Purification method of fibrinogen for producing fibrin glue

Abstract

The invention belongs to the technical field of blood product preparation, and particularly relates to a fibrinogen purification method for producing fibrin glue. The method comprises the following steps in sequence: dissolving a component I precipitate or cryoprecipitate by using a first dissolving solution, and then centrifugally collecting a precipitate part to obtain a centrifuged precipitate; dissolving the centrifuged precipitate by using a first dissolving solution, then centrifugally collecting a supernatant part, and filtering to obtain a centrifuged supernatant; performing S / D inactivation on the centrifuged supernatant to obtain an inactivated product; adjusting the pH and conductivity of the inactivated product, and filtering to obtain an adjusted product; and sequentially carrying out ion exchange chromatography and affinity chromatography on the adjusted product to obtain an affinity chromatography product, and further treating to obtain a fibrinogen finished product. According to the technical scheme, the technical problems that in the prior art, the content of plasminogen in a fibrinogen product is too high, and the content of blood coagulation factors XIII is not ideal can be solved, and the fibrinogen product has ideal popularization and application prospects.

Description

Technical Field

[0001] The invention belongs to the technical field of blood product preparation, and in particular relates to a fibrinogen purification method that can be used to produce fibrin glue. Background Art

[0002] Fibrinogen (Fg), also known as coagulation factor I (FⅠ), has a relative molecular weight of 340 kDa. It is large in molecular weight and easy to precipitate and hydrolyze. It is one of the "central" proteins in the coagulation system. It forms fibrin monomers under the action of thrombin. The monomers are stabilized under the action of coagulation factor XIII and play a role in wound hemostasis. Fibrin is decomposed under the action of plasminogen and other factors to prevent thrombosis. In the human body, coagulation and anticoagulation are in a balanced state, which maintains the flow of blood and can stop bleeding in time when a wound occurs.

[0003] Fibrin glue is a product composed of three proteins: fibrinogen, coagulation factor XIII, and thrombin. When used clinically, it is mixed to form a protein glue, which is used inside and outside the human body such as mucous membranes and wounds to stop bleeding. Since the three products are produced separately, they are mixed when used, which makes the use and production process more cumbersome. And because plasminogen decomposes fibrin glue, the stability time of fibrin glue is shortened. The applicant disclosed a process for preparing fibrinogen based on chromatography in Chinese patent CN115947825A. In this scheme, plasma is treated with ethanol and filtered to obtain component I precipitate or plasma is centrifuged at low temperature to obtain cold precipitate; the precipitate is washed multiple times with a precipitate washing solution to obtain a washed precipitate; a precipitate dissolving solution is added to the washed precipitate and filtered to obtain a filtrate; the filtrate is inactivated to obtain an inactivated filtrate; the pH and conductivity of the inactivated filtrate are adjusted and then filtered; the product is loaded onto an anion exchange chromatography column, and the chromatography buffer, chromatography washing solution and eluent are loaded in sequence; the eluent is ultrafiltered to obtain the target product. However, this process scheme does not consider how to further remove plasminogen, and does not consider how to ensure the purity of fibrinogen and the potency of coagulation factor XIII while removing plasminogen.

[0004] Therefore, in order to overcome the defects of the prior art, it is urgent to develop a product with low plasminogen content and rich in coagulation factor XIII and fibrinogen for producing fibrin glue to meet the needs of practical applications. Summary of the invention

[0005] The present invention is intended to provide a fibrinogen purification method that can be used to produce fibrin glue, so as to solve the technical problems of excessively high plasminogen content and unsatisfactory content of coagulation factor XIII in the prior art fibrinogen products.

[0006] In order to achieve the above object, the present invention adopts the following technical scheme:

[0007] A method for purifying fibrinogen that can be used to produce fibrin glue comprises the following steps performed in sequence:

[0008] S1: using the first dissolving solution to dissolve the component I precipitate or the cold precipitate, and then centrifuging to collect the precipitate to obtain a centrifuged precipitate;

[0009] S2: using the second dissolving solution to dissolve the precipitate after centrifugation, then collecting the supernatant by centrifugation and filtering it to obtain the supernatant after centrifugation;

[0010] S3: performing S / D inactivation on the supernatant after centrifugation to obtain an inactivated product; adjusting the pH and conductivity of the inactivated product, and obtaining an adjusted product after filtration;

[0011] S4: sequentially performing ion exchange chromatography and affinity chromatography on the adjusted product to obtain a product after affinity chromatography;

[0012] S5: The product after affinity chromatography is subjected to ultrafiltration, dialysis, freeze-drying and dry heat inactivation to obtain the finished fibrinogen product.

[0013] Further, in S1, the first dissolving solution is a solution containing 1.0-2.0M glycine and 0.03-0.08M sodium citrate; the mass ratio of the component I precipitate or cryoprecipitate to the first dissolving solution is 1:5-10;

[0014] The dissolution temperature is 20-30°C and the time is 1-3 hours; after dissolution, the temperature is lowered to -2.0-2.0°C, and after reacting for 0.5-2 hours, centrifugation is performed; the centrifugation conditions are 3000-10000 rpm and 5-20 minutes;

[0015] The step S1 is repeated 1 to 3 times.

[0016] Further, in S2, the second dissolving solution is a solution containing 0.03-0.10M tromethamine and having a pH of 8.50-9.50; the mass ratio of the precipitate to the second dissolving solution after centrifugation is 1:5-10;

[0017] The dissolution temperature is 20-30°C and the time is 1-3 hours; the centrifugal conditions are 3000-10000 rpm and 5-20 minutes; and the pore size of the filtration membrane is 0.65-1.0 μm.

[0018] Furthermore, in S3, the pH of the product after adjustment is 8.50 to 9.50, and the conductivity is 0.50 to 1.50 mS / cm.

[0019] Further, in S4, the chromatographic column filler of the ion exchange chromatography is a strong anion exchange filler;

[0020] In the process of ion exchange chromatography, the chromatography column is first treated with a first equilibrium solution; then, the adjusted product is loaded into the chromatography column; the chromatography column is treated with a chromatography buffer solution and a chromatography washing solution in sequence; next, the chromatography column is treated with an eluent, and the effluent after the eluent treatment is collected to obtain the product after ion exchange chromatography;

[0021] The first equilibrium solution is a solution containing 0.02-0.1 M tromethamine, and the pH value is 8.50-9.50;

[0022] The chromatography buffer is a solution containing 0.02-0.1 M tromethamine and has a pH of 8.50-9.50;

[0023] The chromatography washing solution contains 0.02-0.1 M tromethamine, 0.008-0.015 M sodium chloride, and the pH is 8.50-9.50;

[0024] The eluent contains 0.02-0.1 M tromethamine and has a pH of 7.00-8.00.

[0025] Further, in S4, the chromatography column filler for affinity chromatography is a lysine ligand affinity chromatography filler;

[0026] During the affinity chromatography, the chromatography column is treated with the second equilibrium solution; then, the product after the ion exchange chromatography is loaded into the chromatography column, and the flow-through liquid is collected as the product after the affinity chromatography;

[0027] The second balanced solution is a solution containing 0.02-0.1 M tromethamine, and has a pH of 7.00-8.00.

[0028] Further, in S5, the protein content of the product after affinity chromatography is concentrated to 23-27 g / L by ultrafiltration; the dialysis is 3-5 times dialysis and a solution containing arginine, sodium citrate and sodium chloride is used as the dialysate; the parameters of dry heat inactivation are set to 99.5-100.5 boiling water bath inactivation ≥30 min.

[0029] Further, in S1, the component I precipitate is prepared by the following method: the raw plasma is diluted with 2.0-8.0° C. water for injection to a protein content of 40-65 g / L, and then the conductivity is adjusted to 12.0-14.0 mS / cm, the pH is adjusted to 6.80-7.30, and the volume percentage of ethanol is adjusted to 7%-10%, and the reaction is stirred for 1-3 hours and then filtered to obtain the component I precipitate;

[0030] The cryoprecipitate is prepared by the following method: the raw plasma is centrifuged at 12000 rpm / min and 0-2°C, and the precipitate is collected after centrifugation to obtain the cryoprecipitate.

[0031] The technical solution also provides a fibrinogen product obtained by a fibrinogen purification method that can be used to produce fibrin glue.

[0032] Furthermore, in the fibrinogen product, the purity of fibrinogen is ≥95%; the content of coagulation factor XIII is ≥10 IU / ml; and the content of plasminogen is ≤0.5 μg / ml.

[0033] The principle of this scheme is:

[0034] In this technical solution, the precipitate or cryoprecipitate of component I is used as the raw material, the precipitate is washed and dissolved, and then anion exchange chromatography and affinity chromatography are performed to fully enrich and purify the fibrinogen and coagulation factor XIII in the raw plasma, and effectively remove plasminogen. Compared with the prior art, this solution further considers how to effectively remove plasminogen, and fully ensures the purity of fibrinogen and the titer of coagulation factor XIII while removing plasminogen. The obtained product is rich in coagulation factor XIII and fibrinogen, and is particularly suitable for the production and preparation of fibrin glue.

[0035] The beneficial effects of this solution are:

[0036] (1) The plasminogen content is as low as 0.5 μg / ml, which increases the stability time of fibrin glue.

[0037] (2) The factor XIII content reaches 10 IU / ml, which is much higher than the requirement of the Chinese Pharmacopoeia. The Chinese Pharmacopoeia stipulates that the factor XIII content in fibrin glue must reach 1 U / ml.

[0038] (3) The fibrinogen has high purity, good protein activity, and its coagulation activity meets the requirements of the pharmacopoeia.

[0039] (4) The protein yield is high. From the precipitation of component I to the final product, the fibrinogen yield can reach 30% to 40%.

[0040] (5) The entire process is ethanol-free, reducing possible flammable and explosive safety risks. DETAILED DESCRIPTION

[0041] The present invention is further described in detail below with reference to the examples, but the embodiments of the present invention are not limited thereto. Unless otherwise specified, the technical means used in the following examples are conventional means well known to those skilled in the art; the experimental methods used are all conventional methods; the materials, reagents, etc. used can all be obtained from commercial sources.

[0042] The technical solution uses cryoprecipitate or component I precipitate as raw material to prepare fibrinogen.

[0043] The preparation method of component I precipitate is as follows: the raw plasma frozen at -30°C is placed at -5°C overnight after being released from the warehouse, and the surface of the plasma bag is disinfected with 70% to 75% ethanol solution before use, and then the plasma bag is broken and the temperature is controlled to melt at 0 to 4°C. The raw plasma is the supernatant after the blood is centrifuged to remove cells, containing protein, inorganic salts and water, etc., but not blood cells. More specifically, the raw plasma is the human plasma referred to in the "Chinese Pharmacopoeia": human plasma for the production of blood products is healthy human plasma collected by apheresis for the production of plasma protein products. After thawing, the plasma is diluted with 2.0 to 8.0°C injection water to a protein content of 40-65g / L, and the conductivity is adjusted to 12.0 to 14.0mS / cm with sodium chloride. The plasma pH is adjusted to 6.80 to 7.30 with 1.0mol / L acetic acid solution, and the ethanol concentration in the product is adjusted to 7% to 10% (volume percentage). After stirring for 1-3h, the reaction is separated by pressure filtration to obtain component I precipitate.

[0044] The preparation method of cold precipitate is as follows: after the frozen raw plasma is taken out of the warehouse, the surface of the plasma bag is disinfected with 70% to 75% ethanol solution, and then the plasma bag is opened and the temperature is controlled to melt at 0 to 4°C. Then the plasma is centrifuged, and the specific centrifugation parameters are: 12000rpm / min, 0 to 2°C. The precipitate obtained after centrifugation is the cold precipitate collected by centrifugation.

[0045] The method for obtaining the component I precipitate and the cold precipitate is a conventional method in the prior art. The present technical scheme mainly studies how to separate and purify fibrinogen from the component I precipitate or the cold precipitate after obtaining the component I precipitate or the cold precipitate.

[0046] The following is a detailed description of the preparation process of fibrinogen using the precipitation of component I as an example. The same operation method can be used when cold precipitation is used as the raw material.

[0047] (1) Dissolution and reprecipitation of component I precipitate: Take the component I precipitate, add 5 to 10 times the amount of the first dissolving solution (1.0 to 2.0 M glycine + 0.03 to 0.08 M sodium citrate), and stir and dissolve at 20 to 30°C for 1 to 3 hours. Lower the temperature to -2.0 to 2.0°C, and after dissolution, cool and react for 0.5 to 2 hours, then centrifuge at 4000 rpm / 10 min (optional range: 3000 to 10000 rpm, 5 to 20 min) to separate the precipitate and discard the supernatant; repeat this step 1 to 3 times to obtain the precipitate after centrifugation.

[0048] (2) Dissolution of the precipitate after centrifugation: Add 5 to 10 times the amount of the precipitate in the second dissolving solution (0.03 to 0.10 M tromethamine, pH 8.50 to 9.50), stir and dissolve at a temperature of 20 to 30°C for 1 to 3 hours. Centrifuge at 4000 rpm / 10 min (optional range: 3000 to 10000 rpm, 5 to 20 min), collect the supernatant, and filter with a terminal 0.65 to 1.0 μm filter membrane to obtain the supernatant after centrifugation.

[0049] (3) S / D inactivation: Add polysorbate 80 and tributyl phosphate to the supernatant after centrifugation, and the final mass percentages of polysorbate 80 and tributyl phosphate after addition are controlled at 1% and 0.3%, respectively. Then, control the product temperature at 24-26°C and keep it constant for more than 6 hours (implementing the standard requirements) to obtain the inactivated product. The S / D (Solvent / detergent) method of virus inactivation, i.e., using an organic solvent / detergent mixture (S / D) to destroy the lipid membrane of the enveloped virus, is a conventional method in the prior art and will not be described in detail here.

[0050] (4) Product chromatography: Adjust the pH of the inactivated product to 8.50-9.50 and the conductivity to 0.50-1.50 mS / cm, and filter using a 0.2 μm filter membrane to obtain the adjusted product. Load the adjusted product onto the treated anion exchange chromatography column. Alternatively, without going through the anion exchange chromatography step, directly load the adjusted product onto the affinity chromatography column for affinity chromatography.

[0051] Ion exchange chromatography:

[0052] Before using the anion exchange chromatography column, the chromatography column is balanced with the first balancing liquid. The formula of the first balancing liquid is 0.02-0.1M tromethamine, and the pH value is adjusted to 8.50-9.50 using citric acid solution. After loading the product, it is sequentially eluted with 0.02-0.1M tromethamine (the pH is adjusted to 8.50-9.50 using citric acid solution, i.e., chromatography buffer), a solution containing 0.02-0.1M tromethamine and 0.008-0.015M sodium chloride (the pH is adjusted to 8.50-9.50 using citric acid solution, i.e., chromatography washing solution). Then, 0.02-0.1M tromethamine (the pH is adjusted to 7.00-8.00 using citric acid solution, i.e., eluent) is used for elution. The above eluent is collected to obtain the product after ion exchange chromatography for subsequent affinity chromatography steps.

[0053] Affinity chromatography:

[0054] An affinity chromatography filler that can specifically bind to plasminogen can be selected, such as Cytiva's ECH-Lysinesepharose 4Fast Flow. The affinity chromatography column is treated with the second equilibrium solution before use. The formula of the second equilibrium solution is: 0.02-0.1M tromethamine, and the pH is adjusted to 7.00-8.00 using a citric acid solution. Then, the product after ion exchange chromatography is loaded onto the affinity chromatography column, and the flow-through of the product after ion exchange chromatography is collected to obtain the product after affinity chromatography.

[0055] (5) Ultrafiltration preparation: Use a membrane package with a pore size of 100KD to concentrate the protein content to 25g / L (optional range: 23-27g / L), dialyze 3-5 times the volume of the product with a solution containing arginine, sodium citrate and sodium chloride, and then divide it into packages. Dialysis and ultrafiltration are both conventional methods in the prior art, and the dialysate is a conventional formula. Ultrafiltration is mainly for concentrating the product, and dialysis is mainly for removing non-protein small molecule impurities in the product. The specific formula of the dialysate is: 1-3g / L sodium citrate, 5-15.0g / L sodium chloride, 6-10.0g / L arginine, 1-4g / L glycine, 6-10.0g / L sucrose, and adjust its pH to 6.80-7.20.

[0056] (6) Freeze-drying and dry heat inactivation: The packaged products are placed in a freeze-drying cabinet for processing. After freeze-drying, they are inactivated in a boiling water bath at 99.5-100.5°C for ≥30 min to obtain the finished fibrinogen product. The freeze-drying process adopts the conventional method of the prior art: cooling to -45°C and keeping the temperature constant for 10 hours, heating to -25°C and keeping the temperature constant for 30 hours, heating to -20--15°C (preferably -15°C) and controlling the temperature for 5 hours, heating to 0°C and controlling the temperature for 5-8 hours (preferably 7 hours), heating to 10°C and controlling the temperature for 10 hours, heating to 15-20°C (preferably 15°C) and controlling the temperature for 10 hours, heating to 25°C and controlling the temperature for 10 hours.

[0057] Example 1

[0058] In this embodiment, component I precipitation is prepared in the following manner:

[0059] The raw plasma frozen at -30℃ is placed at -5℃ overnight after being released from the warehouse. Before use, the surface of the plasma bag is disinfected with 75% ethanol solution, and then the plasma bag is broken and the temperature is controlled to melt at around 0℃. Among them, raw plasma is the supernatant after the blood is centrifuged to remove cells, containing protein, inorganic salts and water, etc., but no blood cells. More specifically, raw plasma is the human plasma referred to in the "Chinese Pharmacopoeia": human plasma for the production of blood products is healthy human plasma collected by single plasmapheresis for the production of plasma protein products. After thawing, the plasma is diluted with injection water at about 2.0℃ to a protein content of 40g / L, and the conductivity is adjusted to 12.0mS / cm with sodium chloride. The plasma pH is adjusted to 6.80 with 1.0mol / L acetic acid solution, and the ethanol concentration in the product is adjusted to 7% (volume percentage). After stirring for 3 hours, filter press separation is performed to obtain component I precipitate. The above-mentioned component I precipitate is used to prepare fibrinogen. The specific process is as follows:

[0060] (1) Take 5 kg of component I precipitate, add 30 L of the first dissolving solution (1.5 M glycine + 0.06 M sodium citrate), and stir and dissolve at a temperature of about 24°C for 2 h. Lower the temperature to 0±1°C, centrifuge at 4000 rpm / 10 min to separate the precipitate, and discard the supernatant; repeat this step twice to obtain the precipitate after centrifugation.

[0061] (2) Dissolving the precipitate after centrifugation: Add 30 L of the second dissolving solution (0.05 M tromethamine, pH 9.00), stir and dissolve at a temperature of about 24°C for 2 h. Centrifuge at 4000 rpm / 10 min, collect the supernatant, and filter with a 0.65 μm filter membrane to obtain the supernatant after centrifugation (34 L).

[0062] (3) S / D inactivation: Add a mixed solution of polysorbate 80 and tributyl phosphate to the supernatant after centrifugation. The mass percentage of polysorbate 80 is 1%, and the mass percentage of tributyl phosphate is 0.3% based on the final concentration. Control the product temperature at about 25°C and keep the temperature constant for 6 hours to complete the S / D inactivation and obtain the inactivated product.

[0063] (4) Product chromatography: Divide the inactivated product into three parts.

[0064] (4.1) The pH of the first inactivated product was adjusted to 9.00 and the conductivity to 1.00 mS / cm, and the product was filtered using a 0.2 μm filter membrane to obtain an inactivated and adjusted product. This inactivated and adjusted product was used for anion exchange chromatography.

[0065] The filler used in the anion exchange chromatography column is Fractgel EMD TMAE (M), and the specifications of the chromatography column are: 16mm×20cm. Use 3 to 5 column volumes (CV, 3CV is specifically used in this embodiment) of the first equilibrium solution to balance the anion exchange chromatography column. After the equilibrium is completed, the inactivated and adjusted products are loaded into the anion exchange chromatography column, and the loading amount is the filtered product after inactivation: affinity chromatography filler = 10 to 30mL: 1mL (15mL: 1mL is specifically used in this experimental example). Then, use 3 to 5CV (3CV is specifically used in this embodiment) of chromatography buffer and 3 to 5CV (3CV is specifically used in this embodiment) of chromatography washing solution to elute the ion exchange chromatography column. Then, treat the anion exchange chromatography column with 3 to 5CV of eluent (3CV is specifically used in this embodiment). Collect the eluent flowing out of the chromatography column to obtain the product after ion exchange chromatography.

[0066] The formula of the first balanced solution is: 0.05M tromethamine, the pH value is adjusted to 9.00 using citric acid solution;

[0067] The formulation of the chromatography buffer was: 0.05 M tromethamine, pH adjusted to 9.00 using citric acid solution;

[0068] The formula of the chromatography washing solution is: 0.05M tromethamine + 0.010M sodium chloride, and the pH value is adjusted to 9.00 using citric acid solution;

[0069] The formula of the eluent is: 0.05 M tromethamine, adjusted to pH 7.50 using citric acid solution.

[0070] (4.2) The second inactivated product is filtered using a terminal 0.2 μm filter membrane to obtain an inactivated filtered product, which is then subjected to affinity chromatography.

[0071] The filler used in the affinity chromatography column is ECH-Lysine sepharose 4Fast Flow (Cytiva) (in addition, Suzhou Nanovita's WHBP filler can also be used, and the above fillers are all lysine-based affinity chromatography fillers). The column volume is 10mm×20cm. Use 3 to 5 column volumes (CV, 3CV is specifically used in this embodiment) of the second equilibrium liquid to carry out the equilibrium treatment of the affinity chromatography column. Then, the inactivated filtered product is loaded onto the affinity chromatography column, and the loading amount is inactivated filtered product: affinity chromatography filler = 30 to 50mL: 1mL (40mL: 1mL is specifically used in this experimental example). Then, the flow-through liquid of the inactivated filtered product is collected to obtain the affinity chromatography product.

[0072] The formula of the second balanced solution is: 0.05M tromethamine, and the pH is adjusted to 7.5 using citric acid.

[0073] (4.3) The pH of the third inactivated product was adjusted to 9.00 and the conductivity to 1.00 mS / cm, and the product was filtered using a 0.2 μm filter membrane to obtain an inactivated and adjusted product. This inactivated and adjusted product was first subjected to anion exchange chromatography and then to affinity chromatography.

[0074] The anion exchange chromatography step is detailed in (4.1). The product after ion exchange chromatography is obtained. The product after ion exchange chromatography is used for subsequent affinity chromatography. The operation steps can be seen in (4.2), except that the inactivated filtered product is changed to the product after ion exchange chromatography as the starting material for the affinity chromatography operation.

[0075] (5) The ion exchange chromatography products, affinity chromatography products (without ion exchange) and affinity chromatography products (with ion exchange) obtained in (4.1), (4.2) and (4.3) are subjected to ultrafiltration. The protein content is concentrated to 25 g / L using a membrane package with a pore size of 100 KD, and the product volume is dialyzed with a solution containing arginine, sodium citrate and sodium chloride to a volume 5 times that of the product, and then packaged to obtain a semi-finished product. The specific formula of the dialysate is: 2 g / L sodium citrate, 10 g / L sodium chloride, 8 g / L arginine, 3 g / L glycine, 8 g / L sucrose, and its pH is adjusted to 6.80-7.20.

[0076] (6) Freeze-drying and dry heat inactivation: The packaged semi-finished products are placed in a freeze-drying cabinet for processing. After freeze-drying, they are inactivated in a boiling water bath at 99.5-100.5 for 30 minutes to obtain the finished product. The purity of fibrinogen, the potency of factor XIII, and the content of plasminogen are tested for the finished product. The purity and factor XIII are tested in accordance with the requirements of the 2020 edition of the Chinese Pharmacopoeia, and plasminogen is tested by conventional enzyme-linked immunosorbent assay. The results are shown in Table 1. The use of TMAE chromatography + affinity chromatography can ensure that the purity of fibrinogen in the obtained product is ≥95%; the content of coagulation factor XIII is ≥10IU / ml; and the content of plasminogen is <1μg / ml.

[0077] Table 1: Finished product quality characterization results

[0078]

[0079] The fibrinogen prepared in this scheme is mainly used for the production of fibrin glue. Fibrin glue is a product composed of three proteins: fibrinogen, coagulation factor XIII, and thrombin. It is mixed to form a protein glue during clinical use and used in mucous membranes, wounds, and other internal and external parts of the human body to stop bleeding. In the prior art, fibrinogen, coagulation factor XIII, and thrombin are usually separated and purified from raw plasma by different processes. This technical scheme can simultaneously separate and purify a mixed product of fibrinogen and coagulation factor XIII from plasma, in which the fibrinogen purity is high, the coagulation factor XIII titer is ideal, and in this product, the content of plasminogen is very low, which avoids the decomposition of fibrinogen by plasminogen, and further improves the stability of fibrin glue. This technical scheme combines ion exchange chromatography and affinity chromatography, while ensuring the purity of fibrinogen and the titer of coagulation factor XIII, and maintaining plasminogen at a low level.

[0080] Comparative Example 1

[0081] The S1 step of this scheme was explored. In this step, the first dissolving solution (containing 1.0-2.0M glycine and 0.03-0.08M sodium citrate) was used to dissolve the component I precipitate or cold precipitate, and then the precipitate was collected by centrifugation to obtain the centrifuged precipitate. The dissolving temperature used was 20-30°C. This comparative example was carried out with reference to the operating steps of Example 1, except that the stirring and dissolving temperature of step (1) was adjusted (S1 step) to 2, 10, 20, and 30°C. The centrifuged precipitate obtained in step S1 was re-dissolved using the second dissolving solution (refer to Example 1), and the sample of the process site was taken for detection of foreign proteins. The experimental results are shown in Table 2. Foreign proteins include IgA, IgM and fibronectin. If this part of foreign proteins is fully removed in step S1, the interference of these foreign proteins with the subsequent chromatography process can be avoided. From the experimental results, we can see that as the temperature rises, the content of impurity proteins decreases. As the temperature continues to rise, it affects protein binding and activity. Therefore, a dissolution temperature of 20-30°C is most suitable, and 30°C is the highest.

[0082] In previous studies, the inventors used a solution containing glycine to wash and dissolve the component I precipitate or cold precipitate. In order to ensure that the activity of the protein was not negatively affected, a relatively low temperature of 2 to 8°C was used. The research at that time did not notice that the treatment temperature would have such a great impact on the removal effect of protein impurities. It was found that increasing the dissolution temperature can improve the purity of the target protein (reduce the content of impurities), which directly affects the efficiency of the first step of ion exchange chromatography. After the dissolution temperature becomes higher, the removal effect of impurities is better, and the initial removal of plasminogen by the first step of ion exchange is also better, which can increase the affinity chromatography load and enhance the removal effect of affinity chromatography on plasminogen, and further ensure that the content of plasminogen in the final product is lower. The inventors tried to dissolve the component I precipitate at 10°C, according to the operation method of Example 1 (and using the operation process described in (4.1), that is, only anion exchange chromatography). Because the impurities were not fully removed, the removal effect of plasminogen in ion exchange chromatography was not ideal. If affinity chromatography is performed next, its process efficiency will also be negatively affected. After repeated experiments, the concentration of plasmin in the product obtained after anion exchange chromatography increased by about 10-15% (relative to the product obtained by TMAE chromatography (4.1) in Table 1), that is, the impurities that affect the application of the product of this process as fibrin glue increased, which is an undesirable effect. The dissolution temperature of the component I precipitate will affect the removal effect of the plasminogen in the overall process, and the impact on this index is very obvious, which was not predicted by the inventor before the experiment. When using the product of this scheme to prepare fibrin glue, the residual amount of plasminogen is a key factor affecting the quality of fibrin glue. Therefore, the control of the dissolution temperature of the component I precipitate is particularly important. In the inventor's previous research, since there was no need to use the product to directly prepare fibrin glue, the problem of plasminogen residue was not paid attention to, so the effect of the dissolution temperature of the component I precipitate on the plasminogen residue was not found. The discovery of the above rules and their application to the specific preparation process of this scheme have improved product quality and achieved unexpected technical effects.

[0083] Table 2: Results of impurity protein detection at different dissolution temperatures

[0084]

[0085] Comparative Example 2

[0086] The S1 step of this scheme was explored. In this step, the first dissolving solution used was a solution containing 1.0-2.0M glycine and 0.03-0.08M sodium citrate (Example 1 uses a solution containing 1.5M glycine and 0.06M sodium citrate). The inventor studied the solute composition of the first dissolving solution. The 0.06M sodium citrate remained unchanged, and the concentrations of glycine were: 0.5M, 1.0M, 1.5M, 2.0M.

[0087] The first dissolving liquid is used to dissolve the component I precipitate or cold precipitate, and then the precipitate is collected by centrifugation to obtain the centrifuged precipitate. This comparative example is carried out with reference to the operating steps of Example 1 (except for the concentration of glycine). The centrifuged precipitate obtained in step S1 is re-dissolved with the second dissolving liquid (refer to Example 1), and the sample at this process point is taken to detect the yield of fibrinogen. The yield of fibrinogen is: the total amount of fibrinogen sampled by re-dissolving the centrifuged precipitate / the total amount of fibrinogen in the component I precipitate × 100%.

[0088] The experimental results show that the first dissolving solution with glycine concentrations of 0.5M, 1.0M, 1.5M, and 2.0M has a corresponding fibrinogen yield of 50%, 80%, 93%, and 95%, respectively. Therefore, the glycine concentration in the first dissolving solution is preferably 1.5-2.0M.

[0089] The above is only an embodiment of the present invention, and the common knowledge such as the known specific technical solutions and / or characteristics in the solution is not described in detail here. It should be pointed out that for those skilled in the art, without departing from the technical solution of the present invention, several modifications and improvements can be made, which should also be regarded as the protection scope of the present invention, and these will not affect the effect of the implementation of the present invention and the practicality of the patent. The scope of protection required by this application shall be based on the content of its claims, and the specific implementation methods and other records in the specification can be used to interpret the content of the claims.

Claims

1. A method for purifying fibrinogen that can be used to produce fibrin glue, characterized in that: The method includes the following steps: S1: using the first dissolving solution to dissolve the component I precipitate or the cold precipitate, and then centrifuging to collect the precipitate to obtain a centrifuged precipitate; S2: using the second dissolving solution to dissolve the precipitate after centrifugation, then centrifugally collecting the supernatant and filtering it to obtain the supernatant after centrifugation; S3: Perform S / D inactivation on the supernatant after centrifugation to obtain the inactivated product; Adjusting the pH and conductivity of the inactivated product, and obtaining the adjusted product after filtering; S4: sequentially performing ion exchange chromatography and affinity chromatography on the adjusted product to obtain a product after affinity chromatography; S5: The product after affinity chromatography is subjected to ultrafiltration, dialysis, freeze-drying and dry heat inactivation to obtain the finished fibrinogen product.

2. A method for purifying fibrinogen for producing fibrin glue according to claim 1, characterized in that: In S1, the first dissolving solution is a solution containing 1.0-2.0M glycine and 0.03-0.08M sodium citrate; the mass ratio of the component I precipitate or cryoprecipitate to the first dissolving solution is 1:5-10; The dissolution temperature is 20-30°C and the time is 1-3 hours; after dissolution, the temperature is lowered to -2.0-2.0°C, and after reacting for 0.5-2 hours, centrifugation is performed; the centrifugation conditions are 3000-10000 rpm and 5-20 minutes; The step S1 is repeated 1 to 3 times.

3. The method for purifying fibrinogen for producing fibrin glue according to claim 1, characterized in that: In S2, the second dissolving solution is a solution containing 0.03-0.10M tromethamine and having a pH of 8.50-9.50; the mass ratio of the precipitate to the second dissolving solution after centrifugation is 1:5-10; The dissolution temperature is 20-30°C and the time is 1-3 hours; the centrifugal conditions are 3000-10000 rpm and 5-20 minutes; and the pore size of the filtration membrane is 0.65-1.0 μm.

4. The method for purifying fibrinogen for producing fibrin glue according to claim 1, characterized in that: In S3, the pH of the product after adjustment is 8.50 to 9.50, and the conductivity is 0.50 to 1.50 mS / cm.

5. The method for purifying fibrinogen for producing fibrin glue according to claim 1, characterized in that: In S4, the chromatographic column filler of the ion exchange chromatography is a strong anion exchange filler; In the process of ion exchange chromatography, the chromatography column is first treated with a first equilibrium solution; then, the adjusted product is loaded into the chromatography column; the chromatography column is treated with a chromatography buffer solution and a chromatography washing solution in sequence; next, the chromatography column is treated with an eluent, and the effluent after the eluent treatment is collected to obtain the product after ion exchange chromatography; The first equilibrium solution is a solution containing 0.02-0.1 M tromethamine, and the pH value is 8.50-9.50; The chromatography buffer is a solution containing 0.02-0.1 M tromethamine and has a pH of 8.50-9.50; The chromatography washing solution contains 0.02-0.1 M tromethamine, 0.008-0.015 M sodium chloride, and the pH is 8.50-9.50; The eluent contains 0.02-0.1 M tromethamine and has a pH of 7.00-8.

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6. The method for purifying fibrinogen for producing fibrin glue according to claim 1, characterized in that: In S4, the chromatography column filler for affinity chromatography is a lysine ligand affinity chromatography filler; During the affinity chromatography, the chromatography column is treated with the second equilibrium solution; then, the product after ion exchange chromatography is loaded into the chromatography column, and the flow-through liquid is collected as the product after affinity chromatography; The second balanced solution is a solution containing 0.02-0.1 M tromethamine, and has a pH of 7.00-8.

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7. A method for purifying fibrinogen for producing fibrin glue according to claim 1, characterized in that: In S5, the protein content of the product after affinity chromatography is concentrated to 23-27 g / L by ultrafiltration; the dialysis is 3-5 times and a solution containing arginine, sodium citrate and sodium chloride is used as the dialysate; the parameters of dry heat inactivation are set to 99.5-100.5 boiling water bath inactivation ≥30 min.

8. The method for purifying fibrinogen for producing fibrin glue according to claim 1, characterized in that: In S1, the component I precipitate is prepared by the following method: the raw plasma is diluted with 2.0-8.0° C. water for injection to a protein content of 40-65 g / L, the conductivity is adjusted to 12.0-14.0 mS / cm, the pH is adjusted to 6.80-7.30, and the volume percentage of ethanol is adjusted to 7%-10%, and the reaction is stirred for 1-3 hours and then filtered to obtain the component I precipitate; The cryoprecipitate is prepared by the following method: the raw plasma is centrifuged at 12000 rpm / min and 0-2°C, and the precipitate is collected after centrifugation to obtain the cryoprecipitate.

9. A fibrinogen product obtained by the fibrinogen purification method for producing fibrin glue according to any one of claims 1 to 8.

10. The fibrinogen product obtained by the fibrinogen purification method for producing fibrin glue according to claim 9, characterized in that: The fibrinogen purity is ≥95%; the content of coagulation factor XIII is ≥10 IU / ml; and the content of plasminogen is ≤0.5 μg / ml.

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