Preparation method of antithrombin iii quality control product and antithrombin iii quality control product

Abstract

The embodiment of the application belongs to the technical field of inspection and determination, and relates to a preparation method of antithrombin III quality control product and the antithrombin III quality control product, and comprises the following steps: preparing an antithrombin III quality control product mother liquor, wherein the antithrombin III quality control product mother liquor comprises an antithrombin III antibody; taking sucrose and polyethylene glycol, dissolving them in PBS, adding bovine serum albumin, a stabilizer and Proclin300, uniformly mixing, and obtaining a matrix solution; dividing the antithrombin III quality control product mother liquor into at least two parts, diluting one part of the antithrombin III quality control product mother liquor by the matrix solution, obtaining the diluted antithrombin III quality control product mother liquor as a second mother liquor, and taking the other part of the antithrombin III quality control product mother liquor which is not diluted as a first mother liquor; mixing the first mother liquor and the second mother liquor according to a plurality of different volume ratios, and obtaining a high-value antithrombin III quality control product and a low-value antithrombin III quality control product. The antithrombin III quality control product prepared in the application has high stability and can be stored for a long time.

Description

Technical Field

[0001] This application relates to the field of testing and measurement technology, and in particular to the preparation method and quality control of antithrombin III. Background Technology

[0002] Maintaining a dynamic balance between blood coagulation and anticoagulation is a crucial regulatory mechanism for preventing bleeding and thrombosis. Antithrombin III (ATIII), one of the most important anticoagulants in plasma, inhibits coagulation and reduces clot formation by suppressing the activity of thrombin and activated coagulation factors VIIa and IX, thus maintaining the body's coagulation-anticoagulation balance. ATIII is a single-chain α2 glycoprotein with a molecular weight of 58,000. It is a multifunctional serine protease inhibitor, primarily synthesized by liver cells, but can also be synthesized by vascular endothelial cells. Decreased ATIII levels are commonly seen in liver dysfunction, nephrotic syndrome, disseminated intravascular coagulation (DIC), organ failure, acute myocardial infarction, and other infarct-related diseases, or during pregnancy. Elevated ATIII levels are commonly seen in hemophilia A, hemophilia B, and the use of progesterone-based medications.

[0003] Currently, there are few methods for determining the activity and content of antithrombin III (ATIII), including the following two: 1. Chromogenic substrate method: ATIII can form a complex with thrombin, causing it to lose its enzymatic activity in converting fibrinogen into fibrin. After adding an excess of thrombin to the plasma, the thrombin forms a 1:1 complex with the ATIII in the plasma. The remaining thrombin acts on the chromogenic agent, cleaving out the chromogenic group. The degree of color development is negatively correlated with the ATIII activity in the plasma. 2. Immunoassay: Based on the principle of antigen-antibody reaction, the application of monoclonal antibody technology has led to the rapid development of this method. A goat anti-human monoclonal antibody can specifically bind to ATIII, and detection is performed using the enzyme immunoassay principle. This method has good precision and good correlation with other methods.

[0004] Before each assay, each method requires calibration using calibrators, and the measurement system needs quality control using quality control materials. Whole blood calibration and quality control are unsuitable for clinical use due to their short shelf life and poor stability, while lyophilized calibration and quality control offer the advantages of long-term preservation and stability. Traditional ATⅢ quality control materials are mainly prepared from blood products through isolation and purification. This involves lyophilizing and filtering the primary extract obtained through plasma adsorption and affinity chromatography followed by ultrafiltration, then bottling and storing at low temperatures. This method is costly, inefficient, and difficult to control concentration, hindering large-scale application. Modern biotechnology often chooses Pichia pastoris expression and animal mammary gland reactors for recombinant protein expression, which offers high activity and high expression levels, showing broad application prospects.

[0005] Whole blood calibration and quality control suffer from short shelf life and poor stability. The concentration of ATⅢ in normal people is between 19.0 and 35.0 mg / L. The acquisition of different concentration values ​​is random and uncontrollable, which is not conducive to production. Moreover, high concentration ATⅢ samples are very rare, making it difficult to prepare high-value quality control samples. Summary of the Invention

[0006] The purpose of this application is to provide a method for preparing an antithrombin III quality control product and the antithrombin III quality control product itself. The antithrombin III quality control product of this application has high stability and can be stored for a long time.

[0007] To address the aforementioned technical problems, this application provides a method for preparing an antithrombin III quality control product, employing the following technical solution: A method for preparing an antithrombin III quality control sample includes the following steps: Prepare an antithrombin III quality control stock solution, wherein the antithrombin III quality control stock solution comprises antithrombin III; Weigh sucrose and polyethylene glycol, dissolve them in PBS, add bovine serum albumin, stabilizer and Proclin 300, mix well to obtain matrix solution; The antithrombin III quality control stock solution is divided into at least two portions. One portion of the antithrombin III quality control stock solution is diluted with the matrix solution to obtain the diluted antithrombin III quality control stock solution as the second stock solution, and the other undiluted portion of the antithrombin III quality control stock solution is used as the first stock solution. The first mother liquor and the second mother liquor are mixed in multiple preset volume ratios to obtain high-value antithrombin III quality control products and low-value antithrombin III quality control products.

[0008] Furthermore, the concentration of the diluted antithrombin III quality control stock solution is 20 mg / dL.

[0009] Furthermore, the volume ratio of the first mother liquor and the second mother liquor of the high-value antithrombin III quality control product is greater than the volume ratio of the first mother liquor and the second mother liquor of the low-value antithrombin III quality control product.

[0010] Furthermore, after the step of mixing the first mother liquor and the second mother liquor according to a plurality of preset different volume ratios to obtain high-value antithrombin III quality control products and low-value antithrombin III quality control products, the method further includes: Add mannitol to the high-value and low-value antithrombin III quality control products and dissolve them completely. After repackaging, the product is freeze-dried to obtain freeze-dried quality control samples.

[0011] Furthermore, the final concentration of mannitol is 70 g / L.

[0012] Furthermore, the step of performing lyophilization after dispensing to obtain lyophilized quality control products includes: After repackaging, pre-cool for 2 hours, freeze at -45℃ for 10 hours, and then at room temperature for 4 hours to obtain freeze-dried quality control products.

[0013] Furthermore, the stabilizer is prepared by the following steps: The stabilizer was obtained by uniformly mixing 2.5% (w / w) of the compound enzyme stabilizer AES and 100 U / L of aprotinin in a 1:1 volume ratio. To address the aforementioned technical problems, this application also provides an antithrombin III quality control product, prepared using the above-described preparation method, and employing the following technical solution: An antithrombin III quality control product, the antithrombin III quality control product comprising an antithrombin III quality control product stock solution and a matrix solution; The antithrombin III quality control stock solution includes antithrombin III; The matrix solution includes sucrose, polyethylene glycol, PBS, bovine serum albumin, stabilizers, and Proclin 300.

[0014] Furthermore, by mass fraction, the matrix solution comprises 6% sucrose, 4% polyethylene glycol, 0.01M PBS, 1% bovine serum albumin, 1.6% stabilizer, and 0.5% Proclin 300.

[0015] Compared with the prior art, the embodiments of this application have the following main advantages: This application provides a method for preparing antithrombin III quality control products with high and low value ranges. The preparation method simulates the plasma environment to prepare the antithrombin III quality control products, which are stored in lyophilized powder form and have the advantages of stability and long-term preservation. Attached Figure Description

[0016] To more clearly illustrate the solutions in this application, the accompanying drawings used in the description of the embodiments of this application will be briefly introduced below. Obviously, the accompanying drawings described below are some embodiments of this application. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0017] Figure 1 This is a flowchart of an embodiment of the method for preparing the antithrombin III quality control product according to this application; Figure 2 This is a schematic diagram of the stability monitoring results of the antithrombin III quality control product according to this application; Figure 3 This is a schematic diagram of another stability monitoring result of the antithrombin III quality control product according to this application. Detailed Implementation

[0018] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains; the terminology used herein in the specification of the application is for the purpose of describing particular embodiments only and is not intended to be limiting of the application; the terms "comprising" and "having," and any variations thereof, in the specification, claims, and foregoing drawings of this application, are intended to cover non-exclusive inclusion. The terms "first," "second," etc., in the specification, claims, or foregoing drawings of this application are used to distinguish different objects, not to describe a particular order.

[0019] In this document, the term "embodiment" means that a particular feature, structure, or characteristic described in connection with an embodiment may be included in at least one embodiment of this application. The appearance of this phrase in various places throughout the specification does not necessarily refer to the same embodiment, nor is it a separate or alternative embodiment mutually exclusive with other embodiments. It will be explicitly and implicitly understood by those skilled in the art that the embodiments described herein can be combined with other embodiments.

[0020] The following embodiments are provided to facilitate a better understanding of this application, but do not limit the scope of this application. Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods. Unless otherwise specified, the experimental materials used in the following embodiments were purchased from conventional biochemical reagent stores.

[0021] To enable those skilled in the art to better understand the present application, the technical solutions in the embodiments of the present application will be clearly and completely described below with reference to the accompanying drawings.

[0022] Continue to refer to Figure 1 The diagram shows a flowchart of an embodiment of a method for preparing an antithrombin III quality control according to this application. The method for preparing the antithrombin III quality control includes the following steps: S1: Prepare the antithrombin III quality control stock solution, wherein the antithrombin III quality control stock solution includes antithrombin III; S2: Weigh sucrose and polyethylene glycol, dissolve them in PBS, add bovine serum albumin, stabilizer and Proclin 300, mix well to obtain matrix solution; S3: Divide the antithrombin III quality control stock solution into at least two portions, dilute one portion of the antithrombin III quality control stock solution with the matrix solution to obtain the diluted antithrombin III quality control stock solution as the second stock solution, and use the other undiluted portion of the antithrombin III quality control stock solution as the first stock solution. S4: The first mother liquor and the second mother liquor are mixed in multiple preset different volume ratios to obtain high-value antithrombin III quality control products and low-value antithrombin III quality control products.

[0023] As an optional feature of this application, the concentration of the diluted antithrombin III quality control stock solution is 20 mg / dL.

[0024] Optionally, the volume ratio of the first mother liquor and the second mother liquor of the high-value antithrombin III quality control product is greater than the volume ratio of the first mother liquor and the second mother liquor of the low-value antithrombin III quality control product.

[0025] Optionally, after step S4, the following method may be further included: Add mannitol to the high-value and low-value antithrombin III quality control products and dissolve them completely. After repackaging, the product is freeze-dried to obtain freeze-dried quality control samples.

[0026] The final concentration of mannitol is 70 g / L; Optionally, the step of performing lyophilization after dispensing to obtain lyophilized quality control products includes: After repackaging, pre-cool for 2 hours, freeze at -45℃ for 10 hours, and then at room temperature for 4 hours to obtain freeze-dried quality control products.

[0027] Optionally, the stabilizer may be prepared by the following steps: The stabilizer was obtained by uniformly mixing 2.5% (w / w) of the compound enzyme stabilizer AES and 100 U / L of aprotinin in a 1:1 volume ratio.

[0028] In this embodiment, the present application uses purchased antithrombin III for the preparation of quality control products. The present application prepares antithrombin III quality control products in a simulated plasma environment, adds excipients and stabilizers, and freeze-dries them into powder to produce antithrombin III quality control products. Mannitol is used as the excipient in this application.

[0029] The specific preparation process of this application is as follows: 1. Prepare the antithrombin III quality control stock solution Purchase antithrombin III from the SIGMA website, reconstitute it with an appropriate amount of PBS according to the instructions, take 100 μL and perform preliminary determination test using the antithrombin III kit, and use it as the antithrombin III quality control stock solution.

[0030] 2. Stabilizer Preparation Mix 2.5% (w / w) of the compound enzyme stabilizer AES and 100 U / L of aprotinin in a 1:1 volume ratio and store at 2-8°C for later use.

[0031] 3. Preparation of quality control materials Weigh 30g of sucrose and 20g of polyethylene glycol and dissolve them in 500mL of 0.01M PBS. Add 5mL of bovine serum albumin, 8mL of the stabilizer from the previous step, and 2.5mL of Proclin300 to the well-mixed liquid in sequence, mix well, and obtain the matrix solution.

[0032] The mother solution containing antithrombin III was divided into three portions. One portion was reserved for use as the first mother solution (i.e., mother solution A). The remaining two portions of the mother solution containing antithrombin III were diluted with the matrix solution described above so that the concentration of antithrombin III in both portions was 20 mg / dL (determined using an antithrombin III kit), resulting in two second mother solutions (i.e., mother solution B1 and mother solution B2).

[0033] Then, a certain amount of the first mother liquor and the second mother liquor were taken and mixed according to several preset different volume ratios to obtain high-value antithrombin III quality control products and low-value antithrombin III quality control products, respectively. The volume ratio of the first mother liquor and the second mother liquor for the high-value antithrombin III quality control product is greater than that for the low-value antithrombin III quality control product.

[0034] Specifically, the first mother liquor and one portion of the second mother liquor (mother liquor B1) are mixed uniformly at a volume ratio of 20:1 to 18:1 to obtain a high-value antithrombin III quality control product. The preferred volume ratio is 19:1. For example, 950 μl of the first mother liquor and 50 μl of the second mother liquor are mixed uniformly to obtain a high-value antithrombin III quality control product. The first mother liquor and another second mother liquor (mother liquor B2) are mixed evenly at a volume ratio of 1:20 to 1:18 to obtain a low-value antithrombin III quality control product. The preferred volume ratio is 1:19. For example, 50 μl of the first mother liquor and 950 μl of the second mother liquor are mixed evenly to obtain a low-value antithrombin III quality control product.

[0035] 4. Freeze-drying Mannitol was added to the quality control sample and dissolved completely to a final concentration of 70 g / L. The sample was dispensed into 0.5 mL vials and then lyophilized. The lyophilization procedure was: pre-cooling for 2 hours, -45℃ for 10 hours, and then at room temperature for 4 hours. After lyophilization, the vials were sealed and stored for later use to obtain the lyophilized quality control sample.

[0036] 5. Quality control setpoints Take 10 bottles of lyophilized quality control samples, dissolve each in 0.5 mL of purified water, and test each sample using an antithrombin III kit. The target value is the mean of the 10 samples, and the quality control range is ±10% of the target value. Specific value ranges are shown in Table 1 below. Table 1. Antithrombin III values ​​and ranges

[0037] High-value and low-value antithrombin III quality control samples were stored at 2–8°C. One sample was randomly selected each month and tested using an antithrombin III assay kit to monitor its stability. The results are shown in Table 2. Figure 2 and Figure 3 As shown. Figure 2 This is a schematic diagram of the stability monitoring results of the antithrombin III quality control material in this application, specifically a schematic diagram of the stability monitoring results of the low-value antithrombin III quality control material. Figure 3 This is another schematic diagram of the stability monitoring results for antithrombin III quality control products, specifically a schematic diagram of the stability monitoring results for high-value antithrombin III quality control products.

[0038] Table 2 Quality Control Stability Monitoring Results

[0039] The quality control monitoring data shows that the high and low values ​​are within the average range over 15 months, and the antithrombin III prepared according to the above protocol is stable and reliable.

[0040] This application also provides an antithrombin III quality control product, which is prepared using the above-described method for preparing antithrombin III quality control products. By mass fraction, the antithrombin III (ATIII) quality control product includes a matrix solution and an antithrombin III quality control product stock solution. The antithrombin III quality control stock solution includes antithrombin III; The matrix solution comprises, by mass fraction, 6% sucrose, 4% polyethylene glycol, 0.01M PBS, 1% bovine serum albumin, 1.6% stabilizer, and 0.5% Proclin 300.

[0041] The stabilizer is a mixture of 2.5% by mass of the compound enzyme stabilizer AES and 100 U / L of aprotinin in a 1:1 volume ratio.

[0042] The beneficial effects of this application are: This application provides a method for preparing antithrombin III quality control products with high and low value ranges. The preparation method simulates the plasma environment to prepare the antithrombin III quality control products, which are stored in lyophilized powder form and have the advantages of stability and long-term preservation.

[0043] It should be understood that although the steps in the flowcharts of the accompanying figures are shown sequentially as indicated by the arrows, these steps are not necessarily executed in the order indicated by the arrows. Unless explicitly stated herein, there is no strict order restriction on the execution of these steps, and they can be executed in other orders. Moreover, at least some steps in the flowcharts of the accompanying figures may include multiple sub-steps or multiple stages. These sub-steps or stages are not necessarily completed at the same time, but can be executed at different times, and their execution order is not necessarily sequential, but can be performed alternately or in turn with other steps or at least some of the sub-steps or stages of other steps.

[0044] Obviously, the embodiments described above are only some embodiments of this application, not all embodiments. The accompanying drawings show preferred embodiments of this application, but do not limit the patent scope of this application. This application can be implemented in many different forms; rather, the purpose of providing these embodiments is to provide a more thorough and comprehensive understanding of the disclosure of this application. Although this application has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing specific embodiments, or make equivalent substitutions for some of the technical features. Any equivalent structures made using the content of this application's specification and drawings, directly or indirectly applied to other related technical fields, are similarly within the scope of patent protection of this application.

Claims

1. A method for preparing an antithrombin III quality control product, characterized in that, Includes the following steps: Prepare an antithrombin III quality control stock solution, wherein the antithrombin III quality control stock solution comprises antithrombin III; the antithrombin III quality control stock solution is obtained by reconstituted commercially available antithrombin III with PBS and then set using an antithrombin III kit. Weigh sucrose and polyethylene glycol, dissolve them in PBS, add bovine serum albumin, stabilizer, and Proclin 300, mix well to obtain a matrix solution; wherein, the stabilizer is prepared by the following steps: uniformly mix 2.5% by mass of the complex enzyme stabilizer AES and 100 U / L of aprotinin in a 1:1 volume ratio to obtain the stabilizer; the matrix solution includes 6% sucrose, 4% polyethylene glycol, 0.01M PBS, 1% bovine serum albumin, 1.6% stabilizer, and 0.5% Proclin 300; The antithrombin III quality control stock solution is divided into at least two portions. One portion of the antithrombin III quality control stock solution is diluted with the matrix solution to obtain the diluted antithrombin III quality control stock solution as the second stock solution, and the other undiluted portion of the antithrombin III quality control stock solution is used as the first stock solution. The first mother liquor and the second mother liquor are mixed in multiple preset volume ratios to obtain high-value antithrombin III quality control products and low-value antithrombin III quality control products; wherein, when obtaining the high-value antithrombin III quality control product, the volume ratio of the first mother liquor to the second mother liquor is 20:1 to 18:1; and when obtaining the low-value antithrombin III quality control product, the volume ratio of the first mother liquor to the second mother liquor is 1:20 to 1:

18.

2. The method for preparing the antithrombin III quality control product according to claim 1, characterized in that, The concentration of the diluted antithrombin III quality control stock solution was 20 mg / dL.

3. The method for preparing the antithrombin III quality control product according to claim 1, characterized in that, After the step of mixing the first mother liquor and the second mother liquor according to a plurality of preset different volume ratios to obtain high-value antithrombin III quality control products and low-value antithrombin III quality control products, the method further includes: Add mannitol to the high-value and low-value antithrombin III quality control products and dissolve them completely. After repackaging, the product is freeze-dried to obtain freeze-dried quality control samples.

4. The method for preparing the antithrombin III quality control product according to claim 3, characterized in that, The final concentration of mannitol is 70 g / L.

5. The method for preparing the antithrombin III quality control product according to claim 3, characterized in that, The step of performing lyophilization after dispensing to obtain lyophilized quality control samples includes: After repackaging, pre-cool for 2 hours, freeze at -45℃ for 10 hours, and then at room temperature for 4 hours to obtain freeze-dried quality control products.

6. An antithrombin III quality control product, prepared using the preparation method according to any one of claims 1 to 5, characterized in that, The antithrombin III quality control product includes an antithrombin III quality control product stock solution and a matrix solution; The antithrombin III quality control stock solution includes antithrombin III, and the concentration of the diluted antithrombin III quality control stock solution is 20 mg / dL. The matrix solution consists of 6% sucrose, 4% polyethylene glycol, 0.01M PBS, 1% bovine serum albumin, 1.6% stabilizer, and 0.5% Proclin 300.

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