JP2021121411A - Size exclusion chromatography carrier, method for producing the same, and recovery method of micro substance - Google Patents

Abstract

To provide a size exclusion chromatography carrier capable of efficiently recovering a living organism-derived micro substance, particularly extracellular vesicles in a state where physiological activity is not lost, and a method for producing the size exclusion chromatography carrier, and a recovery method of micro substance using the size exclusion chromatography carrier.SOLUTION: There are provided a carrier of size exclusion chromatography having inorganic porous particles and a hydrophilic organic substance layer immobilized on a surface of the inorganic porous particles, in which a ratio of the carbon amount of the hydrophilic organic substance layer with respect to the total amount of the carrier is 1.5 mass% or more and 10 mass% or less, and a method for recovering a micro substance from cell culture supernatant or biological fluid by using the size exclusion chromatography carrier.SELECTED DRAWING: None

Description

The present invention relates to a size exclusion chromatography carrier, a method for producing the size exclusion chromatography carrier, and a method for recovering minute substances.

In recent years, pathological diagnosis using extracellular vesicles such as exosomes as biomarkers has attracted attention. The use of extracellular vesicles requires a technique for isolating and purifying extracellular vesicles from a biological sample, and conventionally, for example, a fractional centrifugation method including an ultracentrifugation step has been used. .. However, the method using ultracentrifugation requires special equipment, takes a long time to recover, is not sufficiently pure, and cannot make a highly accurate diagnosis, so that extracellular vesicles can be efficiently recovered. There is a need for a way to do it. In addition, it is not only necessary to collect the membrane proteins of extracellular vesicles such as exosomes for the purpose of analyzing the membrane proteins of extracellular vesicles, and attempts have been made to collect the membrane proteins of extracellular vesicles without impairing their physiological activity and connect them to pathological diagnosis. Therefore, there is a demand for a method capable of recovering extracellular vesicles without impairing physiological activity.

In Patent Document 1, as a method for easily fractionating extracellular vesicles based on size, cells are obtained by mixing polyethylene glycols having different molecular weights with a biological sample and then centrifuging to obtain a precipitate. A method for fractionating extracellular vesicles has been proposed. In the method described in Patent Document 1, although the amount of extracellular vesicles recovered is large, there is a problem that it is difficult to separate the extracellular vesicles because they are bound to polyethylene glycol and precipitated. rice field.

A method of fractionating extracellular vesicles from a biological sample by size exclusion chromatography is known, but a method using a substrate capable of controlling pore volume and size is not known, and the purity of the target product is unknown. Was inadequate.

On the other hand, porous silica particles are widely used as carriers for various types of chromatography, including size exclusion chromatography. For example, Patent Document 2 describes a technique for subjecting porous silica particles having high alkali resistance to surface treatment according to the type of chromatography, such as affinity chromatography carrier, cation exchange chromatography carrier, and anion exchange chromatography carrier. Examples of a reversed-phase chromatography carrier, a size exclusion chromatography carrier, and the like are given. However, the technique of Patent Document 2 is optimized for protein purification using a method of adsorbing a protein using a ligand and recovering the adsorbed protein, and the technique of Patent Document 2 can be used as it is. , It is not always possible to efficiently recover extracellular vesicles.

International Publication No. 2016/185564 International Publication No. 2016/1634080

The present invention has been made in view of the above circumstances, and is a size exclusion chromatography carrier capable of efficiently recovering biologically-derived micromaterials, particularly extracellular vesicles, without losing physiological activity, and the size thereof. An object of the present invention is to provide a method for producing an exclusion chromatography carrier. Another object of the present invention is to provide a method for recovering microsubstances derived from a living body, particularly extracellular vesicles, which can efficiently recover microsubstances, particularly extracellular vesicles, without losing their physiological activity. ..

The present invention has the following configurations.
[1] A carrier for size exclusion chromatography having inorganic porous particles and a hydrophilic organic substance layer immobilized on the surface of the inorganic porous particles, wherein the amount of carbon in the carrier is 1.5% by mass. A size exclusion chromatography carrier having an amount of 10% by mass or more and 10% by mass or less.
[2] The size exclusion chromatography carrier according to [1], wherein the amount of N in the carrier is 0.5% by mass or less.
[3] The size exclusion chromatography carrier according to [1] or [2], wherein the amount of Cl in the carrier is 0.5% by mass or less.
[4] The size exclusion chromatography carrier according to any one of [1] to [3], wherein the amount of the hydrophilic organic substance immobilized on the unit mass of the inorganic porous particles is 200 μmol / g or more and 1600 μmol / g or less. ..
[5] The size exclusion chromatography carrier according to any one of [1] to [4], wherein the hydrophilic organic layer contains a glycidyl group.
[6] The size exclusion chromatography carrier according to [5], wherein the hydrophilic organic layer does not contain a carbonyl structure derived from an acrylic group or a methacrylic group.
[7] The size exclusion chromatography carrier according to any one of [1] to [6], wherein the inorganic porous particles are porous silica particles.
[8] The size exclusion chromatography carrier according to any one of [1] to [7], wherein the inorganic porous particles have an average particle size of 1 μm or more and 500 μm or less.
[9] The inorganic porous particles have an average pore diameter of 40 nm or more and 115 nm or less and a pore volume of 0.8 mL / g or more and 2.5 mL / g or less, as measured by a mercury intrusion method [1]. ] To [8]. The size exclusion chromatography carrier according to any one of [8].
[10] The method for producing a size exclusion chromatography carrier according to any one of [1] to [9].
The hydrophilic organic substance layer contains a hydrophilic resin, and a composition for forming a hydrophilic organic substance layer containing the hydrophilic resin or a precursor thereof is formed on the surface of the inorganic porous particles, and the amount of carbon in the obtained carrier is the amount of carbon. A method for producing a size exclusion chromatography carrier, which comprises applying and curing the mixture so as to be 1.5% by mass or more and 10% by mass or less.
[11] The production method according to [10], wherein the precursor of the hydrophilic resin contains three or more glycidyl groups and has a mass average molecular weight of 200 or more and 2000 or less.
[12] The production method according to [10] or [11], wherein the precursor of the hydrophilic resin contains at least one selected from glycerol polyglycidyl ether and polyglycerol polyglycidyl ether.
[13] A method for recovering a minute substance from a cell culture supernatant or a biological fluid using the size exclusion chromatography carrier according to any one of [1] to [9].

According to the present invention, there is a size exclusion chromatography carrier capable of efficiently recovering biologically-derived microsubstances, particularly extracellular vesicles, without losing physiological activity, and a method for producing the size exclusion chromatography carrier. Can be provided. The present invention can also provide a method for recovering microsubstances derived from a living body, particularly extracellular vesicles, which can efficiently recover microsubstances, particularly extracellular vesicles, without losing their physiological activity. ..

Hereinafter, embodiments of the present invention will be described. The present invention is not construed as being limited to the following description.

The size exclusion chromatography carrier of the present invention (hereinafter, also simply referred to as “carrier”) has inorganic porous particles and a hydrophilic organic substance layer immobilized on the surface of the inorganic porous particles, and is contained in the carrier. The amount of carbon is 1.5% by mass or more and 10% by mass or less.

The inorganic porous particles contained in the carrier of the embodiment are usually particles having a large number of pores on the surface. The carrier has a hydrophilic organic substance layer on the surface of the inorganic porous particles, and the amount of carbon in the carrier is 1.5% by mass or more and 10% by mass or less. The amount of carbon in the carrier is an index indicating the amount of organic matter in the layer containing organic matter such as the hydrophilic organic matter layer possessed by the carrier. Even when the carrier has a layer containing an organic substance such as a hydrophilic organic substance layer, if the amount of carbon in the carrier is 10% by mass or less, the shape of the surface of the inorganic porous particles is an organic substance such as a hydrophilic organic substance layer. Can be followed by a layer containing. Therefore, the surface of the carrier of the embodiment has a large number of pores substantially similar to the surface of the inorganic porous particles.

Therefore, in size exclusion chromatography, for example, when the carrier of the embodiment is packed in a column and used, when a biological sample containing a biological component of various sizes is passed from a column inlet to an outlet, a component having a small size is used. The more the passage time becomes longer due to the phenomenon of repeating entering and exiting the pores, and the fractionation according to the size of the biological component becomes possible.

Further, if the carrier has a hydrophilic organic substance layer on the surface and the amount of carbon in the carrier is 1.5% by mass or more, the hydrophilic organic substance layer on the surface of the carrier when the biological sample passes through the column By containing water, it is possible to effectively suppress the adhesion of biological components to the surface of the carrier. As a result, it is possible to shorten the column transit time of the biological components of various sizes in the biological sample as compared with the case where the inorganic porous particles are used as they are as the carrier, and it is possible to efficiently fractionate the biological components. It will be possible.

In the carrier of the embodiment, the ratio of the amount of carbon in the hydrophilic organic substance layer to the total amount of the carrier is preferably 1.5% by mass or more, which can effectively suppress the adhesion of biological components to the surface of the carrier. The ratio of the amount of carbon is more preferably 1.75% by mass or more.

From the above viewpoint, the amount of carbon in the carrier is 10% by mass or less, preferably 9% by mass or less. In the carrier of the embodiment, when the carrier does not have a layer containing an organic substance other than the hydrophilic organic substance layer, the ratio of the carbon amount of the hydrophilic organic substance layer to the total amount of the carrier is 10% by mass or less, and 9% by mass or less. preferable. The amount of carbon in the carrier is obtained, for example, by analyzing the carrier by an elemental analysis method. The carbon content of the inorganic porous particles is 0 (zero), and therefore, the carbon content of the organic substance-containing layer such as the hydrophilic organic substance layer is equal to the carbon content of the carrier.

Further, from the viewpoint of increasing the recovery amount of the minute substance, the content of the nitrogen atom (N) in the carrier (hereinafter, also referred to as “N amount”) is preferably 0.5% by mass or less. The amount of N is more preferably 0.1% by mass or less, and further preferably 0.01% by mass or less.

Further, from the viewpoint of increasing the recovery amount of minute substances, the content of chlorine atoms (Cl) in the carrier is preferably 0.5% by mass or less. If it is more than 0.5% by mass, not only the recovered amount may decrease, but also the bioactive function of the minute substance may be impaired. When the Cl content is 0.01% by mass or more and 0.5% by mass or less, it is preferable from the viewpoint of industrial productivity. The amount of N and Cl in the carrier can be obtained by analyzing the carrier by a known elemental analysis method such as fluorescent X-ray analysis or CHN elemental analysis.

The carrier of the embodiment may contain components other than the inorganic porous particles and the hydrophilic organic material layer as long as the effects of the present invention are not impaired. For example, the carrier of the embodiment may have an adhesion layer between the inorganic porous particles and the hydrophilic organic material layer in order to enhance the adhesion of the hydrophilic organic material layer to the inorganic porous particles. Hereinafter, the inorganic porous particles and the hydrophilic organic substance layer constituting the carrier of the embodiment, and the adhesion layer which is an arbitrary component will be described.

(Inorganic porous particles)
The inorganic porous particles are not particularly limited as long as they are porous particles using an inorganic material as a constituent material and having pores on the surface, which are conventionally used in size exclusion chromatography.

Examples of the inorganic material include metal oxides, glass, ceramics and the like. Examples of the metal oxide include silicon oxide (silica), aluminum oxide (alumina), zirconium oxide and the like. These are used alone or in combination of two or more. When two or more kinds are used, it may be a mixture or a composite oxide. The inorganic material preferably contains a metal oxide, particularly silica, as a main component. In this case, the inorganic material may contain an inorganic component other than the metal oxide, for example, phosphate. The fact that the inorganic material contains a metal oxide as a main component means that the inorganic material contains 70% by mass or more of the metal oxide with respect to the total amount of the inorganic material. Further, the inorganic porous particles are preferably spherical. The spherical shape can prevent the pressure loss from becoming high.

As the inorganic porous particles, porous silica particles made of an inorganic material containing silica as a main component are preferable, and silica gel is particularly preferable. The inorganic material containing silica as a main component may contain zirconium oxide, phosphate and the like. As the silica gel, silica gel containing silicon oxide in an amount of 99% by mass or more, preferably 99.9% by mass or more, and more preferably 99.99% by mass or more is used.

The pore volume and pore diameter of the inorganic porous particles can be optimized according to the type of sample targeted by size exclusion chromatography and the size of the target fractionation component. The pore volume and pore diameter of the inorganic porous particles can be measured by a known method, for example, by a mercury intrusion method.

The pore volume of the inorganic porous particles is preferably 0.8 mL / g or more and 2.5 mL / g or less. When the pore volume is 0.8 mL / g or more, in size exclusion chromatography, it is easy to cause a difference in the transit time of the column depending on the size of the component in the sample such as a biological sample. The pore volume is more preferably 1.0 mL / g or more, and even more preferably 1.5 mL / g or more. If the pore volume of the inorganic porous particles is larger than 2.5 mL / g, the particle strength generally decreases, which is not preferable. The pore volume is more preferably 1.95 mL / g or less, further preferably 1.9 mL / g or less.

The pore size of the inorganic porous particles is preferably in the range of 40 nm or more and 115 nm or less, preferably 70 nm or more and 110 nm when fractionating a fine substance having an average particle size of about 50 to 100 nm from a sample. The following is more preferable. This is, for example, an average pore diameter suitable for separating and purifying exosomes from a biological sample. Further, for example, when fractionating a fine substance having an average particle size of about 70 to 150 nm from a sample, the average pore size is preferably in the range of 80 nm or more and 115 nm or less, and more preferably 90 nm or more and 115 nm or less. Further, it is preferable that the pore diameters are the same, and when the pore distribution is obtained by the mercury intrusion method, the distribution is preferably sharp. For example, in the pore distribution based on the mercury intrusion method, the value obtained by dividing the half width of the main peak of the pore distribution curve by the pore diameter of the peak top of the main peak is preferably 0.6 or less, preferably 0.5 or less. Is more preferable.

In application to size exclusion chromatography, the smaller the average particle size of the inorganic porous particles, the better the separation characteristics, but the pressure loss during operation increases, so the value is appropriate considering the specifications of the equipment used. Is adopted. The average particle size of the inorganic porous particles is generally 1 to 500 μm, preferably 7 to 200 μm, and particularly preferably 10 to 160 μm. In the present specification, the average particle size of the inorganic porous particles is the average particle size of the agglomerated particles (secondary particles) in which the primary particles are agglomerated. The average particle size is a 50% integrated value (D50) obtained from the volume-based particle size distribution measured by the laser diffraction / scattering method.

Further, the inorganic porous particles are preferable because the separation performance is improved as the particle size distribution is uniform. Specifically, the particle size is 10% (D10) and the particle size is 90%, and the cumulative amount based on mass or volume is 10% of the total particle size. When the diameter (D90) is set, the value of D90 / D10 is preferably 3 or less. In the present specification, unless otherwise specified, the particle size means a value measured by a laser diffraction / scattering method including an average particle size. However, D90 and D10 when calculating the value of D90 / D10 refer to the values measured by the electric resistance method.

The shape of the inorganic porous particles is not particularly limited. A so-called crushed one can be used, but a spherical one can be preferably used from the viewpoint of filling property into the column used for size exclusion chromatography and suppressing pressure loss during operation. Further, it is preferably spherical including a true sphere and an ellipsoid, and particularly preferably has a shape close to a true sphere.

(Adhesion layer)
The adhesion layer is a layer arbitrarily provided between the inorganic porous particles and the hydrophilic organic substance layer for firmly adhering the two. The adhesion layer is preferably formed using a coupling agent having both the surface of the inorganic porous particles and the reactive group and the hydrophilic organic material layer and the reactive group in one molecule. As the coupling agent, an organometallic coupling agent is preferable. As the organometallic coupling agent, a silane coupling agent, a titanium-based coupling agent, an aluminum-based coupling agent, or the like can be used, and the silane coupling agent is preferable.

The organic metal-based coupling agent is preferably a compound in which a hydrolyzable group is bonded to a metal atom (Si, Ti, Al, etc.) and an organic reactive group having a reactivity with a hydrophilic organic substance or a raw material thereof, and the organic reactive group is preferable. Is appropriately selected depending on the constituent material of the hydrophilic organic substance layer. The hydrophilic organic layer preferably has a glycidyl group as described below. In this case, the organic reactive group is preferably a group having an epoxy group, a carboxy group or a hydroxyl group at the terminal, and an organic reactive group having an epoxy group is preferable.

As the organometallic coupling agent containing an epoxy group, a silane coupling agent having an epoxy group is preferably used. As the epoxy group-containing silane coupling agent, γ-glycidoxypropyltrimethoxysilane, γ-glycidoxypropylmethyldiethoxysilane, β- (3,4-epoxycyclohexyl) ethyltrimethoxysilane and the like can be used.

In order to form an adhesive layer on the surface of the inorganic porous particles, a coupling agent such as an organometallic coupling agent and the inorganic porous particles may be brought into contact with each other under a predetermined temperature condition. As a result, for example, the OH group bonded to the metal atom of the metal oxide or glass reacts with the hydrolyzable group of the organometallic coupling agent on the surface of the inorganic porous particles. The temperature during the reaction is preferably 30 to 400 ° C, more preferably 100 to 300 ° C. The reaction time, that is, the contact time is preferably 0.5 to 40 hours, more preferably 3 to 20 hours. The contact between the coupling agent and the inorganic porous particles is preferably performed in the presence of a solvent.

The solvent can be used without particular limitation as long as it does not react with the coupling agent and is stable at the reaction temperature. From the viewpoint of solubility of the coupling agent, boiling point, and compatibility with other solvents (that is, removability during washing), benzene, toluene, xylene, octane, isooctane, tetrachlorethylene, chlorobenzene, bromobenzene, etc. are usually used. Is preferably used. In addition, it is desirable that the reaction by contact be carried out under reflux of the solvent used.

For the contact between the coupling agent and the inorganic porous particles, a catalyst composed of an amine compound such as triethylamine, pyridine, N, N-diisopropylethylamine may be further used.

The amount of the coupling agent immobilized is preferably an amount that closely covers the entire surface of the inorganic porous particles from the viewpoint of enhancing the adhesion between the inorganic porous particles and the hydrophilic organic substance layer. Specifically, the amount of the coupling agent immobilized per specific surface area of the inorganic porous particles (the value obtained by dividing the amount of the coupling agent immobilized by the specific surface area of the inorganic porous particles) is 1.5 μmol / m ² or more. Is preferable. Immobilization amount of the coupling agent is more preferably ^{1.5~50μmol / m 2, 2.5~50μmol / m} 2 is particularly preferred. The amount of the coupling agent immobilized per unit mass of the inorganic porous particles is preferably 45 μmol / g or more, more preferably 50 to 320 μmol / g, and particularly preferably 100 to 300 μmol / g.

The specific surface area of the inorganic porous particles can be measured by, for example, a mercury intrusion method. Further, the amount of the coupling agent immobilized is determined based on a known method. For example, the amount of carbon contained in one molecule of the coupling agent based on the carbon content measured by the element analysis method for the inorganic porous particles after immobilizing the coupling agent, that is, the inorganic porous particles with an adhesive layer. In addition, the specific surface area of the inorganic porous particles can be used to calculate the amount of the coupling agent immobilized.

After immobilizing the coupling agent on the inorganic porous particles, the organic reactive group of the coupling agent may be further treated to enhance the reactivity with the hydrophilic organic substance. For example, when the organic reactive group contained in the coupling agent has an epoxy group, the inorganic porous particles with an adhesive layer may be treated with an acid such as hydrochloric acid to open the epoxy group to form a diol.

When the carrier of the embodiment has an adhesion layer, the amount of carbon in the adhesion layer should not exceed the amount of carbon in the entire carrier. For example, when the carrier is composed of inorganic porous particles, an adhesion layer and a hydrophilic organic substance layer, the total carbon amount of the adhesion layer and the hydrophilic organic substance layer is adjusted so as to be within the range of the carbon amount of the entire carrier. .. Similarly, it is preferable that the N amount and Cl amount of the adhesion layer do not exceed the N amount and Cl amount of the entire carrier.

(Hydrophilic organic layer)
The hydrophilic organic substance layer is a layer that constitutes the outermost layer of the carrier of the embodiment and has a function of suppressing adhesion of biological components to the surface of the carrier. The hydrophilic organic substance layer is composed of a hydrophilic organic substance having a hydrophilic group, and usually contains a hydrophilic resin having a hydrophilic group. The hydrophilic organic substance may consist of only the hydrophilic resin, may contain an adhesion component for adhering the inorganic porous particles and the hydrophilic organic substance layer, or may contain any other component.

As the hydrophilic group contained in the hydrophilic organic substance, a group having hydrophilicity and not adsorbing a biological component is preferable. Specifically, a glycidyl group, a polyoxyethylene group and the like are preferable, and a polyoxyethylene group and a glycidyl group are particularly preferable. In particular, it is preferable to have a polyoxyethylene group and a glycidyl group on the surface of the hydrophilic organic substance layer from the viewpoint of preventing non-specific adsorption without affecting the surface of the biological sample.

The ratio of the amount of Cl in the hydrophilic organic substance layer to the total amount of the carrier is preferably 0.5% by mass or less from the viewpoint of keeping the amount of Cl in the carrier within the above range. If it is more than 0.5% by mass, the amount of Cl in the carrier may exceed the above range. When the ratio of the Cl content of the hydrophilic organic substance layer to the total amount of the carrier is 0.01% by mass or more and 0.5% by mass or less, it is preferable from the viewpoint of industrial productivity.

The ratio of the N amount of the hydrophilic organic substance layer to the total amount of the carrier is preferably 0.5% by mass or less from the viewpoint of keeping the N amount in the carrier within the above range. The ratio of the N amount of the hydrophilic organic substance layer to the total amount of the carrier is more preferably 0.1% by mass or less, further preferably 0.01% by mass or less. Further, the hydrophilic organic substance has a carbonyl structure (-) derived from an acrylic group or a methacrylic group (CH ₂ = CR-C (= O)-, where R is H or CH _{3) from the viewpoint of improving the recovery amount of minute substances.} It is preferable that C (= O)-) is not contained.

<Hydrophilic resin>
The hydrophilic organic substance usually has a hydrophilic group because the hydrophilic resin contained therein has a hydrophilic group. Examples of the hydrophilic resin include the above-mentioned resins having a hydrophilic group, and an epoxy resin having a glycidyl group is particularly preferable. The epoxy resin having a glycidyl group is preferable because it has a crosslinked structure, so that the hydrophilic organic substance layer can have a dense structure.

The epoxy resin having a glycidyl group is preferably, for example, a cured product of a compound having a plurality of glycidyl groups in one molecule (hereinafter, referred to as “polyepoxyd”). Polyepoxy is a raw material component of an epoxy resin, which is also referred to herein as a "precursor". The number of glycidyl groups in the polyepoxide may be 2 or more, and preferably 3 or more from the viewpoint of increasing the number of cross-linking points. From the viewpoint of availability, the number of glycidyl groups is preferably 10 or less.

When a hydrophilic organic material layer is formed using polyepoxide, in the process of curing the polyepoxide, the glycidyl group or the reactive group contained in the cross-linking agent described later forms a reactive group on the surface of the inorganic porous particles or an adhesive layer. If it has, it reacts with the reactive group on the surface of the adhesion layer and crosslinks between the molecules. As a result, the hydrophilic organic substance layer is immobilized on the surface of the inorganic porous particles directly, or, if it has an adhesive layer, on the surface of the inorganic porous particles via the adhesive layer. The hydrophilic organic substance is immobilized on the surface of the inorganic porous particles when the hydrophilic organic substance is immobilized through the layer existing between the inorganic porous particles and the hydrophilic organic substance layer in this way. including.

Of the plurality of glycidyl groups of the polyepoxide, the glycidyl group that was not used in the reaction remains in the hydrophilic organic substance layer formed by using the polyepoxide as described above. The glycidyl group is also present inside the hydrophilic organic material layer, but is particularly present on the surface and contributes to the property of not adsorbing biological components on the surface of the hydrophilic organic material layer.

Examples of the polyepoxyd include glycidyl ether-based polyepoxydates, glycidyl ester-based polyepoxyds, glycidylamine-based polyepoxyds, and cyclic aliphatic polyepoxyds, which are used as raw material components for ordinary cured epoxy resins.

Among these, polyepoxides (or polyepoxides thereof) having a structure in which the phenolic hydroxyl groups of polyphenols having two or more phenolic hydroxyl groups and the alcoholic hydroxyl groups of polyols having two or more alcoholic hydroxyl groups are replaced with glycidyloxy groups. A glycidyl ether-based polyepoxide which is an oligomer) is preferable.

Further, among the glycidyl ether-based polyepoxides, the glycidyl ether-based polyepoxide obtained from an aliphatic polyol is preferable. Examples of the aliphatic polyol include an alkane polyol, an etheric oxygen atom-containing polyol, a sugar alcohol, a polyoxyalkylene polyol, and a polyester polyol. The polyoxyalkylene polyol is obtained by ring-opening addition polymerization of a monoepoxide such as propylene oxide or ethylene oxide to a polyol having a relatively low molecular weight such as an alkane polyol, an etheric oxygen atom-containing polyol, or a sugar alcohol. Examples of the polyester polyol include a compound having a structure in which an aliphatic diol and an aliphatic dicarboxylic acid are condensed, a compound having a ring-opening polymerization structure in a cyclic ester, and the like.

Specific examples of the glycidyl ether-based polyepoxide derived from an aliphatic polyol include ethylene glycol diglycidyl ether, propylene glycol diglycidyl ether, dipropylene glycol diglycidyl ether, neopentyl glycol diglycidyl ether, and 1,4-butanediol diglycidyl. Ether, 1,6-hexanediol diglycidyl ether, glycerol polyglycidyl ether, diglycerol polyglycidyl ether, trimethylolpropane polyglycidyl ether, sorbitol polyglycidyl ether, pentaerythritol polyglycidyl ether, polyethylene glycol diglycidyl ether, polyoxypropylene Examples thereof include diol diglycidyl ether, polyoxypropylene triol triglycidyl ether, and poly (oxypropylene / oxyethylene) triol triglycidyl ether.

Such polyepoxides include, for example, triol triglycidyl ether, tetraol tetraglycidyl ether, a mixture of triol triglycidyl ether and the same triol diglycidyl ether, tetraol tetraglycidyl ether, triglycidyl ether and di. Examples thereof include a mixture of glycidyl ether and a mixture of triol triglycidyl ether and diol diglycidyl ether.

More specifically, the average number of glycidyl groups per molecule is preferably 3 to 10, glycerol polyglycidyl ether, diglycerol polyglycidyl ether, polyglycerol polyglycidyl ether, trimethylolpropane polyglycidyl ether, penta. Examples thereof include erythritol polyglycidyl ether and sorbitol polyglycidyl ether. Among these, as the polyepoxide which is a raw material component of the epoxy resin having a glycidyl group, glycerol polyglycidyl ether, polyglycerol polyglycidyl ether and the like are preferable, and polyglycerol polyglycidyl ether is particularly preferable.

The mass average molecular weight (Mw) of the polyepoxide is preferably 200 to 2000, more preferably 550 to 1800. When Mw is 200 or more, it is possible to sufficiently cover the surface of the inorganic porous particles, and when it is 2000 or less, surface treatment can be performed while maintaining the shape of the pores on the surface of the inorganic porous particles. It is possible. In addition, Mw in this specification means the mass average molecular weight with respect to polystyrene measured by gel permeation chromatography (GPC). The epoxy equivalent of the polyepoxide (the number of grams [g / eq] of the resin containing 1 gram equivalent of the epoxy group) is preferably 120 to 200 g / eq, more preferably 140 to 185 g / eq.

Commercially available products can be used as the polyepoxide. As commercially available products, specifically, all of Nagase ChemteX's trade names are glycerol polyglycidyl ethers, Denacol EX-313 (Mw: 383), Denacol EX-314 (Mw: 454), and Polyglycerol. Examples of the polyglycidyl ether include Denacol EX-512 (Mw: 630) and Denacol EX-521 (Mw: 1294), and examples of the sorbitol polyglycidyl ether include Denacol EX-622 (Mw: 930).

The epoxy resin having a glycidyl group may be an epoxy resin that is crosslinked and cured by the reaction between the glycidyl groups of the polyepoxyd, and is a crosslinking agent having two or more reactive groups that react with the glycidyl group of the polyepoxyd. It may be an epoxy resin that is crosslinked and cured by the reaction with. Examples of the reactive group contained in the cross-linking agent include an amino group having an active hydrogen, a carboxyl group, a thiol group and the like. Specific examples of the cross-linking agent include polyamines, polycarboxylic acid anhydrides, polyamides, and polythiols.

As the epoxy resin having a glycidyl group used for a hydrophilic organic substance, an epoxy resin that is crosslinked and cured by the reaction between the glycidyl groups of polyepoxide is preferable in consideration of the above N amount and the like. A curing catalyst is usually used to cure the polyepoxy to obtain an epoxy resin. Therefore, the hydrophilic organic substance may contain a curing catalyst together with the epoxy resin.

Examples of the organic substance having a polyoxyethylene group used for the hydrophilic organic substance include 2- [METHOXY (POLYETHYLENEOXY) 6-9PROPYL] TRIMETHOXYSILANE, 2- [METHOXY (POLYETHYLENEOXY) 9-12PROPOYL] TRIMETHOY -24PROPYL] trimethylHOXYSILANE, (both from Gerest) and the like.

Examples of the curing catalyst include curing catalysts such as tertiary amines, imidazoles, Lewis acids, onium salts, dicyandiamides, organic acid dihydrazides, and phosphins. Specific examples of such a catalytic curing agent include 2-methylimidazole, 2-ethyl-4-methylimidazole, tris (dimethylaminomethyl) phenol, boron trifluoride-amine complex, dicyandiamide, and diphenyliodonium hexafluoro. Examples thereof include phosphate, triphenylsulfonium hexafluorophosphate, tris (pentafluorophenyl) borane, boron trifluoride and the like. As the curing catalyst, tris (pentafluorophenyl) borane, boron trifluoride and the like, which do not contain a nitrogen atom, are preferable.

The amount of the curing catalyst with respect to the polyepoxide is preferably 1 to 20 parts by mass with respect to 100 parts by mass of the polyepoxide, or 1 to 20 parts by mass with respect to 100 parts by mass of the total of the polyepoxide and the crosslinking agent when a cross-linking agent is used. Parts are more preferable, and 1 to 5 parts by mass are particularly preferable.

The hydrophilic organic material layer contains a hydrophilic resin, preferably an epoxy resin having a glycidyl group. The hydrophilic organic substance layer may contain various components used in the manufacturing process of the hydrophilic resin such as the above-mentioned curing catalyst as optional components, and other optional components may be contained as long as the effects of the present invention are not impaired. It may be included. It is also conceivable to use the organic substance itself forming the hydrophilic organic substance layer in the present invention as a carrier.

In the carrier of the embodiment, the amount of the hydrophilic organic substance immobilized on the unit mass of the inorganic porous particles is preferably 200 μmol / g or more and 1600 μmol / g or less. When the amount of the hydrophilic organic substance immobilized is 200 μmol / g or more, it is possible to effectively suppress the adhesion of biological components to the surface of the carrier. The amount of the hydrophilic organic substance immobilized is preferably 330 μmol / g or more, more preferably 350 μmol / g or more. Further, when the amount of the hydrophilic organic substance immobilized is 1600 μmol / g or less, the hydrophilic organic substance layer can sufficiently follow the surface shape of the inorganic porous particles. The amount of the hydrophilic organic substance immobilized is preferably 1500 μmol / g or less, and more preferably 1400 μmol / g or less. The amount of immobilization of the hydrophilic organic material layer can be calculated from the amount of carbon in the hydrophilic organic material layer.

When the carrier has an adhesive layer, the amount of carbon obtained by analyzing the carrier by the elemental analysis method is the total amount of the carbon amount derived from the adhesive layer and the carbon amount derived from the hydrophilic organic substance layer. Therefore, when the carrier has an adhesive layer, the amount of carbon derived from the hydrophilic organic layer is obtained by subtracting the amount of carbon derived from the adhesive layer obtained in advance from the total amount of the carbon amount, and the amount of carbon derived from the hydrophilic organic material layer is obtained from the value, and the hydrophilic organic material layer is fixed. The amount of conversion may be calculated.

In the carrier of the embodiment, the carbon content in the hydrophilic organic matter, that is, the carbon content with respect to the total amount of the hydrophilic organic matter constituting the hydrophilic organic matter layer is an amount in which the carbon content in the carrier is within the above range.

(Manufacturing method of carrier)
The carrier of the embodiment is obtained by forming a hydrophilic organic substance layer on the surface of the inorganic porous particles, or on the surface of the adhesion layer when having an adhesion layer. In the following description, the term "surface of inorganic porous particles" includes the surface of the adhesive layer when the adhesive layer is provided.

For example, when the hydrophilic organic substance layer contains a hydrophilic resin, a composition for forming a hydrophilic organic substance layer containing the hydrophilic resin or a precursor thereof is formed on the surface of the inorganic porous particles, and the amount of carbon in the obtained carrier is high. A hydrophilic organic substance layer can be formed on the surface of the inorganic porous particles by applying an amount of 1.5% by mass or more and 10% by mass or less and curing the mixture.

The composition for forming a hydrophilic organic substance layer contains a hydrophilic resin or a precursor thereof. The composition for forming a hydrophilic organic substance layer contains the hydrophilic resin itself when the hydrophilic resin is not reactive, and when the hydrophilic resin is a hydrophilic resin obtained by a curing reaction or a cross-linking reaction, the hydrophilic resin Contains a precursor that is a raw material component of. The hydrophilic resin is preferably the latter, and more preferably an epoxy resin having a glycidyl group. When the hydrophilic resin is an epoxy resin having a glycidyl group, the precursor preferably contains a polyepoxy, and as described above, a cross-linking agent and a curing catalyst may be contained, if necessary.

When the hydrophilic organic substance layer contains other optional components, the composition for forming the hydrophilic organic substance layer contains the other optional components. The composition for forming a hydrophilic organic substance layer may further optionally contain a solvent.

The solvent used in the composition for forming a hydrophilic organic substance layer is a solvent having good solubility in a hydrophilic resin or a compounding component containing a raw material component of the hydrophilic resin and being inert to these compounding components. If there is, the present invention is not particularly limited, and specific examples thereof include alcohols, esters, ethers, ketones, and water.

As a method of applying the composition for forming a hydrophilic organic substance layer to the surface of the inorganic porous particles, a known method can be used, and a method of heating the inorganic porous particles and the hydrophilic organic substance layer forming composition in a solvent. , Dip coating method, spray coating method and the like, but the dip coating method and the method of heating in a solvent are preferable. After the coating treatment, if the composition for forming a hydrophilic organic substance layer contains a solvent, the solvent is removed and a curing treatment is performed. The solvent may be removed by a usual method such as heating and drying.

The conditions of the curing treatment depend on the type of hydrophilic resin. When the hydrophilic resin is an epoxy resin having a glycidyl group, curing treatment at a temperature of 30 to 150 ° C. for 0.5 to 40 hours can be mentioned. The temperature of the curing treatment is preferably 50 to 250 ° C., and the curing time in that case is 0.5 to 40 hours.

The operation of applying and curing the composition for forming the hydrophilic organic substance layer on the surface of the inorganic porous particles should be such that a predetermined amount of the hydrophilic organic substance can be immobilized by one operation, but if it is not possible, the composition is applied. , Repeat the curing operation. Further, when the operations of coating and curing are repeated, for example, when the hydrophilic resin is an epoxy resin having a glycidyl group, if the coating and curing are performed once, the glycidyl group on the surface is formed for each operation. May be diolized by opening the ring by an acid treatment such as hydrochloric acid.

Further, when the precursor of the hydrophilic resin contains a polyepoxide and an arbitrary cross-linking agent and a curing catalyst in the above, a first composition containing the polyepoxide and optionally a cross-linking agent and a second composition containing a curing catalyst. Is prepared, the first composition is applied to the surface of the inorganic porous particles, the second composition is applied, and a curing treatment is performed to form a hydrophilic organic substance layer on the surface of the inorganic porous particles. You may.

In this way, the carrier of the embodiment is obtained. When the carrier of the embodiment has an adhesive layer, the ratio of the total amount of carbon of the adhesive layer and the hydrophilic organic substance layer to the total amount of the carrier is the ratio of the inorganic porous particles to the total amount of the carrier while suppressing the adhesion of biological components to the surface of the carrier. From the viewpoint that the hydrophilic organic substance layer can sufficiently follow the surface shape, 1.5% by mass or more and 10% by mass or less is preferable, and 1.75% by mass or more and 9% by mass or less is more preferable.

Since the hydrophilic organic substance layer is a thin layer that follows the surface shape of the inorganic porous particles, it can be said that the shape and size of the carrier, for example, the average particle size, is substantially the same as the shape and average particle size of the inorganic porous particles. .. That is, the shape of the carrier is preferably spherical, and the average particle size is generally 1 to 500 μm, preferably 7 to 200 μm, and particularly preferably 10 to 160 μm. It can be said that the surface shape of the carrier, for example, the pore volume and the average pore diameter, is substantially the same as the pore volume and the average pore diameter of the inorganic porous particles. That is, the pore volume of the carrier is preferably 0.8 mL / g or more and 2.5 mL / g or less, more preferably 1.0 mL / g or more and 1.95 mL / g or less, and 1.5 mL / g or more and 1.9 mL / g / g. It is more preferably g or less. The average pore diameter of the carrier is preferably in the range of 40 nm or more and 115 nm or less, and more preferably 70 nm or more and 110 nm or less.

(Recovery method for minute substances)
The carrier of the embodiment is used for size exclusion chromatography, which fractionates the micromaterials contained in the sample based on size, usually particle size.

In the size exclusion chromatography using the carrier of the embodiment, if the micro substance to be fractionated is a micro substance derived from a living body, the effect of shortening the transit time of the column tends to be remarkable. The micro substance to which the present invention is applied is not particularly limited as long as it is a micro substance having an average particle size of about 30 μm or less. Specifically, when applied to a fine substance having an average particle size of 1 nm to 10 μm, a remarkable effect can be obtained from the viewpoint of work efficiency. When the average particle size of the fine substance is 1 nm to 500 nm, the effect of the method of the present invention is more remarkable.

The type of the micro substance is not particularly limited as long as it is a dispersion liquid in which extracellular vesicles are dispersed and contained in a liquid medium, and is particularly limited as long as it has a viscosity sufficient to pass through a column. Although not, examples thereof include living cells and various colloidal particles other than living cells. Specific examples thereof include bacteria, yeast cells, fungal cells, erythrocytes, tissues, DNA, RNA, genes, viruses, exosomes, microvesicles, extracellular vesicles such as apoptosis, and proteins such as microRNA and albumin. .. Extracellular vesicles are generally separated from the cytosol by a phospholipid bilayer.

Size exclusion chromatography using the carrier of the present invention is useful for separating and purifying extracellular vesicles, particularly exosomes, from biological samples. The average particle size of exosomes is approximately 40 to 150 nm.

Further, the biological sample containing a minute substance is not limited to a solid or a liquid as long as it contains the minute substance. Size exclusion chromatography usually targets liquid materials. For example, when the biological sample is a solid, the biological sample may be dissolved and dispersed in a liquid medium to make it liquid, and size exclusion chromatography may be performed.

Specific examples of the biological sample containing the minute substance include cell culture supernatant and biological fluid. Biofluids include blood (plasma, serum), urine, saliva, bone marrow, semen, breast milk, amniotic fluid, tears, lymph, and biopsy tissue. Examples of the cell culture supernatant include a cell culture supernatant obtained from human tumor cells cultured in the presence of non-human serum, a cell culture supernatant obtained from mouse tumor cells, and the like.

When the carrier of the embodiment is used for size exclusion chromatography, it is usually used by filling a column for size exclusion chromatography.

When the carrier of the embodiment is used for size exclusion chromatography, for example, exosomes and proteins are fractionated from the same sample, for example, a serum sample containing exosomes and proteins, as compared to the case where inorganic porous particles are used as the carrier. In this case, the processing time can be shortened and the efficiency is improved. Further, as compared with the case where the inorganic porous particles are used as the carrier, the accuracy of fractionation is high, and the purity of the obtained exosome fraction can be improved.

Examples of the carrier of the present invention will be described below. Examples 1 to 3 are examples, and examples 4 and 5 are comparative examples. In addition, Example 6 shows a reference example by the ultracentrifugal separation method.

[Example 1]
As the inorganic porous particles, AGC SI-Tech Co., Ltd.'s trade name, "MS GEL SIL D-75-1000AW", which is a porous silica particle, was used. Hereinafter, the porous silica particles will be referred to as silica gel 1. Silica gel 1 has an average particle size of 73.2 μm, an average pore size of 110.1 nm, a pore volume of 0.85 mL / g, and a specific surface area of 30 m ² / g, as measured by the following methods.

The average particle size of silica gel 1 was measured by a laser light scattering method using LA-950V2 (manufactured by HORIBA, Ltd.). The average pore diameter, pore volume and specific surface area were measured by the mercury press-fitting method using Autopore IV9510 (manufactured by Shimadzu Corporation). The physical characteristics of the following silica gel were also measured by the same method.

To 50 g of silica gel 1, 400 mL of toluene, 11.5 mL of diisopropylethylamine and 14.9 mL of γ-glycidoxypropyltrimethoxysilane were added, and the mixture was refluxed for 4.5 hours. After allowing to cool, the mixture was filtered, washed with 1000 mL of toluene, 500 mL of tetrahydrofuran (THF), and 660 mL of methanol, and dried at 70 ° C. for 24 hours to obtain silica gel 1 having an adhesive layer having an epoxy group treated with a coupling agent. ..

21 g of silica gel 1 treated with a coupling agent was immersed in 84 mL of a 0.5% by mass aqueous hydrochloric acid solution at room temperature for a whole day and night, filtered, washed with 630 mL of distilled water and 630 mL of methanol, and dried at 70 ° C. for a whole day and night. Silica gel 1 with an adhesion layer in which the epoxy group was ring-opened and diolized was obtained.

0.28 g of Denacol EX-521 (trade name, manufactured by Nagase Chemtech Co., Ltd .; polyglycerol polyglycidyl ether, Mw: 1294) and 8.5 mL of methanol are added to 10 g of the obtained diolized silica gel 1 with an adhesive layer. The mixture with and was added, and the mixture was mixed at room temperature for 30 minutes. Then, it was dried at 70 ° C. for a whole day and night.

To this, 40 mL of decane and 9.2 mg of tris (pentafluorophenyl) borane were added, and the mixture was stirred at 100 ° C. for 4 hours. After allowing to cool, the mixture was filtered, washed with 200 mL of toluene, 200 mL of methanol, 200 mL of a 0.5 mass% hydrochloric acid aqueous solution, and 200 mL of a methanol aqueous solution, and dried at 70 ° C. for 24 hours.

To this, a mixed solution of 0.28 g of Denacol EX-521 and 8.5 mL of methanol was added again, and the mixture was mixed at room temperature for 30 minutes. Then, it was dried at 70 ° C. for a whole day and night. Further, 40 mL of decane and 9.2 mg of tris (pentafluorophenyl) borane were added, and the mixture was stirred at 100 ° C. for 4 hours. After allowing to cool, it is filtered, washed with 200 mL of toluene, 200 mL of methanol, and 200 mL of an aqueous methanol solution, and dried at 70 ° C. for 24 hours to obtain a carrier 1 having a hydrophilic organic substance layer on the surface of silica gel 1 via an adhesive layer. Obtained.

As a result of elemental analysis of the obtained carrier 1 using SUMIGRAPH NC-80 (manufactured by Sumitomo Chemical Co., Ltd.), the ratio of the amount of carbon contained in the carrier 1 to the total amount of the carrier 1 was 3.45% by mass. The carbon content in the diolized silica gel 1 with an adhesive layer measured in the same manner was 0.95% by mass. From these, the ratio of the amount of carbon contained in the hydrophilic organic substance layer to the total amount of 1 carrier is determined to be 2.50% by mass. Further, from the obtained amount of carbon, the amount of immobilization of the adhesion layer is determined to be 132 μmol / g, and the amount of immobilization of the hydrophilic organic substance layer is determined to be 463 μmol / g. Further, in the same manner, the ratio (mass%) of the amount of N to the total amount of the carrier was determined. Further, the proportion (mass%) of the amount of Cl was determined using the wavelength dispersive fluorescent X-ray analyzer ZSX100e (manufactured by Rigaku). The results are shown in Table 1.

In Table 1, the amount of carbon is indicated as "amount of C". The ratio of the amount of C in the adhesive layer and the hydrophilic organic material layer is the ratio (mass%) of the amount of C contained in the adhesive layer or the hydrophilic organic material layer to the total amount of the carrier. The ratio of the amount of N (mass%) and the ratio of the amount of Cl (% by mass) in the total carrier are the ratio of the amount of N (% by mass) and Cl in the hydrophilic organic material layer because the adhesive layer does not contain N and Cl. Corresponds to the proportion of quantity (% by mass).

[evaluation]
4.5 g of the obtained carrier 1 was packed in a column (inner diameter; 15 mm, length: 85 mm) to obtain size exclusion chromatography 1. 500 μL of a serum sample containing an exosome having a predetermined amount of CD9 and a protein is injected from the inlet of size exclusion chromatography 1, and fractions sequentially discharged from the outlet are collected every elapsed time (every minute) from the start of discharge. Used for analysis.

The amount of exosome-derived CD9 contained in each fraction obtained at each recovery time was measured by the exoscreen method using an Enspire manufactured by PerkinElmer. Similarly, the total amount of protein was quantified using a fluorometer (product name: QUAIT) manufactured by Thermo Fisher Scientific. For each fraction, the value of the ratio of the amount of exosome CD9 obtained above to the total amount of protein (exosome CD9 amount / total protein amount) was used as an index of purity. The recovery time (elapsed time from the start of excretion) at which the fraction with the largest amount of exosome recovery was obtained was defined as the processing time for exosome recovery. The purity was calculated by taking the ratio of the intensity of exosome CD9 and the total amount of protein of the fraction having the largest amount of exosomes recovered. Table 1 shows the treatment time for exosome recovery, the exosome CD9 intensity and total protein amount of the fraction at the treatment time, and their ratios.

[Example 2]
In the preparation of the carrier 1 according to Example 1 described above, the same applies except that AGC SI-Tech Co., Ltd., trade name, "MS GEL SIL D-50-1000AW" (silica gel 2) was used as the porous silica particles. The carrier 2 was prepared. The average particle size of silica gel 2 is 45.6 μm, the average pore size is 96.8 nm, the pore volume is 1.77 mL / g, and the specific surface area is 71 m ² / g.

The ratio of the amount of carbon contained in the carrier 2 to the total amount of the carrier 2 was 8.07% by mass. The ratio of the carbon content in the diolized silica gel 1 with the adhesive layer measured in the same manner was 2.09% by mass. From these, the ratio of the amount of carbon contained in the hydrophilic organic substance layer to the total amount of the carrier 2 is determined to be 5.98% by mass. Further, from the obtained amount of carbon, the amount of immobilization of the adhesion layer is determined to be 290 μmol / g, and the amount of immobilization of the hydrophilic organic substance layer is determined to be 1108 μmol / g.

[Example 3]
In the preparation of the carrier 1 according to Example 1 described above, the same applies except that AGC SI-Tech Co., Ltd., trade name, "MS GEL SIL D-50-700AW" (silica gel 3) was used as the porous silica particles. The carrier 3 was prepared. The average particle size of silica gel 3 is 45.1 μm, the average pore size is 69.5 nm, the pore volume is 0.81 mL / g, and the specific surface area is 47 m ² / g.

The ratio of the amount of carbon contained in the carrier 3 to the total amount of the carrier 3 was 3.57% by mass. The ratio of the carbon content in the diolized silica gel 3 with the adhesive layer measured in the same manner was 1.44% by mass. From these, the ratio of the amount of carbon contained in the hydrophilic organic substance layer to the total amount of the carrier 3 is determined to be 2.13% by mass. Further, from the obtained amount of carbon, the amount of immobilization of the adhesion layer is determined to be 200 μmol / g, and the amount of immobilization of the hydrophilic organic substance layer is determined to be 394 μmol / g.

[Example 4, Example 5]
Silica gel 1 with an adhesive layer that was diolized in the same manner as in Example 1 described above was prepared and used as a carrier 4. Further, the silica gel 1 used in Example 1 described above was used as the carrier 5.

Using the obtained carriers 2 to 5, evaluation in size exclusion chromatography was performed in the same manner as in carrier 1. The results are shown in Table 1.

[Example 6]
Exosomes were recovered by the ultracentrifugation method using an ultracentrifuge (SCP85H manufactured by Hitachi, Ltd.). The sample was 500 μL of a serum sample containing an exosome and a protein having a predetermined amount of CD9, similar to that used in Example 1. The total processing time in the ultracentrifugator was set to 480 minutes, and for the obtained fraction, the amount of exosome CD9 and the total amount of protein were measured in the same manner as in Example 1, and the value of the ratio (exosome CD9 amount / total protein amount). Was 0.5.

According to the present invention, it is possible to provide a size exclusion chromatography carrier capable of efficiently recovering micromaterials derived from a living body, particularly extracellular vesicles. Such a size exclusion chromatography carrier of the present invention has the potential to separate the desired microsubstances with high purity, and can recover extracellular vesicles without impairing the physiological activity. It can be expected to be used in the fields of testing and diagnosis, such as being able to be used for samples for tests with improved accuracy.

Claims (13)

A carrier for size exclusion chromatography having inorganic porous particles and a hydrophilic organic substance layer immobilized on the surface of the inorganic porous particles, wherein the amount of carbon in the carrier is 1.5% by mass or more and 10% by mass. % Or less size exclusion chromatography carrier. The size exclusion chromatography carrier according to claim 1, wherein the amount of N in the carrier is 0.5% by mass or less. The size exclusion chromatography carrier according to claim 1 or 2, wherein the amount of Cl in the carrier is 0.5% by mass or less. The size exclusion chromatography carrier according to any one of claims 1 to 3, wherein the amount of the hydrophilic organic substance immobilized on the unit mass of the inorganic porous particles is 200 μmol / g or more and 1600 μmol / g or less. The size exclusion chromatography carrier according to any one of claims 1 to 4, wherein the hydrophilic organic layer contains a glycidyl group. The size exclusion chromatography carrier according to claim 5, wherein the hydrophilic organic layer does not contain a carbonyl structure derived from an acrylic group or a methacrylic group. The size exclusion chromatography carrier according to any one of claims 1 to 6, wherein the inorganic porous particles are porous silica particles. The size exclusion chromatography carrier according to any one of claims 1 to 7, wherein the inorganic porous particles have an average particle size of 1 μm or more and 500 μm or less. Claims 1 to 8 of the inorganic porous particles having an average pore diameter of 40 nm or more and 115 nm or less and a pore volume of 0.8 mL / g or more and 2.5 mL / g or less, as measured by a mercury intrusion method. The size exclusion chromatography carrier according to any one of the above. The method for producing a size exclusion chromatography carrier according to any one of claims 1 to 9.
The hydrophilic organic substance layer contains a hydrophilic resin, and a composition for forming a hydrophilic organic substance layer containing the hydrophilic resin or a precursor thereof is formed on the surface of the inorganic porous particles, and the amount of carbon in the obtained carrier is the amount of carbon. A method for producing a size exclusion chromatography carrier, which comprises applying and curing the mixture so as to be 1.5% by mass or more and 10% by mass or less. The production method according to claim 10, wherein the precursor of the hydrophilic resin contains three or more glycidyl groups and has a mass average molecular weight of 200 or more and 2000 or less. The production method according to claim 10 or 11, wherein the precursor of the hydrophilic resin contains at least one selected from glycerol polyglycidyl ether and polyglycerol polyglycidyl ether. A method for recovering a minute substance from a cell culture supernatant or a biological fluid using the size exclusion chromatography carrier according to any one of claims 1 to 9.