CN111848783B - Preparation method of human fibrinogen - Google Patents
Preparation method of human fibrinogen Download PDFInfo
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- CN111848783B CN111848783B CN202010743753.7A CN202010743753A CN111848783B CN 111848783 B CN111848783 B CN 111848783B CN 202010743753 A CN202010743753 A CN 202010743753A CN 111848783 B CN111848783 B CN 111848783B
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Abstract
The invention provides a preparation method of human fibrinogen, which takes cold precipitation as a starting material and comprises the following steps: (1) cold precipitation and dissolution; (2) glycine precipitation; (3) dissolution and filtration of glycine precipitate; (4) S/D virus inactivation treatment; (5) ethanol precipitation; (6) dissolution of ethanol precipitation; (7) Q Sepharose fastflow gel chromatography; (8) ultrafiltration; (9) Preparing stock solution, sterilizing, packaging, freeze-drying, capping and dry heat inactivating to obtain the product. The preparation method of the human fibrinogen adopts glycine and ethanol precipitation combined chromatographic gel purification production technology, has simple and convenient production steps, is easy to operate and low in 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 re-dissolution time of the freeze-dried preparation is less than 5min.
Description
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 coagulation factor and is mainly used for treating acquired fibrinogen deficiency in clinical application: severe liver injury; cirrhosis of the liver; disseminated intravascular coagulation; post partum hemorrhage and coagulation disorder caused by fibrinogen deficiency due to major surgery, trauma or internal hemorrhage, etc. Human fibrinogen is a humanized 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 shortage of medicines in China at present.
The freeze-dried human fibrinogen products sold on the market in China at present have long redissolution time and need more than 20 minutes on average; secondly, the purity is lower, generally about 70-80%. These low purity human fibrinogen preparations are commonly found in clinical applications to have poor appearance, inconvenient use, and thrombogenic risks.
Along with the aggravation of the aging trend of the population of China and the high population of liver disease patients, the domestic clinical demands for the human fibrinogen preparation are increased year by year, and how to develop the high-purity, high-activity and high-safety instant human fibrinogen preparation has positive significance.
Disclosure of Invention
Aiming at the technical problems, the invention aims to provide a preparation method for extracting human fibrinogen by using a simple chromatography, and develops a human fibrinogen preparation which has high purity, high activity, high safety, simple process, easy operation and quick dissolution.
The technical scheme adopted by the invention is as follows:
a preparation method of human fibrinogen, which takes cold precipitation as a starting material, and comprises the following steps:
(1) Cold precipitation dissolution, comprising the steps of:
(a) Pouring the chopped cryoprecipitate into a dissolving solution I with the weight of 6-10 times of that of the cryoprecipitate for dissolving, stirring and dissolving for 2-3 h, and controlling the final temperature of the dissolved product at 24-26 ℃;
(b) Centrifuging the dissolved product in the step (a) to collect a supernatant I; clarifying and filtering, and performing top washing with a dissolving solution I until the weight of the solution is 8-10 times of that of the cryoprecipitate to obtain filtrate;
(2) Glycine precipitation: metering the weight of the filtrate in the step (b), slowly adding glycine in a stirring state, continuously stirring for 1-2 h after the adding, cooling to 5-9 ℃, centrifuging, and collecting glycine precipitate;
(3) Dissolution and filtration of glycine precipitate: accurately weighing the weight of glycine sediment in the step (2), adding the glycine sediment into a dissolving solution II with the weight of 4-6 times that of glycine sediment for dissolving, stirring and dissolving for at least 1h, and finally controlling the temperature of the dissolving solution at 24-26 ℃; centrifuging and collecting supernatant II; clarifying and filtering, and top-washing with a dissolving solution II until the weight of glycine sediment is 5-8 times that of glycine sediment to obtain protein liquid;
(4) S/D virus inactivation treatment: accurately measuring the weight of the protein liquid in the step (3), and slowly adding the prepared S/D solution under stirring to perform virus inactivation treatment;
(5) Ethanol precipitation: adding the inactivated product in the step (4) into a diluent, and cooling to 6-9 ℃; adding 50wt% ethanol below 0 deg.c, controlling the final reaction temperature at 5-8 deg.c, stirring and centrifuging to collect ethanol precipitate;
(6) Dissolution of ethanol precipitation: accurately measuring the weight of the ethanol precipitation in the step (5), adding the solution into the solution III with the weight which is 8 times that of the ethanol precipitation, and dissolving the solution III, wherein the dissolving temperature is controlled to be 24-28 ℃, and stirring and dissolving the solution for not less than 1h; centrifuging to collect supernatant III, clarifying, filtering, and washing the product with solution III;
(7) Q Sepharose fastflow gel chromatography: subjecting the solution clarified and filtered in the step (6) to Q Sepharose fastflow gel adsorption chromatography to obtain a collection liquid;
(8) Ultrafiltration: carrying out ultrafiltration concentration on the collected liquid in the step (7) to obtain a stock solution;
(9) Preparing stock solution, sterilizing, packaging, freeze-drying, capping and dry heat inactivating to obtain the product.
The invention relates to a preparation method of human fibrinogen, wherein the cryoprecipitation in the step (1) is freshly prepared cryoprecipitation or frozen cryoprecipitation after thawing, when the frozen cryoprecipitation is thawed, the frozen cryoprecipitation is naturally thawed between centrifugation, the natural thawing time is not less than 5h, and the temperature between the centrifugation is 6-10 ℃.
The invention relates to a preparation method of human fibrinogen, wherein the adding amount of glycine in the step (2) is 130-170 g/kg of filtrate.
The invention relates to a preparation method of human fibrinogen, wherein the specific steps of the step (4) are as follows: accurately measuring the weight of the protein liquid in the step (3), slowly adding the prepared S/D solution under stirring to ensure that the final concentration of Tween-80 is 0.80-1.20% and the final concentration of tributyl phosphate is 0.24-0.36%, controlling the temperature of the product at 24-26 ℃, and preserving the temperature for 6 hours to perform virus inactivation treatment, wherein the weight ratio of the protein liquid to the S/D solution is 10:1.
The invention relates to a preparation method of human fibrinogen, wherein the specific steps of the step (5) are as follows: adding the inactivated product in the step (4) into a diluent with the weight being 1.2 times that of the product, and cooling to 6-9 ℃; adding 50wt% ethanol below 0deg.C, controlling the final concentration of ethanol at 8wt%, controlling the final temperature of reaction at 5-8deg.C, stirring for 0.5 hr, centrifuging, and collecting ethanol precipitate; according to M 50% ethanol ＝M Inactivating product And (2) calculating the addition rate of 50wt% ethanol below 0 ℃ by 8/(50-8), wherein the addition rate is less than or equal to 60kg/h.
The invention relates to a preparation method of human fibrinogen, wherein the specific steps of the step (7) are as follows: subjecting the solution clarified and filtered in the step (6) to Q Sepharose fastflow gel adsorption chromatography, and collecting a sample flow-through solution; after the sample loading is finished, the chromatographic column is washed by a dissolving solution III with the gel volume of 3-4 times, and the first time of penetrating fluid is collected and combined in the sample loading penetrating fluid; eluting with eluent with the volume of 3-4 times of gel to obtain a collection liquid.
The invention relates to a preparation method of human fibrinogen, wherein the specific steps of the step (8) are as follows: carrying out ultrafiltration concentration on the collected liquid in the step (7) twice by adopting an ultrafilter, wherein the ultrafiltration concentration comprises the first concentration; after the first concentration, adding dialysate with the same weight as the concentrated solution to dialyze for 8 times; second concentration: and (3) starting the second concentration when the dialysis is finished, collecting the concentrated protein liquid into a liquid preparation container, and top-washing the protein liquid to 5 times of 8wt% ethanol precipitation weight by using the dialysis liquid to obtain the stock solution.
The invention relates to a preparation method of human fibrinogen, wherein the specific steps of the step (9) are as follows: accurately measuring the weight of the stock solution in the step (8), stirring and mixing uniformly, 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 a final product is 6.5-7.5 and the protein content is 2.5%; and (5) after the product is sterilized, packaging, freeze-drying, capping and dry heat inactivation are carried out to obtain the product.
The invention relates to a preparation method of human fibrinogen, wherein the formula of a dissolving solution I in the step (1) is as follows: sodium citrate 0.05mol/L, pH 6.8-7.0, temperature less than or equal to 26 ℃; the formula of the dissolving liquid II in the step (3) is as follows: sodium citrate 0.05mol/L, sodium chloride 0.15mol/L, sucrose 0.03mol/L, pH 6.8-7.0, temperature less than or equal to 26 ℃; the formula of the dissolving liquid 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, temperature 24-28 ℃; the formula of the diluent in the step (5) is as follows: sodium citrate 0.05mol/L, sodium chloride 0.15mol/L, sucrose 0.03mol/L, pH 6.8-7.0, temperature 1-5 ℃; the formula of the dialysate in the step (8) is as follows: sodium citrate 0.05mol/L, arginine hydrochloride 0.23mol/L, pH 6.8-7.2, temperature 20-25 ℃.
The invention relates to a preparation method of human fibrinogen, wherein, in the step (b), a tube type centrifuge is used for centrifugation during centrifugation, and the liquid inlet speed is as follows: each centrifuge has no more than 5kg per minute, the temperature of discharged liquid is less than or equal to 26 ℃, and after the centrifugal liquid feeding is finished, the pipeline is top-washed by using the dissolving liquid I; in the step (3), a tube type centrifuge is used for centrifugation during centrifugation, and the liquid inlet speed is as follows: each centrifuge has no more than 5kg per minute, the temperature of discharged liquid is less than or equal to 26 ℃, and after the centrifugal liquid feeding is finished, the pipeline is top-washed by using a dissolving liquid II; centrifuging in the step (2) and the step (5) by using a tube type centrifuge, wherein the liquid inlet speed is as follows: each centrifuge has no more than 5kg per minute, the temperature of discharged liquid is less than or equal to 15 ℃, and after the centrifugal liquid feeding is finished, the centrifugal liquid is used for top-washing the top-washing pipeline; in the step (6), a tube type centrifuge is used for centrifugation, and the liquid inlet speed is as follows: and each centrifuge does not exceed 5kg per minute, the temperature of discharged liquid is less than or equal to 28 ℃, and after the centrifugal liquid feeding is finished, the pipeline is top-washed by using the dissolving liquid III.
The preparation method of the human fibrinogen disclosed by the invention is different from the prior art in that:
1. the preparation method adopting glycine and ethanol precipitation combined with chromatographic gel purification has the advantages of simple and convenient steps, easy operation, lower cost and convenient mass production. Glycine has the function of protecting the activity of human fibrinogen, firstly, glycine is used for precipitation, namely the activity of the human fibrinogen can be protected, secondly, part of factors or enzymes which can activate the human fibrinogen are removed firstly, so that the activity of the human fibrinogen is further protected by the subsequent process, ethanol precipitation is an organic solvent, has a certain influence on the activity of the human fibrinogen, and the ethanol precipitation is rough extraction and can precipitate out protein or enzymes. The final product obtained by extracting and precipitating with glycine precipitation method is obviously superior to the ethanol precipitation process in quality (appearance and redissolution time are obviously improved).
2. The purity of the human fibrinogen finished product prepared by the method reaches more than 90 percent; the re-dissolution time of the freeze-dried preparation is less than 5min.
According to the preparation method of the human fibrinogen, the components and the proportions of the dissolving solution I, the dissolving solution II, the dissolving solution III, the diluent and the dialyzate are all obtained through multiple experimental adjustment confirmation, and the components and the proportions in the application can ensure that the precipitation and the dissolution in each step in the preparation process of the product are more sufficient or the activity of the target protein is protected to the greatest extent.
According to the preparation method of the human fibrinogen, products in the step (b), the step (2), the step (3), the step (5) and the step (6) are centrifuged by a tubular centrifuge, and when the products are centrifuged, the separation and purification of target proteins can be ensured due to different liquid inlet temperatures and different liquid outlet temperatures. The centrifugation will cause the temperature of the reaction solution to rise, and the temperature of the solution is controlled to protect the human fibrinogen from denaturation.
The method for preparing human fibrinogen according to the present invention will be further described with reference to specific examples.
Detailed Description
A preparation method of human fibrinogen, which takes cold precipitation as a starting material, and comprises the following steps:
(1) Cold precipitation dissolution
(a) Cutting the cryoprecipitate into blocks with the size of 1-3 cm, then pouring the minced cryoprecipitate into a dissolving solution I with the weight which is 8 times that of the cryoprecipitate for dissolving, stirring and dissolving for 2 hours, and controlling the final temperature of the dissolved product at 24-26 ℃;
the cryoprecipitation is freshly prepared cryoprecipitation or frozen cryoprecipitation after thawing; when the frozen sediment is thawed, the frozen sediment is naturally thawed in a centrifugal room, the natural thawing time is 5 hours, and the temperature between the centrifugal rooms is 6-10 ℃; production scale: batch casting cryoprecipitation amount is 60+/-10 kg; (see patent application 201510992448.0, "a method for preparing human fibrinogen"), for a specific preparation of the cryoprecipitate;
the formula of the dissolving liquid I is as follows: sodium citrate 0.05mol/L, pH 6.8-7.0, temperature less than or equal to 26 ℃; the dissolving solution I is adopted, the cost is low, and the purposes of protecting and dissolving the cryoprecipitate can be achieved by simple preparation.
(b) Centrifuging the dissolved product in the step (a) to collect a supernatant I; during centrifugation, a tube type centrifuge is used for centrifugation, and the liquid inlet speed is as follows: each centrifuge has no more than 5kg per minute, the temperature of discharged liquid is less than or equal to 26 ℃, and after the centrifugal liquid feeding is finished, a pipeline is top-washed by using a dissolving liquid I (each centrifuge is no less than 3 kg); clarifying and filtering the supernatant I by using a filter I treated by the dissolving liquid I, and performing top washing by using the dissolving liquid I until the weight is 9 times of that of the cryoprecipitate to obtain filtrate; the aperture of the filter I is 0.6-0.45 nm;
(2) Glycine precipitation
Metering the weight of the filtrate in the step (b), slowly adding glycine under stirring, continuing stirring for 1h after adding, cooling to 7+/-2 ℃, centrifuging, and collecting glycine precipitate, wherein the adding amount of glycine is 150g/kg of filtrate (namely 150g of glycine is added into every 1kg of filtrate); during centrifugation, a tube type centrifuge is used for centrifugation, and the liquid inlet speed is as follows: each centrifuge has a liquid outlet temperature of less than or equal to 15 ℃ and is used for top-washing and pipeline-washing (each centrifuge is not less than 3 kg) with centrifugal liquid outlet after the centrifugal liquid inlet is finished;
(3) Dissolution and filtration of glycine precipitate
Accurately weighing the weight of glycine sediment in the step (2), adding the glycine sediment into a dissolution solution II with the weight which is 5 times that of glycine sediment for dissolution, stirring and dissolving for 1h, controlling the temperature of the dissolution solution to be between 24 and 26 ℃ finally, centrifuging, and collecting a supernatant II;
during centrifugation, a tube centrifuge is used for centrifugation, and the liquid inlet speed is as follows: each centrifuge has no more than 5kg per minute, the temperature of discharged liquid is less than or equal to 26 ℃, and after the centrifugal liquid feeding is finished, a dissolving liquid II is used for top washing a pipeline (each centrifuge is no less than 3 kg); clarifying and filtering the supernatant II by using a filter II treated by the dissolving solution II, and top-washing the supernatant II until the weight of glycine sediment is 6.5 times that of the supernatant II to obtain protein liquid; the aperture of the filter II is 0.6-0.45 nm;
the formula of the dissolving liquid II is as follows: sodium citrate 0.05mol/L, sodium chloride 0.15mol/L, sucrose 0.03mol/L, pH 6.8-7.0, temperature less than or equal to 26 ℃;
(4) S/D virus inactivation treatment
Accurately measuring the weight of the protein liquid in the step (3), and slowly adding the prepared S/D solution under stirring to ensure that the final concentration of Tween-80 is 1wt% and the final concentration of tributyl phosphate is 0.3wt%; controlling the temperature of the product at 24-26 ℃, and preserving the temperature for 6 hours to perform virus inactivation treatment; the weight ratio of the protein liquid to the S/D solution is 10:1;
(5) Ethanol precipitation
Adding the inactivated product in the step (4) into a diluent with the weight being 1.2 times that of the product, and cooling to 6-9 ℃; adding 50wt% ethanol below 0deg.C, controlling the final concentration of ethanol at 8wt%, controlling the final temperature of reaction at 5-8deg.C, stirring for 0.5 hr, centrifuging, and collecting ethanol precipitate; according to M 50% ethanol ＝M Inactivating product Calculating the addition rate of 50wt% ethanol below 0 ℃ by 8/(50-8), wherein the addition rate is less than or equal to 60kg/h;
during centrifugation, a tube type centrifuge is used for centrifugation, and the liquid inlet speed is as follows: each centrifuge has a liquid outlet temperature of less than or equal to 15 ℃ and a centrifugal liquid outlet top washing pipeline (each centrifuge is not less than 3 kg) after the centrifugal liquid inlet is finished;
the formula of the diluent is as follows: sodium citrate 0.05mol/L, sodium chloride 0.15mol/L, sucrose 0.03mol/L, pH 6.8-7.0, temperature 1-5 ℃;
according to the original ethanol concentration and the required final ethanol concentration, according to M 50% ethanol ＝M Inactivating product The amount of the ethanol added with 50wt percent is calculated by 8/(50-8), and the ethanol with the original concentration of 50wt percent is adopted instead of 95wt percent, because the ethanol with the original concentration of 50wt percent is added into the reaction liquid, 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 precipitation in the step (5), adding the solution into the solution III with the weight which is 8 times that of the ethanol precipitation for dissolution, controlling the dissolution temperature to be 24-28 ℃, stirring and dissolving for 1h, and centrifugally collecting supernatant III;
during centrifugation, a tube type centrifuge is used for centrifugation, and the liquid inlet speed is as follows: each centrifuge has no more than 5kg per minute, the temperature of discharged liquid is less than or equal to 28 ℃, and after the centrifugal liquid feeding is finished, the pipeline is top-washed by using a dissolving liquid III (each centrifuge is no less than 3 kg); clarifying and filtering the supernatant III by using a filter III treated by the dissolving liquid III, and top-washing products by using not less than 5kg of the dissolving liquid III; the aperture of the filter III is 0.6-0.45 nm;
the formula of the dissolving liquid III is as follows: tris (Tris) 0.2mol/L, sodium chloride 0.25mol/L, calcium chloride 0.01mol/L, pH 6.55-6.65, temperature 24-28deg.C;
(7) Q Sepharose fastflow gel chromatography
Loading the solution filtered in the step (6) by using the regenerated Q Sepharose FF gel, and collecting loading flow-through liquid; after the sample loading is finished, the chromatographic column is washed by a dissolving solution III with the gel volume of 3-4 times, and the first time of penetrating fluid is collected and combined in the sample loading penetrating fluid; eluting with eluent with the volume of 3-4 times of gel to obtain a collection liquid; the pressure of the inlet liquid is less than 0.2MPa during sample loading, washing and eluting, and the flow rate is less than or equal to 2kg/min;
when loading, flushing and eluting, the pressure of the inlet fluid is less than 0.2MPa, which is the safe use pressure of the chromatographic system; the flow rate is less than or equal to 2kg/min, so that the foreign protein can be adsorbed and removed in the preparation process of the product, and the target protein is purified.
(8) Ultrafiltration
Carrying out ultrafiltration concentration on the collected liquid in the step (7) twice by using an ultrafilter, and carrying out concentration dialysis for the first time: an ultrafilter is started to adjust proper pressure, the inlet pressure is less than or equal to 0.4MPa, the outlet pressure is less than or equal to 0.1MPa, and the collected liquid is concentrated for the first time; after the first concentration, adding dialysate with the same weight as the concentrated solution to dialyze for 8 times; second concentration: starting to concentrate for the second time when the dialysis is finished, collecting the concentrated protein liquid into a liquid preparation container, and top-washing the protein liquid to 5 times of 8wt% ethanol precipitation weight by using the dialysis liquid to obtain a stock solution; the molecular weight cut-off of the ultrafilter is 100KD;
the dialysate formula is as follows: sodium citrate 0.05mol/L, arginine hydrochloride 0.23mol/L, pH 6.8-7.2, temperature 20-25 ℃;
(9) Preparing stock solution, sterilizing, packaging, lyophilizing, capping, and inactivating under dry heat to obtain the final product
Accurately measuring the weight of the stock solution in the step (8), stirring and mixing uniformly, 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 a final product is 6.5-7.5 and the protein content is 2.5g/100ml; and (5) after the product is sterilized, packaging, freeze-drying, capping and dry heat inactivation are carried out to obtain the product.
The purity of the human fibrinogen finished product prepared in the embodiment is 90.9%, and the reconstitution time of the freeze-dried preparation is less than 5min.
The above examples are only illustrative of the preferred embodiments of the present invention and are not intended to limit the scope of the present invention, and various modifications and improvements made by those skilled in the art to the technical solution of the present invention should fall within the scope of protection defined by the claims of the present invention without departing from the spirit of the present invention.
Claims (8)
1. A method for preparing human fibrinogen, which takes cold precipitation as a starting material, and is characterized in that: the method comprises the following steps:
(1) Cold precipitation dissolution, comprising the steps of:
(a) Pouring the chopped cryoprecipitate into a dissolving solution I with the weight of 6-10 times of that of the cryoprecipitate for dissolving, stirring and dissolving for 2-3 h, and controlling the final temperature of the dissolved product at 24-26 ℃;
(b) Centrifuging the dissolved product in the step (a) to collect a supernatant I; clarifying and filtering, and performing top washing with a dissolving solution I until the weight of the solution is 8-10 times of that of the cryoprecipitate to obtain filtrate;
(2) Glycine precipitation: metering the weight of the filtrate in the step (b), slowly adding glycine in a stirring state, continuously stirring for 1-2 h after the adding, cooling to 5-9 ℃, centrifuging, and collecting glycine precipitate;
(3) Dissolution and filtration of glycine precipitate: accurately weighing the weight of glycine sediment in the step (2), adding the glycine sediment into a dissolving solution II with the weight of 4-6 times that of glycine sediment for dissolving, stirring and dissolving for at least 1h, and finally controlling the temperature of the dissolving solution at 24-26 ℃; centrifuging and collecting supernatant II; clarifying and filtering, and top-washing with a dissolving solution II until the weight of glycine sediment is 5-8 times that of glycine sediment to obtain protein liquid;
(4) S/D virus inactivation treatment: accurately measuring the weight of the protein liquid in the step (3), and slowly adding the prepared S/D solution under stirring to perform virus inactivation treatment;
(5) Ethanol precipitation: adding the inactivated product in the step (4) into a diluent, and cooling to 6-9 ℃; adding 50wt% ethanol below 0 deg.c, controlling the final reaction temperature at 5-8 deg.c, stirring and centrifuging to collect ethanol precipitate;
(6) Dissolution of ethanol precipitation: accurately measuring the weight of the ethanol precipitation in the step (5), adding the solution into the solution III with the weight which is 8 times that of the ethanol precipitation, and dissolving the solution III, wherein the dissolving temperature is controlled to be 24-28 ℃, and stirring and dissolving the solution for not less than 1h; centrifuging to collect supernatant III, clarifying, filtering, and washing the product with solution III;
(7) Qsehalosefastflow gel chromatography: subjecting the solution clarified and filtered in the step (6) to QEE fastfastflow gel adsorption chromatography to obtain a collection liquid;
(8) Ultrafiltration: carrying out ultrafiltration concentration on the collected liquid in the step (7) to obtain a stock solution;
(9) Preparing stock solution, sterilizing, packaging, freeze-drying, capping and dry heat inactivating to obtain a product;
the formula of the dissolving liquid I in the step (1) is as follows: sodium citrate 0.05mol/L, pH 6.8-7.0, temperature less than or equal to 26 ℃;
the specific steps of the step (7) are as follows: subjecting the solution clarified and filtered in the step (6) to QEE casefastflow gel adsorption chromatography, and collecting a sample flow-through liquid; after the sample loading is finished, the chromatographic column is washed by a dissolving solution III with the gel volume of 3-4 times, and the first time of penetrating fluid is collected and combined in the sample loading penetrating fluid; eluting with eluent with the volume of 3-4 times of gel to obtain a collection liquid;
the formula of the dissolving liquid II in the step (3) is as follows: sodium citrate 0.05mol/L, sodium chloride 0.15mol/L, sucrose 0.03mol/L, pH 6.8-7.0, temperature less than or equal to 26 ℃; the formula of the dissolving liquid 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, temperature 24-28 ℃; the formula of the diluent in the step (5) is as follows: sodium citrate 0.05mol/L, sodium chloride 0.15mol/L, sucrose 0.03mol/L, pH 6.8-7.0, temperature 1-5 ℃; the formula of the dialysate in the step (8) is as follows: sodium citrate 0.05mol/L, arginine hydrochloride 0.23mol/L, pH 6.8-7.2, temperature 20-25 ℃.
2. The method for producing human fibrinogen according to claim 1, wherein: the cryoprecipitation in the step (1) is freshly prepared cryoprecipitation or frozen cryoprecipitation after thawing; when the frozen sediment is thawed, the frozen sediment is naturally thawed in a centrifugal room, the natural thawing time is not less than 5 hours, and the temperature between the centrifugal rooms is 6-10 ℃.
3. The method for producing human fibrinogen according to claim 1, wherein: the adding amount of glycine in the step (2) is 130-170 g/kg of filtrate.
4. The method for producing human fibrinogen according to claim 1, wherein: the specific steps of the step (4) are as follows: accurately measuring the weight of the protein liquid in the step (3), slowly adding the prepared S/D solution under stirring to ensure that the final concentration of Tween-80 is 0.80-1.20% and the final concentration of tributyl phosphate is 0.24-0.36%, controlling the temperature of the product at 24-26 ℃, and preserving the temperature for 6 hours to perform virus inactivation treatment, wherein the weight ratio of the protein liquid to the S/D solution is 10:1.
5. The method for producing human fibrinogen according to claim 1, wherein: the specific steps of the step (5) are as follows: adding the inactivated product in the step (4) into a diluent with the weight being 1.2 times that of the product, and cooling to 6-9 ℃; adding 50wt% ethanol below 0deg.C, controlling the final concentration of ethanol at 8wt%, controlling the final temperature of reaction at 5-8deg.C, stirring for 0.5 hr, centrifuging, and collecting ethanol precipitate; the addition rate of 50wt% ethanol below 0 ℃ is less than or equal to 60kg/h calculated as M50% ethanol = M inactivated product x 8/(50-8).
6. The method for producing human fibrinogen according to claim 1, wherein: the specific steps of the step (8) are as follows: carrying out ultrafiltration concentration on the collected liquid in the step (7) twice by adopting an ultrafilter, wherein the ultrafiltration concentration comprises the first concentration; after the first concentration, adding dialysate with the same weight as the concentrated solution to dialyze for 8 times; second concentration: and (3) starting the second concentration when the dialysis is finished, collecting the concentrated protein liquid into a liquid preparation container, and top-washing the protein liquid to 5 times of 8wt% ethanol precipitation weight by using the dialysis liquid to obtain the stock solution.
7. The method for producing human fibrinogen according to claim 1, wherein: the specific steps of the step (9) are as follows: accurately measuring the weight of the stock solution in the step (8), stirring and mixing uniformly, 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 a final product is 6.5-7.5 and the protein content is 2.5%; and (5) after the product is sterilized, packaging, freeze-drying, capping and dry heat inactivation are carried out to obtain the product.
8. The method for producing human fibrinogen according to any one of claims 1 to 7, wherein: centrifuging in the step (b) by using a tube type centrifuge, wherein the liquid inlet speed is as follows: each centrifuge has no more than 5kg per minute, the temperature of discharged liquid is less than or equal to 26 ℃, and after the centrifugal liquid feeding is finished, the pipeline is top-washed by using the dissolving liquid I; in the step (3), a tube type centrifuge is used for centrifugation during centrifugation, and the liquid inlet speed is as follows: each centrifuge has no more than 5kg per minute, the temperature of discharged liquid is less than or equal to 26 ℃, and after the centrifugal liquid feeding is finished, the pipeline is top-washed by using a dissolving liquid II; centrifuging in the step (2) and the step (5) by using a tube type centrifuge, wherein the liquid inlet speed is as follows: each centrifuge has no more than 5kg per minute, the temperature of discharged liquid is less than or equal to 15 ℃, and after the centrifugal liquid feeding is finished, the centrifugal liquid is used for top-washing the top-washing pipeline; in the step (6), a tube type centrifuge is used for centrifugation, and the liquid inlet speed is as follows: and each centrifuge does not exceed 5kg per minute, the temperature of discharged liquid is less than or equal to 28 ℃, and after the centrifugal liquid feeding is finished, the pipeline is top-washed by using the dissolving liquid III.
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