CN111773188B - Preparation method of urokinase freeze-dried powder - Google Patents
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Abstract
The invention discloses a preparation method of urokinase freeze-dried powder, and relates to the technical field of medicines. The preparation formula comprises urokinase, human serum albumin, sodium chloride, mannitol, konjac glucomannan and water for injection, wherein the mass ratio of mannitol to konjac glucomannan is 15-25:3-6, and the preparation method comprises the steps of preparing an auxiliary material solution, adsorbing and decarburizing active carbon, preparing a main drug, freeze-drying, tamponade and capping. By improving the formula and the freeze-drying process, the addition of human serum albumin is reduced, the cost is reduced, and the effects of improving the stability and the titer are achieved.
Description
Technical Field
The invention relates to the technical field of medicines, and in particular relates to a preparation method of urokinase freeze-dried powder.
Background
Urokinase is a kind of protease separated and purified from fresh human urine, and is usually white or off-white lyophilized cake or powder. Mainly prepared into enzyme thrombolytic drugs. It activates the conversion of plasminogen to plasmin in the body, thereby hydrolyzing fibrin and dissolving the newly formed thrombus.
Urokinase is mainly used for thrombolysis treatment of thromboembolic diseases clinically. Including acute generalized pulmonary embolism, coronary embolism and myocardial infarction with chest pain within 6-12 hours, acute cerebral embolism with symptoms shorter than 3-6 hours, retinal artery embolism and other iliac femoral vein thrombosis with serious peripheral artery embolism symptoms. Also used for preventing thrombosis after artificial heart valve operation, keeping the smoothness of a blood vessel cannula and a thoracic cavity and pericardial cavity drainage tube, and the like. The curative effect of thrombolysis needs to be maintained by subsequent heparin anticoagulation.
The prior freeze drying technology is to remove water from a frozen state without direct sublimation in a liquid state under the conditions of low temperature and low pressure by utilizing a frozen solution to complete drying. The freeze drying technology can well maintain the original physicochemical properties and physiological activity of the medicine, and the loss of the effective components is little. In addition, the specific loose porous structure of the freeze-dried preparation can ensure that the medicine is easy to be rehydrated to recover the activity, and the freeze-dried preparation has low water content and is easy to store. Thus, freeze-drying is one of the best methods for clinical and laboratory preservation of protein products.
Chinese patent application 201510326053.7 discloses a preparation method of a urokinase lyophilized preparation, the formulation of the preparation comprises urokinase, human serum albumin, sodium chloride, mannitol and water for injection, and the preparation steps comprise: preparing an auxiliary material solution: adding sodium chloride and mannitol into water for injection under stirring to dissolve; adding active carbon for adsorption, and cooling to below 10 ℃ after decarbonization; stirring, dissolving human serum albumin, adding urokinase, filtering with filter membrane, bottling, semi-plugging, freeze drying, pressing, slowly vacuumizing, taking out, capping, visual inspection, packaging, and inspecting. The preparation method improves the titer by low-temperature batching, and a special low-temperature batching device is used in the preparation process; the addition of human serum albumin can cause the solution to foam, the yield of the finished product is reduced, the foaming can be reduced through magnetic stirring, and the yield is improved.
Chinese patent application 200410058006.0 discloses a pharmaceutical composition containing active prourokinase, its freeze-drying method and freeze-dried preparation. The composition contains appropriate amount of prourokinase, excipient, buffer solution and salt. Meanwhile, a protective agent can be added to obtain a better freeze-drying effect, and the protective agent can be protein. The application also provides a method for freeze-drying a solution of the composition, comprising the steps of: putting the packaged urokinase raw product into a freeze dryer which is pre-cooled to minus 35 ℃ to minus 45 ℃ for pre-freezing for 2-4h, then vacuumizing, heating to minus 25 ℃ to minus 30 ℃ for the first time, heating for the second time after the water in the bottle is basically sublimated to raise the temperature to 25-35 ℃, keeping the temperature for 0.5-1.5h after stabilization, and finishing freeze drying, wherein the method improves the storage stability.
Urokinase is easy to inactivate when placed at room temperature, and the protection effect of human serum albumin on the thermal denaturation of urokinase at present is widely applied to urokinase products. However, human serum albumin is expensive as a protective agent, and the preparation cost of urokinase products is further increased. In view of this, the application provides a preparation method of urokinase powder, which reduces the addition of human serum albumin, reduces the cost, and achieves the effect of improving the stability.
Disclosure of Invention
The invention aims to provide a preparation method of urokinase powder, which can obviously reduce the addition of human serum albumin, reduce the cost and simultaneously improve the stability and the titer.
In order to achieve the purpose, the technical scheme of the invention is as follows:
a preparation method of urokinase freeze-dried powder comprises the following steps of preparing a preparation formula from urokinase, human serum albumin, sodium chloride, mannitol, konjac glucomannan and water for injection, wherein the mass ratio of the mannitol to the konjac glucomannan is 15-25:3-6, and the preparation method comprises the following steps:
(1) preparing an auxiliary material solution: adding water for injection into a mixing tank, adding sodium chloride, mannitol and konjac glucomannan under stirring, and continuously stirring to obtain an auxiliary material solution;
(2) activated carbon adsorption and decarburization: adding activated carbon moistened with water for injection into the auxiliary material solution, stirring, decarburizing, and sealing and cooling to obtain a feed liquid 1;
(3) preparing a main medicine: adding human serum albumin into the feed liquid 1 under stirring, stirring until the human serum albumin is completely dissolved to obtain a feed liquid 2, then adding urokinase into the feed liquid 2, stirring until the urokinase is completely dissolved, supplementing water for injection, continuously stirring, and filtering with a saturated filter membrane to obtain a main drug solution;
(4) freeze-drying: filling the main medicine solution obtained in the step (3) with half plugs, and freeze-drying, wherein the freeze-drying process comprises the following steps:
keeping the temperature at minus 35 ℃ to minus 25 ℃ for 2 to 3 hours;
keeping the temperature at minus 45 ℃ to minus 40 ℃ for 0.5 to 1.5 hours;
vacuumizing to below 10Pa, and keeping the temperature of-20 to-10 ℃ for 8 to 12 hours;
keeping the temperature of minus 5-0 ℃ for 8-12 h;
keeping the temperature of 3-7 ℃ for 4-6 h;
keeping the temperature of 8-12 ℃ for 0.5-1.5 h.
(5) And (5) plugging and capping.
Preferably, in the formula of the preparation, 8-12 ten thousand units/mL of urokinase, 20-60mg/mL of human serum albumin, 10-15mg/mL of sodium chloride, 0.15-0.25g/mL of mannitol and 0.03-0.06g/mL of konjac glucomannan are added; further preferably, the mass ratio of the mannitol to the konjac glucomannan is 9-11:2, and further preferably 5: 1; most preferably, 10 ten thousand units/mL of urokinase, 35mg/mL of human serum albumin, 12mg/mL of sodium chloride, 0.2g/mL of mannitol and 0.04g/mL of konjac glucomannan are used.
Preferably, the stirring is continued for 8-12min, more preferably for 10min in step (1).
Preferably, the stirring temperature in the step (2) is 80-100 ℃; stirring for 15-25 min.
Preferably, the decarbonization in the step (2) is decarbonization by using a sterilized flat filter which is provided with a 0.22 mu m mixed cellulose ester microporous filter membrane.
Preferably, the closed cooling in the step (2) is performed in a magnetic stirring tank to be closed and cooled to below 10 ℃.
Preferably, the stirring in step (3) is continued for 15-25min, and more preferably for 20 min.
Preferably, the saturated filter membrane in the step (3) is a filter membrane wetted by the feed liquid 2.
Preferably, the freeze-drying process in step (4) is as follows:
holding at-20 deg.C for 2.5 h;
holding at-42 deg.C for 1 h;
vacuumizing to below 10Pa, and keeping at-15 ℃ for 10 h;
holding at-2 ℃ for 10 h;
keeping the temperature at 5 ℃ for 5 h;
the temperature is kept at 10 ℃ for 1 h.
Preferably, the step (5) is specifically: and (5) after freeze-drying, pressing, capping, visually inspecting, packaging and fully inspecting.
Compared with the prior art, the invention has the following beneficial effects:
(1) the addition of human serum albumin is obviously reduced, and the cost is reduced;
(2) improve the stability and the potency.
Detailed Description
The present invention will be further explained with reference to specific embodiments in order to make the technical means, the original characteristics, the achieved objects and the effects of the present invention easy to understand, but the following embodiments are only preferred embodiments of the present invention, and not all embodiments are possible. Based on the embodiments in the implementation, other embodiments obtained by those skilled in the art without any creative efforts belong to the protection scope of the present invention.
The experimental methods in the following examples are conventional methods unless otherwise specified, and materials, reagents and the like used in the following examples are commercially available unless otherwise specified.
Examples 1 to 5
Examples 1-5 the composition and content of lyophilized urokinase preparations per 1000 formulation units are shown in table 1:
table 1.
| Example 1 | Example 2 | Example 3 | Example 4 | Example 5 | |
| Urokinase | 1.0 hundred million units | 1.0 hundred million units | 1.0 hundred million units | 0.8 hundred million units | 1.2 hundred million units |
| Human serum albumin | 35mg | 35mg | 35mg | 20mg | 60mg |
| Sodium chloride | 12g | 12g | 12g | 10g | 15g |
| Mannitol | 200g | 220g | 180g | 250g | 150g |
| Konjac glucomannan | 40g | 40g | 40g | 30g | 60g |
| Water for injection | 1000mL | 1000mL | 1000mL | 1000mL | 1000mL |
Examples 1-5 were all prepared according to the following preparation:
(1) preparing an auxiliary material solution: adding 950mL of fresh water for injection into a mixing barrel, sequentially adding sodium chloride, mannitol and konjac glucomannan under stirring, and continuously stirring for 10min to obtain an adjuvant solution;
(2) activated carbon adsorption and decarburization: adding 3.0g767 type injection active carbon (wetted with water for injection), keeping the temperature above 80 deg.C, stirring for 20min, standing for adsorption for 30 min, decarbonizing with sterilized flat filter equipped with 0.22 μm mixed cellulose ester microporous filter membrane to obtain feed liquid 1;
(3) preparing a main medicine: adding human serum albumin into the feed liquid 1 under stirring, stirring to dissolve completely to obtain feed liquid 2, adding urokinase into the feed liquid 2, stirring to dissolve completely, adding water for injection, stirring for 20min, and filtering with saturated filter membrane wetted with the feed liquid 2 to obtain main medicinal solution;
(4) freeze-drying: filling the main medicine solution obtained in the step (3) with half plugs, and freeze-drying, wherein the freeze-drying process comprises the following steps:
holding at-20 deg.C for 2.5 h;
holding at-42 deg.C for 1 h;
vacuumizing to below 10Pa, and keeping at-15 ℃ for 10 h;
holding at-2 ℃ for 10 h;
keeping the temperature at 5 ℃ for 5 h;
the temperature is kept at 10 ℃ for 1 h.
(5) And (3) plug pressing and capping: and (5) after freeze-drying, pressing, capping, visually inspecting, packaging and fully inspecting.
Example 6
Unlike example 1, the lyophilization process of example 6 is as follows:
holding at-35 ℃ for 2 h;
keeping the temperature at minus 45 ℃ for 0.5 h;
vacuumizing to below 10Pa, and keeping at-20 ℃ for 8 h;
holding at-5 ℃ for 8 h;
keeping the temperature at 3 ℃ for 4 h;
keeping the temperature at 8 ℃ for 0.5h,
the rest is the same.
Example 7
Unlike example 1, the lyophilization process of example 7 is as follows:
holding at-25 deg.C for 3 h;
holding at-40 deg.C for 1.5 h;
vacuumizing to below 10Pa, and keeping at-10 ℃ for 12 h;
keeping the temperature at 0 ℃ for 12 h;
keeping the temperature at 7 ℃ for 6 h;
keeping the temperature at 12 ℃ for 1.5h,
the rest is the same.
Comparative examples 1 to 3
Comparative examples 1-3 the composition of the formulation of the components and contents of the lyophilized urokinase preparation per 1000 formulation units is shown in table 2:
table 2.
Comparative example 4
Unlike example 1, the lyophilization process of comparative example 4 was as follows:
pre-freezing for 3-4 hours at the temperature below minus 40 ℃; sublimation drying at-10 deg.C to 0 deg.C for about 17 hr; then the temperature is raised to 20 ℃, and the solution is resolved and dried for 5 to 6 hours,
the rest is the same.
Comparative example 5
Unlike example 1, the lyophilization process of comparative example 4 was as follows:
holding at-20 deg.C for 2.5 h;
holding at-42 deg.C for 1 h;
vacuumizing to below 10Pa, and keeping at-25 ℃ for 5 h;
holding at-15 deg.C for 10 h;
holding at-10 deg.C for 10h
Holding at-2 ℃ for 10 h;
keeping the temperature at 5 ℃ for 5 h;
keeping the temperature of the mixture at 10 ℃ for 2h,
the rest is the same.
Result detection
1. Potency assay
The products of examples and comparative examples were subjected to a total test according to the test standards, and the results are shown in Table 3, wherein the titer was measured according to the method described in Chinese patent CN 104906054B.
Table 3.
It can be seen that the potency is highest in example 1, and both the formulation and the lyophilization process have an effect on potency.
2. Stability survey
2.1 the freeze-dried preparation vial prepared in example 1 is taken and put in a constant temperature and humidity incubator with the temperature of 25 ℃ plus or minus 2 ℃ and the relative humidity of 65 percent plus or minus 5 percent for 6 months, samples are taken at the end of 1, 2, 3 and 6 months respectively, all indexes of the product are examined, and the results of the accelerated test of the product are shown in Table 4.
Table 4.
| Time (moon) | Traits | Clarity of the product | Visible foreign body | Sterile | Potency (%) |
| 0 | White powder | Clear and colorless | Compliance with regulations | Compliance with regulations | 112.5 |
| 1 | White powder | Clear and colorless | Compliance with regulations | Compliance with regulations | 111.7 |
| 2 | White powder | Clear and colorless | Compliance with regulations | Compliance with regulations | 110.6 |
| 3 | White powder | Clear and colorless | Compliance with regulations | Compliance with regulations | 109.8 |
| 6 | White powder | Clear and colorless | Compliance with regulations | Compliance with regulations | 106.6 |
Example 1 after 6 months of accelerated testing, the titer dropped from 112.5% to 106.6%, and good stability was seen.
2.2 vials of the lyophilized formulations of examples 1-7 and comparative examples 1-5, respectively, were taken and placed in a water bath at 60 deg.C, 70 deg.C, 80 deg.C, and the activity was measured every 24h, as determined by the modified agarose-fibrin lysis loop method described in Xiao C, Huang Z, Lin F, et al. The number of days required for the activity of each sample to decrease by 30% was determined, and the results are shown in Table 5.
Table 5.
| Days (sky) | 60℃ | 70℃ | 80℃ |
| Example 1 | 36 | 20 | 8 |
| Example 2 | 32 | 16 | 6 |
| Example 3 | 30 | 18 | 7 |
| Example 4 | 27 | 15 | 5 |
| Example 5 | 25 | 13 | 4 |
| Example 6 | 31 | 17 | 6 |
| Example 7 | 33 | 17 | 7 |
| Comparative example 1 | 8 | 5 | 1 |
| Comparative example 2 | 22 | 10 | 3 |
| Comparative example 3 | 20 | 8 | 2 |
| Comparative example 4 | 18 | 7 | 2 |
| Comparative example 5 | 21 | 9 | 3 |
As can be seen, the freeze-dried preparation of the embodiment of the invention has good stability, and the maximum time of the activity reduction of each sample reaches 30 percent can reach 25 to 36 days.
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents, improvements and the like that fall within the spirit and principle of the present invention are intended to be included therein.
Claims (8)
1. A preparation method of a urokinase freeze-dried powder is characterized in that a preparation formula of the urokinase freeze-dried powder comprises urokinase, human serum albumin, sodium chloride, mannitol, konjac glucomannan and water for injection, the mass ratio of the mannitol to the konjac glucomannan is 15-25:3-6, in the preparation formula, the urokinase is 8-12 ten thousand units/mL, the human serum albumin is 0.02-0.06mg/mL, the sodium chloride is 10-15mg/mL, the mannitol is 0.15-0.25g/mL, and the konjac glucomannan is 0.03-0.06g/mL, and the preparation method comprises the following steps:
(1) preparing an auxiliary material solution: adding water for injection into a mixing tank, adding sodium chloride, mannitol and konjac glucomannan under stirring, and continuously stirring to obtain an auxiliary material solution;
(2) activated carbon adsorption and decarburization: adding activated carbon moistened with water for injection into the auxiliary material solution, stirring, decarburizing, and sealing and cooling to obtain a feed liquid 1;
(3) preparing a main medicine: adding human serum albumin into the feed liquid 1 under stirring, stirring until the human serum albumin is completely dissolved to obtain a feed liquid 2, then adding urokinase into the feed liquid 2, stirring until the urokinase is completely dissolved, supplementing water for injection, continuously stirring, and filtering with a saturated filter membrane to obtain a main drug solution, wherein the saturated filter membrane is a filter membrane wetted by the feed liquid 2;
(4) freeze-drying: filling the main medicine solution obtained in the step (3) with half plugs, and freeze-drying, wherein the freeze-drying process comprises the following steps:
keeping the temperature at minus 35 ℃ to minus 25 ℃ for 2 to 3 hours;
keeping the temperature at minus 45 ℃ to minus 40 ℃ for 0.5 to 1.5 hours;
vacuumizing to below 10Pa, and keeping the temperature of minus 20 to minus 10 ℃ for 8 to 12 hours;
keeping the temperature of minus 5 ℃ to 0 ℃ for 8 to 12 hours;
keeping the temperature at 3-7 ℃ for 4-6 h;
keeping the temperature at 8-12 ℃ for 0.5-1.5 h;
(5) and (5) plugging and capping.
2. The method according to claim 1, wherein the mass ratio of mannitol to konjac glucomannan is 9-11: 2.
3. The method according to claim 2, wherein the mass ratio of mannitol to konjac glucomannan is 5: 1.
4. The method according to claim 1, wherein the stirring is continued for 8 to 12min in the step (1).
5. The method according to claim 1, wherein the stirring in the step (2) is carried out at a temperature of 80 to 100 ℃ for a period of 15 to 25 min.
6. The process according to claim 1, wherein the step (2) of decarbonizing is carried out by using a sterilized plate filter equipped with a 0.22 μm mixed cellulose ester microporous membrane.
7. The method of claim 1, wherein the stirring in step (3) is continued for 15-25 min.
8. A preparation method of urokinase freeze-dried powder is characterized in that a preparation formula of the urokinase freeze-dried powder comprises urokinase, human serum albumin, sodium chloride, mannitol, konjac glucomannan and water for injection, the mass ratio of the mannitol to the konjac glucomannan is 15-25:3-6, in the preparation formula, the urokinase is 8-12 ten thousand units/mL, the human serum albumin is 0.02-0.06mg/mL, the sodium chloride is 10-15mg/mL, the mannitol is 0.15-0.25g/mL, and the konjac glucomannan is 0.03-0.06g/mL, and the preparation method comprises the following steps:
(1) preparing an auxiliary material solution: adding water for injection into a mixing tank, adding sodium chloride, mannitol and konjac glucomannan under stirring, and continuously stirring to obtain an auxiliary material solution;
(2) activated carbon adsorption and decarburization: adding activated carbon moistened with water for injection into the auxiliary material solution, stirring, decarburizing, and sealing and cooling to obtain a feed liquid 1;
(3) preparing a main medicine: adding human serum albumin into the feed liquid 1 under stirring, stirring until the human serum albumin is completely dissolved to obtain a feed liquid 2, then adding urokinase into the feed liquid 2, stirring until the urokinase is completely dissolved, supplementing water for injection, continuously stirring, and filtering with a saturated filter membrane to obtain a main drug solution, wherein the saturated filter membrane is a filter membrane wetted by the feed liquid 2;
(4) freeze-drying: filling the main medicine solution obtained in the step (3) with half plugs, and freeze-drying, wherein the freeze-drying process comprises the following steps:
holding at-20 deg.C for 2.5 h;
holding at-42 deg.C for 1 h;
vacuumizing to below 10Pa, and keeping at-15 ℃ for 10 h;
holding at-2 ℃ for 10 h;
keeping the temperature at 5 ℃ for 5 h;
keeping the temperature at 10 ℃ for 1 h;
(5) and (5) plugging and capping.
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| CN104648834A (en) * | 2013-11-21 | 2015-05-27 | 李和伟 | Protecting device of freeze-drying excipient containing skeleton support agent and binder and preparation method thereof |
| CN104906054A (en) * | 2015-06-15 | 2015-09-16 | 武汉人福药业有限责任公司 | Preparation method of lyophilized urokinase preparation |
| CN105078906A (en) * | 2015-08-11 | 2015-11-25 | 蚌埠丰原涂山制药有限公司 | Urokinase-containing pharmaceutical lyophilized preparation and preparation method thereof |
| CN107224407A (en) * | 2016-03-25 | 2017-10-03 | 李和伟 | A kind of lyophilized formulations containing biology enzyme and its shield tooth product mix |
| CN107049966A (en) * | 2016-12-11 | 2017-08-18 | 青岛冠龙生物制药有限公司 | A kind of freeze-dried preparation method of urokinase |
| CN109959227A (en) * | 2017-12-14 | 2019-07-02 | 青岛华迈士药业有限公司 | A kind of method of urokinase injection lyophilized preparation freeze-drying curve optimization |
| CN110151705A (en) * | 2018-02-13 | 2019-08-23 | 天士力生物医药股份有限公司 | The freeze drying process of injection recombinant human urokinase zymogen |
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