Purification method and application of human fibrinogen

Abstract

The invention provides a purification method and application of human fibrinogen. The method comprises the following steps: S1) mixing a component I precipitate with a cryoprecipitate, and dissolving to obtain a first liquid; s2) adjusting the pH value of the first liquid to 6.0-6.5, carrying out a first reaction, and carrying out solid-liquid separation to obtain a first precipitate; s3) sequentially performing dissolution, S / D virus inactivation and two times of PEG precipitation on the first precipitate to obtain a second precipitate; and S4) dissolving the second precipitate, and carrying out ion exchange chromatography to obtain purified human fibrinogen. The method can solve the problem of lack of a method for large-scale purification of human fibrinogen in the prior art, and is suitable for the technical field of blood products.

Description

Technical Field

[0001] The present invention relates to the technical field of blood products, and in particular to a method for purifying human fibrinogen and its application. Background Art

[0002] Human fibrinogen is a glycoprotein extracted from healthy human plasma and undergoes a series of purification and virus inactivation treatments. Its core role in the coagulation process of human blood cannot be ignored. It is composed of Aα2, Bβ2 and γ2 peptide chains, carrying 3% to 5% carbohydrates. It is cleaved by thrombin to form fibrin, and synergistically works with platelets to build a stable fibrin thrombus. It has a significant therapeutic effect on congenital and acquired fibrinogen deficiency symptoms, such as severe liver damage, cirrhosis, disseminated intravascular coagulation, postpartum hemorrhage and coagulation disorders caused by major surgery.

[0003] In the current prior art, there is a method for preparing human fibrinogen using cold precipitate as a raw material, as disclosed in patent CN201810054783, by chromatography, the cold precipitate is dissolved and then pH adjustment, centrifugation, S / D virus inactivation, anion exchange chromatography, ultrafiltration dialysis and freeze-drying are performed. Although this process has remarkable performance in terms of product purity and biological activity, its most difficult technical problem lies in the limitation of batch production, that is, the purification of fibrinogen depends on its adsorption on the chromatography filler, which not only leads to limited recovery efficiency of the target protein, but also makes it difficult to meet the needs of large-scale production, and the regeneration and reuse of the filler are highly complex, which increases the production cost and limits the availability and practicality of fibrinogen products. Therefore, the development of a purification method that can significantly improve the production scale and efficiency while ensuring product quality has become a key technical problem to be solved in the field of fibrinogen production. Summary of the invention

[0004] The main purpose of the present invention is to provide a method for purifying human fibrinogen and its application, so as to solve the problem that the prior art lacks a method for purifying human fibrinogen on a large scale.

[0005] In order to achieve the above-mentioned purpose, according to the first aspect of the present invention, a method for purifying human fibrinogen is provided, which comprises: S1) mixing component I precipitate with cold precipitate and dissolving them to obtain a first liquid; S2) adjusting the pH of the first liquid to 6.0-6.5 for a first reaction, and obtaining a first precipitate after solid-liquid separation; S3) dissolving the first precipitate, S / D virus inactivating and PEG precipitating twice in sequence to obtain a second precipitate; S4) dissolving the second precipitate and performing ion exchange chromatography to obtain purified human fibrinogen.

[0006] Further, S1) includes: dissolving Component I and cryoprecipitate with an aqueous solution of sodium heparin to obtain a first liquid; preferably, the content of sodium heparin is 3000 - 6000 IU / L; preferably, the volume ratio of the total volume after mixing Component I and cryoprecipitate to the volume of the aqueous solution of sodium heparin is 1:8 - 15; preferably, the dissolving time is 2 - 3 hours; preferably, the dissolving temperature is 20 - 30 °C.

[0007] Further, in S2), the temperature of the first reaction is 10 °C - 20 °C; preferably, the time of the first reaction is 1 - 2 hours.

[0008] Further, S3) includes, S31) mixing the first precipitate with a first dissolving solution and filtering to obtain a second liquid; S32) subjecting the second liquid to S / D virus inactivation to obtain a third liquid; S33) performing a first PEG precipitation treatment and a second PEG precipitation treatment on the third liquid with a PEG solution to obtain a second precipitate; preferably, the first dissolving solution includes 0.1% - 0.5% by mass of tris(hydroxymethyl)aminomethane, 0.1% - 1.0% of sodium citrate, 0.5% - 2.0% of sucrose, 0.2% - 1.0% of lysine hydrochloride, 0.5% - 2% of sodium chloride, and the pH value is 6.5 - 7.5.

[0009] Further, the first PEG precipitation treatment includes: adjusting the temperature of the third liquid to 10 - 15 °C, then mixing with the PEG solution and performing a first reaction to obtain a first PEG precipitate; preferably, the concentration of the PEG solution is 3.0% - 5.0% w / v; preferably, the time of the first reaction is 30 - 60 min.

[0010] Further, the second PEG precipitation treatment includes: mixing the first PEG precipitate with a second dissolving solution and filtering, adjusting the temperature of the filtrate obtained after filtering to 10 - 15 °C, then mixing with the PEG solution and performing a second reaction to obtain a second precipitate; preferably, the time of the second reaction is 30 - 60 min; preferably, the second dissolving solution includes 0.1% - 1.0% of sodium citrate, 0.5% - 2.0% of sodium chloride, and the pH value is 6.5 - 7.5; preferably, the concentration of the PEG solution is 3.0% - 5.0% w / v; preferably, the PEG solution includes one or more of PEG4000 solution or PEG3350 solution, more preferably PEG4000 solution.

[0011] Further, S4) includes: after mixing the second precipitate with the third dissolution solution and filtering, subjecting the supernatant to first concentration and first dialysis to obtain a first concentrated solution; after subjecting the first concentrated solution to ion exchange chromatography to obtain a chromatographic solution, subjecting the chromatographic solution to second concentration and then mixing it with a first buffer solution for second dialysis to obtain purified human fibrinogen; preferably, the third dissolution solution includes sodium citrate with a mass ratio of 0.1% - 1.0%, sodium chloride with a mass ratio of 0.5% - 2.0%, arginine hydrochloride with a mass ratio of 0.2% - 1.0%, and the pH value is 6.5 - 7.5; preferably, the first buffer solution includes sodium citrate with a mass ratio of 0.1% - 1.0%, sodium chloride with a mass ratio of 0.5% - 2.0%, arginine hydrochloride with a mass ratio of 3.0% - 5.0%, glycine with a mass ratio of 2.0% - 3.0%, and the pH value is 6.5 - 7.5.

[0012] Further, the ion exchange chromatography includes anion exchange chromatography; preferably, the chromatographic packing material for anion exchange chromatography includes TOYOPEARL(R) DEAE 650M, Q Sepharose Fast Flow, or EMD TMAE; preferably, the linear velocity of the anion exchange chromatography is 60 cm / h - 100 cm / h.

[0013] Further, the first dialysis includes: mixing the first concentrated liquid with the third dissolution solution for first dialysis; preferably, the volume ratio of the first concentrated liquid to the third dissolution solution is 1:3 - 5; preferably, in the second dialysis, the volume ratio of the second concentrated liquid to the first buffer solution is 1:3 - 5; preferably, the first concentration includes ultrafiltration concentration; preferably, the second concentration includes ultrafiltration concentration.

[0014] To achieve the above object, according to the second aspect of the present invention, there is provided an application of the above - mentioned purification method of human fibrinogen in the production of human fibrinogen products.

[0015] By applying the technical solution of the present invention, by controlling the pH during the purification process to be 6.0 - 6.5, and performing two - step PEG precipitation and ion exchange chromatography, the human fibrinogen in Component I and cryoprecipitate can be effectively purified, and the human fibrinogen exists in the flow - through solution of the ion exchange chromatography. The recovery efficiency of human fibrinogen is not limited by the adsorption volume of the packing material, and is more suitable for industrial application compared with the prior art and is more suitable for large - scale purification processes of human fibrinogen. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] The specification drawings forming a part of this application are used to provide a further understanding of the present invention. The schematic embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute an improper limitation to the present invention. In the drawings:

[0017] Figure 1The schematic flow chart of the purification method of human fibrinogen according to Embodiment 1 in the description of the present application is shown. Detailed implementation manners

[0018] It should be noted that, without conflict, the embodiments in the present application and the features in the embodiments can be combined with each other. The present invention will be described in detail below in conjunction with the embodiments.

[0019] Term explanation:

[0020] Component I: refers to the first component separated from plasma in the Cohn cold ethanol precipitation method. This component mainly contains fibrinogen, human coagulation factor VIII, fibronectin, and also contains part of human coagulation factor V and human coagulation factor XIII.

[0021] Cold precipitate: refers to the insoluble white flocculent precipitate separated by centrifugation after thawing fresh frozen plasma at 0 - 4°C. Its main components include fibrinogen, coagulation factor VIII, fibronectin, and also contain a small amount of von Willebrand factor, coagulation factor XIII, human albumin, IgG, etc.

[0022] As mentioned in the background art, the purification methods of human fibrinogen in the prior art are not suitable for large-scale purification processes. Based on this, in the present application, the inventors attempt to develop a purification method and application of human fibrinogen.

[0023] In the first typical implementation manner of the present application, a purification method of human fibrinogen is provided. The purification method includes: S1) mixing and dissolving the component I precipitate and the cold precipitate to obtain a first liquid; S2) adjusting the pH of the first liquid to 6.0 - 6.5 (including but not limited to 6.0, 6.1, 6.2, 6.3, 6.4, 6.5) for a first reaction, and obtaining a first precipitate after solid-liquid separation; S3) dissolving the first precipitate in sequence, performing S / D virus inactivation, and two PEG precipitations to obtain a second precipitate; S4) dissolving the second precipitate and then performing ion exchange chromatography to obtain purified human fibrinogen.

[0024] As mentioned in the background art part, since the cold ethanol precipitation method was used to precipitate component I in 1944, and until B BLOMBACK first introduced glycine to purify human fibrinogen in 1958, the extraction and purification technology of fibrinogen has undergone significant development. However, in the 1970s, due to the lack of effective virus inactivation means, the virus transmission risk of fibrinogen products has attracted widespread attention, resulting in the suspension of their production and application in some European and American countries. It was not until the 1990s that with the development and application of the S / D virus inactivation technology, fibrinogen products were able to re-enter clinical use, but the limitations of the production process still exist.

[0025] Subsequently, since it was found that the fibrinogen content in the precipitate of Component I only accounted for 50% of the total plasma fibrinogen, and in addition to human coagulation factor VIII, cryoprecipitate also contains a large amount of fibrinogen, fibronectin, etc. Therefore, in order to increase the extraction amount of human fibrinogen, in the prior art, generally the precipitate of Component I is used as the raw material for the purification of human fibrinogen. In the technology of purifying human fibrinogen using cryoprecipitate as the raw material, for example, patent application CN 201810054783 discloses a method for preparing human fibrinogen by chromatography using cryoprecipitate as the raw material. After dissolving the cryoprecipitate at 24-26°C with stirring for 1-2 hours, the pH is adjusted, and the crude precipitate is obtained by centrifugal separation. After the crude precipitate is dissolved and filtered again, S / D inactivation is carried out. After the inactivation is completed, anion exchange chromatography is carried out, and the collected chromatographic eluate is ultrafiltered and dialyzed and then formulated for lyophilization. Although this process can ensure the quality of the product to a certain extent, since the target protein, fibrinogen, is adsorbed onto the chromatographic packing material, the batch scale cannot be enlarged. Patent CN 116554301 A recovers human fibrinogen by combining the precipitate of Component I with chromatographic waste liquid. These two methods have the following technical limitations:

[0026] 1. Batch production limitation: The efficiency of adsorption chromatography is limited by the adsorption capacity of the packing material and the difficulty of regeneration, which results in limitations in batch scale.

[0027] 2. Protein loss: In the existing process, human fibrinogen is purified from chromatographic waste liquid, and a large amount of human fibrinogen is lost during the dissolution and extraction of cryoprecipitate, thus affecting the yield and cost of the final product.

[0028] In summary, the methods of the prior art are difficult to be applied to the extraction and purification of large-scale human fibrinogen products. By integrating the purification processes of the precipitate of Component I and cryoprecipitate, this application proposes a method for purifying fibrinogen, which can increase the purity and biological activity of human fibrinogen while enlarging the batch scale, so as to solve the above technical problems.

[0029] In the purification method of the present application, component I precipitate and cold precipitate are mixed simultaneously to improve the recovery rates of human coagulation factor VIII and human fibrinogen. The mixed solution is dissolved, and the pH of the mixed solution is precisely regulated for the first reaction. During the first reaction, it is possible to effectively separate human coagulation factor VIII, plasminogen, fibronectin, and albumin impurities contained in component I precipitate and cold precipitate from human fibrinogen. After the first reaction, the human coagulation factor VIII, plasminogen, fibronectin, and albumin impurities contained in component I precipitate and cold precipitate can be removed by simple centrifugation, and a crude precipitate with a relatively high purity of fibrinogen is obtained, which is beneficial for subsequent purification. The regulation of the pH of the above-mentioned first liquid in the present application includes, but is not limited to, adding 0.5 mol / L acetic acid solution to the first liquid for adjustment.

[0030] Moreover, in the present application, the fibrinogen is purified by the PEG precipitation method after S / D virus inactivation. Through two precipitations, the residues of S / D reagents can be removed as much as possible. At the same time, during the centrifugation process, the PEG precipitation method avoids the disadvantage that proteins are prone to denaturation due to heat generated by centrifugation in the traditional cryoethanol method, avoids the use of a large amount of refrigerant, and reduces the cost of the purification process. Moreover, PEG can be removed by simple concentration means during the subsequent purification process, reducing the residue amount of the reagent in human fibrinogen and being beneficial for improving the overall purity of human fibrinogen.

[0031] Finally, the present application further adsorbs and removes the impurity proteins still remaining in the liquid by ion exchange chromatography, enabling the target protein to flow through during the chromatography process. Finally, the flow-through liquid is collected, and high-purity and high-yield fibrinogen can be obtained after certain post-treatment. The purification method of the present application can effectively purify human fibrinogen in component I and cold precipitate. Compared with the prior art, the recovery rate is higher, and a relatively high purity can be maintained, which is beneficial for increasing the scale of the purification batch of human fibrinogen. Moreover, the steps are simple, the production efficiency is high, and it is more suitable for popularization.

[0032] In a preferred embodiment, S1) includes: dissolving Component I and cryoprecipitate with an aqueous solution of sodium heparin to obtain a first liquid; preferably, the content of sodium heparin is 3000 - 6000 IU / L, including but not limited to 3000, 3500, 4000, 4500, 5000, 5500 or 6000 IU / L; preferably, the volume ratio of the total volume after mixing Component I and cryoprecipitate to the volume of the sodium heparin aqueous solution is 1:8 - 15, including but not limited to 1:8, 1:9, 1:10, 1:11, 1:12, 1:13, 1:14 or 1:15; preferably, the dissolution time is 2 - 3 hours, including but not limited to 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9 or 3 hours; preferably, the dissolution temperature is 20 - 30 °C, including but not limited to 20 °C, 21 °C, 22 °C, 23 °C, 24 °C, 25 °C, 26 °C, 27 °C, 28 °C, 29 °C or 30 °C.

[0033] By controlling the content of sodium heparin, the mixing volume and the dissolution conditions of the mixture of Component I precipitate and cryoprecipitate in this application, it is beneficial to fully dissolve Component I precipitate and cryoprecipitate simultaneously, and fully dissolve and extract human coagulation factor VIII, human fibrinogen and other proteins in Component I precipitate and cryoprecipitate. At the same time, as an anticoagulant, sodium heparin can inhibit the agglutination that may be caused by the activation of certain factors contained in these two component precipitates, which is more conducive to the subsequent purification.

[0034] In a preferred embodiment, in S2), the temperature of the first reaction is 10 °C - 20 °C, including but not limited to 10 °C, 11 °C, 12 °C, 13 °C, 14, 15, 16, 17, 18, 19 or 20 °C; preferably, the time of the first reaction is 1 - 2 hours, including but not limited to 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9 or 2 hours.

[0035] By adjusting the pH and temperature in S2) to be within the above ranges in this application, the separation efficiency of human coagulation factor VIII, plasminogen, fibronectin, albumin impurities and human fibrinogen in Component I precipitate and cryoprecipitate in the first liquid can be improved, which is beneficial to concentrating more human fibrinogen in the precipitate, increasing the concentration of the crude precipitate, and further improving the recovery efficiency and the purity of the final product.

[0036] In a preferred embodiment, S3) includes: S31) mixing the first precipitate with a first dissolving solution and filtering to obtain a second liquid; S32) subjecting the second liquid to S / D virus inactivation to obtain a third liquid; S33) performing a first PEG precipitation treatment and a second PEG precipitation treatment on the third liquid using a PEG solution to obtain a second precipitate; preferably, the first dissolving solution includes 0.1% - 0.5% tris(hydroxymethyl)aminomethane (Tris), 0.1% - 1.0% sodium citrate, 0.5% - 2.0% sucrose, 0.2% - 1.0% lysine hydrochloride, 0.5% - 2.0% sodium chloride, and the pH value is 6.5 - 7.5. The purpose of the above first dissolution is to partially dissolve and extract human fibrinogen in the first precipitate using the first dissolving solution and further remove other impurity components.

[0037] The method for S / D virus inactivation in this application includes: diluting the protein content in the above-mentioned second liquid to 1.0% - 2.0%, adding S / D reagent according to 1 / 10 of the weight of the second liquid, so that the concentration of polysorbate 80 in the second liquid is 1.0% ± 0.3% and the concentration of tributyl phosphate is 0.3% ± 0.1%, and the temperature during the S / D virus inactivation process is 24°C - 26°C and maintained for 6 - 8 hours.

[0038] In a preferred embodiment, the first PEG precipitation treatment includes: adjusting the temperature of the third liquid to 10 - 15°C, mixing it with a PEG solution, and performing a first reaction to obtain a first PEG precipitate; preferably, the concentration of the PEG solution is 3.0% - 5.0% w / v, including but not limited to 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, or 5.0%; preferably, the time of the first reaction is 30 - 60 min.

[0039] In a preferred embodiment, the second PEG precipitation treatment includes: mixing the first PEG precipitate with a second dissolution solution and filtering, adjusting the temperature of the filtrate obtained after filtration to 10-15 °C, then mixing with a PEG solution to carry out a second reaction to obtain a second precipitate; preferably, the time of the second reaction is 30-60 min; preferably, the second dissolution solution includes 0.1%-1.0% sodium citrate, 0.5%-2.0% sodium chloride, and the pH value is 6.5-7.5. Preferably, the concentration of the PEG solution is 3.0%-5.0%, w / v, including but not limited to 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9 or 5.0%; preferably, the PEG solution includes one or more of PEG4000 or PEG3350.

[0040] By controlling the conditions of the first PEG precipitation treatment and the second PEG precipitation treatment, the present application can be conducive to improving the removal efficiency of S / D, avoiding its residue in human fibrin, and the components in the second dissolution solution are protective components of human fibrinogen, so as to prevent the structure of human fibrinogen from being damaged, promoting the recovery of more high-purity human fibrinogen, avoiding the problem of protein denaturation caused by the traditional cold ethanol precipitation method in the prior art, being conducive to improving the purification and recovery efficiency of subsequent ion chromatography, and further improving the purity and recovery rate of the final human fibrinogen.

[0041] In a preferred embodiment, S4) includes: mixing the second precipitate with a third dissolution solution and filtering, then subjecting the supernatant to first concentration and first dialysis to obtain a first concentrate; subjecting the first concentrate to ion exchange chromatography to obtain a chromatography solution, subjecting the chromatography solution to second concentration and then mixing with a first buffer solution for second dialysis to obtain purified human fibrinogen; preferably, the third dissolution solution includes 0.1%-1.0% sodium citrate, 0.5%-2.0% sodium chloride, 0.2%-1.0% L-arginine hydrochloride, and the pH value is 6.5-7.5.

[0042] In a preferred embodiment, the ion exchange chromatography includes anion exchange chromatography; preferably, the chromatography packing material for anion exchange chromatography includes but not limited to TOYOPEARL(R)DEAE 650M, Q Sepharose Fast Flow or EMDTMAE; preferably, the linear velocity of anion exchange chromatography is 60 cm / h-100 cm / h, including but not limited to 60, 65, 70, 75, 80, 85, 90, 95 or 100 cm / h.

[0043] By controlling the linear velocity of ion exchange chromatography within the above range, the present application can improve the separation efficiency of impurity proteins and human fibrinogen, as well as the purity of the final human fibrinogen, and ensure the stability of human fibrin in the flow-through solution during ion exchange chromatography, avoiding the structural damage and loss of biological activity of human fibrinogen. Excessively high flow rates may cause impurity proteins to pass through without being effectively retained, while excessively low flow rates may increase the residence time of proteins on the chromatography packing, resulting in unnecessary adsorption and loss of target proteins. Controlling an appropriate linear velocity is beneficial to improving the purity and recovery rate of human fibrinogen in the final flow-through solution.

[0044] In a preferred embodiment, the first concentrated liquid is mixed with the third dissolution solution for the first dialysis; preferably, the volume ratio of the first concentrated liquid to the third dissolution solution is 1:3 - 5; preferably, in the second dialysis, the volume ratio of the second concentrated liquid to the first buffer solution is 1:3 - 5; preferably, the first concentration includes ultrafiltration concentration; preferably, the second concentration includes ultrafiltration concentration.

[0045] The first concentration of the present application includes: adding the third dissolution solution to the second precipitate and stirring for dissolution for more than 1 hour, then performing clarification filtration and collecting the filtrate. After stirring the filtrate evenly, perform ultrafiltration concentration (the first concentration), and according to the concentrated volume, dialyze the concentrated product 3 - 5 times with the third dissolution solution (the first dialysis), wash the ultrafiltration system with the third dissolution solution, and collect the ultrafiltration concentrate, which is the above-mentioned first concentrated liquid.

[0046] The second concentration of the present application includes: stirring the collected chromatography flow-through solution evenly and then performing ultrafiltration concentration (the second concentration), and according to the concentrated volume, dialyze the concentrated product 3 - 5 times with the first buffer solution (the second dialysis), wash the ultrafiltration system with the first buffer solution, and collect the purified human fibrinogen stock solution. Preferably, the first buffer solution used in the second dialysis includes 0.1% - 1.0% sodium citrate, 0.5% - 2.0% sodium chloride, 3.0% - 5.0% arginine hydrochloride, 2.0% - 3.0% glycine, and the pH value is 6.5 - 7.5. Since the obtained liquid after ultrafiltration dialysis is human fibrinogen stock solution (purified human fibrinogen), subsequent lyophilization treatment is required to obtain the finished human fibrinogen. Among them, glycine in the first buffer solution can be used as an excipient during the lyophilization process, and the concentration of arginine hydrochloride is increased compared with the concentration of the dissolution solution because the concentration of fibrinogen is relatively high during the lyophilization process and more arginine hydrochloride is needed to protect it.

[0047] In the second typical implementation manner of the present application, an application of the above purification method of human fibrinogen in the production of human fibrinogen products is provided.

[0048] The human fibrinogen stock solution obtained by the purification method of human fibrinogen of the present application can be diluted and prepared by those skilled in the art according to actual needs, and then subjected to conventional sterilization, aliquoting, and freeze-drying. After freeze-drying, capping and dry heat virus inactivation are carried out to obtain the finished product of human fibrinogen.

[0049] The beneficial effects of the present application will be further explained in detail below in conjunction with specific embodiments.

[0050] Example 1

[0051] 1 Precipitation of Component I and combined dissolution of cryoprecipitate:

[0052] The precipitate of Component I and cryoprecipitate are dissolved using an aqueous solution of sodium heparin. The content of sodium heparin is 3000 IU of sodium heparin added per liter of water for injection. The precipitation dissolution multiple is 12 times, the dissolution time is 3 hours, and the dissolution temperature is 25 °C (the first liquid);

[0053] 2 Adjust pH and centrifuge to obtain the first precipitate:

[0054] The dissolution solution is adjusted to pH 6.2 by adding 0.5 mol / L acetic acid solution. During the reaction process, the temperature is gradually lowered to 20 °C. After continuing the reaction for 1 hour, centrifugation is carried out to collect the precipitate after centrifugation (the first precipitate).

[0055] 3 Dissolution and filtration:

[0056] The precipitate collected by centrifugation is added with the first dissolution solution (the first dissolution solution contains 0.3% tris(hydroxymethyl)aminomethane, 0.5% sodium citrate, 0.5% sucrose, 0.2% lysine hydrochloride, 1.0% sodium chloride, and the pH value is 6.8) and stirred for dissolution for more than 1 hour, and then clarified filtration is carried out to collect the filtrate (the second liquid);

[0057] 4 S / D virus inactivation:

[0058] The protein content of the filtrate is diluted to 1.5%, and the S / D reagent is added according to 1 / 10 of the weight of the filtrate, so that the concentration of polysorbate 80 in the filtrate is 0.7% and the concentration of tributyl phosphate is 0.2%. The temperature is controlled at 25 °C and maintained for 7 hours (the third liquid);

[0059] 5 First PEG precipitation treatment:

[0060] The solution after S / D inactivation is cooled to 15 °C, and PEG4000 is added until the final concentration of PEG4000 is 4.0%. After the addition, the reaction is continued for more than 30 minutes, and then centrifuged to collect the first PEG precipitate;

[0061] 6 Second PEG precipitation treatment:

[0062] Add the first PEG precipitate to the second dissolving solution (the second dissolving solution contains 0.1% sodium citrate, 0.5% sodium chloride, and the pH value is 7.3), stir and dissolve for more than 1 hour, then perform clarification filtration and collect the filtrate. Cool the filtrate to 12°C, add PEG4000 until the final concentration of PEG4000 is 3.0%, continue to react for more than 30 minutes after addition, perform centrifugal separation, and collect the second precipitate;

[0063] 7 Ultrafiltration and dialysis:

[0064] Add the second precipitate to the third dissolving solution (the third dissolving solution contains 0.3% sodium citrate, 2.0% sodium chloride, 0.2% arginine hydrochloride, and the pH value is 7.0), stir and dissolve for more than 1 hour, then perform clarification filtration and collect the filtrate. After stirring the filtrate evenly, perform ultrafiltration concentration (the molecular weight cut-off of the membrane package is 100KD), according to the volume after concentration, dialyze the concentrated product 3 times with the third dissolving solution, wash the ultrafiltration system with the third dissolving solution, and collect the ultrafiltration concentrate (the first concentrate);

[0065] 8 Anion exchange chromatography:

[0066] Perform anion exchange chromatography on the collected ultrafiltration concentrate, and use TOYOPEARL(R) DEAE650M as the chromatography packing material. Control the linear velocity at 60 cm / h during the chromatography process;

[0067] 9 Ultrafiltration:

[0068] After stirring the collected chromatography flow-through liquid evenly, perform ultrafiltration (the molecular weight cut-off of the membrane package is 100KD), according to the volume after concentration, dialyze the concentrated product 4 times with the first buffer solution, wash the ultrafiltration system with the first buffer solution, and collect the stock solution; The first buffer solution contains 1.0% sodium citrate, 1.2% sodium chloride, 3.0% arginine hydrochloride, 2.5% glycine, and the pH value is 6.9.

[0069] 10 Preparation, sterilization and sub-packaging, freeze-drying, dry heat inactivation:

[0070] After diluting and preparing the stock solution, sterilize, sub-package, then perform freeze-drying. After freeze-drying is completed, crimp the vial and perform dry heat virus inactivation to obtain the finished product of human fibrinogen.

[0071] The flow chart of the purification method of human fibrinogen in this example is as Figure 1 shown.

[0072] The quality test results of the human fibrinogen intermediate product are shown in Table 1.

[0073] Table 1

[0074]

[0075] As shown in Table 1, the human fibrinogen finally obtained in Example 1 has a purity of over 90%, a reconstitution time of 15 minutes, a coagulation activity of ≤ 60 seconds, and the product quality meets the "Pharmacopoeia of the People's Republic of China" (Volume III, 2020 Edition, ISBN 978 - 7 - 5214 - 1575 - 9), thus ensuring the product quality and safety.

[0076] Example 2

[0077] 1 Dissolution of the combined precipitate of Component I and cryoprecipitate:

[0078] The precipitate of Component I and cryoprecipitate are dissolved using an aqueous solution of sodium heparin. The content of sodium heparin is 4000 IU of sodium heparin added per liter of water for injection. The dissolution multiple of the precipitate is 10 times, the dissolution time is 2 hours, and the dissolution temperature is 30 °C.

[0079] 2 Adjust the pH and perform centrifugal separation to obtain the first precipitate:

[0080] The dissolution solution is adjusted to pH 6.0 by adding 0.5 mol / L acetic acid solution. During the reaction process, the temperature is gradually lowered to 15 °C. After continuing the reaction for 2 hours, centrifugal separation is carried out to collect the precipitate after centrifugation (the first precipitate).

[0081] 3 Dissolution and filtration:

[0082] The precipitate collected by centrifugation is added with the first dissolution solution (the first dissolution solution contains 0.1% tris(hydroxymethyl)aminomethane, 0.3% sodium citrate, 1.0% sucrose, 0.5% lysine hydrochloride, 0.5% sodium chloride, and the pH value is 7.0) and stirred for dissolution for more than 1 hour, and then clarified filtration is carried out to collect the filtrate.

[0083] 4 S / D virus inactivation:

[0084] The protein content of the filtrate is diluted to 1.8%, and the S / D reagent is added according to 1 / 10 of the weight of the filtrate, so that the concentration of polysorbate 80 in the filtrate is 1.0% and the concentration of tributyl phosphate is 0.3%. The temperature is controlled at 26 °C and maintained for 6 hours.

[0085] 5 First PEG precipitation treatment:

[0086] The solution after S / D inactivation is cooled to 12 °C, and PEG4000 is added until the final concentration of PEG4000 is 3.0%. After the addition, the reaction is continued for more than 30 minutes, and then centrifugal separation is carried out to collect the first PEG precipitate.

[0087] 6 Second PEG precipitation treatment:

[0088] Add the first PEG precipitate to the second dissolution solution (the second dissolution solution contains 0.4% sodium citrate, 1.0% sodium chloride, and the pH value is 6.6), stir and dissolve for more than 1 hour, then perform clarification filtration, and collect the filtrate. Cool the filtrate to 15°C, add PEG4000 until the final concentration of PEG4000 is 5.0%, continue to react for more than 30 minutes after addition, perform centrifugal separation, and collect the second precipitate;

[0089] 7 Ultrafiltration and dialysis:

[0090] Add the second precipitate to the third dissolution solution (the third dissolution solution contains 0.5% sodium citrate, 0.8% sodium chloride, 0.5% arginine hydrochloride, and the pH value is 7.2), stir and dissolve for more than 1 hour, then perform clarification filtration, and collect the filtrate. After stirring the filtrate evenly, perform ultrafiltration concentration. According to the volume after concentration, dialyze the concentrated product 4 times with the third dissolution solution, wash the ultrafiltration system with the third dissolution solution, and collect the ultrafiltration concentrate;

[0091] 8 Anion exchange chromatography:

[0092] Perform anion exchange chromatography on the collected ultrafiltration concentrate. The chromatography packing material is Q Sepharose FastFlow, and the linear velocity during the chromatography process is controlled at 100 cm / h. Collect the chromatography flow-through liquid;

[0093] 9 Ultrafiltration:

[0094] After stirring the collected chromatography flow-through liquid evenly, perform ultrafiltration dialysis and ultrafiltration concentration. According to the volume after concentration, dialyze the concentrated product 5 times with the first buffer solution, wash the ultrafiltration system with the first buffer solution, and collect the stock solution; The first buffer solution contains 0.1% sodium citrate, 0.7% sodium chloride, 4.0% arginine hydrochloride, 2.2% glycine, and the pH value is 6.7.

[0095] 10 Dilution, sterilization and aliquoting, lyophilization, dry heat inactivation:

[0096] After diluting and formulating the stock solution, perform sterilization, aliquoting, and then lyophilization. After lyophilization, crimp the vial and perform dry heat virus inactivation to obtain the finished product of human fibrinogen.

[0097] Example 3:

[0098] 1 Component I precipitate and cryoprecipitate combined dissolution:

[0099] Dissolve the component I precipitate and cryoprecipitate with an aqueous heparin sodium solution. The content of heparin sodium is 5000 IU of heparin sodium added per liter of injection water. The precipitation dissolution multiple is 15 times, the dissolution time is 2.5 hours, and the dissolution temperature is 22°C;

[0100] 2 Adjust the pH, perform centrifugal separation to obtain the first precipitate:

[0101] Adjust the pH of the dissolution solution to 6.4 by adding 0.5 mol / L acetic acid solution. During the reaction process, gradually cool down to 18 °C. After continuing the reaction for 1 hour, perform centrifugal separation and collect the precipitate after centrifugation (the first precipitate).

[0102] 3 Dissolution and filtration:

[0103] Add the first dissolution solution (the first dissolution solution contains 0.5% tris(hydroxymethyl)aminomethane, 0.8% sodium citrate, 1.5% sucrose, 0.8% lysine hydrochloride, 0.7% sodium chloride, and the pH value is 7.3) to the precipitate collected by centrifugation and stir to dissolve for more than 1 hour, then perform clarification filtration and collect the filtrate;

[0104] 4 S / D virus inactivation:

[0105] Dilute the protein content of the filtrate to 2.0%. Calculate and add the S / D reagent according to 1 / 10 of the weight of the filtrate to make the concentration of polysorbate 80 in the filtrate 1.3% and the concentration of tributyl phosphate 0.4%. Control the temperature to 24 °C and maintain for 8 hours;

[0106] 5 First PEG precipitation treatment:

[0107] Cool the solution after S / D inactivation to 10 °C, add PEG4000 until the final concentration of PEG4000 is 3.5%. After the addition, continue the reaction for more than 30 minutes, perform centrifugal separation, and collect the first PEG precipitate;

[0108] 6 Second PEG precipitation treatment:

[0109] Add the second dissolution solution (the second dissolution solution contains 0.7% sodium citrate, 1.5% sodium chloride, and the pH value is 6.9) to the first PEG precipitate and stir to dissolve for more than 1 hour, then perform clarification filtration and collect the filtrate. Cool the filtrate to 12 °C, add PEG4000 until the final concentration of PEG4000 is 4.0%. After the addition, continue the reaction for more than 30 minutes, perform centrifugal separation, and collect the second precipitate;

[0110] 7 Ultrafiltration and dialysis:

[0111] Add the third dissolution solution (the third dissolution solution contains 0.7% sodium citrate, 1.2% sodium chloride, 0.7% arginine hydrochloride, and the pH value is 7.1) to the second precipitate and stir to dissolve for more than 1 hour, then perform clarification filtration and collect the filtrate. After stirring the filtrate evenly, perform ultrafiltration concentration. According to the volume after concentration, dialyze the concentrated product 5 times with the third dissolution solution, wash the ultrafiltration system with the third dissolution solution, and collect the ultrafiltration concentrate;

[0112] 8 Anion exchange chromatography:

[0113] The collected ultrafiltration concentrate was subjected to anion exchange chromatography using TOYOPEARL(R) DEAE650M as the chromatography packing material. The linear velocity during the chromatography process was controlled at 70 cm / h, and the chromatography flow-through was collected.

[0114] 9 Ultrafiltration:

[0115] The collected chromatography flow-through was stirred evenly and then subjected to ultrafiltration dialysis for ultrafiltration concentration. According to the volume after concentration, the concentrated product was dialyzed 5 times with the first buffer solution, and the ultrafiltration system was rinsed with the first buffer solution to collect the stock solution. The first buffer solution contained 0.6% sodium citrate, 1.0% sodium chloride, 3.5% arginine hydrochloride, 3.0% glycine, and the pH value was 6.8.

[0116] 10 Dilution, aseptic filling, lyophilization, dry heat inactivation:

[0117] After the stock solution was diluted and prepared, it was sterilized, filled, and then lyophilized. After lyophilization, it was sealed with a lid and inactivated by dry heat virus inactivation to obtain the finished product of human fibrinogen.

[0118] Example 4:

[0119] 1 Precipitate of Component I and combined dissolution of cryoprecipitate:

[0120] The precipitate of Component I and cryoprecipitate were dissolved using an aqueous solution of sodium heparin. The content of sodium heparin was 6000 IU of sodium heparin added per liter of water for injection. The precipitation dissolution multiple was 8 times, the dissolution time was 3 hours, and the dissolution temperature was 26°C.

[0121] 2 Adjust pH, centrifugally separate to obtain the first precipitate:

[0122] The dissolution solution was adjusted to pH 6.5 by adding 0.5 mol / L acetic acid solution. During the reaction process, the temperature was gradually lowered to 10°C. After continuing the reaction for 2 hours, it was centrifugally separated, and the precipitate after centrifugation was collected.

[0123] 3 Dissolution and filtration:

[0124] The precipitate collected by centrifugation was added with the first dissolution solution (the first dissolution solution contained 0.4% tris(hydroxymethyl)aminomethane, 1.0% sodium citrate, 2.0% sucrose, 1.0% lysine hydrochloride, 1.5% sodium chloride, and the pH value was 6.6) and stirred for dissolution for more than 1 hour, and then subjected to clarification filtration to collect the filtrate.

[0125] 4 S / D virus inactivation:

[0126] The protein content of the filtrate was diluted to 1.0%, and the S / D reagent was added according to 1 / 10 of the weight of the filtrate, so that the concentration of polysorbate 80 in the filtrate was 1.1% and the concentration of tributyl phosphate was 0.3%. The temperature was controlled at 25°C and maintained for 6 hours.

[0127] 5 First PEG precipitation treatment:

[0128] Cool the solution after S / D inactivation to 12°C, add PEG4000 until the final concentration of PEG4000 is 5.0%, continue the reaction for more than 30 minutes after addition, perform centrifugal separation, and collect the first PEG precipitate;

[0129] 6 Second PEG precipitation treatment:

[0130] Add the first PEG precipitate to the second dissolution solution (the second dissolution solution contains 1.0% sodium citrate, 2.0% sodium chloride, and the pH value is 6.7), stir and dissolve for more than 1 hour, then perform clarification filtration and collect the filtrate. Cool the filtrate to 10°C, add PEG4000 until the final concentration of PEG4000 is 3.5%, continue the reaction for more than 30 minutes after addition, perform centrifugal separation, and collect the second precipitate;

[0131] 7 Ultrafiltration and dialysis:

[0132] Add the second precipitate to the third dissolution solution (the third dissolution solution contains 1.0% sodium citrate, 0.7% sodium chloride, 1.0% arginine hydrochloride, and the pH value is 7.3), stir and dissolve for more than 1 hour, then perform clarification filtration and collect the filtrate. After stirring the filtrate evenly, perform ultrafiltration concentration, according to the volume after concentration, dialyze the concentrated product 4 times with the third dissolution solution, wash the ultrafiltration system with the third dissolution solution, and collect the ultrafiltration concentrate;

[0133] 8 Anion exchange chromatography:

[0134] Perform anion exchange chromatography on the collected ultrafiltration concentrate, use EMD TMAE as the chromatography packing material, control the linear velocity during the chromatography process at 80 cm / h, and collect the chromatography flow-through liquid;

[0135] 9 Ultrafiltration:

[0136] After stirring the collected chromatography flow-through liquid evenly, perform ultrafiltration dialysis and ultrafiltration concentration. According to the volume after concentration, dialyze the concentrated product 3 times with the first buffer solution, wash the ultrafiltration system with the first buffer solution, and collect the stock solution; The first buffer solution contains 0.4% sodium citrate, 0.5% sodium chloride, 4.5% arginine hydrochloride, 2.0% glycine, and the pH value is 7.2.

[0137] 10 Dilution, sterilization, aliquoting, lyophilization, dry heat inactivation:

[0138] After diluting and formulating the stock solution, perform sterilization, aliquoting, and then lyophilization. After lyophilization, crimp the vial and perform dry heat virus inactivation to obtain the finished product of human fibrinogen.

[0139] Example 5

[0140] The difference from Example 1 is only that the concentration of the first PEG precipitation treatment and the concentration of the PEG solution in the second PEG precipitation treatment are adjusted to 6% (Steps 5 and 6), and the remaining steps are the same as those in Example 1. The results are shown in Table 2.

[0141] Table 2

[0142]

[0143]

[0144] Example 6

[0145] The difference from Example 1 is only that the concentration of the first PEG precipitation treatment and the concentration of the PEG solution in the second PEG precipitation treatment are adjusted to 2% (Steps 5 and 6), and the remaining steps are the same as those in Example 1. The results are shown in Table 3.

[0146] Table 3

[0147] Example 6 Appearance Light gray or light yellow porous body Vacuum degree Qualified Redissolution time (min) 14 Osmolality (mOsmol / kg) 730 Moisture content (%) 2.0 Purity (%) 86 Coagulation activity (seconds) 45 Tributyl phosphate residue (μg / mL) 4 Polysorbate 80 residue (μg / mL) 62

[0148] Comparative Example 1

[0149] The difference from Example 1 is only that the pH in Step 2 is 5.8, and the remaining steps are the same as those in Example 1. The results are shown in Table 4.

[0150] Table 4

[0151] Comparative Example 1 Appearance Light gray or light yellow porous body Vacuum degree Qualified Redissolution time (min) 18 Osmolality (mOsmol / kg) 780 Moisture content (%) 1.7 Purity (%) 76 Coagulation activity (seconds) 48 Tributyl phosphate residue (μg / mL) 5 Polysorbate 80 residue (μg / mL) 65

[0152] Comparative Example 2

[0153] The difference from Example 1 is only that the pH in Step 2 is 7.0, and the remaining steps are the same as those in Example 1. The results are shown in Table 5.

[0154] Table 5

[0155]

[0156]

[0157] Comparative Example 3

[0158] Prepared by a conventional method, and purified human fibrinogen was carried out by the method described in Patent CN201810054783.

[0159] The finished product indexes of Examples 1 to 6 and Comparative Examples 1 to 3 are shown in Table 6.

[0160] Table 6

[0161]

[0162] As can be seen from the above results, the method of the present invention has good product quality, and each embodiment of the present invention meets the requirements that the reconstitution time ≤ 20 min, the moisture content of the freeze-dried product ≤ 3.0%, the purity ≥ 90%, the coagulation activity ≤ 50 seconds, and the residual amounts of tributyl phosphate and polysorbate 80 are low, indicating that the purity of the human fibrinogen obtained after purification in this application is higher, and it shows that high-quality human fibrinogen products can be obtained by the process of the present invention.

[0163] From the above description, it can be seen that the above embodiments of the present invention achieve the following technical effects: By using the purification method of human fibrinogen in this application and precisely controlling the conditions in the purification steps, each link cooperates with each other, so that the recovery efficiency and purification efficiency of human fibrinogen in this application are both improved. Moreover, the human fibrinogen in this application exists in the flow-through liquid of ion exchange chromatography, and the recovery amount is not limited by the packing material, enabling the batch scale to be enlarged, greatly increasing the recovery amount of human fibrinogen, overcoming the problems existing in the prior art, and finally the recovered human fibrinogen has high purity and high quality. The purification method of this application can promote the further development of the preparation of human fibrinogen products and is conducive to meeting the increasing clinical demand for human fibrinogen products.

[0164] The above are only the preferred embodiments of the present invention and are not intended to limit the present invention. For those skilled in the art, the present invention can have various modifications and changes. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

Claims

1. A method for purifying human fibrinogen, characterized in that, The purification method includes: S1) Mix the component I precipitate with the cold precipitate for dissolution to obtain a first liquid; S2) Adjust the pH of the first liquid to 6.0 - 6.5 for a first reaction, and obtain a first precipitate after solid-liquid separation; S3) Dissolve the first precipitate successively, perform S / D virus inactivation, and perform two PEG precipitations to obtain a second precipitate; S4) Dissolve the second precipitate and then perform ion exchange chromatography to obtain purified human fibrinogen.

2. The purification method according to claim 1, wherein The S1) includes: Dissolve the component I and the cold precipitate with an aqueous solution of sodium heparin to obtain the first liquid; Preferably, the content of sodium heparin is 3000 - 6000 IU / L; Preferably, the volume ratio of the total volume after mixing the component I and the cold precipitate to the volume of the aqueous solution of sodium heparin is 1:8 - 15; Preferably, the dissolution time is 2 - 3 hours; Preferably, the dissolution temperature is 20 - 30 °C.

3. The purification method according to claim 1, wherein In the S2), the temperature of the first reaction is 10 °C - 20 °C; Preferably, the time of the first reaction is 1 - 2 hours.

4. The purification method according to claim 1, wherein The S3) includes, S31) Mix the first precipitate with a first dissolution solution and filter to obtain a second liquid; S32) Perform S / D virus inactivation on the second liquid to obtain a third liquid; S33) Perform a first PEG precipitation treatment and a second PEG precipitation treatment on the third liquid with a PEG solution to obtain the second precipitate; Preferably, the first dissolution solution includes 0.1% - 0.5% by mass of tris(hydroxymethyl)aminomethane, 0.1% - 1.0% by mass of sodium citrate, 0.5% - 2.0% by mass of sucrose, 0.2% - 1.0% by mass of lysine hydrochloride, 0.5% - 2% by mass of sodium chloride, and the pH value is 6.5 - 7.

5.

5. The purification method according to claim 4, characterized in that, The first PEG precipitation treatment includes: After adjusting the temperature of the third liquid to 10 - 15 °C, mix it with a PEG solution for a first reaction to obtain a first PEG precipitate; Preferably, the concentration of the PEG solution is 3.0% - 5.0% w / v; Preferably, the time of the first reaction is 30 - 60 min.

6. The purification method according to claim 5, wherein, The second PEG precipitation treatment includes: Mix the first PEG precipitate with a second dissolution solution and filter. After adjusting the temperature of the filtrate obtained by filtration to 10 - 15 °C, mix it with a PEG solution for a second reaction to obtain the second precipitate; Preferably, the time of the second reaction is 30 - 60 min; Preferably, the second dissolution solution includes 0.1% - 1.0% by mass of sodium citrate, 0.5% - 2.0% by mass of sodium chloride, and the pH value is 6.5 - 7.5; Preferably, the concentration of the PEG solution is 3.0% - 5.0% w / v; Preferably, the PEG solution includes one or more of PEG4000 solution or PEG3350 solution, and more preferably PEG4000 solution.

7. The purification method according to claim 1, wherein The S4) includes: After mixing the second precipitate with a third dissolution solution and filtering, perform a first concentration and a first dialysis on the supernatant to obtain a first concentrate; After subjecting the first concentrated solution to ion exchange chromatography, a chromatographed solution is obtained. The chromatographed solution is secondarily concentrated and then mixed with a first buffer solution for secondary dialysis to obtain the purified human fibrinogen; Preferably, the third dissolving solution comprises sodium citrate with a mass ratio of 0.1% to 1.0%, sodium chloride with a mass ratio of 0.5% to 2.0%, arginine hydrochloride with a mass ratio of 0.2% to 1.0%, and has a pH value of 6.5 to 7.5; Preferably, the first buffer solution comprises sodium citrate with a mass ratio of 0.1% to 1.0%, sodium chloride with a mass ratio of 0.5% to 2.0%, arginine hydrochloride with a mass ratio of 3.0% to 5.0%, glycine with a mass ratio of 2.0% to 3.0%, and has a pH value of 6.5 to 7.

5.

8. The purification method according to claim 7, wherein The ion exchange chromatography includes anion exchange chromatography; Preferably, the chromatographic packing material for the anion exchange chromatography comprises TOYOPEARL(R) DEAE 650M, Q Sepharose Fast Flow, or EMD TMAE; Preferably, the linear velocity of the anion exchange chromatography is 60 cm / h to 100 cm / h.

9. The purification method according to claim 7, characterized in that, The first dialysis includes: mixing the first concentrated liquid with the third dissolving solution for the first dialysis; Preferably, the volume ratio of the first concentrated liquid to the third dissolving solution is 1:3 to 5; Preferably, in the secondary dialysis, the volume ratio of the second concentrated liquid to the first buffer solution is 1:3 to 5; Preferably, the first concentration includes ultrafiltration concentration; Preferably, the second concentration includes ultrafiltration concentration.

10. Use of the method for purifying human fibrinogen according to any one of claims 1 to 9 in the production of human fibrinogen products.

Images