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CN-122005779-A - TLR7/8-lipo+AL freeze-dried vaccine adjuvant and preparation method and application thereof

CN122005779ACN 122005779 ACN122005779 ACN 122005779ACN-122005779-A

Abstract

The invention relates to the technical field of vaccine adjuvants, and discloses a TLR7/8-lipo+AL freeze-dried vaccine adjuvant, a preparation method and application thereof, wherein the freeze-dried vaccine adjuvant comprises a TLR 7/8-coupling-Lipo compound before freeze drying; the invention solves the problems of particle aggregation and structure collapse of the Lipo-AL compound caused by freeze-drying stress in the freeze-drying process by combining sucrose with glycine with the concentration of 2.0 to 3.0 percent, and the invention also provides a preparation method which comprises the steps of preparing the Lipo compound, adsorbing with the aluminum salt adjuvant, adding sucrose and glycine, and finally freeze-drying. The freeze-dried adjuvant prepared by the invention has the characteristics of complete form, quick redissolution and uniform particle size after redissolution, and can still maintain high biological activity under long-term storage.

Inventors

  • ZHAO CE
  • ZHANG QIANG
  • WANG QI
  • YANG BAOCHENG

Assignees

  • 华诺泰生物医药科技(成都)有限公司

Dates

Publication Date
20260512
Application Date
20260414

Claims (10)

  1. 1. A TLR7/8-lipo+al lyophilized vaccine adjuvant comprising: TLR 7/8-coupled-Lipo complex at a concentration of 5.0% to 10.0%; an aluminum salt adjuvant at a concentration of 1.0% to 3.0%; sucrose at a concentration of 2.0% to 3.0%; glycine at a concentration of 1.0% to 2.0%.
  2. 2. A TLR7/8-lipo+al lyophilized vaccine adjuvant according to claim 1, wherein the TLR 7/8-conjugated-Lipo complex comprises: TLR7/8 agonist-lipid conjugate; cationic lipids, helper lipids; wherein the molar ratio of the components TLR7/8 agonist-lipid conjugate, cationic lipid and auxiliary lipid is (5-10): 20-30): 60-75.
  3. 3. A TLR7/8-lipo+al lyophilized vaccine adjuvant according to claim 2, wherein the TLR7/8 agonist-lipid conjugate is an R848-PEG-DSPE conjugate or a loxoribine-PEG-DSPE conjugate; The cationic lipid is DOTAP; The helper lipid comprises DOPC and cholesterol.
  4. 4. A TLR7/8-lipo+al lyophilized vaccine adjuvant according to claim 1, wherein the aluminium salt adjuvant is an aluminium hydroxide adjuvant or an aluminium phosphate adjuvant.
  5. 5. A TLR7/8-lipo+al lyophilized vaccine adjuvant according to claim 1, wherein the TLR 7/8-conjugated-Lipo complex has a Z-average particle size in liquid of 280nm to 300nm.
  6. 6. A method of preparing a TLR7/8-lipo+al lyophilized vaccine adjuvant according to any one of claims 1-5, comprising the steps of: (S1) preparing a suspension of the TLR 7/8-coupled-Lipo complex; (S2) mixing the suspension obtained in the step (S1) with an aluminum salt adjuvant for adsorption; (S3) adding sucrose and glycine to the mixture of step (S2) to obtain a pre-lyophilized composition liquid; (S4) freeze-drying the pre-freeze-dried composition liquid.
  7. 7. The method for preparing a TLR7/8-lipo+al lyophilized vaccine adjuvant according to claim 6, wherein the step (S1) comprises: Dissolving a lipid component comprising the TLR7/8 agonist-lipid conjugate, a cationic lipid and a helper lipid in an organic solvent, and rotary evaporating to form a lipid film; Adding an aqueous buffer solution into the lipid film for hydration; And carrying out sequential extrusion treatment on the suspension obtained after hydration.
  8. 8. The method for preparing a TLR7/8-lipo+al lyophilized vaccine adjuvant according to claim 6, wherein the lyophilization of step (S4) comprises: Pre-freezing at a temperature of-50 ℃ to-30 ℃; Primary drying at a temperature of-30 ℃ to-10 ℃; The secondary drying is performed at a temperature of 20 ℃ to 30 ℃.
  9. 9. The method of claim 6, further comprising the step of preparing the TLR7/8 agonist-lipid conjugate prior to step (S1), the step of preparing the TLR7/8 agonist-lipid conjugate being selected from the group consisting of: coupling R848-COOH with DSPE-PEG2000-Amine by EDC/NHS method; The loxoribine-SH was coupled to DSPE-PEG2000-MALEIMIDE by the maleimide method.
  10. 10. Use of a TLR7/8-lipo+al lyophilized vaccine adjuvant according to any one of claims 1-5 for the preparation of a vaccine for the prevention or treatment of a viral infection.

Description

TLR7/8-lipo+AL freeze-dried vaccine adjuvant and preparation method and application thereof Technical Field The invention relates to the technical field of vaccine adjuvants, in particular to a TLR7/8-lipo+AL freeze-dried vaccine adjuvant and a preparation method and application thereof. Background In order to ensure long-term stability of the vaccine during storage and transport, freeze-drying (lyophilization) is a widely used technological means in biological products, in particular vaccine formulations. Meanwhile, modern vaccine development increasingly relies on complex adjuvant systems, such as the use of Toll-like receptor (TLR) agonists in combination with conventional aluminium salt adjuvants (such as aluminium hydroxide or aluminium phosphate), in order to increase the strength and persistence of the vaccine's immune response. However, such complex adjuvant systems comprising liposomes (Lipo), bioactive molecules (e.g. TLR7/8 agonists) and aluminium salt particles are highly complex in structure and sensitive to the environment. During the freeze-drying process, the system undergoes extreme physical stresses (e.g., ice crystal formation, freeze concentration, dehydration stress) in both freezing and dehydration stages, which can easily lead to disruption of the fine structure of the adjuvant complex. The prior art faces challenges in addressing this problem. Complex adjuvants (e.g., lipo-AL complexes) often undergo irreversible particle aggregation during lyophilization, resulting in a significant increase in particle size and loss of physical uniformity after reconstitution. Such physical disruption directly results in a significant decrease in the biological activity of the adjuvant, for example, its ability to induce key antiviral cytokines (e.g., IFN- α), ultimately affecting the immune efficacy of the vaccine. Currently, although the addition of lyoprotectants (e.g., sugars such as sucrose) is commonly employed, for complex nano-microparticle mixed systems such as Lipo-AL, a single amorphous protectant is often insufficient to provide adequate protection. Under the condition of lacking enough mechanical support, the freeze-dried cake is easy to generate structural collapse in the sublimation drying stage, so that the appearance of the product is influenced, the re-dissolution is difficult, the re-dissolution time is prolonged or the re-dissolution is incomplete, and the use requirement of a vaccine adjuvant is not met. Disclosure of Invention The invention aims to provide a TLR7/8-lipo+AL freeze-dried vaccine adjuvant, and a preparation method and application thereof, which solve the problems of physical instability and loss of biological activity caused by particle aggregation and cake collapse of the TLR7/8-lipo+AL composite adjuvant in the freeze-drying process. In order to achieve the above purpose, the invention is realized by the following technical scheme: in a first aspect, the invention provides a TLR7/8-lipo+AL freeze-dried vaccine adjuvant, which adopts the following technical scheme: A TLR7/8-lipo+al lyophilized vaccine adjuvant which, prior to lyophilization, is a liquid comprising: a TLR 7/8-coupled-Lipo complex at a concentration of 5.0% to 10.0%; an aluminium salt adjuvant at a concentration of 1.0% to 3.0% in elemental aluminium; sucrose at a concentration of 2.0% to 3.0%; glycine at a concentration of 1.0% to 2.0%. By adopting the technical scheme, the invention introduces the specific combination of the sucrose and the glycine as the composite freeze-drying protective agent, and solves the technical problems of particle aggregation, structural damage and loss of biological activity caused by freeze-drying stress (such as ice crystal formation, dehydration and freeze concentration) in the freezing and dehydration processes of the Lipo-AL composite adjuvant. The protection mechanism is as follows: In the pre-freeze stage of freeze-drying, sucrose is used as an amorphous glass body forming agent to form a high-viscosity glassy matrix at low temperature. The substrate embeds and fixes Lipo-AL composite particles, hydrogen bonds are formed through space isolation and replacement of water molecules, growth of ice crystals is effectively inhibited, migration, collision and aggregation of the particles in a freeze concentration phase are prevented, and membrane structures of liposome are stabilized. Meanwhile, glycine is used as a crystallization type framework agent (or filler), and crystallization occurs in the pre-freezing process, so that a porous lattice framework structure is constructed. The glycine backbone provides the necessary mechanical support during the sublimation drying stage, preventing structural collapse of the lyophilized cake due to ice sublimation, ensuring macroscopic morphology of the product. Finally, the glassy matrix formed by sucrose maintains the fine structure and molecular dispersion of the Lipo-AL complex in a dehydrated state, while the glycine backb