Prothrombin activation process and thrombin preparation method

Abstract

The invention provides a prothrombin activation process and a thrombin preparation method, according to the prothrombin activation process, a phospholipid substance is adopted as an activating agent, a calcium chloride solution is used for providing a calcium ion environment, and polysorbate 80 is adopted for assisting dissolution, so that prothrombin generates thrombin, and the invention further discloses the thrombin preparation method. Comprising the following steps: 1) collecting pig blood; 2) centrifugal separation; 3) adsorption separation; (4) eluting prothrombin; (5) carrying out prothrombin activation by adopting the prothrombin activation process; 6) ultrafiltration concentration; according to the prothrombin activation process provided by the invention, phospholipid substances are matched with polysorbate 80 and calcium chloride to serve as thrombin activators, the polysorbate 80 contributes to dissolution of the phospholipid substances, and compared with rabbit brain powder, the method has the advantages that the safety of the product can be remarkably improved, and compared with soybean phospholipid or egg yolk phospholipid, the stability of the product is greatly improved. The prothrombin activation rate can be obviously improved, and the method is suitable for large-scale production.

Description

Technical Field

[0001] The present invention belongs to the technical field of biopharmaceuticals, and particularly relates to a prothrombin activation process and a method for preparing thrombin. Background Art

[0002] Thrombin is a serine protease that plays an important role in the blood coagulation system. It is formed by the activation of prothrombin by an activator and directly acts on the last step of the blood coagulation process, promoting the conversion of soluble fibrinogen in plasma into insoluble fibrin, thereby achieving the purpose of rapid hemostasis.

[0003] Currently in China, prothrombin is mainly prepared from animal plasma and human plasma, and then activated by an activator to become thrombin.

[0004] CN201810585957.5 discloses a preparation process of freeze-dried human thrombin. After obtaining human prothrombin, a calcium chloride solution is added, and then soybean phospholipid or egg yolk lecithin is added as an activator to activate human prothrombin to obtain a crude human thrombin solution. However, the activation effect of using soybean phospholipid or egg yolk phospholipid during the activation process of thrombin is poor.

[0005] CN201510638083.1 discloses a preparation method of porcine thrombin freeze-dried powder. In this method, physiological saline is added to rabbit brain powder, and then prothrombin solution and CaCl 2 are used for zymogen activation to obtain a crude thrombin enzyme solution. The crude enzyme solution is ultrafiltered for desalting and concentrated, and virus inactivation is carried out. The obtained crude enzyme solution passes through a chromatography column, and the target peak is eluted and collected to obtain porcine thrombin. Compared with soybean phospholipid or egg yolk phospholipid, the activation effect of rabbit brain powder is better. However, rabbit brain powder is an exogenous substance, which is likely to introduce viruses and allergenic reactionogens. During the production process, additional virus inactivation of rabbit brain powder is required, increasing the production steps and production cost.

[0006] CN201811040796.8 discloses a preparation method of porcine thrombin, including: 1) preparation of plasma, 2) virus inactivation by S / D method, 3) preparation of crude prothrombin, 4) preparation of thrombin activation solution, 5) purification of thrombin, 6) ultrafiltration desalting, and 7) virus removal by nanofiltration membrane. This method also requires additional virus removal steps, which also increase the production steps and production cost.

[0007] Therefore, there is a need to find a new method for preparing thrombin that is simple to operate and has low cost. Summary of the Invention

[0008] The present invention provides a prothrombin activation process, aiming to solve the problems of poor activation effect, increased production steps, and increased production cost in the prior art.

[0009] The present invention is achieved in this way: a prothrombin activation process uses phospholipid substances as activators, uses calcium chloride solution to provide a calcium ion environment, and uses polysorbate 80 to assist dissolution, so that prothrombin generates thrombin.

[0010] Preferably, the phospholipid substance is one or more of soybean lecithin, phosphatidylcholine, phosphatidylethanolamine, phosphatidylinositol, phosphatidylserine, soybean lecithin, and egg yolk lecithin.

[0011] Preferably, the content of the phospholipid substance is 0.0375‰-0.1% of the total weight of the liquid to be activated, the content of the calcium chloride is 0.03%-0.083% of the total weight of the liquid to be activated, and the content of the polysorbate 80 is 0.3%-2.0% of the total weight of the liquid to be activated, wherein the liquid to be activated includes prothrombin gel eluate, and purified water may also be added.

[0012] The present invention also provides a method for preparing thrombin, comprising the following steps: 1) Pig blood collection: during the pig blood collection process, anticoagulant is added according to the proportion, and the blood is stirred evenly during collection to prevent coagulation. The mass ratio of the pig blood to the anticoagulant can be 8-12:1; 2) Centrifugal separation: centrifuge the pig blood and collect the pig plasma into a collection tank; 3) Adsorption separation: Add the swollen DEAE-dextran gel A-50 into porcine plasma and stir. After stirring, filter the plasma with a filter cloth to obtain a gel containing prothrombin; 4) Prothrombin elution: eluting the prothrombin-containing gel with a 1.9% sodium chloride solution to obtain a prothrombin gel eluate, wherein the mass ratio of the sodium chloride solution to the gel may be 10:3-6; 5) Activating prothrombin using the above-mentioned prothrombin activation process: adding phospholipids, polysorbate 80 and calcium chloride solution to the prothrombin gel eluate, stirring continuously, and reacting at 15-30° C. for 0.5-2 h to obtain an activation solution containing thrombin; 6) Ultrafiltration concentration: Use a 10,000 molecular weight hollow fiber ultrafiltration column to ultrafilter the activation solution. If no white precipitate appears after adding 1% silver nitrate test solution to the ultrafiltrate, stop ultrafiltration. 7) Sterile filtration: The ultrafiltrate is filtered through a microporous membrane in sequence, and the pre-filtered thrombin ultrafiltrate is then filtered through a cartridge filter equipped with a 0.2 μm polyethersulfone filter element to obtain a thrombin solution.

[0013] Compared with the prior art, the embodiments of the present application have the following beneficial effects: The prothrombin activation process provided by the present invention uses phospholipid substances in combination with polysorbate 80 and calcium chloride as thrombin activators. Polysorbate 80 helps dissolve the phospholipid substances. Compared with rabbit brain powder, this method can significantly improve the safety of the product. Compared with soybean phospholipid or egg yolk phospholipid, it can significantly increase the prothrombin activation rate and is suitable for large-scale production. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 is a flowchart of a method for preparing thrombin provided by the present invention. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0015] 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 belongs; the terms used in the specification of this application are only for the purpose of describing specific embodiments and are not intended to limit this application; the terms "including" and "having" and any variations thereof in the specification and claims of this application and the above description of the drawings are intended to cover non-exclusive inclusion. The terms "first", "second", etc. in the specification and claims of this application or the above drawings are used to distinguish different objects and not to describe a specific order.

[0016] Reference to "embodiment" herein means that a particular feature, structure, or characteristic described in connection with the embodiment can be included in at least one embodiment of this application. The phrase appears in various places in the specification and does not necessarily refer to the same embodiment, nor is it an independent or alternative embodiment mutually exclusive with other embodiments. Those skilled in the art will explicitly and implicitly understand that the embodiments described herein can be combined with other embodiments.

[0017] The present invention provides a specific activation process for preparing thrombin, including the following steps: 1) Weighing: Weigh the prothrombin gel eluate, purified water, activator, and calcium chloride respectively according to the ratio. 2) Preparing the solution: Dissolve calcium chloride in an appropriate amount of purified water and cool it. 3) Reacting: Add the activator, polysorbate 80, and the solution to be activated (prothrombin gel eluate, purified water) to the container in sequence according to the ratio. After stirring evenly, add the calcium chloride solution, stir immediately, and react for 1 hour.

[0018] Example 1 This example provides a specific activation process for preparing thrombin, including the following steps: 1) Weighing: Weigh the prothrombin gel eluate, purified water, activator, and calcium chloride respectively according to the ratio. The activator is soy lecithin (phosphatidylcholine 18 - 25%, phosphatidylethanolamine 10 - 16%, phosphatidylinositol 14 - 19%). Weigh 0.0152 g per portion (0.01% of the total weight of the solution to be activated), and weigh 145 g of the prothrombin gel eluate per portion. 2) Solution preparation: Dissolve calcium chloride in an appropriate amount of purified water and cool it. 3) Reaction: Add the activator, polysorbate 80, and the solution to be activated (145 g of prothrombin gel eluate, 0 g of purified water) to the container in proportion successively. After stirring evenly, add the calcium chloride solution, and immediately stir and react for 1 hour. Specifically, the final concentration of polysorbate 80 is 1.0%, and the final concentration of calcium chloride is 0.055%.

[0019] Example 2 This example provides a specific activation process for thrombin preparation, including the following steps: 1) Weighing: Weigh the prothrombin gel eluate, purified water, activator, and calcium chloride respectively according to the ratio. The activator is soy lecithin (phosphatidylcholine 18 - 25%, phosphatidylethanolamine 10 - 16%, phosphatidylinositol 14 - 19%). Weigh 0.0375 g per portion (0.025% of the total weight of the solution to be activated), and weigh 145 g of the prothrombin gel eluate per portion. 2) Solution preparation: Dissolve calcium chloride in an appropriate amount of purified water and cool it. 3) Reaction: Add the activator, polysorbate 80, and the solution to be activated (145 g of prothrombin gel eluate, 0 g of purified water) to the container in proportion successively. After stirring evenly, add the calcium chloride solution, and immediately stir and react for 1 hour. Specifically, the final concentration of polysorbate 80 is 1.0%, and the final concentration of calcium chloride is 0.055%.

[0020] Example 3 This example provides a specific activation process for thrombin preparation, including the following steps: 1) Weighing: Weigh the prothrombin gel eluate, purified water, activator, and calcium chloride respectively according to the ratio. The activator is soy lecithin (phosphatidylcholine 18 - 25%, phosphatidylethanolamine 10 - 16%, phosphatidylinositol 14 - 19%). Weigh 0.0760 g per portion (0.05% of the total weight of the solution to be activated), and weigh 145 g of the prothrombin gel eluate per portion. 2) Solution preparation: Dissolve calcium chloride in an appropriate amount of purified water and cool it. 3) Reaction: Sequentially add the activator, polysorbate 80, and the solution to be activated (145 g of thrombinogen gel eluate and 0 g of purified water) into the container in proportion. After stirring evenly, add the calcium chloride solution, and immediately stir and react for 1 hour. Specifically, the final concentration of polysorbate 80 is 1.0%, and the final concentration of calcium chloride is 0.055%.

[0021] Example 4 This example provides a specific activation process for preparing thrombin, including the following steps: 1) Weighing: Weigh the thrombinogen gel eluate, purified water, activator, and calcium chloride separately in proportion. The activator is soy lecithin (phosphatidylcholine 18 - 25%, phosphatidylethanolamine 10 - 16%, phosphatidylinositol 14 - 19%), weigh 0.1216 g (0.08% of the total weight of the solution to be activated) per g / portion, and weigh 145 g of the thrombinogen gel eluate per g / portion; 2) Prepare the solution: Dissolve calcium chloride in an appropriate amount of purified water and cool it down; 3) Reaction: Sequentially add the activator, polysorbate 80, and the solution to be activated (145 g of thrombinogen gel eluate and 0 g of purified water) into the container in proportion. After stirring evenly, add the calcium chloride solution, and immediately stir and react for 1 hour. Specifically, the final concentration of polysorbate 80 is 1.0%, and the final concentration of calcium chloride is 0.055%.

[0022] Example 5 This example provides a specific activation process for preparing thrombin, including the following steps: 1) Weighing: Weigh the thrombinogen gel eluate, purified water, activator, and calcium chloride separately in proportion. The activator is soy lecithin (phosphatidylcholine 18 - 25%, phosphatidylethanolamine 10 - 16%, phosphatidylinositol 14 - 19%), weigh 0.33 g (0.1% of the total weight of the solution to be activated) per g / portion, and weigh 150 g of the thrombinogen gel eluate per g / portion; 2) Prepare the solution: Dissolve calcium chloride in an appropriate amount of purified water and cool it down; 3) Reaction: Sequentially add the activator and the solution to be activated (150 g of thrombinogen gel eluate and 180 g of purified water) into the container in proportion. After stirring evenly, add the calcium chloride solution, and immediately stir and react for 1 hour. Specifically, the final concentration of calcium chloride is 0.083%.

[0023] Example 6 This example provides a specific activation process for preparing thrombin, including the following steps: 1) Weighing: Weigh the prothrombin gel eluate, purified water, activator, and calcium chloride respectively according to the ratio. The activator is phosphatidylcholine, and weigh 0.0057 g per part (0.0375‰ of the total weight of the solution to be activated), and weigh 145 g of the prothrombin gel eluate per part. 2) Solution preparation: Dissolve calcium chloride in an appropriate amount of purified water and cool it down. 3) Reaction: Add the activator, polysorbate 80, and the solution to be activated (145 g of prothrombin gel eluate and 0 g of purified water) to the container in sequence according to the ratio. After stirring evenly, add the calcium chloride solution, stir immediately and react for 1 hour. Specifically, the final concentration of polysorbate 80 is 1.0%, and the final concentration of calcium chloride is 0.055%.

[0024] Example 7 This example provides a specific activation process for thrombin preparation, including the following steps: 1) Weighing: Weigh the prothrombin gel eluate, purified water, activator, and calcium chloride respectively according to the ratio. The activator is phosphatidylethanolamine, and weigh 0.0070 g per part (0.0375‰ of the total weight of the solution to be activated), and weigh 145 g of the prothrombin gel eluate per part. 2) Solution preparation: Dissolve calcium chloride in an appropriate amount of purified water and cool it down. 3) Reaction: Add the activator, polysorbate 80, and the solution to be activated (145 g of prothrombin gel eluate and 0 g of purified water) to the container in sequence according to the ratio. After stirring evenly, add the calcium chloride solution, stir immediately and react for 1 hour. Specifically, the final concentration of polysorbate 80 is 1.0%, and the final concentration of calcium chloride is 0.055%.

[0025] Example 8 This example provides a specific activation process for thrombin preparation, including the following steps: 1) Weighing: Weigh the prothrombin gel eluate, purified water, activator, and calcium chloride respectively according to the ratio. The activator is phosphatidylethanolamine, and weigh 0.0095 g per part (0.05‰ of the total weight of the solution to be activated), and weigh 145 g of the prothrombin gel eluate per part. 2) Solution preparation: Dissolve calcium chloride in an appropriate amount of purified water and cool it down. 3) Reaction: Add the activator, polysorbate 80, and the solution to be activated (145 g of prothrombin gel eluate and 0 g of purified water) to the container in sequence according to the ratio. After stirring evenly, add the calcium chloride solution, stir immediately and react for 1 hour. Specifically, the final concentration of polysorbate 80 is 1.0%, and the final concentration of calcium chloride is 0.055%.

[0026] Example 9 This embodiment provides a specific activation process for thrombin preparation, including the following steps: 1) Weighing: Weigh prothrombin gel eluate, purified water, activator, and calcium chloride according to the ratio. The activator is phosphatidylinositol, and 0.0152 g is weighed per g portion (0.05‰ of the total weight of the solution to be activated), and 145 g of prothrombin gel eluate is weighed per g portion; 2) Solution preparation: Dissolve calcium chloride in an appropriate amount of purified water and cool it down; 3) Reaction: Add the activator, polysorbate 80, and the solution to be activated (145 g of prothrombin gel eluate and 0 g of purified water) to the container in proportion successively. After stirring evenly, add the calcium chloride solution, stir immediately and react for 1 hour. Specifically, the final concentration of polysorbate 80 is 1.0%, and the final concentration of calcium chloride is 0.055%.

[0027] Example 10 This embodiment provides a specific activation process for thrombin preparation, including the following steps: 1) Weighing: Weigh prothrombin gel eluate, purified water, activator, and calcium chloride according to the ratio. The activator is phosphatidylserine, and 0.0082 g is weighed per g portion (0.05‰ of the total weight of the solution to be activated), and 145 g of prothrombin gel eluate is weighed per g portion; 2) Solution preparation: Dissolve calcium chloride in an appropriate amount of purified water and cool it down; 3) Reaction: Add the activator, polysorbate 80, and the solution to be activated (145 g of prothrombin gel eluate and 0 g of purified water) to the container in proportion successively. After stirring evenly, add the calcium chloride solution, stir immediately and react for 1 hour. Specifically, the final concentration of polysorbate 80 is 1.0%, and the final concentration of calcium chloride is 0.055%.

[0028] Example 11 This embodiment provides a specific activation process for thrombin preparation, including the following steps: 1) Weighing: Weigh prothrombin gel eluate, purified water, activator, and calcium chloride according to the ratio. The activator is 2% soybean lecithin solution, and 3.75 g is weighed per g portion (0.025% of the total weight of the solution to be activated), and 300 g of prothrombin gel eluate is weighed per g portion; 2) Solution preparation: Dissolve soybean lecithin in an appropriate amount of purified water, stir and sonicate to disperse and dissolve it; Dissolve calcium chloride in an appropriate amount of purified water and cool it down; 3) Reaction: Add the activator, polysorbate 80, and the liquid to be activated (300 g of thrombinogen gel eluate and 0 g of purified water) into the container in sequence according to the ratio. After stirring evenly, add the calcium chloride solution, and immediately stir and react for 1 hour. Specifically, the final concentration of polysorbate 80 is 0.3%, and the final concentration of calcium chloride is 0.055%.

[0029] Example 12 This example provides a specific activation process for preparing thrombin, including the following steps: 1) Weighing: Weigh the thrombinogen gel eluate, purified water, activator, and calcium chloride respectively according to the ratio. The activator is a 2% soybean lecithin solution, and 3.8 g is weighed according to g / portion (0.025% of the total weight of the liquid to be activated), and 300 g of thrombinogen gel eluate is weighed according to g / portion; 2) Solution preparation: Dissolve soybean lecithin in an appropriate amount of purified water, stir and sonicate to disperse and dissolve; dissolve calcium chloride in an appropriate amount of purified water and cool it down; 3) Reaction: Add the activator, polysorbate 80, and the liquid to be activated (300 g of thrombinogen gel eluate and 0 g of purified water) into the container in sequence according to the ratio. After stirring evenly, add the calcium chloride solution, and immediately stir and react for 1 hour. Specifically, the final concentration of polysorbate 80 is 0.7%, and the final concentration of calcium chloride is 0.055%.

[0030] Example 13 This example provides a specific activation process for preparing thrombin, including the following steps: 1) Weighing: Weigh the thrombinogen gel eluate, purified water, activator, and calcium chloride respectively according to the ratio. The activator is a 2% soybean lecithin solution, and 3.8 g is weighed according to g / portion (0.025% of the total weight of the liquid to be activated), and 300 g of thrombinogen gel eluate is weighed according to g / portion; 2) Solution preparation: Dissolve soybean lecithin in an appropriate amount of purified water, stir and sonicate to disperse and dissolve; dissolve calcium chloride in an appropriate amount of purified water and cool it down; 3) Reaction: Add the activator, polysorbate 80, and the liquid to be activated (300 g of thrombinogen gel eluate and 0 g of purified water) into the container in sequence according to the ratio. After stirring evenly, add the calcium chloride solution, and immediately stir and react for 1 hour. Specifically, the final concentration of polysorbate 80 is 1.0%, and the final concentration of calcium chloride is 0.055%.

[0031] Example 14 This example provides a specific activation process for preparing thrombin, including the following steps: 1) Weighing: Weigh prothrombin gel eluate, purified water, activator, and calcium chloride respectively according to the proportion. The activator is 2% soybean lecithin solution. Weigh 3.85 g (0.025% of the total weight of the solution to be activated) per part by gram, and weigh 300 g of prothrombin gel eluate per part by gram. 2) Solution preparation: Take an appropriate amount of purified water to dissolve soybean lecithin, stir and ultrasonicate to disperse and dissolve it; take an appropriate amount of purified water to dissolve calcium chloride and cool it. 3) Reaction: Add the activator, polysorbate 80, and the solution to be activated (300 g of prothrombin gel eluate and 0 g of purified water) to the container in proportion successively. After stirring evenly, add the calcium chloride solution, stir immediately and react for 1 hour. Specifically, the final concentration of polysorbate 80 is 2.0%, and the final concentration of calcium chloride is 0.055%.

[0032] Example 15 This example provides a specific activation process for thrombin preparation, including the following steps: 1) Weighing: Weigh prothrombin gel eluate, purified water, activator, and calcium chloride respectively according to the proportion. The activator is soybean lecithin (phosphatidylcholine 18 - 25%, phosphatidylethanolamine 10 - 16%, phosphatidylinositol 14 - 19%). Weigh 0.076 g (0.050% of the total weight of the solution to be activated) per part by gram, and weigh 145 g of prothrombin gel eluate per part by gram. 2) Solution preparation: Take an appropriate amount of purified water to dissolve calcium chloride and cool it. 3) Reaction: Add the activator, polysorbate 80, and the solution to be activated (145 g of prothrombin gel eluate and 0 g of purified water) to the container in proportion successively. After stirring evenly, add the calcium chloride solution, stir immediately and react for 1 hour. Specifically, the final concentration of polysorbate 80 is 1.0%, and the final concentration of calcium chloride is 0.030%.

[0033] Example 16 This example provides a specific activation process for thrombin preparation, including the following steps: 1) Weighing: Weigh prothrombin gel eluate, purified water, activator, and calcium chloride respectively according to the proportion. The activator is soybean lecithin (phosphatidylcholine 18 - 25%, phosphatidylethanolamine 10 - 16%, phosphatidylinositol 14 - 19%). Weigh 0.076 g (0.050% of the total weight of the solution to be activated) per part by gram, and weigh 145 g of prothrombin gel eluate per part by gram. 2) Solution preparation: Take an appropriate amount of purified water to dissolve calcium chloride and cool it. 3) Reaction: Sequentially add an activator, polysorbate 80, and the solution to be activated (145 g of prothrombin gel eluate and 0 g of purified water) into a container in proportion. After stirring evenly, add a calcium chloride solution, immediately stir, and react for 1 hour. Specifically, the final concentration of polysorbate 80 is 1.0%, and the final concentration of calcium chloride is 0.045%.

[0034] Example 17 This example provides a specific activation process for preparing thrombin, including the following steps: 1) Weighing: Weigh prothrombin gel eluate, purified water, activator, and calcium chloride respectively in proportion. The activator is soy lecithin (phosphatidylcholine 18 - 25%, phosphatidylethanolamine 10 - 16%, phosphatidylinositol 14 - 19%), weigh 0.076 g (0.050% of the total weight of the solution to be activated) per g / portion, and weigh 145 g of prothrombin gel eluate per g / portion; 2) Prepare the solution: Dissolve calcium chloride in an appropriate amount of purified water and cool it down; 3) Reaction: Sequentially add an activator, polysorbate 80, and the solution to be activated (145 g of prothrombin gel eluate and 0 g of purified water) into a container in proportion. After stirring evenly, add a calcium chloride solution, immediately stir, and react for 1 hour. Specifically, the final concentration of polysorbate 80 is 1.0%, and the final concentration of calcium chloride is 0.055%.

[0035] Example 18 This example provides a specific activation process for preparing thrombin, including the following steps: 1) Weighing: Weigh prothrombin gel eluate, purified water, activator, and calcium chloride respectively in proportion. The activator is soy lecithin (phosphatidylcholine 18 - 25%, phosphatidylethanolamine 10 - 16%, phosphatidylinositol 14 - 19%), weigh 0.076 g (0.050% of the total weight of the solution to be activated) per g / portion, and weigh 145 g of prothrombin gel eluate per g / portion; 2) Prepare the solution: Dissolve calcium chloride in an appropriate amount of purified water and cool it down; 3) Reaction: Sequentially add an activator, polysorbate 80, and the solution to be activated (145 g of prothrombin gel eluate and 0 g of purified water) into a container in proportion. After stirring evenly, add a calcium chloride solution, immediately stir, and react for 1 hour. Specifically, the final concentration of polysorbate 80 is 1.0%, and the final concentration of calcium chloride is 0.065%.

[0036] Example 19 This example provides a specific activation process for preparing thrombin, including the following steps: 1) Weighing: Weigh the prothrombin gel eluate, purified water, activator, and calcium chloride separately according to the ratio. The activator is soy lecithin (phosphatidylcholine 18 - 25%, phosphatidylethanolamine 10 - 16%, phosphatidylinositol 14 - 19%). Weigh 0.076 g (0.050% of the total weight of the solution to be activated) per portion, and weigh 145 g of the prothrombin gel eluate per portion. 2) Solution preparation: Dissolve calcium chloride in an appropriate amount of purified water and cool it down. 3) Reaction: Add the activator, polysorbate 80, and the solution to be activated (145 g of prothrombin gel eluate, 0 g of purified water) to the container in sequence according to the ratio. After stirring evenly, add the calcium chloride solution, and immediately stir and react for 1 hour. Specifically, the final concentration of polysorbate 80 is 1.0%, and the final concentration of calcium chloride is 0.075%.

[0037] Comparative Example 1 This comparative example provides a specific activation process for thrombin preparation, including the following steps: 1) Weighing: Weigh the prothrombin gel eluate, purified water, activator, and calcium chloride separately according to the ratio. The activator is rabbit brain powder. Weigh 0.03 g (0.02% of the total weight of the solution to be activated) per portion, and weigh 145 g of the prothrombin gel eluate per portion. 2) Solution preparation: Dissolve calcium chloride in an appropriate amount of purified water and cool it down. 3) Reaction: Add the activator and the solution to be activated (145 g of prothrombin gel eluate, 0 g of purified water) to the container in sequence according to the ratio. After stirring evenly, add the calcium chloride solution, and immediately stir and react for 1 hour. Specifically, the final concentration of calcium chloride is 0.073%.

[0038] Experimental test For the activated solutions obtained by stirring for 1 hour after activation in Examples 1 - 19 and Comparative Example 1, refer to the method under "Potency Determination" of thrombin powder in the First Supplementary Edition of Chinese Pharmacopoeia 2020 to determine the potency, and calculate the thrombin potency obtained per milliliter of eluate after activation. The test results of each group and the corresponding activated solution potency are shown in Table 1 below: Table 1 Composition of prothrombin reaction solution and potency of activated solution Test group Type of activator Thrombinogen gel eluate g / portion Purified water g / portion Activator g / portion Concentration of polysorbate 80 % <![CDATA[CaCl 2 Final concentration %]]> Potency U / ml Comparative example 1 Rabbit brain powder 145 / 0.03（0.02%） / 0.073 279.05 Example 1 Soybean lecithin (phosphatidylcholine 18 - 25%, phosphatidylethanolamine 10 - 16%, phosphatidylinositol 14 - 19%) 145 / 0.0152（0.01%） 1.0 0.055 292.84 Example 2 Soybean lecithin (phosphatidylcholine 18 - 25%, phosphatidylethanolamine 10 - 16%, phosphatidylinositol 14 - 19%) 145 / 0.0375（0.025%） 1.0 0.055 424.78 Example 3 Soybean lecithin (phosphatidylcholine 18 - 25%, phosphatidylethanolamine 10 - 16%, phosphatidylinositol 14 - 19%) 145 / 0.0760（0.05%） 1.0 0.055 437.72 Example 4 Soybean lecithin (phosphatidylcholine 18 - 25%, phosphatidylethanolamine 10 - 16%, phosphatidylinositol 14 - 19%) 145 / 0.1216（0.08%） 1.0 0.055 440.16 Example 5 Soybean lecithin (phosphatidylcholine 18 - 25%, phosphatidylethanolamine 10 - 16%, phosphatidylinositol 14 - 19%) 150 180 0.33（0.1%） / 0.083 452.91 Example 6 Phosphatidylcholine 145 / 0.0057（0.0375‰） 1.0 0.055 / Example 7 Phosphatidylethanolamine 145 / 0.0070（0.0375‰） 1.0 0.055 352.12 Example 8 Phosphatidylethanolamine 145 / 0.0095（0.05‰） 1.0 0.055 376.27 Example 9 Phosphatidylinositol 145 / 0.0152（0.05‰） 1.0 0.055 310.76 Example 10 Phosphatidylserine 145 / 0.0082（0.05‰） 1.0 0.055 348.93 Example 11 2% soybean lecithin solution 300 / 3.75（0.025%） 0.3 0.055 621.0 Example 12 2% soybean lecithin solution 300 / 3.8（0.025%） 0.7 0.055 580.6 Example 13 2% soybean lecithin solution 300 / 3.8（0.025%） 1.0 0.055 571.6 Example 14 2% soybean lecithin solution 300 / 3.85（0.025%） 2.0 0.055 654.5 Example 15 Soybean lecithin (phosphatidylcholine 18 - 25%, phosphatidylethanolamine 10 - 16%, phosphatidylinositol 14 - 19%) 145 / 0.0760（0.050%） 1.0 0.030 318.70 Example 16 Soybean lecithin (phosphatidylcholine 18 - 25%, phosphatidylethanolamine 10 - 16%, phosphatidylinositol 14 - 19%) 145 / 0.0760（0.050%） 1.0 0.045 336.44 Example 17 Soybean lecithin (phosphatidylcholine 18 - 25%, phosphatidylethanolamine 10 - 16%, phosphatidylinositol 14 - 19%) 145 / 0.0760（0.050%） 1.0 0.055 349.95 Example 18 Soybean lecithin (phosphatidylcholine 18 - 25%, phosphatidylethanolamine 10 - 16%, phosphatidylinositol 14 - 19%) 145 / 0.0760（0.050%） 1.0 0.065 350.55 Example 19 Soybean lecithin (phosphatidylcholine 18 - 25%, phosphatidylethanolamine 10 - 16%, phosphatidylinositol 14 - 19%) 145 / 0.0760（0.050%） 1.0 0.075 353.63 It can be seen from the above results that the combined use of phospholipid substances and polysorbate 80 can improve the activation effect of prothrombin. Since the polysorbate 80 reagent is much cheaper than activators such as rabbit brain powder and phospholipids, the combined use with phospholipid substances greatly improves the thrombin yield and reduces the cost.

[0039] It should be noted that, for the foregoing embodiments, for the sake of simple description, they are all expressed as a series of action combinations. However, those skilled in the art should know that the present invention is not limited by the described action sequence, because according to the present invention, certain steps may be performed in other sequences or simultaneously. Secondly, those skilled in the art should also know that the embodiments described in the specification are all preferred embodiments, and the actions and modules involved are not necessarily essential to the present invention.

[0040] The above embodiments are only used to illustrate the technical solutions of the present invention, rather than limiting the protection scope of the invention. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all embodiments. Based on these embodiments, all other embodiments obtained by those of ordinary skill in the art without creative efforts belong to the scope of protection of the present invention. Although the present invention has been described in detail with reference to the above embodiments, those of ordinary skill in the art can still, without conflict and without creative efforts, combine, add or delete the features in the embodiments of the present invention according to the situation, or make other adjustments, so as to obtain different technical solutions that essentially do not deviate from the concept of the present invention. These technical solutions also belong to the scope of protection of the present invention.

Claims

1. A prothrombin activation process, characterized in that: Phospholipids are used as activators, calcium chloride solution is used to provide a calcium ion environment, and polysorbate 80 is used as a solubilizer to enable prothrombin to generate thrombin.

2. The prothrombin activation process according to claim 1, characterized in that: The phospholipid substances are one or more of soybean lecithin, phosphatidylcholine, phosphatidylethanolamine, phosphatidylinositol, phosphatidylserine, soybean lecithin, and egg yolk lecithin.

3. The prothrombin activation process according to claim 2, characterized in that: The content of the phospholipid substance is 0.0375‰-0.1% of the total weight of the liquid to be activated, the content of the calcium chloride is 0.03%-0.083% of the total weight of the liquid to be activated, and the content of the polysorbate 80 is 0.3%-2.0% of the total weight of the liquid to be activated.

4. A method for preparing thrombin, characterized in that: The steps include: 1) Collection of pig blood; 2) Centrifugal separation; 3) Adsorption separation; 4) Prothrombin elution; 5) activating prothrombin using the prothrombin activation process according to any one of claims 1 to 3; 6) Ultrafiltration concentration; 7) Sterile filtration.

5. The method for preparing thrombin according to claim 4, characterized in that: The pig blood collection specifically includes: adding an anticoagulant during the pig blood collection process, and stirring the blood evenly while collecting the blood to prevent coagulation.

6. The method for preparing thrombin according to claim 5, characterized in that: The centrifugal separation specifically includes: centrifuging the pig blood and collecting the pig plasma into a collection tank.

7. The method for preparing thrombin according to claim 6, characterized in that: The adsorption separation is specifically as follows: adding the swollen DEAE-dextran gel A-50 into pig plasma and stirring, and after the stirring is completed, filtering the plasma with a filter cloth to obtain a gel containing prothrombin.

8. The method for preparing thrombin according to claim 7, characterized in that: The prothrombin elution is specifically as follows: the prothrombin-containing gel is eluted with a 1.9% sodium chloride solution to obtain a prothrombin gel eluate.

9. The method for preparing thrombin according to claim 8, characterized in that: The prothrombin activation is specifically performed by adding phospholipids, polysorbate 80 and calcium chloride solution to the prothrombin gel eluate, and continuously stirring to react to obtain an activation solution containing thrombin.

10. The method for preparing thrombin according to claim 9, characterized in that: The ultrafiltration concentration is specifically as follows: ultrafiltration is performed on the activation solution using a hollow fiber ultrafiltration column with a molecular weight of 10,000, and when no white precipitate is produced after adding 1% silver nitrate test solution to the ultrafiltrate, the ultrafiltration is stopped; The sterilization filtration is specifically as follows: the ultrafiltrate is filtered through a microporous filter membrane in sequence, and the thrombin ultrafiltrate after pre-filtration is filtered through a cartridge filter equipped with a 0.2 μm polyethersulfone filter element to obtain a thrombin solution.

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