Preparation method of low-resistance aluminum phosphate nano adjuvant

Abstract

The invention discloses a preparation method and application of a low-resistance nano aluminum phosphate adjuvant, and relates to the technical field of vaccine adjuvants. According to the method, a nano aluminum phosphate core is prepared through cooperation of a low-temperature environment and a crystal form directing agent, then surface modification is carried out through chitosan, hyaluronic acid or a polyethylene glycol derivative under mild conditions, the dosage of the crystal form directing agent is 0.5%-2% of the mass of aluminum salt, and the dosage of a polymer is 5%-20% of the mass of the nano core. The adjuvant can be directly used for preparing vaccines such as HPV (human papilloma virus) and the like, meets the industrialization requirements of efficient antigen loading, low safety risk and strong immune response, and has remarkable creativity and practicability.

Description

Technical Field

[0001] This invention relates to the field of vaccine adjuvant technology, and specifically proposes a method for preparing low-resistance aluminum phosphate nanoadjuvants. Background Technology

[0002] Aluminum phosphate adjuvant is one of the most widely used legally mandated vaccine adjuvants globally due to its ability to efficiently elicit humoral immune responses. However, its traditional preparation process and product performance suffer from three major defects, and existing improvement technologies have failed to effectively overcome these limitations, severely restricting its application in high-safety vaccines: Traditional aluminum phosphate adjuvants are mostly prepared using room temperature co-precipitation, relying on "random grain growth" to form amorphous gel particles. This results in two main problems: firstly, a wide particle size distribution, with a polydispersity index (PDI) often >0.4, and antigen adsorption efficiency fluctuating by more than 30%, making it difficult to meet the requirements of standardized vaccine production; secondly, the amorphous structure is prone to crystal transformation, with particle hard agglomeration exceeding 20% ​​during storage, directly leading to adjuvant activity attenuation. Although existing technologies mention the preparation of nano-aluminum phosphate, such as patent CN109432418A, the precise use of crystal-directing agents is not clearly defined, and the dual problems of particle size uniformity and structural stability remain unresolved.

[0003] To reduce the risk of adjuvant antibodies, some studies have attempted to modify the surface of aluminum phosphate particles, but there are two major limitations: First, choosing non-biocompatible polymers such as polyvinylpyrrolidone (PVP) can improve stability, but may introduce new immunogenic impurities; second, uncontrolled modification conditions can lead to weak binding between the polymer and the particles, resulting in a high rate of delamination of the modified layer after 3 months of storage, which prevents it from continuously exerting its immune camouflage effect.

[0004] After repeated vaccinations with traditional aluminum adjuvants, the AAA (antigen-associated antigen) production rate exceeds 60%, with titers reaching over 1:1000. This not only competitively binds to adjuvant surface sites, reducing antigen adsorption, but may also trigger local inflammatory responses. Current technological improvements focus on "enhancing immunogenicity" or "optimizing particle size," failing to prioritize "reducing AAA" as a core objective, and lacking a comprehensive technical system linking "crystal form regulation - polymer modification - AAA inhibition." Consequently, these advancements cannot meet the safety requirements of vaccines requiring multiple booster immunizations.

[0005] In summary, existing technologies lack an integrated solution for precisely controlling the nanostructure, targeting and selecting biocompatible polymers, and addressing AAA risks. Developing a low-resistance aluminum phosphate nanoadjuvant preparation method that meets these requirements is of urgent practical significance for promoting vaccine technology upgrades. Summary of the Invention

[0006] In view of this, the present invention proposes a method for preparing low-resistance aluminum phosphate nano-adjuvants with controllable process and high safety. By precisely controlling the nano-core structure through low temperature environment and specific crystal form directing agent, and then modifying the surface with polysaccharides such as chitosan or hyaluronic acid under mild conditions, the triple technical goals of uniform particle size, stable structure and low AAA level are achieved, providing a safe and efficient adjuvant solution for multiple vaccinations.

[0007] The technical solution of this invention is achieved as follows: This invention provides a method for preparing low-resistance aluminum phosphate nanoadjuvants, comprising the following steps: 1. Preparation of nanoscale aluminum phosphate cores Raw material preparation: Prepare an aluminum salt solution with a concentration of 0.2-0.8 mol / L, preferably aluminum chloride or aluminum nitrate solution, which has high solubility and few impurities; and a phosphate solution with a concentration of 0.1-0.4 mol / L, preferably disodium hydrogen phosphate or sodium dihydrogen phosphate solution, which has strong pH buffering capacity and can avoid sudden changes in the pH of the reaction system. Crystal form regulation reaction: Place the aluminum salt solution in a reaction vessel and maintain the system temperature at 0-10℃ using an ice bath or low-temperature circulation device. Low temperature can inhibit excessively rapid crystal growth and avoid the formation of micron-sized particles. Turn on the stirrer at 600-1000 rpm to ensure uniform mixing of the raw materials. Add citrate or tartrate as crystal form guiding agents to the system. Sodium citrate and potassium sodium tartrate are preferred because they have a weak coordination effect with aluminum ions and can induce the formation of regular spherical crystal nuclei. The amount added is 0.5%-2% of the mass of aluminum salt. This ratio can balance the crystal nucleus induction efficiency and the difficulty of subsequent purification. Excessive amount can easily lead to impurity residue. Then, add phosphate solution dropwise at a rate of 3-8 mL / min. After the addition is complete, continue the reaction for 30-90 minutes to ensure sufficient crystal nucleus growth. Purification: The precipitate was collected by centrifugation at 8000-12000 rpm for 10-20 minutes. High speed can efficiently separate nanoparticles and avoid loss. The supernatant was repeatedly washed with ultrapure water until the conductivity was <50μS / cm to remove unreacted salt ions and residual directing agents to avoid affecting subsequent modification reactions, thus obtaining pure nano-sized aluminum phosphate cores. The core gel was freeze-dried at -50℃ and 0.1Pa for 4 hours and then weighed to ensure accurate polymer dosage calculation. The average particle size was 20-100nm and the PDI was <0.2.

[0008] 2. Biocompatible surface modification Dispersion pretreatment: The nano-sized aluminum phosphate core is redispersed in ultrapure water to make the solid content 1%-3%, preferably 2%. This range can balance the dispersion effect and subsequent modification efficiency. Ultrasonic treatment at 300-500W for 5-15 minutes can break up slight agglomeration and ensure the monodispersity of particles, laying the foundation for the uniformity of modification. Targeted modification reaction: Adjust the pH of the dispersion to 4.0-6.5 using dilute acid (e.g., 0.1M acetic acid) or dilute alkali (e.g., 0.1M sodium hydroxide). (This pH range is the optimal solubility and adsorption range for chitosan / hyaluronic acid: chitosan is cationic at pH < 6.5 and can bind to negatively charged aluminum phosphate particles via electrostatic interactions; hyaluronic acid is structurally stable at pH 4.0-6.5 and is not easily degraded.) Add a biocompatible polymer, preferably chitosan or hyaluronic acid. Natural polysaccharides are non-immunogenic and can... To form a dense "immune camouflage layer"; or polyethylene glycol and its derivatives, which are highly hydrophilic and can reduce the probability of particles being recognized by the immune system), the amount added should be 5%-20% of the mass of the nanocore. If it is less than 5%, the modification layer will be incomplete and unable to play an anti-AAA role; if it is more than 20%, it will easily lead to particle agglomeration. The system should be heated to 40-60℃. The mild temperature can promote the bonding reaction between the polymer and the particles and avoid polymer denaturation caused by high temperature. Stir the reaction at 150-250 rpm for 3-5 hours to ensure uniform coating of the modification layer. Final product purification: Centrifuge again at 8000-12000 rpm for 10-20 minutes to collect the product, wash with ultrapure water 2-4 times to remove unbound free polymers, and obtain low-resistance aluminum phosphate nanoadjuvant.

[0009] The present invention has the following advantages over the prior art: This invention utilizes low-temperature inhibition of crystal growth (0-10℃) and the induction of regular crystal nuclei by specific crystal-directing agents to prepare nanocores with an average particle size strictly controlled at 20-100nm and a PDI < 0.2. The particle size uniformity is improved by more than 50% compared with traditional adjuvants. Furthermore, the spherical crystal structure is stable, and the aggregation rate after 6 months of storage at 4℃ is much lower than that of traditional adjuvants. This invention completely solves the industry pain point of "uneven particle size and easy aggregation" and provides a structural basis for the stability of antigen adsorption efficiency.

[0010] Polysaccharide polymers such as chitosan or hyaluronic acid can form a dense and stable surface modification layer under conditions of pH 4.0-6.5 and 40-60℃. On the one hand, they can shield the immunogenic epitopes of aluminum phosphate particles through "immune camouflage", reducing the AAA production rate from 60% of traditional adjuvants to below 10% and the titer from above 1:1000 to below 1:200. On the other hand, the modification layer has no additional immunogenicity, and the incidence of redness and swelling at the injection site is significantly reduced compared with traditional adjuvants, making it particularly suitable for vaccines that require 3 or more doses.

[0011] All raw materials are pharmaceutical-grade conventional excipients, which are low in cost and easy to obtain; and each step has clear quality control standards, resulting in a significantly improved batch-to-batch product qualification rate compared to traditional processes, which can be directly adapted to the large-scale production needs of small and medium-sized biopharmaceutical companies.

[0012] The high specific surface area of ​​the nanocore enables an antigen adsorption rate of over 90%, and the adsorbed antigen can be slowly released in vivo. Meanwhile, the surface modification layer does not affect the binding site between the antigen and the adjuvant. Animal experiments show that the specific antibody titer induced by the adjuvant of this invention is higher than that of traditional adjuvants, achieving the dual goals of "improved safety" and "enhanced immune efficacy", breaking through the technical bottleneck of "difficulty in balancing safety and efficacy". Detailed Implementation

[0013] The technical solutions of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0014] Example 1 Preparation of nanoscale aluminum phosphate core (1) Preparation of raw material solutions: Prepare 0.5 mol / L aluminum nitrate aqueous solution and 0.25 mol / L disodium hydrogen phosphate aqueous solution respectively, and store at 4℃ for later use; (2) Crystal form regulation reaction: Take 500 mL of aluminum nitrate solution and place it in a 1000 mL three-necked flask. Cool it to 5 °C in an ice bath and turn on the stirrer (800 rpm). Add sodium citrate (calculated as 1% of aluminum nitrate mass, i.e. 0.5 mol / L × 0.5 L × 213 g / mol × 1% ≈ 0.53 g) to the flask and stir to dissolve. Then add 500 mL of disodium hydrogen phosphate solution dropwise at a rate of 5 mL / min. After the addition is complete, maintain the reaction at 5 °C and 800 rpm for 60 min. (3) Purification: Transfer the reaction solution to a centrifuge tube, centrifuge at 10,000 rpm for 15 min, discard the supernatant; add ultrapure water to the precipitate, vortex disperse and centrifuge again, repeat washing 4 times until the conductivity of the supernatant drops to 45 μS / cm, to obtain nano-sized aluminum phosphate core gel.

[0015] Surface finishing (1) Dispersion pretreatment: The nano-core gel was redispersed in 500 mL of ultrapure water to make the solid content 2%, and ultrasonically dispersed for 10 min using a 300 W ultrasonic instrument (ultrasonic interval: 3 s working, 2 s pause). (2) Modification reaction: Adjust the pH of the dispersion to 5.5 with 0.1 mol / L acetic acid, add chitosan (the core gel weighs 8 g after being freeze-dried at -50℃ and 0.1 Pa for 4 h, and the corresponding amount added is about 0.8 g based on the mass of the nano core of 10%), heat to 50℃, and stir at 200 rpm for 4 h. (3) Purification of the final product: Centrifuge the reaction solution at 10,000 rpm for 15 min and discard the supernatant; wash the precipitate three times with ultrapure water, and centrifuge under the same conditions each time; disperse the final precipitate in pH 6.8 PBS, adjust the aluminum concentration to 2 mg / mL, and filter through a 0.22 μm filter membrane to obtain the final product.

[0016] Example 2 Preparation of nanoscale aluminum phosphate core Steps (1) and (3) are the same as in Example 1; in step (2), sodium citrate is replaced with sodium potassium tartrate (calculated based on 1.5% of aluminum nitrate mass, the amount added is 0.8g), and the other reaction conditions (5℃, 800rpm, reaction for 60min) remain unchanged.

[0017] Surface finishing Completely the same as Example 1 (chitosan dosage 10%, pH 5.5, reaction at 50℃ for 4 hours).

[0018] Example 3-1 Preparation of nanoscale aluminum phosphate core: exactly the same as in Example 1 (1% sodium citrate, reaction at 5°C).

[0019] Surface finishing (1) The dispersion pretreatment is the same as in Example 1; (2) Adjust the pH to 5.0 with 0.1 mol / L acetic acid, add hyaluronic acid (calculated as 15% of the nano core mass, the amount added is 1.2 g), heat to 45℃, and stir at 200 rpm for 4 h; (3) The final product purification is the same as in Example 1.

[0020] Example 3-2 Preparation of nanoscale aluminum phosphate core: exactly the same as in Example 1.

[0021] Surface finishing (1) The dispersion pretreatment is the same as in Example 1; (2) Adjust the pH to 6.0 with 0.1 mol / L sodium hydroxide, add mPEG-NH2 (molecular weight 5000, calculated based on 8% of the nano core mass, the amount added is 0.64 g), heat to 55℃, and stir at 200 rpm for 4 h; (3) The final product purification is the same as in Example 1.

[0022] Example 4-1 Preparation of nanoscale aluminum phosphate core Steps (1) and (3) are the same as in Example 1; in step (2), the amount of sodium citrate is 0.5% of the mass of aluminum nitrate (0.27g added), and the other reaction conditions remain unchanged.

[0023] Surface finishing The amount of chitosan used was 5% of the mass of the nanocore (0.4g), and the other conditions were the same as in Example 1.

[0024] Example 4-2 Preparation of nanoscale aluminum phosphate core In step (2), the amount of sodium citrate used is 2% of the mass of aluminum nitrate (1.06g added), and the rest is the same as in Example 1.

[0025] Surface finishing The amount of chitosan used was 20% of the mass of the nanocore (1.6g), and the other conditions were the same as in Example 1.

[0026] Example 5 Preparation of low-resistance aluminum phosphate nanoadjuvant: exactly the same as in Example 1 (chitosan modification + sodium citrate directing).

[0027] Vaccine composition preparation Take 10 mL of the above adjuvant (20 mg of aluminum) and add 1.33 mg of recombinant HPV16L1 protein (aluminum:antigen = 15:1). Incubate at room temperature for 1 hour to obtain the HPV vaccine composition.

[0028] Comparative Example 1 Preparation of nanoscale aluminum phosphate core Steps (1) and (3) are the same as in Example 1; in step (2), sodium citrate is not added, but stirring is maintained at 5°C and 800 rpm, and the reaction is carried out for 60 min after adding disodium hydrogen phosphate solution.

[0029] Surface modification: exactly the same as in Example 1 (chitosan modification).

[0030] Comparative Example 2 Preparation of nanoscale aluminum phosphate core: exactly the same as in Example 1 (sodium citrate directed).

[0031] Surface finishing The dispersion pretreatment was the same as in Example 1; the pH was adjusted to 6.5 with 0.1 mol / L sodium hydroxide, and PVPK30 (calculated as 10% of the nanocore mass, the amount added is approximately 0.8 g) was added. The mixture was stirred at 50°C and 200 rpm for 4 h; the final product purification was the same as in Example 1.

[0032] Comparative Example 3 Preparation of nanoscale aluminum phosphate core: exactly the same as in Example 1 (sodium citrate directed).

[0033] Surface finishing The dispersion pretreatment was the same as in Example 1; the pH was adjusted to 7.5 with 0.1 mol / L sodium hydroxide, chitosan (the amount was the same as in Example 1) was added, the temperature was raised to 70°C, and the mixture was stirred at 200 rpm for 4 hours; the final product purification was the same as in Example 1.

[0034] Comparative Example 4 Preparation of nanoscale aluminum phosphate core: exactly the same as in Example 1 (sodium citrate directed).

[0035] Final product processing: The purified nanocores were directly dispersed in pH 6.8 PBS, the aluminum concentration was adjusted to 2 mg / mL, and filtered through a 0.22 μm filter membrane without surface modification.

[0036] Comparative Example 5 Traditional aluminum phosphate preparation (1) Prepare an aqueous solution of 0.5 mol / L aluminum nitrate and 0.25 mol / L disodium hydrogen phosphate (room temperature 25℃); (2) Take 500 mL of aluminum nitrate solution and place it in a three-necked flask. Stir at room temperature (25℃) and 500 rpm. Add 500 mL of disodium hydrogen phosphate solution directly. After the addition is complete, react at room temperature for 60 min. (3) Centrifuge at 10,000 rpm for 15 min, wash with ultrapure water 3 times to obtain traditional aluminum phosphate gel.

[0037] PVP Modification Traditional aluminum phosphate gel was dispersed in ultrapure water, PVPK30 (calculated as 10% of gel mass) was added, and the mixture was stirred at pH 7.0 and 60℃ for 4 hours. After purification, it was dispersed in PBS (aluminum concentration 2 mg / mL).

[0038] Performance verification methods 1. Physicochemical property determination (particle size, PDI, agglomeration rate) Instrument: Dynamic light scattering instrument (DLS, model Zetasizer Nano ZS90); Particle size and PDI determination: Take the final product adjuvant (concentration 0.1 mg / mL, dispersed in pH 6.8 PBS), equilibrate at 25℃ for 10 min, scattering angle 90°, and measure each sample in parallel 3 times, and take the average value; Agglomeration rate determination after 6 months: The adjuvant was sealed and stored in a refrigerator at 4°C for 6 months. The particle size after storage was determined according to the above method. The agglomeration rate was calculated as: (particle size after storage - initial particle size) / initial particle size × 100%.

[0039] 2. Antigen adsorption rate determination (HBsAg / HPV16L1 protein) Antigens: Recombinant HBsAg (purity ≥95%), recombinant HPV16L1 protein (purity ≥95%); Adsorption method: The adjuvant and antigen were mixed at a ratio of aluminum element mass to antigen mass = 10:1 (15:1 in Example 5), and the mixture was subjected to end-over-end rotary adsorption at room temperature for 1 hour. Measurement procedure: Centrifuge at 12000 rpm for 20 min, collect the supernatant, and use the double antibody sandwich ELISA method to detect the free antigen content in the supernatant; Adsorption rate calculation: (Total initial antigen - Amount of free antigen in supernatant) / Total initial antigen × 100%.

[0040] 3. Immunological and safety verification (animal experiments) Laboratory animals: SPF-grade Balb / c mice (female, 6-8 weeks old, 5 mice per group); Immunization schedule: Subcutaneous primary immunization on day 0 (dose per mouse: 50 μg antigen + 200 μg Al, supplemented with PBS to 200 μL), booster immunization with the same dose on day 14; Sample collection: Blood was collected from the orbital cavity on day 28, incubated at 37°C for 30 min, and then centrifuged at 3000 rpm for 15 min to separate the serum; Antibody titer determination: The titers of anti-adjuvant antibody (AAA) (coating antigen is traditional aluminum phosphate adjuvant), anti-HBsAg IgG (coating antigen is HBsAg), and anti-HPV16L1 IgG (coating antigen is HPV16L1 protein) in serum were detected by indirect ELISA. The highest serum dilution with an OD450nm value ≥0.2 was taken as the antibody titer. Safety observation: Within 72 hours after immunization, the redness and swelling at the injection site of mice were observed daily (redness and swelling diameter ≥2mm was considered positive), and the incidence of redness and swelling was calculated as (number of positive mice / total number of mice in each group × 100%).

[0041] 4. Stability determination of the modified layer (Comparative Example 2 only) Methods: The modified adjuvant was stored at 4℃ for 3 months, centrifuged at 12000 rpm for 20 min, and the supernatant was collected. The content of free polymer (PVP) in the supernatant was determined by Coomassie Brilliant Blue method. Calculation of shedding rate: Amount of free polymer in supernatant / Total amount of initially added polymer × 100%.

[0042] Performance verification results: Physicochemical properties and antigen adsorption rate of each sample

[0043] Immunological and safety results of each sample

[0044] The average particle size of Example 1 was 72 nm (PDI 0.16), and the aggregation rate was 3.2% after 6 months of storage at 4°C, with an antigen adsorption rate of 92%. In Comparative Example 1, the particle size increased to 180 nm (PDI 0.38), the aggregation rate increased to 18%, and the antigen adsorption rate decreased to 72%. This demonstrates that low temperature alone cannot achieve the uniformity and stability of nanonuclei. The synergistic combination of low temperature environment and crystal form guiding agent is a necessary technical means to prepare nanonuclei that meet the requirements. The structural regulation effect produced by this combination cannot be achieved through a single technical feature.

[0045] In Example 1, the anti-adjuvant antibody (AAA) titer was 180, with a 3.2% aggregation rate after 6 months; in Comparative Example 4, the AAA titer surged to 2850, with an aggregation rate of 15%. This indicates that the nanonucleus itself does not possess the function of "resisting the production of anti-adjuvant antibodies." Only through surface modification with "chitosan / hyaluronic acid / polyethylene glycol derivatives" can both "immune camouflage (reducing AAA)" and "steric hindrance (inhibiting aggregation)" be simultaneously achieved. The modification step is crucial for solving the core technical problem of this invention and cannot be omitted.

[0046] Comparative Example 2 showed an AAA titer of 980, an incidence of redness and swelling of 15%, and a 45% desquamation rate of the modified layer after 3 months. This indicates that commonly used non-specific polymers such as PVP in the prior art cannot achieve the "low immunogenicity + high binding stability" effect of the "polysaccharide / polyethylene glycol derivative" as defined in the claims. The choice of polymer type in this invention is non-obvious.

[0047] Comparative Example 5 had a particle size of 1600 nm, an AAA titer of 1320, and a specific antibody titer of 10 × 10⁻⁶. 4 This demonstrates that even with surface modification, traditional adjuvants are still inferior to this invention in terms of particle size, stability, safety, and immunogenicity. This proves that the technical approach of "precise nanonuclei and specific modifications" in this invention is fundamentally different from existing traditional adjuvant technologies and is not a conventional improvement on traditional technologies.

[0048] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for preparing a low-resistance aluminum phosphate nanoadjuvant, characterized in that... This includes the following steps: S1. Preparation of nano-sized aluminum phosphate core: Under stirring conditions at 0-10℃, phosphate solution is added dropwise to aluminum salt solution to carry out the reaction. Citrate or tartrate is added as a crystal directing agent during the reaction. After the reaction is completed, the nano-sized aluminum phosphate core is obtained by purification. S2. Surface modification: The nano-sized aluminum phosphate core is dispersed in an aqueous phase, and a biocompatible polymer is added. The mixture is stirred and reacted for 3-5 hours at pH 4.0-6.5 and temperature 40-60℃. After the reaction is completed, the low-resistance aluminum phosphate nano-adjuvant is obtained by purification. The biocompatible polymer is a polysaccharide polymer or polyethylene glycol and its derivatives, wherein the polysaccharide polymer is chitosan or hyaluronic acid.

2. The method as described in claim 1, characterized in that, In S1, the phosphate solution is a disodium hydrogen phosphate solution or a sodium dihydrogen phosphate solution, and the aluminum salt solution is an aluminum chloride solution or an aluminum nitrate solution.

3. The method as described in claim 1, characterized in that, In S1, the citrate is sodium citrate, and the tartrate is potassium sodium tartrate; the amount of the crystal guiding agent added is 0.5%-2% of the mass of the aluminum salt.

4. The method as described in claim 1, characterized in that, In S1, the purification includes centrifugation and ultrapure water washing. The centrifugation speed is 8000-12000 rpm and the time is 10-20 min. The washing is continued until the conductivity of the supernatant is less than 50 μS / cm.

5. The method as described in claim 1, characterized in that, In S2, the amount of the biocompatible polymer added is 5%-20% of the mass of the nano-sized aluminum phosphate core.

6. The method as described in claim 1, characterized in that, In S2, the nano-sized aluminum phosphate core is dispersed in the aqueous phase, then subjected to ultrasonic treatment before the biocompatible polymer is added. The ultrasonic treatment power is 300-500W and the time is 5-15min.

7. The method as described in claim 1, characterized in that, In S2, the stirring speed is 150-250 rpm and the reaction time is 3-5 hours; the purification includes centrifugation and ultrapure water washing, with centrifugation speed of 8000-12000 rpm and time of 10-20 min, and washing 2-4 times.

8. A low-resistance aluminum phosphate nanoadjuvant, characterized in that, The adjuvant is prepared by the method of any one of claims 1-7, wherein the average particle size is 20-100 nm, the polydispersity index is less than 0.2, and the agglomeration rate is less than 10% after being stored at 4°C for 6 months.

9. A vaccine composition, characterized in that, It comprises the low-resistance aluminum phosphate nanoadjuvant of claim 8 and a pharmaceutically acceptable antigen.

10. The use of the low-resistance aluminum phosphate nanoadjuvant of claim 8 in the preparation of vaccines for the prevention or treatment of viral or bacterial diseases.

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