A method for titering a marine sodium polysaccharide

Abstract

The present application relates to the technical field of medicine, and provides a potency calibration method of haena polysaccharide.The present application adopts p-nitroaniline colorimetric method to test the haena polysaccharide activity curve, wherein the abscissa of the haena polysaccharide activity curve is the reciprocal of the weight of haena polysaccharide, and the ordinate is the logarithm of the residual activity unit of FVIII; the weight of haena polysaccharide for inhibiting the iFXase (endogenous factor X enzyme) activity of 1 IU FVIII is taken as 1 potency unit, and the potency of haena polysaccharide is obtained according to the haena polysaccharide activity curve.The potency calibration method provided by the present application can accurately calibrate the anticoagulant activity intensity of haena polysaccharide, and has important significance in aspects of guaranteeing drug efficacy, drug safety and quality control.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical technology, and in particular to a method for titer determination of sodium hyaluronate polysaccharide. Background Technology

[0002] Sea cucumbers contain abundant polysaccharides in their body walls, mainly including two categories: fucoidan sulfate and fucosylated chondroitin sulfate. Among them, sea cucumber fucosylated chondroitin sulfate exhibits rich biological activities due to its unique structure, such as lowering blood lipids, anti-tumor, antiviral, and anti-inflammatory effects, especially with good anticoagulant and antithrombotic activities.

[0003] Drug potency refers to the dose or concentration of a drug required to achieve a certain effect. It is an important indicator of the strength of a drug's action, reflecting the drug's affinity for its receptor and its intrinsic activity. Different batches of drugs, even those from the same manufacturer, may exhibit variations in actual activity due to factors such as manufacturing processes and raw material sources. Potency calibration accurately determines the intensity of a drug's activity, ensuring that physicians can determine appropriate dosages based on accurate potency in clinical use, thereby enabling patients to receive effective treatment.

[0004] Sea cucumber polysaccharide is a fucoidan sulfate-based natural polysaccharide extracted from the body wall of sea cucumbers. It can target the intrinsic coagulation pathway and has good anticoagulant activity. Currently, there are no marketed drugs with the same target. It is necessary to establish a potency calibration method for sea cucumber polysaccharide standards in order to accurately determine the activity intensity of sea cucumber polysaccharide. Summary of the Invention

[0005] In view of this, the present invention provides a method for potency calibration of sodium hyaluronate. The potency calibration method provided by the present invention can accurately calibrate the anticoagulant activity of sodium hyaluronate, which is of great significance in ensuring drug efficacy, medication safety, and quality control.

[0006] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0007] A method for potency determination of sea sodium polysaccharide includes the following steps:

[0008] The activity curve of sea sodium polysaccharide was obtained by the p-nitroaniline colorimetric method. The abscissa of the sea sodium polysaccharide activity curve is the reciprocal of the weight of sea sodium polysaccharide, and the ordinate is the logarithm of the residual active units of FVIII.

[0009] The potency of sea sodium polysaccharide was determined by the weight of sea sodium polysaccharide that inhibited the activity of iFXase (intrinsic coagulation factor X enzyme) formed by 1 IU FVIII, based on the sea sodium polysaccharide activity curve.

[0010] The sodium thalassemia polysaccharide is a fucoidylated chondroitin sulfate polysaccharide; the weight-average molecular weight of the sodium thalassemia polysaccharide is 90,000 to 130,000, and the monosaccharide composition includes glucuronic acid, N-acetylgalactosamine, and fucose, wherein the molar ratio of glucuronic acid, N-acetylgalactosamine, and fucose is 1:0.8 to 1.2:0.5 to 0.8; the mass percentage of sulfate ester groups in the sodium thalassemia polysaccharide is 25% to 40%.

[0011] Preferably, the method for obtaining the activity curve of the sea sodium polysaccharide includes:

[0012] Prepare working solutions of sodium hyaluronate with gradient concentrations;

[0013] The sodium polysaccharide working solution, FVIII solution, and activating reagent solution are mixed and subjected to a first incubation to obtain a first incubation solution; the components of the activating reagent solution include FIXa, FIIa, phospholipids, buffer solution, and CaCl2.

[0014] The first incubation solution and the FX solution were mixed for a second incubation to obtain a second incubation solution;

[0015] The second incubation solution and the chromogenic substrate solution were mixed for a third incubation, and then a stop solution was added. The absorbance of the resulting mixture at 405 nm was measured to obtain the absorbance value. The chromogenic substrate was SXa-11.

[0016] Substitute the absorbance value into the FVIII complex activity standard curve, calculate the residual FVIII activity units, and plot the sea sodium polysaccharide activity curve with the reciprocal of the sea sodium polysaccharide weight as the x-axis and the logarithm of the residual FVIII activity units as the y-axis; the x-axis of the FVIII complex activity standard curve is the logarithm of the FVIII concentration, and the y-axis is the absorbance value.

[0017] Preferably, the temperature for the first incubation is 37°C and the incubation time is 5 to 20 minutes.

[0018] Preferably, the second incubation temperature is 37°C and the incubation time is 1 to 10 minutes.

[0019] Preferably, the third incubation temperature is 37°C and the incubation time is 10–30 min.

[0020] Preferably, the terminating solution is a citric acid solution with a concentration of 0.01–0.1 g / mL.

[0021] Preferably, the concentration range of the sodium polysaccharide working solution is 328–500 ng / mL.

[0022] Preferably, the buffer solution in the activating reagent solution is Tris-HCl.

[0023] Preferably, the method for obtaining the activity standard curve of the FVIII complex includes:

[0024] Prepare FVIII standard solutions with gradient concentrations;

[0025] The FVIII standard solution, water, and activating reagent solution are mixed for a fourth incubation to obtain a fourth incubation solution; the components of the activating reagent solution include FIXa, FIIa, phospholipids, buffer solution, and CaCl2.

[0026] The fourth incubation solution and the FX solution are mixed for a fifth incubation to obtain the fifth incubation solution;

[0027] The fifth incubation solution and the chromogenic substrate solution were mixed for a sixth incubation, and then a stop solution was added. The absorbance of the resulting mixture at 405 nm was measured to obtain the absorbance value. The chromogenic substrate was SXa-11.

[0028] A standard curve of the FVIII complex activity was plotted with the logarithm of FVIII concentration on the x-axis and absorbance value on the y-axis.

[0029] Preferably, the concentration of the FVIII standard solution is 0.95–3 IU / mL.

[0030] This invention provides a method for titer determination of sodium hyaluronate (SHH) polysaccharide, comprising the following steps: obtaining an activity curve of SHHH polysaccharide using the p-nitroaniline colorimetric method, wherein the abscissa of the activity curve is the reciprocal of the weight of SHHH polysaccharide, and the ordinate is the logarithm of the residual FVIII activity units; taking the weight of SHHH polysaccharide that inhibits the formation of 1 IU FVIII iFXase (intrinsic coagulation factor X enzyme) activity as one titer unit, and obtaining the titer of SHHH polysaccharide based on the activity curve. The pharmacological effect of SHHH polysaccharide is mainly as a non-heparin-like anticoagulant, and its mechanism of action is the selective inhibition of the terminal rate-limiting enzyme in the intrinsic coagulation pathway, "intrinsic factor X enzyme (FIXa-FVIIIa-PL-Ca)". 2+This invention uses the activity of sea sodium polysaccharide to inhibit iFXase (intrinsic coagulation factor X enzyme) to determine its potency. In the presence of phospholipids (PLP) and calcium ions, FVIII is activated by thrombin to form FVIIIa. FVIIIa, FIXa, phospholipids, and calcium ions form iFXase (intrinsic coagulation factor X enzyme). iFXase (intrinsic coagulation factor X enzyme) can activate factor FX to form FXa. FXa can hydrolyze the chromogenic substrate SXa-11 to release p-nitroaniline (pNA). pNA has light absorption at 405 nm, and the amount of pNA is directly proportional to the absorbance at 405 nm. Based on the above principle, this invention uses the p-nitroaniline colorimetric method to test the activity curve of sea sodium polysaccharide and to determine the potency of 1 IU of inhibitory activity. The weight of sodium naphalium polysaccharide with iFXase (intrinsic coagulation factor X enzyme) activity formed by FVIII is defined as one potency unit. The potency of sodium naphalium polysaccharide is calculated through its activity curve. The potency calibration method provided by this invention can accurately calibrate the anticoagulant activity of sodium naphalium polysaccharide, which is of great significance in ensuring drug efficacy, medication safety, and quality control. Attached Figure Description

[0031] Figure 1 This is a schematic diagram of the detection mechanism of the FVIII activity assay kit;

[0032] Figure 2 The standard curve of the activity of the FVIII complex obtained by experimenter A in the first experiment;

[0033] Figure 3 The activity curve of sea sodium polysaccharide obtained by experimenter A in the first experiment;

[0034] Figure 4 The standard curve of the activity of the FVIII complex obtained by experimenter A in the second experiment;

[0035] Figure 5 The activity curve of sea sodium polysaccharide obtained by experimenter A in the second experiment;

[0036] Figure 6 The standard curve of the activity of the FVIII complex obtained by experimenter B in the first experiment;

[0037] Figure 7 The activity curve of sodium hyaluronate polysaccharide obtained by experimenter B in the first experiment;

[0038] Figure 8 The standard curve of the activity of the FVIII complex obtained by experimenter B in the second experiment;

[0039] Figure 9 The image shows the activity curve of sodium hyaluronate obtained by experimenter B in the second experiment. Detailed Implementation

[0040] First, let's explain sea sodium polysaccharide.

[0041] In this invention, the sea cucumber polysaccharide refers to a natural polysaccharide extracted from the body wall of a sea cucumber. The sea cucumber includes, but is not limited to, one or more of the following: Holothurialeucospilota, Rough Sea Cucumber, Spiny Sea Cucumber, Black Sea Cucumber, and Black Milk Sea Cucumber, preferably Holothurialeucospilota (Brandt). The sea cucumber polysaccharide is a fucoidan-based chondroitin sulfate polysaccharide. The weight-average molecular weight of the sea cucumber polysaccharide is 90,000 to 130,000, the polydispersity index is ≤2, and the monosaccharide composition includes glucuronic acid, N-acetylgalactosamine, and fucose. The molar ratio of glucuronic acid, N-acetylgalactosamine, and fucose is 1:0.8–1.2:0.5–0.8. The mass percentage of sulfate groups in the sea cucumber polysaccharide is 25–40%.

[0042] The structural unit of sea sodium polysaccharide is shown in Formula I:

[0043]

[0044] In formula I: R1 represents H and SO3 - Or fucose, where R2 is H or SO3 - R3 is H or SO3 - R4 is H or fucose; at least one of R1 and R4 is fucose; X ⊕ Indicates a cation;

[0045] The structures of the fucosylates in R1 and R4 are shown in Formula I-1:

[0046]

[0047] In Equation I-1: R5 is H or SO 3- R6 is H or SO 3- R7 is H or SO 3- .

[0048] The cation in Formula I is hydrogen ion, alkali metal ion, alkaline earth metal ion, ammonium ion or iron ion, specifically hydrogen ion, potassium ion, sodium ion, calcium ion, ammonium ion or iron ion, preferably sodium ion; when the cation is sodium ion, the mass percentage of sodium ion in the sea sodium polysaccharide is 5-15%.

[0049] Sea sodium polysaccharide is a natural fucoidan sulfated chondroitin polysaccharide extracted from the body wall of sea cucumbers by the inventors. It can target the terminal rate-limiting enzyme iFXase in the intrinsic coagulation pathway, with good anticoagulation effect and little bleeding side effect, thus achieving the effect of anticoagulation and no bleeding.

[0050] The evaluation of the anticoagulant activity of sodium saccharide in mice showed that sodium saccharide significantly prolonged the APTT clotting time in mice and rats, without affecting PT and TT clotting times. These results indicate that sodium saccharide targets the intrinsic coagulation pathway without affecting the extrinsic coagulation pathway.

[0051] Following the heparin bioassay method in General Pharmacopoeia 1208, the activities of sea sodium polysaccharide against factor IIa, factor Xa, ATIII-IIa, and ATIII-Xa were tested. The results showed that at the experimental dose of 500 μg / mL, sea sodium polysaccharide showed no activity against factor IIa, factor Xa, and ATIII-IIa, but only a weak effect on ATIII-Xa (inhibition rate 36.7%). The activity of the rate-limiting enzyme in the intrinsic coagulation pathway, iFXase, was tested using ELISA. The results showed that the test drug significantly inhibited iFXase, with an IC50 concentration of [missing value]. 50 The value was 207.5 ng / mL; the above experiment further clarified the mechanism that sea sodium polysaccharide can selectively inhibit the activity of iFXase, the rate-limiting enzyme of the intrinsic coagulation pathway, and target the intrinsic coagulation pathway.

[0052] The calibration method of the present invention is specifically a method for calibrating the potency of sodium polysaccharide standard. The potency calibration method of the present invention will be described in detail below.

[0053] This invention provides a method for titer determination of sodium polysaccharide, comprising the following steps:

[0054] The activity curve of sea sodium polysaccharide was obtained by the p-nitroaniline colorimetric method. The abscissa of the sea sodium polysaccharide activity curve is the reciprocal of the weight of sea sodium polysaccharide, and the ordinate is the logarithm of the residual active units of FVIII.

[0055] The potency of sea sodium polysaccharide was determined by the weight of sea sodium polysaccharide that inhibits the activity of iFXase (intrinsic coagulation factor X enzyme) formed by 1 IU FVIII, based on the sea sodium polysaccharide activity curve.

[0056] In this invention, the method for obtaining the activity curve of the sea sodium polysaccharide includes:

[0057] Prepare working solutions of sodium hyaluronate with gradient concentrations;

[0058] The sodium polysaccharide working solution, FVIII solution, and activating reagent solution are mixed and subjected to a first incubation to obtain a first incubation solution; the components of the activating reagent solution include FIXa, FIIa, phospholipids, buffer solution, and CaCl2.

[0059] The first incubation solution and the FX solution were mixed for a second incubation to obtain a second incubation solution;

[0060] The second incubation solution and the chromogenic substrate solution were mixed for a third incubation, and then a stop solution was added. The absorbance of the resulting mixture at 405 nm was measured to obtain the absorbance value. The chromogenic substrate was SXa-11.

[0061] Substitute the absorbance value into the FVIII complex activity standard curve, calculate the residual FVIII activity units, and plot the standard curve with the reciprocal of the weight of sea sodium polysaccharide as the x-axis and the logarithm of the residual FVIII activity units as the y-axis; the x-axis of the FVIII complex activity standard curve is the logarithm of the FVIII concentration, and the y-axis is the absorbance value.

[0062] The reagent kit used in this invention for testing the activity curve of sea sodium polysaccharide using the p-nitroaniline colorimetric method is the FVIII Activity Assay Kit (specification: BIOPHEN FVIII:C). The reagents in the FVIII Activity Assay Kit include R1, R2, R3, and R4, where R1 is FX, R2 is an activating reagent containing FIXa, FIIa, phospholipids, buffer, and CaCl2, and the buffer is Tris-HCl; R3 is the chromogenic substrate SXa-11, and R4 is Tris-BSA Buffer, used to dilute the FVIII standard solution.

[0063] In this invention, the preparation methods for each solution used in the p-nitroaniline colorimetric method are as follows:

[0064] Ultrapure water was added to R1, R2, and R3 respectively to obtain an FX solution, an activating reagent solution, and a chromogenic substrate solution. The concentration of the FX solution was preferably 40–60 nM, more preferably 50 nM. The concentration of FIXa in the activating reagent solution was preferably 40–80 nM, more preferably 60 nM. The concentration of FIIa was preferably 30–60 nM, more preferably 47 nM. The concentration of phospholipid was preferably 0.1–0.6 mg / mL, more preferably 0.32 mg / mL. The concentration of CaCl2 was preferably 5–15 mM, more preferably 12 mM. The concentration of the chromogenic substrate solution was preferably 4–12 mM, more preferably 8.4 mM. FVIII was dissolved and diluted using R4 to obtain an FVIII solution.

[0065] This invention first prepares working solutions of sodium hyaluronate with gradient concentrations. In this invention, the concentration range of the sodium hyaluronate working solutions is preferably 328–500 ng / mL, more preferably 328.05–500 ng / mL, specifically 328.05 ng / mL, 364.5 ng / mL, 405 ng / mL, 450 ng / mL, and 500 ng / mL; the solvent used to prepare the sodium hyaluronate working solutions is preferably water, and the water is preferably double-distilled water (ddH2O).

[0066] After obtaining the sodium polysaccharide working solution, the present invention mixes the sodium polysaccharide working solution, FVIII solution, and activating reagent solution for a first incubation to obtain a first incubation solution; the components of the activating reagent solution include FIXa, FIIa, phospholipids, buffer solution, and CaCl2. In the present invention, the concentration of the FVIII solution is preferably 3 IU / mL; the volume ratio of the sodium polysaccharide working solution to the FVIII solution is preferably 1:1; the volume ratio of the sodium polysaccharide working solution to the activating reagent solution is preferably 1:1; the temperature of the first incubation is preferably 37°C, and the incubation time is preferably 5–20 min, specifically 15 min. During the first incubation, in the presence of phospholipids (PLP) and calcium ions, FVIII is activated by FIIa to form FVIIIa, and FVIIIa, FIXa, phospholipids, and calcium ions form endogenous factor X enzyme (iFXase).

[0067] After obtaining the first incubation solution, the present invention mixes the first incubation solution and the FX solution for a second incubation to obtain the second incubation solution. In the present invention, the volume ratio of the sodium saccharide working solution to the FX solution is preferably 1:1; the temperature of the second incubation is 37°C, and the incubation time is 1-10 min, preferably 1 min. During the second incubation, iFXase (endogenous coagulation factor X enzyme) activates FX to form FXa, while the presence of sodium saccharide inhibits the activity of some iFXase (endogenous coagulation factor X enzyme), preventing the activation of FX.

[0068] After obtaining the second incubation solution, the present invention mixes the second incubation solution and the chromogenic substrate solution for a third incubation, then adds a stop solution, and tests the absorbance of the resulting mixture at 405 nm to obtain the absorbance value. In this invention, the chromogenic substrate is SXa-11; the volume ratio of the sodium saccharide working solution to the chromogenic substrate solution is preferably 1:1; the temperature of the third incubation is preferably 37℃, and the incubation time is preferably 10-30 min, specifically 15 min; the stop solution is preferably a citric acid solution, and the concentration of the citric acid solution is preferably 0.01-0.1 g / mL, specifically 0.02 g / mL; the volume ratio of the sodium saccharide working solution to the stop solution is preferably 2:3. The instrument used to test the absorbance is preferably an enzyme-linked immunosorbent assay (ELISA) reader. During the third incubation, FXa hydrolyzes the chromogenic substrate SXa-11, releasing p-nitroaniline (pNA). pNA absorbs light at 405 nm, and the amount of pNA is directly proportional to the absorbance value at 405 nm.

[0069] After obtaining the absorbance value, the present invention substitutes the absorbance value into the FVIII complex activity standard curve, calculates the residual FVIII activity units, and plots the sea sodium polysaccharide activity curve with the reciprocal of the sea sodium polysaccharide weight as the abscissa and the logarithm of the residual FVIII activity units as the ordinate. In the present invention, after obtaining the sea sodium polysaccharide activity curve, the invention also includes obtaining a fitting equation based on the sea sodium polysaccharide activity curve.

[0070] In this invention, the horizontal axis of the FVIII complex activity standard curve is the logarithm of the FVIII concentration, and the vertical axis is the absorbance.

[0071] In this invention, the method for obtaining the activity standard curve of the FVIII complex preferably includes:

[0072] Prepare FVIII standard solutions with gradient concentrations;

[0073] The FVIII standard solution, water, and activating reagent solution are mixed for a fourth incubation to obtain a fourth incubation solution; the components of the activating reagent solution include FIXa, FIIa, phospholipids, buffer solution, and CaCl2.

[0074] The fourth incubation solution and the FX solution are mixed for a fifth incubation to obtain the fifth incubation solution;

[0075] The fifth incubation solution and the chromogenic substrate solution were mixed for a sixth incubation, and then a stop solution was added. The absorbance of the resulting mixture at 405 nm was measured to obtain the absorbance value. The chromogenic substrate was SXa-11.

[0076] A standard curve of the FVIII complex activity was plotted with the logarithm of FVIII concentration on the x-axis and absorbance value on the y-axis.

[0077] In this invention, the kit used to test the activity standard curve of the FVIII complex is the same as the kit used to test the activity curve of sea sodium polysaccharide. The preparation methods of FX solution, activating reagent solution and chromogenic substrate solution are also the same as those used in the sea sodium polysaccharide activity curve test, and will not be repeated here.

[0078] This invention first prepares FVIII standard solutions with gradient concentrations. In this invention, the concentration range of the FVIII standard solutions is preferably 0.95–3 IU / mL, more preferably 0.94922 IU / mL–3 IU / mL, and the specific concentrations of the gradient FVIII standard solutions are 3 IU / mL, 2.25 IU / mL, 1.6875 IU / mL, 1.265625 IU / mL, and 0.94922 IU / mL. In a specific embodiment of this invention, it is preferable to use reagent R4 from the kit to dissolve FVIII to prepare a 4 IU / mL stock solution, and then use reagent R4 to serially dilute the stock solution to obtain the FVIII standard solutions of each gradient.

[0079] After obtaining the FVIII standard solution, the present invention mixes the FVIII standard solution, water, and activating reagent solution for a fourth incubation to obtain a fourth incubation solution; the preferred temperature for the fourth incubation is 37°C, and the preferred incubation time is 5-20 min, specifically 15 min; the specific operating conditions for the fourth incubation are the same as those for the first incubation, except that the sodium polysaccharide working solution is replaced with water and the FVIII solution is replaced with FVIII standard solutions of gradient concentrations.

[0080] After obtaining the fourth incubation solution, the present invention mixes the fourth incubation solution with the FX solution for a fifth incubation to obtain the fifth incubation solution. In the present invention, the temperature of the fifth incubation is preferably 37°C, and the incubation time is preferably 1 to 10 minutes, specifically 1 minute. The specific operating conditions of the fifth incubation are the same as those of the second incubation, and will not be repeated here.

[0081] After obtaining the fifth incubation solution, the present invention mixes the fifth incubation solution with the chromogenic substrate solution for a sixth incubation, then adds a stop solution, and tests the absorbance of the resulting mixture at 405 nm to obtain the absorbance value; the chromogenic substrate is SXa-11. In the present invention, the preferred temperature for the sixth incubation is 37°C, and the preferred incubation time is 10–30 min, specifically 15 min; the specific operating conditions for the sixth incubation are the same as those for the third incubation, and will not be repeated here.

[0082] After obtaining the absorbance values, this invention plots a standard curve of the FVIII complex activity with the logarithm of the FVIII concentration on the x-axis and the absorbance values ​​on the y-axis. In a specific embodiment of this invention, after obtaining the standard curve of the FVIII complex activity, the method further includes deriving a fitting equation based on the standard curve.

[0083] In this invention, the absorbance value obtained from the sodium thalassium polysaccharide activity curve test is substituted into the FVIII complex activity standard curve to calculate the FVIII concentration corresponding to the absorbance value. This FVIII concentration is the residual FVIII concentration in the sodium thalassium polysaccharide activity curve test. The residual FVIII activity units are further calculated based on the residual FVIII concentration. The sodium thalassium polysaccharide activity curve is plotted based on the logarithm of the residual FVIII activity units and the corresponding absorbance value. Then, the weight of sodium thalassium polysaccharide required to inhibit the activity of iFXase (intrinsic coagulation factor X enzyme) forming 1 IU of FVIII is calculated based on the sodium thalassium polysaccharide activity curve, which is one potency unit of sodium thalassium polysaccharide. For ease of representation, the potency of sodium thalassium polysaccharide is ultimately expressed as the number of potency units per unit mass of polysaccharide. The unit mass mentioned in this invention is 1 mg. In a specific embodiment of this invention, the potency of sodium thalassium polysaccharide is tested four times, and the average of the four tests is taken as the potency of sodium thalassium polysaccharide.

[0084] The technical solutions of this invention will be clearly and completely described below with reference to the embodiments thereof. Obviously, the described embodiments are only a part of the embodiments of this invention, and not all of them. Based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this invention.

[0085] Example 1: Extraction of sodium polysaccharide

[0086] After careful separation of the body wall from other tissues, the sea cucumber *Holothurialeucospilota* (Brandt) was immediately immersed in acetone and stored at 4°C for 24 hours. The dried tissue (500g) was ground into powder and suspended in 10L of 0.1mol / L sodium acetate buffer (pH 6) containing 50g papain. The mixture was incubated at 65°C for 24 hours, followed by low-temperature centrifugation (4000rpm, 4°C, 30min) to obtain the enzymatic supernatant. The pH was adjusted to 2.5±0.5 by adding 6mol / L hydrochloric acid solution to the supernatant, and the reaction was carried out with stirring for 2 hours. The mixture was then centrifuged at low temperature (4000rpm, 4°C, 30min) to obtain the acid hydrolysis supernatant. The pH of the acid hydrolysis supernatant was adjusted to 7.0 ± 0.5 by adding 40 wt% sodium hydroxide aqueous solution. While stirring, an equal volume of 95 vol% ethanol was added to the reaction system. After maintaining the temperature at -10℃ for 24 h, the mixture was centrifuged at low temperature (4000 rpm, 4℃ for 30 min) to collect the precipitate. The precipitate was dissolved in 500 mL of distilled water, and 1 L of 95 vol% ethanol was added. After maintaining the temperature at -10℃ for 24 h, the mixture was centrifuged at low temperature (4000 rpm, 4℃ for 30 min) to collect the precipitate. The precipitate was washed with 500 mL of 95 vol% ethanol, centrifuged at low temperature (4000 rpm, 4℃ for 30 min), and the solid was collected. The solid was dissolved in 500 mL of distilled water and freeze-dried to obtain 4 g of crude sodium polysaccharide.

[0087] Four g of crude sodium polysaccharide was placed on a DEAE cellulose column (10 cm × 4 cm) pre-equilibrated with 0.1 mol / L HAc-NaAc buffer (pH 6). The column was washed with 5 L of HAc-NaAc buffer (pH 6) containing 0.5 mol / L NaCl and 2 L of HAc-NaAc buffer (pH 6) containing 1 mol / L NaCl, respectively, at a flow rate of 50 mL / min. 500 mL of each fraction was collected. Molecular weight and molecular weight distribution were determined by HPLC. Fractions with a weight-average molecular weight of 90,000–130,000 were collected, and one volume of 95 vol% ethanol was added to the eluent. The mixture was incubated at -10°C for 24 h, followed by low-temperature centrifugation (4000 rpm, 4°C for 30 min), and the resulting precipitate was collected. The precipitate was dissolved in distilled water and concentrated to half its original volume using an ultrafiltration membrane with a molecular weight cutoff of 10,000. Water was added back to the original volume, and the process was repeated, ultrafiltration to half the original volume, adding water again, and concentrating to half the original volume. The concentrated solution was collected, freeze-dried, and 2.5 g of pure sodium polysaccharide (sodium cation) was obtained. Multiple batches of parallel samples were prepared using the above procedure, and batch 202401 was used in subsequent examples. The samples from batch 202401 had a weight-average molecular weight of 108,791, a PDI value of 1.32, a molar ratio of glucuronic acid, N-acetylgalactosamine, and fucose of 1:1.04:0.67, a sodium ion content of 9.24%, and a sulfate ester group content of 27.3% as determined by ion chromatography.

[0088] Example 2: Establishment of a method for determining the potency of sodium polysaccharide standards

[0089] The pharmacological effects of sodium hyaluronate are mainly as a non-heparin-like anticoagulant, with the mechanism of action being the selective inhibition of the terminal rate-limiting enzyme in the intrinsic coagulation pathway, "intrinsic factor X (FIXa-FVIIIa-PL-Ca)". 2+ (complex, iFXase).

[0090] Since FVIII standards are readily available, the FVIII activity assay kit (FVIII:C) was used to determine the potency of this product by measuring its inhibitory activity against iFXase (intrinsic coagulation factor X enzyme). Under the assay conditions, the amount of sodium naphthoic acid polysaccharide that inhibits the activity of iFXase (intrinsic coagulation factor X enzyme) formed by 1 IU of FVIII was defined as one potency unit of this product.

[0091] The detection mechanism of the FVIII activity assay kit is as follows: In the presence of phospholipids (PLP) and calcium ions, FVIII is activated by thrombin to form FVIIIa. FVIIIa, FIXa phospholipids, and calcium ions form an enzyme complex (iFXase (intrinsic coagulation factor X enzyme)), thereby activating factor FX. The generated factor FXa hydrolyzes the chromogenic substrate SXa-11, releasing p-nitroaniline (pNA). pNA absorbs light at 405 nm, and the amount of pNA is directly proportional to the absorbance value at 405 nm (OD405). The content of p-nitroaniline at 405 nm is detected by a microplate reader to reflect the enzyme complex activity. The logarithm of the FVIII concentration is directly proportional to OD405. The mass of sodium polysaccharide that inhibits the enzyme complex activity formed by 1 IU of FVIII is defined as one potency unit of this product. Figure 1 This is a schematic diagram of the detection mechanism of the FVIII activity assay kit.

[0092] The specific calibration method is as follows:

[0093] 1. Instruments and Materials

[0094] The instruments and reagents required for the experiment are shown in Tables 1 and 2.

[0095] Table 1. Instruments required for the experiment

[0096]

[0097] Table 2. Reagents and reagents required for the experiment

[0098]

[0099] 2. Experimental Methods

[0100] 2.1 Activity standard curve of FVIII complex

[0101] 1) Preparation of reagent solution:

[0102] 2.5 mL of ultrapure water was added to R1 (FX), R2 (FIXa, FIIa, phospholipid, Tris-HCl, CaCl2), and R3 (SXa-11) respectively to obtain solutions R1 (FX), R2 (activator), and R3 (chromogenic substrate); the concentration of R1 solution was 50 nM; the concentration of FIXa in R2 solution was 60 nM, the concentration of FIIa was 47 nM, the concentration of phospholipid was 0.32 mg / mL, and the concentration of CaCl2 was 12 mM; the concentration of R3 solution was 8.4 mM. R4 (Tris-BSA Buffer) was used to dilute the FVIII standard solution.

[0103] 2) Preparation of FVIII standard solution:

[0104] Dissolve 4.9 IU / vial of FVIII in 1.225 mL of R4 reagent to prepare a 4 IU / mL FVIII stock solution; dissolve 1000 μL of the 4 IU / mL FVIII stock solution in 333 μL of R4 to prepare a 3 IU / mL FVIII standard solution; dissolve 600 μL of the 3 IU / mL FVIII standard solution in 200 μL of R4 to prepare a 2.25 IU / mL FVIII standard solution; dissolve 600 μL of the 2.25 IU / mL FVIII standard solution in 200 μL of R4 reagent. Prepare a 1.6875 IU / mL FVIII standard solution by adding 200 μL of R4 to 600 μL of the 1.6875 IU / mL FVIII standard solution; prepare a 1.265625 IU / mL FVIII standard solution by adding 200 μL of R4 to 600 μL of the 1.265625 IU / mL FVIII standard solution; prepare a 0.94922 IU / mL FVIII standard solution by adding 200 μL of R4 to 600 μL of the 1.265625 IU / mL FVIII standard solution.

[0105] 3) Preparation of the reaction termination solution:

[0106] Weigh 1g of citric acid and add 50mL of ddH2O to prepare a citric acid stop solution with a concentration of 0.02g / mL.

[0107] 4) Determination of the activity standard curve of the FVIII complex:

[0108] (1) Add 20 μL of ddH2O to each of the 5 wells of the 96-well plate;

[0109] (2) Add 20 μL of FVIII standard solution of different concentrations to the above test wells in sequence, and add 20 μL of L2 solution to each well. After adding, incubate at 37°C for 15 min.

[0110] (3) Add 20 μL of L1 solution to each well and incubate at 37°C for 1 min;

[0111] (4) Add 20 μL of R3 solution to each well, incubate at 37°C for 15 min, add 30 μL of stop solution, and measure the absorbance (OD405) at 405 nm using an ELISA reader.

[0112] (5) Plot the activity standard curve of the FVIII complex with the logarithm of FVIII concentration as the x-axis and OD405 as the y-axis.

[0113] 2.2 Polysaccharide activity titer determination

[0114] 1) Preparation of reagent solution:

[0115] Add 2.5 mL of ultrapure water to R1 (FX), R2 (FIXa, FIIa, phospholipid, Tris-HCl, CaCl2), and R3 (SXa-11) respectively to obtain R1 (FX) solution, R2 (activator) solution, and R3 (chromogenic substrate) solution. R4 (Tris-BSA Buffer) is used to dilute the FVIII standard solution. The preparation method and concentration are the same as those for the kit solution preparation in section 2.1, FVIII complex activity standard curve section.

[0116] 2) Preparation of 3 IU FVIII solution:

[0117] Take FVIII (4.9 IU / vial), dissolve it with 1.225 mL of R4 reagent to prepare 4 IU / mL FVIII; take 1000 μL of 4 IU / mL FVIII, add 333 μL of R4 to prepare 3 IU / mL FVIII solution.

[0118] 3) Preparation of polysaccharide mother liquor and working solution

[0119] Accurately weigh 25 mg of the polysaccharide reference standard and dissolve it in 10 mL of ddH2O to prepare a 2.5 mg / mL polysaccharide stock solution 1. Take 100 μL of stock solution 1 and add 9900 μL of ddH2O to dilute it to a 25 μg / mL polysaccharide stock solution 2. Take 100 μL of stock solution 2 and add 4900 μL of ddH2O to dilute it to a 500 ng / mL polysaccharide working solution 1. Take 900 μL of polysaccharide working solution 1 and add 100 μL of ddH2O to dilute it. Prepare a polysaccharide working solution 2 with a concentration of 450 ng / mL; take 900 μL of polysaccharide working solution 2, add 100 μL of ddH2O, and dilute to prepare a polysaccharide working solution 3 with a concentration of 405 ng / mL; take 900 μL of polysaccharide working solution 3, add 100 μL of ddH2O, and dilute to prepare a polysaccharide working solution 4 with a concentration of 364.5 ng / mL; take 900 μL of polysaccharide working solution 4, add 100 μL of ddH2O, and dilute to prepare a polysaccharide working solution 5 with a concentration of 328.05 ng / mL.

[0120] 4) Preparation of the reaction termination solution

[0121] Weigh 1g of citric acid and add 50mL of ddH2O to prepare a citric acid stop solution with a concentration of 0.02g / mL.

[0122] 5) Polysaccharide activity assay

[0123] (1) Add 20 μL of ddH2O to the blank test wells of the 96-well plate, and add 20 μL of polysaccharide working solution of different concentrations to the polysaccharide test wells in sequence;

[0124] (2) Add 20 μL of 3 IU / mL FVIII solution to each of the above test wells in sequence, and add 20 μL of L2 to each well. After adding, incubate at 37°C for 15 min.

[0125] (3) Add 20 μL of L1 solution to each well and incubate at 37°C for 1 min;

[0126] (4) Add 20 μL of R3 solution to each well, incubate at 37°C for 15 min, add 30 μL of stop solution, and measure the absorbance (OD405) at 405 nm using an ELISA reader.

[0127] (5) Substitute the absorbance value into the FVIII complex activity standard curve to determine the concentration of residual FVIII. For ease of calculation, use the reciprocal of the polysaccharide sample amount magnified 50 times as the x-axis and the logarithm of the residual FVIII unit magnified 50 times as the y-axis to construct a regression equation.

[0128] (6) The initial concentration of FVIII was 3 IU / mL. The amount of FVIII and polysaccharide solution added was 20 μL. For ease of calculation, the data was amplified 50 times. The calculation was based on the amount of FVIII added being 1 mL, that is, the amount of FVIII added in the reaction system was 3 IU. The amount of sea sodium polysaccharide required to inhibit 1 IU and leave 2 IU was calculated, which is 1 potency unit of sea sodium polysaccharide.

[0129] 3. Experimental Results:

[0130] The following experimental data were obtained by two researchers who independently conducted two tests each. The results are as follows:

[0131] 3.1 Results of the first experiment by experimenter A

[0132] 1) Activity standard curve of FVIII complex

[0133] The activity test results of FVIII complex at different unit concentrations are shown in Table 3.

[0134] Table 3. Results of FVIII complex enzyme activity

[0135] FVIII (IU / mL) 0.94921875 1.265625 1.6875 2.25 3 OD405 0.895 1.005 1.243 1.442 1.643 Log(FVIII) -0.022633692 0.102305045 0.227243782 0.352182518 0.477121255

[0136] Based on the experimental data, a standard curve was plotted with the logarithm of FVIII concentration on the x-axis and absorbance OD405 on the y-axis. The results are as follows: Figure 2 As shown.

[0137] 2) Results of polysaccharide activity assay

[0138] The results of the polysaccharide activity tests at different concentrations are shown in Table 4. Based on the experimental data, the absorbance of the polysaccharide working solution was substituted into the FVIII complex enzyme activity standard curve, and the logarithm of the residual FVIII concentration was calculated. The results are shown in Table 4.

[0139] Table 4. Results of polysaccharide activity testing

[0140]

[0141] The initial concentration of FVIII was 3 IU / mL, and the amounts of both FVIII and polysaccharide solution added were 20 μL. Based on the calculation of 3 IU of FVIII added to the reaction system, the data needs to be amplified 50 times. The results are shown in Table 5, the results of Experimenter A's first experiment.

[0142] Table 5 Valence Measurement Data Table

[0143]

[0144] Based on the addition of 3 IU of FVIII in the reaction system, the weight of the sodium hyaluronate required to inhibit 1 IU and leave a residual 2 IU is defined as one potency unit (i.e., 1 IU) of sodium hyaluronate. According to the results in Table 5, a regression equation was established with the logarithm of residual activity units × 50 as the ordinate and 1 / polysaccharide sample amount × 50 as the abscissa. The results are as follows. Figure 3 As shown.

[0145] according to Figure 3 The regression equation was calculated based on a residual activity unit of 2 IU (i.e., inhibition of 1 IU), resulting in Log 2IU = 0.301029996. Substituting this into the regression equation (i.e., 0.1479X - 0.7626 = 0.301029996), the reciprocal of the sample amount (X) was calculated to be 7.191548 μg. -1 The sample amount was 0.139052115 μg. For ease of representation, the potency of sea sodium polysaccharide is expressed as the number of potency units per unit mass (mg) of polysaccharide. After calculation, the potency of the sea sodium polysaccharide standard is 7192 IU / mg.

[0146] 3.2 Experimenter A - Results of the Second Experiment

[0147] 1) Activity standard curve of FVIII complex

[0148] The activity results of the FVIII complex at different unit concentrations are shown in Table 6.

[0149] Table 6 Results of FVIII complex enzyme activity

[0150]

[0151] Based on the experimental data, a standard curve was plotted with the logarithm of FVIII concentration on the x-axis and absorbance OD405 on the y-axis. The results are as follows: Figure 4 As shown.

[0152] 2) Results of polysaccharide activity assay

[0153] The activity results of polysaccharides at different unit concentrations are shown in Table 7. Based on the experimental data, the absorbance of the polysaccharide working solution was substituted into the FVIII complex enzyme activity standard curve, and the logarithm of the residual FVIII concentration was calculated. The results are shown in Table 7.

[0154] Table 7 Results of polysaccharide activity test

[0155] Polysaccharide working solution (μg / mL) 0.32805 0.3645 0.405 0.45 0.5 OD405 0.418 0.328 0.27 0.227 0.182 LogFVIII concentration logarithm -0.34244 -0.40268 -0.441499 -0.47028 -0.5004

[0156] The initial concentration of FVIII was 3 IU / mL, and the amounts of both FVIII and polysaccharide solution added were 20 μL. Based on the calculation of 3 IU of FVIII added to the reaction system, the data needs to be amplified 50 times. The results are shown in Table 8, the results of Experimenter A's second experiment.

[0157] Table 8 Valence Measurement Data Table

[0158]

[0159]

[0160] Based on the addition of 3 IU of FVIII in the reaction system, the weight of the sodium hyaluronate required to inhibit 1 IU and leave a residual 2 IU is defined as one potency unit (i.e., 1 IU) of sodium hyaluronate. According to the results in Table 8, a regression equation was established with the logarithm of residual activity units × 50 as the ordinate and 1 / polysaccharide sample amount × 50 as the abscissa. The results are as follows: Figure 5 As shown.

[0161] The residual active unit is 2 IU, and Log 2IU = 0.301029996. Substituting this into the regression equation, the reciprocal of the sample amount is calculated to be 7.465604 μg. -1 The sample amount was 0.133948 μg. For ease of representation, the potency of the sodium natriuretic polysaccharide is expressed as the number of potency units per unit mass (mg) of polysaccharide. After calculation, the potency of the polysaccharide reference standard was 7466 IU / mg.

[0162] 3.3 Experimenter B - Results of the First Experiment

[0163] 1) Activity standard curve of FVIII complex

[0164] The activity test results of FVIII complex at different unit concentrations are shown in Table 9.

[0165] Table 9 Results of FVIII complex enzyme activity

[0166] FVIII (IU / mL) 0.94921875 1.265625 1.6875 2.25 3 OD405 0.711 0.891 1.165 1.487 1.749 Log(FVIII) -0.022633692 0.102305045 0.227243782 0.352182518 0.477121255

[0167] Based on the experimental data, a standard curve was plotted with the logarithm of FVIII concentration on the x-axis and absorbance OD405 on the y-axis. The results are as follows: Figure 6 As shown.

[0168] 2) Results of polysaccharide activity assay

[0169] The activity results of polysaccharides at different unit concentrations are shown in Table 10. Based on the experimental data, the absorbance of the polysaccharide working solution was substituted into the FVIII complex enzyme activity standard curve, and the logarithm of the residual concentration of FVIII was calculated. The results are shown in Table 10.

[0170] Table 10 Results of polysaccharide activity testing

[0171] Polysaccharide working solution (ug / mL) 0.32805 0.3645 0.405 0.45 0.5 OD405 value 0.373 0.326 0.238 0.211 0.166 LogFVIII concentration logarithm -0.1597 -0.18167 -0.2228144 -0.23544 -0.25647

[0172] The initial concentration of FVIII was 3 IU / mL, and the amounts of both FVIII and polysaccharide solution added were 20 μL. Based on the calculation of 3 IU of FVIII added to the reaction system, the data needs to be amplified 50 times. The results are shown in Table 11, the results of Experimenter B's first experiment.

[0173] Table 11 Valence Measurement Data Table

[0174]

[0175] Based on the addition of 3 IU of FVIII in the reaction system, the weight of the sodium hyaluronate required to inhibit 1 IU and leave a residual 2 IU is defined as one potency unit (i.e., 1 IU) of sodium hyaluronate. According to the results in Table 11, a regression equation was established with the logarithm of residual activity units × 50 as the ordinate and 1 / polysaccharide sample amount × 50 as the abscissa. The results are as follows: Figure 7 As shown.

[0176] The residual active unit is 2 IU, and Log 2IU = 0.301029996. Substituting this into the regression equation, the reciprocal of the sample amount is calculated to be 7.906336 μg. -1 The sample amount was 0.126481 μg. For ease of representation, the potency of the sodium natriuretic polysaccharide is expressed as the number of potency units per unit mass (mg) of polysaccharide. After calculation, the potency of the polysaccharide reference standard was 7906 IU / mg.

[0177] 3.4 Experimenter B - Results of the Second Experiment

[0178] 1) Activity standard curve of FVIII complex

[0179] The activity test results of FVIII complex at different unit concentrations are shown in Table 12.

[0180] Table 12 Results of FVIII complex enzyme activity

[0181] FVIII (IU / mL) 0.94921875 1.265625 1.6875 2.25 3 OD405 0.783 0.953 1.134 1.356 1.473 Log(FVIII) -0.022633692 0.102305045 0.227243782 0.352182518 0.477121255

[0182] Based on the experimental data, a standard curve was plotted with the logarithm of FVIII concentration on the x-axis and absorbance OD405 on the y-axis. The results are as follows: Figure 8 As shown.

[0183] 2) Results of polysaccharide activity assay

[0184] The results of polysaccharide activity tests at different unit concentrations are shown in Table 13. Based on the experimental data, the absorbance of the polysaccharide working solution was substituted into the FVIII complex enzyme activity standard curve, and the logarithm of the residual FVIII concentration was calculated. The results are shown in Table 13.

[0185] Table 13 Results of polysaccharide activity testing

[0186] Polysaccharide working solution (ug / mL) 0.32805 0.3645 0.405 0.45 0.5 OD405 0.446 0.357 0.291 0.227 0.203 LogFVIII concentration logarithm -0.25893 -0.3213 -0.36755 -0.4124 -0.42922

[0187] The initial concentration of FVIII was 3 IU / mL, and the amounts of both FVIII and polysaccharide solution added were 20 μL. Based on the calculation of 3 IU of FVIII added to the reaction system, the data needs to be amplified 50 times. The results are shown in Table 14, the results of Experimenter B's second experiment.

[0188] Table 14 Valence Measurement Data Table

[0189]

[0190] Based on the addition of 3 IU of FVIII in the reaction system, the weight of the sodium hyaluronate required to inhibit 1 IU and leave a residual 2 IU is defined as one potency unit (i.e., 1 IU) of sodium hyaluronate. According to the results in Table 14, a regression equation was established with the logarithm of residual activity units × 50 as the ordinate and 1 / polysaccharide sample amount × 50 as the abscissa. The results are as follows: Figure 9 As shown.

[0191] Based on the residual activity unit of 2 IU, Log 2IU = 0.301029996. Substituting this into the regression equation, the reciprocal of the sample amount is calculated to be 6.463757 μg. -1 The sample amount was 0.154709 μg. For ease of representation, the potency of the sodium natriuretic polysaccharide is expressed as the number of potency units per unit mass (mg) of polysaccharide. After calculation, the potency of the polysaccharide reference standard was 6464 IU / mg.

[0192] 3.5 Results of sodium polysaccharide potency determination

[0193] Based on the results of four experiments conducted by the two researchers, the potency of sea sodium polysaccharide was 7192 IU / mg, 7466 IU / mg, 7906 IU / mg, and 6464 IU / mg, with an average of 7257 IU / mg. Therefore, the potency of sea sodium polysaccharide was determined to be 7257 IU / mg.

[0194] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A method for potency determination of sodium polysaccharide, characterized in that, Includes the following steps: The activity curve of sea sodium polysaccharide was obtained by the p-nitroaniline colorimetric method. The abscissa of the sea sodium polysaccharide activity curve is the reciprocal of the weight of sea sodium polysaccharide, and the ordinate is the logarithm of the residual active units of FVIII. The potency of sea sodium polysaccharide was determined by the weight of sea sodium polysaccharide that inhibited the formation of iFXase by 1 IU FVIII, based on the sea sodium polysaccharide activity curve. The sodium thalassemia polysaccharide is a fucoidan-based chondroitin sulfate polysaccharide; the weight-average molecular weight of the sodium thalassemia polysaccharide is 90,000 to 130,000, and the monosaccharide composition includes glucuronic acid, N-acetylgalactosamine, and fucose, with a molar ratio of glucuronic acid, N-acetylgalactosamine, and fucose of 1:0.8 to 1.2:0.5 to 0.8; the mass percentage of sulfate ester groups in the sodium thalassemia polysaccharide is 25% to 40%. The method for obtaining the activity curve of the sea sodium polysaccharide includes: Prepare working solutions of sodium hyaluronate with gradient concentrations; The sodium polysaccharide working solution, FVIII solution, and activating reagent solution are mixed and subjected to a first incubation to obtain a first incubation solution; the components of the activating reagent solution include FIXa, FIIa, phospholipids, buffer solution, and CaCl2. The first incubation solution and the FX solution were mixed for a second incubation to obtain a second incubation solution; The second incubation solution and the chromogenic substrate solution were mixed for a third incubation, and then a stop solution was added. The absorbance of the resulting mixture at 405 nm was measured to obtain the absorbance value. The chromogenic substrate was SXa-11. Substitute the absorbance value into the FVIII complex activity standard curve, calculate the residual FVIII activity units, and plot the sea sodium polysaccharide activity curve with the reciprocal of the sea sodium polysaccharide weight as the x-axis and the logarithm of the residual FVIII activity units as the y-axis; the x-axis of the FVIII complex activity standard curve is the logarithm of the FVIII concentration, and the y-axis is the absorbance value.

2. The valence calibration method according to claim 1, characterized in that, The first incubation temperature is 37°C, and the incubation time is 5~20 min.

3. The valence calibration method according to claim 1, characterized in that, The second incubation temperature is 37°C, and the incubation time is 1~10 min.

4. The valence calibration method according to claim 1, characterized in that, The third incubation temperature is 37°C, and the incubation time is 10~30 min.

5. The valence calibration method according to claim 1, characterized in that, The terminating solution is a citric acid solution with a concentration of 0.01~0.1 g / mL.

6. The valence calibration method according to claim 1, characterized in that, The concentration range of the sodium polysaccharide working solution is 328~500 ng / mL.

7. The valence calibration method according to claim 1, characterized in that, The buffer solution in the activating reagent solution is Tris-HCl.

8. The valence calibration method according to claim 1, characterized in that, The method for obtaining the activity standard curve of the FVIII complex includes: Prepare FVIII standard solutions with gradient concentrations; The FVIII standard solution, water, and activating reagent solution are mixed for a fourth incubation to obtain a fourth incubation solution; the components of the activating reagent solution include FIXa, FIIa, phospholipids, buffer solution, and CaCl2. The fourth incubation solution and the FX solution are mixed for a fifth incubation to obtain the fifth incubation solution; The fifth incubation solution and the chromogenic substrate solution were mixed for a sixth incubation, and then a stop solution was added. The absorbance of the resulting mixture at 405 nm was measured to obtain the absorbance value. The chromogenic substrate was SXa-11. A standard curve of the FVIII complex activity was plotted with the logarithm of FVIII concentration on the x-axis and absorbance value on the y-axis.

9. The valence calibration method according to claim 8, characterized in that, The concentration of the FVIII standard solution is 0.95~3 IU / mL.

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