In-vitro plasminogen activator titer determination and quality control method

Abstract

The invention provides a method for measuring the titer of a plasminogen activator in vitro and controlling the quality of the plasminogen activator, which specifically comprises the following steps: measuring the thrombolytic activity of the plasminogen activator by using a full-automatic blood coagulation analyzer, and detecting the change of light conduction penetrating through thrombus clots by using a photoelectric detector to simulate the dissolution process of in-vivo fibrin clots. The method is high in specificity and good in accuracy and precision, can be used as a conventional method for evaluating the titer of the plasminogen activator medicine, and provides technical reserve for improving the quality controllability of products and establishing related standard substances.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technologies, and particularly to a method for in vitro determination of the titer of plasminogen activator and quality control. Background Art

[0002] Thrombosis is the common pathological basis of the three major cardiovascular diseases globally (myocardial infarction, stroke, venous thromboembolism). Cardiovascular diseases are characterized by high incidence, high disability rate, and high mortality rate. Venous thrombolysis is an important means for treating thrombotic diseases. Plasminogen activator thrombolytic drugs can directly or indirectly activate plasminogen into plasmin, thereby dissolving the fibrin clot in the thrombus.

[0003] The rapid development of the domestic plasminogen activator industry has put forward higher requirements for product quality. However, the key quality attribute such as the titer determination method reflecting thrombolytic effectiveness is still the traditional bubble rising method. This method requires a special visual water bath device. The reaction end point (the time when the last bubble rises to the surface of the reaction solution) needs to be judged by the human eye, and a stopwatch is used for timing. Taking the logarithm of the titer of the standard product solution as the abscissa and the logarithm of the reaction end point time as the ordinate for linear regression, and calculating the titer of the plasminogen activator sample through the linear regression equation. Only a single concentration of the standard product or sample can be run at a time, and a single batch determination takes 2 hours, with problems such as being cumbersome and time-consuming, low experimental throughput, and large errors.

[0004] Enzyme titer is a key quality attribute of plasminogen activator products, reflecting the clinical mechanism of action, the integrity of biological functions, and batch-to-batch consistency of such drugs. Currently, there are differences in host cells and production processes used by different manufacturers, resulting in significant differences in the activity units of plasminogen activator products in clinical applications, making it difficult to unify and easily causing clinical medication problems. For example, TNK-A from CSPC Mingfulai (Guangzhou) Co., Ltd. and TNK-B from Boehringer Ingelheim International GmbH in the present invention. Due to their different glycosylation modifications, different activity measurement methods and standards, and different definitions of activity units, their specific activities vary greatly. The specific activity of TNK-A is 6.25×10 5 IU / mg, while that of TNK-B is 200 U / mg. This huge difference may cause potential problems for accurate clinical medication, thereby affecting the clinical treatment of stroke emergency patients. Establishing an accurate, sensitive, and efficient titer evaluation method is of great significance for the quality control of such enzymes. Summary of the Invention

[0005] In view of the above problems, the present invention provides a method for in vitro determination of the potency of plasminogen activators. By performing enzyme activity and potency determination according to this method, a unified reference substance can be used to transfer the same defined enzyme activity unit to different plasminogen activator products, thereby accurately and efficiently unifying the enzyme activity unit and completely avoiding the troubles in clinical medication.

[0006] To achieve the above object, the specific technical solutions provided by the present invention are as follows:

[0007] The first aspect of the present invention provides a method for pharmacodynamic evaluation and quality control of plasminogen activators, and the method realizes pharmacodynamic evaluation and quality control by in vitro determination of the potency of plasminogen activators.

[0008] In the present invention, "potency" or "activity" refers to the efficacy unit of a substance that causes a biological reaction. In some embodiments, the potency refers to the ability of a plasminogen activator to activate plasminogen.

[0009] Furthermore, the in vitro determination of the potency of plasminogen activators is performed by a fully automatic coagulation analyzer.

[0010] In the present invention, plasminogen activator refers to a profibrinolytic protease that activates plasminogen to generate active plasmin. Plasminogen activators include plasmin (PL), tissue-type plasminogen activator (t-PA), urokinase-type plasminogen activator (u-PA), streptokinase (SK), urokinase (UK), high molecular weight kininogen (HMWK), vitronectin (VN), and the second-generation thrombolytic drug recombinant tissue-type plasminogen activator (i.e., alteplase, rt-PA) and the gene-modified variants of the third-generation thrombolytic drug rt-PA, tenecteplase (TNK) and reteplase (rPA) developed in recent years.

[0011] Reteplase is a deletion variant of non-glycosylated tissue-type plasminogen activator (tPA). This protease is obtained from inactive inclusion bodies in Escherichia coli and is converted into an active form after in vitro folding. It can specifically bind to fibrin and convert it into active plasmin, and then degrade fibrin to dissolve the formed thrombus and achieve the purpose of dredging blood vessels.

[0012] TNK is obtained by replacing the amino acids at three sites of the alteplase molecule, that is, threonine at position 103 is replaced by asparagine, asparagine at position 117 is replaced by glutamine, and the amino acids at positions 296-299 are replaced by four alanines. It has high fibrin specificity. It binds to fibrin through lysine residues, activates plasminogen bound to fibrin to be converted into plasmin, thereby dissolving blood clots. This effect is significantly enhanced compared to activating plasminogen in circulation, and selectively activates plasminogen, so it does not produce the common bleeding complications when using streptokinase.

[0013] Further, the plasminogen activator includes any one of tenecteplase, reteplase, urokinase, streptokinase, tissue plasminogen activator, and alteplase.

[0014] Further, the plasminogen activator is any one of tenecteplase, reteplase, and alteplase.

[0015] Further, the steps of the method include:

[0016] Dilute the sample to be tested with a sample diluent to a protein concentration of 5 μg / mL to obtain a sample stock solution; dilute the sample stock solution to a sample dilution series solution, use an automatic coagulation analyzer to measure the clot lysis time of the sample dilution series solution, and use the external standard method to calculate the enzyme titer according to the following formula: Enzyme activity (IU / ml) = mean protein concentration (mg / ml) × specific activity of the standard product (IU / mg), specific activity (IU / mg) = enzyme activity (IU / ml) × protein concentration (mg / ml).

[0017] Further, the sample to be tested is a plasminogen activator.

[0018] In some embodiments, the sample to be tested includes commercial or self-made plasminogen activator products.

[0019] Further, the plasminogen activator includes any one of tenecteplase, reteplase, urokinase, streptokinase, tissue plasminogen activator, and alteplase.

[0020] Further, the plasminogen activator is any one of tenecteplase, reteplase, and alteplase.

[0021] Further, the sample diluent is human albumin diluted with phosphate buffer.

[0022] Further, the pH of the phosphate buffer is 7.4.

[0023] Further, the final concentration of human albumin is 5 mg / mL.

[0024] Further, the preparation method of the sample diluent is as follows: Measure 80 mL of phosphate buffer solution, add 0.5 g of human albumin, dilute it to 100 mL with phosphate buffer solution, and store it at 4°C after fully dissolving.

[0025] Further, the measurement steps of the fully automatic coagulation analyzer are as follows: Using the mixed solution as the starting reagent and the human plasma thrombin solution as the intermediate reagent, measure the clot dissolution time of the sample dilution series solution after setting the parameters.

[0026] Further, the instrument parameters of the fully automatic coagulation analyzer are as follows: The volume of the sample solution is 20 μL, the volume of the starting reagent is 200 μL, the volume of the intermediate reagent is 20 μL, the reaction temperature is 37 ± 1°C, the detection wavelength is 405 nm, the measurement time is 900 s, the delay time is 10 s, the algorithm is the threshold method, the threshold method is the curve percentage, the threshold limit is 10%, and the acquisition direction is reverse.

[0027] Further, the concentrations of the sample dilution series solution are 800 ng / mL, 1000 ng / ml, and 1200 ng / ml.

[0028] Further, the preparation method of the human plasma thrombin solution is as follows: Reconstitute the freeze-dried powder of human plasma thrombin with water and dilute it to 33 U / mL with the sample diluent.

[0029] Further, the mixed solution is a mixed solution of human plasma fibrinogen solution and human plasma plasminogen solution, and the final concentration of the mixed solution is 1:50 (v / v).

[0030] Further, the model of the fully automatic coagulation analyzer is ACL TOP 750.

[0031] In the second aspect of the present invention, a kit for measuring the titer of plasminogen activator is provided, and the kit includes the reagents used in the method of the first aspect of the present invention.

[0032] Further, the kit further includes an instruction manual.

[0033] In the third aspect of the present invention, the application of the method of the first aspect of the present invention and / or the kit of the second aspect of the present invention in the efficacy evaluation and quality control of plasminogen activator is provided.

[0034] 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 the detection efficiency, reduces the manual operation error, has high accuracy and precision, can be used as a conventional method for evaluating the titer of plasminogen activator drugs, and makes technical preparations for improving the quality controllability of products and the establishment of related reference substances. Description of the Drawings

[0035] Figure 1 It is a result graph of the methodological verification for the determination of the titer of plasminogen activator by an automatic blood coagulation analyzer. Among them, 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 prescription buffer in the reteplase blank group, 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 prescription buffer in the tenecteplase blank group.

[0036] Figure 2 It is a standard curve and linear investigation result graph for the determination of the titer of plasminogen activator. Among them, A is the reteplase standard curve, B is the tenecteplase standard curve, C is the linear investigation result graph of reteplase, and D is the linear investigation result graph of tenecteplase.

[0037] Figure 3 It is a result graph of the consistency of the automatic blood coagulation analyzer method and the bubble rising method by Bland-Altman analysis.

[0038] Figure 4 It is a full-sequence mass spectrometry peak graph of four plasminogen activators (the red ones are the matched amino acid sequences). Among them, A is tenecteplase A, B is tenecteplase B, C is alteplase, and D is reteplase.

[0039] Figure 5 It is a HILIC curve graph of tenecteplase standard product A.

[0040] Figure 6 It is a HILIC curve graph of tenecteplase standard product B.

[0041] Figure 7 It is a result graph of the activities of 12 batches of TNK products with two glycosylation modifications measured by the automatic blood coagulation analyzer method. Specific implementation manners

[0042] Next, the technical solutions in the embodiments of the present invention will be clearly and completely described in conjunction with the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative efforts fall within the protection scope of the present invention.

[0043] Embodiment

[0044] I. Experimental materials

[0045] 1. Instruments and consumables

[0046] Electronic balance (METTLER TOLEDO, USA, model: ME166DU); pure water machine (MILLIPORE, USA, model: Milli-Q IQ7000); fully automatic coagulometer (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); centrifugal concentrator (Thermo Fisher Scientific, USA).

[0047] Coagulometer sample cup (WERFEN, Spain, 2 mL, product number: 5575100); Coagulometer ACL TOP colorimetric analysis cup (Werfen, Spain, product number 29400100); Glass reagent bottle for coagulometer (Beijing Jinyuchen Medical Equipment Co., Ltd., 10, 20 mL); Chromatographic column ACQUITY UPLC Peptide BEH C18 (2.1 mm × 100 mm, 1.7 μm, Waters); Chromatographic column ACQUITY UPLC Glycan BEH Amide (2.1 mm × 150 mm, 1.7 μm, Waters); Supelco Supelclean ENVI-Carb SPE tube, (Sigma Aldrich).

[0048] 2. Experimental reagents

[0049] Tenecteplase reference standard A (31100 IU / vial, 0.0527 mg / vial, 0.1 ml / vial), batch number: 20141200102-S, Shijiazhuang Pharmaceutical Group Mingfulo (Guangzhou) Co., Ltd.; Tenecteplase reference standard B (200 U / mg, about 5 mg / ml, 0.25 ml / vial), batch number: 12639515, Boehringer Ingelheim International Gmbh, Germany; Tenecteplase for injection (1.0×10 7IU / vial, 16mg / vial) A, batch numbers: 20230715T, 20230729T, 20230724T, Shijiazhuang Pharmaceutical Group Mingfulo (Guangzhou) Co., Ltd.; Tenecteplase stock solution A, batch numbers: A24072001P, B24080801P, C24080901P, Shijiazhuang Pharmaceutical Group Mingfulo (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, batch numbers: 12630045, 12630050, 12630052; Retaplase reference standard (5 million U / vial, 8.9mg / vial), batch number: 20230401, lyophilized powder, Aide Pharmaceutical Co., Ltd. (Beijing); Retaplase for injection (8.9mg / vial), batch number: 20220401, Aide Pharmaceutical Co., Ltd. (Beijing); Alteplase reference standard (0.98mg / ml, 580,000IU / mg), batch number: WS-03-12213859, Boehringer Ingelheim International Gmbh, Germany; Alteplase for injection (20mg / vial), batch number: 302182, Boehringer Ingelheim International Gmbh, Germany; Human serum albumin, Sigma, USA, batch number: 1003095609; Human plasma thrombin, EMD Millipore, USA, 1000U per vial, batch number: 3468951, lyophilized powder; Human plasma fibrinogen (containing at least 90% clottable protein), EMD Millipore, USA, 500mg per vial, batch number: 3171544; Human plasma plasminogen, EMD Millipore, USA, 100mg per vial, containing 1. per ml.5 mg, batch number: 4037914; RapiGest SF protease surfactant, Waters, USA, batch number: 186001860; Trypsin, Roche, USA, batch number: 54734422; 2-AB Dextran Calibration Ladder, Waters, USA, batch number: 186006841; PNGase F, Aglient Technologies, USA, batch number: DG87-503C; phosphoric acid, sodium dihydrogen phosphate dihydrate, disodium hydrogen phosphate dihydrate were purchased from Sinopharm Chemical Reagent Co., Ltd.; Hemagglutination Analyzer cleaning solution A, Werfen, Spain, batch number: 09831700; Hemagglutination Analyzer cleaning solution B, Werfen, Spain, batch number: 09832700; Hemagglutination Analyzer rinsing solution, Werfen, Spain, batch number: 20302400; acetonitrile, Solvslichro, batch number B2301006017; polysorbate 20, tween 80, L-arginine, formic acid, dithiothreitol, iodoacetamide, isopropanol, 2-aminobenzamide, 25% ammonium hydroxide solution, 2-mercaptoethanol were all purchased from Sigma Aldrich, USA; ammonium bicarbonate, Aladdin, USA, batch number: D2326118; triethylamine, Honeywell, USA, batch number: 65897; sodium cyanoborohydride and acetic acid were purchased from Merck, Germany; PNGase F, New England Biolabs, USA, batch number: P0704L; dimethyl sulfoxide and 20% sodium dodecyl sulfate (SDS) stock solution were purchased from Roth, Germany; 10% NP40, Biorigin, Brazil, batch number: BN24524.

[0050] 3. Reagent preparation

[0051] Phosphate buffer (pH 7.4): Weigh 3.56 g of sodium dihydrogen phosphate dihydrate and 17.30 g of disodium hydrogen phosphate dihydrate, add 1500 ml of ultrapure water to dissolve, add 2.0 ml of 10% tween 80 solution (v / v), adjust the pH value to 7.402 with phosphoric acid, add water to dilute to 2 L, and mix well.

[0052] Sample diluent: Measure 80 ml of phosphate buffer (pH 7.4), add 0.5 g of human serum albumin, dilute to 100 ml with phosphate buffer, and store at 4 °C after fully dissolving.

[0053] Formulation buffer: Weigh 55 g of arginine, add 0.4 ml of 10% tween 80 solution, add 800 ml of water to dissolve fully, then add water to dilute to 1 L, and adjust the pH value to 7.4 with phosphoric acid solution.

[0054] Human plasma thrombin solution: Reconstitute the freeze-dried powder of human plasma thrombin with 1 mL of water, and dilute it with the sample diluent to obtain a solution containing 33 U per 1 mL, and store it at 4°C.

[0055] Human plasminogen solution: Store it at 4°C after equilibrating to room temperature.

[0056] Fibrinogen solution in human plasma: Take 1 vial of fibrinogen in human plasma, and dilute it with the sample diluent to obtain a solution containing 2 mg of clottable fibrinogen per 1 mL, and prepare it immediately before use.

[0057] Mixed solution: Take 35 mL of fibrinogen solution in human plasma, add 700 μL of human plasminogen solution and mix well to obtain a 1:50 (v / v) mixed solution, and prepare it immediately before use.

[0058] RapiGest SF solution: Take 1 vial of RapiGest SF (1 mg / vial), add 1 mL of ultrapure water, mix well to obtain it, and prepare it freshly before use.

[0059] Trypsin solution: Take 1 vial of Trypsin (1 mg / vial), add 1 mL of ultrapure water, mix well to obtain it, and prepare it freshly before use.

[0060] Reducing agent: Take 1 mL of 20% stock solution of sodium dodecyl sulfate (SDS), add 9 mL of water, and then add 558.07 μL of 2-mercaptoethanol and mix well.

[0061] 2-AB labeling solution: Take 700 μL of dimethyl sulfoxide, add 300 μL of glacial acetic acid, cool and add 0.05 g of 2-aminobenzamide, and then transfer all the dissolved 2-aminobenzamide solution to an EP tube containing 0.06 g of sodium cyanoborohydride to obtain it.

[0062] Adjusting solution: Take 400 mL of isopropanol, add 2 mL of triethylamine, add 598 mL of water, and mix well to obtain it, and prepare it freshly before use.

[0063] II. Experimental methods

[0064] 1. Preparation of sample series solutions

[0065] 1.1 Reteplase: Take 1 vial of reteplase for injection, add 5 mL of sterile injection water to dilute it into a solution containing about 1.78 mg of reteplase per mL. Take appropriate amounts of the above reteplase formulated solution and reteplase stock solution, and use an ultraviolet spectrophotometer to perform a maximum absorption wavelength scan in the wavelength range of 240 - 550 nm. Using the formulated buffer as the blank, read the maximum absorption wavelength and the absorption value A at 320 nm max and A 320 , calculate A max and A 320The difference is taken as the extinction coefficient of 1.69 (1 mg / ml recombinant rPA in this buffer is max -A 320 The protein content of the above preparations and stock solutions was calculated by accurately measuring an appropriate amount of the above solutions and gradually diluting them with sample diluent to a protein concentration of 5 μg / ml to obtain a sample stock solution.

[0066] 1.2. Tenecteplase: Take 1 vial of Tenecteplase A for injection and add 3 ml of sterile water for injection to dilute it into a solution containing about 5.5 mg of Tenecteplase per ml. Take appropriate amounts of the prescription solution, the reconstituted Tenecteplase A preparation, and the Tenecteplase A stock solution, and use a UV spectrophotometer with the prescription buffer as the 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 was calculated by taking the extinction coefficient as 1.9 (1 mg / ml recombinant tenecteplase in this buffer under A 280 -A 320 The protein content of the above preparations and stock solutions was calculated by accurately measuring an appropriate amount of the above solutions and gradually diluting them with sample diluent to a protein concentration of 5 μg / ml to obtain a sample stock solution.

[0067] 1.3. Take 800, 1000 and 1200 μl of the 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 sample solutions with protein concentrations of 800 ng / ml, 1000 ng / ml and 1200 ng / ml, respectively. Prepare 2 samples of each concentration in parallel, and measure each solution in parallel 3 times.

[0068] 2. Preparation of standard curve solution

[0069] 2.1. Reteplase: Take 1 vial of Reteplase standard and prepare a standard stock solution with a protein concentration of 5 μg / ml as described in 1.1.

[0070] 2.2 Tenecteplase: Take one vial of Tenecteplase A standard and dilute it with 0.1 ml of sterile water for injection to 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".

[0071] 2.3. Respectively take 600, 800, 1000, 1200, and 1400 μl of reteplase standard stock solution and tenecteplase A standard stock solution, add 4.4, 4.2, 4.0, 3.8, and 3.6 ml of sample diluent, and dilute to 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 2 replicates in parallel for each standard concentration, and measure each standard curve solution in parallel 3 times.

[0072] 3. Determination of enzyme activity by automated coagulometer

[0073] 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 above sample cup, starting reagent, intermediate reagent, and cleaning solution reagent bottle in the appropriate positions on the reagent rack, select the corresponding reagent names and placement positions in the ACL TOP version 5.3.0 software. After the instrument recognizes them, start the determination of clot lysis time.

[0074] Table 1. ACL TOP 750 system parameters

[0075]

[0076]

[0077] Perform linear regression analysis with the logarithm of the clot lysis time (seconds) of the standard curve solution as the ordinate and the logarithm of the standard curve protein concentration (ng / ml) as the abscissa to obtain the standard curve equation. Substitute the logarithm of the clot lysis time (seconds) of the sample series dilution solution into the standard curve equation to obtain the measured protein concentrations of the 3 sample dilution solutions, and calculate the average measured protein concentration before dilution. Calculate the enzyme activity (IU / ml) of the plasminogen activator sample according to the following formula = protein concentration mean (mg / ml) × standard specific activity (IU / mg), and specific activity (IU / mg) = enzyme activity (IU / ml) × protein concentration (mg / ml).

[0078] 4. Methodological verification

[0079] 4.1. Specificity study

[0080] Take the prescription buffer as the sample and measure its clot lysis activity by the method in 3.

[0081] Take an appropriate amount of human serum albumin, dilute it with sample diluent, and prepare a solution with a molar concentration equivalent to that of the reteplase or tenecteplase sample series solution, and measure the clot lysis time by the method in 3.

[0082] 4.2 Accuracy, Precision, Linearity and Range

[0083] Reteplase: Take 1 vial of reteplase reference standard, add 5 ml of the prescription buffer for reconstitution, 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 the sample diluent to a methodological verification stock solution containing 5 μg of reteplase per 1 ml.

[0084] Tenecteplase: Take 5 vials of tenecteplase reference standard A, add 0.1 ml of the prescription buffer for reconstitution to each vial, and then mix the 5 vials well. Determine the protein concentration according to the method under 1.2. Accurately measure an appropriate amount of the above solution, and dilute it with the sample diluent to a methodological verification stock solution containing 5 μg of tenecteplase per 1 ml.

[0085] Respectively take 600, 800, 1000, 1200, 1400 μl of the above stock solutions, add 4.4, 4.2, 4.0, 3.8 and 3.6 ml of the sample diluent, mix well, and the resulting series of solutions with concentrations of 600, 800, 1000, 1200, 1400 ng / ml are used as the test solutions at 5 different potency levels (60%, 80%, 100%, 120%, 140%). Two experimenters measure the potency of the above 5 test solutions at different potency levels 2 times a day for 2 consecutive days, with each potency level measured 8 times in total, and calculate the mean value. Evaluate the accuracy with the relative bias (RB, = measured value of potency / theoretical value of potency × 100%). Calculate the coefficient of variation (CV, %) of the measurement results at each potency level. Perform a linear regression analysis with the measured values of the potency of the 5 test solutions as the abscissa and the theoretical potency as the ordinate.

[0086] 4.3 Repeatability

[0087] Prepare the standard curve according to item "2". Take reteplase for injection (20220401) and tenecteplase for injection (batch number: 20230715T), and parallelly prepare 6 series of sample solutions according to item "1". Perform the potency determination according to the method in item "3", measure each sample solution 2 times, and calculate the average value.

[0088] 4.4 Robustness

[0089] Reteplase: Examine the stability of the solution. Place the above series of reteplase sample solutions in the coagulometer at different times (0 h, 1 h, 3 h), and perform the potency determination respectively.

[0090] Tenecteplase: Examine the stability of the solution. Place the series of tenecteplase samples (batch number: 20230715T) solutions in the coagulometer at different times (0 h, 3 h, 5 h), and perform the activity determination respectively.

[0091] Examine the effect of a slight change in the ratio of fibrinogen to plasminogen in the mixed solution on the method, and determine the titer / biological activity of the same sample at different ratios of fibrinogen to plasminogen (1:45, 1:50, 1:55).

[0092] 5. Determination of the titer of plasminogen activator by the bubble rising method

[0093] 5.1. Retavase

[0094] 5.1.1. Solution preparation

[0095] Sample diluent: 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 well.

[0096] Human plasma plasminogen solution: Take human plasma plasminogen and dilute it with the sample diluent to a solution containing 1 mg per 1 ml.

[0097] For the preparation of human plasma thrombin solution and fibrinogen solution in human plasma, see item "3" of the experimental materials.

[0098] Standard product solution: Take 1 vial of Retavase standard product, add 5 ml of sterile injection water, and prepare solutions with concentrations of 1.00, 0.50, 0.25, 0.125, and 0.0625×10 3 U per 1 ml with the sample diluent.

[0099] Test sample solution: Take 1 vial of Retavase for injection, add 5 ml of sterile injection water for reconstitution, or take the Retavase stock solution and equilibrate it to room temperature, prepare a solution with a concentration of 1×10 6 U / ml with water, and then prepare solutions with concentrations of 1.00, 0.50, 0.25, and 0.125×10 3 U per 1 ml with the sample diluent.

[0100] Mixed solution: Take 150 μl of the test sample solution (standard product solution), add an equal volume of human plasma thrombin solution, mix well, and prepare immediately before use.

[0101] 5.1.2. Determination method

[0102] Take a test tube, add 1ml of human plasma fibrinogen solution and 20μl of human plasma lysozyme solution, mix well, and place in an ice bath for 10 minutes. Add 20μl of mixed solution of each concentration (1 standard and 1 test solution, a total of 9 concentrations) in sequence, shake well immediately and place in a 37±1℃ water bath as the starting point of the reaction, and count the time respectively. The reaction system coagulates within 30 seconds. When the last small bubble in the clot rises to the surface of the reaction solution, it is regarded as the end point of the reaction and the time is counted. Linear regression is performed with the logarithm of the concentration of the reteplase standard (U / ml) as the horizontal axis and the logarithm of the reaction end point time (seconds) as the vertical axis to calculate the test product titer (U / ml).

[0103] 5.2 Tenecteplase

[0104] 5.2.1 Solution preparation

[0105] Sample diluent: weigh 17.406g of disodium hydrogen phosphate dodecahydrate, 1.778g of sodium dihydrogen phosphate dihydrate, add 0.3g of Tween 80 solution, and dilute with water to 1L. After fully mixing, take 80ml of the above buffer, add 0.5g of human albumin, and continue to add the above buffer to dilute to 100ml.

[0106] Human plasma plasminogen solution: Take human plasma plasminogen and add sample diluent to dilute it to a solution containing 1 mg per 1 ml.

[0107] For details on the preparation of human plasma thrombin solution and fibrinogen solution in human plasma, please refer to the experimental materials under item "3".

[0108] Standard solution: Take 1 vial of Tenecteplase Standard A, add 0.1 ml of sterile water for injection, and use sample diluent to prepare solutions with concentrations of 2750, 1375, 687.5, 343.75, 171.88 and 85.938 IU per 1 ml.

[0109] Test solution: Take tenecteplase A for injection and add 3 ml of water to dissolve. Take the stock solution of tenecteplase A and equilibrate it to room temperature. Determine the protein concentration according to the experimental material "3" and then use the sample diluent to make a solution with a concentration of 1 ml containing 1 μg.

[0110] Solution A: Take 150 μl of the test solution (standard solution), add an equal volume of human plasma thrombin solution, mix well, and prepare before use.

[0111] Solution B: Take 1 ml of human plasma fibrinogen solution, add 20 μl of human plasma lysozyme solution, mix well, and prepare before use.

[0112] 5.2.2 Determination method

[0113] Take a test tube, add 0.2 ml of solution A to solution B, blow and mix for 15 seconds, and immediately place in a 37±1°C water bath after bubbles appear on the upper layer, as the starting point of the reaction, and time them separately. The reaction system coagulates within 30 seconds, and when the last small bubble in the coagulation rises to the surface of the reaction solution, it is regarded as the end point of the reaction, and the time is counted. Linear regression is performed with the logarithm of the concentration of the tenecteplase standard (U / ml) as the abscissa and the logarithm of the reaction end point time (seconds) as the ordinate to calculate the enzyme activity (U / ml) of the test sample.

[0114] 6. Determination of amino acid sequences of tenecteplase A and tenecteplase B

[0115] 6.1. Preparation of sample solution

[0116] Dilute tenecteplase standard A and tenecteplase standard B to 1 mg / ml with 50 mM ammonium bicarbonate solution, take 500 μl of the above dilutions into ultrafiltration tubes (pore size 3 kDa), centrifuge at 5°C, 12000 rpm / min for 10 minutes, discard the filtrate and add 500 μl of 50 mM ammonium bicarbonate solution, repeat the ultrafiltration and liquid replacement operation 3 times.

[0117] Take the solution after ultrafiltration and determine the protein concentration according to the method under "3". Take an appropriate amount of the solution containing 100 μg of protein into an EP tube, add 20 μl of RapiGest SF solution respectively, and incubate in a 57°C water bath for 30 minutes.

[0118] After cooling to room temperature, add 2μl of 1mol / L dithiothreitol solution, mix well, and continue incubation at 57℃ water bath for 1 hour. Cool to room temperature, add 2μl of 1mol / L iodoacetamide solution, mix well, and place at 25℃ away from light for 1 hour. Add 2μl of PNGase F solution, mix well, and incubate at 37℃ for 16 hours. After cooling to 25℃, add 2μl of Trypsin solution, mix well, and place in 37℃ water bath for 4 hours, and add 2μl of 10% formic acid solution to terminate the reaction.

[0119] 6.2 Liquid phase and mass spectrometry conditions

[0120] Liquid chromatography conditions: ACQUITY UPLC peptide BEH C18 (100 mm × 2.1 mm, 1.7 um, ) column, with 0.1% formic acid / water solution as mobile phase A, 0.1% formic acid / acetonitrile solution as mobile phase B, 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°C, injection volume 1 μl, sample chamber temperature 4°C,

[0121] Mass spectrometry conditions: Electrospray ionization source (ESI), detection mode Full Scan-ddMS2, positive ion scan mode, mass spectrometry voltage 3600 V, sheath gas flow rate 35 Arb, ion transfer tube temperature 320 °C, evaporation temperature 350 °C, primary mass spectrometry resolution 60000, scanning range (M / Z = 200 - 2000), maximum injection time 100 ms; secondary mass spectrometry resolution 30000, HCD mode collision fragmentation, collision energy 30%, EASY-ICTM turned on.

[0122] 6.3. Data processing method

[0123] Input the theoretical amino acid sequence of tenecteplase into BioPharma Finder. Based on the built-in algorithms and picture visualization functions in the software, match the test results with the data in the database according to the set parameters, and find the matching amino acids in the database through the masses of the primary and secondary fragments of the peptide segments to achieve amino acid identification. Fixed modification is iodoacetamide alkylation (carbamidomethyl-C), and variable modification is deamination (deamidation-N).

[0124] 7. Glycosylation analysis of tenecteplase A and tenecteplase B

[0125] 7.1. Preparation of sample solution

[0126] Dilute tenecteplase standard A and tenecteplase standard B to 1 mg / ml with ultrapure water respectively. Precisely measure 25 μl of each of the above dilutions, add 2 μl of reducing agent, mix well and incubate at 90 °C for 10 minutes. Sequentially add 2 μl of 10% NP-40 solution and 2 μl of PNGase F solution, and incubate at 37 °C for 2 hours. After taking out, add 5 μl of 2-AB labeling solution respectively, incubate at 65 °C for 2 hours, and add 300 μl of water to terminate the reaction.

[0127] Purify the samples after incubation using Supelclean tubes. Add 1 ml of conditioning solution to the Supelclean tube, discard the effluent; add 3 ml of water, discard the effluent, and complete pre-conditioning. Add the samples after incubation to the Supelclean tube, add 6 ml of ethanol to wash the samples, and discard the washing solution. Add 2 ml of conditioning solution and collect the sample eluate.

[0128] Fully concentrate the eluate in a centrifugal concentrator, dissolve it with 100 μl of water, and then add 300 μl of acetonitrile and mix well to obtain the solution.

[0129] 7.2. Liquid phase conditions and mass spectrometry conditions

[0130] Liquid chromatography conditions: Use ACQUITY UPLC Glycan BEH Amide (2.1 mm × 150 mm, 1.7μm) column, with 50mmol / L ammonium formate / 50% acetonitrile solution (pH4.5±0.05) as mobile phase A, acetonitrile as mobile phase B, gradient elution (0~0.3min, 56%B→50%B; 0.3~40min, 50%B→15%B; 40~41min, 15%B→56%B, 40~41min, 56%B), fluorescence detector, excitation wavelength 330nm, emission wavelength 420nm, flow rate 0.6ml / min, column temperature 60℃, injection volume 2μl, sample chamber temperature 5℃,

[0131] Mass spectrometry conditions: electrospray ion source (ESI), detection mode MS, positive ion scan mode, scan range (M / Z=200-2000).

[0132] 7.3 Data Processing Methods

[0133] The sugar library provided by 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.

[0134] 8. Determination of the enzyme activity of different glycosylated tenecteplase (TNK) products using an automatic hemagglutination instrument

[0135] According to the fully automatic coagulometer assay under item "3", tenecteplase standard A was used to assay the activity of 3 batches of TNK-A stock solution samples, 3 batches of TNK-A preparation samples, 3 batches of TNK-B stock solution samples, and 3 batches of TNK-B preparation samples. Similarly, tenecteplase standard B was used to assay the activity of the above 12 batches of samples. Compare whether there is a difference in the activity values ​​of the two glycosylated TNKs, TNK-A and TNK-B samples, measured using the TNK-A standard, and then compare the similarities and differences in the activity assay values ​​of the two glycosylated TNK products when TNK-B was used as the standard.

[0136] 3. Experimental Results

[0137] 1. Exclusive inspection results

[0138] like Figure 1 As shown in BC and EF, no clot dissolution was detected with human serum albumin and formulation buffer (excipient blank) using this method. Figure 1 A in FIG. 1 is the coagulation curve of the reaction solution of the reteplase sample. Figure 1In it, D is the coagulation curve of the reaction solution of TNK sample A. Under the reaction condition of 37°C, fibrinogen forms fibrin clots with a relatively high optical density under the action of thrombin, and the light absorption value increases accordingly. The plasminogen activator promotes the conversion of plasminogen in the reaction system into active plasmin and dissolves the clots, and the light absorption value decreases accordingly. The software calculates the end point of the clot dissolution reaction time using the threshold method, and the threshold = the baseline value of the coagulation curve + ((the maximum absorbance value of the coagulation curve - the baseline value of the coagulation curve) × 10%).

[0139] The above results show that human albumin in the dilution buffer and the formulation buffer do not interfere with the potency determination of rPA, and the specificity of the method is good.

[0140] 2. Results of the investigation of accuracy, precision, linearity and range

[0141] 2.1. Reteplase: The potencies (U / ml) of rPA at 5 different potency levels (60%, 80%, 100%, 120%, 140%) were determined by 2 experimental personnel within 2 days respectively. For each potency level, 2 test solutions were prepared in parallel. The results are shown in Table 2 and Table 3. The relative biases determined within the potency range of 60% - 140% are all within the range of 0.29% - 1.01%, indicating good accuracy of the method. The coefficient of variation (CV, %) of the potency determination values for 8 experiments at each potency level is less than 2.0%, and the corresponding upper 95% confidence limit is less than 4.0%, indicating good intermediate precision of the method. The intermediate precision of the method is good. Taking the theoretical potency levels of the 5 test solutions as the X-axis and the corresponding determined potencies as the Y-axis, as shown in Figure 2 C in it, 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 within the potency range of 60% - 140%.

[0142] Table 2. Detection results of reteplase potency levels (n = 8)

[0143]

[0144]

[0145] Table 3. Accuracy and intermediate precision of the determination of 5 potency levels of reteplase by the automatic blood coagulation analyzer

[0146]

[0147] 2.2 Tenecteplase: The enzyme activity (U / ml) of TNK at 5 different potency levels (60%, 80%, 100%, 120%, 140%) was measured by 2 experimenters within 2 days. Two test solutions were prepared in parallel for each potency level. The results are shown in Tables 4 and 5. The relative bias measured within the potency range of 60% - 140% was within the range of -0.24% - 0.27%, indicating good accuracy of the method. The coefficient of variation (CV, %) of the potency measurement values for 8 experiments at each potency level was less than 2.0%, and the corresponding upper 95% confidence limit was less than 4.0%, indicating good intermediate precision of the method. With the theoretical potency levels of the 5 test solutions as the X-axis and the corresponding measured potencies as the Y-axis, as shown in Figure 2 D in

[0148] Table 4. Detection results of tenecteplase potency levels (n = 8)

[0149]

[0150] Table 5. Accuracy and intermediate precision of five potency levels of tenecteplase measured by an automatic blood coagulation analyzer

[0151]

[0152]

[0153] 3. Results of repeatability investigation

[0154] Retaplase: Take the rPA sample (batch number: 20220401), prepare the sample series solution and the standard curve solution on the same day. Six samples at each concentration (800, 1000, 1200 ng / ml) were prepared in parallel, and each sample was measured 2 times. The average of the 12 measurement results was 1.03×10 6 U / ml, and the CV of the repeatability measurement results was 1.38%.

[0155] Tenecteplase: Take the TNK sample (batch number: 20230715T), prepare the sample series solution and the standard curve solution on the same day. Six samples at each concentration (800, 1000, 1200 ng / ml) were prepared in parallel, and each sample was measured 2 times. The average of the 36 measurement results was 1003320.67 IU / vial, and the CV of the repeatability measurement results was 1.83%.

[0156] The above results all prove that the repeatability of the blood coagulation analyzer method is good.

[0157] 4. Results of robustness investigation

[0158] Reteplase: Take the rPA sample (batch number: 20220401), prepare the sample series solutions and standard curve solutions, dispense the above solutions into sample cups, and place them in an automatic blood coagulation analyzer. After 0 h, 3 h, and 5 h, perform potency determination respectively; use mixed solutions of fibrinogen and plasminogen in different ratios (1:45, 1:50, 1:55), with other conditions remaining unchanged, and determine the potency of the same batch of samples. The determination results are shown in Table 6, and the CVs are all less than 3%, indicating that rPA has good stability within 5 hours in the blood coagulation analyzer. When the ratio of plasminogen to fibrinogen in the mixed solution changes slightly, it has little impact on the determination results, and the durability of the method is good.

[0159] Table 6. Durability Results of Reteplase

[0160]

[0161] Tenecteplase: Take the TNK sample (batch number: 20230715T), prepare the sample series solutions and standard curve solutions, dispense the above solutions into sample cups, and place them in an automatic blood coagulation analyzer. After 0 h, 3 h, and 5 h, perform enzyme activity determination respectively; use mixed solutions of fibrinogen and plasminogen in different ratios (1:45, 1:50, 1:55), with other conditions remaining unchanged, and determine the enzyme activity of the same batch of samples. The determination results are shown in Table 7, and the CVs are all less than 3%, indicating that TNK has good stability within 5 hours in the blood coagulation analyzer. When the ratio of plasminogen to fibrinogen in the mixed solution changes slightly, it has little impact on the determination results, and the durability of the method is good.

[0162] Table 7. Durability Results of Tenecteplase

[0163]

[0164]

[0165] 5. Comparison of Sample Determination Results

[0166] 5.1. Comparison of Results of Determining rPA Samples by Bubble Rise Method and Automatic Blood Coagulation Analyzer Method

[0167] Take 2 batches of injectable rPA and 1 batch of rPA stock solution, prepare the sample series solutions and standard curve solutions, and determine the potency by the automatic blood coagulation analyzer method according to the experimental method in item "3". At the same time, determine the potency by the bubble rise method under item "5". In both methods, each sample is determined in parallel 3 times. The results are shown in Table 8, and the determination results of both methods meet the requirements (for the preparation: it should be 0.9 - 1.2×10 6 U / ml, for the stock solution: it should be not less than 1.0×10 6U / ml). The coefficient of variation (CV) of the samples measured by the fully automatic coagulometer method for 3 times was less than 1%, while that of the bubble rising method was between 5% and 10%. Paired T-tests were performed on the measurement results of the two methods, and the P values were all greater than 0.05, indicating no significant statistical differences. The measurement results of the two methods were in good agreement, and the precision of the coagulometer method was significantly better than that of the bubble rising method.

[0168] Table 8. Comparison of the results of measuring rPA samples by the bubble rising method and the fully automatic coagulometer method

[0169]

[0170] 5.2. Comparison of the results of measuring TNK samples by the bubble rising method and the fully automatic coagulometer method

[0171] The measurement method was the same as that for reteplase, and the potencies of 6 batches of TNK preparations and the stock solution sample A were measured. The results of the Shapiro-Wilk test showed that both groups of data were normally distributed. As shown in Table 9, the p value analyzed by paired t-test was greater than 0.05, indicating no significant differences between the two measurements. The average ratio of the fully automatic coagulometer method to the bubble rising method was 1.01, and basically all points were within the 95% confidence interval (0.7 - 1.2) (as Figure 3 shown). Therefore, the measurement results of the two methods were in good agreement, and the precision of the coagulometer method was significantly better than that of the bubble rising method.

[0172] Table 9. Comparison of the results of measuring TNK samples by the bubble rising method and the fully automatic coagulometer method

[0173]

[0174]

[0175] 6. Amino acid sequence analysis results of TNK samples

[0176] The primary structure of tenecteplase was confirmed by LC-MS / MS complete mass spectrometry analysis of trypsin-digested tenecteplase standard A and tenecteplase standard B. The results showed that the TNK peptide segments from the two manufacturers were the same, with consistent sequences, and the sequence coverage at the amino acid level was 100%, and the results were as Figure 4 shown in Tables 10 - 13.

[0177] Table 10. Mass spectrometry data results of tenecteplase A digested peptide segments

[0178]

[0179]

[0180]

[0181] Table 11. Mass spectrometry data results of tenecteplase B digested peptide segments

[0182]

[0183]

[0184]

[0185] Table 12. Mass spectrometry data results of reteplase digested peptide segments

[0186]

[0187]

[0188] Table 13. Mass spectrometry data results of alteplase digested peptide segments

[0189]

[0190]

[0191]

[0192] 7. Glycosylation profile analysis results of TNK samples

[0193] After reducing two types of tenecteplase standards and digesting the oligosaccharides with peptide 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 profile structure analysis was carried out using LC-MS. The results are shown in Figure 4-5 , the main glycan structures of tenecteplase are associated with terminal galactose or sialic acid (the abundances of F(6)A2G(4)1Ga(3)1, F(6)A2G(4)2S(3)1, and F(6)A2G(4)2S(3,3)2 exceed 10%). However, triantennary and tetraantennary structures also appear in large amounts. Each N-glycosylation site has a unique glycan complexity. Most glycans are fucosylated.

[0194] The glycosylation of tenecteplase A and tenecteplase B was characterized by area normalization method, and the results are as shown in Figure 5-6As shown in Table 14 and Table 15, among the above three main glycoforms, only the content of F(6)A2G(4)2S(3,3)2 is similar in the two tenecteplases. The peak area percentage of F(6)A2G(4)1Ga(3)1 in tenecteplase A is about 8% lower than that in tenecteplase B, while the peak area percentage of F(6)A2G(4)2S(3,3)2 in tenecteplase A is about 16% higher than that in tenecteplase B. In addition, there are also differences in the glycosylation modifications of these two enzymes in other glycoform structures, especially in the glycoforms within the retention time of 30 - 45 min after the F(6)A2G(4)2S(3,3)2 peak, which are significantly different. 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 about 14% of the total glycan content. The glycoforms of tenecteplase B are more diverse, mainly 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, accounting for about 15% of its total glycan content.

[0195] Table 14. Glycosylation Characterization Results of Tenecteplase A

[0196]

[0197] Table 15. Glycosylation Characterization Results of Tenecteplase B

[0198]

[0199]

[0200] 8. Determination of Enzyme Potency of TNK Products with Different Glycosylation Modifications by Automatic Coagulometer

[0201] Take two TNK products with different glycosylation modifications, 3 batches of TNK-A stock solution and preparation samples each, and 3 batches of TNK-B stock solution and preparation samples each. According to the experimental method in item "3" (automatic coagulometer method), using TNK-A and TNK-B as standards respectively, perform clot lysis activity determination, and each sample is measured in parallel 3 times. Calculate the enzyme activities of TNK products from two manufacturers, and the results all meet the requirements of the current quality standards, indicating that the automatic coagulometer method can be used to evaluate TNK products with different glycoforms. As Figure 7As shown, when TNK-A was used as the standard, the paired T-tests were performed on the clot lysis activity measurement values of the two TNK products, and the P-value was 0.83; while when TNK-B was used as the standard, the P-value obtained from the paired T-test was 0.68, both of which were greater than 0.05. This indicates that there is no significant statistical difference in the enzyme activities of the two TNK products with different sugar types but the same amino acid sequence, and they can be used as each other's standards to evaluate the enzyme activity by the automatic blood coagulation analyzer method.

[0202] The description of the above embodiments is only for understanding the method of the present invention and its core idea. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and modifications can be made to the present 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 evaluating the efficacy and quality control of a plasminogen activator, characterized in that: The method achieves drug efficacy evaluation and quality control by measuring the potency of plasminogen activator in vitro; Preferably, the in vitro determination of the plasminogen activator titer is performed by a fully automatic coagulation analyzer.

2. The method according to claim 1, characterized in that The plasminogen activator includes any one of tenecteplase, reteplase, urokinase, streptokinase, tissue plasminogen activator, and alteplase; preferably, the plasminogen activator is any one of tenecteplase, reteplase, and alteplase.

3. The method according to claim 1, characterized in that The steps of the method include: Dilute the sample to be tested with a sample diluent to a protein concentration of 5 μg / mL to obtain a sample stock solution; The sample stock solution was diluted to a sample dilution series solution, and the clot dissolution time of the sample dilution series solution was measured using a fully automatic coagulation analyzer. The enzyme titer was calculated using the external standard method according to the following formula: Enzyme activity (IU / ml) = mean protein concentration (mg / ml) × specific activity of standard (IU / mg), Specific activity (IU / mg) = enzyme activity (IU / ml) x protein concentration (mg / ml).

4. The method according to claim 3, characterized in that The sample to be tested is a plasminogen activator; preferably, the plasminogen activator includes any one of tenecteplase, reteplase, urokinase, streptokinase, tissue plasminogen activator, and alteplase; Preferably, the plasminogen activator is any one of tenecteplase, reteplase and alteplase.

5. The method according to claim 3, characterized in that: The sample diluent is human albumin diluted with phosphate buffer; Preferably, the pH of the phosphate buffer is 7.4; Preferably, the final concentration of human albumin is 5 mg / mL; Preferably, the sample diluent is prepared by measuring 80 mL of phosphate buffer, adding 0.5 g of human albumin, diluting to 100 mL with phosphate buffer, and storing at 4° C. after fully dissolving.

6. The method according to claim 3, characterized in that The measuring steps of the fully automatic coagulation analyzer are: using the mixed solution as the starting reagent and the human plasma thrombin solution as the intermediate reagent, and measuring the clot dissolution time of the sample dilution series solution after setting the parameters; Preferably, the instrument parameters of the fully automatic coagulation analyzer are: the sample solution volume is 20 μL, the starting reagent volume is 200 μL, the intermediate reagent volume is 20 μL, the reaction temperature is 37±1°C, the detection wavelength is 405 nm, the measurement time is 900 s, the delay time is 10 s, the operation rule is the threshold method, the threshold method is the curve percentage, the threshold limit is 10%, and the collection direction is reverse.

7. The method according to claim 6, characterized in that The concentrations of the sample dilution series solutions are 800 ng / mL, 1000 ng / ml and 1200 ng / ml; Preferably, the preparation method of the human plasma thrombin solution is: reconstitute the human plasma thrombin lyophilized powder with water, and dilute it to 33U / mL with a sample diluent; Preferably, the mixed solution is a mixed solution of a human plasma fibrinogen solution and a human plasma plasminogen solution, and the final concentration of the mixed solution is 1:50 (v / v).

8. The method according to claim 1, characterized in that The model of the fully automatic coagulation analyzer is ACL TOP750.

9. A kit for determining the potency of a plasminogen activator, characterized in that: The kit comprises the reagents used in the method according to any one of claims 1 to 8; Preferably, the kit further comprises instructions.

10. Use of the method according to any one of claims 1 to 8 and / or the kit according to claim 9 in the evaluation of the efficacy and quality control of plasminogen activators.

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