Preparation method of aluminum hydroxide adjuvant

Abstract

The invention relates to a preparation method of an aluminum hydroxide adjuvant. Specifically, the method comprises the following steps: simultaneously spraying an alkali solution and an aluminum chloride solution into a reaction pipeline in a vaporific form through two pump bodies by virtue of fluid pressure difference, or spraying the alkali solution into a flowing aluminum chloride solution reaction pipeline in a vaporific form by virtue of fluid pressure difference, and controlling the temperature in the pipeline to be 70-80 DEG C for reaction, the reaction liquid flows out through a pipeline within a certain time to obtain an aluminum hydroxide adjuvant crude product, and then the aluminum hydroxide adjuvant is obtained through centrifugation, ion removal, sterilization and packaging. The quality index of the aluminum hydroxide adjuvant obtained by the method is good, stable and uniform, and the immunoadsorption effect of the adjuvant and the quality, effectiveness and safety of a vaccine are ensured. The method is simple in technological process, the whole process is continuously carried out, the barrier of intermittent operation is broken through, operation is simple, and the method can be applied to large-scale production.

Description

Technical Field

[0001] The invention belongs to the field of vaccine adjuvants, and particularly relates to a method for preparing an aluminum hydroxide adjuvant. Background Art

[0002] Aluminum hydroxide has a history of nearly 100 years as an adjuvant component of vaccines. Aluminum hydroxide adjuvant can improve the effectiveness of vaccines by increasing antibodies. It is currently widely used in dozens of vaccines, including hepatitis B, diphtheria, pertussis and other commonly used vaccines for children.

[0003] The existing aluminum hydroxide adjuvant production methods mainly include sodium hydroxide method and ammonia method. All of them are titration reactions, which belong to the category of intermittent reactions. Specifically, sodium hydroxide solution or ammonia solution is added dropwise to aluminum chloride. The aluminum hydroxide adjuvant obtained is seriously affected by the drop rate, reaction temperature, stirring speed and pH value of the reaction end point; at the same time, the aluminum hydroxide adjuvant solution is prone to agglomeration and flocculation during sterilization and storage, and the adjuvant solution is prone to instability and needs to be re-prepared, which increases the preparation cost. In addition, studies have shown that when the particle size of the adjuvant and antigen conjugate increases, the presentation of antigens by antigen-presenting cells is weakened, affecting the immune effect. The aluminum hydroxide adjuvant produced by the ammonia method has a small particle size of nanometers. However, in the production of ammonia adjuvants, ammonia water is added to the aluminum chloride solution in a stream-added manner. At the moment of contact, more large particulate matter will be produced, and non-hydroxy aluminum oxide precipitation will be formed after dialysis and high pressure, which will lead to large batch differences in adjuvant appearance, particle size, etc., affecting the quality of the adjuvant, which is not conducive to batch consistency control.

[0004] The continuous development of vaccine products has put forward higher requirements on the quality of adjuvants needed for vaccines. A high-quality production method is urgently needed. Summary of the invention

[0005] The object of the present invention is to provide a method for continuously producing aluminum hydroxide adjuvant.

[0006] In a first aspect of the present invention, a method for preparing a continuous aluminum hydroxide adjuvant is provided, wherein the method comprises the following steps:

[0007] (1) introducing 0.05-0.5 mol / L alkaline solution and 0.05-0.3 mol / L aluminum chloride solution into a reaction pipe, reacting at 70-80° C. to obtain a reaction mixture;

[0008] (2) cooling the reaction mixture and centrifuging it to filter the precipitate;

[0009] (3) adding sodium chloride solution to the precipitate for washing, thereby removing residual ions;

[0010] (4) sterilizing and packaging the product obtained in step (3) to obtain a finished aluminum hydroxide adjuvant.

[0011] In another preferred embodiment, in the step (2), the centrifugation condition is a rotation speed of 6000-8000 rpm.

[0012] In another preferred embodiment, in the step (2), the centrifugation time is 8-12 min.

[0013] In another preferred embodiment, in the step (2), the centrifugation condition is a rotation speed of 7000 rpm and a time of 10 min.

[0014] In another preferred embodiment, the alkaline solution and the aluminum chloride solution are atomized by an atomizer before being introduced into the reaction pipeline, and then sprayed into the pipeline reactor in the form of mist.

[0015] In another preferred embodiment, in the step (1), the introduction rate of the alkaline solution is 10-15 ml / min.

[0016] In another preferred embodiment, in the step (1), the introduction rate of the aluminum chloride solution is 10-15 ml / min.

[0017] In another preferred embodiment, in the step (1), the atomized alkaline solution is sprayed in the form of mist into a pipeline reactor into which an atomized aluminum chloride atmosphere is previously introduced.

[0018] In another preferred embodiment, the alkaline solution is selected from the following group: sodium hydroxide solution, or ammonia water.

[0019] In another preferred embodiment, the atomizer is selected from the following group: a high-pressure atomizer, a high-speed shearing machine, or a combination thereof.

[0020] In another preferred embodiment, the molar ratio of sodium hydroxide to aluminum chloride during the reaction of step (1) is 2 to 6:1.

[0021] In another preferred embodiment, the molar ratio of sodium hydroxide to aluminum chloride is 2 to 4:1.

[0022] In another preferred embodiment, the molar ratio of sodium hydroxide to aluminum chloride is 3:1.

[0023] In another preferred embodiment, in the step (1), the alkaline solution enters the tubular reactor in the form of atomization, and the aluminum chloride solution enters the tubular reactor simultaneously in the form of atomization or at a constant rate.

[0024] In another preferred embodiment, during the reaction, the pH of the system is controlled to be 5.5-8.0.

[0025] In another preferred embodiment, the step (1) further comprises: filtering the alkaline solution and the aluminum chloride solution with a 0.45 μm filter element before entering the reaction pipeline.

[0026] In another preferred embodiment, the preparation of the alkaline solution and the aluminum chloride solution is completed in a preparation tank.

[0027] In another preferred embodiment, the preparation tank is equipped with a 0.1-1.0 μm filter element.

[0028] In another preferred embodiment, the method further comprises: repeating step (3) so that the residual ion concentration reaches the allowable limit;

[0029] Among them, chloride ≤0.33wt%, nitrate ≤100ppm, sulfate ≤0.5wt%, ammonium salt ≤50ppm, arsenic ≤1ppm, iron ≤15ppm, heavy metal ≤20ppm.

[0030] In another preferred embodiment, the washing comprises: dissolving the precipitate obtained in step (2) into a suspension with a sodium chloride solution, and then centrifuging to obtain a precipitate.

[0031] In another preferred embodiment, the concentration of the solid phase in the suspension is 3-6 mg / mL.

[0032] In another preferred embodiment, in the step (3), the concentration of the sodium chloride solution is 0.1-0.3 wt %.

[0033] In another preferred embodiment, in the step (3), the temperature of the sodium chloride solution is 30-70°C.

[0034] In another preferred embodiment, in the step (3), the concentration of the sodium chloride solution is 0.1-0.2 wt %.

[0035] In another preferred embodiment, in the step (3), the temperature of the sodium chloride solution is 45-55°C.

[0036] In another preferred embodiment, the washing comprises: adding the precipitate to a sodium chloride solution to form

[0037] In another preferred embodiment, in step (4), the sterilization is performed at a high temperature of 110-130°C.

[0038] In another preferred embodiment, the high temperature sterilization time is 20-40 minutes per time.

[0039] In another preferred embodiment, the high temperature sterilization is performed 1 to 10 times.

[0040] In another preferred embodiment, the sterilization temperature in step (4) is 121° C. and the time is 30 min.

[0041] In the second aspect of the present invention, there is provided an aluminum hydroxide adjuvant obtained by the method as described in the first aspect of the present invention.

[0042] In the third aspect of the present invention, there is provided a use of the aluminum hydroxide adjuvant as described in the second aspect of the present invention for preparing human vaccines or veterinary vaccines.

[0043] In the fourth aspect of the present invention, there is provided an apparatus for preparing aluminum hydroxide using the method described in the first aspect of the present invention, the apparatus comprising: a preparation tank, a disperser, a tubular packed bed and a centrifuge tank.

[0044] It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features specifically described below (such as embodiments) can be combined with each other to form a new or preferred technical solution. Due to space limitations, they will not be described one by one here. BRIEF DESCRIPTION OF THE DRAWINGS

[0045] Figure 1 This is a diagram of the aluminum hydroxide adjuvant production device of the present invention.

[0046] Figure 2 This is the particle size distribution diagram of aluminum hydroxide adjuvant sample 1.

[0047] Figure 3 This is the particle size distribution diagram of aluminum hydroxide adjuvant sample 2.

[0048] Figure 4 This is the particle size distribution diagram of aluminum hydroxide adjuvant sample 3.

[0049] Figure 5 This is the particle size distribution diagram of aluminum hydroxide adjuvant sample 4.

[0050] Figure 6 This is a timeline graph of the weight of mice after plague vaccine injection. DETAILED DESCRIPTION

[0051] The inventors have conducted extensive and in-depth research, and through a large number of screenings and tests, provided a method for continuously producing aluminum hydroxide adjuvants. The method adopts a continuous control mode, and obtains a two-phase mist dispersion liquid by a high-pressure microdispersor or a high-speed shearing machine. The aluminum hydroxide adjuvant obtained by the dispersion liquid reaction has stable suspension, is easy to disperse, and has uniform particles, which is helpful for further producing vaccine products with better uniformity. At the same time, the production process is simple and convenient for large-scale production. The present invention was completed on this basis.

[0052] the term

[0053] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0054] As used herein, when used in reference to a specific recited numerical value, the term "about" means that the value may vary by no more than 1% from the recited value. For example, as used herein, the expression "about 100" includes all values ​​between 99 and 101 (e.g., 99.1, 99.2, 99.3, 99.4, etc.).

[0055] As used herein, the term "comprising" or "including (comprising)" may be open, semi-closed and closed. In other words, the term also includes "consisting essentially of" or "consisting of".

[0056] As used herein, the term "room temperature" or "normal temperature" refers to a temperature of 4-40°C, preferably, 25±5°C.

[0057] As used herein, the terms "tube reactor", "reaction tube", "tubular packed bed" or "tube" are used interchangeably and refer to a tube-shaped reactor for the alkali solution and the aluminum chloride solution.

[0058] The main advantages of the present invention include:

[0059] 1. The aluminum hydroxide adjuvant obtained by the method of the present invention has less particulate matter, stable suspension, easy dispersion, easy control of product parameters, good, stable and uniform quality indicators, small batch differences in appearance, particle size, etc., which is conducive to batch consistency control;

[0060] 2. The aluminum hydroxide adjuvant obtained by the method of the present invention has good immunoadsorption effect, which can further improve the quality, effectiveness and safety of vaccines;

[0061] 3. The method of the present invention is a continuous controlled production method, which is conducive to expanding the production scale and stabilizing the adjuvant production in case of emergencies;

[0062] 4. The method of the present invention can also effectively solve the problems of high production cost, complicated steps, long cycle and the like in the existing intermittent reaction process, greatly improve the operation reaction time, reduce the amount of workers, reduce the enterprise cost, and has the advantages of simple process and high production efficiency.

[0063] The present invention will be further described below in conjunction with specific implementation. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. The experimental methods in the following examples where specific conditions are not specified are usually performed under conventional conditions, such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the conditions recommended by the manufacturer. Unless otherwise stated, percentages and parts are calculated by weight.

[0064] Unless otherwise defined, all professional and scientific terms used herein have the same meanings as those familiar to those skilled in the art. In addition, any methods and materials similar or equivalent to those described herein can be applied to the methods of the present invention. The preferred implementation methods and materials described herein are for demonstration purposes only.

[0065] Example 1

[0066] Preparation of sodium hydroxide solution: prepare 0.3 mol / L sodium hydroxide solution or 20 L of ammonia water in a sodium hydroxide preparation tank, filter the prepared solution through a 0.45 um filter element and set aside.

[0067] Preparation of aluminum chloride solution: prepare 8L of 0.1mol / L aluminum chloride solution in an aluminum chloride preparation tank, filter the prepared solution through a 0.45um filter element and set aside.

[0068] Example 2

[0069] The sodium hydroxide solution was added in the form of spray through the pipeline A spray head by fluid pressure difference, and the aluminum chloride solution was simultaneously added to the pipeline reactor through the symmetrically positioned B spray head. The pipeline temperature was controlled at 70-80° C. for reaction. For specific conditions, see Table 1 below.

[0070] During the reaction, the sodium hydroxide solution and the aluminum chloride solution simultaneously enter the above-mentioned pipeline reactor in an atomized form, and the amount of the sodium hydroxide solution added is adjusted in real time to a pH value set by the system.

[0071] The molar ratio of sodium hydroxide and aluminum chloride consumed in the mixed reaction is about 3.0.

[0072] After the reaction liquid flows out of the tubular packed bed within a certain period of time, it is cooled and centrifuged at a speed of 7000 rpm for 10 minutes to obtain a precipitate, which is dissolved into a 5 mg / ml suspension with a 0.15 wt% NaCl aqueous solution at 50°C, centrifuged, and operated multiple times until the residual ions obtained meet the requirements of the pharmacopoeia. The mixed solution is sterilized at 121°C for 30 minutes, and the subpackage is sterilized again at 121°C for 30 minutes to obtain the finished aluminum hydroxide adjuvant.

[0073] Comparative Example 1 (Changing the feeding method)

[0074] The sodium hydroxide solution is added to the flowing aluminum chloride solution pipeline reactor through pipeline A in the form of constant rate dropwise addition through fluid pressure difference, and the pipeline temperature is controlled at 70-80°C for reaction. For specific conditions, see the table below.

[0075] During the reaction, the sodium hydroxide solution is dripped into the above-mentioned pipeline reactor at a constant rate, and the amount of the sodium hydroxide solution added is adjusted in real time to a pH value set by the system. The molar ratio of the sodium hydroxide and aluminum chloride consumed in the mixed reaction is about 3.0.

[0076] After the reaction liquid flows out of the tubular packed bed within a certain period of time, it is cooled and centrifuged at a speed of 7000 rpm for 10 minutes to obtain a precipitate, which is dissolved into a 5 mg / ml suspension with a 0.15% NaCl aqueous solution at 50°C, centrifuged, and operated multiple times until the residual ions obtained meet the requirements of the pharmacopoeia. The mixed solution is sterilized at 121°C for 30 minutes, and the subpackage is sterilized again at 121°C for 30 minutes to obtain the finished aluminum hydroxide adjuvant.

[0077] Example 2

[0078] The sodium hydroxide solution is added into the flowing aluminum chloride solution pipeline reactor through the pipeline A head in the form of spray or shear through the fluid pressure difference. The pipeline temperature is controlled at 70-80°C for reaction. The specific conditions are shown in Table 1 below.

[0079] During the reaction, the sodium hydroxide solution enters the above-mentioned pipeline reactor in the form of atomization, and the amount of the sodium hydroxide solution added is adjusted in real time to a pH value set by the system.

[0080] The molar ratio of sodium hydroxide and aluminum chloride consumed in the mixed reaction is about 3.0.

[0081] After the reaction liquid flows out of the tubular packed bed within a certain period of time, it is cooled and centrifuged at a speed of 7000 rpm for 10 minutes to obtain a precipitate, which is dissolved into a 5 mg / ml suspension with a 0.15% NaCl aqueous solution at 50°C, centrifuged, and operated multiple times until the residual ions obtained meet the requirements of the pharmacopoeia. The mixed solution is sterilized at 121°C for 30 minutes, and the subpackage is sterilized again at 121°C for 30 minutes to obtain the finished aluminum hydroxide adjuvant.

[0082] Table 1 Reaction parameters and corresponding performance comparison table

[0083]

[0084]

[0085] It can be seen from Table 1 that:

[0086] 1. The parameters such as pH, appearance, precipitation rate, conductivity, adsorption rate, Zeta potential, isoelectric point, particle size DV (90) of the aluminum hydroxide adjuvant prepared by the flow reaction method of the present invention are consistent with those of commercial adjuvants. The quality of the obtained product is even better than that of the current commercial products, and the particle size is uniform.

[0087] 2. The adjuvant particles obtained are related to the pH value and the addition speed. The higher the pH, the larger the particle size, the greater the precipitation performance, and the lower the protein adsorption rate, which does not meet the standard.

[0088] 3. In the reaction system, when the addition rate is too fast or the pH is high, the adsorption rate of the obtained product does not meet the standard and the product appears to be agglomerated. Therefore, it is preferred to react in the range of pH = 5.0-7.3 (more preferably 5.5-7.0).

[0089] 4. Compared with the spraying method, the flow feeding method is easy to produce large particles. Although the adsorbed protein meets the standard, the particle size is large, indicating that the product is easy to aggregate during storage and is not easy to store for a long time. In a preferred approach, at least one of the alkali and alumina material flows is added by spraying, atomizing or shearing. Preferably, the alkali is added by spraying, atomizing or shearing.

[0090] Compared with the prior art, the method described in this aspect greatly improves the uniformity of particle preparation, can well control the size of particles, and keeps the precipitation rate within the range required by the pharmacopoeia. Compared with intermittent reaction, the flow reaction method of the present invention greatly shortens the operation reaction time, improves production efficiency, reduces the amount of workers, reduces enterprise costs, and most importantly, the obtained product parameters are easier to control and the quality is more uniform.

[0091] Example 4: Safety testing of plague vaccine prepared with aluminum hydroxide adjuvant

[0092] 60 mice, half male and half female, were randomly divided into 6 groups, namely sample group 1, sample group 2, sample group 3, sample group 4, commercial adjuvant group 1, and commercial adjuvant group 2, with 10 mice in each group. The corresponding vaccine / adjuvant was injected into the inner thigh muscle of each group of mice, 0.2 ml / mouse, and the mice were marked. The injection site of the mice was observed for 3 consecutive days for whether there were nodules, redness and swelling, and the body temperature and body weight were measured every other day. After 2 weeks, the mice were boosted with immunization once, and the mice were killed in the third week after the initial immunization.

[0093] like Figure 6As shown, the injection sites of mice in each group were observed for 3 consecutive days after the initial immunization, and no hard nodules or redness or swelling occurred in the mice; the body temperature of mice in each group was monitored every 1 day for 3 consecutive weeks, and no fever occurred in the mice; the body weight of mice in each group was monitored every 1 day for 3 consecutive weeks, and the body weight of mice increased steadily with the increase of weeks, and no abnormal phenomenon occurred in the mice. The experimental results show that the vaccine containing the adjuvant obtained by the method of the present invention is highly safe and has no other abnormal reactions.

[0094] All documents mentioned in the present invention are cited as references in this application, just as each document is cited as reference individually. In addition, it should be understood that after reading the above teachings of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the claims attached to this application.

Claims

1. A method for preparing a continuous aluminum hydroxide adjuvant, characterized in that: The method comprises the following steps: (1) introducing 0.05-0.5 mol / L alkaline solution and 0.05-0.3 mol / L aluminum chloride solution into a reaction pipeline, reacting at 70-80° C. to obtain a reaction mixture; (2) cooling the reaction mixture and centrifuging it to filter the precipitate; (3) adding sodium chloride solution to the precipitate for washing, thereby removing residual ions; (4) sterilizing and packaging the product obtained in step (3) to obtain a finished aluminum hydroxide adjuvant.

2. The method according to claim 1, characterized in that In the step (1), the alkaline solution and the aluminum chloride solution are atomized by an atomizer before being introduced into the reaction pipeline, and then sprayed into the pipeline reactor in the form of mist.

3. The method according to claim 1, characterized in that In the step (1), the atomized alkaline solution is sprayed in the form of mist into a pipeline reactor into which an atomized aluminum chloride atmosphere is previously introduced.

4. The method according to claim 1, characterized in that The alkaline solution is selected from the following group: sodium hydroxide solution or ammonia water.

5. The method according to claim 1, characterized in that In the step (1), the alkaline solution enters the tubular reactor in the form of atomization, and the aluminum chloride solution enters the tubular reactor simultaneously in the form of atomization or at a constant rate.

6. The method according to claim 1, characterized in that: The step (1) further comprises: filtering the alkaline solution and the aluminum chloride solution with a 0.45 μm filter element before entering the reaction pipeline.

7. The method according to claim 1, characterized in that: The method further comprises: repeating step (3) so that the residual ion concentration reaches the allowable limit; Among them, chloride ≤0.33wt%, nitrate ≤100ppm, sulfate ≤0.5wt%, ammonium salt ≤50ppm, arsenic ≤1ppm, iron ≤15ppm, heavy metal ≤20ppm.

8. The method according to claim 1, characterized in that In the step (3), the concentration of the sodium chloride solution is 0.1-0.3wt%.

9. An aluminum hydroxide adjuvant obtained by the method according to any one of claims 1 to 7.

10. The use of the aluminum hydroxide adjuvant as claimed in claim 9, characterized in that Used to prepare human vaccines or veterinary vaccines.

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