阅读说明：本技术 一种人纤维蛋白原的制备方法 (Preparation method of human fibrinogen ) 是由 谢长福 胡辉恒 汪模正 邓坤 于 2020-07-29 设计创作，主要内容包括：本发明提供了一种人纤维蛋白原的制备方法,以冷沉淀为起始原料,包括如下步骤：(1)冷沉淀溶解；(2)甘氨酸沉淀；(3)甘氨酸沉淀的溶解及过滤；(4)S/D病毒灭活处理；(5)乙醇沉淀；(6)乙醇沉淀的溶解；(7)Q Sepharose fastflow凝胶层析处理；(8)超滤；(9)原液配制、除菌分装、冻干、轧盖、干热灭活后得到产品。本发明所述的人纤维蛋白原的制备方法,采用甘氨酸和乙醇沉淀结合色谱凝胶纯化生产工艺,生产步骤简便,易操作且成本较低,便于规模化生产；按本方法制备的人纤维蛋白原成品,纯度达到90％以上；冻干制剂的复溶时间小于5min。(The invention provides a preparation method of human fibrinogen, which takes cryoprecipitate as an initial raw material and comprises the following steps: (1) performing cold precipitation and dissolution; (2) precipitating glycine; (3) dissolving and filtering the glycine precipitate; (4) S/D virus inactivation treatment; (5) ethanol precipitation; (6) dissolving the ethanol precipitate; (7) q Sepharose fast flow gel chromatography treatment; (8) ultrafiltration; (9) preparing stock solution, sterilizing, subpackaging, freeze-drying, capping, and performing dry heat inactivation to obtain the product. The preparation method of the human fibrinogen adopts the glycine and ethanol precipitation combined with the chromatographic gel purification production process, has simple and convenient production steps, is easy to operate, has lower cost and is convenient for large-scale production; the purity of the human fibrinogen finished product prepared by the method reaches more than 90 percent; the reconstitution time of the lyophilized preparation is less than 5 min.)

1. A preparation method of human fibrinogen uses cryoprecipitate as a starting material, and is characterized in that: the method comprises the following steps:

(1) the method comprises the following steps:

(a) pouring the chopped cold precipitate into a dissolving solution I with the weight 6-10 times that of the cold precipitate for dissolving, stirring and dissolving for 2-3 hours, and controlling the final temperature of the dissolved product to be 24-26 ℃;

(b) centrifuging the product dissolved in the step (a) and collecting supernatant I; clarifying and filtering, and washing the precipitate with a dissolving solution I until the weight of the precipitate is 8-10 times of that of the precipitate to obtain a filtrate;

(2) and (3) glycine precipitation: measuring the weight of the filtrate obtained in the step (b), slowly adding glycine under a stirring state, continuously stirring for 1-2 h after the glycine is added, cooling to 5-9 ℃, centrifuging, and collecting glycine precipitate;

(3) dissolution and filtration of glycine precipitate: accurately weighing the weight of the glycine precipitate in the step (2), adding the glycine precipitate into a dissolving solution II which is 4-6 times of the weight of the glycine precipitate for dissolving for not less than 1h under stirring, and finally controlling the temperature of the dissolving solution to be 24-26 ℃; centrifuging and collecting supernatant II; clarifying, filtering, and washing with a dissolving solution II until the weight of the dissolving solution is 5-8 times that of glycine precipitate to obtain a protein solution;

(4) S/D virus inactivation treatment: accurately measuring the weight of the protein solution in the step (3), and slowly adding the prepared S/D solution under stirring for virus inactivation treatment;

(5) ethanol precipitation: adding the inactivated product in the step (4) into a diluent, and cooling to 6-9 ℃; adding 50 wt% ethanol below 0 ℃, controlling the final reaction temperature to be 5-8 ℃, continuing stirring after the ethanol is added, and centrifugally collecting ethanol precipitate;

(6) dissolution of ethanol precipitate: accurately measuring the weight of the ethanol precipitate in the step (5), adding the ethanol precipitate into a dissolving solution III which is 8 times of the weight of the ethanol precipitate for dissolving, controlling the dissolving temperature at 24-28 ℃, and stirring for dissolving for not less than 1 hour; centrifuging, collecting supernatant III, clarifying, filtering, and washing the product with solution III;

(7) q Sepharose fast flow gel chromatography treatment: subjecting the solution clarified and filtered in the step (6) to QSepharose fastflow gel adsorption chromatography to obtain a collection liquid;

(8) and (3) ultrafiltration: carrying out ultrafiltration concentration on the collected liquid in the step (7) to obtain a stock solution;

(9) preparing stock solution, sterilizing, subpackaging, freeze-drying, capping, and performing dry heat inactivation to obtain the product.

2. The method for producing human fibrinogen according to claim 1, characterized in that: the cryoprecipitate in the step (1) is freshly prepared cryoprecipitate or frozen cryoprecipitate after unfreezing; and when the cryopreserved cryoprecipitate is thawed, naturally thawing the cryopreserved cryoprecipitate in a centrifugal room, wherein the natural thawing time is not less than 5 hours, and the temperature between centrifuges is 6-10 ℃.

3. The method for producing human fibrinogen according to claim 1, characterized in that: the addition amount of the glycine in the step (2) is 130-170 g/kg of filtrate.

4. The method for producing human fibrinogen according to claim 1, characterized in that: the step (4) comprises the following specific steps: and (3) accurately measuring the weight of the protein solution in the step (3), slowly adding the prepared S/D solution under stirring to ensure that the final concentration of the Tween-80 is 0.80-1.20% and the final concentration of the tributyl phosphate is 0.24-0.36%, controlling the temperature of the product at 24-26 ℃, and keeping the temperature for 6 hours for virus inactivation treatment, wherein the weight ratio of the protein solution to the S/D solution is 10: 1.

5. The method for producing human fibrinogen according to claim 1, characterized in that: the step (5) comprises the following specific steps: adding the inactivated product obtained in the step (4) into a diluent with the weight of 1.2 times, and cooling to 6-9 ℃; adding 50 wt% ethanol with the final concentration of 8 wt% below 0 ℃, controlling the final reaction temperature at 5-8 ℃, continuously stirring for 0.5h, and centrifugally collecting ethanol precipitate after the 50 wt% ethanol is added; according to M_{50% ethanol}＝M_{Inactivated preparation}Calculating the addition of 50 wt% ethanol below 0 ℃ by multiplying 8/(50-8), wherein the addition speed is less than or equal to 60 kg/h.

6. The method for producing human fibrinogen according to claim 1, characterized in that: the step (7) comprises the following specific steps: subjecting the solution clarified and filtered in the step (6) to Q Sepharose fast flow gel adsorption chromatography, and collecting a sample loading flow-through solution; after the sample loading is finished, washing the chromatographic column by using a dissolving solution III with the gel volume being 3-4 times, and collecting and combining the first time flow-through solution into the sample loading flow-through solution; eluting with eluent with the volume 3-4 times that of the gel to obtain a collected solution.

7. The method for producing human fibrinogen according to claim 1, characterized in that: the step (8) comprises the following specific steps: carrying out ultrafiltration concentration twice on the collected liquid in the step (7) by using an ultrafilter, wherein the ultrafiltration concentration comprises first concentration; after the first concentration, adding dialysate with the same weight as the concentrate for dialysis for 8 times; and (3) second concentration: and (3) starting second concentration after dialysis is finished, collecting the concentrated protein liquid into a liquid preparation container, and carrying out top washing on the concentrated protein liquid to 5 times of the weight of 8 wt% ethanol precipitate by using dialysate to obtain the stock solution.

8. The method for producing human fibrinogen according to claim 1, characterized in that: the step (9) comprises the following specific steps: accurately measuring the weight of the stock solution in the step (8), stirring and uniformly mixing the stock solution, detecting the pH value and the protein content of the stock solution, and preparing the solution according to the detection result to ensure that the pH value in the final product is 6.5-7.5 and the protein content is 2.5%; and (4) after the product is sterilized, subpackaging, freeze-drying, capping and carrying out dry heat inactivation to obtain the product.

9. The method for producing human fibrinogen according to claim 1, characterized in that: the formula of the dissolving solution I in the step (1) is as follows: 0.05mol/L of sodium citrate, the pH value of 6.8-7.0 and the temperature of less than or equal to 26 ℃; the formula of the dissolving solution II in the step (3) is as follows: 0.05mol/L of sodium citrate, 0.15mol/L of sodium chloride and 0.03mol/L of cane sugar, wherein the pH value is 6.8-7.0, and the temperature is less than or equal to 26 ℃; the formula of the dissolving solution III in the steps (6) and (7) is as follows: 0.2mol/L Tris, 0.25mol/L sodium chloride, 0.01mol/L calcium chloride, pH 6.55-6.65 and temperature 24-28 ℃; the formula of the diluent in the step (5) is as follows: 0.05mol/L of sodium citrate, 0.15mol/L of sodium chloride and 0.03mol/L of cane sugar, wherein the pH value is 6.8-7.0, and the temperature is 1-5 ℃; the formula of the dialysate in the step (8) is as follows: 0.05mol/L of sodium citrate, 0.23mol/L of arginine hydrochloride, pH of 6.8-7.2 and temperature of 20-25 ℃.

10. The method for producing human fibrinogen according to any of claims 1 to 9, wherein: centrifuging by using a tubular centrifuge in the step (b), wherein the liquid inlet speed is as follows: each centrifuge does not exceed 5kg per minute, the temperature of the liquid outlet is less than or equal to 26 ℃, and the pipeline is top-washed by a dissolving solution I after the centrifugal liquid inlet is finished; and (4) centrifuging by using a tubular centrifuge in the step (3), wherein the liquid inlet speed is as follows: each centrifuge does not exceed 5kg per minute, the temperature of the liquid outlet is less than or equal to 26 ℃, and the pipeline is top-washed by a dissolving solution II after the centrifugal liquid inlet is finished; and (5) centrifuging by using a tubular centrifuge during centrifuging, wherein the liquid inlet speed is as follows: each centrifuge does not exceed 5kg per minute, the liquid outlet temperature is less than or equal to 15 ℃, and the top washing pipeline is top washed by the centrifuged liquid outlet after the centrifuged liquid inlet is finished; and (5) centrifuging by using a tubular centrifuge in the step (6), wherein the liquid inlet speed is as follows: each centrifuge does not exceed 5kg per minute, the liquid outlet temperature is less than or equal to 28 ℃, and the pipeline is top-washed by a dissolving solution III after the centrifugal liquid inlet is finished.

Technical Field

The invention belongs to the technical field of biological pharmacy, and particularly relates to a preparation method of human fibrinogen.

Background

Human fibrinogen is a blood coagulation factor and is mainly used for treating acquired fibrinogen deficiency in clinical application: severe liver damage; cirrhosis of the liver; disseminated intravascular coagulation; postpartum hemorrhage and blood coagulation disorder caused by fibrinogen deficiency due to major surgery, trauma or internal hemorrhage, etc. The human fibrinogen is a human blood preparation, has few clinical side effects, contains a special virus inactivation process in the preparation process, has reliable guarantee on safety, is used as a necessary preparation for treating hemorrhagic surgery, and belongs to a medicine in shortage at present.

The freeze-dried human fibrinogen products sold in the market at present have longer redissolution and dissolution time, which is more than 20 minutes on average; secondly, the purity is low, generally about 70-80%. These low purity human fibrinogen preparations are ubiquitous in clinical applications, having poor appearance, inconvenient use, and risk of thrombogenesis.

Along with the increasing trend of the aging population in China and the high population of patients with liver diseases, the clinical requirement on human fibrinogen preparations in China is increased year by year, and how to develop the human fibrinogen preparation with high purity, high activity, high safety and instant dissolution has positive significance at the present stage.

Disclosure of Invention

In view of the above technical problems, the present invention aims to provide a preparation method for extracting human fibrinogen by using simple chromatography, and to develop a human fibrinogen preparation with high purity, high activity, high safety, simple process, easy operation, and rapid dissolution.

The technical scheme adopted by the invention is as follows:

a preparation method of human fibrinogen uses cryoprecipitate as a starting material, and comprises the following steps:

(1) the method comprises the following steps:

(a) pouring the chopped cold precipitate into a dissolving solution I with the weight 6-10 times that of the cold precipitate for dissolving, stirring and dissolving for 2-3 hours, and controlling the final temperature of the dissolved product to be 24-26 ℃;

(b) centrifuging the product dissolved in the step (a) and collecting supernatant I; clarifying and filtering, and washing the precipitate with a dissolving solution I until the weight of the precipitate is 8-10 times of that of the precipitate to obtain a filtrate;

(2) and (3) glycine precipitation: measuring the weight of the filtrate obtained in the step (b), slowly adding glycine under a stirring state, continuously stirring for 1-2 h after the glycine is added, cooling to 5-9 ℃, centrifuging, and collecting glycine precipitate;

(3) dissolution and filtration of glycine precipitate: accurately weighing the weight of the glycine precipitate in the step (2), adding the glycine precipitate into a dissolving solution II which is 4-6 times of the weight of the glycine precipitate for dissolving for not less than 1h under stirring, and finally controlling the temperature of the dissolving solution to be 24-26 ℃; centrifuging and collecting supernatant II; clarifying, filtering, and washing with a dissolving solution II until the weight of the dissolving solution is 5-8 times that of glycine precipitate to obtain a protein solution;

(4) S/D virus inactivation treatment: accurately measuring the weight of the protein solution in the step (3), and slowly adding the prepared S/D solution under stirring for virus inactivation treatment;

(5) ethanol precipitation: adding the inactivated product in the step (4) into a diluent, and cooling to 6-9 ℃; adding 50 wt% ethanol below 0 ℃, controlling the final reaction temperature to be 5-8 ℃, continuing stirring after the ethanol is added, and centrifugally collecting ethanol precipitate;

(6) dissolution of ethanol precipitate: accurately measuring the weight of the ethanol precipitate in the step (5), adding the ethanol precipitate into a dissolving solution III which is 8 times of the weight of the ethanol precipitate for dissolving, controlling the dissolving temperature at 24-28 ℃, and stirring for dissolving for not less than 1 hour; centrifuging, collecting supernatant III, clarifying, filtering, and washing the product with solution III;

(7) q Sepharose fast flow gel chromatography treatment: subjecting the solution clarified and filtered in the step (6) to QSepharose fastflow gel adsorption chromatography to obtain a collection liquid;

(8) and (3) ultrafiltration: carrying out ultrafiltration concentration on the collected liquid in the step (7) to obtain a stock solution;

(9) preparing stock solution, sterilizing, subpackaging, freeze-drying, capping, and performing dry heat inactivation to obtain the product.

The preparation method of the human fibrinogen comprises the step (1), wherein the cryoprecipitate is freshly prepared cryoprecipitate or thawed cryoprecipitate, when the cryoprecipitate is thawed, the cryoprecipitate is placed in a centrifugal room to be naturally thawed, the natural thawing time is not less than 5 hours, and the temperature between the centrifugal room is 6-10 ℃.

The preparation method of the human fibrinogen comprises the step (2) of adding 130-170 g/kg of glycine into the filtrate.

The preparation method of the human fibrinogen comprises the following specific steps of the step (4): and (3) accurately measuring the weight of the protein solution in the step (3), slowly adding the prepared S/D solution under stirring to ensure that the final concentration of the Tween-80 is 0.80-1.20% and the final concentration of the tributyl phosphate is 0.24-0.36%, controlling the temperature of the product at 24-26 ℃, and keeping the temperature for 6 hours for virus inactivation treatment, wherein the weight ratio of the protein solution to the S/D solution is 10: 1.

The preparation method of the human fibrinogen comprises the following specific steps of (5): adding the inactivated product obtained in the step (4) into a diluent with the weight of 1.2 times, and cooling to 6-9 ℃; adding 50 wt% ethanol with the final concentration of 8 wt% below 0 ℃, controlling the final reaction temperature at 5-8 ℃, continuously stirring for 0.5h, and centrifugally collecting ethanol precipitate after the 50 wt% ethanol is added; according to M_{50% ethanol}＝M_{Inactivated preparation}Calculating the addition of 50 wt% ethanol below 0 ℃ by multiplying 8/(50-8), wherein the addition speed is less than or equal to 60 kg/h.

The preparation method of the human fibrinogen comprises the following specific steps of (7): subjecting the solution clarified and filtered in the step (6) to Q Sepharose fast flow gel adsorption chromatography, and collecting a sample loading flow-through solution; after the sample loading is finished, washing the chromatographic column by using a dissolving solution III with the gel volume being 3-4 times, and collecting and combining the first time flow-through solution into the sample loading flow-through solution; eluting with eluent with the volume 3-4 times that of the gel to obtain a collected solution.

The preparation method of the human fibrinogen comprises the following specific steps of (8): carrying out ultrafiltration concentration twice on the collected liquid in the step (7) by using an ultrafilter, wherein the ultrafiltration concentration comprises first concentration; after the first concentration, adding dialysate with the same weight as the concentrate for dialysis for 8 times; and (3) second concentration: and (3) starting second concentration after dialysis is finished, collecting the concentrated protein liquid into a liquid preparation container, and carrying out top washing on the concentrated protein liquid to 5 times of the weight of 8 wt% ethanol precipitate by using dialysate to obtain the stock solution.

The preparation method of the human fibrinogen comprises the following specific steps of step (9): accurately measuring the weight of the stock solution in the step (8), stirring and uniformly mixing the stock solution, detecting the pH value and the protein content of the stock solution, and preparing the solution according to the detection result to ensure that the pH value in the final product is 6.5-7.5 and the protein content is 2.5%; and (4) after the product is sterilized, subpackaging, freeze-drying, capping and carrying out dry heat inactivation to obtain the product.

The preparation method of the human fibrinogen comprises the following steps of (1) preparing a dissolving solution I: 0.05mol/L of sodium citrate, the pH value of 6.8-7.0 and the temperature of less than or equal to 26 ℃; the formula of the dissolving solution II in the step (3) is as follows: 0.05mol/L of sodium citrate, 0.15mol/L of sodium chloride and 0.03mol/L of cane sugar, wherein the pH value is 6.8-7.0, and the temperature is less than or equal to 26 ℃; the formula of the dissolving solution III in the steps (6) and (7) is as follows: Tris0.2mol/L, sodium chloride 0.25mol/L, calcium chloride 0.01mol/L, pH 6.55-6.65, and temperature 24-28 ℃; the formula of the diluent in the step (5) is as follows: 0.05mol/L of sodium citrate, 0.15mol/L of sodium chloride and 0.03mol/L of cane sugar, wherein the pH value is 6.8-7.0, and the temperature is 1-5 ℃; the formula of the dialysate in the step (8) is as follows: 0.05mol/L of sodium citrate, 0.23mol/L of arginine hydrochloride, pH of 6.8-7.2 and temperature of 20-25 ℃.

The preparation method of the human fibrinogen comprises the following steps of (a) centrifuging by using a tubular centrifuge, wherein the liquid inlet speed is as follows: each centrifuge does not exceed 5kg per minute, the temperature of the liquid outlet is less than or equal to 26 ℃, and the pipeline is top-washed by a dissolving solution I after the centrifugal liquid inlet is finished; and (4) centrifuging by using a tubular centrifuge in the step (3), wherein the liquid inlet speed is as follows: each centrifuge does not exceed 5kg per minute, the temperature of the liquid outlet is less than or equal to 26 ℃, and the pipeline is top-washed by a dissolving solution II after the centrifugal liquid inlet is finished; and (5) centrifuging by using a tubular centrifuge during centrifuging, wherein the liquid inlet speed is as follows: each centrifuge does not exceed 5kg per minute, the liquid outlet temperature is less than or equal to 15 ℃, and the top washing pipeline is top washed by the centrifuged liquid outlet after the centrifuged liquid inlet is finished; and (5) centrifuging by using a tubular centrifuge in the step (6), wherein the liquid inlet speed is as follows: each centrifuge does not exceed 5kg per minute, the liquid outlet temperature is less than or equal to 28 ℃, and the pipeline is top-washed by a dissolving solution III after the centrifugal liquid inlet is finished.

The difference between the preparation method of human fibrinogen and the prior art is that:

1. the preparation method of glycine and ethanol precipitation combined with chromatographic gel purification is simple and convenient in steps, easy to operate, low in cost and convenient for large-scale production. The glycine has the activity of protecting human fibrinogen, firstly glycine is used for precipitation, the activity of the human fibrinogen can be protected, secondly, part of factors or enzymes which can activate the human fibrinogen can be removed in one step, the activity of the human fibrinogen can be further protected by a subsequent process, ethanol precipitation is an organic solvent, the activity of the human fibrinogen has certain influence, and the glycine is extracted in a rough manner, and both proteins or enzymes can be precipitated. The final product obtained by extracting and precipitating by using a glycine precipitation method is obviously superior to the final product obtained by using an ethanol precipitation process in quality (the appearance and the redissolution time are obviously improved).

2. The purity of the human fibrinogen finished product prepared by the method reaches more than 90 percent; the reconstitution time of the lyophilized preparation is less than 5 min.

The components and the proportion of the solution I, the solution II, the solution III, the diluent and the dialysate are obtained by multiple experimental adjustment and confirmation, and the components and the proportion in the method can ensure that the target protein activity is fully or maximally protected by precipitation and dissolution in each step in the preparation process of the product.

According to the preparation method of human fibrinogen, products in the step (b), the step (2), the step (3), the step (5) and the step (6) are centrifuged by using a tubular centrifuge, and during centrifugation, due to different inlet liquid temperatures and different outlet liquid temperatures, separation and purification of target protein can be guaranteed. The reaction liquid is heated during centrifugation, and the temperature of the discharged liquid is controlled to protect human fibrinogen from denaturation.

The method for producing human fibrinogen according to the present invention will be further described with reference to the following examples.

Detailed Description

A preparation method of human fibrinogen uses cryoprecipitate as a starting material, and comprises the following steps:

(1) cold precipitation dissolution

(a) Cutting the cryoprecipitate into blocks of 1-3 cm, then pouring the chopped cryoprecipitate into a dissolving solution I with the weight 8 times that of the cryoprecipitate for dissolving, stirring and dissolving for 2 hours, and controlling the final temperature of a product after dissolving to be 24-26 ℃;

the cryoprecipitate is freshly prepared cryoprecipitate or frozen cryoprecipitate after unfreezing; when the cryopreserved cryoprecipitate is thawed, naturally thawing the cryopreserved cryoprecipitate in a centrifugal room for 5 hours at the temperature of 6-10 ℃ between centrifuges; production scale: the amount of the batch-fed cold precipitation is 60 plus or minus 10 kg; (the specific preparation method of the cryoprecipitate is described in the patent 'a preparation method of human fibrinogen' with the application number of 201510992448.0);

the formula of the dissolving solution I is as follows: 0.05mol/L of sodium citrate, the pH value of 6.8-7.0 and the temperature of less than or equal to 26 ℃; the dissolving solution I is adopted, the cost is low, and the purpose of protecting and dissolving the cryoprecipitate can be achieved by simple preparation.

(b) Centrifuging the product dissolved in the step (a) and collecting supernatant I; the centrifugal process is carried out by a tubular centrifugal machine, and the liquid inlet speed is as follows: each centrifuge does not exceed 5kg per minute, the temperature of the discharged liquid is less than or equal to 26 ℃, and the pipeline is top-washed by a dissolving solution I (not less than 3kg per centrifuge) after the centrifugal liquid feeding is finished; clarifying and filtering the supernatant I by using a filter I treated by the dissolving solution I, and washing the supernatant I by using the dissolving solution I to 9 times of the weight of the cryoprecipitate to obtain a filtrate; the aperture of the filter I is 0.6-0.45 nm;

(2) precipitation of glycine

Weighing the filtrate obtained in the step (b), slowly adding glycine under stirring, continuing to stir for 1h after the addition is finished, cooling to 7 +/-2 ℃, centrifuging, collecting glycine precipitate, wherein the addition amount of glycine is 150g/kg of filtrate (namely 150g of glycine is added into each 1kg of filtrate); the centrifugal process is carried out by a tubular centrifugal machine, and the liquid inlet speed is as follows: each centrifuge does not exceed 5kg per minute, the liquid outlet temperature is less than or equal to 15 ℃, and after the centrifugal liquid inlet is finished, the top washing pipeline is top washed by the centrifugal liquid outlet (not less than 3kg per centrifuge);

(3) dissolving and filtering glycine precipitate

Accurately weighing the weight of the glycine precipitate in the step (2), adding the glycine precipitate into a dissolving solution II which is 5 times of the weight of the glycine precipitate for dissolving, stirring and dissolving for 1h, controlling the temperature of the dissolving solution to be 24-26 ℃ finally, centrifuging, and collecting a supernatant II;

during centrifugation, the centrifugal machine is used for centrifugation, and the liquid inlet speed is as follows: each centrifuge does not exceed 5kg per minute, the temperature of the discharged liquid is less than or equal to 26 ℃, and the pipeline is top-washed by a dissolving solution II (not less than 3kg per centrifuge) after the centrifugal liquid feeding is finished; clarifying and filtering the supernatant II by using a filter II treated by a dissolving solution II, and top-washing the supernatant II by using the dissolving solution II until the weight of the supernatant II is 6.5 times that of the glycine precipitate to obtain a protein solution; the pore diameter of the filter II is 0.6-0.45 nm;

the formula of the dissolving solution II is as follows: 0.05mol/L of sodium citrate, 0.15mol/L of sodium chloride and 0.03mol/L of cane sugar, wherein the pH value is 6.8-7.0, and the temperature is less than or equal to 26 ℃;

(4) S/D virus inactivation treatment

Accurately measuring the weight of the protein solution in the step (3), and slowly adding the prepared S/D solution under stirring to ensure that the final concentration of the Tween-80 is 1 wt% and the final concentration of the tributyl phosphate is 0.3 wt%; controlling the temperature of the product at 24-26 ℃, and carrying out virus inactivation treatment after 6h of heat preservation; the weight ratio of the protein solution to the S/D solution is 10: 1;

(5) ethanol precipitation

Adding the inactivated product obtained in the step (4) into a diluent with the weight of 1.2 times, and cooling to 6-9 ℃; adding 50 wt% ethanol with the final concentration of 8 wt% below 0 ℃, controlling the final reaction temperature at 5-8 ℃, continuously stirring for 0.5h, and centrifugally collecting ethanol precipitate after the 50 wt% ethanol is added; according to M_{50% ethanol}＝M_{Inactivated preparation}Calculating the addition of 50 wt% ethanol below 0 ℃ by multiplying by 8/(50-8), wherein the addition speed is less than or equal to 60 kg/h;

the centrifugal process is carried out by a tubular centrifugal machine, and the liquid inlet speed is as follows: each centrifuge does not exceed 5kg per minute, the liquid outlet temperature is less than or equal to 15 ℃, and the pipeline is top-washed by the centrifuged liquid outlet after the centrifugation liquid inlet is finished (each centrifuge does not exceed 3 kg);

the formula of the diluent is as follows: 0.05mol/L of sodium citrate, 0.15mol/L of sodium chloride and 0.03mol/L of cane sugar, wherein the pH value is 6.8-7.0, and the temperature is 1-5 ℃;

according to the original ethanol concentration and the required final ethanol concentration, according to M_{50% ethanol}＝M_{Inactivated preparation}The theoretical amount of 50 wt% ethanol is calculated by multiplying by 8/(50-8), and the 50 wt% ethanol is adopted instead of the original concentration ethanol of 95 wt%, because the 50 wt% ethanol is added into the reaction solution, the influence on the activity of the human fibrinogen is small, the volume is controllable, and the subsequent operation is easy.

(6) Dissolution of ethanol precipitate

Accurately measuring the weight of the ethanol precipitate in the step (5), adding the ethanol precipitate into a dissolving solution III which is 8 times of the weight of the ethanol precipitate for dissolving, controlling the dissolving temperature at 24-28 ℃, stirring for dissolving for 1 hour, and centrifuging to collect a supernatant III;

the centrifugal process is carried out by a tubular centrifugal machine, and the liquid inlet speed is as follows: each centrifuge does not exceed 5kg per minute, the temperature of the discharged liquid is less than or equal to 28 ℃, and the pipeline is top-washed by a dissolving solution III (each centrifuge does not exceed 3kg) after the centrifugal liquid feeding is finished; clarifying and filtering the supernatant III by using a filter III treated by the dissolving solution III, and washing a product by using not less than 5kg of the dissolving solution III; the pore diameter of the filter III is 0.6-0.45 nm;

the formula of the dissolving solution III is as follows: 0.2mol/L of Tris (hydroxymethyl) aminomethane (Tris), 0.25mol/L of sodium chloride and 0.01mol/L of calcium chloride, wherein the pH value is 6.55-6.65, and the temperature is 24-28 ℃;

(7) q Sepharose fast flow gel chromatography treatment

Carrying out sample chromatography on the solution filtered in the step (6) by using regenerated Q Sepharose FF gel, and collecting sample flow-through liquid; after the sample loading is finished, washing the chromatographic column by using a dissolving solution III with the gel volume being 3-4 times, and collecting and combining the first time flow-through solution into the sample loading flow-through solution; eluting with eluent with the volume 3-4 times that of the gel to obtain a collected solution; the liquid inlet pressure is less than 0.2MPa and the flow rate is less than or equal to 2kg/min during sample loading, washing and elution;

when the sample is loaded, washed and eluted, the liquid inlet pressure is less than 0.2MPa, which is the safe use pressure of the chromatography system; the flow rate is less than or equal to 2kg/min, and the impurity protein can be adsorbed and removed in the preparation process of the product, so that the target protein is purified.

(8) Ultrafiltration

And (3) carrying out ultrafiltration concentration twice on the collected liquid in the step (7) by using an ultrafilter, and carrying out concentration dialysis for the first time: opening an ultrafilter to adjust the pressure to be less than or equal to 0.4MPa and less than or equal to 0.1MPa, and concentrating the collected liquid for the first time; after the first concentration, adding dialysate with the same weight as the concentrate for dialysis for 8 times; and (3) second concentration: starting second concentration after dialysis, collecting the concentrated protein solution into a solution preparation container, and washing the protein solution with the dialysate to 5 times the weight of 8 wt% ethanol precipitate to obtain stock solution; the molecular weight cut-off of the ultrafilter is 100 KD;

the formula of the dialysate is as follows: 0.05mol/L of sodium citrate, 0.23mol/L of arginine hydrochloride, pH of 6.8-7.2 and temperature of 20-25 ℃;

(9) preparing stock solution, sterilizing, packaging, lyophilizing, capping, and inactivating by dry heat to obtain the final product

Accurately measuring the weight of the stock solution in the step (8), stirring and uniformly mixing the stock solution, detecting the pH value and the protein content of the stock solution, and preparing the solution by using water for injection according to the detection result to ensure that the pH value in the final product is 6.5-7.5 and the protein content is 2.5g/100 ml; and (4) after the product is sterilized, subpackaging, freeze-drying, capping and carrying out dry heat inactivation to obtain the product.

The purity of the human fibrinogen finished product prepared in this example was 90.9%, and the reconstitution time of the lyophilized preparation was less than 5 min.

The above-mentioned embodiments are merely illustrative of the preferred embodiments of the present invention, and do not limit the scope of the present invention, and various modifications and improvements of the technical solution of the present invention by those skilled in the art should fall within the protection scope defined by the claims of the present invention without departing from the spirit of the present invention.