A fibrinogen preparation process based on chromatographic methods

Abstract

The present application relates to the technical field of blood product production, and particularly relates to a fibrinogen preparation process based on a chromatography method, comprising the following steps which are sequentially performed: treating blood plasma with ethanol and obtaining component I precipitate through pressure filtration or performing low-temperature centrifugal treatment on the blood plasma to obtain a cold precipitate; washing the precipitate multiple times with a precipitate washing solution to obtain a washed precipitate; adding a precipitate dissolving solution to the washed precipitate and filtering to obtain a filtrate; performing inactivation operation on the filtrate to obtain an inactivated filtrate; adjusting the pH and conductivity of the inactivated filtrate and then filtering; loading the product into an anion exchange chromatography column, sequentially loading a chromatography buffer, a chromatography washing solution and an eluent; and performing ultrafiltration treatment on the eluent to obtain a target product. The technical solution can solve the technical problem that the purity and yield of fibrinogen obtained by the existing preparation process are not ideal, and at the same time, the process flow is simplified, the process safety factor is improved, and the technical solution has a good application and promotion prospect.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of blood product production, and particularly relates to a fibrinogen preparation process based on a chromatographic method. BACKGROUND

[0002] Fibrinogen (Fg), also known as coagulation factor I (F I), is one of the "central" proteins in the blood coagulation system, has a relative molecular weight of 340 kDa, 2964 (6) amino acid residues, a molecular length of about 45 nm, a maximum width of 4.8 nm, a half-life of 96-144 hours, an isoelectric point of 5.1-5.5, and a sedimentation coefficient of 7.7-7.9. Fg is mainly synthesized by the liver, is a glycoprotein containing 3%-5% of carbohydrates, and is composed of two identical parts, each part containing three peptide chains, namely Aa, Bb, and g chains, which are composed of 610 / 461 and 410 amino acid residues, respectively. Each part is connected by 12 disulfide bonds, and the two parts are connected by three disulfide bonds formed by cysteine 8, 9 of the two g chains and cysteine 28 of the Aa chain.

[0003] Most of the Fg exists in the human body plasma, about 15% exists outside the blood vessels, the content of Fg in the normal human plasma is 2.0-4.0 g / L, is the main determinant of plasma viscosity, and the required amount for hemostasis is about 25%-50% of the normal amount, and the plasma level below 1-1.5 g / L can cause coagulation disorders. Fibrinogen is cleaved to release fibrinopeptide A and B under the action of thrombin, forms fibrin, and together with platelets forms a stable fibrin thrombus to complete the hemostasis mechanism. Fg is prepared from the plasma of healthy people by separation, purification and virus inactivation treatment, is mainly used in the treatment of congenital and acquired fibrinogen deficiency or deficiency, such as severe liver damage, cirrhosis, DIC, and postpartum massive hemorrhage, and is one of the essential emergency medicines for massive hemorrhage hemostasis in clinical application. In addition, it is reported in the literature that Fg also has the ability to induce smooth muscle cell proliferation and chemotaxis to promote the repair of damaged tissues. It can be seen that fibrinogen plays an important role in clinical application.

[0004] The raw materials for extracting fibrin mainly include two kinds of component I (F I) precipitate produced by low-temperature ethanol process and plasma cryoprecipitate. Most blood product manufacturers at home and abroad basically use F I precipitate as the raw material for extracting human fibrin. F I precipitate mainly contains fibrinogen, F VIII, fibronectin Fn and other components. Cryoprecipitate is a white flocculent precipitate produced after fresh frozen plasma is thawed at low temperature, which contains rich F VIII, von Willebrand factor, fibrinogen, fibronectin and the like. At the same time, cryoprecipitate is also the main starting raw material for preparing F VIII.

[0005] In terms of purification technology, low-temperature ethanol method is the basis of the world's plasma protein separation industrial production. With the progress of science and technology, the separation and purification methods of blood products are increasing, such as centrifugation method, caprylate precipitation method, pressure filtration method, chromatography method, ultrafiltration method, etc. The methods for extracting and purifying fibrinogen include low-temperature ethanol precipitation method and glycine precipitation method, which are used alone or in combination. Some researchers have further introduced chromatography technology to improve the purity of the product. For example, Chinese patent CN101703763A (Production method of human fibrinogen) reports the application of DEAE Sephadex A50 gel adsorption in the preparation of fibrinogen. Chinese patent CN103351432A (Process for extracting human coagulation factor VIII and human fibrinogen from plasma component precipitate) reports the application of aluminum hydroxide gel in the preparation of fibrinogen. However, the existing methods have various shortcomings: the existing preparation methods have the problems of unsatisfactory fibrinogen yield and purity; the ethanol precipitation method uses a large amount of ethanol, which poses a hidden danger to the safety of process operation; the mechanical strength of DEAE Sephadex A50 gel in the swollen state is poor, and the volume and flow rate may change during column chromatography, resulting in reduced process controllability and affecting product quality; special materials such as DEAE Sephadex A50 and aluminum hydroxide adsorbents may also contaminate the final product and affect product quality.

[0006] In summary, there is an urgent need to develop a new preparation process for fibrinogen to improve the yield and purity of fibrinogen and meet the market demand for high-quality blood products. SUMMARY

[0007] The present application aims to provide a fibrinogen preparation process based on chromatography method to solve the technical problem of unsatisfactory purity and yield of fibrinogen obtained by the existing preparation process.

[0008] To achieve the above-mentioned purpose, the present application adopts the following technical solutions:

[0009] A fibrinogen preparation process based on chromatography method, comprising the following steps in sequence:

[0010] S1 washing precipitate: using precipitate washing liquid to wash the crushed component I precipitate or cryoprecipitate for multiple times to obtain washed precipitate;

[0011] S2 dissolving precipitate: adding precipitate dissolving liquid to the washed precipitate, and after dissolving and filtering, obtaining filtrate;

[0012] S3 inactivation operation: inactivating the filtrate to obtain inactivated filtrate; adjusting the pH and conductivity of the inactivated filtrate, and then filtering to obtain adjusted filtrate;

[0013] S4 chromatography operation: the adjusted filtrate is loaded on an anion exchange chromatography column, and the eluent is collected after loading the chromatography buffer, the chromatography washing liquid and the eluent in sequence.

[0014] The principle and advantages of the present solution are:

[0015] In the present technical solution, fibrinogen is prepared by using component I precipitate or cold precipitate as raw material, and glycine washing and chromatography. The present patent avoids the use of large amounts of ethanol precipitation method, improves the process operation safety factor, and ensures the protein activity. The present technical solution also avoids the use of DEAE Sephadex A50, aluminum hydroxide adsorbent and other special materials, reduces the pollution of materials to the final product. The process of the present patent is simple, convenient to operate, low in equipment investment, and high in purity of the final product. The purity of fibrinogen product is required to be not less than 70% in the third edition of Chinese Pharmacopoeia, and the purity of fibrinogen produced by the present patent is more than 90%. Since fibrinogen is a large molecular long-chain protein, the protein is unstable and is prone to form a precipitate. At the same time, during the preparation of component I precipitate, it is easy to be activated by other coagulation factors to form a fibrin precipitate, so the yield is low. The present patent uses washing and precipitation process to effectively improve the yield and purity of fibrinogen. And the present technical solution can also simplify the operation steps and improve the safety factor in the production process.

[0016] Further, in S1, the component I precipitate is obtained by adjusting the protein content, conductivity, pH value and ethanol content of the plasma to 40-65 g / L, 12.0-14.0 mS / cm, 6.80-7.30 and 7-10% respectively to obtain a precipitation system; and the precipitation system is subjected to stirring reaction for 1-3 h and pressure filtration to obtain the component I precipitate.

[0017] The cold precipitate is obtained by centrifugal treatment of the plasma and taking the precipitate.

[0018] Further, in S1, the precipitate washing liquid is a solution containing 0.06±0.02 mol / L tromethamine and 1.5±0.5 mol / L glycine, and the pH value is 7.00±0.5 and the temperature is 2-8℃.

[0019] Further, in S1, the washing temperature is 0-5℃, the washing times are 2-3 times, and the washing time of each time is 30-60 min.

[0020] Further, in S2, the precipitate dissolving liquid is a buffer solution containing 0.03-0.08 M tromethamine, the pH value is 9.00-9.80, and the temperature is 20-30℃; and the dissolving time is 1-3 h.

[0021] Further, in S3, the inactivation operation adopts S / D inactivation; the S / D solution is a solution containing 110 g / L of polysorbate 80 and 33 g / L of tributyl phosphate, and the temperature is 25±5℃; the dosage of the S / D solution is one-tenth of the volume of the filtrate.

[0022] Further, in S3, the pH value of the adjusted filtrate is 9.00-9.80, and the conductivity is 1.0-3.00 mS / cm.

[0023] Further, in S4, the chromatography buffer is a buffer containing 0.03-0.08 M of tromethamine, and the pH value is 9.00-9.80; the chromatography washing solution is a buffer containing 0.01-0.03 M of sodium chloride and 0.03-0.08 M of tromethamine, and the pH value is 9.00-9.80; and the eluent is a buffer containing 0.03-0.08 M of tromethamine, and the pH value is 7.00-7.80.

[0024] Further, in S4, the chromatography buffer is a buffer containing 0.03-0.08 M of tromethamine, and the pH value is 9.00-9.80; the chromatography washing solution is a buffer containing 0.01-0.03 M of sodium glutamate and 0.03-0.08 M of tromethamine, and the pH value is 9.00-9.80; and the eluent is a buffer containing 0.03-0.08 M of tromethamine, and the pH value is 7.30-8.30, and the conductivity is adjusted to 2.5-3.5 mS / cm using sodium glutamate.

[0025] Further, it further comprises S5 ultrafiltration dialysis: the flow-through of the eluent is subjected to ultrafiltration dialysis treatment to obtain the finished product.

[0026] In summary, the technical scheme uses component I precipitate, cold precipitate as raw materials to produce fibrinogen, first uses the precipitate washing solution composed of tromethamine + glycine to wash the crushed precipitate, promotes the enrichment of fibrinogen in the precipitate, then uses the precipitate dissolving solution composed of tromethamine to dissolve the washed precipitate. After S / D inactivation and filtration, the anion exchange chromatography is used to further enrich the fibrinogen, and finally the finished product with ideal purity and yield is obtained.

[0027] The beneficial effects of the technical scheme are as follows:

[0028] (1) The process sequence of washing and precipitating first and then dissolving and S / D inactivation is adopted, the intermediate product obtained can be treated by anion exchange chromatography in one step, and the finished product meeting the requirements can be obtained. The chromatographic purification of fibrinogen in the prior art needs to adopt multiple steps including anion exchange chromatography, cation exchange chromatography and affinity chromatography. It can be seen that the technical scheme simplifies the process flow and improves the production efficiency. Due to the shortening of the process flow, the probability of introducing impurities or pollutants is reduced, and the product quality is improved.

[0029] (2) The selection of the types of the precipitate washing liquid and the precipitate dissolving liquid is very important for achieving the process purpose. The inventors found through research on a large number of candidate schemes that the precipitate washing liquid composed of tromethamine + glycine and the precipitate dissolving liquid composed of tromethamine greatly improve the product quality and process efficiency compared with other schemes, and unexpected technical effects are obtained.

[0030] (3) The use of the anion exchange chromatography of the present scheme replaces the combination of a large number of chromatography modes in the prior art, which has a relatively strict requirement on the treatment method of the sample to be chromatographed and the conditions of the anion exchange chromatography. The preparation method of the sample to be chromatographed and the types of the chromatography buffer, the chromatography washing liquid and the eluent used in the present scheme ensure that a fibrinogen product with high purity can be obtained by anion exchange chromatography, and the yield is also increased.

[0031] (4) Due to the shortening of the process flow and the special process flow of the present scheme, the activity of the obtained fibrinogen is greatly improved, which is significantly better than similar products.

[0032] (5) The present technical scheme can reduce the frequency of using low-temperature ethanol precipitation method. After obtaining component I precipitate, the method is no longer used, which greatly improves the process safety and reduces the construction cost of the explosion-proof workshop.

[0033] (6) In addition to using conventional sodium chloride as the elution buffer, it is found that replacing sodium chloride with sodium glutamate achieves satisfactory results. As an amino acid buffer salt, the ionic strength of sodium glutamate in solution changes more slowly than sodium chloride. For ion exchange chromatography, the charge change is more moderate. As a step elution salt, sodium glutamate has a better effect than sodium chloride. BRIEF DESCRIPTION OF DRAWINGS

[0034] Figure 1 The figure is the elution spectrum of TAME used as the elution buffer of the chromatography column for experimental example 3 (showing the linear elution peak of conductivity). DETAILED DESCRIPTION

[0035] The application will be further described in detail below with reference to the examples, but the embodiments of the application are not limited thereto. If not specifically indicated, the technical means used in the following examples and experimental examples are conventional means known to those skilled in the art, and the materials, reagents and the like used can be obtained from commercial channels.

[0036] Embodiment

[0037] A fibrinogen preparation process based on chromatography method, the general process is as follows:

[0038] (1) Plasma fusion: select healthy human plasma, thaw for 3-10 h at-5-5℃, disinfect the surface of the plasma bag with 75% ethanol solution, then break the bag, mix and dissolve the plasma at-5-5℃. The plasma used in this step is the human plasma referred to in Chinese Pharmacopoeia: the human plasma for blood plasma product production is the healthy human plasma collected by apheresis for the production of plasma protein products, which is the supernatant after centrifugation of blood to remove cells, containing proteins, inorganic salts and water, etc., and does not contain blood cells.

[0039] (2) Preparation of component I precipitate: after dissolution, the plasma is diluted with 2.0-8.0℃ water for injection to a protein content of 40-65 g / L, and the conductivity is adjusted to 12.0-14.0 mS / cm with sodium chloride particles. The pH of the plasma is adjusted to 6.80-7.30 with 1.0 mol / L acetic acid solution, the ethanol concentration in the product is adjusted to 7%-10% (volume percent), and the reaction is stirred for 1-3 h, then filtered and separated to obtain component I precipitate. The method for obtaining component I precipitate is a conventional means of the prior art, and the present technical solution mainly studies how to separate and purify fibrinogen from component I precipitate after obtaining it.

[0040] (3) Component I precipitate washing: the component I precipitate is crushed, 10-15 times the amount of precipitate (mass) of precipitate washing solution (0.06±0.02 mol / L tris + 1.5±0.5 mol / L glycine, pH 7.00±0.5, 2-8℃) is added, the temperature is controlled at 0-5℃, and the washing is stirred for 30-60 min. After washing, centrifugation is performed at 4000 rpm for 20 min, and the washing is performed for 2-3 times.

[0041] (4) Component I precipitate dissolution: 5-8 times the amount of precipitate (mass) of precipitate dissolution solution (0.03-0.08 M tris, pH 9.00-9.80, 20-30℃) is added to the component I precipitate after washing and centrifugation, and the temperature is maintained for 1-3 h for dissolution.

[0042] (5) Filtration: after dissolution, the product is filtered using a deep filter (the filtration operation is performed to achieve a clear effect, and the pore size is 1 μm) to obtain a filtrate.

[0043] (6) S / D inactivation: according to the volume of the product (filtrate), slowly add one-tenth of the volume of the product with S / D solution (containing 110 g / L polysorbate 80 and 33 g / L of tributyl phosphate, temperature 25±5℃), obtain the inactivated filtrate.

[0044] (7) Product adjustment: adjust the pH of the filtrate to 9.00-9.80 and the conductivity to 1.0-3.00 mS / cm with 0.5M NaOH. Filter the product with a 0.2 μm filter core (pre-filtering with filter plates or large pore filter cores can be used first), and obtain the adjusted filtrate.

[0045] (8) Anion exchange chromatography: anion exchange chromatography fillers such as TMAE chromatography filler, Q chromatography filler (ligand is quaternary amino group, for example: Nanogle 50Q), DEAE chromatography filler (ligand is diethylaminoethyl, for example: Unigel 80DEAE) can be selected, and conventional means in the prior art are used to pack and chromatograph in the chromatography column, and anion exchange chromatography is carried out. As a specific example, in the subsequent examples, experimental examples and comparative examples, if no special instructions are given, Fractgel EMD TMAE(M) gel is used for chromatography purification. The various anion exchange chromatography fillers mentioned above can all achieve the same separation effect. Before loading, the chromatography column is treated with 3-8 column bed volumes of solution (composition: 0.03-0.08M Tris solution, pH 9.00-9.80), and the chromatography column is balanced by conventional means in the prior art, and then the product (i.e. the adjusted filtrate in (7)) is loaded. The subsequent examples and comparative examples use a unified column balancing method, i.e. the chromatography column is balanced with 8 column bed volumes of solution (composition same as the elution buffer). The loading capacity of the product is not higher than 15 g of protein / L of gel, the loading flow rate is not higher than 120 cm / h, 3-8 column bed volumes of buffer (chromatography buffer, elution buffer) (0.03-0.08M Tris, pH 9.00-9.80) are used for elution, 3-8 column bed volumes of chromatography washing solution (0.03-0.08M Tris+0.01-0.03M sodium chloride, pH 9.00-9.80) are used for washing, the elution and washing solutions are discarded, and 3-6 column bed volumes of eluent (0.03-0.08M Tris, pH 7.00-7.80) are used for elution, and the eluent (flow-through) is collected.

[0046] (9) The eluate is subjected to routine ultrafiltration dialysis using a 50KD membrane to concentrate the protein. The eluate protein is ultrafiltrated and concentrated to 15-25 g / L, and then dialyzed against an equal volume of dialysis solution (dialysis solution formula: 0.5-4.0 g / L sodium citrate + 10-17 g / L sodium chloride + 8-13 g / L arginine + 2-6 g / L glycine + 10-15 g / L sucrose, pH 7.20±0.50, adjusted to pH with hydrochloric acid, temperature 20-30°C) for 3-5 times to obtain a fibrinogen sample. The obtained fibrinogen sample is adjusted to the specified protein concentration of the finished product, and then is divided into reagent bottles to obtain a fibrinogen finished product. In the technical solution, the amount of the fibrinogen finished product divided into the reagent bottles is 25 mL, and the protein concentration is 25 g / L. The subsequent examples and comparative examples use uniform ultrafiltration and dialysis conditions, the eluate protein is ultrafiltrated and concentrated to 20 g / L, and then is dialyzed against an equal volume of dialysis solution for 4 times, and the dialysis solution formula is: 3.0 g / L sodium citrate, 15 g / L sodium chloride, 11 g / L arginine, 4 g / L glycine, 12 g / L sucrose, pH 7.20, temperature 25°C.

[0047] (10) Sample inspection:

[0048] The fibrinogen purity is detected according to the third part of the 2020 edition of the Chinese Pharmacopoeia, and the purity calculation method is as follows: the test sample is diluted to contain 2-3 mg of fibrinogen per 1 ml with 0.85%-0.90% physiological saline, the protein content P1 (general rule 0731 first method) is detected; 10 ml of the test sample solution is taken, an equal amount of a thrombin solution (containing 0.05 mmol / L calcium chloride) containing 3 IU / ml is added, and it is placed at 37°C for 20 minutes, and the precipitate is separated by centrifugation or filtration at 2500 revolutions per minute per minute, and then washed with physiological saline for 3 times, and the coagulable protein content P2 is detected, and the fibrinogen purity (%) is calculated as P2 / P1*100.

[0049] Clotting activity: the thrombin solution is diluted with physiological saline to 3 IU / ml, and the test sample solution is diluted to 3 mg / ml for standby. The thrombin solution (3 mg / ml) preheated to 37°C is added to the reaction tube, and shaken uniformly. It is placed at 37°C, and the clotting time is recorded by an automatic detection instrument. The average value of two determination results should not exceed 60 seconds.

[0050] Yield is the total amount of fibrinogen in the chromatography eluate obtained per ton of plasma.

[0051] The fibrinogen is prepared according to the above method, and the specific parameters are selected according to Table 1.

[0052] Table 1: Parameter settings of Groups 1-3 and finished product test results (*: the specification of fibrinogen finished product is: protein content 25 g / L, 25 mL per bottle, at least > 800 bottles per ton of plasma; #: at least > 70%, preferably > 85%)

[0053]

[0054] Experimental Example 1: Screening experiment for the precipitate washing liquid in step (3)

[0055] In order to improve the process stability and improve the quality of fibrinogen, the inventors tested and screened the washing liquid of component I precipitate, and the candidate precipitate washing liquid is shown in Table 2. The preparation process of fibrinogen in this experimental example is shown in Group 1 of Table 1, and the difference between different tests is the type and corresponding parameter setting of the specific precipitate washing liquid.

[0056] The experiment No. 8 is basically the same as Group 1, and the difference from Group 1 is that in "(3) Component I precipitate washing", the component I precipitate is crushed and added to the normal temperature precipitate washing liquid (25℃), then cooled to 5℃, and then stirred and washed, and after washing, the precipitate is obtained by centrifugation. That is, the precipitate washing liquid of the experiment No. 8 is not pre-cooled.

[0057] The experiment No. 9 is basically the same as Group 1, and the difference from Group 1 is that in "(3) Component I precipitate washing", the component I precipitate is added to the precipitate washing liquid, and after stirring and washing, the supernatant is discarded. That is, the solid-liquid separation is not carried out by centrifugation during the washing process.

[0058] Table 2: Candidate precipitate washing liquid, related parameter settings and fibrinogen sample test results

[0059] Number Precipitation wash composition Agitation wash temperature (°C) Fibrinogen purity (%) Fibrinogen yield (vials / ton of plasma) 1 (Group 1) 0.06 mol / L tris; 1.5 mol / L glycine; pH 7.00 5 91 950 2 0.06 mol / L sodium chloride; 1.5 mol / L glycine; pH 7.00 5 80 720 3 0.2 mol / L tris; 3 mol / L glycine; pH 7.00 5 89 1050 4 0.01 mol / L tris; 0.5 mol / L glycine; pH 7.00 5 90 550 5 0.06 mol / L tris; 1.5 mol / L glycine; pH 8.00 5 92 890 6 0.06 mol / L tris; 1.5 mol / L glycine; pH 6.00 5 87 880 7 0.06 mol / L tris; 1.5 mol / L glycine; pH 7.00 10 83 430 8 0.06 mol / L tris; 1.5 mol / L glycine; pH 7.00 5 88 500 9 0.06 mol / L tris; 1.5 mol / L glycine; pH 7.00 5 75 480

[0060] According to the experimental results of Table 2, the precipitation washing solution composed of tris and glycine can ideally ensure the purity and yield of fibrinogen product (Group 1). If the precipitation washing solution is replaced by the combination of sodium chloride and glycine, the purity and yield of fibrinogen will be significantly reduced under the present process conditions (No. 2 in Table 2), and the product will also be turbid before chromatography (i.e., turbid after washing, dissolving and filtering). The concentrations of tris and glycine are also key factors affecting the process results. The high concentration of glycine in No. 3 causes other impurities to be precipitated with fibrinogen, which increases the yield but also leads to a decrease in purity. In addition, the high concentrations of tris and glycine will introduce more impurities and cause waste of reagents, so No. 3 is not an ideal choice compared with Group 1. The low concentration of glycine in No. 4 results in poor precipitation of fibrinogen, which leads to a too low yield of fibrinogen. The pH value of the precipitation washing solution also significantly affects the performance of the finished product. The high pH value in No. 5 leads to a decrease in the yield of fibrinogen, and the low pH value in No. 6 leads to a decrease in both the purity and yield of fibrinogen.

[0061] In addition to the factors of the precipitation washing solution itself, other process operation methods also have a significant impact on the performance of the finished product. The high temperature during stirring and washing in No. 7 leads to a significant decrease in both the yield and purity. The experiment of No. 8 is not pre-cooled, which also leads to a significant decrease in both the yield and purity. The experiment of No. 9 does not use centrifugation to separate the solid and liquid during washing and precipitation, which also leads to a significant decrease in both the yield and purity.

[0062] In summary, through experimental research, we can find that by using appropriate precipitation washing solution and washing method, the fibrinogen component in component I can be fully precipitated and enriched, and other non-target proteins or other components can be removed, so that fibrinogen can be fully enriched, the purity and yield of fibrinogen can be improved, and the product quality can be improved.

[0063] Experimental Example 2: Screening experiment of the precipitation and dissolution solution for step (4)

[0064] To improve the process stability and enhance the quality of fibrinogen, the inventors tested and screened the precipitate dissolution solution of component I. For the purpose of protecting the protein in the raw material, the inventors tried to add some protective substances to the precipitate dissolution solution in order to avoid protein degradation and enhance the product quality. However, it was found through testing that the addition of protective substances would seriously interfere with the chromatography effect, resulting in a decrease in the quality and yield of the obtained fibrinogen product. For example, the inventors tried to replace the precipitate dissolution solution 0.05M tris in group 1 of Table 1 with 15 g / L sodium citrate + 8 g / L sodium chloride + 10 g / L sucrose + 0.05M tris (pH 9.5), or 15 g / L sodium citrate + 0.05M tris (pH 9.5), or 15 g / L sodium citrate + 8 g / L sodium chloride + 10 g / L sucrose (pH 7.0), or 15 g / L sodium citrate + 8 g / L sodium chloride + 8 g / L arginine (pH 7.0), or 10 g / L sodium citrate + 8 g / L sodium chloride + 4 g / L arginine (pH 7.0), with the other conditions being the same as those in group 1. However, the solution formed after the above-mentioned precipitate dissolution solution dissolved the precipitate could not be loaded for chromatography, seriously affecting the efficiency of the chromatography column, and the fibrinogen in the eluate was difficult to enrich and separate from the impure proteins. Therefore, the technical solution finally adopted a scheme without adding additional protein protective agents, and only tris solution was used to dissolve the precipitate.

[0065] In addition, the temperature of the tris solution used to dissolve the precipitate also had a great influence on the performance of the finished product. The inventors adjusted the temperature of the precipitate dissolution solution to 35°C based on group 1. At high temperature, fibrinogen was unstable and easy to precipitate. The dissolution of the precipitate was not completed, and the product was turbid after filtration, which could not proceed to the next step. The inventors adjusted the temperature of the precipitate dissolution solution to 15°C based on group 1. The solubility of fibrinogen in the tris solution was poor, and the fibrinogen precipitate could not be dissolved at this temperature. When the precipitate was dissolved and washed, the precipitate could not be completely dissolved and was still in a precipitated state. The content of fibrinogen in the dissolution solution was low, and the next step was not performed.

[0066] Experimental Example 3: Screening experiment for the chromatography conditions of step (8)

[0067] To improve the process stability and enhance the quality of fibrinogen, the inventors tested and screened various buffers used in anion exchange chromatography. The candidate buffers are shown in Table 3. The preparation process of fibrinogen in this experimental example is shown in group 1 of Table 1, and the difference between different tests is the selection of specific buffer types.

[0068] Table 3: Screening of chromatography buffer, chromatography washing solution and eluent and detection results of fibrinogen samples

[0069] Group Chromatography buffer Chromatography wash Eluate Fibrinogen purity (%) Fibrinogen yield (vials / ton of plasma) 1 (Group 1) 0.05 M Tris pH 9.50 0.05 M tris 0.02 M sodium chloride pH 9.50 0.05 M tris pH 7.50 91 950 2 0.05 M Tris pH 9.50 0.05 M Tris pH 9.50 0.05 M tris pH 7.50 80 1300 3 0.05 M tris 0.02 M sodium chloride pH 9.50 0.05 M tris 0.02 M sodium chloride pH 9.50 0.05 M tris pH 7.50 92 700 4 0.05 M Tris pH 9.50 0.05 M tris 0.02 M sodium chloride pH 9.50 0.01 mol / L sodium phosphate dibasic 0.01 mol / L sodium phosphate monobasic pH 7.50 85 600

[0070] The experiment No. 2 is based on the group 1, the chromatography washing solution is replaced by tris solution without sodium chloride. Although the yield is improved, the purity of fibrinogen is greatly reduced. Therefore, the addition of sodium chloride in the chromatography washing solution is a key factor to ensure the purity of the product. The experiment No. 3 is based on the group 1, the chromatography buffer solution is replaced by tris solution containing sodium chloride. The prepared fibrinogen product has a purity meeting the requirements, but the yield is greatly reduced. The inventor analyzes the reason that the addition of sodium chloride makes the fibrinogen and other proteins unable to be fully adsorbed to the column material, resulting in a large loss of raw materials, so the yield is greatly reduced. The experiment No. 4 adjusts the eluent. Under the condition that the pH value of the eluent is unchanged, the purity and yield of the product are greatly affected. The use of disodium hydrogen phosphate and sodium dihydrogen phosphate as the buffer solution not only introduces new impurities, but also causes the precipitation of proteins in the sample (the precipitated proteins can be observed in the flow-through of the eluent), and the protection effect on fibrinogen is not as good as tris. Therefore, the purity and yield of fibrinogen are both not ideal.

[0071] In addition to the TMAE chromatography filler, the inventor also tried other manufacturers' anion chromatography fillers, including fillers with DEAE (diethylaminoethyl) and Q (quaternary amino group) ligands, which can all achieve the same separation effect. The typical elution curve of the TAME chromatography filler is shown in Figure 1 .

[0072] The inventor also tried to replace sodium chloride in the chromatography washing solution with sodium glutamate (equivalent replacement) based on the group 1, and added sodium glutamate in the eluent, which can also separate fibrinogen and other impure proteins, and achieve a relatively ideal effect. See the experimental data in Table 4. In Table 4, the pH value of the eluent 0.05M tris is adjusted to the specified value using the conventional method, and the conductivity is adjusted to the specified level using sodium glutamate. The other process steps and reagent usage are the same as those in group 1. As can be seen from the experimental data in Table 4, the use of glutamic acid under appropriate conditions can improve the purity of fibrinogen without greatly reducing the yield. That is, the pH value of the eluent is maintained at 7.3-8.3, and the conductivity is maintained at 2.5-3.5 mS / cm, the purity of fibrinogen can reach 94-95%, and the yield can reach more than 850 bottles / ton of plasma.

[0073] Table 4: Purity and yield of sodium glutamate as eluent

[0074]

[0075] Experimental Example 4: Screening experiment for raw materials

[0076] The technical solution uses component I precipitate as raw material for purification of fibrinogen, which improves the yield of fibrinogen compared to using general cold precipitate. The specific preparation method of the cold precipitate is as follows: after the raw material blood plasma is fused, the precipitate is formed under the condition of 0-2°C and the centrifugal speed of more than 10,000 rpm. The supernatant of the centrifuged blood plasma can be used for adjusting the product parameters in the process of preparing component I precipitate and the like in the patent process. The experimental results are shown in Table 5. Using component I precipitate as raw material can improve the yield of fibrinogen, and using the process can ensure a relatively ideal purity of fibrinogen whether component I precipitate or cold precipitate is used as raw material.

[0077] Table 5: Fibrinogen yield and purity under different raw materials

[0078] Starting material Fibrinogen purity (%) Fibrinogen yield (vials / ton of plasma) Fraction I precipitate 91 950 Cryoprecipitate 91 850

[0079] Comparative Example 1

[0080] The comparative example is basically the same as group 1 in Table 1, except that the process for obtaining the product (adjusted filtrate) for chromatography before step (8) TMAE chromatography is different. The specific differences are as follows:

[0081] Referring to steps (1) and (2), component I precipitate is obtained, then referring to step (4), the component I precipitate is dissolved, then referring to steps (5) and (6), the dissolved component I precipitate is subjected to S / D inactivation to obtain inactivated filtrate. The temperature of the inactivated filtrate is adjusted to 3°C, low-temperature ethanol at -25°C is added to a final ethanol concentration of 10%, and the temperature is controlled at 3°C. After stirring for 2h, pressure filtration is performed to separate the low-temperature ethanol precipitate. Referring to steps (3) and (4), the precipitate is washed with tris+glycine and dissolved with tris. Then referring to step (7), the pH and conductivity are adjusted and filtered to obtain the product (adjusted filtrate) for chromatography.

[0082] The process sequence of S / D inactivation and glycine washing is adjusted by using the method of the comparative example, and a low-temperature ethanol precipitation step is added in the middle. The low-temperature ethanol precipitation operation requires the use of a large amount of ethanol, which has safety risks, and ethanol can affect the structure of the protein, thereby affecting the activity of the product. In addition, the change of the operation process sequence also significantly affects the product quality and production efficiency. The fibrinogen sample obtained by the method of the example has a fibrinogen purity of 92% and a fibrinogen yield of less than 500 bottles / ton of blood plasma. The low-temperature ethanol precipitation step is used after obtaining the component I precipitate in the comparative example, which increases the complexity of the process, but does not improve the purity of the finished product, and the yield is reduced.

[0083] The above-mentioned are only embodiments of the present application, and common technical solutions and / or common knowledge of the scheme are not described in detail. It should be pointed out that, for those skilled in the art, without departing from the technical solutions of the present application, a number of modifications and improvements can be made, which should also be considered as the protection scope of the present application, and these will not affect the effect and practicality of the patent. The protection scope claimed in the present application should be subject to the content of its claims, and the specific implementation mode and the like recorded in the specification can be used to explain the content of the claims.

Claims

1. A process for the preparation of fibrinogen based on a chromatographic method, characterized in that: It comprises the following steps in sequence: S1 washing the precipitate: washing the precipitate of component I or cold precipitate after crushing with precipitate washing solution for multiple times to obtain washed precipitate; The precipitate of component I is obtained by adjusting the protein content, conductivity, pH value and ethanol content of the plasma to 40-65 g / L, 12.0-14.0 mS / cm, 6.80-7.30 and 7-10% respectively to obtain a precipitate system; the precipitate system is subjected to stirring reaction for 1-3 h and pressure filtration to obtain the precipitate of component I; The cold precipitate is obtained by centrifugal treatment of the plasma and taking the precipitate; The precipitate washing solution is a solution containing 0.06±0.02 mol / L tromethamine and 1.5±0.5 mol / L glycine, and the pH value is 7.00±0.5 and the temperature is 2-8℃; S2 dissolving the precipitate: adding precipitate dissolving solution to the washed precipitate, and after dissolving and filtering, the filtrate is obtained; The precipitate dissolving solution is a buffer solution containing 0.03-0.08 M tromethamine, the pH value is 9.00-9.80 and the temperature is 20-30℃; the dissolving time is 1-3 h; S3 inactivation operation: inactivating the filtrate to obtain inactivated filtrate; Adjusting the pH and conductivity of the inactivated filtrate, and then filtering to obtain adjusted filtrate; S4 chromatography operation: loading the adjusted filtrate into an anion exchange chromatography column, loading chromatography buffer, chromatography washing solution and eluent in sequence, and collecting the flow-through of the eluent; The chromatography buffer is a buffer solution containing 0.03-0.08 M tromethamine, the pH value is 9.00-9.80; the chromatography washing solution is a buffer solution containing 0.01-0.03 M sodium glutamate and 0.03-0.08 M tromethamine, the pH value is 9.00-9.80; the eluent is a buffer solution containing 0.03-0.08 M tromethamine, the pH value is 7.30-8.30, and the conductivity is adjusted to 2.5-3.5 mS / cm using sodium glutamate.

2. A process for the preparation of fibrinogen based on a chromatographic method according to claim 1, characterized by: In S1, the washing temperature is 0-5℃, the washing times are 2-3 times, and the washing time of each time is 30-60 min.

3. A process for the preparation of fibrinogen based on chromatographic methods according to claim 1, characterized by: In S3, the inactivation operation adopts S / D inactivation; the S / D solution is a solution containing 110 g / L polysorbate 80 and 33 g / L tributyl phosphate, and the temperature is 25±5℃; the amount of S / D solution is one-tenth of the volume of the filtrate.

4. A process for the preparation of fibrinogen based on chromatographic methods according to claim 3, characterized by: In S3, the pH value of the adjusted filtrate is 9.00-9.80, and the conductivity is 1.00-3.00 mS / cm.

5. The process for the preparation of fibrinogen based on chromatographic methods according to claim 1, characterized by the fact that: It further comprises S5 ultrafiltration dialysis: performing ultrafiltration dialysis treatment on the flow-through of the eluent to obtain the finished product.

Images