JPH04256439A - Pyrogen adsorbent - Google Patents

Abstract

PURPOSE:To remove a pyrogen, that is, a substance which causes pyrexia by adsorption when allowed to enter a living body by injection, artificial dialysis or other method. CONSTITUTION:The pore diameter of porous polymer beads contg. glycidyl methacrylate as a constituent is controlled to >=50,000 (expressed in terms of the mol.wt. of dextran) and polyethyleneimine is bonded to the epoxy group of the glycidyl methacrylate.

Description

[Detailed description of the invention]

0001

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to an adsorbent effective for removing a pyrogen, a substance that induces heat generation when administered or eluted into a living body by injection, artificial dialysis, etc. More specifically, the present invention relates to an adsorption/removal agent for pyrogens contained in injection solutions, infusions, dialysates, and the like.

0002

BACKGROUND OF THE INVENTION It has been found that lipopolysaccharide (LPS), which is derived from the cell walls of Gram-negative bacteria such as Escherichia coli, is currently a problematic pyrogen. Various studies have been conducted regarding the removal of pyrogens, and attempts have been made to remove them using activated carbon, ion exchange resins, and various separation membranes, but they have not yet reached the level of practical use. That is, these adsorbents have low selectivity for pyrogen, and a satisfactory amount of adsorption and removal cannot be obtained. Furthermore, there is also the problem of impurity elution during the adsorbent synthesis process, so it is currently impossible to actually use it.

0003

SUMMARY OF THE INVENTION An object of the present invention is to obtain an adsorbent which has high selectivity for pyrodiene and has a high adsorption amount and speed.

0004

[Means for Solving the Problem] The present inventors have conventionally discovered that the phosphoric acid group of LPS interacts with the amino group, and that among the amino groups, the primary amino group type has the weakest interaction with anionic substances. , the selectivity for LPS is high, and when the amino group is bonded to the end of the hydrophobic group or when the amino group is bonded to the hydrophobic carrier, both the phosphoric acid group and the hydrophobic group of LPS interact. It was discovered that high selectivity and adsorption ability can be achieved by using this method, and based on this knowledge, the present invention was completed.

That is, the present invention provides a pyrogen adsorbent made by bonding polyethyleneimine to the epoxy groups of porous polymer beads containing glycidyl methacrylate as a constituent component. Note that it is preferable that the pore diameter of the porous polymer beads containing glycidyl methacrylate as a constituent component is 50,000 or more in terms of the molecular weight of dextran.

Porous polymer beads containing glycidyl methacrylate as a constituent in the present invention include, for example, glycidyl methacrylate or other monomers compatible therewith, such as acrylic acid, methyl acrylate, methacrylic acid, and methyl methacrylate. , acrylic monomers such as hydroxyethyl methacrylate, styrene monomers such as styrene, p-methylstyrene, and α-methylstyrene, and difunctional monomers such as divinylbenzene and methylenebisacrylamide are mixed in any proportion, In addition, solvents that are non-polymerizable and do not react with these monomers, such as aliphatic hydrocarbons such as pentane, hexane, octane, and isooctane, benzene, toluene,
After polymerization by adding an appropriate amount of one or a mixture of two or more of aromatic hydrocarbons such as ethylbenzene and xylene, aliphatic alcohols such as hexanol, octanol, and dodecanol, and aromatic alcohols such as benzyl alcohol, These are porous beads obtained by removing the solvent by a known method such as extraction. By appropriately selecting the type, combination, and amount of these solvents added, the pore diameter can be arbitrarily controlled within the range of 102 to 107 in terms of the molecular weight of dextran.

[0007] Further, during the polymerization, an appropriate initiator or catalyst such as benzoyl peroxide, azobisisobutylnitrile, etc. may be used as necessary. The resulting porous beads contain epoxy groups, and the content thereof is preferably 0.01 meq/g or more, more preferably 0.1 meq/g or more. The dispersion medium for dispersing these monomer solutions may be any material as long as it can stably disperse the monomer solution, and any combination of surfactants, inorganic salts, water-soluble polymers, etc. can be used. By appropriately combining conditions such as the type of surfactant, amount added, speed of stirring and dispersion, etc.
Beads of arbitrary particle size up to several mm can be obtained. The pore size of porous beads can be evaluated by gel permeation chromatography (GPC) using dextran standard molecular weight substances.

That is, porous polymer beads containing glycidyl methacrylate as a constituent are sieved by an appropriate method and packed into a stainless steel column for liquid chromatography. Beads for pore size evaluation are 100
Even if it is less than μm, it is preferable that the size is as uniform as possible. Stainless steel column sizes are usually commercially available (
For example, 4.6mmφ*150mm manufactured by Nippon Seimitsu Co., Ltd.)
A column is sufficient. The standard molecular weight substance used for GPC measurement is preferably one that has as little interaction as possible with the polymer beads, and dextran standard molecular weight substance (manufactured by Sigma Chemical Co., Ltd.) is suitable. Other standard molecular weight substances,
For example, globular proteins, polyethylene glycol, etc.
This is not appropriate because it may not necessarily elute in the order of molecular weight or may be adsorbed. The measurement method may be normal aqueous GPC measurement using standard molecular weight dextran. That is, the elution capacity of dextran of various molecular weights is measured using a liquid chromatography device using distilled water as an eluent.

When the porous polymer beads containing glycidyl methacrylate as a constituent component are used as a pyrogen adsorbent, the molecular weight of dextran is 50,000 or more.
Preferably, it has an exclusion limit molecular weight in the range of 100,000 or more. The molecular weight of polyethyleneimine in the present invention is not particularly limited, but is preferably 500 or more, more preferably 2,000 or more and 1,000,000 or less.

[0010] A method for bonding polyethylene imine to porous polymer beads containing glycidyl methacrylate as a constituent is obtained by dispersing the polymer beads in water, adding polyerylene imine, and heating. At this time, the reaction is carried out in the presence of a basic substance such as caustic soda or triethylamine, if necessary.

[0011]

[Operation] According to the present invention, a technology for adsorbing pyrogens contained in injection solutions, dialysates, infusions, culture chemicals, etc. has been established. Although the mechanism is not clear, LPS is selectively adsorbed by the primary amino group of the adsorbent, the hydrophobic group bonded to it, and the hydrophobic carrier, and the adsorption surface is increased by controlling the pore size of the adsorbent. It is thought that the diffusion of other coexisting proteins to the adsorbed surface can be controlled.

0012

Effects of the Invention The pyrogen adsorbent of the present invention has (1) high selectivity without adsorbing salts, amino acids, and useful proteins other than pyrogens, and (2) high pyrogen adsorption amount and adsorption rate. It has excellent effects.

[0013]

EXAMPLES The present invention will be explained in more detail with reference to Examples below, but the present invention is not limited to these Examples. The quantitative measurement of pyrogens was conducted by Seikagaku Kogyo Co., Ltd. (
A limulus color development test was conducted using an endotoxin quantitative reagent kit manufactured by Co., Ltd.). Production Example 1 Add 0.3 g of benzoyl peroxide to 14.3 g of glycidyl methacrylate, 13.0 g of divinylbenzene, and 50 ml of diethylbenzene, stir well and uniformly, stir and disperse in 300 ml of a 1% partially saponified polyvinyl alcohol aqueous solution, and stir and disperse at 70°C. The polymerization reaction is carried out for 16 hours. After the reaction is complete, the beads are filtered and thoroughly washed with warm water to completely remove polyvinyl alcohol. Furthermore, diethylbenzene and unreacted monomers are completely extracted and removed with methanol at 50°C. When the obtained polymer beads were dried and the content of epoxy groups was measured by the hydrochloric acid dimethylformamide method, it was found that 3 m
It was mol/g. 5g of these beads in 100ml of distilled water
2 g of polyethyleneimine having an average molecular weight of 6,000 was added, and further 0.1 g of triethylamine was added, and the mixture was heated at 80° C. for 8 hours. Filter the reacted beads and
Wash thoroughly with warm water at 0°C. The beads thus obtained are washed with 30 mmol phosphate buffer and 1 mol saline, and the salts are completely removed with endotoxin-free distilled water. The beads were classified and beads having a diameter of 37 to 74 μm were taken out and filled in a stainless steel rack, and GPC measurement was performed. As a result, the exclusion limit molecular weight of these beads was approximately 400,000.

Production Example 2 Production was carried out in the same manner as in Production Example 1 except that 2 g of polyethyleneimine having an average molecular weight of 100,000 was added. The beads were classified and beads having a diameter of 37 to 74 μm were taken out, filled in a stainless steel column, and subjected to GPC measurement. As a result, the exclusion limit molecular weight of these beads was approximately 200,000.

Example 1 1 g of each of the adsorbents obtained in Production Examples 1 and 2 was packed into a stainless steel column (inner diameter 4.5 mm, length 150 mm), and 150 ml of 1 mol saline solution was passed through the column at a rate of 0.5 ml/min. After that, the salt was completely washed away with pyrogen-free distilled water, and then the endotoxin derived from Escherichia coli (LPS Control E, manufactured by Seikagaku Kogyo Co., Ltd.) was removed.
055-B5) 250ml of distilled water adjusted to 1ng/ml
1 was passed through the column at a flow rate of 0.5 ml/min, and the endotoxin contained in the distilled water collected from the column outlet was quantified.

Example 2 L-isoleucine, L-leucine, L-lysine, L-methionine, L-phenylalanine, L-threonine, L
- Distilled water containing 0.1 g/1 each of valine and L-tryptophan was added to Escherichia coli-derived endotoxin (LPS control manufactured by Seikagaku Corporation) E. coli 0
100 ml of a solution prepared to 1 ng/ml with 55-B5)
1 g of each of the adsorbents obtained in Production Examples 1 and 2 was placed inside.
Disperse and adsorb at room temperature for 8 hours with stirring. The endotoxin concentration and amino acid concentration of the adsorbed supernatant were measured, and the endotoxin adsorption rate and amino acid recovery rate were calculated. For amino acid analysis, an amino acid analyzer manufactured by JASCO Corporation was used.

[0017]

Claims (2)

[Claims] 1. A pyrogen adsorbent comprising polyethyleneimine bonded to the epoxy groups of porous polymer beads containing glycidyl methacrylate as a constituent component. 2. The pyrogen adsorbent according to claim 1, wherein the porous polymer beads containing glycidyl methacrylate as a constituent have a pore diameter of 50,000 or more in terms of the molecular weight of dextran.