A preparation method and application of aluminum hydroxide adjuvant

Abstract

The present invention relates to a preparation method and application of an aluminum hydroxide adjuvant, comprising the following steps: a mixing reaction step: adding an aluminum chloride solution and a sodium hydroxide solution into an aqueous system for reaction to obtain an aluminum hydroxide colloidal suspension; preparing a mixed liquor: centrifuging the aluminum hydroxide colloidal suspension to obtain a precipitate, adding water to the precipitate to prepare a suspension, and adding urea solution to obtain a mixed liquor. In the present application, sodium hydroxide and aluminum chloride react under an aqueous system, and their reaction is more sufficient, which is conducive to forming a stable crystal structure, and is conducive to aluminum hydroxide aggregation to form adjuvant particles of 1 5 μm; during autoclaving, the urea thermal hydrolysis product reacts with the adjuvant to generate a crystalline aluminum hydroxide having a structure between pseudo-boehmite and boehmite; the adjuvant of the present application is resistant to acid and alkali adjustment, and has strong adsorption capacity and no violent reaction after mixing with an antigen; the adjuvant of the present application has a large particle size range, and different antigens match adjuvants of a specific particle size range to produce the best effect.

Description

Technical Field

[0001] The present invention relates to the technical field of vaccines, and in particular to a preparation method and application of an aluminum hydroxide adjuvant. Background Art

[0002] Aluminum hydroxide adjuvant, as the longest-used human adjuvant, can effectively adsorb antigens to improve the immunogenicity of vaccines. Aluminum adjuvants have a long history and have been well proven in terms of scale, practicality, and safety. Today, aluminum adjuvants are receiving increasing attention. Currently, there are many production processes for aluminum adjuvants, and the properties and scope of application of aluminum adjuvants are still relatively simple. It is necessary to develop adjuvants with different physical and chemical properties to adapt to various antigens and improve the performance of vaccines. It is generally believed that aluminum hydroxide adjuvants have more than three structural forms, which can be boehmite, behenite, hydrated aluminum hydroxide, etc. During the preparation process or subsequent use of aluminum hydroxide adjuvants, aluminum hydroxide adjuvants can transform into different forms, and their physical and chemical properties are extremely unstable in different environments. This makes it impossible to accurately control the quality of the adjuvant during preparation, processing, sterilization, and use, and it lacks stability and controllability.

[0003] In the selection of adjuvants for use in vaccines, a suitable adjuvant can achieve the optimal adsorption state when added in a small amount, thereby enhancing the induced immune response and improving immunogenicity. There are many factors that affect the properties of aluminum hydroxide adjuvants. For example, factors in the preparation process include the dripping method, process pH control, end point selection, etc., and factors in the post-treatment of adjuvants include pH, conductivity, electron microscopic structure, particle size, etc. Actual studies have found that in mixing with antigens, existing aluminum hydroxide adjuvants have poor stability, are prone to forming colloidal agglomerates, and the precipitate after adsorption is sparse or easy to agglomerate. The pH after adsorption is uncontrollable, and it may be necessary to add acid and alkali twice to adjust the pH of the system, which seriously affects the application of aluminum hydroxide adjuvants. Summary of the Invention

[0004] The present invention provides a method for preparing an aluminum hydroxide adjuvant, comprising the following steps:

[0005] Mixing reaction step: adding aluminum chloride solution and sodium hydroxide solution into a water system to react to obtain aluminum hydroxide colloidal suspension;

[0006] Prepare a mixed solution: centrifuge the aluminum hydroxide colloidal suspension to obtain a precipitate, add water to the precipitate to prepare a suspension, and add urea solution to obtain a mixed solution.

[0007] Preferably, the mixing reaction step comprises: a first stage: adjusting the amount of the sodium hydroxide solution added until the water system reaches a set pH value;

[0008] The second stage: the pH setting value is then maintained by adjusting the amount of the sodium hydroxide solution added, and the aluminum chloride solution is added to carry out the reaction.

[0009] The third stage: aging by adjusting the amount of the sodium hydroxide solution added to maintain the pH set value.

[0010] Preferably, the duration of the first stage is no more than 5 minutes;

[0011] The pH setting value is 5.5-7.0;

[0012] The aging process includes stirring at a speed of 200 rpm for 60-90 minutes.

[0013] Preferably, in the mixing reaction step:

[0014] The ratio of the volume of the water system to the volume of the sodium hydroxide solution consumed in the reaction is 160:6-12;

[0015] The ratio of the volume of the water system to the volume of the aluminum chloride solution consumed in the reaction is 160:3-6;

[0016] The molar ratio of sodium hydroxide and aluminum chloride consumed in the mixed reaction is 2.8-3.0;

[0017] The mixing reaction was carried out at 70° C. with stirring.

[0018] Preferably, in the mixing reaction step:

[0019] The molar concentration of the sodium hydroxide solution is 0.25-0.6 mol / L, and the molar concentration of the aluminum chloride solution is 0.17-0.41 mol / L;

[0020] The sodium hydroxide solution is added through a first spray head located at the center of the top of the reaction tank, and the aluminum chloride solution is added through a second spray head and a third spray head symmetrically arranged at the center of the top of the reaction tank.

[0021] Preferably, in the step of preparing the mixed solution: adding 20-60°C water to the precipitate to prepare a suspension with a mass concentration of 3-10 mg / ml;

[0022] The mass concentration of the urea solution is 0.05%; the added amount of the urea solution is 1 / 40-1 / 30 of the mass of the aluminum hydroxide.

[0023] Preferably, the mixing reaction step further comprises:

[0024] Adjusting the pH of the reaction system: adding aluminum chloride solution to the water system until the pH is 5.0.

[0025] Preferably, the step of preparing the mixed solution further comprises:

[0026] Steps for preparing the adjuvant stock solution: sterilize the mixed solution, dialyze to remove ammonia, and then aliquot and sterilize again to obtain the aluminum hydroxide stock solution.

[0027] The present application also provides a use of an adjuvant prepared by the above method in preparing a vaccine.

[0028] The preparation method of the aluminum hydroxide adjuvant and the adjuvant of the present invention have the following advantages:

[0029] 1. In this application, sodium hydroxide and aluminum chloride react in an aqueous system, resulting in a more complete reaction. Compared with the traditional alkali-in-aluminum solution preparation process, this method can largely avoid the generation of non-aluminum hydroxide substances in a local high-concentration hydroxide ion environment after the alkali solution is added to the aluminum chloride solution. Moreover, as the aluminum hydroxide concentration increases to a certain range, the entry of hydroxide ions easily causes colloidal precipitation, resulting in irreversible passivation of the adjuvant.

[0030] 2. In this application, the reaction in an aqueous environment is more conducive to the aggregation of aluminum hydroxide into adjuvant particles of 1-5 μm. These particles retain the characteristics of pseudo-boehmite, namely, large specific surface area and strong adsorption capacity. The adjuvant particles can interact during the antigen adsorption process, tightly encapsulating the antigen and adjuvant, significantly slowing the release of the antigen in the body and achieving a sustained immune effect.

[0031] 3. During autoclaving in the present application, urea is hydrolyzed by heat to a temperature above 80°C, releasing hydroxide ions. The reaction intensifies as the temperature rises. At this time, the aluminum hydroxide prepared with a pseudo-boehmite structure also undergoes a deprotonation reaction as the temperature rises. The hydroxide ions generated by hydrolysis neutralize the released protons and, to a certain extent, accelerate the deprotonation process. At the same time, the unreacted aluminum oxides in the adjuvant can fully react with the weak base. This uniform reaction promotes an orderly transformation of the adjuvant morphology. This orderly transformation, to a certain extent, generates a crystalline structure between pseudo-boehmite and boehmite. This structure retains a large specific surface area, is resistant to repeated autoclaving, and maintains relatively stable physical and chemical properties.

[0032] 4. The adjuvant of the present application is resistant to acid and alkali adjustments and does not react violently (such as agglomeration, precipitation, caking, etc.) after mixing with the antigen. This can avoid the impact of secondary pH adjustment on the body's immune response or sensitization after adsorption preparation into the stock solution. Existing aluminum hydroxide adjuvant processes have specific pH or particle size and have extremely high requirements for the use environment. Environmental changes during use can easily cause irreversible changes in the properties of aluminum hydroxide adjuvants.

[0033] 5. The adjuvant of the present application has a large particle size range. Different antigens can produce the best effect by matching adjuvants with a specific particle size range. The adjuvants with different physical and chemical properties prepared in the present application can provide the best compatible adjuvants for different antigens. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 The particle size of the suspension obtained in Example 1 (before high pressure) was measured using a Malvern Zetasizer Nano particle size analyzer.

[0035] Figure 2 The particle size of the first mixed solution obtained in Example 1 after one autoclaving (sterilization) was measured using a Malvern Zetasizer Nano particle size analyzer.

[0036] Figure 3 The particle size of the first mixed solution obtained in Example 1 after secondary autoclaving (sterilization) was measured using a Malvern Zetasizer Nano particle size analyzer.

[0037] Figure 4 The particle size of the first mixed solution obtained in Example 1 after three (multiple) autoclaving (sterilization) was measured using a Malvern Zetasizer Nano particle size analyzer.

[0038] Figure 5 The particle size of the second mixed solution obtained in Example 1 after one autoclave (sterilization) was measured using a Malvern Zetasizer Nano particle size analyzer (pH 7.0).

[0039] Figure 6 , for Example 1 Figures 1 to 4 The pH changes of the samples were measured.

[0040] Figure 7 The particle size of the suspension obtained in Example 2 (before high pressure) was measured using a Malvern Zetasizer Nano particle size analyzer.

[0041] Figure 8 The particle size of the first mixed solution obtained in Example 2 after one autoclave (sterilization) was measured using a Malvern Zetasizer Nano particle size analyzer.

[0042] Figure 9 The particle size of the first mixed solution obtained in Example 2 after secondary autoclaving (sterilization) was measured using a Malvern Zetasizer Nano particle size analyzer.

[0043] Figure 10 The particle size of the first mixed solution obtained in Example 2 after three (multiple) autoclaving (sterilization) was measured using a Malvern Zetasizer Nano particle size analyzer.

[0044] Figure 11The particle size of the second mixed solution obtained in Example 2 after one autoclave (sterilization) was measured using a Malvern Zetasizer Nano particle size analyzer (pH 6.05).

[0045] Figure 12 , for Example 2 Figures 7 to 10 The pH changes of the samples were measured.

[0046] Figure 13 The particle size of the suspension obtained in Example 3 (before high pressure) was measured using a Malvern Zetasizer Nano particle size analyzer.

[0047] Figure 14 The particle size of the first mixed solution obtained in Example 3 after one autoclaving (sterilization) was measured using a Malvern Zetasizer Nano particle size analyzer.

[0048] Figure 15 The particle size of the first mixed solution obtained in Example 3 after secondary autoclaving (sterilization) was measured using a Malvern Zetasizer Nano particle size analyzer.

[0049] Figure 16 The particle size of the first mixed solution obtained in Example 3 after three (multiple) autoclaving (sterilization) was measured using a Malvern Zetasizer Nano particle size analyzer.

[0050] Figure 17 The particle size of the second mixed solution obtained in Example 3 after one autoclave sterilization was measured using a Malvern Zetasizer Nano particle size analyzer (pH 7.50).

[0051] Figure 18 , for Example 3 Figures 13 to 16 The pH changes of the samples were measured.

[0052] Figure 19 The particle size of the suspension obtained in Example 4 (before high pressure) was measured using a Malvern Zetasizer Nano particle size analyzer.

[0053] Figure 20 The particle size of the first mixed solution obtained in Example 4 after one autoclave (sterilization) was measured using a Malvern Zetasizer Nano particle size analyzer.

[0054] Figure 21 The particle size of the first mixed solution obtained in Example 4 after secondary autoclaving (sterilization) was measured using a Malvern Zetasizer Nano particle size analyzer.

[0055] Figure 22The particle size of the first mixed solution obtained in Example 4 after three (multiple) autoclaving (sterilization) was measured using a Malvern Zetasizer Nano particle size analyzer.

[0056] Figure 23 The particle size of the second mixed solution obtained in Example 4 after one autoclave (sterilization) was measured using a Malvern Zetasizer Nano particle size analyzer (pH 6.44).

[0057] Figure 24 , for Example 4 Figures 19 to 22 The pH changes of the samples were measured. DETAILED DESCRIPTION

[0058] The technical solution of the present invention will be described in detail below with reference to the accompanying drawings and preferred embodiments.

[0059] Example 1:

[0060] 1. Experimental preparation steps:

[0061] 160 L of water was pre-placed in a reaction tank. 9 L of 0.4 mol / L sodium hydroxide solution was prepared in a sodium hydroxide preparation tank, and 4.5 L of 0.27 mol / L aluminum chloride solution was prepared in an aluminum chloride preparation tank. Both the sodium hydroxide and aluminum chloride preparation tanks were filtered using 0.45 μm filter cartridges. The sodium hydroxide solution was added through a first spray nozzle located at the center of the top of the reaction tank. The aluminum chloride solution was added through second and third spray nozzles symmetrically located at the center of the top of the reaction tank, located 2 / 3 of the radius from the first spray nozzle. The reaction tank was stirred at 160 rpm, the jacket water circulation system was turned on, and the temperature was raised to 70°C and maintained.

[0062] 2. Steps for adjusting the pH of the reaction system:

[0063] Aluminum chloride solution was added to the reaction tank to adjust the pH of the water system to 5.0 at a flow rate of 1 ml / min.

[0064] 3. Mixing reaction steps:

[0065] Stage 1: Sodium hydroxide solution and aluminum chloride solution were added simultaneously, and the amount of sodium hydroxide solution added was adjusted in real time until the water system reached a set pH value of 6.5. The flow rate of the sodium hydroxide solution was 35 ml / min, and the flow rate of the aluminum chloride solution was 18 ml / min. The time for adjusting the pH to the set value was 2 minutes.

[0066] The second stage: then the pH setting value is maintained by adjusting the amount of sodium hydroxide solution added in real time, and aluminum chloride solution is added to react.

[0067] The third stage: after the addition of the aluminum chloride solution is completed, the pH setting value is maintained by adjusting the amount of the sodium hydroxide solution added, that is, stirring at a speed of 200 rpm for 80 minutes.

[0068] After the reaction was completed, 3.58 mol of sodium hydroxide solution and 1.21 mol of aluminum chloride solution were consumed, and the molar ratio of sodium hydroxide and aluminum chloride consumed in the mixed reaction was 3.0.

[0069] 4. Prepare the mixed solution: While stirring, cool, discharge, and centrifuge at 7000 rpm for 10 min to obtain a precipitate, which was dissolved in 50°C water to a 5 mg / ml suspension.

[0070] The suspension was divided into two parts and weighed. In the first part (1), the mass of aluminum hydroxide was 3.2 kg, and 80 g of 0.05% urea solution was added, wherein the amount of urea solution added was 1 / 40 of the mass of the aluminum hydroxide. In the second part (2), the mass of aluminum hydroxide was 3.2 kg, and 105 g of 0.05% urea solution was added, wherein the amount of urea solution added was 1 / 30 of the mass of the aluminum hydroxide.

[0071] 5. Steps for preparing adjuvant stock solution: sterilize the mixed solution at 121°C for 30 minutes, dialyze through a dialysis bag to remove ammonia until no residual ions are left, and then divide the solution into portions and sterilize again at 121°C for 30 minutes to obtain aluminum hydroxide stock solution.

[0072] Example 2:

[0073] 1. Experimental preparation steps:

[0074] Pre-place 160L of water in a reaction tank, prepare 6L of 0.25mol / L sodium hydroxide solution in a sodium hydroxide preparation tank, and 3L of 0.17mol / L aluminum chloride solution in an aluminum chloride preparation tank. Both the sodium hydroxide and aluminum chloride preparation tanks are filtered using 0.45μm filter cartridges. Add the sodium hydroxide solution through a first spray nozzle located at the center of the top of the reaction tank. Add the aluminum chloride solution through second and third spray nozzles symmetrically located at the center of the top of the reaction tank, located 2 / 3 the radius from the first spray nozzle. Stir the reaction tank at 150rpm, start circulating water in the jacket, and raise the temperature to 70°C and maintain it there.

[0075] 2. Steps for adjusting the pH of the reaction system:

[0076] Aluminum chloride solution was added to the reaction tank to adjust the pH of the water system to 5.0 at a flow rate of 1 ml / min.

[0077] 3. Mixing reaction steps:

[0078] Stage 1: Sodium hydroxide solution and aluminum chloride solution were added simultaneously, and the amount of sodium hydroxide solution added was adjusted in real time until the water system reached a pH set value of 5.5. The flow rate of the sodium hydroxide solution was 28 ml / min, and the flow rate of the aluminum chloride solution was 15 ml / min. The pH set value was adjusted for 3 minutes.

[0079] The second stage: then the pH setting value is maintained by adjusting the amount of sodium hydroxide solution added in real time, and aluminum chloride solution is added to react.

[0080] The third stage: after the addition of the aluminum chloride solution is completed, the pH setting value is maintained by adjusting the amount of the sodium hydroxide solution added for aging, that is, stirring at a speed of 200 rpm for 60 minutes.

[0081] After the reaction was completed, 1.48 mol of sodium hydroxide solution and 0.51 mol of aluminum chloride solution were consumed, and the molar ratio of sodium hydroxide and aluminum chloride consumed in the mixed reaction was 2.9.

[0082] 4. Prepare the mixed solution: While stirring, cool, discharge, and centrifuge at 7000 rpm for 10 min to obtain a precipitate, which was dissolved in 20°C water to a 3 mg / ml suspension.

[0083] The suspension was divided into two parts and weighed. In the first part (1), the mass of aluminum hydroxide was 1.5 kg, and 37 g of 0.05% urea solution was added, wherein the amount of urea solution added was 1 / 40 of the mass of the aluminum hydroxide. In the second part (2), the mass of aluminum hydroxide was 1.5 kg, and 50 g of 0.05% urea solution was added, wherein the amount of urea solution added was 1 / 30 of the mass of the aluminum hydroxide.

[0084] 5. Steps for preparing adjuvant stock solution: sterilize the mixed solution at 121°C for 30 minutes, dialyze through a dialysis bag to remove ammonia until no residual ions are left, and then divide the solution into portions and sterilize again at 121°C for 30 minutes to obtain aluminum hydroxide stock solution.

[0085] Example 3:

[0086] 1. Experimental preparation steps:

[0087] 160 L of water was pre-placed in a reaction tank. 12 L of 0.6 mol / L sodium hydroxide solution was prepared in a sodium hydroxide preparation tank, and 6 L of 0.41 mol / L aluminum chloride solution was prepared in an aluminum chloride preparation tank. Both the sodium hydroxide and aluminum chloride preparation tanks were filtered using 0.45 μm filter cartridges. The sodium hydroxide solution was added through a first spray nozzle located at the center of the top of the reaction tank. The aluminum chloride solution was added through second and third spray nozzles symmetrically located at the center of the top of the reaction tank, located 2 / 3 of the radius from the first spray nozzle. The reaction tank was stirred at 180 rpm, the jacket water circulation system was turned on, and the temperature was raised to 70°C and maintained.

[0088] 2. Steps for adjusting the pH of the reaction system:

[0089] Aluminum chloride solution was added to the reaction tank to adjust the pH of the water system to 5.0 at a flow rate of 1 ml / min.

[0090] 3. Mixing reaction steps:

[0091] Stage 1: Sodium hydroxide solution and aluminum chloride solution were added simultaneously, and the amount of sodium hydroxide solution added was adjusted in real time until the water system reached a set pH value of 7.0. The flow rate of the sodium hydroxide solution was 45 ml / min, and the flow rate of the aluminum chloride solution was 23 ml / min. The pH setting time was 2 minutes.

[0092] The second stage: then the pH setting value is maintained by adjusting the amount of sodium hydroxide solution added in real time, and aluminum chloride solution is added to react.

[0093] The third stage: after the addition of the aluminum chloride solution is completed, the pH setting value is maintained by adjusting the amount of the sodium hydroxide solution added, that is, stirring at a speed of 200 rpm for 90 minutes.

[0094] After the reaction was completed, 6.9 mol of sodium hydroxide solution and 2.46 mol of aluminum chloride solution were consumed, and the molar ratio of sodium hydroxide and aluminum chloride consumed in the mixed reaction was 2.8.

[0095] 4. Prepare the mixed solution: While stirring, cool, discharge, and centrifuge at 7000 rpm for 10 min to obtain a precipitate, which was dissolved in 60°C water to form a 10 mg / ml suspension.

[0096] The suspension was divided into two parts and weighed. In the first part (1), the mass of aluminum hydroxide was 3.0 kg, and 75 g of 0.05% urea solution was added, wherein the amount of urea solution added was 1 / 40 of the mass of the aluminum hydroxide. In the second part (2), the mass of aluminum hydroxide was 3.0 kg, and 85 g of 0.05% urea solution was added, wherein the amount of urea solution added was 1 / 35 of the mass of the aluminum hydroxide.

[0097] 5. Steps for preparing adjuvant stock solution: sterilize the mixed solution at 121°C for 30 minutes, dialyze through a dialysis bag to remove ammonia until no residual ions are left, and then divide the solution into portions and sterilize again at 121°C for 30 minutes to obtain aluminum hydroxide stock solution.

[0098] Example 4:

[0099] 1. Experimental preparation steps:

[0100] 160 L of water was pre-placed in a reaction tank. 8 L of 0.25 mol / L sodium hydroxide solution was prepared in a sodium hydroxide preparation tank, and 4 L of 0.17 mol / L aluminum chloride solution was prepared in an aluminum chloride preparation tank. Both the sodium hydroxide and aluminum chloride preparation tanks were filtered using 0.45 μm filter cartridges. The sodium hydroxide solution was added through a first spray nozzle located at the center of the top of the reaction tank. The aluminum chloride solution was added through second and third spray nozzles symmetrically located at the center of the top of the reaction tank, located 2 / 3 of the radius from the first spray nozzle. The reaction tank was stirred at 160 rpm, the jacket water circulation system was turned on, and the temperature was raised to 70°C and maintained.

[0101] 2. Steps for adjusting the pH of the reaction system:

[0102] Aluminum chloride solution was added to the reaction tank to adjust the pH of the water system to 5.0 at a flow rate of 1 ml / min.

[0103] 3. Mixing reaction steps:

[0104] Stage 1: Sodium hydroxide solution and aluminum chloride solution were added simultaneously, and the amount of sodium hydroxide solution added was adjusted in real time until the water system reached a set pH value of 6.0. The flow rate of the sodium hydroxide solution was 36 ml / min, and the flow rate of the aluminum chloride solution was 18 ml / min. The pH setting time was 3 minutes.

[0105] The second stage: then the pH setting value is maintained by adjusting the amount of sodium hydroxide solution added in real time, and aluminum chloride solution is added to react.

[0106] The third stage: after the addition of the aluminum chloride solution is completed, the pH setting value is maintained by adjusting the amount of the sodium hydroxide solution added, that is, stirring at a speed of 200 rpm for 90 minutes.

[0107] After the reaction was completed, 1.95 mol of sodium hydroxide solution and 0.68 mol of aluminum chloride solution were consumed, and the molar ratio of sodium hydroxide and aluminum chloride consumed in the mixed reaction was 2.87.

[0108] 4. Prepare the mixed solution: While stirring, cool, discharge, and centrifuge at 7000 rpm for 10 min to obtain a precipitate, which was dissolved in 50°C water to a 5 mg / ml suspension.

[0109] The suspension was divided into two parts and weighed. In the first part (1), the mass of aluminum hydroxide was 1.7 kg, and 42 g of 0.05% urea solution was added, wherein the amount of urea solution added was 1 / 40 of the mass of the aluminum hydroxide. In the second part (2), the mass of aluminum hydroxide was 1.7 kg, and 56 g of 0.05% urea solution was added, wherein the amount of urea solution added was 1 / 30 of the mass of the aluminum hydroxide.

[0110] 5. Steps for preparing adjuvant stock solution: sterilize the mixed solution at 121°C for 30 minutes, dialyze through a dialysis bag to remove ammonia until no residual ions are left, and then divide the solution into portions and sterilize again at 121°C for 30 minutes to obtain aluminum hydroxide stock solution.

[0111] From the treatment results of the first sample of Examples 1-4 under different conditions, and the treatment results of the second sample, it can be seen that Figure 1-24 The results of the comparison show that:

[0112] 1) Sterilization after adding urea does not cause coagulation of the adjuvant and the formation of large particles, and can stabilize the particle size of the adjuvant within a certain range, i.e., 500nm-5μm.

[0113] 2) After adding urea and undergoing repeated sterilization, the adjuvant's structure, pH and other physical and chemical properties tend to be stable.

[0114] 3) Properly increasing the amount of urea to increase the pH to a certain extent did not cause an irreversible sudden change in the particle size of the adjuvant with the increase of urea decomposition products, nor did it cause agglomeration or change in the crystal properties. It can be used for the preparation of different antigens, which also shows that at 121°C, the urea hydrolysis products can react with the aluminum hydroxide adjuvant.

[0115] In summary, the adjuvant suspension prepared in this application can be treated with urea solution to further stabilize its physical and chemical properties. It can be used as an adjuvant to adsorb different types of antigens within a wide pH range (5.5-7.5) and a wide particle size range (500nm-5μm), adapting to the diversity of antigens.

[0116] Taking the pertussis potency experiment as an example, the first sample ① was prepared according to Examples 1-4 of this application, and the second sample ② was used to prepare the adjuvant. At the same time, the French InvivoGen brand aluminum adjuvant (commercial adjuvant) and the adjuvant prepared by the patent authorization announcement number CN104367998 process (patented adjuvant) were selected. The three adjuvants were adsorbed on the same pertussis toxin to prepare a stock solution, and then mice were immunized for comparative experiments.

[0117] The specific steps are as follows: After the three adjuvants are respectively adsorbed on the same pertussis toxin to prepare a stock solution, three dilutions of pertussis potency standard and test sample (8x, 40x, 200x) are immunized. NIH mice weighing 10-12g are selected, half male and half female, and 20 mice are immunized for each dilution, and 0.5ml is injected intraperitoneally. 21 days after the challenge mice are immunized, each mouse is challenged with 0.03ml of bacterial solution (containing 80,000 bacteria) in the brain cavity. After the challenge, the animals are observed for 14 days, and the number of deaths is recorded daily. Animals with paralysis, head swelling, hunched back and obvious ruffling of hair on the 14th day are also counted as dead. The test sample titer is calculated by the mass reaction parallel line method. The immune titer of each human dose should not be less than 4.0IU. The mouse titer results are shown in Table 1.

[0118] As can be seen from Table 1, the titer of the antigen prepared by this patent is significantly higher than that of the antigens prepared by the other two adjuvants.

[0119] Table 1 The titer of the prepared antigens of each adjuvant

[0120] Samples ① and ② of each embodiment Antigen 1 titer (IU / ml) Antigen 2 titer (IU / ml) Antigen 3 titer (IU / ml) Example 1① (pH 6.52) 12.025 16.611 14.534 Example 1② (pH 7.0) 11.733 12.015 14.636 Example 2① (pH 5.5) 10.043 9.644 11.356 Example 2② (pH 6.05) 10.735 8.014 13.255 Example 3① (pH 7.02) 17.823 11.905 9.184 Example 3② (pH 7.50) 8.685 8.116 7.891 Example 4① (pH 6.05) 9.732 8.116 9.700 Example 4② (pH 6.44) 13.688 16.55 12.325 Patented adjuvant 4.352 8.133 7.098 Commercial adjuvants 8.368 11.885 9.048

[0121] Specific toxicity testing was performed on the antigens prepared in Table 1. The specific steps were as follows: A histamine sensitization test was performed on mice in accordance with pharmacopoeia requirements. Four days after immunization, each mouse was intraperitoneally injected with 0.5 ml of histamine diphosphate (4 mg / ml). Rectal temperature was measured 30 minutes later. Deaths in mice indicated high toxicity in the immunization solution. These deaths were recorded for comparison with histamine sensitization toxicity in mice. The specific toxicity test results are compared in Table 2 below.

[0122] As can be seen from Table 2, the adjuvant prepared by this patent not only enhances the titer of the antigen but also weakens the toxicity of the antigen, and has the advantages of strong adsorption to the antigen and slow antigen release.

[0123] Table 2 Toxicity test results of the prepared antigens of each adjuvant

[0124]

[0125] The antigen stock solution prepared in Table 1 was tested for physical and chemical properties according to the requirements of the European Pharmacopoeia. The results are shown in Table 3.

[0126] Table 3 Results of physical and chemical property tests on the antigen stock solution prepared in Table 1

[0127]

[0128]

[0129] As can be seen from Table 3, the main physical and chemical properties of the adjuvant prepared in this application are close to those of the European commercial adjuvant.

Claims

1. A method for preparing an aluminum hydroxide adjuvant, characterized in that: The steps include: Mixing reaction step: adding aluminum chloride solution and sodium hydroxide solution into a water system to react to obtain aluminum hydroxide colloidal suspension; preparing a mixed solution: centrifuging an aluminum hydroxide colloidal suspension to obtain a precipitate, adding water to the precipitate to prepare a suspension, and adding a urea solution to obtain a mixed solution; The mixing reaction step comprises: a first stage: adjusting the amount of the sodium hydroxide solution added until the water system reaches a set pH value; The second stage: then the pH setting value is maintained by adjusting the amount of the sodium hydroxide solution added, and the aluminum chloride solution is added to react; The third stage: aging by adjusting the amount of the sodium hydroxide solution added to maintain the pH set value; The pH setting value is 5.5-7.

0.

2. the preparation method of aluminum hydroxide adjuvant as claimed in claim 1, is characterized in that, The duration of the first stage is no more than 5 minutes; The aging includes stirring at a speed of 200 rpm for 60 min to 90 min.

3. The preparation method of aluminum hydroxide adjuvant as claimed in claim 1, wherein In the mixing reaction step: The volume ratio of the aqueous system and the volume of the sodium hydroxide solution consumed in the reaction is 160: 6-12; The ratio of the volume of the water system to the volume of the aluminum chloride solution consumed in the reaction is 160:3-6; The molar ratio of sodium hydroxide and aluminum chloride consumed in the mixed reaction is 2.8-3.0; The mixing reaction was carried out at 70° C. with stirring.

4. the preparation method of aluminum hydroxide adjuvant as claimed in claim 1, is characterized in that, In the mixing reaction step: The molar concentration of the sodium hydroxide solution is 0.25-0.6 mol / L, and the molar concentration of the aluminum chloride solution is 0.17-0.41 mol / L; The sodium hydroxide solution is added through a first spray head located at the center of the top of the reaction tank, and the aluminum chloride solution is added through a second spray head and a third spray head symmetrically arranged at the center of the top of the reaction tank.

5. The preparation method of aluminum hydroxide adjuvant as claimed in claim 1, wherein In the step of preparing the mixed solution: adding 20-60°C water to the precipitate to prepare a suspension with a mass concentration of 3-10 mg / ml; The mass concentration of the urea solution is 0.05%; the amount of the urea solution added is 1 / 40-1 / 30 of the mass of the aluminum hydroxide.

6. The preparation method of aluminum hydroxide adjuvant as claimed in claim 1, wherein The mixing reaction step also includes: Adjusting the pH of the reaction system: adding aluminum chloride solution to the water system until the pH reaches 5.

0.

7. The preparation method of aluminum hydroxide adjuvant as claimed in claim 1, wherein After the step of preparing the mixed solution, the following steps are further included: Steps for preparing the adjuvant stock solution: sterilize the mixed solution, dialyze to remove ammonia, and then aliquot and sterilize again to obtain the aluminum hydroxide stock solution.

8. Use of an adjuvant prepared by the method for preparing an aluminum hydroxide adjuvant according to any one of claims 1 to 7 in preparing a vaccine.

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