阅读说明：本技术 一种纤维蛋白原的生产方法 (Production method of fibrinogen ) 是由 于引航 杨莉 闫磊 郝斌 孙婷 樊祥彬 杨永碧 于 2020-03-30 设计创作，主要内容包括：本发明涉及一种纤维蛋白原的生产方法,包括以下步骤：采集血浆和速冻血浆；融浆和去冷沉淀；制取组分I沉淀；组分I沉淀的溶解；采用S/D病毒灭活法灭活病毒；第一次乙醇沉淀、离心、溶解；第二次乙醇沉淀、离心分离沉淀；配制半成品；除菌、分装；冻干；干热病毒灭活。采用本发明所述方法具有以下优点：获得的纤维蛋白原纯度得以极大提升,可达到92％以上；凝固活力高,为18～22s；复融时间短,为5～10min；冻干后外观更加均一、稳定,收率大于2000瓶/吨血浆,收率显著提高；制品长期稳定性好,经过36个月的长期稳定性试验仍能保证很好的内在质量；双重病毒灭活使临床用药更加安全,无不良反应。(The invention relates to a production method of fibrinogen, which comprises the following steps: collecting blood plasma and quick-frozen blood plasma; slurry melting and cold precipitation removal; preparing component I precipitate; dissolving the component I precipitate; inactivating the virus by adopting an S/D virus inactivation method; precipitating with ethanol, centrifuging, and dissolving; precipitating with ethanol for the second time, and centrifuging to separate precipitate; preparing a semi-finished product; sterilizing and subpackaging; freeze-drying; and (4) performing dry heat virus inactivation. The method has the following advantages: the purity of the obtained fibrinogen is greatly improved and can reach more than 92 percent; the solidification activity is high and is 18-22 s; the re-melting time is short and is 5-10 min; the appearance is more uniform and stable after freeze-drying, the yield is more than 2000 bottles/ton of blood plasma, and the yield is obviously improved; the product has good long-term stability, and can still ensure good internal quality after 36-month long-term stability test; the dual virus inactivation ensures that the clinical medication is safer without adverse reaction.)

1. A method for producing fibrinogen, comprising the steps of:

(1) collecting blood plasma and quick-frozen blood plasma;

(2) slurry melting and cryoprecipitation removal: pre-melting the quick-frozen blood plasma; then, slurry is melted and centrifugally separated, and cold precipitation and supernatant are obtained;

(3) preparing a component I precipitate: adjusting the pH value and the ethanol concentration of the supernatant obtained in the step (2), and then centrifuging to obtain a component I precipitate and a supernatant;

(4) dissolution of the component I precipitate: dissolving the component I precipitate obtained in the step (3) in a dissolving buffer solution I, and filtering to obtain a filtrate A;

(5) inactivating viruses in the filtrate A by adopting an S/D virus inactivation method to obtain virus inactivation liquid;

(6) first ethanol precipitation: adding an ethanol solution into the virus inactivation solution obtained in the step (5) to obtain a mixed solution A;

(7) first centrifugal separation and precipitation: centrifuging the mixed solution A obtained in the step (6) to obtain fibrinogen precipitate;

(8) fibrinolysis by first centrifugation: dissolving the fibrinogen precipitate obtained in the step (7) by using a dissolving buffer solution II to obtain a dissolving solution, and filtering the dissolving solution;

(9) and (3) second ethanol precipitation: adding the solution filtered in the step (8) into an ethanol solution to obtain a mixed solution B;

(10) and (3) second centrifugal separation and precipitation: centrifuging the mixed solution B obtained in the step (9) to obtain fibrinogen precipitate;

(11) preparing a semi-finished product: dissolving the fibrinogen precipitate obtained in the step (10) by using a dissolving buffer solution III, and adjusting the content of the components to obtain a semi-finished product;

(12) sterilizing and subpackaging;

(13) freeze-drying;

(14) and (4) performing dry heat virus inactivation.

2. The method for producing fibrinogen according to claim 1, comprising the steps of:

(1) collecting blood plasma and quick-frozen blood plasma;

(2) slurry melting and cryoprecipitation removal: pre-melting the quick-frozen blood plasma for 1-2 hours; then, slurry is melted; performing centrifugal separation after slurry melting, wherein the rotating speed is 5000-5600 rpm, and the liquid inlet amount of a centrifugal machine is 900-1100L/min, so as to obtain cryoprecipitate and supernatant;

(3) preparing a component I precipitate: adjusting the pH value of the supernatant obtained in the step (2) to 7.00-7.20, adjusting the ethanol concentration to 8%, and then centrifuging at the rotating speed of 5000-5600 rpm, wherein the liquid inlet amount of the centrifuge is 700-800L/min to obtain component I precipitate and supernatant;

(4) dissolution of the component I precipitate: dissolving the component I precipitate obtained in the step (3) in a dissolving buffer solution I, wherein the pH value of the dissolving buffer solution I is 7.2-7.4, the weight of the dissolving buffer solution I is 15-18 times of that of the component I precipitate, the dissolving time is 1-1.5 hours, filtering is carried out, and filter elements for filtering are 1 deep filter element of 30SP and 4 deep filter elements of 90SP which are connected in series to obtain a filtrate A;

(5) inactivating viruses in the filtrate A by adopting an S/D virus inactivation method to obtain virus inactivation liquid;

(6) first ethanol precipitation: adding an ethanol solution into the virus inactivation solution obtained in the step (5) to obtain a mixed solution A; the dropping speed of the ethanol solution is 0.5-0.7 kg/min;

(7) first centrifugal separation and precipitation: carrying out continuous flow centrifugation on the mixed liquor A obtained in the step (6), wherein the rotating speed is 6500rpm, the liquid inlet flow of a centrifuge is 1-2L/min, and the liquid outlet temperature is 0-2 ℃, so as to obtain fibrinogen precipitate;

(8) fibrinolysis by first centrifugation: dissolving the fibrinogen precipitate obtained in the step (7) by using a dissolving buffer solution II, wherein the pH value of the dissolving buffer solution II is 7.2-7.4, the temperature is 34-35 ℃, the time is 1 hour, so as to obtain a dissolved solution, and then filtering the dissolved solution by using a filter element, wherein the adopted filter element is a deep filter element of 4 90 SP;

(9) and (3) second ethanol precipitation: adding the solution filtered in the step (8) into an ethanol solution to obtain a mixed solution B, adding the ethanol solution, and then stirring for 30min, wherein the dropping speed of the ethanol solution is 0.5-0.7 kg/min;

(10) and (3) second centrifugal separation and precipitation: performing continuous flow centrifugation on the mixed liquor B obtained in the step (9) to obtain fibrinogen precipitate, wherein the centrifugal rotation speed is controlled to be 6500rpm, the inlet liquid flow of a centrifugal machine is controlled to be 1-2L/min, and the outlet liquid temperature is controlled to be 0-2 ℃;

(11) preparing a semi-finished product: dissolving the fibrinogen precipitate obtained in the step (10) by using a dissolving buffer solution III, wherein the pH of the dissolving buffer solution III is 7.2-7.4, and adjusting the content of each component to obtain a semi-finished product;

(12) sterilizing and subpackaging;

(13) freeze-drying: and (3) freeze-drying the products subpackaged in the step (12), wherein the freeze-drying conditions comprise: keeping the temperature at 0 ℃ for 2 h-3 h, cooling the product at-10 ℃ for 2h, and keeping the temperature at-46 ℃ to-48 ℃ for 4 h-7 h; carrying out sublimation drying at the temperature of minus 5 ℃ to minus 2 ℃ for 50h to 55 h; resolving and drying for 16-20 h at 30-32 ℃;

(14) and (4) performing dry heat virus inactivation.

3. The fibrinogen production method according to claim 1 or 2, wherein in step (1), the collected fresh plasma is quickly frozen within 30min and stored in a freezer at-30 ℃, and the quick freezing technology is a flat plate direct-cooling quick freezing technology.

4. The method for producing fibrinogen according to claim 1 or 2, wherein the pre-melting in step (2) comprises: transferring the quick-frozen plasma from the condition of minus 30 ℃ to the condition of 0-4 ℃ for pre-melting, spraying the plasma with injection water at the temperature of below 10 ℃, spraying and sterilizing with 75% of alcohol, washing the plasma with alcohol with the injection water, and drying the plasma; the slurry melting method comprises the following steps: transferring the pre-melted plasma to a plasma melting tank after breaking the bag, and circularly melting the plasma by using water for injection with the interlayer temperature of 30 ℃; after slurry melting, continuously separating and centrifuging at the speed of 5000-5600 rpm to obtain a cryoprecipitate and a supernatant, and storing at-30 ℃; in the step (3), transferring the supernatant obtained in the step (2) into a reaction tank, controlling the temperature of plasma to be 0-1 ℃, and adjusting the pH value of the plasma to be 7.00-7.20 by using an acetic acid buffer solution with the pH value of 4.0; dripping 53.3% ethanol into the plasma at a speed of less than or equal to 1.5kg/min to ensure that the final ethanol concentration of the plasma is 8%, reducing the temperature in the dripping process, and controlling the temperature of the plasma to be-1 to-3 ℃; centrifuging the plasma at the temperature of-1 to-3 ℃ at the rotating speed of 5000 to 5600rpm to obtain the component I precipitate and supernatant.

5. The fibrinogen production method according to claim 1 or 2, characterized in that in the step (4), the component I precipitate obtained by centrifugal separation in the step (3) is cut into pieces, the weight is weighed, a dissolution buffer I is prepared according to 15-18 times of the weight of the precipitate, the solution is uniformly stirred at 34-35 ℃ until the precipitate is dissolved for 1-1.5 h, and the dissolution solution is filtered by a filter element; the formulation of lysis buffer I included: the concentration of sodium citrate is 1.2%, the concentration of Tris is 0.27%, the concentration of lysine hydrochloride is 0.44%, the concentration of sucrose is 1%, the concentration of sodium chloride is 1%, the solvent is water for injection, and the pH value is 7.2-7.4.

6. The fibrinogen production method according to claim 1 or 2, characterized in that in step (5), an S/D solution is prepared according to 0.1 time of the weight of the filtrate A obtained in step (4), the S/D solution is added dropwise to the filtrate A at a speed of less than or equal to 0.6kg/min to obtain a mixed solution, the temperature of the mixed solution is controlled to be 24-26 ℃, the mixed solution is continuously and slowly stirred, and the temperature is kept for 6 hours to inactivate lipid-enveloped viruses to obtain a virus inactivation solution; the formula of the S/D solution comprises: tween 80, tributyl phosphate and water for injection; in the S/D solution, the mass fraction of the Tween 80 is 11 percent, and the mass fraction of the tributyl phosphate is 3.3 percent; when in use, the S/D solution is dripped into the filtrate A at the speed of less than or equal to 0.6kg/min, so that the final concentration of the Tween 80 in the mixed solution is 1.1 percent, and the final concentration of the tributyl phosphate is 0.33 percent.

7. The fibrinogen production method according to claim 1 or 2, wherein in step (6), the temperature of the virus inactivation solution obtained in step (5) is reduced to 0-1 ℃, a 50% ethanol solution at-25 ℃ is added during stirring to obtain a mixed solution A, the ethanol concentration in the mixed solution A is 8%, and the temperature is controlled to be-2 to-2.5 ℃; continuously stirring for 30min after the dropwise addition is finished; the dropping rate of the 50% ethanol solution is 0.5-0.7 kg/min.

8. The fibrinogen production method according to claim 1 or 2, wherein in step (8), the dissolving buffer solution II is prepared according to the weight of the fibrinogen precipitate obtained by centrifugation in step (7), the precipitate is cut up and put into a dissolving tank, and the precipitate is uniformly stirred at 34-35 ℃ until being dissolved for 1 h; the formula of the dissolving buffer solution II comprises: the concentration of sodium citrate is 1.2%, the concentration of Tris is 0.27%, the concentration of lysine hydrochloride is 0.44%, the concentration of sucrose is 1%, the concentration of sodium chloride is 1%, the solvent is water for injection, and the pH value is 7.2-7.4; in the step (9), the temperature of the solution filtered in the step (8) is reduced to 0-1 ℃, a 50% ethanol solution at-25 ℃ is added in the stirring process to obtain a mixed solution B, the ethanol concentration in the mixed solution B reaches 8%, and the temperature is controlled to be-2 to-2.5 ℃; and continuously stirring for 30min after the ethanol solution is added dropwise.

9. The fibrinogen production method according to claim 1 or 2, characterized in that in step (11), a dissolving buffer solution III is prepared according to the weight of the fibrinogen precipitate obtained by centrifugation in step (10), the precipitate is cut up and put into a dissolving tank, the precipitate is uniformly stirred at 34-35 ℃ until the precipitate is dissolved, the time is 1h, the protein content is measured, and according to the result of the measured protein content, the protein concentration is adjusted by using the dissolving buffer solution III so that the protein concentration in the semi-finished product is 2.7%; adjusting the sucrose content, the glycine content and the pH value to ensure that the sucrose content, the glycine content and the pH value of the semi-finished product are 5%, 1% and 7.2-7.4 respectively; the formulation of lysis buffer III included: the concentration of sodium citrate is 1.5%, the concentration of Tris is 0.27%, the concentration of sodium chloride is 0.82%, the content of sucrose is 5%, the content of glycine is 1%, the solvent is water for injection, and the pH value is 7.2-7.4.

10. The fibrinogen production method according to claim 1 or 2, wherein in step (12), the semi-finished product obtained in step (11) is sterilized and filtered by using a sterilized filter element, and is packaged in a packaging amount of 25 mL/bottle; the adopted filter element is a degerming filter element with the aperture of 1 filter element of 0.22 um; in the step (13), vacuum sealing is carried out after freeze-drying, the temperature of the product does not exceed 34 ℃ in the freeze-drying process, and the whole freeze-drying process lasts for 3-4 days; in the step (14), the freeze-dried fibrinogen product obtained in the step (13) is subjected to heat preservation in a water bath at the temperature of 98-100 ℃ for 30min, and then dry heat virus inactivation is carried out.

Technical Field

The invention belongs to the field of biological pharmacy, and particularly relates to a production method of fibrinogen.

Background

Human fibrinogen (Fg), coagulation factor I, is the highest blood coagulation factor in plasma and is also a "central" protein in the coagulation system. The final stage of coagulation is thrombin formation, which converts fibrinogen to fibrin. Besides being directly involved in the blood coagulation process, fibrinogen can also mediate platelet aggregation to influence blood viscosity, and is an important risk factor for the onset of cardiovascular and cerebrovascular diseases. Also plays an important role in the pathological processes of atherosclerosis formation, tumor metastasis and the like. Defects in the Fg gene can lead to congenital hypo (or non) fibrinogenemia, as well as abnormal fibrinogen.

In the common pathway of blood coagulation, thrombin firstly cleaves amino ends Arg16-Gly17 of two A α chains of fibrinogen to release a pair of fibrinopeptides A to form a fibrin monomer I, and cleaves amino ends Arg14-Gly15 of two B β chains of fibrinogen to release a pair of fibrinopeptides B to form a fibrin monomer II, a polymerization part of the fibrin monomer is exposed, and unstable soluble fibrin monomer is formed through non-covalent bonding²⁺Under the action of the fibrin monomer, the fibrin monomer is mutually cross-linked to generate stable soluble fibrin, and the formed components of blood are wrapped in the stable soluble fibrin to form firm thrombus. Fg has other functions besides being involved in blood coagulation, such as being combined with platelet membrane glycoprotein IIb/IIIa to mediate platelet aggregation reaction, and being involved in atherosclerosis, tumor metastasis and the like; fibrinogen levels also affect blood viscosity, and in particular, in recent years, elevated plasma fibrinogen levels have been found to be an important risk factor for cardiovascular and cerebrovascular, thrombotic disease. Plasma fibrinogen is also an acute phase protein and can be elevated in a short time under many stress conditions, such as infection, severe trauma, etc. Clinically, Fg is mainly used for treating congenital, acquired fibrinogen reduction or deficiency, severe liver injury, liver cirrhosis, disseminated intravascular coagulation and postpartum hemorrhageAnd blood coagulation disorders caused by major surgery, trauma or internal bleeding. In addition, Fg is also a military strategic reserve of drugs for hemostasis, and must be stocked every year.

The purity of the Fg is an important index influencing the intrinsic quality of the Fg, the low purity can influence the coagulation activity of the product, the coagulation activity can be obviously reduced, and the redissolution time can be influenced, and reports show that the purity of the Fg is lower than 80%, the redissolution time can exceed 20 minutes, and simultaneously protein precipitation and protein particle suspension can be easily generated, so that inconvenience is brought to clinical use. Meanwhile, the stability of the Fg is affected by too low purity, which causes problems in storage.

At present, 11 blood product units in China have the production capacity of Fg, most blood product enterprises adopt a low-temperature ethanol method to extract Fg from the component I, and the defects are that the purity of the product is low and generally does not exceed 80%, the appearance is poor, the re-solubility is poor (20-30 minutes), and the yield is low (800-. Cold precipitation is also used as a raw material to extract Fg from the raw material, but the cold precipitation has low Fg content, and the final product contains impurities of the human coagulation factor VIII, so that the Fg is activated to denature the Fg, and the yield of the human coagulation factor VIII is influenced. And coprecipitation is carried out by adopting cryoprecipitation and the component I, namely ethanol with a certain concentration is added into melted plasma, the cryoprecipitate and the component I are precipitated together after the temperature is reduced, and the precipitation contains 8 percent of ethanol, so that the human coagulation factor VIII in the precipitation is denatured and inactivated.

In recent years, it has been reported that Fg is prepared by chromatography, for example, by Q Sepharose Fast Flow anion exchange chromatography in CN105504046A, a preparation method of human fibrinogen; CN107827974A method for preparing human fibrinogen adopts lysine affinity chromatography and cation exchange chromatography; CN107540743A method for preparing human fibrinogen by double-layer chromatography adopts Q Sepharose Fast Flow anion exchange chromatography and heparin affinity chromatography, etc., the chromatography method has certain improvement on Fg purity, but has the problems that the service life of the chromatography filler is prolonged, the purification effect is weakened along with the increase of the use times, whether the long-term stability of the product is influenced remains to be researched, and meanwhile, the price of the chromatography filler is high, the production cost is increased, the price of the medicine is high, and the treatment pressure of patients is increased.

Disclosure of Invention

Aiming at the problems, the invention discloses a production process for greatly improving the purity of fibrinogen, in order to improve the inherent quality and yield of fibrinogen, ensure the long-term stability of products and reduce the production cost. The invention optimizes and improves the traditional fibrinogen production process, so that the yield of the obtained final product is more than 2000 bottles/ton plasma from the original 1000 bottles/ton plasma, and the product purity is more than 92%; the coagulation activity is 18-22 s; the re-melting time is short and is 5-10 min; the appearance is more uniform and stable after freeze-drying.

The technical scheme for solving the technical problems is as follows:

a method for producing fibrinogen, comprising the steps of:

(1) collecting blood plasma and quick-frozen blood plasma;

(2) slurry melting and cryoprecipitation removal: pre-melting the quick-frozen blood plasma; then, slurry is melted and centrifugally separated, and cold precipitation and supernatant are obtained;

(3) preparing a component I precipitate: adjusting the pH value and the ethanol concentration of the supernatant obtained in the step (2), and then centrifuging to obtain a component I precipitate and a supernatant;

(4) dissolution of the component I precipitate: dissolving the component I precipitate obtained in the step (3) in a dissolving buffer solution I, and filtering to obtain a filtrate A;

(5) inactivating viruses in the filtrate A by adopting an S/D virus inactivation method to obtain virus inactivation liquid;

(6) first ethanol precipitation: adding an ethanol solution into the virus inactivation solution obtained in the step (5) to obtain a mixed solution A;

(7) first centrifugal separation and precipitation: centrifuging the mixed solution A obtained in the step (6) to obtain fibrinogen precipitate;

(8) fibrinolysis by first centrifugation: dissolving the fibrinogen precipitate obtained in the step (7) by using a dissolving buffer solution II to obtain a dissolving solution, and filtering the dissolving solution;

(9) and (3) second ethanol precipitation: adding the solution filtered in the step (8) into an ethanol solution to obtain a mixed solution B;

(10) and (3) second centrifugal separation and precipitation: centrifuging the mixed solution B obtained in the step (9) to obtain fibrinogen precipitate;

(11) preparing a semi-finished product: dissolving the fibrinogen precipitate obtained in the step (10) by using a dissolving buffer solution III, and adjusting the content of the components to obtain a semi-finished product;

(12) sterilizing and subpackaging;

(13) freeze-drying;

(14) and (4) performing dry heat virus inactivation.

The invention has the beneficial effects that: the fibrinogen production method provided by the invention is a low-temperature ethanol production method capable of improving the purity of fibrinogen, and overcomes the problems of low fibrinogen purity, low solidification activity, poor appearance and redissolution effect, low yield and relatively poor comprehensive plasma utilization rate caused by the traditional low-temperature ethanol method.

On the basis of the technical scheme, the invention can be further improved as follows.

Further, the method for producing fibrinogen may include the steps of:

(1) collecting blood plasma and quick-frozen blood plasma;

(2) slurry melting and cryoprecipitation removal: pre-melting the quick-frozen blood plasma for 1-2 hours; then, slurry is melted; performing centrifugal separation after slurry melting, wherein the rotating speed is 5000-5600 rpm, and the liquid inlet amount of a centrifugal machine is 900-1100L/min, so as to obtain cryoprecipitate and supernatant;

(3) preparing a component I precipitate: adjusting the pH value of the supernatant obtained in the step (2) to 7.00-7.20, adjusting the ethanol concentration to 8%, and then centrifuging at the rotating speed of 5000-5600 rpm, wherein the liquid inlet amount of the centrifuge is 700-800L/min to obtain component I precipitate and supernatant;

(4) dissolution of the component I precipitate: dissolving the component I precipitate obtained in the step (3) in a dissolving buffer solution I, wherein the pH value of the dissolving buffer solution I is 7.2-7.4, the weight of the dissolving buffer solution I is 15-18 times of that of the component I precipitate, the dissolving time is 1-1.5 hours, filtering is carried out, and filter elements for filtering are 1 deep filter element of 30SP and 4 deep filter elements of 90SP which are connected in series to obtain a filtrate A;

(5) inactivating viruses in the filtrate A by adopting an S/D virus inactivation method to obtain virus inactivation liquid;

(6) first ethanol precipitation: adding an ethanol solution into the virus inactivation solution obtained in the step (5) to obtain a mixed solution A; the dropping speed of the ethanol solution is 0.5-0.7 kg/min;

(7) first centrifugal separation and precipitation: carrying out continuous flow centrifugation on the mixed liquor A obtained in the step (6), wherein the rotating speed is 6500rpm, the liquid inlet flow of a centrifuge is 1-2L/min, and the liquid outlet temperature is 0-2 ℃, so as to obtain fibrinogen precipitate;

(8) fibrinolysis by first centrifugation: dissolving the fibrinogen precipitate obtained in the step (7) by using a dissolving buffer solution II, wherein the pH value of the dissolving buffer solution II is 7.2-7.4, the temperature is 34-35 ℃, the time is 1 hour, so as to obtain a dissolved solution, and then filtering the dissolved solution by using a filter element, wherein the adopted filter element is a deep filter element with 4 SP 90;

(9) and (3) second ethanol precipitation: adding the solution filtered in the step (8) into an ethanol solution to obtain a mixed solution B, adding the ethanol solution, and then stirring for 30min, wherein the dropping speed of the ethanol solution is 0.5-0.7 kg/min;

(10) and (3) second centrifugal separation and precipitation: performing continuous flow centrifugation on the mixed liquor B obtained in the step (9) to obtain fibrinogen precipitate, wherein the centrifugal rotation speed is controlled to be 6500rpm, the inlet liquid flow of a centrifugal machine is controlled to be 1-2L/min, and the outlet liquid temperature is controlled to be 0-2 ℃;

(11) preparing a semi-finished product: dissolving the fibrinogen precipitate obtained in the step (10) by using a dissolving buffer solution III, wherein the pH of the dissolving buffer solution III is 7.2-7.4, and adjusting the content of each component to obtain a semi-finished product;

(12) sterilizing and subpackaging;

(13) freeze-drying: and (3) freeze-drying the products subpackaged in the step (12), wherein the freeze-drying conditions comprise: keeping the temperature at 0 ℃ for 2 h-3 h, cooling the product at-10 ℃ for 2h, and keeping the temperature at-46 ℃ to-48 ℃ for 4 h-7 h; carrying out sublimation drying at the temperature of minus 5 ℃ to minus 2 ℃ for 50h to 55 h; resolving and drying for 16-20 h at 30-32 ℃;

(14) and (4) performing dry heat virus inactivation.

The beneficial effect who adopts above-mentioned scheme does:

in the step (2), the pre-melting time of the raw material plasma at 0-4 ℃ is shortened from traditional 4-6 hours to 1-2 hours, so that the problem that the purity and yield are influenced by activation denaturation of fibrinogen caused by natural melting of traditional frozen plasma at room temperature or too long pre-melting time is avoided, for example, the method described in the invention patent of human fibrinogen production method (application publication number: 103405754A) and the like. The method adopts centrifugal separation after slurry melting, the rotating speed of the centrifugal separation is 5000-5600 rpm, the liquid inlet amount of the centrifugal machine is 900-1100L/min, and the rotating speed and the liquid inlet amount are favorable for improving the quality and the yield of the fibrinogen and the stability of products. The centrifugal speed of 5000-5600 rpm can ensure that cryoprecipitate is fully centrifugally separated, and meanwhile, the structural integrity of fibrinogen can be ensured, so that the biological activity of a fibrinogen product is ensured, a certain effect is realized on reducing the dissolving time and improving the solidification activity, the liquid inlet amount is 900-1100L/min, the cryoprecipitate can be fully centrifugally separated, the flow can ensure that the product is at a low temperature of 0-4 ℃, the biological activity of the fibrinogen is ensured, and the solidification activity of the fibrinogen is improved to a certain extent; if the rotating speed is too low, the cryoprecipitation centrifugal separation is easy to be incomplete, the purity of the final product is influenced, and the like, and if the rotating speed is too high, the centrifugal shearing force is easy to be too large, the structure of the protein is easy to be damaged, the biological activity of the protein is further influenced, the dissolving time of the final product is too long, the coagulation activity is too low, and the like; if the liquid inlet amount is too small, the protein product can be quickly cooled by the centrifugal machine, the liquid medicine is lower than 0 ℃, and then the freezing and icing phenomenon occurs, so that the cryoprecipitation separation is incomplete, the final fibrinogen purity is influenced, and the like, if the liquid inlet amount is too large, the temperature is easily reduced, the protein easily exceeds 4 ℃, and further the protein denaturation influences the dissolution time, the solidification activity and the like of the final product.

In the step (3), the rotating speed of the centrifuge is 5000-5600 rpm, the liquid inlet amount of the centrifuge is 700-800L/min, and the rotating speed and the liquid inlet amount are favorable for improving the quality and the yield of the fibrinogen and the stability of the product. The centrifugal speed of 5000-5600 rpm can ensure that the component I precipitate is fully centrifugally separated, so that the yield of fibrinogen is ensured, the centrifugal speed is favorable for maintaining the biological activity of the fibrinogen, the liquid inlet amount is 700-800L/min, the component I precipitate can be fully centrifugally separated, the flow can ensure that the product is at a low temperature of-1 to-3 ℃, the biological activity of the fibrinogen is ensured, the dissolving time of the final product can be shortened, and the solidification activity of the final product can be improved; if the rotating speed is too low, the component I is easy to precipitate and centrifugally separate incompletely, the yield of a final product is influenced, and if the rotating speed is too high, the centrifugal shearing force is easy to be too large, the structure of protein is easy to be damaged, the biological activity of the protein is further influenced, the dissolving time of the final product is too long, the solidification activity is too low, and the like; if the liquid inlet amount is too small, the protein product can be quickly cooled by the centrifugal machine, the liquid medicine is lower than minus 3 ℃, and then the freezing and icing phenomena occur, so that the component I is not completely precipitated and separated, and the final fibrinogen yield is influenced, and the like, and if the liquid inlet amount is too large, the temperature is not completely reduced, the protein is easily higher than minus 1 ℃, and further the fibrinogen denaturation is caused to influence the dissolution time, the coagulation activity and the like of the final product.

In both the step (2) and the step (3), although the centrifugation step is involved, the centrifugation effect is different, and the set operation parameters are different. The centrifugation in step (2) is a step of centrifuging to remove cryoprecipitate (the cryoprecipitate is an unnecessary protein), the precipitate obtained by the centrifugation in step (3) is a component I precipitate, the precipitate is used for preparing fibrinogen, and the two proteins have different molecular weights and structures and different requirements on environment, so that the parameters set during the centrifugation are different.

In the step (4), the dissolving time of the component I is improved by 15-18 times from the traditional 10-13 times, fibrinogen in the component I is quickly and fully dissolved within 1-1.5 h, and the denaturation and activation of the fibrinogen caused by insufficient dissolving or too long dissolving time are avoided, so that the purity and the yield of the fibrinogen are reduced. The filter elements for filtration are deep filter elements of 1 SP 30 and 4 SP 90 connected in series, and the arrangement is favorable for full filtration of target protein, namely fibrinogen, and retention of foreign protein, and is favorable for improving the final purity of products.

In the steps (4), (8) and (11), the pH value of the dissolving buffer solution is adjusted from 6.6-6.8 to 7.2-7.4, so that the fibrinogen reaches the isoelectric point in the pH range, the fibrinogen is more fully precipitated, and the yield is greatly improved compared with the conventional pH value of 6.6-6.8. The prior art dissolution precipitation pH is typically set to around 6.8.

In the step (6) and the step (9), the speed of dripping the ethanol solution is reduced from 1.0-1.2 kg/min to 0.5-0.7 kg/min, and the fibrinogen is fully precipitated on the premise of ensuring the activity of the fibrinogen. The too fast speed of dropping ethanol can easily cause the too violent change of the alcohol concentration to cause the denaturation of the fibrinogen, and influence the quality and the yield of the fibrinogen, and the too slow speed of dropping ethanol can easily cause the too long preparation time of the fibrinogen, so that the activity of the fibrinogen is influenced. Therefore, the invention ensures that the fibrinogen is more fully precipitated and has higher purity on the premise of ensuring the activity of the fibrinogen, and the optimum dropping speed of the ethanol is determined by groping and is reduced to 0.5-0.7 kg/min from the past 1.0-1.2 kg/min.

In the step (7) and the step (10), the liquid inlet speed of the centrifugal separation sediment is reduced to 1-2L/min from 4-5L/min, so that the sediment can be fully centrifugally settled. The liquid inlet speed of centrifugal separation and precipitation in the prior art is 2.5-3.0L/min, for example, the invention patent of a preparation method of a human fibrinogen preparation (application publication No. 101229367A). The excessive centrifugal speed may cause insufficient centrifugation of fibrinogen to cause loss, so that the liquid inlet speed of centrifugal separation and precipitation adopted by the method is 1-2L/min, the fibrinogen is fully settled, and the purity and yield of the fibrinogen are ensured.

In the step (7) and the step (10), the centrifugal rotating speed is 6500rpm, the effluent temperature is 0-2 ℃, the centrifugal speed which is favorable for 6500rpm can ensure that the refined precipitate of the component I can be fully centrifugally separated, so as to ensure the yield of fibrinogen, meanwhile, the centrifugal rotating speed is favorable for removing foreign protein, so as to ensure the purity of the fibrinogen, and the effluent temperature is 0-2 ℃ so as to ensure that the product is in a low-temperature condition, so as to ensure the biological activity of the fibrinogen, reduce the dissolution time of the final product and improve the solidification activity of the final product. The problem that the purity of the final product is influenced and the like is easily caused by that the foreign protein cannot be sufficiently removed due to too low centrifugal rotating speed, and the problem that the centrifugal shearing force is too large due to too high centrifugal rotating speed, the structure of the protein is easily damaged, the biological activity of the protein is influenced, the dissolving time of the final product is too long, the coagulation activity is too low and the like are easily caused. The protein product can be quickly cooled by the centrifuge due to the excessively low temperature of the outlet liquid, so that the freezing and icing phenomena occur, the component I is not thoroughly refined, precipitated and separated, the final fibrinogen yield is influenced, and the like.

In the step (8), in the dissolving process of the dissolving buffer solution II, the temperature is controlled to be 34-35 ℃, the time is controlled to be 1 hour, and the arrangement is favorable for fully dissolving the refined precipitate of the component I, so that the yield and the purity of the human fibrinogen are improved. If the temperature is too low, the refined precipitate of the component I is not sufficiently dissolved, the insoluble precipitate is easily activated and denatured, and the final yield, purity, dissolving time, coagulating activity and the like of the fibrinogen are affected, and if the temperature is too high, the fibrinogen is easily activated to form the fibrin, and the yield, purity, coagulating activity, dissolving time and the like of the fibrinogen are affected. The problems of insufficient dissolution, easy activation and denaturation of insoluble fibrinogen to form insoluble human fibrin, influence on yield, purity, solidification activity, dissolution time and the like of the fibrinogen are easily caused by too short time, the problems of damage to the structure of the fibrinogen caused by the generated shearing force, denaturation of the fibrinogen, influence on the yield, solidification activity, dissolution time and the like of the fibrinogen are easily caused by too long time when the stirring paddle in the dissolution process contacts with a product for a long time.

Then, the adopted filter element is a deep filter element of 4 SP 90 filters the solution, the adoption of the filter element is beneficial to fully filtering target protein, namely fibrinogen, and intercepting foreign protein, thereby being beneficial to improving the final purity and yield of the product.

In the step (13), the time for pre-freezing, primary drying and analysis drying in the freeze-drying process is adjusted, so that the freeze-drying time of fibrinogen is 3-4 days, the product is stable, the appearance is uniform, the re-melting time is as short as 5-10 min, and the problems of non-uniform structure, poor freeze-drying shape, long re-melting time, poor stability and the like of the final fibrinogen product caused by the traditional freeze-drying time within 3-4 days are solved.

In this step, the lyophilization conditions were set as: keeping the temperature at 0 ℃ for 2 h-3 h, cooling the product at-10 ℃ for 2h, and keeping the temperature at-46 ℃ to-48 ℃ for 4 h-7 h; carrying out sublimation drying at the temperature of minus 5 ℃ to minus 2 ℃ for 50h to 55 h; resolving and drying for 16-20 h at 30-32 ℃. The temperature and time for freeze-drying have the advantages of good shape, uniform appearance, short dissolving time, high solidification activity, good long-term stability of the product, suitability for long-term storage of the product and the like. The first stage is as follows: keeping the temperature of the plate at 0 ℃ for 2-3 hours to ensure that the product is stable and uniform before reaching a metastable state; and a second stage: the plate temperature is set at-10 ℃ for 2 hours, so that the product can pass through a metastable state stably, and the appearance of the product can be better crystallized and is more exquisite; and a third stage: the plate temperature is set to be-46 to-48 ℃ and kept for 4 to 7 hours so as to quickly freeze the product and keep the product for a period of time to stabilize the structure of the product; a fourth stage: the temperature of the plate layer is kept between-5 ℃ and-2 ℃ for 50-55 hours, so that the product is always below the eutectic point in one-time sublimation and the temperature of the product approaches to the plate temperature when the sublimation is finished, and the biological activity of the product is not influenced and the uniformity of the freeze-drying appearance of the product is ensured; the fifth stage: the analysis drying for 16-20 h at 30-32 ℃ is to ensure that the moisture content of the final product reaches about 3 percent, thereby being beneficial to the long-term storage of the product.

The inventors have explored a number of lyophilization conditions in their studies, for example: the temperature of the plate is controlled to be-5-0 ℃ for sublimation in the first sublimation, the temperature of the plate is controlled to be-15 ℃ for sublimation in the first sublimation, the temperature of the plate for secondary sublimation is controlled to be 28 ℃ for 12 hours, and the temperature of the plate for secondary sublimation is controlled to be 32.5 ℃ for 16-20 hours, etc. By comparison, the freeze-drying conditions adopted by the invention are the optimal freeze-drying conditions. When a method of controlling the plate temperature at minus 5-0 ℃ for sublimation by one-time sublimation is adopted, the phenomenon that the appearance of the product is unqualified after the test is carried out. When the sublimation is carried out by controlling the plate temperature at minus 15 ℃ by one-time sublimation, the test shows that the one-time sublimation time is too long and leads to too long redissolution time of the product (the dissolution time is more than 20 minutes), and the quality of the product is reduced. When the temperature of the secondary sublimation plate was controlled at 28 ℃ and maintained for 12 hours, the moisture content of the product was found to be excessively high and the reconstitution time was found to be long (the dissolution time of the product was 20 minutes or more) after the test. After the method of controlling the temperature of the secondary sublimation plate at 32.5 ℃ and keeping the temperature for 16-20 hours is adopted, the moisture content of the product is found to be lower after the test, which is not beneficial to the long-term storage of the product. By adopting the freeze-drying method, the obtained product has high purity (more than 92 percent), high solidification activity (18-21 s), short re-melting time (5-8 min), high yield (more than 2000 bottles/ton of blood plasma) and good stability (the product can be kept stable for a long time after 36 months).

The invention adopts the low-temperature ethanol precipitation method to prepare fibrinogen from human blood plasma, and the purity of the obtained product is high and can reach more than 92 percent by changing the traditional process method; the solidification activity is high and is 18-22 s; the re-melting time is short and is 5-10 min; the appearance is more uniform and stable after freeze-drying, and the defects of fibrinogen extraction by the traditional low-temperature ethanol method are overcome, such as low product purity, generally 70-80%; the stability of the protein is poor; the freeze-dried product has poor appearance and long re-melting time, generally more than 20 minutes. In the prior art, human fibrinogen is extracted from the component I by using a chromatography method, for example, the invention patent of a method for extracting human fibrinogen from the component I by using a column chromatography method (application publication No. 1012212129A), but the cost of the chromatography method is much higher than that of a low-temperature ethanol method, and gel is irreversibly worn after long-term use, so that the yield and quality of a product are affected due to unstable process parameters.

The invention extracts fibrinogen from the plasma component I precipitate with a yield higher than 2000 bottles/ton plasma. Human fibrinogen is also reported to be extracted from cryoprecipitate, for example, in the invention patent of "production method of human fibrinogen" (application publication No. 101703763a), the final yield of the product is not high from component I due to low content of fibrinogen in cryoprecipitate, and cryoprecipitate is used as the starting material of human coagulation factor VIII, if used for producing fibrinogen, the yield of the factor VIII is inevitably influenced, the yield is low, the comprehensive utilization rate of plasma is reduced, and the production cost is increased.

At present, no long-term stability result of fibrinogen prepared by any method is reported, the long-term stability result determines whether the quality of the product can be kept for a long time without any influence, and the long-term stability result has important significance on medicines sold on the market in the future. The long-term stability of the product of the invention is good, and the long-term stability test of 36 months can still ensure good internal quality.

Further, in the step (1), the collected fresh blood plasma is quickly frozen within 30min and stored in a refrigeration house with the temperature of minus 30 ℃, and the quick freezing technology is a flat plate direct cooling quick freezing technology.

The beneficial effect who adopts above-mentioned scheme is: the flat-plate direct-cooling quick-freezing technology is adopted to replace the traditional forced convection quick-freezing technology to quickly freeze the raw material plasma, so that the freezing time of the raw material plasma from 4 hours to 30 minutes is shortened, the activity of coagulation factor products such as fibrinogen can be greatly protected, the activation and denaturation of the fibrinogen caused by long quick-freezing time can be avoided, the protein content and purity of the fibrinogen are greatly improved, the redissolution time is reduced, and the stability is greatly improved.

Further, in the step (2), the pre-melting method comprises: transferring the quick-frozen plasma from the condition of minus 30 ℃ to the condition of 0-4 ℃ for pre-melting, spraying the plasma with injection water at the temperature of below 10 ℃, spraying and sterilizing with 75% of alcohol, washing the plasma with alcohol with the injection water, and drying the plasma; the slurry melting method comprises the following steps: transferring the pre-melted plasma to a plasma melting tank after breaking the bag, and circularly melting the plasma by using water for injection with the interlayer temperature of 30 ℃; and after slurry melting, continuously separating and centrifuging at the speed of 5000-5600 rpm to obtain a cryoprecipitate and a supernatant, and storing at the temperature of-30 ℃.

The beneficial effect who adopts above-mentioned scheme is: the method is favorable for extracting fibrinogen through the pre-melting at the temperature of 0-4 ℃, the purity and the yield of a final product can be improved, the biological activity of the fibrinogen can be ensured by circularly melting the plasma through the injection water with the interlayer temperature of 30 ℃, the plasma can be quickly dissolved, the fibrinogen is prevented from being activated to form denatured fibrin due to overlong melting time, the coagulation activity and the dissolving time of the final product are influenced, and the long-term stability of the product can be improved.

Further, in the step (3), transferring the supernatant obtained in the step (2) into a reaction tank, controlling the temperature of the plasma to be between 0 and 1 ℃, and adjusting the pH value of the plasma to be 7.00 to 7.20 by using an acetic acid buffer solution with the pH value of 4.0; dripping 53.3% ethanol solution into the plasma at a speed of less than or equal to 1.5kg/min to ensure that the final ethanol concentration of the plasma is 8%, reducing the temperature in the dripping process, and controlling the temperature of the plasma to be-1 to-3 ℃; centrifuging the plasma at the temperature of-1 to-3 ℃ at the rotating speed of 5000 to 5600rpm to obtain the component I precipitate and supernatant.

The beneficial effect who adopts above-mentioned scheme is: the pH value of the plasma is 7.00-7.20, the final ethanol concentration is 8%, the full sedimentation and separation of fibrinogen are facilitated, foreign protein cannot be settled, further ensuring the yield and purity of the fibrinogen, ensuring that the product is always in a lower temperature condition when the dripping speed of the ethanol solution is less than or equal to 1.5kg/min, avoiding the denaturation caused by the local temperature rise of the product due to the overhigh dripping speed of the ethanol, reducing the temperature in the dripping process to ensure that the temperature of the plasma is between-1 and-3 ℃, and centrifuging at the same time at the temperature of between-1 and-3 ℃, the temperature range can ensure the biological activity of the fibrinogen, further improve the coagulation activity and the dissolution time of the final product, the centrifugal speed of the rotating speed of 5000-5600 rpm can ensure that the component I precipitate can be fully centrifugally separated, thereby ensuring the yield of the fibrinogen, and simultaneously, the centrifugal rotating speed is beneficial to keeping the biological activity of the human fibrinogen.

Further, in the step (4), the component I precipitate obtained by centrifugal separation in the step (3) is cut into fragments, the weight is weighed, a dissolving buffer solution I is prepared according to 15-18 times of the weight of the precipitate, the solution is uniformly stirred at the temperature of 34-35 ℃ until the precipitate is dissolved for 1-1.5 hours, and a filter element is adopted to filter the dissolving solution; the formulation of lysis buffer I included: the concentration of sodium citrate is 1.2%, the concentration of Tris is 0.27%, the concentration of lysine hydrochloride is 0.44%, the concentration of sucrose is 1%, the concentration of sodium chloride is 1%, the solvent is water for injection, and the pH value is 7.2-7.4.

The beneficial effect who adopts above-mentioned scheme is: the dissolution multiple of 15-18 is beneficial to fully dissolving the component I precipitate, so that the yield and purity of the final product can be improved; the dissolving conditions of 34-35 ℃ and 1-1.5 h of dissolving time are favorable for fully dissolving the component I precipitate, but protein can not be activated and denatured, meanwhile, impurity protein is fully separated out, so that the yield and purity of fibrinogen are improved, the dissolving pH value is 7.2-7.4, the pH value is far away from the isoelectric point of the fibrinogen, the full dissolving of the fibrinogen is favorable, meanwhile, the pH value can fully settle the impurity protein, and the purity and the solidification activity of a product are favorable for being improved.

Further, in the step (5), preparing an S/D solution according to 0.1 time of the weight of the filtrate A obtained in the step (4), dripping the S/D solution into the filtrate A at a speed of less than or equal to 0.6kg/min to obtain a mixed solution, controlling the temperature of the mixed solution to be 24-26 ℃, continuously and slowly stirring, and keeping the temperature for 6 hours to inactivate the lipid-enveloped viruses to obtain a virus inactivated solution; the formula of the S/D solution comprises: tween 80, tributyl phosphate and water for injection; in the S/D solution, the mass fraction of the Tween 80 is 11 percent, and the mass fraction of the tributyl phosphate is 3.3 percent; when in use, the S/D solution is dripped into the filtrate A at the speed of less than or equal to 0.6kg/min, so that the final concentration of the Tween 80 in the mixed solution is 1.1 percent, and the final concentration of the tributyl phosphate is 0.33 percent.

The beneficial effect who adopts above-mentioned scheme is: the method adopts Tween 80 and tributyl phosphate organic solvent as the virus inactivating agent of the coagulation factor product, is a classical virus inactivating method, can fully inactivate lipid-enveloped viruses, improves the safety of products, and can ensure that the biological activity of fibrinogen is not affected.

Further, in the step (6), the temperature of the virus inactivation liquid obtained in the step (5) is reduced to 0-1 ℃, a 50% ethanol solution at-25 ℃ is added in the stirring process to obtain a mixed liquid A, the ethanol concentration in the mixed liquid A reaches 8%, and the temperature is controlled to be-2 to-2.5 ℃; continuously stirring for 30min after the dropwise addition is finished; the dropping rate of the 50% ethanol solution is 0.5-0.7 kg/min.

The beneficial effect who adopts above-mentioned scheme is: adding 50% ethanol solution with the temperature of precooling-25 ℃ to ensure that the final ethanol concentration of the product is 8% and is favorable for fully settling and separating fibrinogen, and foreign proteins can not be settled, so as to ensure the yield and purity of the fibrinogen, the dropping speed of the ethanol is 0.5-0.7 kg/min to ensure that the product is always at a lower temperature condition, the product can not be denatured due to the local temperature rise of the product caused by the overhigh dropping speed of the ethanol, and the product can not be subjected to the shearing of stirring due to the overhigh dropping speed to further cause the activated denaturation of the fibrinogen to influence the coagulation activity and the yield of the final product, the temperature is reduced in the dropping process, the temperature of plasma is-2 to-2.5 ℃, the temperature range can ensure that the fibrinogen can not be frozen due to the overlow temperature, the fibrinogen can not be denatured due to the overhigh temperature, and is favorable for ensuring the biological activity of the fibrinogen, thereby improving the coagulation activity and the dissolution time of the final product.

Further, in the step (8), preparing a dissolving buffer solution II according to the weight of the fibrinogen precipitate obtained by centrifugation in the step (7), chopping the precipitate, putting the chopped precipitate into a dissolving tank, and uniformly stirring the chopped precipitate at the temperature of 34-35 ℃ until the precipitate is dissolved for 1 hour; the formula of the dissolving buffer solution II comprises: the concentration of sodium citrate is 1.2%, the concentration of Tris is 0.27%, the concentration of lysine hydrochloride is 0.44%, the concentration of sucrose is 1%, the concentration of sodium chloride is 1%, the solvent is water for injection, and the pH value is 7.2-7.4.

The beneficial effect who adopts above-mentioned scheme is: the pH value of the dissolving buffer solution is adjusted from 6.6-6.8 to 7.2-7.4, so that the fibrinogen reaches the isoelectric point in the pH range, the fibrinogen is more fully precipitated, and the yield is greatly improved compared with the conventional pH value of 6.6-6.8. The dissolving temperature is 34-35 ℃, so that the component I refined precipitate can be fully dissolved without being activated and denatured, and meanwhile, the impure protein is fully separated out, and the yield and the purity of the fibrinogen are further improved.

Further, in the step (9), the temperature of the filtered solution obtained in the step (8) is reduced to 0-1 ℃, a 50% ethanol solution at-25 ℃ is added in the stirring process to obtain a mixed solution B, the ethanol concentration in the mixed solution B reaches 8%, and the temperature is controlled to be-2-2.5 ℃; and continuously stirring for 30min after the ethanol solution is added dropwise.

The beneficial effect who adopts above-mentioned scheme is: the 50% ethanol solution with the temperature of minus 25 ℃ is added, so that the fibrinogen is fully settled and the foreign protein is removed by controlling the concentration of the ethanol in the low-temperature ethanol extraction method, the purity and the yield of the final product are ensured, the solidification activity of the final product can also be improved, and the continuous stirring for 30min after the dropwise addition of the ethanol solution is finished is favorable for full contact and uniform mixing of the ethanol and the product, so that the full precipitation and purification of the fibrinogen are ensured, and the yield and the purity of the final product are ensured.

Further, in the step (11), preparing a dissolving buffer solution III according to the weight of the fibrinogen precipitate obtained by centrifugation in the step (10), chopping the precipitate, putting the chopped precipitate into a dissolving tank, uniformly stirring the chopped precipitate at the temperature of 34-35 ℃ until the precipitate is dissolved for 1 hour, measuring the protein content, and adjusting the protein concentration by using the dissolving buffer solution III according to the measured protein content result to ensure that the protein concentration in the semi-finished product is 2.7%; adjusting the sucrose content, the glycine content and the pH value to ensure that the sucrose content, the glycine content and the pH value of the semi-finished product are 5%, 1% and 7.2-7.4 respectively; the formulation of lysis buffer III included: the concentration of sodium citrate is 1.5%, the concentration of Tris is 0.27%, the concentration of sodium chloride is 0.82%, the content of sucrose is 5%, the content of glycine is 1%, the solvent is water for injection, and the pH value is 7.2-7.4.

The beneficial effect who adopts above-mentioned scheme is: the pH value of the dissolving buffer solution is adjusted from 6.6-6.8 to 7.2-7.4, so that the fibrinogen reaches the isoelectric point in the pH range, the fibrinogen is more fully precipitated, and the yield is greatly improved compared with the conventional pH value of 6.6-6.8.

The effect of the components of lysis buffer III was: the sodium citrate and the Tris can enhance the buffer capacity of the solution, so that the long-term stability of the product is ensured, the sodium chloride can ensure the osmolarity of the final product, and the sucrose and the glycine are used as the frameworks of the freeze-dried finished product to ensure the final shape of the product. The stability of a buffer system of the product can be ensured by adopting 1.5 percent of sodium citrate and 0.27 percent of Tris, so that the long-term stability of the product is ensured, the concentration of 0.82 percent of sodium chloride is close to the physiological concentration of a human body, the safety of the product is improved, and the salt concentration is favorable for the stability of the final freeze-dried product; the stability of freeze-drying skeleton is guaranteed to the concentration of 5% sucrose content and 1% glycine, is favorable to forming good freeze-drying shape, and this concentration can promote the redissolution time of final freeze-dried product simultaneously.

Further, in the step (12), the semi-finished product obtained in the step (11) is subjected to sterilization filtration by using a sterilized filter element, and is subpackaged, wherein the subpackaging amount is 25 mL/bottle; the adopted filter element is a degerming filter element with the aperture of 1 filter element of 0.22 um.

The beneficial effect who adopts above-mentioned scheme is: the prepared product is sterilized and filtered, so that the sterility and the safety of the product are ensured.

Further, in the step (13), vacuum sealing is carried out after freeze-drying, the temperature of the product does not exceed 34 ℃ in the freeze-drying process, and the whole freeze-drying process lasts for 3-4 days.

The beneficial effect who adopts above-mentioned scheme is: the vacuum sealing is used for ensuring that the product is in a vacuum state, improving the long-term stability of the product and facilitating the storage and transportation of the product.

Further, in the step (14), the human fibrinogen product freeze-dried in the step (13) is subjected to heat preservation in a water bath at the temperature of 98-100 ℃ for 30min, and then dry heat virus inactivation is carried out.

The beneficial effect who adopts above-mentioned scheme is: the heat preservation is carried out in a water bath at the temperature of 98-100 ℃ for 30min so as to inactivate non-lipid enveloped viruses in the product and improve the safety of the product.

Detailed Description

The principles and features of this invention are described below in conjunction with examples which are set forth to illustrate, but are not to be construed to limit the scope of the invention.

The present invention provides a method for producing fibrinogen, which is improved in various ways in view of the disadvantages of the prior art, for example, including but not limited to the following aspects: (1) the plate direct-cooling quick-freezing technology is adopted to replace the traditional forced convection quick-freezing technology to quickly freeze the raw material plasma; (2) pre-melting the plasma at 0-4 ℃ for 1-2 hours; (3) centrifuging to remove the cryoprecipitated FrI with the multiple of dissolving buffer solution of 15-18 times; (4) the pH value of the dissolving buffer solution is 7.2-7.4; (5) the dropping rate of the 50% ethanol solution is 0.5-0.7 kg/min; (6) the liquid inlet speed of the centrifugal separation sediment is 1-2L/min. (7) And (3) improving a freeze-drying process, wherein the freeze-drying time is 3-4 days. (8) In the production, double virus inactivation is carried out by adopting an S/D method and a heat treatment method of heat-preservation water bath at 98-100 ℃, so that lipid-enveloped viruses and non-lipid-enveloped viruses are effectively inactivated, and the safety of products is ensured. The production method provided by the invention has the advantages that: the purity of human fibrinogen is greatly improved to more than 92 percent; the solidification activity is high and is 18-22 s; the re-melting time is short and is 5-10 min; the appearance is more uniform and stable after freeze-drying, and the yield reaches 2000 bottles/ton plasma, which is higher than the yield of the prior art in the field. The dual virus inactivation ensures that the clinical medication is safer without adverse reaction. The product of the invention has good long-term stability, and can still ensure good internal quality after 36-month long-term stability test.

Specifically, the method for producing fibrinogen of the present invention may include the steps of:

(1) plasma collection and quick freezing: quickly freezing the collected fresh plasma by flat plate direct cooling quick freezing technology within 30min, and storing in a freezer at-30 deg.C.

(2) Slurry melting and cryoprecipitation removal: transferring the mixture from a refrigeration house at the temperature of minus 30 ℃ to a pre-melting chamber at the temperature of 0-4 ℃ for pre-melting for 1-2 hours, spraying the mixture with injection water at the temperature of below 10 ℃, spraying and sterilizing the mixture with 75% of alcohol, washing the alcohol with the injection water, and drying the mixture. The method comprises the steps of breaking bags of raw material plasma, transferring the raw material plasma to a plasma melting tank, circularly melting the plasma through injection water with the interlayer temperature of 30 ℃, continuously separating and centrifuging at the speed of 5000-5600 rpm, wherein the liquid inlet amount of a centrifuge is 900-1100L/min, obtaining cryoprecipitate and supernatant, and storing the cryoprecipitate and the supernatant in a refrigeration house with the temperature of-30 ℃.

(3) Preparation of component I precipitate: and (3) transferring the supernatant separated and subjected to the cold precipitation in the step (2) into a reaction tank, controlling the temperature of the plasma to be between 0 and 1 ℃, and adjusting the pH value of the plasma to be 7.00 to 7.20 by using an acetic acid buffer solution with the pH value of 4.0. Dripping 53.3% ethanol solution into plasma at a speed of less than or equal to 1.5kg/min to make the final concentration of ethanol in the plasma be 8%, reducing the temperature during dripping, and controlling the temperature of the plasma to be-1 to-3 ℃. Centrifuging the plasma at the temperature of-1 to-3 ℃, wherein the rotating speed is 5000 to 5600rpm, and the liquid inlet amount of the centrifuge is 700 to 800L/min, so as to obtain component I precipitate and supernatant.

(4) Dissolution of the component I precipitate: and (3) cutting the component I precipitate obtained by centrifugal separation in the step (3) into fragments, weighing, preparing a dissolving buffer solution I according to 15-18 times of the weight of the precipitate, uniformly stirring at 34-35 ℃ until the precipitate is dissolved, wherein the dissolving pH value is 7.2-7.4, the time is 1-1.5 h, and filtering the dissolved solution by serially connecting 1 deep filter element of 30SP and 4 deep filter elements of 90 SP.

(5) S/D virus inactivation: preparing an S/D solution according to 0.1 time of the weight of the filtrate filtered in the step (4), dropwise adding the S/D solution into the filtrate filtered in the step (4) at a speed of less than or equal to 0.6kg/min to obtain a mixed solution, controlling the temperature of the mixed solution to be 24-26 ℃, continuously and slowly stirring, and keeping the temperature for 6 hours to inactivate the lipid-enveloped viruses to obtain a virus inactivation solution.

(6) First ethanol precipitation: and (3) reducing the temperature of the virus inactivation liquid obtained in the step (5) to 0-1 ℃, adding a 50% ethanol solution at the temperature of-25 ℃ in the stirring process, wherein the dropping speed of the ethanol solution is 0.5-0.7 kg/min, so as to obtain a mixed liquid A, wherein the ethanol concentration in the mixed liquid A reaches 8%, and the temperature is controlled to be-2-2.5 ℃. After the dropwise addition, the stirring was continued for 30 min.

(7) First centrifugal separation and precipitation: and (4) connecting a centrifugal machine, performing continuous flow centrifugation on the mixed liquid A obtained in the step (6), controlling the rotating speed of each centrifuge to be 6500rpm, controlling the liquid inlet flow of the centrifugal machine to be 1-2L/min, and controlling the liquid outlet temperature to be 0-2 ℃. Centrifuging to obtain fibrinogen precipitate.

(8) First centrifugation of fibrinogen lysis: preparing a dissolving buffer solution II according to the weight of the fibrinogen precipitate obtained by centrifugation in the step (7), chopping the precipitate, putting the chopped precipitate into a dissolving tank, uniformly stirring the chopped precipitate at the temperature of 34-35 ℃ until the precipitate is dissolved, wherein the dissolving pH value is 7.2-7.4, and the time is 1 h. And filtering the dissolved solution by using 4 deep filter elements of 90SP to obtain filtrate.

(9) And (3) second ethanol precipitation: and (3) reducing the temperature of the filtered solution obtained in the step (8) to 0-1 ℃, adding a 50% ethanol solution at the temperature of-25 ℃ in the stirring process, wherein the dropping speed of the ethanol solution is 0.5-0.7 kg/min, so as to obtain a mixed solution B, wherein the ethanol concentration in the mixed solution B reaches 8%, and the temperature is controlled to be-2-2.5 ℃. After the dropwise addition of the ethanol solution, the stirring is continued for 30 min.

(10) And (3) second centrifugal separation and precipitation: and (4) connecting a centrifugal machine, performing continuous flow centrifugation on the mixed liquid B obtained in the step (9), controlling the rotating speed of each centrifuge to be 6500rpm, controlling the liquid inlet flow of the centrifugal machine to be 1-2L/min, and controlling the liquid outlet temperature to be 0-2 ℃. A fibrinogen precipitate is obtained.

(11) Preparing a semi-finished product: preparing a dissolving buffer solution III according to the weight of the fibrinogen precipitate obtained by centrifugation in the step (10), chopping the precipitate, putting the chopped precipitate into a dissolving tank, uniformly stirring the solution at the temperature of 34-35 ℃ until the precipitate is dissolved, determining the protein content, and adjusting the content of each component to obtain a semi-finished product, wherein the dissolving pH value is 7.2-7.4, and the time is 1 h; according to the measured protein content result, regulating the protein concentration by using a dissolving buffer solution III to ensure that the protein concentration in the semi-finished product is 2.7 percent; and adjusting the sucrose content, the glycine content and the pH value to ensure that the sucrose content, the glycine content and the pH value of the semi-finished product are 5%, 1% and 7.2-7.4 respectively.

(12) And (3) degerming and subpackaging: and (3) performing sterile filtration on the semi-finished product obtained in the step (11) by using a sterilized 1-piece sterilizing filter element with the aperture of 0.22um, and subpackaging the semi-finished product with the subpackaging amount of 25 mL/bottle.

(13) Freeze-drying: preserving the temperature of the product subpackaged in the step (12) at 0 ℃ for 2 h-3 h and cooling the product at-10 ℃ for 2h and cooling at-46 ℃ to-48 ℃ for 4 h-7 h; carrying out sublimation drying at the temperature of minus 5 ℃ to minus 2 ℃ for 50h to 55 h; resolving and drying for 16-20 h at 30-32 ℃. After freeze-drying, the vacuum seal is carried out, and the temperature of the product does not exceed 34 ℃ during freeze-drying.

(14) Dry heat virus inactivation: and (3) preserving the heat of the freeze-dried human fibrinogen product obtained in the step (13) in a water bath at the temperature of 98-100 ℃ for 30min, and performing dry heat virus inactivation.

Specifically, in the step (4), the preparation method of the dissolution buffer I comprises the following steps: mixing sodium citrate, Tris, lysine hydrochloride, sucrose and sodium chloride, adding water for injection to dissolve, uniformly stirring, and adjusting the pH value to 7.2-7.4 by using 12M HCL; in the dissolution buffer I, the concentration of sodium citrate was 1.2%, the concentration of Tris was 0.27%, the concentration of lysine hydrochloride was 0.44%, the concentration of sucrose was 1%, and the concentration of sodium chloride was 1%.

In the step (5), the preparation method of the S/D solution comprises the following steps: adding 11% of Tween 80 and 3.3% of tributyl phosphate, adding injection water, fully dissolving, and then dropwise adding into the filtrate filtered in the step (4) at a speed of less than or equal to 0.6kg/min, so that the final concentration of Tween 80 is 1.1% and the final concentration of tributyl phosphate is 0.33%.

In the step (8), the preparation method of the dissolution buffer solution II comprises the following steps: mixing sodium citrate, Tris, lysine hydrochloride, sucrose and sodium chloride, adding water for injection to dissolve, uniformly stirring, adjusting the pH value to 7.2-7.4 by 12M HCL, and adjusting the concentration of the sodium citrate, the concentration of the Tris to 0.27%, the concentration of the lysine hydrochloride to 0.44%, the concentration of the sucrose to 1% and the concentration of the sodium chloride to 1% in a dissolution buffer solution II.

In the step (11), the preparation method of the dissolution buffer III comprises the following steps: mixing sodium citrate, Tris and sodium chloride, adding water for injection to dissolve, uniformly stirring, and adjusting the pH value to 7.2-7.4 by using 12M HCL; in the lysis buffer III, the concentration of sodium citrate was 1.5%, the concentration of Tris was 0.27% and the concentration of sodium chloride was 0.82%.

The following description is given by way of specific examples.

The percentages in the present invention are mass percentages unless otherwise specified.