JP2002202297A - Organic-inorganic hybrid body for affinity chromatography and its manufacturing method - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a carrier for affinity chromatography having high pressure tightness and the small degree of swelling and contraction to thereby enable high speed separation, sufficient hydrophilicity, and extremely little non-specific adsorption of a protein or the like except material to be separated. SOLUTION: A solution including a hydrophilic monomer and a crosslinking agent is impregnated in inorganic porous material, and thereafter polymerization and bridging are generated, to thereby easily synthesize organic gel inside pores of the inorganic porous material. The hydrophilic organic gel is a polymer having a functional group capable of immobilizing a ligand or a spacer in the affinity chromatography.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to a carrier for affinity chromatography.

[0002]

2. Description of the Related Art Affinity chromatography refers to the separation and separation of a carrier consisting of an insoluble natural or synthetic polymer.
Chromatography using a column in which a substance (ligand) that specifically binds to a substance to be purified is immobilized and packed with the obtained ligand-immobilized carrier, and is particularly used for separation and purification of biologically relevant substances. Conventionally used carriers for affinity chromatography include:
Inorganic carriers include porous silica gel particles. Organic carriers include natural and synthetic polymer carriers,
As carriers derived from natural products, agarose, dextran,
Particles made of a polysaccharide such as cellulose include particles made of polystyrene, polyacrylamide, and the like as synthetic polymer carriers. Among the above-mentioned conventional carriers, inorganic porous silica gel particles have a high physical strength and are well separated carriers, but there is a disadvantage that the degree of non-specific adsorption is high, and examples of those actually used are as follows. Very few.
On the other hand, among organic carriers, as a natural carrier, those obtained by introducing various functional groups into porous agarose particles (trade name: Sepharose) and dextran particles (trade name: Sephadex) are commercially available. Is most commonly used. Agarose gel is said to be suitable as a carrier when handling high molecular weight biological substances because the limit molecular weight of impurities that can be eliminated is larger than that of dextran or acrylamide. However, a drawback common to these hydrophilic carriers is that they have low physical strength and lack pressure resistance, and it is difficult to perform high-speed analysis, that is, an analysis operation in a short time.

[0003]

SUMMARY OF THE INVENTION The present invention has been made to solve the above-mentioned conventional drawbacks, and has as its object to provide a high pressure resistance and a small degree of swelling and shrinkage. An object of the present invention is to provide a carrier for affinity chromatography which has high hydrophilicity and has very little nonspecific adsorption of proteins and the like other than the substance to be separated.

[0004]

The present inventors have conducted intensive studies to solve these problems, and as a result, impregnated an inorganic porous material with a solution containing a hydrophilic monomer and a crosslinking agent, and thereafter polymerized the inorganic porous material. It has been found that an organic gel can be easily synthesized inside the pores of an inorganic porous material by crosslinking, and the present invention has been accomplished.
The hydrophilic organic gel is a polymer having a functional group capable of immobilizing a ligand or a spacer in affinity chromatography.

[0005]

DETAILED DESCRIPTION OF THE INVENTION

The inorganic porous material used as a core material in the present invention includes alkaline earth metal carbonates, silicates, phosphates, sulfates, metal oxides, metal hydroxides, other metal silicates, Alternatively, other metal carbonates and the like can be used.

More specifically, calcium carbonate, barium carbonate, magnesium carbonate and the like are used as the alkaline earth metal carbonate, calcium silicate, barium silicate, magnesium silicate and the like are used as the alkaline earth metal silicate. The earth metal phosphates include calcium phosphate, barium phosphate, magnesium phosphate, and the like, and the alkaline earth metal sulfates include calcium sulfate, barium sulfate, and the like.
And magnesium sulfate.

Further, as metal oxides, silica, titanium oxide, iron oxide, cobalt oxide, zinc oxide, nickel oxide, manganese oxide, aluminum oxide, etc., and as metal hydroxides, iron hydroxide, nickel hydroxide, hydroxide Examples thereof include aluminum, calcium hydroxide, and chromium hydroxide.

The other metal silicates include zinc silicate and aluminum silicate, and the other metal carbonates include zinc carbonate and basic copper carbonate.
The shape may be any shape, and is not particularly limited.
It is preferable to have a spherical porous structure,
JP-A-57-55454, JP-A-61-222791
3. Spherical inorganic porous particles or spherical hollow inorganic porous particles disclosed in JP-A-63-258842 can be exemplified. In order to increase the breaking strength, Japanese Patent Application Laid-Open
It is possible to use particles obtained by encapsulating the inorganic nonporous particles disclosed in JP-A-216256 with an inorganic porous material layer. In the case of natural products, shirasu balloons, perlite, etc. can be used.

[0010] The porosity of these porous substances may be a pore volume of 0.02 to 3.0 ml / g, because if the pore volume is too small, it is difficult to form an organic gel. If the pore volume is too large, the breaking strength will be too small.
g. The pore diameter is 0.1 nm to 1
mm, this is because if the pore diameter is too small, it is difficult to form an organic gel, and if the pore diameter is too large, the breaking strength will be too small,
Preferably, the thickness is about 5 nm to 50 nm. Furthermore,
The size of the porous particles is about 5 nm to 100 mm, preferably about 1 μm to 4 mm.

According to the method of compounding of the present invention, a monomer, an initiator, a crosslinking agent and the like are dissolved in an appropriate solvent or water, and the solution is impregnated with a porous inorganic compound. A composite of an inorganic compound and a polymer gel is easily synthesized by polymerizing with radiation or the like.

The organic gel to be complexed is not particularly limited, and any substance may be used as long as it is a cross-linkable substance, and is capable of gelling from a state in which the spherical inorganic porous particles are impregnated as a liquid. It does not matter if it is. More specifically, the method of crosslinking may be any of covalent bond, ionic bond, and intermolecular force bond, or may be a substance that causes gelation by entanglement.

The monomer used for the covalent cross-linking is not particularly limited as long as it is water-soluble and can carry out ordinary radical polymerization. In general, acrylamide, methacrylamide, N-vinylpyridone, acrylic acid, methacrylic acid, P-styrenesulfonic acid, vinylsulfonic acid, 2-methacryloyloxyethylsulfonic acid, 3
-Methacryloyloxy-2-hydroxypropylsulfonic acid, allylsulfonic acid, methacrylsulfonic acid,
And ammonium salts and alkali metal salts of these acids, dimethylaminoethyl acrylate, dimethylaminoethyl methacrylate, quaternized hydrochloric acid, nitric acid, dimethyl sulfate, diethyl sulfate or ethyl chloride of 2-vinylpyridine and 4-vinylpyridine, acrylic acid Methacrylic acid, 2-hydroxyethyl methacrylate, 2-hydroxyethyl acrylate, 2-acrylamido-2-methylpropanesulfonic acid, N-isopropylacrylamide,
N-vinylformamide is exemplified.

Examples of the crosslinking agent having two or more polymerizable functional groups include ethylene glycol, propylene glycol, trimethylolpropane, glycerin, polyoxyethylene glycol, polyoxypropylene glycol, polyglycerin, N, N'-methylene Bisacrylamide, N, N-methylene-bis-N vinylacetamide, N, N-butylene-bis-Nvinylacetamide,
Tolylene diisocyanate, hexamethylene diisocyanate, allylated starch, allylated cellulose, diallyl phthalate, tetraallyloxyethane, pentaeristol triallyl ether, trimethylolpropane triallyl ether, diethylene glycol diallyl ether, triallyl trimellitate, etc. Can be

The initiator is not particularly limited, and any initiator suitable for gelation may be selected. Examples thereof include hydrogen peroxide, persulfates such as potassium persulfate, sodium persulfate, and the like. An azo initiator such as ammonium persulfate, for example, 2,2'-azobis (2-amidinopropane) dihydrochloride, 2,2'-azobis (N, N'-dimethyleneisobutylamidine) dihydrochloride, 2,2 '-Azobis {2-methyl-N- [1,1, -bis (hydroxymethyl) -2-hydroxyethyl] propionamide}, 2,2'-azobis [2- (2-imidazoline-
2-yl) propane] dihydrochloride, 4,4'-azobis (4-cyanovaleric acid), 2,2'-azobisisobutyronitrile, 2,2'-azobis (2,4'-dimethylvalero Nitrile) and the like. Hydrogen peroxide or persulfate can be used as a redox initiator by combining a reducing substance such as sulfite or L-ascorbic acid or an amine salt.

Crosslinking by ionic bonding is carried out, for example, by gelling a polymer electrolyte having a cation or anion such as an ammonium salt or a carboxyl group by crosslinking with a polyvalent ionic substance such as calcium by ionic bonding. is there. Crosslinking due to intermolecular forces is common in natural polymers and the like, for example, agarose, starch, galactomannan, nitrocellulose, methylcellulose, hydroxypropylmethylcellulose, pectic acid, alginic acid, agar, carrageenan, protecoglycan, glycoprotein, gelatin, actin , Tubulin, hemoglobin S, insulin, fibrin, ovalbumin, serum albumin, myosin, collagen, polypeptide and the like. Synthetic polymers include polyvinyl alcohol and the like.

In order to synthesize an organic gel in spherical inorganic porous particles, the concentration of the monomer may be from 0.1% by weight to 99.9% by weight, and the concentration of the crosslinking agent may be from 0.001% by weight to 50% by weight. . The concentration of the initiator and the polymerization method may be selected according to the method of preparing the gel. For example, if ammonium peroxodisulfate (APS) is well known as an initiator, the monomer and the crosslinking agent may be used. Dissolved in water,
Impregnate the inorganic porous material, and remove the supernatant, if necessary, by filtration or the like. Thereafter, the polymerization may be performed by adding a solution of a polymerization accelerator such as TEMED or maintaining the temperature at an appropriate temperature. In addition, in the case of a substance having physical crosslinking such as a polysaccharide, for example, a crosslinking substance is heated and dissolved in water and impregnated with an inorganic porous substance. If necessary, the inorganic porous substance is settled by centrifugation, or the supernatant is removed by filtration or the like. After that, cooling may be performed.

[0018]

EXAMPLES Next, the present invention will be described specifically by way of examples, but the gist of the present invention is not limited to these examples.

Example 1 2 g of agarose (manufactured by Wako Pure Chemical Industries) was dissolved in 10 ml of a phosphate buffer solution at 90 ° C., and 5 g of spherical porous silica particles (particle size: 1.7 to 4 mm; manufactured by Fuji Silysia Chemical Ltd.) Into the solution. Once the solution had completely penetrated the silica, the solution around the silica was removed and the agarose was cross-linked at low temperature. After washing sufficiently, a complex was obtained.

(Example 2) 4 g of the particles obtained in Example 1 were dispersed in 10 ml of distilled water, and 200 g of cyanogen bromide was added.
Add mg and stir. The pH is maintained at about 10 for 30 minutes by dropwise addition of 5N NaOH. After 30 minutes, filter and wash,
10 μm of 0.2 μmol / ml concanavalin A solution
The reaction was carried out overnight at 4 ° C. After the reaction, the mixture was filtered and washed to obtain a concanavalin A-immobilized complex.

(Embodiment 3) The particles of Embodiment 1 are 6 mm in inner diameter.
And packed in a stainless steel column having a length of 75 mm, and examined for pressure resistance and swelling property with water. Filling is 30m of purified water
2 g of the filler was stirred in for 10 minutes, and then 2.0 ml
This is done by filling at a constant flow rate at / min. The pressure resistance is
The measurement was performed by passing purified water through a column at various flow rates and examining the relationship between the flow rate and pressure loss. The swellability is measured by examining the change in column pressure when flowing liquids having different ionic strengths. In the evaluation of pressure resistance, the particles of Example 1 had a proportional relationship between the pressure and the flow rate up to 4.5 ml / min, but the particles of Comparative Example 1 were too high in pressure at 0.007 ml / min and could not be measured. . In the evaluation of the swellability, the eluate was eluted from a 40 mM phosphate buffer for 200 m.
When the solution was changed to the phosphate buffer of M, no increase in pressure was observed.

Claims (4)

[Claims] 1. An affinity of an organic-inorganic hybrid having a hydrophilic gel held in pores of an inorganic porous substance, wherein the hydrophilic gel has a functional group capable of immobilizing a ligand or a spacer in affinity chromatography. Organic-inorganic hybrid for chromatography. 2. The organic-inorganic hybrid for affinity chromatography according to claim 3, wherein the organic-inorganic hybrid is spherical. 3. An affinity of an organic-inorganic hybrid having a hydrophilic gel held in pores of an inorganic porous substance, wherein the hydrophilic gel has a functional group capable of immobilizing a ligand or a spacer in affinity chromatography. A method for producing an organic-inorganic hybrid for chromatography. 4. A method for producing an organic-inorganic hybrid for affinity chromatography, wherein the organic-inorganic hybrid according to claim 3 is spherical.