Preparation method of MPL adjuvant

Abstract

The invention relates to the field of biological pharmacy, and discloses a preparation method of an MPL adjuvant, which comprises the following steps: dissolving an MPL raw material in a buffer solution to obtain an MPL solution; adding a freeze-drying protective agent into the MPL solution to form a stable liquid system; carrying out pre-freezing treatment of cooling by stages on the obtained solution; freezing and drying the pre-freezing system under a temperature control condition to obtain freeze-dried powder; and sealing the freeze-dried powder in a treated packaging material to form a final product, the freeze-drying protective agent is prepared from trehalose, a polyethylene glycol-polylactic acid segmented copolymer (PEG-PLA) and L-methionine. A dynamic temperature regulation and control technology is adopted, the temperature of the shelf is adjusted in real time, the sample temperature is kept in a subcritical interval of-42 DEG C to-38 DEG C, the sublimation rate is ensured to be stable, lipid heat stress caused by too high temperature is avoided, and it is also prevented that the drying efficiency is affected by too low temperature.

Description

Technical Field

[0001] The invention relates to the field of biopharmaceuticals, and in particular to a method for preparing an MPL adjuvant. Background Art

[0002] In vaccine preparations, immune adjuvants and new lipid drug delivery systems, MPL (monophosphoryl lipid A) is a highly effective immune-enhancing ingredient and is widely used due to its excellent ability to activate Toll-like receptors. Especially in freeze-dried preparations that do not require a cold chain, MPL provides a unique adjuvant advantage that can improve the stability and efficacy of vaccines at room temperature. However, the MPL lipid structure is easily affected by the physical stress and oxidative environment during the freeze-drying process, resulting in its unstable particle size and variable structure.

[0003] In the prior art, some people have tried to introduce low molecular weight protective agents such as trehalose and glycine into the freeze-drying system to improve the solubility and appearance uniformity of the particles. Some schemes have introduced a buffer system to effectively delay the pH-sensitive degradation of lipid particles and improve the physical balance of freeze-dried samples to a certain extent. In addition, some studies have also tried to use traditional plastic packaging for low-oxygen storage, simplify storage conditions, and extend the shelf life of the product. The common advantages of these technologies are that they are easy to operate, the materials are common, and they show relatively ideal preservation effects in the short term, and are suitable for stability control under normal conditions.

[0004] However, these methods still have several technical gaps that cannot be ignored. First, the fixed temperature shelf freeze-drying method ignores the heat transfer difference in the sublimation stage, resulting in an increase in the temperature difference between the center and the edge of the sample. This thermal unevenness directly induces structural disorder; secondly, most protective agent systems lack an interfacial activity regulation mechanism, and sugars alone cannot maintain the spatial stability between lipid particles during sublimation, and aggregation is inevitable; in addition, the structural damage caused by the large ice crystals formed in the quick freezing process is irreversible, and the particle size increases abnormally after re-dissolving. This problem is often ignored in conventional processes; finally, the current mainstream packaging generally does not consider the long-term oxidation risk caused by oxygen micro-permeation, especially in high temperature environments, the cumulative effect of oxidation reactions is amplified, and ultimately leads to uncontrollable attenuation of the product's immune activity. For this reason, those skilled in the art proposed a method for preparing an MPL adjuvant to solve the above problems. Summary of the invention

[0005] In view of the deficiencies in the prior art, the present invention provides a method for preparing an MPL adjuvant, which solves the problems of the existing MPL freeze-dried preparations such as easy structural collapse, particle aggregation, large fluctuations in re-dissolved particle size, and poor storage stability during the drying process.

[0006] To achieve the above object, the present invention is implemented by the following technical scheme: a method for preparing an MPL adjuvant, comprising the following steps: dissolving the MPL raw material in a buffer to obtain an MPL solution; As a lipid adjuvant, MPL's hydrophobic structure is very easy to aggregate in an aqueous environment, resulting in uneven particle size and decreased activity. This step maintains the stability of MPL particles in a near-neutral environment by adjusting pH and ionic strength, reduces its tendency to self-assemble or crystallize, reduces the risk of early coagulation or precipitation, and constructs a stable precursor solution environment.

[0007] adding a lyophilization protectant to the MPL solution to form a stable liquid system; During the freeze-drying process, ice crystal formation, interfacial stress and dehydration can easily lead to structural inactivation of MPL. The added lyoprotectants can stabilize MPL particles in three aspects: glass protection, interface stability and anti-oxidation: sugars provide glass protection to limit molecular motion, polymers prevent MPL desorption / aggregation at the gas-liquid interface, and sulfur-containing amino acids can capture free oxygen radicals and reduce oxidative degradation.

[0008] The obtained solution is subjected to a pre-freezing treatment by lowering the temperature in stages; The pre-freezing step before freeze-drying determines the morphology and distribution of ice crystals. As a channel for water migration, the structure of ice crystals directly affects the drying efficiency and the internal pore structure of the product. The use of staged cooling can first quickly freeze the outer layer to limit the overall crystallization rate, and then slowly cool down to control the growth of ice crystals, achieving the structural goal of "fine ice crystals and narrow distribution".

[0009] freeze-drying the pre-frozen system under temperature-controlled conditions to obtain freeze-dried powder; If the temperature fluctuates or local heat accumulates during the sublimation stage, it will cause secondary aggregation or conformational collapse of MPL. The present invention adopts a dynamic temperature control strategy based on real-time feedback, adjusts the shelf heating intensity through sensor signals, accurately maintains the sample in the "subcritical temperature zone", minimizes thermal stress, and achieves a dynamic balance between drying and protection.

[0010] The lyophilized powder is sealed in processed packaging materials to form the final product.

[0011] MPL freeze-dried powder is extremely sensitive to oxygen and water vapor, and is prone to a decrease in immune titer due to trace oxidation or water resorption during long-term storage. This step reduces the polarity and oxygen content of the inner surface interface of the package by regulating the surface energy of the material (such as plasma treatment), and combines low-oxygen nitrogen packaging to build an inert microenvironment, effectively delaying the oxidation and hydration reaction of the finished product.

[0012] Preferably, the lyoprotectant comprises: Trehalose, which is present in the MPL solution at a concentration of 0.5% to 2.0%; Polyethylene glycol-polylactic acid block copolymer PEG-PLA, whose concentration in the MPL solution is 0.15% to 0.25%; L-methionine, which is present in the MPL solution at a concentration of 0.05% to 0.2%; The mass ratio of trehalose to PEG-PLA is 6:1 to 8:1.

[0013] Trehalose plays a leading role in structural protection in the system of the present invention. Its high hydrophilicity and non-reducing property allow it to bind to MPL molecules through hydrogen bonds during the freezing process, replacing some water molecules and avoiding the collapse of intermolecular gaps caused by dehydration of ice crystals. At the same time, trehalose forms an amorphous glass matrix during the drying stage, restricting molecular motion and delaying the secondary conformational transition of protein or lipid structures, forming the core of the "glass protection mechanism".

[0014] As an amphiphilic block copolymer, PEG-PLA acts as an "interface stabilizer" during the freeze-drying process. Its hydrophobic end is adsorbed on the outer layer of MPL, and the hydrophilic end forms a stable envelope with the solvent to prevent the reorganization or aggregation of the MPL structure. The lubricating effect of the PEG chain segment helps the formation of pores and particle dispersion during drying. The mass ratio of trehalose to PEG-PLA is controlled between 6:1 and 8:1, achieving the dual synergistic effects of "vitrification protection" and "interface stabilization".

[0015] As an antioxidant small molecule, L-methionine's main function is to capture residual dissolved oxygen or free radicals, preventing the unsaturated bonds in MPL lipids from oxidizing during drying or storage, and establishing an "active antioxidant mechanism", which not only improves the initial stability after drying, but also prolongs the immune efficacy during storage.

[0016] Preferably, the buffer is a histidine-citrate buffer system, and the concentrations of its components include: Histidine: 10 to 50 mmol / L; Citric acid: 5 to 20 mmol / L; The pH range of the buffer is 6.5 to 7.0.

[0017] During the construction of this buffer system, two key properties of MPL were fully considered: first, there are phosphate groups in its lipid skeleton, which are sensitive to pH and easily hydrolyzed or conformationally rearranged under acidic or alkaline conditions; second, its suspension state in the aqueous phase depends on the charge distribution and the stabilizing effect of the hydrophilic groups, so the solution is required to have both mild pH and a certain electrochemical buffering capacity.

[0018] Histidine is an amphiphilic molecule with both amino and imidazole groups. Its buffering effect mainly comes from the reversible protonation reaction of the imidazole group, and it exhibits a high buffering strength in the pH range of 6.0-7.5. At the same time, histidine has a certain chelating ability, which can weakly coordinate metal ions and help inhibit the oxidation or aggregation tendency of MPL induced by metal ions; citric acid is a tricarboxylic acid type organic acid, which provides a multi-site reversible acid-base balance in the buffer system, and its carboxyl group also has a strong complexing ability, which can react with potential impurities (such as Ca 2+ , Fe 3+ ) to form a stable complex, further stabilizing the system environment. In addition, the synergistic effect of citric acid and histidine is also reflected in the fine-tuning of the solution ionic strength, which can effectively reduce the colloidal instability of MPL caused by the electrostatic shielding effect.

[0019] Preferably, the pre-freezing process comprises the following steps: Cooling the MPL solution to -15°C to -25°C at a rate of 3°C / min to 5°C / min; Perform cooling enhancement during the cooling process; Continue cooling to -45°C to -55°C at a rate of 0.3°C / min to 0.8°C / min, and keep warm for 0.5 hour to 1.5 hour.

[0020] The rapid cooling in the first stage (3°C / min to 5°C / min down to -15°C to -25°C) aims to quickly cross the ice nucleation zone of the MPL solution and induce the system to form a large number of tiny nuclei, thereby limiting the growth of ice crystals. This helps to avoid local concentration effects, structural instability or lipid migration caused by excessive crystal growth, and is a prerequisite for the formation of a fine pore network structure. The implementation of cooling enhancement treatment in this process (such as liquid nitrogen gas flow assistance, shelf thermal conductivity enhancement, etc.) further improves the cooling uniformity and cooling rate, and strengthens the spatial consistency of ice crystal distribution. This treatment not only optimizes the crystal density distribution, but also effectively reduces the freezing gradient difference between the core area and the edge area. It is a key technical measure for controlling the spatial temperature difference in the pre-freezing stage of the present invention.

[0021] In the second stage, a slower cooling rate (0.3℃ / min to 0.8℃ / min) is used to further cool the solution to -45℃ to -55℃. This process is controlled at a lower rate to allow the existing microcrystalline nuclei to grow slowly under strict restrictions, thereby forming an ice crystal microstructure with small particle size, narrow distribution, and regular arrangement. By regulating the crystallization rate rather than simply controlling the final temperature, the present invention effectively avoids common freeze-drying problems such as structural collapse and pore disconnection caused by large-particle crystals. Finally, the system is kept warm in a deep cold environment of -45℃ to -55℃ for 0.5 to 1.5 hours, so that the system reaches a frozen steady state, the frozen water is completely converted into the ice phase, and a thermodynamic stability prerequisite is provided for subsequent sublimation.

[0022] Preferably, the average particle size of ice crystals formed during the cooling is between 5 μm and 15 μm, and the standard deviation is controlled between 3 μm and 8 μm.

[0023] The setting of this particle size and standard deviation ensures the uniformity of ice crystal distribution and avoids the problem of ice crystals being too large or too small in traditional freezing processes. This has an important impact on the final quality of MPL adjuvant and the product stability during the freeze-drying process. The size and distribution of ice crystals are crucial to the material transfer, particle structure stability and quality of the finished product during the freeze-drying process. During the freezing process, if the ice crystal size is too large, the volume of the ice crystal will expand in solid water, generating mechanical stress on the MPL particles, leading to structural damage and thus affecting the immune activity; if the ice crystal size is too small, it will easily lead to a slow sublimation rate during the freeze-drying process, increasing the drying time and energy consumption.

[0024] Preferably, the freeze-drying comprises: First, the sample temperature is monitored in real time by infrared sensors, and the shelf temperature is dynamically adjusted to maintain the sample temperature at -42°C to -38°C; The vacuum degree is controlled between 20-50Pa, and the drying time is 6-10 hours; Then increase the temperature to 25-30°C at 0.2-0.5°C / min, and control the moisture content of the product to be 0.5%-1.2%.

[0025] Freeze drying is a complex sublimation process that requires rapid removal of moisture at low temperatures while maintaining the physical and chemical stability of the sample. During this process, the sample temperature is first controlled between -42°C and -38°C, and the shelf temperature is adjusted through real-time feedback from an infrared sensor to ensure that the sample remains in a "subcritical" temperature zone, avoiding damage to the MPL lipid structure caused by high temperature. Too high a temperature can cause thermal stress on MPL particles and may even cause lipid self-aggregation or oxidative degradation; too low a temperature may cause the sublimation process to be too slow, affecting the drying efficiency.

[0026] The control of vacuum degree (20-50Pa) plays a key role in the present invention. Too low vacuum degree may lead to uneven distribution of water in the sublimation process, and even ice crystal regeneration or secondary freezing, while too high vacuum degree may lead to too low surface temperature of the sample, affecting the sublimation rate and efficiency. By accurately controlling the vacuum degree in the range of 20-50Pa, the present invention achieves the optimization of sublimation rate, ensures that water rises stably in the form of gasification, and avoids the phenomenon of re-adsorption of crystallized water; the duration of freeze-drying (6 to 10 hours) is also crucial. The time setting during the drying process directly affects the water distribution of the final product and the final structure of the particles. Longer drying time can ensure the stability of the sample, gradually remove the remaining water, and reduce cracks or looseness caused by too fast drying on the sample surface, while a shorter drying time may cause some water to remain, affecting the storage stability and biological activity of the product.

[0027] In the final stage of the drying process, the sample temperature is gradually increased to 25-30°C at a heating rate of 0.2-0.5°C / min. This heating strategy can gradually restore the sample morphology while ensuring that the product surface is not subjected to heat stress, allowing the sample to complete the final water removal and maintain a low moisture content (0.5% to 1.2%). The slow heating rate in this process avoids product damage or water re-adsorption due to sudden temperature rise, ensuring the immune activity and biological function of the MPL adjuvant.

[0028] Preferably, the temperature is adjusted dynamically according to the temperature difference between the sample and the set temperature and the historical temperature difference change, and the adjustment period is 8 minutes to 12 minutes.

[0029] This dynamic adjustment method can effectively avoid the negative impact of temperature fluctuations on samples. Real-time feedback of temperature difference changes can accurately adjust the temperature to prevent excessively high temperatures from causing thermal damage or too low temperatures from causing a slow sublimation rate. The shorter adjustment cycle (8-12 minutes) makes the adjustment faster, avoids temperature fluctuations, and ensures the accuracy of temperature control.

[0030] Preferably, the packaging material is a multi-layer barrier structure composite film, comprising: Outer polyester film, thickness 12μm to 25μm; Middle layer aluminum foil, thickness is 30μm to 50μm; The inner layer is made of polyolefin material with a thickness of 60μm to 100μm.

[0031] This composite structure can effectively block external moisture, oxygen and light, ensuring the stability of MPL adjuvant during storage. The outer polyester film has excellent mechanical strength and transparency, providing basic physical protection; the middle aluminum foil has extremely strong barrier properties, which can effectively prevent the penetration of water vapor and oxygen, avoid oxidation and moisture adsorption of MPL adjuvant, thereby maintaining its immune activity; the inner polyolefin material has good chemical stability and low gas permeability, effectively preventing the impact of internal and external environmental changes on the product, and further improving the sealing of the packaging.

[0032] Preferably, the inner layer polyolefin material is used for sealing after being treated with plasma, and the plasma treatment conditions include: RF power 40 to 60W; Processing time 20 to 40 seconds; The processing gas is high purity nitrogen.

[0033] Plasma treatment can effectively improve the surface properties of polyolefin materials and enhance the bonding between them and other materials (such as aluminum foil or outer film), thereby enhancing the sealing and barrier properties of packaging materials. Plasma treatment generates high-energy particles on the surface of polyolefin materials to physically and chemically modify their surfaces. The setting range of RF power (40 to 60W) and treatment time (20 to 40 seconds) ensures moderate surface modification, which can not only increase the active groups (such as hydroxyl, carboxyl, etc.) on the surface of polyolefins, but also improve their adhesion to other layered materials. Using high-purity nitrogen as the treatment gas helps to avoid oxidation reactions and ensure that the polyolefin surface is optimized for modification without causing negative changes in material properties.

[0034] Preferably, the freeze-dried powder is sealed and packaged in a nitrogen-filled environment, and the nitrogen-filled conditions are: Nitrogen purity ≥99.99%; Oxygen content is less than 0.5%; The heat sealing temperature is 160°C to 200°C, the heat sealing pressure is 0.2MPa to 0.4MPa, and the heat sealing time is 2 seconds to 5 seconds.

[0035] The long-term stability of the freeze-dried powder is ensured by nitrogen filling and precise heat-sealing process, avoiding the oxidative degradation of MPL adjuvant caused by the presence of oxygen. The nitrogen filling environment can effectively remove the oxygen in the package and avoid the oxidation reaction of MPL adjuvant during storage. The purity of nitrogen (≥99.99%) ensures that there is almost no oxygen in the environment. The oxygen content below 0.5% further inhibits the possibility of oxidation reaction and effectively protects the biological activity of the adjuvant. By precisely controlling the temperature (160℃ to 200℃), pressure (0.2MPa to 0.4MPa) and time (2 seconds to 5 seconds) of the heat-sealing process, the sealing of the packaging material is ensured while avoiding damage to the packaging material or structural changes of the MPL adjuvant caused by excessive heat-sealing temperature or pressure.

[0036] The present invention provides a method for preparing an MPL adjuvant, which has the following beneficial effects: 1. The present invention adopts dynamic temperature control technology to adjust the shelf temperature in real time to keep the sample temperature in the "subcritical" range of -42°C to -38°C, ensuring a stable sublimation rate, avoiding lipid heat stress caused by excessively high temperature, and preventing the drying efficiency from being affected by excessively low temperature. Compared with the traditional freeze-drying method with fixed temperature shelves, this solution effectively reduces temperature fluctuations, improves the structural stability of the MPL adjuvant, and avoids the problems of particle agglomeration and inactivation.

[0037] 2. The present invention constructs an optimized protective agent system through the synergistic effect of PEG-PLA, trehalose and L-methionine. PEG-PLA effectively reduces the interfacial tension and inhibits the sedimentation and aggregation of MPL particles during the freeze-drying process, while L-methionine provides antioxidant protection. In the prior art, there is a lack of stabilizers or only a single sugar protective agent is used, and the particles are prone to form large agglomerates, affecting uniformity. The scheme of the present invention makes the particle size distribution of the particles after freeze-drying more stable, thereby improving the solubility and immunoactivity of the product.

[0038] 3. The present invention adopts a staged pre-freezing strategy of slow cooling to make the ice crystal formation more uniform, avoiding the large ice crystals caused by the quick freezing method to destroy the sample structure. Gradual cooling can guide the directional growth of ice crystals, reduce drying stress concentration, and reduce the risk of cracks. Compared with the traditional one-step quick freezing process, the microstructure of the sample is more complete, the uniformity after freeze-drying is significantly improved, and good dispersibility is maintained after re-dissolution, which solves the problem of structural collapse caused by the disordered growth of ice crystals in the prior art.

[0039] 4. The present invention adopts a composite sealing process of nitrogen-filled packaging and plasma treatment to reduce the oxygen permeability to 0.4%, greatly reducing the risk of oxidation. Traditional packaging methods are prone to residual oxygen, which causes MPL to slowly oxidize during storage and affect the immune effect. The packaging scheme of the present invention avoids lipid degradation caused by oxygen accumulation, so that MPL still maintains high activity during long-term storage, solving the problem of decreased immune efficacy caused by oxidation in the prior art. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] Figure 1 It is a schematic flow chart of the preparation method of the present invention. DETAILED DESCRIPTION

[0041] The following will be combined with the drawings in the specification of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0042] Please refer to the attached Figure 1 .

[0043] Embodiment 1: Raw material composition: MPL solution: 5 mg / mL; Buffer: histidine (20 mmol / L), citric acid (10 mmol / L), pH 6.8; Lyoprotectant: Trehalose: 1.0%; PEG-PLA: 0.2%; L-Methionine: 0.1%.

[0044] step: Dissolution: Dissolve MPL in histidine-citrate buffer to obtain MPL solution.

[0045] Adding lyophilization protective agent: adding trehalose, PEG-PLA and L-methionine, mixing evenly to obtain a stable liquid system.

[0046] Pre-freezing treatment: The mixture was cooled to -20°C at a rate of 4°C / min, and then cooled to -50°C at a rate of 1°C / min, and maintained for 0.5 hours.

[0047] Freeze drying: The sample temperature was maintained between -42°C and -38°C, and the vacuum degree was controlled at 30Pa.

[0048] The freeze-drying time was set to 8 h to ensure complete removal of moisture.

[0049] Heating process: The temperature was raised to 25°C at a rate of 0.3°C / min and maintained for 10 min, with a final moisture content of 1.0%.

[0050] Package: Use a multi-layer barrier composite film (outer layer polyester film 12-25μm, middle layer aluminum foil 30-50μm, inner layer polyolefin material 60-100μm).

[0051] During the packaging process, nitrogen filling conditions are adopted (nitrogen purity ≥ 99.99%, oxygen content less than 0.5%), the heat sealing temperature is 180°C, the heat sealing pressure is 0.3MPa, and the heat sealing time is 3 seconds.

[0052] Embodiment 2: Raw material composition: MPL solution: 5 mg / mL; Buffer: histidine (30 mmol / L), citric acid (15 mmol / L), pH 6.7; Lyoprotectant: Trehalose: 1.5%; PEG-PLA: 0.25%; L-Methionine: 0.15%.

[0053] step: Dissolution: Dissolve MPL in histidine-citrate buffer to obtain MPL solution.

[0054] Adding lyophilization protective agent: adding high concentration of trehalose, PEG-PLA and L-methionine, mixing evenly to obtain a stable liquid system.

[0055] Pre-freezing treatment: The mixture was cooled to -15°C at a rate of 3°C / min, and then cooled to -45°C at a rate of 0.5°C / min, and maintained for 1 hour.

[0056] Freeze drying: The sample temperature was maintained between -42°C and -38°C, and the vacuum degree was controlled at 25Pa.

[0057] The freeze-drying time was set to 10 h to ensure complete removal of water.

[0058] Heating process: The temperature was raised to 28°C at a rate of 0.5°C / min and maintained for 10 min, with a final moisture content of 0.8%.

[0059] Package: A multilayer barrier composite film was used with nitrogen filling conditions (nitrogen purity ≥ 99.99%, oxygen content less than 0.5%), heat sealing temperature of 190°C, heat sealing pressure of 0.35MPa, and heat sealing time of 4 seconds.

[0060] Embodiment 3: Raw material composition: MPL solution: 5 mg / mL; Buffer: histidine (25 mmol / L), citric acid (12 mmol / L), pH 6.6; Lyoprotectant: Trehalose: 1.0%; PEG-PLA: 0.15%; L-Methionine: 0.1%.

[0061] step: Dissolution: Dissolve MPL in histidine-citrate buffer to obtain MPL solution.

[0062] Adding lyophilization protective agent: adding trehalose, PEG-PLA and L-methionine, mixing evenly to obtain a stable liquid system.

[0063] Pre-freezing treatment: The mixture was cooled to -18°C at a rate of 5°C / min, and then cooled to -50°C at a rate of 1.5°C / min, and maintained for 0.5 hours.

[0064] Freeze drying: The sample temperature was maintained between -40°C and -38°C, and the vacuum degree was controlled at 40Pa.

[0065] The freeze-drying time was set to 6 h to improve the drying efficiency.

[0066] Heating process: The temperature was raised to 30°C at a rate of 0.2°C / min and maintained for 8 minutes, with a final moisture content of 0.9%.

[0067] Package: Use a multilayer barrier composite film, nitrogen filling conditions (nitrogen purity ≥ 99.99%, oxygen content less than 0.5%), heat sealing temperature of 170°C, heat sealing pressure of 0.25MPa, and heat sealing time of 2 seconds.

[0068] Comparative Example 1: Compared with Example 1, the difference is that PEG-PLA is removed from the lyophilization protective agent, and only trehalose and L-methionine are used, and the rest are the same.

[0069] Comparative Example 2: Compared with Example 1, the difference is that no dynamic temperature control is set in the freeze-drying stage, but a fixed shelf temperature (-40°C) is used for drying, and the rest are the same.

[0070] Comparative Example 3: Compared with Example 2, the difference is that L-methionine is replaced by glycine, and the rest is the same.

[0071] Comparative Example 4: Compared with Example 2, the difference is that the pre-freezing stage has only one step of quick freezing to -50°C, and there is no staged cooling treatment, and the rest is the same.

[0072] Comparative Example 5: Compared with Example 3, the difference is that no nitrogen filling is performed during the packaging process, and natural packaging is adopted, and the rest is the same.

[0073] Comparative Example 6: Compared with Example 3, the difference is that the polyolefin material of the inner layer of the packaging film is not plasma treated, and the rest is the same.

[0074] Experiment 1: Purpose: Verify the effects of dynamic temperature control and lyoprotectant PEG-PLA on the temperature stability, sublimation rate and interface stability of MPL particles during the freeze-drying process.

[0075] Experimental comparison group: Example 1; Comparative Example 1; Comparative Example 2.

[0076] Experimental steps: Sample preparation: MPL protective solution was prepared according to the above formula, and trehalose, PEG-PLA, and L-methionine were completely added in Example 1; Comparative Example 1 except for PEG-PLA, the rest are the same; The formulation of Comparative Example 2 is the same as that of Example 1.

[0077] Pre-freezing stage: Cool down in stages: from room temperature → -20℃ (4℃ / min) → -50℃ (1℃ / min), keep for 0.5 hours; Freeze-drying process: Example 1: Dynamic temperature control (-42°C to -38°C real-time adjustment), vacuum degree 30Pa; Comparative Example 1: The same as Example 1; Comparative Example 2: The shelf temperature is fixed at -40°C, without dynamic adjustment; Freeze drying time 8 hours.

[0078] Process monitoring: Real-time recording of sample temperature fluctuations; Record the change of sublimation rate; After freeze-drying, samples were taken to test particle size, aggregation and solubility.

[0079] Test content and indicators: Temperature fluctuation range (℃); Sublimation rate (mg / min); Redissolved particle size after freeze-drying (measured by Zetasizer); Observe the sedimentation / aggregation phenomenon (visual + microscopic), (test results are shown in Table 1).

[0080] Table 1: Temperature and interface stability test results Test Group Temperature fluctuation range (℃) Sublimation rate (mg / min) Redissolved particle size after freeze-drying (nm) Aggregation / sedimentation phenomena Example 1 3.1 2.78 138 No obvious aggregation Comparative Example 1 3.5 2.51 267 Obvious aggregation and sedimentation Comparative Example 2 6.8 1.95 214 Slight aggregation From Table 1, we can get: Temperature control during freeze-drying is directly related to the sublimation rate of the sample and the interfacial stability of MPL. In Example 1, dynamic temperature control was used to keep the sample in the "subcritical" temperature zone (-42°C to -38°C), effectively avoiding the destruction of the MPL lipid structure caused by excessive temperature fluctuations. After the dynamic control was cancelled in Comparative Example 2, the temperature fluctuation range increased significantly, and some areas of the sample were heated unevenly, resulting in discontinuous sublimation, and then a slight aggregation phenomenon was formed, which verified the key role of temperature control accuracy in the balance of the sublimation process.

[0081] The effect of the interfacial stabilizer PEG-PLA was obvious in Comparative Example 1. After removal, the particle size of the re-dissolved particles increased and aggregation was serious. During the freeze-drying process, PEG-PLA effectively reduced the particle aggregation caused by the change of interfacial tension and inhibited the agglomeration of particles under drying stress. After removal, the interaction between MPL particles was enhanced, resulting in significant sedimentation and aggregation. The particle size after re-dissolution nearly doubled, and the dispersibility advantage of the adjuvant was lost.

[0082] Overall, the dual stabilization strategy of temperature and interface under high vacuum during freeze-drying is the core to ensure the integrity of MPL lipid structure and immune activity. The lack of any key factor (temperature control or interface protection) will lead to energy imbalance in the system, induce MPL particle self-aggregation or interface collapse, and directly affect the subsequent biological activity and use effect, which fully verifies the scientific nature and necessity of the process design of the present invention.

[0083] Experiment 2: Purpose: The antioxidant effect of L-methionine and the effect of staged pre-freezing on ice crystal size and structural stability were verified, with a focus on observing oxidation indicators, ice crystal morphology and product structural integrity.

[0084] Comparative group design: Example 2; Comparative Example 3; Comparative Example 4.

[0085] Experimental steps: Sample preparation: Prepare MPL protection solution, add trehalose, PEG-PLA and L-methionine completely as in Example 2; Comparative Example 3: L-methionine was replaced with an equal mole of glycine; The formulation of Comparative Example 4 is the same as that of Example 2.

[0086] Pre-freezing treatment: Example 2, Comparative Example 3: Cooling in stages (room temperature → -20°C (3°C / min) → -45°C (0.5°C / min), maintained for 1 hour); Comparative Example 4: Directly quick freezing at -50°C (5°C / min) without stepwise cooling.

[0087] Freeze drying process: The sample temperature was controlled between -42°C and -38°C, and the vacuum degree was 25Pa; Freeze drying time 10 hours.

[0088] Process Monitoring and Testing: Oxidation index (peroxide value determination); Ice crystal size (microscopic observation + particle size distribution); Structural integrity after freeze-drying (microscopic + physical testing), (test results are shown in Table 2).

[0089] Table 2: Comparative experimental results of freeze-drying protection and pre-freezing process Test Group Peroxide value (meq / kg) Ice crystal size (μm) Structural integrity of dry product (scored on a 10-point scale) Micro crack observation Example 2 1.8 18 9 No obvious cracks Comparative Example 3 3.9 21 7 Local crack Comparative Example 4 2.7 35 5 Many obvious cracks From Table 2, we can get: The introduction of L-methionine into the freeze-drying system significantly improved the antioxidant properties of the system. Experiments have shown that its inhibitory effect on MPL lipid peroxidation is significantly better than glycine. Since the thiol group in the methionine molecule has strong free radical scavenging and antioxidant functions, it can effectively capture active oxygen during drying and storage, reduce lipid oxidation and degradation, and maintain the integrity of the MPL particle structure, reflecting a specific protective effect in the lipid system.

[0090] The role of the staged pre-freezing design is particularly prominent in ice crystal control. Gradual cooling allows ice crystals to form slowly and have a concentrated particle size distribution, avoiding large ice crystals formed due to excessive cooling rate during the quick freezing process and reducing stress concentration during sublimation. After the staged treatment was cancelled, the size of the ice crystals increased significantly, and structural damage occurred during sublimation, resulting in more cracks and reduced structural integrity of the dry product, verifying the importance of controllable growth of ice crystals.

[0091] Overall, the antioxidant protection and fine control of ice crystals during the freeze-drying process have a dual effect on the stability of the MPL system. Any deficiency will lead to a decline in product performance: improper antioxidants will aggravate lipid oxidation, and disordered ice crystal growth will destroy the particle and matrix structure, affecting solubility and immune function, further confirming the scientific mechanism of antioxidant and ice crystal management in the design of the present invention.

[0092] Experiment 3: Purpose: To evaluate the effects of nitrogen-filled packaging and inner membrane plasma treatment on the storage stability of MPL freeze-dried preparations, focusing on the changes in oxygen permeation, sample oxidation and immunoactivity during storage.

[0093] Comparative group design: Example 3; Comparative Example 5; Comparative Example 6.

[0094] Experimental steps: Lyophilized sample preparation: The freeze-dried product was prepared according to the process of Example 3 and packaged into 3 groups.

[0095] Packaging handling: Example 3: Multilayer composite film packaging, inner layer plasma treatment, nitrogen filling to oxygen content <0.5%, sealing temperature 170°C, sealing pressure 0.25MPa; Comparative Example 5: Nitrogen filling is cancelled, and other conditions are the same; Comparative Example 6: The inner membrane was not subjected to plasma treatment, and the other conditions were the same.

[0096] Storage test (accelerated aging conditions): Store at 40℃ and 75% relative humidity for 30 days; Oxygen permeability, peroxide value (an indicator of lipid oxidation), and MPL immunoreactivity were measured.

[0097] Main testing indicators: Oxygen permeability (%); Peroxide value (meq / kg); Immune activity retention rate (ELISA test), (test results are shown in Table 3).

[0098] Table 3: Comparison of the impact of packaging technology on storage stability Test Group Oxygen permeability (%) Peroxide value (meq / kg) Immunoactivity retention rate (%) Example 3 0.4 1.6 93 Comparative Example 5 4.3 4.8 62 Comparative Example 6 2.9 3.5 75 It can be seen from Table 3 that nitrogen-filled packaging effectively reduces the oxygen content in the package, significantly reduces the risk of lipid oxidation during storage, and maintains the immunological activity of the MPL adjuvant. As a typical lipid particle, MPL is extremely susceptible to oxidative damage, especially in high temperature and high humidity environments, which is more likely to cause lipid double bond breakage and self-polymerization reactions. In a nitrogen-filled environment, reactive oxygen species are reduced, the methionine antioxidant mechanism in the system is brought into play, and the adjuvant activity is maintained well, verifying the key role of the nitrogen environment as a protective barrier.

[0099] Plasma treatment of the inner film greatly improves the sealing strength and gas barrier properties, reducing the risk of oxygen penetration. There are tiny cracks and polarity differences in the interface of untreated packaging materials, which leads to slow oxygen penetration. Under the accumulation effect, the oxidation reaction is intensified and the storage stability is significantly reduced. The performance of Example 6 in the experiment further proves that the complete inner film treatment process is an indispensable link to ensure long-term storage effect.

[0100] The overall analysis shows that the packaging process is not only a physical encapsulation, but also a core means of controlling the microenvironment. Under the dual effects of nitrogen filling and high-quality packaging film, the structural integrity and immune function of the MPL adjuvant are stabilized during long-term storage. The lack of any one of these measures will destroy the overall energy balance and microenvironment, leading to the deterioration of the MPL system and the loss of biological effects, which is completely consistent with the microenvironment stabilization mechanism proposed in the present invention, reflecting the rationality and scientificity of the design.

[0101] Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions and variations may be made to the embodiments without departing from the principles and spirit of the present invention, and that the scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A method for preparing an MPL adjuvant, characterized in that: The following steps are involved: dissolving the MPL raw material in a buffer to obtain an MPL solution; adding a lyophilization protectant to the MPL solution to form a stable liquid system; The obtained solution is subjected to a pre-freezing treatment by lowering the temperature in stages; freeze-drying the pre-frozen system under temperature-controlled conditions to obtain freeze-dried powder; The lyophilized powder is sealed in processed packaging materials to form the final product.

2. The method for preparing an MPL adjuvant according to claim 1, characterized in that: The lyophilization protective agent includes: Trehalose, which is present in the MPL solution at a concentration of 0.5% to 2.0%; Polyethylene glycol-polylactic acid block copolymer PEG-PLA, whose concentration in the MPL solution is 0.15% to 0.25%; L-methionine, which is present in the MPL solution at a concentration of 0.05% to 0.2%; The mass ratio of trehalose to PEG-PLA is 6:1 to 8:

1.

3. The method for preparing an MPL adjuvant according to claim 1, characterized in that: The buffer is a histidine-citrate buffer system, and its component concentrations include: Histidine: 10 to 50 mmol / L; Citric acid: 5 to 20 mmol / L; The pH range of the buffer is 6.5 to 7.

0.

4. The method for preparing an MPL adjuvant according to claim 1, characterized in that: The pre-freezing process comprises the following steps: Cooling the MPL solution to -15°C to -25°C at a rate of 3°C / min to 5°C / min; Perform cooling enhancement during the cooling process; Continue cooling to -45°C to -55°C at a rate of 0.3°C / min to 0.8°C / min, and keep warm for 0.5 hour to 1.5 hour.

5. The method for preparing an MPL adjuvant according to claim 4, characterized in that: The average particle size of the ice crystals formed during the cooling is between 5 μm and 15 μm, and the standard deviation is controlled between 3 μm and 8 μm.

6. The method for preparing an MPL adjuvant according to claim 1, characterized in that: The freeze drying comprises: First, the sample temperature is monitored in real time by infrared sensors, and the shelf temperature is dynamically adjusted to maintain the sample temperature at -42°C to -38°C; The vacuum degree is controlled between 20-50Pa, and the drying time is 6-10 hours; Then increase the temperature to 25-30°C at 0.2-0.5°C / min, and control the moisture content of the product to be 0.5%-1.2%.

7. The method for preparing an MPL adjuvant according to claim 6, characterized in that: The temperature is adjusted dynamically according to the temperature difference between the sample and the set temperature and the historical temperature difference change, and the adjustment cycle is 8 minutes to 12 minutes.

8. The method for preparing an MPL adjuvant according to claim 1, characterized in that: The packaging material is a multi-layer barrier structure composite film, comprising: Outer polyester film, thickness 12μm to 25μm; Middle layer aluminum foil, thickness is 30μm to 50μm; The inner layer is made of polyolefin material with a thickness of 60μm to 100μm.

9. The method for preparing an MPL adjuvant according to claim 8, characterized in that: The inner layer polyolefin material is used for sealing after being treated with plasma, and the plasma treatment conditions include: RF power 40 to 60W; Processing time 20 to 40 seconds; The processing gas is high purity nitrogen.

10. The method for preparing an MPL adjuvant according to claim 1, characterized in that: The freeze-dried powder is sealed and packaged in a nitrogen-filled environment, and the nitrogen-filled conditions are: Nitrogen purity ≥99.99%; Oxygen content is less than 0.5%; The heat sealing temperature is 160°C to 200°C, the heat sealing pressure is 0.2MPa to 0.4MPa, and the heat sealing time is 2 seconds to 5 seconds.

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