Method for determining titer of defibrase of agkistrodon acutus

Abstract

The invention provides a method for determining the titer of an agkistrodon acutus defibrase, and relates to the technical field of defibrase. The method comprises the following steps: preparing a buffer solution, namely mixing 0.05-0.15 mol / L of a Tris-HCl solution, bovine serum albumin and Tween-20 to prepare the buffer solution in which every 100 mL of the Tris-HCl solution contains 0.05-0.15 g of bovine serum albumin, 0.03-0.05 mL of Tween-20 and 2-4 g of trehalose, and the pH value of the buffer solution is 7.2-7.4; preparing a chromogenic substrate solution; dissolving Bz-Phe-Val-Arg-pNA by using DMSO (Dimethylsulfoxide), and then diluting by using a buffer solution; preparing a standard solution and a test solution; and then detecting by using an ultraviolet spectrophotometer. According to the method provided by the invention, the accuracy is remarkably improved.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of defibrase, in particular to a method for determining the titer of Agkistrodon acutus defibrase. BACKGROUND

[0002] Snake venom is rich in proteins and contains a variety of enzymes, most of which have catalytic activity. These enzymes are mainly divided into neurotoxins and hemorrhagic toxins. These enzymes have important significance in biochemical research, biochemical drug development, and clinical application. As a type of hemorrhagic toxin in snake venom, thrombin-like enzyme (TLE) is the most studied and clinically mature biochemical drug.

[0003] Defibrase is a single component proteolytic enzyme extracted from Agkistrodon halys or Agkistrodon acutus venom. It is a thrombin-like snake venom that has significant anticoagulant effects such as defibrillation, viscosity reduction, disaggregation, and thrombolysis. It is widely used in the clinical treatment and prevention of occlusive cardiovascular and cerebrovascular thrombotic diseases. Agkistrodon acutus and Agkistrodon halys are both snakes with high TLE content. Agkistrodon acutus venom contains several enzymes with similar thrombin-like effects. In recent years, it has been found that Agkistrodon acutus defibrase has good efficacy for more than 100 diseases and complications such as cardiovascular disease, diabetes, and cancer. Further research and utilization of Agkistrodon acutus defibrase have received more attention.

[0004] However, the previous production process of defibrase was to purify snake venom and then add excipients for packaging. Most of the production processes are rough, the equipment is outdated, the process and product quality control are poor, the quality standard system is not perfect, there is a lack of snake venom, raw material quality control standards and specificity identification of snake venom in preparations, and the purity, abnormal toxicity and titer determination method need to be optimized. Some methods for determining the titer of defibrase have been disclosed in the prior art. The traditional method uses the fibrin clotting method to determine the titer of defibrase by measuring the time of fibrinogen converted to fibrin by defibrase in vitro. This method is not only subjective, but also affected by the excipient dextrose, which affects the accuracy of defibrase preparation detection.

[0005] "Method for determining the potency of defibrase for injection by blood coagulation instrument method" (Yankun, Kan Weina, Chinese Pharmacist, 2012, Vol. 15, No. 6, 1008-049X (2012) 06-0843-03) uses fibrinogen as a substrate, and uses a blood coagulation instrument to determine the clotting time of defibrase on the substrate. According to the clotting time and potency of the control, the potency of defibrase is calculated. Although this method overcomes the errors caused by subjective factors in the visual observation of initial clotting time, it still essentially investigates the linear correlation between the potency of defibrase and the clotting time within a certain concentration range, and the specificity is not strong, and it is easily affected by dextrose in the preparation.

[0006] Chinese patent application CN 112852917 A discloses a defibrase potency determination device, which comprises a incubator, a culture dish, a purification mechanism, an air inlet pipe and an electric heating plate. The incubator is provided with a placing mechanism for placing the culture dish. An electric heating plate is embedded on the inner wall of the incubator. A uniform heating assembly is arranged on the side wall of the incubator. The top surface of the incubator is provided with a purification mechanism. The outlet of the purification mechanism is connected with the inlet of the air inlet pipe. The outlet of the air inlet pipe extends above the inner cavity of the incubator. The first frame, the second frame, the third frame, the fourth frame, the fifth frame and the sixth frame are arranged side by side. The first frame, the second frame, the third frame, the fourth frame, the fifth frame, the sixth frame, the disinfectant tank and the seventh frame are all provided with matching connecting holes. Connecting bolts are arranged in the connecting holes. The invention avoids the differences in the determination standards of the test personnel, which leads to the differences in the potency of the products. It realizes efficient and accurate determination of thrombin-like enzyme. However, it mainly avoids the errors caused by subjective factors, and does not explore the errors caused by the factors of the preparation itself.

[0007] Therefore, a method for determining the potency of Agkistrodon halsy antivenin is provided, which can significantly improve the accuracy of the determination of the potency of Agkistrodon halsy antivenin and is the research focus of researchers in the field. SUMMARY

[0008] The present application provides a method for determining the potency of Agkistrodon halsy antivenin.

[0009] To achieve the above-mentioned purpose, the technical scheme adopted by the present application is as follows: On the one hand, the present application provides a method for determining the potency of Agkistrodon halsy antivenin, which comprises the following steps: Step 1, preparation of buffer solution: mix 0.05-0.15 mol / L Tris-HCl solution, bovine serum albumin and Tween-20 to prepare a buffer solution containing 0.05-0.15 g bovine serum albumin, 0.03-0.05 mL Tween-20 and 2-4 g trehalose per 100 mL Tris-HCl solution, and the pH is 7.2-7.4; Step 2, preparation of chromogenic substrate solution: dissolve Bz-Phe-Val-Arg-pNA with DMSO, then dilute with buffer; Step 3, preparation of standard solution: take fibrinolysin standard, add buffer, dilute; Step 4, preparation of test sample solution: dissolve fibrinolysin sample to be tested with buffer, dilute; Step 5, calculation of fibrinolysin titer: Incubate the chromogenic substrate solution, then mix the test sample solution or the standard solution with the chromogenic substrate solution at a volume ratio of 1:15-20, detect the absorbance of the hydrolysis product by using ultraviolet spectrophotometer, read the absorbance A0 at 3 minutes and the absorbance A at 6 minutes, obtain the average value of the ΔA value of the standard solution and the test sample solution; determine the protein content W of the test sample according to the protein content determination method; then calculate the fibrinolysin titer according to ΔA and W, wherein W is the mass percentage.

[0010] Preferably, in step 5, the formula according to which the fibrinolysin titer is calculated is: Formula (1).

[0011] Preferably, the concentration of the Tris-HCl solution is 0.1 mol / L.

[0012] Preferably, in step 1, the buffer is a buffer containing 0.1 g bovine serum albumin (BSA), 0.05 mL Tween-20 and 3 g trehalose per 100 mL Tris-HCl solution, and the pH is 7.4.

[0013] Preferably, in step 1, the buffer needs to be stored at 3-5°C; further preferably, in step 1, the buffer needs to be stored at 4°C.

[0014] Preferably, in step 2, the dilution with buffer is to dilute Bz-Phe-Val-Arg-pNA to a concentration of 0.2-0.3 mmol / L with buffer.

[0015] Further preferably, in step 2, the dilution with buffer is to dilute Bz-Phe-Val-Arg-pNA to a concentration of 0.25 mmol / L with buffer.

[0016] Preferably, in step 3, the dilution is to dilute the fibrinolysin standard to a concentration of 3-8 U / mL; Further preferably, in step 3, the dilution is to dilute the fibrinolysin standard to a concentration of 5 U / mL; Preferably, in step 4, the dissolution is centrifugation, and the parameters are: 1000-1200 rpm, 5-10 min; Further preferably, in step 4, the parameters of the centrifugation are: 1100 rpm, 8 min.

[0017] Preferably, in step 4, the test sample solution needs to be filtered with a filter membrane, and the filter membrane is a PVDF filter membrane with a pore size of 0.20-0.24 μm.

[0018] Further preferably, in step 4, the filter membrane is a PVDF filter membrane with a pore size of 0.22 μm.

[0019] Preferably, in step 4, the dilution is to dilute the fibrinolysin sample to be tested to a concentration of 3-8 U / mL.

[0020] Further preferably, in step 4, the dilution is to dilute the fibrinolysin sample to be tested to a concentration of 5 U / mL.

[0021] Preferably, in step 5, the temperature of the incubation is 36.0-37.5℃, and the time is 2-3 min.

[0022] Preferably, in step 5, the detection wavelength for detecting the absorbance of the hydrolysis product by using the ultraviolet spectrophotometer is 405 nm.

[0023] Preferably, in step 5, the volume ratio is 1:15-18.

[0024] Further preferably, in step 5, the volume ratio is 1:18.

[0025] Preferably, in step 5, the determination method of the protein content of the test sample is as follows: Dilute the test sample with water to a concentration of 0.05-0.15 mg / mL, and determine the protein content according to the protein content determination method to calculate the mass percentage of the protein of the test sample.

[0026] Preferably, the protein content determination method is the protein content determination method 0731 recorded in the Chinese Pharmacopoeia.

[0027] Compared with the prior art, the present application has the following beneficial effects: 1. The present application configures a specific buffer solution with BSA, Tween-20, trehalose and Tris-HCl buffer, which can significantly improve the non-specific adsorption of fibrinolysin in the reaction, protect the enzyme activity, inhibit the adsorption of hydrophobic surfaces, and reduce the background interference of the reaction system, thereby significantly improving the accuracy of the determination method.

[0028] 2. The present application uses Bz-Phe-Val-Arg-pNA as a chromogenic substrate, and the specific buffer solution prepared in the present application can significantly improve the accuracy of the determination method. BRIEF DESCRIPTION OF DRAWINGS

[0029] Figure 1 Lineweaver-Burk plot of the affinity kinetics of defibrase with substrate Bz-Phe-Val-Arg-pNA in Example 1. DETAILED DESCRIPTION

[0030] In order to make the technical means, creative features, purposes and effects of the present application easy to understand, the following specific embodiments are further illustrated. However, the following embodiments are only preferred embodiments of the present application, not all. Based on the embodiments in the embodiments, other embodiments obtained by those skilled in the art without creative labor are within the scope of the present application. It is worth mentioning that the raw materials used in the present application are ordinary commercially available products, and their sources are not specifically limited. The technical and scientific terms used in the embodiments have the meanings generally understood by those skilled in the art to which the present application belongs.

[0031] Part of the raw material purchase information: Defibrase standard: purchased from China Food and Drug Inspection Research Institute, batch number: 140620-200802 Agkistrodon acutus venom: purchased from Jilin Aodong Pharmaceutical Group Yanji Co., Ltd.; Defibrase: purchased from Jilin Aodong Pharmaceutical Group Yanji Co., Ltd.; Bz-Phe-Val-Arg-pNA: purchased from Shanghai Dongfeng Biochemical Technology Co., Ltd.

[0032] Example 1 A method for determining the titer of Agkistrodon acutus defibrase, comprising the following steps: Step 1, preparation of buffer solution: mix 0.1 mol / L Tris-HCl solution, bovine serum albumin (BSA), and Tween-20 to prepare a buffer solution containing 0.1 g bovine serum albumin (BSA), 0.05 mL Tween-20, and 3 g trehalose per 100 mL Tris-HCl solution, with a pH of 7.4; store at 4°C for later use.

[0033] Step 2, preparation of chromogenic substrate solution: dissolve Bz-Phe-Val-Arg-pNA in DMSO to 10 mg / mL, then dilute with buffer solution to 0.25 mmol / L; Step 3, preparation of standard solution: take the defibrase standard, add buffer solution, and dilute to a concentration of 5 U / mL; Step 4, preparation of test sample solution: dissolve the defibrase sample to be tested with buffer solution, centrifuge at 1100 rpm for 8 min, take the supernatant, pass through a 0.22 μm PVDF filter membrane, and dilute to a concentration of 5 U / mL; Step 5, determination of ΔA value: the chromogenic substrate solution was incubated in a water bath at 37℃±0.5℃ for 2-3 min, then the test sample solution or standard solution was mixed with the chromogenic substrate solution at a volume ratio of 1:18, the absorbance of the hydrolysis product was detected by ultraviolet spectrophotometer, and the absorbance A0 at 3 min and the absorbance A at 6 min were accurately read, the ΔA value (ΔA=A-A0) of the standard solution and the test sample solution was calculated by taking the average of three parallel determinations; The fibrinolytic enzyme titer was calculated according to the following formula: Formula (1).

[0034] The determination method of the protein content (mass percentage) of the test sample is as follows: About 50 mg of the product was accurately weighed, dissolved and diluted to the mark in a 50 mL volumetric flask with water, shaken well, and 5 mL was accurately taken and diluted to the mark in a 50 mL volumetric flask with water, shaken well, as the test sample solution. According to the protein content determination method recorded in the Chinese Pharmacopoeia, the calculation was made, and the protein content (mass percentage) of the test sample was obtained.

[0035] Example 2 Screening of buffer preparation concentration Preparation of buffer 1: 0.1 mol / L Tris-HCl solution, bovine serum albumin (BSA), and Tween-20 were mixed to prepare a buffer containing 0.05 g bovine serum albumin (BSA), 0.06 mL Tween-20, and 4 g trehalose per 100 mL Tris-HCl solution, with a pH of 7.4; store at 4℃ for later use.

[0036] Preparation of buffer 2: 0.1 mol / L Tris-HCl solution, bovine serum albumin (BSA), and Tween-20 were mixed to prepare a buffer containing 0.15 g bovine serum albumin (BSA), 0.03 mL Tween-20, and 2 g trehalose per 100 mL Tris-HCl solution, with a pH of 7.4; store at 4℃ for later use.

[0037] Steps 2-5 of Example 1 were repeated using buffer 1 and buffer 2, respectively.

[0038] Example 3 Screening of the volume ratio of the test sample solution or standard solution to the chromogenic substrate solution Compared with Example 1, the volume ratio of the test sample solution or standard solution to the chromogenic substrate solution was changed to 1:15 and 1:20, respectively, and the rest of the operation was consistent with Example 1.

[0039] Comparative Example 1 A method for determining the potency of the fibrinolytic enzyme of Agkistrodon acutus, compared with Example 1, changing the chromogenic substrate to S-2238.

[0040] comprising the following steps: Step 1, preparation of buffer solution: take 0.1 mol / L Tris-HCl solution, bovine serum albumin (BSA), Tween-20 mixed, prepared into 100 mL Tris-HCl solution containing 0.1 g bovine serum albumin (BSA), 0.05 mL Tween-20 and 3 g trehalose buffer solution, pH is 7.4; Store at 4℃ for later use.

[0041] Step 2, preparation of chromogenic substrate solution: dissolve S-2238 in DMSO to 10 mg / mL, then dilute with buffer to 0.25 mmol / L; Step 3, preparation of standard solution: take fibrinolytic enzyme standard, add buffer, dilute to a concentration of 5 U / mL; Step 4, preparation of test solution: dissolve the fibrinolytic enzyme sample to be tested with buffer, centrifuge at 1100 rpm for 8 min, take the supernatant, pass through a 0.22 μm PVDF filter membrane, and dilute to a concentration of 5 U / mL; Step 5, determination of ΔA value: incubate the chromogenic substrate solution in a water bath at 37℃±0.5℃ for 2-3 min, then mix the test solution or standard solution with the chromogenic substrate solution at a volume ratio of 1:18, use a UV spectrophotometer to detect the absorbance of the hydrolysis product, accurately read the absorbance A0 at 3 minutes and the absorbance A at 6 minutes, determine in parallel for 3 times, and obtain the average value of ΔA value (ΔA = A-A0) of the standard solution and the test solution; The potency of the fibrinolytic enzyme is calculated according to the following formula: Formula (1).

[0042] The determination method of the protein content (mass percentage) of the test product is as follows: Take about 50 mg of the product, accurately weigh, dissolve and dilute to the mark in a 50 mL volumetric flask with water, shake well, accurately take 5 mL into a 50 mL volumetric flask, dilute to the mark with water, shake well, and use as the test solution. According to the protein content determination method recorded in the Chinese Pharmacopoeia, calculate it, and you will get it.

[0043] Comparative Example 2 A method for determining the potency of the fibrinolytic enzyme of Agkistrodon acutus, changing the buffer to 0.1 mol / L Tri-imidazole buffer, pH 8.5.

[0044] comprising the following steps: Step 1, preparation of buffer solution: Tris 6.055 g, imidazole 3.404 g, 12 mol / L HCl 1.22 mL, constant volume to 500 mL.

[0045] The rest is consistent with example 1.

[0046] Comparative example 3 A method for determining the potency of the defibrase of Agkistrodon acutus, compared with example 1, the concentration of raw materials in the buffer solution in step 1 and the volume ratio in step 5 are changed.

[0047] Comprising the following steps: Step 1, preparation of buffer solution: 0.1 mol / L Tris-HCl solution, bovine serum albumin (BSA), Tween-20 are mixed to prepare a buffer solution containing 0.2 g bovine serum albumin (BSA), 0.06 mL Tween-20 and 1 g trehalose per 100 mL Tris-HCl solution, pH 7.4; Store at 4℃ for later use.

[0048] Step 2, preparation of chromogenic substrate solution: dissolve Bz-Phe-Val-Arg-pNA in DMSO to 10 mg / mL, then dilute with buffer to 0.25 mmol / L; Step 3, preparation of standard solution: take defibrase standard, add buffer, dilute to a concentration of 5 U / mL; Step 4, preparation of test solution: defibrase sample to be tested is dissolved with buffer, centrifuged at 1100 rpm for 8 min, the supernatant is passed through a 0.22 μm PVDF filter membrane, and then diluted to a concentration of 5 U / mL; Step 5, determination of ΔA value: incubate the chromogenic substrate solution in a water bath at 37℃±0.5℃ for 2-3 min, then mix the test solution or standard solution with the chromogenic substrate solution at a volume ratio of 1:12, use ultraviolet spectrophotometer to detect the absorbance of hydrolysis product, accurately read the absorbance A0 at 3 minutes and the absorbance A at 6 minutes, parallel determination for 3 times, and calculate the average value of ΔA value (ΔA= A- A0) of standard solution and test solution; The potency of defibrase is calculated according to the following formula: Formula (1).

[0049] Wherein, the determination method of the protein content (mass percentage) of the test sample is as follows: About 50 mg of the product was precisely weighed, dissolved and diluted to the mark in a 50 mL volumetric flask with water, shaken well, and 5 mL was precisely measured and diluted to the mark in a 50 mL volumetric flask with water, shaken well, as the test sample solution. According to the protein content determination method recorded in the Chinese Pharmacopoeia, the calculation was made, and the result was obtained.

[0050] The results of the Agkistrodon acutus venom titer (specific activity) of Examples 1-3 and Comparative Examples 1-3 are shown in Table 1 below, wherein the standard sample was tested by the method of Example 1.

[0051] Table 1. Agkistrodon acutus venom titer

[0052] From the data in Table 1, it can be seen that the specific determination method of the present application, especially in combination with the specific diluent and chromogenic substrate, has higher accuracy in titer determination. The ΔA mean value of all examples deviates less from the standard sample, and the titer error is small, especially for Examples 1-2. In Comparative Example 1, the chromogenic substrate was changed, in Comparative Example 2, the diluent was changed, and in Comparative Example 3, the concentration of the raw material in the buffer and the volume ratio in Step 5 were changed. The ΔA mean value deviated significantly from the standard sample, and the titer error was significantly increased. It is proved that the accuracy is significantly improved under the specific test method of the present application.

[0053] Finally, it should be noted that the above content is only used to illustrate the technical solutions of the present application, and is not a limitation on the protection scope of the present application. Simple modifications or equivalent replacements of the technical solutions of the present application made by those skilled in the art do not deviate from the essence and scope of the technical solutions of the present application.

Claims

1. A method for determining the potency of batroxobin, characterized in that, The method comprises the following steps: Step 1, preparing buffer solution: mixing 0.05-0.15 mol / L Tris-HCl solution, bovine serum albumin and Tween-20 to prepare a buffer solution containing 0.05-0.15 g bovine serum albumin, 0.03-0.05 mL Tween-20 and 2-4 g trehalose per 100 mL Tris-HCl solution, and the pH is 7.2-7.4; Step 2, preparing chromogenic substrate solution: dissolving Bz-Phe-Val-Arg-pNA with DMSO and then diluting with the buffer solution; Step 3, preparing standard solution: taking fibrinolysin standard product and adding the buffer solution to dilute; Step 4, preparing test sample solution: dissolving the fibrinolysin sample to be tested with the buffer solution to dilute; Step 5, calculating the fibrinolysin titer: Incubating the chromogenic substrate solution, mixing the test sample solution or the standard solution with the chromogenic substrate solution at a volume ratio of 1:15-20, detecting the absorbance of the hydrolysis product by using a UV spectrophotometer, reading the absorbance A0 at 3 minutes and the absorbance A at 6 minutes, obtaining the average value of the ΔA value of the standard solution or the test sample solution; determining the protein content W of the test sample according to the protein content determination method; and then calculating the fibrinolysin titer according to the ΔA and W, wherein W is the mass percentage.

2. The method of claim 1, wherein, In step 5, the formula for calculating the fibrinolysin titer is: Equation (1).

3. The method of claim 1, wherein, In step 1, the buffer solution is a buffer solution containing 0.1 g bovine serum albumin, 0.05 mL Tween-20 and 3 g trehalose per 100 mL Tris-HCl solution, and the pH is 7.

4.

4. The method of claim 1, wherein, In step 2, the dilution with the buffer solution is to dilute Bz-Phe-Val-Arg-pNA to a concentration of 0.2-0.3 mmol / L with the buffer solution.

5. The method of claim 1, wherein, In step 4, the dissolving is centrifugation, and the parameters are: 1000-1200 rpm, 5-10 min.

6. The method of claim 1, wherein, In step 4, the test sample solution needs to be filtered with a filter membrane, and the filter membrane is a PVDF filter membrane with a pore size of 0.2-0.24 μm.

7. The method of claim 1, wherein, In step 4, the dilution is to dilute the fibrinolysin sample to be tested to a concentration of 3-8 U / mL.

8. The method of claim 1, wherein, In step 5, the detection wavelength for detecting the absorbance of the hydrolysis product by using a UV spectrophotometer is 405 nm.

9. The method of claim 1, wherein, In step 5, the volume ratio is 1:

18.

10. The method of claim 1, wherein, In step 5, the determination method of the protein content of the test sample is as follows: Diluting the test sample with water to a concentration of 0.05-0.15 mg / mL, determining according to the protein content determination method, and calculating, so as to obtain the mass percentage of the protein of the test sample.

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