A preparation method of MPL adjuvant

Abstract

This application relates to the field of biopharmaceuticals and discloses a preparation method of an MPL adjuvant, which includes the following steps: dissolving the MPL raw material in a buffer solution to obtain an MPL solution; adding a lyoprotectant to the MPL solution to form a stable liquid system; performing a pre-freezing treatment on the obtained solution by cooling in stages; performing freeze-drying on the pre-frozen system under temperature control conditions to obtain a freeze-dried powder; sealing the freeze-dried powder in a treated packaging material to form a final product; the lyoprotectant includes: trehalose, poly(ethylene glycol)-poly(lactic acid) block copolymer PEG-PLA, L-methionine. The present invention adopts a dynamic temperature control technology to adjust the shelf temperature in real time, so that the sample temperature is maintained in the "subcritical" range of -42°C to -38°C, ensuring a stable sublimation rate, avoiding lipid thermal stress caused by too high temperature, and also preventing too low temperature from affecting the drying efficiency.

Description

Technical Field

[0001] The present invention relates to the field of biopharmaceuticals, and specifically to a preparation method of MPL adjuvant. Background Art

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

[0003] In the prior art, attempts have been made to introduce low-molecular-weight protectants such as trehalose and glycine into the freeze-drying system to improve the redissolvability and appearance homogeneity of particles. In some schemes, a buffer system is introduced, which effectively delays the pH-sensitive degradation of lipid particles and improves the physical equilibrium of freeze-dried samples to a certain extent. In addition, some studies have also tried to use traditional plastic packaging for low-oxygen preservation, simplify storage conditions, and extend the product shelf life. The common advantage of these technologies is that they are easy to operate, the materials are common, and they show relatively ideal preservation effects in the short term, which is suitable for stability control under ordinary conditions.

[0004] However, there are still several technical gaps that cannot be ignored in these methods. First, the fixed-temperature shelf freeze-drying method ignores the heat transfer difference during the sublimation stage, resulting in an enlarged temperature difference between the center and the edge of the sample, and this thermal inhomogeneity directly induces structural disorder. Second, most protectant systems lack an interfacial activity regulation mechanism, and relying solely on sugars cannot maintain the spatial stability between lipid particles during sublimation, and aggregation is inevitable. In addition, the structural damage caused by large ice crystals formed in the quick-freezing process is irreversible, and the particle size increases abnormally after redissolution, which is often ignored in conventional processes. Finally, the current mainstream packaging generally does not consider the long-term oxidation risk brought by oxygen micro-permeation. Especially in high-temperature environments, the cumulative effect of oxidation reactions is amplified, ultimately leading to uncontrollable attenuation of the immunological activity of the product. Therefore, those skilled in the art have proposed a preparation method of MPL adjuvant to solve the above problems. Summary of the Invention

[0005] In view of the deficiencies of the prior art, the present invention provides a preparation method of MPL adjuvant, which solves the problems of easy collapse of the structure, aggregation of particles, large fluctuations in redissolved particle size, and poor storage stability during the drying process of existing MPL freeze-dried preparations.

[0006] To achieve the above object, the present invention is realized through the following technical solutions: A preparation method of MPL adjuvant, comprising the following steps:

[0007] Dissolve the MPL raw material in a buffer solution to obtain an MPL solution;

[0008] As a lipid adjuvant, the hydrophobic structure of MPL is extremely prone to aggregation in an aqueous environment, resulting in uneven particle size and decreased activity. In this step, by adjusting the pH and ionic strength, the particle stability of MPL in a near-neutral environment is maintained, its self-assembly or crystallization tendency is reduced, the risk of early aggregation or precipitation is lowered, and a stable precursor solution environment is constructed.

[0009] Add a lyoprotectant to the MPL solution to form a stable liquid system;

[0010] During the freeze-drying process, MPL is prone to structural inactivation due to ice crystal formation, interfacial stress, and dehydration. The added lyoprotectant stabilizes MPL particles synergistically from three aspects: vitrification protection, interfacial stabilization, and antioxidant protection. Sugars provide vitreous protection to limit molecular movement, polymers prevent the desorption / aggregation of MPL at the gas-liquid interface, and sulfur-containing amino acids can capture free oxygen radicals and reduce oxidative degradation.

[0011] Perform a pre-freezing treatment on the obtained solution by cooling it in stages;

[0012] The pre-freezing step before freeze-drying determines the morphology and distribution of ice crystals. As the water migration channels, the structure of ice crystals directly affects the drying efficiency and the internal pore structure of the product. By cooling in stages, the outer layer can be quickly frozen first to limit the overall crystallization rate, and then the cooling rate can be controlled at a low speed to control the growth of ice crystals, achieving the structural goal of "fine ice crystals and narrow distribution".

[0013] Perform freeze-drying on the pre-frozen system under temperature control conditions to obtain a freeze-dried powder;

[0014] If there are temperature fluctuations or local heat accumulation 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 heating intensity of the shelf through sensing signals, accurately maintains the sample within the "subcritical temperature zone", minimizes heat stress to the greatest extent, and achieves a dynamic balance between drying and protection.

[0015] Seal the freeze-dried powder in a treated packaging material to form a final product.

[0016] MPL freeze-dried powder is extremely sensitive to oxygen and water vapor. During long-term storage, the immune potency is likely to decrease due to trace oxidation or moisture reabsorption. In this step, by regulating the surface energy of the material (such as plasma treatment), the polarity and oxygen content of the inner surface interface of the packaging are reduced. Combined with low-oxygen nitrogen filling packaging, an inert microenvironment is constructed to effectively delay the oxidation and hydration reactions of the finished product.

[0017] Preferably, the lyoprotectant includes:

[0018] Trehalose, with a concentration in the MPL solution ranging from 0.5% to 2.0%;

[0019] Polyethylene glycol - polylactic acid block copolymer PEG - PLA, with a concentration in the MPL solution ranging from 0.15% to 0.25%;

[0020] L - Methionine, with a concentration in the MPL solution ranging from 0.05% to 0.2%;

[0021] wherein the mass ratio of the trehalose to the PEG - PLA is from 6:1 to 8:1.

[0022] Trehalose plays a leading role in structural protection in the system of the present invention. Its high hydrophilicity and non - reducing property enable it to bind to MPL molecules through hydrogen bonds during the freezing process, replacing some water molecules and avoiding the collapse of intermolecular voids caused by ice crystal dehydration. Meanwhile, trehalose forms an amorphous glass matrix during the drying stage, restricting molecular movement, delaying the secondary conformational transformation of protein or lipid structures, and constituting the core of the "glass - state protection mechanism".

[0023] As an amphiphilic block copolymer, PEG - PLA acts as an "interface stabilizer" during the freeze - drying process. Its hydrophobic end adsorbs on the outer layer of MPL, and its hydrophilic end forms a stable coating with the solvent, preventing the reorganization or aggregation of the MPL structure. The lubricating effect of the PEG segment contributes to the formation of pores and particle dispersion during drying. Controlling the mass ratio of trehalose to PEG - PLA between 6:1 and 8:1 realizes the dual synergistic effects of "vitrification protection" and "interface stabilization".

[0024] As an antioxidant small molecule, L - Methionine mainly functions to capture residual dissolved oxygen or free radicals, preventing the oxidation of unsaturated bonds in MPL lipids during drying or storage, establishing an "active antioxidant mechanism", which not only improves the initial stability after drying but also extends the immune efficacy during storage.

[0025] Preferably, the buffer solution is a histidine - citric acid buffer system, and its component concentrations include:

[0026] Histidine: 10 to 50 mmol / L;

[0027] Citric acid: 5 to 20 mmol / L;

[0028] The pH range of the buffer solution is from 6.5 to 7.0.

[0029] In the construction of this buffer system, two key properties of MPL were fully considered: one is that there are phosphate groups in its lipid skeleton, which are sensitive to pH and prone to hydrolysis or conformational rearrangement under acidic or alkaline conditions; the other is that its suspension state in the aqueous phase depends on the charge distribution and the stabilizing effect of hydrophilic groups. Therefore, the solution is required to have a mild pH value and a certain electrochemical buffer capacity.

[0030] Histidine is an amphoteric molecule with both amino and imidazole groups. Its buffering effect mainly comes from the reversible protonation reaction of the imidazole group, showing a relatively high buffering intensity in the pH range of 6.0 - 7.5. At the same time, histidine has a certain chelating ability and can weakly coordinate metal ions to assist in inhibiting 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. At the same time, its carboxyl group also has a strong complexing ability and can form stable complexes with potential impurities (such as Ca 2+ 、Fe 3+ ), further stabilizing the system environment. In addition, the synergistic effect of citric acid and histidine is also reflected in the fine-tuning of the ionic strength of the solution, which can effectively reduce the colloidal instability of MPL caused by the electrostatic shielding effect.

[0031] Preferably, the pre-freezing treatment includes the following steps:

[0032] Cool the MPL solution at a rate of 3°C / min to 5°C / min to -15°C to -25°C;

[0033] Perform cooling enhancement treatment during the temperature reduction process;

[0034] Continue to cool at a rate of 0.3°C / min to 0.8°C / min to -45°C to -55°C and keep warm for 0.5 hour to 1.5 hours.

[0035] The rapid cooling in the first stage (from 3°C / min to 5°C / min to -15°C to -25°C) aims to quickly cross the ice nucleation zone of the MPL solution, induce a large number of tiny ice nuclei to form in the system, thereby restricting the growth of ice crystals, helping to avoid local concentration effects, structural instability or lipid migration caused by excessive crystal growth, which is the premise for forming a fine pore network structure. And during this process, performing cooling enhancement treatment (such as means of assisting with liquid nitrogen gas flow, enhancing the heat conduction efficiency of the shelf, etc.) further improves the cooling uniformity and the temperature reduction rate, and strengthens the spatial consistency of the 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, which is the key technical measure for controlling the spatial temperature difference in the pre-freezing stage of the present invention.

[0036] In the second stage, a reduced cooling rate (0.3 °C / min to 0.8 °C / min) is adopted to further cool the solution to -45 °C to -55 °C. This process is carried out at a lower rate to allow the existing microcrystal nuclei to grow slowly under strict constraints, 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 particles. Finally, it is kept warm for 0.5 to 1.5 hours in a cryogenic environment of -45 °C to -55 °C to make the system reach a frozen steady state, completely converting the frozen water into the ice phase and providing a thermodynamically stable premise for subsequent sublimation.

[0037] Preferably, the average particle size of the ice crystals formed during cooling is between 5 μm and 15 μm, and the standard deviation is controlled between 3 μm and 8 μm.

[0038] The setting of this particle size and standard deviation ensures the uniformity of the ice crystal distribution and avoids the problems of too large or too small ice crystals in the traditional freezing process, which has an important impact on the final quality of the MPL adjuvant and the product stability during the freeze-drying process. The size and distribution of ice crystals are crucial for mass transfer, particle structure stability, and the quality of the finished product during the freeze-drying process. During the freezing process, if the ice crystal particle size is too large, it will cause the volume expansion of ice crystals in solid water, generating mechanical stress on the MPL particles and resulting in structural damage, thus affecting the immunological activity; if the ice crystal particle size is too small, it will easily lead to too slow sublimation rate during the freeze-drying process, increasing the drying time and energy consumption.

[0039] Preferably, the freeze-drying includes:

[0040] First, according to the infrared sensor to monitor the sample temperature in real time, dynamically adjust the shelf temperature to keep the sample temperature at -42 °C to -38 °C;

[0041] And control the vacuum degree between 20 - 50 Pa, and the drying time is 6 - 10 hours;

[0042] Then heat up to 25 - 30 °C at a rate of 0.2 - 0.5 °C / min, and control the moisture content of the product at 0.5% - 1.2%.

[0043] Freeze-drying is a complex sublimation process, which requires rapid removal of moisture at low temperature while maintaining the physical and chemical stability of the sample. During this process, first control the sample temperature between -42 °C and -38 °C, and adjust the shelf temperature in real time through the infrared sensor feedback to ensure that the sample is kept in a "subcritical" temperature zone, avoiding the destruction of the MPL lipid structure caused by high temperature. Too high temperature will cause thermal stress of the MPL particles and may even lead to lipid self-aggregation or oxidative degradation; while too low temperature may lead to too slow sublimation process, affecting the drying efficiency.

[0044] The control of the vacuum degree (20 - 50 Pa) plays a crucial role in the present invention. An excessively low vacuum degree may lead to uneven moisture distribution during the sublimation process, and even the regeneration of ice crystals or secondary freezing may occur. While an excessively high vacuum degree may cause the surface temperature of the sample to be too low, affecting the sublimation rate and efficiency. By precisely controlling the vacuum degree within the range of 20 - 50 Pa, the present invention realizes the optimization of the sublimation rate, ensuring that the moisture steadily rises in the form of gasification, and avoiding the phenomenon of re - adsorption of crystalline moisture; the duration of freeze - drying (6 to 10 hours) is also crucial. The time setting during the drying process directly affects the moisture distribution of the final product and the final structure of the particles. A longer drying time can ensure the stability of the sample, gradually remove the remaining moisture, and reduce cracks or looseness caused by the overly rapid drying of the sample surface. While a shorter drying time may result in residual moisture, affecting the storage stability and biological activity of the product.

[0045] 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 morphology of the sample on the premise of ensuring that the surface of the product is not thermally stressed, enabling the sample to complete the final moisture removal and maintaining a low moisture content (0.5% to 1.2%). The slow heating rate during this process avoids product damage or moisture re - adsorption caused by sudden temperature rise, ensuring the immunological activity and biological function of the MPL adjuvant.

[0046] Preferably, the adjustment of the temperature is dynamically adjusted according to the temperature difference between the sample and the set temperature and the change of the historical temperature difference, and the adjustment period is 8 minutes to 12 minutes.

[0047] This dynamic adjustment method can effectively avoid the negative impact of temperature fluctuations on the sample. The real - time feedback of the temperature difference change can precisely adjust the temperature, preventing thermal damage caused by too high temperature or too slow sublimation rate caused by too low temperature. The shorter adjustment period (8 - 12 minutes) makes the adjustment faster, avoiding temperature oscillation and ensuring the accuracy of temperature control.

[0048] Preferably, the packaging material is a multi - layer barrier - structure composite film, including:

[0049] The outer - layer polyester film with a thickness of 12 μm to 25 μm;

[0050] The middle - layer aluminum foil with a thickness of 30 μm to 50 μm;

[0051] The inner - layer polyolefin material with a thickness of 60 μm to 100 μm.

[0052] This composite structure can effectively block external moisture, oxygen, and light, ensuring the stability of the 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, effectively preventing the penetration of water vapor and oxygen, avoiding the oxidation of the MPL adjuvant and water adsorption, thereby maintaining its immunological activity; the inner polyolefin material has good chemical stability and low gas permeability, effectively preventing the influence of internal and external environmental changes on the product, and further improving the sealing performance of the packaging.

[0053] Preferably, the inner polyolefin material is used for sealing after plasma treatment, and the plasma treatment conditions include:

[0054] Radio frequency power: 40 to 60 W;

[0055] Treatment time: 20 to 40 seconds;

[0056] Treatment gas: high-purity nitrogen.

[0057] Plasma treatment can effectively improve the surface properties of the polyolefin material, enhance the bonding force between it and other materials (such as aluminum foil or the outer film), thereby enhancing the sealing and barrier properties of the packaging material. Plasma treatment generates high-energy particles on the surface of the polyolefin material through excitation, causing physical and chemical modification of its surface. The set range of radio frequency power (40 to 60 W) and treatment time (20 to 40 seconds) ensure moderate surface modification, which can not only increase the active groups (such as hydroxyl groups, carboxyl groups, etc.) on the polyolefin surface but also improve its adhesion to other layer materials. Using high-purity nitrogen as the treatment gas helps to avoid oxidation reactions, ensuring an optimized modification effect on the polyolefin surface without causing negative changes in material properties.

[0058] Preferably, the lyophilized powder is sealed and packaged in a nitrogen-filled environment, and the conditions for filling nitrogen are:

[0059] Nitrogen purity ≥ 99.99%;

[0060] Oxygen content < 0.5%;

[0061] Heat sealing temperature: 160°C to 200°C, heat sealing pressure: 0.2 MPa to 0.4 MPa, heat sealing time: 2 seconds to 5 seconds.

[0062] Through nitrogen filling and precise heat-sealing technology, the long-term stability of the freeze-dried powder is ensured, and the oxidative degradation of the MPL adjuvant caused by the presence of oxygen is avoided. The nitrogen-filled environment can effectively exclude oxygen in the package, preventing the oxidation reaction of the MPL adjuvant during storage. The purity of nitrogen (≥99.99%) ensures that there is almost no oxygen component in the environment, and the oxygen content below 0.5% further inhibits the possibility of oxidation reaction, effectively protecting the biological activity of the adjuvant; by precisely controlling the temperature (160°C to 200°C), pressure (0.2 MPa to 0.4 MPa), and time (2 seconds to 5 seconds) of the heat-sealing process, the sealing performance 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.

[0063] The present invention provides a preparation method of an MPL adjuvant. It has the following beneficial effects:

[0064] 1. The present invention adopts a dynamic temperature regulation technology to adjust the shelf temperature in real time, keeping the sample temperature in the "subcritical" range of -42°C to -38°C, ensuring a stable sublimation rate, avoiding lipid thermal stress caused by too high temperature, and preventing low temperature from affecting the drying efficiency. Compared with the traditional freeze-drying method with a fixed-temperature shelf, this solution effectively reduces temperature fluctuations, improves the structural stability of the MPL adjuvant, and avoids problems such as particle aggregation and inactivation.

[0065] 2. Through the synergistic effect of PEG-PLA, trehalose, and L-methionine, the present invention constructs an optimized protective agent system. PEG-PLA effectively reduces the interfacial tension and inhibits the sedimentation and aggregation of MPL particles during freeze-drying, while L-methionine provides antioxidant protection. In the prior art, due to the lack of stabilizers or the use of only a single sugar-based protective agent, particles are prone to form large aggregates, affecting the uniformity. The solution of the present invention makes the particle size distribution after freeze-drying more stable, improving the redissolvability and immunological activity of the product.

[0066] 3. The present invention adopts a staged pre-freezing strategy with slow cooling to make the ice crystal formation more uniform, avoiding the damage to the sample structure caused by large ice crystals in the quick-freezing method. Gradual cooling can guide the directional growth of ice crystals, reduce the concentration of drying stress, and lower 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 it maintains good dispersibility after redissolution, solving the problem of structural collapse caused by the disordered growth of ice crystals in the prior art.

[0067] 4. The present invention adopts a composite sealing process of nitrogen filling packaging and plasma treatment, reducing the oxygen permeability to 0.4%, greatly reducing the oxidation risk. The traditional packaging method is prone to residual oxygen, resulting in the slow oxidation of MPL during storage and affecting the immune effect. The packaging solution of the present invention avoids lipid degradation caused by oxygen accumulation, enabling MPL to remain highly active during long-term storage and solving the problem of decreased immune efficacy caused by oxidation in the prior art. BRIEF DESCRIPTION OF THE DRAWINGS

[0068] Figure 1 It is a schematic flow chart of the preparation method of the present invention. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0069] The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the protection scope of the present invention.

[0070] Please refer to the attached Figure 1 .

[0071] Example 1:

[0072] Raw material composition:

[0073] MPL solution: 5 mg / mL;

[0074] Buffer solution: histidine (20 mmol / L), citric acid (10 mmol / L), pH 6.8;

[0075] Lyoprotectant:

[0076] Trehalose: 1.0%;

[0077] PEG-PLA: 0.2%;

[0078] L-Methionine: 0.1%.

[0079] Steps:

[0080] Dissolution: Dissolve MPL in the histidine-citric acid buffer solution to obtain an MPL solution.

[0081] Add lyoprotectant: Add trehalose, PEG-PLA and L-methionine, and mix evenly to obtain a stable liquid system.

[0082] Pre-freezing treatment:

[0083] Cool to -20°C at a rate of 4°C / min, and then cool to -50°C at a rate of 1°C / min, and hold for 0.5 hours.

[0084] Lyophilization:

[0085] The sample temperature is maintained between -42°C and -38°C, and the vacuum degree is controlled at 30 Pa.

[0086] The lyophilization time is set to 8 hours to ensure complete removal of moisture.

[0087] Heating process:

[0088] The temperature is raised to 25°C at a rate of 0.3°C / min and maintained for 10 minutes, and the final moisture content is 1.0%.

[0089] Packaging:

[0090] A multi-layer barrier composite film is used (outer layer polyester film 12 - 25μm, middle layer aluminum foil 30 - 50μm, inner layer polyolefin material 60 - 100μm).

[0091] During the packaging process, a nitrogen filling condition is adopted (nitrogen purity ≥99.99%, oxygen content < 0.5%), the heat sealing temperature is 180°C, the heat sealing pressure is 0.3 MPa, and the heat sealing time is 3 seconds.

[0092] Example 2:

[0093] Raw material composition:

[0094] MPL solution: 5 mg / mL;

[0095] Buffer solution: Histidine (30 mmol / L), citric acid (15 mmol / L), pH 6.7;

[0096] Lyoprotectant:

[0097] Trehalose: 1.5%;

[0098] PEG-PLA: 0.25%;

[0099] L-Methionine: 0.15%.

[0100] Steps:

[0101] Dissolution: Dissolve MPL in the histidine-citric acid buffer solution to obtain an MPL solution.

[0102] Add lyoprotectant: Add high concentrations of trehalose, PEG-PLA, and L-methionine, and mix evenly to obtain a stable liquid system.

[0103] Pre-freezing treatment:

[0104] Cool to -15°C at a rate of 3°C / min, and then cool to -45°C at a rate of 0.5°C / min and hold for 1 hour.

[0105] Lyophilization:

[0106] The sample temperature is maintained between -42°C and -38°C, and the vacuum degree is controlled at 25 Pa.

[0107] The lyophilization time is set to 10 hours to ensure complete removal of moisture.

[0108] Heating process:

[0109] The temperature is raised to 28°C at a rate of 0.5°C / min and maintained for 10 minutes, and the final moisture content is 0.8%.

[0110] Packaging:

[0111] Use a multi-layer barrier composite film, under nitrogen filling conditions (nitrogen purity ≥ 99.99%, oxygen content below 0.5%), the heat sealing temperature is 190°C, the heat sealing pressure is 0.35 MPa, and the heat sealing time is 4 seconds.

[0112] Example 3:

[0113] Raw material composition:

[0114] MPL solution: 5 mg / mL;

[0115] Buffer solution: histidine (25 mmol / L), citric acid (12 mmol / L), pH 6.6;

[0116] Lyoprotectant:

[0117] Trehalose: 1.0%;

[0118] PEG-PLA: 0.15%;

[0119] L-Methionine: 0.1%.

[0120] Steps:

[0121] Dissolution: Dissolve MPL in the histidine-citric acid buffer solution to obtain an MPL solution.

[0122] Add lyoprotectant: Add trehalose, PEG-PLA and L-methionine, and mix evenly to obtain a stable liquid system.

[0123] Pre-freezing treatment:

[0124] Cool to -18°C at a rate of 5°C / min, and then cool to -50°C at a rate of 1.5°C / min and hold for 0.5 hours.

[0125] Lyophilization:

[0126] The sample temperature is maintained between -40°C and -38°C, and the vacuum degree is controlled at 40 Pa.

[0127] The freeze-drying time is set to 6 hours to improve the drying efficiency.

[0128] Heating process:

[0129] The temperature is raised to 30°C at a rate of 0.2°C / min, maintained for 8 minutes, and the final moisture content is 0.9%.

[0130] Packaging:

[0131] A multi-layer barrier composite film is used, under nitrogen filling conditions (nitrogen purity ≥ 99.99%, oxygen content below 0.5%), the heat-sealing temperature is 170°C, the heat-sealing pressure is 0.25 MPa, and the heat-sealing time is 2 seconds.

[0132] Comparative Example 1: Compared with Example 1, the difference is that PEG-PLA is removed from the freeze-drying protectant, and only trehalose and L-methionine are used, and the rest are the same.

[0133] 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.

[0134] Comparative Example 3: Compared with Example 2, the difference is that L-methionine is replaced with glycine, and the rest are the same.

[0135] Comparative Example 4: Compared with Example 2, the difference is that in the pre-freezing stage, it is only quickly frozen to -50°C in one step without staged temperature reduction treatment, and the rest are the same.

[0136] Comparative Example 5: Compared with Example 3, the difference is that nitrogen is not filled during the packaging process, and natural packaging is used, and the rest are the same.

[0137] Comparative Example 6: Compared with Example 3, the difference is that the inner layer polyolefin material of the packaging film is not subjected to plasma treatment, and the rest are the same.

[0138] Experiment 1:

[0139] Experiment purpose:

[0140] To verify the effects of dynamic temperature control and the freeze-drying protectant PEG-PLA on the temperature stability, sublimation rate, and MPL particle interface stability during the freeze-drying process.

[0141] Experiment control group:

[0142] Example 1; Comparative Example 1; Comparative Example 2.

[0143] Experiment steps:

[0144] Sample preparation:

[0145] Prepare the MPL protective solution according to the aforementioned formula. In Example 1, trehalose, PEG-PLA, and L-methionine were added completely;

[0146] In Comparative Example 1, PEG-PLA was removed, and the rest was the same;

[0147] The formulation of Comparative Example 2 was the same as that of Example 1.

[0148] Pre-freezing stage:

[0149] Cool down in stages: from room temperature → -20°C (4°C / min) → -50°C (1°C / min), and hold for 0.5 hours;

[0150] Freeze-drying process:

[0151] Example 1: Dynamic temperature control (adjusted in real time from -42°C to -38°C), vacuum degree 30 Pa;

[0152] Comparative Example 1: The same as Example 1;

[0153] Comparative Example 2: The shelf temperature was fixed at -40°C without dynamic adjustment;

[0154] The freeze-drying time was 8 hours.

[0155] Process monitoring:

[0156] Record the temperature fluctuation of the sample in real time;

[0157] Record the change in sublimation rate;

[0158] After freeze-drying, take samples to detect the particle size, aggregation situation, and redissolution property.

[0159] Test contents and indicators:

[0160] Temperature fluctuation range (°C);

[0161] Sublimation rate (mg / min);

[0162] Redissolved particle size after freeze-drying (measured by Zetasizer);

[0163] Observe the sedimentation / aggregation phenomenon (visual + microscopic), (the test results are shown in Table 1).

[0164] Table 1: Experimental results table of temperature and interface stability

[0165] Test Group Temperature Fluctuation Range (°C) Sublimation Rate (mg / min) Redissolved Particle Size after Freeze-drying (nm) Aggregation / Sedimentation Phenomenon 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

[0166] It can be obtained from Table 1 that:

[0167] The temperature control during the freeze-drying process is directly related to the sublimation rate of the sample and the interfacial stability of MPL. In Example 1, dynamic temperature control was adopted to keep the sample always in the "subcritical" temperature range (-42°C to -38°C), effectively avoiding the destruction of the MPL lipid structure caused by excessive temperature fluctuations. After canceling the dynamic regulation in Comparative Example 2, the temperature fluctuation range increased significantly, and uneven heating occurred in some areas of the sample, resulting in discontinuous sublimation and then slight aggregation, verifying the key role of temperature control accuracy in the balance of the sublimation process.

[0168] The role of the interfacial stabilizer PEG-PLA was obvious in Comparative Example 1. After removal, the re-dissolved particle size increased and the aggregation was severe. During the freeze-drying process, PEG-PLA effectively reduced the particle aggregation caused by changes in interfacial tension and inhibited the particle agglomeration under drying stress. After removal, the interaction force between MPL particles increased, resulting in significant sedimentation and aggregation, and the particle size after re-dissolution nearly doubled, losing the dispersibility advantage of the adjuvant.

[0169] Overall, the dual stability strategy of temperature and interface under high vacuum during the freeze-drying process is the core to ensure the integrity of the MPL lipid structure and immunological activity. The lack of any key factor (temperature control or interface protection) will lead to energy imbalance in the system, induce self-aggregation of MPL particles or interface collapse, directly affecting subsequent biological activity and usage effects, fully verifying the scientificity and necessity of the process design of the present invention.

[0170] Experiment 2:

[0171] Experiment purpose:

[0172] Verify the antioxidant effect of L-methionine and the influence of staged pre-freezing on the ice crystal particle size and structural stability, and focus on observing oxidation indexes, ice crystal morphology and product structure integrity.

[0173] Control group design:

[0174] Example 2; Comparative Example 3; Comparative Example 4.

[0175] Experiment steps:

[0176] Sample preparation:

[0177] Prepare the MPL protective solution. In Example 2, trehalose, PEG-PLA, and L-methionine were completely added;

[0178] In Comparative Example 3, L-methionine was replaced with equimolar glycine;

[0179] The formulation of Comparative Example 4 was the same as that of Example 2.

[0180] Pre-freezing treatment:

[0181] Example 2, Comparative Example 3: Stepwise cooling (room temperature → -20°C (3°C / min) → -45°C (0.5°C / min), hold for 1 hour);

[0182] Comparative Example 4: Direct quick-freezing at -50°C (5°C / min), without stepwise cooling.

[0183] Freeze-drying process:

[0184] The sample temperature is controlled between -42°C and -38°C, and the vacuum degree is 25 Pa;

[0185] The freeze-drying time is 10 hours.

[0186] Process monitoring and testing:

[0187] Oxidation index (determination of peroxide value);

[0188] Ice crystal particle size (microscopic observation + particle size distribution);

[0189] The structural integrity after freeze-drying (microscopic + physical detection), (the test results are shown in Table 2).

[0190] Table 2: Comparison experimental results of freeze-drying protection and pre-freezing process

[0191] Test Group Peroxide Value (meq / kg) Ice Crystal Particle Size (μm) Dry Product Structural Integrity (Scored on a 10-point Scale) Observation of Microscopic Cracks Example 2 1.8 18 9 No Obvious Cracks Comparative Example 3 3.9 21 7 Local Cracks Comparative Example 4 2.7 35 5 Many Obvious Cracks

[0192] It can be seen from Table 2 that:

[0193] The introduction of L-methionine into the freeze-drying system significantly improves the antioxidant performance of the system. Experiments prove that its inhibitory effect on the lipid peroxidation of MPL is significantly better than that of glycine. Since the thiol group in the methionine molecule has strong free radical scavenging and antioxidant functions, it can effectively capture the reactive oxygen species during the drying and storage processes, reduce lipid oxidative degradation, and maintain the integrity of the MPL particle structure, demonstrating a specific protective effect in the lipid system.

[0194] The role of the stepwise pre-freezing design is particularly prominent in ice crystal control. The gradual cooling enables the slow formation of ice crystals with a concentrated particle size distribution, avoiding the large-sized ice crystals formed during the quick-freezing process due to too fast cooling rate, and reducing the stress concentration during sublimation. After canceling the segmented treatment, the ice crystal size significantly increases, resulting in structural damage during sublimation, leading to an increase in cracks and a decrease in the structural integrity of the dried product, verifying the importance of controllable ice crystal growth.

[0195] Overall, the antioxidant protection and fine ice crystal control during the freeze-drying process act on the stability of the MPL system in a dual manner. The absence of any one of them will lead to a decline in product performance: improper antioxidants will exacerbate lipid oxidation, and disordered ice crystal growth will damage the particle and matrix structures, affecting the redissolution and immune functions, further verifying the scientific mechanism of antioxidant and ice crystal management in the design of the present invention.

[0196] Experiment 3:

[0197] Purpose of the experiment:

[0198] To evaluate the effects of nitrogen - filled packaging and inner - membrane plasma treatment on the storage stability of MPL freeze - dried preparations, with a focus on observing oxygen permeation, sample oxidation, and changes in immunological activity during storage.

[0199] Control group design:

[0200] Example 3; Comparative Example 5; Comparative Example 6.

[0201] Experimental procedure:

[0202] Preparation of freeze - dried samples:

[0203] Prepare the freeze - dried products according to the process of Example 3 and divide them into 3 groups.

[0204] Packaging treatment:

[0205] Example 3: Multilayer composite film packaging, inner - layer plasma treatment, nitrogen - filled until the oxygen content < 0.5%, sealing temperature 170°C, sealing pressure 0.25 MPa;

[0206] Comparative Example 5: Nitrogen filling is cancelled, and the others are the same;

[0207] Comparative Example 6: The inner - membrane is not treated with plasma, and the others are the same.

[0208] Storage test (accelerated aging conditions):

[0209] Store for 30 days at 40°C and relative humidity of 75%;

[0210] Measure oxygen permeation, peroxide value (lipid oxidation index), and MPL immunological activity.

[0211] Main detection indicators:

[0212] Oxygen permeability rate (%);

[0213] Peroxide value (meq / kg);

[0214] Immunological activity retention rate (detected by ELISA), (test results are shown in Table 3).

[0215] Table 3: Comparison table of the effects of packaging processes on storage stability

[0216] Test Group Oxygen Permeability (%) Peroxide Value (meq / kg) Immune Activity 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

[0217] As can be seen from Table 3, nitrogen - filled packaging effectively reduces the oxygen content inside 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 vulnerable to oxidative damage, especially in high - temperature and high - humidity environments, which are more likely to trigger lipid double - bond cleavage and self - polymerization reactions. In a nitrogen - filled environment, reactive oxygen species are reduced, and the methionine antioxidant mechanism in the system can function, maintaining good adjuvant activity, verifying the key role of the nitrogen environment as a protective barrier.

[0218] Plasma treatment of the inner membrane greatly improves the sealing strength and gas barrier properties, reducing the risk of oxygen penetration. There are tiny cracks and polarity differences at the interface of untreated packaging materials, resulting in slow oxygen penetration. Under the cumulative effect, the oxidation reaction intensifies, and the storage stability significantly decreases. The performance of Comparative Example 6 in the experiment further proves that a complete inner - membrane treatment process is an essential link to ensure long - term storage effects.

[0219] Overall analysis shows that the packaging process is not only physical encapsulation but also a core means of controlling the micro - environment. Under the dual action of nitrogen filling and high - quality packaging film, the structural integrity and immunological function of the MPL adjuvant are stabilized during long - term storage. The lack of either measure will disrupt the overall energy balance and micro - environment, leading to the deterioration of the MPL system and the loss of biological effects, which is completely consistent with the micro - environment stability mechanism proposed in this invention, reflecting the rationality and scientific nature of the design.

[0220] Although the embodiments of the present invention have been shown and described, for those of ordinary skill in the art, it can be understood that various changes, modifications, substitutions, and variations can be made to these embodiments without departing from the principles and spirit of the present invention. The scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A method for preparing a freeze-dried product of MPL adjuvant, characterized in that: The following steps are involved: The MPL raw material is dissolved in a buffer to obtain an MPL solution, wherein the buffer is a histidine-citrate buffer system, and the 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; A lyophilization protectant is added to the MPL solution to form a stable liquid system, wherein the lyophilization protectant 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; The obtained solution is subjected to a pre-freezing treatment of cooling in stages, and the pre-freezing treatment 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; freeze-drying the pre-frozen system under temperature-controlled conditions to obtain freeze-dried powder; The freeze-dried powder is sealed in a treated packaging material to form a final product, wherein the packaging material is a multi-layer barrier structure composite film, including an inner layer of polyolefin material; The inner layer polyolefin material is used for sealing after being treated with plasma, and the plasma treatment conditions include: radio frequency power of 40 to 60W; treatment time of 20 to 40 seconds; treatment gas is high-purity nitrogen; The freeze-dried powder is sealed and packaged in a nitrogen-filled environment.

2. The method for preparing a freeze-dried product of MPL adjuvant according to claim 1, characterized in that: In the pre-freezing process, 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.

3. The method for preparing a freeze-dried product of 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%.

4. The method for preparing a freeze-dried product of MPL adjuvant according to claim 3, 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.

5. The method for preparing a freeze-dried product of 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.

6. The method for preparing a freeze-dried product of 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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