A method for the preparation of a ready-to-use stable 3D-MLA adjuvant solution

Abstract

The application relates to the technical field of biological pharmacy, and discloses a preparation method of a ready-to-use stable 3D-MLA adjuvant solution, which comprises the following steps: dissolving citric acid monohydrate and trisodium citrate dihydrate in water for injection to prepare a citrate buffer; under stirring, adding polysorbate 80 into the citrate buffer to dissolve the polysorbate 80; under stirring, adding 3D-MLA into the obtained solution to dissolve the 3D-MLA; adjusting the pH value of the solution; using water for injection to fix the volume; performing sterilization filtration through a filter membrane, filling the filtrate into a container and sealing the container; and cold storage. Through the citrate buffer and the adjustment of the pH value of the adjuvant solution, the combination of the buffer system and the pH environment effectively inhibits the chemical degradation of the 3D-MLA in the water for injection, the technical problem that the solution is prone to degradation is solved, and a ready-to-use preparation which can be stably stored under cold storage conditions is obtained.

Description

Technical Field

[0001] This invention relates to the field of biopharmaceutical technology, and in particular to a method for preparing a ready-to-use stable 3D-MLA adjuvant solution. Background Technology

[0002] 3D-MLA (3-O-deacyl-4'-monophosphoryllipid A) is an immune adjuvant and a TLR4 agonist. It is commonly used in vaccine formulations to enhance the body's immune response to vaccine antigens and improve the protective effect of vaccines.

[0003] Because the molecular structure of 3D-MLA contains easily hydrolyzed ester bonds and phosphate groups, and its molecules are highly hydrophobic, its stability in aqueous solutions is poor. To circumvent this fundamental stability problem, existing technologies typically require the use of solid dosage forms such as lyophilized formulations to ensure its chemical and physical stability during storage.

[0004] However, developing ready-to-use 3D-MLA adjuvant solutions is an important trend in this field, but this requires first addressing the long-term stability of the formulation in aqueous solutions. 3D-MLA is prone to chemical degradation in aqueous solutions, leading to reduced purity and loss of biological activity of the active ingredient. Simultaneously, the hydrophobic nature of 3D-MLA makes it easily adsorbed onto the inner walls of containers in solution, causing physical loss of the active ingredient and resulting in inaccurate formulation content and dosage uniformity. Similarly, there is a risk of 3D-MLA loss due to adsorption or degradation during formulation preparation and sterile filtration processes.

[0005] Therefore, how to overcome the problems of chemical degradation and physical adsorption of 3D-MLA in aqueous solution, and provide a ready-to-use 3D-MLA adjuvant solution that can be stored under cold for a long time and has stable quality, as well as its preparation method, is a problem that needs to be solved by those skilled in the art. Summary of the Invention

[0006] The purpose of this invention is to provide a method for preparing a ready-to-use stable 3D-MLA adjuvant solution, which solves the problem that 3D-MLA is chemically unstable and prone to degradation in aqueous solution, and is also prone to physical adsorption, thus making its aqueous solution unable to be stored stably for a long time and requiring fresh preparation for use.

[0007] To achieve the above objectives, the present invention provides a ready-to-use stable 3D-MLA adjuvant solution, employing the following technical solution:

[0008] A ready-to-use stable 3D-MLA adjuvant solution, the adjuvant solution comprising the following components:

[0009] 0.5-2.0 mg / mL of 3D-MLA;

[0010] Polysorbate 80 at concentrations of 0.1-1.0 mg / mL;

[0011] 10-110 mM citrate buffer;

[0012] And water for injection.

[0013] By adopting the above technical solution, the inventors discovered that by combining 3D-MLA, polysorbate 80 and citrate buffer in a specific way and controlling the pH value of the solution within a specific range, the two major technical problems of chemical degradation and physical adsorption of 3D-MLA adjuvant in aqueous solution can be solved in a synergistic way, thereby obtaining a ready-to-use solution that can be stably stored for at least 24 months under 2-8℃ conditions.

[0014] The stabilization mechanism of this invention includes two aspects:

[0015] On the one hand, chemical stability is achieved: This invention inhibits the chemical degradation of 3D-MLA through the synergistic effect of a specific buffer system and pH environment. The inventors discovered that the citrate buffer system itself provides better chemical stabilization protection for 3D-MLA compared to conventional acetate buffer. Meanwhile, pH is another key factor. The inventors experimentally confirmed that pH 5.0-6.0 is a specific stability window; within this range, the hydrolysis rate of 3D-MLA is effectively inhibited. Outside this range, degradation is accelerated at pH 4.5 or lower, or at pH 7.0 or higher.

[0016] On the other hand, physical stability is achieved: This invention utilizes polysorbate 80 as a nonionic surfactant to solve the problem of physical adsorption loss of 3D-MLA. 3D-MLA molecules are hydrophobic and easily adsorb onto the inner wall of the container. Polysorbate 80 works through preferential adsorption or micellar mechanisms, effectively preventing the physical adsorption of the active ingredient of 3D-MLA to the container surface, ensuring dosage uniformity and content recovery rate of the solution during storage.

[0017] Therefore, through the above-mentioned dual stabilization mechanism, the present invention provides a ready-to-use 3D-MLA adjuvant solution that can be industrially produced, does not require immediate preparation, and can maintain long-term chemical, physical, and biological stability under refrigeration conditions.

[0018] Preferably, the pH value of the adjuvant solution is 5.0-6.0.

[0019] Preferably, the concentration of 3D-MLA is 1.0 mg / mL, the concentration of polysorbate 80 is 0.5 mg / mL, and the concentration of citrate buffer is 50-60 mM.

[0020] Preferably, the citrate buffer solution is prepared from citrate monohydrate and trisodium citrate dihydrate in water for injection.

[0021] Preferably, the weight ratio of the citric acid monohydrate to the trisodium citrate dihydrate is in the range of 1:1.23 to 1:5.31.

[0022] By adopting the above-mentioned preferred technical solutions, the present invention provides a specific formulation range verified by embodiments. This range, in particular the specific weight ratio of citric acid monohydrate to trisodium citrate dihydrate, exhibits excellent stability while meeting pH and buffer concentration requirements, and has high industrial application value.

[0023] This invention provides a method for preparing a ready-to-use stable 3D-MLA adjuvant solution, using the following technical solution:

[0024] A method for preparing a ready-to-use stable 3D-MLA adjuvant solution as described above includes the following steps:

[0025] S1. Dissolve citrate monohydrate and trisodium citrate dihydrate in water for injection to prepare citrate buffer;

[0026] S2. Under stirring, add polysorbate 80 to the citrate buffer solution to dissolve it;

[0027] S3. Add 3D-MLA to the solution obtained in step S2 while stirring, and dissolve it.

[0028] S4. Adjust the pH value of the solution;

[0029] S5. Adjust the volume to the target volume using water for injection;

[0030] S6. The solution obtained in step S5 is filtered through a filter membrane with sterilization pores for sterilization, and the filtrate is filled into a container and sealed.

[0031] S7. Store the sealed container from step S6 at a temperature of 2°C to 8°C.

[0032] By employing the above technical solution, this invention provides a clear, stable, and controllable preparation process. This method ensures the complete dissolution and uniform dispersion of each component through a specific order of addition. The method first prepares a citrate buffer solution with a stable pH environment, then adds polysorbate 80 to pre-establish a stable system, and finally adds the easily degradable and easily adsorbed 3D-MLA active ingredient. This order helps protect 3D-MLA during the formulation process, preventing degradation or adsorption loss during the dissolution stage. Subsequent pH adjustment, filtration, and refrigerated storage steps further ensure that the final product meets the requirements for sterile injections and can be stably stored for a long period.

[0033] Preferably, in step S4, the solution is cooled to room temperature before adjusting the pH value.

[0034] Preferably, in step S4, the pH value is adjusted to 5.0-6.0 using hydrochloric acid solution or sodium hydroxide solution.

[0035] Preferably, in step S6, the filter membrane is a PVDF low-adsorption filter membrane.

[0036] Preferably, in step S6, the container is a vial.

[0037] By adopting the above-mentioned preferred technical solutions, the present invention further refines the process control parameters. Cooling to room temperature before pH adjustment ensures the accuracy of pH measurement; using a PVDF low-adsorption filter membrane further reduces the loss of active ingredients during filtration; and using vials provides a stable and sealed storage environment for the adjuvant, which, together with the refrigeration step in S7, ensures the long-term stability of the product.

[0038] In summary, the present invention has at least one of the following beneficial technical effects:

[0039] 1. This invention utilizes a citrate buffer solution prepared from citrate monohydrate and trisodium citrate dihydrate, and adjusts the pH of the adjuvant solution to a predetermined range. By combining the buffer system with the pH environment, the chemical degradation of 3D-MLA in water for injection is effectively inhibited. This solves the technical problem that 3D-MLA is easily degraded in aqueous solution and cannot be stored for a long time. As a result, the adjuvant solution can maintain stable chemical and biological activity under refrigerated storage conditions, and a ready-to-use formulation that does not require immediate preparation is obtained.

[0040] 2. This invention, by including polysorbate 80 in the adjuvant solution, utilizes the effect of this component as a nonionic surfactant to effectively prevent the physical adsorption of 3D-MLA to the inner wall of the container, thus solving the technical problems of reduced content and uneven dosage of active ingredients due to adsorption, and ensuring the content recovery rate of the adjuvant solution during long-term storage.

[0041] 3. This invention employs a feeding sequence of first adding polysorbate 80 to dissolve it, followed by adding 3D-MLA to dissolve it, after preparing the citrate buffer solution. This ensures that 3D-MLA is under the combined protection of polysorbate 80 and the citrate buffer solution during dissolution. Simultaneously, by using a PVDF low-adsorption filter membrane for sterilization filtration, the chemical degradation and physical adsorption loss of 3D-MLA during the preparation and filtration processes are synergistically reduced, ensuring the quality and component content of the adjuvant solution finally filled into the container. Detailed Implementation

[0042] The technical solutions of the present invention will be clearly and completely described below with reference to embodiments, comparative examples, and test examples. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0043] Examples 1-5:

[0044] Example 1:

[0045] This embodiment provides a method for preparing a ready-to-use stable 3D-MLA adjuvant solution, including the following steps:

[0046] S1. Preparation of citrate buffer: Weigh 2.56g of citrate monohydrate and 13.59g of trisodium citrate dihydrate, add them to 800mL of water for injection, and start magnetic stirring until the buffer salts are completely dissolved.

[0047] S2. Adding and dissolving the stabilizer: Slowly add 0.5 g of polysorbate 80 to the citrate buffer while stirring continuously. Continue stirring until the polysorbate 80 is completely dissolved and the solution is clear;

[0048] S3. Dissolving the active ingredient: Accurately weigh 1.0g of 3D-MLA and slowly add the 3D-MLA powder to a buffer solution containing polysorbate 80. Continue stirring until the 3D-MLA is completely dissolved.

[0049] S4. Adjust pH: Cool the solution to room temperature (20°C). Accurately measure the pH of the solution using a calibrated pH meter. Adjust the pH of the solution to 5.5 by slow titration with 1M hydrochloric acid or 1M sodium hydroxide solution while stirring.

[0050] S5. Volume adjustment and mixing: Transfer the pH-adjusted solution to a 1000mL volumetric flask, dilute to the mark with water for injection, tighten the stopper, and then invert to mix.

[0051] S6. Sterilization Filtration and Filling: The final prepared adjuvant solution is sterilized by passing it through a PVDF low-adsorption membrane filter with sterile pore size. Under aseptic conditions, the filtrate is filled into pre-sterilized vials, 1.0 mL per vial, and sealed with sterile rubber stoppers and aluminum caps.

[0052] S7. Storage: Store the sealed adjuvant solution at 2℃-8℃.

[0053] Example 2:

[0054] This embodiment provides a method for preparing a ready-to-use stable 3D-MLA adjuvant solution, including the following steps:

[0055] S1. Preparation of citrate buffer: Weigh 1.31g of citrate monohydrate and 1.62g of trisodium citrate dihydrate, add them to 800mL of water for injection, and start magnetic stirring until the buffer salts are completely dissolved.

[0056] S2. Adding and dissolving the stabilizer: Slowly add 0.1 g of polysorbate 80 to the citrate buffer while stirring continuously. Continue stirring until the polysorbate 80 is completely dissolved and the solution is clear;

[0057] S3. Dissolving the active ingredient: Accurately weigh 0.5g of 3D-MLA and slowly add the 3D-MLA powder to a buffer solution containing polysorbate 80. Continue stirring until the 3D-MLA is completely dissolved.

[0058] S4. Adjust pH: Cool the solution to room temperature (20°C). Accurately measure the pH of the solution using a calibrated pH meter. Adjust the pH of the solution to 5.0 by slow titration with 1M hydrochloric acid or 1M sodium hydroxide solution while stirring.

[0059] S5. Volume adjustment and mixing: Transfer the pH-adjusted solution to a 1000mL volumetric flask, dilute to the mark with water for injection, tighten the stopper, and then invert to mix.

[0060] S6. Sterilization Filtration and Filling: The final prepared adjuvant solution is sterilized by passing it through a PVDF low-adsorption membrane filter with sterile pore size. Under aseptic conditions, the filtrate is filled into pre-sterilized vials and sealed with sterile rubber stoppers and aluminum caps.

[0061] S7. Storage: Store the sealed adjuvant solution at 2℃-8℃.

[0062] Example 3:

[0063] This embodiment provides a method for preparing a ready-to-use stable 3D-MLA adjuvant solution, including the following steps:

[0064] S1. Preparation of citrate buffer: Weigh 3.89g of citrate monohydrate and 26.85g of trisodium citrate dihydrate, add them to 800mL of water for injection, and start magnetic stirring until the buffer salts are completely dissolved.

[0065] S2. Adding and dissolving the stabilizer: Slowly add 1.0 g of polysorbate 80 to the citrate buffer while stirring continuously. Continue stirring until the polysorbate 80 is completely dissolved and the solution is clear;

[0066] S3. Dissolving the active ingredient: Accurately weigh 2.0g of 3D-MLA and slowly add the 3D-MLA powder to a buffer solution containing polysorbate 80. Continue stirring until the 3D-MLA is completely dissolved.

[0067] S4. Adjust pH: Cool the solution to room temperature (20°C). Accurately measure the pH of the solution using a calibrated pH meter. Using 1M hydrochloric acid or 1M sodium hydroxide solution, slowly titrate with stirring to precisely adjust the pH of the solution to 6.0.

[0068] S5. Volume adjustment and mixing: Transfer the pH-adjusted solution to a 1000mL volumetric flask, dilute to the mark with water for injection, tighten the stopper, and then invert to mix.

[0069] S6. Sterilization Filtration and Filling: The final prepared adjuvant solution is sterilized by passing it through a PVDF low-adsorption membrane filter with sterile pore size. Under aseptic conditions, the filtrate is filled into pre-sterilized vials and sealed with sterile rubber stoppers and aluminum caps.

[0070] S7. Storage: Store the sealed adjuvant solution at 2℃-8℃.

[0071] Example 4:

[0072] This embodiment provides a method for preparing a ready-to-use stable 3D-MLA adjuvant solution, including the following steps:

[0073] S1. Preparation of citrate buffer: Weigh 6.57g of citrate monohydrate and 8.10g of trisodium citrate dihydrate, add them to 800mL of water for injection, and start magnetic stirring until the buffer salts are completely dissolved.

[0074] S2. Adding and dissolving the stabilizer: Slowly add 0.5 g of polysorbate 80 to the citrate buffer while stirring continuously. Continue stirring until the polysorbate 80 is completely dissolved and the solution is clear;

[0075] S3. Dissolving the active ingredient: Accurately weigh 1.0g of 3D-MLA and slowly add the 3D-MLA powder to a buffer solution containing polysorbate 80. Continue stirring until the 3D-MLA is completely dissolved.

[0076] S4. Adjust pH: Cool the solution to room temperature (20°C). Accurately measure the pH of the solution using a calibrated pH meter. Using 1M hydrochloric acid or 1M sodium hydroxide solution, slowly titrate with stirring to precisely adjust the pH of the solution to 5.0.

[0077] S5. Volume adjustment and mixing: Transfer the pH-adjusted solution to a 1000mL volumetric flask, dilute to the mark with water for injection, tighten the stopper, and then invert to mix.

[0078] S6. Sterilization Filtration and Filling: The final prepared adjuvant solution is sterilized by passing it through a PVDF low-adsorption membrane filter with sterile pore size. Under aseptic conditions, the filtrate is filled into pre-sterilized vials and sealed with sterile rubber stoppers and aluminum caps.

[0079] S7. Storage: Store the sealed adjuvant solution at 2℃-8℃.

[0080] Example 5:

[0081] This embodiment provides a method for preparing a ready-to-use stable 3D-MLA adjuvant solution, including the following steps:

[0082] S1. Preparation of citrate buffer: Weigh 0.51g of citrate monohydrate and 2.72g of trisodium citrate dihydrate, add them to 800mL of water for injection, and start magnetic stirring until the buffer salts are completely dissolved.

[0083] S2. Adding and dissolving the stabilizer: Slowly add 0.5 g of polysorbate 80 to the citrate buffer while stirring continuously. Continue stirring until the polysorbate 80 is completely dissolved and the solution is clear;

[0084] S3. Dissolving the active ingredient: Accurately weigh 1.0g of 3D-MLA and slowly add the 3D-MLA powder to a buffer solution containing polysorbate 80. Continue stirring until the 3D-MLA is completely dissolved.

[0085] S4. Adjust pH: Cool the solution to room temperature (20°C). Accurately measure the pH of the solution using a calibrated pH meter. Using 1M hydrochloric acid or 1M sodium hydroxide solution, slowly titrate with stirring to precisely adjust the pH of the solution to 5.5.

[0086] S5. Volume adjustment and mixing: Transfer the pH-adjusted solution to a 1000mL volumetric flask, dilute to the mark with water for injection, tighten the stopper, and then invert to mix.

[0087] S6. Sterilization Filtration and Filling: The final prepared adjuvant solution is sterilized by passing it through a PVDF low-adsorption membrane filter with sterile pore size. Under aseptic conditions, the filtrate is filled into pre-sterilized vials and sealed with sterile rubber stoppers and aluminum caps.

[0088] S7. Storage: Store the sealed adjuvant solution at 2℃-8℃.

[0089] Comparative Examples 1-5:

[0090] Comparative Example 1:

[0091] Compared to Example 1, the differences are as follows: in step S1, the 50mM citrate buffer is replaced with 1xPBS buffer (containing 10mM phosphate and 137mM sodium chloride); in step S4, the pH is adjusted to 7.4. The remaining steps are the same as in Example 1.

[0092] Comparative Example 2:

[0093] The difference from Example 1 is that in step S4, the pH of the solution is precisely adjusted to 7.0. The remaining steps are the same as in Example 1.

[0094] Comparative Example 3:

[0095] The difference from Example 1 is that in step S4, the pH of the solution is precisely adjusted to 4.5. The remaining steps are the same as in Example 1.

[0096] Comparative Example 4:

[0097] The difference from Example 1 is that step S2 is omitted, i.e., polysorbate 80 is not added to the citrate buffer. The remaining steps are the same as in Example 1.

[0098] Comparative Example 5:

[0099] The difference from Example 1 is that in step S1, the 50 mM citrate buffer is replaced with 50 mM acetate buffer (prepared by weighing appropriate amounts of sodium acetate and glacial acetic acid). The remaining steps are the same as in Example 1.

[0100] Test Examples 1-4:

[0101] Test Example 1:

[0102] Experimental methods:

[0103] The adjuvant solutions prepared in Examples 1-5 (initial samples with T=0) and a representative sample (Example 1) were subjected to the following basic property tests:

[0104] Visual inspection: Place the sample against a white and black background and observe the clarity, color, and presence of insoluble matter under light conditions.

[0105] pH determination: The pH values ​​of the solutions in each example were determined at room temperature (20°C) using a precision pH meter calibrated with standard buffer solution.

[0106] Osmotic molar concentration: The osmotic molar concentration of the solutions in each example was determined using an ice-point osmometer.

[0107] Sterility test: Take the sample from Example 1 and filter it using a membrane filtration method with a sterile pore size. Place the membrane in thioglycolate fluid medium and tryptic soy peptone liquid medium respectively, and incubate for 14 days under the specified conditions. Observe for sterile growth.

[0108] Bacterial endotoxins: The sample from Example 1 was tested using the Limulus Amebocyte Lysate (LAL) gel electrophoresis method, and the endotoxin limit (L) was set at 2.5 EU / mL.

[0109] Experimental data:

[0110] Table 1: Initial physicochemical properties and sterility / endotoxin test results of adjuvant solutions

[0111] Example Appearance pH (T=0) Osmotic pressure (mOsmol / kg) Aseptic examination Bacterial endotoxins (EU / mL) Example 1 Clarity, micro-opalescent 5.48 141 Compliant <0.1 Example 2 Clarity, micro-opalescent 5.03 38 Not detected Not detected Example 3 Clarity, micro-opalescent 5.97 279 Not detected Not detected Example 4 Clarity, micro-opalescent 5.01 137 Not detected Not detected Example 5 Clarity, micro-opalescent 5.52 41 Not detected Not detected

[0112] in conclusion:

[0113] The initial solutions of Examples 1-5 were all clear, slightly opalescent liquids, indicating that 3D-MLA has good apparent solubility or dispersibility in the citrate buffer system and in the presence of polysorbate 80.

[0114] The pH values ​​(5.01-5.97) were close to the target pH (5.0-6.0) of each embodiment. The osmolality (38-279 mOsmol / kg) showed a regular change with the buffer salt concentration (approximately 40 mOsmol / kg for 10 mM buffer salt concentration and approximately 280 mOsmol / kg for 100 mM buffer salt concentration), which was consistent with the expected formulation.

[0115] Representative Example 1 passed the sterility test, and the bacterial endotoxin content (<0.1 EU / mL) was below the set limit (2.5 EU / mL).

[0116] In summary, this formulation (citrate buffer, specific pH 5.0-6.0, and polysorbate 80) can prepare a ready-to-use adjuvant solution that meets the basic physicochemical and sterility requirements for injectable preparations.

[0117] Test Example 2:

[0118] Experimental methods:

[0119] Stability study: The adjuvant solution samples of Examples 1-5 and Comparative Examples 1-5 were subjected to stability studies under the following conditions:

[0120] Long-term stability: 2℃-8℃.

[0121] Accelerated stability testing: Temperature 25℃, relative humidity 60%RH.

[0122] Detection method (HPLC determination of 3D-MLA purity): Samples were taken at T=0 and at specified time points (long-term: 6, 12, 24 months; accelerated: 1, 3, 6 months). The chemical purity of 3D-MLA was determined by high-performance liquid chromatography (HPLC).

[0123] Chromatographic conditions:

[0124] Column: Waters XBridge C18 (4.6 x 250 mm, 5 μm);

[0125] Mobile phase A: 0.1% aqueous solution of trifluoroacetic acid;

[0126] Mobile phase B: 0.1% trifluoroacetic acid in acetonitrile solution;

[0127] Flow rate: 1.0 mL / min;

[0128] Column temperature: 35°C;

[0129] Detection wavelength: 210nm;

[0130] Injection volume: 20 μL;

[0131] Gradient elution procedure:

[0132] Table 2: HPLC gradient elution program

[0133] Time (min) Mobile phase A (%) Mobile phase B (%) 0 90 10 5 90 10 30 10 90 35 10 90 35.1 90 10 40 90 10

[0134] Purity calculation: Peak area normalization method was used.

[0135] 3D-MLA purity (%) = (3D-MLA main peak area / (3D-MLA main peak area + total impurity peak area)) * 100%.

[0136] Experimental data:

[0137] Table 3: 3D-MLA purity (%) during long-term stability (2℃-8℃)

[0138] Group T=0 T=6m T=12m T=24m Example 1 99.7 99.5 99.3 98.9 Example 2 99.6 99.4 99.2 98.8 Example 3 99.5 99.3 99.0 98.6 Example 4 99.7 99.6 99.3 99.0 Example 5 99.6 99.5 99.1 98.7 Comparative Example 1 99.6 96.1 92.4 85.3 Comparative Example 2 99.7 97.5 95.0 90.1 Comparative Example 3 99.5 98.0 96.2 91.5 Comparative Example 4 99.7 99.4 99.2 98.8 Comparative Example 5 99.6 97.1 94.8 92.3

[0139] Table 4: 3D-MLA purity (%) in accelerated stability (25℃ / 60%RH)

[0140] Group T=0 T=1m T=3m T=6m Example 1 99.7 99.1 98.4 97.5 Example 2 99.6 99.0 98.2 97.2 Example 3 99.5 98.8 98.0 96.9 Example 4 99.7 99.2 98.5 97.7 Example 5 99.6 99.1 98.3 97.3 Comparative Example 1 99.6 90.2 81.5 70.4 Comparative Example 2 99.7 93.4 87.2 80.1 Comparative Example 3 99.5 94.5 88.9 82.3 Comparative Example 4 99.7 99.0 98.3 97.3 Comparative Example 5 99.6 95.1 91.3 88.5

[0141] in conclusion:

[0142] 3D-MLA, as a lipid molecule, is prone to hydrolytic degradation in aqueous solutions. The core mechanism of this invention lies in inhibiting this degradation pathway through a specific buffer system and pH environment.

[0143] The importance of pH range:

[0144] Under both long-term (Table 3) and accelerated (Table 4) conditions, the 3D-MLA purity of Examples 1-5 (pH 5.0-6.0) remained at a high level (>98.5% after 24 months of refrigeration; >96.5% after 6 months of accelerated treatment).

[0145] In contrast, the degradation rates of Comparative Example 2 (pH 7.0) and Comparative Example 1 (pH 7.4) were accelerated. This indicates that a neutral to slightly alkaline environment (pH ≥ 7.0) accelerates the hydrolysis of 3D-MLA.

[0146] Meanwhile, the degradation rate of Comparative Example 3 (pH 4.5) was also faster than that of Example Group (pH 5.0-6.0), indicating that an overly acidic environment is also not conducive to the stability of 3D-MLA.

[0147] Data confirms that the pH range of 5.0-6.0 helps maintain the chemical stability of 3D-MLA.

[0148] The criticality of buffer components:

[0149] Comparative Example 5 (acetate buffer, pH 5.5) has the same pH value as Example 1 (citate buffer, pH 5.5).

[0150] However, the degradation rate of Comparative Example 5 was faster than that of Example 1. This indicates that the citrate buffer system itself provides chemical stabilization protection different from that of the acetate buffer system.

[0151] Other factors:

[0152] Comparative Example 4 (without polysorbate 80) showed no significant difference in chemical purity (purity of the unadsorbed portion in solution) compared to Example 1 under both long-term and accelerated conditions. This indicates that polysorbate 80 does not primarily contribute to inhibiting the chemical degradation of 3D-MLA in this formulation.

[0153] In summary, the data from Test Example 2 confirm that a specific combination of the citrate buffer system and the pH range of 5.0-6.0 helps to achieve long-term chemical stability of 3D-MLA in ready-to-use aqueous solutions.

[0154] Test Example 3:

[0155] Experimental methods:

[0156] Stability Study: This test was used to evaluate the inhibitory effect of polysorbate 80 on the physical adsorption loss of 3D-MLA on the inner wall of the container. Samples from Example 1 (containing 0.05% polysorbate 80) and Comparative Example 4 (without polysorbate 80) were selected. All samples were prepared according to their respective methods and filled into vials, sealed, and stored at 2℃-8℃.

[0157] Detection method (HPLC determination of 3D-MLA content): Samples were taken at T=0 (initial) and T=7 days. The HPLC chromatographic conditions in Test Example 2 were used, but the external standard method was used for quantification.

[0158] Sample preparation: When taking samples at T=7 days, to avoid disturbing the drug already adsorbed on the bottle wall, the solution sample is taken from the center of the vial for testing.

[0159] Content calculation: The content recovery rate was calculated using the initial concentration at T=0 (C0, theoretical concentration) and the measured concentration of the solution at T=7 days (C7).

[0160] Calculation formula: Content recovery rate (%) = (C7 / C0) * 100%.

[0161] Experimental data:

[0162] Table 5: Recovery rate of 3D-MLA content in adjuvant solution after storage at 2℃-8℃ for 7 days

[0163] Group <![CDATA[Initial concentration C0 (mg / mL)]]> <![CDATA[Concentration C7 (mg / mL) after 7 days]]> Content recovery rate (%) Example 1 1.02 0.99 97.1 Comparative Example 4 1.01 0.72 71.3

[0164] in conclusion:

[0165] 3D-MLA molecules are hydrophobic and can be easily physically adsorbed onto the surfaces of hydrophobic or hydrophilic containers (such as the inner glass wall of a vial).

[0166] In Comparative Example 4 (without polysorbate 80), the recovery rate of 3D-MLA in the solution after 7 days of storage was only 71.3%, indicating that approximately 28.7% of the active ingredient was lost from the solution and adsorbed onto the inner wall of the container. This loss of content resulted in the actual dosage being lower than the labeled amount, failing to meet the requirements for drug uniformity.

[0167] In Example 1, polysorbate 80, as a nonionic surfactant, preferentially interacts with the inner wall of the container or inhibits the physical adsorption of the 3D-MLA active ingredient to the container surface through mechanisms such as micelle formation.

[0168] Table 5 shows that the content recovery rate of Example 1 (97.1%) was higher than that of Comparative Example 4. This indicates that polysorbate 80 is one of the components that maintains the physical stability of 3D-MLA in ready-to-use solutions, helping to maintain the accuracy and uniformity of the active ingredient content during storage.

[0169] Test Example 4:

[0170] Experimental methods:

[0171] Stability samples: Samples from Test Example 2 were taken for long-term stability studies at 2℃-8℃, with initial values ​​at T=0 and T=24 months (T=24m). The test group included Examples 1-5 and Comparative Examples 1-5.

[0172] Detection method (in vitro TLR4 reporter gene cell line method):

[0173] Cell preparation: The HEK-Blue™ TLR4 reporter gene cell line was used. This cell line stably expresses human TLR4, MD2, and CD14 and contains an NF-κB-induced secretory alkaline phosphatase (SEAP) reporter gene.

[0174] Sample preparation: Dilute the samples from T=0 and T=24m groups to the same 3D-MLA working concentration (e.g., 100 ng / mL, based on the theoretical concentration at T=0) with HEK-Blue™ detection medium (DMEM, 10% FBS).

[0175] Cell culture: The diluted sample was added to pre-coated HEK-BlueTMTLR4 cells, with a blank control culture medium provided. The cells were incubated at 37°C in a 5% CO2 incubator for 20-24 hours.

[0176] Activity assay: After incubation, collect the cell culture supernatant. Add SEAP assay reagent (e.g., QUANTI-Blue™ solution) to the supernatant and incubate at 37°C in the dark for 1-3 hours.

[0177] Reading: The absorbance (OD value) at 620 nm was measured using a microplate reader. The OD value is directly proportional to the TLR4 agonist activity (i.e., adjuvant biological activity).

[0178] Activity retention rate calculation: Activity retention rate (%) = (OD value of sample at T=24m / OD value of sample at T=0) * 100%;

[0179] Experimental data:

[0180] Table 6: Retention of biological activity of adjuvant solutions after 24 months of storage at 2℃-8℃

[0181] Group T=0mOD620 T=24mOD620 Activity retention rate (%) Example 1 1.88 1.81 96.3 Example 2 1.85 1.77 95.7 Example 3 1.91 1.79 93.7 Example 4 1.89 1.83 96.8 Example 5 1.86 1.74 93.5 Comparative Example 1 1.87 0.35 18.7 Comparative Example 2 1.90 0.58 30.5 Comparative Example 3 1.85 0.69 37.3 Comparative Example 4 1.88 1.78 94.7 Comparative Example 5 1.86 0.81 43.5

[0182] in conclusion:

[0183] Relationship between activity and chemical structure: The adjuvant activity (TLR4 agonist ability) of 3D-MLA depends on its intact molecular structure.

[0184] Data correlation analysis:

[0185] Table 6 shows that after 24 months of storage at 2℃-8℃, the biological activity retention rate of Examples 1-5 (citrate, pH 5.0-6.0) was all above 93%. This result is consistent with the chemical purity (>98.5%) observed in Test Example 2 (Table 3).

[0186] Comparative Examples 1, 2, 3, and 5 all showed a loss of biological activity after 24 months of storage (activity retention rate was only 18.7%-43.5%). This result correlates with the chemical degradation observed in Test Example 2 (Table 3) (purity decreased to 85.3%-92.3%). This indicates that chemical hydrolysis of 3D-MLA under neutral, slightly alkaline, or excessively acidic conditions (pH 4.5, 7.0, 7.4) or using acetate buffer (pH 5.5) leads to a reduction in its biological function.

[0187] The solution of Comparative Example 4 retained biological activity (94.7%), which was close to that of Example 1 (96.3%). This further confirms that the function of polysorbate 80 is to inhibit physical adsorption (Test Example 3), while the stability of chemical and biological activity is determined by the buffer system and pH environment.

[0188] In summary, the combination of the citrate buffer system and pH 5.0-6.0 helps to maintain the chemical integrity and biological functionality of 3D-MLA in ready-to-use aqueous solutions over a long period of time.

Claims

1. A process for the preparation of a ready-to-use stable 3D-MLA adjuvant solution, characterized in that, The method comprises the following steps: S1, dissolving citric acid monohydrate and trisodium citrate dihydrate in water for injection to prepare a citrate buffer solution; S2, under stirring, adding polysorbate 80 to the citrate buffer solution to dissolve the polysorbate 80, to obtain a polysorbate 80-containing buffer solution; S3, under stirring, adding 3D-MLA to the polysorbate 80-containing buffer solution to dissolve the 3D-MLA, to obtain a 3D-MLA mixed solution; S4, adjusting the pH value of the 3D-MLA mixed solution to 5.0-6.0, to obtain a pH-adjusted mixed solution; S5, using water for injection to make up the volume of the pH-adjusted mixed solution, to obtain a volume-adjusted mixed solution; S6, performing sterilization filtration on the volume-adjusted mixed solution through a filter membrane with a sterilization pore size, and filling the filtrate into a container and sealing the container; S7, storing the container at 2-8℃ under refrigeration.

2. A process for the preparation of a ready-to-use stable 3D-MLA adjuvant solution according to claim 1, characterized in that, In the S1 step, the weight ratio of the citric acid monohydrate to the trisodium citrate dihydrate ranges from (1:1.23) to (1:5.31).

3. A process for the preparation of a ready-to-use stable 3D-MLA adjuvant solution according to claim 1, characterized in that, In the S5 step, the concentration of 3D-MLA in the volume-adjusted mixed solution is 0.5-2.0 mg / mL, the concentration of polysorbate 80 is 0.1-1.0 mg / mL, and the concentration of the citrate buffer solution is 10-110 mM.

4. A process for the preparation of a ready-to-use stable 3D-MLA adjuvant solution as claimed in claim 1, wherein, In the S4 step, the solution is cooled to room temperature before adjusting the pH value.

5. A process for the preparation of a ready-to-use stable 3D-MLA adjuvant solution according to claim 4, characterized in that, In the S4 step, hydrochloric acid solution or sodium hydroxide solution is used to adjust the pH value.

6. A process for the preparation of a ready-to-use stable 3D-MLA adjuvant solution according to claim 1, characterized in that, In the S6 step, the filter membrane is a PVDF low-adsorption filter membrane.

7. A process for the preparation of a ready-to-use stable 3D-MLA adjuvant solution according to claim 6, characterized in that, In the S6 step, the container is a vial.