JP2008206915A - Sterilization method for compound and manufacturing method of medical material using it - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a sterilization method for a compound being labile in relation to radiation, which suppresses functional decline due to the degeneration or the like of the compound by radiation sterilization, does not need processing such as freezing because of few generation of deleterious by-products, and causes little degeneration of the compound by drying. <P>SOLUTION: The radiation is emitted in the state of dissolving and/or dispersing the compound labile in relation to the radiation in a solution containing a buffer solution whose pH is ≥3 and <10. <P>COPYRIGHT: (C)2008,JPO&INPIT

Description

  The present invention relates to a compound sterilization technique suitably used in fields such as medicine, medicine, proteome analysis and food production.

  When pharmaceuticals and medical devices are used in the medical setting, they must be sterilized. That is, it is necessary to finally sterilize when producing pharmaceuticals and medical instruments. In the Japanese Pharmacopoeia, a heating method, a gas method, and an irradiation method are shown as final sterilization methods for pharmaceuticals and medical devices.

  Irradiation methods are broadly classified into radiation methods and high-frequency methods. Among them, the radiation method is a sterilization method which has been widely used in recent years because it hardly causes modification of an object to be sterilized by heat and causes less problem of toxicity due to residual gas. High-energy radiation is used for irradiation, and γ-ray is used as standard. When γ-ray energy is absorbed by a substance, the molecule of the substance is ionized or excited. Then, a dissociation reaction occurs, and as a result, highly reactive radicals are generated and react one after another with ordinary molecules. When this series of reactions occurs with the most important nucleic acid to maintain the life of a microorganism, the nucleic acid molecule is cleaved and directly kills the microorganism.

  However, radicals generated by γ-ray irradiation may react not only with microorganisms but also with molecules constituting pharmaceuticals and medical materials, thereby deteriorating their functions and physical properties. In recent years, the surface of substrates has been chemically or biochemically modified to give specific adsorption, reactivity, or biocompatibility. When chemical or biochemical modification is performed, the surface characteristics obtained by these methods have a problem that their functions are lowered by γ-ray irradiation.

  With respect to such problems, a method of radiation sterilization with a base material impregnated with an antioxidant such as sodium pyrosulfite or ascorbic acid (see Patent Document 1), or a method of radiation sterilization in a frozen state (Patent Document 2). And a method of radiation sterilization in a state in which the amount of residual solvent is reduced (see Patent Document 3). In recent years, a method of radiation sterilization in a state where the organic solvent content is increased and the moisture content is decreased (see Patent Document 4) has also been proposed.

  In the method of impregnating the base material with the above-mentioned antioxidant, since the antioxidant is a compound unstable to radiation, there is a problem that it becomes a harmful substance by being modified by irradiation. For example, when sodium pyrosulfite is used, harmful substances such as sulfurous acid and sulfur dioxide are generated. Therefore, it is not preferable to use it for final sterilization of pharmaceuticals and medical devices. In addition to the cost of raw materials for antioxidants, it is also disadvantageous in terms of cost because the number of processes for adding antioxidants increases and the associated equipment is required.

  In addition, the method of irradiating with radiation in a frozen state has a big problem that the physiological activity is lost when a compound having physiological activity is frozen. Further, there is a problem of cost increase such as equipment necessary for freezing, electric power necessary for the freezing, and transportation in a frozen state.

  In addition, the method of radiation sterilization in a state where the residual solvent is lowered is that the moisture content is 2% or less, and it cannot be used for a compound that needs to be kept in a wet state. For example, hemodialysis membranes and the like depend on the microporous structure of the material, and there is a problem that the originally planned performance cannot be obtained because these structures change due to drying.

  Further, in the method of radiation sterilization in a state where the organic solvent content is increased and the moisture content is reduced, the moisture content is reduced to 20% by weight or less, so when this method is used for the production of medical materials, etc. There is no description or suggestion that the organic solvent residue after washing affects biocompatibility or the performance of the material deteriorates, for example, there is no consideration about the decrease in graft efficiency when used for radiation grafting. For this reason, it may not be suitable for practical use.

Furthermore, compounds having physiological activity are in a solution state because there is a concern that the activity may be greatly impaired due to fluctuations in the pH of the solution, and the performance of coexisting materials may be reduced due to fluctuations in the pH of the solution. Sometimes it is necessary to suppress pH fluctuations.
Japanese Patent No. 3432240 U.S. Pat. No. 4,620,908 Special table 2003-527210 gazette JP 2006-289071 A

  The object of the present invention is to suppress functional degradation due to denaturation of the compound by radiation sterilization in a compound unstable to radiation, and there is little generation of harmful by-products, and no treatment such as freezing is required. It is another object of the present invention to provide a method for sterilizing a compound with less modification of the compound by drying and a method for producing a medical material using the method.

In order to achieve the above object, the present invention has the following configuration.
1. A method for sterilizing a compound, comprising irradiating a compound unstable to radiation with radiation in a state of being dissolved and / or dispersed in a solution containing a buffer solution having a pH of 3 or more and less than 10.
2. 2. The method for sterilizing a compound according to 1 above, wherein the solution containing the buffer contains an organic solvent.
3. 3. The method for sterilizing a compound according to 1 or 2 above, wherein the pH of the buffer solution is 4 or more and 9 or less.
4). 4. The method for sterilizing a compound according to any one of 1 to 3, wherein the buffer solution has a pH of 5 or more and 8 or less.
5. 5. The method for sterilizing a compound according to any one of 1 to 4 above, wherein the organic solvent contains a hydroxyl group, and at least one is a secondary or tertiary hydroxyl group.
6). 6. The method for sterilizing a compound according to any one of 1 to 5 above, wherein the radiation labile compound comprises an amino acid as a constituent element.
7). 7. The method for sterilizing a compound according to any one of 1 to 6 above, wherein the compound unstable to radiation has blood anticoagulant activity.
8). 8. The method for sterilizing a compound according to any one of 1 to 7 above, wherein the compound having blood anticoagulant activity comprises a portion having blood anticoagulant activity and a polymer chain portion.
9. The polymer chain portion contains at least one selected from polyethylene glycol residues, polyvinyl pyrrolidone residues, polypropylene glycol residues, polyvinyl alcohol residues, and residues of any copolymer thereof, 9. A method for sterilizing the compound according to 8.
10. 10. The method for sterilizing a compound according to any one of 7 to 9, wherein the compound unstable to radiation has antithrombin activity.
11. A method for producing a medical material, wherein the compound unstable to radiation is immobilized on a substrate using the method for sterilizing a compound according to any one of 1 to 10 above.
12 A method for producing a medical device, wherein the medical material according to 11 is incorporated in a container.
13. 13. The method for producing a medical device as described in 12 above, wherein the medical device is an artificial kidney.

  According to the present invention, when a compound unstable to radiation is sterilized by radiation, the pH of the solution can be suppressed by dissolving and / or dispersing the compound in a buffer solution. It is possible to prevent sterilization, prevent generation of harmful by-products, do not require a treatment such as freezing, and reduce the compound denaturation by drying. Further, when a medical material is produced by immobilizing the compound on a substrate by, for example, radiation grafting, it is possible to immobilize the compound while maintaining the activity of the compound by suppressing the change in pH of the solution.

  The radiation used in the present invention refers to high-energy particle beams and electromagnetic waves, and examples include α rays, β rays, γ rays, X rays, ultraviolet rays, electron beams, and neutron rays. Of these, γ-rays, electron beams and X-rays are suitable for sterilization because they can kill microorganisms efficiently. The radiation dose is preferably 5 kGy or more, more preferably 10 kGy or more, and further preferably 15 kGy or more. However, irradiation with excessive radiation not only denatures the compound, but also increases the time required for irradiation and decreases productivity, so 5000 kGy or less is preferable, 1000 kGy or less is more preferable, and 100 kGy or less is more preferable.

  In the present invention, a compound that is unstable to radiation means that when irradiated with radiation, at least a part of the molecular structure in the compound is changed, and a physical function and / or a chemical function and / or an organism It refers to a compound that changes its functional function. A compound unstable to radiation may be sterilized alone or together with a substrate such as a medical material. It is also possible to modify the substrate by immobilizing the compound unstable to the radiation on the substrate by irradiation. Thus, in the method for producing a medical material according to the present invention, since the compound can be immobilized and the medical material can be sterilized at the same time, the number of production steps can be reduced. In this case, it is also possible to chemically graft a compound unstable to radiation onto the substrate surface.

  The base material used in the present invention refers to a material to which a compound unstable to radiation is to be imparted, and a polymer compound material is preferably used. Examples of the polymer compound constituting the polymer compound material include, for example, polymethyl methacrylate, polypropylene, polyethylene, polyvinyl chloride, polyvinylidene chloride, polyvinylidene fluoride, polyvinyl acetate, polycarbonate, cellulose, cellulose acetate, and cellulose triacetate. , Polyacrylonitrile, polysulfone, polyethersulfone, polystyrene, polyurethane and the like, but are not limited thereto.

  In the present invention, a medical base material can be used as the base material. Examples of medical base materials include artificial blood vessels, catheters, blood bags, contact lenses, intraocular lenses, and surgical aids. Biological component separation modules and blood purification modules, preferably medical devices such as artificial kidneys In addition, a separation membrane that is used by being incorporated in a device is also included. Here, the separation membrane for separating biological components refers to a membrane for separating a biological material by filtration or dialysis and collecting a part thereof.

  The buffer used in the present invention is a solution in which the pH does not change even when a small amount of acid or base is added or the concentration slightly changes. For example, phosphate buffer, trishydroxy Examples include methylaminomethane buffer, bis (2-hydroxyethyl) iminotris (hydroxymethyl) methane buffer, acetate buffer, citrate buffer, and borate buffer. Among these, phosphate buffer, trishydroxymethylaminomethane buffer and bis (2-hydroxyethyl) iminotris (hydroxymethyl) methane buffer have a buffering action in neutral and / or acidic regions, and have physiological activity. This is preferable because it is often added as a solvent for a compound having the above. However, under strongly acidic conditions such as less than pH 3 or strongly basic conditions such as pH 10 or more, some molecular structures in the compound having physiological activity change, resulting in physical functions and / or chemical properties. Since it is considered that the function and / or biological function is changed or the performance of the material itself is lowered when, for example, a graft-immobilized material is created, the buffering range is pH 3 or more, preferably 5 or more. Moreover, as an upper limit, it is less than pH10 and 8 or less is preferable. The glass electrode method is used for measuring the pH, but it is not limited to this as long as it can be measured with the same accuracy.

  In addition, the solution containing the buffer solution used in the present invention means an aqueous solution of the above buffer solution, other solvent, or a solution containing the solution. It does not mean anything that fluctuates beyond the pH range, and preferably the pH of the buffer solution does not fluctuate.

  In the present invention, the following organic solvent is preferably contained in the solution containing the buffer solution, and examples of the organic solvent that is suitably used include a solvent containing a carbon atom and / or a silicon atom in the molecule. Hydroxyl groups are highly effective in stabilizing radicals generated by radiation irradiation, are nonionic functional groups, have little interaction with compounds with strong surface charges, have little redox power, and have little modification of compounds. Therefore, an organic solvent having a hydroxyl group in the molecule, for example, methanol, ethanol, 1-propanol, 1-butanol, 1,4-butanediol and the like are preferably used. In particular, secondary and tertiary hydroxyl groups are more effective in stabilizing radicals, so organic solvents having secondary and / or tertiary hydroxyl groups such as glycerin, propylene glycol, isopropanol, 2-butanol, 2, 3-butanediol, 1,3-butanediol and the like are preferably used. However, an organic solvent having only a primary hydroxyl group such as ethylene glycol or polyethylene glycol is not included in the organic solvent referred to in the present invention because the effect of stabilizing radicals is low. In addition, when the method for sterilizing a compound of the present invention is used for the production of a medical material or a medical device in which the compound is incorporated, it is necessary to consider its safety. Therefore, a non-aqueous solvent having low toxicity is preferably used. It is done.

  Moreover, the upper limit of the moisture content in the solution containing the buffer solution according to the present invention is 90 vol%, and if it exceeds 90 vol%, the effect of radical stabilization of the organic solvent cannot be sufficiently obtained. In the range of 90 vol% or less, it is preferable that the moisture content is larger. On the other hand, when the moisture content is low, for example, when used for medical devices, there is a concern that the residual organic solvent after washing may affect the biocompatibility, and for example, when used for radiation grafting, the grafting efficiency decreases. Can be considered. Therefore, the water content is preferably 25 vol% or more, more preferably 50 vol% or more of the total solvent amount.

The moisture content in the present invention is defined by the following equation.
(Volume of water contained in a solution containing a radiation labile compound and a buffer solution in which the compound is dissolved and / or dispersed) / (Radiation labile compound and the compound dissolved and / or dispersed) Solution volume containing buffer solution) x 100 (%)
In the present invention, dissolution of a compound unstable to radiation means that the compound dissolves in a solution containing a buffer solution to form a uniform mixture, that is, a solution. In addition, the dispersion of a compound means that the compound is scattered in a solution containing a buffer solution. The concentration of the compound is not particularly limited, but depending on the compound, if the concentration is too high, the cross-linking reaction proceeds between the compounds and the original physical properties of the compound may be lost due to gelation or the like. It is preferably 50% by weight or less, more preferably 30% by weight or less, and still more preferably 20% by weight or less in an aqueous solution of a solution containing a buffer solution.

  The radiation-labile compound in the present invention preferably contains an amino acid as a constituent element. A compound containing an amino acid as a constituent element has high biocompatibility, and therefore has high safety when used for medical purposes. Furthermore, compounds containing amino acids as a constituent element have physiological activity and are effective even when used on their own, but are also effective when immobilized on the surface of medical materials, and their uses are limited. is not. A compound having an amino acid as a constituent element is a compound containing a naturally occurring amino acid, such as a compound composed only of an amino acid such as a protein or a peptide, a glycoprotein, an amino acid complex, an aminoacyl adenylic acid, or the like. Although what is comprised from a thing other than these amino acids and amino acids is mentioned, It is not limited to these.

  The compound having blood anticoagulant activity refers to a compound in which the prothrombin time is prolonged by 30% or more when the compound is added to blood so as to have a concentration of 10 μg / ml as compared with unadded blood. The prothrombin time can be measured by the method described in the following document.

Masamitsu Kanai et al., “Proposal for Clinical Examination Revised 30th Edition”, Kanehara Publishing, 1993, pp. 416-418
That is, mix 1 volume of 3.2 vol% sodium citrate and 9 volumes of blood, and take 0.1 ml of citrated plasma collected in a small test tube (inner diameter 8 mm, length 7.5 cm) in a 37 ° C. constant temperature water bath. Heat for about 3 minutes. At the same time as adding 0.2 ml of tissue thromboplastin / calcium reagent kept at the same temperature, start the second clock, shake gently, and measure the time until fibrin precipitates while tilting.

In addition, the radiation labile compound in the present invention is preferably a compound having blood anticoagulant activity. In general, when blood comes into contact with a material, a coagulation reaction proceeds. Therefore, when a material that comes into contact with blood, particularly a material that comes into contact with blood for a long time, is used for medical purposes, it is necessary to suppress blood coagulation. Since the present invention considers the production of a material to be used in contact with blood, the compound is preferably a compound having blood anticoagulant activity. Examples of the compound having anticoagulant activity, heparin, nafamostat mesylate, sodium citrate, sodium oxalate, alpha ₁ antitrypsin, alpha ₂ macroglobulin, C1 inhibitor, but thrombomodulin and protein C, etc., in these It is not limited. Examples of the compound having blood anticoagulant activity include a compound having a portion having blood anticoagulant activity and a polymer chain portion. Such a polymer chain portion preferably includes a polyethylene glycol residue, a polyvinylpyrrolidone residue, Examples thereof include a polymer chain selected from the group consisting of a polypropylene glycol residue, a polyvinyl alcohol residue, and a copolymer residue containing them.

  In addition, among compounds having blood anticoagulant activity, there are compounds that exhibit strong blood anticoagulant activity by suppressing thrombin activity. Accordingly, the compound is more preferably a compound having antithrombin activity. The compound having antithrombin activity in the present invention is a compound that suppresses the activity of thrombin, which is a coagulation-related substance in blood, and plasma of HaemoSys "ECA-Tkit" added with the compound at a concentration of 10 µg / ml. Refers to a compound whose measured value increases by 50% or more compared to that of plasma without compound. The measuring device is not limited to this as long as it can measure with the same accuracy. As examples of compounds having antithrombin activity, 4-methoxy-benzenesulfonyl-Asn (PEG2000-Ome) -Pro-4-amidinobenzylamide (hereinafter abbreviated as Compound A) represented by the following chemical formula, antithrombin III , And hirudin.

(In the formula, PEG represents a polyethylene glycol residue, and Me represents a methyl group.)
EXAMPLES Hereinafter, although an Example is given and this invention is demonstrated, this invention is not limited by these examples.

(Measurement of antithrombin activity value)
Since compound A was used as a radiation labile compound, the residual ratio after irradiation of the antithrombin activity value was used as an index indicating the activity of antithrombin. For measurement of the antithrombin activity value, an ECA-T kit manufactured by HaemoSys was used as a reagent, and COATRON M1 (code 80 800 000) manufactured by TECO Medical Instruments Production was used as an apparatus. First, 20 μl of the solution to be measured was added to 80 μl of human plasma (Cosmo Bio Human Plasma 12271210, lot.16878) and stirred. This solution is used as a sample solution. The sample solution is cooled on an ice bath until immediately before the measurement. 100 μl of ECA prothrombin buffer, 30 μl of sample solution and 25 μl of ECA-T substrate were mixed, incubated at a temperature of 37 ° C. for 60 seconds, and set in the apparatus. To this, 50 μl of ECA ecarin reagent was added for measurement. Measurement was performed on a sample prepared using ultrapure water as a blank measurement target solution. The residual rate was calculated by the following formula (1).

(Preparation of buffer)
The pH 5 buffer solution is obtained by dissolving bis (2-hydroxyethyl) iminotris (hydroxymethyl) methane (manufactured by Dojin Chemical) and sodium chloride (manufactured by Sigma Aldrich) in ultrapure water, and dropping 6N hydrochloric acid (manufactured by Sigma Aldrich) dropwise. The solution was adjusted to pH 5. The glass electrode method was used for measuring pH, and the apparatus was measured using a HORIBA pH meter Castany LAB F-22. As a result, the pH was 5.0. In addition, an acetate buffer was also prepared as another pH 5 buffer. Acetic acid and sodium acetate were dissolved in ultrapure water and prepared. It was 5.0 as a result of measuring pH by the same method as the above.

  The pH 7.8 and 10 buffer solutions were prepared by dissolving trishydroxymethylaminomethane (manufactured by Katayama Chemical Co., Ltd.) and sodium chloride (manufactured by Sigma-Aldrich) in ultrapure water, and dropping 6N hydrochloric acid (manufactured by Sigma-Aldrich) dropwise. The pH was adjusted. The pH was measured using the same method as the pH 5 buffer. As a result, the pH was 7.8 and 10.0, respectively.

(Example 1)
Compound A was dissolved in ultrapure water to prepare a Compound A aqueous solution having a concentration of 5000 ppm by weight. The aqueous solution of Compound A, ultrapure water, isopropanol (manufactured by Sigma-Aldrich), and a buffer solution having a double concentration of pH 5 were mixed in the volumes shown in Table 1. The compound A buffer was irradiated with γ rays. At this time, the absorbed dose of γ rays was 25 kGy. The antithrombin activity values of the compound A buffer before irradiation and the compound A buffer after irradiation were measured, and the residual ratio of the antithrombin activity value was calculated from the following equation (1). As a result, when the isopropanol fraction was 0.1, 1, 10, and 50%, the antithrombin residual ratios were 15.3, 19.4, 37.8, and 76.8%, respectively.
A = 100 × (BD) / (CD) Formula (1)
Symbol in formula (1) A: Antithrombin activity remaining rate (%)
B: Sample measured value after irradiation (sec)
C: Sample measurement value before irradiation (sec)
D: Blank measurement value (sec).

(Example 2)
Compound A was dissolved in ultrapure water to prepare a Compound A aqueous solution having a concentration of 5000 ppm by weight. The aqueous solution of Compound A, ultrapure water, isopropanol (manufactured by Sigma Aldrich), and a buffer solution of pH 7.8 having a double concentration were mixed in the volumes shown in Table 1. The compound A buffer was irradiated with γ rays. At this time, the absorbed dose of γ rays was 25 kGy. The antithrombin activity values of the compound A buffer before irradiation and the compound A buffer after irradiation were measured, and the residual ratio of the antithrombin activity value was calculated from the above formula (1). As a result, when the isopropanol fraction was 0.1, 1, 10, and 50%, the antithrombin residual rates were 12.9, 13.6, 17.6, and 66.4%, respectively.

(Comparative Example 1)
Compound A was dissolved in ultrapure water to prepare a Compound A aqueous solution having a concentration of 5000 ppm by weight. The aqueous solution of Compound A, ultrapure water, isopropanol (manufactured by Sigma Aldrich), and a buffer solution having a double concentration of pH 10 were mixed in the volumes shown in Table 1. The compound A buffer was irradiated with γ rays. At this time, the absorbed dose of γ rays was 25 kGy. The antithrombin activity values of the compound A buffer before irradiation and the compound A buffer after irradiation were measured, and the residual ratio of the antithrombin activity value was calculated from the above formula (1). As a result, when the isopropanol fraction was 0.1, 1, 10, 50%, the antithrombin residual ratios were 2.1, 2.4, 1.5, and 4.8%, respectively.

  The present invention is a method of radiation sterilizing a compound having physiological activity unstable to radiation while maintaining its activity, and can be sterilized without using an antioxidant and is a harmful antioxidant decomposition product. It can be expected to reduce the risk. Moreover, since the pH fluctuation can be suppressed, it is possible to suppress a decrease in physiological activity of a compound having physiological activity unstable to radiation. Thus, according to the present invention, it is possible to sterilize physiologically active substances that are unstable to various radiations while suppressing denaturation.

Claims (13)

  A method for sterilizing a compound, comprising irradiating a compound unstable to radiation with radiation in a state of being dissolved and / or dispersed in a solution containing a buffer solution having a pH of 3 or more and less than 10.   2. The method for sterilizing a compound according to claim 1, wherein the solution containing the buffer contains an organic solvent.   The method for sterilizing a compound according to claim 1 or 2, wherein the pH of the buffer solution is 4 or more and 9 or less.   The method for sterilizing a compound according to any one of claims 1 to 3, wherein the pH of the buffer solution is 5 or more and 8 or less.   The method for sterilizing a compound according to any one of claims 1 to 4, wherein the organic solvent contains a hydroxyl group, and at least one is a secondary or tertiary hydroxyl group.   The method for sterilizing a compound according to any one of claims 1 to 5, wherein the compound unstable to radiation comprises an amino acid as a constituent element.   The method for sterilizing a compound according to any one of claims 1 to 6, wherein the compound unstable to radiation has blood anticoagulant activity.   The method for sterilizing a compound according to any one of claims 1 to 7, wherein the compound having blood anticoagulant activity comprises a portion having blood anticoagulant activity and a polymer chain portion.   The polymer chain portion contains at least one selected from a polyethylene glycol residue, a polyvinyl pyrrolidone residue, a polypropylene glycol residue, a polyvinyl alcohol residue, and a residue of any copolymer thereof. Item 9. A method for sterilizing a compound according to Item 8.   10. The method for sterilizing a compound according to any one of claims 7 to 9, wherein the compound unstable to radiation has antithrombin activity.   A method for producing a medical material, wherein the compound unstable to radiation is immobilized on a substrate using the method for sterilizing a compound according to any one of claims 1 to 10.   A method for producing a medical device, comprising incorporating the medical material according to claim 11 in a container.   The method for producing a medical device according to claim 12, wherein the medical device is an artificial kidney.