A preparation method and product of human fibrinogen

Abstract

The present invention provides a preparation method and product of human fibrinogen, belonging to the field of protein production technology. In the preparation method provided by the present invention, component I is precipitated as a raw material, and a specific dissolving liquid is combined with a low-temperature ethanol method to obtain a human fibrinogen stock solution. The preparation method provided by the present invention combines the S / D virus inactivation method and the dry heat virus inactivation method to inactivate the virus, which can further ensure the safety of human fibrinogen. The purity of human fibrinogen prepared by the preparation method provided by the present invention is as high as 87.4%-98.3%. The re-dissolution speed is fast, and it only takes 4-5 minutes to be re-dissolved. The re-dissolution solution is clear and only slightly opalescent. The coagulation time is short, and it only takes 19-22s.

Description

Technical Field

[0001] The invention belongs to the technical field of protein production, and specifically relates to a preparation method and product of human fibrinogen. Background Art

[0002] Human fibrinogen (Fg) is a protein component in plasma, with a content of up to 2-4g. It plays a key role in the human coagulation system. The final stage of coagulation is that Fg is converted into fibrin under the action of thrombin. Fibrin and other blood cell components aggregate into insoluble masses to achieve the purpose of hemostasis. In addition to directly participating in the coagulation process, Fg also plays an important role in platelet aggregation and blood viscosity. Common diseases related to Fg deficiency include: congenital fibrinogen reduction or deficiency; severe liver damage, cirrhosis, disseminated intravascular coagulation, postpartum hemorrhage, and coagulation disorders caused by fibrinogen deficiency due to major surgery, trauma or internal bleeding.

[0003] At present, there are two main methods for extracting raw materials of fibrinogen: one is to use cold precipitation as raw material, and obtain it through extraction, adsorption, centrifugation, chromatography and other manufacturing processes. The human fibrinogen precipitate obtained by using cold precipitation as raw material and extraction, adsorption, centrifugation, chromatography and other manufacturing processes has low solubility and is difficult to dissolve. The other is to use the low-temperature ethanol process to produce component I precipitation, but this process mainly has the problem of low purity. The purity of the products produced is less than 80%, and the impurity content of the products is high. Patients are prone to adverse reactions when using them for injection. On the other hand, the product of this process takes a long time to re-dissolve. Since the product also contains a small amount of fibronectin, the thermal stability of fibronectin is poor, especially when it is easily denatured during dry heat inactivation, which leads to easy protein precipitation during re-dissolution.

[0004] Patent CN108017710B discloses a method for preparing human fibrinogen by double-layer chromatography, the preparation process includes precipitation, dissolution and filtration of component I; S / D virus inactivation; Q Sepharose Fast Flow anion exchange chromatography; primary ultrafiltration concentration; heparin affinity chromatography; secondary ultrafiltration concentration; detection preparation; packaging and freeze-drying; rolling and dry heat inactivation. The protein content of the prepared product is 2.2-2.4%, and glycine and sodium citrate are used as protective agents. The purity of human fibrinogen prepared by this method is increased to 96.2%, and the re-dissolution time is 9 minutes. However, the improvement of product stability effect is still not involved.

[0005] In view of the current market preparation technology and clinical application of human fibrinogen, it is particularly necessary to develop a new human fibrinogen extraction process with high purity, good solubility and good stability. Summary of the invention

[0006] In view of the above shortcomings, the present invention provides a preparation method and product of human fibrinogen. In the preparation method provided by the present invention, component I is precipitated as a raw material, and a human fibrinogen stock solution is prepared by using a specific dissolving liquid combined with a low-temperature ethanol method. The preparation method provided by the present invention combines the S / D virus inactivation method and the dry heat virus inactivation method to inactivate the virus. The safety of human fibrinogen can be further guaranteed. The purity of human fibrinogen prepared by the preparation method provided by the present invention is as high as 87.4%-98.3%. The re-dissolution speed is fast, and it can be re-dissolved in only 4-5 minutes. The re-dissolution solution is clear with only a slight opalescence. The coagulation time is short, and it only takes 19-22s.

[0007] In order to achieve the above-mentioned object of the invention, the technical solution of the present invention is as follows:

[0008] In one aspect, the present invention provides a method for preparing human fibrinogen, characterized in that the preparation method comprises the following steps:

[0009] S1, dissolving the precipitate of component I and filtering it to obtain filtrate A;

[0010] S2, filtrate A is inactivated by S / D to obtain an inactivated solution;

[0011] S3. The inactivated solution is precipitated with low-temperature ethanol twice to obtain a human fibrinogen stock solution.

[0012] Specifically, the preparation method of the component I precipitate described in step S1 is: removing the plasma after the cold precipitate and using the low-temperature ethanol precipitation method to obtain the component I precipitate.

[0013] More specifically, the removal of cold precipitate comprises: soaking the raw plasma in 75% alcohol for 2-3 minutes, then washing the surface with water for injection, breaking the bag of the raw plasma and transporting it to a melting tank, using 10-37°C water for interlayer circulation melting, stopping the circulation after melting, controlling the plasma temperature between 0-4°C, centrifuging and separating the cold precipitate, and transporting the plasma after removing the cold precipitate to a reaction tank for extraction and separation.

[0014] More specifically, the low-temperature ethanol precipitation method includes adding a buffer to the plasma after removing the cold precipitate to adjust the pH, adding low-temperature ethanol, and centrifuging to obtain component I precipitate.

[0015] Preferably, the temperature of the plasma after removing the cryoprecipitate is controlled between 0-4°C.

[0016] Preferably, the low-temperature ethanol is 90%-95% ethanol at -15 to -20°C.

[0017] More preferably, the low-temperature ethanol is 95% ethanol at -15°C.

[0018] Preferably, the volume ratio of ethanol to plasma is 10%, and the flow rate of adding ethanol is no more than 1.0 L / min.

[0019] Further preferably, the flow rate of adding ethanol includes 0.5 L / min, 0.6 L / min, 0.7 L / min, 0.8 L / min, 0.9 L / min, and 1.0 L / min.

[0020] Preferably, the pH is adjusted by adding an acetate buffer of pH 4.0 to adjust the pH to 6.8-7.2.

[0021] Specifically, the dissolving solution used in step S1 is dissolving solution A, and the dissolving solution A contains 1.6%-2.0% sodium citrate and 0.9%-1.1% sodium chloride;

[0022] Preferably, the dissolving solution A contains 1.85% sodium citrate and 0.9% sodium chloride.

[0023] Preferably, the preparation method of the dissolving solution A is to dissolve sodium citrate and sodium chloride in water for injection, stir evenly, and add 1 mol / L HCl to adjust the pH to 6.6-7.0.

[0024] Specifically, the ratio of the component I precipitate to the dissolving solution A in step S1 is 1:15-20 w / v.

[0025] Preferably, the ratio of the component I precipitate to the dissolving solution A in step S1 is 1:15 w / v.

[0026] Specifically, the filtration in step S1 is performed using a plate and frame filter.

[0027] Preferably, the balancing liquid of the plate and frame filter is balancing liquid A, and the balancing liquid A contains 3.2%-4.0% calcium chloride and 0.9%-1.1% sodium chloride.

[0028] Further preferably, the filter plate used in the plate and frame filter is a K700 filter plate.

[0029] Further preferably, the balancing liquid of the plate and frame filter is balancing liquid A, and the balancing liquid A contains 3.7% calcium chloride and 0.9% sodium chloride.

[0030] Further preferably, the balancing solution A is prepared by dissolving calcium chloride and sodium chloride in water for injection, stirring evenly, and adding 1 mol / L HCl to adjust the pH to 6.6-7.0.

[0031] Further preferably, the ratio of sodium citrate in the dissolving solution A to calcium chloride in the balancing solution A is 1:1-3.

[0032] Still more preferably, the ratio of sodium citrate in the dissolving solution A to calcium chloride in the balancing solution A is 1:2.

[0033] Specifically, the filtration flow rate in step S1 is no more than 1.5 L / min.

[0034] Preferably, the filtration flow rate in step S1 is 1.0-1.5 L / min.

[0035] Specifically, the S / D inactivation described in step S2 is to add S / D inactivation liquid to the filtrate A at a ratio of 100 mL of S / D inactivation liquid per liter of filtrate for inactivation.

[0036] Preferably, the inactivation temperature is maintained at 24°C and the inactivation is carried out for 6 hours.

[0037] Specifically, the two low-temperature ethanol precipitations described in step S3 include:

[0038] (1) adding ethanol to obtain a first precipitate, dissolving the first precipitate with dissolving solution B, filtering, and collecting the filtrate; (2) adding ethanol to the filtrate to obtain a second precipitate, dissolving the second precipitate with dissolving solution C, filtering, and obtaining a human fibrinogen stock solution.

[0039] Preferably, the dissolving solution B contains 0.85%-1.2% sodium citrate, 0.9%-1.1% sodium chloride, and 0.35%-0.4% arginine hydrochloride.

[0040] More preferably, the dissolving solution B contains 1.15% sodium citrate, 0.9% sodium chloride, and 0.37% arginine hydrochloride.

[0041] Preferably, the preparation method of the dissolving solution B is to dissolve sodium citrate, sodium chloride and arginine hydrochloride in water for injection, stir evenly, and add glycine-hydrochloric acid buffer with pH=3.6 to adjust the pH to 6.6-7.0.

[0042] Preferably, the ratio of the first precipitate to the dissolving solution B is 1:15-20 w / v.

[0043] Further preferably, the ratio of the first precipitate to the dissolving solution B is 1:15 w / v.

[0044] Preferably, the ethanol in step S3(1) is 45%-55% ethanol at -15 to -20°C.

[0045] More preferably, the low-temperature ethanol is 50% ethanol at -15°C.

[0046] More preferably, the volume ratio of ethanol to plasma is 10%, and the flow rate of adding ethanol is no more than 1.0 L / min.

[0047] Preferably, the dissolving solution C contains 1.35%-1.75% sodium citrate and 3.5%-4.0% arginine hydrochloride.

[0048] More preferably, the dissolving solution C contains 1.5% sodium citrate and 3.7% arginine hydrochloride.

[0049] Preferably, the preparation method of the dissolving solution C is to dissolve sodium citrate and arginine hydrochloride in water for injection, stir evenly, and add 1 mol / L HCl to adjust the pH to 6.8-7.0.

[0050] Preferably, the ratio of the second precipitate to the dissolving solution C is 1:5-6 w / v.

[0051] Further preferably, the ratio of the second precipitate to the dissolving solution C is 1:5 w / v.

[0052] Preferably, the filtration in step S3(1) is performed using a plate and frame filter.

[0053] Further preferably, the filter plate used in the plate and frame filter is a SUPRA EK 1P filter plate.

[0054] More preferably, the filter uses a dissolving liquid B equilibrium filter.

[0055] Preferably, the ethanol in step S3(2) is 45%-55% ethanol at -15 to -20°C.

[0056] More preferably, the low-temperature ethanol is 50% ethanol at -15°C.

[0057] More preferably, the volume ratio of ethanol to plasma is 10%, and the flow rate of adding ethanol is no more than 1.0 L / min.

[0058] Preferably, the filtration in step S3(2) is performed using a plate and frame filter.

[0059] Further preferably, the filter plate used in the plate and frame filter is a SUPRA EK 1P filter plate.

[0060] More preferably, the filter uses a dissolving liquid C equilibrium filter.

[0061] Preferably, the flow rate of the filtration in steps S3(1) and (2) is no greater than 1.5 L / min.

[0062] Further preferably, the flow rate of the filtration in steps S3(1) and (2) is 1.0-1.5 L / min.

[0063] Specifically, the preparation method further comprises:

[0064] S4, after adjusting the protein concentration, filtering, packaging, freeze-drying and capping to obtain human fibrinogen freeze-dried powder;

[0065] S5. Inactivate the virus by dry-heat method using human fibrinogen lyophilized powder to obtain the finished human fibrinogen preparation.

[0066] Preferably, the solution used to adjust the protein concentration in step S4 is dissolving solution C.

[0067] Preferably, the protein concentration is adjusted in step S4 to 2.5%-3.5%.

[0068] Preferably, the filtration in step S4 is performed using a 0.45 μm filter element.

[0069] Preferably, the freeze-drying process described in step S4 is as follows:

[0070] The products are put into the cabinet at room temperature, dropped to -6℃, and kept warm for 180 minutes for pre-freezing; dropped to -40℃, kept warm for 180 minutes for freezing; raised to -15℃, kept warm for 120 minutes for annealing; dropped to -45℃, kept warm for 360 minutes to continue freezing; start to vacuum to 10Pa, and maintain 10Pa±5Pa; raised to -40℃, kept warm for 60 minutes; raised to -15℃, kept warm for 120 minutes; raised to 0℃ for low-temperature sublimation, and kept warm for 2220 minutes; raised to 10℃ for vacuum drying, and kept warm for 120 minutes; raised to 33℃ for vacuum drying, and kept warm for 240 minutes; draw the ultimate vacuum, maintain 33℃ until the vacuum is qualified, and the maximum temperature of the product during the freeze-drying process shall not exceed 35℃.

[0071] Preferably, the dry heat method for inactivating the virus in step S5 is to inactivate the virus at a temperature of 99.5±5°C for 30 minutes.

[0072] Further preferably, the dry heat inactivation of the virus in step S5 is performed at 100° C. for 30 minutes.

[0073] In another aspect, the present invention provides human fibrinogen, which is prepared by the above-mentioned preparation method.

[0074] The beneficial effects of the present invention are:

[0075] (1) The purity of human fibrinogen prepared by the preparation method provided by the present invention is as high as 87.4%-98.3%.

[0076] (2) The human fibrinogen prepared by the preparation method provided by the present invention has a fast resolubility speed, which takes only 4-5 minutes to be resolubilized, and the resolubility solution is clear with only a slight opalescence. The coagulation time is short, which takes only 19-22 seconds. DETAILED DESCRIPTION

[0077] The technical solution of the present invention will be clearly and completely described below in conjunction with specific embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all of the embodiments; based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention.

[0078] The preparation method of the basic solution used in the present invention is as follows:

[0079] 1. pH 4.0 acetate buffer: Take 65.6g of anhydrous sodium acetate, add 244.9mL of glacial acetic acid, fully dissolve with appropriate amount of water for injection, add water for injection to make up to 1L and mix well;

[0080] 2. Glycine-HCl buffer solution with pH=3.6: Take 250mL 0.2mol / L glycine, add 25mL 0.2mol / L HCl, and dilute to 1L with water for injection.

[0081] 3. Preparation of S / D inactivation solution: S / D inactivation solution contains 10% Tween80 and 3.3% tributyl phosphate (TNBP). When preparing, first dissolve Tween80 with warm water for injection, and then add TNBP after it is completely dissolved. At this time, the inactivation solution will become clear. The temperature is controlled at 25°C and inactivated for 8 hours to obtain S / D inactivation solution.

[0082] Example 1

[0083] 1. Preparation of dissolving solution:

[0084] Preparation of Dissolving Solution A: Dissolving Solution A contains 1.85% sodium citrate and 0.9% sodium chloride. The preparation method of Dissolving Solution A is to dissolve sodium citrate and sodium chloride in water for injection, stir evenly, and add 1 mol / L HCl to adjust the pH to 6.6-7.0.

[0085] Preparation of balanced solution A: Balanced solution A contains 3.7% calcium chloride and 0.9% sodium chloride. The preparation method of balanced solution A is to dissolve calcium chloride and sodium chloride in water for injection, stir evenly, and add 1 mol / L HCl to adjust the pH to 6.6-7.0.

[0086] Preparation of Dissolving Solution B: Dissolving Solution B contains 1.15% sodium citrate, 0.9% sodium chloride, and 0.37% arginine hydrochloride. The preparation method of Dissolving Solution B is to dissolve sodium citrate, sodium chloride, and arginine hydrochloride in water for injection, stir evenly, and add glycine-hydrochloric acid buffer with pH=3.6 to adjust the pH to 6.6-7.0.

[0087] Preparation of Dissolving Solution C: Dissolving Solution C contains 1.5% sodium citrate and 3.7% arginine hydrochloride. The preparation method of Dissolving Solution C is to dissolve sodium citrate and arginine hydrochloride in water for injection, stir evenly, and add 1 mol / L HCl to adjust the pH to 6.8-7.0.

[0088] 2. Preparation method of human fibrinogen

[0089] Raw plasma collection and inspection shall be carried out strictly in accordance with the provisions of "Human Plasma for Blood Product Production" in Part III of the 2020 edition of the "Chinese Pharmacopoeia" and the requirements of the "Technical Guidelines for Implementing the Quarantine Management of Raw Plasma".

[0090] (1) Soak the raw plasma in 75% alcohol for 23 minutes, then rinse the surface with injection water, then break the bag of the raw plasma and transfer it to a melting tank, use 1037°C water to melt the plasma in an interlayer circulation, stop the circulation after melting, control the plasma temperature between 0.4°C, centrifuge and separate the cold precipitate, and transfer the plasma after removing the cold precipitate to a reaction tank for extraction and separation;

[0091] (2) Preparation of component I precipitate: The plasma temperature was controlled between 0 and 4°C, and pH 4.0 acetate buffer was added to adjust the pH to 6.8-7.2. 95% ethanol at -15°C was added to make the volume ratio of ethanol to plasma 10%. The flow rate of ethanol addition was 1 L / min, and the final temperature of the reaction solution was controlled at -1 to -3°C. After adding ethanol, the reaction solution was centrifuged continuously at 10,000 rpm to obtain component I precipitate;

[0092] (3) Dissolution and filtration of component I precipitate: Add dissolving solution I to the component I precipitate at a ratio of component I precipitate: dissolving solution A = 1:15 (w / v), control the temperature at 30-37°C, and stir for 1 hour to completely dissolve the component I precipitate to obtain component I precipitate dissolving solution. Install the K700 filter plate, use the balancing solution A to balance the filter, and after balancing for 20 minutes, start filtering the component I precipitate dissolving solution at a flow rate of 1.5 L / min to obtain filtrate A;

[0093] (4) S / D inactivation: 100 mL of S / D inactivation solution was added to filtrate A at a rate of 0.5 L / min under stirring, the temperature was maintained at 24° C., and the inactivation was performed for 6 h to obtain an inactivation solution;

[0094] (5) First precipitation: the inactivation solution was cooled to between 0 and 1°C, 50% ethanol at -15°C was added, the volume ratio of ethanol to plasma was 10%, the flow rate of ethanol addition was 1 L / min, the final temperature of the reaction solution was controlled at -1 to -3°C, and after the addition of ethanol, the solution was centrifuged continuously at 10,000 rpm to obtain the first precipitate;

[0095] (6) Dissolution and filtration of the first precipitate: Add solution B to the first precipitate at a ratio of first precipitate: solution B = 1:15 (w / v), stir for 1 hour, and control the temperature at 30-37°C to completely dissolve the first precipitate to obtain the first precipitate solution. Install the SUPRA EK 1P filter plate, and use solution B to balance the filter. After balancing for 20 minutes, start filtering the first precipitate solution at a flow rate of 1.5 L / min to obtain filtrate B;

[0096] (7) Second precipitation: Filtrate B was cooled to between 0 and 1°C, and 50% ethanol at -15°C was added to make the volume ratio of ethanol to plasma 10%. The flow rate of ethanol addition was 1 L / min, and the final temperature of the reaction solution was controlled at -1 to -3°C. After adding ethanol, the mixture was centrifuged continuously at 10,000 rpm to obtain a second precipitate.

[0097] (8) Preparation of human fibrinogen stock solution: Add the dissolving solution C to the second precipitate at a ratio of second precipitate: dissolving solution C = 1:5 (w / v), stir for 30 min, control the temperature at 30-37°C, and completely dissolve the second precipitate to obtain the second precipitate dissolving solution. Install the SUPRA EK 1P filter plate, use the dissolving solution C to balance the filter, and after balancing for 20 min, start filtering the second precipitate dissolving solution at a flow rate of 1.5 L / min to obtain the human fibrinogen stock solution;

[0098] (9) Liquid preparation: adjusting the protein concentration of human fibrinogen stock solution to 2.5% using dissolving solution C, and filtering using a 0.45 μm filter element to obtain a liquid preparation semi-finished product;

[0099] (10) Packaging and lyophilization: sterilize the semi-finished liquid preparation into 25 mL injection bottles, freeze-dry, and cap after lyophilization to obtain human fibrinogen lyophilized powder;

[0100] (11) Dry heat method for inactivating viruses: The human fibrinogen lyophilized powder is placed in a dry heat sterilizer and inactivated at 100° C. for 30 minutes to obtain a finished human fibrinogen preparation.

[0101] Example 2

[0102] 1. Preparation of dissolving solution:

[0103] Preparation of Dissolving Solution A: Dissolving Solution A contains 1.6% sodium citrate and 1.1% sodium chloride. The preparation method of Dissolving Solution A is to dissolve sodium citrate and sodium chloride in water for injection, stir evenly, and add 1 mol / L HCl to adjust the pH to 6.6-7.0.

[0104] Preparation of balanced solution A: Balanced solution A contains 3.2% calcium chloride and 1.1% sodium chloride. The preparation method of balanced solution A is to dissolve calcium chloride and sodium chloride in water for injection, stir evenly, and add 1 mol / L HCl to adjust the pH to 6.6-7.0.

[0105] Preparation of Dissolving Solution B: Dissolving Solution B contains 1.2% sodium citrate, 1.1% sodium chloride, and 0.35% arginine hydrochloride. The preparation method of Dissolving Solution B is to dissolve sodium citrate, sodium chloride, and arginine hydrochloride in water for injection, stir evenly, and add glycine-hydrochloric acid buffer with pH=3.6 to adjust the pH to 6.6-7.0.

[0106] Preparation of Dissolving Solution C: Dissolving Solution C contains 1.35% sodium citrate and 3.5% arginine hydrochloride. The preparation method of Dissolving Solution C is to dissolve sodium citrate and arginine hydrochloride in water for injection, stir evenly, and add 1 mol / L HCl to adjust the pH to 6.8-7.0.

[0107] 2. Preparation method of human fibrinogen

[0108] Raw plasma collection and inspection shall be carried out strictly in accordance with the provisions of "Human Plasma for Blood Product Production" in Part III of the 2020 edition of the "Chinese Pharmacopoeia" and the requirements of the "Technical Guidelines for Implementing the Quarantine Management of Raw Plasma".

[0109] (1) Soak the raw plasma in 75% alcohol for 2-3 minutes, then rinse the surface with injection water, then break the bag of the raw plasma and transfer it to a melting tank, use 10-37°C water for interlayer circulation melting, stop the circulation after melting, control the plasma temperature between 0-4°C, centrifuge and separate the cold precipitate, and transfer the plasma after removing the cold precipitate to a reaction tank for extraction and separation;

[0110] (2) Preparation of component I precipitate: The plasma temperature was controlled between 0 and 4°C, and pH 4.0 acetate buffer was added to adjust the pH to 6.8-7.2. 95% ethanol at -15°C was added to make the volume ratio of ethanol to plasma 10%. The flow rate of ethanol addition was 1 L / min, and the final temperature of the reaction solution was controlled at -1 to -3°C. After adding ethanol, the reaction solution was centrifuged continuously at 10,000 rpm to obtain component I precipitate;

[0111] (3) Dissolution and filtration of component I precipitate: Add dissolving solution I to the component I precipitate at a ratio of component I precipitate: dissolving solution A = 1:20 (w / v), control the temperature at 30-37°C, stir for 1 hour, and completely dissolve the component I precipitate to obtain component I precipitate dissolving solution. Install the K700 filter plate, use the balancing solution A to balance the filter, and after balancing for 20 minutes, start filtering the component I precipitate dissolving solution at a flow rate of 1.5 L / min to obtain filtrate A;

[0112] (4) S / D inactivation: 100 mL of S / D inactivation solution was added to filtrate A at a rate of 0.5 L / min under stirring, the temperature was maintained at 24° C., and the inactivation was performed for 6 h to obtain an inactivation solution;

[0113] (5) First precipitation: the inactivation solution was cooled to between 0 and 1°C, 50% ethanol at -15°C was added, the volume ratio of ethanol to plasma was 10%, the flow rate of ethanol addition was 1 L / min, the final temperature of the reaction solution was controlled at -1 to -3°C, and after the addition of ethanol, the solution was centrifuged continuously at 10,000 rpm to obtain the first precipitate;

[0114] (6) Dissolution and filtration of the first precipitate: Add solution B to the first precipitate at a ratio of first precipitate: solution B = 1:20 (w / v), stir for 1 hour, and control the temperature at 30-37°C to completely dissolve the first precipitate to obtain the first precipitate solution. Install the SUPRA EK 1P filter plate, use solution B to balance the filter, and after balancing for 20 minutes, start filtering the first precipitate solution at a flow rate of 1.5 L / min to obtain filtrate B;

[0115] (7) Second precipitation: Filtrate B was cooled to between 0 and 1°C, and 50% ethanol at -15°C was added to make the volume ratio of ethanol to plasma 10%. The flow rate of ethanol addition was 1 L / min, and the final temperature of the reaction solution was controlled at -1 to -3°C. After adding ethanol, the mixture was centrifuged continuously at 10,000 rpm to obtain a second precipitate.

[0116] (8) Preparation of human fibrinogen stock solution: Add the dissolving solution C to the second precipitate at a ratio of second precipitate: dissolving solution C = 1:6 (w / v), stir for 30 min, control the temperature at 30-37°C, and completely dissolve the second precipitate to obtain the second precipitate dissolving solution. Install the SUPRA EK 1P filter plate, use the dissolving solution C to balance the filter, and after balancing for 20 min, start filtering the second precipitate dissolving solution at a flow rate of 1.5 L / min to obtain the human fibrinogen stock solution;

[0117] (9) Liquid preparation: adjusting the protein concentration of human fibrinogen stock solution to 2.5% using dissolving solution C, and filtering using a 0.45 μm filter element to obtain a liquid preparation semi-finished product;

[0118] (10) Packaging and lyophilization: sterilize the semi-finished liquid preparation into 25 mL injection bottles, freeze-dry, and cap after lyophilization to obtain human fibrinogen lyophilized powder;

[0119] (11) Dry heat method for inactivating viruses: The human fibrinogen lyophilized powder is placed in a dry heat sterilizer and inactivated at 100° C. for 30 minutes to obtain a finished human fibrinogen preparation.

[0120] Example 3

[0121] The difference between Example 3 and Example 1 is that the preparation method of the dissolving solution is different.

[0122] Preparation of Dissolving Solution A: Dissolving Solution A contains 2.0% sodium citrate and 0.9% sodium chloride. The preparation method of Dissolving Solution A is to dissolve sodium citrate and sodium chloride in water for injection, stir evenly, and add 1 mol / L HCl to adjust the pH to 6.6-7.0.

[0123] Preparation of balanced solution A: Balanced solution A contains 4.0% calcium chloride and 0.9% sodium chloride. The preparation method of balanced solution A is to dissolve calcium chloride and sodium chloride in water for injection, stir evenly, and add 1 mol / L HCl to adjust the pH to 6.6-7.0.

[0124] Preparation of Dissolving Solution B: Dissolving Solution B contains 0.85% sodium citrate, 0.9% sodium chloride, and 0.4% arginine hydrochloride. The preparation method of Dissolving Solution B is to dissolve sodium citrate, sodium chloride, and arginine hydrochloride in water for injection, stir evenly, and add glycine-hydrochloric acid buffer with pH=3.6 to adjust the pH to 6.6-7.0.

[0125] Preparation of Dissolving Solution C: Dissolving Solution C contains 1.75% sodium citrate and 4.0% arginine hydrochloride. The preparation method of Dissolving Solution C is to dissolve sodium citrate and arginine hydrochloride in water for injection, stir evenly, and add 1 mol / L HCl to adjust the pH to 6.8-7.0.

[0126] Comparative Example 1

[0127] The only difference between Comparative Example 1 and Example 1 is that the balancing solution A in step (3) is replaced by the dissolving solution A.

[0128] Comparative Example 2

[0129] The difference between Comparative Example 2 and Example 1 is that the preparation method of dissolving solution B is different.

[0130] Preparation of Dissolving Solution B: Dissolving Solution B contains 1.2% sodium citrate, 1.1% sodium chloride, and 0.35% arginine hydrochloride. The preparation method of Dissolving Solution B is to dissolve sodium citrate, sodium chloride, and arginine hydrochloride in water for injection, stir evenly, and add 1 mol / L HCl to adjust the pH to 6.6-7.0.

[0131] Comparative Example 3

[0132] The difference between Comparative Example 3 and Example 1 is only that:

[0133] 1. Replace the balancing solution A in step (3) with the dissolving solution A.

[0134] 2. The preparation method of dissolving solution B is different.

[0135] Preparation of Dissolving Solution B: Dissolving Solution B contains 1.2% sodium citrate, 1.1% sodium chloride, and 0.35% arginine hydrochloride. The preparation method of Dissolving Solution B is to dissolve sodium citrate, sodium chloride, and arginine hydrochloride in water for injection, stir evenly, and add 1 mol / L HCl to adjust the pH to 6.6-7.0.

[0136] Comparative Example 4

[0137] The difference between Comparative Example 4 and Example 1 is that the preparation method of dissolving solution A is different.

[0138] Preparation of Dissolving Solution A: Dissolving Solution A contains 1.4% sodium citrate, 0.9% sodium chloride, and 1.1% sucrose. The preparation method of Dissolving Solution A is to dissolve sodium citrate, sodium chloride, and sucrose in water for injection, stir evenly, and add 1 mol / L HCl to adjust the pH to 6.6-7.0.

[0139] Comparative Example 5

[0140] The difference between Comparative Example 5 and Example 1 is that the preparation method of the dissolving solution C is different.

[0141] Preparation of Dissolving Solution C: Dissolving Solution C contains 0.9% sodium citrate and 0.36% arginine hydrochloride. The preparation method of Dissolving Solution C is to dissolve sodium citrate and arginine hydrochloride in water for injection, stir evenly, and add 1 mol / L HCl to adjust the pH to 6.8-7.0.

[0142] Experimental Example 1

[0143] The pH, purity, coagulation activity, reconstitution time, visible foreign matter, stability, and water content of Examples 1-3 and Comparative Examples 1-5 were measured. The measurement method is as follows:

[0144] pH determination: refer to General Chapter 0631 of the 2020 edition of the Chinese Pharmacopoeia.

[0145] Purity determination: refer to the first method of general chapter 0731 of the 2020 edition of the Chinese Pharmacopoeia.

[0146] Coagulation activity: dilute the thrombin solution to 3IU / mL and the test solution to 3mg / mL with 0.9% sodium chloride solution. Add an appropriate amount of thrombin solution (3IU / mL) preheated to 37°C to the reaction tube, then add an equal amount of the test solution (3mg / mL), shake well. Set at 37°C and record the coagulation time using an automatic detection instrument.

[0147] Determination of reconstitution time: Equilibrate the test sample to 37°C, add 37°C sterile water for injection according to the labeled amount (25mL), shake gently, and it should be completely dissolved within 30 minutes.

[0148] Determination of visible foreign matter: refer to General Chapter 0904 of the 2020 edition of the Chinese Pharmacopoeia.

[0149] Stability determination: After re-dissolving the test sample, keep it in a 30-37℃ water bath for 60 minutes and observe whether there is clot or fibrin precipitation.

[0150] Moisture determination: refer to General Chapter 0832 of the 2020 edition of the Chinese Pharmacopoeia.

[0151] The test results of Examples 1-3 are shown in Table 1, and the test results of Comparative Examples 1-5 are shown in Table 2.

[0152] Table 1 Test results of Examples 1-3

[0153]

[0154] Table 2 Comparative Examples 1-5 Test Results

[0155]

[0156] The above detailed description is a specific description of one of the feasible embodiments of the present invention, and the embodiment is not intended to limit the scope of the present invention. It should be pointed out that any equivalent implementation or change that does not deviate from the present invention should be included in the scope of the technical solution of the present invention. Therefore, the protection scope of the patent of the present invention shall be based on the attached requirements.

Claims

1. A method for preparing human fibrinogen, characterized in that: The preparation method comprises the following steps: S1, the precipitate of component I is dissolved and filtered to obtain filtrate A; S2, filtrate A is inactivated by S / D to obtain an inactivated solution; S3, the inactivated solution is precipitated twice with low-temperature ethanol to prepare a human fibrinogen stock solution; The dissolving solution used in step S1 is dissolving solution A, and the dissolving solution A contains 1.6%-2.0% sodium citrate and 0.9%-1.1% sodium chloride; The filtration described in step S1 is performed by using a plate-and-frame filter, the balancing solution of the plate-and-frame filter is balancing solution A, and the balancing solution A contains 3.2%-4.0% calcium chloride and 0.9%-1.1% sodium chloride; the two low-temperature ethanol precipitations described in step S3 include adding ethanol to obtain a first precipitate, dissolving the first precipitate with a dissolving solution B and filtering, collecting the filtrate, adding ethanol to the filtrate to obtain a second precipitate, dissolving the second precipitate with a dissolving solution C, and filtering to obtain a human fibrinogen stock solution; The dissolving solution B contains 0.85%-1.2% sodium citrate, 0.9%-1.1% sodium chloride, and 0.35%-0.4% arginine hydrochloride; the preparation method of the dissolving solution B is to dissolve sodium citrate, sodium chloride, and arginine hydrochloride in water for injection, stir evenly, and add glycine-hydrochloric acid buffer with pH = 3.6 to adjust the pH; The dissolving solution C contains 1.35%-1.75% sodium citrate and 3.5%-4.0% arginine hydrochloride; The ratio of sodium citrate in the dissolving solution A to calcium chloride in the balancing solution A is 1:

2.

2. The preparation method according to claim 1, characterized in that: The ratio of component I precipitate to dissolving solution A described in step S2 is 1:15-20 w / v.

3. The preparation method according to claim 1, characterized in that: The ratio of the first precipitate to the dissolving solution B in step S3 is 1:15-20 w / v.

4. The preparation method according to claim 1, characterized in that: The ratio of the second precipitate to the dissolving solution C in step S3 is 1:5-6 w / v.

5. The preparation method according to claim 1, characterized in that: The preparation method further comprises: S4, after adjusting the protein concentration, filtering, packaging, freeze-drying and capping to obtain human fibrinogen freeze-dried powder; S5. Inactivate the virus by dry-heat method using human fibrinogen lyophilized powder to obtain the finished human fibrinogen preparation.

6. The preparation method according to claim 5, characterized in that: The protein concentration adjustment described in step S4 is to use dissolution solution C to adjust the protein concentration to 2.5%-3.5%.

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