A stable adenovirus vector lyophilized formulation and use thereof

Abstract

The present application relates to a kind of stable adenovirus vector freeze-dried preparation and its application, the freeze-dried preparation, for stabilizing the biological activity of preventive vaccine or and therapeutic biological medicine preparation, while also can reduce the damage of freeze-drying process to biological product activity, the preparation is prepared from effective component, salt buffer, saccharide freeze-drying protective agent, protein protective agent, surfactant, excipient, pH is 6.0-7.0.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to a freeze-dried formulation for solving the long-term stability of active ingredients in biological products. Background Technology

[0002] The instability of biological products limits their wider use to some extent. For example, the COVID-19 pandemic highlighted the importance of vaccines in transportation, storage, and distribution, while vaccine instability often results in short shelf lives and dependence on low temperatures. Improving vaccine stability and extending its shelf life has been a cutting-edge issue for decades, especially for active biological products. Researching how to ensure good stability over a relatively long period is of great significance. Many outstanding formulation scientists have continuously developed numerous formulations and optimized process parameters to maintain the activity of biopharmaceuticals. Their research goal is to provide products that maintain sufficient quality (potency, activity, and immunogenicity) during storage and after accidental exposure to abnormal conditions until the drug is used. It is well known that the stability of biological drugs such as viruses and proteins in aqueous media above 8°C is limited, and their thermal stability has always been challenged. Coupled with the uncertainty and inconsistency of cold chain transportation and storage conditions, liquid formulations, although convenient to prepare, also limit the potential for long-term drug use. Freeze-drying has a wide range of applications in the biopharmaceutical and food industries. By removing free water through freeze-drying (making the final water content 1% to 3% of the total product weight), the stability of viruses or proteins can be significantly increased and their shelf life extended by effectively inhibiting or slowing down unstable pathways such as degradation that may occur in aqueous media.

[0003] Based on existing relevant technical knowledge, this invention screened key components in freeze-drying formulations through a series of experiments. Summary of the Invention

[0004] The present invention aims to develop and provide a lyophilized formulation that utilizes lyophilization, which on the one hand can improve the problem of reduced potency, activity and immunogenicity of biological products after lyophilization, and on the other hand can protect and maintain the long-term stability of their biological quality.

[0005] This invention also utilizes this formulation to develop a freeze-drying process for recombinant adenovirus preparations, which is used for freeze-drying recombinant adenovirus preparations.

[0006] Specific embodiments of the present invention

[0007] This invention provides a lyophilized formulation prepared from an active ingredient, a salt buffer solution, a sugar lyophilization protectant, a protein protectant, a surfactant, and an excipient, with a pH of 6.0 to 7.0.

[0008] The active ingredient is a recombinant adenovirus vaccine.

[0009] The salt buffer solution is selected from: citric acid, PB, Tris-HCl, physiological saline, DPBS and KPBS buffer;

[0010] The sugar freeze-drying protectant is selected from: sucrose, trehalose, raffinose, or cyclodextrin;

[0011] The protein-based protective agent is selected from: human serum albumin, bovine serum albumin, gelatin, hydrolyzed whey protein, and hydrolyzed casein.

[0012] The surfactant is selected from: Tween 20, Tween 80, Proxam F12, Proxam F68, and Span 20;

[0013] The excipients are selected from: glycine, dextran, polyvinylpyrrolidone, mannitol, sorbitol, and trehalose.

[0014] In the liquid formulation, the active ingredient is selected from: respiratory syncytial virus recombinant adenovirus vaccine, varicella-zoster virus vaccine.

[0015] The vaccines include recombinant adenovirus vaccines for herpes simplex virus (HSV), recombinant adenovirus vaccines for novel coronavirus, recombinant adenovirus vaccines for metapneumovirus, recombinant adenovirus vaccines for vesicular stomatitis virus (VSDV), recombinant adenovirus vaccines for herpes simplex virus type I, recombinant adenovirus vaccines for herpes simplex virus type II, recombinant adenovirus vaccines for Helicobacter pylori, and therapeutic recombinant adenovirus vaccines for hepatitis B. Preferably, the active ingredient is a recombinant adenovirus vaccine for varicella-zoster virus (VZV). Most preferably, the active ingredient, respiratory syncytial virus (RSV) recombinant adenovirus vaccine, contains virus particles (vp) with a density of 1.0 × 10⁻⁶. 9 ~5.0×10 13 A quantity of vp / ml is present.

[0016] The respiratory syncytial virus recombinant adenovirus vaccine is prepared as follows:

[0017] (1) Construct and synthesize shuttle plasmids containing protective antigenic nucleotides;

[0018] (2) The shuttle plasmid described in step (1) was recombined with the backbone plasmid using a recombinant kit to obtain a plasmid containing protective antigen.

[0019] Adenoviral recombinants of protein genes;

[0020] (3) Transfect the adenovirus recombinant described in step (2) into HEK-293 or HEK-293.2sus;

[0021] (4) The packaging cells described in step (3) are HEK-293 or HEK-293.2sus;

[0022] (5) Harvest the replication-defective recombinant adenovirus released from the packaging cells described in step (4);

[0023] (6) Harvest plaque purification from the replication-defective recombinant adenovirus described in step (5);

[0024] (7) Expand the culture of the replication-defective recombinant adenovirus from step (6);

[0025] (8) Repeatedly freeze and thaw the replication-defective recombinant adenovirus cultured in step (7), and remove the supernatant after centrifugation;

[0026] (9) The freeze-thaw product in step (8) is physically purified and then concentrated by ultrafiltration;

[0027] (10) The culture product from step (9) was purified by column chromatography.

[0028] Preferably, the salt buffer is selected from KPBS or PB buffer, and its molar concentration is 5 mmol / L to 100 mmol / L.

[0029] Preferably, the sugar freeze-drying protectant is selected from sucrose, trehalose, or raffinose, and its concentration is 5% to 30%.

[0030] Preferably, the protein-based protective agent is human serum albumin, with a concentration of 0.1-5%.

[0031] Preferably, the surfactant is Tween 80, with a concentration of 0.001% to 0.8%.

[0032] Preferably, the excipient is selected from dextran, mannitol, or PVP, and the concentration is 2% to 12%.

[0033] The salt buffer solution described in this invention is selected from KPBS or PB buffer, and its molar concentration is 10 mmol / L to 50 mmol / L;

[0034] The sugar freeze-drying protectant is selected from sucrose, trehalose, or raffinose, and its concentration is 5% to 20%.

[0035] The protein-based protective agent is human serum albumin, with a concentration of 0.2% to 3%.

[0036] The surfactant is Tween 80, with a concentration of 0.01% to 0.6% or 0.015% to 0.4%.

[0037] The excipient is selected from dextran, mannitol, or PVP, and the concentration is 3% to 8%.

[0038] This invention further provides a method for preparing the lyophilized formulation, the method comprising the following steps: weighing each component according to a mass fraction or molar amount of 2×, dissolving them in water for injection and making up to a final volume, each preparation being freshly prepared and used immediately, adjusting to a specified pH value, filtering for sterilization using a 0.22μm filter, taking the biological product stock solution and appropriately diluting it, mixing it thoroughly with the lyophilization formulation at a 1:1 volume ratio, placing it in the sample chamber of a lyophilizer, placing the temperature electrodes, closing the chamber door, setting the lyophilization process flow and parameters, and performing lyophilization; after lyophilization, filling with nitrogen to break the vacuum, and performing stoppering and capping; the lyophilization process flow mainly includes the following steps: shelf precooling, freezing, annealing, primary drying and secondary drying; the lyophilization process... The main process parameters are as follows: shelf pre-cooling temperature is -10 to 20°C, lasting at least 30 minutes; freezing temperature is -60 to -40°C, lasting 3 to 8 hours; annealing temperature is -30 to -10°C, lasting 0.5 to 5 hours; the first drying stage is -60 to -45°C, lasting 4 to 6 hours, and the second stage is -40 to -25°C, lasting at least 12 hours, with a vacuum pressure of 0.08 to 0.5 mbar during this stage; the secondary drying temperature is 5 to 20°C, lasting 6 to 12 hours, with a vacuum pressure of 0.001 to 0.3 mbar during this stage; wherein, the heating rate during temperature changes is 0.01 to 1.5°C / min, and the cooling rate is 0.5 to 5°C / min. Preferably, the heating rate is 0.1 to 1°C / min, and the cooling rate is 0.6 to 2°C / min.

[0039] The determination of the formulation components in the final lyophilized formulation described in this invention is based on screening key excipients by analyzing the changes in titers before and after lyophilization, and comprehensively evaluating the scores of the lyophilized cake to determine the usable formulation.

[0040] The viral infectivity is determined by measuring the viral titer, using the half-cell infectious dose (CCID). 50 The endpoint dilution method was used to determine the infectivity of recombinant adenovirus in HEK-293T cells. The specific steps are as follows:

[0041] (1) Take 50 μl of virus, add 450 μl of maintenance solution, mix well, and mark the dilution as 10⁻¹; take 50 μl of virus dilution with a dilution of 10⁻¹, add 450 μl of maintenance solution, mix well, and mark the dilution as 10⁻²; and so on, dilute the virus to 10⁻¹⁰.

[0042] (2) HEK-293T cells were digested and diluted to a concentration of 2.0 × 10⁻⁶. 5Cell suspension per mL. Mix the cell suspension with an equal volume of graded virus dilution buffer and set aside.

[0043] (3) Seed cells in 96-well cell culture plates, 10 cells per well. 4 For each cell, columns 1A-1H contain 100 μl / well of a mixture of virus dilution (10⁻⁶) and cells; columns 2A-2H contain 100 μl / well of a mixture of virus dilution (10⁻⁷) and cells, and so on. Columns 5A-5H contain 100 μl / well of a mixture of virus dilution (10⁻¹⁰) and cells; columns 6A-6H contain 100 μl / well of maintenance buffer as a negative control. Reproducible cells are set up in columns 7H-12H.

[0044] (4) Calculate CCID using the Reed-Muench method or the Karer method. 50 .

[0045] The evaluation of the shaping effect of the freeze-dried cake after freeze-drying was conducted by three people unrelated to this experiment. The freeze-dried cake was evaluated and scored with a maximum score of 3 points. The average score was calculated, and the higher the score, the better the shaping effect.

[0046] In the experiments of this invention, the virus used was recombinant chimpanzee adenovirus type 63, but ChAd3, ChAd83, and ChAd155 could also be used.

[0047] Other chimpanzee adenoviruses such as ChAd157, ChAdOx1, and ChAdOx2, as well as one or more human adenoviruses of serotypes 1 to 57, such as serotypes 5, 7, 26, 35, or 48, do not affect the results of the experiments in this invention.

[0048] Compared with the prior art, the advantages of the present invention are as follows:

[0049] (1) By using different types and concentrations of excipients alone or in combination, the shape of freeze-dried cakes can be kept stable.

[0050] (2) The combined use of sugar and protein freeze-drying protectants can more effectively maintain the stability of biological products while reducing damage during the freeze-drying process.

[0051] (3) By optimizing the freeze-drying process, the quality of the freeze-dried cake is guaranteed, while the loss of biological product quality (potency, activity and immunogenicity) during the freeze-drying process can be reduced. Detailed Implementation

[0052] The product and its preparation method according to the present invention will be further described below with reference to specific embodiments, but the present invention is not limited to the following embodiments. Unless otherwise specified, the methods described are conventional methods. Unless otherwise specified, the raw materials can be obtained from publicly available commercial sources.

[0053] Example 1: Screening of Excipients

[0054] The various excipients are present alone or in combination with other components. The different excipient formulations described below all contain 20 mmol / L PB buffer and do not contain biological products. The excipient #1 is 3% mannitol; excipient #2 is 3% PVP; excipient #3 is 3% dextran; excipient #4 is 3% glycine; excipient #5 is 3% sorbitol; excipient #6 consists of 2% PVP and 2% glycine; excipient #7 consists of 2% PVP and 2% mannitol; excipient #8 consists of 2% PVP and 2% dextran; excipient #9 consists of 2% PVP and 2% sorbitol; excipient #10 consists of 2% mannitol and 2% dextran; excipient #11 consists of 2% mannitol and 2% sorbitol; excipient #12 consists of 2% dextran and 2% sorbitol; and excipient #13 consists of 2% trehalose and 2% sucrose. After freeze-drying, the shape-enhancing effect of the freeze-dried cake is scored. The following are the ratings for different formulas.

[0055] serial number 1 2 3 4 5 6 7 8 9 10 11 12 13 freeze-dried biscuit rating 2.98 2.80 2.78 2.33 0.8 2.06 3.00 2.20 1.50 2.00 2.78 2.5 2.00

[0056] Based on the above results, it can be seen that when only one excipient is added, sorbitol has the worst excipient effect, mannitol has the best, and PVP and dextran are the next best. Among the combinations of excipients, 2% PVP and 2% mannitol, as well as 2% mannitol and 2% sorbitol have better excipient effects.

[0057] Example 2: Experiment on freeze-drying shaping effect and its protective effect

[0058] Formula 1 contains 20 mmol / L PB buffer, 3% mannitol, and 0.1 mol / L sucrose; Formula 2 contains 20 mmol / L PB buffer, 3% mannitol, and 0.2 mol / L sucrose; Formula 3 contains 20 mmol / L PB buffer, 3% mannitol, and 0.3 mol / L sucrose; Formula 4 contains 20 mmol / L PB buffer, 3% mannitol, and 0.4 mol / L sucrose; Formula 5 contains 20 mmol / L PB buffer, 4% mannitol, and 0.1 mol / L sucrose; Formula 6 contains 20 mmol / L PB buffer, 4% mannitol, and 0.1 mol / L sucrose; Formula 7 contains 20 mmol / L PB buffer, 4% mannitol, and 0.3 mol / L sucrose; Formula 8 contains 20 mmol / L PB buffer, 4% mannitol, and 0.4 mol / L sucrose; Formula 9 contains 20 mmol / L PB buffer, 5% mannitol, and 0.1 mol / L sucrose; Formula 10 contains 20 mmol / L PB buffer, 5% mannitol, and 0.2 mol / L sucrose; Formula 11 contains 20 mmol / L PB buffer, 5% mannitol, and 0.3 mol / L sucrose. After freeze-drying, the excipient effect of the freeze-dried cake was scored. The scores and titers before and after freeze-drying for different formulas are shown below.

[0059]

[0060] The results above indicate that while increasing sucrose concentration improves the protective effect against the virus, it also reduces the excipient properties of the freeze-dried cake. Therefore, to ensure good excipient properties, the required excipient concentration also needs to be appropriately increased with increasing sucrose concentration.

[0061] Example 3: The Influence of Different Buffer Systems on the Freeze-Drying Protection Effect

[0062] Formula 1 contains 20 mmol / L KPBS, 0.02% Tween 80, 0.1 mol / L trehalose, and 5% mannitol; Formula 2 contains physiological saline, 0.02% Tween 80, 0.1 mol / L trehalose, and 5% mannitol; Formula 3 contains 20 mmol / L Tris-HCl, 0.02% Tween 80, 0.1 mol / L trehalose, and 5% mannitol; Formula 4 contains 20 mmol / L... DPBS, 0.02% Tween 80, 0.1 mol / L trehalose, 5% mannitol; Formula 5 contains 20 mmol / L DPBS, 0.02% Tween 80, 0.1 mol / L trehalose, 5% mannitol; Formula 6 contains 20 mmol / L citric acid, 0.02% Tween 80, 0.1 mol / L trehalose, 5% mannitol; The following are the scores and titers before and after lyophilization for different formulas.

[0063]

[0064]

[0065] Based on the above results, it can be concluded that different buffer systems have no effect on the lyophilization excipient effect, and DPBS and PB buffer systems are better buffers in lyophilization formulations.

[0066] Example 4: Combination of different amorphous sugars

[0067] Formula 1 contains 10 mmol / L Tris-HCl, 0.04% Tween 80, 0.1 mol / L sucrose, 5% cyclodextrin, and 5% mannitol; Formula 2 contains 10 mmol / L Tris-HCl, 0.04% Tween 80, 0.1 mol / L sucrose, 0.1 mol / L trehalose, and 5% mannitol; Formula 3 contains 10 mmol / L Tris-HCl, 0.04% Tween 80, 0.1 mol / L sucrose, 0.1 mol / L raffinose, and 5% mannitol; Formula 4 contains 10 mmol / L Tris-HCl, 0.04% Tween 80, 0.1 mol / L trehalose, 5% cyclodextrin, and 5% mannitol; Formula 5 contains 10 mmol / L Tris-HCl, 0.04% Tween 80, 0.1 mol / L trehalose, 5% cyclodextrin, and 5% mannitol; Formula 5 contains 10 mmol / L Tris-HCl, 0.04% Tween 80, 0.1 mol / L trehalose, 5% cyclodextrin, and 5% mannitol; Tris-HCl, 0.04% Tween 80, 0.1 mol / L trehalose, 0.1 mol / L raffinose, 5% mannitol. Formula 6 contains 10 mmol / L Tris-HCl, 0.04% Tween 80, 5% cyclodextrin, 0.1 mol / L raffinose, and 5% mannitol; Formula 7 contains 10 mmol / L Tris-HCl, 0.04% Tween 80, 0.1 mol / L sucrose, and 5% mannitol; Formula 8 contains 10 mmol / L Tris-HCl, 0.04% Tween 80, 5% cyclodextrin, and 5% mannitol; Formula 9 contains 10 mmol / L Tris-HCl, 0.04% Tween 80, 0.1 mol / L raffinose, and 5% mannitol; Formula 10 contains 10 mmol / L Tris-HCl, 0.04% Tween 80, 0.1 mol / L trehalose, and 5% mannitol. The following are the scores and titers before and after lyophilization for the different formulas.

[0068]

[0069] To improve the lyophilization protection of the formulation against the virus, different sugar protectants were combined. The results show that while different sugar combinations did improve the protective effect to some extent, the excipient effect remained poor; sugars used alone showed better excipient effects.

[0070] Example 5: Comparison of different protein-based protective agents

[0071] Formula 1 contains 20 mmol / L KPBS, 0.025% Tween 80, 0.1 mol / L sucrose, 4.5% mannitol, and 0.5% hydrolyzed whey protein; Formula 2 contains 20 mmol / L KPBS, 0.025% Tween 80, 0.1 mol / L sucrose, 4.5% mannitol, and 0.5% hydrolyzed casein; Formula 3 contains 20 mmol / L KPBS, 0.025%... Formula 1 contains Tween 80, 0.1 mol / L sucrose, 4.5% mannitol, and 0.5% BSA; Formula 4 contains 20 mmol / L KPBS, 0.025% Tween 80, 0.1 mol / L sucrose, 0.5% HSA, and 4.5% mannitol; Formula 5 contains 20 mmol / L KPBS, 0.025% Tween 80, 0.1 mol / L sucrose, 4.5% mannitol, and 0.5% gelatin. The following are the scores and titers before and after lyophilization for the different formulas.

[0072]

[0073] Based on the above results, it can be concluded that the addition of protein-based protective agents resulted in better protection against the virus and lower titer loss. BSA and HSA showed good protective effects, while gelatin had the worst protective effect and poor excipient properties.

[0074] Example 6: Protective effects of different concentrations of surfactants and protein protectants

[0075] Formula 1 contains 5 mmol / L PB, 0.02% Tween 80, 0.1 mol / L sucrose, and 5% mannitol; Formula 2 contains 5 mmol / L PB, 0.04% Tween 80, 0.1 mol / L sucrose, and 5% mannitol; Formula 3 contains 5 mmol / L PB, 0.06% Tween 80, 0.1 mol / L sucrose, and 5% mannitol; Formula 4 contains 5 mmol / L PB, 0.02% Tween 80, 0.1 mol / L sucrose, 0.25% HSA, and 5% mannitol; Formula 5 contains 5 mmol / L PB, 0.02% Tween 80, 0.1 mol / L sucrose, 0.5% HSA, and 5% mannitol; Formula 6 contains 5 mmol / L PB, 0.02% Tween 80, 0.1 mol / L sucrose, 1% HSA, and 5% mannitol. The following are the scores and titers before and after freeze-drying for different formulations.

[0076]

[0077] Based on the above results, it can be concluded that the specific amount of surfactant has no significant effect on virus protection, and the protective effect is best when the amount of HSA is 1%.

[0078] Example 7: Screening of different carbohydrates and different protein protective agents

[0079] Formula 1 contains 5 mmol / L PB, 0.02% Tween 80, 0.15 mol / L sucrose, 0.5% HSA, and 5% mannitol; Formula 2 contains 5 mmol / L PB, 0.02% Tween 80, 0.15 mol / L raffinose, 0.5% HSA, and 5% mannitol; Formula 3 contains 5 mmol / L PB, 0.02% Tween 80, 5% cyclodextrin, 0.5% HSA, and 5% mannitol; Formula 4 contains 5 mmol / L PB, 0.02% Tween 80, 0.15 mol / L trehalose, 0.5% HSA, and 5% mannitol. The following are the scores and titers before and after lyophilization for the different formulas.

[0080]

[0081]

[0082] The results above indicate that the combined use of carbohydrate and protein-based protective agents significantly enhances the protection against viruses.

[0083] Example 8: Long-term stability test data

[0084] Four lyophilized formulations were prepared, and their long-term stability was determined. The titer monitoring results for the four different lyophilized formulations are shown below.

[0085]

[0086] in,

[0087] Prescription 1 is as follows: 5 mmol / L LPB, 0.02% Tween 80, 0.15 mol / L sucrose, and 5% mannitol.

[0088] Prescription No. 2 is as follows: 5 mmol / L PB, 0.02% Tween 80, 0.15 mol / L sucrose, 0.25% HSA, and 5% mannitol.

[0089] Prescription No. 3 is as follows: 5 mmol / L LPB, 0.02% Tween 80, 0.15 mol / L sucrose, 0.5% HSA, and 5% mannitol.

[0090] Prescription No. 4 is as follows: 5 mmol / L LPB, 0.02% Tween 80, 0.15 mol / L sucrose, 1% HSA, and 5% mannitol.

[0091] The results showed that, of the four formulations, formulation 1 was the control formulation without a protectant, and after storage at 2–8°C for 360 days, formulation 3 showed the best protective effect, with the viral titer decreasing by only 0.0375 lg values. The titer loss values ​​of formulations 2 and 4 were less than 0.5 lg values.

[0092] It should be noted that optimizations and improvements can be easily made by those skilled in the art, and all of these improvements should be within the scope of protection of this invention. The various technical features of the technical solutions described in this application can be appropriately combined as needed.

[0093] The above description is merely a preferred embodiment of the present invention. It should be noted that the embodiments of the present invention are not limited to the described embodiments. Various changes, modifications, substitutions, combinations, and simplifications can be made without departing from the principle of the present invention, and all such substitutions should be considered equivalent replacements and should also be considered within the scope of protection of the present invention.

Claims

1. A lyophilized formulation characterized in that: The recombinant adenovirus vaccine is prepared by 20 mM KPBS, 0.025% Tween 80, 0.1 mol / L sucrose, 4.5% mannitol and 0.5% protein protective agent, pH 6.0-7.0; the protein protective agent is selected from hydrolyzed casein, BSA or HSA.

2. A lyophilized formulation characterized in that: The recombinant adenovirus vaccine is prepared by 5 mM PB, 0.02% Tween 80, 0.1 mol / L sucrose, 5% mannitol and 0.25%-1% HSA, pH 6.0-7.

0.

3. A lyophilized formulation characterized in that: The recombinant adenovirus vaccine is prepared by 5 mM PB, 0.02% Tween 80, 0.1 mol / L sucrose, 5% mannitol and 0.25%-1% HSA, pH 6.0-7.

0.

4. The freeze-dried preparation according to any one of claims 1-3, characterized in that: The recombinant adenovirus vaccine is: respiratory syncytial virus recombinant adenovirus vaccine, varicella-zoster virus recombinant adenovirus vaccine, novel coronavirus recombinant adenovirus vaccine, metapneumovirus recombinant adenovirus vaccine, vesicular stomatitis virus recombinant adenovirus vaccine, herpes simplex virus type I recombinant adenovirus vaccine, herpes simplex virus type II recombinant adenovirus vaccine, Helicobacter pylori recombinant adenovirus vaccine or therapeutic hepatitis B recombinant adenovirus vaccine.

5. Process for the preparation of a lyophilized formulation according to any one of claims 1 to 4, characterized in that, The method comprises the following steps: each component is weighed according to a mass fraction or a molar substance amount of 2x, dissolved in water for injection and then diluted to a constant volume, each preparation is prepared for immediate use, adjusted to a specified pH value, filtered and sterilized by using a 0.22 μm filter, a biological product stock solution is appropriately diluted, a freeze-drying formula is mixed uniformly according to a volume ratio of 1:1, a temperature electrode is placed, a door is closed tightly, a freeze-drying process and parameters are set, freeze-drying is performed, after freeze-drying is completed, nitrogen is filled to break the vacuum, and plugging and capping are performed; the freeze-drying process mainly comprises the following steps: The shelf precooling temperature is -10-20℃, the duration is at least 30 min; the freezing temperature is -60--40℃, the duration is 3-8 h; the annealing temperature is -30--10℃, the duration is 0.5-5 h; the first-stage primary drying temperature is -60--45℃, the duration is 4-6 h, the second-stage primary drying temperature is -40--25℃, the duration is at least 12 h, and the vacuum pressure value in this stage is 0.08-0.5 mbar; the secondary drying temperature is 5-20℃, the duration is 6-12 h, and the vacuum pressure value in this stage is 0.001-0.3 mbar; wherein, the temperature increasing rate is 0.01-1.5℃ / min, and the temperature decreasing rate is 0.5-5℃ / min.

6. The production method according to claim 5, characterized by The temperature increasing rate is 0.1-1℃ / min, and the temperature decreasing rate is 0.6-2℃ / min. ​

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