A method for in vitro determination of the potency of a plasminogen activator

Abstract

The application provides a method for determining the titer of plasminogen activator in vitro, specifically determining the thrombolytic activity of the plasminogen activator by using a full-automatic coagulation analyzer, comprising: diluting the sample to be detected to a protein concentration of 5 μg / mL by using a sample diluent to obtain a sample stock solution; diluting the sample stock solution to a sample dilution series solution, and determining the clot lysis time by using the full-automatic coagulation analyzer; the sample diluent is a phosphate buffer diluted human blood albumin, the pH of the phosphate buffer is 7.4, and the final concentration of the human blood albumin is 5 mg / mL. The application also provides a corresponding kit and application thereof. The method has high specificity, good accuracy and precision, and makes technical reserves for improving the controllability of product quality and the establishment of related standard substances.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to a method for in vitro determination of plasminogen activator potency. Background Technology

[0002] Thrombosis is the common pathological basis of the three major cardiovascular diseases worldwide (myocardial infarction, stroke, and venous thromboembolism). Cardiovascular diseases are characterized by high incidence, high disability rate, and high mortality rate. Intravenous thrombolysis is an important means of treating thrombotic diseases. Plasminogen activators (PAA) can directly or indirectly activate plasminogen into plasmin, thereby dissolving fibrin clots in thrombi. The rapid development of the domestic PAA industry has placed higher demands on product quality. However, key quality attributes, such as the potency assay reflecting thrombolytic effectiveness, still rely on the traditional bubble-rising method. This method requires a special visual water bath device, and the reaction endpoint (the time it takes for the last bubble to rise to the surface of the reaction solution) must be determined visually using a stopwatch. A linear regression is performed with the logarithm of the standard solution potency as the x-axis and the logarithm of the reaction endpoint time as the y-axis. The potency of the PAA sample is calculated using the linear regression equation. Only a single concentration of standard or sample can be tested at a time, and a single batch test takes 2 hours, resulting in problems such as being cumbersome, time-consuming, having low throughput, and large errors.

[0003] Enzyme potency is a key quality attribute of plasminogen activator products, reflecting the integrity of their clinical mechanism of action, biological function, and batch-to-batch consistency. Currently, differences in host cells and manufacturing processes used by different manufacturers result in significant variations in the active units of plasminogen activator products in clinical applications, making standardization difficult and easily causing confusion in clinical medication. This substantial difference may pose potential problems for precise clinical medication, thereby affecting the clinical treatment of stroke patients. Establishing accurate, sensitive, and efficient potency evaluation methods is of great significance for the quality control of this type of enzyme.

[0004] In existing technologies, bovine serum albumin (BSA) is commonly used as a stabilizer in dilution buffers for determining the potency or activity of plasminogen activator. However, human serum albumin (HSA) and BSA differ substantially in amino acid sequence (homology is only about 76%), three-dimensional conformation, subunit structure (HSA is a single subunit, BSA is a tri-subunit), physicochemical properties (such as differences in stability under acidic conditions), and drug binding affinity. Previous studies have shown that the structural and functional differences between serum albumins from different species cannot be resolved by simple equivalent substitution. Therefore, choosing a specific albumin as a stabilizer for dilution systems of plasminogen activator is not a conventional approach in this field, but rather requires experimental validation based on specific application scenarios. Summary of the Invention

[0005] To address the aforementioned problems, this invention provides a method for in vitro determination of plasminogen activator potency. By using this method to determine enzyme activity and potency, a unified standard substance can be used to transfer enzyme activity units with the same definition to different plasminogen activator products, thereby accurately and efficiently unifying enzyme activity units and completely avoiding the difficulties in clinical medication.

[0006] To achieve the above objectives, the specific technical solution provided by the present invention is as follows: The first aspect of this invention provides a method for determining the potency of plasminogen activator in vitro.

[0007] In this invention, "potency" or "activity" refers to the unit of efficacy of a substance in evoking a biological response. In some embodiments, potency refers to the ability of a plasminogen activator to activate plasminogen.

[0008] Furthermore, the in vitro determination of plasminogen activator potency was performed using a fully automated coagulation analyzer.

[0009] In this invention, plasminogen activator refers to a plasminogen activator that generates active plasmin. Plasminogen activators include plasmin (PL), tissue plasminogen activator (t-PA), urokinase plasminogen activator (u-PA), streptokinase (SK), urokinase (UK), high molecular weight kininogen (HMWK), glass connexin (VN), and recently developed second-generation thrombolytic drugs such as recombinant tissue plasminogen activator (i.e., alteplase, rt-PA) and genetically modified variants of the third-generation thrombolytic drug rt-PA, tenecteplase (TNK) and reteplase (rPA).

[0010] Reteplase is a deletion variant of non-glycosylated tissue plasminogen activator (tPA). This protease is obtained from inactive inclusion bodies in Escherichia coli. After folding in vitro, it transforms into its active form, which can specifically bind to fibrin and convert it into an active plasmin, thereby degrading fibrin, dissolving existing thrombi, and achieving the purpose of clearing blood vessels.

[0011] TNK is derived by replacing three amino acid sites in the alteplase molecule: threonine at position 103 is replaced by asparagine, asparagine at position 117 is replaced by glutamine, and amino acids 296-299 are replaced by four alanine residues. It exhibits high fibrin specificity. It binds to fibrin via lysine residues, activating plasminogen bound to fibrin to convert into plasmin, thereby dissolving blood clots. This effect is significantly enhanced compared to activation of circulating plasminogen, and it selectively activates plasminogen, thus avoiding the bleeding complications commonly associated with streptokinase administration.

[0012] Furthermore, the plasminogen activator is any one of tenecteplase, reteplase, and alteplase.

[0013] Furthermore, the steps of the method include: The sample to be tested was diluted with sample diluent to a protein concentration of 5 μg / mL to obtain a sample stock solution. The sample stock solution was then diluted to a series of sample dilution solutions. The clot dissolution time of the series of sample dilution solutions was measured using an automated coagulation analyzer. The enzyme titer was calculated using the external standard method according to the following formula: Enzyme activity (IU / ml) = average protein concentration (mg / ml) × specific activity of standard (IU / mg), Specific activity (IU / mg) = enzyme activity (IU / ml) × protein concentration (mg / ml).

[0014] Furthermore, the sample to be tested is a plasminogen activator.

[0015] In some embodiments, the sample to be tested includes a commercially available or self-made plasminogen activator product.

[0016] Furthermore, the plasminogen activator includes any one of tenecteplase, reteplase, urokinase, streptokinase, tissue plasminogen activator, and alteplase.

[0017] Furthermore, the plasminogen activator is any one of tenecteplase, reteplase, and alteplase.

[0018] Furthermore, the sample diluent is human serum albumin diluted with phosphate buffer.

[0019] Furthermore, the pH of the phosphate buffer solution is 7.4.

[0020] Furthermore, the final concentration of the human serum albumin is 5 mg / mL.

[0021] Furthermore, the sample diluent is prepared as follows: 80 mL of phosphate buffer is taken, 0.5 g of human serum albumin is added, and the solution is diluted to 100 mL with phosphate buffer. After being fully dissolved, it is stored at 4°C.

[0022] Furthermore, the measurement steps of the fully automated coagulation analyzer are as follows: using a mixed solution as the starting reagent and human plasma thrombin solution as the intermediate reagent, the clot dissolution time of the sample dilution series solutions is measured after setting the parameters.

[0023] Furthermore, the instrument parameters of the fully automated coagulation analyzer are as follows: sample solution volume is 20 μL, initial reagent volume is 200 μL, intermediate reagent volume is 20 μL, reaction temperature is 37±1℃, detection wavelength is 405 nm, measurement time is 900 s, delay time is 10 s, the calculation algorithm is threshold method, the threshold method is curve percentage, the threshold limit is 10%, and the acquisition direction is reverse.

[0024] Furthermore, the concentrations of the sample dilution series solutions are 800 ng / mL, 1000 ng / mL, and 1200 ng / mL.

[0025] Furthermore, the method for preparing the human plasma thrombin solution is as follows: the human plasma thrombin lyophilized powder is reconstituted with water and diluted to 33 U / mL with sample diluent.

[0026] Furthermore, the mixed solution is a mixture of human plasma fibrinogen solution and human plasma plasminogen solution, and the final concentration of the mixed solution is 1:50 (v / v).

[0027] Furthermore, the fully automated coagulation analyzer is model ACL TOP 750.

[0028] A second aspect of the present invention provides a kit for determining the potency of plasminogen activator, the kit comprising the reagents used in the method described in the first aspect of the present invention.

[0029] Furthermore, the kit also includes instructions.

[0030] The third aspect of the present invention provides the application of the method described in the first aspect of the present invention and / or the kit described in the second aspect of the present invention in determining the potency of plasminogen activator.

[0031] Advantages and beneficial effects of the present invention: The present invention provides a method for in vitro determination of plasminogen activator, which is simple to operate, improves detection efficiency, reduces human operation error, and has high accuracy and precision. It can be used as a routine method for determining the potency of plasminogen activator drugs, and provides technical reserves for improving the quality controllability of products and establishing relevant standard substances. Attached Figure Description

[0032] Figure 1 The figure shows the methodological validation results for determining plasminogen activator titer using a fully automated coagulation analyzer. In the figure, A is the coagulation curve of the reteplase sample reaction solution, B is the coagulation curve of human serum albumin in the reteplase control group, C is the coagulation curve of the reteplase blank group prescription buffer, D is the coagulation curve of the tenecteplase sample A reaction solution, E is the coagulation curve of human serum albumin in the tenecteplase control group, and C is the coagulation curve of the tenecteplase blank group prescription buffer.

[0033] Figure 2 The figures show the standard curve and linearity test results for plasminogen activator potency determination. In the figure, A is the standard curve for reteplase, B is the standard curve for tenecteplase, C is the linearity test result for reteplase, and D is the linearity test result for tenecteplase.

[0034] Figure 3 Figure showing the consistency results between the Bland-Altman analysis of the fully automated coagulation analyzer method and the bubble rise method.

[0035] Figure 4 The images show the full-sequence mass spectra of four plasminogen activators (red indicates the matched amino acid sequences), where A is tenecteplase A, B is tenecteplase B, C is alteplase, and D is reteplase.

[0036] Figure 5 The HILIC curve for tenecteplase standard A.

[0037] Figure 6 The HILIC curve for tenecteplase standard B.

[0038] Figure 7 The graph shows the activity results of 12 batches of TNK products with two glycosylation modifications, as measured by a fully automated coagulation analyzer. Detailed Implementation

[0039] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0040] Example I. Experimental Materials 1. Instrument Consumables Electronic balance (METTLER TOLEDO, USA, model: ME166DU); Pure water system (MILLIPORE, USA, model: Milli-Q IQ7000); Automated coagulation analyzer (WERFEN, Spain, model: ACLTOP750); Orbitrap Exploris 480 mass spectrometer (Thermo Fisher Scientific, USA); Vanquish liquid chromatograph (Thermo Fisher Scientific, USA); BioPharma Finder data processing software (Thermo Fisher Scientific, USA); UPLC I-Class / Synapt G2-Si liquid chromatography-mass spectrometry (Waters, USA); UNIFI workstation (Waters, USA); Ultraviolet spectrophotometer (SHIMADZU, Japan, model: UV-2700); pH meter (METTLER TOLEDO, USA, model: S470-K); Low-temperature high-speed centrifuge ST8R (Thermo Fisher Scientific, USA); Constant temperature water bath (Thermo Fisher Scientific, USA). Scientific Corporation; centrifugal concentrator (Thermo Fisher Scientific, USA).

[0041] Blood coagulation analyzer sample cups (WERFEN, Spain, 2 mL, catalog number: 5575100); ACL TOP colorimetric analysis cups for blood coagulation analyzers (WERFEN, Spain, catalog number: 29400100); glass reagent bottles for blood coagulation analyzers (Beijing Jinyuchen Medical Equipment Co., Ltd., 10 mL and 20 mL); chromatographic column ACQUITY UPLC Peptide BEH C18 (2.1 mm × 100 mm, 130 Å, 1.7 μm, Waters); chromatographic column ACQUITY UPLC Glycan BEH Amide (2.1 mm × 150 mm, 130 Å, 1.7 μm, Waters); Supelco Supelclean ENVI-Carb SPE tubes (Sigma Aldrich).

[0042] 2. Experimental reagents Tenecteplase Standard A (31100 IU / vial, 0.0527 mg / vial, 0.1 ml / vial), batch number: 20141200102-S, CSPC Mingfule (Guangzhou) Co., Ltd.; Tenecteplase Standard B (200 U / mg, approximately 5 mg / ml, 0.25 ml / vial), batch number: 12639515, Boehringer Ingelheim International GmbH, Germany; Tenecteplase for injection (1.0 × 10⁻⁶ IU / vial, approximately 5 mg / ml, 0.25 ml / vial), batch number: 12639515, Boehringer Ingelheim International GmbH, Germany; 7Tenecteplase stock solution A, batch numbers: 20230715T, 20230729T, 20230724T, CSPC Mingfule (Guangzhou) Co., Ltd.; Tenecteplase for injection (5000U / vial, 25mg / vial) B, Boehringer Ingelheim International GmbH, Germany, batch numbers: 205504, 205505, 205851; Tenecteplase stock solution B, Boehringer Ingelheim International GmbH, Germany. GmbH, batch numbers: 12630045, 12630050, 12630052; Reteplase standard (5 million U / vial, 8.9 mg / vial), batch number: 20230401, lyophilized powder, AIDE Pharmaceutical (Beijing) Co., Ltd.; Reteplase for injection (8.9 mg / vial), batch number: 20220401, AIDE Pharmaceutical (Beijing) Co., Ltd.; Alteplase standard (0.98 mg / ml, 580,000 IU / mg), batch number: WS-03-12213859, Boehringer Ingelheim International GmbH, Germany; Alteplase for injection (20 mg / vial), batch number: 302182, Boehringer Ingelheim International GmbH, Germany; Human serum albumin, Sigma-Aldrich, USA, batch number: 1003095609; Human plasma thrombin, EMD, USA Millipore, 1000 U per vial, batch number: 3468951, lyophilized powder; Human plasma plasminogen (containing at least 90% coagulable protein), EMD Millipore, USA, 500 mg per vial, batch number: 3171544; Human plasma plasminogen, EMD Millipore, USA, 100 mg per vial, containing 1 mg per ml.5 mg, batch number: 4037914; RapiGest SF protease surfactant, Waters Laboratories, USA, batch number: 186001860; Trypsin, Roche Laboratories, USA, batch number: 54734422; 2-AB Dextran Calibration Ladder, Waters Laboratories, USA, batch number: 186006841; PNGase F, Aglient Technologies, USA, batch number: DG87-503C; Phosphate, sodium dihydrogen phosphate dihydrate, and disodium hydrogen phosphate dihydrate were purchased from China National Pharmaceutical Group Chemical Reagent Co., Ltd.; Coagulation analyzer cleaning solution A, Werfen, Spain, batch number: 09831700; Coagulation analyzer cleaning solution B, Werfen, Spain, batch number: 09832700; Coagulation analyzer rinsing solution, Werfen, Spain, batch number: 20302400; Acetonitrile, Solvslichro The following ingredients were purchased from Sigma-Aldrich (USA): polysorbate 20, Tween 80, L-arginine, formic acid, dithiothreitol, iodoacetamide, isopropanol, 2-aminobenzamide, 25% ammonium hydroxide solution, and 2-mercaptoethanol; ammonium bicarbonate (USA), Aladdin (USA), batch number: D2326118; triethylamine (USA), Honeywell (USA), batch number: 65897; sodium cyanoborohydride and acetic acid (Germany), Merck (Germany); PNGase F (USA), New England Biolabs (USA), batch number: P0704L; dimethyl sulfoxide and 20% sodium dodecyl sulfate (SDS) stock solution (Germany), Roth (Germany); and 10% NP40 (Brazil), Biorigin (Brazil), batch number: BN24524.

[0043] 3. Reagent preparation Phosphate buffer (pH 7.4): Weigh 3.56 g of sodium dihydrogen phosphate dihydrate and 17.30 g of disodium hydrogen phosphate dihydrate, dissolve in 1500 ml of ultrapure water, add 2.0 ml of 10% Tween 80 solution (v / v), adjust the pH to 7.402 with phosphate, dilute with water to 2 L, and mix thoroughly.

[0044] Sample dilution buffer: Measure 80 ml of phosphate buffer (pH 7.4), add 0.5 g of human serum albumin, dilute with phosphate buffer to 100 ml, and store at 4°C after complete dissolution. In this example, the final concentration of human serum albumin in the sample dilution buffer was preferably determined to be 5 mg / mL. Under this concentration condition, the determination results of each batch of samples showed good repeatability and stability.

[0045] Prescription buffer: Weigh 55 g of arginine, add 0.4 ml of 10% Tween 80 solution, add 800 ml of water to dissolve completely, then dilute with water to 1 L, and adjust the pH to 7.4 with phosphate solution.

[0046] Human plasma thrombin solution: Redissolve human plasma thrombin lyophilized powder in 1 mL of water, dilute with sample diluent to obtain a solution containing 33 U per 1 mL, and store at 4 °C.

[0047] Human plasma plasminogen solution: After equilibration to room temperature, store at 4°C.

[0048] Human plasma fibrinogen solution: Take one vial of human plasma fibrinogen and dilute it with sample diluent to obtain a solution containing 2 mg of coagulating fibrinogen per 1 ml. Prepare fresh before use.

[0049] Mixed solution: Take 35 ml of human plasma fibrinogen solution and add 700 µl of human plasma plasminogen solution and mix well to obtain a 1:50 (v / v) mixed solution. Prepare fresh before use.

[0050] RapiGest SF solution: Take one vial of RapiGest SF (1mg / vial), add 1ml of ultrapure water, mix well and it is ready for use. Prepare fresh before use.

[0051] Trypsin solution: Take one vial of Trypsin (1mg / vial), add 1ml of ultrapure water, mix well and it is ready to use. Prepare fresh before use.

[0052] Reducing agent: Take 1 ml of 20% sodium dodecyl sulfate (SDS) stock solution, add 9 ml of water, then add 558.07 μl of 2-mercaptoethanol and mix well.

[0053] 2-AB labeled solution: Take 700 μl of dimethyl sulfoxide, add 300 μl of concentrated acetic acid, cool, add 0.05 g of 2-aminobenzamide, and then transfer the entire dissolved 2-aminobenzamide solution to an EP tube containing 0.06 g of sodium cyanoborohydride.

[0054] Adjust the solution: Take 400ml of isopropanol, add 2ml of triethylamine, add 598ml of water, mix well and it is ready to use. Prepare fresh before use.

[0055] II. Experimental Methods 1. Preparation of sample series solutions 1.1 Reteplase: Take one vial of reteplase for injection and dilute it with 5 mL of sterile water for injection to prepare a solution containing approximately 1.78 mg of reteplase per mL. Take an appropriate amount of the above reteplase formulation solution and reteplase stock solution, and use a UV spectrophotometer to scan for the maximum absorption wavelength in the range of 240–550 nm. Using the formulation buffer as a blank, read the absorbance value A at the maximum absorption wavelength and at a wavelength of 320 nm. max and A 320 Calculate A max With A 320 The difference, with an extinction coefficient of 1.69 (1 mg / ml recombinant rPA in this buffer solution A) max -A 320 Calculate the protein content of the above formulation and stock solution using the absorbance value. Accurately measure an appropriate amount of the above solution and gradually dilute it with sample diluent to a protein concentration of 5 µg / ml to obtain the sample stock solution.

[0056] 1.2 Tenecteplase: Take one vial of tenecteplase A for injection and dilute it with 3 ml of sterile water for injection to a solution containing approximately 5.5 mg of tenecteplase per ml. Take appropriate amounts of the prescription solution, the reconstituted tenecteplase A preparation, and the stock tenecteplase A solution, and use a UV spectrophotometer with the prescription buffer as a blank to read the absorbance values ​​at wavelengths of 280 nm and 320 nm. 280 and A 320 Calculate A 280 With A 320 The difference, with an extinction coefficient of 1.9 (1 mg / ml recombinant tenecteplase in this buffer solution A) 280 -A 320 Calculate the protein content of the above formulation and stock solution using the absorbance value. Accurately measure an appropriate amount of the above solution and gradually dilute it with sample diluent to a protein concentration of 5 µg / ml to obtain the sample stock solution.

[0057] 1.3 Take 800, 1000, and 1200 µl of reteplase sample stock solution and tenecteplase sample stock solution respectively, add 4.2, 4.0, and 3.8 ml of sample diluent, and dilute to a protein concentration of 800 ng / ml, 1000 ng / ml, and 1200 ng / ml respectively. Prepare two copies of each concentration, and measure each solution in three parallel trials.

[0058] 2. Preparation of standard curve solutions 2.1 Reteplase: Take one vial of reteplase standard and prepare a standard stock solution with a protein concentration of 5 µg / ml as described in 1.1.

[0059] 2.2 Tenecteplase: Take one vial of tenecteplase A standard and dilute it with 0.1 ml of sterile water for injection to prepare a solution containing approximately 0.527 mg of tenecteplase per ml. Prepare a standard stock solution with a protein concentration of 5 µg / ml according to the method in "1.2".

[0060] 2.3 Take 600, 800, 1000, 1200, and 1400 µl of reteplase standard stock solution and tenecteplase A standard stock solution, respectively, and add 4.4, 4.2, 4.0, 3.8, and 3.6 ml of sample diluent to obtain linear standard solutions with protein concentrations of 600 ng / ml, 800 ng / ml, 1000 ng / ml, 1200 ng / ml, and 1400 ng / ml, respectively. Prepare two replicates for each standard concentration, and perform three replicates for each standard curve solution.

[0061] 3. Determination of enzyme activity using a fully automated blood coagulation analyzer Using the mixed solution as the starting reagent and human plasma thrombin solution as the intermediate reagent, set the instrument parameters according to Table 1. Add 1 mL of the standard curve (sample) series solution to the sample cup. Place the sample cup, starting reagent, intermediate reagent, and washing solution bottle in the appropriate positions on the reagent rack. In the ACL TOP version 5.3.0 software, select the corresponding reagent name and placement position. After the instrument recognizes the reagent, start the clot dissolution time measurement.

[0062] Table 1. ACL TOP 750 System Parameters

[0063] A linear regression analysis was performed with the logarithm of the dissolution time (seconds) of the standard curve solution as the ordinate and the logarithm of the protein concentration (ng / ml) as the abscissa to obtain the standard curve equation. Substituting the logarithms of the dissolution time (seconds) of the sample series of diluted solutions into the standard curve equation, the measured protein concentrations of the three sample diluted solutions were obtained, and the average measured protein concentration before dilution was calculated. The enzyme activity (IU / ml) of the plasminogen activator sample was calculated using the following formula: average protein concentration (mg / ml) × specific activity of the standard (IU / mg), and specific activity (IU / mg) = enzyme activity (IU / ml) × protein concentration (mg / ml).

[0064] 4. Methodological Validation 4.1 Specificity Research The prescription buffer solution was used as a sample, and its clot-dissolving activity was determined using the method described in section 3.

[0065] Take an appropriate amount of human serum albumin and dilute it with sample diluent to prepare a solution with a molar concentration equivalent to that of reteplase or tenecteplase sample series solutions. Then, determine the clot dissolution time using the method described in section 3. Human serum albumin, as a protein protectant, helps maintain the conformational stability of plasminogen activator during dilution, thereby ensuring the accuracy of the activity assay.

[0066] 4.2 Accuracy, Precision, Linearity, and Range Reteplase: Take one vial of reteplase standard, add 5 ml of prescription buffer to reconstitute, and mix well. Determine the protein concentration according to the method in 1.1. Accurately measure an appropriate amount of the above solution and dilute it with sample diluent to a method validation stock solution containing 5 µg reteplase per ml.

[0067] Tenecteplase: Take 5 vials of tenecteplase standard A, add 0.1 ml of prescription buffer to each to reconstitute, and mix the 5 vials well. Determine the protein concentration according to section 1.2, accurately measure an appropriate amount of the above solution, and dilute it with sample diluent to a method validation stock solution containing 5 µg tenecteplase per ml.

[0068] Take 600, 800, 1000, 1200, and 1400 µl of the above stock solution, respectively, and add 4.4, 4.2, 4.0, 3.8, and 3.6 ml of sample diluent. After mixing, these are the series solutions with concentrations of 600, 800, 1000, 1200, and 1400 ng / ml, serving as the test solutions for five different potency levels (60%, 80%, 100%, 120%, and 140%). Two researchers measured the potency of the test solutions at each of the five different potency levels daily for two consecutive days, for a total of eight measurements at each potency level. The mean was calculated. Accuracy was evaluated using relative bias (RB, = measured potency / theoretical potency × 100%). The coefficient of variation (CV, %) of the measurement results for each potency level was calculated. Linear regression analysis was performed with the measured potency values ​​of the five test solutions on the x-axis and the theoretical potency on the y-axis.

[0069] 4.3 Repeatability Prepare a standard curve according to section “2”. Take reteplase for injection (20220401) and tenecteplase for injection (batch number: 20230715T) and prepare 6 parallel sample series solutions according to section “1”. Determine the potency according to the method in section “3”. Measure each sample solution twice and calculate the average value.

[0070] 4.4 Durability Reteplase: To investigate the stability of the solution, the above reteplase sample series solutions were placed in a blood coagulation analyzer for different times (0h, 1h, 3h) and the potency was determined.

[0071] Tenecteplase: To investigate the stability of the solution, a series of tenecteplase samples (batch number: 20230715T) were placed in a coagulation analyzer for different times (0h, 3h, 5h) and the activity was measured.

[0072] The effect of minute changes in the ratio of fibrinogen to plasminogen in the mixed solution on the method was investigated, and the potency / biological activity of the same sample was determined at different ratios of fibrinogen to plasminogen (1:45, 1:50, 1:55).

[0073] 5. Determination of plasminogen activator potency using the bubble rising method 5.1 Reteplase 5.1.1 Solution Preparation Sample dilution solution: Weigh 7.10 g of disodium hydrogen phosphate and 1.38 g of sodium dihydrogen phosphate, add 0.1 g of Tween 80 solution, dilute with water to 1 L, and mix thoroughly.

[0074] Human plasma plasminogen solution: Take human plasma plasminogen and dilute it with sample diluent to a solution containing 1 mg per 1 ml.

[0075] For details on the preparation of human plasma thrombin solution and human plasma fibrinogen solution, please refer to item "3" of Experimental Materials.

[0076] Standard solutions: Take one vial of reteplase standard, add 5 ml of sterile water for injection, and dilute with sample diluent to prepare solutions with concentrations of 1.00, 0.50, 0.25, 0.125, and 0.0625 × 10⁻⁶ per ml. 3 A solution of U.

[0077] Test solution: Dissolve one vial of reteplase for injection in 5 ml of sterile water for injection, or bring the reteplase stock solution to room temperature and dilute with water to a concentration of 1×10⁻⁶. 6 Solutions with concentrations of U / ml were prepared using sample diluent to obtain concentrations of 1.00, 0.50, 0.25, and 0.125 × 10⁻⁶ U / ml. 3 A solution of U.

[0078] Mixed solution: Take 150 μl of the test solution (standard solution), add an equal volume of human plasma thrombin solution, mix well, and prepare fresh before use.

[0079] 5.1.2 Determination Method Take a test tube and add 1 ml of human plasma fibrinogen solution and 20 μl of human plasma lysozyme solution respectively. Mix well and place in an ice bath for 10 minutes. Then, add 20 μl of each concentration (a total of 9 concentrations, consisting of 1 part standard solution and 1 part test solution) of the mixed solution sequentially, shake immediately, and place in a 37±1℃ water bath as the reaction start point, timing each step. The reaction system should coagulate within 30 seconds. The reaction endpoint should be defined as the moment when the last small bubble in the coagulated solution rises to the surface, timing the step. Perform a linear regression with the logarithm of the reteplase standard concentration (U / ml) on the x-axis and the logarithm of the reaction endpoint time (seconds) on the y-axis to calculate the test sample potency (U / ml).

[0080] 5.2 Tenecteplase 5.2.1 Solution Preparation Sample dilution buffer: Weigh 17.406 g of disodium hydrogen phosphate dodecahydrate and 1.778 g of sodium dihydrogen phosphate dihydrate, add 0.3 g of Tween 80 solution, and dilute with water to 1 L. After thorough mixing, take 80 ml of the above buffer solution, add 0.5 g of human serum albumin, and continue adding the above buffer solution to dilute to 100 ml.

[0081] Human plasma plasminogen solution: Take human plasma plasminogen and dilute it with sample diluent to a solution containing 1 mg per 1 ml.

[0082] For details on the preparation of human plasma thrombin solution and human plasma fibrinogen solution, please refer to item "3" of Experimental Materials.

[0083] Standard solutions: Take one vial of tenecteplase standard A1, add 0.1 ml of sterile water for injection, and prepare solutions with sample diluent containing 2750, 1375, 687.5, 343.75, 171.88, and 85.938 IU per ml, respectively.

[0084] Test solution: Dissolve tenecteplase A for injection in 3 ml of water. Take the stock solution of tenecteplase A and equilibrate to room temperature. Measure the protein concentration according to section "3" of the experimental materials, and then prepare solutions with a concentration of 1 µg per ml using sample diluent.

[0085] Solution A: Take 150 μl of the test solution (standard solution), add an equal volume of human plasma thrombin solution, mix well, and prepare fresh before use.

[0086] Solution B: Take 1 ml of human plasma fibrinogen solution, add 20 μl of human plasma lysozyme solution, mix well, and prepare fresh before use.

[0087] 5.2.2, Determination Method Take a test tube and add 0.2 ml of solution A to solution B. Mix by pipetting for 15 seconds. Once bubbles form on the upper layer, immediately place the tube in a water bath at 37±1℃ as the starting point of the reaction and start timing. The reaction system will solidify within 30 seconds. The reaction endpoint is defined as the moment when the last small bubble in the solidified mass rises to the surface of the reaction solution, and the timing is also defined. Perform a linear regression with the logarithm of the tenecteplase standard concentration (U / ml) on the x-axis and the logarithm of the reaction endpoint time (seconds) on the y-axis to calculate the enzyme activity (U / ml) of the test sample.

[0088] 6. Determination of the amino acid sequences of tenecteplase A and tenecteplase B 6.1 Preparation of Sample Solution Tenecteplase standard A and tenecteplase standard B were diluted to 1 mg / ml with 50 mM ammonium bicarbonate solution. 500 µl of the diluted solution was placed in an ultrafiltration tube (3 kDa pore size) and centrifuged at 5 °C and 12000 rpm / min for 10 minutes. After discarding the filtrate, 500 µl of 50 mM ammonium bicarbonate solution was added. The ultrafiltration solution replacement operation was repeated 3 times.

[0089] Take the solution after ultrafiltration and measure the protein concentration according to the method in section “3”. Take an appropriate amount of the solution containing 100µg of protein into an EP tube, add 20 µl of RapiGest SF solution, and incubate in a water bath at 57℃ for 30 minutes.

[0090] After cooling to room temperature, add 2 µl of 1 mol / L dithiothreitol solution, mix well, and incubate in a 57°C water bath for 1 hour. After cooling to room temperature, add 2 µl of 1 mol / L iodoacetamide solution, mix well, and incubate at 25°C in the dark for 1 hour. Add 2 µl of PNGase F solution, mix well, and incubate at 37°C for 16 hours. After cooling to 25°C, add 2 µl of Trypsin solution, mix well, incubate in a 37°C water bath for 4 hours, and finally add 2 µl of 10% formic acid solution to terminate the reaction.

[0091] 6.2 Liquid Chromatography and Mass Spectrometry Conditions Liquid chromatography conditions: An ACQUITY UPLC peptide BEH C18 column (100 mm × 2.1 mm, 1.7 μm, 300 Å) was used with 0.1% formic acid / water solution as mobile phase A and 0.1% formic acid / acetonitrile solution as mobile phase B, using gradient elution (0–85 min, 3% B → 32% B; 85–90 min, 32% B → 90% B; 90.1–100 min, 3% B), flow rate 0.2 ml / min, column temperature 60 ℃, injection volume 1 µl, and sample chamber temperature 4 ℃. Mass spectrometry conditions: Electrospray ionization (ESI) source, detection mode Full Scan-ddMS2, positive ion scan mode, mass spectrometry voltage 3600V, sheath gas flow rate 35Arb, ion transmission tube temperature 320℃, evaporation temperature 350℃, primary mass spectrometry resolution 60000, scan range (M / Z=200-2000), maximum injection time 100ms; secondary mass spectrometry resolution 30000, using HCD mode collision fragmentation, collision energy 30%, EASY-ICTM enabled.

[0092] 6.3 Data Processing Methods Input the theoretical amino acid sequence of tenecteplase into BioPharma Finder. Based on the software's built-in algorithm and image visualization capabilities, the detection results are matched with data in the database according to the set parameters. Matching amino acids are found in the database based on the quality of primary and secondary fragments of the peptide, thus achieving amino acid identification. The fixed modification is iodoacetamide alkylation (carbamidomethyl-C), and the variable modification is deamination (deamidide-N).

[0093] 7. Glycogram analysis of tenecteplase A and tenecteplase B 7.1 Preparation of Sample Solution Tenecteplase standard A and tenecteplase standard B were diluted to 1 mg / ml with ultrapure water. 25 μl of each diluted solution was accurately measured, and 2 μl of reducing agent was added. After mixing, the mixture was incubated at 90°C for 10 minutes. Then, 2 μl of 10% NP-40 solution and 2 μl of PNGase F solution were added sequentially, and the mixture was incubated at 37°C for 2 hours. After incubation, 5 μl of 2-AB labeled solution was added, and the mixture was incubated at 65°C for 2 hours. The reaction was terminated by adding 300 μl of water.

[0094] Purify the incubated sample using Supelclean tubes. Add 1 ml of conditioning solution to the Supelclean tube and discard the eluent; add 3 ml of water and discard the eluent to complete the pre-conditioning. Add the incubated sample to the Supelclean tube, add 6 ml of ethanol to wash the sample, and discard the wash solution. Add 2 ml of conditioning solution and collect the sample eluent.

[0095] Concentrate the eluent thoroughly in a centrifuge, dissolve it in 100 μl of water, add 300 μl of acetonitrile, and mix well to obtain the final product.

[0096] 7.2 Liquid chromatography and mass spectrometry conditions Liquid chromatography conditions: An ACQUITY UPLC Glycan BEH Amide column (2.1 mm × 150 mm, 130 Å, 1.7 μm) was used. Mobile phase A was 50 mmol / L ammonium formate / 50% acetonitrile solution (pH 4.5 ± 0.05), and mobile phase B was acetonitrile. Gradient elution was used (0–0.3 min, 56% B → 50% B; 0.3–40 min, 50% B → 15% B; 40–41 min, 15% B → 56% B; 40–41 min, 56% B). A fluorescence detector was used with an excitation wavelength of 330 nm and an emission wavelength of 420 nm. The flow rate was 0.6 ml / min, the column temperature was 60 ℃, the injection volume was 2 μl, and the sample chamber temperature was 5 ℃. Mass spectrometry conditions: electrospray ionization source (ESI), detection mode MS, positive ion scan mode, scan range (M / Z=200~2000).

[0097] 7.3 Data Processing Methods The built-in sugar library of UNIFI software was used to analyze and match each oligosaccharide by mass-to-charge ratio and GU value, and the area normalization method was used for quantification.

[0098] 8. Determine the enzyme activity of different glycosylated modified tenecteplase (TNK) products using a fully automated coagulation analyzer. Following the fully automated coagulation analyzer assay method under section "3", tinexerase standard A was used to determine the activity of 3 batches of TNK-A stock solution samples, 3 batches of TNK-A formulation samples, 3 batches of TNK-B stock solution samples, and 3 batches of TNK-B formulation samples. Similarly, tinexerase standard B was used to determine the activity of the above 12 batches of samples. The activity values ​​of the two glycosylated modified TNKs, TNK-A and TNK-B, measured using TNK-A standard, were compared to determine if there were any differences. Furthermore, the activity values ​​of the two glycosylated modified TNK products were compared when TNK-B was used as the standard.

[0099] III. Experimental Results 1. Results of specificity investigation like Figure 1 As shown in BC and EF, no clot dissolution was detected in human serum albumin and prescription buffer (excipient blank) when using this method. Figure 1 In the figure, A represents the coagulation curve of the reteplase sample reaction solution. Figure 1In the diagram, D represents the coagulation curve of TNK sample A. Under reaction conditions of 37℃, fibrinogen forms a fibrin clot with high optical density under the action of thrombin, resulting in an increase in light absorbance. Plasminogen activator promotes the conversion of plasminogen in the reaction system into active plasmin, which dissolves the clot, leading to a decrease in light absorbance. The software uses a threshold method to calculate the endpoint of the clot dissolution reaction time: Threshold = Baseline value of coagulation curve + ((Maximum absorbance of coagulation curve - Baseline value of coagulation curve) × 10%).

[0100] The above results indicate that human serum albumin in the dilution buffer and the prescription buffer do not interfere with the determination of rPA potency, and the method has good specificity.

[0101] 2. Results of the assessment of accuracy, precision, linearity, and range 2.1 Reteplase: Two researchers measured the potency (U / ml) of rPA at five different potency levels (60%, 80%, 100%, 120%, and 140%) over two days. Two replicates were prepared for each potency level. The results are shown in Tables 2 and 3. The relative bias of the measurements within the 60%–140% potency range was within the range of 0.29%–1.01%, indicating good accuracy of the method. The coefficient of variation (CV, %) of the potency measurements at each potency level across eight experiments was less than 2.0%, and the corresponding 95% confidence upper limit was less than 4.0%, indicating good intermediate precision of the method. The theoretical potency levels of the five test solutions were plotted as the X-axis, and the corresponding measured potency as the Y-axis. Figure 2 As shown in C, the linear regression equation is Y=0.9974X+4.8440, r=0.9992, and the slope is close to 1.0, indicating that this method has good linearity in the valence level range of 60%~140%.

[0102] Table 2. Results of reteplase titer test (n=8)

[0103] Table 3. Accuracy and intermediate precision of reteplase titers at five levels using a fully automated coagulation analyzer

[0104] 2.2 Tenecteplase: Two researchers measured the enzyme activity (U / ml) of TNK at five different potency levels (60%, 80%, 100%, 120%, and 140%) over two days. Two replicates were prepared for each potency level. The results are shown in Tables 4 and 5. The relative bias of the measurements within the 60%–140% potency range was within -0.24% to 0.27%, indicating good accuracy of the method. The coefficient of variation (CV, %) of the potency measurements at each potency level across eight experiments was less than 2.0%, and the corresponding 95% confidence upper limit was less than 4.0%, indicating good intermediate precision of the method. The theoretical potency levels of the five test solutions were plotted on the X-axis, and the corresponding measured potency on the Y-axis, as shown in Tables 4 and 5. Figure 2 As shown in D, the linear regression equation is Y=0.9990X+0.5328, r=0.9999, and the slope is close to 1.0, indicating that this method has good linearity in the valence level range of 60%~140%.

[0105] Table 4. Results of tenepoilase titer test (n=8)

[0106] Table 5. Accuracy and intermediate precision of the fully automated coagulation analyzer for determining five potency levels of tenecteplase.

[0107] 3. Results of repeatability testing Reteplase: Take rPA sample (batch number: 20220401), prepare sample series solutions and standard curve solutions on the same day. Prepare 6 parallel aliquots for each concentration sample (800, 1000, 1200 ng / ml), and measure each sample twice. The average value of the 12 results is 1.03 × 10⁻⁶. 6 U / ml, the CV of the repeatability test results was 1.38%.

[0108] Tenecteplase: Take TNK sample (batch number: 20230715T), prepare sample series solutions and standard curve solutions on the same day, prepare 6 parallel aliquots for each concentration sample (800, 1000, 1200 ng / ml), and measure each sample twice. The average value of 36 measurement results is 1003320.67 IU / vial, and the CV of the repeatability measurement result is 1.83%.

[0109] All the above results demonstrate that the blood coagulation assay has good repeatability.

[0110] 4. Durability test results Reteplase: Take rPA sample (batch number: 20220401), prepare sample series solutions and standard curve solutions, dispense the above solutions into sample cups, and place them in a fully automated blood coagulation analyzer for 0h, 3h, and 5h for titer determination. Using mixed solutions of fibrinogen and plasminogen at different ratios (1:45, 1:50, 1:55), with other conditions unchanged, the titer of the same batch of samples was determined. The results are shown in Table 6. The CV was less than 3% for all samples, indicating that rPA has good stability in the blood coagulation analyzer within 5 hours. Small changes in the ratio of plasminogen to fibrinogen in the mixed solution have little impact on the test results, indicating good robustness of the method.

[0111] Table 6. Results of reteplase durability

[0112] Tenecteplase: Take TNK sample (batch number: 20230715T), prepare sample series solutions and standard curve solutions, dispense the above solutions into sample cups, and place them in a fully automated blood coagulation analyzer for 0h, 3h, and 5h for enzyme activity determination. Using mixed solutions of fibrinogen and plasminogen at different ratios (1:45, 1:50, 1:55), with other conditions unchanged, the enzyme activity of the same batch of samples was measured. The results are shown in Table 7. The CV was less than 3% for all samples, indicating that TNK has good stability in the blood coagulation analyzer within 5 hours. Small changes in the ratio of plasminogen to fibrinogen in the mixed solution have little impact on the measurement results, indicating good robustness of the method.

[0113] Table 7. Results of tenepase durability

[0114] 5. Comparison of sample measurement results 5.1 Comparison of results from the bubble rise method and the fully automated coagulation analyzer method for determining rPA samples Two batches of injectable rPA and one batch of rPA stock solution were taken to prepare a series of sample solutions and a standard curve solution. The potency was determined using the fully automated coagulation analyzer method according to experimental method "3". Simultaneously, the potency was determined using the bubble rise method under item "5". For both methods, each sample was measured in triplicate. The results are shown in Table 8. The results of both methods met the requirements (preparation: should be within 0.9~1.2×10). 6 U / ml, stock solution: should not be less than 1.0×10 6 (U / ml). The CV of the three measurements using the fully automated coagulation analyzer was less than 1%, while that using the bubble rise method was 5%–10%. Paired t-tests were performed on the results of the two methods, and the p-values ​​were all greater than 0.05, indicating no statistically significant difference. The results of the two methods showed good consistency, and the precision of the coagulation analyzer method was significantly better than that of the bubble rise method.

[0115] Table 8. Comparison of results of rPA sample determination by the bubble rise method and the fully automated coagulation analyzer method.

[0116] 5.2 Comparison of results from the bubble rise method and the fully automated coagulation analyzer for the determination of TNK samples The assay method was the same as for reteplase, and the potency of 6 batches of TNK preparations and stock solution sample A was measured. The Shapiro-Wilk test results showed that both sets of data were normally distributed. As shown in Table 9, the p-value of the paired t-test analysis was greater than 0.05, indicating no significant difference between the two assays. The mean ratio of the fully automated coagulation analyzer method to the bubble rise method was 1.01, and almost all points were within the 95% confidence interval (0.7-1.2). Figure 3 (As shown). Therefore, the results from the two methods are in good agreement, and the precision of the coagulation analyzer method is significantly better than that of the bubble rise method.

[0117] Table 9. Comparison of results of TNK sample determination by the bubble rise method and the fully automated coagulation analyzer method.

[0118] 6. Amino acid sequence analysis results of TNK samples The primary structure of tenecteplase was confirmed by LC-MS / MS intact mass spectrometry analysis of trypsin-digested tenecteplase standards A and B. The results showed that the TNK peptides from the two manufacturers were identical, with consistent sequences and 100% amino acid sequence coverage. Figure 4 As shown in Table 10-13.

[0119] Table 10. Mass spectrometry data of tenecteplase A digested peptides

[0120] Table 11. Mass spectrometry data of tenecteplase B digested peptides

[0121] Table 12. Mass spectrometry data of reteplase-digested peptides

[0122] Table 13. Mass spectrometry data of alteplase-digested peptides

[0123] 7. Glycosylation analysis results of TNK samples After reducing two types of tenecteplase standards and digesting oligosaccharides with peptidyl N-glycosidase F (PNGase F), the purified oligosaccharides were fluorescently labeled with 2-aminobenzamide (2-AB). Separation was performed by hydrophilic interaction liquid chromatography (HILIC), and the oligosaccharide structure was analyzed by LC-MS. The results are shown in the figure. Figure 4-5 The major glycan structures of tenecteplases are those with terminal galactose or sialic acid (F(6)A2G(4)1Ga(3)1, F(6)A2G(4)2S(3)1, and F(6)A2G(4)2S(3,3)2, with abundances exceeding 10%. However, tri-antenna and tetra-antenna structures also appear in large numbers. Each N-glycosylation site exhibits unique glycan complexity. Most glycans are fucosylated.

[0124] The glycosylation of tenecteplase A and tenecteplase B was characterized using the area normalization method, and the results are as follows: Figure 5-6 As shown in Tables 14 and 15, among the three main glycoforms mentioned above, only the content of F(6)A2G(4)2S(3,3)2 is similar in the two tenecteplases. The peak area % of F(6)A2G(4)1Ga(3)1 in tenecteplase A is about 8% lower than that in tenecteplase B, while the peak area % of F(6)A2G(4)2S(3,3)2 in tenecteplase A is about 16% higher than that in tenecteplase B. In addition, the glycosylation modification of these two enzymes also differs in other glycoform structures, especially after the F(6)A2G(4)2S(3,3)2 peak, there are significant differences in the glycoforms within the retention time of 30-45 min. The main glycoforms of tenecteplase A in this region are A3S(6)1G(4,4,3)3S(3,3)2, F(6)A3G(4)3S(3,3,3)3, and F(6)A4G(4)4Lac1S(3,3)2, accounting for approximately 14% of the total polysaccharide content. Tenecteplase B exhibits a more diverse range of glycoforms, primarily including F(6)A3G(4)3S(3,3)2, A4F(3)2G(4)3S(6)1, and F(6)A3G(4)3S(3,3,6)3, which account for approximately 15% of the total polysaccharide content.

[0125] Table 14. Characterization results of tenecteplase A glycosylation

[0126] Table 15. Characterization results of tenecteplase B glycosylation

[0127] 8. Determination of enzyme titers of TNK products with different glycosylation modifications using a fully automated coagulation analyzer. Three batches each of TNK-A stock solution and formulation samples, and three batches each of TNK-B stock solution and formulation samples, with different glycosylation modifications, were taken. Clot dissolving activity was determined using the fully automated coagulation analyzer method (Experimental Method "3"), with TNK-A and TNK-B as standards. Each sample was measured in triplicate. The enzyme activity of the TNK products from the two manufacturers was calculated. The results all met the current quality standards, indicating that the fully automated coagulation analyzer method can be used to evaluate TNK products with different glycosylation forms. Figure 7 As shown, when TNK-A was used as the standard, a paired t-test was performed on the clot-dissolving activity assays of the two TNK products, and the p-value was 0.83; while when TNK-B was used as the standard, the p-value of the paired t-test was 0.68, both greater than 0.05. This indicates that there is no statistically significant difference in enzyme activity between the two TNK products with different sugar forms but the same amino acid sequence, and they can be used as standards for each other for enzyme activity evaluation using the fully automated coagulation analyzer method.

[0128] The above description of the embodiments is only for understanding the method and core ideas of the present invention. It should be noted that those skilled in the art can make various improvements and modifications to the present invention without departing from the principles of the invention, and these improvements and modifications will also fall within the protection scope of the claims of the present invention.

Claims

1. A method for in vitro determination of plasminogen activator potency, characterized in that, The method uses a fully automated coagulation analyzer for measurement and includes the following steps: Dilute the sample to be tested with sample diluent to a protein concentration of 5 μg / mL to obtain a sample stock solution; The sample stock solution was diluted to a series of sample dilution solutions. The clot dissolution time of the sample dilution series solutions was measured using a fully automated coagulation analyzer. The enzyme titer was calculated using the external standard method according to the following formula: Enzyme activity (IU / ml) = average protein concentration (mg / ml) × specific activity of standard (IU / mg). Specific activity (IU / mg) = enzyme activity (IU / ml) × protein concentration (mg / ml); The sample diluent was human serum albumin diluted with phosphate buffer.

2. The method according to claim 1, characterized in that, The sample diluent was prepared as follows: 80 mL of phosphate buffer solution with pH 7.4 was taken, 0.5 g of human serum albumin was added, and the solution was diluted to 100 mL with phosphate buffer solution. After being fully dissolved, the solution was stored at 4°C.

3. The method according to claim 1, characterized in that, The final concentration of the human serum albumin was 5 mg / mL.

4. The method according to claim 1, characterized in that, The plasminogen activator is any one of tenecteplase, reteplase, and alteplase.

5. The method according to claim 1, characterized in that, The steps of the fully automated coagulation analyzer are as follows: using a mixed solution as the starting reagent and human plasma thrombin solution as the intermediate reagent, the clot dissolution time of the sample dilution series solutions is measured after setting the instrument parameters. The instrument parameters are as follows: sample solution volume is 20 μL, initial reagent volume is 200 μL, intermediate reagent volume is 20 μL, reaction temperature is 37±1℃, detection wavelength is 405 nm, measurement time is 900 s, delay time is 10 s, the algorithm is threshold method, the threshold method is curve percentage, the threshold limit is 10%, and the acquisition direction is reverse.

6. The method according to claim 1, characterized in that, The concentrations of the sample dilution series solutions were 800 ng / mL, 1000 ng / mL, and 1200 ng / mL.

7. The method according to claim 6, characterized in that, The method for preparing the human plasma thrombin solution is as follows: reconstitute the human plasma thrombin lyophilized powder with water, and dilute it to 33 U / mL with sample diluent; The mixed solution is a mixture of human plasma fibrinogen solution and human plasma plasminogen solution, and the final concentration of the mixed solution is 1:50 (v / v).

8. A kit for determining the potency of plasminogen activator, characterized in that, The kit includes the reagents used in the method according to any one of claims 1-7, wherein the plasminogen activator is any one of tenecteplase, reteplase, and alteplase.

9. The use of the method according to any one of claims 1-7 and / or the kit according to claim 8 in determining the potency of plasminogen activator.

10. The application according to claim 9, characterized in that, The plasminogen activator is any one of tenecteplase, reteplase, and alteplase.

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