Method for filtering, separating and purifying human fibrinogen from plasma

Abstract

The invention belongs to the technical field of blood product preparation, and particularly relates to a method for filtering, separating and purifying human fibrinogen from plasma. The method sequentially comprises the following steps: balancing cellulose, mixing the balanced cellulose with raw material plasma, adsorbing and filtering to obtain cellulose precipitate; leaching the cellulose precipitate, soaking the cellulose precipitate in the precipitate dissolving solution, and filtering to obtain filtrate; carrying out first ion exchange chromatography on the filtrate; and carrying out secondary ion exchange chromatography to obtain a protein solution with fibrinogen purity of more than 80%. According to the technical scheme, the technical problems that in the prior art, the process steps of purifying and enriching the human fibrinogen from the plasma cryoprecipitate are tedious, and the utilization rate of the human fibrinogen is not ideal can be solved. According to the cryoprecipitated protein solution prepared by the method, VIII factor, vWF factor and fibrinogen products can be separated at the same time through chromatography, the utilization rate of plasma protein is improved, and the method has ideal popularization and application prospects.

Description

Technical Field

[0001] This invention belongs to the field of blood product preparation technology, specifically relating to a method for filtering, separating, and purifying human fibrinogen from plasma. Background Technology

[0002] Cryoprecipitate refers to the cold-insoluble precipitate formed in plasma under low-temperature conditions. In 1959, American physician J.P. Pool first observed a small amount of cold-insoluble precipitate appearing when frozen plasma thawed at 4°C. Pool, along with Shannon et al., further developed a method in 1965 for preparing cryoprecipitate preparations from single plasma samples using a closed, sterile system. Cryoprecipitate is mainly rich in human coagulation factor VIII, human fibrinogen, and fibronectin. Simultaneously, cryoprecipitate preparations are also used to prepare coagulation factor VIII preparations for treating hemophilia A. The main disadvantages of using cryoprecipitate in clinical treatment are the variable potency of the preparation, the large volume required for each transfusion, the low purity of the preparation, the presence of particulate matter and blood type antibodies, the occasional occurrence of allergic reactions, and the fact that non-lyophilized cryoprecipitate preparations prepared by blood banks and central blood stations generally require low-temperature (-20°C) refrigeration.

[0003] Human fibrinogen (Fg) is a protein component in blood plasma, with a concentration as high as 2-4 g / L. Fg plays a crucial role in the coagulation system; in the final stage of coagulation, Fg is converted into fibrin by thrombin. Fibrin then aggregates with other blood cell components to form insoluble clumps, thus achieving hemostasis. Traditional human fibrinogen production processes currently mainly employ cryogenic precipitation and FI precipitation (using acid precipitation and low-temperature ethanol precipitation), which suffer from drawbacks such as low solubility, long reconstitution time, and numerous impurities. Currently, the domestic supply of processed human fibrinogen remains insufficient.

[0004] Currently, cryoprecipitates separated by centrifugation are generally used in the production of coagulation factor VIII, while fibrinogen is treated as waste during production. This results in the underutilization of plasma raw materials. Traditional human fibrinogen production processes, involving acid precipitation and low-temperature ethanol precipitation, suffer from low solubility and low dissolution efficiency. Therefore, it is evident that human fibrinogen in plasma is not being fully utilized, and the production efficiency of human fibrinogen is unsatisfactory. There is an urgent need to optimize the process for purifying and enriching human fibrinogen from plasma to meet market demand for human fibrinogen products. Summary of the Invention

[0005] The present invention aims to provide a method for filtering, separating and purifying human fibrinogen from plasma, in order to solve the technical problems of the cumbersome process steps and unsatisfactory utilization rate of human fibrinogen in the existing technology for purifying and enriching human fibrinogen from plasma cryoprecipitate.

[0006] To achieve the above objectives, the present invention adopts the following technical solution:

[0007] A method for filtering and purifying human fibrinogen from plasma, comprising the following steps performed sequentially:

[0008] S1: After equilibration, cellulose is mixed with raw plasma, stirred, and then filtered to obtain cellulose precipitate; after rinsing the cellulose precipitate, it is soaked in the precipitate dissolving solution and filtered to obtain the filtrate;

[0009] S2: Perform the first ion exchange chromatography on the filtrate;

[0010] S3: Perform a second ion exchange chromatography on the product of S2 to obtain a protein solution with fibrinogen purity >80%.

[0011] Furthermore, in S2, the chromatography column used for the first ion exchange chromatography is filled with DEAE chromatography packing material;

[0012] First, the chromatography column is treated with the first equilibration solution, then the filtrate is loaded into the chromatography column, and the flow-through is collected to obtain fibrinogen intermediate material I; fibrinogen intermediate material I is filtered, ultrafiltered, dialyzed and pH adjusted to obtain fibrinogen intermediate material II.

[0013] Furthermore, in S2, the first equilibration solution contains 0.015M-0.025M tris(hydroxymethyl)aminomethane, 0.08-0.12M alanine, 0.10-0.14M glycine, and 0.09-0.15M sodium chloride, with a pH of 6.70-7.00 and a conductivity of 14mS / cm-16mS / cm.

[0014] Furthermore, in S2, the pH of fibrinogen intermediate II is 8.5-9.5.

[0015] Furthermore, in S3, the chromatographic column used for the second ion exchange chromatography is TMAE chromatographic packing material;

[0016] First, the chromatography column is treated with the second equilibration buffer, and then the fibrinogen intermediate material II is loaded into the chromatography column; then the chromatography column is treated with the second equilibration buffer and washing buffer, and finally the chromatography column is eluted with elution buffer to collect fibrinogen intermediate material III; fibrinogen intermediate material III is filtered, ultrafiltered and dialyzed to obtain fibrinogen product.

[0017] Furthermore, in S3, the second equilibration solution contains 0.05M tris(hydroxymethyl)aminomethane, with a pH of 8.50-9.50; the washing solution contains 0.05M tris(hydroxymethyl)aminomethane and 0.05M sodium chloride, with a pH of 8.5-9.5; and the eluent contains 0.05M tris(hydroxymethyl)aminomethane, with a pH of 6.5-7.50.

[0018] Furthermore, in S1, the amount of cellulose used is 2-4 g cellulose / L plasma;

[0019] The method for rinsing cellulose precipitate is as follows: the cellulose precipitate is rinsed sequentially with a first washing solution and a second washing solution;

[0020] The first washing solution is a 10mM sodium citrate solution with a pH of 7.50-7.80; the second washing solution is a solution containing 10mM sodium citrate and 2M glycine with a pH of 7.10-7.30.

[0021] Furthermore, the precipitate solution contains 0.015-0.025M tris(hydroxymethyl)aminomethane, 10-20mM sodium citrate, 0.05-0.15M alanine, and 0.09M-0.15M sodium chloride, with a pH of 6.60-7.00 and a conductivity of 10.0mS / cm-17.0mS / cm.

[0022] Furthermore, the cellulose precipitate after rinsing is soaked in a precipitation dissolving solution at 31-35℃ for 10-50 minutes; the ratio of the amount of cellulose precipitate after rinsing to the amount of precipitation dissolving solution is 1:8-12.

[0023] This technical solution also provides a protein solution rich in human fibrinogen obtained according to a method for filtering, separating, and purifying human fibrinogen from plasma.

[0024] The principle behind this solution is:

[0025] This technical solution involves adding cellulose to cryogenic plasma and separating the cellulose precipitate adsorbed with plasma cryoprecipitate using methods such as filtration or pressure filtration. This yields a cellulose precipitate adsorbed with cryoprecipitates such as factor VIII, vWF factor, fibrinogen, and fibronectin. After dissolution, the cellulose precipitate can be further purified and enriched for fibrinogen. The dissolved precipitate is then separated from coagulation factor VIII and fibrinogen using 650M chromatography media, and further purified using chromatography to obtain the human fibrinogen product.

[0026] This technical solution provides a simple and easy method for separating and obtaining cryoprecipitate from raw plasma, which significantly reduces costs compared to conventional low-temperature centrifugation. This separation method eliminates the need for a low-temperature centrifuge, reducing equipment investment, and recognizing that centrifuges are high-speed rotating devices with inherent operational risks.

[0027] Factor VIII, vWF, fibrinogen, and fibronectin, all cold-insoluble proteins adsorbed on cellulose, exhibit good solubility in the precipitate dissolution solution of this method. In contrast, plasma cryoprecipitate obtained by low-temperature centrifugation has very poor solubility, requiring pre-melting, pulverization, washing, and dissolution steps to achieve dissolution. The cryoprecipitate separated by this method, due to the addition of cellulose, forms loose channels during its formation, making it easy to disperse during dissolution. Fiber precipitates containing coagulation factor VIII do not require pre-melting or pulverization, simplifying the operation, providing a more controllable production environment, reducing the possibility of microbial introduction, and ensuring product quality. Cellulose is widely distributed in nature, insoluble in water and common organic solvents, and is a major component of plant cell walls. Cellulose is the most abundant natural organic compound in the world. It is a fibrous, capillary-rich linear polymer composed of many β-D-glucose groups linked by 1,4-glycosidic bonds. Its porous structure, large surface area, and amphiphilic nature give it a certain degree of adsorption capacity. Cellulose is relatively inexpensive to obtain, making it particularly suitable for industrial production. The cellulose referred to in this invention is not limited to any one type of cellulose, nor is it limited to any particular form of cellulose.

[0028] Compared with the prior art, the present invention has the following advantages:

[0029] (1) This separation method does not require the use of a centrifuge, reducing equipment investment and saving costs.

[0030] (2) The production of fiber precipitates containing cryoprecipitates is simple, safer, and easier to mass-produce.

[0031] (3) The cryoprecipitate fibers produced by this method can be directly dissolved without pre-melting and crushing, making the operation simple, the production environment more controllable, the possibility of introducing microorganisms low, and the product quality more guaranteed. The new process can effectively improve the complex steps of acid precipitation and at the same time, improve the utilization rate of cryoprecipitate proteins.

[0032] (4) The cryoprecipitate protein solution prepared by this method can be separated from the products of factor VIII, factor vWF and fibrinogen by chromatography, thereby improving the utilization rate of plasma proteins. Through further purification of fibrinogen, its purity can reach more than 81%. Attached Figure Description

[0033] Figure 1 This is a photograph of a typical cellulose precipitation (cellulose adsorption precipitation) of Example 1.

[0034] Figure 2 This is a photograph of a typical cellulose precipitate dispersed in a precipitate dissolution solution, as shown in Example 1.

[0035] Figure 3 This is a photograph of the solution state after typical cellulose precipitation dissolution and filtration in Example 1.

[0036] Figure 4 This is a photograph of typical plasma centrifugation cryoprecipitate cryopreservation in Comparative Example 1. Detailed Implementation

[0037] The present invention will be further described in detail below with reference to embodiments, but the implementation of the present invention is not limited thereto. Unless otherwise specified, the technical means used in the following embodiments are conventional means well known to those skilled in the art; the experimental methods used are all conventional methods; and the materials, reagents, etc. used are all commercially available.

[0038] Example 1

[0039] A process for producing fibrinogen from blood plasma, the general process flow is as follows:

[0040] (1) Raw material plasma processing: Raw material plasma stored at -30℃ is placed at -5℃ overnight after being taken out of storage. Before use, the surface of the plasma bag is disinfected with 70%-75% ethanol solution, and then the plasma bag is broken and thawed at 0-4℃. The thawed plasma (hereinafter referred to as mixed plasma) is taken for testing of plasma coagulation factor VIII, fibrinogen, fibronectin, and von Willebrand factor (vWF).

[0041] Raw plasma is the supernatant after centrifuging blood to remove cells. It contains proteins, inorganic salts, and water, but no blood cells. More specifically, raw plasma refers to human plasma as defined in the Chinese Pharmacopoeia: human plasma used in the production of blood products is plasma from healthy individuals collected using apheresis to produce plasma protein products.

[0042] (2) Lower the plasma temperature to -2℃-2℃, add the cellulose that has been balanced with the fiber balancing solution (this technical solution specifically uses ordinary paper fiber filter plates produced by Shenyang Great Wall Fiber Filter) while stirring, and stir for 10-30 minutes (the specific stirring time in subsequent experimental studies is 15 minutes). Stir to make the cellulose evenly dispersed in the plasma, and maintain the temperature at -2℃-2℃ during the stirring process.

[0043] The fiber balancing solution is formulated as follows: 10mM-20mM sodium citrate + 0.1M-0.14M sodium chloride, pH 7.50-7.90 (adjusted with citric acid), conductivity 11mS / cm-14mS / cm. The cellulose to plasma ratio is 2-4g cellulose / L plasma (preferably 3g cellulose / L plasma).

[0044] Cellulose is widely distributed in nature. It is a fibrous, capillary-rich linear polymer composed of many β-D-glucose groups linked by 1,4-glycosidic bonds. It possesses porous structure and a large surface area, and contains many hydrophilic hydroxyl groups, thus exhibiting a certain degree of adsorption. Cellulose is insoluble in water and common organic solvents, is a major component of plant cell walls, and is the most abundant natural organic compound in the world. Cellulose is relatively inexpensive to obtain, making it particularly suitable for industrial production. The cellulose referred to in this invention is not limited to any particular type or form.

[0045] (3) Filter the cellulose-containing plasma using a cellulose filter plate or membrane. The component that cannot pass through the filter membrane after separation (named cellulose precipitate) undergoes further processing. The filter membrane material is not limited (polyethersulfone, cellulose acetate, etc., can be used; cellulose acetate membranes were specifically used in subsequent experiments). The pore size of the filter membrane can be 1-10 μm. A typical cellulose precipitate (fiber adsorption precipitate) is photographed as an example; see [link to example]. Figure 1 This image is only used to show the general appearance of the cellulose precipitate. After filtration, the portion of the cellulose precipitate that could not pass through the filter membrane was rinsed using the first and second wash solutions sequentially. Samples were then taken for fibrinogen and fibronectin detection.

[0046] The first wash solution is used at a volume of 150-300 mL per 1 L of raw plasma, with a flow rate of 200-400 mL / min. The solution consists of 10 mM sodium citrate, and the pH is adjusted to 7.50-7.80 using citric acid. The solution is pre-cooled in a refrigerator at 2-8℃ before use.

[0047] The second wash solution is used at a volume of 200-450 mL per 1 L of raw plasma, with a flow rate of 200-400 mL / min. The composition is 10 mM sodium citrate + 2 M glycine. The pH value is adjusted to 7.10-7.30 (at 25℃) using sodium hydroxide. The solution is pre-cooled in a refrigerator at 2-8℃ before use.

[0048] (4) Remove the cellulose precipitate after top washing and dissolve the soluble components in the cellulose precipitate using a precipitate dissolving solution. The dissolving temperature is 31-35℃ (water bath), and the solution is stirred continuously for 10-50 minutes (45 minutes for subsequent experimental studies). The volume of the precipitate dissolving solution is 8-12 times the weight of the precipitate, and the formula is: 0.015-0.025M tris(hydroxymethyl)aminomethane + 10-20mM sodium citrate + 0.05-0.15M alanine + 0.09M-0.12M sodium chloride, pH 6.60-7.00, conductivity 10.0mS / cm-17.0mS / cm. See typical images of cellulose adsorbing plasma and then dissolving. Figure 2This image is only used to show the approximate appearance of the fibrin after it has adsorbed plasma and then been dissolved in a precipitate-dissolving solution. After the above process is completed, the fibrin is removed by silk filtration. The filtrate is a protein solution rich in coagulation factor VIII and human fibrinogen (see typical image). Figure 3 The image shows the filtrate after silk filtration (this image is only used to show the general appearance of the filtrate) and is used for further separation and enrichment of human fibrinogen.

[0049] (5) First Ion Exchange Chromatography: The filtrate obtained in (4) was loaded onto a TOYOPEARL DEAE 650M column (anion exchange chromatography column, DEAE chromatography packing material, ligand: diethylaminoethyl). In the first ion exchange chromatography, the formulation of the first equilibration solution was 0.015M-0.025M tris(hydroxymethyl)aminomethane + 0.08-0.12M alanine + 0.10-0.14M glycine, the pH was adjusted to 6.70-7.00 with hydrochloric acid, the preferred conductivity was 14mS / cm-16mS / cm, and 0.09-0.15M sodium chloride. After the first ion exchange chromatography, the flow-through liquid after loading the filtrate was collected as fibrinogen intermediate raw material I.

[0050] (6) The collected flow-through (fibrinogen intermediate material I) is filtered through a 0.22 μm filter cartridge, then concentrated 5-8 times using a 100 KD ultrafiltration membrane, and dialyzed 5 times with dialysis buffer. The dialysis buffer used in this step is a 0.05 M tris(hydroxymethyl)aminomethane solution, with the pH adjusted to 6.5-7.5 by hydrochloric acid. After the conventional filtration, ultrafiltration, and dialysis processes described above, the material is further adjusted for pH and diluted. Specifically, a diluent is used to further dilute the ultrafiltration and dialysis material to 3-6 times its original concentration. The diluent is a 0.5 M tris(hydroxymethyl)aminomethane solution, with the pH adjusted to 9.20-9.50 by citric acid. The pH of the diluted material (referred to as fibrinogen intermediate material II) is controlled at 8.5-9.5 for the next step of chromatography.

[0051] (7) Second ion exchange chromatography: Fibrinogen intermediate II was loaded onto a Fractgel EMD TMAE column (anion exchange chromatography column). In the second ion exchange chromatography, the second equilibration buffer was formulated with 0.05M tris(hydroxymethyl)aminomethane, and the pH was adjusted to 8.50-9.50 with citric acid. The washing buffer was formulated with 0.05M tris(hydroxymethyl)aminomethane + 0.05M sodium chloride, and the pH was adjusted to 8.5-9.5 with citric acid; the fibrinogen eluent was formulated with 0.05M tris(hydroxymethyl)aminomethane, and the pH was adjusted to 6.5-7.50 with citric acid. After elution, the eluent was collected to obtain fibrinogen intermediate III.

[0052] (8) Ultrafiltration Dialysis: The fibrinogen eluent (fibrinogen intermediate material III) was collected and concentrated 5-8 times using a 100KD ultrafiltration membrane, followed by 5-fold dialysis. The dialysis solution used in this step was a conventional formula containing sodium citrate, sodium chloride, arginine, and glycine, with a pH of 6.5-7.5. This step was mainly used to remove small molecule impurities and purify the protein. After completing the above process, a fibrinogen sample was obtained for fibrinogen purity testing, i.e., detecting the percentage of coagulated fibrinogen in the fibrinogen sample relative to the total protein.

[0053] Fibrinogen was isolated and enriched using the following specific method (hereinafter referred to as: specific experimental study):

[0054] S1: Preparation of a protein solution rich in coagulation factor VIII and human fibrinogen:

[0055] The raw plasma was thawed using the above-mentioned conventional method. A sample of the thawed raw plasma was retained for testing of coagulation factor VIII, human fibrinogen, and von Willebrand factor (vWF). The plasma was measured into a container, and the container containing the plasma product was placed in an ice bath for water bath stirring and temperature control. The temperature control range for adsorption and filtration was within -2℃ to 2℃. The amount of cellulose added was 2g cellulose / L plasma (or 3g cellulose / L plasma, or 4g cellulose / L plasma). The cellulose (paper fiber filter plate) was thoroughly wetted with cellulose equilibration solution to ensure uniform dispersion. The equilibration solution was then drained, and the cellulose from the drained equilibration solution was added to the plasma. The mixture was stirred at low temperature for 15 minutes for adsorption. The mixture of cellulose and plasma was then filtered through a 10μm filter membrane to separate the cellulose precipitate and the filtered liquid fraction. The precipitate was washed with a first wash buffer (260 mL per 1 L of raw plasma, flow rate 300 mL / min) and a second wash buffer (430 mL per 1 L of raw plasma, flow rate 300 mL / min). The filtered plasma was retained for testing of human fibrinogen content. The cellulose precipitate after washing was removed and dissolved in 10 times its weight of precipitate dissolving solution in a 34°C water bath with stirring for 45 min. The cellulose was removed by silk filtration. The filtrate was a protein solution rich in coagulation factor VIII and human fibrinogen, which was used for further fibrinogen separation.

[0056] The specific details of the solutions used in the above process are as follows:

[0057] The specific formula for the fiber balancing solution is: 15mM sodium citrate, 0.12M sodium chloride, with citric acid used to adjust the pH to 7.70 and the conductivity to approximately 13mS / cm, at a temperature of 2℃ (refrigerated).

[0058] The specific formula for the first wash solution is: 10mM sodium citrate, with the pH adjusted to 7.70 using citric acid, and the temperature set at 2°C (refrigerated).

[0059] The specific formula for the second wash solution is: 10mM sodium citrate, 2M glycine, pH adjusted to 7.20 (at 25℃) with sodium hydroxide, and the temperature is 2℃ (refrigerated).

[0060] The specific formulation of the precipitation solution is as follows: 0.020M tris(hydroxymethyl)aminomethane, 10mM sodium citrate, 0.1M alanine, and 0.1M sodium chloride, with a pH of 6.80 and a conductivity of approximately 14.0 mS / cm. Since the focus of this scheme is on the enrichment of fibrinogen, the above composition of the precipitation solution can further optimize the subsequent enrichment of fibrinogen.

[0061] S2: First ion exchange chromatography

[0062] In the specific operation process, the specifications of the TOYOPEARL DEAE 650M column are: 26mm / 40cm, and the amount of the first equilibration solution is 4-6 column volumes (CV, 5CV is used in subsequent specific experimental studies). The loading amount of the filtrate rich in human fibrinogen obtained in step (4) is: the mass ratio of filtrate to packing material is 1:16-35 (1:25 is used in subsequent specific experimental studies). After loading, the flow-through liquid is collected to obtain fibrinogen intermediate raw material I.

[0063] The first equilibration solution consists of 0.02M tris(hydroxymethyl)aminomethane, 0.1M alanine, and 0.12M glycine, with the pH adjusted to 6.60-7.00 by hydrochloric acid, and a conductivity of 14-16 mS / cm (0.09-0.15M sodium chloride). For details on the determination of the specific conductivity and pH, please refer to Table 2.

[0064] After conventional filtration, ultrafiltration for 6-fold concentration and dialysis (see step (6) above, dialysate: 0.05M tris(hydroxymethyl)aminomethane solution, pH 7.0), fibrinogen intermediate material I was diluted 5-fold with diluent (0.5M tris(hydroxymethyl)aminomethane, pH 9.4) and its pH was adjusted to 9.4 and its conductivity to 1.5 mS / cm for the next chromatography experiment to obtain fibrinogen intermediate material II.

[0065] S3: Second ion exchange chromatography

[0066] In the specific operation, the Fractgel EMD TMAE column specifications are: 50mm / 40cm, and the volume of the second equilibration buffer is 4-6 column volumes (CV, 5CV was used in subsequent experimental studies). The loading amount of fibrinogen intermediate material II obtained in the previous step is: the mass ratio of fibrinogen intermediate material II to packing material is 1:4-20 (1:15 was used in subsequent experimental studies). After loading, it is first washed with 2 column volumes of the second equilibration buffer, then washed with 2 column volumes of washing buffer, and then eluted with 5 column volumes of elution buffer. After elution, the eluent is collected to obtain fibrinogen intermediate material III.

[0067] The second equilibration solution is: 0.05M tris(hydroxymethyl)aminomethane, with citric acid used to adjust the pH to 9.30.

[0068] The washing solution consists of 0.05M tris(hydroxymethyl)aminomethane + 0.05M sodium chloride, with citric acid used to adjust the pH to 9.3.

[0069] The eluent is specifically composed of 0.05M tris(hydroxymethyl)aminomethane, with the pH adjusted to 7.4 using citric acid.

[0070] Fibrinogen intermediate material III was subjected to conventional ultrafiltration dialysis to obtain fibrinogen samples, which were then used for fibrinogen purity testing.

[0071] Experimental Example 1

[0072] Fibrinogen samples were prepared under the aforementioned parameters of 2 g cellulose / L plasma, 3 g cellulose / L plasma, and 4 g cellulose / L plasma. Fibrinogen was detected using an ELISA kit, and the fibrinogen content in the mixed plasma, filtered plasma, and precipitate solution was measured separately. The proportion of fibrinogen in the filtered plasma and precipitate solution was calculated.

[0073] The proportion of fibrinogen in filtered plasma is calculated as follows: the ratio of fibrinogen in filtered plasma to the total amount of fibrinogen in mixed plasma.

[0074] The fibrinogen content in the precipitate solution is calculated as follows: the ratio of fibrinogen in the precipitate solution to the amount of fibrinogen in the mixed plasma.

[0075] The mixed plasma is formed by melting the raw plasma and then determining its fibrinogen content. The filtered plasma includes the portion of plasma mixed with cellulose that passes through the filter membrane (the filtered liquid portion), and the portion that passes through the filter membrane after two head washes. These portions are collected and combined, and the amount of fibrinogen in the combined liquid is detected and calculated. The cellulose precipitate is removed after head washing, placed in a precipitate dissolving solution for a period of time, and then filtered through silk. The filtrate portion is used for component analysis, specifically determining the amount of fibrinogen (i.e., the amount of fibrinogen in the precipitate dissolving solution).

[0076] The fibrinogen content in the 650M flow-through solution is calculated as follows: the percentage of fibrinogen in the fibrinogen intermediate raw material I relative to the amount of fibrinogen in the filtrate (as mentioned above: a protein solution rich in coagulation factor VIII and human fibrinogen).

[0077] After the second ion exchange chromatography, the fibrinogen purity of the fibrinogen sample was determined (by integrating the chromatographic UV spectrum, the fibrinogen purity was calculated; purity is the percentage of coagulated fibrinogen in the fibrinogen sample relative to the total protein).

[0078] The experimental results for different fiber dosages are shown in Table 1 (the filtered plasma and precipitate solution obtained in step S1 were tested). All experiments were conducted in accordance with the description in the "Specific Experimental Studies" section above. The data in Table 1 mainly focus on the study of step S1. Except for the change in cellulose dosage, all other technical parameters were performed in accordance with the description in the "Specific Experimental Studies" section.

[0079] Table 1: Percentage of fibrinogen in filtered plasma and precipitate lysate with different cellulose addition levels

[0080] Experiment number Cellulose usage percentage Fibrinogen percentage in filtered plasma Precipitated solution fibrinogen content 1 2g cellulose / L plasma 59％ 40％ 2 3g cellulose / L plasma 56％ 42％ 3 4g cellulose / L plasma 61％ 37％

[0081] The experimental results above show that the preparation method described in this technical solution can enrich fibrinogen in the precipitate solution. Furthermore, the process is simple, the equipment used is basic, and it is safer and easier for large-scale, batch production. A cellulose dosage of 2-3 g cellulose / L plasma is recommended. In particular, using 3 g cellulose / L plasma allows fibrinogen in the plasma to be more efficiently adsorbed onto the cellulose, rather than remaining largely in the filtered plasma. Moreover, with a cellulose dosage of 3 g cellulose / L plasma, the proportion of fibrinogen in the precipitate solution is higher after the cellulose precipitate dissolves.

[0082] The results of the first ion exchange chromatography experiment are shown in Table 2 (using cellulose at a concentration of 3 g / L plasma). The effects of different conductivity and pH values ​​of the first equilibration solution on the purification and enrichment of fibrinogen were investigated. All experiments were conducted according to the methods described in the "Specific Experimental Studies" section above. The data in Table 2 mainly focus on step S2 (first equilibration solution). Except for the changes in pH and conductivity of the first equilibration solution, all other technical parameters were performed as described in the "Specific Experimental Studies." It is evident that a combination of conductivity and pH of 15.5 mS / cm and 7.0, or a combination of 16.0 mS / cm and 6.5, is optimal for the second equilibration solution.

[0083] Table 2: Fibrinogen content in 650M eluate after the first ion exchange chromatography

[0084] Experiment number pH value of equilibrium solution Equilibrium fluid conductivity (mS / cm) 650M flow-through fluid fibrinogen percentage 1 6.8 14.0 98％ 2 6.8 15.3 95％ 3 6.8 16.0 99％ 4 6.6 15.3 94％ 5 7.0 15.5 100％ 6 6.5 15.5 90％ 7 6.5 16.0 100％

[0085] The fibrinogen preparation experiment was repeated three times according to the method described in the "Specific Experimental Study" section above. Each time, cellulose was used at a concentration of 3 g fiber / L plasma, and the first equilibration solution had a conductivity of 15.5 mS / cm and a pH of 7.0. The purity of the fibrinogen in the final products was tested and found to be 81%, 86%, and 84%, respectively. This indicates that the final products have relatively high purity, meeting the requirements for subsequent applications.

[0086] Comparative Example 1

[0087] This comparative example uses centrifugation to collect cryoprecipitate. The specific procedure is as follows: After the frozen raw plasma is removed from storage, the surface of the plasma bag is sterilized with 70%-75% ethanol solution. Then, the plasma bag is broken open, and the temperature is controlled at 0-4℃ for thawing. The thawed plasma is then used to detect coagulation factor VIII, fibrinogen, fibronectin, and von Willebrand factor (vWF). The plasma is then centrifuged at 12000 rpm / min at 0-2℃. The precipitate collected after centrifugation is the cryoprecipitate collected by centrifugation. Unlike the cryoprecipitate collected by the cellulose-assisted adsorption method in Example 1, the cryoprecipitate obtained in this comparative example is characterized by a non-loose precipitate and poor solubility. A typical photograph of centrifuged cryoprecipitate from frozen plasma can be found here. Figure 4 .

[0088] To compare the performance of the cold precipitate obtained in this comparative example with that in the aforementioned examples, the cold precipitate obtained in this comparative example was added to the precipitation dissolving solution used in the "Specific Experimental Study" of Example 1 at a weight ratio of 1:10. After dissolving in a 34°C stirred water bath for 45 minutes, the dissolution of the cold precipitate was observed. The results showed that the precipitate was not fully dissolved and remained in large particles in the precipitation dissolving solution. These results indicate that cold precipitates obtained using traditional methods are difficult to dissolve simply by mixing with the dissolving solution; pre-melting, crushing, washing, and dissolving are necessary to ensure proper dissolution. In Example 1, however, the cellulose precipitate after top washing could be fully dissolved in the precipitation dissolving solution after dissolving in a 34°C stirred water bath for 45 minutes. The precipitation dissolving solution showed better dissolution of fibrinogen obtained from the cellulose precipitate in this scheme, reducing the complexity of the process.

[0089] In addition, this technical solution is the first to use cellulose to enrich fibrinogen. Prior to the development of this process, those skilled in the art were unaware that cellulose has an affinity for these cold-insoluble proteins (fibrinogen, factor VIII, vWF factor, etc., proteins enriched in cryoprecipitates). The results of this study exceeded the inventors' expectations. Using cellulose, the yield of fibrinogen can reach a certain level, which can be used for further purification and enrichment.

[0090] Furthermore, the two top-washing processes are crucial for removing contaminating proteins under the conditions of this process. The top-washing process effectively removes albumin and IgG (considered contaminating proteins relative to the target protein in this scheme). Through top-washing, albumin and IgG from the cellulose precipitate can be eluted, each accounting for approximately 4% of the total albumin and IgG in the raw plasma. Removing these contaminating proteins, such as albumin / IgG, through top-washing allows for further purification, including fibrinogen. Using cellulose can enrich cold-insoluble proteins precipitated in plasma and, under certain elution conditions, elute IgG / albumin without affecting the yield of the main products, IgG and albumin (further enrichment and purification of the eluted IgG and albumin can also be performed, but these process steps are beyond the scope of this scheme), achieving the enrichment and separation of cold-insoluble proteins. This phenomenon was first discovered in this scheme, and cold-insoluble proteins were successfully enriched using these findings.

[0091] Comparative Example 2

[0092] This comparative example was conducted according to the "Specific Experimental Study" in Example 1, using a cellulose dosage of 3 g fiber / L plasma, and the first equilibration solution had a conductivity of 15.5 mS / cm and a pH of 7.0. The specific changes compared to the "Specific Experimental Study" in Example 1 are as follows:

[0093] Test 1: Following the "Specific Experimental Study" in Example 1, after steps S1 and S2, fibrinogen intermediate material I was obtained. Conventional ultrafiltration and dialysis were performed according to step (8) of Example 1 to obtain a fibrinogen sample. This test did not include the S3 chromatography step, and the purity of the fibrinogen sample was 48%. The purity of fibrinogen was not ideal; therefore, a second ion exchange chromatography step (S3) was required to significantly improve the purity of fibrinogen.

[0094] Test 2: Step S1 was performed according to the "Specific Experimental Study" in Example 1, but the DEAE chromatography step in S2 was omitted. The filtrate obtained in S1 (a protein solution rich in coagulation factor VIII and human fibrinogen) was processed according to the filtration, ultrafiltration, and dialysis methods in S2 of the "Specific Experimental Study." A diluent (0.5M tris(hydroxymethyl)aminomethane, pH 9.4) was added for a 5-fold dilution, adjusting the pH to 9.4 for the next chromatography experiment, yielding fibrinogen intermediate material II. Then, fibrinogen intermediate material II was used for step S3 of the "Specific Experimental Study" to obtain a fibrinogen sample with a purity of 75%. The purity of fibrinogen was not ideal; a DEAE ion exchange chromatography step was needed before TMAE ion exchange chromatography to sufficiently improve the purity of fibrinogen in the product.

[0095] In the inventor's prior patented technology, fibrinogen was prepared by precipitating or cryoprecipitating component I (CN115947825A). This method used a single anion exchange chromatography (e.g., using a Fractgel EMD TMAE column) to obtain the fibrinogen product. However, this solution does not target the separation and enrichment of fibrinogen from component I precipitation or cryoprecipitation. Therefore, the method in the prior patent is not applicable to this technical solution, and the inventor needs to reconsider the method for enriching and purifying fibrinogen. The fibrinogen in this solution comes from plasma processed in step S1, not from component I precipitation or cryoprecipitation. This test also demonstrates that, given the specific characteristics of the S1 process in this solution, using only a single TMAE ion exchange chromatography step cannot effectively guarantee the purity of the target substance in the final product. Adding a step of directly collecting the flow-through using DEAE chromatography can effectively improve the purity of the final product. Furthermore, the DEAE chromatography step is simpler than the TMAE chromatography step, requiring only passing the sample to be purified through the column and collecting the flow-through, without the need for multiple rinsing and elution steps. This technical solution can significantly improve product purity by adding a simple chromatography step, which was something the inventors did not anticipate before the research and development was completed.

[0096] The above descriptions are merely embodiments of the present invention, and common knowledge such as specific technical solutions and / or characteristics are not described in detail here. It should be noted that those skilled in the art can make various modifications and improvements without departing from the technical solutions of the present invention, and these should also be considered within the scope of protection of the present invention. These modifications and improvements will not affect the effectiveness of the implementation of the present invention or the practicality of the patent. The scope of protection claimed in this application should be determined by the content of its claims, and the specific embodiments described in the specification can be used to interpret the content of the claims.

Claims

1. A method for the isolation and purification of human fibrinogen from plasma by filtration, characterized in that: The method comprises the following steps in sequence: S1: mixing cellulose with raw material blood plasma after balance, stirring and filtering to obtain cellulose precipitate; after elution of the cellulose precipitate, soaking in precipitate dissolving solution and filtering to obtain filtrate; S2: performing first ion exchange chromatography on the filtrate; S3: performing second ion exchange chromatography on the product of S2 to obtain a protein solution with fibrinogen purity of >80%.

2. A method for the purification of human fibrinogen from plasma by filtration according to claim 1, characterized in that: In S2, the filler of the chromatography column used in the first ion exchange chromatography is DEAE chromatography filler; The chromatography column is first treated with first balance solution, then the filtrate is loaded into the chromatography column, and flow-through is collected to obtain fibrinogen intermediate material I; after filtering, ultrafiltration, dialysis and pH adjustment of the fibrinogen intermediate material I, fibrinogen intermediate material II is obtained.

3. A method of purifying human fibrinogen from plasma by filtration according to claim 2, characterized in that: In S2, the first balance solution contains 0.015M-0.025M tris, 0.08-0.12M alanine, 0.10-0.14M glycine and 0.09-0.15M sodium chloride, has a pH of 6.70-7.00 and a conductivity of 14mS / cm-16mS / cm.

4. The method of claim 3, wherein the method is for purifying human fibrinogen from plasma. In S2, the pH of the fibrinogen intermediate material II is 8.50-9.

50.

5. The method of claim 1, wherein the method is for purifying human fibrinogen from plasma. In S3, the filler of the chromatography column used in the second ion exchange chromatography is TMAE chromatography filler; The chromatography column is first treated with second balance solution, then the fibrinogen intermediate material II is loaded into the chromatography column; then the chromatography column is treated with second balance solution and washing solution, and finally the chromatography column is eluted with eluent to collect fibrinogen intermediate material III; after filtering, ultrafiltration and dialysis of the fibrinogen intermediate material III, fibrinogen product is obtained.

6. A method of purifying human fibrinogen from plasma by filtration according to claim 5, characterized in that: In S3, the second balance solution contains 0.05M tris, has a pH of 8.50-9.50; the washing solution contains 0.05M tris and 0.05M sodium chloride, has a pH of 8.5-9.5; and the eluent contains 0.05M tris, has a pH of 6.5-7.

50.

7. A method of purifying human fibrinogen from plasma by filtration separation according to claim 3, characterized in that: In S1, the amount of cellulose is 2-4g cellulose / L blood plasma; The method for eluting the cellulose precipitate comprises sequentially using first and second elution solutions to elute the cellulose precipitate; The first elution solution is 10mM sodium citrate solution with a pH of 7.50-7.80; and the second elution solution is a solution containing 10mM sodium citrate and 2M glycine with a pH of 7.10-7.

30.

8. The method of claim 7, wherein the method is for purifying human fibrinogen from plasma. The precipitate dissolving solution contains 0.015-0.025M tris, 10-20mM sodium citrate, 0.05-0.15M alanine and 0.09M-0.15M sodium chloride, has a pH of 6.60-7.00 and a conductivity of 10.0mS / cm-17.0mS / cm.

9. A method of purifying human fibrinogen from plasma by filtration according to claim 8, characterized in that: The eluted cellulose precipitate is soaked in the precipitate dissolving solution at 31-35℃ for 10-50min; and the use amount ratio of the eluted cellulose precipitate to the precipitate dissolving solution is 1:8-12.

10. A human fibrinogen enriched protein solution obtained by a method for the purification of human fibrinogen from plasma according to any one of claims 1 to 9.

Images