WO2012029171A1 - Method for measuring physiologically active substance of biological origin - Google Patents

Abstract

A technique is provided in which measurement precision can be increased or measurement time reduced in measurement of a physiologically active substance of biological origin, such as an endotoxin or ß-D-glucan, in hyaluronic acid. A hyaluronic acid preparation and water for injection are heated to a temperature of 30-40°C, and the hyaluronic acid preparation is then diluted by a factor of 10-200 with the water for injection. The diluted specimen thus obtained is further brought to 36-40°C and mixed with AL reagent, and the concentration of an endotoxin or ß-D-glucan is measured using a laser diffraction particle analyzer.

Description

Method for measuring biologically active substances derived from living organisms

The present invention relates to a biologically active substance derived from an organism having a property of gelling by a reaction with a blood cell extract of horseshoe crab, such as endotoxin and β-D-glucan, in particular for detecting or measuring the concentration thereof in hyaluronic acid. Regarding the method.

Endotoxin is a lipopolysaccharide present in the cell wall of Gram-negative bacteria and is the most typical pyrogenic substance. If an infusion solution, injection drug, blood, or the like contaminated with this endotoxin enters the human body, it may cause serious side effects such as fever and shock. For this reason, it is obliged to manage the above drugs so that they are not contaminated by endotoxin.

By the way, there is a serine protease activated by endotoxin in the blood cell extract of horseshoe crab (hereinafter also referred to as “AL: Amoebocyte lysate”). Then, when AL reacts with endotoxin, coagulogen present in AL is hydrolyzed into coagulin and associated by an enzyme cascade by serine protease activated according to the amount of endotoxin, and an insoluble gel is formed. The If such AL characteristics are used, endotoxin can be detected with high sensitivity.

Β-D-glucan is a polysaccharide (polysaccharide) that forms a cell membrane characteristic of fungi. By measuring β-D-glucan, it is possible to detect not only common clinical fungi such as Candida, Spergillus, and Cryptococcus, but also the presence of rare fungi. It becomes possible.

Also in this case, β-D-glucan can be detected with high sensitivity by utilizing the property that the blood cell extract component AL of horseshoe crab coagulates (gel coagulation) with β-D-glucan.

Specific methods for detecting or measuring the concentration of biologically active substances derived from organisms such as endotoxin and β-D-glucan (hereinafter also referred to as predetermined physiologically active substances) using the blood cell extract component AL of horseshoe crab include the following: Can be exemplified. First, a sample mixture to be detected and concentration measurement (hereinafter also simply referred to as “measurement of a predetermined bioactive substance”) and AL mixed with the sample is allowed to stand, and after a certain time, the container is placed. There is a semi-quantitative gelation method that determines whether gelation has occurred based on the presence or absence of dripping of the admixture and whether a sample contains a predetermined physiologically active substance at a certain concentration or higher. In addition, the sample turbidity is measured using a turbidimetric method that measures and analyzes the turbidity of the sample that accompanies the formation of a gel due to the reaction between AL and a predetermined physiologically active substance, or a synthetic substrate that is hydrolyzed by an enzyme cascade to develop a color. There are colorimetric methods that measure color changes.

When measuring a predetermined physiologically active substance by the turbidimetric method described above, a mixed solution of the measurement sample and AL is generated in a glass measurement cell that has been subjected to dry heat sterilization. Then, the gelation of the mixed solution is optically measured from the outside. On the other hand, as a method capable of measuring a predetermined physiologically active substance in a shorter time, gel particles are generated by stirring a mixed solution of a measurement sample and AL using, for example, a magnetic stirrer. A light scattering method (laser light scattering particle measurement method) for detecting the number of peaks of laser light scattered by the laser beam has been proposed. Similarly, a stirring turbidimetric method has been proposed in which turbidity of a sample due to gel particles generated by stirring a mixed solution of a measurement sample and AL is detected based on the intensity of light transmitted through the mixed solution.

Incidentally, in cataract surgery, intraocular lens insertion surgery, and the like, it is known to inject a hyaluronic acid preparation into the anterior chamber in order to maintain the tension of the anterior chamber of the eyeball during the operation. When this hyaluronic acid preparation is contaminated with endotoxin, a situation called TASS (Toxic Anterior Segment Syndrome) in which the anterior chamber is inflamed after surgery may be caused.

On the other hand, since the regulation of the endotoxin allowable value in the hyaluronic acid preparation is 1.2 EU / mL, it is necessary to precisely measure the concentration of endotoxin contained in the hyaluronic acid preparation. However, the viscosity of hyaluronic acid is very high, and even if it is attempted to measure the concentration of endotoxin by reacting with AL, it may be difficult to mix AL and the hyaluronic acid preparation uniformly. In addition, a component that promotes or suppresses the reaction between endotoxin and AL is contained in hyaluronic acid, which may affect the measurement result (hereinafter also referred to as “measurement interference occurs”). Furthermore, since the viscosity of hyaluronic acid is high, the amount of hyaluronic acid collected varies, which may cause variations in measurement results. For these reasons, it may be difficult to measure endotoxin in a hyaluronic acid preparation with high accuracy.

JP 2004-061314 A JP-A-10-293129 International Publication No. WO2008 / 038329 Pamphlet

The present invention has been devised in view of the above-mentioned problems, and the object of the present invention is to increase measurement accuracy or shorten measurement time in the measurement of biologically active substances derived from organisms in hyaluronic acid. It is to provide technology that is possible.

In the present invention, the aggregation start time in a mixed solution of hyaluronic acid, which is the target of the predetermined physiologically active substance measurement, and the AL reagent is detected, and the predetermined physiologically active substance is detected or the concentration is measured based on this aggregation start time. The greatest feature of the present invention is that it has a dilution step of preparing a diluted specimen by adding hyaluronic acid and dilution water, and the temperature of the hyaluronic acid and dilution water at that time is 30 ° C. or more and 40 ° C. or less. It is a point to do.

More specifically, by measuring the concentration of the physiologically active substance in the hyaluronic acid by reacting the biologically active substance derived from the organism present in hyaluronic acid with AL which is a blood cell extract of horseshoe crab. A method for measuring a physiologically active substance,
Having a dilution step of preparing a diluted specimen by adding hyaluronic acid and dilution water not contaminated with the biologically active substance derived from the organism,
In the dilution step, hyaluronic acid and dilution water are set to a temperature of 30 ° C. or higher and 40 ° C. or lower.

Here, since the viscosity of hyaluronic acid is very high, when it is mixed with the AL reagent at the same concentration, it becomes difficult to mix the two uniformly. In addition, hyaluronic acid may contain a substance that promotes or suppresses the reaction between a predetermined physiologically active substance and AL, which may cause measurement interference. Furthermore, since the viscosity of hyaluronic acid is high, the amount of hyaluronic acid collected varies and the measurement results may vary.

On the other hand, since the reaction between the predetermined physiologically active substance and AL is very sensitive, the reaction between the predetermined physiologically active substance and AL in hyaluronic acid is promoted by diluting the hyaluronic acid to be measured. It has been found that the effects of the substances to be suppressed can be relatively suppressed. Accordingly, in the present invention, a diluted specimen is prepared by adding hyaluronic acid and dilution water, and the concentration of a predetermined physiologically active substance is measured for the diluted specimen. In addition, it has been found that hyaluronic acid is polymerized by heating to 30 ° C. or higher and its viscosity is lowered. On the other hand, it is known that a predetermined physiologically active substance is inactivated under a temperature environment exceeding 40 ° C. Therefore, in the present invention, the diluted specimen is prepared after setting the hyaluronic acid and the dilution water to a temperature of 30 ° C. or higher and 40 ° C. or lower.

This makes it possible to prepare a more homogeneous diluted sample, and further suppress the influence of substances contained in hyaluronic acid that promote or suppress the reaction between a predetermined physiologically active substance and AL to suppress measurement interference. It becomes possible. In addition, inactivation of the predetermined physiologically active substance in hyaluronic acid can be suppressed. This makes it possible to measure the predetermined physiologically active substance in hyaluronic acid with higher accuracy.

In the dilution step, the temperature of hyaluronic acid and dilution water may be 37 ° C. ± 1 ° C. Here, the Japanese Pharmacopoeia stipulates that the measurement of endotoxin should be performed under a temperature condition of 37 ° C. ± 1 ° C. That is, when measuring a predetermined physiologically active substance in a diluted sample, the temperature of the diluted sample needs to be 37 ° C. ± 1 ° C. Therefore, by setting hyaluronic acid and dilution water at 37 ° C. ± 1 ° C. at the time of the dilution step, measurement of the predetermined physiologically active substance in the diluted specimen can be started earlier.

Further, the dilution rate in the dilution step may be in the range of 10 to 200 times. As mentioned above, hyaluronic acid contains substances that promote or suppress the reaction between a predetermined physiologically active substance and AL, and may interfere with the measurement. Therefore, it is possible to measure the concentration of a predetermined physiologically active substance without dilution. Doing so increases measurement variation. On the other hand, measurement interference can be suppressed by diluting hyaluronic acid. Moreover, by diluting hyaluronic acid, the viscosity can be lowered, and hyaluronic acid and the AL reagent can be mixed more uniformly. As a result, it is possible to improve the accuracy of measurement of the predetermined physiologically active substance with respect to the diluted specimen, and to suppress measurement variations.

In the dilution step, the total weight of the added hyaluronic acid and dilution water is measured,
The amount of added hyaluronic acid is obtained from the change in the total weight before and after the hyaluronic acid is added,
The dilution factor of the diluted specimen may be specified from the amount of the added hyaluronic acid and the amount of the diluted water added.

Here, since the viscosity of hyaluronic acid is high, the collected amount of hyaluronic acid varies in the dilution step, and the diluted specimen may not be prepared with good reproducibility. In such a case, the change in the total weight of hyaluronic acid and diluting water before and after the addition of hyaluronic acid is measured with a weighing scale such as an electronic balance, and the specific gravity of hyaluronic acid is 1 g / mL, and the amount of hyaluronic acid is determined. An estimate should be made. When hyaluronic acid is collected in advance and diluted with dilution water, the dilution water is not added at the time of collection of hyaluronic acid, so the weight including the container before and after collecting hyaluronic acid. What is necessary is just to measure the change of a with a weight meter. Thereby, the dilution rate of the diluted specimen can be specified more accurately, and the measurement of the predetermined physiologically active substance in hyaluronic acid can be performed with higher accuracy. In the dilution step, the diluted specimen obtained by measuring the weight change and having the known dilution rate may be further diluted to a desired dilution rate. According to this diluting operation, it has been known that the viscosity of hyaluronic acid is sufficiently lowered when diluted to about 5 times, so that it is possible to more reliably suppress variations in the amount of diluted specimen collected and the dilution rate. As a result, it is possible to improve the reproducibility of measurement of biologically active substances derived from living organisms with an easy operation.

In the present invention, the method further comprises a mixing step of mixing the diluted specimen with AL.
In the mixing step, the temperature of the diluted specimen may be 36 ° C. or higher and 40 ° C. or lower. Here, as described above, in the Japanese Pharmacopoeia, endotoxin measurement should be performed under the temperature condition of 37 ° C. ± 1 ° C. Therefore, regardless of the turbidimetric method, stirring turbidimetric method, colorimetric method, or laser light scattering particle measurement method, the mixture of the diluted specimen and the AL reagent was transferred to the measurement cell at the time of measurement. Thereafter, the measurement cell is kept at 37 ° C. ± 1 ° C. with a heater.

Therefore, if the temperature of the diluted sample is set to 36 ° C. or more and 40 ° C. or less in the mixing step, mixing of the diluted sample and the AL reagent is completed, and the work such as transfer to the measuring cell and setting to the measuring device is being performed. Even if the temperature of the diluted sample slightly decreases, the temperature of the mixed solution can be quickly brought to the range of 37 ° C. ± 1 ° C. after the measurement is started. According to this, it becomes possible to measure a predetermined physiologically active substance in hyaluronic acid more quickly or with higher accuracy.

Here, in the dilution step, hyaluronic acid and dilution water are kept at a temperature of 30 ° C. or higher and 40 ° C. or lower (of course, 37 ° C. ± 1 ° C. may be used), and the diluted specimen is temporarily stored at room temperature or cooled and stored for a predetermined period. In addition, a new method of heating to a temperature not lower than 36 ° C. and not higher than 40 ° C. may be taken in the next mixing step, and this does not exclude taking such a method.

In the mixing step, the temperature of the diluted specimen may be 37 ° C. ± 1 ° C. If it does so, the temperature of a liquid mixture can be made into the range of 37 degreeC +/- 1 degreeC more reliably. According to this, the predetermined physiologically active substance in hyaluronic acid can be measured more quickly or with high accuracy.

In this case as well, in the dilution step, hyaluronic acid and dilution water are kept at a temperature of 30 ° C. or higher and 40 ° C. or lower, and the diluted specimen is temporarily stored at room temperature or cooled and stored for a predetermined period, and then in the next mixing step. A new method of heating to a temperature of 37 ° C. ± 1 ° C. may be taken, and this does not exclude taking such a method. .

In the present invention, the method further comprises a mixing step of mixing the diluted specimen with AL.
The temperature of the diluted specimen prepared in the dilution step is 37 ° C. ± 1 ° C.,
The mixing step may be performed while maintaining the temperature of the diluted specimen.

That is, in the dilution step, the temperature of the diluted specimen is set in the range of 37 ° C. ± 1 ° C., and the measurement is started by mixing with the AL reagent while maintaining the temperature. Then, the mixed solution can be brought into the temperature range of 37 ° C. ± 1 ° C. more reliably and more quickly. For example, the viscosity of hyaluronic acid is automatically adjusted by performing all steps of the dilution process, the mixing process, and the measurement of the predetermined physiologically active substance in the same heat-retaining apparatus set at a temperature of 37 ° C. ± 1 ° C. It is possible to reduce the interference of the measurement and suppress the measurement interference, and it is possible to immediately shift to the measurement of the predetermined physiologically active substance.

In the present invention, the biologically active substance derived from the organism may be endotoxin or β-D-glucan.

Then, endotoxin, which is the most typical pyrogen, can be detected or measured more accurately, and hyaluronic acid contaminated with endotoxin can enter the human body and cause side effects. Similarly, β-D-glucan can be detected or measured more accurately, and a wide range of fungal infections including rare fungi can be screened more accurately.

In addition, about the means to solve the subject of the above-mentioned this invention, it can use combining as much as possible.

In the present invention, when detecting or measuring the concentration of the physiologically active substance in hyaluronic acid using the reaction between biologically active substance derived from organisms such as endotoxin and β-D-glucan and AL, It is possible to improve the measurement accuracy or shorten the measurement time.

It is a figure which shows schematic structure of the laser light scattering measuring apparatus in the Example of this invention. It is a flowchart which shows the content of the measurement routine in Example 1 of this invention. It is a flowchart which shows the content of the measurement routine 2 in Example 2 of this invention. It is a flowchart which shows the content of the measurement routine 3 in Example 3 of this invention. It is a flowchart which shows the content of the measurement routine 4 in Example 4 of this invention. FIG. 2 is a schematic diagram for explaining the process of AL gelation by endotoxin or β-D-glucan and the detection method thereof.

Hereinafter, the best mode for carrying out the present invention will be described in detail with reference to the drawings.

[Example 1]
The process by which AL and endotoxin react to form a gel is well examined. That is, as shown in FIG. 6, when endotoxin binds to factor C, which is a serine protease in AL, factor C is activated to become active factor C. Active factor C is another serine protease in AL. A certain factor B is hydrolyzed and activated to obtain an activated factor B. This activated factor B immediately hydrolyzes the precursor of the clotting enzyme in AL to form a clotting enzyme, and this clotting enzyme hydrolyzes the coagulogen in AL to produce coagulin. And it is thought that the produced coagulin associates with each other to further generate an insoluble gel, and the entire AL is involved and gelled.

Similarly, when β-D-glucan binds to factor G in AL, factor G is activated to become active factor G. Active factor G hydrolyzes the precursor of coagulation enzyme in AL. Coagulation enzyme. As a result, similarly to the reaction between endotoxin and AL, coagulin is produced, and the produced coagulin associates with each other to further produce an insoluble gel.

This series of reactions is similar to the fibrin gel formation process mediated by serine proteases such as Christmas factors and thrombin found in mammals. Such an enzyme cascade reaction has a very strong amplification action because even a very small amount of activator is activated by linking the subsequent cascade. Therefore, according to the method for measuring a predetermined physiologically active substance using AL, it is possible to detect a very small amount of the predetermined physiologically active substance on the order of subpicogram / mL.

As a measuring method capable of quantifying a predetermined physiologically active substance, as described above, a turbidimetric method and a laser light scattering particle measuring method can be mentioned. As shown in FIG. 5, these measurement methods detect the coagulin aggregate produced by the enzyme cascade reaction of AL as turbidity of the sample and the latter as fine particles of gel generated in the system. Highly sensitive measurement is possible.

In particular, the laser light scattering particle measurement method directly measures the gel fine particles generated in the system, so it has higher sensitivity than the turbidimetric method and generally forcibly stirs the sample consisting of AL and the specimen. Therefore, the formation of gel can be detected in a shorter time compared to the turbidimetric method.

Next, a specific example of the effect of endotoxin on the human body will be described. In surgery that requires removal of the anterior chamber water of the eyeball, such as cataract surgery and intraocular lens insertion surgery, a hyaluronic acid preparation is injected into the anterior chamber in order to maintain the tension of the anterior chamber of the eyeball during the operation. It is known. If this hyaluronic acid preparation is contaminated with endotoxin, TASS (Toxic Anterior Segment Syndrome) may develop, and the anterior chamber may become inflamed after surgery.

On the other hand, since the regulation of the endotoxin allowable value in the hyaluronic acid preparation is 1.2 EU / mL, it is necessary to precisely measure the concentration of endotoxin contained in the hyaluronic acid preparation. However, the viscosity of hyaluronic acid is very high, and it was difficult to uniformly mix AL and the hyaluronic acid preparation even when trying to measure the endotoxin concentration using AL. In addition, there is a possibility that measurement variation may occur due to measurement interference caused by a component that promotes or suppresses the reaction between endotoxin and AL. For these reasons, it may be difficult to measure endotoxin in a hyaluronic acid preparation with high accuracy.

Therefore, in this example, the hyaluronic acid preparation was diluted 200 times with water for injection before measurement. As a result, the viscosity of hyaluronic acid decreases, and the AL reagent and the diluted specimen of the hyaluronic acid preparation can be mixed more uniformly. In addition, since measurement interference can be suppressed at that time, it is possible to suppress variations in endotoxin concentration measurement.

In this example, the hyaluronic acid preparation and water for injection were heated to 37 ° C. ± 1 ° C. when diluting the hyaluronic acid preparation. As for hyaluronic acid, it is known that when heated to about 30 ° C., the polymerization is dissolved and the viscosity is lowered. Further, it is known that endotoxin in hyaluronic acid is inactivated when the temperature exceeds 40 ° C. Therefore, by heating the hyaluronic acid preparation and water for injection to a range of 37 ° C. ± 1 ° C., the viscosity of hyaluronic acid can be reduced without deactivating endotoxin.

Next, the configuration of the light scattering particle measuring apparatus 1 used for measuring endotoxin in this embodiment will be described with reference to FIG. A laser light source is used as the light source 12 used in the light scattering particle measuring apparatus 1 (other than that, an ultra-bright LED or the like may be used). The light emitted from the light source 12 is narrowed by the incident optical system 13 and enters the sample cell 14. The sample cell 14 contains a mixture of a hyaluronic acid preparation for which endotoxin is to be measured and an AL reagent. The sample cell 14 is housed in a heater-equipped holder 23 that incorporates an electric heater and keeps the sample cell 14 at 37 ° C. ± 1 ° C.

The holder with heater 23 is provided with an incident window 23a for allowing the light from the incident optical system 13 to pass therethrough and an emission window 23b for emitting the transmitted light that has passed through the sample cell 14. The light that has entered the sample cell 14 through the incident window 23a is scattered by particles (coagulogen monomer, measurement object such as coagulogen oligomer) in the mixture. The holder with heater 23 is provided with a scattered light window 23c for emitting scattered light from the sample in the sample cell 14 to the side of the optical axis of the incident light.

Further, an emission optical system 15 is arranged on the extension of the direction of the scattered light window 23c with respect to the center of the sample cell 14. In order to further extend the optical axis of the emission optical system 15, the light receiving element 16 that receives the scattered light scattered by the particles in the mixed liquid in the sample cell 14 and constricted by the emission optical system 15 and converts it into an electrical signal. Is arranged. The light receiving element 16 includes an amplifying circuit 17 that amplifies the electric signal photoelectrically converted by the light receiving element 16, a filter 18 for removing noise from the electric signal amplified by the amplifying circuit 17, and the electricity after the noise is removed. An arithmetic unit 19 that calculates the number of gel particles from the number of signal peaks, further determines the reaction start time and derives the endotoxin concentration, and a display 20 that displays the result are electrically connected.

The sample cell 14 is provided with a stirrer 21 that rotates by applying an electromagnetic force from the outside and stirs the mixed liquid as a sample, and a stirrer 22 is provided outside the sample cell 14. ing. Thus, it is possible to adjust the presence / absence of stirring and the stirring speed.

Next, the procedure for measuring endotoxin of the hyaluronic acid preparation in this example will be described with reference to FIG. FIG. 2 is a flowchart showing the measurement routine 1 in this embodiment. First, in this example, as shown in S101, the hyaluronic acid preparation to be measured and the water for injection as a diluent are both heated to 37 ° C. ± 1 ° C. Here, it is desirable that the hyaluronic acid preparation and the water for injection are kept warm for a sufficient time to reach a sufficient temperature equilibrium state after reaching 37 ° C. ± 1 ° C. For example, the temperature may be kept at 37 ° C. ± 1 ° C. for about 30 minutes. This dissolves the polymerization of hyaluronic acid, lowers the viscosity of the hyaluronic acid preparation, and makes it easy to stir and dilute.

Next, as shown in S102, the hyaluronic acid preparation is diluted 200 times with water for injection. As a result, the viscosity of the hyaluronic acid preparation is further reduced and it becomes easier to stir, and the influence of a component that promotes or suppresses the reaction between endotoxin and AL can be reduced, thereby suppressing measurement interference. Thereby, the measurement accuracy of endotoxin can be improved.

Next, as shown in S103, the diluted specimen is heated again to 37 ° C. ± 1 ° C. This is because the temperature of the diluted specimen may be partially lowered during the operation of S102, and the temperature of the diluted specimen is again 37 ° C. ± 1 ° C. Here again, it is desirable that the diluted specimen is kept warm for a sufficient time to reach a sufficient temperature equilibrium state after reaching 37 ° C. ± 1 ° C. For example, the temperature may be kept at 37 ° C. ± 1 ° C. for about 30 minutes. When the temperature of the diluted specimen does not decrease during the operation of S102 and the temperature of the diluted specimen is maintained at 37 ° C. ± 1 ° C., it is not always necessary to heat again.

Next, as shown in S104, the diluted specimen of 37 ° C. ± 1 ° C. and the AL reagent are mixed. Then, preparation for measurement of laser light scattering particles, such as transferring the mixed solution to the sample cell 14, is performed. In this case, it is natural that the AL reagent may be heated to 37 ° C. ± 1 ° C.

Then, as shown in S105, measurement is started by the laser light scattering particle measuring apparatus 1. Specifically, the sample cell 14 into which the mixed liquid has been transferred is set in the holder 23 with a heater, and stirring is started by the stirrer 22 and the stirrer 21. Here, the heater set temperature of the heater-equipped holder 23 is 37 ° C. ± 1 ° C. as defined by the Japanese Pharmacopoeia. Further, the temperature of the mixed solution is already about 37 ° C. ± 1 ° C. Therefore, it is possible to sufficiently stir immediately after setting the sample cell 14 into which the mixed solution has been transferred to the holder 23 with a heater, and measurement can be started immediately.

As described above, in this example, the hyaluronic acid preparation to be measured and water for injection were first heated to a temperature of 37 ° C. ± 1 ° C. and then mixed to dilute the hyaluronic acid preparation 200 times. Thereby, the viscosity of hyaluronic acid itself can be reduced and it can be made into the state which can fully be stirred. As a result, the hyaluronic acid preparation can be diluted more uniformly. Moreover, by diluting the hyaluronic acid preparation 200 times, scattering by hyaluronic acid itself when incident light is incident on the hyaluronic acid can be suppressed. Thereby, the background noise in the laser light scattering particle measurement method can be reduced, and the measurement accuracy can be improved.

Furthermore, since the diluted specimen is reheated to 37 ° C. ± 1 ° C. and mixed with the AL reagent and measurement is started with the laser light scattering particle measuring apparatus 1, it can be easily stirred again immediately before the start of measurement. The measurement can be started immediately after being set in the apparatus. In this example, the sample temperature at the time of diluting the hyaluronic acid preparation, mixing the diluted specimen with the AL reagent, and measuring can be made equal, so that the conditions are the same from the start of sample preparation to the end of measurement. Therefore, more stable measurement can be performed.

[Example 2]
Next, the procedure for measuring endotoxin of the hyaluronic acid preparation according to Example 2 of the present invention will be described. FIG. 3 is a flowchart showing the measurement routine 2 in the present embodiment.

In S201 of measurement routine 2, both the hyaluronic acid preparation to be measured and water for injection are raised to a temperature range of 30 ° C. or higher and 40 ° C. or lower. Here, it is desirable that the hyaluronic acid preparation and water for injection are kept warm in a temperature range of 30 ° C. to 40 ° C. for a sufficient time to reach a sufficient temperature equilibrium state. For example, the temperature may be kept in a temperature range of 30 ° C. or more and 40 ° C. or less for about 30 minutes as in the first embodiment.

If the temperature of the hyaluronic acid preparation and the temperature of the dilution water at the time of diluting the hyaluronic acid preparation are about 30 ° C. or higher at which the polymerization in hyaluronic acid can be dissolved, the viscosity of the hyaluronic acid can be lowered. Moreover, it can suppress that the endotoxin in a hyaluronic acid deactivates by making the temperature of a hyaluronic acid formulation into 40 degrees C or less. Therefore, by setting the hyaluronic acid preparation to be measured and the water for injection as dilution water within the temperature range of 30 ° C. or higher and 40 ° C. or lower, the viscosity of hyaluronic acid is reduced without affecting the concentration of endotoxin. It is possible.

Next, as shown in S202, the hyaluronic acid preparation is diluted with dilution water to a range of 10 to 200 times. It has been found that the viscosity of hyaluronic acid is sufficiently low when diluted to about 5 times. Therefore, by diluting the hyaluronic acid preparation to a range of 10 times or more and 200 times or less, the viscosity of the hyaluronic acid preparation can be sufficiently lowered to facilitate stirring. Even if the dilution rate is not necessarily 200 times, the effect of reducing the influence of the component that promotes or suppresses the reaction between endotoxin and AL can be expected. Therefore, the measurement accuracy of endotoxin can be further improved by the process of S202.

Next, as shown in S203, the diluted specimen is heated again to 36 ° C. or higher and 40 ° C. or lower. This is because the temperature of the diluted specimen may be partially lowered during the operation of S202, and the temperature of the diluted specimen is again set to a temperature of 36 ° C. or higher and 40 ° C. or lower. Again, it is desirable that the diluted specimen is kept warm for a sufficient time to reach a sufficient temperature equilibrium state after reaching 36 ° C. or higher and 40 ° C. or lower. For example, the temperature may be maintained at 36 ° C. or higher and 40 ° C. or lower for about 30 minutes. If the temperature of the diluted specimen does not decrease during the operation of S202 and the temperature of the diluted specimen is maintained at 36 ° C. or higher and 40 ° C. or lower, it is not always necessary to heat again.

Since steps S104 and S105 are the same as the measurement routine shown in the first embodiment, detailed description thereof is omitted. In this embodiment, in S203, the temperature of the diluted specimen is set to 36 ° C. or more and 40 ° C. or less, slightly higher than 37 ° C. ± 1 ° C., which is the temperature condition for measuring endotoxin defined by the Japanese Pharmacopoeia. Therefore, even if the temperature of the diluted specimen is somewhat lowered during the process of S104, the temperature of the diluted specimen is set to 37 ° C. ± 1 ° C. early when set in the laser light scattering particle measuring apparatus 1 in S105. Can be.

Example 3
Next, Embodiment 3 of the present invention will be described. In this example, hyaluronic acid and diluting water were first charged in an environment of 37 ° C. ± 1 ° C., and all of the dilution of the hyaluronic acid preparation, mixing of the diluted specimen with the AL reagent, and measurement of endotoxin in the mixed solution An example in the environment will be described.

FIG. 4 shows a flowchart of the measurement routine 3 in the present embodiment. In S301 of this routine, the hyaluronic acid preparation to be measured and the water for injection as dilution water are both put in an environment of 37 ° C. ± 1 ° C. For example, the hyaluronic acid preparation and the liquid for injection may be stored in a thermostatic bath set at 37 ° C. ± 1 ° C. At that time, the AL reagent and the laser light scattering particle measuring apparatus 1 shown in FIG. 1 are also stored in the same thermostatic chamber.

In S302, a diluted specimen is prepared by diluting a hyaluronic acid preparation 200 times with water for injection in a thermostatic chamber. Furthermore, in S303, the diluted specimen prepared in S302 and the AL reagent stored in the high-temperature tank are mixed in the constant-temperature tank, and the mixed liquid is transferred to the sample cell 14. In S304, the sample cell 14 in which the mixed liquid is stored is set in the holder 23 with a heater (in this case, the heater does not need to be operated) of the laser light scattering particle measuring apparatus 1 stored in the thermostat as described above. To start measurement.

As described above, in this example, the dilution of the hyaluronic acid preparation with the injection solution, the mixing of the diluted specimen with the AL reagent, and the measurement with the laser light scattering particle measuring apparatus 1 are all in the environment of 37 ° C. ± 1 ° C. I decided to do it below. Therefore, since the sample containing hyaluronic acid is always maintained at 37 ° C. ± 1 ° C., the operation can be performed under a certain condition from the start of sample preparation to the end of measurement, and more stable measurement can be performed. . Moreover, useless time such as heating for temperature adjustment can be omitted, and endotoxin in the hyaluronic acid preparation can be measured earlier or more reliably.

In this example, the hyaluronic acid preparation, the injection solution, the AL reagent, and the laser light scattering particle measuring device 1 were stored in a thermostat set at 37 ° C. ± 1 ° C. In the sample cell 14 in which the dilution of the hyaluronic acid preparation with water for injection and the mixing of the diluted specimen and the AL reagent are all set in the holder 23 with a heat retaining heater of the laser light scattering particle measuring apparatus 1 and temperature controlled at 37 ° C. ± 1 ° C. You may make it carry out. Also by this, the temperature of the sample containing hyaluronic acid is always maintained at 37 ° C. ± 1 ° C., so that useless time such as heating for temperature adjustment can be omitted, and hyaluronic acid can be prevented earlier or more reliably. Endotoxin in the formulation can be measured.

Example 4
Next, a fourth embodiment of the present invention will be described. In this example, a diluted specimen is prepared by adding a hyaluronic acid preparation to water for injection. Then, by measuring the weight of the added hyaluronic acid preparation, an intermediate diluted sample whose dilution factor is accurately known is prepared. Then, the endotoxin is measured by mixing a diluted sample obtained by further diluting the above-mentioned intermediate diluted sample so as to obtain a desired dilution ratio and the AL reagent. FIG. 5 shows a flowchart of the measurement routine 4 in the present embodiment.

In S401 of this routine, a hyaluronic acid preparation to be measured is added to a known amount of water for injection, and an intermediate diluted specimen is prepared by diluting the hyaluronic acid preparation to 5 to 200 times. At this time, the weight change of the diluted solution due to the addition of the hyaluronic acid preparation is measured with a weight scale such as an electronic balance. By doing so, since the addition amount of the hyaluronic acid preparation can be calculated (with a specific gravity of 1 g / mL), the dilution rate of the diluted specimen can be accurately estimated.

Next, in S402, a diluted sample is prepared by further diluting the above-mentioned intermediate diluted sample with water for injection so as to obtain a desired dilution ratio (10 to 200 times). Here, since it has been known that the viscosity of hyaluronic acid is sufficiently low when diluted to about 5 times, preparing a diluted specimen having a desired dilution ratio according to the dilution ratio of an intermediate diluted specimen is not possible. Easy. When the process of S402 is completed, the processes of S104 and S105 are executed. Since steps S104 and S105 are equivalent to the measurement routine shown in the first embodiment, detailed description thereof is omitted.

In this routine, even when it is difficult to accurately collect a desired amount of the hyaluronic acid preparation due to the high viscosity of the hyaluronic acid preparation, the dilution factor of the diluted specimen can be specified more accurately. In addition, since it has been known that the viscosity of hyaluronic acid is sufficiently reduced when diluted to about 5 times, as described above, a diluted sample having a desired dilution factor according to the dilution factor of the intermediate diluted sample is used. It is easy to prepare. Therefore, preparation of a diluted specimen can be performed with higher reproducibility, and good reproducibility can be obtained with respect to the endotoxin measurement result.

In S105 of this routine, the temperature of the mixed solution is about 37 ° C. ± 1 ° C., but in S401 and S402, the hyaluronic acid preparation, water for injection, intermediate diluted sample and diluted sample are not necessarily 30 ° C. or higher and 40 ° C. The following temperature range is not necessary. In this example, the hyaluronic acid formulation was added to a certain amount of water for injection, but it is needless to say that the water for injection may be added to a certain amount of hyaluronic acid formulation to achieve a desired dilution ratio. In addition, in the process of S401 of this example, the process of S402 may be omitted when the dilution ratio of the intermediate diluted sample becomes a desired dilution ratio by accurately controlling the amount of hyaluronic acid added in advance. It doesn't matter.

In the above examples, the endotoxin measurement in the hyaluronic acid preparation has been described with respect to the measurement by the laser light scattering particle measurement method. However, the present invention includes the turbidimetric method, the stirring turbidimetric method, and the colorimetric method. Of course, it may be applied to other measurement methods. In that case, what is necessary is just to change the process of S105 and S304 in a measurement routine and measurement routine 2 thru | or 4 to each measuring method.

Here, as described above, there is currently a regulation that sets the endotoxin permissible value in the hyaluronic acid preparation to 1.2 EU / mL. When the hyaluronic acid preparation containing 1.2 EU / mL endotoxin is diluted 200-fold, the endotoxin concentration is 0.006 EU / mL. It can also be measured by nephelometry, diffusion nephelometry, or colorimetry.

However, there is an active movement to regulate the endotoxin admixture value in hyaluronic acid preparations to a lower level. Then, when measuring the endotoxin concentration with high accuracy for a diluted specimen further diluted 200-fold, it may be difficult to use the turbidimetric method, diffusion turbidimetric method, and colorimetric method. Therefore, by applying the present invention to the laser light scattering particle measurement method as in the above-described example, even if the endotoxin contamination allowable value in the hyaluronic acid preparation is regulated to be lower, the endotoxin concentration is highly accurate. Can be measured. This is because the laser light scattering particle measurement method can measure a lower concentration of endotoxin than other methods.

In the present invention, hyaluronic acid is diluted with water for injection and the temperature is controlled by a very simple method to improve the accuracy of endotoxin measurement and shorten the measurement time. Therefore, according to the present invention, it is not necessary to add a special substance other than the AL reagent to hyaluronic acid, and there is little possibility that endotoxin is further mixed or reduced during the operation. This is a more appropriate method for measuring endotoxin concentrations.

Further, in the above-described embodiment, the case where endotoxin is measured as an example of the predetermined physiologically active substance has been described. However, the above-described embodiment is directed to other predetermined physiologically active substances including β-D-glucan. Is also applicable. In the above examples, the expression is based on the premise that the hyaluronic acid preparation, water for injection, AL reagent, etc. are heated to the target temperature range. Needless to say, there may be no need.

DESCRIPTION OF SYMBOLS 1 ... Laser light scattering particle measuring device 12 ... Light source 13 ... Incident optical system 14 ... Sample cell 15 ... Output optical system 16 ... Light receiving element 17 ... Amplifying circuit 18 ... · Filter 19 ··· Arithmetic device 20 ··· Display device 21 ··· Stirrer 22 · · · Stirrer 23 · · · Holder 23a with heater · · · Entrance window 23b · · · Light window

Claims (8)

A biologically active substance derived from a living organism that measures the concentration of the physiologically active substance in the hyaluronic acid by reacting a biologically active substance derived from an organism present in hyaluronic acid with AL, which is a blood cell extract of horseshoe crab. A measuring method,
Having a dilution step of preparing a diluted specimen by adding hyaluronic acid and dilution water not contaminated with the biologically active substance derived from the organism,
In the dilution step, hyaluronic acid and dilution water are set to a temperature of 30 ° C. or higher and 40 ° C. or lower, and a biologically active substance measuring method derived from a living body is characterized. In the dilution step, the temperature of hyaluronic acid and dilution water is set to 37 ° C ± 1 ° C, and the method for measuring a biologically-derived biologically active substance according to claim 1, wherein 3. The method for measuring a biologically active substance derived from an organism according to claim 1 or 2, wherein a dilution ratio in the dilution step is in a range of 10 to 200 times. In the dilution step, the total weight of the added hyaluronic acid and dilution water is measured,
The amount of added hyaluronic acid is obtained from the change in the total weight before and after the hyaluronic acid is added,
The organism according to any one of claims 1 to 3, wherein the dilution rate of the diluted specimen is specified from the amount of the added hyaluronic acid and the amount of the diluted water added. A method for measuring a physiologically active substance derived therefrom. And further comprising a mixing step of mixing the diluted specimen with AL.
The method for measuring a biologically active substance derived from a living body according to any one of claims 1 to 4, wherein in the mixing step, the temperature of the diluted specimen is set to 36 ° C or higher and 40 ° C or lower. In the mixing step, the temperature of the diluted specimen is set to 37 ° C. ± 1 ° C. The method for measuring a biologically active substance derived from a living organism according to claim 5. And further comprising a mixing step of mixing the diluted specimen with AL.
The temperature of the diluted specimen prepared in the dilution step is 37 ° C. ± 1 ° C.,
The method for measuring a biologically active substance derived from an organism according to claim 1 or 2, wherein the mixing step is performed while maintaining the temperature of the diluted specimen. The method for measuring a biologically active substance of biological origin according to any one of claims 1 to 7, wherein the biologically active substance of biological origin is endotoxin or β-D-glucan.

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