Porous particles and method for producing the same

Abstract

This application relates to porous particles and a method for producing the same. The porous particles provided by this application have properties suitable for protein purification, particularly affinity chromatography.

Description

Technical Field

[0001] [Cross-reference to Related Applications] This application claims the benefit of priority based on Korean Patent Application No. 10-2023-0013569 filed on February 1, 2023 and Korean Patent Application No. 10-2023-0061331 filed on May 11, 2023, and all contents disclosed in the documents of the Korean patent applications are included as part of this specification.

[0002] The present invention relates to porous particles and a method for manufacturing the same. Specifically, the present invention relates to porous particles for affinity chromatography and a method for manufacturing the same.

Background Art

[0003] As the fields of biopharmaceuticals and regenerative medicine expand, the demand for systems capable of efficiently separating and purifying cells, tissues, microorganisms, etc. is increasing.

[0004] In conventional affinity chromatography technology, purification resins using hydrophilic agarose as a raw material have been mainly used. However, such resins swell due to the buffer during the purification process and their strength decreases. As a result, there has been a problem that the purification rate and purification efficiency decrease. Therefore, attempts have been made to manufacture synthetic polymers with improved strength.

[0005] When using purification resins, non-specific binding of impurities mainly occurs through hydrophobic interaction. The adsorbed impurities are fouled on the purification resin particles, which may adversely affect the activity of the Protein A ligand immobilized on the resin and the binding of the target substance. In addition, the impurities may be eluted together with the purified target substance, resulting in a decrease in the purity of the final product. If the impurities are administered to patients, it may also induce an immune reaction.

[0006] Therefore, there is a need for technological development related to particles capable of reducing non-specific adsorption and a method for manufacturing the same.

Summary of the Invention

Problems to be Solved by the Invention

[0007] One object of the present invention is to provide porous particles of an open pore system.

[0008] Another object of the present invention is to provide porous particles with little non-specific adsorption.

[0009] Another object of the present invention is to provide particles for protein purification (e.g., column resin).

[0010] Another object of the present invention is to provide porous particles suitable for affinity chromatography.

[0011] Another object of the present invention is to provide a method for manufacturing the porous particles as described above.

[0012] The above objects and other objects of the present invention can all be solved by the present invention described in detail below.

Means for Solving the Problems

[0013] According to a specific example of the present invention, porous particles and a method for manufacturing the same are provided. The particles can be used as particles for protein purification (column resin), specifically for affinity chromatography. At this time, the "porous particles" mean particles having voids inside and / or on the surface of the particles. More specifically, the porous particles mean particles having an open pore system.

[0014] Particles for protein purification (column resin) are exposed to a solution containing antibody proteins and other impurities derived from cell culture. Therefore, a situation occurs where impurities are non-specifically adsorbed onto the particle surface. In order to ensure the binding between the particles and the target substance and increase the purity of the final product, non-specific adsorption of impurities must be reduced. The inventor of the present invention completed the present invention after confirming through experiments that the production method described below and the particles produced thereby can reduce such non-specific adsorption.

[0015] Specifically, in affinity chromatography, for porous particles to have sufficient purification ability, the target substance (e.g., IgG) must be able to bind as much as possible to the protein A ligand. In order to provide as much such binding as possible, since the specific surface area of the particles is high, it is necessary to immobilize a large amount of the ligand on the particles. In order to increase the specific surface area, the pores of the particles must be sufficiently large and the number of pores (or porosity) must also be ensured at a high level. This is because when the pores of the particles are small, the ligand may not be able to move to the inside of the particles during ligand immobilization, and the target substance may not be able to move inside during purification of the target substance. Also, for smooth progress of purification, the morphology of the particles needs to be uniform.

[0016] And in the purification process, the adsorption between the impurity protein and the purification particles mainly occurs through the hydrophobic bond between the impurity protein and the purification particles. Therefore, the more hydrophilic groups the particles have, the more the hydrophobic bond (non-specific adsorption) can be reduced.

[0017] Considering such points, the inventor of the present invention repeated research on the substances used in the production of porous particles, their content, reaction conditions, and surface characteristics of the particles, and completed the present invention.

[0018] Hereinafter, the present invention will be described in more detail.

[0019] In an example related to the present invention, the present invention relates to a method for producing porous particles. The porous particles produced by the method of the present invention can have properties suitable for protein purification applications, specifically affinity chromatography.

[0020] Specifically, the method includes a step of performing a suspension polymerization reaction after mixing a dispersed phase composition (B) and a continuous phase composition (A) in a reactor. At this time, the "dispersed phase composition" means a composition that can form a dispersed phase (or droplets) after being mixed with the continuous phase composition, and the "continuous phase composition" means a composition that can form a continuous phase after being mixed with the dispersed phase composition.

[0021] In a specific example of the present invention, the dispersed phase composition includes a polymerizable monomer (b1) containing an epoxy group, a crosslinking agent (b2), and a porogen (b3). In a specific example of the present invention, the dispersed phase composition can additionally include an initiator (b4) in addition to the polymerizable monomer (b1) containing an epoxy group, the crosslinking agent (b2), and the porogen (b3). In another specific example, the dispersed phase composition may be a mixture composed of the above-described components.

[0022] A suspension polymer obtained containing units derived from the polymerizable monomer (b1) and the crosslinking agent (b2) can be obtained through suspension polymerization of the dispersed phase composition. More specifically, a suspension polymer containing units derived from the polymerizable monomer (b1) and the crosslinking agent (b2) through the suspension polymerization, or a reaction product of the polymerizable monomer (b1) and the crosslinking agent (b2) (performed by an initiator) can be obtained. The suspension polymer may be particles or a group of particles containing the same, and each individual particle can have the properties of the particles described below.

[0023] In one example, the polymerizable monomer (b1) can be a monomer containing an unsaturated carbon-carbon bond and an epoxy group (or glycidyl group). The type of such a polymerizable monomer is not particularly limited. For example, it may be a (meth)acrylate monomer having an epoxy group. More specifically, examples include glycidyl (meth)acrylate, 4,5-epoxybutyl (meth)acrylate, or 9,10-epoxystearyl (meth)acrylate. However, the types of polymerizable monomers included in the dispersed phase composition of the present invention are not specified by the monomers listed above.

[0024] The dispersed phase composition further includes a crosslinking agent (b2). In a specific example of the present invention, as the crosslinking agent, a compound containing at least two carbon-carbon double bonds (e.g., vinyl groups) capable of participating in a crosslinking reaction can be used.

[0025] Examples of crosslinking agents that can be used according to specific examples of the present invention include polyfunctional (meth)acrylates such as ethylene glycol di(meth)acrylate, trimethylolpropane tri(meth)acrylate, pentaerythritol tri(meth)acrylate, dipentaerythritol hexa(meth)acrylate, dipentaerythritol hexamethacrylate, or trimethylpropane trimethacrylate. Or one or more components among the listed crosslinking agents can also be used together.

[0026] In one example, the crosslinking agent compound may not contain a hydrophilic group (e.g., a hydroxy group). As a result of experimental confirmation, when the crosslinking agent contains a hydrophilic group in addition to the carbon-carbon double bond, the reaction rate of the crosslinking agent may decrease, and conditions may be formed where it is difficult to create sufficient pores in the particles.

[0027] The dispersed phase composition further includes a porogen (b3). A porogen is a component that does not participate in the polymerization reaction but can escape from the dispersed phase droplets during the suspension polymerization reaction to form voids (pores) in the polymer particles.

[0028] In a specific example of the present application, the porogen (b3) is a porogen having a difference in Hansen solubility parameter from the epoxy group-containing polymerizable monomer (b1) of 1.30 to 4.70, or can contain such a porogen. Specifically, the difference in Hansen solubility parameter between the epoxy group-containing polymerizable monomer (b1) and the porogen can be 1.40 or more, 1.50 or more, 1.60 or more, 1.70 or more, 1.80 or more, 1.90 or more, 2.00 or more, 2.10 or more, 2.20 or more, 2.30 or more, 2.40 or more, 2.50 or more, 2.60 or more, 2.70 or more, 2.80 or more, 2.90 or more, 3.00 or more, 3.10 or more, 3.20 or more, 3.30 or more, 3.40 or more, 3.50 or more, 3.60 or more, 3.70 or more, 3.80 or more, 3.90 or more, or 4.00 or more. And the upper limit can be, for example, 4.60 or less, 4.50 or less, 4.40 or less, 4.30 or less, 4.20 or less, 4.10 or less, 4.00 or less, 3.90 or less, 3.80 or less, 3.70 or less, 3.60 or less, 3.50 or less, 3.40 or less, 3.30 or less, 3.20 or less, 3.10 or less, or 3.00 or less. As a result of experimental confirmation, when the difference in Hansen solubility parameter as described above is satisfied, it is possible to provide particles having a porous structure with surface pores of a sufficient size through which the target substance can flow into the interior of the particles and reach even the center of the particles.

[0029] In one example, as the porogen, a non-aromatic substance can be used. For example, the porogen can be or can include an acetate-based substance (porogen). In a specific example, as the porogen component, one or more selected from the group consisting of butyl acetate, isobutyl acetate, amyl acetate, isoamyl acetate, hexyl acetate, isohexyl acetate, and heptyl acetate can be used. When using an acetate-based porogen that satisfies the above-described solubility parameter-related numerical values, it is advantageous for the particles to ensure sufficient purification ability in affinity chromatography applications. Specifically, when other types of porogens (e.g., aromatic porogens such as toluene and mesitylene) are used instead of the above-described acetate-based porogens, it has been experimentally confirmed that there is a large deviation regarding the degree of generation of cracked particles. That is, when manufacturing particles as in the present invention, the ratio of cracked particles can be significantly reduced (stable formation of non-cracked particles), and particles having sufficiently large pores can be provided, and as a result, it is advantageous for reducing non-specific adsorption.

[0030] In a specific example of the present invention, the dispersion phase composition can further include an initiator (b4). The type of initiator is not particularly limited as long as it does not hinder the securing of the particle characteristics required in the present application. For example, initiators such as organic peroxide initiators and azo group initiators can be used. Specifically, compounds such as benzoyl peroxide, di-t-amyl peroxide, t-butyl peroxybenzoate, 2,5-dimethyl-2,5-di-(t-butylperoxy)hexane, 2,5-dimethyl-2,5-di-(t-butylperoxy)hexyne-3, di-cumyl peroxide, lauryl peroxide, 2,2'-azobis(2,4-dimethylvaleronitrile), and mixtures thereof can be used, but are not limited thereto.

[0031] In a specific example of the present invention, the dispersed phase composition can contain 50 parts by weight or more of the polymerizable monomer (b1) based on 100 parts by weight of the crosslinking agent (b2). For example, based on 100 parts by weight of the crosslinking agent (b2), the dispersed phase composition can contain 60 parts by weight or more, 70 parts by weight or more, 80 parts by weight or more, 90 parts by weight or more, 100 parts by weight or more, 110 parts by weight or more, 120 parts by weight or more, 130 parts by weight or more, 140 parts by weight or more, 150 parts by weight or more, 160 parts by weight or more, 170 parts by weight or more, 180 parts by weight or more, 190 parts by weight or more, or 200 parts by weight or more of the polymerizable monomer (b1). The upper limit of the content of the polymerizable monomer (b1) in the dispersed phase composition can be, for example, 400 parts by weight or less, 350 parts by weight or less, 300 parts by weight or less, 250 parts by weight or less, 200 parts by weight or less, 150 parts by weight or less, or 100 parts by weight or less. Further, in a specific example of the present invention, based on 100 parts by weight of the crosslinking agent (b2), the polymerizable monomer (b1) can be contained in an amount of 100 parts by weight or more, 150 parts by weight or more, or 200 parts by weight or more. When the above-described content is satisfied, an emulsion can be stably formed, cracking of particles during polymerization can be significantly suppressed, and an appropriate level of particle strength can be ensured without making the pore size of the final particles excessively large or excessively small. In particular, when the contents of the components (b1) and (b2) satisfy the above range, the ratio of cracked particles can be reduced (non-cracked particles can be stably formed), and particles having sufficiently large pores can be provided, and as a result, it is advantageous for reducing non-specific adsorption.

[0032] In a specific example of the present invention, the dispersed phase composition can contain 45% by weight or more of the porogen (b3) based on 100% by weight of the total content of the polymerizable monomer (b1), the crosslinking agent (b2), and the porogen (b3). For example, when based on 100% by weight of the total content, the content of the porogen (b3) (which can be, for example, an acetate-based porogen) can be 50% by weight or more, 55% by weight or more, or 60% by weight or more. And the upper limit of its content can be, for example, 70% by weight or less, 65% by weight or less, 60% by weight or less, or 55% by weight or less. When the above-mentioned content is satisfied, it is advantageous for stably forming an emulsion and ensuring particle characteristics suitable for use (e.g., characteristics of few particle cracks and high spheroidization rate). In particular, when the contents of components (b1) and (b2) satisfy the above range, the ratio of cracked particles can be reduced (stably forming non-cracked particles), and particles having sufficiently large pores can be provided, and as a result, it is advantageous for reducing non-specific adsorption.

[0033] The content of the initiator (b4) is not particularly limited. An appropriate amount of the initiator can be used at a level that does not hinder ensuring the characteristics of the target particles. For example, the dispersed phase composition can contain 0.1 part by weight or more, specifically 0.2 part by weight or more, 0.3 part by weight or more, 0.4 part by weight or more, 0.5 part by weight or more, 0.6 part by weight or more, 0.7 part by weight or more, 0.8 part by weight or more, 0.9 part by weight or more, or 1.0 part by weight or more of the initiator based on 100 parts by weight of the combined weight of the polymerizable monomer and the crosslinking agent. And the upper limit of the content of the initiator can be, for example, 5.0 parts by weight or less, specifically 4.5 parts by weight or less, 4.0 parts by weight or less, 3.5 parts by weight or less, 3.0 parts by weight or less, 2.5 parts by weight or less, 2.0 parts by weight or less, 1.5 parts by weight or less, or 1.0 part by weight or less.

[0034] In one example, the continuous phase composition (A) can contain water and a polymeric surfactant.

[0035] Although not particularly limited, the water can be distilled water or deionized water (DIW).

[0036] In one example, the continuous phase composition can be a mixture of water and a polymeric surfactant. That is, the continuous phase composition can consist only of water and a polymeric surfactant.

[0037] In a specific example of the present invention, the polymeric surfactant can have a weight average molecular weight (MW) within a predetermined range and / or a hydrolyzed degree within a predetermined range.

[0038] In one example, the weight average molecular weight of the polymeric surfactant can be 60,000 or more. Specifically, the lower limit of the weight average molecular weight can be, for example, 65,000 or more, 70,000 or more, 80,000 or more, 85,000 or more, 90,000 or more, 95,000 or more, 100,000 or more, 105,000 or more, 110,000 or more, 115,000 or more, 120,000 or more, 125,000 or more, 130,000 or more, 135,000 or more, 140,000 or more. And its upper limit can be 200,000 or less. Specifically, the upper limit of the weight average molecular weight can be, for example, 190,000 or less, 185,000 or less, 180,000 or less, 175,000 or less, 170,000 or less, 165,000 or less, 160,000 or less, 155,000 or less, 150,000 or less, 145,000 or less, 140,000 or less, 135,000 or less, 130,000 or less, 125,000 or less, 120,000 or less, 115,000 or less, or 110,000 or less. The weight average molecular weight can be measured using GPC.

[0039] In one example, the polymeric surfactant can have a degree of hydration in the range of 80 to 99%. More specifically, the lower limit of the degree of hydration can be, for example, 81% or more, 82% or more, 83% or more, 84% or more, or 85% or more, and the upper limit can be, for example, 98% or less, 97% or less, 96% or less, 95% or less, 94% or less, 93% or less, 92% or less, 91% or less, or 90% or less. As the surfactant having the degree of hydration as described above, for example, polyvinyl alcohol can be used. The degree of hydration can be measured using 1H-NMR.

[0040] Examples of polymeric surfactants that can be used include poly(vinyl alcohol) (PVA) or polyvinylpyrrolidone.

[0041] In one example, in the continuous phase composition, the concentration of the polymeric surfactant can be 1.0% or more. For example, when a continuous phase composition that is a mixture of water and a polymeric surfactant is used, based on the total weight (100% by weight) of the continuous phase composition, the content of the polymeric surfactant can be 1.0% by weight or more. Specifically, the lower limit of the concentration of the polymeric surfactant in the continuous phase composition (A) can be 1.5% by weight or more, 2.0% by weight or more, 2.5% by weight or more, 3.0% by weight or more, or 3.5% by weight or more. And the upper limit of the concentration of the polymeric surfactant can be less than 4.0%, specifically, for example, 3.5% or less, 3.0% or less, 2.5% or less, 2.0% or less, or 1.5% or less. When using a polymeric surfactant that satisfies the above concentration, uniform and stable droplets can be formed when the continuous phase and the dispersed phase are mixed, which is advantageous for obtaining the shape of the final particles targeted in the present invention.

[0042] In one example, the weight ratio of the dispersed phase composition to the continuous phase composition (weight ratio of the dispersed phase composition: weight ratio of the continuous phase composition) can be in the range of 1:2 to 1:15, 1:4 to 1:12, or 1:6 to 1:10.

[0043] The suspension polymerization can be carried out under conditions that do not interfere with ensuring the particle characteristics by the production method of the present invention. For example, the suspension polymerization reaction can be carried out at a temperature of 100°C or lower, more specifically, at a temperature of 40 to 95°C. And during the polymerization reaction at the above temperature, stirring can be carried out at a speed of 600 to 1,000 rpm.

[0044] In one example, the method can further include a washing step after the completion of the suspension polymerization reaction. By such washing, impurities unrelated to the particles that are the suspension polymer can be removed. The washing method is not particularly limited, and known washing methods can be used. For example, the washing can be carried out by putting the suspension polymer into a washing liquid containing alcohol (e.g., alcohol containing ethanol) and / or water at a high temperature (e.g., 40 to 60°C) (e.g., distilled water) and stirring. Although not particularly limited, such washing can be repeated, for example, two or more times.

[0045] In one example, the method can further include a drying step after the above washing. Through drying, solvent residues and the like can be removed. The drying method is not particularly limited, and known drying methods can be used. For example, the drying can be carried out using an oven or under normal temperature conditions. Also, although not particularly limited, the drying can be carried out under vacuum.

[0046] In one example, the method can further include the steps of centrifuging, washing, and drying the suspension polymer after the completion of the suspension polymerization reaction. The explanations regarding washing and drying are the same as above.

[0047] The suspension polymer, that is, the particles produced by the above method including suspension polymerization can have characteristics suitable for protein purification or separation applications.

[0048] In one example, the particles produced by the method of the present invention can have a size within the range of 30 to 70 μm. At this time, the particle size refers to the particle size (D 50) means. And D 50 means the size of the particles corresponding to 50% volume accumulation of the particle size distribution. Specifically, the particles produced according to the present invention may have a lower limit of the size of 35 μm or more, 40 μm or more, or 45 μm or more, and the upper limit may be 65 μm or less, 60 μm or less, 55 μm or less, 50 μm or less, or 45 μm or less. When exceeding the particle range, the number of particles that can be filled in the purification column decreases, and the content of the ligand attached to the particle surface decreases, resulting in a decrease in purification efficiency in some cases. And when the size of the particles is smaller than the particle size, the density of the packed beads filled in the purification column becomes excessively large, making it difficult for the solution to be purified to escape, resulting in a decrease in purification efficiency in some cases.

[0049] In one example, the method of the present invention can provide a group of particles with a low ratio of cracked particles. For example, the number ratio of cracked particles among all the produced particles can be 20% or less, 15% or less, 10% or less, or 5% or less. That is, according to the present invention, particles with a stable shape are provided. When the ratio of cracked particles is high, the production efficiency can be considered low. Furthermore, when the particles are used for protein purification, the cracked particles may clog the passage through which the solution flows, resulting in a decrease in purification efficiency, and the cracked particles may escape from the column outlet, reducing the product purity in some cases.

[0050] In one example, the particles can exhibit a high spheroidization rate. For example, the spheroidization rate calculated by the ratio of the size (D 50 ) of the particles to the length (L) of the particles having the longest dimension length (L) among all the produced particles can be 80% or more, 85% or more, or 90% or more. That is, according to the present invention, uniform particles with a high degree of spheroidization are provided. When the spheroidization rate is low, the particles may not be sufficiently filled in the column, resulting in a decrease in purification efficiency in some cases.

[0051] In one example, the particles can have a pore size of 30 nm or more when measuring the pore size by mercury intrusion porosimetry. Mercury intrusion porosimetry is a measurement method that converts the pore size through the amount of mercury flowing in from the surface to the inside of the particles by pressure. According to a specific example of the present invention, the data measured in relation to mercury intrusion porosimetry can be displayed in a histogram, and the size of the pores of the particles can be calculated from the measurement of the total intrusion volume and the ratio of the total pore area at the maximum mercury injection pressure (at this time, the size of the pores of the particles can be regarded as the average size, and the average means the arithmetic mean). If the pores of the particles are excessively small, only impurities smaller in size than the target protein may flow into and be adsorbed inside the particles, resulting in an increase in non-specific adsorption and a possible decrease in purification efficiency. For example, the pore size of the particles can be 35 nm or more, 40 nm or more, 45 nm or more, 50 nm or more, 55 nm or more, or 60 nm or more. Also, the upper limit of the average pore size of the particles can be 500 nm or less. If the average pores of the particles are excessively large, the purification efficiency may decrease due to an increase in non-specific adsorption caused by an increase in the residence time of impurities in the interparticle voids smaller than the particle pores, and the purification efficiency may decrease due to a decrease in the specific surface area inside the particles and a decrease in the content of the attached ligand. For example, the pore size of the particles can be 400 nm or less, 300 nm or less, 200 nm or less, or 100 nm or less.

[0052] In one example, the average surface pore size of the particles can be in the range of 110 to 240 nm. In a specific example of the present invention, the average surface pore size is related to the degree of openness of the particle surface, and means the (arithmetic) average value calculated based on measuring the pore sizes of 20 or more (wherein the pore size means the length of the largest dimension among the shapes of the pores) on the SEM image of the particle surface. The degree of openness of the particle surface cannot be confirmed only by the pore size of the particles by mercury intrusion method. Considering the flow of the target substance or fluid through the particles, the specific surface area of the particles, and the mechanical strength of the particles, etc., such average surface pore size can be, for example, 120 nm or more, 130 nm or more, 140 nm or more, 150 nm or more, 160 nm or more, 170 nm or more, 180 nm or more, 190 nm or more or 200 nm or more, and the upper limit can be, for example, 230 nm or less, 220 nm or less, 210 nm or less, 200 nm or less, 190 nm or less, 180 nm or less, 170 nm or less, 160 nm or less or 140 nm or less.

[0053] In one example, the porosity of the particles can satisfy the range of 15 to 40%. Specifically, the porosity of the particles can be, for example, 16% or more, 17% or more, 18% or more, 19% or more, 20% or more, 21% or more, 22% or more, 23% or more, 24% or more or 25% or more, and can be 39% or less, 38% or less, 37% or less, 36% or less, 35% or less, 34% or less, 33% or less, 32% or less, 31% or less or 30% or less. When the porosity is smaller than the aforementioned range, it is difficult to expect high purification efficiency because the specific surface area of the particles is low, and when the porosity exceeds the aforementioned range, the mechanical strength may deteriorate.

[0054] In a specific example of the present invention, the method further includes a predetermined surface treatment step for the suspension polymer. Specifically, the method further includes a step of hydrophilizing the surface of the suspension polymer through epoxy group deactivation.

[0055] The particles obtained through the suspension polymerization described above have epoxy groups derived from the epoxy group-containing polymerizable monomer. In the present invention, an epoxy inactivator (e.g., a compound having an epoxy reactive group and a hydrophilic group different therefrom) is reacted with the particles formed by the suspension polymerization to inactivate the epoxy groups on the particle surface and introduce a hydrophilic group onto the surface of the particles. Through the introduction of a hydrophilic group by epoxy group inactivation, non-specific adsorption caused by hydrophobic binding between impurity proteins and purified particles can be reduced.

[0056] As described above, in a specific example of the present invention, at least 50 parts by weight or more of the epoxy group-containing polymerizable monomer (b1) is used with respect to 100 parts by weight of the crosslinking agent (b2), so that the particles which are suspension polymers have a sufficient amount of epoxy groups on their surfaces. Since a sufficient amount of epoxy groups on the particle surface is ensured, when the epoxy inactivator and the particles react with each other, the surface hydrophilization of the particles by epoxy inactivation can also be sufficiently achieved.

[0057] The hydrophilization can be carried out by reacting an epoxy inactivator having an epoxy reactive group and a hydrophilic group different therefrom with the suspension polymer having the epoxy groups. For example, such epoxy group inactivation can be carried out through a method of injecting the particles produced into an epoxy inactivator or a solution containing the same. At this time, the solvent used is not particularly limited. Further, the reaction for surface hydrophilization can be carried out, for example, within the range of 30 to 50 °C for 10 to 30 hours.

[0058] In one example, the epoxy inactivator can be a compound having an epoxy reactive group and a hydrophilic group different therefrom. The epoxy reactive group can be, for example, a thiol group or an amine group, and the hydrophilic group different from the epoxy reactive group can be, for example, a hydroxy group.

[0059] For example, the inactivator may contain one or more selected from 1-thioglycerol, ethanolamine, and 2-mercaptoethanol. For example, when 1-thioglycerol is used as the inactivator, epoxy group inactivation occurs by an epoxy-thiol reaction, and when ethanolamine is used as the inactivator, epoxy group inactivation occurs by an epoxy-amine reaction. However, the former case is more advantageous in terms of reaction efficiency. Therefore, it is preferable to use an epoxy inactivator that contains a thiol group as an epoxy reaction group and a hydroxy group as a hydrophilic group different from the epoxy group, such as 1-thioglycerol.

[0060] In a specific example of the present invention, a suspension polymerization reaction is carried out after mixing a continuous phase composition (A) and a dispersed phase composition (B) to produce a suspension polymer containing units derived from the polymerizable monomer (b1) and the crosslinking agent (b2); and a method including the step of hydrophilizing the surface of the suspension polymer through epoxy group inactivation can provide porous particles showing a low non-specific adsorption rate. For example, the method can provide the porous particles having a non-specific adsorption rate of 1.5% or less with respect to BSA (Bovine serum albumin). Specifically, the particles produced by the method can satisfy a non-specific adsorption rate of 1.0% or less, more specifically 0.5% or less, 0.45% or less, 0.4% or less, 0.35% or less, 0.3% or less, 0.25% or less, 0.2% or less, 0.15% or less, 0.1% or less, 0.05% or less, or 0.01% or less with respect to BSA (Bovine serum albumin). The non-specific adsorption rate is measured with respect to a mixture containing the porous particles, BSA, and water, and is calculated as the content (weight) ratio of the BSA adsorbed on the porous particles to the total content (weight) of BSA introduced into the mixture. According to a specific example of the present invention, in the mixture for measuring the non-specific adsorption rate, the porous particles and the BSA can be contained at a weight ratio of porous particles 1 to 3:BSA 1.

[0061] In one example, the method can provide particles that meet a predetermined hydrophilic group content. Specifically, the method can provide particles in which the content of hydrophilic groups per unit weight of the particles (μmol / g) is 500 μmol / g or more, specifically 1000 μmol / g or more. At this time, the content of hydrophilic groups per unit weight of the particles (μmol / g) can be calculated by multiplying the epoxy group content (μmol / g) of the suspension polymer obtained by suspension polymerization before the surface hydrophilization treatment by the number of hydrophilic groups in the epoxy inactivator. And the epoxy group content (μmol / g) of the suspension polymer obtained by the suspension polymerization can be calculated by calculating the epoxy group content (μmol) by titration using 0.1 M NaOH with respect to a predetermined weight of the particles and dividing this by the weight of the particles. More specifically, it can be calculated by the method described in the experiments described later. For example, the content of hydrophilic groups (e.g., hydroxy groups) per unit weight of the particles provided by the method can be 1500 μmol / g or more, 2000 μmol / g or more, 2500 μmol / g or more, 3000 μmol / g or more, 3500 μmol / g or more, 4000 μmol / g or more, or 4500 μmol / g or more. And the upper limit of the content of hydrophilic groups per unit weight of the particles can be, for example, 5000 μmol / g or less, specifically 4500 μmol / g or less, 4000 μmol / g or less, 3500 μmol / g or less, or 3000 μmol / g or less.

[0062] Through the treatment using the epoxy inactivator as described above, particles having hydrophilic groups within the above range can provide excellent purification efficiency. Specifically, in the purification process, the adsorption between the impurity protein and the purified particles mainly occurs through the hydrophobic bond between the impurity protein and the purified particles. Therefore, the more hydrophilic groups the particles have at an appropriate level, the more the hydrophobic bond (non-specific adsorption) can be reduced. Such a tendency is also confirmed in the examples described later.

[0063] In one example, as a result of the epoxy deactivation and hydrophilic treatment as described above, the method can provide porous particles in which the epoxy group content on the surface is adjusted to a predetermined value or less. Specifically, the epoxy group content (μmol) per unit weight (g) of the epoxy deactivated particles can be 50 μmol / g or less. Such an epoxy group content (μmol / g) can be calculated by calculating the epoxy group content (μmol) by titration with 0.1 M NaOH for a predetermined weight of particles and dividing this by the weight of the particles. More specifically, it can be calculated by the method described in the experiments described below. At this time, such an epoxy group content (μmol / g) can be calculated by calculating the epoxy group content (μmol) by titration with 0.1 M NaOH for a predetermined weight of particles and dividing this by the weight of the particles. More specifically, it can be calculated by the method described in the experiments described below. Specifically, the epoxy content of the epoxy deactivated particles can be, for example, 45 μmol / g or less, 40 μmol / g or less, 35 μmol / g or less, 30 μmol / g or less, 25 μmol / g or less, 20 μmol / g or less, 15 μmol / g or less, 10 μmol / g or less, or 5.0 μmol / g or less. The epoxy groups of the particles in the present application are derived from a polymerizable substance for particle formation, that is, a polymerizable monomer having an epoxy group. However, on the premise that the particles are actually used for the purification of the target substance, if an excessive amount of epoxy groups remain on the particle surface, these epoxy groups may react or bind with substances other than the target substance (e.g., impurities), thereby reducing the purification efficiency. In consideration of this, particles in which the epoxy groups are deactivated are used in the present invention. Also, the lower limit of the epoxy group content (μmol) per unit weight of the epoxy deactivated particles can be 1 μmol / g or more, 5 μmol / g or more, 10 μmol / g or more, 15 μmol / g or more, 20 μmol / g or more, 25 μmol / g or more, 30 μmol / g or more, 35 μmol / g or more, or 40 μmol / g or more.For reference, when the epoxy group content on the particle surface before inactivation is excessively small, the surface hydrophilic treatment through epoxy inactivation may not be sufficient, and as a result, when the particles are actually used as column resin, they may not be able to bind sufficiently to the substance to be bound to the particles (e.g., rProteinA). Therefore, as described above, it is necessary to use a sufficient content of the polymerizable monomer containing an epoxy group.

[0064] Although not particularly limited, after the epoxy inactivation and hydrophilic treatment are carried out, the particles can be washed and / or dried. The washing and drying can be carried out in the same manner as described above.

[0065] In an example related to the present invention, the present invention relates to porous particles. The porous particles can be produced by the method described above and can be used, for example, as column resin for protein purification and separation.

[0066] In an example of the present invention, the particles contain polymerized units derived from a polymerizable monomer having an epoxy group and a cross-linking agent. The types and properties of the polymerizable monomer having an epoxy group and the cross-linking agent are the same as described above.

[0067] In one example, the polymerizable monomer can be a monomer containing an unsaturated carbon-carbon bond and an epoxy group (or glycidyl group). The types of such polymerizable monomers are not particularly limited. For example, examples of the polymerizable monomer include (meth)acrylate-based monomers having an epoxy group, more specifically glycidyl (meth)acrylate, 4,5-epoxybutyl (meth)acrylate, or 9,10-epoxystearyl (meth)acrylate. However, the types of polymerizable monomers contained in the dispersion composition of the present invention are not specified by these monomers.

[0068] In one example, a polyfunctional (meth)acrylate can be used as the crosslinking agent. Specifically, polyfunctional (meth)acrylates such as ethylene glycol di(meth)acrylate, trimethylolpropane tri(meth)acrylate, pentaerythritol tri(meth)acrylate, dipentaerythritol hexa(meth)acrylate, dipentaerythritol hexamethacrylate or trimethylolpropane trimethacrylate can be used, or one or more components of the listed crosslinking agents can also be used together. However, the crosslinking agent is not limited to the listed substances.

[0069] Other particles of the present invention can further contain substances derived from the manufacturing method described above and the like.

[0070] In one example, the porous particles can have a particle size (D 50 ) within the range of 30 to 70 μm. Since the specific description is the same as above, it is omitted here.

[0071] In one example, the porous particles can have a ratio of the number of cracked particles to the total number of particles of 20% or less. Since the specific description is the same as above, it is omitted here.

[0072] In one example, the porous particles can satisfy a spheroidization rate of 80% or more. Since the specific description is the same as above, it is omitted here.

[0073] In one example, the porous particles can have a pore size of 30 nm or more. Since the specific description is the same as above, it is omitted here.

[0074] In one example, the porous particles can have a surface pore size in the range of 110 to 240 nm. Since the specific description is the same as above, it is omitted here.

[0075] In one example, the porous particles can have a porosity in the range of 15 to 40%. Since the specific description is the same as above, it is omitted here.

[0076] In one example, the particles can be surface-treated with a hydrophilic treatment, that is, an epoxy deactivator having an epoxy reactive group and a hydrophilic group different from the epoxy reactive group.

[0077] Epoxy deactivation and hydrophilic treatment of the particles would mean that they are surface-treated with an epoxy deactivator having an epoxy reactive group and a hydrophilic group different from the epoxy reactive group. Refer to the above for such surface treatment.

[0078] In one example, the porous particles can be particles having a non-specific adsorption rate for BSA (Bovine serum albumin) of 1.5% or less. Since the specific description is the same as above, it is omitted here.

[0079] In one example, as a result of the epoxy deactivation and hydrophilic treatment as described above, the porous particles can satisfy a predetermined hydrophilic group content. Specifically, the porous particles can satisfy a content of hydrophilic groups (μmol / g) per unit weight of the epoxy deactivated particles of 500 μmol / g or more, specifically 1000 μmol / g or more. Since the specific description is the same as above, it is omitted here.

[0080] In one example, as a result of the epoxy deactivation and hydrophilic treatment as described above, the porous particles can satisfy a predetermined epoxy group content. Specifically, the porous particles can have an epoxy group content (μmol) per unit weight (g) of the epoxy deactivated particles of 50 μmol / g or less. Since the specific description is the same as above, it is omitted here.

Advantages of the Invention

[0081] According to a specific example of the present invention, when used for protein purification particle (column resin) applications, particles are provided that can improve the non-specific adsorption of impurities to the particles. Thereby, the binding capacity of the antibody protein to the particles can be made sufficient, and a high purity of the final product can be ensured.

Brief Description of the Drawings

[0082] Figures 1 to 7 are photographs of the surfaces of particles (base resins) produced in the examples and comparative examples.

[0083] [Figure 1] Figure 1 is a photograph of the surface of the particles (base resin) of Example 1 taken using an SEM apparatus. [Figure 2] Figure 2 is a photograph of the surface of the particles (base resin) of Example 2 taken using an SEM apparatus. [Figure 3] Figure 3 is a photograph of the surface of the particles (base resin) of Example 3 taken using an SEM apparatus. [Figure 4] Figure 4 is a photograph of the surface of the particles (base resin) of Comparative Example 5 taken using an SEM apparatus. [Figure 5] Figure 5 is a photograph of the surface of the particles (base resin) of Comparative Example 6 taken using an SEM apparatus. [Figure 6] Figure 6 is a photograph of the surface of the particles (base resin) of Comparative Example 7 taken using an SEM apparatus. [Figure 7] Figure 7 is a photograph of the surface of the particles (base resin) of Comparative Example 11 taken using an SEM apparatus.

[0084] In each drawing, the white bar means a size of 100 nm, and the black portions observed from the particles are pores.

[0085] Comparing the drawings of the examples and the comparative examples, the black parts in the image of the examples, that is, the parts where more pores can be seen, are darker than the image of the comparative examples. This means that the pores or surface pores of the particles in the examples are larger and more numerous than those of the particles in the comparative examples. This is consistent with the following experimental results (see Tables 5 - 6) which state that in the case of the comparative examples (e.g., Comparative Examples 5 - 7), the pore size measured by MIP of the particles is at the level of 10 - 11 nm, and in the case of the examples (e.g., Examples 1 - 2), the pore size measured by MIP of the particles is at the level of 35 - 40 nm. And looking at the experimental results in Tables 5 - 6, it is confirmed that there is also a clear difference in the average surface pore size between the examples (at the level of 120 - 160 nm) and the comparative examples (at the level of 105 nm or less, especially, Comparative Example 11 is confirmed to be 45 nm).

Mode for Carrying Out the Invention

[0086] Hereinafter, the actions and effects of the invention will be described more specifically through specific examples of the invention. However, this is presented as an exemplification of the invention, and the scope of the rights of the invention is not limited in any sense thereby.

[0087] Examples and Comparative Examples The particle - forming components, their contents, and process conditions are as described in the following table, and the conditions for manufacturing each particle in other examples and comparative examples are as follows. In particular, when there is no explanation regarding temperature, the temperature at which the manufacturing process is carried out (or each manufacturing stage) or the temperature at which the properties of the manufactured particles are calculated or measured means normal temperature (the temperature in a state where neither cooling nor heating is performed, and the temperature within the range of about 15 - 30 °C).

[0088] Production of the Dispersed Phase After stirring the polymerizable monomer (M), the cross - linking agent (C), and the porogen (P), BPO (about 1.25 parts by weight with respect to 100 parts by weight of the total mass of the polymerizable monomer and the cross - linking agent), which is a thermal initiator, was additionally charged, and stirred at normal temperature for about 5 minutes to produce a dispersion phase.

[0089] *GMA: Glycidyl methacrylate *EGDMA: Ethylene glycol dimethacrylate *TMPTMA: Trimethylolpropane trimethacrylate

[0090] Production of the Continuous Phase PVA (weight average molecular weight (M w ) is 85,000 - 124,000 and the hydrolysis rate satisfies 87 - 89%) was dissolved in distilled water at a concentration of 2% by weight to prepare a continuous phase.

[0091] Production of Particles (Base Resin) by Suspension Polymerization 720 g of the continuous phase was added to the reactor, 90 g of the dispersed phase was added, and the mixture was stirred at about 800 rpm at room temperature until a homogeneous dispersion was obtained. Then, the temperature of the reactor was raised, and polymerization was carried out at a temperature of about 80°C, about 800 rpm, and nitrogen purging conditions for about 6 hours.

[0092] Obtaining of Particles The polymerized particles were collected and washed twice with distilled water at 50°C and five times with ethanol. Then, they were dried in an oven at about 80°C to obtain the particles.

[0093] [Table 1]

[0094] [Table 2]

[0095] [Table 3]

[0096] [Table 4]

[0097] Evaluation 1: Evaluation of the Characteristics of the Base Resin The particles produced in the examples and comparative examples were evaluated according to the following content.

[0098] 1. Particle (base resin) size The produced particles were dried, and the dried particles were dispersed in ethanol at a level of about 10%. Then, the particle size (D 50 ) was measured using a PSA device. At this time, D 50 means the size of the particles corresponding to 50% of the volume cumulative of the particle size distribution.

[0099] 2. Spheroidization rate (%) of particles (base resin) The spheroidization rate was confirmed by checking the length (L) of the longest dimension among the produced particles through SEM, and calculating the percentage ratio (D 50 ) of the length (L) to the particle size (D 50 ) measured in item 1.

[0100] 3. Crack ratio (%) of particles (base resin) The crack ratio of the particles was determined by checking through SEM the number of particles with defects on the surface or cracked without maintaining a spherical shape among the produced particles, and calculating the percentage ratio of the number of particles maintaining a spherical shape to the total number of particles.

[0101] 4. Pore shape of particles (base resin) For the dried particles, pores formed on the surface and inside were observed using an SEM (JSM7610F-plus from JEOL) device.

[0102] 5. Pore size of particles (base resin) and porosity of particles The pore size of the particles was measured using mercury intrusion porosimetry (MIP) (Autopore V9600, from Micromeritics).

[0103] 6. Surface pore size On the SEM image of the particle surface, more than 20 pore sizes (at this time, the pore size means the length of the largest dimension among the pore shapes) were measured, and the arithmetic mean value thereof was calculated. Through the calculated surface pore size, the degree of openness of the particle surface (which cannot be confirmed by MIP) can be indirectly confirmed.

[0104]

Table 5

[0105]

Table 6

[0106] Evaluation 2: Evaluation of the Non-Specific Adsorption Rate The particles produced in the examples and comparative examples were processed according to the following content, and the related content was evaluated.

[0107] 1. Epoxy content, SBC, and non-specific adsorption rate of particles (base resin) (1) Analysis of epoxy content (μmol / g) of particles (base resin) 1) 0.1 g of particles (deionized water (DI water) is used for the control group) is mixed with 1 mL of HCl / acetone solution (1 / 40 (v / v)) and sonicated for 4 minutes (UCP-02 of Jeiotech). 2) An indicator solution (0.1% cresol red and 0.1% thymol blue are mixed at a volume ratio of 1:3 and adjusted to pH 7.0) is added to the mixture in 1). 3) Titrate with 0.1 M NaOH until the color of the solution becomes purple-blue. 4) Calculate the epoxy content using the following formula. [Formula] Epoxy content (mmol) =[V0 (ml) - V (ml)] × CNaOH (mol / L) In the above formula, V0: The amount of NaOH (mL) added to the control group sample using deionized water (DI water) instead of particles V: The amount of NaOH (mL) added to the experimental group sample containing epoxy CNaOH: The concentration of NaOH used in the titration 5) Divide the calculated epoxy content by the weight of the above particles (using 0.1 g in item (1) above), and confirm the epoxy content per unit weight of the particles (base resin).

[0108] (2) Measurement of static binding capacity (SBC, mg / mL) (References: Ind. Eng. Chem. Res. 2013, 52, 26, 8800 - 8811, Biochemical Engineering Journal 145 (2019) 53 - 61) 1) Prepare 50 μL of the base resin slurry. After thawing the antibody solution (IgG from human serum, 5 mg / mL PBS, pH 7.4), add 1xPBS to prepare an antibody solution at 3 mg / mL. 2) After mixing the resin slurry and the antibody solution, use a mixer at room temperature for 1 hour to bind IgG to the resin. 3) After precipitating the resin, remove the supernatant (or store it if necessary), and inject 5 mL of 1xPBS for washing. 4) After precipitating the resin, remove the washing solution (or store it if necessary), and inject 500 μL of 100 mM glycine - HCl (pH 2.5) eluent to elute IgG. 5) After precipitating the resin, collect the eluate and store it at 4°C. 6) Measure the IgG concentration in the eluate at 280 nm using a UV-vis spectrophotometer (Agilent's 8453 UV-visible spectrophotometer). After IgG binding, measure the IgG content in each of the supernatant, washing solution, and eluate, and calculate the IgG content in each solution through an IgG calibration curve.

[0109] (3) Nonspecific adsorption rate (%) during SBC measurement Based on 100% of the total IgG content (weight) in the supernatant, washing solution, and eluate confirmed in the above 2)-6), substitute the IgG content (%) confirmed in the washing solution into the nonspecific adsorption rate (%) during SBC measurement.

[0110] 2. Epoxy content (μmol / g), hydrophilic functional group content (μmol / g), and nonspecific adsorption rate (%) after epoxy deactivation of the particles (base resin) (1) Epoxy deactivation 1) Inject the base resin into a 1-thioglycerol solution (0.5 M 1-thioglycerol + 0.1 M sodium sulfate + 0.1 M EDTA (pH 8.5)), and react at 37°C for 17 hours. For Comparative Example 1, ethanol amine was used instead of 1-thioglycerol. 2) After precipitating the particles by methods such as centrifugation, remove the supernatant. Wash three times with DIW.

[0111] (2) Epoxy content (μmol / g) after epoxy deactivation Epoxy deactivated particles, that is, particles surface-treated with 1-thioglycerol were prepared by drying at room temperature, and the epoxy content of the particles surface-treated with 1-thioglycerol was measured in the same manner as described above.

[0112] (3) Content of hydrophilic functional groups (μmol / g) The content of hydrophilic functional groups of the particles surface-treated with 1-thioglycerol was measured as shown in the following <Formula>. <Formula> Content of hydrophilic functional groups after 1-thioglycerol surface treatment = (Epoxy content of the particles before 1-thioglycerol surface treatment) × (Number of hydroxyl functional groups in 1-thioglycerol)

[0113] (4) Non-specific adsorption rate (%) The epoxy deactivated particles were dried. Then, a 10 mg / mL solution of BSA (A9418 from Sigma) dissolved in DIW and the particles were mixed at a concentration of 31.3 mg / mL. Then, the mixture was stirred at room temperature for 18 hours, washed 5 times with DIW, and dried at room temperature. The non-specific adsorption rate was calculated through the ratio of the content of BSA adsorbed on the particles to the total content of BSA introduced during stirring at room temperature. At this time, the content of BSA adsorbed on the particles was analyzed using the Pierce (trademark) BCA Protein Assay Kit (Thermo Fischer).

[0114]

Table 7

[0115]

Table 8

[0116] ※In the case of Comparative Example 2, since it was confirmed that the non-specific adsorption rate of the base resin itself was a high value, subsequent repeated non-specific adsorption rate tests (i.e., related experiments after the Epoxy deactivation treatment) were not conducted.

[0117] ※In Comparative Example 4 where the epoxy content of the base resin itself was very low, the non-specific adsorption rate test for the base resin was not conducted. Similarly, the non-specific adsorption rate test was not conducted for the base resins of Comparative Examples 5 to 7.

Claims

1. A continuous phase composition (A); and a dispersion phase composition (B) containing a polymerizable monomer (b1) containing an epoxy group, a crosslinking agent (b2), and a porogen (b3); after mixing, a suspension polymerization reaction is carried out to produce a suspension polymer containing units derived from the polymerizable monomer (b1) and the crosslinking agent (b2); comprising the dispersion phase composition contains 50 parts by weight or more of the epoxy group-containing polymerizable monomer (b1) based on 100 parts by weight of the crosslinking agent (b2), the porogen (b3) contains a porogen whose difference in Hansen solubility parameter from the epoxy group-containing polymerizable monomer (b1) satisfies the range of 1.30 to 4.70, A method for producing porous particles.

2. the dispersion phase composition contains 150 parts by weight or more of the epoxy group-containing polymerizable monomer (b1) based on 100 parts by weight of the crosslinking agent (b2), The method for producing porous particles according to Claim 1.

3. the porogen (b3) contains a porogen whose difference in Hansen solubility parameter from the epoxy group-containing polymerizable monomer (b1) is 2.50 to 4.50, The method for producing porous particles according to Claim 1.

4. including an acetate-based porogen as the porogen (b3), The method for producing porous particles according to Claim 1.

5. the dispersion phase composition contains 45% by weight or more of the porogen (b3) based on 100% by weight of the total content of the epoxy group-containing polymerizable monomer (b1), the crosslinking agent (b2), and the porogen (b3), The method for producing porous particles according to Claim 1.

6. the crosslinking agent (b2) includes a crosslinking agent that does not contain a hydroxyl group (-OH), or is a crosslinking agent that does not contain a hydroxyl group (-OH), The method for producing porous particles according to Claim 1.

7. the continuous phase composition contains water and a polymeric surfactant, The method for producing porous particles according to Claim 1.

8. the suspension polymer has a particle shape, The particles have a D within the range of 30 to 70 μm ５０ size (at this time, D ５０ means the size of the particles corresponding to 50% of the volume cumulative of the particle size distribution), The method for producing porous particles according to Claim 1.

9. the suspension polymer has a particle shape, the pores of the particles have a pore size measured by mercury intrusion porosimetry in the range of 30 to 500 nm, and the average surface pore of the particles, which is the arithmetic average of 20 or more pore sizes observed on the particle surface, is in the range of 110 to 240 nm, The method for producing porous particles according to Claim 1.

10. The suspension polymer has a particulate shape, and the particles have a porosity in the range of 15 to 40%, A method for producing the porous particles according to claim 1.

11. Further comprising the step of hydrophilizing the surface of the suspension polymer through epoxy group deactivation; A method for producing the porous particles according to claim 1.

12. The suspension polymer has an epoxy group derived from the epoxy group-containing polymerizable monomer (b1), An epoxy deactivator having an epoxy reactive group and a hydrophilic group different therefrom is reacted with the suspension polymer having the epoxy group to hydrophilize the surface of the suspension polymer, A method for producing the porous particles according to claim 11.

13. The epoxy deactivator contains one or more of a thiol group and an amine group as an epoxy reactive group, and contains a hydroxy group as a hydrophilic group different from the epoxy group, A method for producing the porous particles according to claim 12.

14. Containing polymer units derived from a polymerizable monomer having an epoxy group and a crosslinking agent, Particle size (D within the range of 30 to 70 μm ５０ ) and The pore size measured by the mercury intrusion method is in the range of 30 to 500 nm, The average particle surface pore, which is the arithmetic mean of 20 or more pore sizes observed on the particle surface, is in the range of 110 to 240 nm, The porosity satisfies 15 to 40%, Porous particles.

15. The porous particles are surface-treated with an epoxy deactivator having an epoxy reactive group and a hydrophilic group different from the epoxy reactive group, The porous particles according to claim 14.

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