In vitro detection method for coagulation promoting properties of recombinant human factor IX for injection

Abstract

This invention relates to an in vitro detection method for the procoagulant properties of recombinant human coagulation factor IX for injection. The detection method includes: adding FIXa solution and antithrombin III-heparin mixed solution to the wells of a multi-well plate, reacting at room temperature, and then adding FVIIIa solution and FX solution; then adding a chromogenic substrate solution; measuring the absorbance (OD405) of the mixture at room temperature and a wavelength of 405 nm; and calculating the inhibition rate of ATIII against FIXa using the absorbance value to complete the in vitro detection of the procoagulant properties of recombinant human coagulation factor IX for injection; wherein the inhibition rate % = (OD405) / (FVIIIa) = 1 / 2 * ... v ―OD405 s ) / (OD405 v ―OD405 b )×100%; OD405 s OD values ​​of the sample treatment group; OD405 v OD value of the solvent control group; OD405 b : OD value of the blank group.

Description

Technical Field

[0001] This invention belongs to the field of bioanalytical detection, specifically relating to an in vitro detection method for the procoagulant properties of recombinant human coagulation factor IX for injection. Background Technology

[0002] Hemophilia B is a hereditary X-chromosome-related disorder primarily caused by a deficiency of functional clotting factor IX, leading to impaired blood clotting. Patients may experience spontaneous bleeding or bleeding after injury. Over time, repeated bleeding in joints and muscles can cause severe arthritis. Furthermore, due to the relatively short half-life of clotting factor IX, hemophilia B patients require frequent infusions of clotting factor IX (FIX) to treat or prevent bleeding.

[0003] Recombinant human coagulation factor IX is completely identical to human FIX. In the extrinsic coagulation pathway, after coagulation factor IX (FIX) is activated, it can further bind with coagulation factor FVIIIa to form a Tenase complex, activating coagulation factor X, and subsequently accelerating the generation of thrombin and fibrin in the enzymatic reaction cascade of the coagulation system.

[0004] The measurement of human plasma coagulation factor IX procoagulant activity (FIX:C) is commonly used in the diagnosis of bleeding disorders caused by coagulation abnormalities and is of great significance in the diagnosis of coagulation disorders. Currently, the most commonly used clinical methods for detecting the procoagulant properties of coagulation factor IX often rely on FIX-deficient plasma (FIXDP). Summary of the Invention

[0005] To address the aforementioned problems, this invention provides an in vitro detection method for the procoagulant properties of injectable recombinant human coagulation factor IX based on a chromogenic substrate method. By simulating the process of FIXa (activated FIX product) binding with FVIIIa to form a tenase complex and activating FX in vitro, and observing the formation of FXa, the inhibitory effect of ATIII on the above activation reaction process is examined. This allows for the evaluation of the procoagulant mechanism and properties of injectable recombinant human coagulation factor IX at different concentrations of heparin and different ATIII contents. This method offers high sensitivity, good accuracy, short detection time, and applicability to various automated analytical instruments.

[0006] Specifically, this invention provides an in vitro detection method for the procoagulant properties of recombinant human coagulation factor IX for injection, the detection method comprising the following steps:

[0007] Add FIXa solution and antithrombin III-heparin mixed solution to the wells of a multi-well plate and react at room temperature; then add FVIIIa solution and FX solution and react at 36-38℃ for 5-6 min; then add chromogenic substrate solution; measure the absorbance (OD405) of the mixture at room temperature and 405 nm wavelength; use the absorbance to determine the inhibition rate of ATIII against FIXa, and complete the in vitro detection of the procoagulant properties of recombinant human coagulation factor IX for injection.

[0008] Wherein, the inhibition rate % = (OD405) v ―OD405 s ) / (OD405 v ―OD405 b )×100%;

[0009] OD405 s : OD value of the sample processing group, which is processed according to the above detection steps;

[0010] OD405 v : OD value of the solvent control group, which, compared to the sample treatment group, did not undergo the preparation and addition of antithrombin III-heparin mixed solution;

[0011] OD405 b : OD value of the blank group, which, compared to the sample treatment group, did not undergo the preparation and addition of FVIIIa solution, FIXa solution, and antithrombin III-heparin mixed solution.

[0012] Preferably, the preparation process of the FVIIIa solution is as follows: incubate 1.5-2.5 nM FVIII solution and 3.5-4.5 nM α-thrombin solution at room temperature for 5-8 min, and then add 7-9 nM hirudin solution and 45-55 μM phospholipid solution to obtain the FVIIIa solution.

[0013] Preferably, the preparation process of the FIXa solution is as follows: incubating 45-55 nM FIX solution with 20-30 pM FXIa solution at room temperature for 12-14 h to obtain the FIXa solution.

[0014] Preferably, in the antithrombin III-heparin mixed solution, the concentration of antithrombin III is 5-250 nM and the concentration of heparin is 0.01-0.1 U / mL;

[0015] Preferably, in the antithrombin III-heparin mixed solution, when the heparin concentration is 0.1 U / mL, the concentration of antithrombin III is 5 nM, 10 nM, 50 nM or 250 nM; when the heparin concentration is 0.01 U / mL, the concentration of antithrombin III is 10 nM, 50 nM or 250 nM.

[0016] Preferably, the concentration of the FX solution is 90-110 nM, and more preferably 100 nM.

[0017] Preferably, the following amounts are added to each well of the multi-well plate: 8-12 μL of FIXa solution; 8-12 μL of antithrombin III-heparin mixed solution; 8-12 μL of FVIIIa solution; and 8-12 μL of FX solution; preferably, the multi-well plate is a 384-well plate.

[0018] Preferably, the chromogenic substrate is selected as S2765, with the molecular formula N-α-ZD-Arg-Gly-Arg-pNA·2HCl; the concentration of the chromogenic substrate in each well is controlled to be 190-210 μM, preferably 200 μM.

[0019] Preferably, the absorbance is measured every 2-3 minutes, more preferably every 2 minutes, for a total measurement time of 2 hours.

[0020] Beneficial effects

[0021] This invention can be used as a method for in vitro detection of the procoagulant properties of injectable recombinant human coagulation factor IX. It has advantages such as high sensitivity and simple operation. The reaction time is short, and the generation of FXa is detected by chromogenic substrate method, thereby judging the procoagulant effect of injectable recombinant human coagulation factor IX. Attached Figure Description

[0022] Figure 1 This is a graph showing the change of OD405nm values ​​over time at different ATIII and heparin concentrations in Example 1. Detailed Implementation

[0023] The present invention will be further illustrated by the following embodiments. It should be understood that the following embodiments are for illustrative purposes only and are not intended to limit the present invention.

[0024] Recombinant human coagulation factor IX is completely identical to human FIX. In the extrinsic coagulation pathway, after activation, coagulation factor IX (FIX) can further bind to FVIIIa to form a tenase complex, activating coagulation factor X and subsequently accelerating the generation of thrombin and fibrin in the enzymatic reaction cascade of the coagulation system. Furthermore, in the presence of heparin, the activity of FIXa is negatively regulated by the serine protease inhibitor antithrombin III (ATIII).

[0025] This invention provides an in vitro detection method for the procoagulant properties of injectable recombinant human coagulation factor IX based on a chromogenic substrate method. By simulating the process of FIXa (activated FIX product) binding with FVIIIa to form a Tenase complex and activating FX in vitro, and observing the formation of FXa, the inhibitory effect of ATIII on the above activation reaction process is investigated. Furthermore, the procoagulant mechanism and procoagulant properties of injectable recombinant human coagulation factor IX at different concentrations of heparin and different ATIII contents are evaluated.

[0026] The following is an exemplary description of an in vitro detection method for the procoagulant properties of recombinant human coagulation factor IX for injection provided by the present invention. The detection method may include the following steps:

[0027] (1) Prepare FVIIIa solution (solution A), FIXa solution (solution B), FX solution, antithrombin III-heparin mixed solution, and chromogenic substrate solution respectively;

[0028] (2) Add FIXa solution and antithrombin III-heparin mixed solution to the wells of the multi-well plate and mix and react at room temperature for 25-35 min, preferably 30 min;

[0029] (3) Continue to add FVIIIa solution and FX solution, and react at 36-38℃ for 5-6 min, preferably at 37℃ for 5 min;

[0030] (4) Then, add the color-developing substrate solution;

[0031] (5) Measure the absorbance of the mixture (OD405nm or OD405) at room temperature and 405nm wavelength. Measure once every 2-3 minutes, preferably once every 2 minutes, for a total measurement time of 2 hours.

[0032] (6) Using the absorbance values ​​measured above and combined with graphing software (such as Graphpad Prism 6), the inhibition rate of ATIII against FIXa was calculated to evaluate the procoagulant properties of recombinant human coagulation factor IX for injection in vitro. The inhibition rate of each well was calculated using the full-activity wells and the background signal wells. Wherein, the inhibition rate % = (OD405) / (F405) v ―OD405 s ) / (OD405 v ―OD405 b )×100%; OD405 s : OD value of the sample processing group, which is processed according to the above detection steps; OD405 v: OD value of the solvent control group, which, compared to the sample treatment group, did not undergo the preparation and addition of the antithrombin III-heparin mixed solution; OD405 b : OD value of the blank group, which, compared to the sample treatment group, did not undergo the preparation and addition of FVIIIa solution, FIXa solution, and antithrombin III-heparin mixed solution.

[0033] Recombinant human coagulation factor IX is partially identical to human FIX. In the extrinsic coagulation pathway, after activation, coagulation factor IX (FIX) can further bind with coagulation factor FVIIIa to form a tenase complex, activating coagulation factor X and subsequently accelerating the generation of thrombin and fibrin in the enzymatic reaction cascade of the coagulation system. In the presence of heparin, FIXa forms a 1:1 complex with AT-III in plasma. The remaining FIXa activates FX, and the resulting FXa acts on the substrate, cleaving out a chromogenic group. The degree of color development is positively correlated with the amount of remaining FIXa and negatively correlated with the activity of AT-III in plasma. Among them, (OD405) s ―OD405 b ) / (OD405 v ―OD405 b ) × 100% represents the signal intensity ratio of the sample treatment group to the solvent control group after removing the influence of background values ​​in both the sample treatment group and the solvent control group, 100% — (OD405) s ―OD405 b ) / (OD405 v ―OD405 b )×100% represents the inhibition rate of ATIII against FIXa, 100%―(OD405) s ―OD405 b ) / (OD405 v ―OD405 b ) × 100% = (OD405) v ―OD405 s ) / (OD405 v ―OD405 b )×100%, therefore (OD405) v ―OD405 s ) / (OD405 v ―OD405 b The value of ATIII × 100% represents the inhibition rate of FIXa.

[0034] In some embodiments, the preparation process of the FVIIIa solution (solution A) can be as follows: incubate 1.5-2.5 nM FVIII solution and 3.5-4.5 nM α-thrombin solution at room temperature for 5-8 min, then add 7-9 nM hirudin solution and 45-55 μM cephalin solution to obtain the FVIIIa solution. The concentrations mentioned above are the final concentrations of each substance in the FVIIIa solution (solution A). Preferably, the solvent for the FVIIIa solution can be an aqueous solution containing 50 mM Tris (pH 7.4), 100 mM NaCl, 5 mM CaCl2, and 0.2% BSA.

[0035] In some embodiments, the preparation process of the FIXa solution (solution B) can be as follows: incubating 45-55 nM FIX solution with 20-30 pM FXIa solution at room temperature for 12-14 h to obtain the FIXa solution. The concentrations mentioned above are the final concentrations of each substance in the FIXa solution (solution B). Preferably, the solvent for the FIXa solution can be an aqueous solution containing 50 mM Tris (pH 7.4), 100 mM NaCl, 5 mM CaCl2, and 0.2% BSA.

[0036] In some embodiments, the antithrombin III-heparin mixture can be obtained by diluting it with a buffer solution; as an example, the buffer solution may include 50 mM Tris (pH 7.4), 100 mM NaCl, 10 mM CaCl2, and 0.2% BSA.

[0037] In the antithrombin III-heparin mixed solution, the concentration of antithrombin III can be 5-250 nM, and the concentration of heparin can be 0.01-0.1 U / mL. Preferably, in the antithrombin III-heparin mixed solution, when the heparin concentration is 0.1 U / mL, the concentration of antithrombin III is 5 nM, 10 nM, 50 nM, or 250 nM; when the heparin concentration is 0.01 U / mL, the concentration of antithrombin III is 10 nM, 50 nM, or 250 nM.

[0038] In some embodiments, the concentration of the FX solution can be 90-110 nM, preferably 100 nM.

[0039] In some embodiments, the amount of FIXa solution added to each well of the multi-well plate can be controlled to be 8-12 μL; the amount of antithrombin III-heparin mixed solution added to be 8-12 μL; the amount of FVIIIa solution added to be 8-12 μL; and the amount of FX solution added to be 8-12 μL. Preferably, the multi-well plate can be a 384-well plate.

[0040] In some embodiments, the chromogenic substrate can be S2765 (molecular formula N-α-ZD-Arg-Gly-Arg-pNA·2HCl); the amount added per well can be 19-21 μL, preferably 20 μL; and the concentration of the chromogenic substrate in each well is controlled to be 190-210 μM, preferably 200 μM.

[0041] This invention provides a method for in vitro detection of the procoagulant properties of recombinant human coagulation factor IX for injection. It features a short reaction time, detects the generation of FXa using a chromogenic substrate method, and thus determines the procoagulant effect of recombinant human coagulation factor IX for injection. It has advantages such as high sensitivity and simple operation.

[0042] The following examples further illustrate the present invention in detail. It should also be understood that the following examples are only for further explanation of the present invention and should not be construed as limiting the scope of protection of the present invention. Any non-essential improvements and adjustments made by those skilled in the art based on the above description of the present invention are within the scope of protection of the present invention. The specific process parameters, etc., in the following examples are merely examples within a suitable range; that is, those skilled in the art can make appropriate selections within the appropriate range based on the description herein, and are not intended to be limited to the specific values ​​in the examples below.

[0043] Example 1

[0044] The in vitro detection of the procoagulant properties of recombinant human coagulation factor IX for injection was conducted by studying the inhibitory effects of different concentrations of ATIII and heparin on FXa production.

[0045] (1) Preparation of FVIIIa solution (Solution A). Incubate 2 nM FVIII (source: GLPBIO) solution and 4 nM α-thrombin (source: abcam) solution at room temperature for 5 min, then add 8 nM hirudin (source: GLPBIO) solution and 50 μM cephalin (source: aladdin) to obtain the FVIIIa solution.

[0046] Prepare the FIXa solution (Solution B). Incubate 50 nM FIX (source: Pfizer) solution with 25 pM FXIa (source: HTI) solution at room temperature for 13 h to obtain the FIXa solution.

[0047] Prepare antithrombin III-heparin mixed solutions. A series of antithrombin III-heparin mixed solutions were obtained by dilution with buffer solution; wherein the concentrations of the series of antithrombin III-heparin mixed solutions were: 250 nMATIII + 0.1 U / mL heparin, 50 nMATIII + 0.1 U / mL heparin, 10 nMATIII + 0.1 U / mL heparin, solvent control group + 0.1 U / mL heparin (used for comparison with the solvent control group, no inhibition rate calculation required), solvent control group, blank group; 250 nMATIII + 0.01 U / mL heparin, 50 nMATIII + 0.01 U / mL heparin, 10 nMATIII + 0.01 U / mL heparin, solvent control group + 0.01 U / mL heparin (used for comparison with the solvent control group, no inhibition rate calculation required).

[0048] The concentration of the FX (source: abcam) solution is 100 nM.

[0049] (2) Add 10 μL of FIXa solution and 10 μL of antithrombin III-heparin mixed solution to the wells of the 384-well plate and mix and react at room temperature for 30 min.

[0050] (3) Continue to add 10 μL of FVIIIa solution and 10 μL of FX solution, and react at 37°C for 5 min;

[0051] (4) Then, add 20 μL of chromogenic substrate S2765 (source: Chromogenix) solution; control the concentration of the chromogenic substrate in each well to be 200 μM;

[0052] (5) Measure the absorbance at room temperature and 405 nm wavelength, once every 2 min, for a total of 2 h.

[0053] (6) The inhibition rate of ATIII on FIXa was obtained by using the absorbance values ​​measured above and combined with GraphpadPrism6, thereby completing the in vitro evaluation of the procoagulant properties of recombinant human coagulation factor IX for injection.

[0054] Table 1 shows the measured OD405 and the inhibition rate of ATIII against FIXa at different concentrations of ATIII and heparin. As can be seen from Table 1, in the presence of heparin, 250 nM and 50 nM ATIII, when combined with 0.1 U / mL or 0.01 U / mL heparin, showed comparable inhibitory effects on FIXa. However, at 10 nM ATIII, 0.1 U / mL heparin exhibited relatively better inhibitory effects on FIXa than 0.01 U / mL heparin. Therefore, using 0.1 U / mL heparin in combination with ATIII can achieve better inhibitory effects on FIXa.

[0055] Table 1. OD405 and the inhibition rate of ATIII on FIXa at different concentrations of ATIII and heparin.

[0056]

[0057] Figure 1 The graph shows the changes in OD405nm values ​​over time at different ATIII and heparin concentrations in Example 1. As can be seen from the graph, the trends of OD values ​​in each group change with increasing reaction time. The curves for the solvent control group, the solvent control group + 0.1 U / mL heparin, and the solvent control group + 0.01 U / mL heparin show similar trends, indicating that the presence of heparin alone has almost no inhibitory effect on FIXa. Furthermore, the curve shows a linear trend within 2 hours of the reaction, indicating that the reaction time is reasonable.

[0058] Example 2

[0059] The detection method in this embodiment is the same as in Example 1, mainly to further test the inhibitory effect of ATIII on FIXa at 250 nM, 50 nM, and 5 nM under 0.1 U / mL heparin conditions. In the presence of heparin, ATIII and FIXa were co-incubated, and then FVIIIa was added to form a tenase complex, activating FX to FXa, which interacts with the added specific chromogenic substrate S2765. The absorbance at 405 nm was detected using a microplate reader, and the test was repeated three times. The experimental results are shown in Tables 2-4 below:

[0060] Table 2. Effect of ATIII on FIXa activity (Test 1)

[0061]

[0062] Table 3. Effect of ATIII on FIXa activity (Test 2)

[0063]

[0064] Table 4. Effect of ATIII on FIXa activity (Test 3)

[0065]

[0066] As shown in Table 2-4, ATIII inhibits the activity of activated FIX in a concentration-dependent manner. At an ATIII concentration of 250 nM, the activity of activated FIX is almost completely inhibited. Therefore, it can be inferred that, in vivo, at a physiological ATIII concentration of 2.3 μM, activated FIX can be inhibited by negative feedback from the body.

[0067] Although the present invention has been described in detail through the preferred embodiments above, it should be understood that the above description should not be considered as a limitation of the present invention. Various modifications and substitutions to the present invention will be apparent to those skilled in the art after reading the above description. Therefore, the scope of protection of the present invention should be defined by the appended claims.

Claims

1. An in vitro detection method for the procoagulant properties of recombinant human coagulation factor IX for injection, characterized in that, The detection method includes the following steps: Add FIXa solution and antithrombin III-heparin mixed solution to the wells of a multi-well plate and react at room temperature; then add FVIIIa solution and FX solution and react at 36-38℃ for 5-6 min; then add chromogenic substrate solution; measure the absorbance (OD405) of the mixture at room temperature and 405 nm wavelength; use the absorbance to determine the inhibition rate of ATIII against FIXa, and complete the in vitro detection of the procoagulant properties of recombinant human coagulation factor IX for injection. The preparation process of the FIXa solution is as follows: incubate 45-55 nM FIX solution with 20-30 pM FXIa solution at room temperature for 12-14 h to obtain the FIXa solution. In the antithrombin III-heparin mixed solution, the concentration of antithrombin III is 5-250 nM, and the concentration of heparin is 0.01-0.1 U / mL; The preparation process of the FVIIIa solution is as follows: incubate 1.5-2.5 nM FVIII solution and 3.5-4.5 nM α-thrombin solution at room temperature for 5-8 min, and then add 7-9 nM hirudin solution and 45-55 µM cephalin solution to obtain the FVIIIa solution. The concentration of the FX solution is 90-110 nM; The chromogenic substrate was selected as S2765, with the molecular formula N-α-ZD-Arg-Gly-Arg-pNA·2HCl; the concentration of the chromogenic substrate in each well was controlled to be 190-210 μM. In each well of the multi-well plate, the following amounts were added: FIXa solution 8-12 μL; antithrombin III-heparin mixed solution 8-12 μL; FVIIIa solution 8-12 μL; FX solution 8-12 μL; and chromogenic substrate solution 19-21 μL. The inhibition rate % = (OD405) v ―OD405 s ) / (OD405) v ―OD405 b ) × 100%; OD405 s : OD value of the sample processing group, which is processed according to the above detection steps; OD405 v : OD value of the solvent control group, which, compared to the sample treatment group, did not undergo the preparation and addition of antithrombin III-heparin mixed solution; OD405 b : OD value of the blank group, which, compared to the sample treatment group, did not undergo the preparation and addition of FVIIIa solution, FIXa solution, and antithrombin III-heparin mixed solution.

2. The detection method according to claim 1, characterized in that, In the antithrombin III-heparin mixed solution, when the heparin concentration is 0.1 U / mL, the concentration of antithrombin III is 5 nM, 10 nM, 50 nM or 250 nM; when the heparin concentration is 0.01 U / mL, the concentration of antithrombin III is 10 nM, 50 nM or 250 nM.

3. The detection method according to claim 1, characterized in that, The concentration of the FX solution is 100 nM.

4. The detection method according to claim 1, characterized in that, The perforated plate is a 384-hole plate.

5. The detection method according to claim 1, characterized in that, The concentration of the chromogenic substrate in each well was controlled at 200 μM.

6. The detection method according to claim 1, characterized in that, The absorbance was measured every 2-3 minutes for a total of 2 hours.

7. The detection method according to claim 6, characterized in that, The absorbance was measured every 2 minutes.

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