Squalene-containing oil-in-water adjuvant, and methods of making and using same

Abstract

The application relates to the technical field of vaccine preparation, and discloses an oil-in-water squalene-containing adjuvant as well as a preparation method and application thereof. The adjuvant comprises the following components: squalene, polysorbate 80, sorbitan trioleate, a temperature-sensitive nonionic block copolymer, an amino acid CMT regulator, a polyhydric alcohol CMT regulator, citric acid, sodium citrate, and the rest is injection water. The preparation method comprises the following steps: preparing an oil phase; dissolving the temperature-sensitive nonionic block copolymer, the CMT regulator, the citric acid and the sodium citrate in low-temperature injection water, then pre-treating by temperature rising to obtain an aqueous phase; shearing and emulsifying the aqueous phase and the oil phase at a specific temperature; and finally, controlling the cooling speed of the hot emulsion and filtering. Through the composite formula and the specific process, the long-term storage stability of the adjuvant is remarkably improved, the immunocompetence of the vaccine is guaranteed, the process reproducibility is good, and the production cost is reduced.

Description

Technical Field

[0001] This invention relates to the field of vaccine formulation technology, and in particular to an oil-in-water adjuvant containing squalene, its preparation method, and its application. Background Technology

[0002] Vaccine adjuvants are key components for enhancing vaccine immunogenicity. Among them, oil-in-water (O / W) emulsion adjuvants have received widespread attention in modern vaccine development because they can effectively induce cellular and humoral immune responses, and the oil phase usually has good biocompatibility.

[0003] To ensure the physical stability of such adjuvants, existing technologies typically employ small-molecule surfactants (such as polysorbates) in combination to reduce the oil-water interfacial tension, and prepare them through high-shear emulsification processes. To further improve long-term storage performance, some approaches introduce high-molecular-weight polymers as steric stabilizers, aiming to form a physical barrier on the oil droplet surface and inhibit droplet aggregation.

[0004] However, existing technologies still face challenges. Simply mixing polymeric stabilizers with small-molecule emulsifiers does not ensure efficient and orderly adsorption and arrangement of the polymer at the oil droplet interface during emulsification. This leads to the risk of increased droplet size and wider distribution in the prepared adjuvants during long-term storage or temperature changes, indicating insufficient physical stability. This structural instability not only directly affects the efficacy of adjuvants in delivering antigens and activating the immune system, but also makes the reproducibility of the preparation process difficult to control, increasing the complexity of quality control and production costs.

[0005] Therefore, this invention proposes a squalene-containing oil-in-water adjuvant, its preparation method, and its application to address the shortcomings of existing technologies. Summary of the Invention

[0006] The purpose of this invention is to provide an oil-in-water adjuvant containing squalene, its preparation method and application. In the prior art, oil-in-water adjuvants have problems such as poor long-term storage stability, insufficient protection of immune activity, and high cost due to poor reproducibility of the preparation process.

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

[0008] In a first aspect, the present invention provides an oil-in-water adjuvant containing squalene, employing the following technical solution:

[0009] An oil-in-water adjuvant containing squalene comprises the following components in 1000 parts by weight: squalene: 25-100 parts; polysorbate 80: 1-20 parts; sorbitan trioleate: 1-20 parts; thermosensitive nonionic block copolymer: 0.5-10 parts; amino acid CMT modifier: 1-15 parts; polyol CMT modifier: 10-50 parts; citric acid: 0.9-2.1 parts; sodium citrate: 0.6-18.5 parts; the remainder being water for injection: to bring the total to 1000 parts.

[0010] The innovation of this invention lies in constructing a multi-component synergistic composite stable system by adopting the above technical solution. The stability mechanism of this system is explained as follows:

[0011] Construction of the basic emulsion layer: Polysorbate 80 (hydrophilic) and sorbitan trioleate (lipophilic) in the formulation are used as a combination of small molecule surfactants. The synergistic effect of the two can effectively reduce the interfacial tension between the squalene oil phase and the water for injection aqueous phase, so that squalene can be efficiently dispersed under mechanical shearing, forming a water-in-oil (O / W) emulsion basic structure with small particle size.

[0012] Formation of the steric stabilizing layer: Thermosensitive nonionic block copolymers (such as poloxamer 188 and / or poloxamer 407) are introduced into the formulation as polymer stabilizers. These copolymers have a polyoxyethylene-polyoxypropylene-polyoxyethylene (PEO-PPO-PEO) structure. The hydrophobic PPO central block adsorbs and anchors to the surface of the squalene oil droplets, while the two hydrophilic PEO segments extend into the aqueous phase. These extended PEO chains form a dense polymer hydration layer on the oil droplet surface, i.e., a steric hindrance layer. This steric hindrance layer provides strong steric repulsion through physical barrier action, effectively preventing droplets from approaching each other and coalescing, thereby endowing the emulsion with excellent thermodynamic stability and long-term physical stability.

[0013] Precise control of stabilizing layer performance: The formulation further incorporates amino acid-based CMT modifiers and polyol-based CMT modifiers. The core function of these two components is as "performance modifiers" for the thermosensitive nonionic block copolymer. By altering the solvent environment of the aqueous phase, they influence the interaction (i.e., solvation ability) between water molecules and PEO and PPO blocks, thereby precisely controlling the critical micelle temperature and critical micelle concentration of the thermosensitive nonionic block copolymer. This control ensures that the polymer stabilizer exhibits optimal interfacial adsorption conformation and efficiency at specific preparation process temperatures (e.g., temperature activation and shear emulsification stages), enabling it to anchor more quickly and firmly to the oil droplet surface, forming a uniform and complete steric hindrance layer.

[0014] In summary, this technical solution combines the rapid emulsification capability of small-molecule emulsifiers with the steric stability of high-molecular polymers, and innovatively uses an amino acid and polyol composite system to optimize the interfacial adsorption efficiency of the polymeric stabilizer. This high physical stability, combined with the precise pH control of the buffer system, jointly ensures the integrity of the adjuvant emulsion structure and the chemical stability of the antigen. The structural integrity of the adjuvant is the basis for its immune activation function (e.g., promoting antigen presentation and creating a microenvironment for recruiting immune cells at the injection site). Therefore, the squalene-containing oil-in-water adjuvant obtained by this invention not only has the characteristics of smaller particle size and narrower particle size distribution, exhibiting significantly improved storage stability, thermal stability, and resistance to temperature fluctuations, but more importantly, it ensures the stability and high efficiency of the vaccine adjuvant's immune activity during storage and use.

[0015] Preferably, the thermosensitive nonionic block copolymer is poloxamer 188 and / or poloxamer 407. Poloxamer 188 has a high PEO content of approximately 80%, providing excellent hydrophilicity and steric barrier thickness. Poloxamer 407 has a PEO content of approximately 70% and a different molecular weight. By employing the above technical solution, the physical properties of the stable layer (such as thickness and density) can be finely adjusted, either alone or in combination, to match different formulation requirements.

[0016] Preferably, the amino acid-based CMT modulator is L-arginine or L-lysine. By adopting the above technical solution, both L-arginine and L-lysine are pharmaceutical-grade amino acids with excellent biocompatibility and hydration capacity, and can be used as highly efficient CMT modulators.

[0017] Preferably, the polyol-based CMT modulator is sorbitol or mannitol. By adopting the above technical solution, sorbitol and mannitol, in addition to acting synergistically as CMT modulators, can also act as isotonic modulators of the formulation, ensuring that the osmotic pressure of the adjuvant is compatible with physiological conditions and reducing irritation at the injection site.

[0018] Preferably, the squalene-containing oil-in-water adjuvant has a pH value of 6.0-7.5. By employing the above technical solution, citric acid and sodium citrate form a buffer pair, stabilizing the pH value of the formulation within the weakly acidic to neutral range. This pH environment not only meets the physiological requirements of pharmaceutical injections but also helps improve the compatibility stability of the adjuvant when mixed with antigens (especially protein or peptide antigens), preventing antigen degradation or denaturation.

[0019] Secondly, the present invention provides a method for preparing an oil-in-water adjuvant containing squalene, using the following technical solution:

[0020] A method for preparing a squalene-containing oil-in-water adjuvant includes the following steps:

[0021] a) Mix squalene, polysorbate 80 and sorbitan trioleate and heat to 60-70°C to obtain the oil phase;

[0022] b) Dissolve the thermosensitive nonionic block copolymer, amino acid CMT modifier, polyol CMT modifier, citric acid and sodium citrate in water for injection at 4-10°C to obtain a low-temperature aqueous solution; then heat the low-temperature aqueous solution to 50-60°C and maintain it to obtain a pretreated aqueous phase;

[0023] c) The pretreated aqueous phase is added to the oil phase and shear emulsified to obtain a hot emulsion;

[0024] d) Cool the hot emulsion to 25°C to obtain a cooled emulsion; add water for injection to the cooled emulsion to a total of 1000 parts by weight, mix, and filter through a 0.22 μm filter membrane to obtain the squalene-containing oil-in-water adjuvant.

[0025] By adopting the above technical solution, the preparation method of the present invention aims to solve the problem in the existing preparation process that the polymeric stabilizer fails to be effectively adsorbed at the oil droplet interface, resulting in stability defects in the emulsion after formation and during storage.

[0026] The innovation of the preparation method of this invention lies in the fact that it is not a simple mixing and emulsification process, but rather a complete process for controlling the interfacial adsorption and conformational arrangement of polymer stabilizers by combining the "low-temperature dissolution and heating pretreatment" in step b) with the "controlled cooling" in step d).

[0027] The principle of this process is explained in detail below:

[0028] Complete dissolution and hydration of the polymer (first half of step b):

[0029] In step b), the thermosensitive nonionic block copolymers (poloxamer 188 and / or poloxamer 407) are first dissolved in cryogenic water for injection at 4-10°C. Thermosensitive polymers exhibit the highest solubility at this low temperature, with the strongest hydration of their polyoxyethylene (PEO) hydrophilic segments, allowing for full chain extension and complete molecular-level dissolution. This step ensures the absence of undissolved polymer aggregates in the aqueous phase, providing a foundation for subsequent uniform and efficient interfacial adsorption.

[0030] Polymer pre-activation and conformation preparation (second half of step b):

[0031] Subsequently, the low-temperature aqueous solution is heated to 50-60°C and maintained for a specific time. This temperature is typically higher than or close to the critical micelle temperature (CMT) of the polymer. During this stage, the hydrophobic blocks of the polyoxypropylene (PPO) polymer undergo dehydration, enhancing hydrophobicity; simultaneously, the amino acid and polyol CMT modifiers in the formulation work synergistically to finely regulate this transformation process. This "pretreatment" step transforms the polymer molecules from a fully hydrated dissolved state to a high-energy, more hydrophobically anchored "activated" state, preparing the conformation for subsequent contact with the oil phase.

[0032] Efficient emulsification and rapid interfacial anchoring of polymers (step c):

[0033] In step c), the "activated" pretreated aqueous phase and the heated oil phase (60-70℃) are emulsified under high shear at a high temperature (58-62℃). The high temperature significantly reduces the viscosity of both the oil and water phases, and the high shear rate provides enormous mechanical energy, breaking the oil phase into tiny droplets and instantly generating a large new oil-water interface. At this point, the polymer molecules in the "activated" state (PPO blocks are extremely hydrophobic) have extremely high interfacial adsorption kinetics, enabling them to immediately and firmly "capture" and anchor onto the surface of the newly formed squalene oil droplets.

[0034] Orderly construction and solidification of the stabilizing layer (step d):

[0035] The controlled-rate cooling (specific cooling rate) in step d) is a crucial step in achieving long-term adjuvant stability. During the cooling of the hot emulsion to 25°C, the slow cooling allows sufficient time for the polymer molecules already anchored to the oil droplet surface to undergo interfacial rearrangement. The PEO hydrophilic segments extending into the aqueous phase undergo ordered rehydration and spatial extension during this process, ultimately forming a dense, well-organized, and highly sterically hindered stable layer on the droplet surface. If rapid cooling (such as quenching) is used, the polymer chains will be disorderedly "frozen" at the interface, failing to form an effective steric barrier.

[0036] In summary, the method of the present invention ensures that the polymer stabilizer exerts its steric stabilizing effect to the maximum extent by precisely controlling the dissolution, thermal activation, adsorption and cooling rearrangement of the polymer stabilizer in the aqueous phase, as well as the adsorption and cooling rearrangement process at the oil-water interface. This results in the preparation of an oil-in-water adjuvant containing squalene with uniform particle size and extremely high physical stability (resistant to long-term storage and temperature fluctuations).

[0037] Preferably, in step b), the holding time is 15-30 minutes. By adopting the above technical solution, this holding time ensures that the temperature-sensitive nonionic block copolymer can fully complete the transition from the dissolved state to the activated state in the presence of the CMT regulator, so that the aqueous phase system reaches thermodynamic equilibrium, preparing for subsequent efficient emulsification.

[0038] Preferably, in step c), the shear emulsification conditions are: emulsification at a temperature of 58-62°C and a shear rate of 5,000-15,000 rpm for 10-15 minutes. By adopting the above technical solution, this temperature range (58-62°C) matches the pretreatment temperature (50-60°C) in step b), maintaining the activated state of the polymer; at the same time, the high shear rate and duration provide sufficient mechanical energy to overcome the oil-water interfacial tension and efficiently form submicron-sized fine droplets.

[0039] Preferably, in step d), the cooling rate is 1-5°C / minute. By adopting the above technical solution, this specific slow cooling rate is a process guarantee for achieving the ordered arrangement and full hydration of the polymer stable layer, which is crucial for obtaining the long-term storage stability of the adjuvant.

[0040] Thirdly, the present invention provides the application of a squalene-containing oil-in-water adjuvant in the preparation of vaccines or immunomodulators, employing the following technical solution:

[0041] The squalene-containing oil-in-water adjuvant described in the first aspect is mixed with one or more antigens to prepare a vaccine or immunizing agent.

[0042] By adopting the above technical solution, the application of the adjuvant of the present invention in vaccines or immunizing agents aims to solve the technical problem that existing adjuvants, due to physical or chemical instability, result in reduced vaccine potency, poor immunization effect, or poor batch-to-batch consistency after mixing with antigens.

[0043] The mechanism by which the adjuvant of this invention can effectively enhance the immune activity of vaccines is as follows:

[0044] Highly efficient antigen delivery and immune cell activation:

[0045] The adjuvant provided by this invention is essentially a highly stable oil-in-water (O / W) emulsion. Its submicron-sized, homogeneous squalene oil droplets serve as ideal antigen delivery carriers. After injection, these droplets carrying or adsorbing antigens can be efficiently recognized and phagocytosed by antigen-presenting cells (APCs), such as dendritic cells and macrophages. Due to the excellent physical stability of the adjuvant, its droplet size remains unchanged during storage and use, ensuring maximized and highly consistent surface area for interaction with APCs, thereby inducing a stronger and more reliable release of cytokine signals and effectively activating subsequent specific T-cell and B-cell immune responses.

[0046] Maintaining antigen integrity and ensuring immunogenicity:

[0047] The conformational integrity of antigens (especially protein and polypeptide antigens) is fundamental to inducing an effective immune response. The adjuvant of this invention utilizes a built-in citrate-sodium citrate buffer system to precisely control the pH within a physiologically compatible range of 6.0-7.5. When the adjuvant is mixed with the antigen solution to prepare the final vaccine formulation, this buffer system effectively resists pH fluctuations that may be caused by external environmental factors or the original antigen solution, providing a mild chemical environment for the antigen. This maximizes the protection of the antigen's spatial structure and key epitopes from damage or degradation, thereby fully preserving its original immunogenicity.

[0048] In summary, the adjuvant of this invention, through its complex stabilizing system, ensures both the physical structural stability of the adjuvant and its chemical buffering capacity against the external environment. This allows it to function not only as a highly efficient immune activation signal and antigen delivery system when used as a vaccine component, but also as an antigen protectant. The synergistic effect of physical and chemical stability jointly ensures that the final vaccine product exhibits higher potency, stronger immunogenicity, and superior safety throughout its entire lifecycle.

[0049] In summary, the present invention has at least one of the following beneficial technical effects:

[0050] 1. This invention constructs a multi-component synergistic composite stable system by compounding small-molecule emulsifiers, thermosensitive nonionic block copolymers, and amino acid and polyol CMT modifiers. The key to this invention lies in utilizing CMT modifiers to optimize the adsorption conformation of the polymer at the squalene oil droplet interface, forming a dense and efficient steric hindrance layer that effectively inhibits droplet aggregation and Austmann ripening. Therefore, this invention significantly improves the physical stability of the squalene-containing oil-in-water adjuvant under long-term storage and temperature fluctuations.

[0051] 2. This invention ensures the physical stability of the emulsion structure through the aforementioned composite stabilizing system, and maintains the chemical stability of the formulation by combining a buffer system composed of citric acid and sodium citrate. The key to this invention is that the uniform and stable droplet size ensures efficient and consistent uptake by antigen-presenting cells; simultaneously, the precise pH environment ensures the integrity of the antigen conformation. This synergy of physical and chemical stability ultimately ensures the stability and efficiency of the vaccine adjuvant's immunomodulatory activity during storage and use.

[0052] 3. This invention achieves thermodynamic and kinetic control over the interfacial adsorption and conformational arrangement of polymer stabilizers by employing a preparation process combining low-temperature dissolution, heated pretreatment, and controlled-rate cooling. The key advantage lies in ensuring high efficiency of the emulsification process and high homogeneity of the final product through pre-activation and ordered rearrangement steps. This highly reproducible preparation process significantly improves batch-to-batch consistency of squalene-containing oil-in-water adjuvants, substantially reduces product scrap rates, and thus simplifies quality control processes and lowers overall production costs. Detailed Implementation

[0053] The main raw materials and reagents used in the following examples and comparative examples have the following sources and specifications. Reagents not specifically mentioned are all commercially available analytical grade or higher grade products.

[0054] Squalene: Molecular formula C 30 H 50 CAS No.: 111-02-4. This invention uses pharmaceutical grade specifications.

[0055] Polysorbate 80: CAS No.: 9005-65-6. This invention uses pharmaceutical grade excipients.

[0056] Sorbitan trioleate: CAS No.: 26266-58-0. This invention uses pharmaceutical grade excipients.

[0057] Temperature-sensitive nonionic block copolymers: poloxamer 188, poloxamer 407.

[0058] Poloxamer 188: One of the thermosensitive nonionic block copolymers used in this invention. It is a polyoxyethylene-polyoxypropylene-polyoxyethylene (PEO-PPO-PEO) triblock copolymer, CAS No.: 9003-11-6. The polymer has a hydrophobic polyoxypropylene (PPO) block at the center and hydrophilic polyoxyethylene (PEO) blocks on both sides. The average molecular weight of Poloxamer 188 ranges from 7680 to 9510 Da, with PEO content accounting for approximately 80% of the total mass. This invention uses pharmaceutical grade specifications, and it is a white flaky or powdery solid at room temperature.

[0059] Poloxamer 407: One of the thermosensitive nonionic block copolymers used in this invention. Its structure is also a polyoxyethylene-polyoxypropylene-polyoxyethylene (PEO-PPO-PEO) triblock copolymer, CAS number: 9003-11-6. The average molecular weight of poloxamer 407 ranges from 9840 to 14600 Da, with PEO content accounting for approximately 70% of the total mass. This invention uses pharmaceutical grade excipients, which are white flakes or powder solids at room temperature.

[0060] Amino acid CMT regulators: L-arginine, L-lysine.

[0061] L-Arginine: CAS No.: 74-79-3. This invention uses injection grade specifications.

[0062] L-Lysine: CAS No.: 56-87-1. This invention uses injection grade specifications.

[0063] Polyol-based CMT modifiers: sorbitol or mannitol.

[0064] Sorbitol, CAS number: 50-70-4, is a pharmaceutical excipient grade.

[0065] Mannitol, CAS number: 69-65-8, is a pharmaceutical excipient grade.

[0066] Citric acid: CAS No.: 77-92-9 (anhydrous). This invention uses pharmaceutical grade specifications.

[0067] Sodium citrate: CAS No.: 6132-04-3. This invention uses pharmaceutical grade specifications.

[0068] Water for injection: Water for injection that meets the standards of the Chinese Pharmacopoeia or the United States Pharmacopeia.

[0069] Model antigen: Ovalbumin (OVA), CAS number: 9006-59-1.

[0070] Example 1 - Implementation 3.

[0071] Example 1:

[0072] This embodiment provides an oil-in-water adjuvant containing squalene, which comprises the following components per 1000 parts by weight:

[0073] Squalene: 50 parts; Polysorbate 80: 10 parts; Sorbitan trioleate: 10 parts; Thermosensitive nonionic block copolymer (poloxam 188): 5 parts; Amino acid CMT modifier (L-arginine): 7 parts; Polyol CMT modifier (sorbitol): 30 parts; Citric acid: 2.1 parts; Sodium citrate: 7.3 parts; The remainder is water for injection: make up to 1000 parts. The pH value of this oil-in-water adjuvant is 6.5.

[0074] The preparation method of this oil-in-water adjuvant includes the following steps:

[0075] a) Weigh 50 parts of squalene, 10 parts of polysorbate 80 and 10 parts of trioleate sorbitan, mix them and heat to 65°C to obtain the oil phase.

[0076] b) Weigh 5 parts of poloxamer 188, 7 parts of L-arginine, 30 parts of sorbitol, 2.1 parts of citric acid and 7.3 parts of sodium citrate, add them to 800 parts of water for injection that has been cooled to 8°C, and stir until the components are completely dissolved to obtain a low-temperature aqueous solution; heat the low-temperature aqueous solution to 55°C and maintain the temperature for 20 minutes to obtain a pretreated aqueous phase.

[0077] c) Add the pretreated aqueous phase obtained in step b) to the oil phase obtained in step a), and emulsify at a shear rate of 10,000 rpm at a system temperature of 60°C for 12 minutes to obtain a hot emulsion.

[0078] d) Cool the hot emulsion obtained in step c) to 25°C at a cooling rate of 3°C / min to obtain a cooled emulsion; add water for injection to the cooled emulsion to a total of 1000 parts by weight, mix and filter through a 0.22 μm filter membrane to obtain an oil-in-water adjuvant containing squalene.

[0079] Example 2:

[0080] This embodiment provides an oil-in-water adjuvant containing squalene, which comprises the following components per 1000 parts by weight:

[0081] Squalene: 25 parts; Polysorbate 80: 1 part; Sorbitan trioleate: 1 part; Thermosensitive nonionic block copolymer (poloxam 407): 0.5 parts; Amino acid CMT modifier (L-lysine): 1 part; Polyol CMT modifier (mannitol): 10 parts; Citric acid: 1.3 parts; Sodium citrate: 0.6 parts; The remainder is water for injection: make up to 1000 parts. The pH value of this oil-in-water adjuvant is 6.0.

[0082] The preparation method of this oil-in-water adjuvant includes the following steps:

[0083] a) Weigh 25 parts of squalene, 1 part of polysorbate 80 and 1 part of trioleate sorbitan, mix them and heat to 60°C to obtain the oil phase.

[0084] b) Weigh 0.5 parts of poloxamer 407, 1 part of L-lysine, 10 parts of mannitol, 1.3 parts of citric acid and 0.6 parts of sodium citrate, add them to 900 parts of water for injection that has been cooled to 10°C, stir until the components are completely dissolved to obtain a low-temperature aqueous solution; heat the low-temperature aqueous solution to 50°C and maintain the temperature for 15 minutes to obtain a pretreated aqueous phase.

[0085] c) Add the pretreated aqueous phase obtained in step b) to the oil phase obtained in step a), and emulsify at a shear rate of 5,000 rpm at a system temperature of 58°C for 15 minutes to obtain a hot emulsion.

[0086] d) Cool the hot emulsion obtained in step c) to 25°C at a cooling rate of 5°C / min to obtain a cooled emulsion; add water for injection to the cooled emulsion to a total of 1000 parts by weight, mix and filter through a 0.22 μm filter membrane to obtain an oil-in-water adjuvant containing squalene.

[0087] Example 3:

[0088] This embodiment provides an oil-in-water adjuvant containing squalene, which comprises the following components per 1000 parts by weight:

[0089] Squalene: 100 parts; Polysorbate 80: 20 parts; Sorbitan trioleate: 20 parts; Thermosensitive nonionic block copolymer (Poloxamer 188: 5 parts; Poloxamer 407: 5 parts); Amino acid CMT modifier (L-arginine): 15 parts; Polyol CMT modifier (sorbitol): 50 parts; Citric acid: 0.9 parts; Sodium citrate: 18.5 parts; The remainder is water for injection: make up to 1000 parts. The pH value of this oil-in-water adjuvant is 7.5.

[0090] The preparation method of this adjuvant includes the following steps:

[0091] a) Weigh 100 parts of squalene, 20 parts of polysorbate 80 and 20 parts of trioleate sorbitan, mix them and heat to 70°C to obtain the oil phase.

[0092] b) Weigh 5 parts of poloxamer 188, 5 parts of poloxamer 407, 15 parts of L-arginine, 50 parts of sorbitol, 0.9 parts of citric acid and 18.5 parts of sodium citrate, add them to 700 parts of water for injection that has been cooled to 4°C, and stir until the components are completely dissolved to obtain a low-temperature aqueous solution; heat the low-temperature aqueous solution to 60°C and maintain the temperature for 30 minutes to obtain a pretreated aqueous phase.

[0093] c) Add the pretreated aqueous phase obtained in step b) to the oil phase obtained in step a), and emulsify at a shear rate of 15,000 rpm at a system temperature of 62°C for 10 minutes to obtain a hot emulsion.

[0094] d) Cool the hot emulsion obtained in step c) to 25°C at a cooling rate of 1°C / min to obtain a cooled emulsion; add water for injection to the cooled emulsion to a total of 1000 parts by weight, mix and filter through a 0.22 μm filter membrane to obtain an oil-in-water adjuvant containing squalene.

[0095] Comparative Examples 1-7.

[0096] Comparative Example 1:

[0097] Compared to Example 1, the difference is that the formulation does not contain poloxamer 188, L-arginine, and sorbitol. In the aqueous phase preparation step b), only citric acid and sodium citrate are dissolved in water for injection and heated to 55°C; no low-temperature dissolution or heating and holding treatment is performed. All other steps are the same.

[0098] Comparative Example 2:

[0099] Compared to Example 1, the difference is that the formulation does not contain L-arginine and sorbitol. In the aqueous phase preparation step b), poloxamer 188, citric acid, and sodium citrate are dissolved in water for injection and heated to 55°C, without low-temperature dissolution and heating treatment. All other steps are the same.

[0100] Comparative Example 3:

[0101] Compared to Example 1, the difference lies in that, in the aqueous phase preparation step b), poloxamer 188, L-arginine, sorbitol, citric acid, and sodium citrate were directly dissolved in water for injection at room temperature (25°C), and then directly heated to 55°C without low-temperature dissolution and heating and holding treatment. All other steps were the same.

[0102] Comparative Example 4:

[0103] The difference from Example 1 is that the formulation does not contain L-arginine. Everything else is the same.

[0104] Comparative Example 5:

[0105] The difference from Example 1 is that the formulation does not contain sorbitol. Everything else is the same.

[0106] Comparative Example 6:

[0107] The difference from Example 1 is that the emulsification temperature in step c) is set to 70°C. All other conditions are the same.

[0108] Comparative Example 7:

[0109] Compared to Example 1, the difference lies in that, in step b) of the aqueous phase preparation, the heating temperature of the pretreated aqueous phase is set to 30°C, and the temperature is maintained at this temperature for 20 minutes. All other aspects are the same.

[0110] Test case

[0111] Test Example 1: Determination of basic physicochemical properties.

[0112] 1. Experimental Samples:

[0113] Oil-in-water adjuvants containing squalene prepared in Examples 1, 2 and 3.

[0114] 2. Experimental steps:

[0115] (1) Appearance and pH value determination:

[0116] Take 5 mL of each sample from Examples 1-3 and place them in a clean, transparent glass bottle. Under room temperature (25°C) and natural light conditions, visually observe their color and state, and record whether there is stratification, flocculation, or visible particulate matter.

[0117] Take 10 mL of each sample from Examples 1-3 and measure the pH value of the samples using a laboratory pH meter calibrated with standard buffer solutions (pH 4.01, 7.00) at a constant temperature of 25°C. Each sample was measured three times, and the average value was recorded.

[0118] (2) Particle size distribution and zeta potential measurement:

[0119] The samples from Examples 1-3 were diluted 100-fold with water for injection and gently mixed. A dynamic light scattering (DLS) particle size analyzer was used, with the detection temperature set to 25°C and the equilibration time to 120 seconds. The diluted samples were placed in the instrument, and their average hydrated particle size (Z-average), polydispersity index (PDI), and zeta potential were measured. Each sample was measured three times, and the average value was recorded.

[0120] 3. Experimental data:

[0121] The basic physicochemical characterization data of Examples 1-3 are shown in Table 1.

[0122] Table 1: Basic physicochemical property characterization data of Examples 1-3

[0123] Sample number Appearance pH value Average particle size (nm) PDI Zeta potential (mV) Example 1 A homogeneous milky white liquid, without stratification or visible particulate matter. 6.52 135.2 0.163 -21.5 Example 2 A homogeneous milky white liquid, without stratification or visible particulate matter. 6.03 98.7 0.211 -18.3 Example 3 A homogeneous milky white liquid, without stratification or visible particulate matter. 7.48 161.4 0.185 -24.7

[0124] The experimental data in Table 1 show that the adjuvant samples prepared in Examples 1, 2, and 3 all appeared as homogeneous milky white liquids, without any observed layering, flocculation, or visible particulate matter. The measured pH values ​​(6.03 to 7.48) were all within the range of 6.0 to 7.5. Dynamic light scattering results showed that the average particle size of the three examples was in the nanometer range (98.7 nm to 161.4 nm), and the polydispersity index (PDI) values ​​were all below 0.25 (0.163 to 0.211). The measured Zeta potential values ​​were all negative (-18.3 mV to -24.7 mV), indicating that the droplet surface was charged.

[0125] The above results confirm the feasibility of the technical solution of the present invention. This solution involves a specific aqueous phase preparation procedure, namely step b), in which the thermosensitive nonionic block copolymer, amino acid-based CMT modifier, and polyol-based CMT modifier are first fully dissolved and hydrated in water for injection at a low temperature (4°C to 10°C). This low-temperature aqueous solution is then heated to 50°C to 60°C and held at this temperature to obtain a pretreated aqueous phase. This pretreated aqueous phase is then mixed with the oil phase at a set temperature (58°C to 62°C) in the subsequent emulsification step c). This series of controlled process steps forms the basis for the formation of the aforementioned uniform nanoemulsion with controlled particle size.

[0126] Examples 1, 2, and 3 cover combinations of component mass fractions and process parameters within different ranges. Data from Test Example 1 consistently demonstrates that the technical solutions of this invention are reproducible within the stated parameter range, enabling the preparation of oil-in-water adjuvants that meet predetermined physical properties (nanoscale particle size, low PDI, uniform appearance). These uniform physical properties are prerequisites for achieving long-term storage stability of the adjuvant.

[0127] Test Example 2: Accelerated stability test (evaluating long-term storage stability).

[0128] 1. Experimental Samples:

[0129] Oil-in-water adjuvants containing squalene prepared in Examples 1, 2, and 3.

[0130] Oil-in-water adjuvants containing squalene were prepared according to Comparative Examples 1, 2, 3, 4, 5, 6, and 7.

[0131] 2. Experimental steps:

[0132] (1) The samples of Examples 1-3 and Comparative Examples 1-7 were aseptically dispensed into 5mL vials and sealed with caps.

[0133] (2) Place all the above samples in a constant temperature incubator at 40℃±2℃ and store them in the dark.

[0134] (3) Take out the corresponding batch of samples in the 0th month, the 1st month, the 3rd month and the 6th month respectively.

[0135] (4) After sampling, first equilibrate at room temperature (25℃) for 30 minutes, visually observe the appearance of the sample, and record whether there is stratification, flocculation, oil droplet precipitation or demulsification.

[0136] (5) Using the dynamic light scattering (DLS) method described in Test Example 1 (2), determine the average hydrated particle size (Z-average) and polydispersity index (PDI) of each sample at different time points. If the sample shows visible layering or demulsification, mark it as "N / A" (not applicable) or record the appearance, and do not perform DLS measurement again.

[0137] 3. Experimental data:

[0138] The stability data of Examples 1-3 and Comparative Examples 1-7 under accelerated storage conditions at 40°C are shown in Table 2.

[0139] Table 2: Changes in particle size and PDI during accelerated stability testing (at 40℃)

[0140] Sample number Detection time Average particle size (nm) PDI Appearance Example 1 October 135.2 0.163 Uniform emulsion January 139.1 0.169 Uniform emulsion March 141.8 0.177 Uniform emulsion June 146.3 0.185 Uniform emulsion Example 2 October 98.7 0.211 Uniform emulsion January 101.5 0.219 Uniform emulsion March 105.8 0.228 Uniform emulsion June 110.4 0.236 Uniform emulsion Example 3 October 161.4 0.185 Uniform emulsion January 164.3 0.19 Uniform emulsion March 169.1 0.197 Uniform emulsion June 173.9 0.203 Uniform emulsion Comparative Example 1 October 141.5 0.315 Uniform emulsion January 388.2 0.622 Uniform emulsion March 915 0.904 Obvious flocculation June N / A N / A Severe oil-water separation Comparative Example 2 October 150.3 0.288 Uniform emulsion January 327.4 0.521 Uniform emulsion March 540.1 0.71 Visible oil droplets June N / A N / A Breast breaking Comparative Example 3 October 165.7 0.354 Uniform emulsion January 310.8 0.513 Uniform emulsion March 604.5 0.781 Visible aggregation June N / A N / A Oil droplet precipitation Comparative Example 4 October 138.1 0.201 Uniform emulsion January 255.6 0.347 Uniform emulsion March 451.9 0.503 Uniform emulsion June 715.3 0.611 Bottom sedimentation Comparative Example 5 October 140.4 0.196 Uniform emulsion January 279.1 0.385 Uniform emulsion March 488 0.537 Uniform emulsion June 758 0.642 Bottom sedimentation Comparative Example 6 October 181.2 0.309 Uniform emulsion January 366.5 0.463 Uniform emulsion March 612 0.608 Visible aggregation June 805.4 0.722 obvious clustering Comparative Example 7 October 177.6 0.294 Uniform emulsion January 341.3 0.435 Uniform emulsion March 570.8 0.581 Visible aggregation June 763.1 0.69 obvious clustering

[0141] The accelerated stability test data in Table 2 show that the adjuvants prepared in Examples 1, 2, and 3 maintained a homogeneous emulsion appearance after being stored at 40°C for 6 months. The average particle size and polydispersity index (PDI) remained stable without significant increase. For example, the average particle size of Example 1 increased from 135.2 nm to 146.3 nm, and the PDI increased from 0.163 to 0.185, demonstrating the physical stability of the adjuvant under these accelerated conditions.

[0142] In contrast, samples from Comparative Examples 1-7 all exhibited physical instability, showing a sharp increase in average particle size and PDI during storage, and noticeable changes in appearance such as flocculation, oil-water separation, demulsification, or oil droplet precipitation at the end of storage (3 or 6 months). In particular, the comparison between Comparative Example 3 (whose composition was the same as Example 1, but the preparation process was different, with the aqueous phase component dissolved at 25°C and without pre-treatment) and Example 1 (comparative Example 3 showed oil droplet precipitation at 6 months, while Example 1 was stable) confirms that the specific aqueous phase treatment procedure in step b), namely low-temperature (4°C to 10°C) dissolution and hydration followed by pre-treatment at higher temperatures (50°C to 60°C), is crucial for obtaining high-temperature storage stability.

[0143] Furthermore, comparisons of Comparative Example 4 (L-arginine deficiency) and Comparative Example 5 (sorbitol deficiency) with Example 1 (particle sizes increased to 715.3 nm and 758.0 nm, respectively, after 6 months) confirmed the synergistic effect of amino acid-based CMT modifiers and polyol-based CMT modifiers in the system. The technical solution of this invention, through a ternary synergistic system (temperature-sensitive nonionic block copolymer, amino acid-based CMT modifier, and polyol-based CMT modifier), combined with specific low-temperature hydration, temperature-pre-activation, and temperature-controlled emulsification processes, forms a physically stable interface layer on the surface of squalene oil droplets. This interface layer effectively inhibits the aggregation or coalescence of droplets under high-temperature storage conditions of 40°C, thereby solving the technical problem of insufficient physical stability of adjuvants.

[0144] Test Example 3: Freeze-thaw stability test.

[0145] 1. Experimental Samples:

[0146] Oil-in-water adjuvants containing squalene prepared in Examples 1, 2, and 3.

[0147] Oil-in-water adjuvants containing squalene prepared in Comparative Examples 1, 2, 3, 4, 5, 6, and 7. (Note: The initial data for each sample at cycle 0 are taken from month 0 data in Test Example 2.)

[0148] 2. Experimental steps:

[0149] (1) The samples of Examples 1-3 and Comparative Examples 1-7 were aseptically dispensed into 5mL vials and sealed with caps.

[0150] (2) Place all the above samples in a freezer at -20℃±2℃ and freeze for 24 hours.

[0151] (3) Take out the sample and thaw it at room temperature (25℃±2℃) for 24 hours.

[0152] (4) Steps (2) and (3) above constitute a freeze-thaw cycle. Repeat this cycle for a total of 5 cycles.

[0153] (5) After completing 5 cycles, equilibrate the sample at room temperature (25°C) for 30 minutes. First, visually observe the appearance of the sample and record whether there is any layering, flocculation, oil droplet precipitation or demulsification.

[0154] (6) Determine the average hydrated particle size (Z-average) and polydispersity index (PDI) of each sample according to the dynamic light scattering (DLS) method described in Test Example 1 (2). If the sample shows visible demulsification or severe stratification, mark it as "N / A" (not applicable).

[0155] 3. Experimental data: The stability data of Examples 1-3 and Comparative Examples 1-7 after 5 freeze-thaw cycles are shown in Table 3.

[0156] Table 3: Changes in particle size and PDI before and after freeze-thaw stability test (5 cycles)

[0157] Sample number Freeze-thaw cycle count Average particle size (nm) PDI Appearance Example 1 0 135.2 0.163 Uniform emulsion 5 151.6 0.198 Uniform emulsion Example 2 0 98.7 0.211 Uniform emulsion 5 119.3 0.245 Uniform emulsion Example 3 0 161.4 0.185 Uniform emulsion 5 179 0.213 Uniform emulsion Comparative Example 1 0 141.5 0.315 Uniform emulsion 5 N / A N / A Severe demulsification, oil-water separation Comparative Example 2 0 150.3 0.288 Uniform emulsion 5 N / A N / A Obvious oil phase precipitation Comparative Example 3 0 165.7 0.354 Uniform emulsion 5 N / A N / A Demulsification, flocculent material Comparative Example 4 0 138.1 0.201 Uniform emulsion 5 934.7 0.761 A large amount of visible particulate matter Comparative Example 5 0 140.4 0.196 Uniform emulsion 5 981 0.793 A large amount of visible particulate matter Comparative Example 6 0 181.2 0.309 Uniform emulsion 5 1055.2 0.83 flocculation Comparative Example 7 0 177.6 0.294 Uniform emulsion 5 1102 0.854 flocculation

[0158] Table 3 shows the freeze-thaw stability test data, indicating that the samples from Examples 1, 2, and 3 maintained a homogeneous emulsion appearance after five freeze-thaw cycles (-20°C to 25°C), without any demulsification, flocculation, or oil-water separation. DLS measurements showed only a slight increase in average particle size and PDI. For example, the average particle size of Example 1 increased from 135.2 nm to 151.6 nm, and the PDI increased from 0.163 to 0.198. These results demonstrate that the adjuvant prepared according to the technical solution of this invention possesses freeze-thaw stability.

[0159] In contrast, Comparative Examples 1, 2, and 3 all exhibited severe physical failure (demulsification, oil phase precipitation, or flocculent formation) after 5 cycles, making DLS measurements impossible. The complete failure of Comparative Example 3 (whose composition was the same as Example 1, but prepared using an aqueous phase process with dissolution at 25°C and without preheating) confirms that the specific aqueous phase treatment procedure in step b)—namely, low-temperature hydration and preheating activation—is essential for constructing a freeze-thaw resistant structure. This process allows the thermosensitive nonionic block copolymer to form a specific hydration state and prepolymer structure in the aqueous phase before emulsification.

[0160] The particle sizes of Comparative Example 4 (lacking L-arginine) and Comparative Example 5 (lacking sorbitol) increased sharply after freeze-thaw cycles (reaching 934.7 nm and 981.0 nm, respectively), and the PDI increased significantly, indicating that the emulsion structure had been disrupted. This data, compared with Example 1, confirms that the ternary synergistic system (thermosensitive nonionic block copolymer, amino acid-based CMT modifier, and polyol-based CMT modifier) ​​is essential to resist the mechanical stress generated by ice crystal formation and melting during freeze-thaw cycles. The technical solution of this invention, through the combination of this ternary synergistic system and a specific aqueous pretreatment process, forms a physically stable composite interface layer at the oil droplet interface. This interface layer maintains structural integrity during low-temperature freezing and room-temperature thawing cycles, thereby ensuring the physical stability of the adjuvant.

[0161] Test Example 4: Evaluation of basic immune activity.

[0162] 1. Experimental Samples:

[0163] Oil-in-water adjuvant containing squalene prepared in Example 1.

[0164] Oil-in-water adjuvant containing squalene prepared in Comparative Example 1.

[0165] Oil-in-water adjuvant containing squalene prepared in Comparative Example 3.

[0166] Physiological saline (blank control).

[0167] 2. Experimental steps:

[0168] (1) Preparation of immunomodulators:

[0169] The squalene-containing oil-in-water adjuvant samples from Examples 1, 1, and 3 were aseptically mixed with ovalbumin (OVA) solution, and vortexed for 30 seconds. The final concentration was adjusted so that each immunization dose (100 μL) contained 10 μg of OVA. The saline group was prepared by mixing with OVA in the same manner.

[0170] (2) Animal immunization:

[0171] Female BALB / c mice aged 6-8 weeks were randomly divided into 4 groups of 8 mice each. On day 0 and day 14 of the experiment, mice in each group were immunized by subcutaneous injection at multiple sites on their backs, with each mouse receiving 100 μL of the corresponding immunizing agent.

[0172] (3) Serum sample collection and processing:

[0173] On day 28 of the experiment (14 days after the second immunization), whole blood was collected from mice in each group using the orbital venous plexus sampling method. Blood samples were allowed to stand at room temperature for 1 hour, then centrifuged at 4°C and 3000 rpm for 15 minutes. The supernatant was separated and collected as serum. Serum samples were stored at -80°C for later use.

[0174] (4) Antibody titer detection:

[0175] The titer of total OVA-specific IgG antibodies in serum was detected using an indirect ELISA method. Serum samples were serially diluted, and the highest dilution with an OD value greater than 2.1 times that of the negative control was used as the antibody titer for that serum sample. Data were converted and recorded using log10.

[0176] 3. Experimental data:

[0177] The results of detecting the titer of OVA-specific IgG antibodies in the serum of mice in each group are shown in Table 4.

[0178] Table 4: OVA-specific IgG antibody titers in the serum of mice in each group (log10)

[0179] Group Animal number 1 Animal number 2 Animal No. 3 Animal number 4 Animal number 5 Animal number 6 Animal number 7 Animal number 8 Mean ± Standard Deviation physiological saline <2.0 <2.0 <2.0 <2.0 <2.0 <2.0 <2.0 <2.0 <2.0 Comparative Example 1 3.15 3.41 3.28 3.09 3.33 3.52 3.2 3.18 3.27±0.15 Comparative Example 3 3.98 4.21 4.05 4.3 3.89 4.16 4.25 4.08 4.11±0.14 Example 1 5.01 5.24 4.95 5.31 5.18 5.09 5.22 5.1 5.14±0.12

[0180] The immunogenicity evaluation data in Table 4 show that, compared with the saline control group (IgG antibody titer log10 < 2.0), all adjuvant groups induced OVA-specific IgG antibody responses. The mean antibody titer (log10) of the Example 1 group was 5.14. The mean antibody titer of Comparative Example 1 (lacking the ternary synergistic system and specific process) was 3.27. The mean antibody titer of Comparative Example 3 (with the same components as Example 1, but using an aqueous phase preparation process dissolved at 25°C without pretreatment) was 4.11.

[0181] The antibody titer of Example 1 (5.14) was higher than that of Comparative Example 1 (3.27). This data comparison shows that the combination of the ternary synergistic system (thermosensitive nonionic block copolymer, amino acid CMT modulator, and polyol CMT modulator) and the specific preparation process used in this invention is necessary to obtain the antibody titer level.

[0182] The comparison between Example 1 (5.14) and Comparative Example 3 (4.11) confirms that the aqueous phase preparation process has a direct impact on the level of immune response. Comparative Example 3, which used 25°C dissolution without preheating, induced a lower antibody titer than Example 1, which used the specific aqueous phase treatment procedure in step b) (low-temperature hydration, preheating activation). This data confirms that this specific aqueous phase treatment procedure, combined with the adjuvant nanoemulsion structure formed by the ternary synergistic system, is the reason for achieving this level of immune response, thus solving the technical problem of insufficient adjuvant immunostimulatory activity.

[0183] Test Example 5: Evaluation of post-storage immune activity preservation.

[0184] 1. Experimental Samples:

[0185] Accelerated aging group: Taken from test example 2, the oil-in-water adjuvant containing squalene from example 1 after being stored at 40°C for 6 months.

[0186] Freeze-thaw aging group: Taken from test example 3, the oil-in-water adjuvant containing squalene from example 1 after 5 freeze-thaw cycles.

[0187] Blank control group: physiological saline.

[0188] Fresh control group (data cited): This test case uses the initial immunogenicity data of the corresponding sample in Test Case 4 (Table 4) for benchmark comparison. Here, "fresh" means that the sample has not undergone any accelerated storage or freeze-thaw cycles after preparation (i.e., storage time is 0).

[0189] The “fresh” data cited (from Table 4) are as follows: the average IgG antibody titer (log10) in the fresh control group of Example 1 is 5.14 ± 0.12; the average in the fresh control group of Comparative Example 1 is 3.27 ± 0.15; and the average in the fresh control group of Comparative Example 3 is 4.11 ± 0.14.

[0190] Note: Based on the data from Test Case 2 and Test Case 3, the samples from Comparative Example 1 and Comparative Example 3, after being stored under accelerated conditions for 6 months or undergoing 5 freeze-thaw cycles, all experienced severe demulsification, oil-water separation, or oil droplet precipitation, losing their basic physical form as emulsion adjuvants and therefore could not be used for subsequent animal immune evaluation.

[0191] 2. Experimental steps:

[0192] (1) Preparation of immunomodulators:

[0193] The squalene-containing oil-in-water adjuvant samples from the "accelerated aging group" (Example 1, 40℃ / 6M) and the "freeze-thaw aging group" (Example 1, 5x F / T) were mixed with the model antigen ovalbumin (OVA) solution under aseptic conditions, using the same method as in Test Example 4. The blank control group (physiological saline group) was also mixed with OVA.

[0194] (2) Animal immunization:

[0195] Female BALB / c mice aged 6-8 weeks were randomly divided into 3 groups (accelerated aging group, freeze-thaw aging group, and saline group), with 8 mice in each group. They were immunized according to the same immunization program (subcutaneous injection on day 0 and day 14) and dosage described in Test Example 4.

[0196] (3) Serum sample collection and processing:

[0197] Similar to test case 4, serum was collected and processed on day 28 of the experiment.

[0198] (4) Antibody titer detection:

[0199] Similar to test example 4, the titer of total OVA-specific IgG antibodies in serum was detected using an indirect ELISA method.

[0200] 3. Experimental data:

[0201] The results of detecting the titer of OVA-specific IgG antibodies in the serum of mice in the aging sample immunization group are shown in Table 5.

[0202] Table 5: OVA-specific IgG antibody titers in the serum of mice immunized with aged samples (log10)

[0203] Group Animal number 1 Animal number 2 Animal No. 3 Animal number 4 Animal number 5 Animal number 6 Animal number 7 Animal number 8 Mean ± Standard Deviation physiological saline <2.0 <2.0 <2.0 <2.0 <2.0 <2.0 <2.0 <2.0 <2.0 Example 1 (40℃ / 6M) 4.98 5.15 5.03 5.2 4.9 5.06 5.11 4.95 5.05±0.11 Example 1 (5x F / T) 4.92 5.08 4.99 5.13 5.05 4.96 5.17 4.89 5.02±0.10

[0204] The test data in Table 5 show that the average OVA-specific IgG antibody titer (log10) induced in mice immunized with the squalene-containing oil-in-water adjuvant of Example 1 (accelerated aging group) stored at 40°C for 6 months was 5.05. The average antibody titer induced in mice immunized with the Example 1 adjuvant after 5 freeze-thaw cycles (freeze-thaw aging group) was 5.02. Comparison of this data with the fresh control group of Example 1 adjuvant in Test Example 4 (average value 5.14) showed no significant difference among the three groups. This result confirms that the ability of the Example 1 adjuvant to induce an immune response did not significantly decrease after exposure to physical stress conditions.

[0205] Meanwhile, the accelerated aging sample data and freeze-thaw aging sample data from Example 1 (5.05 and 5.02) were compared with the “fresh” comparative example data from Test Example 4. These data (5.05 and 5.02) were significantly higher than the mean antibody titer of the fresh control group in Comparative Example 1 (3.27) and the mean antibody titer of the fresh control group in Comparative Example 3 (4.11). In contrast, the squalene-containing oil-in-water adjuvants in Comparative Examples 1 and 3 underwent physical structural degradation (demulsification or delamination) under the same storage conditions (as shown in Test Examples 2 and 3), rendering them unsuitable for evaluating the immunomodulatory activity of this test example.

[0206] The two technical problems of physical stability and immunomodulatory activity addressed by the present invention are verified in a unified manner. Test Examples 2 and 3 confirm that by combining the ternary synergistic system (thermosensitive nonionic block copolymer, amino acid CMT modulator, and polyol CMT modulator) with a specific aqueous phase preparation procedure (low-temperature hydration and pre-activation at elevated temperature), the adjuvant achieves physical stability against high temperatures and freeze-thaw cycles. Data from Test Example 5 further confirms that this physical stability ensures that the adjuvant's function of inducing antibody responses is maintained during storage, and that its activity level after aging is still higher than that of the unaged comparative sample, thus solving the technical problem of decreased immunomodulatory activity due to physical structural damage of the adjuvant.

Claims

1. An oil-in-water squalene-containing adjuvant, characterized in that, It includes the following components per 1000 parts by weight: Squalene: 25-100 parts; Polysorbate 80: 1-20 parts; Sorbitan trioleate: 1-20 parts; Temperature-sensitive nonionic block copolymer: 0.5-10 parts; Amino acid-based CMT modulator: 1-15 parts; Polyol-based CMT modifier: 10-50 parts; Citric acid: 0.9-2.1 parts; Sodium citrate: 0.6-18.5 parts; The remainder is water for injection: bring the total to 1000 parts; The preparation method of the squalene-containing oil-in-water adjuvant includes the following steps: a) Mix squalene, polysorbate 80 and sorbitan trioleate and heat to 60-70°C to obtain the oil phase; b) Dissolve the thermosensitive nonionic block copolymer, amino acid CMT modifier, polyol CMT modifier, citric acid, and sodium citrate in water for injection at 4-10°C to obtain a low-temperature aqueous solution; then heat the low-temperature aqueous solution to 50-60°C and maintain it to obtain a pretreated aqueous phase; c) The pretreated aqueous phase is added to the oil phase, and shear emulsification is performed at a temperature of 58-62°C and a shear rate of 5,000-15,000 rpm for 10-15 minutes to obtain a hot emulsion. d) Cool the hot emulsion to 25°C to obtain a cooled emulsion; add water for injection to the cooled emulsion to a total of 1000 parts by weight, mix, and filter through a 0.22 μm filter membrane to obtain an oil-in-water adjuvant containing squalene; The thermosensitive nonionic block copolymer is poloxamer 188 and / or poloxamer 407; the amino acid CMT modulator is L-arginine or L-lysine; and the polyol CMT modulator is sorbitol or mannitol.

2. An oil-in-water squalene-containing adjuvant according to claim 1, characterised in that, The pH value of the squalene-containing oil-in-water adjuvant is 6.0-7.

5.

3. A process for the preparation of an oil-in-water squalene-containing adjuvant as claimed in any one of claims 1-2, characterized in that, Includes the following steps: a) Mix 25-100 parts by weight of squalene, 1-20 parts by weight of polysorbate 80 and 1-20 parts by weight of trioleate sorbitan and heat to 60-70°C to obtain an oil phase; b) Dissolve 0.5-10 parts by weight of a thermosensitive nonionic block copolymer, 1-15 parts by weight of an amino acid-based CMT modifier, 10-50 parts by weight of a polyol-based CMT modifier, 0.9-2.1 parts by weight of citric acid, and 0.6-18.5 parts by weight of sodium citrate in water for injection at 4-10°C to obtain a low-temperature aqueous solution; then heat the low-temperature aqueous solution to 50-60°C and maintain it to obtain a pretreated aqueous phase; c) The pretreated aqueous phase is added to the oil phase, and shear emulsification is performed at a temperature of 58-62°C and a shear rate of 5,000-15,000 rpm for 10-15 minutes to obtain a hot emulsion. d) Cool the hot emulsion to 25°C to obtain a cooled emulsion; add water for injection to the cooled emulsion to a total of 1000 parts by weight, mix, and filter through a 0.22 μm filter membrane to obtain an oil-in-water adjuvant containing squalene; The thermosensitive nonionic block copolymer is poloxamer 188 and / or poloxamer 407; the amino acid CMT modulator is L-arginine or L-lysine; and the polyol CMT modulator is sorbitol or mannitol.

4. A process for the preparation of an oil-in-water squalene-containing adjuvant according to claim 3, characterized in that, In step b), the holding time is 15-30 minutes.

5. A process for the preparation of an oil-in-water squalene-containing adjuvant according to claim 3, characterized in that, In step d), the cooling rate is 1-5°C / minute.

6. The use of a squalene-containing oil-in-water adjuvant according to any one of claims 1-2 in the preparation of vaccines or immunizing agents.