WO2019187377A1 - Base material for filler, manufacturing method of base material for filler, filler, and protein purification method - Google Patents

Abstract

This base material for a filler comprises a natural polymer immobilized on the surface of a porous organic polymer carrier, the porous organic polymer carrier has a crosslinking degree of 50-85 mol%, the exclusion limit molecular weight is 200,000-800,000, the ratio of pore volume is 96-99 vol%, and the ratio of the mass of groups derived from the natural polymer to the mass of the base material for a filler is 200-300 mg/g.

Description

Filler base material, filler base material manufacturing method, filler and protein purification method

The present invention relates to a filler substrate, a method for producing a filler substrate, a filler, and a protein purification method.
This application claims priority on March 29, 2018 based on Japanese Patent Application No. 2018-063418 filed in Japan, the contents of which are incorporated herein by reference.

Conventionally, chromatography is used for the adsorption, separation and purification of biopolymers such as proteins. Chromatographic fillers include inorganic fillers such as silica gel, natural polymer fillers such as agarose, dextran, cellulose, chitosan, and organics such as polystyrene, poly (meth) acrylate, poly (meth) acrylamide, etc. Polymeric fillers are known. These fillers are used as they are or with various interactive functional groups added as necessary in order to enable use in various separation modes.

When a column filled with a filler is operated multiple times, the column is usually washed with an alkaline solution. Natural polymer fillers and organic polymer fillers are superior in stability to alkaline solutions used for column cleaning compared to inorganic fillers.
However, the natural polymer filler has insufficient mechanical strength. For this reason, a column packed with a natural polymer filler is difficult to use under high flow rate processing conditions. In addition, natural polymer fillers have insufficient mechanical strength, and thus are difficult to use for protein separation and purification on large scale columns.

Therefore, conventionally, organic polymer fillers such as polystyrene, poly (meth) acrylate, poly (meth) acrylamide and the like are desirable for the column filler used under high flow rate processing conditions.

Biopolymers such as proteins have a large molecular weight. In a column used under high flow rate processing conditions, in order to increase the adsorption capacity of the separation target having a large molecular weight, it is necessary to increase the diffusion efficiency of the separation target into the pores of the carrier used as the filler. . In order to increase the diffusion efficiency of the separation object into the pores, it is necessary to increase the pore diameter and pore volume of the support.
However, in general, a carrier having a large pore diameter has a small specific surface area, so that it is difficult to obtain a sufficient adsorption capacity for the separation target. In addition, since the filler having a large pore volume has poor mechanical strength, the performance under a high flow rate treatment condition may not be sufficiently obtained.

It is known that a spacer made of an organic polymer is introduced into a base material by graft polymerization as one method for increasing the adsorption capacity under a high flow rate processing condition in a filler having a large pore volume. For example, Patent Document 1 and Non-Patent Document 1 succeed in improving the protein adsorption capacity by using a filler in which an organic polymer is covalently bonded to a base material by graft polymerization. However, the introduction of the organic polymer to the base material by graft polymerization has a drawback that it is difficult to obtain reproducibility.

Also, a technique using a polysaccharide such as dextran or pullulan as a spacer is widely known. For example, in Patent Document 2 and Non-Patent Documents 2 to 4, by successfully immobilizing dextran having an average molecular weight of about 40,000 on an agarose carrier and introducing ion exchange groups, the adsorption capacity was successfully improved. Yes.

In Patent Document 3, the inside of the pore is filled and fixed with a water-soluble linear organic polymer containing a polysaccharide, and an affinity functional group is bound to the water-soluble linear organic polymer site. Fillers that are porous particles are described. Patent Document 3 describes a filler that has a large amount of protein that can be purified at a time because of its high adsorption amount, and can significantly improve the purification rate.
Moreover, it is described that the filler of patent document 3 is harder than the conventional filler, and is excellent in durability.

Patent Document 4 describes a porous carrier having a porosity of 80% or more and a column pressure loss of 0.02 MPa or more and less than 0.2 MPa.

European Patent No. 0722360 Japanese Patent No. 5826180 Japanese Patent No. 5250985 JP 2012-141212 A

Journal of Chromatography A, 1006 (2003), 229-240 J. et al. Sep. Sci. 2011, 34, 2950-2959 Journal of Chromatography A, 1217 (2010) 5084-5091 Journal of Chromatography A, 1146 (2007) 202-215

In order to purify a large amount of biopolymers such as proteins in a short time, it has sufficient mechanical strength as a packing material to be packed in the column, and the adsorption capacity of biopolymers is high even under high flow rate processing conditions. A high one is required.
However, the conventional fillers have sufficient mechanical strength and the biopolymer adsorption capacity is not sufficiently high. For this reason, in the filler, it is required to further increase the adsorption capacity of the biopolymer while ensuring the mechanical strength.

The present invention has been made in view of the above circumstances, and is provided with a filler base material and a filler base material from which a filler having sufficient mechanical strength and a high biopolymer adsorption capacity can be obtained. It is an object to provide a manufacturing method.
Another object of the present invention is to provide a filler having a sufficient mechanical strength using the above-mentioned filler base material and having a high biopolymer adsorption capacity, and a protein purification method using the filler. To do.

In order to solve the above-mentioned problems, the present inventor has conducted extensive research as described below.
As a result, the following findings (1) to (5) were obtained. The inventor has conceived the present invention based on the findings (1) to (5) above.
(1) A porous organic polymer carrier having a degree of cross-linking of 50 mol% to 85 mol% has good mechanical strength, and a filler using a base material for filler using the polymer may be a protein or the like. Even if the adsorption capacity of the biopolymer is increased, the mechanical strength can be secured.

(2) A porous organic polymer carrier having an exclusion limit molecular weight of 200,000 to 800,000 and a pore volume ratio of 96 volume% to 99 volume% has a size and number of pores in each pore. Is appropriate. For this reason, the filler using the base material for fillers using this becomes a thing with high adsorption capacity of a biopolymer.
(3) For fillers on which the natural polymer is immobilized on the surface so that the ratio of the mass of the group derived from the natural polymer to the mass of the base material for filler is 200 mg / g to 300 mg / g The base material can sufficiently exhibit the effect as a spacer due to the immobilization of the group derived from the natural polymer, and the biopolymer based on the group derived from the natural polymer while ensuring the ratio of the pore volume. The effect of improving the adsorption capacity is sufficiently obtained.

(4) A filler in which an interactive functional group is covalently bonded to a filler base material in which a natural polymer is immobilized on the surface of a porous organic polymer carrier of (1) to (3), By selecting the type of the interactive functional group, a filler according to the application can be obtained.
(5) Since the filler of (4) has a high biopolymer adsorption capacity even under high flow rate processing conditions, a large amount of biopolymer such as protein can be purified in a short time.

Further, the present inventor polymerizes a raw material monomer containing a monofunctional monomer having an epoxy group and a predetermined amount of a crosslinkable monomer in the presence of a predetermined amount of a diluent and a polymerization initiator by the following method. Then, the epoxy group derived from the monofunctional monomer of the polymer is ring-opened and the epoxy group is introduced using epichlorohydrin and secondary alcohol, and 200 mg / g to 300 mg / g It discovered that said base material for fillers was obtained by fix | immobilizing the group derived from a natural polymer, and came to complete this invention.
That is, the first aspect of the present invention provides a substrate described in the following [1].

[1] A filler base material in which a natural polymer is immobilized on the surface of a porous organic polymer carrier,
The porous organic polymer carrier has a cross-linking degree of 50 mol% to 85 mol%, an exclusion limit molecular weight of 200,000 to 800,000, and a pore volume ratio of 96 vol% to 99 vol%.
A filler base material having a ratio of a mass of a group derived from a natural polymer to a mass of the filler base material of 200 mg / g to 300 mg / g.

The base material of the first aspect of the present invention can preferably include the following features.
[2] The filler base material according to [1], wherein the natural polymer is a polysaccharide.
[3] The filler base material according to [2], wherein the polysaccharide is dextran.

The second aspect of the present invention provides the filler described in [4] below.
[4] A filler in which an interactive functional group is covalently bonded to the filler substrate according to any one of [1] to [3].
The second aspect of the present invention can preferably include the following features.
[5] The filler according to [4], wherein the interactive functional group is a cation exchange group.
[6] The filler according to [4], wherein the interactive functional group is an anion exchange group.

The third aspect of the present invention is a method for producing a filler substrate described in [7] below.
[7] A method for producing a filler substrate according to any one of [1] to [3],
A step of polymerizing a raw material monomer containing a monofunctional monomer having an epoxy group and a crosslinkable monomer in the presence of a diluent and a polymerization initiator to obtain a polymer α,
A step (A) in which the concentration of the crosslinkable monomer in the raw material monomer is 50 mol% to 85 mol%, and the diluent is used in a volume of 3.8 times to 5.8 times the raw material monomer;
A step (B) of obtaining a carrier β comprising a porous organic polymer by opening an epoxy group derived from a monofunctional monomer of the polymer α;
A step (C) of obtaining a carrier γ by introducing an epoxy group into the carrier β using epichlorohydrin and a secondary alcohol;
A step of obtaining a carrier δ by immobilizing a natural polymer on the carrier γ, wherein the ratio of the mass of the group derived from the natural polymer to the mass of the carrier δ is 200 mg / g to 300 mg / g (D The manufacturing method of the base material for fillers which has these.

The third aspect of the present invention can preferably include the following features.
[8] The monofunctional monomer having an epoxy group is glycidyl methacrylate,
The crosslinkable monomer is ethylene glycol dimethacrylate;
The diluent is chlorobenzene,
[7] The method for producing a filler base material according to [7], wherein the secondary alcohol is 2-propanol.
[9] The method for producing a filler base material according to [7] or [8], wherein the natural polymer is dextran having a weight average molecular weight of 100,000 to 1,000,000.
[10] The method for producing a filler base material according to any one of [7] to [9], wherein the density of the epoxy group of the carrier γ is 350 μmol to 650 μmol per gram of carrier γ.

The fourth aspect of the present invention is a protein purification method described in [11] below.
[11] A step of passing a solution containing protein through a column packed with the packing material according to any one of [4] to [6];
And a step of eluting the protein adsorbed on the filler.

The filler in which the interactive functional group is covalently bonded to the filler base material of the present invention has sufficient mechanical strength and a high adsorption capacity for biopolymers such as proteins. The filler of the present invention has a large amount of biopolymer that can be purified at one time per unit volume, can contribute to an improvement in purification rate, and can be suitably used under high flow rate treatment conditions.

Hereinafter, preferred examples of the filler substrate, the filler substrate production method, the filler, and the protein purification method of the present invention will be described in detail. The following embodiments are specifically described for better understanding of the gist of the invention, and do not limit the present invention unless otherwise specified. Additions, omissions, substitutions, combinations, and other modifications can be made without departing from the spirit of the present invention.
In the present specification, “(meth) acryl” means one or both of “acryl” and “methacryl”.

[Base material for filler]
The base material for filler of the present embodiment is one in which a natural polymer is immobilized on the surface of a porous organic polymer carrier.
The porous organic polymer carrier has a crosslinking degree of 50 mol% to 85 mol%, an exclusion limit molecular weight of 200,000 to 800,000, and a pore volume ratio of 96 vol% to 99 vol%.

When the degree of cross-linking of the porous organic polymer carrier is 50 mol% to 85 mol%, the filler base material using the porous organic polymer carrier has a high adsorption capacity for biopolymers such as proteins and sufficient mechanical strength. It will have. The degree of crosslinking of the porous organic polymer carrier is preferably 55 mol% to 70 mol%. When the degree of cross-linking of the porous organic polymer carrier is less than 50 mol%, the mechanical strength is insufficient, and a filler including a filler base material using the porous organic polymer carrier cannot be used under high flow rate processing conditions. If the degree of cross-linking of the porous organic polymer carrier is more than 85 mol%, the group derived from the natural polymer is not sufficiently introduced to the surface. Run short.

The exclusion limit molecular weight of the porous organic polymer carrier is 200,000 to 800,000, more preferably 300,000 to 500,000. When the exclusion limit molecular weight of the porous organic polymer carrier is 200,000 or more, the base material for filler using the porous organic polymer carrier has a high adsorption capacity for biopolymers such as proteins. When the exclusion limit molecular weight is 800,000 or less, the filler base material using the molecular weight has sufficient mechanical strength.

The ratio of the pore volume of the porous organic polymer carrier (ratio of the volume of the pores to the total volume of the filler) is 96% to 99% by volume. When the proportion of the pore volume is 96% by volume or more, the base material for filler using this has a high adsorption capacity for biopolymers such as proteins. In addition, when the proportion of the pore volume is 99% by volume or less, a filler having sufficient mechanical strength can be obtained from the filler base material using the pore volume.

The natural polymer immobilized on the surface of the porous organic polymer carrier is not particularly limited as long as it is soluble in water, and a linear organic polymer having a hydroxyl group and an amino group in the skeleton is preferable.
As the linear organic polymer, for example, a highly hydrophilic polysaccharide having a plurality of hydroxyl groups in the molecule is preferably used. Specific examples of the polysaccharide include agarose, dextran, pullulan, starch, cellulose, and derivatives thereof. Among these polysaccharides, dextran is preferably used because of its high water solubility and relatively easy introduction into a porous organic polymer carrier.

The weight average molecular weight of the natural polymer is not particularly limited, but is preferably 5,000 to 5,000,000, and more preferably 100,000 to 1,000,000. When the weight average molecular weight of the natural polymer is 5,000 or more, the effect of improving the adsorption amount by the group derived from the immobilized natural polymer becomes more remarkable. Further, when the molecular weight of the natural polymer is 5,000,000 or less, the group derived from the immobilized natural polymer occupies the majority of the pore internal space of the base material for filler, thereby Thus, it is possible to prevent the room for separation and penetration of the high molecular weight separation object such as the like into the space in the pore.

The ratio of the mass of the group derived from the natural polymer to the mass of the base material for filler is 200 mg / g to 300 mg / g, preferably 210 mg / g to 280 mg / g. When the mass ratio of the group derived from the natural polymer is 200 mg / g or more, the effect of improving the adsorption capacity of the biopolymer by the group derived from the natural polymer is sufficiently obtained in the filler base material. When the mass ratio of the group derived from the natural polymer is 300 mg / g or less, the group derived from the natural polymer immobilized on the filler base material is in the pore space of the porous organic polymer carrier. By occupying the majority, it is possible to prevent a room for high-molecular-weight separation objects such as proteins from diffusing and penetrating into the pore space.

[Manufacturing method of base material for filler]
Next, the manufacturing method of the base material for fillers of this embodiment is demonstrated.
The method for producing a filler base material of the present embodiment includes the following steps (A) to (D).
Step (A) is a step of obtaining a polymer α by polymerizing a raw material monomer containing a monofunctional monomer having an epoxy group and a crosslinkable monomer in the presence of a diluent and a polymerization initiator. In the step (A), the concentration of the crosslinkable monomer in the raw material monomer is set to 50 mol% to 85 mol%, and the diluent is used in a volume of 3.8 times to 5.8 times that of the raw material monomer.

Step (B) is a step of obtaining a carrier β made of a porous organic polymer by opening an epoxy group derived from a monofunctional monomer of the polymer α.
Step (C) is a step of obtaining a carrier γ by introducing an epoxy group into the carrier β using epichlorohydrin and a secondary alcohol.
Step (D) is a step of obtaining the base material for filler of the present embodiment (hereinafter sometimes referred to as “carrier δ”) by immobilizing the natural polymer on the carrier γ. In the step (D), the ratio of the mass of the group derived from the natural polymer to the mass of the carrier δ is set to 200 mg / g to 300 mg / g.

"Process (A)"
In the step (A), a raw material monomer containing a monofunctional monomer having an epoxy group and a crosslinkable monomer is polymerized in the presence of a diluent and a polymerization initiator to obtain a polymer α.
The monofunctional monomer having an epoxy group used when synthesizing the polymer α is a compound having only one ethylenic carbon-carbon double bond (referred to as a polymerizable functional group). The crosslinkable monomer is a compound having a plurality of polymerizable functional groups. The raw material monomer may contain a monofunctional monomer having an epoxy group and another monomer that is not a crosslinkable monomer, if necessary.

(Monofunctional monomer having an epoxy group)
The monofunctional monomer having an epoxy group is not particularly limited. For example, glycidyl (meth) acrylate, 4,5-epoxypentyl (meth) acrylate, 4- (2,3-epoxypropyl) -n -Epoxy compounds such as butyl (meth) acrylate, 9,10-epoxystearyl acrylate, 4- (2,3-epoxypropyl) cyclohexylmethyl acrylate, allyl glycidyl ether, 3,4-epoxycyclohexylmethyl (meth) acrylate, Examples include alicyclic epoxy compounds such as 3,4-epoxycyclohexylethyl (meth) acrylate, 3,4-epoxycyclohexylpropyl (meth) acrylate, and vinylbenzyl glycidyl ether. These monofunctional monomers having an epoxy group may be used alone or in combination of two or more.

The monofunctional monomer having an epoxy group preferably includes one or both of glycidyl (meth) acrylate and 3,4-epoxycyclohexylmethyl (meth) acrylate among the above-mentioned compounds. It is preferable to use glycidyl methacrylate because it preferably contains (meth) acrylate and is readily available commercially.

(Crosslinkable monomer)
The crosslinkable monomer is not particularly limited, and a monomer having a plurality of polymerizable functional groups can be used. Examples of the bifunctional monomer include divinyl aromatic compounds such as divinylbenzene, alkylene glycol-di (meth) acrylates such as ethylene glycol di (meth) acrylate and polyethylene glycol di (meth) acrylate, alkylene (carbon number) 1-11) Bis (meth) acrylates, N, N′-methylenebis (meth) acrylamide, N, N′-ethylene-bis (meth) acrylamide, N, N′-hexamethylene-bis (meth) acrylamide, etc. Examples include alkylene (having 1 to 11 carbon atoms) bis (meth) acrylamides. Examples of the trifunctional monomer include trivinylbenzene and glycerin tri (meth) acrylate. These crosslinkable monomers may be used alone or in combination of two or more.
Among the above-mentioned compounds, the crosslinkable monomer preferably contains ethylene glycol dimethacrylate, and it is preferable to use ethylene glycol dimethacrylate alone, which can easily form pores having a volume sufficient to adsorb proteins. Most preferred.

The crosslinkable monomer concentration in the raw material monomer corresponds to the degree of crosslinking of the polymer α, and corresponds to the degree of crosslinking of the base material for filler (carrier δ) obtained in the step (D) described later.
That is, the degree of cross-linking of the polymer α is a value indicating the mole percentage (mol%) of the cross-linkable monomer in the raw material monomer used for the polymerization of the polymer α, and is obtained by the following formula.
Degree of crosslinking of polymer α (mol%) = (number of moles of crosslinking monomer / number of moles of raw material monomer) × 100

In this embodiment, the mole percentage of the crosslinkable monomer in the raw material monomer is 50 mol% to 85 mol%. As a result, a polymer α having a crosslinking degree of 50 mol% to 85 mol% is obtained, and in the step (D), a filler base material (carrier δ) having a crosslinking degree of 50 mol% to 85 mol% is obtained. The molar percentage of the crosslinkable monomer in the raw material monomer is preferably 55 mol% to 70 mol% so that a filler base material (carrier δ) having a crosslinking degree of 55 mol% to 70 mol% can be obtained.

(Other monomers)
As the monofunctional monomer having an epoxy group and the other monomer that is not a crosslinkable monomer, a monomer having an unsaturated functional group can be used and is not particularly limited. Examples of other monomers include aromatic monomers such as styrene, methylstyrene, ethylstyrene, hydroxystyrene, and chlorostyrene, methyl (meth) acrylate, ethyl (meth) acrylate, hydroxyethyl (meth) acrylate, hydroxypropyl ( (Meth) acrylate, hydroxybutyl (meth) acrylate, hydroxypentyl (meth) acrylate, 2-chloroethyl (meth) acrylate, (meth) acrylates such as polyethylene glycol (meth) acrylate, dimethyl (meth) acrylamide, diethyl (meth) (Meth) acrylamides such as acrylamide, hydroxyethyl (meth) acrylamide, hydroxypropyl (meth) acrylamide, and hydroxybutyl (meth) acrylamide Haloalkyl (1-4 carbon atoms) vinyl ether, hydroxyalkyl (1-4 carbon atoms) vinyl ether, vinyl acetate and the like.

(Diluent)
In this embodiment, a diluent is used when polymerizing a raw material monomer composed of a monofunctional monomer having an epoxy group, a crosslinkable monomer, and other monomers contained as necessary. By using a diluent, a porous polymer α can be obtained.
As the diluent, the raw material monomer can be dissolved, and can be uniformly mixed with the raw material monomer and the polymerization initiator under the polymerization reaction conditions. The polymer α obtained by polymerizing the raw material monomer is not dissolved, and the polymerization reaction is not performed. An active organic solvent can be used. Such an organic solvent varies depending on the type of raw material monomer and can be arbitrarily selected.

Examples of the diluent include aromatic hydrocarbons such as chlorobenzene, toluene, xylene, diethylbenzene and dodecylbenzene, saturated hydrocarbons such as hexane, heptane and decane, alcohols such as isoamyl alcohol, hexyl alcohol and octyl alcohol. Can be used. These diluents may be used alone or in combination of two or more.
Among the diluents described above, chlorobenzene is preferably used because a pore size sufficient to adsorb the biopolymer can be obtained.

Diluent is used in a volume of 3.8 to 5.8 times the volume of the raw material monomer. When calculating the ratio between the volume of the diluent and the volume of the raw material monomer, the values obtained by measuring the volume (volume) of the raw material monomer as a mixture and the volume (volume) of the diluent, respectively, at room temperature are used. . By adjusting the use amount of the diluent, the exclusion limit molecular weight and the ratio of the pore volume in the polymer α and the filler base material (carrier δ) can be changed.

By setting the volume of the diluent to be used to be 3.8 times or more of the volume of the raw material monomer, a porous organic high molecular weight having an exclusion limit molecular weight of 200,000 or more and a pore volume ratio of 96% by volume or more. A polymer α which is a molecule is obtained. The amount of the diluent used is preferably 4.0 or more times the volume of the raw material monomer in order to further increase the exclusion limit molecular weight and pore volume ratio of the polymer α. By setting the amount of the diluent used to a volume of 5.8 times or less that of the raw material monomer, it is possible to prevent the hardness of the polymer α from becoming too low, and a polymer α having sufficient mechanical strength can be obtained. The amount of the diluent used is preferably 5.0 times or less the volume of the raw material monomer in order to obtain a polymer α having better mechanical strength.

(Polymerization initiator)
As the polymerization initiator used for the polymerization of the raw material monomers, various known radical polymerization initiators such as organic peroxides and azo initiators can be used.
Examples of the organic peroxide used as the polymerization initiator include tert-butylperoxyneodecanoate, t-butylperoxy-2-ethylhexanoate, and t-butylperoxyisobutyrate in the case of butyl peroxide. In the case of amyl peroxide systems, such as rate, 2,5-dimethyl-2,5-di (benzoylperoxy) hexane, t-butylperoxyacetate, t-butylperoxybenzoate, etc., t-amylperoxy-2-ethyl Hexanoate, t-amyl peroxy-n-octoate, t-amyl peroxyacetate, t-amyl peroxybenzoate, etc., in peroxy carbonate type, t-butyl peroxyisopropyl carbonate, t-butyl peroxy-2 -Ethylhexyl carbonate, t-amyl peroxy In dialkyl peroxides such as -2-ethylhexyl carbonate, di (2-ethylhexyl) peroxydicarbonate, di (sec-butyl) peroxydicarbonate, dicumyl peroxide, 2,5-dimethyl-2,5- In the peroxyketal type such as di (t-butylperoxy) hexane, di-t-butylperoxide, di-t-amylperoxide, etc., 1,1-di (t-butylperoxy) cyclohexane, 2, Examples include 2-di (t-butylperoxy) butane, ethyl-3,3-di (t-butylperoxy) butyrate, 1,1-di (t-amylperoxy) cyclohexane, and the like.

Examples of the azo initiator used as the polymerization initiator include 2,2′-azobis (4-methoxy-2,4-dimethylvaleronitrile) and 2,2′-azobis (2,4-dimethylvalero) in the azonitrile series. Nitrile), 2,2′-azobis (2-methylpropionitrile), 2,2′-azobis (2-methylbutyronitrile), 1,1′-azobis (cyclohexane-1-carbonitrile), etc. In the system, 2,2′-azobis [N- (2-propenyl) -2-methylpropionamide], 2,2′-azobis [N-butyl-2-methylpropionamide], 2,2′-azobis [ Other azo compounds such as N-cyclohexyl-2-methylpropionamide], 2,2′-azobis (2-methylpropionamide oxime), dimethyl- , 2'-azobis (2-methyl propionate), 4,4'-azobis (4-cyanovaleric acid), 2,2'-azobis (2,4,4-trimethylpentane) and the like.

These polymerization initiators may be used alone or in combination of two or more.
The polymerization initiator can be used in any amount. For example, the total amount of raw material monomers consisting of a monofunctional monomer having an epoxy group, a crosslinkable monomer, and other monomers contained as necessary is 100 parts by mass. On the other hand, it is used in an amount of 0.01 to 15 parts by mass, preferably 0.05 to 10 parts by mass.

As a method for polymerizing the raw material monomer, a known method such as a suspension polymerization method, an emulsion method, a method disclosed in JP-B-58-058026 and JP-A-53-090991 can be used. . Specifically, a raw material monomer composed of a monofunctional monomer having an epoxy group, the above-described mole percentage of the crosslinkable monomer, and other monomers contained as necessary, a polymerization initiator, and a volume of the above A method of preparing a mixed solution with a diluent and polymerizing by a suspension polymerization method or the like can be used.

In this embodiment, a polymer α having a crosslinking degree of 50 mol% to 85 mol% is obtained by performing the above polymerization. Since the degree of crosslinking of the polymer α is 50 mol% or more, a filler base material having sufficiently high mechanical strength can be obtained. In addition, since the degree of crosslinking of the polymer α is 85 mol% or less, it is possible to sufficiently ensure the density of the epoxy groups that serve as a foothold for modifying the group derived from the natural polymer on the surface in the step (D). As a result, in the step (D), a filler base material in which a group derived from a natural polymer is sufficiently introduced on the surface is obtained.

In the step (A), the polymer α synthesized by polymerization is a porous organic polymer, and is preferably in the form of particles.
The particle size of the particulate polymer α is not particularly limited, and can be appropriately determined according to the particle size of the target filler base material. The particle size of the particulate polymer α can be adjusted by the following method.
For example, the particle diameter of the polymer α polymerized by the suspension polymerization method can be adjusted by a method of classifying the polymer α using a target sieve. For example, when polymerizing the polymer α by the emulsion method, the particle size of the polymer α can be adjusted by a method of controlling the stirring speed and the like during the polymerization.

"Process (B)"
Next, in the step (B), the epoxy group derived from the monofunctional monomer of the polymer α is opened to obtain a carrier β made of a porous organic polymer.
In the present embodiment, ring opening of the epoxy group of the polymer α means that water is added to the epoxy group of the polymer α, and the epoxy group is converted to a 1,2-diol group (—CH (OH) —CH ₂ —OH). ).

The method for ring-opening the epoxy group of the polymer α is not particularly limited, and any method may be used. Examples thereof include a method using an acidic aqueous solution containing hydrochloric acid, sulfuric acid, phosphoric acid and the like, and a method using a basic aqueous solution containing sodium hydroxide, potassium hydroxide and the like.

The presence of 1,2-diol groups in the carrier β can be confirmed and quantified by a mass change from the polymer α to the carrier β.
Specifically, the mass of the polymer α is measured in advance, the epoxy group of the polymer α is opened to form the carrier β, the mass of the carrier β is measured, and the mass of the carrier β is determined from the mass of the carrier β. Subtract the mass of
This makes it possible to quantify the amount of water added to the epoxy group.
In addition, by observing the intensity of the absorption peak before and after the ring opening treatment of the epoxy group from the spectrum of the epoxy group contained in the carrier β obtained by an infrared spectrophotometer, the progress of the ring opening treatment can be qualitatively determined. It is also possible to grasp.

The ratio of the exclusion limit molecular weight and the pore volume in the carrier β corresponds to the ratio of the exclusion limit molecular weight and the pore volume of the porous organic polymer support (carrier γ) obtained in the step (C) described later, and the steps described later. This corresponds to the exclusion limit molecular weight and the ratio of the pore volume of the porous organic polymer carrier (carrier γ) forming the filler base material (carrier δ) obtained in (D).

"Process (C)"
Step (C) is a step of obtaining a carrier γ (porous organic polymer carrier) by using epichlorohydrin as an epoxidizing agent and using a secondary alcohol as a reaction solvent and introducing an epoxy group into the carrier β. is there.
Epoxidation of the carrier β means that epichlorohydrin is reacted with the hydroxyl group of the 1,2-diol group generated on the surface of the polymer α by ring opening of the epoxy group in the step (B), so that the carrier surface It means introducing an epoxy group.

The method for obtaining the carrier γ by introducing an epoxy group into the carrier β is not particularly limited, and any method may be used. Specifically, for example, after mixing the carrier β, a mixed liquid of epichlorohydrin and a secondary alcohol in a reaction vessel and reacting, mixing of the basic substance and the secondary alcohol in the reaction vessel. The method of adding a liquid and making it react is mentioned.
In this embodiment, since a secondary alcohol is used as a reaction solvent in the reaction for introducing an epoxy group into the carrier β, a carrier γ into which a preferable amount of epoxy is introduced is obtained.

Examples of secondary alcohols include 2-propanol, 2-butanol, 3-methyl-2-butanol, 2-pentanol, and 3-pentanol. As the secondary alcohol, it is preferable to use 2-propanol which is economically excellent.
Further, the base used in the reaction for introducing the epoxy group into the carrier β to obtain the carrier γ is not particularly limited, and sodium hydride, potassium t-butoxide, sodium methoxide and the like can be suitably used.

The density of the epoxy group of the carrier γ is preferably 350 μmol to 650 μmol per gram of carrier γ. When the density of the epoxy group of the carrier γ is 350 μmol / g or more, the amount of the group derived from the natural polymer immobilized on the carrier γ in the step (D) described later is sufficiently increased, and the protein or the like A base material for a filler having a high biopolymer adsorption capacity can be obtained. The density of the epoxy group of the carrier γ is more preferably 440 μmol / g or more. On the other hand, when the density of the epoxy group is 650 μmol / g or less, a plurality of epoxy groups react with one molecule of the natural polymer in immobilization of the group derived from the natural polymer in the step (D) described later. Can be prevented. As a result, it is difficult to immobilize groups derived from natural polymers in a form that adheres to the carrier, and the effect as a spacer of groups derived from natural polymers is likely to be exerted, and the adsorption capacity of biopolymers such as proteins is increased. A high filler substrate is obtained. The density of the epoxy group of the carrier γ is more preferably 570 μmol / g or less.

The method for confirming the density of the epoxy group of the carrier γ is not particularly limited. For example, the following method can be used. First, the carrier γ obtained by introducing an epoxy group into the carrier β is reacted with diethylamine to form a carrier ζ. The carrier ζ is dispersed in an appropriate aqueous solution, and titration with an acid is performed to determine the amount of diethylamino groups of the carrier ζ. The number of moles of diethylamino group contained in the carrier ζ is determined from the titration value, and this is used as the number of moles of the epoxy group of the carrier γ to calculate the density of the epoxy group.

"Process (D)"
Step (D) is a step in which a natural polymer is immobilized on a carrier γ (porous organic polymer carrier) to obtain a filler base material (carrier δ).
As the natural polymer immobilized on the carrier γ, it is particularly preferable to use dextran having a weight average molecular weight of 100,000 to 1,000,000. Dextran having a weight average molecular weight of 100,000 to 1,000,000 is preferable because it easily dissolves in water and allows easy introduction of a group derived from a natural polymer into a porous organic polymer carrier.

As a method of immobilizing a group derived from a natural polymer on the carrier γ, any method may be used as long as it is a method used in a particle surface modification method with a polymer. For example, an epoxy group previously introduced into the carrier γ and / or a reactive functional group (for example, unreacted glycidyl group) present on the surface of the carrier γ itself is reacted with a natural polymer dissolved in water. There is a way to fix it.
Among these, the method of immobilizing a group derived from a natural polymer using an epoxy group introduced into the carrier γ is relatively stable against water, and the reaction with the natural polymer It is preferable from the viewpoint of excellent selectivity.

The amount of the group derived from the natural polymer in the carrier δ (filler base material) can be appropriately adjusted depending on the concentration of the natural polymer during the reaction with the carrier γ. In step (D), the ratio of the mass of the group derived from the natural polymer to the mass of the carrier δ (immobilization amount) is 200 mg / g to 300 mg / g.
The amount of the group derived from the natural polymer immobilized on the carrier δ can be confirmed and quantified by, for example, the mass difference between the carrier γ and the carrier δ. Specifically, the dry mass of the carrier γ before immobilizing the group derived from the natural polymer is calculated by subtracting from the dry mass of the carrier δ.

When the natural polymer is a polysaccharide, the phenol-sulfuric acid method can be used as a method for calculating the amount of the group derived from the natural polymer of the carrier δ (filler base material). Specifically, phenol and concentrated sulfuric acid are brought into contact with the carrier δ, and the concentration in the supernatant of the furfural derivative generated by decomposition of the polysaccharide-derived component is calculated by measuring the absorbance at a wavelength of 490 nm.
This makes it possible to measure the amount of immobilized groups derived from the polysaccharide.

[filler]
In the filler of this embodiment, an interactive functional group is covalently bonded to the filler base material described above.
The packing material of this embodiment is an organic polymer-based packing material, and is suitably used as a packing material packed in a column when purifying a biopolymer such as protein using chromatography.

The interactive functional group that forms the filler is a functional group that interacts with the protein. Specifically, examples of the interactive functional group include an ion exchange group, a hydrophobic group, and an affinity functional group. The interactive functional group can be appropriately selected according to the use of the filler. For example, when a cation exchange group is selected as the interactive functional group, the filler is a cation exchange resin, and when an anion exchange group is selected, the filler is an anion exchange resin.

As an ion exchange group, well-known things, such as a weak anion exchange group, a strong anion exchange group, a weak cation exchange group, a strong cation exchange group, can be used, for example.
Examples of the hydrophobic group include, but are not particularly limited to, a butyl group, a heptyl group, an octyl group, and a phenyl group.
Examples of the affinity functional group include biochemically active substances having affinity for proteins. Examples thereof include Protein A, Protein G, antibody, lectins, and pseudo peptide ligands thereof.

The filler is preferably in the form of particles. There is no special restriction | limiting in the average particle diameter of a filler, It can select as needed. When the filler is used as a filler for liquid chromatography, it is preferably 10 μm to 300 μm, more preferably 15 μm to 200 μm, and particularly preferably 20 μm to 100 μm. The average particle size of the filler of the present invention may be selected according to the purpose, and may be, for example, in the range of 10 to 20 μm, 20 to 40 μm, 40 to 80 μm, 80 to 150 μm, and the like. If the particle size of the filler is not too small, consolidation is unlikely to occur and use under high flow rate conditions is not difficult. Moreover, if the particle size of the filler is not too large, it is easy to increase the adsorption capacity of the purification object.

The average particle diameter is a volume-converted average particle diameter in a wet state. The volume-converted average particle diameter is measured with a Coulter counter or an image analysis type particle size distribution measuring device. Of these, an image analysis type particle size distribution measurement device is preferable. As the image analysis type particle size distribution measurement device, for example, a flow type particle image analysis device (trade name: FPIA-3000, manufactured by Sysmex Corporation) can be used.

[Method for producing filler]
Next, the manufacturing method of the filler of this embodiment is demonstrated.
The manufacturing method of the filler of this embodiment has the process (E) shown below.
"Process (E)"
Step (E) is a step of obtaining a carrier ε (filler) in which an interactive functional group is covalently bonded to the carrier δ by introducing an interactive functional group into the carrier δ (substrate for filler). is there.

The method of introducing an interactive functional group into the carrier δ and making the carrier ε covalently bound to the interactive functional group is not particularly limited, and the type of the carrier ε and the interactive functional group is not limited. Will be decided accordingly. The amount of the interactive functional group introduced into the carrier δ can be adjusted by the amount of the reagent to be reacted.
The introduction of the interactive functional group is preferably performed by a reaction with a hydroxyl group and / or an amino group because it is simple. In this case, the reaction between the group derived from the natural polymer of the carrier δ and the interactive functional group is usually used. For example, when a reaction between a hydroxyl group and an interactive functional group is utilized, the hydroxyl group derived from the carrier γ (porous organic polymer carrier) is considered to be probabilistically small, but may be utilized.

When the interactive functional group is an ion exchange group, for example, the method described below can be used.
That is, when the base point of the reaction in the group derived from the natural polymer of the carrier δ is a hydroxyl group and / or an amino group, bromoethylsulfonic acid, monochloroacetic acid, chlorohydroxypropanesulfonic acid, 2,3-epoxysulfonic acid, Cation exchange in which a cation exchange group is covalently bonded to a group derived from a natural polymer by reacting 1,3-propane sultone, 1,4-butane sultone, etc. with the hydroxyl group and / or amino group of the carrier δ A carrier ε, which is a resin, is obtained.
Similarly, by reacting diethylaminoethyl chloride hydrochloride, glycidyltrimethylammonium hydrochloride, etc. with the hydroxyl group and / or amino group of the carrier δ, an anion exchange group is covalently bonded to the group derived from the natural polymer. A carrier ε, which is an anion exchange resin, is obtained.

When the interactive functional group is a hydrophobic group, for example, bromobutane, chlorobutane, octyl chloride, phenyl glycidyl ether, etc. are reacted with the hydroxyl group and / or amino group of the group derived from the natural polymer of the carrier δ. Thus, a hydrophobic carrier ε into which a hydrophobic group has been introduced is obtained.
When the interactive functional group is an affinity functional group, the affinity functional group is immobilized on a group derived from the natural polymer of the carrier δ using a generally used method for immobilizing a ligand of a biochemical substance. A carrier ε is obtained.

[Protein purification method]
The protein purification method of the present embodiment includes a step of passing a solution containing the protein through a column packed with the above-described packing material and a step of eluting the protein adsorbed on the packing material.
In the protein purification method of the present embodiment, a known method can be used except that the above filler is used.
In the protein purification method of the present embodiment, since the column packed with the above-mentioned packing is used, the amount of biopolymer that can be purified at a time per unit volume is large, and the protein can be efficiently purified under high flow rate processing conditions. .

Hereinafter, the present invention will be described more specifically with reference to examples and comparative examples. In addition, this invention is not limited only to a following example.
[Example 1]
“Step (A) Synthesis of Polymer α-1”
A raw material monomer consisting of 14.5 g of glycidyl methacrylate, which is a monofunctional monomer having an epoxy group, and 24.5 g of ethylene glycol dimethacrylate, which is a crosslinkable monomer, 195.5 g of chlorobenzene, which is a diluent, and a polymerization initiator An oil phase was prepared by dissolving 2.4 g of 2,2′-azobis (2,4-dimethylvaleronitrile) and bubbling nitrogen gas for 30 minutes.
The mole percentage (crosslinking degree) of the crosslinkable monomer in the raw material monomer was 55 mol%. Moreover, since the diluent was 177.6 ml and the raw material monomer was 35.5 ml, the volume of the diluent was 5.0 times that of the raw material monomer.

Next, apart from the oil phase, 15.0 g of PVA-224 (manufactured by Kuraray Co., Ltd., polyvinyl alcohol having a saponification degree of 87.0% to 89.0%), 500 g of ion-exchanged water, and salting out An aqueous phase in which 5.0 g of sodium chloride as an agent was dissolved was prepared.

The aqueous phase and the oil phase were transferred to a separable flask and dispersed with a stirring rod equipped with a half-moon stirring blade at a rotational speed of 430 rpm for 20 minutes. . Thereafter, the aqueous phase was removed by centrifugation, and the resulting polymer was transferred onto a glass filter and washed thoroughly in the order of hot water, denatured alcohol, and water. After washing, sieving classification was performed in a water bath, and water was removed by vacuum filtration for 5 minutes or more to obtain 190 g of a water-wet polymer having a particle size of 25 μm to 75 μm. The mass of polymer α-1 obtained by drying the obtained water-wet polymer was 37 g.

"Step (B) Ring opening of epoxy group"
100 g of water-wet polymer (dry weight 19.5 g of polymer α-1) was transferred to a separable flask, 500 mL of 0.5 mol / L sulfuric acid aqueous solution was added, and the mixture was heated to 60 ° C. and reacted for 6 hours. Thereafter, the carrier was transferred onto a glass filter and washed with 2000 mL or more of water. Thus, a porous organic polymer carrier (carrier β-1) in which an epoxy group was opened and a 1,2-diol group was introduced was obtained.
The completion of the reaction was carried out by infrared spectroscopy using a Thermo Scientific (registered trademark) Nicolet (registered trademark) iS (registered trademark) 10 FT-IR spectrometer (trade name, manufactured by Thermo Fisher Scientific). This was confirmed by measuring the disappearance of epoxy group absorption on -1.

With respect to the carrier β-1, the exclusion limit molecular weight and the ratio of the pore volume were determined by the following method.
Carrier β-1 was packed in a chromatography housing having an inner diameter of 4.6 mm and a length of 100 mm to obtain a column. Using the obtained column, the eluent is water, the differential refractive index detector is used as the detector, the flow rate is 0.30 mL / min (linear flow rate: 108 cm / hr), and the size using pullulan of each molecular weight. Exclusion chromatography was performed to generate a calibration curve. In the calibration curve, the molecular weight of the smallest pullulan among the pullulans having the same elution of the pullulan on the polymer side was defined as the exclusion limit molecular weight. As a result, the exclusion limit molecular weight of the carrier β-1 of Example 1 was 400,000.

The amount of eluent obtained from the retention time of pullulan with the molecular weight of the exclusion limit and the multiplier of the flow rate of the eluent was defined as the exclusion limit capacity. The permeation limit capacity was determined from the elution time and flow rate multiplier of ethylene glycol when ethylene glycol was measured. The difference between the exclusion limit capacity and the penetration limit capacity was defined as the pore volume. The ratio of the pore volume was determined by the following formula.
Pore volume ratio (%) = {(permeation limit capacity) − (exclusion limit capacity)} / {(volume in column) − (exclusion limit capacity)} × 100
As a result, the ratio of the pore volume of the carrier β-1 of Example 1 was 98% by volume.

“Process (C) Introduction of epoxy group”
20 g of a water-wet carrier (carrier β-1) having an epoxy group opened was weighed on a glass filter and thoroughly washed with 2-propanol. After washing, the carrier β-1 is transferred to a separable flask, 15.6 g of 2-propanol and 10 g of epichlorohydrin are added, stirred at room temperature, and 2.5 g of potassium t-butoxide is further added to 15.6 g of 2-propanol. The solution dissolved in was added, heated to 30 ° C., and stirred for 3 hours. After completion of the reaction, the carrier was transferred onto a glass filter and thoroughly washed with water, acetone and water in this order to obtain a water-wet carrier (carrier γ-1).

The density of introduction of epoxy groups in the obtained carrier γ-1 was measured by the following procedure.
As a result of collecting 5.0 g of water-wet carrier (carrier γ-1) and determining the dry mass, it was 0.80 g. Next, the same amount of water-wet carrier γ (carrier γ-1) is weighed into a separable flask, dispersed in 40 g of water, 12 g of diethylamine is added with stirring at room temperature, heated to 50 ° C., and heated to 4 ° C. Stir for hours. After completion of the reaction, the obtained carrier was transferred onto a glass filter, thoroughly washed with water and acetone in that order, and dried under reduced pressure to obtain dry carrier ζ-1.

The obtained dry carrier ζ-1 was transferred to a beaker, dispersed in 150 mL of a 0.5 mol / L aqueous potassium chloride solution, and titrated with 0.1 mol / L hydrochloric acid at the time when the pH reached 4.0. Based on this, the amount of diethylamine introduced into the carrier ζ-1 was calculated, and the density of the epoxy group of the carrier γ-1 was calculated according to the following formula. As a result, the density of the epoxy group was 520 μmol / g.
Epoxy group density (μmol / g) = {0.1 × hydrochloric acid volume at neutralization (μl) / dry weight of carrier γ (g)}

“Process (D) Introduction of natural polymer”
In a separable flask, 5.5 g of natural polymer dextran 200,000 (manufactured by Wako Pure Chemical Industries, Ltd., average weight molecular weight 180,000-210,000) and 7.2 g of water are measured and stirred at room temperature until dissolved. did. To the obtained dextran solution, 10 g of water-wet carrier (carrier γ-1) (dry weight 0.16 g) was added and stirred at 40 ° C. for 1 hour. Thereafter, 40 mg of tetrabutylammonium iodide, 10 mg of sodium borohydride, and 1.7 g of a 48% aqueous sodium hydroxide solution were added, and the mixture was stirred at 40 ° C. for 16 hours. After completion of the reaction, the carrier was transferred onto a glass filter and thoroughly washed with water to obtain a water wet carrier δ-1.

The amount of dextran immobilized on the obtained carrier δ-1 (substrate for filler) was calculated by the following procedure.
20 mg of carrier δ-1 (substrate for filler) dried from water-wet carrier δ-1 is weighed into a sample tube, 0.5 mL of water, 0.5 mL of 5% phenol aqueous solution, 2.5 mL of concentrated sulfuric acid, And left to stand for 1 hour. After standing, the absorbance at a wavelength of 490 nm of the 20-fold diluted solution was measured with Infinite M200PRO (trade name, manufactured by Tecan), and the amount of dextran introduced was calculated from the concentration of the furfural derivative contained in the solution. As a result, the amount of dextran immobilized was 250 mg / g based on the mass of the carrier δ-1.

"Step (E) Introduction of interactive functional group"
4.0 g (dry weight 0.72 g) of a water-wet carrier into which dextran was introduced (carrier δ-1) was weighed into a separable flask and dispersed in 4.8 g of water and 5.6 g of 1,3-propane sultone. Heated to 40 ° C. While stirring at 40 ° C., 4.0 g of a 48% aqueous sodium hydroxide solution was added, followed by stirring at 40 ° C. for 3 hours.
After completion of the reaction, the carrier was transferred onto a glass filter and washed with water, acetone and water in this order. After washing, the carrier was transferred to a beaker, 200 mL of water was added and sufficiently dispersed, and then allowed to stand for 45 minutes. After removing the supernatant, the carrier was washed with water on a glass filter to obtain water- filled filler 1 (carrier ε-1) into which a sulfo group as a cation exchange group was introduced.

The obtained filler 1 was packed into a PEEK chromatographic housing (manufactured by Sakai Seisakusho Co., Ltd.) having an inner diameter of 4.0 mm and a length of 50 mm. Filler 1 was filled using a 0.3 mol / L sodium sulfate aqueous solution as a slurry preparation solution at a flow rate of 4.2 mL / min over 20 minutes. Using the obtained column, the adsorption / elution test was performed by the method shown below, and the adsorption capacity of IgG (iminoglobulin G) was measured.
Table 1 shows the measured IgG adsorption capacity. The adsorbed IgG was recovered by fractionating the eluate from the elution step.

“Measurement of IgG Adsorption Capacity of Packing Material Introduced with Cation Exchange Group” <Adsorption Step>
Immunoglobulin G (IgG) concentration: 0.9 mg / mL to 1.1 mg / mL Adsorption buffer: 20 mM citrate buffer (pH 5.3)
・ Liquid feeding speed: 1000 cm / hr (flow rate 2.1 mL / min)

<Washing step>
Wash buffer: 20 mM citrate buffer (pH 5.3)
・ Liquid feeding amount: 10 CV
<Elution step>
-Eluent: 20 mM citrate buffer solution (pH 5.3) and 1.0 M NaCl-Feed volume: 10 CV
(CV is an abbreviation for column volume and corresponds to 0.63 mL in the above experiment.)

In the adsorption step, when 10% of IgG contained in the solution leaked from the column, the capacity of IgG adsorbed on the packing material was defined as the IgG adsorption capacity. The adsorption capacity was calculated by the following equation (1).
Adsorption capacity (mg / mL-gel) = (Amount of liquid delivered at the time of 10% leakage (mL) × IgG concentration (mg / mL)) / Gel volume (mL-gel) (1)
In equation (1), the amount of liquid delivered at the time of 10% leakage is the absorption of the ultraviolet (UV) absorption observed by the detector when the IgG solution was delivered when the column was not installed, and the absorption. This is the total amount of liquid delivered from the beginning of liquid delivery when a 10% UV absorption value was observed. The gel volume is the volume of the packing material, and is the value of the volume when the packing material in the column is transferred to the measuring cylinder and sufficiently settled.

The pressure loss at a flow rate of 1000 cm / hour (flow rate 2.1 mL / min) was measured with the column having the adsorption capacity measured installed in the apparatus. Next, an empty column containing no filler was installed, and the pressure loss at 1000 cm / hour of the liquid feeding system was measured. From the two pressure loss values, the pressure loss of the column under a high flow rate condition of 1000 cm / hour was calculated. The results are shown in Table 1.
In the experiment for measuring the adsorption capacity, no increase in the back pressure was observed, indicating that the obtained column can be used without any trouble even under high flow rate conditions. From this, it was found that the filler 1 has sufficient strength.

[Example 2]
"Step (E) Introduction of interactive functional group"
4.0 g (dry weight 0.72 g) of a water-wet carrier (carrier δ-1) introduced with dextran obtained in the same manner as in Example 1 was weighed into a separable flask, and 5 mol / L aqueous sodium hydroxide solution was added. The mixture was dispersed in 0 g and stirred at room temperature for 30 minutes. 5.0 g of water in which 3.0 g of diethylaminoethyl chloride hydrochloride was dissolved was added, the temperature was raised to 70 ° C., and the mixture was stirred for 2 hours.
After completion of the reaction, the carrier was transferred onto a glass filter and washed with water. After washing, the carrier was transferred to a beaker, 200 mL of water was added and sufficiently dispersed, and then allowed to stand for 45 minutes. After removing the supernatant, the carrier was washed with water on a glass filter to obtain a filler 2 (carrier ε-2) into which a diethylamino group as an anion exchange group was introduced.

The obtained packing material 2 is filled into a chromatographic housing made of PEEK having an inner diameter of 4.0 mm and a length of 50 mm, and an adsorption / elution test is performed by the following method to measure the adsorption capacity of BSA (bovine serum albumin). did. The adsorption capacity of BSA was determined in the same manner as the method for determining the adsorption capacity of IgG in Example 1.
Table 1 shows the adsorption capacity of BSA in Example 2. The adsorbed BSA was recovered by separating the eluate from the elution step.

“Measurement of BSA adsorption capacity of fillers with anion exchange groups” <Adsorption step>
Bovine serum albumin (BSA) concentration: 0.9 mg / mL to 1.1 mg / mL Adsorption buffer: 50 mM Tris hydrochloride buffer (pH 8.5) Feed rate: 1000 cm / hour (flow rate 2.1 mL / Min)

<Washing step>
Washing buffer: 50 mM Tris hydrochloride buffer (pH 8.5) Feeding volume: 10 CV
<Elution step>
-Eluent: 50 mM Tris hydrochloride buffer solution (pH 8.5) and 1.0 M NaCl-Feed volume: 10 CV

Further, in the same manner as in Example 1, the pressure loss of the column under the high flow rate condition was calculated. The results are shown in Table 1.
Moreover, also in Example 2, the phenomenon which a back pressure raises was not seen in the experiment which measures adsorption capacity, and it turned out that the filler 2 has sufficient intensity | strength.

[Example 3]
"Step (E) Introduction of interactive functional group"
4.0 g (dry weight 0.72 g) of a water-wet carrier (carrier δ-1) introduced with dextran obtained in the same manner as in Example 1 was weighed into a separable flask, 8.0 g of water, and glycidyltrimethylammonium hydrochloride. 4.5g was added and it stirred at 40 degreeC for 8 hours.
After completion of the reaction, the carrier was transferred onto a glass filter and washed with water. After washing, the carrier was transferred to a beaker, 200 mL of water was added and sufficiently dispersed, and then allowed to stand for 45 minutes. After removing the supernatant, the carrier was washed with water on a glass filter to obtain a filler 3 (carrier ε-3) into which a trimethylammonium group as an anion exchange group was introduced.

Using the obtained filler 3, the adsorption capacity of BSA was measured in the same manner as in Example 2. The results are shown in Table 1.
Further, in the same manner as in Example 1, the pressure loss of the column under the high flow rate condition was calculated. The results are shown in Table 1.
Moreover, also in Example 3, the phenomenon which a back pressure raises was not seen in the experiment which measures adsorption capacity, and it turned out that the filler 3 has sufficient intensity | strength.

[Example 4]
In the step of introducing an epoxy group, the amount of epichlorohydrin used was 4.0 g, the amount of potassium t-butoxide was 1.0 g, and the stirring time after heating to 30 ° C. was 6 hours. Except for this, a filler 4 (carrier ε-4) was obtained in the same manner as in Example 1.
The introduction density of the epoxy group of the carrier γ-4 obtained in the course of manufacturing the filler 4 and the amount of dextran immobilized on the carrier δ-4 (substrate for filler) were obtained in the same manner as in Example 1. It was. The results are shown in Table 1.

Using the filler 4, the IgG adsorption capacity was measured in the same manner as in Example 1. The results are shown in Table 1.
Further, in the same manner as in Example 1, the pressure loss of the column under the high flow rate condition was calculated. The results are shown in Table 1.
Moreover, also in Example 4, the phenomenon which a back pressure raises was not seen in the experiment which measures adsorption capacity, and it turned out that the filler 4 has sufficient intensity | strength.

[Example 5]
In the step of introducing an epoxy group, filler 5 was used in the same manner as in Example 1 except that the amount of epichlorohydrin used was 20.0 g and the amount of potassium tert-butoxide was 5.0 g. (Carrier ε-5) was obtained.
The introduction density of the epoxy group of the carrier γ-5 obtained in the course of manufacturing the filler 5 and the amount of dextran immobilized on the carrier δ-5 (substrate for filler) were determined in the same manner as in Example 1. It was. The results are shown in Table 1.

The adsorption capacity of IgG was measured in the same manner as in Example 1 using the filler 5. The results are shown in Table 1.
Further, in the same manner as in Example 1, the pressure loss of the column under the high flow rate condition was calculated. The results are shown in Table 1.
Moreover, also in Example 5, the phenomenon which raises back pressure was not seen in the experiment which measures adsorption capacity, and it turned out that the filler 5 has sufficient intensity | strength.

[Example 6]
In the step of synthesizing polymer α, filler 6 (carrier) was the same as in Example 1 except that the amount of glycidyl methacrylate used was 10.9 g and the amount of ethylene glycol dimethacrylate was 28.2 g. ε-6) was obtained.
With respect to the carrier β-6 obtained in the process of producing the filler 6, the exclusion limit molecular weight and the ratio of the pore volume were determined in the same manner as in Example 1. The results are shown in Table 1.
Further, the introduction density of the epoxy group of the carrier γ-6 and the amount of dextran immobilized on the carrier δ-6 (substrate for filler) were determined in the same manner as in Example 1. The results are shown in Table 1.

Using the filler 6, the IgG adsorption capacity was measured in the same manner as in Example 1. The results are shown in Table 1.
Further, in the same manner as in Example 1, the pressure loss of the column under the high flow rate condition was calculated. The results are shown in Table 1.
Moreover, also in Example 6, the phenomenon which a back pressure raises was not seen in the experiment which measures adsorption capacity, and it turned out that the filler 6 has sufficient intensity | strength.

[Comparative Example 1]
In the step of synthesizing the polymer α, the filler of Comparative Example 1 was obtained in the same manner as in Example 1 except that the amount of chlorobenzene used as the diluent was 40.6 g. The volume of the diluent in Comparative Example 1 was 1.0 times that of the raw material monomer.
For the carrier β (ratio 1) obtained in the process of producing the filler of Comparative Example 1, the exclusion limit molecular weight and the ratio of the pore volume were determined in the same manner as in Example 1. The results are shown in Table 1.
In addition, the introduction density of the epoxy group of the carrier γ (ratio 1) obtained in the course of manufacturing the filler of Comparative Example 1 and the amount of dextran immobilized on the carrier δ (ratio 1) (substrate for filler) This was determined in the same manner as in Example 1. The results are shown in Table 1.

The adsorption capacity of IgG was measured in the same manner as in Example 1 using the filler of Comparative Example 1. The results are shown in Table 1.
Further, in the same manner as in Example 1, the pressure loss of the column under the high flow rate condition was calculated. The results are shown in Table 1.
Moreover, also in the comparative example 1, the phenomenon which a back pressure raises in the experiment which measures adsorption capacity was not seen, but it turned out that the filler of the comparative example 1 has sufficient intensity | strength.

[Comparative Example 2]
A filler of Comparative Example 2 was obtained in the same manner as in Example 1 except that dimethyl sulfoxide was used instead of 2-propanol in the step of introducing an epoxy group.
The introduction density of the epoxy group of the carrier γ (ratio 2) obtained in the process of manufacturing the filler of Comparative Example 2 and the amount of dextran immobilized on the carrier δ (ratio 2) (substrate for filler) were carried out. Determined in the same manner as in Example 1. The results are shown in Table 1.

The adsorption capacity of IgG was measured in the same manner as in Example 1 using the filler of Comparative Example 2. The results are shown in Table 1.
Further, in the same manner as in Example 1, the pressure loss of the column under the high flow rate condition was calculated. The results are shown in Table 1.
Moreover, also in the comparative example 2, the phenomenon which a back pressure raises was not seen in the experiment which measures adsorption capacity, and it turned out that the filler of the comparative example 2 has sufficient intensity | strength.

[Comparative Example 3]
In the step of introducing an epoxy group, a filler of Comparative Example 3 was obtained in the same manner as in Example 1 except that ethanol was used instead of 2-propanol.
The introduction density of the epoxy group of the carrier γ (ratio 3) obtained in the process of manufacturing the filler of Comparative Example 3 and the amount of dextran immobilized on the carrier δ (ratio 3) (substrate for filler) were carried out. Determined in the same manner as in Example 1. The results are shown in Table 1.

The adsorption capacity of IgG was measured in the same manner as in Example 1 using the filler of Comparative Example 3. The results are shown in Table 1.
Further, in the same manner as in Example 1, the pressure loss of the column under the high flow rate condition was calculated. The results are shown in Table 1.
Also in Comparative Example 3, the phenomenon of increasing the back pressure was not observed in the experiment for measuring the adsorption capacity, and it was found that the filler of Comparative Example 3 had sufficient strength.

[Comparative Example 4]
In the step of introducing the natural polymer, the filler of Comparative Example 4 was obtained in the same manner as in Example 1 except that the amount of water used for dissolving dextran 200,000 was 14.4 g.
The amount of dextran immobilized on the carrier δ (ratio 4) (base material for filler) obtained in the course of manufacturing the filler of Comparative Example 4 was determined in the same manner as in Example 1. The results are shown in Table 1.

The adsorption capacity of IgG was measured in the same manner as in Example 1 using the filler of Comparative Example 4. The results are shown in Table 1.
Further, in the same manner as in Example 1, the pressure loss of the column under the high flow rate condition was calculated. The results are shown in Table 1.
Moreover, also in the comparative example 4, the phenomenon which raises back pressure was not seen in the experiment which measures adsorption capacity, and it turned out that the filler of the comparative example 4 has sufficient intensity | strength.

[Comparative Example 5]
In the step of synthesizing the polymer α, the polymer α is synthesized in the same manner as in Example 1 except that the amount of glycidyl methacrylate used is 27.8 g and the amount of ethylene glycol dimethacrylate is 11.3 g. did. As a result, the oil phase did not solidify and the polymer α was not obtained.

[Comparative Example 6]
In the step of synthesizing the polymer α, the filling of Comparative Example 6 was performed in the same manner as in Example 1 except that the amount of glycidyl methacrylate used was 18.3 g and the amount of ethylene glycol dimethacrylate was 20.8 g. An agent was obtained.
For the carrier β (ratio 6) obtained in the process of producing the filler of Comparative Example 6, the exclusion limit molecular weight and the ratio of the pore volume were determined in the same manner as in Example 1. The results are shown in Table 1.
Further, the introduction density of the epoxy group of the carrier γ (ratio 6) and the amount of dextran immobilized on the carrier δ (ratio 6) (substrate for filler) were determined in the same manner as in Example 1. The results are shown in Table 1.

The IgG adsorption capacity was measured in the same manner as in Example 1 using the filler of Comparative Example 6. However, the column back pressure increased and the sample could not be fed. In addition, the pressure loss could not be calculated because the column back pressure increased. From this, it can be seen that the filler of Comparative Example 6 has insufficient strength.

Table 1 shows the molar percentage (crosslinking degree) of the crosslinkable monomer in the raw material monomers of Examples 1 to 6 and Comparative Examples 1 to 6, the volume ratio of the diluent to the raw material monomer, and the exclusion limit molecular weight of the porous organic polymer carrier. The ratio of the pore volume, the ratio of the mass of the group derived from the natural polymer to the mass of the filler substrate (dextran immobilization amount), the adsorption capacity of the filler and the pressure loss are shown. (IgG) described in the column of adsorption capacity in Table 1 means that it is the adsorption capacity of IgG, and (BSA) means that it is the adsorption capacity of BSA.

As shown in Table 1, the fillers of Examples 1 to 6 had a higher adsorption capacity than Comparative Examples 1 to 4.
In Comparative Example 1, since the diluent volume ratio was small, the ratio of the pore volume of the base material for filler was small, and the amount of dextran immobilized was low. As a result, the adsorption capacity of the filler of Comparative Example 1 was low.

In Comparative Example 2, since dimethyl sulfoxide was used instead of 2-propanol, the amount of dextran immobilized on the base material for filler increased. As a result, the adsorption capacity of the filler of Comparative Example 2 was low.
In Comparative Example 3, since ethanol was used instead of 2-propanol, the amount of dextran immobilized on the filler base material was low. As a result, the adsorption capacity of the filler of Comparative Example 3 was low.
In Comparative Example 4, since the amount of dextran immobilized on the filler base material was low, the adsorption capacity of the filler was low.

In Comparative Example 5, a polymer (porous organic polymer) could not be obtained because the degree of crosslinking was insufficient.
In Comparative Example 6, since the degree of cross-linking of the filler base material was insufficient, the adsorption capacity of the filler was low.

Since the filler of the present invention exhibits a high adsorption capacity for proteins under high flow rate treatment conditions, it is preferably used in the purification of biopolymers. The present invention provides a filler having a sufficient mechanical strength and a high biopolymer adsorption capacity, and a method for producing the filler.

Claims (11)

It is a base material for filler in which a natural polymer is immobilized on the surface of a porous organic polymer carrier,
The porous organic polymer carrier is
The degree of crosslinking is from 50 mol% to 85 mol%,
Exclusion limit molecular weight is 200,000 to 800,000,
The proportion of the pore volume is 96 vol% to 99 vol%,
A filler base material having a ratio of a mass of a group derived from a natural polymer to a mass of the filler base material of 200 mg / g to 300 mg / g. The base material for filler according to claim 1, wherein the natural polymer is a polysaccharide. The base material for filler according to claim 2, wherein the polysaccharide is dextran. A filler in which an interactive functional group is covalently bonded to the filler base material according to any one of claims 1 to 3. The filler according to claim 4, wherein the interactive functional group is a cation exchange group. The filler according to claim 4, wherein the interactive functional group is an anion exchange group. A method for producing a filler base material according to any one of claims 1 to 3,
A step of polymerizing a raw material monomer containing a monofunctional monomer having an epoxy group and a crosslinkable monomer in the presence of a diluent and a polymerization initiator to obtain a polymer α,
A step (A) in which the concentration of the crosslinkable monomer in the raw material monomer is 50 mol% to 85 mol%, and the diluent is used in a volume of 3.8 times to 5.8 times the raw material monomer;
Opening the epoxy group derived from the monofunctional monomer of the polymer α to obtain a carrier β made of a porous organic polymer (B);
A step (C) of obtaining a carrier γ by introducing an epoxy group into the carrier β using epichlorohydrin and a secondary alcohol;
A step of obtaining a carrier δ by immobilizing a natural polymer on the carrier γ, wherein the ratio of the mass of the group derived from the natural polymer to the mass of the carrier δ is 200 mg / g to 300 mg / g (D The manufacturing method of the base material for fillers which has these. The monofunctional monomer having an epoxy group is glycidyl methacrylate,
The crosslinkable monomer is ethylene glycol dimethacrylate;
The diluent is chlorobenzene,
The method for producing a filler base material according to claim 7, wherein the secondary alcohol is 2-propanol. The method for producing a filler base material according to claim 7 or 8, wherein the natural polymer is dextran having a weight average molecular weight of 100,000 to 1,000,000. 10. The method for producing a filler base material according to claim 7, wherein the density of the epoxy group of the carrier γ is 350 μmol to 650 μmol per gram of carrier γ. Passing a protein-containing solution through a column filled with the packing material according to any one of claims 4 to 6,
And a step of eluting the protein adsorbed on the filler.