Zinc-aluminum composite adjuvant and preparation method thereof

Abstract

The application relates to the technical field of biological vaccines, in particular to a zinc-aluminum composite adjuvant and a preparation method thereof. The preparation method comprises the following steps: mixing a zinc salt solution and a specific complexing agent solution, adjusting pH, and forming a mixed solution A; under stirring, simultaneously adding an aluminum salt solution and a mixed alkali solution containing a phosphate and a hydroxide into the mixed solution A, controlling the pH of a reaction system in a range of 6.5-7.5, and obtaining a zinc-aluminum composite suspension after reaction; and finally, washing and sterilizing to obtain the zinc-aluminum composite adjuvant. The complexing agent is preferably a compound of L-histidine and carnosine succinamide. The zinc-aluminum composite particles with uniform structure and high stability are prepared through step-by-step coprecipitation and synergistic control of the preferred organic ligand, the problems of easy agglomeration and large batch difference of the traditional adjuvant in the terminal sterilization process are effectively solved, and the stability and protein adsorption capacity of the adjuvant are significantly improved.

Description

Technical Field

[0001] This invention relates to the field of biological vaccine technology, specifically to a zinc-aluminum composite adjuvant and its preparation method. Background Technology

[0002] Vaccine adjuvants are key components for enhancing antigen-specific immune responses and play an indispensable role in the development of novel vaccines. Among them, aluminum adjuvants, as the oldest and most widely used inorganic salt adjuvants in clinical practice, are widely recognized for their good safety record and ability to induce strong humoral immunity (Th2 response).

[0003] However, traditional aluminum adjuvants also have significant limitations. To overcome these shortcomings, zinc-aluminum composite adjuvants have emerged, aiming to combine the immunomodulatory advantages of zinc ions and aluminum salts. However, existing zinc-aluminum composite adjuvants are mostly prepared using simple co-precipitation methods, which involve initial mixing of zinc and aluminum salts with complexing agents, resulting in non-uniform particles. The lack of precise control over the nucleation and precipitation processes of zinc and aluminum ions leads to poor uniformity in the chemical composition, spatial structure, and particle size of the resulting complex, resulting in poor batch-to-batch stability. This structural disorder further affects its adsorption efficiency with antigens and the reliability of its immunomodulatory effects.

[0004] Secondly, existing zinc-aluminum composite adjuvant systems lack an effective organic protective layer under moist heat sterilization conditions at the formulation stage. Particles are prone to severe aggregation and crystal rearrangement at 121°C, resulting in a significant increase in particle size, dispersibility, and sedimentation speed. This makes it difficult to balance aseptic protection and physicochemical stability, thus affecting the physical stability and immunogenicity of the formulation. In addition, existing technologies mostly rely on traditional chelating agents such as citrate, which have limited ability to regulate immune response types and cannot achieve precise control of Th1 / Th2 immune balance, failing to meet the urgent need for comprehensive immune protection in next-generation vaccines. Summary of the Invention

[0005] To address the shortcomings of the existing technology, this invention provides a zinc-aluminum composite adjuvant and its preparation method.

[0006] To solve the above-mentioned technical problems, the technical solution adopted by the present invention is as follows:

[0007] A method for preparing a zinc-aluminum composite adjuvant includes the following steps:

[0008] (1) Mix the zinc salt solution and the complexing agent solution, adjust the pH to 4.5-6.5, stir the reaction to obtain mixture A;

[0009] (2) Prepare aluminum salt solution and mixed alkaline solution respectively, wherein the mixed alkaline solution contains phosphate and hydroxide;

[0010] (3) Under stirring conditions, the aluminum salt solution and the mixed alkaline solution are simultaneously added dropwise to the mixture.

[0011] In solution A, the pH of the reaction solution is controlled within the range of 6.5-7.5; after the addition is complete, stirring is continued to obtain a zinc-aluminum composite suspension.

[0012] (4) The zinc-aluminum composite suspension is washed and sterilized to obtain the zinc-aluminum composite adjuvant.

[0013] This invention addresses the aforementioned problems by exploring different combinations of organic ligands and employing stepwise precipitation control in the preparation method: First, under pH 5.5 conditions, a zinc salt solution and a complexing agent are pre-mixed and stirred to form a stable zinc-multi-site organic complexing system mixture A. This allows zinc ions to preferentially coordinate with imidazole groups, peptide bonds, and carboxyl groups, forming zinc-rich organically coordinated organic zinc. Subsequently, under conditions of 25°C and 500 rpm, an aluminum salt solution and a mixed alkaline solution are simultaneously added dropwise. By controlling the pH, the reaction system is stabilized at pH 7.0, allowing aluminum ions to preferentially react with OH- in the organic zinc of the zinc and complexing agent mixture. - / HPO4 2- An aluminum hydroxyl / aluminum phosphate shell is generated, thus forming zinc-aluminum composite particles; then, free ions and small molecule impurities are removed by washing with a 100 kDa ultrafiltration membrane, and the particles are sterilized before storage.

[0014] Preferably, the complexing agent is selected from at least one of sodium organic acid, L-histidine, carnosine, and carnosine succinate; the sodium organic acid is selected from at least one of sodium citrate, sodium L-glutamate, and sodium gluconate.

[0015] Furthermore, the complexing agent is a mixture of L-histidine and carnosine succinate in a mass ratio of (1.5-2.8):1.

[0016] The reason for using a 2:1 combination of L-histidine and carnosine succinamide as the complexing agent is that one has a gentle coordination and the other has a strong multidentate coordination and a peptide structure, which can synergistically form a stable zinc-rich organic prenuclear layer and a dense organic protective layer, significantly reducing particle aggregation after sterilization; using inexpensive L-histidine as the main component, only a small amount of expensive derivatives are added to achieve the same or even better stability; the resulting particles have more uniform particle size and better dispersibility, which is beneficial to subsequent antigen adsorption and immune response enhancement.

[0017] Preferably, the zinc salt in the zinc salt solution in step (1) is selected from at least one of zinc chloride, zinc sulfate, and zinc nitrate; and the aluminum salt in the aluminum salt solution in step (2) is selected from at least one of aluminum chloride, aluminum sulfate, and aluminum nitrate.

[0018] Preferably, the concentration of the zinc salt solution in step (1) is 5-20 mM, and the concentration of the complexing agent solution is 20-70 g / L.

[0019] Preferably, the concentration of the aluminum salt solution in step (2) is 18-30 mM; the mixed alkaline solution contains 50-150 mM disodium hydrogen phosphate and 150-250 mM sodium hydroxide.

[0020] Preferably, in steps (1)-(3), the ratio of zinc salt solution to aluminum salt solution is controlled such that the molar ratio of aluminum to zinc in the final zinc-aluminum composite adjuvant is 1.5-3.5:1, more preferably 2.0-3.0:1, and most preferably about 2.5:1.

[0021] Preferably, in step (3), the dropping rate of the aluminum salt solution and the mixed alkaline solution are each independently 0.5-2 mL / min, and the stirring speed is 300-700 rpm.

[0022] Preferably, the washing in step (4) is performed using an ultrafiltration membrane with a molecular weight cutoff of 50-200 kDa; the sterilization conditions are sterilization at 115-125°C for 20-40 minutes.

[0023] Preferably, the preparation method of the carnosine succinate amide includes: dissolving carnosine in a buffer solution with a pH of 7.5-8.5, adding succinic anhydride, stirring and reacting at 0-10℃ for 1-3 hours, adjusting the pH to neutral, dialysis, and then lyophilizing to obtain carnosine succinate amide; wherein the molar ratio of carnosine to succinic anhydride is 1:0.8-1.2.

[0024] A zinc-aluminum composite adjuvant is obtained using the aforementioned method for preparing zinc-aluminum composite adjuvants.

[0025] A vaccine composition comprising the zinc-aluminum complex adjuvant and an antigen selected from protein antigens, polypeptide antigens, or combinations thereof.

[0026] The zinc-aluminum composite adjuvant prepared by this invention has beneficial technical effects such as controllable structure, high stability, and optimized immunological performance: First, it can stably obtain zinc-aluminum composite particles with nanoscale particle size, narrow distribution, and uniform composition, significantly improving the problems of large particle size and large batch-to-batch variation in traditional zinc-aluminum precipitates; Second, due to the formation of a multi-site organic protective layer on the particle surface by L-histidine and carnosine succinate, the aggregation of adjuvant during sterilization is significantly reduced, and the particle size and dispersibility are well maintained before and after sterilization, ensuring both terminal sterilization and the physicochemical stability of the formulation; Third, the synergistic effect of zinc-rich organic matter and the outer aluminum hydroxyl / aluminum phosphate shell improves the adsorption and sustained release capacity of protein or polypeptide antigens, which is conducive to efficient antigen presentation, and enhances Th1 / cellular immune responses while maintaining the humoral immune advantages of traditional aluminum adjuvants, thus making it more suitable for vaccine preparation that requires a combination of humoral and cellular immunity.

[0027] The beneficial effects of this invention are:

[0028] 1. This invention provides a zinc-aluminum composite adjuvant and its preparation method. By employing a specific complexing agent combination of L-histidine and carnosine succinate amide, and combining it with a stepwise co-precipitation process, the nucleation and growth process of the zinc-aluminum complex is precisely controlled, resulting in the preparation of zinc-aluminum composite particles. The prepared zinc-aluminum composite adjuvant has a small particle size, with minimal particle size increase over 60 days, and a suspension stability index of 16.5%, far lower than the comparative example. This fundamentally solves the technical problems of disordered structure, uneven particle size, large batch-to-batch variability, and poor storage stability of zinc-aluminum adjuvants prepared by traditional simple co-precipitation methods.

[0029] 2. The zinc-aluminum composite adjuvant prepared in this invention not only maintains a high protein adsorption rate, but its unique structure and surface properties also promise to synergistically activate a stronger Th1-type cellular immune response during antigen presentation, compensating for the shortcomings of traditional aluminum adjuvants that favor Th2-type humoral immunity. This combination of highly efficient adsorption and the potential to balance immune responses makes the adjuvant of this invention particularly suitable for the design of novel vaccines that require the simultaneous mobilization of humoral and cellular immunity, expanding its application potential in the prevention of complex infectious diseases or tumor vaccines. Detailed Implementation

[0030] The invention will now be described in further detail with reference to specific embodiments, but it should not be construed as limiting the scope of the invention to the following embodiments.

[0031] Example 1

[0032] A method for preparing a zinc-aluminum composite adjuvant includes the following steps:

[0033] (1) Dissolve 0.68g ZnCl2 in water and make up to 500mL to obtain a 10mM zinc salt solution; dissolve 5.88g complexing agent in water and make up to 100mL to obtain a complexing agent solution; mix 100mL of zinc salt solution and 20mL of complexing agent solution, adjust the pH to 5.5 with 1M NaOH, stir for 30min to obtain mixture A;

[0034] (2) Take Dissolve in water and bring to a final volume of 1 L to obtain a 25 mM aluminum salt solution; prepare a mixed alkaline solution containing 100 mM... and 200 mM NaOH;

[0035] (3) Place the mixture A in a 1L reactor and add 100mL of aluminum salt solution at a flow rate of 1mL / min under the stirring conditions of 25℃ and 500rpm. At the same time, add the mixed alkali solution at a flow rate of 1mL / min to keep the pH of the reaction solution at 7.0. After the addition is completed, continue stirring for 70min to obtain a zinc-aluminum composite suspension.

[0036] (4) The zinc-aluminum composite suspension was washed with a 100 kDa ultrafiltration membrane to remove free ions, then sterilized at 121°C for 30 min, cooled, and stored at 4°C to obtain the zinc-aluminum composite adjuvant.

[0037] The complexing agent is a mixture of L-histidine and carnosine succinate in a mass ratio of 2:1.

[0038] The preparation method of the carnosine succinate is as follows: 60 mmol / L carnosine is dissolved in a tris(hydroxymethyl)aminomethane buffer solution at pH 8.0. An equimolar amount of succinic anhydride is added while stirring at 400 rpm. The mixture is stirred and reacted at 4°C for 2 h. The pH is adjusted to neutral. The mixture is dialyzed for 48 h through a dialysis bag with a molecular weight cutoff of 2000 Da, with the dialysate being changed every 6 h during the process to remove small molecule impurities. The mixture is then lyophilized to obtain carnosine succinate.

[0039] Example 2

[0040] The process is basically the same as in Example 1, except that the volume of aluminum salt solution added in step (3) is 80 mL. The specific steps of step (3) are as follows: place the mixture A in a 1L reactor, and add 80 mL of aluminum salt solution at a flow rate of 1 mL / min under the conditions of stirring at 25°C and 500 rpm; at the same time, add the mixed alkaline solution at a flow rate of 1 mL / min to keep the pH of the reaction solution at 7.0; after the addition is completed, continue stirring for 70 min to obtain a zinc-aluminum composite suspension.

[0041] Example 3

[0042] The process is basically the same as in Example 1, except that the volume of aluminum salt solution added in step (3) is 120 mL. The specific steps of step (3) are as follows: place the mixture A in a 1L reactor, and add 120 mL of aluminum salt solution at a flow rate of 1 mL / min under the conditions of stirring at 25°C and 500 rpm; at the same time, add the mixed alkaline solution at a flow rate of 1 mL / min to keep the pH of the reaction solution at 7.0; after the addition is completed, continue stirring for 70 min to obtain a zinc-aluminum composite suspension.

[0043] Example 4

[0044] A method for preparing a zinc-aluminum composite adjuvant includes the following steps:

[0045] (1) Dissolve 0.68g ZnCl2 in water and make up to 500mL to obtain a 10mM zinc salt solution; dissolve 5.88g complexing agent in water and make up to 100mL to obtain a complexing agent solution; mix 100mL of zinc salt solution and 20mL of complexing agent solution, adjust the pH to 5.5 with 1M NaOH, stir for 30min to obtain mixture A;

[0046] (2) Take Dissolve in water and bring to a final volume of 1 L to obtain a 25 mM aluminum salt solution; prepare a mixed alkaline solution containing 100 mM... and 200 mM NaOH;

[0047] (3) Place the mixture A in a 1L reactor and add 100mL of aluminum salt solution at a flow rate of 1mL / min under the stirring conditions of 25℃ and 500rpm. At the same time, add the mixed alkali solution at a flow rate of 1mL / min to keep the pH of the reaction solution at 7.0. After the addition is completed, continue stirring for 70min to obtain a zinc-aluminum composite suspension.

[0048] (4) The zinc-aluminum composite suspension was washed with a 100 kDa ultrafiltration membrane to remove free ions, then sterilized at 121°C for 30 min, cooled, and stored at 4°C to obtain the zinc-aluminum composite adjuvant.

[0049] The complexing agent is L-histidine.

[0050] Example 5

[0051] A method for preparing a zinc-aluminum composite adjuvant includes the following steps:

[0052] (1) Dissolve 0.68g ZnCl2 in water and make up to 500mL to obtain a 10mM zinc salt solution; dissolve 5.88g complexing agent in water and make up to 100mL to obtain a complexing agent solution; mix 100mL of zinc salt solution and 20mL of complexing agent solution, adjust the pH to 5.5 with 1M NaOH, stir for 30min to obtain mixture A;

[0053] (2) Take Dissolve in water and bring to a final volume of 1 L to obtain a 25 mM aluminum salt solution; prepare a mixed alkaline solution containing 100 mM... and 200 mM NaOH;

[0054] (3) Place the mixture A in a 1L reactor and add 100mL of aluminum salt solution at a flow rate of 1mL / min under the stirring conditions of 25℃ and 500rpm. At the same time, add the mixed alkali solution at a flow rate of 1mL / min to keep the pH of the reaction solution at 7.0. After the addition is completed, continue stirring for 70min to obtain a zinc-aluminum composite suspension.

[0055] (4) The zinc-aluminum composite suspension was washed with a 100 kDa ultrafiltration membrane to remove free ions, then sterilized at 121°C for 30 min, cooled, and stored at 4°C to obtain the zinc-aluminum composite adjuvant.

[0056] The complexing agent is carnosine succinate.

[0057] The preparation method of the carnosine succinate is as follows: 60 mmol / L carnosine is dissolved in a tris(hydroxymethyl)aminomethane buffer solution at pH 8.0. An equimolar amount of succinic anhydride is added while stirring at 400 rpm. The mixture is stirred and reacted at 4°C for 2 h. The pH is adjusted to neutral. The mixture is dialyzed for 48 h through a dialysis bag with a molecular weight cutoff of 2000 Da, with the dialysate being changed every 6 h during the process to remove small molecule impurities. The mixture is then lyophilized to obtain carnosine succinate.

[0058] Example 6

[0059] It is basically the same as Example 1, except that the complexing agent is a mixture of L-histidine and carnosine succinate in a mass ratio of 1.5:1.

[0060] The preparation method of the carnosine succinate is the same as that in Example 1.

[0061] Example 7

[0062] It is basically the same as Example 1, except that the complexing agent is a mixture of L-histidine and carnosine succinate in a mass ratio of 2.8:1.

[0063] The preparation method of the carnosine succinate is the same as that in Example 1.

[0064] Comparative Example 1

[0065] A method for preparing a zinc-aluminum composite adjuvant includes the following steps:

[0066] (1) Dissolve 0.68g ZnCl2 in water and make up to 500mL to obtain a 10mM zinc salt solution; dissolve 5.88g complexing agent in water and make up to 100mL to obtain a complexing agent solution; mix 100mL of zinc salt solution and 20mL of complexing agent solution, adjust the pH to 5.5 with 1M NaOH, stir for 30min to obtain mixture A;

[0067] (2) Take Dissolve in water and bring to a final volume of 1 L to obtain a 25 mM aluminum salt solution; prepare a mixed alkaline solution containing 100 mM... and 200 mM NaOH;

[0068] (3) Place the mixture A in a 1L reactor and add 100mL of aluminum salt solution at a flow rate of 1mL / min under the stirring conditions of 25℃ and 500rpm. At the same time, add the mixed alkali solution at a flow rate of 1mL / min to keep the pH of the reaction solution at 7.0. After the addition is completed, continue stirring for 70min to obtain a zinc-aluminum composite suspension.

[0069] (4) The zinc-aluminum composite suspension was washed with a 100 kDa ultrafiltration membrane to remove free ions, then sterilized at 121°C for 30 min, cooled, and stored at 4°C to obtain the zinc-aluminum composite adjuvant.

[0070] The complexing agent is a mixture of L-histidine and carnosine in a mass ratio of 2:1.

[0071] Comparative Example 2

[0072] A method for preparing a zinc-aluminum composite adjuvant includes the following steps:

[0073] (1) Dissolve 0.68g ZnCl2 in water and make up to 500mL to obtain a 10mM zinc salt solution; dissolve 5.88g complexing agent in water and make up to 100mL to obtain a complexing agent solution; mix 100mL of zinc salt solution and 20mL of complexing agent solution, adjust the pH to 5.5 with 1M NaOH, stir for 30min to obtain mixture A;

[0074] (2) Take Dissolve in water and bring to a final volume of 1 L to obtain a 25 mM aluminum salt solution; prepare a mixed alkaline solution containing 100 mM... and 200 mM NaOH;

[0075] (3) Place the mixture A in a 1L reactor and add 100mL of aluminum salt solution at a flow rate of 1mL / min under the stirring conditions of 25℃ and 500rpm. At the same time, add the mixed alkali solution at a flow rate of 1mL / min to keep the pH of the reaction solution at 7.0. After the addition is completed, continue stirring for 70min to obtain a zinc-aluminum composite suspension.

[0076] (4) The zinc-aluminum composite suspension was washed with a 100 kDa ultrafiltration membrane to remove free ions, then sterilized at 121°C for 30 min, cooled, and stored at 4°C to obtain the zinc-aluminum composite adjuvant.

[0077] The complexing agent is sodium citrate.

[0078] Comparative Example 3

[0079] A method for preparing a zinc-aluminum composite adjuvant includes the following steps:

[0080] (1) Dissolve 0.68g ZnCl2 in water and make up to 500mL to obtain a 10mM zinc salt solution; dissolve 5.88g complexing agent in water and make up to 100mL to obtain a complexing agent solution; mix 100mL of zinc salt solution and 20mL of complexing agent solution, adjust the pH to 5.5 with 1M NaOH, stir for 30min to obtain mixture A;

[0081] (2) Take Dissolve in water and bring to a final volume of 1 L to obtain a 25 mM aluminum salt solution; prepare a mixed alkaline solution containing 100 mM... and 200 mM NaOH;

[0082] (3) Place the mixture A in a 1L reactor and add 100mL of aluminum salt solution at a flow rate of 1mL / min under the stirring conditions of 25℃ and 500rpm. At the same time, add the mixed alkali solution at a flow rate of 1mL / min to keep the pH of the reaction solution at 7.0. After the addition is completed, continue stirring for 70min to obtain a zinc-aluminum composite suspension.

[0083] (4) The zinc-aluminum composite suspension was washed with a 100 kDa ultrafiltration membrane to remove free ions, then sterilized at 121°C for 30 min, cooled, and stored at 4°C to obtain the zinc-aluminum composite adjuvant.

[0084] The complexing agent is L-glutamate sodium.

[0085] Comparative Example 4

[0086] A method for preparing a zinc-aluminum composite adjuvant includes the following steps:

[0087] (1) Dissolve 0.68g ZnCl2 in water and make up to 500mL to obtain a 10mM zinc salt solution; dissolve 5.88g complexing agent in water and make up to 100mL to obtain a complexing agent solution; mix 100mL of zinc salt solution and 20mL of complexing agent solution, adjust the pH to 5.5 with 1M NaOH, stir for 30min to obtain mixture A;

[0088] (2) Take Dissolve in water and bring to a final volume of 1 L to obtain a 25 mM aluminum salt solution; prepare a mixed alkaline solution containing 100 mM... and 200 mM NaOH;

[0089] (3) Place the mixture A in a 1L reactor and add 100mL of aluminum salt solution at a flow rate of 1mL / min under the stirring conditions of 25℃ and 500rpm. At the same time, add the mixed alkali solution at a flow rate of 1mL / min to keep the pH of the reaction solution at 7.0. After the addition is completed, continue stirring for 70min to obtain a zinc-aluminum composite suspension.

[0090] (4) The zinc-aluminum composite suspension was washed with a 100 kDa ultrafiltration membrane to remove free ions, then sterilized at 121°C for 30 min, cooled, and stored at 4°C to obtain the zinc-aluminum composite adjuvant.

[0091] The complexing agent is sodium phosphate.

[0092] Comparative Example 5

[0093] A method for preparing a zinc-aluminum composite adjuvant includes the following steps:

[0094] (1) Dissolve 0.68g of ZnCl2 in water and dilute to 500mL to obtain a 10mM zinc salt solution; take Dissolve in water and bring to a final volume of 1 L to obtain a 25 mM aluminum salt solution; prepare a mixed alkaline solution containing 100 mM... Add 200 mM NaOH; dissolve 6.5 g of complexing agent in water and bring the volume to 100 mL to obtain a complexing agent solution; mix 100 mL of zinc salt solution, 100 mL of aluminum salt solution, and 20 mL of complexing agent solution, adjust the pH to 5.5 with 1 M NaOH, and stir for 30 min to obtain mixture A;

[0095] (2) Place the mixture A in a 1L reactor and add the mixed alkaline solution dropwise at a flow rate of 1mL / min under the conditions of stirring at 25℃ and 500rpm to keep the pH of the reaction solution at 7.0; after the addition is completed, continue stirring for 70min to obtain a zinc-aluminum composite suspension.

[0096] (3) The zinc-aluminum composite suspension was washed with a 100 kDa ultrafiltration membrane to remove free ions, then sterilized at 121°C for 30 min, cooled, and stored at 4°C to obtain the zinc-aluminum composite adjuvant.

[0097] The complexing agent is a mixture of L-histidine and carnosine succinate in a mass ratio of 2:1.

[0098] The preparation method of the carnosine succinate is as follows: 60 mmol / L carnosine is dissolved in a tris(hydroxymethyl)aminomethane buffer solution at pH 8.0. An equimolar amount of succinic anhydride is added while stirring at 400 rpm. The mixture is stirred and reacted at 4°C for 2 h. The pH is adjusted to neutral. The mixture is dialyzed for 48 h through a dialysis bag with a molecular weight cutoff of 2000 Da, with the dialysate being changed every 6 h during the process to remove small molecule impurities. The mixture is then lyophilized to obtain carnosine succinate.

[0099] Comparative Example 6

[0100] The method is basically the same as in Example 1, except that aluminum salt is added first, followed by zinc salt; the preparation method of the zinc-aluminum composite adjuvant includes the following steps:

[0101] (1) Take Dissolve in water and bring to a final volume of 1 L to obtain a 25 mM aluminum salt solution; dissolve 5.88 g of complexing agent in water and bring to a final volume of 100 mL to obtain a complexing agent solution; mix 100 mL of the aluminum salt solution and 20 mL of the complexing agent solution, adjust the pH to 5.5 with 1 M NaOH, and stir for 30 min to obtain mixture A;

[0102] (2) Dissolve 0.68g ZnCl2 in water and dilute to 500mL to obtain a 10mM zinc salt solution; prepare a mixed alkaline solution containing 100mM zinc salt. and 200 mM NaOH;

[0103] (3) Place the mixture A in a 1L reactor and add 100mL of zinc salt solution at a flow rate of 1mL / min under the stirring conditions of 25℃ and 500rpm. At the same time, add the mixed alkaline solution at a flow rate of 1mL / min to keep the pH of the reaction solution at 7.0. After the addition is completed, continue stirring for 70min to obtain a zinc-aluminum composite suspension.

[0104] (4) The zinc-aluminum composite suspension was washed with a 100 kDa ultrafiltration membrane to remove free ions, then sterilized at 121°C for 30 min, cooled, and stored at 4°C to obtain the zinc-aluminum composite adjuvant.

[0105] The complexing agent is a mixture of L-histidine and carnosine succinate in a mass ratio of 2:1.

[0106] The preparation method of the carnosine succinate is the same as in Example 1.

[0107] Comparative Example 7

[0108] The method is basically the same as in Example 1, except that only zinc salt is added; the preparation method of the zinc complex adjuvant includes the following steps:

[0109] (1) Dissolve 0.68g ZnCl2 in water and bring the volume to 500mL to obtain a 10mM zinc salt solution; dissolve 5.88g complexing agent in water and bring the volume to 100mL to obtain a complexing agent solution; mix 100mL of zinc salt solution and 20mL of complexing agent solution, adjust the pH to 5.5 with 1M NaOH, and stir for 30min to obtain mixed solution A; prepare mixed alkaline solution containing 100mM and 200 mM NaOH;

[0110] (2) Place the mixture A in a 1L reactor and add the mixed alkaline solution dropwise at a flow rate of 1mL / min under stirring conditions of 25℃ and 500rpm to keep the pH of the reaction solution at 7.0; after the addition is complete, continue stirring for 70min to obtain a zinc composite suspension.

[0111] (3) The zinc composite suspension was washed with a 100 kDa ultrafiltration membrane to remove free ions, then sterilized at 121°C for 30 min, cooled, and stored at 4°C to obtain the zinc composite adjuvant.

[0112] The complexing agent is a mixture of L-histidine and carnosine succinate in a mass ratio of 2:1.

[0113] The preparation method of the carnosine succinate is the same as in Example 1.

[0114] Comparative Example 8

[0115] The method is basically the same as in Example 1, except that only aluminum salt is added; the preparation method of the aluminum composite adjuvant includes the following steps:

[0116] (1) Take Dissolve in water and bring to a final volume of 1 L to obtain a 25 mM aluminum salt solution; dissolve 5.88 g of the complexing agent in water and bring to a final volume of 100 mL to obtain a complexing agent solution; mix 100 mL of the aluminum salt solution and 20 mL of the complexing agent solution, adjust the pH to 5.5 with 1 M NaOH, and stir for 30 min to obtain mixture A; prepare a mixed alkaline solution containing 100 mM... and 200 mM NaOH;

[0117] (2) Place the mixture A in a 1L reactor and add the mixed alkaline solution dropwise at a flow rate of 1mL / min under the conditions of 25℃ and 500rpm stirring to keep the pH of the reaction solution at 7.0; after the addition is completed, continue stirring for 70min to obtain an aluminum composite suspension.

[0118] (3) The above aluminum composite suspension was washed with a 100 kDa ultrafiltration membrane to remove free ions, then sterilized at 121°C for 30 min, cooled, and stored at 4°C to obtain the aluminum composite adjuvant.

[0119] The complexing agent is a mixture of L-histidine and carnosine succinate in a mass ratio of 2:1.

[0120] The preparation method of the carnosine succinate is as follows: 60 mmol / L carnosine is dissolved in a tris(hydroxymethyl)aminomethane buffer solution at pH 8.0. An equimolar amount of succinic anhydride is added while stirring at 400 rpm. The mixture is stirred and reacted at 4°C for 2 h. The pH is adjusted to neutral. The mixture is dialyzed for 48 h through a dialysis bag with a molecular weight cutoff of 2000 Da, with the dialysate being changed every 6 h during the process to remove small molecule impurities. The mixture is then lyophilized to obtain carnosine succinate.

[0121] Test Example 1

[0122] Protein adsorption rate: determined using the Lowry method. Specifically, lysozyme protein solutions of different concentrations were first prepared, and denoted as the initial protein concentration (C0). 初始蛋白 The absorbance at 650 nm was measured using an enzyme-linked immunosorbent assay (ELISA) reader, and a standard curve was plotted. The mass ratio of aluminum to protein was 1:4, 1:1, and 2:1. The appropriate amount of the zinc-aluminum composite adjuvant of this invention was mixed with the protein solution, vortexed, and then placed on a universal shaker for 2 hours. 1 mL of the mixture was centrifuged. 0.5 mL of the supernatant was placed in a centrifuge tube, and an equal volume of 0.5 mL of colorimetric reagent was added. The mixture was vortexed for 10 minutes, and then 2 mL of Folin-Ciocalteu reagent was added under dark conditions. After vortexing and standing for 30 minutes, the absorbance at 650 nm was measured using an ELISA reader. The protein concentration in the supernatant, i.e., the concentration of unadsorbed protein C, was calculated using the aforementioned standard curve. 未吸附蛋白 The protein adsorption rate is calculated using the following formula: Protein adsorption rate = (C 初始蛋白 -C 未吸附蛋白 ) / C 初始蛋白 ×100%. Each group was tested 4 times, and the average value was taken. The results are shown in Table 1.

[0123] Table 1 Results of protein adsorption rate test

[0124]

[0125] Test Example 2

[0126] Stability test: The particle size of the zinc-aluminum composite adjuvants prepared in the examples and comparative examples was measured on day 0 (d0) and day 60 (d60), respectively, in nm. Each group was tested 5 times and the average value was taken. The results are shown in Table 2 below.

[0127] Suspension stability test: Take 4 ml each of the zinc-aluminum composite adjuvant prepared in the above examples and comparative examples (0.5 mg / ml) into 15 ml centrifuge tubes, add 1.5 mg of green fluorescent protein to each, and bring the volume to 5 ml. Mix well, then place vertically to allow natural sedimentation. After the aluminum adjuvant adsorbs the green fluorescent protein, the sedimentation of the aluminum adjuvant can be clearly seen. Observe and record for 1.5 h. The volume of adjuvant after sedimentation / total volume is recorded as the suspension stability index. Each group is measured 4 times, and the average value is taken. The results are shown in Table 2.

[0128] Table 2 Stability performance test results

[0129]

[0130] The results above show that the zinc-aluminum composite adjuvant prepared by this invention possesses high stability and strong protein adsorption capacity. Specific analysis is as follows: Example 1 exhibits the best performance in terms of high protein adsorption rate (92%) at an aluminum-to-protein ratio of 2:1, relatively stable physicochemical properties, initial particle size of 162.8 nm, PDI of 0.113, and particle size growth to only 171.6 nm after 60 days, with a suspension stability index of 16.5%. Examples 1-3 show that different ratios of aluminum and zinc salts result in less than ideal particle structure and surface charge distribution, leading to slightly lower protein adsorption rates and suspension stability compared to Example 1. Examples 1 and 4-5 show that L-histidine mildly coordinates to form a stable pre-nucleus, while carnosine succinate strongly anchors and forms a dense protective layer. The combination of these two components optimizes particle surface properties and structural stability, significantly outperforming single components, thus achieving synergistic effects in sterilization tolerance, protein adsorption, and storage stability. In contrast, Comparative Examples 1-4, using traditional complexing agents such as sodium citrate, L-glutamate, sodium phosphate, or changing the key component to carnosine instead of carnosine succinate, showed a significant decrease in both adsorption and stability. Comparative Example 5, employing an initial blending process of zinc-aluminum salts and complexing agents, even using the same preferred complexing agent, exhibited performance far inferior to Example 1. Further comparison of Example 1 and Comparative Examples 6-8 revealed that the order of zinc and aluminum addition significantly impacted the results. Adding only zinc or aluminum salts also yielded poor results. This is because Comparative Example 8, with only aluminum salt, demonstrated strong adsorption due to the mature ligand exchange mechanism of aluminum adjuvant, but its structure was unstable. Comparative Example 7, with only zinc salt, could form a stable core with the dedicated complexing agent but lacked adsorption function. Both had fatal shortcomings when used alone. This invention, through stepwise co-precipitation, allows zinc to construct a stable core and aluminum to form a functional shell, achieving a balance between structural stability and efficient adsorption. Therefore, Example 1 performed best in both tests, fully demonstrating the synergistic importance of the specific complexing agent combination and stepwise preparation process employed in this invention.

Claims

1. A method for preparing a zinc-aluminum composite adjuvant, characterized in that, Includes the following steps: (1) Mix the zinc salt solution and the complexing agent solution, adjust the pH to 4.5-6.5, stir the reaction to obtain mixture A; (2) Prepare aluminum salt solution and mixed alkaline solution respectively, wherein the mixed alkaline solution contains phosphate and hydroxide; (3) Under stirring conditions, the aluminum salt solution and the mixed alkaline solution are simultaneously added dropwise to the mixture A, and the pH of the reaction solution is controlled within the range of 6.5-7.5; After the addition is complete, continue stirring to obtain a zinc-aluminum composite suspension; (4) The zinc-aluminum composite suspension is washed and sterilized to obtain the zinc-aluminum composite adjuvant; The complexing agent is selected from at least one of L-histidine and carnosine succinate; The concentration of the zinc salt solution in step (1) is 5-20 mM, and the concentration of the complexing agent solution is 20-70 g / L.

2. The preparation method of the zinc-aluminum composite adjuvant as described in claim 1, characterized in that, The zinc salt in the zinc salt solution in step (1) is selected from at least one of zinc chloride, zinc sulfate, and zinc nitrate; the aluminum salt in the aluminum salt solution in step (2) is selected from at least one of aluminum chloride, aluminum sulfate, and aluminum nitrate.

3. The preparation method of the zinc-aluminum composite adjuvant as described in claim 1, characterized in that, The concentration of the aluminum salt solution in step (2) is 18-30 mM; the mixed alkaline solution contains 50-150 mM disodium hydrogen phosphate and 150-250 mM sodium hydroxide.

4. The preparation method of the zinc-aluminum composite adjuvant as described in claim 1, characterized in that, In step (3), the dropping rate of the aluminum salt solution and the mixed alkaline solution is 0.5-2 mL / min, and the stirring speed is 300-700 rpm.

5. The preparation method of the zinc-aluminum composite adjuvant as described in claim 1, characterized in that, The washing in step (4) is performed using an ultrafiltration membrane with a molecular weight cutoff of 50-200 kDa; the sterilization conditions are sterilization at 115-125℃ for 20-40 min.

6. The method for preparing the zinc-aluminum composite adjuvant as described in claim 1, characterized in that, The preparation method of the carnosine succinate amide includes: dissolving carnosine in a buffer solution with a pH of 7.5-8.5, adding succinic anhydride, stirring and reacting at 0-10℃ for 1-3 hours, adjusting the pH to neutral, dialysis, and then lyophilizing to obtain carnosine succinate amide; wherein the molar ratio of carnosine to succinic anhydride is 1:0.8-1.

2.

7. A zinc-aluminum composite adjuvant, characterized in that, It is obtained by the preparation method of zinc-aluminum composite adjuvant as described in any one of claims 1-6.

8. A vaccine composition, characterized in that, The vaccine composition comprises the zinc-aluminum complex adjuvant of claim 7 and an antigen, wherein the antigen is selected from protein antigens, polypeptide antigens, or combinations thereof.

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