Porous polymer particles and protein purification columns using the same

Abstract

The present invention relates to porous polymer particles comprising: a polymer matrix; and pores dispersed in the polymer matrix and having a diameter of 50 nm to 500 nm; wherein the epoxy content contained in 1 g of the porous polymer particles is 500 μmol or more and less than 5,000 μmol; and a protein purification column using the same.

Description

[Technical Field]

[0001] Cross-reference of related applications This application claims priority rights under Korean Patent Application No. 10-2022-0117461 dated September 16, 2022, and Korean Patent Application No. 10-2023-0121985 dated September 13, 2023, and all content disclosed in the documents of said Korean Patent Applications is incorporated herein by reference.

[0002] This invention relates to porous polymer particles and a protein purification column using the same. [Background technology]

[0003] With the expansion of the biopharmaceutical and regenerative medicine fields, there is a growing need for systems that can efficiently separate or purify cells, tissues, microorganisms, and other microorganisms.

[0004] Conventional techniques primarily used purification resins made from hydrophilic agarose. However, such resins swell with buffer during the purification process, reducing their strength and resulting in problems with low purification speed and efficiency.

[0005] In contrast, while using synthetic polymers as resins for protein purification offers the advantage of higher strength than agarose, if the synthetic polymer exhibits low sphericity and large particle size deviation, it can lead to differences in the migration paths of separated (or purified) materials, resulting in reduced separation efficiency. Furthermore, the surface properties of the synthetic polymer can cause non-specific adsorption of proteins, further reducing purification efficiency.

[0006] Therefore, technologies for producing polymer particles suitable for use in protein purification resins are being actively researched, and recently, research is progressing on emulsification devices that can form fine emulsion droplets for producing polymer particles. Fine emulsion droplets are formed by dispersing one type of fluid within another, using two fluids that have low affinity and do not mix with each other, such as water and oil. Representative emulsions include water-in-oil (W / O) emulsions, in which water droplets are dispersed within oil, and oil-in-water (O / W) emulsions, in which oil is dispersed within water.

[0007] An emulsifying device for generating such emulsion droplets is made by bonding two substrates together, with a channel of a predetermined shape formed on one substrate and an inlet for injecting oil and water into the channel formed on the other substrate.

[0008] However, with conventionally manufactured polymer particles, the viscosity of the pore-forming material injected for pore formation is high, making reuse difficult due to adsorption within the emulsifier, which reduces the production rate. [Overview of the Initiative] [Problems that the invention aims to solve]

[0009] The present invention provides porous polymer particles that are manufactured by a highly efficient production method and have particle size and pore size suitable for achieving high protein purification performance in protein purification columns.

[0010] Furthermore, the present invention provides a protein purification column containing the porous polymer particles. [Means for solving the problem]

[0011] To solve the above problems, this specification provides porous polymer particles comprising a polymer matrix; and pores dispersed in the polymer matrix having a diameter of 50 nm to 500 nm; wherein the epoxy content in 1 g of porous polymer particles, as measured by the following formula 1, is 500 μmol or more and less than 5000 μmol.

[0012] [Formula 1] Epoxy content (μmol / g)=[(V0-V)×C 塩基 [×1000] / W

[0013] In the above formula 1, V is the amount of base (mL) used to titrate the experimental group sample containing porous polymer particles, V0 is the amount of base (mL) used to titrate the control group sample in which distilled water was used instead of porous polymer particles in the experimental group sample, and C 塩基 is the concentration (M) of the base used in the titration, and W is the mass (g) of the porous polymer particles used in the titration.

[0014] This specification also provides a protein purification column comprising the porous polymer particles.

[0015] The following describes in more detail porous polymer particles and protein purification columns using them according to specific embodiments of the invention.

[0016] Unless otherwise expressly stated herein, technical terms are used solely to refer to specific embodiments and are not intended to limit the invention.

[0017] As used herein, the singular form includes the plural form unless the wording explicitly indicates the opposite.

[0018] As used herein, "includes" embodies a particular characteristic, domain, integer, stage, operation, element and / or component, and does not exclude the presence or addition of other particular characteristics, domains, integers, stages, operations, elements, components and / or groups.

[0019] And in this specification, terms including ordinal numbers such as "first" and "second" are used for the purpose of distinguishing one component from other components and are not limited by said ordinal numbers. For example, within the scope of the rights of the present invention, the first component may be named the second component, and similarly, the second component may be named the first component.

[0020] In this specification, "porous particles" can mean particles having pores inside and / or on the surface of the particles.

[0021] In this specification, (meth)acrylate means including all acrylates and methacrylates.

[0022] In this specification, unless otherwise specifically defined or explained, the temperature at which the manufacturing process is carried out (or each manufacturing stage) or the temperature at which the numerical characteristics of the manufactured particles are calculated or measured is normal temperature. Specifically, in this application, "normal temperature" means the temperature in a state where there is no particular heating or cooling, and for example, it can mean a temperature in the range of 15°C to 30°C, or 20°C to 30°C.

[0023] In this specification, examples of substituents are described below, but are not limited thereto.

[0024] In this specification, the term "substitution" means that another functional group is bonded in place of a hydrogen atom in a compound, and the position of substitution is not limited as long as it is the position where the hydrogen atom is substituted, that is, the position where the substituent can be substituted. When two or more substitutions occur, the two or more substituents may be the same or different from each other.

[0025] In this specification, the term "substituted or unsubstituted" means that a molecule is substituted or unsubstituted with one or more substituents selected from the group consisting of deuterium; halogen groups; cyano groups; nitro groups; hydroxyl groups; carbonyl groups; ester groups; imide groups; amide groups; primary amino groups; carboxyl groups; sulfonic acid groups; sulfonamide groups; phosphine oxide groups; alkoxy groups; aryloxy groups; alkylthiooxy groups; arylthiooxy groups; alkylsulfoxy groups; arylsulfoxy groups; silyl groups; boron groups; alkyl groups; cycloalkyl groups; alkenyl groups; aryl groups; aralkyl groups; aralkenyl groups; alkylaryl groups; alkoxysilylalkyl groups; arylphosphine groups; or heterocyclic groups containing one or more N, O, and S atoms, or that a molecule is substituted or unsubstituted with two or more substituents linked together from the exemplified substituents. For example, "substituents with two or more substituents linked together" may be a biphenyl group. That is, a biphenyl group may be an aryl group and may be interpreted as a substituent with two phenyl groups linked together.

[0026] In this specification, aromatic is a property that satisfies Huckels' rule, and according to Huckels' rule, it can be defined as an aromatic substance that satisfies all three of the following conditions.

[0027] 1) There must be 4n+2 electrons that are completely conjugated by a hollow p-orbital, an unsaturated bond, a hole electron pair, etc. 2) The 4n+2 electrons must form a planar isomer and must have a ring structure. 3) All atoms in the ring must be able to participate in the conjugation.

[0028] In this specification, "aliphatic" refers to organic compounds remaining after removing the aromatic compounds mentioned above.

[0029] In this specification, an alkyl group is a monovalent functional group derived from an alkane, which may be linear or branched. The number of carbon atoms in the linear alkyl group is not particularly limited, but is preferably 1 to 20. The number of carbon atoms in the branched alkyl group is 3 to 20. Specific examples of alkyl groups include, but are not limited to, methyl, ethyl, propyl, n-propyl, isopropyl, butyl, n-butyl, isobutyl, tert-butyl, sec-butyl, 1-methyl-butyl, 1-ethyl-butyl, pentyl, n-pentyl, isopentyl, neopentyl, tert-pentyl, hexyl, n-hexyl, 1-methylpentyl, 2-methylpentyl, 4-methyl-2-pentyl, 3,3-dimethylbutyl, 2-ethylbutyl, heptyl, n-heptyl, 1-methylhexyl, octyl, n-octyl, tert-octyl, 1-methylheptyl, 2-ethylhexyl, 2-propylpentyl, n-nonyl, 2,2-dimethylheptyl, 1-ethyl-propyl, 1,1-dimethyl-propyl, isohexyl, 2-methylpentyl, 4-methylhexyl, 5-methylhexyl, and 2,6-dimethylheptan-4-yl. The alkyl group may be substituted or unsubstituted, and if substituted, examples of substituents are as described above.

[0030] The present invention will be described in more detail below.

[0031] 1.Porous polymer particles According to one embodiment of the invention, porous polymer particles can be provided that comprise a polymer matrix and pores dispersed in the polymer matrix having a diameter of 50 nm to 500 nm, wherein the epoxy content in 1 g of porous polymer particles, as measured by the following formula 1, is 500 μmol or more and less than 5000 μmol.

[0032] [Formula 1] Epoxy content (μmol / g)=[(V o -V)×C 塩基 [×1000] / W

[0033] In the above formula 1, V is the amount (mL) of base used in the titration of the experimental group sample containing porous polymer particles, and V o This is the amount (mL) of base used in the titration of the control group sample in which distilled water was used instead of porous polymer particles in the experimental group sample, and C 塩基 is the concentration (M) of the base used in the titration, and W is the mass (g) of the porous polymer particles used in the titration.

[0034] The inventors of the present invention have completed their invention by confirming through experiments that the porous polymer particles of the above embodiment have a pore diameter adjusted to 50 nm to 500 nm, providing a large surface area, and are suitable for application as protein purification particles used in the production of pharmaceuticals through processes such as ligand attachment.

[0035] In particular, the pores are induced from a single-pore-inducing substance, as described later, which simplifies the particle manufacturing process. Furthermore, the low viscosity of the single-pore-inducing substance allows it to pass through the step emulsifier at a high speed, resulting in increased process efficiency.

[0036] Furthermore, the single-pore-inducing substance that forms the pores has suitable miscibility with monomers, crosslinking agents, and polymers that form the polymer matrix, and can stably form pores of the appropriate size range described above during polymerization of porous polymer particles.

[0037] On the other hand, through experiments, we confirmed that it is possible to achieve a protein purification efficiency of an equivalent or higher level compared to conventional methods of mixing two or more stomatal-inducing substances, thus completing the invention.

[0038] Furthermore, when the pores are induced from a pore-inducing substance containing a mixture of alcohol-based compounds and aromatic hydrocarbon compounds, it is possible to introduce pores with completely different pore characteristics than those obtained when using only one type of alcohol-based compound or one type of aromatic hydrocarbon compound, thereby achieving optimal performance for application to protein purification particles used in the production of pharmaceuticals through processes such as ligand attachment.

[0039] Furthermore, the porous polymer particles of the above embodiment can be manufactured using a step emulsification apparatus, enabling the production of particles of uniform size, thereby maximizing particle performance.

[0040] Specifically, the porous polymer particles of the above embodiment may include a polymer matrix. The matrix acts as a substrate, host, or binder for the porous polymer particles, and pores can be dispersed inside or outside the matrix.

[0041] The polymer matrix may include repeating units derived from monomers. These monomer-derived repeating units are those contained in a polymer formed by the polymerization of monomer compounds, and the monomer is not particularly limited, but for example, the monomer may be an epoxy group-containing monomer, and glycidyl methacrylate, glycidyl acrylate, or glycidyl acrylamide can be used alone or in combination with an epoxy group-free monomer.

[0042] Examples of monomers that do not contain the epoxy group include methyl methacrylate, methyl acrylate, ethyl acrylate, ethyl hexyl acrylate, butyl acrylate, hydroxyethyl methacrylate, trimethylolpropane triacrylate, N-isopropylacrylamide, and N-butyl acrylate.

[0043] The polymer matrix may further contain repeating units derived from the crosslinking agent. The type of crosslinking agent is not particularly limited, but for example, polyfunctional (meth)acrylates such as ethylene glycol dimethyl (meth)acrylate, ethylene glycol di(meth)acrylate, trimethylolpropane tri(meth)acrylate, pentaerythritol tri(meth)acrylate, or dipentaerythritol hexa(meth)acrylate can be used. Alternatively, one or more components of the listed crosslinking agents may be used together. When considering the formation of a hydrophilic surface of the particles, it is preferable to use ethylene glycol di(meth)acrylate.

[0044] On the other hand, the porous polymer particles of the above embodiment may contain pores. These pores are induced from one or two pore-inducing substances described later. The pore-inducing substances (pologens) are contained in droplets, which are particle precursors used in the process of manufacturing porous polymer particles, and are converted into pores during the pore formation process in which porous polymer particles are formed from the droplets.

[0045] Furthermore, when the pores are induced from a pore-inducing substance containing a mixture of alcohol-based compounds and aromatic hydrocarbon compounds, it is possible to introduce pores with completely different pore characteristics than those obtained when using only one type of alcohol-based compound or one type of aromatic hydrocarbon compound, thereby achieving optimal performance for application to protein purification particles used in the production of pharmaceuticals through processes such as ligand attachment.

[0046] The pores can be dispersed in the polymer matrix. The pores can be dispersed inside or outside the matrix.

[0047] The pores may have diameters of 50 nm to 500 nm, 50 nm to 400 nm, 50 nm to 300 nm, 50 nm to 250 nm, 50 nm to 200 nm, or 100 nm to 400 nm. In particular, the pore diameter may be a value measured for pores formed inside or on the surface of the porous polymer particles, and the measurement method is not limited, but it can be measured by SEM, for example.

[0048] Specifically, the porous polymer particles may have pore diameters on the particle surface measured by SEM of 50 nm to 500 nm, or 50 nm to 400 nm, or 50 nm to 300 nm, or 50 nm to 250 nm, or 50 nm to 200 nm, or 100 nm to 400 nm.

[0049] The pore diameter can be defined as the distance between two points where a straight line passing through the centroid of the pore intersects the boundary line of the pore, for each type of pore with diverse shapes. In other words, the statement that a pore has a diameter of 50 nm to 500 nm means that the pore diameter is at most 500 nm and at least 50 nm.

[0050] As a result, the porous polymer particles can provide a large surface area and can be applied to particles for protein purification used in the production of pharmaceuticals through processes such as ligand attachment. On the other hand, if the pore diameter is excessively reduced to less than 50 nm, there may occur a problem that sufficient space for ligand and target protein attachment is not ensured and the purification performance deteriorates. Also, if the pore diameter is excessively increased to more than 500 nm, there may occur a problem that the strength of the particles becomes weak, the specific surface area of the particles becomes small, and the purification performance deteriorates.

[0051] On the other hand, for the porous polymer particles of the one embodiment, the epoxy content contained in 1 g of the porous polymer particles measured by the following formula 1 may have an upper numerical range of less than 5000 μmol, or less than 4000 μmol, or less than 3000 μmol, or less than 2700 μmol, or less than 2500 μmol, or less than 2000 μmol, or less than 1500 μmol, or less than 1300 μmol, or less than 1150 μmol, or less than 1000 μmol, and the lower numerical range may be 500 μmol or more, or 800 μmol or more. By combining the upper numerical range and the lower numerical range, the numerical range between the lower limit and the upper limit can also be satisfied. For example of the numerical range between the lower limit and the upper limit, it may be 500 μmol or more and less than 5000 μmol.

[0052] The epoxy content means the content of epoxy functional groups contained in the polymer matrix contained in the porous polymer particles and can be measured by the titration method of the following formula 1.

[0053] [Formula 1] Epoxy content (μmol) = [(V o - V) × C 塩基 × 1000] / W

[0054] In the formula 1, V is the amount (mL) of the base used for titration of the experimental group sample containing the porous polymer particles, V oThis is the amount (mL) of base used in the titration of the control group sample in which distilled water was used instead of porous polymer particles in the experimental group sample, and C 塩基 is the concentration (M) of the base used in the titration, and W is the mass (g) of the porous polymer particles used in the titration.

[0055] In the above formula 1, the experimental group sample containing porous polymer particles corresponds to a sample obtained by dissolving porous polymer particles in a solvent and then adding an indicator. There are no special restrictions on the type of solvent or indicator that can be used. However, to give an example of the experimental group sample containing porous polymer particles, one example is a solution obtained by mixing 0.1 g of porous polymer particles with 1 mL of HCl / acetone solution (volume ratio 1 / 40) and performing sonication for 4 minutes, to which an indicator solution (0.1% cresol red and 0.1% thymol blue mixed in a volume ratio of 1:3, and then adjusting the pH to 7.0 using NaOH in a concentration range of 1 to 1000 mM) is added.

[0056] In the above formula 1, the control group sample, in which distilled water is used instead of porous polymer particles in the experimental group sample, is identical to the experimental group sample except that distilled water is dissolved in the solvent instead of porous polymer particles. An example of the control group sample is a solution obtained by mixing 0.1 g of distilled water with 1 mL of HCl / acetone solution (volume ratio 1 / 40) and performing sonication for 4 minutes, to which an indicator solution (0.1% cresol red and 0.1% thymol blue mixed in a volume ratio of 1:3, and then adjusting the pH to 7.0 using NaOH in a concentration range of 1 to 1000 mM) is added.

[0057] In the aforementioned formula 1, the examples of bases used in the titration are not particularly limited, and any base commonly used in the field of acid-base titration can be applied without restriction. However, one example of a base used in the titration is NaOH.

[0058] The porous polymer particles of the above embodiment have the advantage of being able to stably form a large number of pores within the appropriate range described above, by satisfying the epoxy content in 1 g of porous polymer particles measured by the above formula 1, which is 500 μmol or more and less than 5000 μmol.

[0059] If the epoxy content in 1 g of porous polymer particles, as measured by the above formula 1, increases excessively to 5000 μmol or more, the epoxy-containing monomers, which have low affinity for pore-inducing substances, may prevent sufficient formation of a polymer matrix by monomers (or crosslinking agents). This can lead to an excessive increase in pore size, weakening of particle strength, and a decrease in specific surface area, resulting in reduced purification performance.

[0060] Furthermore, if the epoxy content in 1 g of porous polymer particles, as measured by the above formula 1, decreases excessively to less than 500 μmol, excessive formation of a polymer matrix by monomers other than epoxy-containing monomers (or crosslinking agents) with low affinity for pore-inducing substances may occur. This can lead to an excessive reduction in pore size, resulting in insufficient space for ligands and target proteins to attach, and consequently, a decrease in purification performance.

[0061] On the other hand, the porous polymer particles may have particle sizes of 10 μm to 150 μm, 30 μm to 50 μm, or 35 μm to 45 μm as measured by an optical microscope. When purifying proteins, the inside of the column is packed with protein purification particles and the substance to be purified is passed through it. However, if the size of the protein purification particles is smaller than the above range, it is difficult for reactants such as protein particles to pass through the column. Also, if the size exceeds the above range, that is, if the size of the packing material in the column is excessively large, a lot of empty space (space between particles) is created in the column, so the substance to be purified can pass through the column without adhering to the particle surface, which can reduce the purification efficiency.

[0062] The particle diameter can refer to the distance between two points where a straight line passing through the center of gravity of a particle intersects the particle's boundary line, for each particle having a diverse shape. In other words, when the porous polymer particles are said to have a diameter of 10 μm to 150 μm, it means that the diameter of the porous polymer particles is at most 150 μm and at least 10 μm.

[0063] Furthermore, the porous polymer particles may have a coefficient of variation of particle size measured by an optical microscope of 20% or less, or 15% or less, or 10% or less, or 7% or less, or 6.8% or less, or 0.1% or more, or 0.1% to 20%, or 0.1% to 15%, or 0.1% to 10%, or 0.1% to 7%, or 0.1% to 6.8%.

[0064] Examples of methods for measuring the coefficient of variation of the porous polymer particles are not limited to those described above, but for example, it can be obtained by the following equation 2.

[0065] [Formula 2] Coefficient of variation (%) = (Standard deviation of particle size / Average particle size) × 100

[0066] By reducing the coefficient of variation of the particle size of the porous polymer particles to the range described above, it becomes possible to produce porous polymer particles of uniform size, thereby maximizing particle performance.

[0067] In contrast, if the coefficient of variation of the particle size of the porous polymer particles increases excessively, such as exceeding 20%, the particle size deviation becomes large and non-uniform, resulting in the generation of large particles, which may make them unsuitable for application as protein purification particles used in the production of pharmaceuticals.

[0068] On the other hand, the porous polymer particles may also be spherical. Spherical refers to a shape that is close to a sphere, which can be confirmed with the naked eye, but for example, it can mean that the value obtained by dividing the length of the longest dimension of the particle's shape by the average diameter is approximately 0.80 or more. When the particles are spherical, a large surface area can be secured, which can improve the protein purification performance.

[0069] On the other hand, the porous polymer particles may also be particles for protein purification.

[0070] On the other hand, the porous polymer particles may be polymerization reaction products of a dispersed phase solution and a continuous phase aqueous solution containing one or two pore-inducing substances, a monomer, and a crosslinking agent. Porous polymer particles can be produced by this polymerization. In this case, the polymerization takes place at the interface between the continuous phase and the dispersed phase (i.e., the surface of the dispersed phase droplet) and / or inside the dispersed phase, which is formed after mixing the dispersed phase solution and the continuous phase aqueous solution.

[0071] In other words, the examples of the method for producing porous polymer particles according to the above embodiment are not particularly limited, but as an example, a method for producing porous polymer particles can be used that includes the step of mixing a dispersed phase solution containing one or two types of pore-inducing substances, a monomer, and a crosslinking agent, and a continuous phase aqueous solution, and carrying out a polymerization reaction.

[0072] The dispersed phase solution refers to a composition that can form a dispersed phase (or droplets) after being mixed with a continuous phase aqueous solution. The continuous phase aqueous solution refers to a composition that can form a continuous phase after being mixed with the dispersed phase solution.

[0073] The continuous phase may be an aqueous phase. The aqueous solution that can be used as the continuous phase may be, but is not limited to, a solution containing water and an ionic or nonionic surfactant. For example, the ionic surfactant may be sodium dodecyl sulfate (SDS), and the nonionic surfactant may be Tween 20, Tween 40, Tween 60, Tween 80, Triton X-100, polyvinyl alcohol (PVA), polyethylene glycol (PEG), etc. In one example, a 0.5% SDS solution can be used as the continuous phase. The water may be distilled water or deionized water, although this is not particularly limited.

[0074] On the other hand, the dispersed phase is not particularly limited, but may be a substance that is insoluble in the continuous phase and polymerizable by a subsequent crosslinking reaction. The dispersed phase solution may contain one or two pore-inducing substances, a monomer, and a crosslinking agent.

[0075] The monomer is not particularly limited, but for example, the monomer may be an epoxy group-containing monomer, such as glycidyl methacrylate, glycidyl acrylate, or glycidyl acrylamide, which can be used alone or in combination with an epoxy group-free monomer.

[0076] Examples of monomers that do not contain the epoxy group include methyl methacrylate, methyl acrylate, ethyl acrylate, ethyl hexyl acrylate, butyl acrylate, hydroxyethyl methacrylate, trimethylolpropane triacrylate, N-isopropylacrylamide, and N-butyl acrylate.

[0077] In the dispersed phase solution, the monomer content may be 10% to 60% by weight, or 10% to 50% by weight, or 10% to 40% by weight, or 17% to 33% by weight, based on 100% by weight of the dispersed phase solution.

[0078] In the aforementioned dispersed phase solution, if the monomer content increases excessively, such as to more than 60% by weight based on 100% by weight of the dispersed phase solution, the epoxy content in 1g of porous polymer particles, as measured by Equation 1, increases excessively to 5000 μmol or more. As a result, the epoxy-containing monomers, which have low affinity for pore-inducing substances, do not adequately form a polymer matrix by monomers (or crosslinking agents). This can lead to an excessive increase in pore size, weakening of particle strength, and a decrease in specific surface area, resulting in reduced purification performance.

[0079] Furthermore, if the monomer content in the dispersed phase solution decreases excessively, such as to less than 10% by weight based on 100% by weight of the dispersed phase solution, the epoxy content in 1 g of porous polymer particles, as measured by Equation 1, decreases excessively to less than 500 μmol. This can lead to excessive formation of a polymer matrix by monomers other than epoxy-containing monomers (or crosslinking agents) that have low affinity for pore-inducing substances, resulting in an excessive reduction in pore size. This can cause problems such as a decrease in purification performance because sufficient space for ligands and target proteins to attach cannot be secured.

[0080] The type of crosslinking agent is not particularly limited, but for example, polyfunctional (meth)acrylates such as ethylene glycol dimethyl (meth)acrylate, ethylene glycol di(meth)acrylate, trimethylolpropane tri(meth)acrylate, pentaerythritol tri(meth)acrylate, or dipentaerythritol hexa(meth)acrylate can be used. Alternatively, one or more components of the listed crosslinking agents may be used together. When considering the formation of a hydrophilic surface of the particles, it is preferable to use ethylene glycol di(meth)acrylate.

[0081] In the dispersed phase solution, the content of the crosslinking agent may be 10% to 60% by weight, or 10% to 50% by weight, or 10% to 40% by weight, or 17% to 37% by weight, based on 100% by weight of the dispersed phase solution.

[0082] In one example, the dispersed phase may be a solution of glycidyl methacrylate (GMA) and ethylene glycol dimethacrylate (EGDMA) mixed in a mass ratio of 5:1 to 1:5, 4:1 to 1:4, 3:1 to 1:3, or 2:1 to 1:2.

[0083] In other words, the content ratio of the crosslinking agent to 1 part by weight of the monomer may be 0.1 to 10 parts by weight, or 0.5 to 2 parts by weight. When the above content is satisfied, it is advantageous to stably form an emulsion and to ensure the desired level of particle strength and morphological characteristics through crosslinking.

[0084] On the other hand, the pore-inducing substance may include one single compound or a mixture of two compounds. In the dispersed phase solution, the content of one to two pore-inducing substances may be 10% to 80% by weight, or 20% to 80% by weight, or 40% to 80% by weight, or 45% to 80% by weight, or 44% to 55% by weight, based on 100% by weight of the dispersed phase solution.

[0085] If the content of the pore-inducing substance is excessively reduced to less than 10% by weight, the average diameter of the pores within the porous polymer particles decreases excessively, making it difficult to secure sufficient space for protein binding. Conversely, if the content of the pore-inducing substance increases excessively to more than 80% by weight, the average diameter of the pores within the porous polymer particles increases excessively, making it difficult to secure the strength of the particles.

[0086] The pore-inducing substance is characterized by having a viscosity of 0.5 cP to 15 cP, or 0.8 cP to 15 cP, or 0.8 cP to 10 cP, or 0.8 cP to 5 cP, or 0.8 cP to 1.5 cP, or 0.8 cP to 1.2 cP, as measured at 20°C. The low viscosity of the single pore-inducing substance allows it to pass through the step emulsifier at a high speed, resulting in increased process efficiency.

[0087] The examples of methods for measuring viscosity are not particularly limited, and conventional viscosity measurement methods can be applied without restriction. However, to give one example, viscosity can be measured at a temperature of 20-25°C using a Brookfield viscometer DV2T instrument equipped with Spindle 61 for low viscosity measurement, and with a fixed RPM of 200. However, it is not necessarily limited to this, and generally, viscosity can be measured with other measuring devices as long as the RPM value is the same and the viscosity range of the sample being measured (e.g., solution) is at a level of about 10-1000 cp.

[0088] The aforementioned pore-inducing substance may include one compound selected from the group consisting of aromatic hydrocarbon compounds, ketone compounds, and acetate compounds. In other words, the aforementioned pore-inducing substance may include one of the following compounds: one aromatic hydrocarbon compound, one ketone compound, or one acetate compound.

[0089] The aromatic hydrocarbon compound may include an aromatic hydrocarbon compound or a derivative compound in which some of the hydrogen atoms contained in the aromatic hydrocarbon compound are substituted with other functional groups. Specifically, the aromatic hydrocarbon compound may include an aromatic hydrocarbon compound having 6 to 20 carbon atoms that is substituted with one or more C1 to C10 alkyl groups. In the aromatic hydrocarbon compound having 6 to 20 carbon atoms that is substituted with one or more C1 to C10 alkyl groups, the C1 to C10 alkyl groups may be substituted with at least one, or 1 to 6, or 1 to 5, or 1 to 4 atoms. The examples of the aromatic hydrocarbon compound are not particularly limited, but one example is that it may include mesitylene.

[0090] The ketone compound may include a ketone compound or a derivative compound in which some of the hydrogen atoms contained in the ketone compound are substituted with other functional groups. Specifically, the ketone compound may include an aliphatic ketone compound having 5 to 20 carbon atoms. The examples of the ketone compound are not particularly limited, but one example is diisobutyl ketone.

[0091] The acetate compound may include an acetate compound or a derivative compound in which some of the hydrogen atoms contained in the acetate compound are substituted with other functional groups. Specifically, the acetate compound may include an aliphatic acetate compound having 6 to 20 carbon atoms. The examples of the acetate compound are not particularly limited, but one example is that it may include amyl acetate or hexyl acetate.

[0092] On the other hand, the pore-inducing substance may include a mixture of alcohol-based compounds and aromatic hydrocarbon-based compounds. In the dispersed phase solution, the content of the pore-inducing substance, which includes a mixture of alcohol-based compounds and aromatic hydrocarbon-based compounds, may be 10% to 80% by weight. If the content of the pore-inducing substance is excessively reduced to less than 10% by weight, the average diameter of the pores in the porous polymer particles decreases excessively, making it difficult to secure sufficient space for protein binding. Conversely, if the content of the pore-inducing substance increases excessively to more than 80% by weight, the average diameter of the pores in the porous polymer particles increases excessively, making it difficult to secure the strength of the particles.

[0093] On the other hand, the content of the aromatic hydrocarbon compound may be 10 to 1000 parts by weight per 100 parts by weight of the alcohol compound. Within this range, it is possible to satisfy pore size and particle size suitable for application to protein purification particles.

[0094] The alcohol compound may have 11 or fewer carbon atoms, 10 or fewer carbon atoms, 1 or more carbon atoms, 1 to 11 carbon atoms, or 1 to 10 carbon atoms. If the number of carbon atoms in the alcohol compound increases excessively, such as to more than 11 carbon atoms, phase separation may occur excessively quickly in the dispersed phase during particle polymerization, potentially forming core-shell particles that are not porous. During protein purification, ligands to which the protein to be purified can adhere are attached to the particle surface. However, core-shell particles have fewer ligand attachment sites compared to porous particles, making them unsuitable for protein purification.

[0095] More specifically, the alcoholic compound may include an aliphatic alcohol having 1 to 11 carbon atoms. The aliphatic alcohol having 1 to 11 carbon atoms may be substituted or unsubstituted, and if substituted, examples of substituents are as described above.

[0096] The specific examples of the aliphatic alcohols having 1 to 11 carbon atoms are not particularly limited, but for example, they may include one or more compounds selected from the group consisting of 1-decanol, 1-butanol, 1-pentanol, 1-hexanol, 1-heptanol, 1-octanol, and 1-nonanol.

[0097] The aromatic hydrocarbon compound may have 11 or fewer carbon atoms, or 10 or fewer carbon atoms, or 6 or more carbon atoms, or 6 to 11 carbon atoms, or 6 to 10 carbon atoms. When an aliphatic hydrocarbon compound is used instead of the aromatic hydrocarbon compound, phase separation occurs excessively quickly in the dispersed phase during particle polymerization, which can result in the formation of core-shell particles that are not porous. During protein purification, ligands to which the protein to be purified can adhere are attached to the particle surface. However, core-shell particles have fewer sites to which ligands can adhere compared to porous particles, making them unsuitable for protein purification.

[0098] Furthermore, the aromatic hydrocarbon compound may include an aromatic hydrocarbon compound having 6 to 8 carbon atoms substituted with an alkyl group having 1 to 3 carbon atoms. The aromatic hydrocarbon compound may also include toluene.

[0099] In one example, the dispersed phase solution may contain 10 to 1000 parts by weight, or 60 to 500 parts by weight, of the pore-inducing substance per 100 parts by weight of the monomer. Specifically, the lower limit of the content of the pore-inducing substance per 100 parts by weight of the monomer may be, for example, 10 parts by weight or more, 60 parts by weight or more, 200 parts by weight or more, 250 parts by weight or more, 300 parts by weight or more, 350 parts by weight or more, 400 parts by weight or more, 450 parts by weight or more, or 500 parts by weight or more. Furthermore, the upper limit of the content of the pore-inducing substance per 100 parts by weight of the monomer may be, for example, 1000 parts by weight or less, 550 parts by weight or less, 500 parts by weight or less, 450 parts by weight or less, 400 parts by weight or less, 350 parts by weight or less, 300 parts by weight or less, 250 parts by weight or less, or 200 parts by weight or less. When the above-mentioned content is satisfied, it is advantageous for stably forming an emulsion and ensuring the desired level of particle strength and morphological characteristics through crosslinking.

[0100] The dispersed phase solution may further contain an initiator. The type of initiator is not particularly limited, such as a photopolymerization initiator or a thermal polymerization initiator, as long as it does not hinder the securing of particle properties by the manufacturing method of this invention. A wide variety of examples widely used in the conventional field of polymer particle manufacturing can be applied without limitation.

[0101] To give an example, initiators such as ketone initiators, organic peroxide initiators, and azo initiators can be used. Specifically, compounds such as 2,2-dimethoxy-2-phenylacetophenone, 2-Hydroxy-4'-(2-hydroxyethoxy)-2-methylpropiophenone, 1-Hydroxycyclohexyl phenyl ketone, benzoyl peroxide, di-t-amyl peroxide, t-butyl peroxybenzoate, 2,5-dimethyl-2,5-di-(t-butyl peroxy)hexane, 2,5-dimethyl-2,5-di-(t-butyl peroxy)hexine-3, or dicumyl peroxide, and mixtures thereof can be used, but are not limited to these.

[0102] The amount of the initiator is not particularly limited. Only an appropriate amount of initiator can be used at a level that does not hinder the securing of the desired particle properties. For example, 0.01 parts by weight or more, specifically 0.05 parts by weight or more, 0.10 parts by weight or more, 0.15 parts by weight or more, or 0.20 parts by weight or more of initiator can be included per 100 parts by weight of the dispersed phase solution. The upper limit of the initiator content may 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.

[0103] The polymerization is carried out under conditions that do not hinder the securing of particle properties. For example, the temperature and stirring speed at which polymerization is carried out are appropriately controlled by those skilled in the art.

[0104] On the other hand, the porous polymer particles may be polymerization reaction products of droplets obtained by passing a dispersed phase solution and a continuous phase aqueous solution containing one or two types of pore-inducing substances, monomers, and a crosslinking agent through a step emulsifier. Various types of O / W droplets can be produced in a uniform size using the step emulsifier. The description of the dispersed phase solution and continuous phase aqueous solution containing the pore-inducing substances, monomers, and crosslinking agents is the same as described above.

[0105] In contrast, with existing O / W droplet manufacturing equipment that is not a step emulsifier (for example, batch suspension polymerization), the coefficient of variation of particle size can increase sharply, leading to problems of large and non-uniform particle size deviations.

[0106] The step emulsification apparatus may include a first inlet for injecting the dispersed phase solution; a second inlet for injecting the continuous phase solution; a dispersed phase channel through which the dispersed phase solution flows; a continuous phase channel through which the continuous phase solution flows; and fine channels through which the dispersed phase solution flows into the continuous phase channel and forms droplets.

[0107] In particular, when the viscosity of the aforementioned single-pore-inducing substance is low and it passes through a step emulsifier to produce droplets, the maximum droplet generation rate is increased, droplets do not adhere to the inside of the step emulsifier, droplet generation is smooth even during repeated use, and process efficiency is improved.

[0108] Furthermore, the low viscosity of the pore-inducing substance containing the mixture of alcohol-based and aromatic hydrocarbon compounds resulted in a high maximum droplet generation rate when passing through the step emulsifier to obtain droplets. The droplets did not adhere to the inside of the step emulsifier, droplet generation was smooth even during repeated use, and process efficiency was improved.

[0109] The velocity of the dispersed phase flowing through the dispersed phase channel is not limited to this, but may be between 1 μl / min and 10 ml / min. The velocity of the continuous phase flowing through the continuous phase channel is not limited to this, but may be between 0.1 μl / min and 100 ml / min.

[0110] Specifically, the porous polymer particles are obtained by a manufacturing method that involves passing a dispersed phase solution and a continuous phase aqueous solution containing one to two types of pore-inducing substances, monomers, and a crosslinking agent through a step emulsifier, the steps of which include: injecting the dispersed phase solution into a first inlet; injecting the continuous phase solution into a second inlet; and allowing the dispersed phase flowing through the dispersed phase channel to flow through the microchannels into the continuous phase channel to form droplets.

[0111] More specifically, referring to Figures 10 and 11 below, the step emulsification apparatus is manufactured by bonding a silicon substrate 1 on which a set emulsification pattern 10 is formed and a glass substrate 2 on which first and second inlet ports 52 and 54 and an outlet port 56 are formed, and then thermally oxidizing the bonded silicon substrate 1 and glass substrate 2.

[0112] Once the emulsification device is prepared, the dispersed phase solution is injected into the dispersed phase supply unit 22 of the dispersed phase channel 20 through the first inlet 52 (S210). Here, the dispersed phase may contain monomers, crosslinking agents, initiators, etc., depending on its purpose. The dispersed phase can be injected into the first inlet 52 by a syringe pump, but is not limited to this. The dispersed phase injected into the dispersed phase supply unit 22 through the first inlet 52 flows through the dispersed phase channel 24. At this time, the velocity of the dispersed phase is not limited to this, but may be 1 μl / min to 10 ml / min. The dispersed phase flowing through the dispersed phase channel 24 flows into the continuous phase channel 34 through a plurality of fine channels 40.

[0113] Furthermore, the continuous phase is injected into the continuous phase supply section 32 of the continuous phase channel 30 through the second injection port 54 (S220). The continuous phase can be injected into the second injection port 54 by a syringe pump, but is not limited to this. The continuous phase injected into the continuous phase supply section 32 through the second injection port 54 flows through the continuous phase flow path 34. At this time, the velocity of the continuous phase is not limited to this, but may be 0.1 μl / min to 100 ml / min.

[0114] The dispersed phase flowing through multiple fine channels 40 into the continuous phase channel 34 encounters the continuous phase flowing in the continuous phase channel 34, penetrates the continuous phase, and forms O / W droplets (S230).

[0115] The O / W droplets flow along the continuous phase channel 34 with the continuous phase to the discharge section 36, and are discharged to the outside of the emulsifier through the discharge port 56.

[0116] On the other hand, in the step of polymerizing the droplets obtained in the step emulsification apparatus, the polymerization can be either photopolymerization or thermal polymerization, and the polymerization reaction is carried out by adding a photopolymerization initiator or a thermal polymerization initiator to the dispersed phase described above. The specific examples of the photopolymerization initiator or thermal polymerization initiator, and the specific conditions for photopolymerization or thermal polymerization are not particularly limited, and a wide variety of examples and conditions widely used in the conventional polymer particle manufacturing field can be applied without restriction.

[0117] 2. Column for protein purification According to another embodiment of the invention, a protein purification column is provided which includes the porous polymer particles of the first embodiment. The description of the porous polymer particles includes all of the content described above in the first embodiment.

[0118] The examples of the aforementioned proteins are not particularly limited; for example, antibody drugs can be used. The porous polymer particles can be used as beads for the protein purification column, and the other components and structures of the protein purification column can be any variety of components and structures widely known in the existing field of purification columns without limitation. [Effects of the Invention]

[0119] According to the present invention, porous polymer particles that are manufactured by a highly efficient production method and have particle size and pore size suitable for achieving high protein purification performance in a protein purification column, and a protein purification column using the same can be provided. [Brief explanation of the drawing]

[0120] [Figure 1] This figure shows SEM images of the porous polymer particles obtained in Example 1 (from left to right: particle surface, cross-section of the outer part of the particle, and cross-section of the center of the particle). [Figure 2] This figure shows SEM images of the porous polymer particles obtained in Example 2 (from left to right: particle surface, cross-section of the outer part of the particle, and cross-section of the center of the particle). [Figure 3] This figure shows SEM images of the porous polymer particles obtained in Example 3 (from left to right: particle surface, cross-section of the outer part of the particle, and cross-section of the center of the particle). [Figure 4] This figure shows an SEM image (particle surface) of the porous polymer particles obtained in Example 4. [Figure 5] This figure shows an SEM image of the porous polymer particles obtained in Example 5. [Figure 6] This figure shows an SEM image of the porous polymer particles obtained in Example 6. [Figure 7] This figure shows an SEM image of the porous polymer particles obtained in Example 7. [Figure 8] This figure shows SEM images of the porous polymer particles obtained in Comparative Example 1 (from left to right: particle surface, cross-section of the outer part of the particle, and cross-section of the center of the particle). [Figure 9] This figure shows an SEM image (particle surface) of the porous polymer particles obtained in Comparative Example 2. [Figure 10] This is a plan view of the first substrate of an emulsifying apparatus according to an embodiment of the present invention. [Figure 11] This is a plan view of the second substrate of an emulsifying apparatus according to an embodiment of the present invention. [Modes for carrying out the invention]

[0121] The invention will be described in more detail by the following embodiments. However, the following embodiments are merely illustrative of the present invention, and the content of the present invention is not limited to the following embodiments.

[0122] <Examples and Comparative Examples: Production of Porous Polymer Particles> Examples 1-7 1) Production of the dispersed phase As shown in Table 1 below, glycidyl methacrylate (GMA) as a monomer, ethylene glycol dimethacrylate (EGDMA) as a crosslinking agent, and porogen as a pore-inducing agent were stirred in a vial. Subsequently, irgacure651 was added to the vial at a concentration of 1% by weight relative to the weight of the stirred solution as a photoinitiator, and the mixture was stirred at room temperature for about 5 minutes to produce the dispersed phase solution.

[0123] 2) Manufacturing of continuous phase A continuous phase solution was prepared by dissolving sodium dodecyl sulfate (SDS) at a concentration of 0.5% in DI water.

[0124] 3) Production of porous polymer particles A connector was connected to a step emulsification microfluidic device. The dispersed phase was injected into the first inlet 52 and the continuous phase into the second inlet 54 using a syringe pump. The dispersed phase flowed through the dispersed phase channel 24 at a rate of 20 μl / min, and the continuous phase flowed through the continuous phase channel 34 at a rate of 80 μl / min, generating fine droplets with a diameter of approximately 37 μm, as measured by an optical microscope. (See Figures 10 and 11 below.)

[0125] The generated droplets were collected in a 3% PVA (molecular weight 85,000-125,000) aqueous solution and shaken to evenly disperse the droplets in the solution. Then, photopolymerization was carried out using a spot UV curing device to produce porous polymer particles.

[0126] Comparative Examples 1-2 1) Production of the dispersed phase The dispersed phase solution was prepared in the same manner as in the above examples, except that glycidyl methacrylate (GMA) was used as the monomer, ethylene glycol dimethacrylate (EGDMA) as the crosslinking agent, and a porogen as the pore-inducing agent, as shown in Table 1 below (content basis: 100 wt% of dispersed phase solution).

[0127] 2) Manufacturing of continuous phase A continuous phase solution was prepared using the same method as in the above example.

[0128] 3) Production of porous polymer particles Porous polymer particles were produced in the same manner as in the above-mentioned examples, except that the dispersed phase obtained in the above-mentioned comparative example was used.

[0129] Comparative Examples 3-4 As shown in Table 1 below, glycidyl methacrylate (GMA), ethylene glycol dimethacrylate (EDGMA), and initiator were dissolved in the pore-inducing substance solution in a volume ratio of 1:0.5 to the total volume of the glycidyl methacrylate (GMA), ethylene glycol dimethacrylate (EDGMA), and initiator, and stirred to form a clear, homogeneous mixed solution to prepare the oil phase. The pore-inducing substance solution was prepared by mixing n-hexane and dodecanol in a 1:1 volume ratio.

[0130] Then, polyvinyl alcohol (PVA) with a molecular weight of 1800 was dissolved in deionized water as a stabilizer, and sodium dodecyl sulfate (SDS) was dissolved as a surfactant to form a colorless, transparent, homogeneous mixed solution, and an aqueous phase with a stabilizer concentration of 0.1% and a surfactant concentration of 3.0% was prepared.

[0131] Then, the oil phase was added to the aqueous phase under mechanical stirring conditions to allow the polymerization reaction to proceed. The reaction solution was then sequentially washed with deionized water and ethanol to remove unreacted residues, and finally vacuum-dried at 70°C to produce porous polymer particles.

[0132] Reference example 1 1) Production of the dispersed phase A dispersed phase solution was prepared in the same manner as in Example 5.

[0133] 2) Manufacturing of continuous phase A continuous phase solution was prepared using the same method as in Example 5.

[0134] 3) Production of porous polymer particles Porous polymer particles were produced using an existing batch suspension polymerization method without using the aforementioned step emulsification microfluidic device.

[0135] <Example of experiment> The properties of the porous polymer particles obtained in the above examples, comparative examples, and reference examples were measured using the following method, and the results are shown in Table 1.

[0136] 1. Epoxy content (1) Titration of experimental group samples 0.1 g of porous polymer particles obtained in the above examples, comparative examples, and reference examples were mixed with 1 mL of HCl / acetone solution (volume ratio 1 / 40) and subjected to sonication for 4 minutes. An indicator solution (0.1% cresol red and 0.1% thymol blue mixed in a 1:3 volume ratio, and then adjusted to pH 7.0 with 0.01 M NaOH) was added to the mixture. Titration with 0.1 M NaOH was carried out until the solution turned purple-blue.

[0137] (2) Titration of control group sample Except for using distilled water instead of the porous polymer particles obtained in the above-mentioned examples, comparative examples, and reference examples, the titration was carried out on the control group sample in the same manner as the experimental group sample.

[0138] (3) Measurement of epoxy content The epoxy content in 1g of porous polymer particles was calculated using the following formula 1.

[0139] [Formula 1] Epoxy content (μmol / g)=[(V0-V)×C NaOH [×1000] / W

[0140] In the above formula 1, V is the amount (mL) of NaOH used in the titration of the experimental group sample containing porous polymer particles. V0 is the amount (mL) of NaOH used in the titration of the control group sample, in which distilled water was used instead of porous polymer particles in the experimental group sample. C NaOH This is the concentration (M) of NaOH used in the titration. W is the mass (g) of the porous polymer particles used in the titration.

[0141] 2. Particle size and coefficient of variation For the porous polymer particles obtained in the above examples, comparative examples, and reference examples, the particles were observed at room temperature using an optical microscope, and the particle diameter and its coefficient of variation (coefficient of variant, CV) were measured. Specifically, measurements were taken for 100 particle samples at room temperature using an optical microscope, and the values ​​were calculated using the following formula 2.

[0142] [Formula 2] Coefficient of variation (%) = (Standard deviation of particle size / Average particle size) × 100

[0143] 3. Pore diameter For the porous polymer particles obtained in the above-mentioned examples, comparative examples, and reference examples, the pore diameter range of the particle surface, outer cross-section, and central cross-section was confirmed using SEM imaging (see Figures 1 to 9 below).

[0144] [Table 1]

[0145] - Mesitylene: Viscosity: 1.0 cP (20℃) - Diisobutyl ketone: Viscosity: 0.9 cP (20℃) - Amyl acetate: Viscosity: 0.9 cP (20℃) - Hexyl acetate: Viscosity: 1.2 cP (20℃) -1-Decanol: Viscosity: 12.0 cP (20℃) - Toluene: Viscosity: 0.56 cP (25℃) -n-butyl acetate: Viscosity: 0.685 cP (25℃) [Explanation of symbols]

[0146] 1: First circuit board, 2: Second circuit board 10: Emulsification pattern, 20: Dispersed phase channel 22: Dispersed phase supply section, 24: Dispersed phase flow path 30: Continuous phase channel, 32: Continuous phase supply unit 34: Continuous phase flow path, 36: Discharge section 40: Fine channel, 52: First injection port 54: 2nd inlet, 56: outlet

Claims

1. Polymer matrix; and Porous polymer particles comprising pores having a diameter of 50 nm to 500 nm, dispersed in the polymer matrix; The porous polymer particles are polymerization reaction products of a dispersed phase solution and a continuous phase aqueous solution containing a pore-inducing substance, monomers, and a crosslinking agent, which include one single compound or a mixture of two compounds. The pore-inducing substance containing the aforementioned single compound comprises one compound selected from the group consisting of aromatic hydrocarbon compounds having 6 to 20 carbon atoms substituted with one or more alkyl groups having 1 to 10 carbon atoms, aliphatic ketone compounds having 5 to 20 carbon atoms, and aliphatic acetate compounds having 6 to 20 carbon atoms. The pore-inducing substance containing the above two mixtures comprises a mixture of aliphatic alcohols having 1 to 11 carbon atoms and aromatic hydrocarbon compounds having 6 to 8 carbon atoms substituted with alkyl groups having 1 to 3 carbon atoms. The monomer includes a monomer containing an epoxy group, The continuous phase aqueous solution comprises water and an ionic surfactant or a nonionic surfactant. In the dispersed phase solution, the content of a pore-inducing substance containing one single compound or a mixture of two compounds is 44% to 55% by weight, based on 100% by weight of the dispersed phase solution. Porous polymer particles in which the epoxy content per 1 g is 500 μmol or more and less than 5000 μmol, as measured by the following formula 1: [Formula 1] Epokishi content (μmol / g) = [(V ０ -V)×C 塩基 [×1000] / W In the above formula 1, V is the amount (mL) of base used in the titration of the experimental group sample containing porous polymer particles. V ０ This is the amount (mL) of base used in the titration of the control group sample, in which distilled water was used instead of porous polymer particles in the experimental group sample. C 塩基 This is the concentration (M) of the base used in the titration. W is the mass (g) of the porous polymer particles used in the titration.

2. The porous polymer particles according to claim 1, wherein the porous polymer particles have a pore diameter of 50 nm to 500 nm on the particle surface as measured by SEM.

3. The porous polymer particles according to claim 1, wherein the porous polymer particles have a particle diameter of 10 μm to 150 μm as measured by an optical microscope.

4. The porous polymer particles according to claim 1, wherein the coefficient of variation of the particle size measured by an optical microscope is 20% or less.

5. The porous polymer particles according to claim 1, wherein the porous polymer particles are particles for protein purification.

6. The porous polymer particles according to claim 1, wherein the aromatic hydrocarbon compound having 6 to 20 carbon atoms substituted with one or more alkyl groups having 1 to 10 carbon atoms includes mesitylene.

7. The porous polymer particles according to claim 1, wherein the aliphatic ketone compound having 5 to 20 carbon atoms includes diisobutyl ketone.

8. The porous polymer particles according to claim 1, wherein the aliphatic acetate compound having 6 to 20 carbon atoms includes amyl acetate or hexyl acetate.

9. The porous polymer particles according to claim 1, wherein the pore-inducing substance containing the aforementioned single compound has a viscosity of 0.5 cP to 15 cP as measured at 20°C.

10. The porous polymer particles according to claim 1, wherein the aliphatic alcohol having 1 to 11 carbon atoms comprises one or more compounds selected from the group consisting of 1-decanol, 1-butanol, 1-pentanol, 1-hexanol, 1-heptanol, 1-octanol, and 1-nonanol.

11. The porous polymer particles according to claim 1, wherein the aromatic hydrocarbon compound having 6 to 8 carbon atoms substituted with an alkyl group having 1 to 3 carbon atoms contains toluene.

12. The porous polymer particles according to claim 1, wherein the monomer content in the dispersed phase solution is 10% to 60% by weight based on 100% by weight of the dispersed phase solution.

13. The porous polymer particles according to claim 1, wherein the content of the crosslinking agent in the dispersed phase solution is 10% to 60% by weight based on 100% by weight of the dispersed phase solution.

14. The porous polymer particles are The process includes a step of passing a dispersion phase solution and a continuous phase aqueous solution containing a pore-inducing substance, monomer, and crosslinking agent, which contain one single compound or a mixture of two, through a step emulsifier to obtain a polymerization reaction product of droplets. The pore-inducing substance containing the aforementioned single compound comprises one compound selected from the group consisting of aromatic hydrocarbon compounds having 6 to 20 carbon atoms substituted with one or more alkyl groups having 1 to 10 carbon atoms, aliphatic ketone compounds having 5 to 20 carbon atoms, and aliphatic acetate compounds having 6 to 20 carbon atoms. The pore-inducing substance containing the above two mixtures comprises a mixture of aliphatic alcohols having 1 to 11 carbon atoms and aromatic hydrocarbon compounds having 6 to 8 carbon atoms substituted with alkyl groups having 1 to 3 carbon atoms. The monomer includes a monomer containing an epoxy group, The method for producing porous polymer particles according to claim 1, wherein the continuous phase aqueous solution comprises water and an ionic surfactant or a nonionic surfactant.

15. The aforementioned step emulsification apparatus is First inlet for injecting the dispersed phase solution; A second inlet for injecting the continuous phase solution; A dispersed phase channel through which a dispersed phase solution flows; A continuous phase channel through which a continuous phase solution flows; and A method for producing porous polymer particles according to claim 14, comprising: a fine channel through which a dispersed phase solution flows into a continuous phase channel to form droplets;

16. A protein purification column comprising porous polymer particles as described in claim 1.

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