阅读说明：本技术 一种凝血酶的制备方法 (Preparation method of thrombin ) 是由 孙丽 于 2019-12-04 设计创作，主要内容包括：本发明提供了一种凝血酶的制备方法,其包括以下步骤：将包含凝血酶的提取液冻干,并且使得包含凝血酶的冻干产物按重量计的含水量不高于3％,优选不高于2.5％,更优选不高于2％,和/或将包含凝血酶的冻干产物的温度升高至101～107℃,再在100±3℃的温度下进行水喷淋式干热处理。该制备方法不仅进一步降低了凝血酶产品中的杂质含量,并且显著地提高了凝血酶产品的效价和有效地去除了病毒,非常适合凝血酶产品的大规模工业生产。(The invention provides a preparation method of thrombin, which comprises the following steps: freeze-drying the extract containing thrombin, and making the water content of the freeze-dried product containing thrombin not higher than 3%, preferably not higher than 2.5%, more preferably not higher than 2% by weight, and/or raising the temperature of the freeze-dried product containing thrombin to 101-107 ℃, and then carrying out water spraying type dry heat treatment at the temperature of 100 +/-3 ℃. The preparation method not only further reduces the content of impurities in the thrombin product, but also obviously improves the titer of the thrombin product and effectively removes viruses, and is very suitable for large-scale industrial production of the thrombin product.)

1. A method of preparing thrombin, comprising the steps of:

the extract liquid containing thrombin is lyophilized, and the water content of the lyophilized product containing thrombin is not higher than 3%, preferably not higher than 2.5%, more preferably not higher than 2% by weight.

2. A method of preparing thrombin, comprising the steps of:

raising the temperature of the freeze-dried product containing thrombin to 101-107 ℃, preferably 103-105 ℃, and then 100 DEG C+3 ℃ and preferably 100 ℃+Carrying out water spraying type dry heat treatment at the temperature of 1 ℃.

3. A method of preparing thrombin, comprising the steps of:

(1) lyophilizing the extract containing thrombin, and making the water content of the lyophilized product containing thrombin not higher than 3%, preferably not higher than 2.5%, more preferably not higher than 2% by weight;

(2) raising the temperature of the freeze-dried product containing thrombin to 101-107 ℃, preferably 103-105 ℃, and then 100 DEG C+3 ℃ and preferably 100 ℃+Carrying out water spraying type dry heat treatment at the temperature of 1 ℃.

4. The method for producing thrombin according to claim 2 or 3, wherein said water-spray dry heat treatment is a freeze-dried product containing thrombinPlacing the product in a water spray purification box, increasing the temperature to 101-107 deg.C, preferably 103-105 deg.C, and then 100 deg.C+3 ℃ and preferably 100 ℃+The freeze-dried product comprising thrombin was continuously sprayed with hot water at a temperature of 1 ℃ for 30 to 60 minutes.

5. The method for producing thrombin according to any one of claims 1, 3 and 4, wherein the extract solution containing thrombin is subjected to virus inactivation and removal treatment with an aqueous solution of tributyl phosphate having a concentration of 0.3% by volume and an aqueous solution of Tween-80 having a concentration of 1% by weight, or an aqueous solution of tributyl phosphate having a concentration of 0.3% by volume and an aqueous solution of TritonX-100 having a concentration of 1% by weight.

6. The method for producing thrombin according to any one of claims 1 and 3 to 5, wherein the thrombin-containing extract is subjected to an enzyme activator activation treatment; preferably, the enzyme activator is N-distearoylphosphatidylacetamide-polyethylene glycol amine.

7. The process for the preparation of thrombin according to any one of claims 1 to 6, comprising the steps of:

(1) adding an adsorbent into the impurity-removed anticoagulated pig plasma to adsorb pig prothrombin, and filtering the adsorbent;

(2) eluting the adsorbent by using an eluent to obtain porcine prothrombin eluent;

(3) performing virus inactivation and removal treatment on the porcine prothrombin eluate by using an aqueous solution of tributyl phosphate with the volume ratio concentration of 0.3% and an aqueous solution of Tween-80 with the weight ratio concentration of 1%, or an aqueous solution of tributyl phosphate with the volume ratio concentration of 0.3% and an aqueous solution of TritonX-100 with the weight ratio concentration of 1%, and performing dialysis;

(4) adding N-distearoyl phosphatidyl acetamide-polyethylene glycol amine to the dialysate to obtain an extract containing porcine thrombin;

(5) lyophilizing an extract comprising porcine thrombin such that the water content of the lyophilized product comprising thrombin is not higher than 3%, preferably not higher than 2.5%, more preferably not higher than 2% by weight;

(6) raising the temperature of the freeze-dried product containing thrombin to 101-107 ℃, preferably 103-105 ℃, and then 100 DEG C+3 ℃ and preferably 100 ℃+The water spray type dry heat treatment is carried out at a temperature of 1 ℃ for 30 minutes to 60 minutes.

8. The method of preparing thrombin according to claim 7, comprising the steps of:

(1) adding an adsorbent into the anticoagulated pig plasma after impurity removal, stirring and standing to adsorb pig prothrombin, filtering the adsorbent, performing suction filtration on the pig plasma adsorbed by the adsorbent, and discarding the filtrate;

(2) washing the adsorbent with a detergent, carrying out suction filtration, discarding filtrate, and then eluting the adsorbent with an eluent to obtain a porcine prothrombin eluent;

(3) performing virus inactivation and removal treatment on the porcine prothrombin eluate by using an aqueous solution of tributyl phosphate with the volume ratio concentration of 0.3% and an aqueous solution of Tween-80 with the weight ratio concentration of 1%, or an aqueous solution of tributyl phosphate with the volume ratio concentration of 0.3% and an aqueous solution of TritonX-100 with the weight ratio concentration of 1%, and dialyzing the eluate subjected to the virus inactivation and removal treatment by using dialysate;

(4) adding N-distearoyl phosphatidyl acetamide-polyethylene glycol amine into the dialysate to ensure that the concentration of the N-distearoyl phosphatidyl acetamide-polyethylene glycol amine in the dialysate is 0.5-1 mol/L, controlling the temperature to be 33-36 ℃, fully stirring for 10-30 minutes, cooling to 1-7 ℃, preferably 4 ℃, standing for 30 minutes to 2 hours to obtain an extracting solution containing the porcine thrombin;

(5) the extracting solution containing the porcine thrombin is subjected to impurity removal, ultrafiltration and sterilization in sequence and then is subjected to freeze-drying, and the water content of the freeze-dried product containing the thrombin is not higher than 3%, preferably not higher than 2.5% and more preferably not higher than 2% by weight;

(6) raising the temperature of the freeze-dried product containing the porcine thrombin to 103-105 ℃, and then 100 DEG C+The water spray type dry heat treatment is carried out at a temperature of 1 ℃ for 30 minutes to 60 minutes.

9. The process for preparing thrombin according to claim 8, wherein in the step 1), the process for preparing anticoagulated porcine plasma comprises: adding an anticoagulant into fresh pig blood, standing, centrifuging, and collecting upper plasma;

preferably, the preparation method of the anticoagulated porcine plasma comprises the following steps: according to the volume ratio of the anticoagulant to the fresh pig blood of 1: 9, adding an anticoagulant into fresh pig blood, slightly stirring, standing at 0-10 ℃, centrifuging at 19000r/min within 5-8 hours at 8-15 ℃ for 15-20 minutes, and collecting upper plasma;

more preferably, the anticoagulant is 38 grams/liter aqueous trisodium citrate;

further preferably, the anticoagulated pig plasma is stored for 0-6 hours at 0-10 ℃ or stored frozen below-18 ℃ for less than one year for later use;

preferably, the impurity removal is step-by-step filtration by using 70-mesh, 100-mesh and 200-mesh nylon filter screens;

preferably, the adsorbent is selected from sephadex G10, 15, 25, 50, 75, 100, 150 and 200; a-25 and 50; q-25 and 50; and C-25 and 50; preferably, the adsorbent is medium particle size sephadex A-50;

preferably, the mass ratio of the anticoagulated pig plasma to the adsorbent is 1000: 1-3, more preferably 1000: 2;

preferably, the stirring time is 30-60 minutes, and more preferably 45 minutes; the standing time is 15-45 minutes, and more preferably 30 minutes.

10. The process for preparing thrombin according to claim 8 or 9, wherein in step (2), the detergent is an aqueous solution comprising 11.7 g/l of sodium chloride and 3 g/l of trisodium citrate;

preferably, the ratio of the detergent to the anticoagulated porcine plasma is 100-200 ml: 1000 g, more preferably 160 ml: 1000 g;

preferably, the eluent is an aqueous solution comprising 117 g/l sodium chloride and 3 g/l trisodium citrate;

preferably, the ratio of the eluent to the anticoagulated pig plasma is 10-30 ml: 1000 g, more preferably 20 ml: 1000 g.

11. The method for preparing thrombin according to any one of claims 8 to 10, wherein in the step (3), the virus inactivation and removal treatment of the porcine prothrombin eluate is carried out by treating the porcine prothrombin eluate at 22-26 ℃ for 4-8 hours using an aqueous solution of tributyl phosphate with a volume ratio concentration of 0.3% and an aqueous solution of tween-80 with a weight ratio concentration of 1%, or an aqueous solution of tributyl phosphate with a volume ratio concentration of 0.3% and an aqueous solution of triton x-100 with a weight ratio concentration of 1%; more preferably, the porcine prothrombin eluate is treated at 24 ℃ for 6 hours;

preferably, the dialysate is 2.94 g/l trisodium citrate in water;

preferably, the ratio of the dialysate to the anticoagulated pig plasma is 2500-3500 ml: 1000 g, more preferably 3000 ml: 1000 g;

preferably, the dialysis time is 4-6 hours, more preferably 5 hours;

preferably, the dialysis temperature is 0-20 ℃.

12. The method for producing thrombin according to any one of claims 8 to 11, wherein in step (5), said removing impurities comprises: adjusting the pH of an extracting solution containing the porcine thrombin to 5.2-5.6 by using 1M glacial acetic acid, standing for 30 minutes at 4 ℃, centrifuging to collect a supernatant, adjusting the pH of the supernatant to 6.0-6.5 by using a saturated sodium bicarbonate aqueous solution, standing for 30 minutes at 4 ℃, centrifuging, and collecting the supernatant;

preferably, the ultrafiltration is ultrafiltration using an ultrafiltration column with a molecular weight of 8000;

preferably, the sterilization is performed by filtering through filter membranes with the pore diameters of 0.8 μm, 0.45 μm and 0.22 μm in sequence, and then filtering through a filter element with the pore diameter of 0.22 μm.

13. The thrombin prepared by the method for preparing thrombin according to any one of claims 1 to 12.

Technical Field

The invention belongs to the field of biochemical pharmacy, relates to a preparation method of thrombin, and particularly relates to a preparation method of thrombin, which can improve the titer, remove viruses and reduce impurities and is suitable for large-scale industrial production.

Background

Thrombin is a serine protease widely present in human and animal blood, a major effector protease in the blood coagulation cascade, exhibiting procoagulant and anticoagulant properties. When the circulating coagulation factor comes into contact with tissue factor in the exposed extravascular tissue, thrombin accumulates on the tissue. Thrombin catalyzes fibrinogen to fibrin by activating platelets, and promotes the stabilization of blood clots, thereby playing a central role in the initiation and development of thrombotic diseases. Since thrombin can directly act on fibrinogen in blood to promote the fibrinogen to be converted into fibrin and accelerate the blood coagulation to stop bleeding, thrombin can be clinically used for stopping bleeding of trauma, operation, oral cavity, ear, nose and throat, urinary, obstetrics and gynecology department and digestive tract bleeding, particularly for stopping bleeding of small blood vessels which are not easy to ligate in the operation, digestive tract bleeding and traumatic bleeding and the like.

At present, thrombin which is clinically applied is mainly an aseptic freeze-dried product of thrombin which is obtained by separating and extracting human blood and animal blood of pigs, cows and the like and is activated by a prothrombin activator, has higher specificity, is a quick-acting local hemostatic, and is widely applied to gastrointestinal bleeding and surgical hemostasis (Songhongxin, Mayongyang, Liminokang, research progress of a thrombin separation and purification method, food research and development, 2004, 25 (6): 65-67). Wherein, because of the factors of sufficient pig blood source, low raw material cost and the like, a large amount of thrombin products are prepared from pig blood, and a small amount of thrombin products are prepared from blood of human blood, bovine blood and the like. The molecular weight of the porcine thrombin is about 35-39 kDa, the porcine thrombin is formed by connecting two polypeptide chains through a disulfide bond, the molecular weight of one chain is about 34kDa, the molecular weight of the other chain is about 5kDa, the isoelectric point (pI) is 5.0-5.5, IEF electrophoresis shows two bands, and the pIs are 5.0 and 5.4 respectively (Xuchang method, Wangyixian, Liudefu and the like, separation, purification and identification of porcine thrombin, university report of Beijing university, 1994, 8 (1): 44-49).

The process of separating and purifying the porcine thrombin roughly comprises the steps of pretreatment of porcine plasma, extraction of prothrombin, activation of prothrombin, preparation of crude thrombin, purification of thrombin and the like. Because the thrombin product is a multi-component biochemical medicine prepared by extracting pig blood, the potential hidden danger of pig-derived virus pollution is caused, and in order to improve the safety of the product, the research on the thrombin virus inactivation process is necessary to be carried out. Further, the existing preparation methods, whether the thrombin product is prepared from animal blood or human blood, have the following problems: (1) the thrombin product has unstable titer; (2) the concentration of impurities and foreign proteins in the product is higher; (3) there is a risk of introducing various viruses, which can limit the clinical use of thrombin products and pose a safety hazard.

The chinese patent application CN103160486 discloses a method for preparing porcine thrombin, which comprises the following steps: firstly, separating pig plasma; secondly, chemical virus inactivation; thirdly, adsorbing prothrombin; fourthly, collecting prothrombin; fifthly, nano-filtration; sixthly, activating prothrombin; and seventhly, freeze-drying and storing the thrombin. The patent application does not disclose the kind of reagent and nanomembrane used for virus inactivation, and the product obtained by the method has high impurity content, and the titer of the product is to be further improved.

In conclusion, there is an urgent need for thrombin products and methods for their production that have higher potency, lower production costs, and are safer and more reliable.

Disclosure of Invention

In order to overcome the above defects of the prior art, the present invention aims to provide a novel thrombin preparation method, which uses an S/D method (i.e. using an aqueous solution of tributyl phosphate with a volume ratio concentration of 0.3% and an aqueous solution of tween-80 with a weight ratio concentration of 1%, or an aqueous solution of tributyl phosphate with a volume ratio concentration of 0.3% and an aqueous solution of triton x-100 with a weight ratio concentration of 1%) in combination with a dry heat method for virus inactivation, and uses N-distearoyl phosphatidyl acetamide-polyethylene glycol amine as an enzyme activator, so that the method not only further reduces the impurity content in the thrombin product, but also significantly improves the titer of the thrombin product and effectively removes viruses, and is very suitable for large-scale industrial production of the thrombin product.

In one aspect, the present invention provides a method for preparing thrombin, comprising the steps of:

the extract liquid containing thrombin is lyophilized, and the water content of the lyophilized product containing thrombin is not higher than 3%, preferably not higher than 2.5%, more preferably not higher than 2% by weight.

In another aspect, the invention provides a method of preparing thrombin, comprising the steps of:

raising the temperature of the freeze-dried product containing the thrombin to 101-107 ℃, preferably 103-105 ℃, and then carrying out water spraying type dry heat treatment at the temperature of 100 +/-3 ℃, preferably 100 +/-1 ℃.

In yet another aspect, the present invention provides a method of preparing thrombin, comprising the steps of:

(1) lyophilizing the extract containing thrombin, and making the water content of the lyophilized product containing thrombin not higher than 3%, preferably not higher than 2.5%, more preferably not higher than 2% by weight;

(2) raising the temperature of the freeze-dried product containing the thrombin to 101-107 ℃, preferably 103-105 ℃, and then carrying out water spraying type dry heat treatment at the temperature of 100 +/-3 ℃, preferably 100 +/-1 ℃.

In the above-mentioned method for preparing thrombin, the water spray type dry heat treatment may be carried out by placing the freeze-dried product containing thrombin in a water spray purification box, raising the temperature to 101 to 107 ℃, preferably 103 to 105 ℃, and then continuously spraying the freeze-dried product containing thrombin with hot water at a temperature of 100 ± 3 ℃, preferably 100 ± 1 ℃ for 30 to 60 minutes.

Preferably, in the above method for preparing thrombin, the extract solution containing thrombin is subjected to virus inactivation and removal treatment with an aqueous solution of tributyl phosphate having a volume ratio concentration of 0.3% and an aqueous solution of tween-80 having a weight ratio concentration of 1%, or an aqueous solution of tributyl phosphate having a volume ratio concentration of 0.3% and an aqueous solution of triton x-100 having a weight ratio concentration of 1%.

Preferably, in the above-mentioned method for producing thrombin, the extract solution containing thrombin is subjected to an enzyme activator activation treatment; preferably, the enzyme activator is N-distearoylphosphatidylacetamide polyethylene glycol-amine.

Preferably, the preparation method of thrombin provided by the invention comprises the following steps:

(1) adding an adsorbent into the impurity-removed anticoagulated pig plasma to adsorb pig prothrombin, and filtering the adsorbent;

(2) eluting the adsorbent by using an eluent to obtain porcine prothrombin eluent;

(3) performing virus inactivation and removal treatment on the porcine prothrombin eluate by using an aqueous solution of tributyl phosphate with the volume ratio concentration of 0.3% and an aqueous solution of Tween-80 with the weight ratio concentration of 1%, or an aqueous solution of tributyl phosphate with the volume ratio concentration of 0.3% and an aqueous solution of TritonX-100 with the weight ratio concentration of 1%, and performing dialysis;

(4) adding N-distearoyl phosphatidyl acetamide-polyethylene glycol amine to the dialysate to obtain an extract containing porcine thrombin;

(5) lyophilizing an extract comprising porcine thrombin such that the water content of the lyophilized product comprising thrombin is not higher than 3%, preferably not higher than 2.5%, more preferably not higher than 2% by weight;

(6) the temperature of the freeze-dried product containing the thrombin is increased to 101-107 ℃, preferably 103-105 ℃, and then water spraying type dry heat treatment is carried out at the temperature of 100 +/-3 ℃, preferably 100 +/-1 ℃ for 30-60 minutes.

More preferably, the preparation method of thrombin provided by the invention comprises the following steps:

(1) adding an adsorbent into the anticoagulated pig plasma after impurity removal, stirring and standing to adsorb pig prothrombin, filtering the adsorbent, performing suction filtration on the pig plasma adsorbed by the adsorbent, and discarding the filtrate;

(2) washing the adsorbent with a detergent, carrying out suction filtration, discarding filtrate, and then eluting the adsorbent with an eluent to obtain a porcine prothrombin eluent;

(3) performing virus inactivation and removal treatment on the porcine prothrombin eluate by using an aqueous solution of tributyl phosphate with the volume ratio concentration of 0.3% and an aqueous solution of Tween-80 with the weight ratio concentration of 1%, or an aqueous solution of tributyl phosphate with the volume ratio concentration of 0.3% and an aqueous solution of TritonX-100 with the weight ratio concentration of 1%, and dialyzing the eluate subjected to the virus inactivation and removal treatment by using dialysate;

(4) adding N-distearoyl phosphatidyl acetamide-polyethylene glycol amine into the dialysate to ensure that the concentration of the N-distearoyl phosphatidyl acetamide-polyethylene glycol amine in the dialysate is 0.5-1 mol/L, controlling the temperature to be 33-36 ℃, fully stirring for 10-30 minutes, cooling to 1-7 ℃, preferably 4 ℃, standing for 30 minutes to 2 hours to obtain an extracting solution containing the porcine thrombin;

(5) the extracting solution containing the porcine thrombin is subjected to impurity removal, ultrafiltration and sterilization in sequence and then is subjected to freeze-drying, and the water content of the freeze-dried product containing the thrombin is not higher than 3%, preferably not higher than 2.5% and more preferably not higher than 2% by weight;

(6) raising the temperature of the freeze-dried product containing the porcine thrombin to 103-105 ℃, and then carrying out water spraying type dry heat treatment at the temperature of 100 +/-1 ℃ for 30-60 minutes.

Preferably, in the step (1), the preparation method of anticoagulated porcine plasma comprises: adding an anticoagulant into fresh pig blood, standing, centrifuging, and collecting upper plasma;

preferably, the preparation method of the anticoagulated porcine plasma comprises the following steps: according to the volume ratio of the anticoagulant to the fresh pig blood of 1: 9, adding an anticoagulant into fresh pig blood, slightly stirring, standing at 0-10 ℃, centrifuging at 19000r/min within 5-8 hours at 8-15 ℃ for 15-20 minutes, and collecting upper plasma;

more preferably, the anticoagulant is 38 grams/liter aqueous trisodium citrate;

further preferably, the anticoagulated pig plasma is stored for 0-6 hours at 0-10 ℃ or stored frozen below-18 ℃ for less than one year for later use;

preferably, the impurity removal is step-by-step filtration by using 70-mesh, 100-mesh and 200-mesh nylon filter screens.

Preferably, the adsorbent is selected from sephadex G10, 15, 25, 50, 75, 100, 150 and 200; a-25 and 50; q-25 and 50; and C-25 and 50; preferably, the adsorbent is medium particle size sephadex A-50;

preferably, the mass ratio of the anticoagulated pig plasma to the adsorbent is 1000: 1-3, more preferably 1000: 2;

preferably, the stirring time is 30-60 minutes, and more preferably 45 minutes; the standing time is 15-45 minutes, and more preferably 30 minutes.

Preferably, in the above step (2), the detergent is an aqueous solution containing 11.7 g/l of sodium chloride and 3 g/l of trisodium citrate;

preferably, the ratio of the detergent to the anticoagulated porcine plasma is 100-200 ml: 1000 g, more preferably 160 ml: 1000 g;

preferably, the eluent is an aqueous solution comprising 117 g/l sodium chloride and 3 g/l trisodium citrate;

preferably, the ratio of the eluent to the anticoagulated pig plasma is 10-30 ml: 1000 g, more preferably 20 ml: 1000 g.

Preferably, in the step (3), the virus inactivation and removal treatment of the porcine prothrombin eluate is to treat the porcine prothrombin eluate at 22-26 ℃ for 4-8 hours by using an aqueous solution of tributyl phosphate with a volume concentration of 0.3% and an aqueous solution of tween-80 with a weight concentration of 1%, or an aqueous solution of tributyl phosphate with a volume concentration of 0.3% and an aqueous solution of triton x-100 with a weight concentration of 1%; more preferably, the porcine prothrombin eluate is treated at 24 ℃ for 6 hours.

Preferably, the dialysate is 2.94 g/l trisodium citrate in water;

preferably, the ratio of the dialysate to the anticoagulated pig plasma is 2500-3500 ml: 1000 g, more preferably 3000 ml: 1000 g;

preferably, the dialysis time is 4-6 hours, more preferably 5 hours;

preferably, the dialysis temperature is 0-20 ℃.

Preferably, in the step (5) above, the removing impurities includes: adjusting the pH of an extracting solution containing the porcine thrombin to 5.2-5.6 by using 1M glacial acetic acid, standing for 30 minutes at 4 ℃, centrifuging to collect supernatant, adjusting the pH of the supernatant to 6.0-6.5 by using saturated sodium bicarbonate, standing for 30 minutes at 4 ℃, centrifuging, and collecting supernatant.

Preferably, the ultrafiltration is ultrafiltration using an ultrafiltration column with a molecular weight of 8000;

preferably, the sterilization is performed by filtering through filter membranes with the pore diameters of 0.8 μm, 0.45 μm and 0.22 μm in sequence, and then filtering through a filter element with the pore diameter of 0.22 μm.

The invention also provides thrombin prepared by the preparation method of the thrombin.

According to a specific technical scheme of the invention, the preparation method of the thrombin comprises the following steps:

1) collecting pig blood and preparing anticoagulated pig plasma, sucking the anticoagulated pig plasma into a high-level storage tank, flowing into a tubular centrifuge through a lower discharge valve of the tank body, starting the tubular centrifuge for centrifugation, and filtering and removing impurities step by step through 70-mesh, 100-mesh and 200-mesh filter screens;

2) and (3) mixing the filtered pig plasma with an adsorbent according to the mass ratio of the pig plasma to the adsorbent of 1000: 2, adding a pretreated adsorbent, stirring for 45 minutes, standing for 30 minutes to adsorb porcine prothrombin, filtering the adsorbed porcine plasma and the precipitated adsorbent by using a 200-mesh nylon filter cloth, carrying out suction filtration on the porcine plasma in the adsorbent, and discarding the filtrate;

3) the adsorbent was washed with an aqueous solution containing 11.7 g/l sodium chloride, 3 g/l trisodium citrate, in which the ratio of detergent to porcine plasma was 160 ml: 1000 g, suction filtration, discarding the filtrate, and then adding an aqueous solution containing 117 g/l sodium chloride, 3 g/l trisodium citrate to the drained adsorbent, wherein the ratio of eluent to porcine plasma is 20 ml: 1000 g, 30 minutes at room temperature, suction filtration and collection of the filtrate, and then the addition of a new aqueous solution containing 117 g/l sodium chloride, 3 g/l trisodium citrate, to the drained adsorbent, where the ratio of eluent to porcine plasma is 20 ml: 1000 g, soaking for 20 minutes at room temperature, carrying out suction filtration, collecting filtrate, and combining the filtrate to obtain porcine prothrombin eluate;

4) treating the porcine prothrombin eluate obtained in the step 3) for 6 hours at 24 ℃ by using an aqueous solution of tributyl phosphate with a volume ratio concentration of 0.3% and tween-80 with a weight ratio concentration of 1%, or an aqueous solution of tributyl phosphate with a volume ratio concentration of 0.3% and TritonX-100 with a weight ratio concentration of 1%;

5) putting the eluent subjected to virus inactivation/removal in the step 4) into a dialysis bag with the molecular weight of 7000, and dialyzing for 5 hours by using 2.94 g/L trisodium citrate water solution, wherein the temperature of the dialysate is controlled to be 0-20 ℃ to obtain dialysate; wherein, the ratio of the dialysate to the pig plasma is 3000 ml: 1000 g;

6) adding N-distearoyl phosphatidyl acetamide-polyethylene glycol Amine, namely DSPE-PEG-Amine, into the dialysate obtained in the step 5), so that the final concentration of the DSPE-PEG-Amine is 0.8mol/L, controlling the temperature to be 35 ℃, fully stirring for 20 minutes, cooling to 4 ℃, and standing for 2 hours to obtain an extracting solution of the porcine thrombin;

7) adjusting the pH value of the extracting solution of the porcine thrombin obtained in the step 6) to 5.4 by using 1M glacial acetic acid, standing for 30 minutes at 4 ℃, centrifuging to collect supernatant, adjusting the pH value of the supernatant to 6.3 by using saturated sodium bicarbonate, standing for 30 minutes at 4 ℃, centrifuging to collect supernatant, and ultrafiltering the supernatant by using an ultrafiltration column with the molecular weight of 8000; sequentially filtering the ultrafiltered extractive solution with sterilizing filter membranes with pore diameters of 0.8 μm, 0.45 μm and 0.22 μm, filtering with filter core of 0.22 μm, collecting supernatant, and preparing lyophilized product of porcine thrombin;

8) processing the porcine thrombin freeze-dried product obtained in the step 7) by using a dry-heat method, and packaging to obtain a thrombin freeze-dried product finished product;

wherein, the dry heating method conditions are as follows: raising the temperature to 103-105 ℃, and then carrying out water spraying type dry heat treatment at 100 +/-1 ℃ for 30-60 minutes.

Compared with the prior art, the invention provides at least the following advantages:

1. one of the objectives of the present invention is to improve the thrombin product titer, and studies have shown that controlling the conditions of lyophilization parameters is an important means to achieve this objective. The dry heating method can remove lipid enveloped virus and non-lipid enveloped virus in the thrombin product, but because the dry heating method uses higher treatment temperature (80 ℃ or 100 ℃), the stability of protein is affected, and the titer of the thrombin product is reduced. The inventor of the application surprisingly finds that the protein can be invariable in the dry heat treatment process by controlling the moisture content of the freeze-dried product to be less than 3% by weight, thereby achieving the effect of stable and controllable titer. The method is very suitable for the requirements of production process and industrial production.

2. In addition, the inventor of the application also finds that the temperature of the freeze-dried product is increased to 101-107 ℃ firstly in the dry heating method treatment process, the stability of heat conduction and the heating uniformity can be guaranteed, the dry heating treatment is continuously guaranteed to be carried out for 30-60 minutes at the temperature of 100 +/-3 ℃, and the dry heating mode adopts a water spraying type dry heating method, so that the heat is more uniform in heating compared with the traditional dry heating box method, and the virus removing/inactivating effect is better.

3. The present inventors have long been working on research and development of a thrombin preparation method, and in a thrombin preparation method (see chinese patent ZL201410591661.6 entitled "a thrombin preparation method") developed before, virus inactivation/removal was performed using an S/D method in combination with a membrane filtration method, wherein the membrane filtration method can effectively retain viruses larger than the pore size of a filter membrane without destroying the structure of active ingredients, and has wide requirements for temperature, pH, etc. of a sample, and good applicability and selectivity. However, the membrane filtration method has high production cost and low production efficiency. The S/D method is combined with the dry heat method to inactivate/remove viruses, and freeze-drying parameter conditions are mainly controlled, wherein the S/D method is good in production cost and operability, the treatment temperature is 24 ℃, the influence on products is small, the treatment time is appropriate, the method is widely applied, and the technical maturity and the recognition degree are high.

4. In order to reduce the risk of bringing in new viruses, the enzyme activator used in the preparation method is DSPE-PEG-Amine. The Chinese patent application with the application number of 201510638083.1 provides a preparation method of a porcine thrombin freeze-dried powder, which uses rabbit brain powder as an animal-derived enzyme activator, and equivalently, a new animal-derived substance is introduced. According to the basic technical requirements of multi-component biochemical drug injection, the process control of the multi-component biochemical drug should be started directly from the source, and the control of the raw materials should include comprehensive information such as animal feeding environment, disease control condition, raw material collection method, necessary virus control and the like. Lecithin is selected in the preparation method and the production system of the porcine thrombin provided by the Chinese patent application with the application number of 201710319423.3 and other patent documents, but the lecithin selected in the actual production process at the present stage is all lecithin extracted from egg yolk, so that the risk of bringing new animal-derived viruses exists. The N-distearoyl phosphatidyl acetamide-polyethylene glycol Amine (DSPE-PEG-Amine) adopted by the application is a pure chemically synthesized enzyme activator, and does not bring new animal source new virus risk.

Detailed Description

The invention is illustrated below with reference to specific examples. It will be understood by those skilled in the art that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention in any way.

The experimental procedures in the following examples are conventional unless otherwise specified. The raw materials and reagent materials used in the following examples are all commercially available products unless otherwise specified.