TLR7 / 8-Lipo+ AL freeze-dried vaccine adjuvant as well as preparation method and application thereof

Abstract

The invention relates to the technical field of vaccine adjuvants, and discloses a TLR7 / 8-Lipo + AL freeze-dried vaccine adjuvant as well as a preparation method and application thereof, and the freeze-dried vaccine adjuvant comprises a TLR7 / 8-coupling-Lipo compound, a TLR7 / 8-Lipo + AL freeze-dried vaccine adjuvant, a TLR7 / 8-Lipo + AL freeze-dried vaccine adjuvant and a TLR7 / 8-Lipo + AL freeze-dried vaccine adjuvant, an aluminum salt adjuvant; sucrose at a concentration of 2.0% to 3.0%; the invention provides a Lipo-AL compound freeze-drying agent and a preparation method thereof, the Lipo-AL compound freeze-drying agent comprises sucrose and glycine, the concentration of the sucrose is 1.0-2.0%, the sucrose and the glycine are specifically combined for use, the problems of particle aggregation and structure collapse caused by freeze-drying stress in the Lipo-AL compound freeze-drying process are synergistically solved, the invention also provides a preparation method, and the preparation method comprises the following steps: preparing a Lipo compound, adsorbing with an aluminum salt adjuvant, adding the sucrose and the glycine, and preparing the Lipo-AL compound freeze-drying agent. And finally, carrying out freeze drying. The freeze-drying adjuvant prepared by the invention has the characteristics of complete form, quick redissolution and uniform particle size after redissolution, and can still keep high biological activity after long-term storage.

Description

Technical Field

[0001] This invention relates to the field of vaccine adjuvant technology, and in particular to a TLR7 / 8-Lipo+AL freeze-dried vaccine adjuvant, its preparation method, and its application. Background Technology

[0002] To ensure the long-term stability of vaccines during storage and transportation, freeze-drying is a widely used process for biological products, especially vaccine formulations. Meanwhile, to enhance the strength and durability of the immune response, modern vaccine development increasingly relies on combined adjuvant systems, such as combining Toll-like receptor (TLR) agonists with traditional aluminum salt adjuvants (e.g., aluminum hydroxide or aluminum phosphate).

[0003] However, these complex adjuvant systems, which include liposomes (Lipo), bioactive molecules (such as TLR7 / 8 agonists), and aluminum salt particles, are highly complex in structure and sensitive to the environment. During freeze-drying, the system undergoes extreme physical stresses in both freezing and dehydration stages (such as ice crystal formation, freeze concentration, and dehydration stress), which can easily lead to the destruction of the fine structure of the adjuvant complex.

[0004] Existing technologies face challenges in addressing this issue. Complex adjuvants (such as the Lipo-AL complex) often undergo irreversible particle aggregation during lyophilization, leading to a significant increase in particle size and loss of physical homogeneity upon reconstitution. This disruption of physical structure directly results in a significant decrease in the adjuvant's biological activity, such as a reduced ability to induce key antiviral cytokines (e.g., IFN-α), ultimately affecting the immunization efficacy of vaccines.

[0005] Currently, although the common approach is to add lyophilization protectants (such as sugars like sucrose), for complex nano- to micron-particle mixtures like Lipo-AL, a single amorphous protectant is often insufficient to provide adequate protection. Without sufficient mechanical support, the lyophilized cake is prone to structural collapse during the sublimation drying stage, affecting not only the product's appearance but also leading to difficulties in reconstitution, prolonged reconstitution time, or incomplete reconstitution, which does not meet the requirements for vaccine adjuvants. Summary of the Invention

[0006] The purpose of this invention is to provide a TLR7 / 8-Lipo+AL freeze-dried vaccine adjuvant, its preparation method, and its application, which solves the problem of physical instability and loss of biological activity caused by particle aggregation and cake collapse during the freeze-drying process of the TLR7 / 8-Lipo+AL composite adjuvant.

[0007] To achieve the above objectives, the present invention provides the following technical solution:

[0008] In a first aspect, the present invention provides a TLR7 / 8-Lipo+AL freeze-dried vaccine adjuvant, which adopts the following technical solution:

[0009] A TLR7 / 8-Lipo+AL lyophilized vaccine adjuvant, which is a liquid containing the following components before lyophilization:

[0010] A TLR7 / 8-coupled-Lipo complex at a concentration of 5.0% to 10.0%;

[0011] An aluminum salt adjuvant with a concentration of 1.0% to 3.0%, calculated as elemental aluminum;

[0012] Sucrose with a concentration of 2.0% to 3.0%;

[0013] Glycine at a concentration of 1.0% to 2.0%.

[0014] By adopting the above technical solution, this invention introduces a specific combination of sucrose and glycine as a composite freeze-drying protectant, which solves the technical problem of particle aggregation, structural damage and loss of bioactivity caused by freeze-drying stress (such as ice crystal formation, dehydration and freeze concentration) in the freezing and dehydration process of Lipo-AL composite adjuvant.

[0015] Its protection mechanism is as follows:

[0016] During the pre-freezing stage of freeze-drying, sucrose acts as an amorphous glass-forming agent, forming a high-viscosity glassy matrix at low temperatures. This matrix encapsulates and immobilizes the Lipo-AL complex particles, effectively inhibiting ice crystal growth and preventing particle migration, collision, and aggregation in the freeze-concentrated phase by spatial isolation and the substitution of water molecules for hydrogen bonding, thus stabilizing the membrane structure of the liposomes.

[0017] Meanwhile, glycine, as a crystalline framework agent (or filler), crystallizes during the pre-freezing process, forming a porous lattice framework structure.

[0018] During the sublimation drying stage, the glycine skeleton provides the necessary mechanical support, preventing the freeze-dried cake from collapsing due to ice sublimation and ensuring the macroscopic morphology of the product.

[0019] Ultimately, the glassy matrix formed by sucrose maintained the fine structure and molecular dispersion of the Lipo-AL complex under dehydration, while the glycine backbone ensured the rapid resolubility of the product.

[0020] Therefore, the freeze-dried composition prepared by the present invention has the characteristics of intact morphology and rapid reconstitution. After undergoing freeze-drying and reconstitution cycles, its key physical properties (such as Z-average particle size and polydispersity) and biological activities (such as the ability to induce IFN-α) are highly preserved, which improves the storage stability and shelf life of the adjuvant.

[0021] Preferably, the TLR7 / 8-conjugated-Lipo complex comprises: a TLR7 / 8 agonist-lipid conjugate; a cationic lipid; and an auxiliary lipid.

[0022] The molar ratio of the components TLR7 / 8 agonist-lipid conjugate, cationic lipid and auxiliary lipid is (5-10):(20-30):(60-75).

[0023] Preferably, the TLR7 / 8 agonist-lipid conjugate is an R848-PEG-DSPE conjugate or a loxoribin-PEG-DSPE conjugate; the cationic lipid is DOTAP; and the auxiliary lipid comprises DOPC and cholesterol.

[0024] Preferably, the aluminum salt adjuvant is an aluminum hydroxide adjuvant or an aluminum phosphate adjuvant.

[0025] Preferably, the Z-average particle size of the TLR7 / 8-coupled-Lipo complex (a) in the liquid is 280 nm to 300 nm.

[0026] By adopting the above technical solution, the specific components, ratios, and key physical parameters of the Lipo complex were further defined. Cationic lipids facilitate the formation of the Lipo complex and its subsequent binding with aluminum salt adjuvants, while the defined particle size range ensures that the adjuvant complex has uniform and suitable physicochemical properties before freeze-drying. This is the process basis for achieving stable freeze-drying and functional retention.

[0027] Secondly, the present invention provides a method for preparing the TLR7 / 8-Lipo+AL lyophilized vaccine adjuvant as described above, using the following technical solution:

[0028] A method for preparing a TLR7 / 8-Lipo+AL lyophilized vaccine adjuvant includes the following steps:

[0029] Prepare a suspension of the TLR7 / 8-coupled-Lipo complex;

[0030] The suspension obtained in step (S1) is mixed with the aluminum salt adjuvant for adsorption;

[0031] The sucrose and glycine are added to the mixture in step (S2) to obtain a pre-lyophilized liquid composition.

[0032] The pre-lyophilized liquid composition is freeze-dried.

[0033] By adopting the above technical solution, the method has clear steps. In particular, the order of adding the protective agent in step (S3) after the formation of the Lipo-AL complex ensures that the protective agent component can be uniformly dispersed in the complex suspension, rather than interfering with the adsorption process in S2. This provides a process guarantee for achieving uniform glassy embedding and framework support in the subsequent freeze-drying process.

[0034] Preferably, step (S1) includes: dissolving the lipid component containing the TLR7 / 8 agonist lipid conjugate, cationic lipid and auxiliary lipid in an organic solvent, and rotary evaporating to form a lipid film; adding an aqueous buffer to the lipid film for hydration; and performing sequential extrusion treatment on the suspension obtained after hydration.

[0035] By adopting the above technical solution and using the methods of thin film dispersion, hydration, and extrusion, a Lipo complex with uniform and controllable particle size can be prepared. This is a prerequisite for achieving effective and repeatable adsorption in step (S2) and ensuring the uniformity of the final product.

[0036] Preferably, the freeze-drying in step (S4) includes:

[0037] Pre-freeze at temperatures ranging from -50°C to -30°C;

[0038] Drying is carried out once at a temperature of -30℃ to -10℃;

[0039] A secondary drying process was carried out at a temperature of 20°C to 30°C. By adopting the above technical solution, optimized freeze-drying process parameters were established. A pre-freezing temperature of -50°C to -30°C ensured complete freezing of the system and allowed glycine to fully crystallize and form a framework; a primary drying temperature of -30°C to -10°C was below the system's critical collapse temperature, ensuring the stability of the sublimation process; and a secondary drying temperature of 20°C to 30°C helped remove residual bound water, further improving the long-term storage stability of the product.

[0040] Preferably, prior to step (S1), the method further includes a step of preparing the TLR7 / 8 agonist-lipid conjugate, which is selected from:

[0041] R848-COOH was coupled with DSPE-PEG2000-Amine via the EDC / NHS method;

[0042] Loxoribin-SH was coupled to DSPE-PEG2000-Maleimide via the maleimide method.

[0043] Thirdly, the present invention provides the application of the TLR7 / 8-Lipo+AL lyophilized vaccine adjuvant as described above in the preparation of vaccines for the prevention or treatment of viral infections.

[0044] In summary, the present invention has at least one of the following beneficial technical effects:

[0045] 1. This invention uses a specific concentration ratio of sucrose and glycine as a composite freeze-drying protectant. This combination works synergistically during freeze-drying. Glycine crystallizes to form a porous framework to prevent the collapse of the cake-like material, while sucrose forms an amorphous matrix embedding complex with a high glass transition temperature. This effectively inhibits particle aggregation and particle size increase of the Lipo-AL complex during freeze-drying, storage, and reconstitution, ensuring the physical homogeneity and stability of the adjuvant suspension.

[0046] 2. By maintaining the nanoscale particle size and structural integrity of the Lipo-AL complex, the TLR7 / 8 agonist coupled to it can maintain the correct delivery characteristics and biological activity. The ability of the composition of the present invention to induce key cytokines such as IFN-α remains at a high level after lyophilization cycles and accelerated storage, which is superior to the comparative example using only a single protectant.

[0047] 3. By introducing glycine as a crystalline framework agent, this invention solves the problem of structural collapse of freeze-dried cakes that is prone to occur in traditional amorphous protective agents, providing excellent mechanical support and giving the final product a complete and full macroscopic morphology. At the same time, this porous structure also significantly improves the resolubility of the product, enabling it to be quickly and completely redispersed after being added to water for injection, which meets the requirements for use as a vaccine adjuvant. Detailed Implementation

[0048] Preparation Examples 1-2:

[0049] Preparation Example 1: Preparation of R848-PEG-DSPE conjugate (conjugate 1) (EDC / NHS method)

[0050] This preparation example is used to prepare a coupling of R848-COOH with 1,2-distearyl-sn-glycerol-3-phosphatidylethanolamine-N-[amino(polyethylene glycol)-2000] (DSPE-PEG2000-Amine).

[0051] Add 10 mg of R848-COOH (molecular formula C) 21 H 27 N5O3 was dissolved in 2 mL of anhydrous DMSO.

[0052] Add 15 mg EDC·HCl (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride) and 9 mg Sulfo-NHS (sodium N-hydroxysulfosuccinimide) to the above solution, stir at room temperature (20°C to 25°C) in the dark, and activate the carboxyl group for 30 min.

[0053] Dissolve another 150 mg of DSPE-PEG2000-Amine in 5 mL of HEPES buffer (50 mM, pH 8.0).

[0054] The activated R848-COOH solution from step (2) was slowly added dropwise to the lipid solution from step (3), and the reaction was continued at room temperature (20°C to 25°C) in the dark for 4 to 6 hours with stirring.

[0055] After the reaction was completed, the reaction mixture was transferred to a dialysis bag (MWCO 3.5kDa 3.5kDa) and dialyzed with water for injection (WFI) at 4°C for 48 hours, during which the external dialysis fluid was changed 6 times.

[0056] The liquid in the dialysis bag was collected, filtered through a 0.22 μm filter membrane for sterilization, and then freeze-dried to obtain a white to slightly yellow powdery solid, which is conjugate 1 (R848-PEG-DSPE).

[0057] Preparation Example 2: Preparation of loxoribin-PEG-DSPE conjugate (conjugate 2) (maleimide method)

[0058] This preparation example is used to prepare a coupling of loxoribin-SH and 1,2-distearyl-sn-glycerol-3-phosphatidylethanolamine-N-[maleimide (polyethylene glycol)-2000] (DSPE-PEG2000-Maleimide).

[0059] 15 mg of loxoribin-SH (molecular formula C) was added. 20 H 28 N6O5S was dissolved in 5 mL of phosphate buffer (PBS, pH 7.0, containing 2 mM EDTA).

[0060] Dissolve another 160 mg of DSPE-PEG2000-Maleimide in 5 mL of the same PBS buffer.

[0061] Under room temperature (20°C to 25°C) and nitrogen protection, the lipid solution from step (2) was slowly added dropwise to the loxoribin solution from step (1).

[0062] The mixed solution was stirred and reacted at room temperature (20°C to 25°C) in the dark for 2 to 4 hours.

[0063] After the reaction was completed, the reaction mixture was transferred to a dialysis bag (MWCO 3.5 kDa) and dialyzed with water for injection (WFI) at 4°C for 48 h, during which the external dialysis fluid was changed 6 times.

[0064] The liquid in the dialysis bag was collected, filtered through a 0.22 μm filter membrane for sterilization, and then freeze-dried to obtain a white to slightly yellow powdery solid, which is conjugate 2 (loxoribin-PEG-DSPE).

[0065] Examples 1-5:

[0066] Example 1:

[0067] This embodiment provides a lyophilized adjuvant composition comprising coupling compound 1, aluminum phosphate adjuvant, sucrose, and glycine, wherein the sucrose concentration is 2.5% and the glycine concentration is 1.5%. Specific preparation steps include:

[0068] Preparation of TLR7 / 8-Conjugated-Lipo Complex: Conjugate 1 (R848-PEG-DSPE), DOTAP, DOPC, and cholesterol prepared in Preparation Example 1 were accurately weighed to a molar ratio of 5:20:45:30. The lipid components were dissolved together in a chloroform / methanol (v / v=3:1) mixed solvent. This organic phase was placed in a round-bottom flask and rotary evaporated (60 rpm) in a 40°C water bath to remove the organic solvent, forming a uniform lipid film. The flask was dried in a vacuum desiccator for 4 hours. 10 mM histidine buffer (pH 6.5) was added to the lipid film, and the film was hydrated in a 50°C water bath for 1 hour. The resulting multi-compartment liposome suspension was extruded sequentially through 400 nm and 100 nm polycarbonate membranes, 10 times each. The homogenized suspension was filtered through a 0.22 μm filter membrane for sterilization to obtain a concentrated TLR7 / 8-conjugated-Lipo complex solution for later use.

[0069] Preparation of the compound adjuvant suspension: Under aseptic conditions, the concentrated Lipo complex obtained in step (1) was diluted with 10 mM histidine buffer (pH 6.5). Subsequently, the aseptic suspension of aluminum phosphate adjuvant was slowly added, and the mixture was slowly stirred (100 rpm) for 60 min at room temperature (20°C to 25°C) for adsorption.

[0070] Preparation of the pre-lyophilized composition: Add sucrose and glycine to the composite adjuvant suspension obtained in step (2), and bring the volume up with water for injection (WFI) to make the final concentrations of each component as follows: TLR7 / 8-coupled-Lipo complex (based on total lipids) 7.5%, aluminum salt (based on elemental aluminum) 2.0%, sucrose 2.5%, and glycine 1.5%. Adjust the pH to 6.8.

[0071] Freeze-drying: Dispense the pre-freeze-dried liquid composition prepared in step (3) into vials at a rate of 0.5 mL / vial, and partially stopper them. Transfer the vials to a freeze dryer and perform the following procedure:

[0072] (1) Pre-freezing stage: The shelf temperature is reduced to -40℃ and maintained for 3 hours.

[0073] (2) First drying stage: The chamber pressure drops to 10Pa and the shelf temperature rises to -20℃, and is kept constant for 30h.

[0074] (3) Secondary drying stage: Maintain the chamber pressure, raise the shelf temperature to 25°C at a rate of 0.2°C / min, keep it constant for 10 hours, after drying, fill with sterile nitrogen gas, seal the cap, and obtain the freeze-dried adjuvant composition of Example 1.

[0075] Example 2:

[0076] This embodiment provides a lyophilized adjuvant composition comprising coupling compound 1, aluminum phosphate adjuvant, sucrose, and glycine, wherein the sucrose concentration is 2.0% and the glycine concentration is 1.0%. Specific preparation steps include:

[0077] Preparation of TLR7 / 8-coupled-Lipo complex: Same as step (1) in Example 1.

[0078] Preparation of the compound adjuvant suspension: Same as step (2) in Example 1.

[0079] Preparation of the pre-lyophilized composition: Add sucrose and glycine to the composite adjuvant suspension obtained in step (2), and bring the volume up with water for injection (WFI) to make the final concentrations of each component as follows: TLR7 / 8-coupled-Lipo complex (based on total lipids) 7.5%, aluminum salt (based on elemental aluminum) 2.0%, sucrose 2.0%, and glycine 1.0%. Adjust the pH to 6.8.

[0080] Freeze-drying: Same as step (4) in Example 1.

[0081] Example 3:

[0082] This embodiment provides a lyophilized adjuvant composition comprising coupling compound 1, aluminum phosphate adjuvant, sucrose, and glycine, wherein the sucrose concentration is 3.0% and the glycine concentration is 2.0%. Specific preparation steps include:

[0083] Preparation of TLR7 / 8-coupled-Lipo complex: Same as step (1) in Example 1.

[0084] Preparation of the compound adjuvant suspension: Same as step (2) in Example 1.

[0085] Preparation of the pre-lyophilized composition: Add sucrose and glycine to the composite adjuvant suspension obtained in step (2), and bring the volume up with water for injection (WFI) to make the final concentrations of each component as follows: TLR7 / 8-coupled-Lipo complex (based on total lipids) 7.5%, aluminum salt (based on elemental aluminum) 2.0%, sucrose 3.0%, glycine 2.0%. Adjust the pH to 6.8.

[0086] Freeze-drying: Same as step (4) in Example 1.

[0087] Example 4:

[0088] This embodiment provides a lyophilized adjuvant composition comprising coupling compound 2, aluminum phosphate adjuvant, sucrose, and glycine, wherein the sucrose concentration is 2.5% and the glycine concentration is 1.5%. Specific preparation steps include:

[0089] Preparation of TLR7 / 8-conjugated-Lipo complex: Accurately weigh conjugate 2 (loxoribin-PEG-DSPE), DOTAP, DOPC and cholesterol obtained in Preparation Example 2, so that their molar ratio is 5:20:45:30. Subsequent preparation steps are the same as step (1) in Example 1.

[0090] Preparation of the compound adjuvant suspension: Under aseptic conditions, the concentrated Lipo complex obtained in step (1) was diluted with 10 mM histidine buffer (pH 6.5). Subsequently, the aseptic suspension of aluminum phosphate adjuvant was slowly added, and the mixture was slowly stirred (100 rpm) for 60 min at room temperature (20°C to 25°C) for adsorption.

[0091] Preparation of the pre-lyophilized composition: Add sucrose and glycine to the composite adjuvant suspension obtained in step (2), and bring the volume up with water for injection (WFI) to make the final concentrations of each component as follows: TLR7 / 8-coupled-Lipo complex (based on total lipids) 7.5%, aluminum salt (based on elemental aluminum) 2.0%, sucrose 2.5%, and glycine 1.5%. Adjust the pH to 6.8.

[0092] Freeze-drying: Same as step (4) in Example 1.

[0093] Example 5:

[0094] This embodiment provides a lyophilized adjuvant composition comprising conjugate 1, aluminum phosphate adjuvant, sucrose, and glycine, wherein the lipid molar ratio of the Lipo complex varies. Specific preparation steps include:

[0095] Preparation of TLR7 / 8-conjugated-Lipo complex: Accurately weigh conjugate 1 (R848-PEG-DSPE), DOTAP, DOPC and cholesterol obtained in Preparation Example 1, so that their molar ratio is 10:30:40:20. Subsequent preparation steps are the same as step (1) in Example 1.

[0096] Preparation of the compound adjuvant suspension: Same as step (2) in Example 1.

[0097] Preparation of the pre-lyophilized composition: Add sucrose and glycine to the composite adjuvant suspension obtained in step (2), and bring the volume up with water for injection (WFI) to make the final concentrations of each component as follows: TLR7 / 8-coupled-Lipo complex (based on total lipids) 7.5%, aluminum salt (based on elemental aluminum) 2.0%, sucrose 2.5%, and glycine 1.5%. Adjust the pH to 6.8.

[0098] Freeze-drying: Same as step (4) in Example 1.

[0099] Comparative Examples 1-6:

[0100] The difference between Comparative Example 1 and Example 1 is that sucrose and glycine are not added in the preparation of the pre-freeze-dried composition in step (3), while the rest are the same.

[0101] Compared with Example 1, Comparative Example 2 differs in that: in the preparation of the pre-freeze-dried composition in step (3), glycine is not added, and the final concentration of sucrose is adjusted to 4.0% (i.e., the same as the total concentration of the protectant in Example 1), while the rest are the same.

[0102] Compared with Example 1, Comparative Example 3 differs in that: in the preparation of the pre-freeze-dried composition in step (3), sucrose is not added, and the final concentration of glycine is adjusted to 4.0% (that is, the same as the total concentration of the protectant in Example 1), while the rest are the same.

[0103] Compared with Example 1, Comparative Example 4 differs in that in the preparation of the pre-freeze-dried composition in step (3), 2.5% sucrose and 1.5% glycine are replaced with 4.0% trehalose, while the rest are the same.

[0104] Compared with Example 1, Comparative Example 5 differs in that in the preparation of the pre-freeze-dried composition in step (3), 2.5% sucrose and 1.5% glycine are replaced with 4.0% mannitol, while the rest are the same.

[0105] Compared with Example 1, Comparative Example 6 differs in that in the preparation of the pre-freeze-dried composition in step (3), 2.5% sucrose and 1.5% glycine are replaced with 2.5% trehalose and 1.5% arginine, while the rest are the same.

[0106] Test Examples 1-4:

[0107] Test Example 1: Reconstitution and Antigen Adsorption Capacity Test of Lyophilized Adjuvants

[0108] This test case is used to evaluate the basic physical properties and functional feasibility of the lyophilized adjuvant compositions prepared in the examples.

[0109] Experimental steps:

[0110] (1) Appearance and Reconstitution: Remove the lyophilized vials from Examples 1-5 from the freeze dryer and visually inspect the appearance and integrity of the lyophilized cake at room temperature, recording any collapse or shrinkage. Then, inject 1.0 mL of water for injection (WFI) as the reconstitution medium into the vial, gently shake, and use a stopwatch to record the time required for the lyophilized cake to completely dissolve or disperse (reconstitution time). Observe the appearance of the reconstituted suspension, recording whether it is uniform and whether there is any visible aggregation or precipitation.

[0111] (2) pH value determination: After the sample is completely reconstituted, the pH value of the reconstituted suspension is measured using a calibrated pH meter.

[0112] (3) Antigen adsorption rate determination:

[0113] Take 0.5 mL of the reconstituted adjuvant suspension (Examples 1-5).

[0114] Add 0.5 mL of histidine buffer containing 100 μg / mL recombinant gE antigen to the suspension and mix well.

[0115] Incubate the antigen-adjuvant mixture at 4°C for 30 minutes.

[0116] After incubation, the mixture was heated at 10000× Centrifuge for 5 minutes under the specified conditions to precipitate the adjuvant-antigen complex.

[0117] Carefully aspirate the supernatant.

[0118] The protein concentration in the supernatant was determined using the BCA protein quantification kit according to standard operating procedures. Simultaneously, the concentration of the original antigen solution was measured. ).

[0119] The formula for calculating antigen adsorption rate (%) is:

[0120] .

[0121] Experimental data:

[0122] Table 1. Physical properties and antigen adsorption capacity of the lyophilized adjuvants in the examples.

[0123]

[0124] in conclusion:

[0125] The data from Test Example 1 (Table 1) show that the lyophilized adjuvants prepared in Examples 1-5 all yielded intact lyophilized cakes without severe collapse, indicating that the selected protective agent formulation provided sufficient mechanical support.

[0126] The innovative mechanism of this invention lies in the specific combination of sucrose as a glass-forming agent (lyophilization protectant) and glycine as a crystalline framework agent (filler). During the freezing stage, this combination inhibits excessive ice crystal growth; during the sublimation drying stage, the porous lattice structure formed by glycine provides physical support, while the amorphous glass formed by sucrose embeds the TLR7 / 8-coupled-Lipo complex and aluminum salt matrix within it, restricting molecular migration and stabilizing the liposome membrane structure.

[0127] Table 1 shows that the reconstitution time for all examples was less than 45 seconds, and the pH value stabilized at around 6.8 after reconstitution. The antigen adsorption rate data indicates that the physicochemical properties (such as charge and surface area) of the adjuvant complex were effectively maintained after lyophilization and reconstitution cycles, without structural damage or loss of functional sites due to lyophilization stress.

[0128] Test Example 2: Comparison of physical stability before and after freeze-drying (particle size and PDI)

[0129] This test example is used to quantitatively evaluate the ability of the lyophilized adjuvant compositions of the present invention (Examples 1-5) and comparative examples (Comparative Examples 1-6) to maintain their particle physical stability after undergoing freeze-drying and reconstitution cycles.

[0130] Experimental steps:

[0131] Sample preparation:

[0132] Samples before freeze-drying: Take the pre-freeze-dried liquids of Examples 1-5 and Comparative Examples 1-6 after the preparation of the pre-freeze-dried composition in step (3) and before dispensing for freeze-drying.

[0133] Freeze-dried samples: Take the freeze-dried vials finally prepared in Examples 1-5 and Comparative Examples 1-6 respectively, add 0.5 mL of water for injection (WFI) to each vial for reconstitution, and shake until homogeneous.

[0134] Sample dilution: Take an appropriate amount of the sample before or after lyophilization and dilute it with 10mM histidine buffer (pH 6.5) to the optimal detection concentration range of the dynamic light scattering (DLS) instrument.

[0135] DLS determination: Using a dynamic light scattering instrument (e.g., Malvern Zetasizer Nano ZS), the samples were equilibrated at 25°C for 120 s. The Z-average particle size (Z-Average Size, nm) and polydispersity index (PDI) of the samples were determined. Each sample was measured in triplicate, and the average value was taken.

[0136] Data processing: Calculate the particle size change rate (Δ%) using the formula: ((particle size after reconstitution - particle size before freeze-drying) / particle size before freeze-drying) × 100%.

[0137] Experimental data:

[0138] Table 2 Comparison of particle size and PDI before and after freeze-drying in the examples and comparative examples

[0139]

[0140] in conclusion:

[0141] The mechanism of this invention lies in the specific combination of sucrose as an amorphous glass-forming agent (providing cryogenic protection) and glycine as a crystalline framework agent (providing mechanical support). During freezing, sucrose forms a high-viscosity glassy matrix that embeds and fixes the Lipo-AL complex, inhibiting ice crystal growth and particle migration; while the crystallization of glycine constructs a porous framework, preventing the collapse of the freeze-dried cake.

[0142] The DLS data in Table 2 support this mechanism. In Examples 1-5, after freeze-drying and rehydration, the Z-mean particle size change rate was controlled below 12%, and the PDI value remained at a low level, indicating that the physical stability of the particles was effectively maintained and no significant aggregation occurred.

[0143] In contrast, Comparative Example 1 (without a protective agent) exhibited uncontrolled aggregation (Δ% > 400%), demonstrating the destructive effects of lyophilization stress (freezing and dehydration) on the Lipo-AL complex. Comparative Examples 3 (glycine only) and 5 (mannitol only) also showed severe aggregation, indicating that crystalline matrix agents alone cannot provide sufficient cryoprotection (amorphous embedding) to prevent liposome membrane fusion. While the aggregation levels in Comparative Examples 2 (sucrose only) and 4 (trehalose only) were lower than those in Comparative Examples 1, 3, and 5, they were still significantly higher than in the examples. This suggests that vitreous forming agents alone, in the absence of matrix support, may lead to micro-collapse during lyophilization or insufficient interparticle spacing upon reconstitution, inducing aggregation.

[0144] Test Example 3: Comparison of adjuvant bioactivity before and after freeze-drying (in vitro activity)

[0145] This test case is used to evaluate the retention of key biological activities (induction of TLR7 / 8 pathway) of the lyophilized adjuvant compositions of the present invention (Examples 1-5) and comparative examples (Comparative Examples 1-6) after undergoing lyophilization and reconstitution cycles.

[0146] Experimental steps:

[0147] (1) Isolation of human PBMCs: Human peripheral blood mononuclear cells (PBMCs) were isolated from heparin-anticoagulated peripheral blood obtained from healthy volunteers using the Ficoll-Paque density gradient centrifugation method.

[0148] (2) Cell culture: PBMCs were resuspended in RPMI-1640 medium containing 10% FBS, and the cell density was adjusted to 1.0 × 10⁶ cells / year. 6 Cells / mL. Seed the cell suspension (200 μL / well) into 96-well cell culture plates.

[0149] (3) Sample preparation and stimulation:

[0150] Take the lyophilized vials from Examples 1-5 and Comparative Examples 1-6, and add 0.5 mL of water for injection (WFI) to reconstitute them.

[0151] Another sample of the pre-lyophilized liquid from Example 1 (i.e. the original liquid before lyophilization) was taken as a 100% activity control (hereinafter referred to as Example 1 - before lyophilization).

[0152] All the above samples were diluted with RPMI-1640 medium to make the final concentration of TLR7 / 8 agonists in the samples (after adding cells) 1.0 μg / mL.

[0153] Set up a blank control culture medium (Medium).

[0154] Add the diluted sample to the corresponding 96-well plate, 20 μL per well.

[0155] (4) Incubation and Collection: The cell culture plates were incubated at 37°C, 5% CO2 for 24 hours. After incubation, the 96-well plates were incubated at 500× Centrifuge for 5 minutes under the specified conditions and collect the cell culture supernatant.

[0156] (5) ELISA detection: The concentration (pg / mL) of IFN-α in the supernatant was detected by using the human IFN-α ELISA kit and following the instructions.

[0157] Experimental data:

[0158] Table 3. Levels of IFN-α induced in PBMCs after reconstitution in the examples and comparative examples.

[0159]

[0160] in conclusion:

[0161] The mechanism of this invention involves the use of a specific combination of sucrose (a glass-forming agent) and glycine (a crystalline framework agent) to stabilize the TLR7 / 8-coupled-Lipo complex with an aluminum salt matrix during freeze-drying. This combination aims to maintain the physical structure of the complex during dehydration and resolvation through amorphous encapsulation and porous framework support.

[0162] Data from Test Example 3 assessed whether this physical protection translated into retention of biological function. Using the IFN-α level before lyophilization in Example 1 (2150.0 pg / mL) as a baseline, Examples 1-5 retained their biological activity after lyophilization and reconstitution (e.g., Example 1 at 2045.7 pg / mL). This indicates that the combination of sucrose and glycine effectively maintains the fine structure of the Lipo-AL complex, ensuring that the TLR7 / 8 agonist remains effectively recognized by cells after reconstitution, thereby initiating downstream signaling pathways.

[0163] In contrast, all comparative examples showed a significant decrease in bioactivity. The most severe loss of activity was observed in Comparative Example 1 (no protectant), Comparative Example 3 (glycine only), and Comparative Example 5 (mannitol only), which is highly consistent with the severe physical aggregation observed in Test Example 2. This confirms that the absence of cryoprotection from a glassy matrix and the presence of a crystalline framework agent alone leads to irreversible structural damage and loss of function in the complex during lyophilization.

[0164] Comparative Examples 2 (sucrose only) and 4 (trehalose only) retained some activity (approximately 780-850 pg / mL), but still showed a loss of activity compared to the examples. This indicates that while vitreous forming agents alone can provide some cryogenic protection, in the absence of the mechanical support framework provided by glycine, the microstructural collapse during freeze-drying or the localized high concentration stress during reconstitution is still sufficient to impair the biological functions of the Lipo-AL complex.

[0165] Test Example 4: Accelerated Stability Test

[0166] This test case is used to evaluate the long-term physical and functional stability of the lyophilized adjuvant compositions of the present invention (represented by Example 1) and key comparative examples (Comparative Example 1 and Comparative Example 2) under accelerated conditions (25°C / 60%RH).

[0167] Experimental steps:

[0168] (1) Sample storage: Take the final freeze-dried sealed vials of Example 1, Comparative Example 1 and Comparative Example 2 and place them in a constant temperature and humidity chamber at 25°C and 60% relative humidity (RH).

[0169] (2) Sampling time points: Samples were taken out at 0 months (initial), 1 month, 3 months and 6 months of storage (n=3 / group / time point).

[0170] (3) Sample analysis:

[0171] At each time point, the extracted samples were reconstituted with 0.5 mL of water for injection (WFI).

[0172] Physical stability: Following the experimental steps (2)-(4) of Test Example 2, the Z-average particle size (nm) and PDI of the reconstituted sample were determined using DLS.

[0173] Functional stability: Following the experimental steps (1)-(5) of Test Example 3, the concentration (pg / mL) of IFN-α induced by the reconstituted sample was determined using PBMC and ELISA.

[0174] Experimental data:

[0175] Table 4 Accelerated stability (25℃ / 60%RH) test data

[0176]

[0177] in conclusion:

[0178] (The N / A marking here indicates that the physical stability of the sample has been completely lost, and its PDI value cannot be obtained because it exceeds the effective detection limit of the instrument).

[0179] The adjuvant stabilization mechanism of this invention relies on a combination of sucrose (a glass-forming agent) and glycine (a crystalline framework agent). This combination aims to form an amorphous protective matrix with a high glass transition temperature (Tg), while the glycine crystals provide a mechanical framework to prevent structural collapse and molecular migration of the freeze-dried cake during storage, especially at high temperatures.

[0180] The data in Table 4 validates this mechanism. Example 1 (of the present invention) was stored at 25°C for 6 months under accelerated conditions, and its Z-average particle size and PDI did not show a significant increase. This indicates that the Tg of this formulation is sufficiently high to effectively fix the physical structure of the Lipo-AL complex at 25°C, inhibiting its degradation and aggregation.

[0181] Comparative Example 1 (without protectant) was already ineffective at 0 months (physical aggregation and low activity) and further degraded during storage.

[0182] In Comparative Example 2 (sucrose only), this formulation already exhibited suboptimal physical state (particle size 649.0 nm) and function (IFN-α 780.5 pg / mL) at 0 months, indicating that the lack of a glycine backbone may lead to micro-collapse during lyophilization. During storage at 25°C, Comparative Example 2 showed a clear trend towards physical aggregation and loss of function. This suggests that the Tg of the sucrose vitreous matrix alone may be insufficient to maintain stability at 25°C, or its mechanical strength may be insufficient to prevent the slow aggregation and structural rearrangement of the Lipo-AL complex over time.

Claims

1. A TLR7 / 8-Lipo+AL lyophilized vaccine adjuvant, characterized in that, include: TLR7 / 8-coupled-Lipo complexes at concentrations ranging from 5.0% to 10.0%; Aluminum salt adjuvants with concentrations ranging from 1.0% to 3.0%; Sucrose with a concentration of 2.0% to 3.0%; Glycine at a concentration of 1.0% to 2.0%.

2. The TLR7 / 8-Lipo+AL lyophilized vaccine adjuvant according to claim 1, characterized in that, The TLR7 / 8-coupled-Lipo complex comprises: TLR7 / 8 agonist-lipid conjugates; cationic lipids; cofactor lipids; The molar ratio of the components TLR7 / 8 agonist-lipid conjugate, cationic lipid and auxiliary lipid is (5-10):(20-30):(60-75).

3. The TLR7 / 8-Lipo+AL lyophilized vaccine adjuvant according to claim 2, characterized in that, The TLR7 / 8 agonist-lipid conjugate is either an R848-PEG-DSPE conjugate or a loxoribin-PEG-DSPE conjugate. The cationic lipid is DOTAP; The auxiliary lipids include DOPC and cholesterol.

4. The TLR7 / 8-Lipo+AL lyophilized vaccine adjuvant according to claim 1, characterized in that, The aluminum salt adjuvant is either aluminum hydroxide adjuvant or aluminum phosphate adjuvant.

5. The TLR7 / 8-Lipo+AL lyophilized vaccine adjuvant according to claim 1, characterized in that, The TLR7 / 8-coupled-Lipo complex has a Z-average particle size of 280 nm to 300 nm in liquid.

6. A method for preparing the TLR7 / 8-Lipo+AL lyophilized vaccine adjuvant as described in any one of claims 1-5, characterized in that, Includes the following steps: (S1) Prepare a suspension of the TLR7 / 8-coupled-Lipo complex; (S2) The suspension obtained in step (S1) is mixed with aluminum salt adjuvant for adsorption; (S3) Add sucrose and glycine to the mixture in step (S2) to obtain a pre-lyophilized liquid composition; (S4) Freeze-dry the pre-freeze-dried liquid composition.

7. The method for preparing a TLR7 / 8-Lipo+AL lyophilized vaccine adjuvant according to claim 6, characterized in that, Step (S1) includes: The lipid component containing the TLR7 / 8 agonist-lipid conjugate, cationic lipids, and auxiliary lipids was dissolved in an organic solvent and then rotary evaporated to form a lipid film. Aqueous buffer solution was added to the lipid membrane for hydration; The suspension obtained after hydration is subjected to sequential extrusion treatment.

8. The method for preparing a TLR7 / 8-Lipo+AL lyophilized vaccine adjuvant according to claim 6, characterized in that, The freeze-drying in step (S4) includes: Pre-freeze at temperatures ranging from -50°C to -30°C; Dry once at a temperature of -30℃ to -10℃; Secondary drying is carried out at a temperature of 20℃ to 30℃.

9. The method for preparing a TLR7 / 8-Lipo+AL lyophilized vaccine adjuvant according to claim 6, characterized in that, Prior to step (S1), the method further includes a step of preparing the TLR7 / 8 agonist-lipid conjugate, wherein the step of preparing the TLR7 / 8 agonist-lipid conjugate is selected from: R848-COOH was coupled with DSPE-PEG2000-Amine via the EDC / NHS method; Loxoribin-SH was coupled to DSPE-PEG2000-Maleimide via the maleimide method.

10. The use of the TLR7 / 8-Lipo+AL lyophilized vaccine adjuvant as described in any one of claims 1-5 in the preparation of a vaccine for the prevention or treatment of viral infections.

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