Preparation method of high-adsorbability aluminum hydroxide adjuvant for livestock

Abstract

The invention discloses a preparation method of a high-adsorbability aluminum hydroxide adjuvant for livestock, which comprises the following steps: when an aluminum chloride solution reacts with alkali liquor, generating a colloidal suspension by combining pH two-step adjustment with dispersant addition; and after instant high-speed shearing and room-temperature aging, performing ultrafiltration purification to remove salts, and finally performing moist heat sterilization to obtain the suspension morphological adjuvant. The adjuvant is high in antigen adsorption rate, low in 72-hour desorption rate, large in specific surface area, uniform in particle and suitable for vaccines of pigs, cattle and poultry, and the problems that a traditional adjuvant is low in adsorption rate and poor in stability are solved.

Description

Technical Field

[0001] The invention relates to the technical field of veterinary vaccine adjuvants, and specifically provides a method for preparing a veterinary high-adsorptive aluminum hydroxide adjuvant. Background Art

[0002] Aluminum hydroxide adjuvant is a widely used adjuvant in veterinary vaccines due to its high safety and good biocompatibility. Its mechanism of action is to form a stable complex by adsorbing antigens and slowly release antigens to enhance the body's immune response.

[0003] Traditional preparation methods mainly use sodium hydroxide method or ammonia method, which have the following defects: Low antigen adsorption rate: The traditional method uses a single pH adjustment, which leads to insufficient formation of aluminum hydroxide colloidal core and insufficient exposure of surface adsorption sites. At the same time, the filter cake formed by centrifugal filtration causes the particles to be tightly packed, the mesoporous structure is destroyed, and the specific surface area is generally less than 200m 2 / g, which cannot provide sufficient antigen binding sites. This results in the adsorption rate of traditional adjuvants to vaccine antigens usually being ≤70%, resulting in the loss of free antigens and reduced immune effects. Poor adsorption stability: Traditional processes do not control the charge balance on the colloid surface. When the pH is adjusted alone, the absolute value of the colloid Zeta potential is usually less than 20mV, resulting in insufficient repulsion between particles. Aggregation and desorption are likely to occur during storage, leading to a 72h desorption rate greater than 20%, which can easily reduce vaccine potency. Uneven particle distribution: Traditional methods rely on natural stirring and dispersion, lacking active shear control, and primary colloidal particles tend to form rigid aggregates. Furthermore, the mechanical extrusion of centrifugal filtration further exacerbates agglomeration, resulting in a wide range of particle size distribution and a high risk of local inflammation after injection. Limitations of the purification process: Centrifugal filtration will cause the aluminum hydroxide colloidal particles to form a dense filter cake due to centrifugal force, destroying the original porous structure. The filter cake needs to be vigorously stirred when resuspended, which will introduce new agglomerates. Centrifugal filtration is prone to form a filter cake, causing particle agglomeration, destroying the porous structure and further reducing the adsorption performance.

[0004] In the prior art, there is no process solution that can solve the above problems at the same time. Therefore, there is an urgent need to develop a method for preparing an aluminum hydroxide adjuvant with high adsorption, high stability and uniform particles. Summary of the Invention

[0005] In view of this, the present invention proposes a method for preparing veterinary highly adsorbable aluminum hydroxide with high antigen adsorption rate, uniform particles and high specific surface area.

[0006] The technical solution of the present invention is achieved as follows: The present invention provides a method for preparing highly adsorbable aluminum hydroxide for veterinary use, comprising the following steps: Colloid formation: Take an aluminum salt solution, slowly add an alkaline solution dropwise, first adjust the pH to 9.0-10.0, then adjust it back to 6.0-7.5, and simultaneously add a dispersant in an amount of 0.5-2wt% of the total mass of the reaction system, and stir to form a colloidal suspension; the aluminum salt solution is a soluble aluminum salt solution such as aluminum chloride or aluminum nitrate, and the alkaline solution is a sodium hydroxide solution or an ammonia solution; the dispersant is selected from one of sodium carboxymethyl cellulose, sodium citrate, polyethylene glycol, sodium alginate, sodium lauryl sulfate, and Tween 80; Particle control: The colloidal suspension is immediately subjected to high-speed shearing for 10-30 minutes at a shear rate of 800-4000 rpm, and then aged at room temperature for 2-6 hours; Purification: Ultrafiltration technology is used with a pressure of 0.2-0.6 MPa. The membrane material is an organic membrane or a ceramic membrane to remove impurities and retain aluminum hydroxide colloidal particles. Sterilization: Sterilize the purified suspension to obtain an aluminum hydroxide adjuvant suspension.

[0007] The above pH callback is achieved by adding acid solution.

[0008] Step 1: Under a strong alkaline environment, aluminum ions rapidly hydrolyze to form a large number of tiny colloidal cores. This step ensures a sufficient number of colloidal cores, laying the foundation for the subsequent porous structure.

[0009] Step 2: Neutralize excess OH with dilute hydrochloric acid or other acid - , so that the absolute value of the zeta potential of the colloid surface rises to ≥30mV. At this point, the charge distribution on the surface of the colloid particles is uniform, and the repulsive and attractive forces are balanced, avoiding agglomeration and retaining an open mesoporous structure.

[0010] The essential difference from traditional technology: Traditional single pH adjustment cannot take into account both colloidal nucleus formation and charge stabilization, while two-step adjustment realizes the step-by-step control of "nucleus formation-structure stabilization", which is the key to improving adsorption rate.

[0011] High-speed shearing: Mechanically breaking up primary aggregates before the colloidal cores form rigid aggregates, controlling the particle size to 80-120nm (D50). Precisely matching shear rate with time prevents excessive shearing that could lead to particle dissolution.

[0012] Room temperature aging: After shearing, the surface charge of the particles is redistributed, and the dispersant consolidates the dispersion effect through the steric effect, so that the Zeta potential is stabilized at ≥30mV, ensuring long-term storage without agglomeration.

[0013] Ultrafiltration purification: the key to preserving porous structure Separation Mechanism: A 50-100nm membrane intercepts colloidal particles, allowing only small salt molecules to pass through, avoiding the mechanical extrusion of centrifugal filtration. Pressure control of 0.2-0.6MPa ensures moderate water retention, while a "water washing-ultrafiltration cycle" removes residual impurities (permeate conductivity ≤ 50μS / cm).

[0014] Structural protection: During the ultrafiltration process, the colloidal particles are always in a dispersed state, while traditional centrifugal filtration will reduce the mesopore volume.

[0015] The present invention has the following beneficial effects compared to the prior art: High adsorption and stability: antigen adsorption rate ≥94%, 72h desorption rate ≤5.1%, far superior to traditional methods; Particle uniformity: colloidal particles with a D50 of 80-120 nm, avoiding inflammation at the injection site; Excellent porous structure: specific surface area ≥300m 2 / g, mesopore volume 0.296-0.409cm 3 / g, providing sufficient antigen adsorption sites; Process adaptability: The suspension form is directly adapted to the vaccine mixing process, eliminating the need for secondary dispersion and simplifying the production process. DETAILED DESCRIPTION

[0016] The following will be combined with the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the embodiments described are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0017] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which embodiments of the present invention belong. If the definitions set forth in this section are contrary to or otherwise inconsistent with definitions set forth in the patents, patent applications, published patent applications, and other publications incorporated herein by reference, the definitions listed in this section take precedence over the definitions incorporated herein by reference.

[0018] Unless otherwise specified, the methods used in the following examples are conventional methods. The materials, reagents, and instruments used are conventional materials, reagents, and instruments in the art, unless otherwise specified, and can be obtained commercially by those skilled in the art.

[0019] When an amount, concentration or other value or parameter is expressed as a range, a preferred range or a range defined by a series of upper preferred values ​​and lower preferred values, this should be understood as specifically disclosing all ranges formed by any pairing of any upper range limit or preferred value with any lower range limit or preferred value, regardless of whether the range is disclosed alone. For example, when a range "1 to 5" is disclosed, the described range should be interpreted as including the ranges "1 to 4", "1 to 3", "1 to 2", "1 to 2 and 4 to 5", "1 to 3 and 5", etc. When a numerical range is described herein, unless otherwise stated, the range is intended to include its endpoints and all integers and fractions within the range. In the present specification and claims, range definitions may be combined and / or interchanged, and if not otherwise stated, such ranges include all subranges contained therein.

[0020] Example 1 Raw materials preparation: Aluminum salt solution: 100 g 0.2 M aluminum chloride solution (AlCl3・6H2O dissolved in ultrapure water, concentration calibrated by titration); Alkaline solution: ammonia solution (analytical grade, diluted to a volume ratio of 1:3, i.e. 1 part 25% ammonia water + 3 parts ultrapure water, concentration of about 6.25%); Dispersant: sodium carboxymethyl cellulose (analytical grade); Callback reagent: 0.2 M hydrochloric acid solution (analytical grade, concentration ensured by standardization).

[0021] Colloid formation: Add 100 g of aluminum chloride solution to a 500 mL three-necked flask, start stirring (500 rpm), and insert a pH meter (accuracy ±0.01); Slowly add 1:3 ammonia solution (drop rate 2 mL / min), monitor the pH in real time, and stop adding when the pH rises to 9.5 (the total amount of ammonia added at this time is about 35 g); Immediately switch to adding 0.2M hydrochloric acid solution (drip rate 1mL / min) to adjust the pH to 6.5 (stirring throughout to avoid local over-acidification), which takes about 12 minutes; Add dispersant simultaneously: add 1.35g sodium carboxymethyl cellulose (1wt% based on the total mass of 100g aluminum chloride solution + 35g ammonia water = 135g) to the system, continue stirring and reacting for 30min to generate a light grayish white colloidal suspension.

[0022] Granularity Control: Stop stirring and transfer the colloidal suspension to a high shear homogenizer (working chamber gap 0.3 mm) and shear for 20 min (speed 2000 rpm); After shearing, transfer to a beaker and age at room temperature (25±2℃) for 4h.

[0023] Ultrafiltration purification: Using 50nm alumina ceramic membrane, ultrafiltration at 0.4MPa pressure; During this period, 4 times the volume of ultrapure water (based on the volume of the colloidal suspension) was added for cyclic washing until the conductivity of the permeate was ≤50μS / cm (measured by a conductivity meter), and the retained colloidal suspension was collected.

[0024] Sterilization: The suspension was transferred to a sterilized bottle and sterilized by moist heat at 121°C for 1 h (autoclave pressure 0.1 MPa), and then cooled to obtain the adjuvant suspension.

[0025] Example 2 Raw materials preparation: Aluminum salt solution: 100g 0.2M aluminum chloride solution; Alkali solution: 25% ammonia solution (undiluted, used directly); Dispersant: sodium citrate (analytical grade); Callback reagent: 0.2M hydrochloric acid solution.

[0026] Colloid formation: Add 100 g of aluminum chloride solution to a 500 mL three-necked flask, stir (500 rpm), and insert a pH meter; Add 1.2g of sodium citrate (dispersant, 1wt% based on 100g of aluminum chloride solution + 20g of 25% ammonia water = 120g total mass) to the aluminum chloride solution and stir to dissolve. Slowly add 25% ammonia solution (drop rate 1.5 mL / min) and stop adding when the pH reaches 9.5 (the total amount of ammonia added at this time is about 20 g); 0.2 M hydrochloric acid solution was added dropwise (drop rate 1 mL / min) to adjust the pH to 7.0, which took about 10 min. The reaction was stirred and continued for 30 min to produce a light grayish white colloidal suspension.

[0027] Particle size control: Same as Example 1 (shearing for 20 min, 2000 rpm; aging for 4 h).

[0028] Ultrafiltration purification: same as in Example 1 (50 nm ceramic membrane, 0.4 MPa).

[0029] Sterilization: Same as Example 1 (sterilization at 121°C for 1 h).

[0030] Example 3 Raw materials preparation: Aluminum salt solution: 100g 0.2M aluminum chloride solution; Alkali solution: 0.5 M sodium hydroxide solution (analytical grade, standardized concentration); Dispersant: Tween 80 (analytical grade); Callback reagent: 0.2M hydrochloric acid solution.

[0031] Colloid formation: Add 100 g of aluminum chloride solution to the flask, stir (500 rpm), and insert a pH meter; Add 0.5M sodium hydroxide solution dropwise (drop rate 1.5mL / min) until the pH rises to 9.5 (about 18g of sodium hydroxide added); Add Tween 80 (0.8 g, 0.5 wt%, based on 100 g aluminum chloride + 18 g sodium hydroxide solution = 118 g total mass) and stir to dissolve; 0.2 M hydrochloric acid was added dropwise to adjust the pH to 6.8 (taking about 8 min) and the reaction was continued for 30 min.

[0032] Particle control: shearing for 20 min (2500 rpm); aging for 4 h.

[0033] Ultrafiltration purification: 50 nm PP organic membrane, 0.3 MPa; washing until the conductivity is ≤ 50 μS / cm.

[0034] Sterilization: 121℃ for 2h.

[0035] Comparative Example 1 Colloid formation: Add 1:3 ammonia solution (concentration of about 6.25%) to 100g of 0.2M aluminum chloride solution, adjust the pH directly to 6.8 (without passing through 9.5), add 1wt% sodium carboxymethyl cellulose, and stir the reaction for 30min; Particle control: same as Example 1 (shearing + aging); Ultrafiltration and sterilization: same as Example 1.

[0036] Comparative Example 2 Colloid formation: Same as Example 1 (pH adjusted to 9.5 first and then to 6.5, and dispersant added); Particle control: Omit high-speed shearing and only age at room temperature for 4 h; Ultrafiltration and sterilization: same as Example 1.

[0037] Comparative Example 3 Colloid formation: Same as Example 1 (pH adjustment + dispersant); Particle control: same as Example 1 (shearing + aging); Purification: centrifuge at 8000 rpm for 10 min, discard the supernatant, and resuspend the precipitate in ultrapure water (concentration is the same as in Example 1); Sterilization: Same as Example 1.

[0038] The performance of the aluminum hydroxide reagents prepared in the above examples and comparative examples was tested respectively: 1. Antigen adsorption rate detection: Sample treatment: 10 mL of the adjuvant suspension was taken and centrifuged at 8000 rpm for 10 min. The precipitate was washed three times with ultrapure water and freeze-dried to obtain aluminum hydroxide solid particles. Adsorption rate: Weigh 10 mg of freeze-dried particles, add 1 mL of 1 mg / mL BSA solution (PBS pH 7.4), and incubate at 25°C with constant shaking (150 rpm) for 2 h. Centrifuge at 4°C and 12,000 rpm for 10 min, and collect the supernatant. Measure the supernatant protein concentration using a BCA protein quantification kit (ThermoFisher) and calculate the adsorption rate: Adsorption rate = (initial protein mass - supernatant protein mass) / initial protein mass × 100%; 2. Desorption rate: After adsorption, the particles were washed three times with PBS, resuspended in 1 mL of PBS, and allowed to stand at 37°C for 72 h. The supernatant was centrifuged at 12,000 rpm for 10 min, and the supernatant protein concentration was measured. Desorption rate = amount of desorbed protein / total amount of adsorbed protein × 100%.

[0039] 3. Specific surface area and mesopore volume: Instrument: Fully automatic specific surface area and pore size analyzer; Sample pretreatment: 50 mg of freeze-dried particles were degassed at 300 °C in vacuum for 3 h; Test method: The BET multi-point method was used to calculate the specific surface area, and the BJH method was used to analyze the mesopore volume (pore size range 2-50nm).

[0040] 4. D50: Instrument: Laser particle size analyzer; Sample treatment: dilute the adjuvant suspension directly 10 times (ultrapure water) and ultrasonically disperse for 30 seconds; Test: Determine D50 (median particle size) and measure three times in parallel to obtain the average value.

[0041] 5. Zeta potential: Instrument: Zeta potential instrument; Sample processing: The adjuvant suspension was directly diluted to a transmittance of 60%-80%; Test: Zeta potential was measured at 25°C, and the average value was obtained by 5 parallel measurements.

[0042]

[0043] Adsorption performance: The antigen adsorption rates of the embodiments were all ≥95%, and the 72h desorption rates were ≤5.1%, which were significantly better than those of the comparative examples (adsorption rate ≤85.7%, desorption rate ≥10.6%), demonstrating that the synergistic effects of pH adjustment, high-speed shearing, and ultrafiltration can improve adsorption stability.

[0044] Structural characteristics: The specific surface area of ​​the embodiment is ≥310m 2 / g, mesopore volume ≥ 0.296 cm 3 / g, better than the comparative example (specific surface area ≤256.89 2 / g), indicating that this process can retain a richer porous structure and provide sufficient adsorption sites.

[0045] Dispersibility: The D50 (80-100 nm) of the embodiment is smaller and more uniform, the absolute value of the Zeta potential is ≥30 mV, the dispersion stability is better than that of the comparative example (D50 ≥150 nm, the absolute value of the Zeta potential ≤25.8 mV), and the particle agglomeration is reduced.

[0046] 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 in the scope of protection of the present invention.

Claims

1. A method for preparing a highly adsorbable aluminum hydroxide adjuvant for veterinary use, characterized in that: The steps include: (1) Take aluminum salt solution, add alkali solution dropwise, adjust the pH to 9-10 first, then adjust it back to 6.0-7.5, and add 0.5-2wt% dispersant at the same time, stir and react to form a light grayish white colloidal suspension; (2) The colloidal suspension obtained in step (1) is immediately subjected to high-speed shear treatment for 10-30 min at a shear rate of 800-4000 rpm, followed by aging at room temperature for 2-6 h; (3) Purifying the aged suspension using ultrafiltration technology, wherein the ultrafiltration pressure is 0.2-0.6 MPa, the membrane material is PVDF organic membrane, PES organic membrane, PP organic membrane or alumina ceramic membrane, and the membrane pore size is 50-100 nm; (4) Sterilize the purified suspension by wet heat sterilization at 121°C for 1-2 hours to obtain an aluminum hydroxide adjuvant suspension.

2. The method for preparing a highly adsorbable aluminum hydroxide adjuvant for animals as claimed in claim 1, wherein The dispersant includes one of sodium carboxymethyl cellulose, sodium citrate, polyethylene glycol, sodium alginate, sodium lauryl sulfate, and Tween 80.

3. The preparation method of the veterinary high-adsorbability aluminum hydroxide adjuvant as claimed in claim 1, wherein The aluminum salt solution is aluminum chloride or aluminum nitrate solution, and the alkali solution is sodium hydroxide solution or ammonia solution.

4. The preparation method of the veterinary high-adsorbability aluminum hydroxide adjuvant as claimed in claim 1, wherein In step (1), the pH is first adjusted to 9.3-9.7 and then adjusted back to 6.5-7.

0.

5. The preparation method of the veterinary high-adsorbability aluminum hydroxide adjuvant as claimed in claim 1, wherein The high-speed shearing rate in step (2) is 1000-3000 rpm, the shearing time is 20 min, and the aging time is 4 h.

6. The method for preparing the veterinary highly adsorbable aluminum hydroxide adjuvant according to claim 1, wherein The ultrafiltration membrane material in step (3) is an alumina ceramic membrane with a pressure of 0.4 MPa.

7. A highly adsorbable aluminum hydroxide adjuvant for veterinary use prepared by the method for preparing the highly adsorbable aluminum hydroxide adjuvant for veterinary use according to any one of claims 1 to 6, characterized in that: The adjuvant is in the form of a suspension, and the antigen adsorption rate of the aluminum hydroxide solid particles in the adjuvant is ≥94%, the desorption rate within 72 hours is ≤5.1%, and the specific surface area is ≥300m 2 / g.

8. The adjuvant according to claim 7, wherein The mesoporous volume of aluminum hydroxide solid particles in the adjuvant is 0.25-0.45 cm 3 / g, D50 is 80-120nm.

9. Use of the aluminum hydroxide adjuvant described in any one of claims 7-8 in the preparation of veterinary inactivated vaccines.

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