阅读说明：本技术 一种人纤维蛋白原的制备方法 (Preparation method of human fibrinogen ) 是由 谢来峰 孙培森 张学成 李光飞 杨保平 赵东生 于 2021-07-01 设计创作，主要内容包括：本发明提供了一种人纤维蛋白原的制备方法,包括：S1、向组分I的沉淀物中加入3.0-5.0倍(V/M)第一溶解液,溶解1.0-3.0h后过滤,加入灭活剂后调pH至6.80-7.20,搅拌灭活至少6h；S2、经醇沉后用第二溶解液复溶或超滤透析过程中用第二溶解液进行溶液置换,使氨丁三醇浓度至0.01～0.1mol/L,pH8.0-9.0,电导率0.1～2ms/cm,澄清过滤待用,所述第二溶解液为0.01-0.1mol/L、pH为8.0-9.0的氨丁三醇溶液；S3、用阴离子交换层析凝胶介质进行层析纯化；S4、用聚醚砜超滤膜包(截留分子量：100KD)超滤透析至蛋白含量≥20g/L,用3-5倍(V/V)的透析液等体积超滤透析；S5、分装、入柜冻干、轧盖后干热灭活病毒处理。本发明利用阴离子交换层析凝胶,单步层析即可有效去除多种关键杂质成分,工艺精简,得到的人纤维蛋白原制品纯度高、杂质少、灭活剂残留含量低。(The invention provides a preparation method of human fibrinogen, which comprises the following steps: s1, adding 3.0-5.0 times (V/M) of first dissolving solution into the precipitate of the component I, dissolving for 1.0-3.0h, filtering, adding an inactivating agent, adjusting pH to 6.80-7.20, stirring and inactivating for at least 6 h; s2, carrying out solution replacement by using a second dissolving solution after alcohol precipitation in a redissolution or ultrafiltration dialysis process to ensure that the concentration of tromethamine is 0.01-0.1mol/L, the pH value is 8.0-9.0, the conductivity is 0.1-2 ms/cm, clarification and filtration are carried out for standby, wherein the second dissolving solution is a tromethamine solution with the concentration of 0.01-0.1mol/L, pH of 8.0-9.0; s3, carrying out chromatographic purification by using an anion exchange chromatography gel medium; s4, performing ultrafiltration dialysis by using a polyether sulfone ultrafiltration membrane (molecular weight cut-off: 100KD) until the protein content is more than or equal to 20g/L, and performing equal-volume ultrafiltration dialysis by using 3-5 times (V/V) of dialysate; s5, subpackaging, freeze-drying in a cabinet, and performing dry heat inactivation virus treatment after rolling a cover. The invention utilizes anion exchange chromatography gel, can effectively remove various key impurity components by single-step chromatography, has simple process, and obtains the human fibrinogen product with high purity, less impurities and low content of residual inactivator.)

1. A method for preparing human fibrinogen, comprising:

s1, adding 3.0-5.0 times (V/M) of first dissolving solution into the precipitate of the component I, dissolving for 1.0-3.0h, filtering, adding an inactivating agent, adjusting pH to 6.80-7.20, stirring and inactivating for at least 6 h;

s2, carrying out alcohol precipitation, redissolving with a second solution or carrying out solution replacement with the second solution in the ultrafiltration dialysis process to ensure that the concentration of tromethamine is 0.01-0.1mol/L and the pH value is 8.0-9.0, clarifying and filtering for later use, wherein the second solution is a tromethamine solution with the concentration of 0.01-0.1mol/L, pH of 8.0-9.0;

s3, carrying out chromatographic purification by using an anion exchange chromatography gel medium;

s4, performing ultrafiltration dialysis by using a polyether sulfone ultrafiltration membrane (molecular weight cut-off: 100KD) until the protein content is more than or equal to 20g/L, and performing equal-volume ultrafiltration dialysis by using 3-5 times (V/V) of dialysate;

s5, subpackaging, freeze-drying in a cabinet, and performing dry heat inactivation virus treatment after rolling a cover.

2. The method for preparing human fibrinogen according to claim 1, wherein the first solution is 0.01 to 0.1mol/L sodium citrate +7.0 to 9.0g/L sodium chloride, and the pH of the solution is 6.8 to 7.2; the inactivator consists of polysorbate 80 and tributyl phosphate, and the final concentrations of the inactivator and the tributyl phosphate are respectively 9-11g/L and 2-4 g/L; the dialysate is 0.01-0.1mol/L sodium citrate, 7-9g/L sodium chloride and 15-20g/L arginine, and the pH value of the solution is 6.5-7.5.

3. The method for preparing human fibrinogen of claim 1, wherein the alcohol precipitation operation in step S2 is: cooling to 0 + -2 deg.C, adding pre-cooled 50% (V/V) ethanol solution to make the ethanol content in the solution be 6-9% (V/V); adjusting pH to 6.8-7.0, cooling to 0-0.5 deg.C, centrifuging, and collecting precipitate; the redissolution operation is as follows: re-dissolving the precipitate with 15-25 times (V/M) of second dissolving solution.

4. The method for preparing human fibrinogen according to claim 1, wherein the anion exchange chromatography gel medium is DEAE650M, FRACTGELEMDTMAE(M) or EshmunoQ.

5. The method for producing human fibrinogen according to claim 2, wherein the solution replacement in step S2 is specifically: performing ultrafiltration dialysis 4-8 times with 100KD membrane, and removing polysorbate 80 and tributyl phosphate by dialysis.

6. The method for preparing human fibrinogen according to claim 1, wherein the step S3 includes the steps of:

s31, sequentially pretreating gel with 3-6CV of 0.5mol/L sodium hydroxide solution, 3-6CV of water for injection, 1-10CV of 0.05-0.5mol/L tromethamine buffer solution with pH of 8.0-9.0, wherein the column volume of the gel is 1CV, and the gel of anion exchange chromatography is DEAE650M, FRACTGELEMDTMAE(M) or EshmunoQ;

s32, loading the filtrate on a column, wherein the loading amount is less than or equal to 100g of protein/L gel;

s33, eluting with 0.01-0.1mol/Lmol/L tromethamine solution with pH of 7.0-8.0, and collecting eluate.

Technical Field

The invention relates to the technical field of biological pharmacy, in particular to a preparation method of human fibrinogen.

Background

Human fibrinogen (molecular weight 340kD, i.e., factor I) is the highest blood coagulation factor in plasma and is also the "central" protein in the blood coagulation system; human fibrinogen is synthesized by the liver and has a symmetrical dimeric structure, with two monomeric amino acids forming a central E domain and a carboxyl end forming two enlarged terminal D domains. Under the action of thrombin, the E, D domain is activated and exposes the binding site; each E structure domain is combined with the D structures of two adjacent human fibrinogen to finally form a net structure, so that the blood coagulation function is realized. Is suitable for congenital fibrinogen reduction or deficiency, severe liver injury, liver cirrhosis, disseminated intravascular coagulation, postpartum hemorrhage, and blood coagulation disorder caused by fibrinogen deficiency caused by major operation, trauma, internal hemorrhage, etc., and is a necessary emergency medicine for major hemorrhage and hemostasis in clinic.

At present, the production of human fibrinogen mainly adopts a low-temperature ethanol method (CN102286095A), blood plasma is taken as a raw material, and the human fibrinogen is obtained by ethanol precipitation for 3-4 times. The method has poor effect of removing key impurities such as plasminogen, fibronectin and the like, and causes poor product stability. For this reason, chromatography is becoming one of the commonly used methods for preparing human fibrinogen. For example, chinese patent application No. 200910237204.6 discloses a method for producing human fibrinogen, which utilizes the molecular properties of fibrinogen and adopts the mode of fibrinogen flow through for preparation; however, a multi-step precipitation process is required to remove the organic solvent added during virus inactivation, and the process is relatively complicated. Chinese patent application No. 201711104891.5 discloses a method for preparing human fibrinogen, which uses affinity chromatography to remove plasminogen to obtain human fibrinogen product with high purity and good stability; however, because the affinity chromatography medium is expensive, the low-temperature ethanol precipitation component is easy to block the filter plate and has small filtration amount, only a high-speed centrifuge can be used for separation, and the equipment and material consumption cost is high; and the process steps are complicated and long in time.

In view of the above, the present invention is particularly proposed.

Disclosure of Invention

The invention solves the problem of how to simultaneously and efficiently remove protein impurities and obtain the human fibrinogen with high purity and good stability by adding the inactivating agent in the preparation process.

In order to solve the above problems, the present invention provides a method for preparing human fibrinogen, comprising: s1, adding 3.0-5.0 times (V/M) of first dissolving solution into the precipitate of the component I, dissolving for 1.0-3.0h, filtering, adding an inactivating agent, adjusting the pH value to 6.80-7.20, and stirring for inactivating for more than or equal to 6 h; s2, precipitating with ethanol, redissolving with a second solution or performing solution replacement with the second solution in the ultrafiltration dialysis process to ensure that the concentration of tromethamine is 0.01-0.1mol/L and the pH value is 8.0-9.0, and clarifying and filtering for later use; the second dissolving solution is tromethamine solution with the concentration of 0.01-0.1mol/L, pH of 8.0-9.0, and is used for preparing a sample for chromatography and keeping the pH value of a product stable; s3, purifying by chromatography with anion exchange chromatography gel; s4, performing ultrafiltration dialysis by using a polyether sulfone ultrafiltration membrane (molecular weight cut-off: 100KD) until the protein content is more than or equal to 20g/L, and performing equal-volume ultrafiltration dialysis by using 3-5 times (V/V) of dialysate; s5, subpackaging, freeze-drying in a cabinet, and performing dry heat inactivation virus treatment after rolling a cover.

Preferably, the pH value of the first solution is 0.01-0.1mol/L sodium citrate +7.0-9.0g/L sodium chloride solution and is 6.8-7.2; the function is to dissolve and precipitate to extract fibrinogen under the condition of high impurity content, and simultaneously keep the stability of fibrinogen; the inactivator is polysorbate 80 and tributyl phosphate, and the final concentrations of the inactivator and the tributyl phosphate are respectively 9-11g/L and 2-4 g/L; the dialysate is 0.01-0.1mol/L sodium citrate, 7-9g/L sodium chloride and 15-20g/L arginine, and the pH value of the solution is 6.5-7.5. For example, the first solution is 17.8g/L sodium citrate +8.5g/L sodium chloride, and the pH of the solution is 6.97; the inactivator is polysorbate 80 and tributyl phosphate, and the final concentrations of the inactivator and the tributyl phosphate in the solution are respectively 10g/L and 3 g/L; the dialysate is 1.5g/L sodium citrate, 9.0g/L sodium chloride and 17.5g/L arginine, and the pH value of the solution is 7.00.

Preferably, the alcohol precipitation operation in step S2 is: cooling to 0 + -2 deg.C, adding pre-cooled 50% (V/V) ethanol solution to make the ethanol content in the solution be 6-9% (V/V); adjusting pH to 6.8-7.0, cooling to 0-0.5 deg.C, centrifuging, and collecting precipitate; the redissolution operation is as follows: re-dissolving the precipitate with 15-25 times (V/M) of second dissolving solution. For example, cooling to 1.0 deg.C, adding 50% (V/V) ethanol solution pre-cooled to-5 deg.C or below to make ethanol content in the solution be 8% (V/V); adjusting the pH value to 6.97, stirring at 0 ℃ and 200rpm for 1h, standing for 1h, centrifuging and collecting precipitate; the mixture was dissolved in a 20-fold (V/M) second solution at 25 ℃ for 1 hour. Preferably, the centrifugation is performed by using a cup centrifuge at 3500rpm for 20min, and the operation temperature is-2.0 ℃.

Preferably, the anion exchange chromatography gel medium is DEAE650M, FRACTGEL EMD TMAE (M) or Eshmuno Q. Preferably, the anion exchange chromatography gel medium is Fractgel EMD TMAE (M) gel.

Preferably, the solution replacement in step S2 is specifically: ultrafiltration dialysis with a 100KD membrane pack was performed at 4-8 volumes to remove polysorbate 80 and tributyl phosphate. .

Preferably, the chromatographic purification operation in step S3 includes: s31, sequentially using 3-6CV of 0.5mol/L sodium hydroxide solution, 3-6CV of water for injection, 1-10CV of 0.05-0.5mol/L tromethamine buffer solution with pH of 8.0-9.0 to pretreat the gel, wherein the flow rate is 80-120cm/h, and the column volume of the gel is 1 CV; s32, loading the filtrate on a column at a flow rate of 80-120cm/h and a loading amount of 19.8-21.4g protein/L gel; s33, after the reaction is finished, eluting the mixture by using 0.05mol/L tromethamine solution with pH7.0-7.5; when the eluent was at OD2_80nmCollecting 4-5CV eluent under less than or equal to 50 mAU.

As an example of the present invention, the chromatography comprises S31, pretreating a gel with 4CV of 0.5mol/L sodium hydroxide solution, 4CV of water for injection, 1.1CV of 0.5mol/L tromethamine buffer solution, pH9.0, 2CV of 0.05mol/L tromethamine buffer solution, pH9.0, in sequence, at a flow rate of 100cm/h, wherein the column volume of the gel is 1 CV; s32, loading the filtrate on a column at a flow rate of 100cm/h and a loading amount of 20.8g protein/L gel; s33, after the reaction is finished, eluting the mixture by using 0.05mol/L tromethamine solution (pH7.37); collecting 4CV when OD value of the eluate at 280nm is 0-50mAU, filtering, and collecting.

Preferably, the step S33 further includes: the gel was top washed with 2-6CV of 0.05mol/L tromethamine buffer solution, pH 8.0-9.0. For example, the gel is top-washed with 4CV of 0.05mol/L tromethamine buffer solution, pH 9.00.

Preferably, the lyophilization conditions of step S5 are as follows: s51, cooling to-2-3 ℃, pre-cooling for 4-6h, cooling to-43-50 ℃, and keeping for 10-12 h; s52, vacuumizing to below 18Pa and keeping for 2.1-2.5 h; s53, maintaining the vacuum degree below 18Pa, heating to-20 to-24 ℃ and keeping for 18-20 h; then heating to 8-10 ℃ and keeping for 25-29 h; heating to 31-35 ℃ and keeping for 15-17 h; s54, controlling the vacuum degree in the box body to be less than or equal to 7Pa when the constant temperature keeping is finished; and (5) pressing, plugging and sealing and deflating.

The freeze-drying process is divided into five stages of precooling, freezing, sublimation drying, primary analysis drying and secondary analysis drying, so that the severe change of the freeze-drying condition of the product is avoided, the product is stabilized and the moisture content is reduced. Specifically, the freeze point of the human fibrinogen prepared herein is about-3 deg.C and the eutectic temperature is about-18 deg.C. And in the pre-cooling stage, the temperature is kept at-2 to 3 ℃ and is pre-cooled slightly higher than the freezing point, and then the temperature is rapidly reduced to-43 to-50 ℃, which is far lower than the eutectic point of-18 ℃, so that the aim of rapidly reducing the temperature can be fulfilled, the number and the size of ice crystals are reduced, the damage of the ice crystals to protein is reduced, and the aim of protecting products is fulfilled. After the product is frozen, the temperature is raised to-20 to-24 ℃ and is lower than the eutectic point, and the crystal water is removed by sublimation. Heating to 8-10 deg.C, and removing the combined water; then heating to 31-35 ℃, and further removing residual moisture; the combined moisture is removed by gradient temperature rise, the temperature change of the product tends to be mild, the stability of the product is improved, the moisture content of the product is less than 1 percent, and the moisture removal is more thorough.

As an example of the invention, the freeze-drying condition is S51, the temperature is reduced to-2 to 3 ℃ after subpackaging and pre-cooled for 4h, and the temperature is reduced to-43 to-50 ℃ and kept for 10 h; s52, vacuumizing to below 18Pa and keeping for 2.1 h; s53, maintaining the vacuum degree below 18Pa, heating to-20 ℃ and keeping the temperature for 20 h; then heating to 10 ℃ and keeping for 29 h; heating to 31-35 ℃ and keeping for 17 h; s54, controlling the vacuum degree in the box body to be less than or equal to 7Pa when the constant temperature keeping is finished; and (5) pressing, plugging and sealing and deflating.

Preferably, the precipitation of component I in step S1 is prepared by the following method: s11, fusing blood plasma of healthy people at 0-4 ℃, centrifuging to remove cryoprecipitate, and collecting supernatant; s12, stirring and adsorbing with DEAES EPHADEX A50 gel for 20-60 min; s13, adjusting the temperature of the supernatant after adsorption to 1.0-1.5 ℃, dripping 95% ethanol (V/V) at the speed of 200-400ml/min, adjusting the temperature to-2.4-2 ℃ at the same time, enabling the final concentration of the ethanol to be 8% (V/V), keeping the temperature for 2-4h, and collecting the precipitate by plate-and-frame filtration.

For example: s11, fusing blood plasma of a healthy person at 0.3 ℃, centrifuging to remove cryoprecipitate, and collecting supernatant; s12, adding DEAE SEPHADEX A50 gel into the supernatant obtained in the step S11, and stirring and adsorbing for 40 min; s13, adjusting the temperature of the supernatant after adsorption to 1.0 ℃, dripping 95% ethanol (V/V) at the speed of 200ml/min, adjusting the temperature to-2.4 ℃ at the same time, keeping the final concentration of the ethanol at 8% (V/V), filtering by a plate frame after 2h of heat preservation, and collecting the precipitate.

Removing cryoprecipitate by centrifugation, wherein the cryoprecipitate is used for producing human coagulation factor VIII; meanwhile, DEAE-SEPHADEX-A-50 gel adsorption is used for removing impurity protein prothrombin. After the pretreated product is separated from the component I and precipitated, the product can be quickly filtered through a plate frame without adding a filter aid which has an original activation side effect on human fibrin.

Compared with the prior art, the preparation method of the human fibrinogen has the following beneficial effects: (1) the invention utilizes Fractgel EMD TMAE (M) and other gels to remove plasminogen, fibronectin and an inactivating agent by single-step chromatography, and obtains a human fibrinogen product with high purity (more than or equal to 95 percent), low plasminogen (less than or equal to 5 mu g/mg), low inactivating agent (polysorbate 80 less than or equal to 100 mu g/mg; tributyl phosphate less than or equal to 3 mu g/mg) and good stability, and the process is simplified; (2) after the human fibrinogen prepared by the invention and the thrombin are gelled and coagulated, the human fibrinogen can be stabilized at 37 ℃ for at least 3 days without hydrolysis; (3) the invention improves the unit filter plate filtration capacity of the component I sediment separated by the plate-and-frame filtration process by pretreating human plasma, solves the problem of filter plate blockage during the component I sediment separation by the plate-and-frame filtration process, is suitable for large-scale production, is suitable for the component I sediment separated by the current mainstream plate-and-frame filtration method, does not need to additionally purchase a centrifuge, and reduces the production cost of enterprises.

Drawings

FIG. 1 is a schematic flow chart of a process for preparing human fibrinogen according to the present invention;

FIG. 2 is a reduced electrophoretogram of the article before and after chromatography in example 1 of the present invention;

FIG. 3 is a reduced electrophoretogram of the article before and after chromatography in example 2 of the present invention;

FIG. 4 is a reduction electrophoretogram of the product before and after chromatography in example 3 of the present invention.

Detailed Description

In order to make the aforementioned objects, features and advantages of the present invention comprehensible, embodiments accompanied with figures are described in detail below.

The extracted human fibrinogen exists in a solution state and is usually subjected to freeze-drying; the prior freeze-drying process is to freeze the solution and then carry out vacuum drying in a low-temperature environment, and has the defects of poor product quality, such as high moisture residue of a freeze-dried finished product, long re-dissolving time and the like. To this end, the applicant optimized the lyophilization process of human fibrinogen.

Researches show that the freezing point of the fibrinogen prepared by the production process is-3 ℃, and the eutectic point is about-18 ℃; as can be seen from Table 1, the product is protected by pre-cooling at a temperature slightly higher than the freezing point, and then rapidly cooling to-50 ℃ which is far lower than the eutectic temperature, so that the number and size of ice crystals are reduced, the damage of the ice crystals to protein is reduced; after the product is frozen, heating to-20 to-24 ℃, wherein the temperature is slightly lower than the eutectic point, so that the product is convenient to sublimate and remove crystallization moisture; then heating to 8-10 deg.C, and removing the combined water; then heating to 31-35 ℃, and further removing residual moisture; the combined moisture is removed by gradient temperature rise, the temperature change of the product tends to be mild, the stability of the product is improved, the moisture content of the product is low, and the moisture removal is more thorough. When the traditional process is adopted for preparation, the temperature is directly reduced to minus 45 ℃ and the vacuum pumping is carried out, so that the bound water is difficult to remove, and simultaneously, a large amount of crystals formed can damage the activity of the protein and even have certain degradation; the freeze-drying is divided into five stages of precooling, freezing, sublimation drying, primary analysis drying and secondary analysis drying, the precooling is carried out at a temperature slightly higher than the approach point, the freezing is carried out at a low temperature quickly, and the quantity and the size of ice crystals are reduced, so that the damage of the freeze-drying to the activity of the protein is reduced; meanwhile, by drying in stages, the crystallization water, the bound water and the residual water are respectively removed, the water content of the product is reduced, the stability is improved, and the redissolution effect is good.

Example 1

As shown in fig. 1, a method for preparing human fibrinogen includes:

s1, taking 35.4kg of the component I, adding 3.5 times (V/M) of the first dissolving solution, dissolving for 1.5 hours at 25.5 ℃, clarifying, filtering, adding 3000g of polysorbate 80 and 900g of tributyl phosphate, adjusting the pH value to 6.89, stirring at constant temperature of 25 ℃, and inactivating for 6 hours.

The first solution is 25.8g/L sodium citrate and 9.0g/L sodium chloride, and the pH value is 6.85.

The component I is a general name in the technical field, and comprises the main components of 80% of fibrinogen, 10% of gamma-globulin, 5% of prothrombin, 2% of beta-globulin, 2% of iodine binding protein and the like. Preferably, the component I is prepared by the following method:

s11, collecting 2015.53kg of healthy human plasma, fusing at 2 ℃, centrifuging at 14000rpm to remove cryoprecipitate, and collecting supernatant;

s12, collecting DEAE SEPHADEX A50 gel 48kg, adding S11 supernatant 1918L into the gel, and stirring and adsorbing at 6.0 deg.C for 40 min.

S13, cooling the supernatant after adsorption to 3.0 ℃, dropping 95% ethanol (V/V) at the speed of 300 ml/min and cooling to 0 ℃ at the same time to make the final concentration of ethanol in the solution be 8% (V/V), keeping the temperature for 3h, filtering by a plate frame, and collecting 11.5kg of the precipitate of the component I. The above operation was repeated twice to obtain 11.3kg and 12.6kg of precipitates, respectively. Preferably, the plate frame filter material is a 773 × 773mm type E1000 filter plate.

S2, cooling to-1.0 ℃, adding a 50% (V/V) ethanol solution precooled to below-5 ℃ to ensure that the final content of ethanol in the solution is 9% (V/V); adjusting the pH value to 6.97, stirring at 0.5 ℃ for 1h at 200rpm, standing for 2h, centrifuging and collecting precipitates.

The centrifugal cup-type centrifuge is adopted for centrifugation for 20min at the rotating speed of 3500rpm and the temperature of-2.0 ℃.

S3, dissolving the precipitate obtained in the step S2 with 20 times (V/M) of second dissolving solution at 25 ℃ for 1h, clarifying, filtering, and purifying by chromatography with Fractgel EMD TMAE (M) gel; preferably, the second solution is 0.1mol/L tromethamine solution with pH of 9.00.

The chromatographic purification comprises:

s31, sequentially using a 4CV of 0.5mol/L sodium hydroxide solution, 4CV of water for injection, 1CV of 0.5mol/L tromethamine buffer solution with pH9.0 and 1CV of 0.5mol/L tromethamine buffer solution with pH9.0 to pretreat the gel, wherein the flow rate is 120cm/h, and the column volume of the Fractgel EMD TMAE (M) gel is 1 CV;

s32, loading the filtrate on a column at a flow rate of 120cm/h and a loading amount of 20.8g protein/L gel;

s33, after the completion, carrying out top washing on the gel by using a 4CV 0.5mol/L tromethamine buffer solution with the pH value of 9.00, and then eluting by using a 0.5mol/L tromethamine solution with the pH value of 8.00; when OD of the eluent is_280nm4CV was collected for 28mAU and filtered through a 0.22 μm cartridge.

The purification effect of chromatography is shown in table 2 and fig. 2, wherein fig. 2 represents the fibronectin removal effect. As can be seen from Table 1, the purity of human fibrinogen in the eluate was 95.8%, the residual amount of plasminogen was 0.08. mu.g/mg protein, the residual amount of tributyl phosphate was < 3. mu.g/mL, and the residual amount of polysorbate 80 was 8. mu.g/mL.

TABLE 1 comparison of purity and impurity content of target before and after chromatography

S4, performing ultrafiltration dialysis by using a polyether sulfone ultrafiltration membrane (molecular weight cut-off: 100KD) until the protein content is 25g/L, performing equal-volume ultrafiltration dialysis by using 4 times (V/V) of dialysate, and performing aseptic filtration;

the dialysate is: 15g/L sodium citrate, 9.0g/L sodium chloride and 20g/L arginine, and the pH value is 7.00. Preferably, the ultrafiltration dialysis is performed at room temperature, such as 25 ℃.

S5, subpackaging, freeze-drying in a cabinet, and performing dry heat inactivation virus treatment after rolling a cover. The dry heat inactivation is the prior art, such as heat preservation for 31min at 99.5 +/-0.5 ℃ in a boiling water bath, and is not described herein in detail.

Preferably, the lyophilization conditions are: s51, subpackaging, cooling to-2 ℃, precooling for 4h, cooling to-45 ℃, and keeping for 10 h; s52, vacuumizing to below 18Pa and keeping for 2.1 h; s53, maintaining the vacuum degree below 18Pa, heating to-20 ℃ at the speed of 1 ℃/h, and keeping the temperature for 20 h; then heating to 10 ℃ at the speed of 2 ℃/h and keeping for 29 h; heating to 31-35 ℃ at the speed of 4-5 ℃/h and keeping for 17 h; s54, controlling the vacuum degree in the box body to be less than or equal to 7Pa when the constant temperature keeping is finished; and (5) pressing, plugging and sealing and deflating.

The quality indexes of freeze-dried samples meet pharmacopeia standards through verification, wherein the purity of human fibrinogen is 96.2%, the fibrinogen and thrombin can be kept for 5 days after gelling without hydrolysis, the plasminogen residual quantity is 1.3 mug/mL, the tributyl phosphate residual quantity is less than 3 mug/mL, and the polysorbate 80 residual quantity is 45 mug/mL.

Example 2

A method for preparing human fibrinogen comprises:

s1, taking 33.9kg of the precipitate of the component I, adding 3.0 times (V/M) of first dissolving solution to dissolve for 1.0h, filtering by using a 30LP filter stack combined with a 1.0um filter element, ejecting the first dissolving solution until the total volume is 300L, adding 3000g of polysorbate 80 and 900g of tributyl phosphate, adjusting the pH value to 6.93, and inactivating for 6 h.

Preferably, all of the above operations are carried out at room temperature, e.g. 25 ℃. The first solution is 12.4g/L sodium citrate and 7.0g/L sodium chloride, and the pH value is 6.97.

Preferably, the component I is prepared by the following method:

s11, fusing 2015.97kg of blood plasma of a healthy person at 0 ℃, centrifuging to remove cryoprecipitate, and collecting supernatant;

s12, collecting DEAE SEPHADEX A50 gel 48kg, adding the supernatant of S11, stirring and adsorbing for 40 min.

S13, adjusting the temperature of the supernatant after adsorption to 1.0 ℃, dripping 95% ethanol (V/V) at the speed of 200ml/min and adjusting the temperature to-2.4 ℃ at the same time to ensure that the final concentration of the ethanol is 8% (V/V), keeping the temperature for 2h, filtering by a plate frame, and collecting 12.5kg of component I precipitate. The above operations S11 to S13 were repeated to obtain 10.6kg and 10.8kg of precipitates, respectively.

S2, performing ultrafiltration dialysis with 100KD membrane, such as replacing with 3-5 times (V/V) of second solution to make tromethamine concentration to 0.05mol/L and pH value to 8.5, and clarifying and filtering with 60LP filter;

s3, performing chromatographic purification by Eshmuno Q gel;

specifically, the method comprises the following steps:

s31, sequentially using 3.9CV of 0.5mol/L sodium hydroxide solution, 3.9CV of water for injection, 1.5CV of 0.5mol/L tromethamine buffer solution with pH8.5, 2CV of 0.05mol/L tromethamine buffer solution with pH8.5 to pretreat the gel, wherein the flow rate is 100cm/h, and the column volume of the Eshmuno Q gel is 1 CV;

s32, performing sample loading chromatography by using filtrate, wherein the sample loading speed is 100cm/h, and the loading capacity of the sample is 21.4g of protein/L gel;

s33, after the completion, carrying out top washing on the gel by using a 2CV tromethamine buffer solution with 0.05mol/L and pH8.5, and carrying out waste treatment on the flow-through liquid and the top-washed buffer solution; then eluting with 0.05mol/L tromethamine (pH7.50) to 50mAU (OD)_280nm) Collecting 4.5CV eluate, and filtering to 300L ultrafiltration tank;

the effect of chromatographic purification is shown in Table 3 and FIG. 3, wherein FIG. 3 represents the effect of fibronectin removal. As can be seen from Table 2, the purity of human fibrinogen in the eluate was 95.5%, the plasminogen-remaining amount was 0.09. mu.g/mg protein, the tributyl phosphate-remaining amount was < 3. mu.g/mL, and the polysorbate-80-remaining amount was 7. mu.g/mL.

TABLE 2 comparison of purity and impurity content before and after chromatography

S4, performing ultrafiltration dialysis with polyethersulfone ultrafiltration membrane (cut-off molecular weight of 100KD) until the protein content is 25g/L, performing equal-volume ultrafiltration dialysis with 4 times (V/V) dialysate, sterilizing, and adjusting the protein content to 21 g/L.

The dialysate is 25.8g/L sodium citrate, 7.0g/L sodium chloride, 17.5g/L arginine, and has a pH value of 7.5. Preferably, the ultrafiltration dialysis is performed at room temperature, such as 25 ℃.

S5, subpackaging, freeze-drying in a cabinet, capping, and performing dry heat inactivation virus treatment. The dry heat inactivated virus is the prior art and is not described in detail herein.

Preferably, the lyophilization conditions are: s51, cooling to-2-3 ℃, pre-cooling for 4h, cooling to-43-50 ℃, and preserving heat for 10 h; s52, after vacuumizing to below 18Pa, keeping for 2.1 h; preferably, the water catcher is vacuumized after the temperature is reduced to below-40 ℃; s53, maintaining the vacuum degree below 18Pa, heating to-20 ℃, and keeping for 20 h; then heating to 10 ℃ and keeping for 29 h; finally, heating to 31-35 ℃, and keeping for 17 h; and S54, controlling the vacuum degree in the box body to be less than or equal to 7Pa when the constant temperature keeping is finished, and then pressing, plugging, sealing and deflating.

The quality indexes of the freeze-dried samples meet pharmacopeia standards through verification, wherein the purity of human fibrinogen is 98.0%, the plasminogen residue is 1.4 mug/mL, the fibrinogen and thrombin can be kept unhydrolyzed for 4 days after being gelatinized, the tributyl phosphate residue is less than 3 mug/mL, and the polysorbate 80 residue is 62 mug/mL.

Example 3

A method for preparing human fibrinogen comprises:

s1, taking 30.6kg of the precipitate of the component I, adding 5 times (V/M) of first dissolving solution, dissolving for 3.0h at 25.0 ℃, and filtering by using a 30LP filter stack combined with a 1.0um filter element; the first dissolution was ejected to 276L, 2760g polysorbate 80 and 828g tributyl phosphate were added, the pH was adjusted to 7.2, and inactivation was performed at 25 ℃ for 6 h.

Wherein the first solution is 2.59g/L sodium citrate and 8.5g/L sodium chloride, and the pH value is 7.2.

The component I is a general name in the technical field, and comprises the main components of 80% of fibrinogen, 10% of gamma-5 globulin, 5% of prothrombin, 2% of beta-globulin, 2% of iodine binding protein and the like. Preferably, the component I is prepared by the following method:

s11, fusing 2015.65kg of healthy human plasma at 4 ℃, centrifuging to remove cryoprecipitate, and collecting supernatant;

s12, adding 48kg of DEAE SEPHADEX A50 gel into the supernatant obtained in the step S11, and stirring and adsorbing for 60 min;

wherein the titer of the factor VII after adsorption is 35IU/ml, and the titer of thrombin is 51IU/ml, and the factor VII after adsorption is suitable for being used as a raw material for preparing a prothrombin compound.

S13, adjusting the supernatant after adsorption to 1.0 ℃, dripping 95% ethanol (V/V) at the speed of 400ml/min and simultaneously cooling to 2.0 ℃ to ensure that the ethanol concentration in the solution is 8% (V/V), preserving the temperature for 4h, filtering by a plate frame, and collecting 10.2kg of component I precipitate.

The above operation was repeated twice to obtain 9.0kg and 11.4kg of the precipitate of component I, respectively. Preferably, the plate frame filter material is a 773 × 773mm type E1000 filter plate.

S2, cooling to 1.2 ℃, adding a 50% (V/V) ethanol solution precooled to below-5 ℃ to ensure that the ethanol content in the solution is 7 percent; adjusting the pH value to 6.8, stirring at 100rpm at 0 ℃ for reaction for 2h, standing for 1h, centrifuging and collecting precipitate.

Preferably, the centrifugation is performed by a cup centrifuge at the rotation speed of 4000rpm and the temperature of-2.0 ℃ for 20 min.

S3, dissolving the precipitate obtained in the step S2 with 25 times (V/M) of second dissolving solution at 25 ℃ for 1h, filtering the solution through a 0.25-micron filter membrane, and performing chromatographic purification by DEAE650M gel;

preferably, the second solution is 0.01mol/L tromethamine solution with pH of 8.0. Specifically, the chromatographic purification comprises the following genera:

s31, pretreating the gel with 3CV of 0.5mol/L sodium hydroxide solution, 3CV of water for injection, 2.0CV of 0.5mol/L tromethamine buffer solution (pH8.0) and 2CV of 0.01mol/L tromethamine buffer solution (pH8.0) in sequence, wherein the flow rate is 80cm/h, and the column volume of the DEAE650M gel is 1 CV;

s32, performing column chromatography on the filtrate at the flow rate of 80cm/h, and loading the sample with 19.8g of protein/L gel;

s33, after the completion, carrying out top washing on the gel by using 6CV tromethamine buffer solution with 0.01mol/L and pH8.0, and carrying out waste treatment on the flow-through liquid and the top-washed buffer solution; eluting with 0.01mol/L tromethamine solution (pH 7.0) to obtain 50mAU (OD)_280nm) The collection of the eluent of 5CV was started and filtered into a 300L ultrafiltration tank.

The chromatographic effect of DEAE650M gel is shown in Table 4 and FIG. 4, where FIG. 4 represents the fibronectin removal effect. As can be seen from Table 3, the purity of human fibrinogen in the eluate was 97.7%, the plasminogen-remaining amount was 0.09. mu.g/mg protein, the tributyl phosphate-remaining amount was < 3. mu.g/mL, and the polysorbate-80-remaining amount was 8. mu.g/mL.

TABLE 3 comparison of purity and impurity content before and after chromatography

S4, performing ultrafiltration dialysis with polyethersulfone ultrafiltration membrane (cut-off molecular weight of 100KD), adjusting protein content to 25g/L, performing isovolumetric ultrafiltration dialysis with 4 times of dialysate, sterilizing, and adjusting protein content to 20 g/L.

Preferably, the dialysate is 3.5g/L sodium citrate +8.0g/L sodium chloride +17.5g/L arginine, pH 6.5. Preferably, the ultrafiltration dialysis is performed at room temperature, such as 25 ℃.

S5, subpackaging, freeze-drying in a cabinet, and performing dry heat inactivation virus treatment after rolling a cover. The dry heat inactivation is the prior art, such as heat preservation for 31min at 99.5 +/-0.5 ℃ in a boiling water bath, and is not described herein in detail.

Preferably, the lyophilization conditions are:

s51, cooling to-2-3 ℃ after subpackage, pre-cooling for 4h, and then cooling to-43-50 ℃ for 10 h;

s52, after vacuumizing to below 18Pa, keeping for 2.1 h; preferably, the water catcher is vacuumized after the temperature is reduced to below-40 ℃;

s53, maintaining the vacuum degree below 18Pa, heating to-20 ℃, and keeping for 20 h; then heating to 10 ℃ and keeping for 29 h; heating to 31-35 ℃, and keeping for 17 h;

and S54, controlling the vacuum degree in the box body to be less than or equal to 7Pa when the constant temperature keeping is finished, and then pressing, plugging, sealing and deflating.

The quality indexes of the prepared freeze-dried samples all accord with pharmacopeia standards through verification, wherein the purity of human fibrinogen is 97.8%, the plasminogen residue is 1.3 mug/mL, the fibrinogen and thrombin can be kept for 5 days without hydrolysis after being gelatinized, the tributyl phosphate residue is less than 3 mug/mL, and the polysorbate 80 residue is 63 mug/mL.

Comparative example 1

3 samples were prepared separately by using the low temperature ethanol process in the existing production, i.e. the preparation method of the example in chinese patent application No. CN 02103825.2.

The samples of examples 1, 2, 3 and comparative examples 1, 2 were analyzed by comparison using one-way variance, and the results are shown in Table 5. It should be noted that: the gelling method is that the fibrinogen component and the thrombin component are mixed in a ratio of 1:1 and then gelling and coagulating, which belongs to the prior art and is not described herein again.

TABLE 5 residual and stability data for different groups of inactivators

As can be seen from Table 5, the fibrinogen purity, the plasminogen residual quantity and the stabilization time after thrombin gelation in the embodiment of the invention are all superior to those of the multi-step low-temperature ethanol process, the polysorbate 80 residual quantity and the tributyl phosphate residual quantity are not obviously different from those of the multi-step low-temperature ethanol process through single-step anion adsorption chromatography, and the effect of single-step anion adsorption chromatography on the removal of the inactivator is the same as that of multi-step ethanol precipitation.

Although the present invention is disclosed above, the present invention is not limited thereto. Various changes and modifications may be effected therein by one skilled in the art without departing from the spirit and scope of the invention as defined in the appended claims.