CN111375052A - Freeze-drying method of freeze-dried preparation of azadirachtin for injection - Google Patents
Freeze-drying method of freeze-dried preparation of azadirachtin for injection Download PDFInfo
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Abstract
The invention belongs to the technical field of freeze-dried preparations, and particularly relates to a freeze-drying method of an azadirachtin freeze-dried preparation for injection. The freeze-drying method of the freeze-dried preparation of the azadirachtin for injection is characterized by comprising the following steps: (1) pre-freezing: the temperature of the heat conducting oil reaches 0 to-10 ℃ in 20 to 60 minutes at the first stage, and the heat conducting oil is kept for 1 to 2 hours; the temperature of the heat conducting oil in the second stage reaches-30 to-45 ℃ within 30-60 minutes, and is kept for 2-4 hours; (2) carrying out primary sublimation; (3) resolving and drying; (4) and finishing the freeze-drying process after the pressure rise test is qualified. The freeze-drying method greatly improves the properties of the freeze-dried product, ensures the stability of the amitraz, and ensures that the quality standard of the amitraz meets all indexes specified by the national drug standard; meanwhile, the obtained freeze-dried product has the advantages of beautiful appearance, high perfectness rate, stable properties and biological activity and the like.
Description
Technical Field
The invention belongs to the technical field of freeze-dried preparations, and particularly relates to a freeze-drying method of an azadirachtin freeze-dried preparation for injection.
Background
The peptide (HM-3) is chemically synthesized eighteen peptides which are synthesized polypeptides of integrin blockers, and researches show that the peptide can effectively inhibit the migration of endothelial cells, but has no influence on the proliferation of tumor cells, Matrigel experiments show that the peptide has a remarkable inhibiting effect on the generation of new blood vessels, and the analysis of the influence of the polypeptide on in-vivo transplanted tumors also shows that the peptide can effectively inhibit the capillary vessels of tumor tissues, so that the polypeptide has no cytotoxicity, and has an antitumor effect by inhibiting the migration and angiogenesis of endothelial cells, an integrin ligand sequence is added in the polypeptide sequence design, some tumors can highly express certain integrin subtypes in the processes of occurrence and development, such as α v β, and the combination of the amphetamine with the peptide inhibits the expression and downstream molecules thereof, so that the peptide can inhibit the occurrence and development of tumors, further, gene chip analysis shows that the amphetamine SL1, KiSS-1, Syk, R2, and VEGF receptor binding with the inhibitory effect of transcriptional signal of inhibiting VEGF receptor kinase 11-6, and VEGF receptor binding with the transcriptional inhibition of intracellular expression of VEGF receptor kinase 11, so that the intracellular transcriptional signal of the intracellular transcriptional kinase-6, the intracellular transcriptional protein expression of the peptide can be detected by the intracellular transcriptional protein kinase-11, the transcriptional protein expression of the inhibitory effect of the inhibitory protein kinase-6, the inhibitory effect of the inhibitory protein of the transcriptional protein kinase-6, the transcriptional protein.
The vacuum freeze drying technology is a technology for freezing aqueous solution at low temperature and then directly sublimating and drying the water in the aqueous solution without passing through a liquid state under a vacuum state, and the dried substance has the advantages of basically unchanged physical and chemical properties, basically unchanged shape, small loss of effective components, good rehydration and long sealing and storage period. The vacuum freeze drying technology provides a stable preservation method for the polypeptide drug with poor stability.
CN102178656A discloses an HM-3 polypeptide freeze-dried powder preparation, which comprises 5-100 parts by weight of HM-3 polypeptide, 20-90 parts by weight of auxiliary materials and 1-10 parts by weight of pH regulators, wherein the pH of the HM-3 polypeptide freeze-dried powder preparation is 5-7; the auxiliary materials are dextran and mannitol; the pH regulator is sodium hydroxide, sodium carbonate, potassium hydroxide, calcium hydroxide, disodium hydrogen phosphate or sodium acetate. The freeze-drying process comprises the following steps:
pre-freezing: setting the temperature of a plate layer to be-60 ℃ to-20 ℃, reducing the temperature of a sample to be-30 ℃ to-10 ℃ in one hour, and preserving the heat for 2-5 hours;
primary drying: setting the temperature of a plate layer to be-20 to-5 ℃ under the vacuum degree of 0 to 30Pa, and preserving heat for 10 to 20 hours;
secondary drying: setting the temperature of a plate layer to be 10-30 ℃, preserving the heat for more than 5 hours, and freeze-drying until the end point judgment of freeze-drying is met; this is HM-3 polypeptide lyophilized powder preparation.
Although the freeze-drying method improves the stability of the freeze-drying preparation to a certain degree, the obtained product has low appearance goodness, has high probability of cracks, atrophy, unevenness and the like, increases the loss rate and improves the production cost.
Disclosure of Invention
In order to overcome the technical problems, the invention provides a freeze-drying method of an amphetamine freeze-dried preparation for injection. The freeze-drying method greatly improves the properties of the freeze-dried product, ensures the stability of the amitraz, and ensures that the quality standard of the amitraz meets all indexes specified by the national drug standard; meanwhile, the obtained freeze-dried product has the advantages of beautiful appearance, high perfectness rate, stable properties and biological activity and the like.
The freeze-drying method of the freeze-dried preparation of the azadirachtin for injection comprises the following steps:
(1) prefreezing
The temperature of the heat conducting oil reaches 0 to-10 ℃ in 20 to 60 minutes at the first stage, and the heat conducting oil is kept for 1 to 2 hours;
the temperature of the heat conducting oil in the second stage reaches-30 to-45 ℃ within 30-60 minutes, and is kept for 2-4 hours;
(2) carrying out primary sublimation;
(3) resolving and drying;
(4) and finishing the freeze-drying process after the pressure rise test is qualified.
The freeze-drying technology is generally divided into three steps of pre-freezing, primary sublimation and analytic drying. Prefreezing is the first step in the lyophilization process and is also a very important step. Studies have shown that the rate of prefreezing affects the ice crystal size of the product, and thus affects the later sublimation rate, resolubility, appearance, bioactivity, etc. of the product, and thus control of prefreezing is critical. During the prefreezing process, the number and size of ice crystals are affected by two factors, namely the nucleation rate and the crystal growth rate, which are both affected by temperature. Generally, the larger the cooling rate is, the larger the supercooling degree and supersaturation degree of the solution are, the smaller the critical crystallization particle size is, the faster the nucleation speed is, and the more and smaller-sized fine ice crystals are easily formed. Therefore, after the ice crystals are sublimated, the size of pores formed in the material is small, the drying rate is low, and the rehydration performance after drying is good. And the ice crystals with large particles are easily formed by slow cooling, and a water vapor escape channel formed after the ice crystals are sublimated has larger size, which is beneficial to improving the drying rate, but the rehydration performance after freeze-drying is poor. The size of the ice crystals affects the drying rate and the dissolution rate and product quality of the dried product. Large ice crystals are beneficial to sublimation, and small ice crystals are not beneficial to sublimation; large ice crystals dissolve slowly, small ice crystals dissolve quickly; the smaller the ice crystal, the more the ice crystal can reflect the original structure of the product after drying. However, it is still not easy for those skilled in the art to solve the above problems in actual production because some theoretical knowledge is provided under the condition of uniform and ideal heat, and the environment is difficult to control and realize the ideal condition in actual production or experiment.
The invention optimizes the pre-freezing step, reduces the temperature by stages and reasonably controls the heat preservation time, thereby forming a uniform crystal structure with proper particle size in the frozen body, so that ice crystals at the upper part in the frozen body are sublimated and a uniform and fine porous structure is left, water vapor sublimated by the ice crystals at the lower part can smoothly escape, and the sublimation process of the whole frozen body is completed. The obtained lyophilized preparation has the advantages of beautiful appearance, high perfection rate, stable properties and biological activity, and the like.
Researches show that the phenomenon of uneven heating of the bottle jelly is obvious after the freeze-drying method of the bottle jelly is adopted. According to the research on the pre-freezing process of the bottled product plate layer, the initial temperature of the sample is higher, the temperature gradient of the upper part and the lower part of the feed liquid is higher, and the ice crystal growth speed is slower. If the cooling rate is slow, the ice crystals formed by the solution are coarse, the speed of the ice interface advancing from bottom to top is slow, the migration time of the solute in the solution is sufficient, and the accumulation of the solute in the frozen layer on the surface of the solution is relatively dense. Therefore, the upper surface layer tends to have more solute and higher density, while the lower bottom layer has smaller density and loose structure. This delamination phenomenon is most pronounced in poorly framed articles, either as bottom atrophy, or as mid-fault, or as top protrusions, or as top desquamation with a crust. But the cooling rate can not be too fast, and ice crystals formed too fast are fine, so that the sublimation resistance is increased, the drying time is prolonged, and the production cost is increased. Therefore, a suitable prefreezing rate is particularly important for a product.
According to some embodiments of the invention, the one time sublimation comprises: the temperature of the heat conducting oil reaches-25-0 ℃ within 2-10 hours, the heat conducting oil is kept for 8-24 hours, and the vacuum degree is 10-50 Pa. The purpose of the first sublimation is to allow free water in the lyophilisate to sublime out. The sublimation process must be heated, otherwise the lack of heat available to the product reduces the sublimation rate and prolongs the drying time, but if too much heat is applied, the temperature of the product exceeds the eutectic or disintegration point temperature and the product melts, resulting in failure of lyophilization. The plate layer of freeze-drying case is the total heat source of sublimation product, and the heat that the product of subliming obtained by radiation, conduction and three kinds of minutes formulas of convection all comes from the plate layer, consequently through the temperature of control plate layer alright control sublimation product. The temperature of the product must not exceed the eutectic temperature or the product will foam or melt as if it were not frozen. The temperature of the product must not exceed the temperature of the disintegration point, otherwise the product disintegrates, blocks the sublimation channel and is also melted. But the temperature of the lamina may well exceed the eutectic or disintegration point temperature. Because only the temperature difference exists between the temperature of the plate layer and the temperature of the product, the heat can be transferred to promote sublimation.
In addition to this, the convective heat transfer pattern is influenced by the vacuum, which depends on the pressure inside the lyophilization chamber, and experiments have shown that the convective heat transfer is negligibly small if the pressure is less than 0.1 mbar and increases significantly if the pressure is greater than 0.1 mbar. Thus, the higher the degree of vacuum of the product during sublimation, i.e. the lower the pressure, the better, but a suitable pressure is required, i.e. a pressure which is capable of generating convection without affecting the sublimation.
According to the principle, the invention optimizes one sublimation to two stages, including: in the first stage, the temperature of the heat conducting oil reaches-25 to-10 ℃ within 2 to 5 hours, the heat conducting oil is kept for 6 to 12 hours, and the vacuum degree is 10 to 50 Pa; and in the second stage, the temperature of the heat conduction oil reaches-5-0 ℃ within 2-5 hours, the heat conduction oil is kept for 1-120 minutes, and the vacuum degree is 10-50 Pa. The optimized sublimation for one time has the advantages of shortening the sublimation time, improving the sublimation efficiency and reducing the production cost.
According to some embodiments of the invention, the desorption drying comprises: the temperature of the heat conducting oil reaches 5-40 ℃ within 4-16 hours, the heat conducting oil is kept for 2-8 hours, and the vacuum degree is 0-50 Pa. The purpose of the desorption drying (i.e. the second sublimation) is to remove part of the bound water.
It has been found that although the foregoing rational control of prefreezing, one-time sublimation, has been effective in improving the properties of lyophilized formulations, there is room for improvement. Through tests, technicians further confirm the optimized conditions of secondary sublimation, particularly divide the analysis drying conditions into 2-3 stages, and control the vacuum degree in different stages, so that the crystallized water and the free water can be removed to the maximum degree, and the phenomena of cracks, atrophy, unevenness and the like caused by the over-high temperature rise operation speed can be further avoided.
The further optimized desorption drying step comprises the following steps: in the first stage, the temperature of the heat conducting oil reaches 20-40 ℃ within 3-6 hours, the temperature is maintained for 1-60 minutes, and the vacuum degree is 10-50 Pa; in the second stage, the temperature of the heat conducting oil reaches 20-40 ℃ within 1-60 minutes, the heat conducting oil is kept for 2-6 hours, and the vacuum degree is 0-5 Pa. The vacuum degree is controlled to be 10-50 Pa in the first stage, so that good heat convection can be guaranteed, and the principle is the same as that in the sublimation stage. In the second stage, because the temperature of the material is raised to the final desorption drying temperature, the desorption drying efficiency is improved through decompression, and the moisture is further removed.
In order to obtain a better effect, the analytic drying further comprises a temperature rise stage before the first stage, the temperature of the heat conduction oil in the temperature rise stage reaches 5-10 ℃ within 3-5 hours, the heat conduction oil is kept for 1-60 minutes, and the vacuum degree is 10-50 Pa.
According to some embodiments of the invention, in the pressure rise test, the pressure rise is set under the condition that the pressure does not exceed 3Pa within 5 minutes, and the pressure rise is judged to be qualified and then ended.
As one embodiment of the present invention, the lyophilization method of the lyophilized injectable azadirachtin preparation comprises:
(1) prefreezing
The temperature of the heat conducting oil reaches 0 to-10 ℃ in 20 to 60 minutes at the first stage, and the heat conducting oil is kept for 1 to 2 hours;
the temperature of the heat conducting oil in the second stage reaches-30 to-45 ℃ within 30-60 minutes, and is kept for 2-4 hours;
(2) one time sublimation
In the first stage, the temperature of the heat conducting oil reaches-25 to-10 ℃ within 2 to 5 hours, the heat conducting oil is kept for 6 to 12 hours, and the vacuum degree is 10 to 50 Pa;
in the second stage, the temperature of the heat conducting oil reaches-5-0 ℃ within 2-5 hours, the heat conducting oil is kept for 1-120 minutes, and the vacuum degree is 10-50 Pa;
(3) drying by desorption
In the temperature rise stage, the temperature of the heat conducting oil reaches 5-10 ℃ within 3-5 hours, the heat conducting oil is kept for 1-60 minutes, and the vacuum degree is 10-50 Pa;
in the first stage, the temperature of the heat conducting oil reaches 20-40 ℃ within 3-6 hours, the temperature is maintained for 1-60 minutes, and the vacuum degree is 10-50 Pa;
in the second stage, the temperature of the heat conducting oil reaches 20-40 ℃ within 1-60 minutes, the heat conducting oil is kept for 2-6 hours, and the vacuum degree is 0-5 Pa;
(4) and (3) finishing the freeze-drying process after the pressure rise test is qualified: the pressure rise is set under conditions such that the pressure does not exceed 3Pa within 5 minutes.
The invention also provides an amphetamine freeze-dried preparation for injection prepared by the method.
Further, the prescription of the freeze-dried preparation of the tiazamiin for injection is as follows: calculated according to the mass portion, 1 to 10 portions of antipeglutide, 1 to 30 portions of auxiliary material dextran and 0.1 to 5 portions of pH buffer system; the pH of the lyophilized preparation is 4-8.
Preferably, the pH buffer system is one or more of acetic acid-sodium acetate, citric acid-sodium citrate or phosphoric acid-phosphate.
Compared with the prior art such as patent CN102178656A, the preparation formula disclosed by the invention uses a pH buffer system, and compared with a common pH regulator, the preparation formula disclosed by the invention can play a role in stabilizing the pH of a product, and is more favorable for improving the stability of a medicine.
The beneficial effects obtained by the invention are as follows:
the invention selects reasonable pre-freezing speed and temperature, reasonable sublimation temperature and desorption drying temperature, reasonable heating rate and reasonable vacuum degree, and the obtained freeze-dried preparation of the tiargin for injection has the advantages of beautiful appearance of freeze-dried products, high perfectness rate, stable properties and biological activity, and the like. Compared with the freeze-dried amphetamine preparation for injection in the prior art, the freeze-dried amphetamine preparation for injection produced by the method has the advantages that the stability is 20% -30% higher, and the perfection rate is higher than 50%. In addition, the freeze-drying process is not only suitable for the preparation prescription used in the invention, but also can improve the product integrity of the prescription in the prior art.
Detailed Description
The following examples are intended to illustrate the invention but are not intended to limit the scope of the invention.
Example 1
The present example provides a lyophilization method for an injectable lyophilized amphetamine formulation, which is formulated as follows:
the preparation method of the freeze-dried preparation of the amitriptyline for injection comprises the following steps:
firstly, preparing a solution, namely adding an HM-3 polypeptide raw material into 400mL of water for injection to prepare a solution, and performing sterile filtration by using a 0.22 mu m membrane for later use to obtain a polypeptide solution;
weighing dextran and sodium acetate as adjuvants, adding into 400mL water for injection to obtain solution, adding needle activated carbon, heating, filtering, removing carbon, filtering with 0.22 μm membrane to obtain pyrogen-free sterile adjuvant water solution, and cooling;
mixing the polypeptide solution and the adjuvant solution, and adding appropriate amount of acetic acid to adjust pH to 4.0; adding water for injection to 1000mL, filling the mixed solution into a clean sterile penicillin bottle, performing batch 1000 bottles, performing half-buckling with a rubber plug, and freeze-drying;
the process of lyophilization comprises:
(1) prefreezing
The temperature of the heat conducting oil reaches 0 ℃ in 60 minutes at the first stage, and the heat conducting oil is kept for 2 hours;
the temperature of the heat conducting oil in the second stage reaches-40 ℃ within 60 minutes, and the heat conducting oil is kept for 4 hours;
(2) one time sublimation
The temperature of the heat conducting oil reaches-20 ℃ within 2 hours, the vacuum degree is about 10Pa, and the heat conducting oil is kept for 24 hours;
(3) drying by desorption
The temperature of the heat conducting oil reaches 40 ℃ within 16 hours, the vacuum degree is about 0Pa, and the heat conducting oil is kept for 6 hours;
(4) pressure rise test
Setting the pressure not to exceed 3Pa within 5 minutes, and finishing after the pressure rise is judged to be qualified.
Example 2
The present example provides a lyophilization method for an injectable lyophilized amphetamine formulation, which is formulated as follows:
the preparation method of the freeze-dried preparation of the amitriptyline for injection comprises the following steps:
firstly, preparing a solution, namely adding an HM-3 polypeptide raw material into 400mL of water for injection to prepare a solution, and performing sterile filtration by using a 0.22 mu m membrane for later use to obtain a polypeptide solution;
weighing adjuvants including dextran, citric acid and sodium citrate, adding into 400mL water for injection to obtain solution, adding needle activated carbon, heating, filtering, removing carbon, filtering with 0.22 μm membrane to obtain pyrogen-free sterile adjuvant water solution, and cooling;
mixing the polypeptide solution and the adjuvant solution, adding water for injection to 1000mL, filling the mixed solution into a clean sterile penicillin bottle, performing batch 1000 pieces, half-buckling a plug by using a rubber plug, and freeze-drying;
wherein the process of lyophilization comprises:
(1) prefreezing
The temperature of the heat conducting oil reaches-5 ℃ in 20 minutes at the first stage, and the heat conducting oil is kept for 1 hour;
the temperature of the heat conducting oil in the second stage reaches minus 40 ℃ within 30 minutes, and the heat conducting oil is kept for 2 hours;
(2) one time sublimation
The temperature of the heat-conducting oil in the first stage reaches-20 ℃ within 4 hours, the heat-conducting oil is kept for 12 hours, and the vacuum degree is 20 Pa;
(3) drying by desorption
In the temperature rise stage, the temperature of the heat conducting oil reaches 8 ℃ within 5 hours, the heat conducting oil is kept for 60 minutes, and the vacuum degree is 20 Pa;
the temperature of the heat-conducting oil in the first stage reaches 30 ℃ within 6 hours, the heat-conducting oil is kept for 60 minutes, and the vacuum degree is 20 Pa;
the temperature of the second stage heat-conducting oil reaches 30 ℃ within 1 minute, the temperature is kept for 6 hours, and the vacuum degree is 0 Pa.
(4) Pressure rise test
Setting the pressure not to exceed 3Pa within 5 minutes, and finishing after the pressure rise is judged to be qualified.
Example 3
The present example provides a lyophilization method for an injectable lyophilized amphetamine formulation, which is formulated as follows:
the preparation method of the freeze-dried preparation of the amitriptyline for injection comprises the following steps:
firstly, preparing a solution, namely adding an HM-3 polypeptide raw material into 400mL of water for injection to prepare a solution, and performing sterile filtration by using a 0.22 mu m membrane for later use to obtain a polypeptide solution;
weighing adjuvants including dextran, disodium hydrogen phosphate and sodium dihydrogen phosphate, adding into 400mL water for injection to obtain solution, adding needle activated carbon, heating, filtering, removing carbon, filtering with 0.22 μm membrane to obtain pyrogen-free sterile adjuvant water solution, and cooling;
mixing the polypeptide solution and the adjuvant solution, adding water for injection to 1000mL, filling the mixed solution into a clean and sterile penicillin bottle, half-buckling the penicillin bottle by using a rubber plug, and freeze-drying;
wherein the process of lyophilization comprises:
(1) prefreezing
The temperature of the heat conducting oil reaches-10 ℃ in 40 minutes at the first stage, and the heat conducting oil is kept for 1.5 hours;
the temperature of the heat conducting oil in the second stage reaches-35 ℃ within 45 minutes, and is kept for 3 hours;
(2) one time sublimation
The temperature of the heat-conducting oil in the first stage reaches-15 ℃ within 5 hours, the heat-conducting oil is kept for 8 hours, and the vacuum degree is 50 Pa;
the temperature of the second stage heat-conducting oil reaches-5 ℃ within 5 hours, the second stage heat-conducting oil is kept for 1 minute, and the vacuum degree is 50 Pa.
(3) Drying by desorption
In the temperature rise stage, the temperature of the heat conducting oil reaches 5 ℃ within 3 hours, the heat conducting oil is kept for 1 minute, and the vacuum degree is 50 Pa;
the temperature of the heat-conducting oil in the first stage reaches 35 ℃ within 3 hours, the heat-conducting oil is kept for 60 minutes, and the vacuum degree is 50 Pa;
the temperature of the second stage heat-conducting oil reaches 35 ℃ within 1 minute, the temperature is kept for 2 hours, and the vacuum degree is 0 Pa.
(4) Pressure rise test
Setting the pressure not to exceed 3Pa within 5 minutes, and finishing after the pressure rise is judged to be qualified.
Comparative example 1
This comparative example provides the lyophilization process and formulation of example 2 in CN 102178656A.
The prescription of the lyophilized preparation of the anizakii peptide is as follows:
the specific freeze-drying process is as follows:
(1) pre-freezing: setting the temperature of the plate layer to-40 ℃, reducing the temperature of the sample to-20 ℃ within one hour, and preserving the temperature for 3 hours;
(2) primary drying: setting the temperature of a plate layer to be-5 ℃ under the vacuum degree of 15Pa, and preserving the heat for 15 hours;
(3) secondary drying: setting the temperature of the plate layer to be 20 ℃, preserving the heat for more than 5 hours, and freeze-drying until the end point judgment of freeze-drying is met.
Comparative example 2
The comparative example provides a lyophilized formulation of amphetamine for injection. The specific prescription is the prescription of the freeze-dried preparation of the azadirachtin for injection, which is described in the embodiment 2 of the invention, and is prepared by adopting the freeze-drying process of the embodiment 2 in CN 102178656A.
The lyophilized injectable azadirachtin formulation of example 2 was formulated as follows:
the lyophilization process steps of example 2 in CN102178656A are as follows:
(1) pre-freezing: setting the temperature of the plate layer to-40 ℃, reducing the temperature of the sample to-20 ℃ within one hour, and preserving the temperature for 3 hours;
(2) primary drying: setting the temperature of a plate layer to be-5 ℃ under the vacuum degree of 15Pa, and preserving the heat for 15 hours;
(3) secondary drying: setting the temperature of the plate layer to be 20 ℃, preserving the heat for more than 5 hours, and freeze-drying until the end point judgment of freeze-drying is met.
Effect verification
The lyophilized formulations obtained in example 1, example 2, example 3 and comparative examples 1-2 were tested and the results were as follows:
TABLE 1
As can be seen from Table 1, the lyophilized preparations obtained in examples 1 to 3 had the advantages of good appearance, high perfection rate, stable properties and biological activity, etc. of the lyophilized products. In contrast, comparative example 1 has the disadvantages of low perfection ratio and large pH change. Comparative example 2 has a disadvantage of low perfection rate, although the pH value meets the standard.
Although the invention has been described in detail hereinabove with respect to a general description and specific embodiments thereof, it will be apparent to those skilled in the art that modifications or improvements may be made thereto based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.
Claims (10)
1. A freeze-drying method of an amphetamine freeze-dried preparation for injection is characterized in that,
the method comprises the following steps:
(1) prefreezing
The temperature of the heat conducting oil reaches 0 to-10 ℃ in 20 to 60 minutes at the first stage, and the heat conducting oil is kept for 1 to 2 hours;
the temperature of the heat conducting oil in the second stage reaches-30 to-45 ℃ within 30-60 minutes, and is kept for 2-4 hours;
(2) carrying out primary sublimation;
(3) resolving and drying;
(4) and finishing the freeze-drying process after the pressure rise test is qualified.
2. The method for lyophilizing a lyophilized injectable azadirachtin formulation according to claim 1, wherein the one-time sublimation comprises: the temperature of the heat conducting oil reaches-25-0 ℃ within 2-10 hours, the heat conducting oil is kept for 8-24 hours, and the vacuum degree is 10-50 Pa.
3. The method for lyophilizing a lyophilized injectable azadirachtin formulation according to claim 2, wherein the one-time sublimation comprises:
in the first stage, the temperature of the heat conducting oil reaches-25 to-10 ℃ within 2 to 5 hours, the heat conducting oil is kept for 6 to 12 hours, and the vacuum degree is 10 to 50 Pa;
and in the second stage, the temperature of the heat conduction oil reaches-5-0 ℃ within 2-5 hours, the heat conduction oil is kept for 1-120 minutes, and the vacuum degree is 10-50 Pa.
4. The method for lyophilizing the lyophilized injectable azadirachtin formulation of any one of claims 1-3, wherein the resolution drying comprises: the temperature of the heat conducting oil reaches 5-40 ℃ within 4-16 hours, the heat conducting oil is kept for 2-8 hours, and the vacuum degree is 0-50 Pa.
5. The method for lyophilizing a lyophilized injectable azadirachtin formulation according to claim 4, wherein the resolution drying comprises:
in the first stage, the temperature of the heat conducting oil reaches 20-40 ℃ within 3-6 hours, the temperature is maintained for 1-60 minutes, and the vacuum degree is 10-50 Pa;
in the second stage, the temperature of the heat conducting oil reaches 20-40 ℃ within 1-60 minutes, the heat conducting oil is kept for 2-6 hours, and the vacuum degree is 0-5 Pa.
6. The method for lyophilizing a lyophilized injectable azadirachtin formulation according to claim 5, wherein the resolution drying further comprises: and adding a temperature rise stage before the first stage, wherein the temperature of the heat conduction oil in the temperature rise stage reaches 5-10 ℃ within 3-5 hours, the heat conduction oil is kept for 1-60 minutes, and the vacuum degree is 10-50 Pa.
7. The lyophilization method for the lyophilized injectable azadirachtin preparation according to any one of claims 1 to 6, wherein the pressure rise is set under such conditions that the pressure does not exceed 3Pa within 5 minutes, and the lyophilization is terminated when the pressure rise is judged to be acceptable.
8. The method for lyophilizing the lyophilized injectable azadirachtin formulation of claim 1, comprising:
(1) prefreezing
The temperature of the heat conducting oil reaches 0 to-10 ℃ in 20 to 60 minutes at the first stage, and the heat conducting oil is kept for 1 to 2 hours;
the temperature of the heat conducting oil in the second stage reaches-30 to-45 ℃ within 30-60 minutes, and is kept for 2-4 hours;
(2) one time sublimation
In the first stage, the temperature of the heat conducting oil reaches-25 to-10 ℃ within 2 to 5 hours, the heat conducting oil is kept for 6 to 12 hours, and the vacuum degree is 10 to 50 Pa;
in the second stage, the temperature of the heat conducting oil reaches-5-0 ℃ within 2-5 hours, the heat conducting oil is kept for 1-120 minutes, and the vacuum degree is 10-50 Pa;
(3) drying by desorption
In the temperature rise stage, the temperature of the heat conducting oil reaches 5-10 ℃ within 3-5 hours, the heat conducting oil is kept for 1-60 minutes, and the vacuum degree is 10-50 Pa;
in the first stage, the temperature of the heat conducting oil reaches 20-40 ℃ within 3-6 hours, the temperature is maintained for 1-60 minutes, and the vacuum degree is 10-50 Pa;
in the second stage, the temperature of the heat conducting oil reaches 20-40 ℃ within 1-60 minutes, the heat conducting oil is kept for 2-6 hours, and the vacuum degree is 0-5 Pa;
(4) and (3) finishing the freeze-drying process after the pressure rise test is qualified: the pressure rise is set under conditions such that the pressure does not exceed 3Pa within 5 minutes.
9. The method for lyophilizing the lyophilized injectable azadirachtin formulation of any one of claims 1-8, wherein the formulation of the lyophilized injectable azadirachtin formulation is: calculated according to the mass portion, 1 to 10 portions of antipeglutide, 1 to 30 portions of auxiliary material dextran and 0.1 to 5 portions of pH buffer system; the pH of the lyophilized preparation is 4-8.
10. The freeze-drying method of the freeze-dried preparation of the atengium for injection according to claim 9, wherein the pH buffer system is one or more of acetic acid-sodium acetate, citric acid-sodium citrate or phosphoric acid-phosphate.
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