TLR9+HA201 composite adjuvant as well as preparation method and application thereof

Abstract

The invention relates to the technical field of vaccine adjuvants, and discloses a TLR9 + HA201 composite adjuvant as well as a preparation method and application thereof, the composite adjuvant is a freeze-dried preparation, and is prepared by freeze-drying a suspension containing lipidosome, saponin, a TLR9 ligand, HA201 molecules, trehalose and glutamic acid; the lipidosome comprises distearoyl phosphatidylcholine and cholesterol in a molar ratio of 1: 0.5 to 1: 2, the content of saponin accounts for 1.0%-3.0% of the total lipid weight in the lipidosome, the concentration of trehalose is 5%-15%, and the concentration of glutamic acid is 0.5%-3%. According to the invention, trehalose and glutamic acid are adopted as a composite freeze-drying protective agent, and the protective effect of a vitrification matrix formed by trehalose and the pH buffer effect of glutamic acid in the freezing process are utilized, so that the problem that multiple active components are easy to degrade, aggregate or leak when coexisting in a liquid phase is solved; therefore, the long-term storage stability of the preparation is improved.

Description

Technical Field

[0001] This invention relates to the field of vaccine adjuvant technology, and in particular to a TLR9+HA201 composite adjuvant, its preparation method and application. Background Technology

[0002] Vaccines are essential biological products for the prevention and control of infectious diseases. Many modern vaccines, such as subunit vaccines, recombinant protein vaccines, or nucleic acid vaccines, have relatively weak autoimmunogenicity. Therefore, they rely on vaccine adjuvants to enhance and modulate the body's immune response to specific antigens, inducing rapid, effective, and long-lasting protective immunity. Currently, various types of adjuvants, including liposomes, Toll-like receptor agonists, and saponin adjuvants, have shown significant application value in the development of novel vaccines.

[0003] To induce stronger or more comprehensive immune activation, existing technologies have attempted to combine adjuvants with different mechanisms of action. However, these combinations face significant formulation challenges. On one hand, integrating multiple chemically diverse active components into a homogeneous delivery system is extremely difficult; simple physical mixing of the components cannot guarantee that different adjuvant components can be simultaneously delivered to the same antigen-presenting cell, hindering their synergistic effects. On the other hand, such complex systems containing multiple biomolecules exhibit poor physicochemical stability in liquid suspensions, with active components prone to degradation, leakage, or aggregation. Although freeze-drying is a common method to improve stability, existing freeze-drying protectant formulations are generally insufficient to effectively protect such multi-component complex systems, still leading to particle aggregation and loss of activity during freezing and reconstitution.

[0004] Furthermore, when using particulate carriers such as liposomes to deliver adjuvants, their physicochemical properties, especially particle size and uniformity, are crucial to the delivery efficiency and cellular uptake of the adjuvant. In the preparation of complex liposomes encapsulating multiple active components, existing processes struggle to precisely control the particle size distribution of the final product. Poor formulation uniformity or inappropriate particle size directly reduces its efficiency in being effectively taken up by antigen-presenting cells, thereby affecting the bioavailability of the adjuvant and the stability of the final immune-enhancing effect. Summary of the Invention

[0005] The purpose of this invention is to provide a TLR9+HA201 composite adjuvant, its preparation method and application, which solves the problems of unstable physicochemical properties and difficulty in long-term storage when multiple bioactive components coexist in existing composite adjuvant systems, and the difficulty in co-delivering each adjuvant component through a uniform carrier, resulting in poor synergistic activation effect and insufficient immune response spectrum.

[0006] To achieve the above objectives, the first aspect of the present invention provides a TLR9+HA201 composite adjuvant, wherein the composite adjuvant is a lyophilized formulation obtained by freeze-drying a suspension, and the suspension comprises the following components:

[0007] Liposomes comprising DSPC and cholesterol, wherein the molar ratio of DSPC to cholesterol is 1:0.5 to 1:2;

[0008] QS-21, wherein the content of QS-21 is 1.0% to 3.0% of the total lipid weight in the liposomes;

[0009] TLR9 ligand;

[0010] HA201 molecule;

[0011] Trehalose;

[0012] Glutamic acid;

[0013] Buffer solution;

[0014] Prior to freeze-drying, the concentration of trehalose is 5% (w / v) to 15% (w / v) and the concentration of glutamic acid is 0.5% (w / v) to 3% (w / v) based on the total volume of the suspension.

[0015] By employing the above technical solution, this invention provides a composite adjuvant system comprising three immunostimulants: TLR9 ligand, HA201 molecule, and QS-21. To address the technical problem that multiple bioactive components and liposome carriers are prone to degradation, aggregation, or leakage when coexisting in an aqueous medium, resulting in poor physicochemical stability and short storage periods, this invention establishes a specific lyophilized formulation.

[0016] The innovation of this invention lies in identifying a specific combination of trehalose and glutamic acid as freeze-drying protectants and defining their concentration range in the suspension before freeze-drying. This specific combination has a synergistic effect on maintaining the stability of complex systems containing DSPC / cholesterol liposomes, TLR9 ligands, HA201 molecules, and QS-21 under freeze-drying stress, and the mechanism is as follows:

[0017] During the freezing stage, trehalose at a specific concentration acts as a highly efficient vitrification matrix forming agent. By replacing water molecules, it forms hydrogen bonds with the liposome membrane surface and biomolecules, resulting in a high-viscosity amorphous vitrified matrix at low temperatures. This matrix effectively inhibits the growth and recrystallization of ice crystals, preventing mechanical damage to the liposome vesicle structure caused by ice crystals.

[0018] During the drying stage, the glassy network structure formed by trehalose provides mechanical support, preventing the liposomes from collapsing, fusing, or aggregating during the dehydration process.

[0019] In this system, glutamate not only acts as an auxiliary lyophilization protectant but also serves as a crucial pH buffer and stabilizer. During freezing, when the buffer components crystallize, causing a drastic pH shift, glutamate effectively buffers this change. Simultaneously, during dehydration and reconstitution, it helps maintain the system's pH within the optimal range, preventing hydrolysis or conformational changes in the TLR9 ligand or HA201 molecules due to pH variations.

[0020] Through the synergistic effect of the above components, the lyophilized compound adjuvant formulation of the present invention solves the instability problem of complex adjuvant systems in the liquid state and achieves excellent long-term storage stability. Upon reconstitution, the formulation can rapidly return to a homogeneous suspension state, maintaining the original particle size, intact structure, and biological activity of the adjuvant complex.

[0021] Preferably, in the above-mentioned compound adjuvant, the concentration of trehalose is 10% (w / v), the concentration of glutamic acid is 1% (w / v), and the molar ratio of DSPC to cholesterol is 1:1.

[0022] By adopting the above-mentioned preferred technical solution, the optimal formulation parameters of the compound adjuvant of the present invention can be obtained. Under these parameters, the stability, resolubility and adjuvant activity of the lyophilized formulation can achieve the optimal balance.

[0023] Preferably, in the above-mentioned compound adjuvant, before freeze-drying, the total lipid concentration of the liposomes in the suspension is 15 mg / mL to 25 mg / mL, the concentration of TLR9 ligand is 0.8 mg / mL to 1.2 mg / mL, and the concentration of HA201 molecules is 0.4 mg / mL to 0.6 mg / mL.

[0024] By adopting the above-mentioned preferred technical solution, the concentration range of each active component is limited, ensuring that an effective immune stimulation dose is achieved while maintaining the good physical state of the suspension before freeze-drying.

[0025] Preferably, in the above-mentioned compound adjuvant, before freeze-drying, the Z-average diameter of the liposomes in the suspension is 100 nm to 150 nm.

[0026] By employing the above-mentioned preferred technical solution, the particle size of the liposome complex is defined. This particle size range is the ideal size for the effective uptake of the adjuvant by antigen-presenting cells, while ensuring the homogeneity and stability of the suspension.

[0027] Preferably, in the above-mentioned compound adjuvant, the buffer solution is a citrate buffer solution, and the pH value of the suspension before freeze-drying is 6.0 to 7.0.

[0028] By adopting the above-mentioned preferred technical solution, the buffer system and optimal pH range were determined. Citrate buffer is a pharmaceutically acceptable buffer, and the slightly acidic environment of pH 6.0 to 7.0 helps maintain the stability of liposomes and reduces the hydrolysis rate of components such as TLR9 ligand.

[0029] A second aspect of this invention provides a method for preparing a TLR9+HA201 composite adjuvant, comprising the following steps:

[0030] S1. Provides a liposome complex suspension containing liposomes, TLR9 ligands and HA201 molecules;

[0031] S2. Add trehalose and glutamic acid to the liposome complex suspension to make the final concentration of trehalose reach 5% (w / v) to 15% (w / v) and the final concentration of glutamic acid reach 0.5% (w / v) to 3% (w / v) to obtain a suspension to be dried.

[0032] S3. Adjust the pH value of the suspension to be dried;

[0033] S4. Freeze-dry the suspension obtained after step S3.

[0034] By employing the above technical solution, the method of the present invention provides a clear, controllable, and logically rigorous process flow for preparing the aforementioned highly stable lyophilized compound adjuvant formulation. The innovative principle of this method lies in its specific sequence of steps:

[0035] Step S1 prepares a complex suspension containing all active components and the liposome carrier. This step ensures that the adjuvant components (TLR9, HA201, QS-21) form the desired binding, encapsulation, or association with the liposome carrier, constituting the core structure for adjuvant function.

[0036] Step S2 involves adding lyophilization protectants (trehalose and glutamic acid) to the formed complex suspension. This subsequent addition order is a key process design feature, ensuring that the protectants are primarily distributed in the external aqueous phase of the liposome complex, rather than being encapsulated within the liposomes. This allows the protectants to provide the most direct and effective protection to the outer surface of the liposomes and the biomolecules in the suspension medium during freeze-drying.

[0037] Step S3 performs a final pH adjustment of the suspension before freeze-drying. This step ensures that the final suspension containing all components (complexes and protectants) has its pH precisely controlled within the range most favorable to its chemical and physical stability (6.0 to 7.0).

[0038] Step S4 removes moisture by freeze-drying, preserving the optimal stable state in the solid dosage form.

[0039] By following this process sequence of first forming the complex, then adding the protective agent, and finally adjusting the pH and lyophilizing, the method of the present invention ensures the controllability of the intermediate processes, as well as the uniformity, stability, and structural integrity of the adjuvant complex after reconstitution of the final lyophilized product.

[0040] Preferably, in the above preparation method, the freeze-drying in step S4 includes:

[0041] Freezing stage: The temperature of the suspension to be dried is lowered to -50°C to -40°C;

[0042] First drying stage: Under a pressure of 50 mTorr to 150 mTorr, the plate temperature is raised to -25°C to -15°C;

[0043] Secondary drying stage: Raise the temperature of the plate to 20°C to 30°C.

[0044] By adopting the above-mentioned preferred technical solution, an optimized freeze-drying curve is provided, which ensures that moisture is effectively removed by sublimation in the first drying stage, avoiding product collapse, and removes residual moisture by desorption in the second drying stage, ultimately obtaining stable freeze-dried cakes with low moisture content.

[0045] Preferably, in the above preparation method, the preparation of the liposome complex suspension in step S1 includes:

[0046] S11. DSPC, cholesterol, and QS-21 are dissolved in an organic solvent to form a lipid solution, and a lipid film is formed by evaporating the organic solvent;

[0047] S12. Hydrate the lipid film using an aqueous buffer containing TLR9 ligand and HA201 molecules to form a crude liposome suspension.

[0048] S13. The crude liposome suspension is subjected to high-pressure homogenization to obtain the liposome complex suspension.

[0049] By adopting the above-mentioned preferred technical solution, the specific process for preparing the core composite was clarified, namely, thin film dispersion combined with high-pressure homogenization.

[0050] In S11, the lipid-soluble DSPC, cholesterol, and amphiphilic QS-21 are co-soluble in an organic solvent, ensuring their uniform distribution in the subsequently formed lipid bilayer.

[0051] S12 uses water containing water-soluble adjuvants (TLR9, HA201) to hydrate the lipid membrane, achieving initial encapsulation of water-soluble components.

[0052] The high-pressure homogenization step in S13 shears and restructures the crude liposomes, ultimately obtaining a liposome complex suspension with uniform particle size (100 to 150 nm).

[0053] More preferably, in the above preparation method, the hydration in step S12 is carried out at a temperature higher than the phase transition temperature (Tc) of the DSPC, wherein the hydration temperature is 60°C to 70°C.

[0054] By adopting the above-mentioned preferred technical solution, the key process parameters of the hydration step are defined. DSPC has a high phase transition temperature (approximately 55°C). Hydration is carried out at a temperature higher than its Tc (60-70°C), and the lipid bilayer is in a more fluid liquid crystal state. This greatly benefits the hydration and dispersion of the lipid film and improves the encapsulation efficiency of water-soluble components (TLR9, HA201).

[0055] The third aspect of this invention provides the application of a TLR9+HA201 compound adjuvant in the preparation of vaccines.

[0056] By adopting the above technical solution, the core innovation of this invention lies in providing a novel composite adjuvant platform that can induce a stronger and more durable immune response during vaccine preparation. The composite adjuvant platform uniquely combines three adjuvant components (TLR9 ​​ligand, HA201 molecule, and QS-21) with different immunostimulatory mechanisms, and achieves co-delivery via a liposome carrier.

[0057] Its immune activation mechanism includes:

[0058] TLR9 ligand: As an agonist of Toll-like receptor 9 (TLR9), it is mainly recognized in the endosomes of antigen-presenting cells (APCs). Its function is to activate plasmacytoid dendritic cells (pDCs) and B cells, strongly induce the production of type I interferon (IFN-I), and drive the body to produce a Th1 immune response.

[0059] HA201 molecule: As a second immunostimulatory molecule, it works synergistically with TLR9 ligand to further enhance dendritic cell (DC) maturation, co-stimulatory molecule expression and antigen presentation by activating another or complementary immune signaling pathway (including but not limited to the STING pathway, NOD-like receptor pathway, etc.).

[0060] QS-21: As a saponin adjuvant, its mechanism of action includes inducing strong cytotoxic T lymphocyte (CTL) responses (Th1 bias) and antibody responses (Th2 bias), and promoting antigen transmembrane delivery into the cytoplasm, thereby enhancing antigen presentation of MHC-I class molecules.

[0061] The composite adjuvant of this invention co-delivers three components (TLR9, HA201, QS-21) with different mechanisms of action and intracellular targets, along with an antigen (if co-encapsulated), to the same antigen-presenting cells (APCs) via a liposome carrier. This co-delivery mechanism ensures that APCs can simultaneously receive multiple activation signals mediated by the TLR9 pathway, the HA201 pathway, and QS-21.

[0062] This multi-pathway, synergistic activation effect, compared to a single adjuvant or a simple physical mixture of multiple adjuvants, can more effectively promote the maturation and migration of APCs, enhance antigen presentation efficiency, and induce a stronger, broader-spectrum (including both Th1-type cellular immunity and Th2-type humoral immunity) and more durable specific immune response. Therefore, the composite adjuvant of the present invention can improve the immunogenicity and protective efficacy of vaccines when used in vaccine preparation.

[0063] In summary, the present invention has at least one of the following beneficial technical effects:

[0064] 1. This invention uses trehalose and glutamic acid as a composite freeze-drying protectant and limits their specific concentration range. It utilizes the protective effect of the vitrified matrix formed by trehalose and the pH buffering effect of glutamic acid during the freezing process. This combination effectively maintains the structural integrity of the liposome complex during freeze-drying and solves the problem of easy degradation, aggregation or leakage of multiple active components when they coexist in the liquid phase, thereby improving the long-term storage stability of the formulation.

[0065] 2. This invention achieves co-delivery of multiple adjuvants by combining two components with different immune activation mechanisms, TLR9 ligand and HA201 molecule, in a DSPC / cholesterol liposome carrier. This design enables antigen-presenting cells to simultaneously receive activation signals from multiple signaling pathways, achieving synergistic effects among the components and helping to induce a broader and more durable specific immune response.

[0066] 3. This invention employs a thin-film dispersion method combined with high-pressure homogenization, and controls the hydration temperature to be higher than the DSPC phase transition temperature, to obtain liposome complexes with uniform particle size and Z-average diameter in the range of 100 nm to 150 nm. This particle size range not only ensures the physical homogeneity of the suspension before freeze-drying, but also facilitates the effective uptake of the adjuvant complex by antigen-presenting cells, providing a physical basis for subsequent synergistic immune activation effects. Detailed Implementation

[0067] The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the preparation examples, examples, comparative examples, and test examples. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0068] Preparation example:

[0069] This preparation example aims to prepare a TLR9+HA201 liposome complex suspension, including the following steps:

[0070] Accurately weigh 5.00 g of distearate phosphatidylcholine (DSPC) and 2.46 g of cholesterol, making their molar ratio 1:1. Separately accurately weigh 0.15 g of saponin (QS-21) (approximately 2.0% (w / w) of the total lipid weight). Place all the above lipid components (DSPC, cholesterol, QS-21) in a clean beaker, add 100 mL of a mixed solvent of chloroform and methanol (9:1 volume ratio), and magnetically stir at room temperature (25°C) until completely dissolved, forming a clear and transparent lipid solution.

[0071] The lipid solution was transferred to a 1L round-bottom flask and mounted on a rotary evaporator. Rotary evaporation was carried out at a water bath temperature of 60°C, a rotation speed of 100 rpm, and a vacuum pressure of 100 mbar for approximately 30 minutes, until the solvent was completely evaporated and a uniform, dense lipid film formed on the inner wall of the flask. The flask was then removed and placed in a vacuum drying oven at room temperature (<10 mbar) for 4 hours to thoroughly remove any residual organic solvent.

[0072] Weigh appropriate amounts of citric acid and trisodium citrate, dissolve them in water for injection (WFI) to prepare a 10 mM citrate buffer, and adjust and confirm the pH value to 6.5. Dissolve TLR9 ligand (CpGODN1826) and HA201 molecules separately in the above hydration buffer to achieve hydration buffer concentrations of 1.0 mg / mL and 0.5 mg / mL, respectively. Based on a total lipid concentration of 20 mg / mL, precisely add 373 mL of the above hydration buffer containing the active ingredients to the lipid membrane flask. Seal the flask and place it in a constant temperature water bath at 65°C (above the phase transition temperature of DSPC), and hydrate by shaking at 120 rpm for 60 minutes to ensure full hydration of the lipid membrane, forming a milky white crude liposome suspension.

[0073] The crude liposome suspension was cooled to room temperature. It was then homogenized 10 times using a high-pressure homogenizer under ice bath cooling conditions at 20,000 psi.

[0074] The homogenized suspension was sterilized by passing it through a 0.22 μm polyethersulfone (PES) sterile filter membrane. The filtrate was collected, which was the TLR9+HA201 liposome complex suspension.

[0075] Examples 1-5:

[0076] Example 1:

[0077] This embodiment provides a TLR9+HA201 composite adjuvant, and the specific preparation steps are as follows:

[0078] S1. Prepare the liposome adjuvant complex suspension: Take 100 mL of the TLR9+HA201 liposome complex suspension obtained in the preparation example. The pH of this suspension is 6.52, and the average Z-diameter is 125 nm.

[0079] S2. Add freeze-drying protectant: Accurately weigh 10.0 g of D-(+)-trehalose dihydrate and 1.0 g of L-glutamic acid, and dissolve them in 100 mL of the above liposome adjuvant complex suspension. Stir until the solids are completely dissolved to form a homogeneous solution.

[0080] S3. pH adjustment: Check the pH of the solution. If it exceeds the range of 6.0-7.0, adjust it with 0.5M citric acid or 0.5M trisodium citrate solution.

[0081] S4. Dispensing and Freeze-drying: Dispense the above solution into 2mL sterile vials at a volume of 1.0mL per vial, partially stopper them, and transfer the dispensed vials to a freeze dryer. Perform the following procedure:

[0082] Freezing stage: The plate temperature is reduced to -50°C at a rate of 1°C / min and held for 3 hours.

[0083] First drying stage: Start the vacuum system and reduce the chamber pressure to 100 mTorr. At the same time, raise the plate temperature to -20°C at a rate of 0.5°C / min and maintain it for 36 hours.

[0084] Secondary drying stage: The plate temperature is increased to 25°C at a rate of 0.2°C / min and maintained for 8 hours. After drying, the chamber is filled with sterile nitrogen to atmospheric pressure, the vials are automatically stoppered and sealed, and then removed and capped.

[0085] Example 2:

[0086] This embodiment aims to prepare a lyophilized compound adjuvant, including the following steps:

[0087] S1. Preparation of a low-cholesterol-ratio liposome suspension: Follow the steps in the preparation example, but adjust the ratio in the lipid solution preparation stage: accurately weigh 5.00 g (approximately 6.35 mmol) of DSPC and 1.23 g (approximately 3.17 mmol) of cholesterol, making their molar ratio 1:0.5. The QS-21 content is maintained at 1.0% (0.06 g) of the total lipid weight. Subsequent lipid film formation, hydration and encapsulation, homogenization, and sterile filtration steps are the same as in the preparation example, yielding a low-cholesterol-ratio TLR9+HA201 liposome complex suspension. The pH value, Z-mean diameter, and other quality indicators of this suspension should meet expectations.

[0088] S2. Add freeze-drying protectant: Accurately weigh 10.0g D-(+)-trehalose dihydrate and 1.0g L-glutamic acid, and dissolve them in 100mL of the low-cholesterol ratio liposome adjuvant complex suspension prepared above.

[0089] S3. pH adjustment: Check the pH of the solution. If it exceeds the range of 6.0-7.0, adjust it with 0.5M citric acid or 0.5M trisodium citrate solution.

[0090] S4. Packaging and freeze-drying: Follow step S4 of Example 1 for packaging and freeze-drying.

[0091] Example 3:

[0092] This embodiment aims to prepare a lyophilized compound adjuvant, including the following steps:

[0093] S1. Preparation of a high-cholesterol-ratio liposome suspension: Follow the steps in the preparation example, but adjust the ratio in the lipid solution preparation stage: accurately weigh 5.00 g (approximately 6.35 mmol) of DSPC and 4.93 g (approximately 12.70 mmol) of cholesterol to a molar ratio of 1:2. Maintain the QS-21 content at 3.0% (0.30 g) of the total lipid weight. Subsequent lipid film formation, hydration and encapsulation, homogenization, and sterile filtration steps are the same as in the preparation example, yielding a high-cholesterol-ratio TLR9+HA201 liposome complex suspension. The pH value, Z-mean diameter, and other quality indicators of this suspension should meet expectations.

[0094] S2. Add freeze-drying protectant: Accurately weigh 10.0g D-(+)-trehalose dihydrate and 1.0g L-glutamic acid, and dissolve them in 100mL of the high cholesterol ratio liposome adjuvant complex suspension prepared above.

[0095] S3. pH adjustment: Check the pH of the solution. If it exceeds the range of 6.0-7.0, adjust it with 0.5M citric acid or 0.5M trisodium citrate solution.

[0096] S4. Packaging and freeze-drying: Follow step S4 of Example 1 for packaging and freeze-drying.

[0097] Example 4:

[0098] This embodiment aims to prepare a composite adjuvant for lyophilization protection, including the following steps:

[0099] S1. Prepare the liposome adjuvant complex suspension: Take 100 mL of the TLR9+HA201 liposome complex suspension obtained in the preparation example.

[0100] S2. Add freeze-drying protectant: Accurately weigh 5.0g D-(+)-trehalose dihydrate and 0.5g L-glutamic acid, and dissolve them in the above 100mL liposome adjuvant complex suspension.

[0101] S3. pH adjustment: Check the pH of the solution. If it exceeds the range of 6.0-7.0, adjust it with 0.5M citric acid or 0.5M trisodium citrate solution.

[0102] S4. Packaging and freeze-drying: Follow step S4 of Example 1 for packaging and freeze-drying.

[0103] Example 5:

[0104] This embodiment aims to prepare a composite adjuvant for lyophilization protection, including the following steps:

[0105] S1. Prepare the liposome adjuvant complex suspension: Take 100 mL of the TLR9+HA201 liposome complex suspension obtained in the preparation example.

[0106] S2. Add freeze-drying protectant: Accurately weigh 15.0g D-(+)-trehalose dihydrate and 3.0g L-glutamic acid, and dissolve them in the above 100mL liposome adjuvant complex suspension.

[0107] S3. pH adjustment: Check the pH of the solution. If it exceeds the range of 6.0-7.0, adjust it with 0.5M citric acid or 0.5M trisodium citrate solution.

[0108] S4. Packaging and freeze-drying: Follow step S4 of Example 1 for packaging and freeze-drying.

[0109] Comparative Examples 1-6:

[0110] Comparative Example 1: Contains no lyophilization protectant

[0111] Compared with Example 1, the difference is that the suspension obtained in the preparation example was directly dispensed and freeze-dried without adding any freeze-drying protectant, while the rest were the same.

[0112] Comparative Example 2: Contains only trehalose as a freeze-drying protectant

[0113] Compared with Example 1, the difference is that the freeze-drying protectant contains only 10.0g of D-(+)-trehalose dihydrate and does not contain L-glutamic acid, while the rest are the same.

[0114] Comparative Example 3: Contains only glutamic acid as a lyophilization protectant

[0115] The difference from Example 1 is that the freeze-drying protectant contains only 1.0 g of L-glutamic acid and does not contain D-(+)-trehalose dihydrate, while the rest are the same.

[0116] Comparative Example 4: Using conventional sugars (sucrose) to replace trehalose

[0117] Compared with Example 1, the difference is that 10.0g of D-(+)-trehalose dihydrate in the freeze-drying protectant was replaced with an equal weight (10.0g) of sucrose, and all other aspects are the same.

[0118] Comparative Example 5: Glutamic acid was replaced with a conventional amino acid (glycine).

[0119] Compared with Example 1, the difference is that 1.0 g of L-glutamic acid in the freeze-drying protectant is replaced with an equimolar amount of glycine, and all other aspects are the same.

[0120] Comparative Example 6: Non-liposome delivery systems

[0121] The difference from Example 1 is that liposomes were not prepared in this comparative example. The TLR9 ligand and HA201 molecule were directly dissolved in 10 mM, pH 6.5 citrate buffer to achieve the same concentration as in the preparation example. Then, the same amount of trehalose and glutamic acid as in Example 1 were added and freeze-dried. All other steps were the same.

[0122] Test Examples 1-5:

[0123] Test Example 1: Characterization of basic physicochemical properties before and after freeze-drying

[0124] This test case aims to evaluate the key physicochemical properties of the lyophilized adjuvant prepared in Example 1 (baseline parameters) of the present invention before lyophilization (i.e., the suspension of the preparation example) and during reconstitution after lyophilization, in order to verify the process feasibility and basic properties of the product of the present invention.

[0125] Experimental methods:

[0126] Appearance and reconstitution properties:

[0127] Visually observe the shape, color, and integrity of the freeze-dried cake obtained in Example 1.

[0128] Reconstitution procedure: At room temperature (25°C), precisely draw 1.0 mL of water for injection (WFI) using a 1 mL syringe and inject it into the lyophilized vial from Example 1. Start timing immediately, and assist dissolution with a steady, gentle circular shaking motion. Visually observe until no visible lyophilized solid residue remains, then stop timing and record the reconstitution time (s). Simultaneously observe the appearance of the reconstituted solution, recording its clarity, color, and presence of insoluble foreign matter.

[0129] Particle size and polydispersity index (PDI) determination:

[0130] Accurately pipette appropriate amounts of the suspension (before lyophilization) from the preparation example and the reconstituted solution (after lyophilization) from Example 1, and dilute them with water for injection to a suitable concentration.

[0131] Dynamic light scattering (DLS) particle size analyzer was used, with the detector angle set to 90° and the equilibrium temperature set to 25°C. The Z-mean diameter and polydispersity index (PDI) of the samples were measured. Each sample was measured three times, and the average value was taken.

[0132] pH value measurement:

[0133] Take appropriate amounts of the suspension from the preparation example and the reconstituted solution from Example 1, and measure their pH values ​​at 25°C using a laboratory pH meter calibrated with standard buffer solution.

[0134] Encapsulation efficiency (EE) determination of active ingredients:

[0135] Ultracentrifugation was used to separate free active ingredients from liposome-encapsulated active ingredients.

[0136] Total amount determination: Accurately pipette 0.1 mL of sample (suspended before lyophilization or reconstituted after lyophilization), add 9.9 mL of a mixed solution of methanol and water (90:10, v / v), sonicate (40 kHz, 100 W) for 15 minutes to demulsify and fully release the active ingredients, and then make up to volume.

[0137] Determination of free liposomes: Accurately pipette 1.0 mL of the sample into an ultracentrifuge tube and centrifuge at 100,000 g and 4 °C for 60 minutes. Liposomes precipitate, and the supernatant contains the free active ingredient. Accurately pipette 0.5 mL of the supernatant and dilute to volume.

[0138] HPLC analysis: The total active ingredient concentration in (a) was determined by high performance liquid chromatography and denoted as C. t The concentration of the free active ingredient in (b) is denoted as C. f .

[0139] Calculate the encapsulation ratio:

[0140] EE=[(C t -C f ) / Ct ]×100%;

[0141] Experimental data:

[0142] Table 1. Comparison of key physicochemical properties before and after freeze-drying in Example 1

[0143]

[0144] Note: N / A in the table means that the preparation example was a liquid suspension before lyophilization and did not involve reconstitution.

[0145] in conclusion:

[0146] The data from Test Example 1 (Table 1) show that the technical solution adopted in this invention has good process feasibility.

[0147] The core of the conclusion lies in the comparison of critical quality attributes (CQA) before and after freeze-drying:

[0148] Structural integrity maintained: The Z-mean diameter (131.7 nm) of the lyophilized and reconstituted sample showed only a slight increase compared to before lyophilization (125.3 nm), and the PDI value (0.203) remained at a low level. This confirms that the specific combination of trehalose and glutamic acid used in this invention can effectively inhibit irreversible fusion, aggregation, or rupture of liposome vesicles caused by stress (such as ice crystal growth and osmotic pressure changes) during freezing and dehydration in a citrate buffer system.

[0149] Retention of active ingredients: Before and after lyophilization, the encapsulation efficiency of TLR9 ligand and HA201 molecules remained at a high level (92.8% and 89.1%, respectively). This further demonstrates that the lyophilization protectant system effectively maintained the integrity of the liposome membrane and prevented leakage of active ingredients during lyophilization-reconstitution cycles.

[0150] Product form: The freeze-dried cakes are intact, reconstituted rapidly (32s), and the solution after reconstitution is homogeneous, indicating that the protective agent system forms an amorphous matrix with a high glass transition temperature and good support, which meets the basic requirements of freeze-dried formulations.

[0151] In summary, the data from Test Example 1 confirms that the specific combination of freeze-drying protectants selected in this invention can effectively solve the stability problem of the TLR9+HA201 liposome complex during the freeze-drying process.

[0152] Test Example 2: Comparison of Appearance and Reconstitution Performance of Lyophilized Products

[0153] This test case aims to visually evaluate the effects of different freeze-drying protectant schemes on the morphological characteristics and reconstitution properties of the TLR9+HA201 composite adjuvant freeze-dried product, so as to highlight the superiority of the specific combination protectant of the present invention.

[0154] Experimental methods:

[0155] Appearance observation of freeze-dried products:

[0156] The lyophilized adjuvant samples prepared in Examples 1-5 and Comparative Examples 1-6 were visually observed. The macroscopic morphology, color, and presence of crystals or insoluble foreign matter were recorded for each sample cake.

[0157] Reconstitution performance test:

[0158] At room temperature (25°C), accurately draw 1.0 mL of water for injection (WFI) and inject it into the lyophilized vial of each sample. Start timing immediately after injection, and assist dissolution with a steady, gentle circular shaking motion. Record the reconstitution time (in seconds) from the addition of water until no visible lyophilized solid residue remains. After reconstitution, immediately observe the appearance of the reconstituted solution and record its clarity, color, presence of turbidity or visible precipitates, flocculent matter, etc.

[0159] Experimental data:

[0160] Table 2. Comparison of appearance and reconstitution properties of freeze-dried products from the examples and comparative examples

[0161]

[0162] in conclusion:

[0163] The macroscopic observations of this test case (Table 2) clearly demonstrate the necessity and superiority of the specific lyophilization protectant combination and its concentration range of the present invention, as well as its importance in solving the lyophilization stability of liposome adjuvants.

[0164] As shown in Examples 1-5, the lyophilized samples under the system of this invention all exhibited intact, loose, and normally colored lyophilized cakes. Their reconstitution time was between 30-45 seconds, demonstrating rapid and thorough reconstitution. The reconstituted solution had a uniform appearance, with no visible aggregates or precipitates, indicating that the liposome structure was effectively protected during the lyophilization-reconstitution process without significant physical damage or aggregation. This is consistent with the mechanism by which the lyophilizing protectant forms a glassy matrix, replaces the interaction of water molecules with the liposome membrane, and provides steric stabilization.

[0165] In summary, the macroscopic performance data of this test case strongly support the invention (Examples 1-5) in the lyophilized formulation of TLR9+HA201 composite adjuvant. By selecting a specific combination of trehalose and glutamic acid, the appearance and reconstitution properties of the lyophilized product can be improved, thus providing an effective solution for the stabilization of complex liposome adjuvants.

[0166] Test Example 3: Comparison of Particle Size Stability After Resolution

[0167] This test case aims to quantitatively evaluate the changes in particle size and uniformity of liposome vesicles in the examples and comparative samples after freeze-drying and reconstitution using the dynamic light scattering (DLS) method, in order to assess the protective effect of different freeze-drying protectant schemes on the physical structural integrity of liposomes.

[0168] Experimental methods:

[0169] Sample preparation: Take the lyophilized samples from Examples 1-5 and Comparative Examples 1-5 (Comparative Example 6 is a non-liposome system and is not applicable to this test). Reconstitute the samples using 1.0 mL of water for injection (WFI) according to the method in Test Example 2. Immediately after reconstitution, accurately pipette an appropriate amount (approximately 20-50 μL) of the reconstituted suspension and add it to a cuvette containing 1 mL of water for injection filtered through a 0.22 μm membrane. Gently invert and mix to dilute the sample to a suitable count rate range for the DLS detector.

[0170] DLS determination: A dynamic light scattering particle size analyzer was used, with the detector angle set to 173° and the sample cell temperature kept constant at 25°C. Each diluted sample was measured, and its Z-mean diameter and polydispersity index (PDI) were recorded. To ensure data accuracy, each sample was measured in triplicate, and the average result was taken.

[0171] Data Analysis: The measured Z-mean diameter was compared with the data from the preparation example (starting material before freeze-drying, Z-mean diameter 125.3 nm, PDI 0.184) to calculate the particle size growth rate. Particle size growth rate (%) = [(Z-mean diameter after reconstitution - Z-mean diameter before freeze-drying) / Z-mean diameter before freeze-drying] × 100%.

[0172] Experimental data:

[0173] Table 3. Comparison of particle size (Z-mean diameter) after reconstitution with PDI in the Examples and Comparative Examples

[0174]

[0175] in conclusion:

[0176] The DLS quantitative data shown in Table 3, from the perspective of microstructural stability, confirm the advanced nature of the present invention:

[0177] Effectiveness of the present invention:

[0178] After reconstitution, the Z-mean diameter of all samples showed only a slight change compared to before lyophilization (125.3 nm) (the growth rate was controlled between 3.4% and 11.8%), and the PDI value remained at a low level (<0.25). This strongly demonstrates that the trehalose + glutamic acid combination used in this invention can effectively maintain the structural integrity of liposome vesicles and inhibit irreversible vesicle fusion or aggregation under the extreme physicochemical environment of lyophilization (such as ice crystal formation, dehydration stress, and osmotic pressure changes).

[0179] The necessity of protective agents:

[0180] Comparative Example 1 (without protectant) exhibited extremely large particle size and a very high PDI value after reconstitution, indicating that the liposome structure was completely destroyed and severe aggregation occurred. This confirms that a lyophilization protectant is absolutely essential for the liposome adjuvant system of the present invention.

[0181] Synergistic effect of combined protective agents:

[0182] The particle size growth rates (100.0% and 147.6%) and PDI values ​​(0.452 and 0.581) of Comparative Example 2 (trehalose only) and Comparative Example 3 (glutamate only) were both higher than those of Example 1 (5.1%, 0.203). This comparison clearly shows that neither component (trehalose nor glutamate) can effectively protect the liposome structure. This indicates a synergistic protective effect between trehalose and glutamate, rather than a simple functional additive effect. The mechanism involves trehalose forming a protective glassy matrix on the membrane surface through the water substitution hypothesis, while glutamate stabilizes the interfacial charge or local pH environment of the liposomes during lyophilization through its zwitterionic properties or buffering capacity. Together, they maintain the stability of the vesicles.

[0183] Specificity of component selection:

[0184] The particle size growth rates of Comparative Example 4 (sucrose instead of trehalose) and Comparative Example 5 (glycine instead of glutamic acid) (58.3% and 76.9%) were significantly higher than those of Example 1. This indicates that while conventional sugars (sucrose) and amino acids (glycine) possess some freeze-drying protection capabilities, their effectiveness is inferior to the specific combination selected in this invention.

[0185] In summary, this invention successfully solves the technical problem of unstable physical structure and easy aggregation of complex liposome adjuvants during freeze-drying by using a specific combination of trehalose and glutamic acid.

[0186] Test Example 4: Comparison of Retention Rate of Active Ingredients after Reconstitution

[0187] This test case aims to quantitatively determine the encapsulation efficiency of the active ingredients (TLR9 ​​ligand and HA201 molecule) inside the liposomes of lyophilized adjuvants prepared by different schemes after reconstitution using high performance liquid chromatography (HPLC), in order to evaluate the effectiveness of different lyophilization protectant systems in preventing leakage of active ingredients during lyophilization-reconstitution cycles.

[0188] Experimental methods:

[0189] Sample preparation: Take the lyophilized samples of Examples 1-5 and Comparative Examples 1-5 (Comparative Example 6 is a non-liposome system with no encapsulation concept and is not included in this test). Reconstitute all samples with 1.0 mL of water for injection (WFI) according to the method of Test Example 2.

[0190] Encapsulation efficiency (EE) determination: The encapsulation efficiency of the active ingredient in each reconstituted sample was determined by ultracentrifugation combined with HPLC analysis. Refer to Test Example 1 for specific procedures.

[0191] For each sample, the total active ingredient concentration C was determined. t and free active ingredient concentration C f .

[0192] The encapsulation efficiency of each active ingredient is calculated using the following formula:

[0193] EE=[(C t -C f ) / C t ×100%;

[0194] Each sample was measured in triplicate, and the average value of the results was taken.

[0195] Experimental data:

[0196] Table 4. Comparison of encapsulation efficiency (EE) of active ingredients after reconstitution between the examples and comparative examples

[0197]

[0198] in conclusion:

[0199] Table 4 presents encapsulation efficiency data that quantitatively reveals the differences in the ability of different lyophilization protectant regimens to maintain liposome membrane integrity and prevent content leakage. The conclusions are as follows:

[0200] The efficient retention effect of the solution of this invention:

[0201] After rehydration, the samples in Examples 1-5 exhibited high encapsulation rates for both active ingredients (TLR9 ​​ligand maintained at 88.1%-94.2%, and HA201 molecules maintained at 85.2%-90.3%). This demonstrates that the trehalose and glutamic acid combination protectant used in this invention can effectively maintain the structural integrity of the liposome phospholipid bilayer during dehydration and rehydration, inhibiting transient pore formation or membrane rupture caused by membrane phase transitions or ice crystal damage, thereby controlling the leakage of active ingredients to an extremely low level.

[0202] Synergistic effect of combined protective agents:

[0203] The encapsulation efficiency of Comparative Example 2 (trehalose only) and Comparative Example 3 (glutamic acid only) was significantly lower than that of Example 1. This difference demonstrates that trehalose and glutamic acid play a synergistic protective role in this system. Trehalose or glutamic acid alone cannot provide sufficient protection, but the combination of the two effectively prevents the leakage of active ingredients. The mechanism is that trehalose mainly immobilizes liposomes by forming an amorphous matrix with a high glass transition temperature, while glutamic acid stabilizes the membrane interface through specific interactions with phospholipid heads or its buffering capacity. Together, they maintain the membrane's compactness.

[0204] Specific advantages of component selection:

[0205] The encapsulation efficiency data for Comparative Example 4 (sucrose) and Comparative Example 5 (glycine) were also lower than those for Example 1. This indicates that while conventional lyophilization protectants have some effect, they cannot achieve the protective level of the specific combination of the present invention. This result confirms that the selection of trehalose and glutamic acid in the present invention is not a simple combination of any sugars and amino acids, but a specific optimized selection for this liposome adjuvant system, and this selection is superior.

[0206] The necessity of protective agents:

[0207] The encapsulation efficiency of Comparative Example 1 (without protectant) was extremely low (<10%), indicating that in the absence of protectant, the physical stress of the freeze-drying process caused catastrophic and irreversible structural damage to the liposomes, resulting in the leakage of most of the active ingredients.

[0208] In summary, this solution effectively addresses the key technical issue of easy leakage of active ingredients in liposome adjuvants during freeze-drying through a specific synergistic combination of trehalose and glutamic acid, ensuring that the product retains its expected composition and biological efficacy after reconstitution.

[0209] Test Example 5: Compatibility Test of Adjuvant Reconstituted Solution Mixed with Antigen

[0210] This test case aims to verify the physical compatibility and short-term stability of the lyophilized adjuvant of Example 1 of the present invention when mixed with the reconstituted herpes zoster gE protein after reconstitution, so as to confirm its feasibility in vaccine preparation.

[0211] Experimental methods:

[0212] Sample preparation:

[0213] Adjuvant solution: Take the lyophilized sample of Example 1 and reconstitute it with water for injection (WFI) according to the method of Test Example 2 to prepare the reconstituted solution of Example 1.

[0214] Antigen solution: Prepare a stock solution of recombinant herpes zoster gE protein and dilute it to the working concentration (e.g., 1.0 mg / mL) using PBS buffer (pH 7.4).

[0215] Mixed solution: At room temperature (25°C), the adjuvant solution in (a) and the antigen solution in (b) are mixed at a volume ratio (v / v) of 1:1 and gently inverted to mix.

[0216] Detection method:

[0217] Visual observation: Immediately after mixing (T=0h) and after standing at room temperature for 2 hours (T=2h), observe the appearance of the mixed solution and record whether there is turbidity, precipitation or flocculent matter.

[0218] DLS assay: At T=0h and T=2h, appropriate amounts of the mixed solution were taken, diluted, and then the Z-mean diameter and PDI were determined by dynamic light scattering (DLS). The adjuvant solution and antigen solution were measured separately as controls.

[0219] Antigen integrity (SEC-HPLC): At T=0h and T=2h, appropriate amounts of the mixed solution were taken and the integrity of gE protein was analyzed by size exclusion high performance liquid chromatography (SEC-HPLC), and the peak area percentage of the main peak (monomer) was recorded.

[0220] Experimental data:

[0221] Table 5: Compatibility and stability data of the reconstituted solution and gE antigen in Example 1

[0222]

[0223] Note: N / A in the table refers to the fact that the individual reconstituted solution of Example 1 does not contain gE protein antigen, so there is no such test data.

[0224] in conclusion:

[0225] The data from this test case confirms the good compatibility of the freeze-dried adjuvant of the present invention in the target application field.

[0226] Physical compatibility:

[0227] As shown in Table 5, after mixing the reconstituted solution of Example 1 with the gE protein solution, the mixture maintained a homogeneous milky white suspension appearance at both T=0h and T=2h, with no precipitation or flocculent matter observed. DLS data showed that the Z-mean diameter of the mixture (135.8 nm) was not significantly different from that of the adjuvant solution alone (131.7 nm), and the slight increase in PDI (0.245) was consistent with the expectation of mixing two different particle size components (liposomes and protein). After standing at room temperature for 2 hours, the particle size and PDI of the mixture remained stable (139.1 nm, 0.258), showing no tendency to aggregate due to incompatibility.

[0228] Antigen integrity: SEC-HPLC data showed that the purity of the main peak of gE protein after mixing with the adjuvant reconstitution solution (98.1%) was essentially consistent with that of the antigen solution alone (98.5%). After standing at room temperature for 2 hours, the purity of the main peak remained at a high level of 97.5%. This indicates that the adjuvant reconstitution solution of the present invention (containing liposomes, QS-21, and residual lyophilization protectant components) does not cause degradation or aggregation of gE protein in the short term.

[0229] In summary, the results of Test Example 5 demonstrate that the TLR9+HA201 composite adjuvant prepared by the present invention using a specific lyophilization protectant scheme (trehalose + glutamic acid) can mix well with the representative antigen (gE protein) after reconstitution, and maintains physical and chemical compatibility and stability within the simulated preparation and use time (2 hours). This confirms the applicability of the present invention and provides feasibility support for its use as a vaccine adjuvant in co-preparation with antigens.

Claims

1. A TLR9+HA201 composite adjuvant, characterized in that, The compound adjuvant is a lyophilized formulation, obtained by freeze-drying a suspension, wherein the suspension contains the following components: Liposomes comprising DSPC and cholesterol, wherein the molar ratio of DSPC to cholesterol is 1:0.5 to 1:2; QS-21, wherein the content of QS-21 is 1.0% to 3.0% of the total lipid weight in the liposomes; TLR9 ligand; HA201 molecule; Trehalose; Glutamic acid; Buffer solution; Prior to freeze-drying, the concentration of trehalose is 5% (w / v) to 15% (w / v) and the concentration of glutamic acid is 0.5% (w / v) to 3% (w / v) based on the total volume of the suspension.

2. The TLR9+HA201 composite adjuvant according to claim 1, characterized in that, The concentration of trehalose is 10% (w / v), the concentration of glutamic acid is 1% (w / v), and the molar ratio of DSPC to cholesterol is 1:

1.

3. The TLR9+HA201 composite adjuvant according to claim 1, characterized in that, Prior to freeze-drying, the total lipid concentration of the liposomes in the suspension was 15 mg / mL to 25 mg / mL, the concentration of TLR9 ligand was 0.8 mg / mL to 1.2 mg / mL, and the concentration of HA201 molecules was 0.4 mg / mL to 0.6 mg / mL.

4. The TLR9+HA201 composite adjuvant according to claim 1, characterized in that, Before freeze-drying, the Z-average diameter of the liposomes in the suspension was 100 nm to 150 nm.

5. The TLR9+HA201 composite adjuvant according to claim 1, characterized in that, The buffer solution is a citrate buffer solution, and the pH of the suspension before freeze-drying is 6.0 to 7.

0.

6. A method for preparing the TLR9+HA201 composite adjuvant according to any one of claims 1-5, characterized in that, Includes the following steps: S1. Provides a liposome complex suspension containing liposomes, TLR9 ligands and HA201 molecules; S2. Add trehalose and glutamic acid to the liposome complex suspension to make the final concentration of trehalose reach 5% (w / v) to 15% (w / v) and the final concentration of glutamic acid reach 0.5% (w / v) to 3% (w / v) to obtain a suspension to be dried. S3. Adjust the pH value of the suspension to be dried; S4. Freeze-dry the suspension obtained after step S3.

7. The method for preparing a TLR9+HA201 composite adjuvant according to claim 6, characterized in that, The freeze-drying in step S4 includes: Freezing stage: The temperature of the suspension to be dried is lowered to -50°C to -40°C; First drying stage: Under a pressure of 50 mTorr to 150 mTorr, the plate temperature is raised to -25°C to -15°C; Secondary drying stage: Raise the temperature of the plate to 20°C to 30°C.

8. The method for preparing a TLR9+HA201 composite adjuvant according to claim 6, characterized in that, The preparation of the liposome complex suspension in step S1 includes: S11. DSPC, cholesterol, and QS-21 are dissolved in an organic solvent to form a lipid solution, and a lipid film is formed by evaporating the organic solvent; S12. Hydrate the lipid film using an aqueous buffer containing TLR9 ligand and HA201 molecules to form a crude liposome suspension. S13. The crude liposome suspension is subjected to high-pressure homogenization to obtain the liposome complex suspension.

9. The method for preparing a TLR9+HA201 composite adjuvant according to claim 8, characterized in that, The hydration in step S12 is carried out at a temperature higher than the phase transition temperature of the DSPC, specifically 60°C to 70°C.

10. The use of the TLR9+HA201 compound adjuvant according to any one of claims 1-5 in the preparation of vaccines.

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