Preparation method of aluminum hydroxide adjuvant with pH regulated and controlled by stages

Abstract

The invention discloses a preparation method of an aluminum hydroxide adjuvant capable of regulating and controlling pH in stages, and aims to solve the problems of non-uniform particle size, unstable adsorption and large inter-batch difference in the traditional process. According to the method, phosphate radical modified hydroxyapatite nanocrystal seeds are taken as an induction template, and three-stage accurate pH regulation and control are carried out: a pre-crystal seed stage activates crystal seed active sites, a main crystallization stage guides directional growth of aluminum hydroxide, and an aluminum phosphate coating layer is formed in a stabilization stage. Compared with a traditional process, the finished product yield is improved, energy consumption is reduced, and the method is suitable for vaccine industrial production.

Description

Technical Field

[0001] This invention relates to the field of vaccine formulation technology, and specifically proposes a method for preparing aluminum hydroxide adjuvant with staged pH control. Background Technology

[0002] Aluminum hydroxide adjuvant is widely used in the vaccine field due to its good biocompatibility and antigen adsorption capacity. Its performance depends on particle size uniformity, adsorption efficiency stability, and batch-to-batch consistency. However, traditional preparation processes have three major pain points: 1. Particle size control challenges: Single-stage pH endpoint control allows aluminum ions to spontaneously nucleate in a homogeneous manner, resulting in a wide particle size distribution. Some micron-sized particles can easily trigger local immune responses, while the low proportion of nano-sized particles affects antigen loading efficiency; 2. Poor adsorption stability: Without seed induction, the aluminum hydroxide crystal structure is prone to amorphous to boehmite phase transition due to local over-alkaliness, leading to fluctuations in the number of surface active sites, reduced antigen adsorption rate, and a high adsorption rate loss after autoclaving, failing to meet the stability requirements of vaccine industrialization; 3. Significant batch-to-batch differences: Traditional processes rely on manual pH adjustment with an accuracy of only ±0.2, and lack dynamic reaction kinetic control. Different batches of products exhibit large differences in Zeta potential (±5mV fluctuation) and sedimentation rate (20-30% difference), resulting in a finished product qualification rate of only 75%, increasing industrial production costs.

[0003] Existing improvement schemes have obvious limitations: for example, the "single-stage pH control method" proposed in the existing technology does not introduce seed crystals and still cannot solve the problem of homogeneous nucleation; although the "reverse microemulsion method" of Southeast University can prepare nanoparticles, it relies on organic phases, which not only increases the post-processing cost, but also poses the risk of organic residues and does not meet the requirements of green process; conventional seed crystal methods mostly use unmodified Al2O3 seed crystals, which lack active sites on the surface and cannot effectively guide directional growth, resulting in limited improvement in particle size uniformity.

[0004] In summary, there is an urgent need to develop an aluminum hydroxide adjuvant preparation technology that does not involve organic phases, allows for precise control of crystal growth, and balances performance with industrial feasibility, in order to solve the core pain points of traditional processes. Summary of the Invention

[0005] In view of this, in order to address the problems of uneven particle size, unstable adsorption, large batch-to-batch differences and difficulty in scaling up the process of aluminum hydroxide adjuvant in the existing technology, a staged pH-seed synergistic preparation method is provided. Through the deep coupling of seed interface engineering and dynamic pH control, the product performance can be precisely controlled and industrialized.

[0006] The technical solution of this invention is achieved as follows: This invention provides a method for preparing aluminum hydroxide adjuvant with staged pH regulation, the core of which lies in the synergistic effect of phosphate-modified hydroxyapatite seed induction and three-stage dynamic pH regulation, specifically including the following key steps: Pre-seed activation stage: Construct a reaction system using deionized water, add phosphate-modified hydroxyapatite nanocrystals at a concentration of 0.5-2 g / L, adjust the pH to 3.5-4.0 with 1-3M hydrochloric acid or sulfuric acid, and stir at 65-75℃ and 800-1200 rpm for 30-60 min to protonate and activate the ≡P—OH groups on the seed surface, forming Al 3+ Adsorption active sites; Main crystal growth stage: 20-30% AlCl3 (or 15-25% KAl(SO4)2) solution is added dropwise at a rate of 0.5-1.5 mL / (min・L reaction system), while 2-4M NaOH is injected simultaneously by spraying. The alkali addition rate is dynamically adjusted by a ruthenium oxide coated micro-nano pH sensor (accuracy ±0.03) and PID algorithm to control the pH to be stable at 6.0-6.8, so that aluminum hydroxide grows epitaxially along the seed crystal surface in a directional manner, avoiding homogeneous nucleation; Surface stabilization stage: 0.5-1M Na2HPO4 solution was added dropwise at a rate of 0.3-0.8mL / (min·L reaction system) to adjust the pH to 7.5-8.0, and a 5nm thick AlPO4 coating layer was generated in situ. At the same time, 0.1% polysorbate 80 was added and the mixture was aged at 35℃ for 30min to optimize the surface charge and colloidal stability. Post-processing stage: Centrifuge at 8000-10000g to collect particles, wash with deionized water until conductivity <10μS / cm, suspend in physiological saline (Al content 10mg / mL), sterilize at 121℃ for 15-20min to obtain the target product.

[0007] In some embodiments, the particle size of the phosphate-modified hydroxyapatite nanocrystals is 50-100 nm.

[0008] In some embodiments, the preparation method of the phosphate-modified hydroxyapatite nanocrystals is as follows: hydroxyapatite nanoparticles are pre-immersed in a 0.05-0.2M NaH2PO4 solution and ultrasonically treated at 300-500W for 20-40 minutes to form ≡P—O on the surface. - Active sites; in industrial production, Ca can be extracted from hydroxyapatite seed crystals through phosphate rock slag. 2+ Synthesized by reacting with NaH2PO4, the cost is reduced by 60%.

[0009] In some embodiments, to achieve process scale-up, the present invention also designs a three-stage series continuous flow reactor: an acidic zone static mixer, a neutral zone fluidized bed aging tank, and an alkaline zone membrane dispersion microreactor.

[0010] In some implementations, the residence time of AlCl3 and seed crystals in the acidic region is <10s.

[0011] In some implementations, the upward flow velocity in the neutral zone is 2 cm / s.

[0012] In some embodiments, phosphate-modified hydroxyapatite nanocrystals are used, through surface ≡P-O - With Al 3+ The coordination bonding inhibits spontaneous homogeneous nucleation, guides directional growth, and solves the problem of uneven particle size.

[0013] In some implementations, the single endpoint control is broken, and a three-stage pH trajectory is established from acid activation to neutral growth to alkaline stability, with pH changes to 3.5-4.0, 6.0-6.8, and 7.5-8.0, respectively.

[0014] The present invention has the following advantages over the prior art: The particle size uniformity is significantly improved, and the particle size distribution is more concentrated; the adsorption efficiency is significantly increased, and the activity is better maintained after autoclaving; the colloidal stability is significantly enhanced, and it is not prone to sedimentation during long-term storage. It is compatible with a variety of vaccine antigen types, has no organic residues, complies with GMP standards, and can be directly used for industrial production. Detailed Implementation

[0015] 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.

[0016] Example 1 100L-scale staged preparation of pH-phosphate modified HAP seed crystals Preparation equipment description This embodiment uses intermittent production equipment, specifically including: the main reaction equipment is a 100L 316L stainless steel reactor (equipped with a Mettler Toledo online pH probe and turbidity probe, pH detection accuracy ±0.03), and a 1000rpm variable frequency stirring device; the post-treatment equipment is a GEAWestfalia disc centrifuge (g value ≥8000), a rotary water bath sterilizer, and an ultrapure water preparation system (outlet water conductivity <5μS / cm).

[0017] Preparation steps Step 1: Pre-seed activation. Take 50g of commercially available hydroxyapatite (HAP) nanocrystals (particle size 50-100nm), disperse them in 1L of 0.1M sodium dihydrogen phosphate solution (pH 4.0), and sonicate at 300W for 30min to complete seed modification. After filtration and drying, add the modified seed to a 100L 316L stainless steel reactor, and add ultrapure water to make up to 100L. Adjust the pH of the system to 3.8 with 1M sulfuric acid solution, set the reaction temperature to 70℃, and the stirring speed to 1000rpm, stirring continuously for 20min to activate the active sites on the seed surface.

[0018] Step 2: Main crystal growth. A 20% aluminum chloride solution was added dropwise to the reactor at a rate of 120 mL / min (corresponding to a reaction system volume of 1.2 mL / (min·L)), while a 2M sodium hydroxide solution was simultaneously sprayed in. The pH of the system was monitored in real time using a ruthenium oxide-coated micro / nano pH sensor (integrated PID algorithm), and the pH was stabilized within the range of 6.5 ± 0.1. The reaction was maintained at 70°C for 60 min. If the turbidity value dropped below 50 NTU during the reaction, 2 g of modified HAP seed crystals were added.

[0019] Step 3: Surface stabilization. Add 0.5 M disodium hydrogen phosphate solution dropwise to the reaction system at a rate of 50 mL / min (corresponding to 0.5 mL / (min·L reaction system)) to adjust the pH of the system to 7.8. Then lower the system temperature to 35 °C and mature for 30 min. During this period, add 0.1% of the total mass of polysorbate 80.

[0020] Step 4: Post-processing. The reaction product was centrifuged for 15 min using a GEAWestfalia disc centrifuge (8000g), and the precipitate was collected. The precipitate was washed three times with ultrapure water prepared by an ultrapure water preparation system, and the conductivity of the washing solution was measured after each wash until the conductivity was below 10 μS / cm. The washed precipitate was then suspended in physiological saline, and the aluminum content in the suspension was adjusted to 10 mg / mL. Finally, it was sterilized in a rotary water bath sterilizer at 121℃ for 15 min to obtain aluminum hydroxide adjuvant.

[0021] Performance verification steps Particle size distribution (D50, PDI): A laser particle size analyzer was used. An appropriate amount of adjuvant suspension was taken and tested according to the instrument operating procedures. The D50 and PDI values ​​were recorded.

[0022] BSA adsorption rate: Referring to Pharmacopoeia General Chapter 3106, the adjuvant and bovine serum albumin (BSA) solution were mixed in proportion, adsorbed by shaking, and then separated by centrifugation. The concentration of residual BSA in the supernatant was detected, and the adsorption rate was calculated.

[0023] 24-hour sedimentation rate: According to GB / T16497-2003, a certain volume of adjuvant suspension is placed in a stoppered graduated cylinder, and after standing for 24 hours, the volume of the supernatant is measured and the sedimentation rate is calculated.

[0024] Adsorption rate loss after sterilization: After sterilizing the adjuvant at 121℃ for 15 min, the adsorption rate was retested according to the BSA adsorption rate test method described above, and the percentage of the difference in adsorption rate before and after sterilization to the adsorption rate before sterilization was calculated.

[0025] The performance results are shown in the table below:

[0026] Example 2 10L small-scale, staged preparation of pH-phosphate modified HAP seed crystals Preparation equipment description This embodiment is a small-scale test using intermittent equipment, specifically including: a 10L 316L stainless steel reactor (with a small online pH probe, accuracy ±0.05), a 1000rpm variable frequency stirrer; the post-processing equipment includes a laboratory benchtop high-speed centrifuge (maximum speed 8000g), a small vertical sterilizer (121℃ temperature control), and a 300W ultrasonic cleaner (for seed crystal modification).

[0027] Preparation steps Step 1: Pre-seed activation. Take 5g of commercially available HAP nanocrystals (particle size 50-100nm), disperse them in 0.1L of 0.1M sodium dihydrogen phosphate solution (pH 4.0), and sonicate them for 30min using a 300W ultrasonic cleaner. After filtration and drying, add the modified seed crystals to a 10L 316L stainless steel reactor. Add ultrapure water to the reactor to make up the total volume to 10L. Adjust the pH to 3.8 with 1M sulfuric acid solution. Set the reaction temperature to 70℃ and the stirring speed to 1000rpm, and continue stirring for 20min.

[0028] Step 2: Main crystal growth. Add 20% aluminum chloride solution dropwise to the reactor at a rate of 10 mL / min (corresponding to a reaction system volume of 1.0 mL / (min·L)), while simultaneously injecting 2M sodium hydroxide solution via spray. Maintain the pH at 6.5 ± 0.1 using a small online pH probe (integrated PID algorithm), and keep the reaction at 70°C for 60 min. If the turbidity value drops below 50 NTU during the reaction, add 0.2 g of modified HAP seed crystals.

[0029] Step 3: Surface stabilization. Add 0.5M disodium hydrogen phosphate solution dropwise to the reaction system at a rate of 4 mL / min (corresponding to 0.4 mL / (min·L reaction system)) to adjust the pH to 7.8, lower the system temperature to 35℃ and mature for 30 min, during which 0.1% of the total mass of polysorbate 80 is added.

[0030] Step 4: Post-processing. The reaction product was centrifuged for 15 min using a laboratory benchtop high-speed centrifuge (8000g), and the precipitate was collected. The precipitate was washed three times with ultrapure water, and the conductivity of the washing solution was measured after each wash until the conductivity was below 10 μS / cm. The washed precipitate was then resuspended in physiological saline, and the aluminum content was adjusted to 10 mg / mL. The solution was then sterilized in a small vertical autoclave at 121°C for 15 min to obtain aluminum hydroxide adjuvant.

[0031] Performance verification steps The performance verification steps are the same as in Example 1.

[0032] The performance results are shown in the table below:

[0033] Example 3 100L scale self-produced HAP seed crystals - staged pH-coordinated preparation Preparation equipment description In this embodiment, HAP seed synthesis was performed using a 50L enamel-lined reactor (with a 600rpm stirrer and an 85℃ constant temperature system) and an acid extraction tower (used for extracting Ca from phosphate rock slag). 2+ The main equipment for adjuvant preparation is the same as in Example 1 (100L 316L stainless steel reactor + Mettler Toledo online pH probe and turbidity probe + 1000rpm variable frequency stirrer), and the post-treatment equipment is the same as in Example 1 (GEAWestfalia disc centrifuge, rotary water bath sterilizer, ultrapure water preparation system).

[0034] Preparation steps Step 1: Preparation of self-produced HAP seed crystals. Take phosphate rock slag and extract calcium ions using an acid extraction tower to achieve a final calcium ion solution concentration of 0.5M. Add this calcium ion solution and 0.3M disodium hydrogen phosphate solution to a 50L enamel-lined reactor in a specific ratio. The calcium ion solution and disodium hydrogen phosphate solution are reacted according to the following formula: Ca... 2+ :PO4 3- The mixture was prepared in a molar ratio of 10:6, and the reaction was carried out at a temperature of 85℃ and a stirring speed of 600 rpm for 2.5 h. After the reaction was completed, the product was collected by centrifugation to obtain HAP nanocrystals with a particle size of 50-100 nm.

[0035] Step 2: Pre-seed activation. Take 50g of self-produced HAP seeds and disperse them in 1L of 0.1M sodium dihydrogen phosphate solution (pH 4.0). Modify the seeds by ultrasonic treatment at 300W for 30min, then filter and dry. Add the modified seeds to a 100L 316L stainless steel reactor. Add ultrapure water to the reactor to make up to 100L of system. Adjust the pH to 3.8 with 1M sulfuric acid solution. Set the reaction temperature to 70℃ and the stirring speed to 1000rpm, and continue stirring for 20min to activate the active sites on the seed surface.

[0036] Step 3: Main crystal growth. Add 20% aluminum chloride solution dropwise to the reactor at a rate of 120 mL / min, simultaneously injecting 2M sodium hydroxide solution via spray. Maintain the pH at 6.5 ± 0.1 using a Mettler Toledo online pH probe (integrated PID algorithm), and keep the reaction at 70°C for 60 min. If the turbidity value drops below 50 NTU during the reaction, add 2 g of self-produced modified HAP seed crystals.

[0037] Step 4: Surface stabilization. Add 0.5M disodium hydrogen phosphate solution dropwise to the reaction system at a rate of 50 mL / min to adjust the pH to 7.8. Lower the system temperature to 35℃ and mature for 30 min. During this period, add 0.1% of the total mass of polysorbate 80.

[0038] Step 5: Post-processing. The precipitate was collected by centrifugation for 15 min using a GEAWestfalia disc centrifuge (8000g). It was washed three times with ultrapure water until the conductivity was <10μS / cm. The precipitate was then suspended in physiological saline (Al content 10mg / mL) and sterilized in a rotary water bath sterilizer at 121℃ for 15 min to obtain aluminum hydroxide adjuvant.

[0039] Performance verification steps Similar to the performance verification steps in Example 1, an additional seed cost calculation is added: the raw material procurement, energy consumption and labor costs in the preparation process of self-produced HAP seeds and commercially available HAP seeds are statistically analyzed, and the cost difference is calculated.

[0040] The performance results are shown in the table below:

[0041] Example 4 100L scale KAl(SO4)2 aluminum salt-staged pH-coordinated preparation Preparation equipment description The main reaction equipment in this embodiment is the same as in Embodiment 1 (100L 316L stainless steel reactor + Mettler Toledo online pH probe and turbidity probe + 1000rpm variable frequency stirrer); an additional laboratory constant temperature dialysis device (30℃ temperature control, dialysis bag molecular weight cutoff of 10kDa) is provided for K⁺ removal, and the post-processing equipment is the same as in Embodiment 1 (GEAWestfalia disc centrifuge, rotary water bath sterilizer, ultrapure water preparation system).

[0042] Preparation steps Step 1: Pre-seed activation. Take 50g of commercially available HAP seed crystals (particle size 50-100nm), disperse them in 1L of 0.1M sodium dihydrogen phosphate solution (pH 4.0), and modify them by ultrasonic treatment at 300W power for 30min. Add the modified seed crystals to a 100L 316L stainless steel reactor, add ultrapure water to make up to 100L of system, adjust the pH to 3.8 with 1M sulfuric acid solution, set the reaction temperature to 70℃ and the stirring speed to 1000rpm, and continue stirring for 20min to activate the active sites on the seed crystal surface.

[0043] Step 2: Main crystal growth. Add 25% potassium aluminum sulfate solution dropwise to the reactor at a rate of 120 mL / min, simultaneously injecting 2M sodium hydroxide solution via spray. Maintain the pH at 6.5 ± 0.1 using a Mettler Toledo online pH probe (integrated PID algorithm), and keep the reaction at 70°C for 60 min. If the turbidity value drops below 50 NTU during the reaction, add 2 g of modified HAP seed crystals. After the reaction, transfer the system to a laboratory isothermal dialysis apparatus (30°C controlled), use a dialysis bag with a molecular weight cutoff of 10 kDa, and dialyze for 5 h using ultrapure water as the dialysate to remove potassium ions.

[0044] Step 3: Surface stabilization. The dialyzed system was reintroduced into a 100L reactor, and 0.5M disodium hydrogen phosphate solution was added dropwise at a rate of 50mL / min to adjust the pH to 7.8. The system temperature was lowered to 35℃ and aged for 30min. During this period, 0.1% of the total mass of polysorbate 80 was added.

[0045] Step 4: Post-processing. The precipitate was collected by centrifugation for 15 min using a GEAWestfalia disc centrifuge (8000g). It was washed three times with ultrapure water until the conductivity was <10μS / cm. The precipitate was then suspended in physiological saline (Al content 10mg / mL) and sterilized in a rotary water bath sterilizer at 121℃ for 15 min to obtain aluminum hydroxide adjuvant.

[0046] Performance verification steps The performance verification steps are the same as in Example 1.

[0047] The performance results are shown in the table below:

[0048] Example 5 Preparation of a 100L / h capacity three-stage series continuous flow reactor Preparation equipment description This embodiment uses a three-stage series continuous flow reactor, and the specific equipment configuration includes: First stage (acid zone): turbine blade-shaped static mixer (5L capacity), equipped with a feed pump (connecting modified seed solution and aluminum salt solution) and a 1M sulfuric acid inlet; Second stage (neutral zone): fluidized bed aging tank (volume 20L), built with 1-3mm inert ceramic balls, temperature control system (70℃), upward flow rate controller (2cm / s), and 2M sodium hydroxide spray device; The third stage (alkaline zone): a polyvinylidene fluoride (PVDF) membrane dispersion microreactor (volume 10L), equipped with a 0.5M disodium hydrogen phosphate feed pump and a ruthenium oxide coated online pH monitor (accuracy ±0.03). The post-processing equipment includes a GEAWestfalia disc centrifuge (8000g), a rotary water bath sterilizer, and an ultrapure water preparation system.

[0049] Preparation steps Step 1: Preparation of pre-seedling modification solution. Prepare a 50 g / L phosphate-modified HAP seed solution according to the method in Example 1. Take 2 L of the phosphate-modified HAP seed solution and dilute it with 3 L of deionized water to obtain 5 L of 20 g / L phosphate-modified HAP seed solution for later use.

[0050] Step 2: Primary acidic zone mixing and activation. A 20 g / L phosphate-modified HAP seed solution and a 20% AlCl3 solution were injected into the first-stage turbine blade static mixer at a volume ratio of 5:1 via a feed pump. The total feed rate was set to 6 L / h (5 L / h of seed solution, 1 L / h of AlCl3 solution, and 0.1 L / h of 1M sulfuric acid). Based on this rate, the seed concentration in the final reaction system (total volume of the three-stage reactor 35 L) stabilized at 0.8 g / L, ensuring the directional epitaxial growth of aluminum hydroxide. Simultaneously, 1M sulfuric acid was injected to adjust the pH of the mixing system to 3.8. The residence time of the system in the mixer was controlled by the feed pump flow rate to 8 s, completing the rapid contact activation between the seed crystals and the aluminum salt.

[0051] Step 3: Primary crystal growth in the secondary neutral zone. The first-stage effluent is continuously introduced into the second-stage fluidized bed aging tank. The upward flow rate controller (2 cm / s) is turned on to fluidize the system. Simultaneously, 2M sodium hydroxide is injected through a spray device. The pH of the system is controlled to be stable at 6.5 ± 0.1 using a PID algorithm. The residence time of the system in the aging tank is controlled by the effluent flow rate to 30 min, thus completing the directional epitaxial growth of aluminum hydroxide.

[0052] Step 4: Surface stabilization in the tertiary alkaline zone. The effluent from the second stage is introduced into the third-stage PVDF membrane dispersion microreactor. 0.5M disodium hydrogen phosphate solution is precisely injected through the membrane module to adjust the pH of the system to 7.8. The residence time of the system in the microreactor is controlled at 15 min. Then, 0.1% polysorbate 80 is added, and the system is allowed to remain in the microreactor for another 15 min to complete the aging process.

[0053] Step 5: Post-processing. The third-stage effluent was continuously introduced into a GEAWestfalia disc centrifuge (8000g). After centrifugation, it was washed three times with ultrapure water prepared by the ultrapure water preparation system (conductivity <10μS / cm), suspended in physiological saline (Al content 10mg / mL), and sterilized in a rotary water bath sterilizer (121℃ / 15min) to obtain aluminum hydroxide adjuvant with a total production capacity of 100L / h.

[0054] Note: In this embodiment, step 2 corresponds to the pre-seedling activation stage of claim 1, step 3 corresponds to the main crystal growth stage, and step 4 corresponds to the surface stabilization stage.

[0055] Performance verification steps Basic performance (D50, PDI, BSA adsorption rate, 24h sedimentation rate, adsorption rate loss after sterilization): Same as Example 1; Continuous production stability: After 8 hours of continuous operation, samples were taken every hour to detect the adsorption rates of D50 and BSA, and the coefficient of variation of 8 batches of data was calculated. Production efficiency: The product output per unit time (1 hour) was statistically analyzed and compared with the production time of a traditional batch reactor (Example 1); Energy consumption and labor costs: The unit product energy consumption (kW·h / L) and the frequency of manual operation (times / h) of the continuous flow equipment and the batch reactor of Example 1 were statistically analyzed.

[0056] The performance results are shown in the table below:

[0057] Comparative Example 1 Traditional single-stage pH seedless preparation Preparation equipment description The reactor used was a 100L ordinary carbon steel batch reactor (with 500rpm stirring and pH test paper), a laboratory benchtop centrifuge (5000g), and a small sterilizer, but without online monitoring and precise temperature control equipment.

[0058] Preparation steps Step 1: Add ultrapure water to a 100L ordinary carbon steel batch reactor until the volume reaches 100L. Add 20% aluminum chloride solution dropwise at a rate of 120mL / min. At the same time, adjust the pH of the system with 2M sodium hydroxide solution. Detect the pH value using ordinary pH test paper and control the final pH to 6.5 (accuracy ±0.2). Set the reaction temperature to 70℃, the stirring speed to 500rpm, and continue the reaction for 60min.

[0059] Step 2: After the reaction was completed, the precipitate was centrifuged for 15 min using a laboratory benchtop centrifuge (5000g) and collected. It was washed three times with ultrapure water, and the conductivity of the washing solution was measured after each wash until the conductivity was below 10 μS / cm. The precipitate was then suspended in physiological saline (Al content 10 mg / mL) and sterilized in a small autoclave at 121°C for 15 min to obtain aluminum hydroxide adjuvant.

[0060] Performance verification steps The performance verification steps are the same as in Example 1.

[0061] The performance results are shown in the table below:

[0062] Comparative Example 2 Unmodified HAP seed crystals + three-stage pH preparation Preparation equipment description Same as Example 1 (100L 316L stainless steel reactor + Mettler Toledo online pH probe and turbidity probe + 1000rpm variable frequency stirrer, GE Awessfalia disc centrifuge, rotary water bath sterilizer, ultrapure water preparation system).

[0063] Preparation steps Step 1: Pre-seed activation. Take 10g of commercially available HAP nanocrystals (particle size 50-100nm, without sodium dihydrogen phosphate modification), disperse them directly in 100L of ultrapure water, add them to a 100L 316L stainless steel reactor, adjust the pH of the system to 3.8 with 1M sulfuric acid solution, set the reaction temperature to 70℃ and the stirring speed to 1000rpm, and stir continuously for 20min.

[0064] Step 2: Main crystal growth. Add 20% aluminum chloride solution dropwise to the reactor at a rate of 120 mL / min, simultaneously injecting 2M sodium hydroxide solution via spray. Maintain the pH at 6.5 ± 0.1 using a Mettler Toledo online pH probe (integrated PID algorithm), and keep the reaction at 70°C for 60 min. If the turbidity value drops below 50 NTU during the reaction, add 2 g of unmodified HAP seed crystals.

[0065] Step 3: Surface stabilization. Add 0.5M disodium hydrogen phosphate solution dropwise to the reaction system at a rate of 50 mL / min to adjust the pH to 7.8. Lower the system temperature to 35℃ and mature for 30 min. During this period, add 0.1% of the total mass of polysorbate 80.

[0066] Step 4: Post-processing. The precipitate was collected by centrifugation for 15 min using a GEAWestfalia disc centrifuge (8000g). It was washed three times with ultrapure water until the conductivity was <10μS / cm. The precipitate was then suspended in physiological saline (Al content 10mg / mL) and sterilized in a rotary water bath sterilizer at 121℃ for 15 min to obtain aluminum hydroxide adjuvant.

[0067] Performance verification steps The performance verification steps are the same as in Example 1.

[0068] The performance results are shown in the table below:

[0069] Comparative Example 3 Preparation by reverse microemulsion method Preparation equipment description The equipment used was a 50L glass reactor (with a 500rpm stirrer and a 70℃ constant temperature system), a laboratory benchtop centrifuge (8000g), a rotary evaporator (for organic phase recovery), and a small autoclave. There were no stainless steel reactors or continuous flow equipment.

[0070] Preparation steps Step 1: Preparation of reverse microemulsion. Measure cyclohexane (oil phase), 20% aluminum chloride solution (aqueous phase), and Span80 (emulsifier) ​​in a volume ratio of 7:3:1, place them in a 50L glass reactor, and sonicate at 300W for 30 minutes to form a reverse microemulsion system.

[0071] Step 2: Reaction and Demulsification. Add 2M sodium hydroxide solution dropwise to the above microemulsion system to adjust the pH to 6.5. Set the reaction temperature to 70℃ and the stirring speed to 500 rpm, and continue the reaction for 60 min. After the reaction is complete, add ethanol to the system to demulsify. After standing and separating the layers, collect the lower aqueous phase product.

[0072] Step 3: Post-processing. The precipitate was collected by centrifugation for 15 min using a laboratory benchtop centrifuge (8000g). It was washed three times with ultrapure water until the conductivity was <10μS / cm. The precipitate was then suspended in physiological saline (Al content 10mg / mL) and sterilized in a small autoclave at 121℃ for 15 min to obtain aluminum hydroxide adjuvant.

[0073] Performance verification steps Particle size distribution, BSA adsorption rate, 24h sedimentation rate, and adsorption rate loss after sterilization: same as the performance verification steps in Example 1; Organic residues: Gas chromatography was used to detect the amount of cyclohexane residues in the adjuvant; Process cost: The procurement costs of oil phase and emulsifier, as well as the costs of demulsification and organic phase recovery and treatment, are calculated and compared with those of Example 1; Wastewater treatment difficulty: Detect the oil content of the washing wastewater and assess the difficulty of treatment.

[0074] The performance results are shown in the table below:

[0075] Comparative Example 4 Three-stage pH + Al2O3 seed preparation Preparation equipment description Same as Example 1 (100L 316L stainless steel reactor + Mettler Toledo online pH probe and turbidity probe + 1000rpm variable frequency stirrer, GE Awessfalia disc centrifuge, rotary water bath sterilizer, ultrapure water preparation system).

[0076] Preparation steps Step 1: Pre-seed activation. Take 10g of Al2O3 nanocrystals with a particle size of 50-100nm, disperse them in 1L of 0.1M sodium dihydrogen phosphate solution (pH 4.0), and sonicate them at 300W for 30min. Add the treated Al2O3 nanocrystals to a 100L 316L stainless steel reactor, add ultrapure water to make up to 100L of system, adjust the pH to 3.8 with 1M sulfuric acid solution, set the reaction temperature to 70℃ and the stirring speed to 1000rpm, and stir continuously for 20min.

[0077] Step 2: Main crystal growth. Add 20% aluminum chloride solution dropwise to the reactor at a rate of 120 mL / min, simultaneously injecting 2M sodium hydroxide solution via spray. Maintain the pH at 6.5 ± 0.1 using a Mettler Toledo online pH probe (integrated PID algorithm), and keep the reaction at 70°C for 60 min. If the turbidity value drops below 50 NTU during the reaction, add 2 g of Al₂O₃ seed crystals.

[0078] Step 3: Surface stabilization. Add 0.5M disodium hydrogen phosphate solution dropwise to the reaction system at a rate of 50 mL / min to adjust the pH to 7.8. Lower the system temperature to 35℃ and mature for 30 min. During this period, add 0.1% of the total mass of polysorbate 80.

[0079] Step 4: Post-processing. The precipitate was collected by centrifugation for 15 min using a GEAWestfalia disc centrifuge (8000g). It was washed three times with ultrapure water until the conductivity was <10μS / cm. The precipitate was then suspended in physiological saline (Al content 10mg / mL) and sterilized in a rotary water bath sterilizer at 121℃ for 15 min to obtain aluminum hydroxide adjuvant.

[0080] Performance verification steps The performance verification steps are the same as in Example 1.

[0081] The performance results are shown in the table below:

[0082] Comparative Example 5 Traditional batch reactor (100L scale, corresponding to Example 1) Preparation equipment description Same as Example 1 (100L 316L stainless steel reactor + Mettler Toledo online pH probe and turbidity probe + 1000rpm variable frequency stirrer, GE Awessfalia disc centrifuge, rotary water bath sterilizer, ultrapure water preparation system).

[0083] Preparation steps The preparation steps are the same as in Example 1, with the core difference being the use of a 100L batch reactor. Each stage requires manual switching of feed and adjustment of parameters, and the total time for a single batch is 90 minutes (including feeding, reaction, and discharging).

[0084] Performance verification steps Basic performance: Same as in Example 1; Inter-batch stability: Eight batches were prepared consecutively, and the adsorption rates of D50 and BSA in each batch were measured to calculate the coefficient of variation. Production efficiency: Statistics on the time consumed per batch and the output per unit time; Energy consumption and labor costs: Statistics on energy consumption per unit product and frequency of manual operation in batch reactors.

[0085] The performance results are shown in the table below:

[0086] 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 staged pH-controlled aluminum hydroxide adjuvant, characterized in that, Includes the following steps: (1) Pre-seed activation stage: The reaction system is constructed based on deionized water. Phosphate-modified hydroxyapatite nanocrystals are dispersed in the reaction system at a concentration of 0.5-2 g / L. The pH of the system is adjusted to 3.5-4.0 using 1-3M sulfuric acid solution. The mixture is stirred at a stirring rate of 800-1200 rpm for 30-60 min at a temperature of 65-75℃ to activate the active sites on the seed surface. (2) Main crystal growth stage: Add aluminum salt solution dropwise to the system in step (1). The aluminum salt solution is an AlCl3 solution with a mass fraction of 20-30% or a KAl(SO4)2 solution with a mass fraction of 15-25%. Simultaneously, use 2-4M NaOH solution to adjust the pH of the system to 6.0-6.8 so that aluminum hydroxide grows epitaxially along the seed crystal surface. (3) Surface stabilization stage: Slowly add 0.5-1M Na2HPO4 solution to the system in step (2), and continue to adjust the pH to 7.5-8.0 with 2-4M NaOH solution to form an aluminum phosphate coating layer on the surface of aluminum hydroxide particles in situ. The time for adding Na2HPO4 solution is controlled at 30-60 min. (4) Post-processing stage: Collect the particles obtained in step (3), centrifuge and wash them with deionized water until the conductivity of the wastewater after washing is <10μS / cm, then suspend the washed particles in physiological saline, adjust the Al content to 10mg / mL, and finally sterilize at 121℃ for 15-20min to obtain aluminum hydroxide adjuvant.

2. The method as described in claim 1, characterized in that, The preparation method of the phosphate-modified hydroxyapatite nanocrystal seeds is as follows: hydroxyapatite nanoparticles are pre-soaked in a 0.05-0.2M sodium dihydrogen phosphate solution at a concentration of 1-3 g / L, and then ultrasonically treated at an ultrasonic power of 300-500 W for 20-40 min.

3. The method as described in claim 1, characterized in that, The hydroxyapatite nanocrystals have a particle size of 50-100 nm.

4. The method as described in claim 1, characterized in that, During the main crystal growth stage, 2-4M NaOH solution is injected by spraying to adjust the pH, and the pH is monitored in real time by a pH sensor, with a pH control accuracy of ±0.03-±0.

1. During the surface stabilization stage, 0.5-1M Na2HPO4 solution is added in a pulsed manner to fine-tune the pH, with a pH fluctuation range of ≤±0.

05.

5. The method as described in claim 1, characterized in that, During the pre-seedling activation stage, the temperature of the reaction system is 65-75℃, and the stirring rate is 800-1200rpm; the concentration of the sulfuric acid solution is 1-3M.

6. The method as described in claim 1, characterized in that, If KAl(SO4)2 solution is used as the aluminum salt, after the main crystal growth stage, dialysis to remove K should be performed. + The procedure involves dialysis for 4-6 hours, using deionized water as the dialysate, and maintaining the temperature at 25-30℃ during the dialysis process.

7. The method as described in claim 1, characterized in that, Steps (1)-(3) are achieved using a three-stage series continuous flow reactor: The first stage is a static mixer in the acid zone, whose internal mixing element is structured like turbine blades, so that the residence time of the aluminum salt solution and the activated seed crystals is less than 10s. The second stage is a neutral zone fluidized bed aging tank, with an upward flow velocity of 1-3 cm / s inside the tank, and the fluidized bed is filled with inert ceramic balls with a diameter of 1-3 mm; The third stage is an alkaline zone membrane dispersion microreactor, with polyvinylidene fluoride as the membrane material, used for the precise addition of phosphate reagents.

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