JP2014169250A - Liquid preparation sterilization method - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a liquid preparation sterilization method that can satisfy the sterility assurance level 10, can sufficiently suppress a decrease in the concentration of active ingredients, and is applicable to a wide range of liquid preparations.SOLUTION: A liquid preparation sterilization method comprises the step of irradiating a liquid preparation (excluding a protein preparation and a synthetic polymer preparation) in a frozen state with radiation.

Description

  The present invention relates to a method for sterilizing a liquid preparation.

  Examples of pharmaceutical sterilization methods include high-pressure steam sterilization, filtration sterilization, aseptic operation, and radiation sterilization.

However, high-pressure steam sterilization may cause a decrease in active ingredient concentration due to deterioration of components, alteration, odor, and coloring, and cannot be widely used. In addition, the sterility assurance level (SAL) of filtration sterilization and aseptic operation methods is 10 ⁻³ , and 10 ⁻⁶ (1 product per 1 million products) is required for original sterilization. The level that may remain is not reached. Moreover, when a liquid formulation is irradiated with radiation, problems such as a decrease in active ingredient concentration due to decomposition of the active ingredient and coloring may occur. For this reason, there are few cases where the liquid preparation is irradiated with radiation for the purpose of sterilization.

  For example, Patent Document 1 describes a povidone iodine preparation that is sterilized by irradiating a povidone iodine solution prepared by mixing povidone iodine, water, and iodide with radiation of 10 kGy to 50 kGy. According to Patent Document 1, attenuation of effective iodine due to radiation irradiation is suitably suppressed by mixing predetermined iodide with povidone iodine.

JP 2011-26248 A

  However, in the method of Patent Document 1, suppression of the decrease in the active ingredient concentration may be insufficient. Moreover, the method of patent document 1 may not be applied to liquid formulations other than povidone iodine.

Accordingly, the present invention provides a method for sterilizing a liquid preparation that can satisfy a sterility assurance level of ^10-6 , can sufficiently suppress a decrease in the concentration of an active ingredient, and can be applied to a wide range of liquid preparations. With the goal.

  The present invention provides a method for sterilizing a liquid preparation, comprising a step of irradiating a frozen liquid preparation (excluding protein preparation and synthetic polymer preparation) with radiation.

According to the sterilization method of the present invention, the sterility assurance level 10 ⁻⁶ can be satisfied. Moreover, the fall of the active ingredient density | concentration of a liquid formulation can fully be suppressed. Furthermore, it can be applied to a wide range of liquid preparations.

  The radiation is preferably radiation exceeding 8 kGy, and preferably radiation exceeding 12 kGy.

  The sterilization effect can be enhanced as the dose of radiation to be irradiated increases. Generally, when the radiation dose is increased, the decrease in the active ingredient concentration becomes larger. On the other hand, according to this invention, even if it irradiates the radiation of the dose exceeding 12 kGy, the fall of an active ingredient density | concentration can fully be suppressed.

FIG. 1 is a diagram illustrating a reaction when a substance is irradiated with radiation. FIG. 2 is a graph showing the relationship between temperature and the concentration of chlorhexidine gluconate during irradiation of a chlorhexidine gluconate formulation. FIGS. 3a and 3b are diagrams for explaining the influence when radiation is applied to ice crystals. FIG. 4 is a diagram showing a behavior when radiation is irradiated to ice. FIG. 5 is a chromatograph showing the HPLC results of chlorhexidine gluconate. FIG. 6 is a graph showing the measurement results of ultraviolet absorption of chlorhexidine gluconate.

(Action of radiation)
FIG. 1 is a diagram illustrating a reaction when a substance is irradiated with radiation. When radiation enters a substance, a random and local reaction occurs in an orbit (spar) due to energy emitted from accelerated particles or electromagnetic waves.

For example, when an aqueous solution having a low concentration is irradiated with an electron beam at room temperature, various active species (radicals and stable molecules) are generated by the most quantitative decomposition of water. Specifically, it is known that six kinds of active species are finally produced by the reaction shown in the following formula (I). Most of these radicals complete the intra-spar reaction in about 10 ⁻⁹ seconds, and the diffusion ends in 10 ⁻⁸ seconds. For example, among the generated active species, the highly reactive OH radical has a short lifetime of 70 nanoseconds. For this reason, even if the movement speed of the OH radical is 10 ⁹ m / sec, the moving distance is as short as about 20 nm. Therefore, OH radicals react with those present around radicals inside the spar. Thus, the radical reacts with the solute (RH) and the solvent that are close to each other within a short time after generation, and then deactivates by the reaction between the radicals shown in the following formulas (II) to (V) or the solute. .

  On the other hand, stable molecules such as hydrogen peroxide produced at the same time have a long life and spread into the system by diffusion and react. Specifically, it is considered that stable molecules diffused in the system due to the reaction represented by the following formula (VI) or the like act on the solute over time. As described above, in an aqueous solution, it is considered that decomposition of a solute by active species generated by radiation occurs, and a decrease in active ingredient concentration occurs.

H ₂ O → e _aq ⁻ + · H + · OH + H ₂ + H ₂ O ₂ + H ₃ O ⁺ (I)
e _aq ⁻ + H ₃ O ⁺ → H + H ₂ O (II)
・ OH + H ₂ → H ₂ O + · H (III)
e _aq ⁻ + H ₂ O ₂ → OH + OH ⁻ (IV)
RH + .OH → R + H ₂ O (V)
RH + H ₂ O ₂ → R + H ₂ O + OH ⁻ (VI)

  As shown in Table 1 below, it is known that OH radicals and OOH radicals are generated as a result of electron spin resonance (ESR) measurement when γ-rays are irradiated with ice at 77K.

  However, the generated radicals are restricted in movement because they exist in ice crystals. As shown in Experimental Example 12 to be described later, as a result of irradiating the ice frozen with purified water with radiation, it became clear that the ice was colored gray. This indicates that when the ice is irradiated with radiation, a color center is generated in the ice crystal and, as a result, electrons are collected in the ice crystal. As a result, it is considered that the amount of radicals generated is reduced and the action of accelerated electrons and radicals on the solute is alleviated. This is considered to have contributed to suppression of the fall of an active ingredient density | concentration.

(Liquid preparation)
Examples of liquid preparations include aqueous solutions, suspensions, gels and the like. More specifically, an injection, an infusion, an eye drop, a dialysis solvent, a liquid for external use and the like can be mentioned. Here, the active ingredient of the liquid preparation is preferably a low molecular compound having a molecular weight of about 10,000 or less because it has a high effect of suppressing a decrease in the concentration of the active ingredient due to irradiation. As shown in Experimental Example 13, which will be described later, a low molecular compound in a liquid preparation irradiated with radiation in a frozen state is energetically stabilized by hydration. Such behavior of low molecular weight compounds is considered to be different from proteins and synthetic polymers which are higher polymers. For this reason, it is thought that a low molecular weight compound has the effect of suppressing the fall of the active ingredient density | concentration by the radiation irradiation in a frozen state higher than protein and a synthetic polymer.

(Frozen state)
The frozen state is a state where the solvent (water) of the liquid preparation is frozen, and is a state of about 0 ° C. or less under atmospheric pressure. Although there is no lower limit to the preferred freezing temperature, about −80 ° C. may be set as the lower limit from the viewpoint that the lower the temperature, the more expensive the freezing. For freezing the liquid preparation, a general freezer may be used, or a cooling medium such as liquid nitrogen may be used. The liquid preparation to be frozen is preferably contained in a sealed container from the viewpoint of ensuring sterility.

  The material of the sealed container is not particularly limited as long as it has a shape retaining property, chemical resistance, and a barrier property capable of maintaining a sterilized state, and examples thereof include glass and plastic. Examples of the plastic include polystyrene, polyethylene, polypropylene, polyethylene terephthalate, polyamide, cyclic polyolefin, and polyvinyl chloride. The sealed container may have a multilayer structure made of these materials.

(Radiation irradiation)
As the radiation, electron beam, γ-ray, X-ray or the like can be used. As a radiation irradiation apparatus, what is used by the radiation irradiation of a normal pharmaceutical can be used. The dose of radiation to be irradiated is preferably more than 5 kGy, more preferably more than 8 kGy, and still more preferably more than 12 kGy. The higher the radiation dose, the higher the sterilization effect. However, the upper limit of the radiation dose is preferably about 100 kGy, for example, because the decrease in active ingredient concentration becomes larger at higher doses.

  Irradiate a frozen liquid formulation. For example, when the radiation irradiation apparatus does not have a cooling function, radiation may be irradiated by installing a frozen liquid preparation on a cooling medium such as dry ice. The liquid preparation after irradiation with radiation can be stored at normal temperature or low temperature. However, as shown in Experimental Example 11 to be described later, from the viewpoint of suppressing the reaction of radicals, the frozen liquid preparation is preferably melted at a low temperature of, for example, 10 ° C. or lower, and stored at a low temperature of, for example, 5-10 ° C. It is preferable to carry out with. Thereby, the fall of an active ingredient density | concentration can further be suppressed.

[Experimental Example 1]
(Radiation irradiation to 0.5 (w / v)% chlorhexidine gluconate preparation)
(Preparation of chlorhexidine gluconate formulation)
Purified water was added to 2.5 mL of a 20.0 (w / v)% chlorhexidine gluconate aqueous solution to make a total volume of 100 mL. Then, 0.5 mL of 1N sodium hydroxide aqueous solution was added for pH adjustment, and the 0.5 (w / v)% chlorhexidine gluconate formulation was prepared.

(Freezing process)
The prepared chlorhexidine gluconate preparation was left to stand in a freezer at −80 ° C. or −20 ° C. for 24 hours or more to freeze.

(Radiation irradiation)
A chlorhexidine gluconate preparation frozen at −80 ° C. or −20 ° C. was uniformly irradiated with an electron beam of 30 or 60 kGy under the following apparatus and conditions. Moreover, the electron beam was irradiated also to the 20 degreeC chlorhexidine gluconate formulation as a control | contrast.
Electron accelerator: Dynamitron electron accelerator Acceleration voltage: 5 MeV
Current: 20 mA

(Component analysis)
Each chlorhexidine gluconate preparation after irradiation was appropriately diluted, and the concentration of chlorhexidine gluconate was quantified by high performance liquid chromatography (HPLC) under the following conditions.
Column: 5 μm octadecyl silylated silica gel, inner diameter 4.6 mm, length 250 mm
Column temperature: 50 ° C
Mobile phase: methanol: 5% aqueous sodium lauryl sulfate solution: phosphoric acid = 800: 200: 1
Flow rate: 2.0 mL / min Detector: UV absorptiometer (measurement wavelength 260 nm, sensitivity 0.04 aufs)

  Table 2 shows the concentration (%) of chlorhexidine gluconate when the concentration of chlorhexidine gluconate in the chlorhexidine gluconate preparation not irradiated with radiation is 100%. The chlorhexidine gluconate formulation at 20 ° C. decreased in concentration of chlorhexidine gluconate as the dose of radiation applied increased. In contrast, the chlorhexidine gluconate preparation in a frozen state was found to have little decrease in the concentration of chlorhexidine gluconate even when the radiation dose to be irradiated was increased.

[Example 2]
(Radiation irradiation to 0.1 (w / v)% chlorhexidine gluconate preparation)
The relationship between the temperature of the aqueous solution during irradiation and the concentration of chlorhexidine gluconate was investigated. Specifically, the 0.1 (w / v)% chlorhexidine gluconate preparation was uniformly irradiated with an electron beam of 20 kGy at 25, 5, -5, -20 and -80 ° C in the same manner as in Experimental Example 1. . Subsequently, in the same manner as in Experimental Example 1, the concentration of chlorhexidine gluconate in each chlorhexidine gluconate preparation after irradiation was quantified.

  Table 3 shows the concentration (%) of chlorhexidine gluconate when the concentration of chlorhexidine gluconate in the chlorhexidine gluconate preparation not irradiated with radiation is 100%. FIG. 2 shows a graph of the results. In FIG. 2, the vertical axis indicates the concentration (%) of chlorhexidine gluconate when the concentration of chlorhexidine gluconate in the chlorhexidine gluconate preparation not irradiated with radiation is 100%. The horizontal axis indicates the temperature (° C.) of the chlorhexidine gluconate preparation irradiated with radiation.

  As a result, a difference was observed in the amount of decrease in the concentration of chlorhexidine gluconate when the chlorhexidine gluconate formulation irradiated with radiation was in a liquid state and in a frozen state. When the chlorhexidine gluconate preparation irradiated with radiation was in a frozen state, the suppression of the decrease in the concentration of chlorhexidine gluconate was remarkably observed.

[Experimental Example 3]
(Radiation irradiation to 10.0 (w / v)% ascorbic acid preparation)
(Preparation of ascorbic acid preparation)
10.0 g of ascorbic acid was dissolved in purified water to make a total volume of 100 mL, and a 10.0 (w / v)% ascorbic acid preparation was prepared.

(Radiation irradiation)
In the same manner as in Experimental Example 1, an ascorbic acid preparation frozen at −80 ° C. or −20 ° C. was uniformly irradiated with an electron beam of 0, 10, 20 or 30 kGy. As a control, the ascorbic acid preparation at 20 ° C. was also irradiated with an electron beam.

(Component analysis)
Each ascorbic acid preparation after irradiation was appropriately diluted, and the concentration of ascorbic acid was quantified by high performance liquid chromatography (HPLC) under the following conditions.
Column: 5 μm octadecyl silylated silica gel, inner diameter 4.6 mm, length 250 mm
Column temperature: 30 ° C
Mobile phase: purified water: phosphoric acid = 1000: 2
Flow rate: 1.0 mL / min Detector: UV absorptiometer (measurement wavelength 275 nm, sensitivity 0.04 aufs)

  Table 4 shows the concentration (%) of ascorbic acid when the concentration of ascorbic acid in the ascorbic acid preparation not irradiated with radiation is 100%. The ascorbic acid formulation at 20 ° C. decreased in the concentration of ascorbic acid as the dose of radiation applied increased. In addition, ascorbic acid concentration decreased and coloring occurred over time. The coloration of ascorbic acid is generally considered that ascorbic acid is oxidized and colored through dehydroascorbic acid.

  On the other hand, it has been clarified that the ascorbic acid preparation in the frozen state has little decrease in the concentration of ascorbic acid even when the dose of radiation to be irradiated is increased. In addition, the ascorbic acid preparation irradiated in the frozen state was not colored even after being stored at 5 ° C. for 1 month.

[Experimental Example 4]
(Radiation irradiation to 1.0 (w / v)% lidocaine formulation)
(Preparation of lidocaine formulation)
Lidocaine 1.0g was melt | dissolved in dilute hydrochloric acid 2mL. To this solution, purified water was added to make up a total volume of 100 mL. Subsequently, a 0.1N aqueous sodium hydroxide solution was added to adjust the pH to 5.0 to 7.0 to prepare a 1.0 (w / v)% lidocaine formulation.

(Radiation irradiation)
In the same manner as in Experimental Example 1, an ascorbic acid preparation frozen at −80 ° C. or −20 ° C. was uniformly irradiated with an electron beam of 0, 15 or 30 kGy. As a control, the ascorbic acid preparation at 20 ° C. was also irradiated with an electron beam.

(Component analysis)
Each lidocaine preparation after irradiation was appropriately diluted, and the concentration of lidocaine was quantified by high performance liquid chromatography (HPLC) under the following conditions.
Column: 5 μm octadecyl silylated silica gel, inner diameter 4.6 mm, length 250 mm
Column temperature: 30 ° C
Mobile phase: 0.02 mol / L phosphate buffer (pH 3.0): A solution of 2.88 g of sodium lauryl sulfate in 1000 mL of acetonitrile = 11: 9 Flow rate: 1.0 mL / min Detector: UV absorption spectrophotometry Meter (measurement wavelength 254nm, sensitivity 0.04aufs)

  Table 5 shows the concentration (%) of lidocaine when the concentration of lidocaine in the lidocaine preparation not irradiated with radiation is 100%. The lidocaine formulation at 20 ° C. decreased the concentration of lidocaine as the dose of radiation applied increased. On the other hand, it was revealed that the lidocaine preparation in the frozen state has little decrease in the concentration of lidocaine even after irradiation with 30 kGy.

[Experimental Example 5]
(Radiation irradiation to 1.0 (w / v)% dipotassium glycyrrhizinate preparation)
(Preparation of dipotassium glycyrrhizinate preparation)
1.0 g of dipotassium glycyrrhizinate was dissolved by heating in purified water to a total volume of 100 mL to prepare a 1.0 (w / v)% dipotassium glycyrrhizinate preparation.

(Radiation irradiation)
In the same manner as in Experimental Example 1, a dipotassium glycyrrhizinate preparation frozen at −80 ° C. or −20 ° C. was uniformly irradiated with an electron beam of 0, 15 or 30 kGy. In addition, as a control, a 20 ° C. dipotassium glycyrrhizinate preparation was also irradiated with an electron beam.

(Component analysis)
Each dipotassium glycyrrhizinate preparation after irradiation was appropriately diluted, and the concentration of dipotassium glycyrrhizinate was determined by high performance liquid chromatography (HPLC) under the following conditions.
Column: 5 μm octadecyl silylated silica gel, inner diameter 4.6 mm, length 250 mm
Column temperature: 40 ° C
Mobile phase: 0.02 mol / L phosphate buffer (pH 3.0): acetonitrile = 65: 35
Flow rate: 0.5 mL / min Detector: UV absorptiometer (measurement wavelength 250 nm, sensitivity 0.04 aufs)

  Table 6 shows the concentration (%) of dipotassium glycyrrhizinate when the concentration of dipotassium glycyrrhizinate in the dipotassium glycyrrhizinate preparation not irradiated with radiation is 100%. In the dipotassium glycyrrhizinate preparation at 20 ° C., the concentration of dipotassium glycyrrhizinate decreased as the dose of radiation applied increased. On the other hand, it was revealed that the dipotassium glycyrrhizinate preparation in a frozen state showed little decrease in the concentration of dipotassium glycyrrhizinate even after irradiation with 30 kGy.

[Experimental Example 6]
(Examination of degradation products by irradiation of 0.5 (w / v)% chlorhexidine gluconate preparation)
When an electron beam is irradiated to a chlorhexidine gluconate preparation, chlorhexidine gluconate is decomposed due to the direct action of the electron beam or the influence of active species. In general, chlorhexidine gluconate preparations are known to decompose by light or heat to produce p-chloroaniline over time. As decomposition products of chlorhexidine gluconate, 1- (4-chlorophenyl) guanidine and p-chlorophenylbiguanide are considered in addition to p-chloroanilinin. Therefore, an electron beam of 30 kGy was applied to 0.5 (w / v)% chlorhexidine gluconate preparation frozen at −20 ° C. and 0.5 (w / v)% chlorhexidine gluconate preparation at room temperature in the same manner as in Experimental Example 1. After the irradiation, decomposition products were detected mainly by p-chloroaniline by HPLC. In addition, degradation products were also detected for a 0.5 (w / v)% chlorhexidine gluconate preparation not irradiated with an electron beam.

  As a result, p-chloroaniline and p-chlorophenylbiguanide were not detected in the 0.5 (w / v)% chlorhexidine gluconate formulation that had not been irradiated. On the other hand, in the 0.5 (w / v)% chlorhexidine gluconate formulation irradiated at room temperature, the production amount of p-chloroaniline was about 2.4 ppm and the production amount of p-chlorophenyl biguanide was about 1.1 ppm. . In addition, p-chloroaniline and p-chlorophenylbiguanide were not detected in the 0.5 (w / v)% chlorhexidine gluconate preparation irradiated at −20 ° C. In addition, p-chloraniline is allowed to be present up to 12.5 ppm in a chlorohexidine gluconate preparation in terms of pharmaceutical raw material standards.

[Experimental Example 7]
(Confirmation of sterilization effect)
It is necessary to set the radiation sterilization dose of pharmaceuticals or the like in accordance with ISO11137-2: 2006. Here, it was examined by the VDmax ¹⁵ method that SAL (guarantee level of sterilization) = 10 ⁻⁶ (level at which one product per 1 million products may cause bacteria to remain) was secured.

According to the VDmax ¹⁵ method, the verification dose for sterilization subjects with an average bioburden of 0.8 at a sterilization dose of 15 kGy is 2.5 kGy. Here, bioburden means the number of microorganisms mixed in the sample. Therefore, 10 samples to be sterilized are uniformly irradiated with 2.5 kGy of radiation, and the sterility test of the sample after irradiation is performed (inspected for the presence of bacteria). If the number of positive samples is 1 or less, The sterilization dose satisfying SAL = 10 ⁻⁶ is determined to be 15 kGy.

Twenty samples were prepared by inoculating an average of 0.8 spores of Bacillus pumilus in 10 mL of sterile purified water. Of these, 10 were frozen at −20 ° C. and 10 were stored at room temperature (15 ° C.). Subsequently, each sample was uniformly irradiated with 2.5 kGy of radiation by the same method as in Experimental Example 1. After irradiation, the survival of the inoculum for each sample was confirmed by sterility testing. As a result, as shown in Table 7, the sterility test result was positive in one sample at normal temperature and one sample in a frozen state at −20 ° C., but the standard of ISO11137-2: 2006 was cleared and sterilization was performed. It was shown that SAL = 10 ⁻⁶ is secured at a dose of 15 kGy.

[Experimental Example 8]
(Confirmation of sterilization effect 2)
Although not specified in ISO11137-2: 2006, a challenge test by spore inoculation was performed for confirmation. Ten samples were prepared by inoculating 1000 Bacillus pumilus spores in 10 mL of sterile purified water. Of these, 5 were frozen at −20 ° C. and 5 were stored at room temperature (15 ° C.). Subsequently, each sample was uniformly irradiated with 15 kGy of radiation in the same manner as in Experimental Example 1. After irradiation, a sterility test was performed on each sample. As a result, as shown in Table 8, the sterility test results were negative in all samples, and it was confirmed that the samples were sterile.

[Experimental Example 9]
(Effect of addition of radical scavenger on reduction of active ingredient concentration by irradiation)
As a method for suppressing the degradation of chlorhexidine gluconate in water by irradiation, a method of adding a radical scavenger to the chlorhexidine gluconate preparation can be considered. Then, after adding potassium iodide or paraben as a radical scavenger to a chlorhexidine gluconate preparation, it was irradiated at room temperature to examine whether the decomposition of chlorhexidine gluconate could be suppressed.

  As a result, chlorhexidine gluconate produced a precipitate with potassium iodide, so potassium iodide could not be added to the chlorhexidine gluconate formulation. Paraben did not produce a precipitate even when added to a chlorhexidine gluconate formulation, but paraben does not dissolve sufficiently in water, so the addition of paraben has the effect of suppressing the degradation of chlorhexidine gluconate by radiation. Not enough.

  As a result of the same examination, ascorbic acid preparations and lidocaine preparations were not sufficiently effective in suppressing the reduction of the active ingredient concentration due to irradiation depending on the method of adding the radical scavenger.

[Experimental Example 10]
(Product and pH change due to irradiation of water and ice)
Generally, radicals generated in ice by electron beam irradiation are present in the crystal and cannot be diffused because they are stabilized by radical transfer or the like, resulting in a longer life than water. For this reason, solutes present in ice are not affected unless they are close to radicals. However, when ice melts into a liquid, the radicals in the crystals are released and react with solutes or other radicals. As a result, many radicals are deactivated after the ice melts, resulting in a stable molecule.

  Here, purified water at normal temperature (20 ° C.) and ice at −80 ° C. were irradiated with a 100 kGy electron beam and analyzed by HPLC to quantify the product. In addition, pH was measured. For ice, HPLC analysis and pH measurement were performed using water after melting.

  Table 9 shows the results. As a result of irradiating purified water at room temperature with an electron beam of 100 kGy, the production of trace amounts of hydrogen peroxide and nitrate ions was confirmed. In addition, as a result of irradiating 100 kGy electron beam to ice at -80 ° C., it was confirmed that trace amounts of hydrogen peroxide and nitrate ions were generated as in normal temperature purified water. And there was little. In general, the chemical species generated by irradiation with water are hydrogen and hydrogen peroxide, but it is considered that nitrate ions are generated from nitrogen in the air. In addition, nitrate ions are considered to cause a decrease in pH after irradiation with hydrogen ions. In addition, since the pH of ice is larger than that of ice, it is considered that ice produces more hydrogen ions than purified water at room temperature. The pH of the purified water that was not irradiated with radiation was 5.26.

[Experimental Example 11]
(Production amount of active species by irradiation and change over time)
The liquid cerium dosimeter is a tetravalent cerium sulfate aqueous solution, and changes to trivalent cerium by a radical reaction in water by irradiation. Therefore, the liquid cerium dosimeter in a liquid state (20 ° C.) and a frozen state (−80 ° C.) was irradiated with radiation, the amount of radicals generated was measured, and the absorbed dose was calculated.

  Specifically, first, a liquid cerium dosimeter at 20 ° C. and a liquid cerium dosimeter frozen at −80 ° C. were irradiated with electron beams of 7, 15, and 30 kGy. Subsequently, the absorbance of 320 nm ultraviolet region of the irradiated liquid cerium dosimeter was measured, and the amount of trivalent cerium produced (mol / L) was calculated. For the frozen liquid cerium dosimeter, the absorbance in the ultraviolet region of 320 nm was measured after thawing.

  The results are shown in Table 10. It was revealed that the amount of trivalent cerium generated by irradiation with radiation in a frozen state was about 1/5 of the amount of trivalent cerium generated by irradiation with liquid. Since trivalent cerium and radicals react equivalently, it was shown that the final radical generation amount in ice is smaller when water and ice are irradiated with the same dose of radiation. This suggests that freezing the aqueous solution decreases the absorbed dose of radiation and can suppress solute decomposition.

  It is known that radicals can be held at a low temperature for a long time. Therefore, if radicals are stored in ice, the effect on the solute will be further reduced. In addition, since the radical reaction is affected by temperature, it is considered that the lower the temperature during melting of ice, the more the reaction rate of the radical can be suppressed. Therefore, in order to reduce the influence on the solute in ice, it is important to store at low temperature as long as possible after irradiation and to melt ice at low temperature.

[Experimental Example 12]
(Investigation of trapping electrons and active species by ice)
Purified water and 0.5 (w / v)% chlorhexidine gluconate formulation were frozen at −80 ° C., and the resulting ice was irradiated with radiation. As a result, the ice was colored gray. The color disappeared when the ice melted. As a result of measuring the reflected light on the ice surface with a color difference meter immediately after radiation irradiation, it became clear that a clear color difference was produced and color was developed as compared with ice not irradiated with radiation. Table 11 shows the results of color difference measurement. As shown in Table 11, since ice (purified water) irradiated with radiation has increased values of a ^* and b ^{* in} the color scales L ^* , a ^* , b ^* , red and yellow The system was strongly colored, indicating that ΔE ^* was 3.5 and the yellow index (YI) was 7.0. The same tendency was observed when the 0.5 (w / v)% chlorhexidine gluconate formulation ice was irradiated with radiation. Since the color tone of the entire color center varies depending on the crystal structure, crystal periodicity, and electrons entering the hole, the color tone of ice was thought to change depending on the ice temperature, crystal structure, solute, and type of solvent.

Usually, when water is irradiated with water at room temperature, water decomposes and various radicals and stable molecules are generated. The lifetime of hydrated electrons in which electrons are hydrated is about 10 ⁻¹³ to 10 ⁻¹¹ seconds at room temperature and has high activity, and thus reacts with other short-lived radicals and solutes in a short time. Therefore, when irradiation of water at room temperature with water, electrons become hydrated electrons or become free electrons and contribute to the chemical reaction and deactivate, so that they are rarely trapped in water.

  On the other hand, it has been reported that ice has an Ih-type hexagonal crystal structure, and takes an amorphous or other crystal form depending on cooling conditions. FIGS. 3a and 3b are diagrams for explaining the influence when radiation is applied to ice crystals. When the ice crystal is irradiated with radiation as shown in FIG. 3a, the electrons of the negatively charged oxygen atom in the ice crystal are excited by accelerated electrons, and the electron is separated from the oxygen atom as shown in FIG. Lattice defects (holes) are generated in the portion. Free electrons enter the lattice defects and stabilize. On the other hand, holes generated by the removal of electrons move in the crystal, and the generated holes and traps become a color center having absorption from the ultraviolet region to the visible region, and as a result, ice is considered to develop color. It is considered that the above-mentioned ice coloring is due to this phenomenon.

  From the results of Experimental Examples 11 and 12, it is considered that in ice, some of the accelerated electrons irradiated on the ice are taken in and stabilized in the ice crystals, and the action of the accelerated electrons is more relaxed than in water. As a result, the amount of radicals generated is considered to be low, and the decomposition of solutes by radicals is suppressed.

  In Experimental Example 12, the color disappeared when the ice was melted. From this, it is considered that the color center collapsed due to melting of ice, and some hydrated electrons and radicals disappeared. FIG. 4 is a diagram showing a behavior when radiation is irradiated to ice. As shown in FIG. 4, it is considered that some hydrated electrons and radicals react with other active species and disappear after the ice melts.

  Further, as shown in Experimental Example 13 below, it was shown that the solute taken into the ice after melting of the ice becomes an energetically stable hydrate.

[Experimental Example 13]
(Hydrate formation by irradiation in the frozen state)
A 0.5 (w / v)% chlorhexidine gluconate formulation was frozen at −80 ° C. and irradiated with 30 kGy radiation. Subsequently, the chlorhexidine gluconate preparation after irradiation was thawed, chlorhexidine gluconate was quantified by HPLC, and ultraviolet absorption was measured. FIG. 5 shows the HPLC results, and FIG. 6 shows the ultraviolet absorption measurement results. As a result, as shown in FIG. 5, an increase in the peak of chlorhexidine gluconate was observed in the measurement by HPLC. Further, as shown in FIG. 6, in the measurement of ultraviolet absorption, an increase in ultraviolet absorption over a range of 200 to 300 nm was observed. When irradiated, the peak of UV absorption at 260 nm of chlorhexidine gluconate should be lower, but after irradiation at −80 ° C., it becomes higher, and UV absorption is observed over the entire 200 to 300 nm. It became clear that it was getting higher. Subsequently, after heating the sample to disrupt hydration, the sample was frozen again and irradiated, resulting in an increase in the peak of chlorhexidine gluconate in HPLC and an increase in UV absorption over the 200-300 nm range. Admitted.

  From the above results, it was considered that a part of chlorhexidine gluconate became a hydrate after irradiation in a frozen state. In general, hydrates are more energetically stable than anhydrides.

Claims (3)

  A method for sterilizing a liquid preparation, comprising a step of irradiating a frozen liquid preparation (excluding a protein preparation and a synthetic polymer preparation) with radiation.   The sterilization method according to claim 1, wherein the radiation is radiation exceeding 8 kGy.   The sterilization method according to claim 1 or 2, wherein the radiation is radiation exceeding 12 kGy.

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