Separation and purification method of fibrinogen with low impure protein
By combining PEG precipitation, ethanol precipitation, and glycine precipitation, the problem of high impurity protein content in fibrinogen purification was solved, improving product purity and activity, simplifying the operation steps, and reducing costs.
Patent Information
- Application Number
- CN202511773045.7
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-11-28
- Publication Date
- 2026-02-06
AI Technical Summary
Existing methods for purifying fibrinogen suffer from problems such as high levels of impurities, low purity, complex procedures, and high costs, which affect product quality and clinical applications.
The formulation of component I solution was screened, and S/D virus inactivation was performed by low temperature ethanol combined with dry heat method. Through a combination of purification steps of PEG precipitation, ethanol precipitation and glycine precipitation, impurities such as plasminogen and fibronectin were removed to improve purity and activity.
It effectively removes plasminogen and fibronectin, improves the purity and coagulation activity of fibrinogen, simplifies operation steps, and reduces production costs.
Smart Images
Figure CN121471340A_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of blood products technology, specifically relating to a method for separating and purifying fibrinogen, a low-contamination protein. Background Technology
[0002] Human fibrinogen (Fg) is a protein component in blood plasma, with a concentration as high as 2-4 g / L. It plays a crucial role in the coagulation system. The final stage of coagulation involves fibrinogen being converted into fibrin monomers by thrombin, which then cross-link with coagulation factor XIII to form an insoluble fibrin clot, achieving hemostasis. Patients with congenital fibrinogen deficiency or hypofibrinogenemia and those with acquired fibrinogen deficiency due to disease are prone to coagulation dysfunction; fibrinogen supplementation is currently the only reliable treatment.
[0003] Human fibrinogen is a blood product with national standards, included in the 2025 edition of the Chinese Pharmacopoeia, Volume III. It has been used clinically in China for many years. The raw materials for extracting human fibrinogen mainly include two types: component I (F1) precipitation produced by low-temperature ethanol processing and plasma cryoprecipitate. Currently, human fibrinogen can be produced on a large scale both domestically and internationally. Methods for extracting and purifying human fibrinogen include low-temperature ethanol precipitation, glycine precipitation, and chromatography; however, the yield and quality of the products vary significantly.
[0004] Patent CN118344463B discloses a method for preparing human fibrinogen. After component I is precipitated and dissolved, it is first inactivated by S / D, and then the stock solution is prepared by two low-temperature ethanol precipitations. The purity of the product prepared by this method is 87.4% to 98.3%, the reconstitution time is 4 to 5 min, and the coagulation time is 19 to 22 s. However, the determination of the content of impurities in the product is not involved.
[0005] Existing methods for preparing fibrinogen still have several drawbacks. First, in traditional fibrinogen purification processes, the S / D inactivation step can lead to the activation and degradation of fibrinogen, affecting product quality and stability. Second, the final product often contains high levels of impurities such as plasminogen, fibronectin, and heparin. These impurities not only affect product purity but may also cause adverse reactions during clinical use. Furthermore, existing purification methods often require complex procedures and expensive equipment, increasing production costs and technical complexity.
[0006] Therefore, there is an urgent need to develop a fibrinogen separation and purification method that can effectively reduce the content of impurities, improve the purity and activity of fibrinogen, and simplify the operation steps to meet the needs of clinical applications. Summary of the Invention
[0007] The purpose of this invention is to design a preparation process for extracting human fibrinogen from component I. This process involves screening the formulation of the solution for component I, inactivating the virus with S / D at low temperature using ethanol, and preparing the product using a dry heat method. This process can yield a high-purity fibrinogen product with low content of impurities such as plasminogen and fibronectin.
[0008] The technical solution adopted by this invention to solve its technical problem is: a method for separating and purifying fibrinogen, which contains low-contamination proteins, comprising the following steps:
[0009] (1) Raw material preparation: Take freshly prepared component I or frozen component I. If frozen component I is used, first cut frozen component I into small cubes of about 1 cm, then thaw it to obtain FI precipitate.
[0010] (2) Dissolution and filtration of FI precipitate: Take the FI precipitate from step (1) and add 10 to 12 times the weight of the precipitate to dissolution solution A. Control the dissolution temperature to 20 to 30°C. After dissolving for 0.5 to 2 hours, filter with a 30SP filter cartridge and collect the filtrate A.
[0011] (3) Polyethylene glycol (PEG) purification and separation: First, adjust the pH of the filtrate A obtained in step (2) to 6.8-7.2, add PEG solution to filtrate A to a final concentration of 5%, stir for 0.5-1h at 10-20℃, centrifuge, with a maximum processing capacity of ≤2.0Kg per minute, and control the temperature of the centrifuged liquid at 10-17℃, and collect the PEG precipitate; add dissolving solution B at 10-15 times the weight of the PEG precipitate, stir at 20-30℃, avoid generating bubbles and precipitate accumulation during stirring, filter after dissolving for 1-2 hours, filter with a 60SP filter cartridge, and collect filtrate B;
[0012] (4) S / D inactivation: Adjust the pH of the filtrate B from step (3) to 6.8-7.2. Under continuous stirring, slowly add the prepared S / D solution at a mass flow rate of ≤3 kg / min until the content of polysorbate 80 is 1% and the content of tributyl phosphate is 0.3%. Stop adding the S / D solution, stir it to make it uniform, filter it, and transfer the filtrate to a special virus inactivation tank. Inactivate at 24-26℃ for no less than 6 hours to obtain the inactivated solution.
[0013] (5) Ethanol purification and separation: Adjust the pH of the inactivation solution in step (4) to 6.8-7.2 and cool it to 0-2℃. Slowly add 50% ethanol solution below -15℃ until the alcohol content is 8%. After adding the ethanol, cool it to -2.5-0℃. Stir for 40-60 min and then centrifuge. The maximum processing capacity is ≤2.0Kg per minute. The temperature of the centrifuged liquid is controlled at -2.5-0℃. Collect the ethanol precipitate. Dissolve the ethanol precipitate with 12-17 times the amount of dissolving solution B at 20-30℃ for 1-2 hours. Avoid the generation of bubbles and the accumulation of precipitate during the dissolution process. Filter the dissolved ethanol precipitate with a 60SP filter cartridge to obtain filtrate C.
[0014] (6) Glycine purification and separation: Adjust the pH of the filtrate C from step (5) to 7.1-7.3, then add glycine to a final concentration of 1.8 mol / L. After it is fully dissolved, cool the solution to 2-10℃, stir for at least 60 min, and centrifuge. The maximum processing capacity per minute is ≤2.0 kg. The temperature of the centrifuged liquid is controlled at 0-4℃. Collect the glycine precipitate. Dissolve the glycine precipitate with 4-8 times the amount of dissolving solution C at room temperature for 1-2 h. Avoid generating bubbles and precipitate accumulation during the dissolution process. Filter the dissolved glycine precipitate with a 0.45 μm filter and collect the filtrate D.
[0015] (7) Ultrafiltration dialysis: The filtrate D from step (6) is dialyzed 3 to 4 times by constant weight with the dissolving solution C, and then concentrated by ultrafiltration to a protein concentration of more than 30 g / L to obtain the original solution;
[0016] (8) Preparation: Based on the protein content of the stock solution in step (7) and the target protein amount per unit dose, calculate the volume of the final preparation required. Dilute the stock solution to 25-28 g / L with dialysis buffer, stir evenly, confirm that the pH value is 6.8-7.2, and obtain the semi-finished product after sterilization by filtration with a sterile filter cartridge.
[0017] (9) Finished product: The semi-finished product is packaged and freeze-dried. The freeze-dried product is then capped and sealed, and then subjected to dry heat inactivation treatment at 100±1℃ for 30 minutes to obtain the finished product.
[0018] As a preferred embodiment of the present invention, the formulation of the solution A includes: sodium citrate concentration of 100.0 mmol / L, lysine hydrochloride concentration of 49.8 mmol / L, sodium heparin concentration of 6 IU / mL, water for injection as solvent, and pH value of 6.8-7.0.
[0019] As a preferred embodiment of the present invention, the formulation of solution B includes: sodium citrate concentration of 51.0 mmol / L, sodium chloride concentration of 153.8 mmol / L, lysine hydrochloride concentration of 19.2 mmol / L, sucrose concentration of 29.2 mmol / L, water for injection as solvent, and pH value of 6.8-7.2.
[0020] As a preferred embodiment of the present invention, the formulation of the solution C includes: sodium citrate concentration of 34.0 mmol / L, sodium glutamate concentration of 11.8 mmol / L, arginine hydrochloride concentration of 94.9 mmol / L, water for injection as solvent, and pH value of 6.8-7.2.
[0021] As a preferred embodiment of the present invention, the S / D reagent formulation includes: 11% polysorbate 80 and 3.3% tributyl phosphate.
[0022] As a preferred technical solution of the present invention, in step (4), the temperature of the virus inactivation tank is controlled at 24-26°C and the S / D virus inactivation time is ≥6h.
[0023] As a preferred technical solution of the present invention, in step (7), the ultrafiltration process has an inlet pressure of ≤3.0 bar and a reflux pressure of ≤1.0 bar.
[0024] The beneficial effects of adopting the above plan are as follows:
[0025] (1) The main steps in the purification process include component I dissolution and filtration + PEG precipitation + S / D inactivation + ethanol precipitation + glycine precipitation. Compared with the traditional component I dissolution + S / D inactivation + ethanol precipitation + glycine precipitation, the first step of PEG precipitation removes most of the coagulation factors II, VII, IX, X and plasminogen and other impurities, preventing the activation and degradation of fibrinogen when S / D inactivation is performed at 24-26℃. The plasminogen removal rate can reach 93%, and the fibronectin removal rate can reach 74%. Ethanol precipitation + glycine precipitation can effectively remove fibronectin (a high content of fibronectin will affect the freeze-drying and reconstitution time of the product) than simple ethanol precipitation + ethanol precipitation. The plasminogen activity in the finished product is less than 3.8%, the fibronectin content can be less than 0.2 g / L, and the heparin content can be less than 0.2 IU / ml.
[0026] (2) Separation and purification by PEG purification-S / D virus inactivation-ethanol precipitation-glycine precipitation can prevent the activation and degradation of fibrinogen by impurities during S / D inactivation, and can improve product purity and coagulation activity.
[0027] (3) By screening the formulation of the dissolving solution of component I, plasminogen and fibrinogen (fibrin) can be dissociated, and plasminogen can be removed to a great extent by combining with the subsequent PEG precipitation.
[0028] (4) Plasminogen can degrade fibrinogen or fibrin. Therefore, adding a certain concentration of lysine hydrochloride to the solution A can competitively bind to the glutamic acid residues of fibrinogen or fibrin. During the separation process, plasminogen and fibrinogen can be prevented from precipitating together, thus achieving effective separation and inhibiting the degradation of fibrinogen or fibrin. Attached Figure Description
[0029] Figure 1 The results of the finished product testing of this invention;
[0030] Figure 2 These are the test results of the intermediate products in Examples 1-3 of the present invention;
[0031] Figure 3 The test results are for Comparative Examples 1-4 of the present invention compared with Example 1. Detailed Implementation
[0032] The technical solution of the present invention will now be clearly and completely described with reference to the accompanying drawings and embodiments. Obviously, the described embodiments are merely some embodiments of the present invention, and not all embodiments. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are within the scope of protection of the present invention.
[0033] In this invention, Example 1 uses the complete preparation steps, Example 2 omits the PEG purification and separation before the S / D inactivation step, and Example 3 performs two ethanol purification and separation steps. The results of the intermediate and finished products of Examples 1-3 are measured.
[0034] Comparative Examples 1-4 were added. The only difference between Comparative Examples 1-4 and Example 1 was the concentration of lysine hydrochloride in solution A. After dissolving component I, Comparative Examples 1-4 were tested for coagulation activity and plasminogen activity.
[0035] The formulation of solution A includes: sodium citrate concentration of 100.0 mmol / L, lysine hydrochloride concentration of 49.8 mmol / L, sodium heparin concentration of 6 IU / mL, water for injection as solvent, and pH value of 6.8–7.0.
[0036] The formulation of solution B includes: sodium citrate concentration of 51.0 mmol / L, sodium chloride concentration of 153.8 mmol / L, lysine hydrochloride concentration of 19.2 mmol / L, sucrose concentration of 29.2 mmol / L, water for injection as solvent, and pH value of 6.8–7.2.
[0037] The formulation of solution C includes: sodium citrate concentration of 34.0 mmol / L, monosodium glutamate concentration of 11.8 mmol / L, arginine hydrochloride concentration of 94.9 mmol / L, water for injection as solvent, and pH value of 6.8–7.2.
[0038] The S / D reagent formulation includes: 11% polysorbate 80 and 3.3% tributyl phosphate.
[0039] Example 1
[0040] This embodiment provides a method for isolating and purifying fibrinogen, which contains low-contamination proteins, and includes the following steps:
[0041] (1) Raw material preparation
[0042] Take freshly prepared component I or frozen component I. If frozen component I is used, first cut frozen component I into small cubes of about 1 cm, then thaw it to obtain FI precipitate. Component I is a component containing fibrinogen that is separated from plasma.
[0043] (2) Dissolution and filtration of FI precipitate
[0044] Take 1 kg of FI precipitate from step (1), add 10 kg of dissolving solution A, control the dissolving temperature at 25°C, and after dissolving for 1 hour, filter with a 30 SP filter element to obtain 11 kg of filtrate A.
[0045] (3) Purification and separation
[0046] First, adjust the pH of the 11 kg filtrate A obtained in step (2) to 7.0. Then, add 2.079 kg of PEG solution to filtrate A to a final concentration of 5%. After stirring for 45 min at 15°C, centrifuge at a maximum processing capacity of ≤2.0 kg per minute and control the temperature of the centrifuged liquid at 13.5°C. Collect 0.8 kg of PEG precipitate. Add 8 kg of dissolving solution B at 10 times the weight of the PEG precipitate. Stir at 25°C at a stirring speed of 100 rpm to avoid the generation of bubbles and the accumulation of precipitate. After dissolving for 1.5 hours, filter using a 60 SP filter cartridge and collect 8.8 kg of filtrate B.
[0047] (4) S / D inactivation
[0048] Adjust the pH of the 8.8 kg filtrate B from step (3) to 7.0. While stirring continuously, slowly add 0.88 kg of the prepared S / D solution at a mass flow rate of ≤3 kg / min until the content of polysorbate 80 is 1% and the content of tributyl phosphate is 0.3%. Stop adding the S / D solution, stir it to make it uniform, filter it, and transfer the filtrate to a special virus inactivation tank to obtain 9.68 kg of inactivation solution.
[0049] (5) Ethanol purification and separation
[0050] Adjust the pH of the 9.68 kg inactivation solution in step (4) to 7.0 and cool it to 1℃. Slowly add 1.71 kg of 50% ethanol solution below -15℃ until the alcohol content is 8%. After adding the alcohol, cool it to -1.25℃, stir for 50 min and centrifuge. The maximum processing capacity is ≤2.0 kg per minute. The temperature of the centrifuged liquid is controlled at -1.25℃. Collect 0.8 kg of ethanol precipitate. Dissolve the ethanol precipitate with 15 times the amount of 12 kg of dissolving solution B at 25℃ for 1.5 hours. Stir at 100 rpm during the dissolution process to avoid the generation of bubbles and the accumulation of precipitate. Filter the dissolved ethanol precipitate with a 60 SP filter cartridge to obtain 12.8 kg of filtrate C.
[0051] (6) Glycine purification and separation
[0052] Adjust the pH of the 12.8 kg filtrate C from step (5) to 7.2, then add 1729.61 g glycine to a final concentration of 1.8 mol / L. After it is fully dissolved, cool the solution to 6°C, stir for at least 60 min, and then centrifuge. The maximum processing capacity is ≤2.0 kg per minute. The temperature of the centrifuged liquid is controlled at 2°C. Collect 1 kg of glycine precipitate. Dissolve the glycine precipitate with 4 times the amount of 4 kg of dissolving solution C at 25°C for 1.5 h. During the dissolution process, the stirring speed is 100 rpm to avoid the generation of bubbles and the accumulation of precipitate. Filter the dissolved glycine precipitate with a 0.45 μm filter and collect 5 kg of filtrate D.
[0053] (7) Ultrafiltration dialysis
[0054] The filtrate D from step (6) was dialyzed three times by constant weight using 10 kg of dissolving solution C, and then concentrated by ultrafiltration to a protein concentration of more than 30 g / L to obtain 3.5 L of stock solution.
[0055] (8) Preparation
[0056] Based on the protein content of the stock solution of 30 g / L and the target protein amount per unit dose in step (7), the required volume of the final preparation was calculated. The stock solution was diluted to 26.5 g / L with 0.46L dialysis buffer, stirred evenly, and the pH value was adjusted to 7.0. After sterilization and filtration with a sterile filter cartridge, 3.96L of semi-finished product was obtained.
[0057] (9) Finished product
[0058] The semi-finished product is packaged and freeze-dried. The freeze-dried product is then subjected to dry heat inactivation treatment at 100±1℃ for 30 minutes. The treated product is then capped and sealed to obtain the finished product.
[0059] Example 2
[0060] This embodiment provides a method for isolating and purifying fibrinogen, which contains low-contamination proteins, and includes the following steps:
[0061] (1) Raw material preparation
[0062] Take freshly prepared component I or frozen component I. If frozen component I is used, first cut frozen component I into small cubes of about 1 cm, then thaw it to obtain FI precipitate. Component I is a component containing fibrinogen that is separated from plasma.
[0063] (2) Dissolution and filtration of FI precipitate
[0064] Take 1 kg of FI precipitate from step (1), add 10 kg of dissolving solution A, control the dissolving temperature at 25°C, and after dissolving for 1 hour, filter with a 30 SP filter element to obtain 11 kg of filtrate A.
[0065] (3) S / D inactivation
[0066] Adjust the pH of the 11 kg filtrate A from step (2) to 7.0. While stirring continuously, slowly add 1.1 kg of the prepared S / D solution at a mass flow rate of ≤3 kg / min until the content of polysorbate 80 is 1% and the content of tributyl phosphate is 0.3%. Stop adding the S / D solution, stir it to make it uniform, filter it, and transfer the filtrate to a special virus inactivation tank to obtain 12.1 kg of inactivation solution.
[0067] (4) Ethanol purification and separation
[0068] Adjust the pH of the 12.1 kg inactivation solution in step (3) to 7.0 and cool it to 1℃. Slowly add 2.14 kg of 50% ethanol solution below -15℃ to the alcohol content of 8%. After adding the alcohol, cool it to -1.25℃, stir for 50 min and centrifuge. The maximum processing capacity is ≤2.0 kg per minute. The temperature of the centrifuged liquid is controlled at -1.25℃. Collect 0.7 kg of ethanol precipitate. Dissolve the ethanol precipitate with 15 times the amount of 10.5 kg of dissolving solution B at 25℃ for 1.5 hours. Stir at 100 rpm during the dissolution process to avoid the generation of bubbles and the accumulation of precipitate. Filter the dissolved ethanol precipitate with a 60 SP filter cartridge to obtain 11.2 kg of filtrate C.
[0069] (5) Glycine purification and separation
[0070] Adjust the pH of the 11.2 kg filtrate C from step (4) to 7.2, then add 1513.4 g of glycine to a final concentration of 1.8 mol / L. After it is fully dissolved, cool the solution to 6°C, stir for at least 60 min, and then centrifuge. The maximum processing capacity is ≤2.0 kg per minute. The temperature of the centrifuged liquid is controlled at 2°C. Collect 0.8 kg of glycine precipitate. Dissolve the glycine precipitate with 4 times the amount of 3.2 kg of dissolving solution C at 25°C for 1.5 h. During the dissolution process, the stirring speed is 100 rpm to avoid the generation of bubbles and the accumulation of precipitate. Filter the dissolved glycine precipitate with a 0.45 μm filter and collect 4 kg of filtrate D.
[0071] (7) Ultrafiltration dialysis
[0072] The filtrate D from step (6) was dialyzed three times by constant weight using 9 kg of dissolving solution C, and then concentrated by ultrafiltration to a protein concentration of more than 30 g / L to obtain 2.9 L of stock solution.
[0073] (8) Preparation
[0074] Based on the protein content of the stock solution of 30 g / L in step (7) and the target protein amount per unit dose, the required volume of the final preparation was calculated. The stock solution was diluted to 26.5 g / L with 0.38 L dialysis buffer, stirred evenly, and the pH value was adjusted to 7.0. After sterilization and filtration with a sterile filter cartridge, 3.28 L of semi-finished product was obtained.
[0075] (9) Finished product
[0076] The semi-finished product is packaged and freeze-dried. The freeze-dried product is then subjected to dry heat inactivation treatment at 100±1℃ for 30 minutes. The treated product is then capped and sealed to obtain the finished product.
[0077] Example 3
[0078] This embodiment provides a method for isolating and purifying fibrinogen, which contains low-contamination proteins, and includes the following steps:
[0079] (1) Raw material preparation
[0080] Take freshly prepared component I or frozen component I. If frozen component I is used, first cut frozen component I into small cubes of about 1 cm, then thaw it to obtain FI precipitate. Component I is a component containing fibrinogen that is separated from plasma.
[0081] (2) Dissolution and filtration of FI precipitate
[0082] Take 1 kg of FI precipitate from step (1), add 10 kg of dissolving solution A, control the dissolving temperature at 25°C, and after dissolving for 1 hour, filter with a 30 SP filter element to obtain 11 kg of filtrate A;
[0083] (3) PEG purification and separation
[0084] First, adjust the pH of the 11 kg filtrate A obtained in step (2) to 7.0. Then, add 2.079 kg of PEG solution to filtrate A to a final concentration of 5%. After stirring for 45 min at 15°C, centrifuge at a maximum processing capacity of ≤2.0 kg per minute. Control the centrifuged liquid temperature at 13.5°C and collect 0.8 kg of PEG precipitate. Add 8 kg of dissolving solution B at 10 times the weight of the PEG precipitate. Stir at 25°C at a stirring speed of 100 rpm to avoid generating bubbles and precipitate accumulation. After dissolving for 1.5 hours, filter using a 60 SP filter cartridge and collect 8.8 kg of filtrate B.
[0085] (4) S / D inactivation
[0086] Adjust the pH of the 8.8 kg filtrate B from step (3) to 7.0. While stirring continuously, slowly add 0.88 kg of the prepared S / D solution at a mass flow rate of ≤3 kg / min until the content of polysorbate 80 is 1% and the content of tributyl phosphate is 0.3%. Stop adding the S / D solution, stir it to make it uniform, filter it, and transfer the filtrate to a special virus inactivation tank to obtain 9.68 kg of inactivation solution.
[0087] (5) Ethanol purification and separation
[0088] Adjust the pH of the 9.68 Kg inactivation solution in step (4) to 7.0 and cool it to 1℃. Slowly add 1.71 Kg of 50% ethanol solution below -15℃ to the alcohol content of 8%. After adding the alcohol, cool it to -1.25℃. After stirring for 50 min, centrifuge with a maximum processing capacity of ≤2.0Kg per minute. Control the temperature of the centrifuged liquid at -1.25℃ and collect 0.8Kg of ethanol precipitate. Dissolve the ethanol precipitate with 15 times the amount of 12 Kg dissolving solution B at 25℃ for 1.5 hours. Stir at 100rpm during the dissolution process to avoid the generation of bubbles and the accumulation of precipitate. Filter the dissolved ethanol precipitate with a 60SP filter cartridge to obtain 12.8 Kg of filtrate C.
[0089] (6) Ethanol purification and separation
[0090] Adjust the pH of the 12.8 kg filtrate C from step (5) to 7.0 and cool it to 1℃. Slowly add 2.27 kg of 50% ethanol solution below -15℃ until the alcohol content is 8%. After adding the alcohol, cool it to -1.25℃, stir for 50 min and centrifuge. The maximum processing capacity is ≤2.0 kg per minute. The temperature of the centrifuged liquid is controlled at -1.25℃. Collect 0.64 kg of ethanol precipitate. Dissolve the ethanol precipitate with 15 times 9.6 kg of dissolving solution B at 25℃ for 1.5 hours. Stir at 100 rpm during the dissolution process to avoid the generation of bubbles and the accumulation of precipitate. Filter the dissolved ethanol precipitate with a 60SP filter cartridge to obtain 10.24 kg of filtrate.
[0091] (7) Glycine purification and separation
[0092] Adjust the pH of the 10.24 kg filtrate from step (6) to 7.2, then add 1460.56 g of glycine to a final concentration of 1.8 mol / L. After it is fully dissolved, cool the solution to 6°C, stir for at least 60 min, and then centrifuge. The maximum processing capacity per minute is ≤2.0 kg. The temperature of the centrifuged liquid is controlled at 0-4°C. Collect 0.7 kg of glycine precipitate. Dissolve the glycine precipitate with 4 times the amount of 2.8 kg of dissolving solution C at room temperature for 1.5 h. During the dissolution process, the stirring speed is 100 rpm to avoid the generation of bubbles and the accumulation of precipitate. Filter the dissolved glycine precipitate with a 0.45 μm filter and collect 3.5 kg of filtrate D.
[0093] (8) Ultrafiltration dialysis
[0094] The filtrate D from step (7) was dialyzed three times by constant weight using 7.9 kg of dissolving solution C, and then concentrated by ultrafiltration to a protein concentration of more than 30 g / L to obtain 2.58 L of stock solution.
[0095] (9) Preparation
[0096] Based on the protein content of the stock solution of 30 g / L and the target protein amount per unit dose in step (8), the required volume of the final preparation was calculated. The stock solution was diluted to 26.5 g / L with dialysis buffer, stirred evenly, and the pH value was adjusted to 7.0. After sterilization and filtration with a sterile filter cartridge, 2.92 L of semi-finished product was obtained.
[0097] (10) Finished product
[0098] The semi-finished product is packaged and freeze-dried. The freeze-dried product is then subjected to dry heat inactivation treatment at 100±1℃ for 30 minutes. The treated product is then capped and sealed to obtain the finished product.
[0099] The intermediate product results of the samples from Examples 1-3 are as follows: S / D sample Total protein content (g / L) Fibrinogen content (g / L) purity(%) Solidification vitality (s) Plasminogen activity (%) Example 1 14.19 14.77 96.04 19.2 5.8 Example 2 22.63 15.47 68.40 36.6 59.2 Sample before ultrafiltration Total protein content (g / L) Fibrinogen content (g / L) purity(%) Solidification vitality (s) Fibronectin content (g / L) Example 1 12.37 11.12 89.91 20.1 0.110 Example 3 17.04 14.55 85.36 40.2 0.205
[0100] The finished product test results are as follows: sample purity(%) Solidification vitality (s) Plasminogen activity (%) Fibronectin content (g / L) Heparin content (IU / ml) Example 1 95.6 22 3.8 0.158 ＜0.2 Example 2 95.0 44.6 37.9 0.311 ＜0.2 Example 3 96.6 30.7 4.10 0.228 ＜0.2 Pharmacopoeia Standard ≥70 ≤60 — — —
[0101] The results showed that adding a PEG precipitation step before S / D inactivation could remove most of the impurities and avoid affecting product quality; after S / D inactivation, the classic ethanol precipitation was used to remove the S / D reagent; the combination of ethanol-glycine precipitation had better product purity and coagulation activity than the combination of ethanol-ethanol precipitation.
[0102] Comparative Examples 1-4 differ from Example 1 only in the concentration of lysine hydrochloride in solution A; the method steps will not be repeated here.
[0103] The coagulation activity and plasminogen activity of the solutions of component I in Comparative Examples 1-4 were detected, and the results are as follows: sample Lysine hydrochloride concentration (mmol / L) Solidification vitality (s) Plasminogen activity (%) Comparative Example 1 10 38.2 37.3 Comparative Example 2 30 32.2 11.6 Comparative Example 3 50 25.8 6.2 Comparative Example 4 70 25.0 5.7
[0104] The results showed that the higher the concentration of lysine hydrochloride, the better the removal effect on plasminogen, and the coagulation activity was also improved.
Claims
1. A method for isolating and purifying fibrinogen, a low-contamination protein, characterized in that, Includes the following steps: (1) Raw material preparation Take freshly prepared component I or frozen component I. If frozen component I is used, first cut frozen component I into small cubes of about 1 cm, then thaw it to obtain FI precipitate. (2) Dissolution and filtration of FI precipitate Take the FI precipitate from step (1), add 10 to 12 times the weight of the precipitate to dissolve in solution A, control the dissolving temperature at 20 to 30°C, and after dissolving for 0.5 to 2 hours, filter with a 30SP filter cartridge and collect the filtrate A. (3) Purification and separation of polyethylene glycol (PEG) First, adjust the pH of the filtrate A obtained in step (2) to 6.8-7.2, add PEG solution to filtrate A to a final concentration of 5%, stir for 0.5-1h at 10-20℃, centrifuge, with a maximum processing capacity of ≤2.0Kg per minute, and control the temperature of the centrifuged liquid at 10-17℃, and collect the PEG precipitate; Add solution B at 10 to 15 times the weight of the PEG precipitate, stir at 20 to 30°C, avoiding the generation of bubbles and the accumulation of precipitate during stirring, filter after dissolving for 1 to 2 hours, filter with a 60SP filter cartridge, and collect filtrate B. (4) S / D inactivation Adjust the pH of filtrate B from step (3) to 6.8-7.
2. While stirring continuously, slowly add the prepared S / D solution at a mass flow rate of ≤3 kg / min until the content of polysorbate 80 is 1% and the content of tributyl phosphate is 0.3%. Stop adding the S / D solution, stir it to make it uniform, filter it, and transfer the filtrate to a special virus inactivation tank. Inactivate it at 24-26℃ for no less than 6 hours to obtain the inactivated solution. (5) Ethanol purification and separation Adjust the pH of the inactivation solution in step (4) to 6.8-7.2 and cool it to 0-2℃. Slowly add 50% ethanol solution below -15℃ until the ethanol content is 8%. After adding the ethanol, cool it to -2.5-0℃. Stir for 40-60 min and centrifuge. The maximum processing capacity is ≤2.0Kg per minute. Control the temperature of the centrifuged liquid at -2.5-0℃ and collect the ethanol precipitate. Dissolve the ethanol precipitate with 12-17 times the amount of dissolving solution B at 20-30℃ for 1-2 hours. Avoid generating bubbles and precipitate accumulation during the dissolution process. Filter the dissolved ethanol precipitate with a 60SP filter cartridge to obtain filtrate C. (6) Glycine purification and separation Adjust the pH of filtrate C from step (5) to 7.1-7.3, then add glycine to a final concentration of 1.8 mol / L. After it is fully dissolved, cool the solution to 2-10°C, stir for at least 60 min, and then centrifuge. The maximum processing capacity per minute is ≤2.0 kg. The temperature of the centrifuged liquid is controlled at 0-4°C. Collect the glycine precipitate. Dissolve the glycine precipitate with 4-8 times the amount of dissolving solution C at room temperature for 1-2 h. Avoid generating bubbles and precipitate accumulation during the dissolution process. Filter the dissolved glycine precipitate with a 0.45 μm filter and collect the filtrate D. (7) Ultrafiltration dialysis The filtrate D from step (6) was dialyzed 3 to 4 times by constant weight using solution C, and then concentrated by ultrafiltration to a protein concentration of more than 30 g / L to obtain the original solution. (8) Preparation Based on the protein content of the stock solution in step (7) and the target protein amount per unit dose, the volume of the final preparation required is calculated. The stock solution is diluted to 25-28 g / L with dialysis buffer, stirred evenly, and the pH value is confirmed to be 6.8-7.
2. After sterilization and filtration with a sterile filter cartridge, a semi-finished product is obtained. (9) Finished product The semi-finished product is packaged and freeze-dried. The freeze-dried product is then capped and sealed, and subjected to dry heat inactivation treatment at 100±1℃ for 30 minutes to obtain the finished product.
2. The method for isolating and purifying fibrinogen, a low-contamination protein, according to claim 1, is characterized in that: The formulation of the solution A includes: sodium citrate concentration of 100.0 mmol / L, lysine hydrochloride concentration of 49.8 mmol / L, sodium heparin concentration of 6 IU / mL, water for injection as solvent, and pH value of 6.8–7.
0.
3. The method for isolating and purifying fibrinogen, a low-contamination protein, according to claim 1, is characterized in that: The formulation of solution B includes: sodium citrate concentration of 51.0 mmol / L, sodium chloride concentration of 153.8 mmol / L, lysine hydrochloride concentration of 19.2 mmol / L, sucrose concentration of 29.2 mmol / L, water for injection as solvent, and pH value of 6.8–7.
2.
4. The method for isolating and purifying fibrinogen, a low-contamination protein, according to claim 1, is characterized in that: The formulation of solution C includes: sodium citrate concentration of 34.0 mmol / L, monosodium glutamate concentration of 11.8 mmol / L, arginine hydrochloride concentration of 94.9 mmol / L, water for injection as solvent, and pH value of 6.8–7.
2.
5. The method for isolating and purifying fibrinogen, a low-contamination protein, according to claim 4, is characterized in that: The S / D reagent formulation includes: 11% polysorbate 80 and 3.3% tributyl phosphate.
6. The method for isolating and purifying fibrinogen, a low-contamination protein, according to claim 1, is characterized in that: In step (4), the temperature of the virus inactivation tank is controlled at 24-26℃, and the S / D virus inactivation time is ≥6h.
7. The method for isolating and purifying fibrinogen, a low-contamination protein, according to claim 1, is characterized in that: In step (7), the ultrafiltration process requires an ultrafiltration inlet pressure of ≤3.0 bar and a reflux pressure of ≤1.0 bar.