JPH04256437A - 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 is made of polyamino acid contg. at least one of glutamic acid and aspartic acid and having a long chain aliphatic hydrocarbon group bonded to the side chain. The pore diameter of this adsorbent is <=1,000 (expressed in terms of the mol.wt. of dextran) and this adsorbent is practically nonporous.

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 use it in practice.

0003

OBJECTS OF THE INVENTION It is an object of the present invention to efficiently remove a substance (pyrogiene) that induces heat generation when administered or eluted into a living body by injection, artificial dialysis, etc.

0004

[Means for Solving the Problems] The present inventors discovered that the polyamino acid skeleton has excellent biocompatibility and is difficult to denature physiologically active proteins and sugars present in the adsorption system.As a result of intensive research, we found that LPS In addition to the chain, it has a phosphoric acid group and a hydrophobic group, so both ionic adsorption and hydrophobic adsorption are possible.Ionic adsorption is greatly influenced by the coexisting ions and pH of the adsorption system, and the conditions for adsorption. In the case of hydrophobic adsorption, these effects are small, and in fact, it can be adsorbed well even under high ionic strength. Among hydrophobic groups, long-chain alkyl groups have excellent LPS adsorption performance and are non-specific for other coexisting substances. The present inventors have completed the present invention by discovering that there is less adsorption and that in order to increase the recovery rate of coexisting substances, it is better to eliminate pores through which coexisting substances can diffuse.

That is, the present invention provides an adsorbent material comprising a polyamino acid containing at least one of polyglutamic acid and polyaspartic acid, which is characterized in that a long-chain aliphatic hydrocarbon group is bonded to the side chain. The object of the present invention is to provide a pyrogen adsorbent that is substantially non-porous and has a pore size of 1000 or less based on the molecular weight of dextran. This long-chain aliphatic hydrocarbon group is, for example, an amide bond or an ester bond of the polyamino acid -(CH2)m-COOH (m=1
or 2) can be attached to the carboxyl site of the side chain.

[0006] The polyamino acid in the present invention is a polymerized product of one or two derivatives of glutamic acid or aspartic acid in which a long-chain aliphatic hydrocarbon group is bonded to the side chain, and a polyamino acid containing these amino acid derivatives. If so, it may be a copolymer with other amino acids.

[0007] Furthermore, the long-chain aliphatic hydrocarbon group may be introduced after polymerization. The long-chain aliphatic hydrocarbon group in the present invention refers to a saturated or unsaturated long-chain aliphatic hydrocarbon group, and examples thereof include alkyl groups such as straight-chain alkyl groups such as ethyl group, hexyl group, and lauryl group; isobutyl group,
A branched alkyl group such as an isohexyl group, an alkenyl group, an unsaturated group such as an allyl group having 2 carbon atoms
The above-mentioned groups preferably have 4 or more, more preferably 6 or more, and further include alkynyl groups.

The content of these long-chain aliphatic hydrocarbon groups is 1% or more, preferably 5% or more, and more preferably 10% or more of the amino acid residues constituting the polyamino acid.

A method for introducing these long-chain aliphatic hydrocarbon groups into the above-mentioned polyamino acids is to combine glutamic acid or aspartic acid with an alcohol having a long-chain aliphatic hydrocarbon group, such as hexanol or lauryl alcohol, in the presence of an acid catalyst. A method of ester bonding followed by polymerization, or methyl esterification of glutamic acid or aspartic acid with methanol followed by polymerization, followed by transesterification with an alcohol having a long chain aliphatic hydrocarbon group using para-toluenesulfonic acid. can do. Furthermore, an amide bond can be introduced by reacting a primary amine having a long-chain aliphatic hydrocarbon group, such as monohexylamine, octylamine, laurylamine, etc., with methyl ester of glutamic acid or aspartic acid.

In the polymerization of glutamic acid and aspartic acid, the carboxyl group at the γ or β position is protected with a derivative such as methyl ester, and phosgene is reacted to form NCA (
After producing N-carboxy-α-amino acid anhydride), it can be polymerized using triethylamine or the like. At this time, it can also be copolymerized with other amino acids such as NCA such as leucine and phenylalanine.

[0011] The shape of the adsorbent according to the present invention may be such that the polyamino acid containing an amino group and a carboxyl group described above may be formed into a film, a fiber, or a bead. Moreover, it may be coated on various carriers. The carrier used for these may be an inorganic substance such as glass, metal salts, or carbon, or an organic substance such as a polymer. In addition, various shapes of these carriers can be used, such as powder, beads, membranes, fibers, and hollow fibers, and they can be selected according to the module such as the adsorption column or adsorption cartridge. .

[0012] The pore diameter of the adsorbent used in the present invention is required to be 1000 or less in terms of the molecular weight of dextran, and it is required to be substantially non-porous. The pore size of the adsorbent can be evaluated by gel permeation chromatography (GPC) using dextran standard molecular weight substances. That is, the adsorbent is sieved by an appropriate method and packed into a stainless steel column for liquid chromatography. The beads for pore size evaluation are preferably 100 μm or less, and are preferably as uniform in size as possible. The size of the stainless steel column is usually commercially available (for example, 4.6 mmφ*150 manufactured by Nippon Seimitsu Co., Ltd.
mm) column 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.) is considered 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.

[0013]

[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, pyrogens are efficiently adsorbed by the hydrophobic group of the adsorbent regardless of the effects of pH or ionic strength, and by making the adsorbent substantially non-porous, other coexistence substances can be absorbed. It is thought that the diffusion of proteins etc. into the interior of the adsorbent can be controlled.

[0014]

Effects of the Invention The pyrogen adsorbent of the present invention (1) has high selectivity without adsorbing salts other than pyrogens, amino acids, and useful proteins; (2) adsorbs pyrogens over a wide pH and ionic strength range; The adsorbent (3) with a large amount and adsorption rate exhibits excellent effects such as biocompatibility.

[0015]

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 25
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, 3 g of the polyamino acid spherical particles were dispersed in methanol, 12 g of hexanol and 1.5 g of para-toluenesulfonic acid were added, and the mixture was reacted at 60° C. for 5 hours, followed by thorough washing with distilled water. A part of the obtained particles was dried and subjected to elemental analysis, and the results were as shown in the table below. From this result, it was found that about 35% of the methyl ester moiety was substituted with hexyl ester.

Production Example 2 Production Example 1 was repeated except that poly-β-methyl-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. Particles were produced in exactly the same manner. The exclusion limit molecular weight of the obtained particles was 1000. 3 g of these particles were dispersed in methanol, 15 g of laurylamine was added, and the mixture was reacted with stirring at 60° C. for 7 hours, followed by thorough washing with methanol and distilled water. A part of the obtained particles was dried and subjected to elemental analysis, and the results were as shown in the table below. From this result, it was found that about 40% of the methyl ester moiety was substituted with laurylamide.

Example 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. Thoroughly wash off the salt with pyrogen-free distilled water, then remove endotoxin derived from Escherichia coli (LPS Control E, manufactured by Seikagaku Corporation).
055-B5) Albumin solution adjusted to 1510 ng/ml (albumin concentration 25 mg/ml pH = 5.
6) 250 ml of the solution was passed through the column at a flow rate of 0.5 ml/min, and the endotoxin concentration and albumin recovery rate contained in the solution collected from the column outlet were quantified.

Claims (3)

[Claims] Claim 1: An adsorbent comprising a polyamino acid containing at least one of polyglutamic acid and polyaspartic acid, characterized in that a long-chain aliphatic hydrocarbon group is bonded to the side chain, has a pore diameter of A pyrogen adsorbent having a molecular weight of dextran of 1000 or less and being substantially non-porous. 2. The long-chain aliphatic hydrocarbon group is an amide bond, and the polyamino acid has -(CH2) m -COOH(m
=1 or 2) The pyrogen adsorbent according to claim 1, wherein the pyrogen adsorbent is bonded to a carboxyl site of a side chain. 3. The long-chain aliphatic hydrocarbon group is an ester bond of the polyamino acid -(CH2) m -COOH(m
=1 or 2) The pyrogen adsorbent according to claim 1, wherein the pyrogen adsorbent is bonded to a carboxyl site of a side chain.