阅读说明：本技术 一种适合规模化生产的高浓度纳米级氢氧化铝佐剂配制方法 (Preparation method of high-concentration nano-scale aluminum hydroxide adjuvant suitable for large-scale production ) 是由 姚雷 周振发 洪禹 李米杰 杨飞鸽 杜琳 朱卫华 于 2021-09-23 设计创作，主要内容包括：本发明是一种适合规模化配制高浓、纳米级粒径氢氧化铝佐剂的方法。使用氯化铝与氢氧化钠反应,准确控制反应速度,监控体系pH值及反应产物粒径大小,生成粒径小于1微米氢氧化铝胶体粒径,将氢氧化铝胶体在80～90℃环境陈化稳定后无菌分装保存,并取样检测理化指标。该方法简单易操作,适用于规模化生产氢氧化铝佐剂,所生产佐剂粒径小于1微米,同时佐剂铝离子浓度可达4.0mg/ml以上,并可依据实际需要适当调整。(The invention relates to a method suitable for preparing an aluminum hydroxide adjuvant with high concentration and nano-scale particle size in a large scale. The method comprises the steps of reacting aluminum chloride with sodium hydroxide, accurately controlling the reaction speed, monitoring the pH value of a system and the particle size of a reaction product to generate an aluminum hydroxide colloid with the particle size of less than 1 micron, aging and stabilizing the aluminum hydroxide colloid at 80-90 ℃, then performing aseptic subpackage storage, and sampling to detect physical and chemical indexes. The method is simple and easy to operate, is suitable for large-scale production of the aluminum hydroxide adjuvant, the particle size of the produced adjuvant is less than 1 micron, and the concentration of aluminum ions of the adjuvant can reach more than 4.0mg/ml, and can be properly adjusted according to actual requirements.)

1. The preparation method of the aluminum hydroxide adjuvant is characterized by comprising the following steps of:

the method comprises the following steps: cleaning a material tank and a reaction tank required by adjuvant preparation, sterilizing, cooling to a certain temperature, and maintaining the pressure for later use;

step two: weighing solid NaOH, placing the solid NaOH in an alkali tank to prepare 0.1-0.5 mol/L alkali liquor, sterilizing at high pressure, and cooling to 60-80 ℃ for later use; weighing AlCl₃·6H₂O, placing the mixture in an alkaline tank to prepare 0.15-0.5 mol/L solution, and cooling the solution for later use after autoclaving;

step three: pumping an aluminum chloride solution into a customized reaction tank, opening a stirrer and adjusting the rotating speed to enable the solution to be uniformly stirred, keeping the temperature of the aluminum chloride solution to be 60-80 ℃, dropwise adding a sodium hydroxide solution at multiple points by adopting a customized multi-point nozzle at the dropping speed of 100-200 ml/min, simultaneously detecting the change of pH, dropwise adding the aluminum chloride solution and the sodium hydroxide solution when the pH reaches 4.0-4.5, keeping the dropping speed of the sodium hydroxide unchanged, and keeping the pH to be 4.0-4.5 by adjusting the dropping speed of the aluminum chloride;

step four: after the aluminum chloride solution is dripped, continuously dripping the sodium hydroxide solution until the pH value is 5.0-5.9, and stopping dripping;

step five: maintaining the reaction system at 80-90 ℃, keeping the tank ventilated and at constant pressure, continuously stirring, cooling to room temperature when the reaction system in the tank is concentrated to the target volume, and performing sterile subpackage and preservation for later use.

2. The method of claim 1, comprising the steps of:

the method comprises the following steps: cleaning a material tank and a reaction tank required for preparing the adjuvant, sterilizing, cooling to 60-80 ℃, and maintaining the pressure for later use;

step two: weighing solid NaOH, placing the solid NaOH in an alkali tank to prepare 0.1-0.5 mol/L alkali liquor, sterilizing at high pressure, and cooling to 60-80 ℃ for later use; weighing AlCl₃·6H₂O, placing the mixture in an alkaline tank to prepare 0.15-0.5 mol/L solution, and cooling to 60-80 ℃ for later use after autoclaving;

step three: pumping an aluminum chloride solution into a customized reaction tank, opening a stirrer and adjusting the rotating speed to enable the solution to be uniformly stirred, keeping the temperature of the aluminum chloride solution to be 60-80 ℃, dropwise adding a sodium hydroxide solution at multiple points by adopting a customized multi-point nozzle at the dropping speed of 100-200 ml/min, simultaneously detecting the change of pH, dropwise adding the aluminum chloride solution and the sodium hydroxide solution when the pH reaches 4.0-4.5, keeping the dropping speed of the sodium hydroxide unchanged, and keeping the pH to be 4.0-4.5 by adjusting the dropping speed of the aluminum chloride;

step four: after the aluminum chloride solution is dripped, continuously dripping the sodium hydroxide solution until the pH value is 5.0-5.9, and stopping dripping;

step five: maintaining the reaction system at 80-90 ℃, keeping the tank ventilated and at constant pressure, continuously stirring, cooling to room temperature when the reaction system in the tank is concentrated to the target volume, and performing sterile subpackage and preservation for later use.

3. The method of claim 1,

wherein, the first step: and (3) circularly cleaning a material tank and a reaction tank which are required by adjuvant preparation by using 0.3-0.5M alkali liquor for 30-60 min, then discharging the alkali liquor, then washing the alkali liquor by using purified water, detecting that the pH of the washing liquor is neutral, adding a proper amount of purified water, sterilizing at 121 ℃, cooling to 60-80 ℃ after 30min, maintaining the pressure at 0.05-0.06 MPa, and keeping the ventilation volume at 50-70L/min for later use.

4. The method of claim 1,

wherein, the step two: weighing solid NaOH, placing the solid NaOH in an alkali tank to prepare 0.1-0.5 mol/L alkali liquor, carrying out autoclaving at the temperature of 121 ℃ for 20-30 min, then cooling to 60-80 ℃, and maintaining the pressure at 0.05-0.06 MPa for later use; weighing AlCl₃·6H₂And O, placing the mixture into a material tank to prepare a 0.15-0.5 mol/L solution, carrying out autoclaving at 121 ℃ for 20-30 min, then cooling to 60-80 ℃, maintaining the pressure at 0.05-0.06 MPa, keeping the ventilation volume at 50-70L/min for later use, connecting the material tank with a reaction tank through a pipeline at 121 ℃, and carrying out sterilization for 20-30 min.

5. The method of claim 1,

wherein, the third step: setting the pressure in the material tank to be 0.07-0.08 Mpa, setting the ventilation volume to be 50-70L/min, opening a valve for connecting the material tank and the reaction tank, pumping the aluminum chloride solution into the reaction tank, opening a stirrer, setting the rotating speed to be 100-300 rpm, enabling the solution to be uniformly stirred, and keeping the temperature of the aluminum chloride solution to be 60-80 ℃. The method comprises the steps of dropwise adding a sodium hydroxide solution at multiple points by using a customized multi-point nozzle at a dropping speed of 50-200 ml/min, monitoring the change of pH, dropwise adding aluminum chloride and the sodium hydroxide solution at the same time when the pH reaches 4.0-4.5, keeping the dropping speed of the sodium hydroxide unchanged, and keeping the pH between 4.0-4.5 by adjusting the dropping speed of the aluminum chloride.

6. The method of claim 1,

wherein, the step four: and after the aluminum chloride solution is added, closing a connecting valve of the material tank and the reaction tank, and continuously adding the sodium hydroxide solution until the pH value is about 5.0-5.9 (the end point is determined by observing the state of the solution).

7. The method of claim 1,

wherein, the step five: maintaining the temperature at 80-90 ℃, the pressure at 0.05-0.06 Mpa and the ventilation volume at 50-200L/min, continuously stirring for 24 hours, cooling to room temperature when the reaction system is concentrated to 10-30% of the initial volume, aseptically sampling, subpackaging and storing for later use.

Technical Field

The invention belongs to the field of biological medicine, and relates to a preparation method of an aluminum hydroxide adjuvant with high concentration and nano-scale particle size, which is suitable for large-scale production.

Background

Adjuvants are substances that can nonspecifically enhance or alter the immune response of the body to an antigen. There are many substances with adjuvant effect, but only a few are currently approved for use in human vaccines. Aluminum adjuvants are common immunopotentiators, mainly aluminum hydroxide and aluminum phosphate, with aluminum hydroxide being the most widely used and having been used for nearly a hundred years.

The aluminum hydroxide adjuvant is weak crystal boehmite (PCB), which is an amphoteric compound and has positive charge when used under physiological pH value. Binding to antigen by electrostatic attraction, hydrophobic interaction, etc. The aluminum hydroxide adjuvant particles are uniform and fine, the specific surface area is large, and the adsorption rate is high. The preparation method is generally that alkali liquor is added into aluminum salt solution to generate fine aluminum compound particles_，Are mutually aggregated and precipitated. In the preparation process of the aluminum hydroxide adjuvant particles, the particles with at least three structures, namely amorphous hydrated aluminum hydroxide, boehmite (also called boehmite) and bayerite can be formed in the reaction process under the influences of the temperature, the pH value, the reactant dropping speed and other factors of a reaction system. Wherein, the amorphous hydrated aluminum hydroxide particles are loose, have very large specific surface area, have high adsorption rate to the antigen, but have poor structural stability, and are easily influenced by the antigen and the solution environment to generate structural change. Bayerite is an octahedral structure and has good structural stability, so that large particles are easily formed, the specific surface area of the bayerite is much smaller than that of amorphous hydrated aluminum hydroxide, and the adsorption rate of the bayerite to an antigen is much lower. Boehmite, also called boehmite, has a structure of an orthorhombic plate shape, has properties and adsorption capacity to antigens between those of amorphous hydrated aluminum hydroxide and bayerite, and aluminum hydroxide particles with a boehmite structure are more suitable for adsorbing antigen particles.

Vaccine preparations are continuously developed, and higher requirements are also made on the quality of adjuvants required by vaccines. The patent describes a method for preparing a high-concentration nano-scale aluminum hydroxide adjuvant by accurately controlling reaction conditions, which is suitable for large-scale production. The adjuvant prepared by the method has relatively single composition, stable and uniform properties, relatively concentrated particle size and good adsorption effect on antigen particles.

Disclosure of Invention

The invention provides a preparation method of an aluminum hydroxide adjuvant with nano-scale particle size and high concentration, which is suitable for large scale.

The invention relates to a method suitable for preparing an aluminum hydroxide adjuvant with high concentration and nano-scale particle size in a large scale. The method comprises the steps of reacting aluminum chloride with sodium hydroxide, accurately controlling the reaction speed, monitoring the pH value of a system and the particle size of a reaction product to generate an aluminum hydroxide colloid with the particle size of less than 1 micron, aging and stabilizing the aluminum hydroxide colloid at 80-90 ℃, then performing aseptic subpackage storage, and sampling to detect physical and chemical indexes. The method is simple and easy to operate, is suitable for large-scale production of the aluminum hydroxide adjuvant, the particle size of the produced adjuvant is less than 1 micron, and the concentration of aluminum ions of the adjuvant can reach more than 4.0mg/ml, and can be properly adjusted according to actual requirements.

In order to achieve the purpose, the technical scheme adopted by the invention is as follows:

the method comprises the following steps: cleaning a material tank and a reaction tank required by adjuvant preparation, sterilizing, cooling to a certain temperature, and maintaining the pressure for later use;

step two: weighing solid NaOH, placing the solid NaOH in an alkali tank to prepare 0.1-0.5 mol/L alkali liquor, sterilizing at high pressure, and cooling to 60-80 ℃ for later use; weighing AlCl₃·6H₂O, placing in an alkali tank for preparationPreparing 0.15-0.5 mol/L solution, sterilizing at high pressure, and cooling for later use;

step three: pumping an aluminum chloride solution into a customized reaction tank, opening a stirrer and adjusting the rotating speed to enable the solution to be uniformly stirred, keeping the temperature of the aluminum chloride solution to reach 60-80 ℃, dropwise adding a sodium hydroxide solution at multiple points by adopting a customized multi-point nozzle at a dropping speed of 100-200 ml/min, simultaneously detecting the change of pH, and when the pH reaches 4.0-4.5, dropwise adding the aluminum chloride solution and the sodium hydroxide solution at the same time, keeping the dropping speed of the sodium hydroxide unchanged, and keeping the pH between 4.0-4.5 by adjusting the dropping speed of the aluminum chloride.

Step four: and after the aluminum chloride solution is dripped, continuously dripping the sodium hydroxide solution until the pH value is between 5.0 and 5.9 (the end point is determined by observing the state of the solution), and stopping dripping.

Step five: maintaining the reaction system at 80-90 ℃, keeping the tank ventilated and at constant pressure, continuously stirring, cooling to room temperature when the reaction system in the tank is concentrated to the target volume, and performing sterile subpackage and preservation for later use.

Wherein the multi-point nozzle is customized, and the style diagram is shown in figure 1.

Preferably, the preparation method of the aluminum hydroxide adjuvant comprises the following steps:

the method comprises the following steps: cleaning a material tank and a reaction tank required for preparing the adjuvant, sterilizing, cooling to 60-80 ℃, and maintaining the pressure for later use;

step two: weighing solid NaOH, placing the solid NaOH in an alkali tank to prepare 0.1-0.5 mol/L alkali liquor, sterilizing at high pressure, and cooling to 60-80 ℃ for later use; weighing AlCl₃·6H₂O, placing the mixture in an alkaline tank to prepare 0.15-0.5 mol/L solution, and cooling to 60-80 ℃ for later use after autoclaving;

step three: pumping an aluminum chloride solution into a customized reaction tank, opening a stirrer and adjusting the rotating speed to enable the solution to be uniformly stirred, keeping the temperature of the aluminum chloride solution to be 60-80 ℃, dropwise adding a sodium hydroxide solution at multiple points by adopting a customized multi-point nozzle at the dropping speed of 100-200 ml/min, simultaneously detecting the change of pH, dropwise adding the aluminum chloride solution and the sodium hydroxide solution when the pH reaches 4.0-4.5, keeping the dropping speed of the sodium hydroxide unchanged, and keeping the pH to be 4.0-4.5 by adjusting the dropping speed of the aluminum chloride;

step four: after the aluminum chloride solution is dripped, continuously dripping the sodium hydroxide solution until the pH value is between 5.0 and 5.9 (the end point is determined by observing the state of the solution), and stopping dripping;

step five: maintaining the reaction system at 80-90 ℃, keeping the aeration and the constant pressure in the tank, continuously stirring (about 24 hours), cooling to room temperature when the reaction system in the tank is concentrated to the target volume, and aseptically subpackaging and storing for later use.

Wherein, the first step: circularly cleaning a material tank and a reaction tank which are required by adjuvant preparation by using 0.3-0.5M alkali liquor for 30-60 min, then discharging the alkali liquor, then washing the alkali liquor by using purified water, detecting that the pH of the washing liquor is neutral, adding a proper amount of purified water, sterilizing at 121 ℃, cooling to 60-80 ℃ after 30min, maintaining the pressure at 0.05-0.06 Mpa and the ventilation volume at 50-70L/min for later use;

wherein, the step two: weighing solid NaOH, placing the solid NaOH in an alkali tank to prepare 0.1-0.5 mol/L alkali liquor, carrying out autoclaving at the temperature of 121 ℃ for 20-30 min, then cooling to 60-80 ℃, and maintaining the pressure at 0.05-0.06 MPa for later use; weighing AlCl₃·6H₂Placing the O in a material tank to prepare 0.15-0.5 mol/L solution, sterilizing the solution at 121 ℃ for 20-30 min under high pressure, cooling the solution to 60-80 ℃, maintaining the pressure at 0.05-0.06 MPa and the ventilation volume at 50-70L/min for later use, connecting the material tank with a reaction tank through a pipeline at 121 ℃, and sterilizing the solution for 20-30 min;

wherein, the third step: setting the pressure in the material tank to be 0.07-0.08 Mpa, setting the ventilation volume to be 50-70L/min, opening a valve for connecting the material tank and the reaction tank, pumping the aluminum chloride solution into the reaction tank, opening a stirrer, setting the rotating speed to be 100-300 rpm, enabling the solution to be uniformly stirred, and keeping the temperature of the aluminum chloride solution to be 60-80 ℃. The method comprises the steps of dropwise adding a sodium hydroxide solution at multiple points by using a customized multi-point nozzle at a dropping speed of 50-200 ml/min, monitoring the change of pH, dropwise adding aluminum chloride and the sodium hydroxide solution at the same time when the pH reaches 4.0-4.5, keeping the dropping speed of the sodium hydroxide unchanged, and keeping the pH between 4.0-4.5 by adjusting the dropping speed of the aluminum chloride.

Wherein, the step four: and after the aluminum chloride solution is added, closing a connecting valve of the material tank and the reaction tank, and continuously adding the sodium hydroxide solution until the pH value is about 5.0-5.9 (the end point is determined by observing the state of the solution).

Wherein, the step five: maintaining the temperature at 80-90 ℃, the pressure at 0.05-0.06 Mpa and the ventilation volume at 50-200L/min, continuously stirring for 24 hours, cooling to room temperature when the reaction system is concentrated to 10-30% of the initial volume, aseptically sampling, subpackaging and storing for later use.

The invention is different from the existing preparation method of the aluminum hydroxide adjuvant, and has the innovation points that the forming conditions of three reaction products (amorphous hydrated aluminum hydroxide, boehmite and bayerite) of sodium hydroxide and aluminum chloride solution are utilized, and the reaction conditions, such as the system temperature, the change tendency of the system pH value, the reaction process time and other conditions are finely controlled, so as to prepare the high-concentration nano-scale aluminum hydroxide adjuvant with stable property.

Compared with the existing preparation method, the invention has the following advantages:

1. the invention only relates to a customized multipoint nozzle, other production equipment is not increased, and the production cost is hardly influenced;

2. the particle size of the adjuvant particles prepared by the invention is in a nanometer level, so that the immunogenicity of certain antigens can be effectively improved;

3. the adjuvant prepared by the invention has stable property, and can keep the size of nano-scale particles in a wider pH range;

4. the adjuvant prepared by the invention has high concentration which can reach 4.0-10.0 mg/ml, and the specific concentration of the product can be adjusted by the preparation process. Can solve the antigen adsorption problem that the antigen concentration is low and can not be increased again.

Drawings

FIG. 1 is a schematic view of a multi-point nozzle

FIG. 2 shows the results of particle size measurements of 2006091 batches of adjuvants

FIG. 3 shows the results of particle size detection of 2007211 adjuvants

FIG. 4 shows the results of particle size detection of 2007271 adjuvant batches

FIG. 5 shows the particle size detection results of a European imported adjuvant

Detailed description of the invention

In order to make the objects, technical solutions and advantages of the present invention more apparent, the technical solutions of the present invention will be clearly and completely described below with reference to the specific embodiments of the present invention, but the scope of the present invention is not limited to the following description.

Example 1:

the method comprises the following steps: and (3) circularly cleaning a material tank and a reaction tank which are required by adjuvant preparation by using 0.3-0.5M alkali liquor for 30-60 min, then discharging the alkali liquor, then washing the alkali liquor by using purified water, and detecting that the pH value of the washing liquor is neutral. Adding a proper amount of purified water, sterilizing at 121 ℃ for 30min, cooling to 60-80 ℃, maintaining the pressure at 0.05-0.06 MPa, and keeping the ventilation volume at 50-70L/min for later use;

step two: weighing solid NaOH, preparing 0.1-0.5 mol/L alkali liquor in an alkali tank, sterilizing at 121 ℃ for 20-30 min under high pressure, cooling to 60-80 ℃, and maintaining the pressure at 0.05-0.06 MPa for later use; weighing AlCl₃·6H₂O, preparing 0.15-0.5 mol/L solution in a material tank, sterilizing the solution at 121 ℃ for 20-30 min under high pressure, cooling the solution to 60-80 ℃, maintaining the pressure at 0.05-0.06 MPa and the ventilation volume at 50-70L/min for later use, connecting the material tank with a reaction tank through a pipeline at 121 ℃, and sterilizing the solution for 20-30 min;

step three: setting the pressure in the material tank to be 0.07-0.08 Mpa, setting the ventilation volume to be 50-70L/min, opening a valve for connecting the material tank and the reaction tank, pumping the aluminum chloride solution into the reaction tank, opening a stirrer, setting the rotating speed to be 100-300 rpm, enabling the solution to be uniformly stirred, and keeping the temperature of the aluminum chloride solution to be 60-80 ℃. The method comprises the steps of dropwise adding a sodium hydroxide solution at multiple points by using a customized multi-point nozzle at a dropping speed of 50-200 ml/min, monitoring the change of pH, dropwise adding aluminum chloride and the sodium hydroxide solution at the same time when the pH reaches 4.0-4.5, keeping the dropping speed of the sodium hydroxide unchanged, and keeping the pH between 4.0-4.5 by adjusting the dropping speed of the aluminum chloride.

Step four: and after the aluminum chloride solution is added, closing a connecting valve of the material tank and the reaction tank, and continuously adding the sodium hydroxide solution until the pH value is about 5.0-5.9 (the end point is determined by observing the state of the solution).

Step five: maintaining the temperature at 80-90 ℃, the pressure at 0.05-0.06 Mpa and the ventilation volume at 50-200L/min, continuously stirring for 24 hours, cooling to room temperature when the reaction system is concentrated to 10-30% of the initial volume, aseptically sampling, subpackaging and storing for later use.

Step six: the sampled aluminum hydroxide adjuvant is subjected to physical and chemical index detection according to corresponding items of 'tentative aluminum hydroxide adjuvant manufacturing and verification rules'.

1. Partial physicochemical detection result of adjuvant

2. Detection result of particle size of adjuvant colloid

See fig. 2-4.

Example 2 partial parameter comparison of the aluminium hydroxide adjuvant produced according to the invention with a certain european imported adjuvant:

1. partial physicochemical detection result of adjuvant

2. Particle size detection result of certain European imported adjuvant

See fig. 5.