CN113577295B - Human fibrinogen dry heat treatment stabilizer and application thereof - Google Patents
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Abstract
The invention provides a dry heat treatment stabilizer for human fibrinogen, which comprises the following raw materials in parts by weight: 0.03-0.05 molar part of sodium citrate, 0.12-0.15 molar part of sodium chloride and 20-40 parts of arginine hydrochloride. The fibrinogen dry heat treatment stabilizer is suitable for a dry heat inactivation mode of 80 ℃ for 72 hours, can effectively maintain the activity index of human fibrinogen, does not influence the inactivation of viruses by dry heat virus treatment, uses a small number of amino acids, has low dosage and low cost, and is obviously superior to the conventional dry heat treatment stabilizer for human fibrinogen preparations. The human fibrinogen preparation prepared by the dry heat treatment stabilizer meets the requirements in appearance, has good stability under the conventional storage condition and high temperature condition and excellent coagulation activity, is a new, safe and effective human fibrinogen preparation with low cost, and has good industrial application prospect.
Description
Technical Field
The invention belongs to the field of blood product processing, and particularly relates to a human fibrinogen dry heat treatment stabilizer and application thereof.
Background
The human fibrinogen is soluble plasma glycoprotein, has the molecular weight of 340kDa, has the content in plasma second to albumin and immunoglobulin, and is about 2-4.5g/L, is synthesized and secreted by the liver of a human body, has the half-life period of 3-4 days, and is the key for realizing and maintaining hemostasis of the human body.
The human fibrinogen blood product is prepared by separating and freeze-drying healthy human plasma, has a good improvement effect on blood coagulation obstruction caused by fibrinogen deficiency, excessive consumption or excessive fibrinolysis, and is clinically used for blood coagulation disorder caused by fibrinogen deficiency caused by pregnancy poisoning, stillbirth, postpartum hemorrhage, placenta early stripping, major surgery, severe hemorrhage and the like.
The current common preparation method of human fibrinogen is as follows: fibrin or plasma cryoprecipitate is taken as a raw material, amino acid or ethanol is used for repeated precipitation and purification after dissolution and/or a fibrin flow-through method is used for purification by a chromatography method, the operation is complex, the purification effect of the obtained human fibrinogen is unstable, and the removal effect of the human fibrinogen to a common virus inactivator is unstable; furthermore, the most common condition for virus dry heat inactivation in the currently common preparation method of human fibrinogen is treatment at 100 ℃ for 30min, and meanwhile, due to the instability of human fibrinogen, a stabilizer needs to be added during separation and purification, so that the product can tolerate freeze drying and dry heat treatment without affecting the virus inactivation effect of dry heat treatment. Therefore, the stabilizer used in the existing preparation process of human fibrinogen is also suitable for the dry heat treatment method at 100 ℃ for 30 min. For example, the method for extracting human fibrinogen disclosed in patent CN102212129B adopts repeated precipitation of glycine, and mixing and preparing solutions of sodium citrate, arginine hydrochloride and sodium chloride, and finally inactivates viruses by adopting a dry heat treatment method at 100 ℃ for 30min to form a human fibrinogen preparation product containing a stabilizer system of glycine, sodium citrate, arginine hydrochloride and sodium chloride.
However, the research shows that the virus inactivation effect of the treatment at 80 ℃ for 72 hours is better than that of the treatment at 100 ℃ for 30min, and the temperature controllability is stronger. Therefore, the research on a new human fibrinogen stabilizer and a method for preparing the human fibrinogen preparation by means of dry heat inactivation at 80 ℃ for 72 hours has important significance.
Disclosure of Invention
The invention aims to provide a novel human fibrinogen dry heat treatment stabilizer and a novel human fibrinogen preparation method.
The invention provides a dry heat treatment stabilizer for human fibrinogen, which comprises the following raw materials in parts by weight: 0.03-0.05 molar part of sodium citrate, 0.12-0.15 molar part of sodium chloride and 20-40 parts of arginine hydrochloride.
Further, the feed consists of the following raw material components: 0.03-0.05 molar part of sodium citrate, 0.12-0.15 molar part of sodium chloride and 20-40 parts of arginine hydrochloride.
Furthermore, the feed consists of the following raw material components: 0.03 molar part of sodium citrate, 0.12 molar part of sodium chloride and 40 weight parts of arginine hydrochloride.
The invention also provides the application of the dry heat treatment stabilizer in a human fibrinogen preparation.
The invention also provides a human fibrinogen preparation, which comprises the dry heat treatment stabilizer and human fibrinogen, preferably, the proportion of the raw materials of the human fibrinogen and the dry heat treatment stabilizer in the preparation is as follows: 28-38 parts of human fibrinogen, 0.03-0.05mol part of sodium citrate, 0.12-0.15mol part of sodium chloride and 20-40 parts of arginine hydrochloride.
Further, the proportion of the raw materials of the human fibrinogen and the dry heat treatment stabilizer is as follows: 28-38 parts of human fibrinogen, 0.03 mol part of sodium citrate, 0.12 mol part of sodium chloride and 40 mol parts of arginine hydrochloride.
Further, the human fibrinogen is prepared by the following method:
removing impurities from cryoprecipitated solution adsorbent prepared from fresh frozen plasma, inactivating virus, and purifying by ion exchange chromatography.
The invention also provides a preparation method of the human fibrinogen preparation, which comprises the following steps:
(1) Dissolving cryoprecipitate prepared from fresh frozen plasma in a buffer solution to obtain a dissolving solution containing human fibrinogen;
(2) Adding an adsorbent into the dissolved solution for adsorption and impurity removal, and filtering to obtain a clarified solution;
(3) Performing virus inactivation on the clarified solution by using an S/D inactivator according to an S/D method;
(4) Performing column chromatography on the inactivated clarified liquid to obtain a purified sample;
(5) Adding the purified sample into the dry heat treatment stabilizer for ultrafiltration preparation, sterilization and freeze-drying;
(6) And (4) carrying out virus inactivation by dry heat treatment.
Further, the buffer solution in the step (1) is a tris buffer solution; the adsorbent in the step (2) is aluminum hydroxide powder or gel;
the S/D inactivator in the step (3) is a mixture of an organic solvent and a surfactant, wherein the organic solvent is tributyl phosphate, and the surfactant is polysorbate 80;
the column chromatography in the step (4) is ion exchange chromatography, and the column chromatography medium is an ion exchange medium;
the ultrafiltration preparation in the step (5) refers to: preparing raw materials containing a dry heat treatment stabilizer: sodium citrate and sodium chloride in the stabilizer are exchanged into the purified sample to reach the target concentration by a dialysis mode, and arginine hydrochloride in the stabilizer is added to reach the target concentration;
the dry heat treatment in the step (6) is carried out for 72 to 73 hours at the temperature of more than 80 ℃.
Furthermore, the adding amount of the buffer solution in the step (1) is 2-5 times of the mass of the cryoprecipitate;
the addition amount of the adsorbent in the step (2) is as follows: adding 15-30 g of adsorbent into each kilogram of cryoprecipitate;
the virus inactivation step in the step (3) is to inactivate for 4 to 8 hours at 25 +/-3 ℃, preferably for 6 hours at 25 +/-1 ℃;
purifying the ion exchange chromatography in the step (4) by adopting hybrid protein, polysorbate 80 and tributyl phosphate flow-through;
the sterilization in the step (5) is filter sterilization by a filter membrane;
the dry heat treatment in the step (6) is carried out at 80 ℃ for 72 hours.
The dry heat treatment stabilizer for fibrinogen is suitable for a dry heat treatment mode of 80 ℃ and 72 hours, can effectively maintain the activity of human fibrinogen, does not influence the virus inactivation effect of dry heat treatment, and has few component types and low cost. The fibrinogen preparation of the invention has the advantages of simple preparation method, good stability, high purity, safety, effectiveness, low cost and good clinical application prospect.
The invention relates to a 'sodium citrate 0.03-0.05mol part, sodium chloride 0.12-0.15mol part, arginine hydrochloride 20-40 weight parts' which refers to: in the dry heat treatment stabilizer consisting of the sodium citrate, the sodium chloride and the arginine hydrochloride, the dosage of the sodium chloride corresponding to every 0.03-0.05mmol of the sodium citrate is 0.12-0.15mmol, and the dosage of the corresponding arginine hydrochloride is 20-40mg. The proportional relationship is not limited to "a combination of 0.03 to 0.05mmol of sodium citrate, 0.12 to 0.15mmol of sodium chloride and 20 to 40mg of arginine hydrochloride", but also includes a combination of the amounts of raw materials which are multiplied or reduced, for example, "a combination of 0.03 to 0.05mol of sodium citrate, 0.12 to 0.15mol of sodium chloride and 20 to 40g of arginine hydrochloride", "a combination of 0.06 to 0.10mmol of sodium citrate, 0.24 to 0.30mmol of sodium chloride and 40 to 80mg of arginine hydrochloride", "a combination of 0.015 to 0.025mmol of sodium citrate, 0.06 to 0.075mmol of sodium chloride and 10 to 20mg of arginine hydrochloride", and the like.
Similarly, the mixture ratio of the raw materials of the human fibrinogen and the dry heat treatment stabilizer is as follows: 28-38 parts of human fibrinogen, 0.03-0.05 part of sodium citrate, 0.12-0.15 part of sodium chloride and 20-40 parts of arginine hydrochloride by weight refer to the following components: in the fibrinogen preparation of the inventor, the dosage of sodium citrate corresponding to each 28-38 mg of human fibrinogen is 0.03-0.05mmol, the dosage of sodium chloride corresponding to each 28-38 mg of human fibrinogen is 0.12-0.15mmol, and the dosage of arginine hydrochloride corresponding to each 28-40 mg of human fibrinogen is 20-40mg. The proportional relation is not limited to the combination of 28-38 mg of human fibrinogen, 0.03-0.05mmol of sodium citrate, 0.12-0.15mmol of sodium chloride and 20-40mg of arginine hydrochloride, but also includes the combination of the quantity of the raw materials which are enlarged or reduced by times, and so on.
The "human fibrinogen preparation" according to the present invention refers to a preparation containing human fibrinogen and a stabilizer in a specific amount ratio according to the present invention, and includes various forms such as solid preparations or liquid preparations, for example, lyophilized powders, solutions, and the like.
Obviously, many modifications, substitutions, and variations are possible in light of the above teachings of the invention, without departing from the basic technical spirit of the invention, as defined by the following claims.
The present invention will be described in further detail with reference to the following examples. This should not be understood as limiting the scope of the above-described subject matter of the present invention to the following examples. All the technologies realized based on the above contents of the present invention belong to the scope of the present invention.
Drawings
FIG. 1 is a flow chart of fibrinogen preparation by the present inventors.
FIG. 2 is a PPV viral inactivation curve of dry heat viral inactivation of a fibrinogen preparation of the present invention.
FIG. 3 is a Sindbis virus inactivation curve for dry heat virus inactivation of a fibrinogen preparation of the present invention.
FIG. 4 is an EMCV virus inactivation curve for dry heat virus inactivation of a fibrinogen preparation of the present invention.
Detailed Description
The raw materials and equipment used in the invention are known products and are obtained by purchasing commercial products.
Example 1 preparation of fibrinogen preparation by the inventors
As shown in fig. 1, the preparation method comprises the following steps:
1. preliminary purification
I. Adding 0.2M tris buffer solution into a cryoprecipitate prepared from fresh frozen plasma for dissolving, wherein the proportion of the added tris buffer solution is 3 times of the amount of the cryoprecipitate, and obtaining a human fibrinogen-rich dissolved solution;
adding aluminum hydroxide powder into the tris solution for adsorption treatment, wherein the adding proportion of the aluminum hydroxide is that 20g of the aluminum hydroxide powder is added into each kg of the cold precipitation;
III, centrifuging the solution adsorbed by the aluminum hydroxide, and then clarifying and filtering the supernatant by 0.45 um;
2. inactivation of viruses
Performing virus inactivation on the clarified liquid by using an S/D (S/D) inactivating agent according to an S/D method, wherein the S/D inactivating agent is a mixture of an organic solvent and a surfactant, the surfactant is polysorbate 80, the final concentration of the clarified liquid after being added is 1%, the organic solvent is tributyl phosphate, and the final concentration of the clarified liquid after being added is 0.3%; inactivating at 25 + -1 deg.C for 6 hr;
3. chromatographic purification
Purifying the sample by ion exchange chromatography; the ion exchange chromatography medium is Fractogel EMD DEAE (M), the product is adsorbed onto the chromatography medium, and the product is purified by flowing through the product containing hetero-protein (such as albumin, globulin, von Willebrand factor, human blood coagulation factor VIII or plasminogen), polysorbate 80 and tributyl phosphate.
4. Formulation of
The purified sample is ultrafiltered by the stabilizer formula of the invention, namely, the stabilizer in the dialysis buffer solution of the product is exchanged into the product by a dialysis mode to reach the target concentration, arginine hydrochloride is added to reach the target concentration, and sterilization is carried out, in particular, a filter membrane with the specification of less than 0.22um is adopted for pressure filtration to remove possible bacteria. And finally, subpackaging and freeze-drying, wherein the highest temperature of the product in the freeze-drying process is not more than 35 ℃.
5. Inactivation of viruses
And performing virus inactivation on the freeze-dried preparation by adopting a mode of 80 ℃ and 72 hours to obtain the human fibrinogen preparation.
The beneficial effects of the present invention are demonstrated by the following experimental examples.
Experimental example 1 formulation screening test of the present invention
1. Carrying out a formula combination experiment by using 20-40mg/ml arginine hydrochloride on the basis of 30-50mmol/L sodium citrate and 120-150mmol/L sodium chloride;
1.1 the judgment index 1 of the quality of the formula is that whether the appearance of the human fibrinogen is collapsed or withered after freeze-drying and dry-heating, and the specification of Chinese pharmacopoeia is as follows: it should be gray or yellowish loose body, and clear solution after redissolution with slight opalescence.
The judgment index 2 of the quality of the formula is the redissolution time, which is specified in Chinese pharmacopoeia: should dissolve completely within 30 minutes.
The judgment index 3 of the quality of the formula is the coagulation activity, and the specification of the Chinese pharmacopoeia is as follows: not more than 60 seconds.
1.2 formulation combination test results are shown in Table 1.
TABLE 1 combination of formulations test results
As can be seen from table 1, the human fibrinogen preparation (example 1) obtained by using the formula 4 of the present invention as a human fibrinogen stabilizer and performing dry heat treatment at 80 ℃ for 72 hours has an appearance, a reconstitution time and a coagulation activity that all meet the requirements of the "chinese pharmacopoeia", and the reconstitution time and the coagulation activity far exceed the requirements of the "chinese pharmacopoeia".
2. In contrast to the stabilizer system in patent CN 102212129B:
referring to the fibrinogen preparation method of example 1, only the stabilizer was replaced with the following system:
1) The formula A is as follows: 0.5% glycine, 1.4% sodium citrate (0.0476M), 3% arginine hydrochloride, 0.7% sodium chloride (0.12M);
2) And a formula B:0.5% glycine, 1.52% sodium citrate (0.0517M), 4.3% arginine hydrochloride, 0.8% sodium chloride (0.14M);
3) And a formula C:0.5% glycine, 1.65% sodium citrate (0.0561M), 5.5% arginine hydrochloride, 0.9% sodium chloride (0.1538M);
after subpackaging and freeze-drying, respectively carrying out dry heating at 80 ℃ and 100 ℃, and obtaining the results as shown in table 2:
TABLE 2 comparative test results of formula A of patent CN102212129B and the stabilizer of the invention
TABLE 3. Patent CN102212129B formulation B and inventive stabilizers comparative test results
TABLE 4 comparative test results of formulation C of patent CN102212129B and stabilizer of the invention
As shown in the comparative test results in tables 2, 3 and 4, the stabilizer system in patent CN102212129B contains glycine and sodium citrate with high concentration, and although the lyophilized product has a qualified appearance, after being subjected to dry heat treatment at 80 ℃ for 72 hours, the product has an atrophied appearance, which does not meet the requirements of the "chinese pharmacopoeia", and the redissolution time of the product is long, which indicates that the stabilizer system in patent CN102212129B is not suitable for the human fibrinogen prepared in this example to be subjected to dry heat virus inactivation treatment at 80 ℃ for 72 hours, and has a unqualified appearance and a prolonged redissolution time.
Experimental example 2 Virus inactivation Effect of the present invention
The fibrinogen preparation of example 1 was used for dry heat virus inactivation test, and the results are shown in Table 5, and the virus inactivation curves are shown in FIGS. 2 to 4.
TABLE 5 verification of viral inactivation (n = 3)
| Indicative of virus | PPV | Sindbis | EMCV |
| Inactivating effect | ＞4.0log | ＞4.0log | ＞4.0log |
As can be seen from Table 5, the virus inactivation effect of the fibrinogen preparation of the present inventors is > 4.0log, which completely reaches the national regulation standard.
Experiments show that the dry heat treatment stabilizer can not influence the inactivation effect of dry heat treatment on viruses while protecting the quality of products, and the inactivation titer of the dry heat treatment stabilizer is more than 4.0log.
Experimental example 3 stability of the preparation of the present invention
The human fibrinogen preparation of example 1 was taken and examined for stability under storage conditions of 2-8 ℃ (conventional storage conditions) and 25 ℃ (accelerated stability), respectively.
According to the regulation of Chinese pharmacopoeia, the redissolution time of human fibrinogen should not exceed 30 minutes, the water content should not exceed 5 percent, and the coagulation activity should not exceed 60 seconds.
The results are shown in tables 6 and 7.
TABLE 6.25 ℃ accelerated stability test results (n = 3)
| Redissolution time (min) | Moisture (%) | Coagulation activity(s) | |
| 1 month | / | / | 36 |
| 2 month | / | / | 27 |
| 3 month | 12 | 1.5 | 27 |
| 6 month | 15 | 1.7 | 30 |
| 9 month | 16 | 1.7 | 30 |
| 12 |
6 | 1.7 | 30 |
As can be seen from Table 6, the fibrinogen of the present invention is preserved for 12 months at 25 ℃, the redissolution time, the moisture content and the coagulation activity are all kept at good levels, and each index has no obvious attenuation, and the fibrinogen meets the standard of Chinese pharmacopoeia.
TABLE 7.2-8 ℃ stability test results (n = 3)
| Redissolution time (min) | Moisture (%) | Coagulation activity(s) | |
| 3 month | 12 | 1.3 | 26 |
| 6 month | 15 | 1.9 | 29 |
| 9 month | 15 | 1.8 | 32 |
| 12 |
6 | 1.5 | 27 |
| 18 |
7 | 1.5 | 27 |
| 24 months old | 6 | 1.5 | 27 |
As can be seen from Table 7, the human fibrinogen is stored for 24 months at the temperature of 2-8 ℃, the redissolution time, the moisture content and the coagulation activity of the human fibrinogen are all kept at good levels, and all indexes are not obviously attenuated, thereby meeting the standards of Chinese pharmacopoeia.
The above results demonstrate that the fibrinogen preparation of the present invention is excellent in stability.
In conclusion, the fibrinogen dry heat treatment stabilizer is suitable for a dry heat inactivation mode of 80 ℃ for 72 hours, can effectively maintain the activity index of human fibrinogen, does not influence the inactivation effect of dry heat virus treatment on viruses, uses a small number of amino acids, is low in dosage and cost, and is obviously superior to the conventional dry heat treatment stabilizer of a human fibrinogen preparation. The fibrinogen preparation of the invention has the advantages of qualified appearance, good stability under the conventional storage condition and high temperature condition, excellent coagulation activity, half of the required coagulation time required by Chinese pharmacopoeia, novel, safe, effective and low-cost human fibrinogen preparation, and good industrial application prospect.
Claims (11)
1. A stabilizer for dry heat treatment of human fibrinogen is characterized by comprising the following raw material components: 0.03-0.05mol portion of sodium citrate, 0.12-0.15mol portion of sodium chloride and 20-40 weight portions of arginine hydrochloride.
2. The dry heat treatment stabilizer according to claim 1, which consists of the following raw material components: 0.03 molar part of sodium citrate, 0.12 molar part of sodium chloride and 40 weight parts of arginine hydrochloride.
3. Use of the dry heat treatment stabilizer of claim 1 or 2 in a human fibrinogen preparation.
4. A human fibrinogen preparation comprising the dry heat treatment stabilizer of claim 1 or 2 and human fibrinogen.
5. The fibrinogen preparation of claim 4 wherein the formulation comprises the following raw materials in the ratio of human fibrinogen to the dry heat treatment stabilizer: 28-38 parts of human fibrinogen, 0.03-0.05 part of sodium citrate, 0.12-0.15 part of sodium chloride and 20-40 parts of arginine hydrochloride.
6. The human fibrinogen preparation of claim 5, wherein the ratio of the raw materials of human fibrinogen and the dry heat treatment stabilizer is: 28-38 parts of human fibrinogen, 0.03 mol part of sodium citrate, 0.12 mol part of sodium chloride and 40 mol parts of arginine hydrochloride.
7. The human fibrinogen preparation of any of claims 4-6 wherein said human fibrinogen is prepared by:
removing impurities from cryoprecipitated solution adsorbent prepared from fresh frozen plasma, inactivating virus, and purifying by ion exchange chromatography.
8. A process for the preparation of a human fibrinogen preparation according to any of claims 4 to 7, comprising the steps of:
(1) Dissolving cryoprecipitate prepared from fresh frozen plasma in a buffer solution to obtain a dissolving solution containing human fibrinogen;
(2) Adding an adsorbent into the dissolved solution for adsorption and impurity removal, and filtering to obtain a clarified solution;
(3) Performing virus inactivation on the clarified solution by using an S/D inactivator according to an S/D method;
(4) Performing column chromatography on the inactivated clarified liquid to obtain a purified sample;
(5) Adding the dry heat treatment stabilizer into the purified sample for ultrafiltration preparation, degerming and freeze-drying;
(6) The dry heat treatment is used for virus inactivation.
9. The method according to claim 8, wherein the buffer in step (1) is a tris buffer; the adsorbent in the step (2) is aluminum hydroxide powder or gel;
the S/D inactivator in the step (3) is a mixture of an organic solvent and a surfactant, wherein the organic solvent is tributyl phosphate, and the surfactant is polysorbate 80;
the column chromatography in the step (4) is ion exchange chromatography, and a column chromatography medium is an ion exchange medium;
the ultrafiltration preparation in the step (5) is as follows: preparing raw materials containing a dry heat treatment stabilizer: the sodium citrate and the sodium chloride in the purified sample reach the target concentration through a dialysis mode, and then the arginine hydrochloride in the stabilizing agent is added to reach the target concentration;
and (6) the dry heat treatment is carried out at the temperature of more than 80 ℃ for 72 to 73 hours.
10. The method according to claim 9, wherein the buffer in step (1) is added in an amount of 2 to 5 times the mass of the cryoprecipitate;
the addition amount of the adsorbent in the step (2) is as follows: adding 15-30 g of adsorbent into each kilogram of cryoprecipitate;
the virus inactivation step in the step (3) is to inactivate for 4 to 8 hours at 25 +/-3 ℃;
purifying the ion exchange chromatography in the step (4) by adopting hybrid protein, polysorbate 80 and tributyl phosphate flow-through;
the sterilization in the step (5) is filter sterilization by a filter membrane;
the dry heat treatment in the step (6) is carried out at 80 ℃ for 72 hours.
11. The method of claim 10, wherein the virus inactivation in step (3) is performed at 25 ℃ ± 1 ℃ for 6 hours.
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