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CN-121987804-A - Vaccine freeze-drying protective agent and preparation method and application thereof

CN121987804ACN 121987804 ACN121987804 ACN 121987804ACN-121987804-A

Abstract

The invention relates to a vaccine freeze-drying protective agent, a preparation method and application thereof, and belongs to the technical field of biological medicines. The vaccine freeze-drying protective agent comprises the following raw materials of trehalose, mannitol, arginine, pullulan, 2-hydroxypropyl-beta-cyclodextrin, phospholipid, sodium sulfite and a protective additive, wherein the protective additive is at least one of hydroxy-ectoin, betaine, dodecyl dimethyl betaine and polycarboxylic acid betaine. The preparation method comprises the steps of shearing and mixing 2-hydroxypropyl-beta-cyclodextrin and phospholipid at high speed, sequentially adding the rest components, regulating pH and osmotic pressure, performing sterile filtration, and freeze-drying. The protective agent can effectively inhibit phase separation, sugar recrystallization and interfacial adsorption in the freeze-drying process, remarkably improve the activity retention rate and physical stability of the vaccine in the freeze-drying and storage periods, and is suitable for the preparation of freeze-drying preparations of various vaccines such as virus vaccines, bacterial vaccines, recombinant protein vaccines and the like.

Inventors

  • LI JUN
  • QI SHUYA
  • ZHAO BIN

Assignees

  • 江苏华诺泰生物医药科技有限公司

Dates

Publication Date
20260508
Application Date
20260408

Claims (10)

  1. 1. A vaccine freeze-drying protective agent is characterized by comprising the following raw materials of trehalose, mannitol, arginine, pullulan, 2-hydroxypropyl-beta-cyclodextrin, phospholipid, sodium sulfite and a protective additive, wherein the protective additive is at least one of hydroxy-ectoin, betaine, dodecyl dimethyl betaine and polycarboxylic acid betaine.
  2. 2. The vaccine freeze-drying protective agent according to claim 1, wherein the mass concentration of each component is 60-90 g/L of trehalose, 25-40 g/L of mannitol, 3-8 g/L of arginine, 1-3 g/L of pullulan, 4-8 g/L of 2-hydroxypropyl-beta-cyclodextrin, 0.1-0.3 g/L of phospholipid, 0.05-0.2 g/L of sodium sulfite and 5-12 g/L of protective additive.
  3. 3. The vaccine lyoprotectant of claim 2, wherein the protective additive is a mixture of hydroxyectoine and polycarboxylic acid betaine in a mass ratio of 1:1-5:1.
  4. 4. A vaccine lyoprotectant according to any one of claims 1-3, wherein the phospholipid is selected from soy lecithin or egg yolk lecithin.
  5. 5. A vaccine lyoprotectant according to any one of claims 1-3, wherein the polycarboxylic acid betaine is prepared by the following method: reacting N- (3-dimethylaminopropyl) methacrylamide with tert-butyl bromoacetate in an organic solvent under an inert atmosphere to obtain an intermediate product; the intermediate is polymerized in the presence of a chain transfer agent and an initiator, and then deprotected and purified to yield the polycarboxylic betaine.
  6. 6. The vaccine freeze-drying protective agent according to claim 5, wherein the preparation method of the polycarboxylic acid betaine is characterized in that 3-8g N- (3-dimethylaminopropyl) methacrylamide and 40-80mL of anhydrous acetonitrile are uniformly mixed under the protection of nitrogen, 8-11g of tert-butyl bromoacetate is added, stirring is continued for 20-60min, and the reaction is carried out for 18-36h at 50-65 ℃ to obtain an intermediate product; Under the protection of nitrogen, 0.05-0.2g of 4-cyano-4- (phenylthioformyl thio) valeric acid, 0.01-0.03g of benzoyl peroxide, 8-12g of the intermediate product and N, N-dimethylformamide are uniformly stirred, bubbling deoxidized, reacted for 18-36 hours at 60-80 ℃ and 400-800rpm, diethyl ether is added, white precipitate is separated out, trifluoroacetic acid is added, reacted for 1-4 hours at room temperature and 200-500rpm, dialyzing, purifying, filtering and freeze-drying are carried out, and the polycarboxylic acid betaine is obtained.
  7. 7. A method of preparing a vaccine lyoprotectant according to any one of claims 1-6, comprising the steps of: (1) Mixing 2-hydroxypropyl-beta-cyclodextrin with phospholipid and performing high-speed shearing treatment to obtain a mixture A; (2) Adding trehalose, pullulan, sodium sulfite, mannitol and arginine into the mixture A, stirring and mixing; (3) Adding a protective additive, and continuing stirring; (4) Adjusting the pH to 6.0-7.8, adjusting the osmotic pressure to 400-600 mosm/kg, performing sterile filtration after constant volume, and freeze-drying to obtain the vaccine freeze-drying protective agent.
  8. 8. The method according to claim 7, wherein the high-speed shearing treatment in the step (1) is carried out at a rotational speed of 8000 to 12000 rpm for a time of 2 to 5 min, and the stirring speeds in the step (2) and the step (3) are 400 to 800 rpm.
  9. 9. Use of a vaccine lyoprotectant according to any one of claims 1-6 for the preparation of a vaccine lyoprotectant.
  10. 10. The use of claim 9, wherein the vaccine comprises at least one of a viral vaccine, a bacterial vaccine, or a recombinant protein vaccine.

Description

Vaccine freeze-drying protective agent and preparation method and application thereof Technical Field The invention relates to the technical field of biological medicines, in particular to a vaccine freeze-drying protective agent and a preparation method and application thereof. Background The vaccine is used as a preventive biological product, and is often influenced by various unstable factors such as temperature fluctuation, repeated freeze thawing, drying stress, interfacial adsorption and the like in the storage, transportation and use processes, so that the activity of antigen is easy to be reduced, the potency is reduced, and the immune effect and the inoculation safety are directly influenced. The freeze drying technology can greatly improve the long-term stability of the vaccine, but the freeze concentration, the mechanical damage of ice crystals, the dehydration stress, the re-dissolution impact and other problems which are accompanied by the freeze drying process can also cause irreversible damage to the activity of the vaccine. Therefore, it is often desirable to add suitable lyoprotectants to the lyophilized formulation to maintain the structural integrity and bioactivity of the vaccine. The commonly used lyoprotectant mainly comprises non-reducing saccharides (such as sucrose and trehalose), excipients (such as mannitol and glycine), amino acids (such as arginine), surfactants, buffer systems and the like. These ingredients alleviate the loss of activity during lyophilization to some extent through mechanisms such as glassy solidification, water substitution, inhibition of ice crystal growth, reduction of interfacial tension, etc. However, the existing protection systems still have the following disadvantages in practical applications: However, existing protection systems may still suffer from the following disadvantages: (1) The physical stability is insufficient, namely, under the condition of higher temperature or humidity, the glassy system of the saccharides is easy to generate phase separation or sugar recrystallization, so that the solid structure is uneven, and the long-term stable retention of active ingredients of the vaccine is further influenced; (2) In order to obtain good freeze-drying appearance and re-dissolution performance, skeleton support is often needed to be provided by depending on easily crystallized components such as mannitol and the like, and glass state protection is maintained by amorphous components such as trehalose, so that contradiction exists between formula design and process control, and collaborative optimization is difficult; (3) The adaptability of the system is limited, the sensitivity difference of different types of vaccines (such as virus vaccines, bacterial vaccines, recombinant protein vaccines and the like) to freezing, drying, oxidation, interfacial adsorption and aggregation is obvious, the traditional protection system mainly comprising single saccharides or excipients is difficult to be universally applied, and the protection effect of the vaccine is especially not ideal for the vaccine which is easy to aggregate or is sensitive to the interface. Therefore, a novel freeze-drying protective agent which can synchronously inhibit freeze-drying phase separation and recrystallization, has freeze-drying formability and re-solubility and wide vaccine suitability is developed, and has important significance in improving the full-cycle stability of vaccine preparations, prolonging the shelf life and guaranteeing the inoculation effect. Disclosure of Invention Aiming at the defects in the prior art, the invention provides a vaccine freeze-drying protective agent, and a preparation method and application thereof. In order to solve the technical problems, the invention adopts the following technical scheme: a freeze-drying protective agent for vaccine comprises trehalose, mannitol, arginine, pullulan, 2-hydroxypropyl-beta-cyclodextrin, phospholipid, sodium sulfite and a protective additive, wherein the protective additive is at least one of hydroxy-ectoine, betaine, dodecyl dimethyl betaine and polycarboxylic acid betaine. Trehalose is used as a typical non-reducing sugar, and can stabilize the microenvironment around antigens or virus particles through water substitution/vitrification in the freeze-drying stage, reduce conformational changes and inactivation caused by dehydration, and help to improve activity retention during reconstitution after freeze-drying. Mannitol is mainly used as a forming agent and a framework support, is easy to crystallize to form a stable freeze-dried block structure, improves the appearance, reduces the collapse risk, and promotes the formation of a re-dissolution channel, thereby improving the re-dissolution speed and the process suitability of the preparation. Arginine as an amino acid stabilizer and an anti-aggregation component can reduce the nonspecific interaction between proteins/particles, inhibit aggregation and coal