Method for rapidly detecting activity of human thrombin in human prothrombin complex

Abstract

The invention relates to a method for rapidly detecting the activity of human thrombin in a human prothrombin complex, and belongs to the technical field of biological product detection. The method comprises the following steps: diluting a human thrombin solution into a plurality of gradients in equal proportion, then adding a fibrinogen solution, standing for a period of time, observing whether clots or fibrin are separated out, and determining the corresponding human thrombin activity titer limit when the clots or fibrin are separated out; mixing the human prothrombin complex with a fibrinogen solution, and detecting the refractive index under the wavelength of 589 nm; and determining the activity of the human thrombin in the human prothrombin complex by comparing the refractive index values. According to the method, the original 24-hour test time is shortened to be within 1 hour, and the problem that the time of an existing method for testing the activity of the human thrombin in the human prothrombin complex is too long is solved.

Description

Technical Field

[0001] This invention relates to the technical field of biological product testing, and specifically to a method for rapidly detecting human thrombin activity in human prothrombin complex. Background Technology

[0002] Human prothrombin complex concentrate (PCC) is a biological product prepared from the plasma of healthy individuals for the treatment or prevention of certain bleeding disorders (such as vitamin K-dependent coagulation factor deficiency). It is purified by low-temperature ethanol protein separation or other approved separation methods, and then subjected to virus removal and inactivation treatment, followed by freeze drying. Its main components are human coagulation factor II (i.e., prothrombin), human coagulation factor VII, human coagulation factor IX, and coagulation factor X.

[0003] Human thrombin activity assays are of great significance for quality control in the production process of human prothrombin complex. (1) Production process control: Ensuring that the produced human prothrombin complex meets the predetermined standards is crucial for the safety and efficacy of clinical use. (2) Product purity: Testing can help determine the purity of the product, i.e., whether the human thrombin activity meets expectations, and whether there is contamination or other byproducts. (3) Therapeutic effect evaluation: Before clinical application, it is necessary to understand the level of human thrombin activity in order to adjust the dosage appropriately and ensure that patients receive appropriate treatment.

[0004] Abnormal human thrombin activity may lead to the following risks, thereby affecting the effectiveness and safety of human prothrombin complex: (1) Thrombosis: High thrombin activity promotes thrombus formation, which may lead to deep vein thrombosis (DVT), pulmonary embolism (PE), myocardial infarction, or stroke. (2) Microvascular thrombosis: Thrombus formation in microvessels may affect organ function, such as causing microvascular thrombosis in the kidneys, liver, or retina. (3) Disseminated intravascular coagulation (DIC): In some cases, excessive thrombin activity may lead to DIC, a serious coagulation disorder that can cause widespread thrombosis and bleeding. (4) Consumptive coagulation disorder: Excessive consumption of coagulation factors may lead to decreased levels of coagulation factors, resulting in a tendency to bleed. (5) Organ damage: Thrombus formation may obstruct the blood supply to organs, leading to organ damage or even failure. (6) Bleeding complications: Although high thrombin activity is usually associated with thrombosis, excessive coagulation may lead to an imbalance in the coagulation system, which may cause bleeding. (7) Treatment difficulties: For patients who need anticoagulation therapy, if the thrombin activity is too high, a higher dose of anticoagulant may be required, which increases the difficulty of treatment management and the risk of bleeding.

[0005] Therefore, accurate detection of thrombin activity in human prothrombin complex is of great significance for ensuring drug quality and patient safety.

[0006] Currently, the method for detecting human thrombin activity in human prothrombin complex concentrate is provided in Chapter 3422, "Human Thrombin Activity Test," of the 2020 edition of the Chinese Pharmacopoeia, Part III. This method is based on the principle that thrombin can coagulate human fibrinogen. The test sample is mixed with human fibrinogen, and the presence or absence of clots or fibrin precipitation is observed to determine whether the thrombin activity of the test sample exceeds the limit. This method is time-consuming, with a single test operation taking approximately one hour and an observation period of 24 hours. This may affect the accuracy of the experimental results, experimental efficiency, and clinical decision-making. The following are the main drawbacks of this method: (1) Sample stability issues: Long experimental processes may lead to sample degradation or deterioration, affecting the accuracy of experimental results. Environmental conditions need to be considered to maintain sample stability, increasing the complexity of the experiment. (2) Resource consumption: Long-duration experiments usually require more reagents, instrument usage time and laboratory space, increasing experimental costs. At the same time, more human resources are needed to monitor the experimental process, especially when continuous monitoring is required. (3) Low time efficiency: Long experimental cycles will delay data analysis, limit the flexibility of experimental design and the throughput of a single experiment, and make it difficult to conduct large-scale experiments. Small changes in experimental conditions may require restarting the entire experiment, resulting in a large waste of time. (4) Poor data synchronization: Long-duration experiments may lead to poor data synchronization, increasing the difficulty of data analysis. When multiple sets of experiments are conducted simultaneously, long-duration experiments may lead to difficulties in data collection and integration. (5) Increased experimental risks: Unexpected experimental failures may occur during long-duration experiments, such as instrument failure and reagent contamination. These risks increase with time. On the other hand, environmental changes during the experimental process (such as temperature and humidity) may also affect the experimental results.

[0007] Therefore, it is necessary to design a method for detecting human thrombin activity in human prothrombin complexes. This method should optimize the experimental procedure, reduce unnecessary steps, and adopt more stable processing methods to ensure the accuracy and reliability of the experimental results. Summary of the Invention

[0008] To address the problems of long detection times leading to poor accuracy and data synchronization in existing methods for detecting human thrombin activity in human prothrombin complex, this invention provides a rapid method for detecting human thrombin activity in human prothrombin complex to solve these problems.

[0009] The technical solution of this invention is as follows: A rapid method for detecting human thrombin activity in human prothrombin complex concentrate involves diluting human thrombin solution into multiple gradients in equal proportions, then adding fibrinogen solution, and observing for clots or fibrin precipitation after a period of time to determine the potency limit of human thrombin activity. The human prothrombin complex concentrate is then mixed with the fibrinogen solution, and the refractive index is measured at a wavelength of 589 nm for 0.5 hours. The human thrombin activity in the human prothrombin complex concentrate is determined by comparing the refractive index values.

[0010] Specifically, the following steps are included: (1) Preparation of positive control 1 and negative control 1 Human thrombin solution was diluted to 0.5 IU / ml with physiological saline solution. Then, human thrombin solution was diluted in multiple gradients according to a human thrombin solution: physiological saline ratio of 1:4 (volume ratio). 0.2 ml of each gradient of human thrombin solution was taken, and 0.2 ml of 0.5% fibrinogen solution was added. The mixture was incubated at 37 ℃ for 24 hours, and the presence or absence of clots or fibrin precipitation was observed. The sample with the lowest concentration of clots or fibrin precipitation was used as positive control one, and the sample with the highest concentration of no clots or fibrin precipitation was used as negative control one. (2) Human thrombin activity standard: The relative deviation of the refractive index of positive control 1 from that of negative control 1 should be greater than 5%; the relative deviation of the refractive index of the test sample from that of negative control 1 should not be greater than 5%. (3) Take 0.5~1.5 ml of human prothrombin complex solution, add an equal volume of 0.5% human fibrinogen solution, place at 37 ℃ for 25~35 minutes, and then use a refractometer in total solids mode to detect the refractive index at a wavelength of 589 nm and an accuracy of ±0.00001 nD.

[0011] Furthermore, in step (1), the human thrombin solution is diluted into 10 gradients.

[0012] Furthermore, when the human prothrombin complex solution contains heparin, protamine sulfate solution is added for neutralization.

[0013] Furthermore, the protamine sulfate solution was prepared by dissolving protamine sulfate in Tris buffer at pH 7.5 and then diluting it.

[0014] Furthermore, 1 IU heparin was neutralized with 10 μg of protamine sulfate.

[0015] Furthermore, in step (3), the amount of human prothrombin complex solution added is 1.0 ml, and the amount of 0.5% human fibrinogen solution added is 1.0 ml.

[0016] Furthermore, in step (3), after adding an equal volume of 0.5% human fibrinogen solution, the mixture is placed at 37°C for 30 minutes.

[0017] The beneficial effects of this invention are as follows: The present invention provides a method for rapid detection of human thrombin activity in human prothrombin complex, which shortens the original 24-hour test time to within 1 hour and can rapidly and quantitatively detect human thrombin activity in human prothrombin complex, thus solving the problem of excessively long test time in existing tests for human thrombin activity in prothrombin complex. Detailed Implementation

[0018] To enable those skilled in the art to better understand the technical solutions of this invention, the technical solutions in the embodiments of this invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of this invention, and not all embodiments. Based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without creative effort should fall within the scope of protection of this invention.

[0019] Example 1 Human thrombin activity gradient test (1) Take human thrombin solution and dilute it to 0.5 IU / ml with physiological saline. Then, dilute it to 10 gradients at a human thrombin solution: physiological saline ratio of 1:4 (volume ratio). The human thrombin titers for each gradient are 0.5 IU / ml, 0.1 IU / ml, 0.02 IU / ml, 0.004 IU / ml, 0.0008 IU / ml, and 1.6 × 10⁻⁶ IU / ml. -4 IU / ml, 3.2×10 -5 IU / ml, 6.4×10 -6 IU / ml, 1.28×10 -6 IU / ml, 2.56×10 -7 IU / ml.

[0020] (2) Take 0.2 ml of each gradient of human thrombin solution, add 0.2 ml of 0.5% fibrinogen solution, and incubate at 37°C for 24 hours. Observe whether clots or fibrin precipitation occur. The sample with the lowest concentration of clots or fibrin precipitation is used as positive control 1, and the sample with the highest concentration of no clots or fibrin precipitation is used as negative control 1. The results of the human thrombin activity gradient test are shown in Table 1 below: Table 1 - Results of Human Thrombin Activity Gradient Test

[0021] Based on the test results in Table 1, the minimum concentration with clots or fibrin precipitation is 0.004 IU / ml, and this sample serves as positive control one. The maximum concentration without clots or fibrin precipitation is 0.0008 IU / ml, and this sample serves as negative control one.

[0022] Human thrombin activity standard: The relative deviation of the refractive index of positive control 1 from that of negative control 1 should be greater than 5%; the relative deviation of the refractive index of the test sample from that of negative control 1 should not be greater than 5%.

[0023] Example 2 Detection of human thrombin activity in human prothrombin complex (1) Preparation of 0.5% human fibrinogen solution: Take lyophilized human fibrinogen, equilibrate to room temperature and then reconstitute it. Dilute the human fibrinogen solution to 5 mg / ml with physiological sodium chloride solution.

[0024] (2) Preparation of human thrombin solution: Take lyophilized human thrombin, equilibrate to room temperature and then reconstitute it, and dilute the human thrombin to 0.5 IU / ml with physiological sodium chloride solution.

[0025] (3) Preparation of test solution: After equilibrating the lyophilized human prothrombin complex to room temperature, reconstitute it. Based on the heparin content of the human prothrombin complex, neutralize 1 ml of human prothrombin complex with 150 μg of protamine sulfate.

[0026] (4) Negative control 2: 0.2 ml physiological sodium chloride solution; Positive control 2: 0.2 ml human thrombin solution (0.5 IU / ml).

[0027] (5) Test method: Take 1 ml of human prothrombin complex solution as the test solution, add 1 ml of 0.5% human fibrinogen solution to it, place at 37 ℃ for 30 minutes, and then use a refractometer in total solids mode to detect its refractive index at a wavelength of 589 nm with an accuracy of ±0.00001 nD. The negative control and positive control were performed in the same way as the test solution.

[0028] (6) Result judgment: The relative deviation between the refractive index of positive control 2 and negative control 2 should be greater than 5%, otherwise the experiment is not valid; the relative deviation between the refractive index of the test sample and negative control 2 should not be greater than 5%, otherwise it is judged as unqualified.

[0029] Test case 1. Testing the robustness of the detection method in Example 2. Test objective: To confirm the degree to which test results are unaffected by minor changes in measurement conditions; the relative standard deviation of the three refractive index measurements in each test group should not exceed 5%.

[0030] 1. Effects of different amounts of protamine sulfate on the results of human thrombin activity assay in human prothrombin complex. Three tests were conducted using the same batch of human prothrombin complex, wherein steps (1), (2), (4) and (5) were the same as in Example 2, except that in step (3), 140 μg of protamine sulfate, 150 μg of protamine sulfate and 160 μg of protamine sulfate were added to the test solution to neutralize the heparin in the test solution.

[0031] The results of the three batches of experiments are shown in Table 2 below: Table 2 - Test results of different amounts of protamine sulfate added

[0032] 2. Effect of incubation time after adding 0.5% human fibrinogen solution on the results of human thrombin activity assay in human prothrombin complex. Three tests were conducted using the same batch of human prothrombin complex. Steps (1) to (4) were the same as in Example 2. The difference was that in step (5), after adding 0.5% human fibrinogen solution, the mixture was placed at 37 °C for 25 minutes, 30 minutes and 35 minutes respectively.

[0033] The results of the three batches of experiments are shown in Table 3 below: Table 3 - Test results of standing time after adding 0.5% human fibrinogen solution

[0034] 3. Effect of the addition amount of human prothrombin complex concentrate and 0.5% human fibrinogen solution on the results of human thrombin activity assay in human prothrombin complex concentrate. Three tests were conducted using the same batch of human prothrombin complex. Steps (1) to (4) were the same as in Example 2. The difference was that in step (5), the amounts of human prothrombin complex and 0.5% human fibrinogen solution added were 0.5 ml:0.5 ml, 1.0 ml:1.0 ml, and 1.5 ml:1.5 ml, respectively.

[0035] The results of the three batches of experiments are shown in Table 4 below: Table 4 - Test results of different amounts of prothrombin complex solution added to 0.5% human fibrinogen solution

[0036] It can be seen that the test results of this invention are not affected by minor changes in the measurement conditions.

[0037] II. Test the repeatability of the detection method in Example 2. 1. Repeatability of test results Test objective: To confirm that, under the same experimental conditions, there is no significant difference in human prothrombin complex thrombin activity measured by the same test personnel.

[0038] Human prothrombin complex samples were collected, and the same researcher performed six human thrombin activity tests on each sample. The testing method was the same as in Example 2. The results of the six batches of experiments are shown in Table 5 below: Table 5 - Test Results of Repeated Trials

[0039] 2. Intermediate precision Test objective: To confirm that there are no significant differences in the results of multiple sampling tests on different dates when using this method to detect human thrombin activity.

[0040] The same batch of human prothrombin complex concentrate national standard was tested on six different dates. The test method was the same as in Example 2. The results of the six batches are shown in Table 6 below: Table 6 - Test Results of Human Prothrombin Complex Standards with Different Production Dates

[0041] It can be seen that the test method of the present invention is repeatable.

[0042] Although the present invention has been described in detail by way of preferred embodiments, the present invention is not limited thereto. Various equivalent modifications or substitutions can be made to the embodiments of the present invention by those skilled in the art without departing from the spirit and essence of the invention, and such modifications or substitutions should all be within the scope of the present invention. Any variations or substitutions that can be easily conceived by those skilled in the art within the technical scope disclosed in the present invention should also be covered within the protection scope of the present invention.

Claims

1. A method for rapid detection of human thrombin activity in human prothrombin complex, characterized in that, Human thrombin solution was diluted in multiple gradients, and then fibrinogen solution was added. After standing for a period of time, the presence of clots or fibrin precipitation was observed to determine the potency limit of human thrombin activity. Then, the refractive index of the human prothrombin complex was measured at a wavelength of 589 nm after mixing with the fibrinogen solution for 0.5 hours. The human thrombin activity in the human prothrombin complex was determined by comparing the refractive index values.

2. The method for rapid detection of human thrombin activity in human prothrombin complex as described in claim 1, characterized in that, Includes the following steps: (1) Preparation of positive control 1 and negative control 1 Human thrombin solution was diluted to 0.5 IU / ml with physiological saline solution. Then, human thrombin solution was diluted in multiple gradients according to a human thrombin solution: physiological saline ratio of 1:4 (volume ratio). 0.2 ml of each gradient of human thrombin solution was taken, and 0.2 ml of 0.5% fibrinogen solution was added. The mixture was incubated at 37 ℃ for 24 hours, and the presence or absence of clots or fibrin precipitation was observed. The sample with the lowest concentration of clots or fibrin precipitation was used as positive control one, and the sample with the highest concentration of no clots or fibrin precipitation was used as negative control one. (2) Human thrombin activity standard: The relative deviation of the refractive index of positive control 1 from that of negative control 1 should be greater than 5%; the relative deviation of the refractive index of the test sample from that of negative control 1 should not be greater than 5%. (3) Take 0.5~1.5 ml of human prothrombin complex solution, add an equal volume of 0.5% human fibrinogen solution, place at 37℃ for 25~35 minutes, and then use a refractometer in total solids mode to detect the refractive index at a wavelength of 589 nm with an accuracy of ±0.00001 nD.

3. The method for rapid detection of human thrombin activity in human prothrombin complex as described in claim 2, characterized in that, In step (1), the human thrombin solution is diluted into 10 gradients.

4. The method for rapid detection of human thrombin activity in human prothrombin complex as described in claim 2, characterized in that, When human prothrombin complex solution contains heparin, protamine sulfate solution is added for neutralization.

5. The method for rapid detection of human thrombin activity in human prothrombin complex as described in claim 4, characterized in that, The protamine sulfate solution was prepared by dissolving protamine sulfate in Tris buffer at pH 7.5 and then diluting it.

6. The method for rapid detection of human thrombin activity in human prothrombin complex as described in claim 4, characterized in that, 1 IU heparin was neutralized with 10 μg protamine sulfate.

7. The method for rapid detection of human thrombin activity in human prothrombin complex as described in claim 2, characterized in that, In step (3), the amount of human prothrombin complex solution added is 1.0 ml, and the amount of 0.5% human fibrinogen solution added is 1.0 ml.

8. The method for rapid detection of human thrombin activity in human prothrombin complex as described in claim 2, characterized in that, In step (3), after adding an equal volume of 0.5% human fibrinogen solution, the mixture is placed at 37°C for 30 minutes.

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