvinyl alcohol are commonly used. Polyvinyl alcohol is generally preferred. The concentration of the dispersant and stabilizer is preferably 0.1% to 5% by mass relative to the aqueous medium.

[0084] Aqueous media, besides water, can also contain salts and other water-soluble components. Examples of salts include commonly used substances such as sodium chloride and calcium chloride. Since solubility varies depending on the salt used, concentrations cannot be specified generally; for example, it may be used when sodium chloride can dissolve 0.1–15% by mass, and calcium chloride can dissolve 1–40% by mass. Salts are added for salting out.

[0085] After nitrogen purging, the suspension polymerization reaction is typically carried out under stirring and heated to 40–100°C for 5–16 hours at atmospheric pressure. During this process, the monomers contained in each oil droplet polymerize in the presence of a diluent, and the polymer network grows. Porous particles are then obtained by removing the diluent. After the reaction, the porous particles can be easily separated from the aqueous medium by filtration. Further, the diluent is removed by washing with solvents such as acetone, methanol, or diethylene glycol dimethyl ether. After drying, the resulting porous particles with glycidyl groups are classified using a sieve and an air classifier.

[0086] The copolymer obtained in step (A) by this operation is a porous organic polymer particle with glycidyl groups derived from glycidyl methacrylate, having the average particle size and pores as described above.

[0087] [Process (B)]

[0088] Next, the glycidyl groups of the porous organic polymer carrier α, derived from glycidyl methacrylate, are subjected to a ring-opening reaction with the block copolymer shown in Formula 1 above.

[0089] Thus, a filler β is obtained in which one end of the block copolymer shown in Formula 1 is bonded to the porous organic polymer carrier α via an ether bond derived from the terminal hydroxyl group.

[0090] As such block copolymers, the Pullonnic (registered trademark) L, P, and F series manufactured by ADEKA Co., Ltd. and BASF Co., Ltd. can be used.

[0091] The proportion of m and the total of l, m, and n in the above block copolymer are as described above.

[0092] The preferred amount of block copolymer used is 100 to 8000 parts by mass relative to 100 parts by mass of the porous organic polymer carrier α100. Using it within this range yields a filler with high alkali resistance and suppression of non-specific adsorption.

[0093] In the ring-opening reaction, boron trifluoride diethyl ether coordination compounds, zinc boron fluoride, trimethylsilyl trifluoromethanesulfonic acid, sulfuric acid, trifluoromethanesulfonic acid, trifluoroacetic acid, dichloroacetic acid, etc., can be used as catalysts. The catalyst is preferably 0.1 to 100 parts by weight, more preferably 0.5 to 20 parts by weight, relative to 100 parts by weight of the porous organic polymer support α. Within this range, the introduction of the block copolymer can be carried out, and reactions such as ester group reactions of the porous organic polymer support α can be prevented. The ring-opening reaction is usually carried out in the presence of a solvent. Diethylene glycol dimethyl ether, etc., can be used as a solvent.

[0094] The resulting porous particles incorporating the block copolymer (i.e., filler β) are washed with dimethyl sulfoxide or the like to remove excess block copolymer, catalyst, etc.

[0095] Furthermore, unreacted glycidyl groups sometimes remain in the resulting filler β. If these glycidyl groups remain, the hydrophobicity of the filler increases, potentially leading to the hydrophobic adsorption of highly hydrophobic water-soluble compounds such as proteins. Therefore, to further improve hydrophilicity, it is desirable to ring-open the remaining glycidyl groups with an inorganic acid.

[0096] Examples of inorganic acids include sulfuric acid, nitric acid, and hydrochloric acid. Sulfuric acid is particularly preferred. The concentration of the inorganic acid used only needs to be around 0.01M to 1.0M, and is particularly preferred to be around 0.1M to 0.5M. If the inorganic acid concentration is above 0.01M, ring-opening is sufficiently achieved. Furthermore, if the inorganic acid concentration is below 1.0M, the ester groups in the support are hydrolyzed, and ionic functional groups are not generated. The resulting particles are washed with water to easily remove excess inorganic acid.

[0097] The packing material of this embodiment has suitable hydrophilicity and size exclusion limit molecular weight. Therefore, by filling this packing material into a liquid chromatography shell, a high-performance size exclusion chromatography column can be obtained, and further, a chromatography apparatus equipped with this size exclusion chromatography column can be obtained. Furthermore, by using this size exclusion chromatography column, a method for separating and sorting biological polymers with high precision using an aqueous eluent can be provided.

[0098] In addition, the filler is highly alkali-resistant, so it can be washed and reused, and can be used continuously for a long time.

[0099] Example

[0100] The effects of the present invention will be made clearer through the following embodiments. Furthermore, the present invention is not limited to the following embodiments and can be implemented with appropriate modifications without altering its spirit.

[0101] [Example 1]

[0102] <Synthesis of porous organic polymer carrier α: copolymer with glycidyl groups>

[0103] The oil phase was prepared by dissolving 33.2 g of glycidyl methacrylate (trade name: Bremmar G (registered trademark) Nippon Oil Co., Ltd.), 5.9 g of ethylene glycol dimethacrylate (trade name: NK Ester 1G, Shin-Nakamura Chemical Industry Co., Ltd.), 11.7 g of chlorobenzene, and 1.9 g of 2,2'-azobis(2,4-dimethylpentanonitrile) in 47.0 g of butyl acetate as a diluent, and bubbling with nitrogen for 30 minutes.

[0104] Next, separately from the oil phase, an aqueous phase was prepared in which 10.0 g of PVA-224 (polyvinyl alcohol manufactured by Clare Co., Ltd., with a saponification degree of 87.0%-89.0%) as a dispersion stabilizer and 10.0 g of sodium chloride as a salting-out agent were dissolved in 480 g of water.

[0105] The aqueous and oil phases were transferred to a detachable flask and dispersed for 20 minutes using a stir bar equipped with a crescent-shaped stirring blade at a rotation speed of 430 rpm. The reactor was then purged with nitrogen and reacted at 60°C for 16 hours. The resulting polymer was then transferred to a glass filter and thoroughly washed in the following order: warm water (approximately 50–80°C), acetone, and water, yielding 125 g of porous organic polymer carrier α1 as porous organic polymer particles. The amount of glycidyl methacrylate used relative to the total monomer content was 88.7 mol%, and the amount of ethylene glycol dimethacrylate used relative to the total monomer content was 11.3 mol%.

[0106] <Ring-opening reaction of glycidyl group>

[0107] The porous organic polymer carrier α1 was measured onto a 100g glass filter and thoroughly washed with diethylene glycol dimethyl ether. After washing, the porous organic polymer carrier α1 was transferred to a detachable flask, and 400g of diethylene glycol dimethyl ether and HO-(C2H4O) were added. l -(C3H6O) m -(C2H4O) n 400g of the block copolymer shown in -H (ADEKA Manufacturing Co., Ltd. (registered trademark) L35, l = 11, m = 16, n = 11, l+m+n = 38, m / (l+m+n) = 43%) was transferred to a 1L detachable flask and dispersed by stirring. Then, 2.0ml of boron trifluoride diethyl ether coordination compound was added, and the mixture was heated to 80°C while stirring at 200rpm for 4 hours to open the glycidyl group, obtaining filler β1, in which one end of the above block copolymer was bound to the porous organic polymer carrier α1 via an ether bond derived from the terminal hydroxyl group. After cooling, the obtained filler β1 was filtered and separated, and then thoroughly washed with water to obtain 138g of filler β1.

[0108] The ring-opening reaction was confirmed through the following steps. A portion of the filler β1, into which the above block copolymer was introduced, was mixed with potassium bromide, granulated under pressure, and then measured using FT-IR (trade name: Nicolet iS10, manufactured by Servo Semiconductor Co., Ltd.). The 908 cm⁻¹ of the infrared absorption spectrum obtained from the glycidyl group was confirmed. -1 The peak height of absorbance was determined. As a result, the 908 cm⁻¹ peak was not observed based on FT-IR. -1 The absorbance peak.

[0109] The obtained filler β1 was classified into particle sizes of 16–37 μm using a sieve, yielding 117.3 g of filler β1.

[0110] <Evaluation of Alkali Resistance>

[0111] The alkali resistance is evaluated by calculating the amount of carboxyl groups generated from the hydrolysis of sodium hydroxide using the following steps.

[0112] First, 4g of the filler was dispersed in 150mL of a 0.5mol / L potassium chloride aqueous solution. The point at which the pH reached 7.0 was set as the neutralization point, and titration was performed using a 0.1mol / L sodium hydroxide aqueous solution. Therefore, the amount of carboxyl groups in the filler before hydrolysis was calculated using the following formula.

[0113] Amount of carboxyl groups (μmol / mL) = 0.1 × Volume of sodium hydroxide aqueous solution at neutralization point (μL) / Apparent volume of filler (mL)

[0114] Here, the apparent volume of the filler is the volume measured after 4g of filler is dispersed in water to prepare the slurry, the slurry is transferred in the vector cylinder, and allowed to stand for a sufficient time.

[0115] Next, 4g of the packing material was measured into a detachable flask, and 20mL of 5mol / L sodium hydroxide aqueous solution was added. The mixture was stirred at 200rpm and subjected to a hydrolysis reaction at 50°C for 20 hours. After cooling, the sodium hydroxide-treated packing material was filtered and separated, then washed in the order of 0.1mol / L HCl aqueous solution and water. The amount of carboxyl groups in the resulting packing material was calculated using the same method as before. The amount of carboxyl groups generated by the reaction with the 5mol / L sodium hydroxide aqueous solution was calculated from the difference in the amount of carboxyl groups before and after the reaction. The result showed that the amount of carboxyl groups generated was 24.0μmol / mL.

[0116] In addition, if the amount of carboxyl group generated is below 40 μmol / mL, the alkali resistance is rated as high.

[0117] <Non-specific adsorption evaluation>

[0118] The obtained filler was filled into a stainless steel column (Sugiyama Corporation) with an inner diameter of 8 mm and a length of 300 mm using the equilibrium slurry method. Non-specific adsorption tests were conducted using the obtained column according to the method shown below.

[0119] The column filled with the above-mentioned packing material was connected to a Shimadzu HPLC system (pump (trade name: LC-10AT, manufactured by Shimadzu Corporation), autosampler (trade name: SIL-10AF, manufactured by Shimadzu Corporation), and photodiode array detector (trade name: SPD-M10A, manufactured by Shimadzu Corporation)). Water was circulated at a flow rate of 0.6 mL / min using 50 mmol / L sodium phosphate buffer as the mobile phase. Using the same sodium phosphate aqueous solution as the mobile phase, 0.7 mg / mL of thyroglobulin (Mw 6.7 × 10⁻⁶) was prepared. 5 ), 0.6 mg / mL γ-globulin (Mw 1.6 × 10), 5 ), 0.96mg / mL BSA (Mw6.65×10 4 ), 0.7 mg / mL ribonuclease (Mw 1.3 × 10), 4 ), 0.4 mg / mL aprotinin (Mw 6.5 × 10), 3 Each sample solution containing 0.02 mg / mL uridine (Mw 244) (made by Merck Sigma Aldrich) was injected in 10 μL via an autosampler. The elution times of each sample were compared using a photodiode array detector at a wavelength of 280 nm to confirm that the order of elution capacity did not contradict the order of molecular weight. Furthermore, when the order of molecular weight and elution capacity did not contradict each other, no nonspecific adsorption occurred, and this was indicated as 0 in Table 1. In cases where there was a contradiction, nonspecific adsorption was induced, and therefore indicated as ×.

[0120] As a result, the elution capacities of each sample from the column packed with packing agent β1 were 11.437 mL, 11.841 mL, 11.883 mL, 12.094 mL, 12.262 mL, and 12.986 mL, respectively, confirming that there was no contradiction between the molecular weight of the samples and the order of elution capacity, and that no non-specific adsorption was induced.

[0121] [Example 2]

[0122] In the ring-opening reaction of the glycidyl group, it acts as HO-(C2H4O). l -(C3H6O) m -(C2H4O) n The block copolymer shown in -H was obtained by using 400g of Adicapur Ronic (registered trademark) L44 (l is 10, m is 23, n is 10, l+m+n is calculated to be 43, and m / (l+m+n) is 53%), except that the filler β2 was obtained by operating in the same manner as in Example 1.

[0123] The alkali resistance of the obtained filler β2 was evaluated in the same manner as in Example 1. The results showed that the carboxyl group formation amount was 23.2 μmol / mL, confirming that filler β2 has excellent alkali resistance.

[0124] Furthermore, the non-specific adsorption evaluation of the obtained packing agent β2 was performed in the same manner as in Example 1. The results showed that the elution capacities of each sample were 11.188 mL, 11.570 mL, 11.687 mL, 11.906 mL, 12.077 mL, and 13.310 mL, confirming that the order of molecular weight and elution capacity of the samples was consistent and did not induce non-specific adsorption.

[0125] [Example 3]

[0126] In the ring-opening reaction of the glycidyl group, HO-(C2H4O) was used instead of Adegapuronic (registered trademark) L35. l -(C3H6O) m -(C2H4O) n -H shows the block copolymer Adicapurn (registered trademark) P123 (l is 20, m is 69, n is 20, l+m+n is 109, m / (l+m+n) is 64%) 400g, except that filler β3 was obtained by operating in the same manner as in Example 1.

[0127] The alkali resistance of the obtained filler β3 was evaluated in the same manner as in Example 1. The results confirmed that the carboxyl group formation amount was 22.3 μmol / mL, and that filler β3 exhibited excellent alkali resistance.

[0128] Furthermore, the non-specific adsorption of the obtained packing agent β3 was evaluated in the same manner as in Example 1. The results showed that the elution capacities for each sample were 11.345 mL, 11.671 mL, 11.770 mL, 12.044 mL, 12.160 mL, and 13.235 mL, confirming that the order of molecular weight and elution capacity did not contradict each other and did not induce non-specific adsorption.

[0129] [Example 4]

[0130] In the ring-opening reaction of the glycidyl group, HO-(C2H4O) was used instead of Adegapuronic (registered trademark) L35. l -(C3H6O) m -(C2H4O) n-H shows the block copolymer (Adegapurnock (registered trademark) F68 (l is 76, m is 29, n is 76, l+m+n is 182, m / (l+m+n) is 16%) 800g, except that filler β4 was obtained by operating in the same manner as in Example 1.

[0131] The alkali resistance of the obtained filler β4 was evaluated using the same procedure as in Example 1. The results showed that the carboxyl group formation amount was 27.6 μmol / mL, confirming that filler β4 possesses excellent alkali resistance.

[0132] Furthermore, the non-specific adsorption of the obtained packing agent β4 was evaluated in the same manner as in Example 1. The results showed that the elution capacities for each sample were 10.71 mL, 11.089 mL, 11.240 mL, 11.584 mL, 11.840 mL, and 13.249 mL, confirming that the order of molecular weight and elution capacity did not contradict each other and did not induce non-specific adsorption.

[0133] [Example 5]

[0134] A porous organic polymer carrier α5 was synthesized using 21.5 g of glycidyl methacrylate (trade name: Bremmar G (registered trademark) Nippon Oil Co., Ltd.), 17.6 g of ethylene glycol dimethacrylate (trade name: NK Ester 1G, Shin-Nakamura Chemical Industry Co., Ltd.), 47.0 g of butyl acetate, 11.7 g of chlorobenzene, and 1.9 g of 2,2'-azobis(2,4-dimethylpentanonitrile) as the oil phase, and the procedure was the same as in Example 1. Filler β5 was obtained by the same procedure as in Example 3. The amount of glycidyl methacrylate used was 63.0 mol% of the total monomers, and the amount of ethylene glycol dimethacrylate used was 37.0 mol% of the total monomers.

[0135] The alkali resistance of the obtained filler β5 was evaluated using the same procedure as in Example 1. The results showed that the carboxyl group formation amount was 19.3 μmol / mL, confirming that filler β5 possesses excellent alkali resistance.

[0136] Furthermore, the non-specific adsorption evaluation of the obtained packing agent β5 was performed in the same manner as in Example 1. The results showed that the elution capacities of each sample were 11.387 mL, 11.734 mL, 11.775 mL, 12.043 mL, 12.44 mL, and 12.945 mL, confirming that the order of molecular weight and elution capacity of the samples was consistent and did not induce non-specific adsorption.

[0137] [Example 6]

[0138] In preparing the porous organic polymer carrier α, 27.8 g of glycidyl methacrylate (trade name: Bremmill G (registered trademark) Nippon Oil Co., Ltd.), 11.3 g of glycerol-1,3-dimethacrylate (trade name: NK Ester 701, Shin-Nakamura Chemical Industry Co., Ltd.), 58.7 g of diethyl succinate, and 1.9 g of 2,2'-azobis(2,4-dimethylpentanonitrile) were used as the oil phase. Otherwise, the procedure was the same as in Example 1 to obtain filler β6. The amount of glycidyl methacrylate used was 79.8 mol% of the total monomers, and the amount of glycerol-1,3-dimethacrylate used was 20.2 mol% of the total monomers.

[0139] The alkali resistance of the obtained filler β6 was evaluated using the same procedure as in Example 1. The results showed that the carboxyl group formation amount was 25.0 μmol / mL, confirming that filler β6 possesses excellent alkali resistance.

[0140] Furthermore, the non-specific adsorption evaluation of the obtained packing agent β6 was performed in the same manner as in Example 1. The results showed that the elution capacities of each sample were 7.58 mL, 8.232 mL, 8.465 mL, 9.075 mL, 9.531 mL, and 12.011 mL, confirming that the order of molecular weight and elution capacity of the samples was consistent and did not induce non-specific adsorption.

[0141] [Comparative Example 1]

[0142] The ring-opening reaction of the glycidyl group used HO-(C2H4O). l -(C3H6O) m -(C2H4O) n -H shows a block copolymer (Pluronic (registered trademark) L121 (l is 5, m is 68, n is 5, l+m+n is 78, m / (l+m+n) is 87%), manufactured by BASF) 400g, except that filler β7 was obtained by operating in the same manner as in Example 1.

[0143] The nonspecific adsorption evaluation of the obtained packing agent β7 was performed in the same manner as in Example 1. The results showed that the elution capacities for each sample were 11.557 mL, 11.584 mL, 11.585 mL, 11.866 mL, 14.750 mL, and 12.487 mL, confirming a discrepancy between the molecular weight and elution capacity, which induced nonspecific adsorption.

[0144] The alkali resistance of the obtained filler β7 was evaluated using the same procedure as in Example 1. The result showed that the carboxyl group formation amount was 11.2 μmol / mL.

[0145] [Comparative Example 2]

[0146] The ring-opening reaction of the glycidyl group used 400g of PEG2000 (manufactured by Kanto Chemical Co., Ltd., average molecular weight 1900-2100) as polyethylene glycol. Otherwise, the procedure was the same as in Example 1 to obtain filler β8. In this Comparative Example 2, it did not contain the substance equivalent to -(C3H6O). m - a block structure.

[0147] The nonspecific adsorption evaluation of the obtained packing agent β8 was performed in the same manner as in Example 1. The results showed that the elution capacities of the samples were 11.1702 mL, 11.5425 mL, 11.6449 mL, 11.9073 mL, 12.0849 mL, and 13.1952 mL, confirming that the order of molecular weight and elution capacity was consistent and that no nonspecific adsorption was induced.

[0148] The alkali resistance of the obtained filler β8 was evaluated in the same manner as in Example 1. The results showed that the carboxyl group formation amount of filler β8 was 148.3 μmol / mL.

[0149] [Comparative Example 3]

[0150] In preparing the polymer α with glycidyl groups, 37.1 g of glycidyl methacrylate (trade name: Bremmill G (registered trademark), manufactured by Nippon Oil Co., Ltd.), 2.0 g of ethylene glycol dimethacrylate (trade name: NK Ester 1G, Shin-Nakamura Chemical Industry Co., Ltd.), 47.0 g of butyl acetate, 11.7 g of chlorobenzene, and 1.9 g of 2,2'-azobis(2,4-dimethylpentanonitrile) were used as the oil phase. Otherwise, the procedure was the same as in Example 1 to obtain filler β9. The amount of glycidyl methacrylate used was 96.3 mol% of the total monomers, and the amount of ethylene glycol dimethacrylate used was 3.7 mol% of the total monomers.

[0151] The alkali resistance of the obtained packing agent β9 was evaluated using the same procedure as in Example 1. The result showed a carboxyl group formation amount of 20.5 μmol / mL. The obtained packing agent β9 was then used to attempt packing a stainless steel column. However, high back pressure and difficulty in liquid delivery prevented packing. Therefore, non-specific adsorption evaluation could not be performed.

[0152] [Comparative Example 4]

[0153] 13.7 g of glycidyl methacrylate (trade name: Bremmar G (registered trademark), manufactured by Nippon Oil Co., Ltd.), 25.4 g of ethylene glycol dimethacrylate (trade name: NK Ester 1G, Shin-Nakamura Chemical Industry Co., Ltd.), 47.0 g of butyl acetate, 11.7 g of chlorobenzene, and 1.9 g of 2,2'-azobis(2,4-dimethylpentanonitrile) were used in the oil phase. Otherwise, the procedure was the same as in Example 1 to obtain filler β10. The amount of glycidyl methacrylate used was 42.9 mol% of the total monomers, and the amount of ethylene glycol dimethacrylate used was 57.1 mol% of the total monomers.

[0154] The alkali resistance of the obtained filler β10 was evaluated in the same manner as in Example 1. The result showed that the carboxyl group formation amount was 44.3 μmol / mL.

[0155] The nonspecific adsorption evaluation of the obtained packing agent β10 was performed in the same manner as in Example 1. The results showed that the elution capacities for each sample were 11.437 mL, 12.930 mL, 11.883 mL, 12.094 mL, 13.866 mL, and 12.986 mL, confirming a discrepancy between the molecular weight and elution capacity, which induced nonspecific adsorption.

[0156] In addition, it was confirmed that the exclusion limit molecular weight of the fillers obtained in Examples 1-6 and Comparative Examples 1, 2 and 4 was all above 1 million.

[0157] Based on the above results, by adopting the configuration of the present invention, a filler with high alkali resistance and suppressed non-specific adsorption can be obtained.

[0158] Comparative Example 1, which demonstrates that any ratio of l, m, and n exceeds the scope of this invention, induced nonspecific adsorption and did not contain the equivalent of -(C3H6O). m In Comparative Example 2, the alkali resistance of the - block decreased.

[0159] Furthermore, Comparative Example 3, which showed that the amount of repeating units derived from polyfunctional monomers was too small, resulted in high back pressure on the size exclusion chromatography column, rendering it unusable. In Comparative Example 4, which showed that the amount of repeating units derived from polyfunctional monomers was too large, nonspecific adsorption was induced.

[0160] surface

[0161]

[0162] 1) PEG2000: Polyethylene glycol #2000 is expressed as a total of 1+n since it does not contain m.

[0163] 2): ○ No non-specific adsorption, ×: Non-specific adsorption exists, -: Cannot be measured.

Claims

1. A filler which is obtained by binding one end of a block copolymer represented by the following Formula 1 to a porous organic polymer carrier via an ether bond derived from a terminal hydroxyl group, the porous organic polymer carrier containing 60 to 95 mol% of a repeating unit derived from glycidyl methacrylate and 5 to 40 mol% of a repeating unit derived from a multifunctional monomer, Formula 1 ###0001### (wherein, in Formula 1, 1 is an integer of 5 to 140, m is an integer of 15 to 75, and n is an integer of 5 to 140; the proportion of m in the total of 1, m, and n is 5 to 85%). Formula 1 HO-(C2H4O) l -(C3H6O) m -(C2H4O) n -H In Formula 1, 1 is an integer of 5 to 140, m is an integer of 15 to 75, and n is an integer of 5 to 140; the proportion of m in the total of 1, m, and n is 5 to 85%, The multifunctional monomer is a (meth)acryl-based monomer containing two or more (meth)acryl groups.

2. The filler according to claim 1, wherein the total of 1, m, and n of the block copolymer is 30 to 330.

3. The filler according to claim 1, wherein the multifunctional monomer is any of ethylene glycol dimethacrylate and glycerol-1, 3-dimethacrylate.

4. The filler according to claim 1, which has an exclusion limit molecular weight of 1 million or more.

5. A method for producing the filler according to any one of claims 1 to 4, comprising the following steps: Step (A) which is a step of polymerizing raw monomers containing glycidyl methacrylate and a multifunctional monomer in the presence of a diluent and a polymerization initiator to obtain a porous organic polymer carrier α, wherein the concentration of the multifunctional monomer in the raw monomers is 5 to 40 mol%, and the diluent is used in a volume of 0.8 to 4.0 times relative to the total volume of the raw monomers; and Step (B) which is a step of ring-opening the glycidyl group derived from glycidyl methacrylate of the porous organic polymer carrier α with the block copolymer represented by Formula 1 to obtain a filler β in which one end of the block copolymer represented by Formula 1 is bound to the porous organic polymer carrier α via an ether bond derived from a terminal hydroxyl group, Formula 1 ###0002### (wherein, in Formula 1, 1 is an integer of 5 to 140, m is an integer of 15 to 75, and n is an integer of 5 to 140; the proportion of m in the total of 1, m, and n is 5 to 85%). Formula 1 wherein In Formula 1, 1 is an integer of 5 to 140, m is an integer of 15 to 75, and n is an integer of 5 to 140; the proportion of m in the total of 1, m, and n is 5 to 85%. The filler according to any one of claims 1 to 4 is filled in a housing for liquid chromatography. ​ HO-(C2H4O) l -(C3H6O) m -(C2H4O) n -H ​ 6. A column for size exclusion chromatography, wherein, ​

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