JPH1135068A - Container for endotoxin - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a container for endotoxin which has little possibility of being broken, and in using endotoxin, with which the endotoxin solution of predetermined concentration can be obtained even after dissolution, with which the adsorption of endotoxin is little, and with which endotoxin solution whose concentration is stable with the passage of time can be prepared. SOLUTION: The material of this container for endotoxin is a high-molecular compound having ester bond, and the endotoxin content of this container in extract, when endotoxin free water equal in amount to a liquid amount to be stored inside when in use is stored and extracted, is made less than 5/100 of endotoxin stored. Polyethylene terephthalate is preferred for the high molecular compound having ester bond.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to an endotoxin container for containing an endotoxin such as a standard endotoxin product.

[0002]

2. Description of the Related Art Endotoxin is a lipopolysaccharide (Lipopolysac) present on the outer membrane of the cell wall of Gram-negative bacteria.
harid, LPS), which has a hydrophilic portion and a hydrophobic portion in the same molecule. For this reason, when an endotoxin aqueous solution is contained in a glass vial, there is a problem that endotoxin is adsorbed on glass or endotoxin activity is reduced due to the influence of metal ions eluted from the glass vial. Further, even when the container is contained in a container made of a hydrophobic material such as polyethylene or polystyrene, the endotoxin is adsorbed to the container and the endotoxin activity decreases.

Due to these problems, a commercially available endotoxin standard used in the measurement of endotoxin contains a known amount of endotoxin in a glass vial, and mannitol and a metal ion chelating agent as stabilizers. Powder.

[0004] As for the method of stabilizing endotoxin, an aqueous solution containing endotoxin has an affinity for endotoxin and does not have the property of inhibiting or promoting the reaction of endotoxin with the hemocyte component liquid of horseshoe crab. A method for stabilizing endotoxin in an aqueous solution, which is characterized by coexistence of a protein, has been proposed (JP-A-7-151760). Further, an endotoxin stabilizer containing one or more selected from the group consisting of amino alcohols, polyhydric alcohols, nonionic surfactants, polysucrose and chelating agents as active ingredients has also been proposed ( JP-A-8-75751).

However, even if these stabilizing methods are used, a container made of polyethylene or polystyrene may not be able to stably hold an endotoxin aqueous solution. In addition, in the case of a glass vial, the use of these proposed stabilization methods can stably retain an endotoxin aqueous solution, but has a disadvantage that it is easily broken.

[0006]

SUMMARY OF THE INVENTION It is an object of the present invention to provide an endotoxin aqueous solution having a predetermined concentration even after dissolution when using endotoxin. It is an object of the present invention to provide an endotoxin container capable of preparing an endotoxin aqueous solution having a low concentration and stable over time.

[0007]

According to the first aspect of the present invention, there is provided a container for endotoxin which contains an amount of endotoxin-free water equal to the amount of liquid to be contained therein during use, and is used for extraction. Is characterized in that the endotoxin content is 5/100 or less of the contained endotoxin, and the material is a polymer compound having an ester bond.

[0008] The container for endotoxin according to claim 2 is
The container for endotoxin according to claim 1, wherein the polymer compound having an ester bond is polyethylene terephthalate.

Hereinafter, the present invention will be described.

The endotoxin container of the present invention is characterized in that the endotoxin content in the extract when the extraction is carried out by containing an amount of endotoxin-free water equal to the amount of the liquid to be contained therein when used is the endotoxin contained therein. Of 5
/ 100 or less. When the above-mentioned endotoxin content exceeds this value, when this container is used as a container for an endotoxin standard having a predetermined concentration, the concentration of the endotoxin standard becomes higher than the predetermined value, and the dispersion increases. . The endotoxin content is preferably not more than 3/100 of the contained endotoxin.

The above-mentioned "amount of liquid to be accommodated inside at the time of use" is the amount of the aqueous solution of endotoxin when the aqueous solution of endotoxin is used in the container of the present invention. Thereafter, the endotoxin is lyophilized to be in a powdered state, and in the case where water is added and redissolved in use to use the endotoxin, the amount of the aqueous solution after the redissolution is used.

The above-mentioned extraction conditions are such that an amount of endotoxin-free water determined as described above is contained and the temperature is set to 37 ° C. for 1 hour under stirring.

There are various methods for measuring the endotoxin content. The method for measuring the endotoxin content in the present invention is based on the synthetic chromogenic substrate method.
The endotoxin content can be measured by the synthetic chromogenic substrate method using Endospecy (trade name), an endotoxin measurement kit manufactured by Seikagaku Corporation.

[0014] Endotoxin in the container can be reduced by washing with endotoxin-free water. Further, if the container of the present invention is molded in a sufficiently clean atmosphere and the opening is sealed in a sufficiently clean atmosphere, a container having a sufficiently low endotoxin content can be produced.

The material of the container of the present invention is limited to a polymer compound having an ester bond. The ester bond may be in the main chain or side chain of the polymer. Further, a monomer for producing a polymer compound shown below may be copolymerized or block copolymerized.

Examples of the high molecular compound having an ester bond in the main chain include polyethylene terephthalate and polybutylene terephthalate.

The high molecular compound having an ester bond in the side chain includes, for example, polyethyl acrylate, polybutyl acrylate, polypropyl acrylate, polymethyl acrylate, polyvinyl acetate, polyethyl methacrylate, polyethyl methacrylate, and the like. Examples thereof include butyl methacrylate and polypropyl methacrylate.

Among these polymer compounds having an ester bond, polyethylene terephthalate is particularly preferred.

These polymers have an ester bond,
It has weak polarity due to the presence of oxygen atoms. Therefore, it is considered that the interaction with the hydrophobic portion of the endotoxin molecule is small, and the adsorption of endotoxin is reduced.

Although the shape of the container of the present invention is not particularly limited, for example, a bottomed tube having one end opened and the other end closed is preferable, and the opening can be closed by a stopper. Is preferred. In the case of the above-mentioned bottomed tubular body, its size is preferably about 5 to 30 mm in outer diameter and about 20 to 150 mm in height.

The plug of the container of the present invention is not particularly limited as long as it can close the opening of the container and is endotoxin-free. The plug is preferably made of rubber, plastic, or the like. Examples of the rubber include butyl rubber, chlorinated butyl rubber, and thermoplastic elastomer. As the plastic, for example, any of a thermoplastic resin and a thermosetting resin can be used. As the thermoplastic resin, for example, polyethylene, polypropylene, polystyrene,
Polymethyl methacrylate, polyvinyl chloride, polyethylene terephthalate, styrene-acrylonitrile copolymer, styrene-maleic anhydride copolymer, styrene-
An acrylic acid copolymer, a styrene-methyl methacrylate copolymer, an ethylene-acrylic acid ester copolymer and the like can be mentioned. Examples of the thermosetting resin include an unsaturated polyester resin, an epoxy resin, and an epoxy-acrylate resin.

For the production of the container of the present invention, a molding method of a high molecular compound is used, for example, extrusion molding, injection molding, blow molding, injection blow molding and the like are used.

When polyethylene terephthalate is used as the material of the container of the present invention, extrusion molding is performed using polyethylene terephthalate heated to 280 to 330 ° C.
It can be performed by any of injection molding, blow molding, and injection blow molding.

Using the endotoxin container of the present invention,
An example of producing an endotoxin standard is described below.
A commercially available endotoxin drug substance (for example, E. coli 01)
1, E. E. coli 055) is dissolved in endotoxin-free water, diluted to a predetermined concentration with endotoxin-free water, placed in the container of the present invention, and lyophilized to produce an endotoxin standard product.

[0025] If necessary, an endotoxin stabilizer may be contained. The amount of the endotoxin stabilizer used is preferably such that the reaction between the endotoxin and the blood cell component solution of horseshoe crab is not inhibited or promoted.

The stabilizers for the endotoxin include:
For example, proteins such as albumin, immunoglobulin, and lysozyme, amino alcohols, polyhydric alcohols, nonionic surfactants, polysucrose, chelating agents, and the like may be used, and one or more of these may be used in combination. .

As the amino alcohol, for example,
Examples include triethanolamine, diethanolamine, and 2-aminoethanol. Examples of the polyhydric alcohol include polyvinyl alcohol (molecular weight: 500 to 3).
500), mannitol, xylitol, glycerol and the like. Examples of the nonionic surfactant include polyoxyethylene ethers (such as a bridish surfactant), polyoxyethylene sorbitan alkyl esters (such as a Tween surfactant), and polyoxyethylene pt-octyl. Examples include phenyl ethers (such as triton-based surfactants) and alkyl glucosides (such as n-octylglucopyranoside and n-dodecylglucopyranoside). Examples of the polysucrose include Ficoll 400 (molecular weight 400,000), Ficoll 7
0 (molecular weight 70,000). Examples of the chelating agent include EDTA (ethylenediaminetetraacetic acid), G
EDTA (bis (2-aminoethyl) ethylene glycol tetraacetic acid; glycol ether diamino tetraacetic acid), BA
PTA (aminophenylethylene glycol tetraacetic acid),
Citric acid, sodium citrate, NTA (nitrilo triacetic acid) and the like.

[0028]

Now, the present invention will be described in further detail with reference to Examples and Comparative Examples of the present invention.

Example 1 A polyethylene terephthalate resin (manufactured by Histo Chemical Co., Ltd., 9921) heated to 280 ° C. was injection molded to form a bottomed tubular container of the present invention (diameter 10 mm × height 70 mm).
Was manufactured.

Next, 3 ml of endotoxin-free water (manufactured by Otsuka Pharmaceutical Co., Ltd., water for injection) was placed in the above container,
For 1 hour under stirring. The endotoxin content in the obtained extract was measured with Endospecy (trade name, manufactured by Seikagaku Corporation) which is an endotoxin measuring reagent. The results are shown in Table 1. The endotoxin content is the endotoxin content to be accommodated [180 EU
(International endotoxin unit) / ml of endotoxin will be accommodated in 3 ml].

COMPARATIVE EXAMPLE 1 A glass vial (Iuchi Seieido, Steri Vial, capacity 5 ml, body diameter 23 mm × height 42 mm) was placed at 250 ° C. for 2 hours.
The mixture was sterilized by dry heat for hours to obtain a container for endotoxin.

Using this container, endotoxin was extracted in the same manner as in Example 1, and the endotoxin content was measured. The results are shown in Table 1. The endotoxin content is the endotoxin content to be accommodated [180 EU
(International endotoxin unit) / ml of endotoxin will be accommodated in 3 ml].

Comparative Example 2 A polyethylene container (manufactured by Iwaki Glass Co., Ltd., snap vial, capacity: 5 ml, diameter: 18 mm × height: 40 mm) was treated with endotoxin-free water (manufactured by Otsuka Pharmaceutical Co., water for injection) for 10 minutes.
It was washed twice and dried to obtain a container for endotoxin.

Using this container, endotoxin was extracted in the same manner as in Example 1, and the endotoxin content was measured. The results are shown in Table 1. The endotoxin content is the endotoxin content to be accommodated [180 EU
(International endotoxin unit) / ml of endotoxin will be accommodated in 3 ml].

[0035]

[Table 1]

Performance Test Next, E.I. 5 mg of E. coli 055 (manufactured by LBL) was diluted with endotoxin-free water (manufactured by Otsuka Pharmaceutical Co., Ltd., water for injection) to prepare a 180 EU / ml endotoxin aqueous solution.

The container used in Example 1 (hereinafter referred to as Test Example 1); manufactured in the same manner as in Example 1, and the endotoxin content was measured in the same manner, and the value was 5/100 of the endotoxin content to be accommodated. In the container that was
Container containing 20 μl of W / V% mannitol (Nihon Pharmaceutical) (Test Example 2); container used in Comparative Example 1 (Comparative Test Example 1); and container used in Comparative Example 2 (Comparative Test Example) 2), 3 ml of the above 180 EU / ml endotoxin aqueous solution was stored and stored at 4 ° C. Immediately after, 24 hours, and 48 hours after dispensing the endotoxin aqueous solution thus stored, the endotoxin activity was measured using Endospecy (trade name, manufactured by Seikagaku Corporation), which is an endotoxin measuring reagent, and the results are shown in Table 2. It was shown to.

[0038]

[Table 2]

From Table 2, it is apparent that the endotoxin activity of the glass vial and the container made of polyethylene rapidly decreases upon storage, and thus is not suitable as a container for storing an endotoxin standard product. However, in the container of the present invention, the endotoxin activity immediately after dispensing could be stably maintained.

[0040]

The structure of the container for endotoxin of the present invention is as described above, and since it is a container manufactured from a high molecular compound, there is little possibility of cracking. Also, when used as a container for endotoxin standard products, even if endotoxin-free water is added to the contained dried endotoxin and redissolved, the adsorption of endotoxin to the container is low, and metal ions such as glass vials are used. Does not affect the reaction between the horseshoe crab blood cell component and endotoxin used for endotoxin measurement, and contains and extracts an amount of endotoxin-free water equal to the amount of liquid to be stored inside during use. Since the endotoxin content in the extract at the time of carrying out the step is 5/100 or less of the contained endotoxin, it is possible to obtain an endotoxin aqueous solution having a predetermined concentration and to stabilize the concentration (activity) over time. An endotoxin aqueous solution excellent in quality can be obtained. Further, when an endotoxin stabilizer is contained, the stability over time of the aqueous endotoxin solution can be further improved.

Claims (2)

[Claims] 1. An extract containing endotoxin-free water in an amount equal to the amount of endotoxin-free water to be contained therein during use, and having an endotoxin content in the extract of 5/100 or less of the contained endotoxin. An endotoxin container made of a polymer compound having an ester bond. 2. The container for endotoxin according to claim 1, wherein the polymer compound having an ester bond is polyethylene terephthalate.