JPH04256438A - Pyrogen adsorbent - Google Patents

Abstract

PURPOSE:To efficiently remove a pyrogen, that is, a substance which causes pyrexia when allowed to enter a living body by injection, artificial dialysis or other method. CONSTITUTION:This pyrogen adsorbent contains polyamino acid having both amino and carboxyl groups as functional groups for adsorption.

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 efficiently remove the above-mentioned exothermic substances.

0004

[Means for Solving the Problems] The present inventors have discovered that the amino group of the adsorbent interacts with the phosphoric acid group of LPS, but also interacts with the functional groups such as carboxyl groups of coexisting substances to cause adsorption; In addition, in order to increase the recovery rate of coexisting substances, it is better to eliminate pores that allow coexisting substances to diffuse, and when using only amino groups as adsorption functional groups, the ionic strength and pH conditions of the adsorption system It was discovered that the scope of use was narrow. However, it was discovered that an adsorbent containing a carboxyl group in addition to an amino group has a small adsorption of coexisting proteins, etc., and is capable of adsorbing in a relatively wide pH range and high ionic strength.Based on this knowledge, the present invention was completed. I ended up doing it.

That is, the present invention provides a pyrogen adsorbent containing a polyamino acid characterized by having both an amino group and a carboxyl group as functional groups for adsorption. Preferably, the adsorbent has a pore diameter of 1000 or less based on the molecular weight of dextran, and is substantially non-porous.

[0006] The polyamino acid in the present invention is a polymer of one or two of glutamic acid, aspartic acid, and hydrophobic ester derivatives of these amino acids, and other amino acids containing these amino acids or amino acid derivatives. It may also be a copolymer with an amino acid.

[0007] As the amino group used in the present invention, a primary, secondary, tertiary or quaternary amino group is used; however, quaternary amino groups can cause ion exchange and significantly change the pH of the adsorption system. Undesirable. In addition, the content of these amino groups is 0.01 meq/g or more, preferably 0.1 m
It is equal to or more than eq/g.

The method for introducing these amino groups into the above-mentioned polyamino acids is to introduce primary amino groups into the hydrophobic ester groups of polyamino acids containing hydrophobic ester derivatives such as methyl ester, ethyl ester, and benzyl ester of glutamic acid and/or aspartic acid. polyamines including hydrazine, ethylenediamine, hexamethylenediamine, hexamethylenetriamine, ethylenetriamine,
1-(N,N-dimethylamino)-2-aminoethane,
1-(N,N-diethylamino)-2-aminoethane,
The primary amino group of alkyl polyamines such as 1-(N,N-dimethylamino)-6-aminohexane and 1-(N,N-diethylamino)-6-aminohexane, and aromatic polyamines such as diaminobenzene and diaminotoluene. There is a method of forming an amide bond using an aminolysis reaction. Further, the carboxyl group can be introduced by alkaline hydrolysis of the above-mentioned hydrophobic ester. The amount of carboxyl group introduced is 0.01 meq/g or more, preferably 0.01 meq/g or more.
It is 1 meq/g or more.

The shape of the adsorbent according to the present invention may be such that the polyamino acid containing amino groups and carboxyl groups described above may be formed into a film, fibers, or beads. Furthermore, it may be coated or chemically bonded onto various carriers. The carrier used for these may be an inorganic substance such as glass, metal salts, carbon, etc.
It may also be an organic substance such as a polymer. Furthermore, various shapes of these carriers can be used, such as powder, beads, membranes, fibers, and hollow fibers, and they can be selected depending on the module such as an adsorption column or an adsorption cartridge.

[0010] The chemical bonding method utilizes the functional groups of the carrier and uses a suitable bonding reagent. For example, aminopropyl silica gel is reacted with a bifunctional reagent such as toluene diisocyanate or terephthalic acid chloride; Examples include a method of bonding amino groups, carboxyl groups, hydroxyl groups, etc. of the polyamino acid having both amino groups and carboxyl groups. As a method for spheroidizing the polyamino acid itself, for example, the polyamino acid is dissolved in a suitable solvent, dispersed in a medium incompatible with this, the solvent of the polyamino acid is removed by evaporation, and the polyamino acid is precipitated. 1986
-1728) and amino acid NCA (N-
There is a method in which the polymerization is carried out in a medium which is compatible with the carboxy-α-amino acid anhydride, but which becomes incompatible upon polymerization. The adsorbent in the present invention must have a pore diameter of 1000 or less in terms of the molecular weight of dextran, and be substantially non-porous.

The pore size of the adsorbent can be determined by gel permeation chromatography using a standard molecular weight dextran substance (
GPC). That is, the adsorbent is sieved by an appropriate method and packed into a stainless steel column for liquid chromatography. Beads for pore size evaluation are 100
It is preferable that the size be less than μm and as uniform as possible. As for the size of the stainless steel column, a commercially available column (for example, 4.6 mmφ*150 mm manufactured by Nippon Seimitsu Co., Ltd.) is sufficient. The standard molecular weight substance used for GPC measurement is preferably one that has as little interaction as possible with the adsorbent, and dextran standard molecular weight substance (manufactured by Sigma Chemical Co., Ltd.) is suitable. Other standard molecular weight substances, such as globular proteins and polyethylene glycol, are not suitable because they do not necessarily elute in the order of molecular weight or are 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.

0012

[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 amino and carboxyl groups of the adsorbent, and by controlling the pore size of the adsorbent, the diffusion of other coexisting proteins into the interior of the adsorbent can be controlled. considered to be a thing.

[0013]

Effects of the Invention The pyrogen adsorbent of the present invention (1) has high selectivity without adsorbing salts, amino acids, and useful proteins other than pyrogens; and (2) generates heat in a relatively wide pH range and high ionic strength. (3) The adsorbent is capable of adsorbing substances and has excellent effects such as being biocompatible.

[0014]

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 2.5% dichloroethane solution of poly-γ-methyl-L-glutamate (hereinafter referred to as PMLG) with an average degree of polymerization of 500 15
Dichloroethane was removed by evaporation at 50° C. while stirring and dispersing 0 g of the solution in 500 ml of a 2.5% aqueous solution of partially saponified polyvinyl alcohol. The obtained polyamino acid spherical particles are filtered and thoroughly washed with warm water to completely remove polyvinyl alcohol. This spherical particle was classified and 37 to 74
Take out the μm one and fill it into a stainless steel column.
GPC measurement was performed. As a result, the exclusion limit molecular weight was approximately 5
It was 00. Further, the polyamino acid spherical particles were dispersed in methanol, 2 g of ethylenediamine was added, and the mixture was heated at 60°C.
After reacting for 5 hours, the mixture was thoroughly washed with distilled water. The obtained particles were left to stand at 30° C. for 1 hour in a 1N aqueous solution of caustic soda, and then the caustic soda was completely washed away with distilled water. When a part of these particles was dried and the amino groups and carboxyl groups were determined by acid-alkali titration, the amino groups were 0.8 meq/g.
, carboxyl group was 0.6 meq/g.

Production Example 2 Production Example 1 except that poly-β-benzyl-L-aspartate with an average degree of polymerization of 450 was used instead of poly-γ-methyl-L-glutamate with an average degree of polymerization of 500. A porous body was produced in exactly the same manner as in Example 1. The exclusion limit molecular weight of the obtained particles was 1000. This was reacted with 4.5 g of hexamethylene diamine in the same manner as in Production Example 1, and then treated with caustic soda. The amino groups and carboxyl groups were determined to be 0.6 meq/g and 0.3 meq/g for carboxyl groups. Ta.

Comparative Example 1 A porous body was produced in exactly the same manner as in Production Example 1, except that the caustic soda treatment was not performed.

Comparative Example 2 In Production Example 2, a porous body was produced in exactly the same manner as Production Example 1, except that the caustic soda treatment was not performed.

1 g of each of the adsorbents obtained in Production Examples 1 and 2 and Comparative Examples 1 and 2 was placed in a stainless steel column (inner diameter 4.5
mm, length 150 mm) and fill with 1 mol saline solution 150 mm.
After passing through the solution at a rate of 0.5 ml/min, the salt is completely washed away with pyrogen-free distilled water, and then endotoxin derived from Escherichia coli (LPS Control E, coli 055-B5, manufactured by Seikagaku Corporation) is removed. )151
250 ml of albumin solution adjusted to 0 ng/ml (albumin concentration 25 mg/ml pH = 5.6) was passed through at a flow rate of 0.5 ml/min and collected from the column outlet. Endotoxin concentration and albumin recovery rate contained in the solution. was quantified.

Claims (2)

[Claims] 1. A pyrogen adsorbent containing a polyamino acid, which has both an amino group and a carboxyl group as functional groups for adsorption. 2. The pyrogen adsorbent according to claim 1, wherein the adsorbent has a pore diameter of 1000 or less based on the molecular weight of dextran and is substantially non-porous.