Thrombin powder capable of being stored at normal temperature and preparation method thereof

Abstract

The invention provides thrombin powder capable of being stored at normal temperature and a preparation method of the thrombin powder. The preparation method comprises the following steps: weighing hydroxyl-beta cyclodextrin, then taking a thrombin solution subjected to cryopreservation, and unfreezing in a water bath for later use; the preparation method comprises the following steps: weighing a proper amount of water for injection, mixing the water for injection with hydroxyl-beta cyclodextrin, stirring to a dissolved state, weighing a thawed thrombin solution, adding the thawed thrombin solution into the dissolved hydroxyl-beta cyclodextrin solution, stirring for 60 minutes by using a stirring motor to enable the hydroxyl-beta cyclodextrin to fully wrap thrombin protein, then supplementing the water for injection to a full dose, and stirring and mixing uniformly; the method comprises the following steps: firstly, carrying out sterilization filtration on a prepared solution by using a polyethersulfone cylindrical filter with the aperture of 0.45 mu m, then filtering and sterilizing by using a sterilized polyethersulfone cylindrical filter with the aperture of 0.22 mu m under the local A condition of a C-grade environment, and sampling and checking visible foreign matters after sterilization is completed. The long-term stability of the thrombin solution at normal temperature and the stability in the transportation process can be effectively ensured by adopting a technology of coating thrombin protein molecules with hydroxyl-beta cyclodextrin.

Description

Technical Field

[0001] The invention relates to the technical field of pharmaceutical preparations, in particular to a thrombin powder capable of being stored at room temperature and a preparation method thereof. Background Art

[0002] Thrombin is a serine protease that plays a key role in the blood coagulation process. The inactive prothrombin produced by the liver is activated into active thrombin through a series of enzymatic reactions in the blood. It can convert fibrinogen into fibrin monomers, thereby promoting the formation of blood clots to prevent bleeding and promote wound healing. In addition, thrombin is also involved in regulating inflammatory responses and other physiological processes. In medicine, the function and activity of thrombin is also one of the important indicators for evaluating the health of blood.

[0003] All lyophilized thrombin powders on the market need to be transported and stored in a refrigerated environment. They are greatly affected by temperature, have poor stability, and are prone to potency decline, which affects product quality and results in poor hemostatic effects. Summary of the invention

[0004] 1. Technical issues to be resolved

[0005] In view of the shortcomings of the prior art, the present invention provides a thrombin powder that can be stored at room temperature and a preparation method thereof, so as to solve the problems raised in the above background technology.

[0006] (II) Technical solution

[0007] To achieve the above objectives, the present invention is implemented through the following technical scheme: a thrombin powder that can be stored at room temperature, comprising: thrombin protein and hydroxy-β-cyclodextrin, wherein the hydroxy-β-cyclodextrin has a three-dimensional ring structure of "hydrophobic inside and hydrophilic outside", and a cavity is formed inside the three-dimensional ring structure, and the cavity is used to enclose the thrombin protein.

[0008] As a preferred embodiment of this embodiment, the ratio of the thrombin protein to hydroxy-β-cyclodextrin is 200-10000 units: 15-80 mg.

[0009] As a preferred embodiment of this embodiment, the ratio of the thrombin protein to hydroxy-β-cyclodextrin is 2000 units: 40 mg.

[0010] As a preferred embodiment of this invention, a method for preparing thrombin powder that can be stored at room temperature comprises the following steps:

[0011] Step 1, weighing hydroxy-β-cyclodextrin, and then taking the frozen thrombin solution, thawing it in a water bath for later use;

[0012] Step 2, measuring an appropriate amount of water for injection and mixing it with hydroxy-β-cyclodextrin, and stirring until dissolved, measuring the thrombin solution that has been thawed and adding it to the dissolved hydroxy-β-cyclodextrin solution, stirring with a stirring motor for 60 minutes to allow the hydroxy-β-cyclodextrin to fully wrap the thrombin protein, and then adding water for injection to the full amount and stirring to mix;

[0013] Step 3, first use a polyethersulfone cartridge filter with a pore size of 0.45 μm to sterilize the prepared solution, and then use a sterilized 0.22 μm polyethersulfone cartridge filter to filter and sterilize under the local A conditions of the Class C environment. After the sterilization is completed, take a sample to check for visible foreign matter. After the inspection is qualified, it is packaged under the local A conditions of the Class B environment;

[0014] Step 4, freeze-drying the packaged thrombin solution using a freeze dryer;

[0015] Step 5: The dried product is automatically plugged in the freeze dryer and transferred to the capping track for capping after being unpacked. Finally, the product is formed.

[0016] As a preferred embodiment of this embodiment, the water bath temperature in step 1 is ≤30° C., and the thawing time of the frozen thrombin solution is ≤2 h.

[0017] As a preferred embodiment of this embodiment, after adding water for injection to the full amount in step 2, the ratio of water for injection, thrombin protein and hydroxy-β-cyclodextrin is 2.5g: 200~10000 units: 15~80mg, and the temperature of the water for injection is 15~25°C.

[0018] As a preferred embodiment of this embodiment, after adding the injection water to the full amount in step 2, the ratio of injection water, thrombin protein and hydroxy-β-cyclodextrin is 2.5g:2000 units:40mg, and the temperature of the injection water is 15~25°C.

[0019] As a preference of this embodiment, in step 2, the stirring frequency of the stirring motor is set at 30 Hz, and the stirring time is 5 minutes.

[0020] As a preferred embodiment of this invention, the freeze-drying process of the thrombin solution in step 4 includes:

[0021] Place the packaged thrombin solution into a freeze dryer, pre-freeze the freeze dryer to -40°C, and maintain it at this temperature for 1 to 2 hours;

[0022] Gradually increase the temperature to 2°C and maintain it at this temperature for 9 to 10 hours;

[0023] The temperature was further raised to 20°C and maintained at this temperature for 6 h;

[0024] The temperature was raised again to 25°C and maintained at this temperature for 2 h;

[0025] Finally the temperature was raised to 35°C and maintained at this temperature for 6 h.

[0026] (III) Beneficial effects

[0027] The present invention provides a thrombin powder that can be stored at room temperature and a preparation method thereof, which has the following beneficial effects:

[0028] The present invention can effectively ensure the long-term stability of the thrombin solution at room temperature and the stability during transportation by using the technology of encapsulating the thrombin protein molecule with hydroxy-β-cyclodextrin, thereby solving the instability and transportation problems that may be encountered in traditional thrombin preparations. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 The present invention is a flow chart of a method for preparing thrombin powder that can be stored at room temperature;

[0030] Figure 2 This is a schematic diagram of the thrombin nanoparticles of the present invention observed under a transmission electron microscope. DETAILED DESCRIPTION

[0031] The embodiments of the present invention are described in detail below, and examples of the embodiments are shown in the accompanying drawings, wherein the same or similar reference numerals throughout represent the same or similar elements or elements having the same or similar functions. The embodiments described below with reference to the accompanying drawings are exemplary and are only used to explain the present invention, and cannot be understood as limiting the present invention.

[0032] In the present invention, unless otherwise clearly specified and limited, a first feature being "above" or "below" a second feature may include that the first and second features are in direct contact, or may include that the first and second features are not in direct contact but are in contact through another feature between them. Moreover, a first feature being "above", "above" and "above" a second feature includes that the first feature is directly above and obliquely above the second feature, or simply indicates that the first feature is higher in level than the second feature. A first feature being "below", "below" and "below" a second feature includes that the first feature is directly below and obliquely below the second feature, or simply indicates that the first feature is lower in level than the second feature.

[0033] The disclosure below provides many different embodiments or examples to implement different structures of the present invention. In order to simplify the disclosure of the present invention, the parts and settings of specific examples are described below. Of course, they are only examples, and the purpose is not to limit the present invention. In addition, the present invention can repeat reference numbers and / or reference letters in different examples, and this repetition is for the purpose of simplicity and clarity, which itself does not indicate the relationship between the various embodiments and / or settings discussed. In addition, the present invention provides various specific examples of processes and materials, but those of ordinary skill in the art can appreciate the application of other processes and / or the use of other materials.

[0034] Please refer to Figure 1-2 This embodiment provides a thrombin powder that can be stored at room temperature and a preparation method thereof.

[0035] Step 1, first weigh hydroxy-β-cyclodextrin, then measure the prescribed amount of frozen thrombin solution, and then thaw the thrombin solution in a water bath. Since thrombin is an enzyme drug, the enzyme is sensitive to temperature. High temperature may cause its structure to change, thereby affecting its biological activity. Even a relatively mild temperature may have an impact on it. Therefore, the water bath temperature needs to be ≤30°C. Since long-term thawing may cause the substances inside to gradually degrade, the thrombin solution needs to be thawed within 2 hours to ensure the best effect of the thrombin solution.

[0036] Step 2, measure an appropriate amount of water for injection, add the water for injection to hydroxy-β-cyclodextrin, stir until the solution is in the state, and then measure the thawed prescription amount of thrombin solution and add it to the dissolved hydroxy-β-cyclodextrin solution. It is explained here that since hydroxy-β-cyclodextrin has a special three-dimensional ring structure of "hydrophobic inside and hydrophilic outside", this structure enables a hydrophobic cavity to be formed inside, so that the thrombin protein molecules can be included, isolating the thrombin protein molecules from the external environment (such as light, heat, oxygen, etc.), thereby preventing the thrombin protein molecules from being attacked by external aggressive molecules and undergoing hydrolysis, enzymolysis, oxidation, polymerization and isomerization reactions, thereby improving the stability of thrombin at room temperature, extending its shelf life and simplifying logistics and storage requirements;

[0037] The mixed hydroxy-β-cyclodextrin solution and thrombin solution are stirred by using a stirring motor. However, too fast a stirring speed of the stirring motor often causes liquid splashing and a large number of bubbles. The presence of bubbles interferes with the full contact between the liquids, resulting in uneven local mixing, thereby affecting the uniformity and quality consistency of the final product. If the bubbles are directly broken, the shear force generated during the rupture may cause changes in the protein structure, affecting its biological activity and stability. If the stirring speed is too slow, the reaction time will be prolonged. For this reason, the stirring frequency of the stirring motor is set to 30 Hz. In order to ensure that all the ingredients can be fully combined to form a stable mixture, the stirring time of the stirring motor is set to 60 minutes, and then water for injection is added to the full amount, and then the stirring motor is set to 30 Hz for stirring for 5 minutes to mix evenly.

[0038] It should be noted that the temperature of water for injection needs to be controlled between 15 and 25°C. Since thrombin solution is sensitive to temperature, it is easy to be inactivated at high temperatures before it is fully wrapped by hydroxy-β-cyclodextrin. If production is carried out at a lower temperature, in order to achieve and maintain low temperature conditions, high-efficiency refrigeration equipment and insulation facilities are required, which will incur higher production costs. Therefore, using water for injection between 15 and 25°C can better dissolve hydroxy-β-cyclodextrin and thrombin solution, which helps to maintain the stability and solubility of the drug ingredients.

[0039] The preparation amount of auxiliary materials in step 1 and step 2 is as follows

[0040] Specifically, each bottle of thrombin powder requires 2000 units of thrombin solution, 40 mg of hydroxy-β-cyclodextrin, and 2.5 ml of water for injection. Note that when dissolving hydroxy-β-cyclodextrin, only 2.5 ml of water for injection needs to be added. After the thrombin solution and hydroxy-β-cyclodextrin solution are evenly stirred, the remaining amount can be added.

[0041] Step 3: The prepared thrombin solution first needs to be pre-filtered through a 0.45μm pore size polyethersulfone (PES) cartridge filter. The main purpose of this step is to remove larger particulate matter and some microorganisms in the solution, laying the foundation for subsequent sterile filtration. The 0.45μm filter can effectively capture and remove large particles that may affect the clarity of the solution, while reducing the risk of filter clogging and ensuring a smooth filtration process.

[0042] After pre-filtration, the solution needs to be transferred to a Class C environment under local Class A conditions for further sterile filtration to minimize the deposition of microorganisms and particulate matter. Under Class C environment and local Class A conditions, a sterilized 0.22μm pore size polyethersulfone (PES) cartridge filter is used for final sterilization filtration. The 0.22μm pore size can effectively block most bacteria and fungal spores, ensuring the purity of the solution under sterile conditions. Through this process, small particles and microorganisms that may remain can be further removed, thereby achieving a highly sterile state, which not only ensures the clarity and sterility of the thrombin solution, but also meets the quality control standards of the pharmaceutical industry, ensuring the safety and effectiveness of the final product.

[0043] After completing the sterilization filtration of the 0.22 μm pore size polyethersulfone cartridge filter, the thrombin solution needs to be sampled and checked for visible foreign matter. Sampling should also be performed under sterile conditions to ensure that no new contaminants are introduced during the sampling process to ensure the purity of the sample.

[0044] After the sampling is completed, a certain amount of thrombin solution sample is extracted according to the standard operating procedures, and then the thrombin solution sample is placed in a clean glass container for observation. Before observation, the container should be checked with the naked eye for cracks or other defects. A clarity tester should be used. The tested thrombin solution sample must not contain metal chips, glass chips, fibers longer than 2mm, lumps with a maximum particle size of more than 2mm, and smoke-like particle deposits visible to the naked eye when gently rotated after standing for a certain period of time, uncountable particle groups or precipitates that cannot be shaken off, and protein flocs that are difficult to count, and other obviously visible foreign matter, as well as fine visible foreign matter such as dots, short fibers less than 2mm and lumps. If foreign matter is detected, it means that the packaging is unsuccessful. If the inspection is qualified, these qualified solutions will be moved to the local A conditions of the Class B environment for subpackaging.

[0045] Step 4, adding heat transfer oil to the freeze dryer, using the freeze dryer to freeze dry according to a set temperature freeze drying curve to form freeze dried powder, the freeze drying step in the freeze dryer includes:

[0046] Pre-freezing stage: pre-freeze the freeze dryer to -40°C, put the packaged thrombin solution into the freeze dryer for freeze drying, and maintain this temperature for about 1 to 2 hours to fully freeze the solution and form a uniform and stable ice crystal structure;

[0047] Sublimation drying stage: The temperature is gradually raised to about 2°C and maintained at this temperature for about 9 to 10 hours, so that the thrombin solution with an ice crystal structure is directly transformed from a solid state to a gaseous state, i.e., sublimation, thereby effectively removing most of the water in the thrombin solution;

[0048] Secondary drying stage: the temperature is further raised to about 20°C and maintained at this temperature for about 6 hours to further remove trace moisture that may be left over during the sublimation process;

[0049] Final drying stage: Raise the temperature again to about 25°C and maintain it at that temperature for about 2 hours to ensure that as much water as possible is removed;

[0050] Final drying confirmation: Finally, the temperature is raised to about 35°C and maintained at this temperature for about 6 hours. This step is to ensure that the thrombin product has reached the required final dryness, reduce the residual moisture content as much as possible, and ensure product stability and quality;

[0051] The thrombin solution is freeze-dried to maintain its biological activity and stability, and finally a dry powder form is obtained, which is convenient for storage and transportation.

[0052] It should be noted that during the entire freeze-drying process, an appropriate vacuum degree needs to be maintained to ensure that the water can sublime effectively, and the gradual increase in temperature needs to be precisely controlled to avoid denaturation of the drug components due to excessively high temperatures.

[0053] Step 5: The dried product is automatically plugged in the freeze dryer and transferred to the capping track for capping after being unpacked. Finally, the product is formed.

[0054] Step 6: Conduct accelerated tests and long-term stability tests on the product.

[0055] According to the stability test requirements of the Chinese Pharmacopoeia 2020 edition, accelerated tests and long-term stability studies were conducted on thrombin powder to test whether it can show good stability under different conditions.

[0056] The results of the acceleration experiment are as follows:

[0057] Accelerated test conditions (test temperature: 40±2℃, humidity: 75±5%), the specific results are shown in the following table:

[0058] Properties: During the entire experimental period, the sample appearance remained stable and no changes that did not meet the requirements were observed.

[0059] Moisture: The moisture content increased slightly from 3.4% initially to 3.6% at 6 months, always below the specified maximum of 5.0%.

[0060] Visible Foreign Matter: No foreign matter was detected.

[0061] Titer: The initial titer was 100%, remained constant during the first two months, and dropped to 98% between the third and sixth months.

[0062] Milligram potency: decreased from 40 U / mg initially to 39 U / mg at month 6.

[0063] Sterility test: The samples at all time points met the sterility requirements.

[0064] Properties: During the entire experimental period, the sample appearance remained stable and no changes that did not meet the requirements were observed.

[0065] Moisture content: increased from 3.0% at the beginning to 3.2% after 6 months, still meeting the specified requirements.

[0066] Visible foreign matter: No foreign matter was found.

[0067] Titer: decreased from 102% initially to 98% at month 6.

[0068] Milligram potency: decreased from 41 U / mg to 39 U / mg.

[0069] Sterility test: The samples at all time points met the sterility requirements.

[0070] Properties: During the entire experimental period, the sample appearance remained stable and no changes that did not meet the requirements were observed.

[0071] Moisture: Increased from 3.0% to 3.2%, remaining below the regulatory limit.

[0072] Visible foreign matter: No foreign matter was found.

[0073] Titer: Gradually decreased from the initial 103% to 98% at the 6th month.

[0074] Milligram potency: decreased from 41 U / mg to 39 U / mg.

[0075] Sterility test: The samples at all time points met the sterility requirements.

[0076] Accelerated test conclusion: All three batches of thrombin solution showed good stability in the accelerated stability test. All indicators such as properties, moisture content, visible foreign matter, potency and milligram potency met the standard requirements, and the sterility was maintained. As time went by, the potency and milligram potency showed a slight downward trend, but these changes were still within an acceptable range, and the product still met the quality standards overall. This shows that despite a certain decrease in activity under extreme conditions, the product can maintain its performance and quality as a whole.

[0077] The long-term stability test results are as follows:

[0078] Long-term stability test conditions (test temperature: 30±2℃, humidity: 60±10%), the specific results are shown in the following table:

[0079] Properties: During the entire experimental period, the sample appearance remained stable and no changes that did not meet the requirements were observed.

[0080] Moisture: changed from 3.4% at the beginning to 3.4% at 36 months, always below the specified 5.0%.

[0081] Visible Foreign Matter: No visible foreign matter was detected.

[0082] Titer: changed from 100% initially to 98% at 36 months and remained in the range of 80% to 150%.

[0083] Milligram titer: changed from 40 U / mg initially to 39 U / mg at 36 months, always above the prescribed 10 U / mg.

[0084] Sterility: Samples at all time points met the sterility requirements.

[0085] Properties: During the entire experimental period, the sample appearance remained stable and no changes that did not meet the requirements were observed.

[0086] Moisture: changed from 3.0% at the beginning to 3.3% at 36 months, always below the specified 5.0%.

[0087] Visible Foreign Matter: No visible foreign matter was detected.

[0088] Titer: changed from 102% initially to 98% at 36 months and remained in the range of 80% to 150%.

[0089] Milligram titer: changed from 41 U / mg initially to 40 U / mg at 36 months, always above the prescribed 10 U / mg.

[0090] Sterility: Maintain compliance at all times.

[0091] Properties: During the entire experimental period, the sample appearance remained stable and no changes that did not meet the requirements were observed.

[0092] Moisture: changed from 3.0% at the beginning to 3.5% at 36 months, always below the specified 5.0%.

[0093] Visible Foreign Matter: No visible foreign matter was detected.

[0094] Titer: changed from 103% initially to 100% at 36 months and remained in the range of 80% to 150%.

[0095] Milligram titer: changed from 41 U / mg initially to 39 U / mg at 36 months, always above the prescribed 10 U / mg.

[0096] Sterility: Maintain compliance at all times.

[0097] Conclusion on long-term stability: The three batches of thrombin solutions showed good stability within 36 months. All batches of samples met the standard requirements in terms of properties, moisture content, visible foreign matter, potency and milligram potency, and the sterility was also maintained continuously. With the passage of time, the potency and milligram potency showed a slight downward trend, but overall these changes did not affect the overall quality and effectiveness of the product, and the product still met the quality standards. Therefore, it can be considered that these batches of thrombin solution have a longer shelf life under the recommended storage conditions.

[0098] The results of the accelerated experiment and the long-term stability experiment show that under the accelerated test conditions (40±2℃, humidity 75±5%), the various indicators of thrombin powder remain stable within 6 months, and under the long-term stability experiment conditions (30±2℃, humidity 60±10%), the various indicators of thrombin powder remain stable within 36 months. This shows that the technology of using hydroxy-β-cyclodextrin including thrombin protein molecules can effectively ensure the long-term stability of thrombin powder at room temperature and the stability during transportation, solving the instability and transportation problems that may be encountered by traditional thrombin preparations.

[0099] Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions and variations may be made to the embodiments without departing from the principles and spirit of the present invention, and that the scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A thrombin powder that can be stored at room temperature, characterized in that: include: Thrombin protein and hydroxy-β-cyclodextrin, wherein the hydroxy-β-cyclodextrin has a three-dimensional ring structure of "hydrophobic inside and hydrophilic outside", and a cavity is formed inside the three-dimensional ring structure, and the cavity is used to include the thrombin protein.

2. The thrombin powder that can be stored at room temperature according to claim 1, characterized in that: The ratio of the thrombin protein to hydroxy-β-cyclodextrin is 200-10000 units: 15-80 mg.

3. A method for preparing thrombin powder that can be stored at room temperature, characterized in that: The following steps are involved: Step 1, weighing hydroxy-β-cyclodextrin, and then taking the frozen thrombin solution, thawing it in a water bath for later use; Step 2, measuring an appropriate amount of water for injection and mixing it with hydroxy-β-cyclodextrin, and stirring until dissolved, measuring the thrombin solution that has been thawed and adding it to the dissolved hydroxy-β-cyclodextrin solution, stirring with a stirring motor for 60 minutes to allow the hydroxy-β-cyclodextrin to fully wrap the thrombin protein, and then adding water for injection to the full amount and stirring to mix; Step 3, first use a polyethersulfone cartridge filter with a pore size of 0.45 μm to sterilize the prepared solution, and then use a sterilized 0.22 μm polyethersulfone cartridge filter to filter and sterilize under the local A conditions of the Class C environment. After the sterilization is completed, take a sample to check for visible foreign matter. After the inspection is qualified, it is packaged under the local A conditions of the Class B environment; Step 4, freeze-drying the packaged thrombin solution using a freeze dryer; Step 5: The dried product is automatically plugged in the freeze dryer and transferred to the capping track for capping after being unpacked. Finally, the product is formed.

4. The method for preparing a thrombin powder that can be stored at room temperature according to claim 3, characterized in that: The water bath temperature in step 1 is ≤30° C., and the thawing time of the frozen thrombin solution is ≤2 h.

5. The method for preparing a thrombin powder that can be stored at room temperature according to claim 3, characterized in that: After adding the injection water to the full amount in step 2, the ratio of injection water, thrombin protein and hydroxy-β-cyclodextrin is 2.5g: 200~10000 units: 15~80mg, and the temperature of the injection water is 15~25°C.

6. The method for preparing a thrombin powder that can be stored at room temperature according to claim 3, characterized in that: In step 2, the stirring frequency of the stirring motor is set at 30 Hz, and the stirring time is 5 min.

7. The method for preparing a thrombin powder that can be stored at room temperature according to claim 3, characterized in that: The process of freeze-drying the thrombin solution in step 4 comprises: Place the packaged thrombin solution into a freeze dryer, pre-freeze the freeze dryer to -40°C, and maintain it at this temperature for 1 to 2 hours; Gradually increase the temperature to 2°C and maintain it at this temperature for 9 to 10 hours; The temperature was further raised to 20°C and maintained at this temperature for 6 h; The temperature was raised again to 25°C and maintained at this temperature for 2 h; Finally the temperature was raised to 35°C and maintained at this temperature for 6 h.

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