阅读说明：本技术 一种高纯人凝血酶的制备方法 (Preparation method of high-purity human thrombin ) 是由 庾昌文 何甜 王振国 刘国维 于 2021-09-12 设计创作，主要内容包括：本发明公开了一种高纯人凝血酶的制备方法,属于生物制药领域。本发明从去冷沉淀人血浆中提取人凝血酶的方法,包括新鲜冰冻人血浆去除冷沉淀；DEAE Sephadex A-50凝胶吸附；超滤后料液激活；S/D病毒灭活；离子交换层析；除菌过滤；纳米膜除病毒过滤；分装；冷冻干燥。本发明通过改善人凝血酶的提取工艺,制得的人凝血酶,稳定性好,产品纯度高,安全可靠,比活可达2600IU/mg蛋白质以上,满足市场的需求,也提高了珍贵血浆资源的利用价值。(The invention discloses a preparation method of high-purity human thrombin, belonging to the field of biological pharmacy. The invention relates to a method for extracting human thrombin from human plasma without cryoprecipitate, which comprises the steps of removing cryoprecipitate from fresh frozen human plasma; DEAE Sephadex A-50 gel adsorption; activating the feed liquid after ultrafiltration; S/D virus inactivation; ion exchange chromatography; sterilizing and filtering; removing viruses by using the nano membrane and filtering; subpackaging; and (5) freeze drying. The human thrombin prepared by the method provided by the invention through improving the extraction process of the human thrombin has the advantages of good stability, high product purity, safety and reliability, the specific activity can reach more than 2600IU/mg protein, the market demand is met, and the utilization value of precious plasma resources is also improved.)

1. The preparation method of the high-purity human thrombin is characterized by comprising the following steps of:

(1) fresh frozen human plasma to remove cryoprecipitate: thawing and uniformly mixing fresh frozen human plasma, and removing cryoprecipitate through a flow centrifuge to obtain centrifuged plasma supernatant;

(2) DEAE Sephadex A-50 gel adsorption: adding DEAE Sephadex A-50 gel into the supernatant obtained in the step (1), wherein the addition amount of the xerogel is 0.5-1.5g/kg of feed liquid, the xerogel is swelled with hot injection water at more than 70 ℃, then cooled with cold injection water at less than 25 ℃, finally balanced by a balance buffer solution, stirred at a proper rotation speed (ensuring that the gel is uniformly mixed and distributed in the solution and is not too fast), for 1.0-2.0 hours, collecting the gel, washing for about 5 times by the balance buffer solution, eluting by the elution buffer solution, collecting the eluent and filtering by a 0.45 mu m filter element, and storing the gel after regeneration for reuse;

(3) and (3) ultrafiltration: ultrafiltering and concentrating the filtrate obtained in the step (2), adjusting the factor titer of the eluent II to 5-20IU/mL, and adjusting the conductance to 0.5-4.5ms/cm through dialysis;

(4) activating: sterilizing and filtering the feed liquid obtained in the step (3), adjusting the pH value to 6.5-7.5, then adding the prepared calcium chloride mother liquid into the feed liquid in proportion after sterilizing and filtering, keeping the concentration of calcium ions at 30-80mM, and activating for 5-15 days at 2-8 ℃;

(5) and (3) filter pressing: adding diatomite and one third of diatomite into a pre-paved plate frame to the activated suspension obtained in the step (4) according to the proportion of 5-20g of diatomite per kg of feed liquid, adding the rest into the feed liquid, performing filter pressing to collect supernatant, and filtering by using a filter element with the diameter of 0.45 mu m;

(6) S/D virus inactivation: slowly adding the S/D inactivation solution into the filtrate obtained in the step (5) under mild stirring, stirring for 15-30min after the addition is finished, wherein the addition time is less than or equal to 30min, adjusting the temperature of the solution to 24-26 ℃, inactivating for 6 hours, and recording the temperature once every 30 min;

(7) ion exchange chromatography: fully balancing the ion exchange chromatography column with a balance solution, filtering the feed liquid obtained in the step (6) by using a 0.45-micrometer filter element, then loading, flushing about 10 column volumes with a washing buffer solution after loading, then eluting with an elution buffer solution, collecting the eluent, and then adjusting the thrombin titer to be about 500IU/mL by using a diluent;

(8) and (3) degerming and filtering: sterilizing and filtering the product by using a 0.22 mu m filter element;

(9) virus removal and filtration by a nano membrane: firstly, prefiltering by using a filter element with the diameter of 0.1 mu m, and then performing virus removal filtration by using a filter element with the diameter of 15 nanometers;

(10) subpackaging;

(11) and (3) freeze drying: precooling, quick-freezing, annealing, primary drying and secondary drying are carried out, and finally the human thrombin freeze-dried powder is obtained.

2. The DEAE Sephadex A-50 gel adsorption in the step (2) adopts a plasma adsorption and filtration device (Chinese invention patent CN201810248696.8, a plasma adsorption and filtration device for the production of blood products of coagulation factors).

3. The formula of the equilibrium buffer solution in the step (2) is as follows: 0.01-0.02M sodium citrate, 0.15-0.25M sodium chloride, and pH of 7.0-8.0.

4. The formula of the S/D virus inactivation solution in the step (6) is as follows: the solubility of Tween-80 was 1% (w/w), and the concentration of TNBP was 0.3% (w/w).

5. The formula of the equilibrium buffer solution in the step (7) is as follows: 0.1-0.5M alanine, 0.05-0.1M sodium chloride, pH 6.5-7.5; the formula of the washing buffer solution is as follows: 0.1-0.5M alanine, 0.1-0.3M sodium chloride, pH 6.5-7.5; the formula of the elution buffer solution is as follows: 0.1-0.5M alanine, 0.3-0.5M sodium chloride, pH 6.5-7.5; the formula of the diluent is as follows: 0.1-0.5M alanine, 0.05-0.15M sodium chloride, pH 6.5-7.5, wherein alanine also acts as a protective agent.

6. The human thrombin product prepared by the method for preparing the high-purity human thrombin.

Technical Field

The invention belongs to the field of biological pharmacy, relates to an extraction process of blood products, and particularly relates to an extraction method of human thrombin.

Background

Human thrombin is a serine protease with a molecular weight of about 37kd, a white amorphous powder, soluble in water and insoluble in organic solvents. Comprises two chains connected by disulfide bonds, the light chain and the heavy chain have molecular weights of 6000 and 31000 respectively, and the active site is on the heavy chain. Because antithrombin exists in high concentration in plasma, thrombin exists only for a few minutes, and exists in the form of prothrombin in the plasma, the content of the prothrombin is very small, and the content of the prothrombin in normal plasma is 0.11-0.30 mg/mL. The factor Xa, V, calcium ion and phospholipid form a "prothrombinase complex" which is formed by the cleavage of prothrombin in sequence at the peptide bonds arginine 274-threonine 275 and arginine 323-isoleucine 324, respectively, under the action of the prothrombinase complex. Factor Xa is the active center of this complex and factor Xa alone also slowly cleaves prothrombin. Factor V is activated to Va by thrombin and factor Xa, is more active than factor V, and acts as a cofactor in the process of activating prothrombin. The calcium ions link factor Xa and prothrombin to phospholipids, Va also links to phospholipids through their hydrophobic regions, the main role of which is to provide an active surface.

Thrombin is a blood stopping agent, and is mainly suitable for stopping bleeding of small blood vessels, capillaries and parenchymal viscera which are difficult to stop bleeding clinically; can be used for stopping bleeding of trauma, operation, oral cavity, ear, nose, throat, urinary system, burn, orthopedics, neurosurgery, ophthalmology, gynecology and obstetrics, and digestive tract. The thrombin directly acts on the last step of the blood coagulation process to promote the conversion of soluble fibrinogen in the plasma into insoluble fibrin, thereby achieving the purpose of quick-acting hemostasis. But also can promote the mitosis of epithelial cells and accelerate wound healing, and is a quick-acting local hemostatic. Thrombin is suitable for the bleeding of small blood vessels, capillaries, parenchymal organs and other various kinds of bleeding which are difficult to be stopped by ligation.

The thrombin has good local hemostatic effect and no side effect. At present, the application range of thrombin is gradually expanded, bleeding and hemostasis of parts such as surgical operation, ear, nose and throat, oral cavity, gynecological, urinary and digestive tracts and the like are developed from simple local external application, and the thrombin can also be used as an important raw material of various external hemostatic medicaments and is widely applied to clinic. Meanwhile, in addition to the traditional blood coagulation effect, the effect of thrombin in cerebrovascular diseases is increasingly emphasized, and the current research shows that thrombin not only has toxic effect in cerebral hemorrhage and cerebral ischemia, but also has important effect in normal development and injury protection of the central nervous system. Clinical studies have shown that small doses produce neuroprotective effects, whereas large doses have cytotoxic effects. Therefore, the clinical application of the medicine is gradually extensive, so that the thrombin which is very much in short supply at present is more scarce, and the production prospect is good.

The current common process for extracting human thrombin from plasma is chromatography, and the raw material is cryoprecipitated plasma or Cohn component III. The two raw material extraction processes of the prior related invention patents in China are related, for example, the process of the invention is a preparation method of human thrombin (application number 201610614283.8), and the process comprises the steps of dissolving Cohn component III again, inactivating viruses, carrying out anion exchange chromatography, adding calcium ions for activation, carrying out cation exchange chromatography and carrying out nanofiltration; the patent discloses a method for preparing human thrombin from cold glue-removed plasma (application No. 201510586400.X), which comprises the steps of adsorbing prothrombin in the cold glue-removed plasma by anion resin, eluting and filtering to obtain a prothrombin solution, adding a calcium chloride solution to activate the prothrombin solution to obtain a thrombin solution, inactivating S/D virus, carrying out cation chromatography to obtain a purified thrombin solution, carrying out ultrafiltration, dialysis and concentration, carrying out nanofiltration, carrying out sterilization and filtration, carrying out split charging and freeze drying, and carrying out dry heat inactivation to obtain a human thrombin finished product; the patent application No. 201510520812.3 discloses a method for preparing freeze-dried human thrombin, which comprises the steps of using Cohn component III as a raw material, activating human prothrombin and calcium chloride by an isoelectric precipitation method to obtain a human thrombin crude product, inactivating S/D virus, carrying out SP-Sephadex C-50 chromatography purification to obtain refined human thrombin, carrying out ultrafiltration concentration, adding a stabilizer, carrying out nanofiltration, carrying out sterilization filtration, freeze-drying and dry heating to obtain a target product.

The invention develops a new optimized process for extracting human thrombin, develops a preparation process of a human thrombin preparation suitable for industrial production scale, and has the characteristics of good stability, high purity and better virus removal effect.

Disclosure of Invention

The invention aims to overcome the defects of the prior art and provide the extraction method of the human thrombin, and the prepared human thrombin preparation has high purity, high stability and good virus inactivation effect.

In order to achieve the purpose, the invention adopts the following technical scheme:

a preparation method of high-purity human thrombin comprises the following steps:

(1) fresh frozen human plasma to remove cryoprecipitate: thawing and uniformly mixing fresh frozen human plasma, and removing cryoprecipitate through a flow centrifuge to obtain centrifuged plasma supernatant;

(2) DEAE Sephadex A-50 gel adsorption: adding DEAE Sephadex A-50 gel into the supernatant obtained in the step (1), wherein the addition amount of the xerogel is 0.5-1.5g/kg of feed liquid, the xerogel is swelled with hot injection water at more than 70 ℃, then cooled with cold injection water at less than 25 ℃, finally balanced by a balance buffer solution, stirred at a proper rotation speed (ensuring that the gel is uniformly mixed and distributed in the solution and is not too fast), for 1.0-2.0 hours, collecting the gel, washing for about 5 times by the balance buffer solution, eluting by the elution buffer solution, collecting the eluent and filtering by a 0.45 mu m filter element, and storing the gel after regeneration for reuse;

(3) and (3) ultrafiltration: ultrafiltering and concentrating the filtrate obtained in the step (2), adjusting the factor titer of the eluent II to 5-20IU/mL, and adjusting the conductance to 0.5-4.5ms/cm through dialysis;

(4) activating: sterilizing and filtering the feed liquid obtained in the step (3), adjusting the pH value to 6.5-7.5, then adding the prepared calcium chloride mother liquid into the feed liquid in proportion after sterilizing and filtering, keeping the concentration of calcium ions at 30-80mM, and activating for 5-15 days at 2-8 ℃;

(5) and (3) filter pressing: adding diatomite and one third of diatomite into a pre-paved plate frame to the activated suspension obtained in the step (4) according to the proportion of 5-20g of diatomite per kg of feed liquid, adding the rest into the feed liquid, performing filter pressing to collect supernatant, and filtering by using a filter element with the diameter of 0.45 mu m;

(6) S/D virus inactivation: slowly adding the S/D inactivation solution into the filtrate obtained in the step (5) under mild stirring, stirring for 15-30min after the addition is finished, wherein the addition time is less than or equal to 30min, adjusting the temperature of the solution to 24-26 ℃, inactivating for 6 hours, and recording the temperature once every 30 min;

(7) ion exchange chromatography: fully balancing the ion exchange chromatography column with a balance solution, filtering the feed liquid obtained in the step (6) by using a 0.45-micrometer filter element, then loading, flushing about 10 column volumes with a washing buffer solution after loading, then eluting with an elution buffer solution, collecting the eluent, and then adjusting the thrombin titer to be about 500IU/mL by using a diluent;

(8) and (3) degerming and filtering: sterilizing and filtering the product by using a 0.22 mu m filter element;

(9) virus removal and filtration by a nano membrane: firstly, prefiltering by using a filter element with the diameter of 0.1 mu m, and then performing virus removal filtration by using a filter element with the diameter of 15 nanometers;

(10) subpackaging;

(11) and (3) freeze drying: precooling, quick-freezing, annealing, primary drying and secondary drying are carried out, and finally the human thrombin freeze-dried powder is obtained.

The DEAE Sephadex A-50 gel adsorption in the step (2) adopts a plasma adsorption and filtration device (Chinese invention patent CN201810248696.8, a plasma adsorption and filtration device for the production of blood products of coagulation factors).

The formula of the equilibrium buffer solution in the step (2) is as follows: 0.01-0.02M sodium citrate, 0.15-0.25M sodium chloride, and pH of 7.0-8.0.

The formula of the S/D virus inactivation solution in the step (6) is as follows: the solubility of Tween-80 was 1% (w/w), and the concentration of TNBP was 0.3% (w/w).

The formula of the equilibrium buffer solution in the step (7) is as follows: 0.1-0.5M alanine, 0.05-0.1M sodium chloride, pH 6.5-7.5; the formula of the washing buffer solution is as follows: 0.1-0.5M alanine, 0.1-0.3M sodium chloride, pH 6.5-7.5; the formula of the elution buffer solution is as follows: 0.1-0.5M alanine, 0.3-0.5M sodium chloride, pH 6.5-7.5; the formula of the diluent is as follows: 0.1-0.5M alanine, 0.05-0.15M sodium chloride, pH 6.5-7.5.

The invention has the advantages that:

(1) DEAE Sephadex A-50 gel adsorption, the pilot scale used in this company's plasma adsorption filtration device (Chinese invention patent CN201810248696.8 a blood plasma adsorption filtration device for the production of blood products of coagulation factors). The automatic filtering and collecting device can realize the streamlined treatment of automatic filtering and collecting of gel, greatly reduce the labor intensity of workers, simultaneously stabilize and control the pressure difference in the whole filtering process, play a better role in protecting the blood coagulation factor components and gel particles which are subsequently manufactured, and is suitable for industrial production scale.

(2) The extraction method of human thrombin provided by the invention has uncomplicated flow and easy operation, and reduces the influence on products and the possibility of introducing more impurities compared with other processes such as adding a stabilizer or an impurity removal agent (such as polyethylene glycol) additionally.

(3) And virus is removed by adopting nano filtration of 15 nanometers, so that a better virus filtering effect is ensured, and the safety is higher.

(4) The human thrombin prepared by the process adopts alanine as a stabilizer, has good stability, high product purity, safety and reliability, has the titer recovery rate of about 50 percent per ton of thrombin compared with the domestic extraction process data of cryoprecipitated human plasma, and has the specific activity of more than 2600IU/mg protein.

Detailed Description

The present invention will be described in further detail with reference to examples, but the embodiments of the present invention are not limited thereto.

Example 1

(1) Fresh frozen human plasma to remove cryoprecipitate: melting and uniformly mixing 10kg of fresh frozen human plasma, and removing cryoprecipitate through a flow centrifuge to obtain centrifuged plasma supernatant;

(2) DEAE Sephadex A-50 gel adsorption: adding DEAE Sephadex A-50 gel into the supernatant obtained in the step (1), wherein the addition amount of xerogel is 0.5g/kg of feed liquid, the xerogel is swelled with hot injection water at more than 70 ℃, cooled with cold injection water at less than 25 ℃, finally balanced with balance buffer solution, stirred for 1.0 hour at proper rotation speed (ensuring that the gel is uniformly mixed and distributed in the solution and is not too fast), collecting the gel, washing for 5 times by the balance buffer solution, eluting by the elution buffer solution, collecting the eluent and filtering by a 0.45 mu m filter element, collecting 1L of filtrate, regenerating the gel and storing for reuse, and the formula of the balance buffer solution is as follows: 0.02M sodium citrate, 0.2M sodium chloride, pH 7.0;

(3) and (3) ultrafiltration: ultrafiltering and concentrating the filtrate obtained in the step (2), adjusting the titer of a factor II of the eluent to 18IU/mL, and adjusting the conductance to 3ms/cm through dialysis;

(4) activating: sterilizing and filtering the feed liquid obtained in the step (3), adjusting the pH value to 6.8, then adding the prepared calcium chloride mother liquid into the feed liquid in proportion after sterilizing and filtering, keeping the concentration of calcium ions at 60mM, and activating for 7 days at 2-8 ℃;

(5) and (3) filter pressing: adding 10g of diatomite into the activated suspension obtained in the step (4), pre-paving a plate frame with 5g of diatomite, performing pressure filtration to collect supernatant, and filtering with a 0.45-micrometer filter element;

(6) S/D virus inactivation: slowly adding the S/D inactivation solution into the filtrate obtained in the step (5) under mild stirring, stirring for 15min after the addition is finished, adjusting the temperature of the solution to 25 ℃, inactivating for 6 hours, and recording the temperature every 30 min;

(7) ion exchange chromatography: fully balancing the ion exchange chromatography column with a balancing solution, filtering the feed liquid obtained in the step (6) by using a 0.45-micrometer filter element, then loading, flushing 10 column volumes with a washing buffer solution, then eluting with an elution buffer solution, obtaining a human thrombin titer of about 6.2 ten thousand units per kilogram of plasma according to the total conversion of the eluent, then adjusting the thrombin titer by using a diluent to be about 500IU/mL, wherein the formula of the balancing buffer solution is as follows: 0.3M alanine, 0.07M sodium chloride, pH 6.8; the formula of the washing buffer solution is as follows: 0.3M alanine, 0.2M sodium chloride, pH 6.8; the formula of the elution buffer solution is as follows: 0.3M alanine, 0.5M sodium chloride, pH 6.8; the formula of the diluent is as follows: 0.3M alanine, 0.1M sodium chloride, pH 6.8;

(8) and (3) degerming and filtering: sterilizing and filtering the product by using a 0.22 mu m filter element;

(9) virus removal and filtration by a nano membrane: firstly, prefiltering by using a filter element with the diameter of 0.1 mu m, and then performing virus removal filtration by using a filter element with the diameter of 15 nanometers;

(10) subpackaging;

(11) and (3) freeze drying: precooling, quick-freezing, annealing, primary drying and secondary drying are carried out, and finally the human thrombin freeze-dried powder is obtained.

Example 2

(1) Fresh frozen human plasma to remove cryoprecipitate: melting and uniformly mixing 15kg of fresh frozen human plasma, and removing cryoprecipitate through a flow centrifuge to obtain centrifuged plasma supernatant;

(2) DEAE Sephadex A-50 gel adsorption: adding DEAE Sephadex A-50 gel into the supernatant obtained in the step (1), wherein the addition amount of xerogel is 1g/kg of feed liquid, the xerogel is swelled with hot injection water at more than 70 ℃, cooled with cold injection water at less than 25 ℃, balanced with balance buffer solution, stirred at proper rotation speed (ensuring that the gel is uniformly mixed and distributed in the solution and is not too fast), collecting the gel, washing for 4 times by the balance buffer solution, eluting by the elution buffer solution, collecting the eluent, filtering by a 0.45 mu m filter element, collecting 1.3L of filtrate, regenerating the gel and storing for reuse, and the formula of the balance buffer solution is as follows: 0.015M sodium citrate, 0.2M sodium chloride, pH 7.5;

(3) and (3) ultrafiltration: ultrafiltering and concentrating the filtrate obtained in the step (2), adjusting the titer of a factor II of the eluent to 8IU/mL, and adjusting the conductance to 4ms/cm through dialysis;

(4) activating: sterilizing and filtering the feed liquid obtained in the step (3), adjusting the pH value to 7.0, then adding the prepared calcium chloride mother liquid into the feed liquid in proportion after sterilizing and filtering, keeping the concentration of calcium ions at 55mM, and activating for 15 days at 2-8 ℃;

(5) and (3) filter pressing: adding 12g of diatomite into the activated suspension obtained in the step (4), pre-paving a plate frame with 6g of diatomite, performing pressure filtration to collect supernatant, and filtering with a 0.45-micrometer filter element;

(6) S/D virus inactivation: slowly adding the S/D inactivation solution into the filtrate obtained in the step (5) under mild stirring, stirring for 30min after the addition is finished, adjusting the temperature of the solution to 24 ℃, inactivating for 6 hours, and recording the temperature every 30 min;

(7) ion exchange chromatography: fully balancing the ion exchange chromatography column with a balancing solution, filtering the feed liquid obtained in the step (6) by using a 0.45-micrometer filter element, then loading, flushing 10 column volumes with a washing buffer solution, then eluting with an elution buffer solution, obtaining a human thrombin titer of about 6.1 ten thousand units per kilogram of plasma according to the total conversion of the eluent, then adjusting the thrombin titer by using a diluent to be about 500IU/mL, wherein the formula of the balancing buffer solution is as follows: 0.3M alanine, 0.08M sodium chloride, pH 7.0; the formula of the washing buffer solution is as follows: 0.3M alanine, 0.1M sodium chloride, pH 7.0; the formula of the elution buffer solution is as follows: 0.3M alanine, 0.4M sodium chloride, pH 7.0; the formula of the diluent is as follows: 0.3M alanine, 0.12M sodium chloride, pH 7.0;

(8) and (3) degerming and filtering: sterilizing and filtering the product by using a 0.22 mu m filter element;

(9) virus removal and filtration by a nano membrane: firstly, prefiltering by using a filter element with the diameter of 0.1 mu m, and then performing virus removal filtration by using a filter element with the diameter of 15 nanometers;

(10) subpackaging;

(11) and (3) freeze drying: precooling, quick-freezing, annealing, primary drying and secondary drying are carried out, and finally the human thrombin freeze-dried powder is obtained.

Example 3

(1) Fresh frozen human plasma to remove cryoprecipitate: melting and uniformly mixing 20kg of fresh frozen human plasma, and removing cryoprecipitate through a flow centrifuge to obtain centrifuged plasma supernatant;

(2) DEAE Sephadex A-50 gel adsorption: adding DEAE Sephadex A-50 gel into the supernatant obtained in the step (1), wherein the addition amount of xerogel is 1.5g/kg of feed liquid, the xerogel is swelled with hot injection water at more than 70 ℃, cooled with cold injection water at less than 25 ℃, balanced with balance buffer solution, stirred at proper rotation speed (ensuring that the gel is uniformly mixed and distributed in the solution and is not too fast), collecting the gel, washing for 5 times by the balance buffer solution, eluting by the elution buffer solution, collecting the eluent, filtering by a 0.45 mu m filter element, collecting 1.8L of filtrate, and storing the gel after regeneration for reuse, wherein the formula of the balance buffer solution is as follows: 0.015M sodium citrate, 0.2M sodium chloride, pH 7.5;

(3) and (3) ultrafiltration: ultrafiltering and concentrating the filtrate obtained in the step (2), adjusting the titer of a factor II of the eluent to 20IU/mL, and adjusting the conductance to 3.5ms/cm through dialysis;

(4) activating: sterilizing and filtering the feed liquid obtained in the step (3), adjusting the pH value to 7.2, then adding the prepared calcium chloride mother liquid into the feed liquid in proportion after sterilizing and filtering, keeping the concentration of calcium ions at 65mM, and activating for 8 days at 2-8 ℃;

(5) and (3) filter pressing: adding 18g of diatomite into the activated suspension obtained in the step (4), pre-paving a plate frame with 9g of diatomite, performing pressure filtration to collect supernatant, and filtering with a 0.45-micrometer filter element;

(6) S/D virus inactivation: slowly adding the S/D inactivation solution into the filtrate obtained in the step (5) under mild stirring, stirring for 25min after the addition is finished, adjusting the temperature of the solution to 24 ℃, inactivating for 6 hours, and recording the temperature every 30 min;

(7) ion exchange chromatography: fully balancing the ion exchange chromatography column with a balancing solution, filtering the feed liquid obtained in the step (6) by using a 0.45-micron filter element, then loading, flushing 10 column volumes with a washing buffer solution, then eluting with an elution buffer solution, obtaining a human thrombin titer of about 6.4 ten thousand units per kilogram of plasma according to the total conversion of the eluent, then adjusting the thrombin titer by using a diluent to be about 500IU/mL, wherein the formula of the balancing buffer solution is as follows: 0.25M alanine, 0.06M sodium chloride, pH 7.2; the formula of the washing buffer solution is as follows: 0.25M alanine, 0.15M sodium chloride, pH 7.2; the formula of the elution buffer solution is as follows: 0.25M alanine, 0.45M sodium chloride, pH 7.2; the formula of the diluent is as follows: 0.25M alanine, 0.08M sodium chloride, pH 7.2.

(8) And (3) degerming and filtering: sterilizing and filtering the product by using a 0.22 mu m filter element;

(9) virus removal and filtration by a nano membrane: firstly, prefiltering by using a filter element with the diameter of 0.1 mu m, and then performing virus removal filtration by using a filter element with the diameter of 15 nanometers;

(10) subpackaging;

(11) and (3) freeze drying: precooling, quick-freezing, annealing, primary drying and secondary drying are carried out, and finally the human thrombin freeze-dried powder is obtained.

The above-mentioned embodiment is only one of the preferred embodiments of the present invention, and the protection scope of the present invention is not limited thereby, so: all equivalent changes made by the process of the present invention are within the scope of the present invention, and are within the scope of the present invention.