CN108066759B - Aluminum hydroxide adjuvant and preparation method and application thereof - Google Patents
Aluminum hydroxide adjuvant and preparation method and application thereof Download PDFInfo
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Abstract
The invention provides an aluminum hydroxide adjuvant and a preparation method and application thereof. The aluminum salt solution and the sodium hydroxide solution are mixed and then are washed and exchanged by 0.75-0.95% of sodium chloride solution to prepare the aluminum salt solution. The invention improves the existing preparation process of the aluminum hydroxide adjuvant, and the method can realize that the surface charge and the particle size of the aluminum hydroxide adjuvant tend to be stable and the pH value is closer to the pH value of human body fluid, is suitable for the adsorption of one or more antigens, and meets the application requirements of various vaccines in preparations.
Description
Technical Field
The invention belongs to the field of vaccine preparations, and particularly relates to an aluminum hydroxide adjuvant, and a preparation method and application thereof.
Background
Aluminum adjuvants have been used for 90 years as immunological adjuvants for vaccines. It is also the only human vaccine adjuvant approved by FDA in the united states because of its high adsorption capacity and better adsorption to some proteinaceous antigens than other adjuvants. As early as 1926, glenny et al found that suspending the aluminum adjuvant with Diphtheria Toxin (DT) significantly increased the immunogenicity of diphtheria toxin. The aluminum adjuvants currently in use are mainly aluminum hydroxide, aluminum phosphate, aluminum hydroxide-aluminum phosphate mixtures.
The aluminum hydroxide adjuvant is widely used for preparing vaccines, and the adsorption capacity of the aluminum hydroxide adjuvant is one of key indexes in the preparation of the vaccines. The aluminum hydroxide adjuvant particles are uniform and fine, and have large specific surface area, so that the condition of high adsorption rate is met. In the vaccine preparation, the higher the adsorption rate, the more remarkable the immune effect will be.
The aluminum hydroxide adjuvant is prepared by adding alkali liquor to aluminum salt solution to generate neutral uncharged Al (H) 2 O 3 )(OH) 3 Aggregate with each other and precipitate. Aluminum hydroxide is an amphoteric compound having an isoelectric point of 11.4, and therefore, when used at physiological pH, is positively charged and binds to an antigen by electrostatic attraction, hydrophobic interaction, or the like.
Disclosure of Invention
The invention provides an aluminum hydroxide adjuvant and a preparation method and application thereof, and the method ensures that the physicochemical properties of the prepared aluminum hydroxide adjuvant, such as particle size, adsorption capacity and the like, are consistent with those of commercialization.
The technical scheme of the invention is as follows:
a preparation method of an aluminum hydroxide adjuvant comprises the following steps:
1) Simultaneously dripping 1-14 percent (mass fraction) of aluminum salt solution and 0.4-4 percent (mass fraction) of sodium hydroxide solution into 0.85-0.95 percent (mass fraction) of sodium chloride solution under the condition of stirring, and maintaining the pH value of a reaction system to be 5.5-6.4 by adjusting the dripping speed of the aluminum salt solution and the sodium hydroxide solution; stopping dropping the aluminum salt solution when the concentration of aluminum ions in the reaction system reaches 2-10 mg/mL; continuously dropwise adding the sodium hydroxide solution until the pH value of a reaction system is 6.4-7.2, namely a titration end point;
the aluminum salt is potassium aluminum sulfate (KAl (SO) 4 )·12H 2 O)；
2) Stopping stirring, removing supernatant after the reaction system is static, and changing the solution by adopting 0.75-0.95% (mass fraction) of sodium chloride solution to thoroughly remove free potassium ions and sulfate ions;
3) Performing damp-heat sterilization;
the wet heat sterilization can adopt a pharmacopeia method, generally adopts the temperature of 121 ℃, and lasts for 30-60 minutes;
4) After moist heat sterilization, cooling to room temperature under the condition of stirring; thus obtaining the aluminum hydroxide adjuvant.
The room temperature according to the invention is generally from 15 to 25 ℃.
Further, the stirring speed in the step 1) is 100-1000rpm, preferably 300-700rpm.
Further, the concentration of the sodium chloride solution in the step 1) is 0.9% (mass fraction).
Further, the concentration of the aluminum salt solution in the step 1) is 8-10% (mass fraction).
Further, the concentration of the sodium hydroxide solution in the step 1) is 1-4% (mass fraction), preferably 2%.
Further, step 1) maintains the pH value of the reaction system in the range of 5.9-6.2 by adjusting the dropping speed of the aluminum salt solution and the sodium hydroxide solution.
Further, after the aluminum salt solution is dripped in the step 1), continuously dripping the sodium hydroxide solution until the pH value of the reaction system is 6.5-6.8, namely the titration end point.
Further, in the step 2), 0.8-0.9% (mass fraction) of sodium chloride solution is adopted for liquid change. Generally, the substitution is carried out 2 to 6 times.
Further, the stirring speed in the step 4) is 100-1000rpm, preferably 300-700rpm.
On the basis of the common knowledge in the field, the above preferred conditions can be combined with each other to obtain the preferred embodiments of the invention.
A preferred embodiment of the present invention is: a preparation method of an aluminum hydroxide adjuvant comprises the following steps:
1) Simultaneously dripping 10 percent (mass fraction) of aluminum salt solution and 2 percent (mass fraction) of sodium hydroxide solution into 0.9 percent (mass fraction) of sodium chloride solution under the condition of stirring at 500rpm, and maintaining the pH value range of a reaction system to be 6.0 by adjusting the dripping speed of the aluminum salt solution and the sodium hydroxide solution; stopping dropping the aluminum salt solution when the concentration of aluminum ions in the reaction system reaches 2-10 mg/mL;
continuously dropwise adding the sodium hydroxide solution until the pH value of the reaction system is 6.8, namely the titration end point;
the aluminum salt is aluminum potassium sulfate;
2) Stopping stirring, removing supernatant after the reaction system is static, and adopting 0.9% (mass fraction) of sodium chloride solution to change the solution so as to thoroughly remove free potassium ions and sulfate ions;
3) Moist heat sterilization: adopting the temperature of 121 ℃ for 30-60 minutes;
4) After moist heat sterilization, the temperature is reduced to room temperature under the stirring condition of 100-1000rpm, and the aluminum hydroxide adjuvant can be prepared.
The invention also comprises the aluminum hydroxide adjuvant prepared by the method.
The particle size of the aluminum hydroxide adjuvant prepared by the invention is 1.4-12.6nm, and preferably 2.5-8.9nm.
A preferred embodiment of the present invention is: an aluminum hydroxide adjuvant has particle diameter of 2.5-8.9nm, particle size distribution shown in figure 1, and isoelectric point of 10.2.
The invention also comprises the application of the aluminum hydroxide adjuvant in the preparation of vaccines; the vaccine is preferably a human vaccine, and includes but is not limited to virus adsorption vaccines and protein adsorption vaccines.
Preferably, the aluminum concentration of the aluminum hydroxide adjuvant in the vaccine is 0.2-0.6mg/mL; it is further preferred that the final adsorbed concentration of aluminium in the vaccine is 0.3-0.5mg/mL.
The invention improves the existing preparation process of the aluminum hydroxide adjuvant, and the method can realize that the surface charge and the particle size of the aluminum hydroxide adjuvant tend to be stable and the pH value is closer to the pH value of human body fluid, is suitable for the adsorption of one or more antigens, and meets the application requirements of various vaccines in preparations.
Drawings
FIG. 1 is a particle size diagram of an aluminum hydroxide adjuvant prepared according to the present invention.
Detailed Description
The following examples are intended to illustrate the invention, but are not intended to limit the scope of the invention. The examples do not show the specific techniques or conditions, according to the technical or conditions described in the literature in the field, or according to the product specifications. The reagents or instruments used are conventional products available from regular distributors, not indicated by the manufacturer.
Example 1 preparation of an aluminum hydroxide adjuvant
(1) Weigh KAl (SO) 4 )·12H 2 Dissolving O in water for injection to prepare 10% aluminum salt solution; sodium hydroxide was weighed and dissolved in water for injection to prepare a 2% sodium hydroxide solution.
(2) 0.9% sodium chloride solution was added to the reactor and stirring was turned on at 500rpm.
(3) Simultaneously dripping the aluminum salt solution and the sodium hydroxide solution into a reactor, maintaining the pH value of the reaction system to be 6.0 by adjusting the speed of a peristaltic pump, and stopping dripping the aluminum salt solution when the concentration of aluminum ions in the reaction system reaches 2-10 mg/mL; the sodium hydroxide solution was continuously added dropwise until the end point reached pH 6.8, and stirring was continued for 30 minutes.
(4) Standing, removing supernatant, adding 0.9% sodium chloride solution, and repeatedly replacing for 4 times to remove free potassium ions and sulfate ions;
(5) Collecting aluminum hydroxide adjuvant, and performing wet heat sterilization at 121 ℃ for 30min. After the sterilization is finished, when the temperature is reduced to 80 ℃, stirring is carried out, the stirring speed is 500rpm, the stirring is carried out until the temperature is room temperature (20-25 ℃), and the aluminum hydroxide adjuvant is collected.
Example 2 detection of adsorption Capacity of aluminum hydroxide adjuvant
The adsorption capacity was examined by the method of European pharmacopoeia using the aluminum hydroxide adjuvant prepared in example 1 and Denmark Alhydrogel adjuvant.
(1) Experimental methods
The aluminum hydroxide adjuvant (sample group) and the Denmark Alhydrogel adjuvant (control group) prepared in example 1 were diluted to an aluminum content of 5mg/mL and a pH of 6.0, respectively. BSA (bovine serum albumin) solutions with pH of 6.0 and a content of 10mg/mL were prepared, diluted to concentrations of 1mg/mL, 2mg/mL, 3mg/mL, 5mg/mL, 8mg/mL and 10mg/mL, respectively, and the pH was adjusted to 6.0. An aluminum hydroxide adjuvant was mixed with the above series of BSA solutions in a volume ratio of 1. And centrifuging to collect supernatant, and detecting the protein content of the supernatant by adopting a lowry method.
(2) Results of the experiment
The results are shown in Table 1 (the results are the BSA content in the supernatant mg/mL)
TABLE 1
The results show that: no protein is detected in the supernatant of three BSA solutions with low concentration content, three proteins with high concentration present increasing trend, and the sample group is consistent with the control group and meets the qualified standard.
Example 3 determination of particle size distribution of aluminum hydroxide adjuvant
The physical and chemical parameters of the aluminum hydroxide adjuvant and the Denmark Alhydrogel adjuvant prepared in the example 1 are detected;
(1) Detecting the isoelectric point of the sample aluminum hydroxide adjuvant by adopting a Marvens Zetaszer potentiometer and an MPT-2 automatic titrator;
(2) The particle size of the sample aluminum hydroxide adjuvant was measured using a malvern mastersizer 3000 laser particle sizer.
The results of the above assays are shown in table 2 below and in fig. 1:
table 2: detection result of physical and chemical parameters of aluminum hydroxide adjuvant
As can be seen from the results of table 2 and fig. 1, the aluminum hydroxide adjuvant prepared by the method of the present invention is consistent with the particle size distribution of the commercial product.
Although the invention has been described in detail with respect to the general description and the specific embodiments thereof, it will be apparent to those skilled in the art that modifications and improvements can be made based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.
Claims (3)
1. The preparation method of the aluminum hydroxide adjuvant is characterized by comprising the following steps of:
1) Simultaneously dripping 10% of aluminum potassium sulfate solution and 2% of sodium hydroxide solution into 0.9% of sodium chloride solution under the condition of stirring rotation speed of 500rpm, and maintaining the pH value range of a reaction system to be 6.0 by adjusting the dripping speeds of the aluminum potassium sulfate solution and the sodium hydroxide solution; stopping dripping the aluminum potassium sulfate solution when the concentration of aluminum ions in the reaction system reaches 2-10 mg/mL; continuously dropwise adding the sodium hydroxide solution until the pH value of the reaction system is 6.8, namely the titration end point;
2) Stirring for 30 minutes after reaching the titration end point, stopping stirring, removing a supernatant after a reaction system is static, replacing the solution by adopting 0.9% sodium chloride solution, repeatedly replacing for 4 times, and collecting an aluminum hydroxide adjuvant;
3) Performing damp-heat sterilization;
4) After moist heat sterilization, cooling to room temperature under the condition that the stirring speed is 100-1000 rpm; thus obtaining the aluminum hydroxide adjuvant.
2. The method according to claim 1, wherein the moist heat sterilization in step 3) is performed at 121 ℃ for 30min.
3. The method according to claim 1, wherein the stirring speed in the step 4) is 500rpm.
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| CN112569350B (en) * | 2019-09-30 | 2024-02-09 | 成都迈科康生物科技有限公司 | Aluminum hydroxide adjuvant and preparation method and application thereof |
| CN111175348A (en) * | 2019-12-27 | 2020-05-19 | 深圳康泰生物制品股份有限公司 | Detection method of isoelectric point of aluminum hydroxide adjuvant and application of detection method in preparation of vaccine |
| CN111184860B (en) * | 2020-02-21 | 2022-05-06 | 中牧实业股份有限公司 | Improved aluminum hydroxide adjuvant and preparation method thereof |
| CN113666404B (en) * | 2021-09-23 | 2023-07-28 | 北京智飞绿竹生物制药有限公司 | Preparation method of high-concentration nanoscale aluminum hydroxide adjuvant suitable for large-scale production |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102167372A (en) * | 2010-11-30 | 2011-08-31 | 汪晋强 | Method for preparing aluminum hydroxide and iron hydroxide coproduced with potassium hydroxide and sodium sulfate by using vanadium slurry |
| CN104367998A (en) * | 2014-11-12 | 2015-02-25 | 武汉生物制品研究所有限责任公司 | Preparation method of aluminum hydroxide adjuvant |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102167372A (en) * | 2010-11-30 | 2011-08-31 | 汪晋强 | Method for preparing aluminum hydroxide and iron hydroxide coproduced with potassium hydroxide and sodium sulfate by using vanadium slurry |
| CN104367998A (en) * | 2014-11-12 | 2015-02-25 | 武汉生物制品研究所有限责任公司 | Preparation method of aluminum hydroxide adjuvant |
Non-Patent Citations (1)
| Title |
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| 氢氧化铝佐剂配制工艺的优化;李德娟 等;《中国生物制品学杂志》;20101031;第23卷(第10期);第1135-1137页 全文，尤其是摘要，第2节和第3节 * |
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