Aluminum phosphate adjuvant and preparation method thereof
Micron-scale aluminum phosphate adjuvant was prepared by chemical precipitation method and the particle size was adjusted using different buffer systems, which solved the problems of poor process stability and difficult particle size regulation in the prior art, and achieved uniformity of micro-nano-size aluminum phosphate adjuvant Sex and stability.
Patent Information
- Application Number
- CN202411593791.3
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-11-08
- Publication Date
- 2025-02-18
AI Technical Summary
In the prior art, the preparation process of micro-nano-sized aluminum phosphate adjuvants has poor stability and complex operation, making it difficult to achieve uniform particle size regulation.
Micron-scale aluminum phosphate adjuvant was prepared by chemical precipitation method, and its particle size was adjusted by a replacement buffer system, including the use of Cit, PB, Tris and PBS buffer systems to adjust the pH value to achieve particle size regulation.
Effective control of the particle size of aluminum phosphate adjuvant is achieved, and micro-nano-sized aluminum phosphate adjuvant with uniform particle size distribution and good stability is obtained, which simplifies the preparation process and improves the convenience of operation.
Smart Images
Figure CN119454929A_ABST
Abstract
Description
Technical Field
[0001] The invention belongs to the field of immunotechnology, and particularly relates to an aluminum phosphate adjuvant and a preparation method thereof. Background Art
[0002] Vaccination is one of the most important means of preventing infectious diseases. Adjuvants are basic ingredients to improve the effectiveness of vaccination. Aluminum salts mixed with antigens and injected into the body can induce more antibodies than antigens alone. Aluminum salt adjuvants have been used for more than 90 years. Despite the rapid development of new adjuvants, most of them are still in the experimental stage. Since the only adjuvants approved by the Food and Drug Administration for human vaccines are aluminum adjuvants, they have a long history of safe and effective use, so aluminum adjuvants still account for a large proportion of the current market.
[0003] The particle size of the commercial aluminum phosphate (AP) adjuvant is 3-4 μm, the isoelectric point is pH 5, and it is negatively charged under physiological conditions, making it a good cationic protein adsorbent. Studies have shown that the immune effect of aluminum phosphate adjuvants depends largely on the adjuvant's ability to adsorb proteins. Different aluminum adjuvants have different particle sizes, isoelectric points, and morphological characteristics, which may lead to differences in the adjuvant's affinity for antigens and vaccine efficacy. Immunodominance can be achieved by reducing the size of aluminum adjuvant particles to produce more effective adjuvants to stimulate cellular and humoral immune responses. Compared with aluminum adjuvants of micron size (aluminum phosphate particles of 3-4 μm), aluminum adjuvant particles of micron size (aluminum phosphate particles of 300nm-600nm) are smaller, have a larger specific surface area, a larger binding area with antigens, and better biocompatibility, which can enhance the body's immune response and significantly reduce the side effects of adjuvants. Therefore, the production of aluminum phosphate adjuvants of different particle sizes through particle size regulation is the focus of aluminum phosphate adjuvant research.
[0004] However, the physical and chemical properties of micro-nano-sized aluminum phosphate adjuvants are greatly affected by the preparation conditions, and the preparation process has poor repeatability. Therefore, the art still needs a method with stable process and simple and convenient operation to achieve the preparation of micro-nano-sized aluminum phosphate adjuvants. Summary of the invention
[0005] In order to solve the above problems existing in the prior art, the present invention prepares a micron-sized aluminum phosphate adjuvant by using a chemical precipitation method, and then adjusts the particle size of the aluminum phosphate adjuvant by replacing the buffer system, thereby inventing a simple and efficient method for adjusting the particle size of the aluminum phosphate adjuvant, as well as a method for preparing aluminum phosphate adjuvants in the micro-nano scale range.
[0006] The first aspect of the present invention provides a method for adjusting the particle size of an aluminum phosphate adjuvant, the method comprising resuspending micron-sized aluminum phosphate in any of the following ways:
[0007] 1) When using Cit buffer system as resuspension solution:
[0008] Resuspending the micron-sized aluminum phosphate in a Cit buffer system at a pH of 6-8, so that the particle size of the aluminum phosphate in the aluminum phosphate adjuvant is 400nm-430nm;
[0009] 2) When using PB buffer system as resuspension solution:
[0010] Resuspending micron-sized aluminum phosphate in a PB buffer system with a pH of 6.5-8, so that the particle size of the aluminum phosphate in the aluminum phosphate adjuvant is 445nm-585nm;
[0011] 3) When using Tris buffer system as resuspension solution:
[0012] Resuspending the micron-sized aluminum phosphate in a Tris buffer system at a pH of 7.8-8.2, so that the particle size of the aluminum phosphate in the aluminum phosphate adjuvant is 420nm-460nm;
[0013] Resuspending micron-sized aluminum phosphate in a Tris buffer system at a pH of 6.0-7.5, so that the particle size of aluminum phosphate in the aluminum phosphate adjuvant is 3000nm-3500nm;
[0014] 4) When using PBS buffer system as resuspension solution:
[0015] Resuspending micron-sized aluminum phosphate in a PBS buffer system at a pH of 6-6.5, so that the particle size of aluminum phosphate in the aluminum phosphate adjuvant is 3300nm-4200nm;
[0016] The micron-sized aluminum phosphate is resuspended in a PBS buffer system with a pH of 6.8-8, so that the particle size of the aluminum phosphate in the aluminum phosphate adjuvant is 2600nm-3300nm.
[0017] The second aspect of the present invention provides a method for preparing an aluminum phosphate adjuvant, the method comprising resuspending micron-sized aluminum phosphate in any of the following ways:
[0018] 1) When using Cit buffer system as resuspension solution:
[0019] Resuspending the micron-sized aluminum phosphate in a Cit buffer system at a pH of 6-8, so that the particle size of the aluminum phosphate in the aluminum phosphate adjuvant is 400nm-430nm;
[0020] 2) When using PB buffer system as resuspension solution:
[0021] Resuspending micron-sized aluminum phosphate in a PB buffer system at a pH of 6.5-8, so that the particle size of the aluminum phosphate in the aluminum phosphate adjuvant is 445-585 nm;
[0022] 3) When using Tris buffer system as resuspension solution:
[0023] The micron-sized aluminum phosphate is resuspended in a Tris buffer system with a pH of 7.8-8.2, so that the particle size of the aluminum phosphate in the aluminum phosphate adjuvant is 420nm-460nm.
[0024] In a preferred embodiment, the micron-sized aluminum phosphate is prepared by chemical precipitation;
[0025] The chemical precipitation method for preparing micron-sized aluminum phosphate comprises: uniformly mixing an aluminum salt solution and a phosphate solution at a P / Al molar ratio of 1:1 to obtain an initial aluminum phosphate adjuvant, and then performing a precipitation treatment, collecting the precipitate to obtain a micron-sized aluminum phosphate precipitate;
[0026] Preferably, the chemical precipitation method for preparing micron-sized aluminum phosphate is operated at 60°C;
[0027] Preferably, the aluminum salt is at least one of aluminum chloride, potassium aluminum sulfate, and aluminum nitrate;
[0028] Preferably, the phosphate is at least one of trisodium phosphate, disodium hydrogen phosphate, sodium dihydrogen phosphate, tripotassium phosphate, dipotassium hydrogen phosphate, and potassium dihydrogen phosphate.
[0029] In a preferred embodiment, the resuspension treatment is: resuspending the micron-sized aluminum phosphate precipitate in the resuspension solution at a ratio of 1:(0.5-5), preferably 1:(1-3), and further preferably 1:1, of the volume of the micron-sized aluminum phosphate before precipitation to the volume of the resuspension solution.
[0030] In a preferred embodiment, the method for preparing the aluminum phosphate adjuvant further comprises: washing the micron-sized aluminum phosphate precipitate with water and then resuspending it;
[0031] Preferably, the water washing is performed 2-3 times.
[0032] In a preferred embodiment, the method for preparing the aluminum phosphate adjuvant further comprises: sterilizing the micron-sized aluminum phosphate precipitate and then resuspending it;
[0033] Preferably, the sterilization treatment is any one of ozone sterilization, radiation sterilization, and high-temperature steam sterilization.
[0034] In a preferred embodiment, the Cit buffer system is a citrate buffer; it is prepared using citric acid, sodium citrate and water, and the pH of the Cit buffer system is adjusted by adjusting the relative amounts of citric acid and sodium citrate;
[0035] The PB buffer system is a sodium dihydrogen phosphate / disodium hydrogen phosphate buffer; it is prepared using sodium dihydrogen phosphate, disodium hydrogen phosphate and water, and the pH of the PB buffer system is adjusted by adjusting the relative amounts of sodium dihydrogen phosphate and disodium hydrogen phosphate;
[0036] The Tris buffer system is a Tris-HCl buffer; it is prepared using Tris, HCl and water, and the pH of the Tris buffer system is adjusted by adjusting the relative amounts of Tris and HCl;
[0037] The PBS buffer system is a phosphate buffered saline solution, which is prepared from sodium dihydrogen phosphate, disodium hydrogen phosphate, sodium chloride and water. The pH of the PBS buffer system is adjusted by adjusting the relative amounts of sodium dihydrogen phosphate and disodium hydrogen phosphate.
[0038] The third aspect of the present invention provides an aluminum phosphate adjuvant prepared according to the method for preparing the aluminum phosphate adjuvant provided by the second aspect of the present invention.
[0039] In a preferred embodiment, when the Cit buffer system is used, when the pH of the buffer is 6, the particle size, potential and pH of AP are 405nm, -45.53mV, and 6.22, respectively; when the pH of the buffer is 6.5, the particle size, potential and pH of AP are 403nm, -46.58mV, and 6.50, respectively; when the pH of the buffer is 7, the particle size, potential and pH of AP are 426nm, -48.58mV, and 6.65, respectively; when the pH of the buffer is 7.5, the particle size, potential and pH of AP are 417nm, -48.91mV, and 6.67, respectively; when the pH of the buffer is 8, the particle size, potential and pH of AP are 409nm, -47.12mV, and 6.69, respectively;
[0040] When the PB buffer system was used, when the pH of the buffer was 6.5, the particle size, potential and pH of AP were 581nm, -30.2mV and 6.18, respectively; when the pH of the buffer was 7, the particle size, potential and pH of AP were 489nm, -34.79mV and 6.56, respectively; when the pH of the buffer was 7.5, the particle size, potential and pH of AP were 450nm, -37.40mV and 6.76, respectively; when the pH of the buffer was 8, the particle size, potential and pH of AP were 450nm, -39.42mV and 6.9, respectively;
[0041] When the Tris buffer system was used and the pH of the buffer was 8, the particle size, potential and pH of AP were 442 nm, -27.37 mV and 6.42, respectively.
[0042] The fourth aspect of the present invention provides a vaccine containing an aluminum phosphate adjuvant, wherein the aluminum phosphate adjuvant is the aluminum phosphate adjuvant provided in the second aspect of the present invention.
[0043] The preparation method of the aluminum phosphate adjuvant provided by the present invention has simple process, easy operation and good repeatability. The particle size of the aluminum phosphate adjuvant can be controlled by simply replacing the buffer system and adjusting the pH of the buffer system, so as to obtain a micro-nano-sized aluminum phosphate adjuvant with uniform particle size distribution and good stability. BRIEF DESCRIPTION OF THE DRAWINGS
[0044] Figure 1 The particle size distribution diagram of the initial aluminum phosphate adjuvant (a) and the Zeta potential under different pH conditions (b) in the preparation example.
[0045] Figure 2 The microscopic morphology of the initial aluminum phosphate adjuvant characterized by scanning electron microscopy in the preparation example.
[0046] Figure 3 The X-ray diffraction pattern of the initial aluminum phosphate adjuvant in the preparation example.
[0047] Figure 4 This is the infrared spectrum of the initial aluminum phosphate adjuvant in the preparation example. DETAILED DESCRIPTION
[0048] To make the technical solution, purpose and advantages of the present invention clearer, the present invention is further described in detail below through specific implementation examples. It should be understood that the specific implementation methods described herein are only used to illustrate and explain the present invention, and are not used to limit the present invention.
[0049] The first aspect of the present invention provides a method for adjusting the particle size of an aluminum phosphate adjuvant, the method comprising resuspending micron-sized aluminum phosphate in any of the following ways:
[0050] 1) When using Cit buffer system as resuspension solution:
[0051] Resuspending the micron-sized aluminum phosphate in a Cit buffer system at a pH of 6-8, so that the particle size of the aluminum phosphate in the aluminum phosphate adjuvant is 400nm-430nm;
[0052] 2) When using PB buffer system as resuspension solution:
[0053] Resuspending micron-sized aluminum phosphate in a PB buffer system with a pH of 6.5-8, so that the particle size of the aluminum phosphate in the aluminum phosphate adjuvant is 445nm-585nm;
[0054] 3) When using Tris buffer system as resuspension solution:
[0055] Resuspending the micron-sized aluminum phosphate in a Tris buffer system at a pH of 7.8-8.2, so that the particle size of the aluminum phosphate in the aluminum phosphate adjuvant is 420nm-460nm;
[0056] Resuspending micron-sized aluminum phosphate in a Tris buffer system at a pH of 6.0-7.5, so that the particle size of aluminum phosphate in the aluminum phosphate adjuvant is 3000nm-3500nm;
[0057] 4) When using PBS buffer system as resuspension solution:
[0058] Resuspending micron-sized aluminum phosphate in a PBS buffer system at a pH of 6-6.5, so that the particle size of aluminum phosphate in the aluminum phosphate adjuvant is 3300nm-4200nm;
[0059] The micron-sized aluminum phosphate is resuspended in a PBS buffer system with a pH of 6.8-8, so that the particle size of the aluminum phosphate in the aluminum phosphate adjuvant is 2600nm-3300nm.
[0060] According to the first aspect of the present invention, the micron-sized aluminum phosphate refers to aluminum phosphate particles with a particle size of 3-4 μm; wherein the particle size of the aluminum phosphate particles is measured using a laser particle size analyzer (eg, Bettersize 2600 purchased from Bettersize).
[0061] The micron-sized aluminum phosphate can be prepared by a chemical precipitation method.
[0062] Specifically, the chemical precipitation method for preparing micron-sized aluminum phosphate may include: uniformly mixing an aluminum salt solution and a phosphate solution at a P / Al molar ratio of 1:1 to obtain an initial aluminum phosphate adjuvant, then performing a precipitation treatment, and collecting the precipitate to obtain a micron-sized aluminum phosphate precipitate.
[0063] Furthermore, the chemical precipitation method for preparing micron-sized aluminum phosphate can be operated at 60°C; for example, the water used to dissolve the aluminum salt and the phosphate is preheated to 60°C.
[0064] Furthermore, the chemical precipitation method for preparing micron-sized aluminum phosphate can be to use a peristaltic pump to control the flow rate at 5 mL / min, control the stirring speed at 450 rpm, and add the aluminum salt solution and the phosphate solution to the reaction container at the same time, and keep the temperature at 60° C. during the whole process. After the reaction solution is added, the solution changes from transparent to milky white, and the heating and stirring are stopped, and it is allowed to cool naturally at room temperature.
[0065] The aluminum salt may be at least one of soluble aluminum salts such as aluminum chloride (eg aluminum chloride hexahydrate), potassium aluminum sulfate, and aluminum nitrate, and is preferably aluminum chloride.
[0066] The phosphate may be at least one of trisodium phosphate (eg, trisodium phosphate dodecahydrate), disodium hydrogen phosphate, sodium dihydrogen phosphate, tripotassium phosphate, dipotassium hydrogen phosphate, and potassium dihydrogen phosphate, preferably trisodium phosphate.
[0067] According to the first aspect of the present invention, the resuspension treatment can be a volume ratio of the volume of the micron-sized aluminum phosphate before the precipitation treatment (i.e., the volume of the initial aluminum phosphate adjuvant) to the resuspension solution of 1: (0.5-5), preferably 1: (1-3), and more preferably 1: 1, and the micron-sized aluminum phosphate precipitate is resuspended in the resuspension solution. After the initial aluminum phosphate adjuvant prepared by the aforementioned chemical precipitation method is precipitated, the obtained micron-sized aluminum phosphate precipitate is resuspended in the resuspension solution, and the mixture of the micron-sized aluminum phosphate precipitate and the resuspension solution is shaken, or the mixture of the micron-sized aluminum phosphate precipitate and the resuspension solution is treated by ultrasound, so that the micron-sized aluminum phosphate precipitate is evenly mixed in the resuspension solution.
[0068] Specifically, the specific operation of the resuspension treatment includes: centrifuging the micron-sized aluminum phosphate stock solution (i.e., the initial aluminum phosphate adjuvant) at a speed of 4500 rpm, discarding the supernatant, resuspending in water for the first two times according to the volume before resuspension: the volume of the resuspension solution = 1:1, mixing the solution evenly by ultrasound at an ultrasonic power of 400w, and resuspending in different buffers after the third centrifugation.
[0069] According to the first aspect of the present invention, the method further comprises washing the micron-sized aluminum phosphate precipitate with water and then resuspending it. The washing may be performed 2-3 times.
[0070] According to the first aspect of the present invention, the method may further include sterilizing the micron-sized aluminum phosphate precipitate and then resuspending it. The sterilization treatment may be any conventional method in the art, such as ozone sterilization, irradiation sterilization, high-temperature steam sterilization, etc.
[0071] The Cit buffer system is a citrate buffer; for example, it can be a citric acid / sodium citrate buffer, which is prepared using citric acid, sodium citrate and water. The pH of the Cit buffer system can be adjusted by adjusting the relative amounts of citric acid and sodium citrate.
[0072] The PB buffer system (phosphate buffer solution, PB) is a phosphate buffer solution; for example, it can be a sodium dihydrogen phosphate / disodium hydrogen phosphate buffer solution, which is prepared using sodium dihydrogen phosphate, disodium hydrogen phosphate and water. The pH of the PB buffer system can be adjusted by adjusting the relative amounts of sodium dihydrogen phosphate and disodium hydrogen phosphate.
[0073] The Tris buffer system is a Tris-HCl (tris (hydroxymethyl)aminomethane hydrochloride or Tris hydrochloride-hydrochloric acid) buffer, which is prepared using Tris (tris (hydroxymethyl)aminomethane hydrochloride), HCl and water. The pH of the Tris buffer system can be adjusted by adjusting the relative amounts of Tris and HCl.
[0074] The PBS buffer system (phosphate buffered saline, PBS) is a phosphate buffered saline solution; it is prepared with sodium dihydrogen phosphate, disodium hydrogen phosphate, sodium chloride and water, and the pH of the PBS buffer system can be adjusted by adjusting the relative amounts of sodium dihydrogen phosphate and disodium hydrogen phosphate.
[0075] The second aspect of the present invention provides a method for preparing an aluminum phosphate adjuvant, the method comprising resuspending micron-sized aluminum phosphate in any of the following ways:
[0076] 1) When using Cit buffer system as resuspension solution:
[0077] Resuspending the micron-sized aluminum phosphate in a Cit buffer system at a pH of 6-8, so that the particle size of the aluminum phosphate in the aluminum phosphate adjuvant is 400nm-430nm;
[0078] 2) When using PB buffer system as resuspension solution:
[0079] Resuspending micron-sized aluminum phosphate in a PB buffer system at a pH of 6.5-8, so that the particle size of the aluminum phosphate in the aluminum phosphate adjuvant is 445-585 nm;
[0080] 3) When using Tris buffer system as resuspension solution:
[0081] The micron-sized aluminum phosphate is resuspended in a Tris buffer system with a pH of 7.8-8.2, so that the particle size of the aluminum phosphate in the aluminum phosphate adjuvant is 420nm-460nm.
[0082] Among them, in the aluminum phosphate adjuvant, the size of the aluminum phosphate particles is 300nm-600nm (also called "micro-nano particles"), and the particle size is detected by a dynamic light scattering instrument (such as Zetasizer pro purchased from Malvern Panalytical).
[0083] Wherein, the micron-sized aluminum phosphate is as described in the first aspect of the present invention. The resuspension treatment is as described in the first aspect of the present invention.
[0084] The third aspect of the present invention provides an aluminum phosphate adjuvant, which is prepared by the preparation method of the aluminum phosphate adjuvant provided by the second aspect of the present invention.
[0085] According to the third aspect of the present invention, in the aluminum phosphate adjuvant, the particle size of the aluminum phosphate particles is 400nm-600nm, the PDI is 0.1-0.3 (obtained by dynamic light scattering), the Zeta potential of the aluminum phosphate adjuvant is -25mV~-50mV, and the pH of the aluminum phosphate adjuvant is 6.0-7.0.
[0086] Furthermore, when the Cit buffer system is used, when the pH of the buffer is 6, the particle size, potential and pH of AP are 405nm, -45.53mV and 6.22 respectively; when the pH of the buffer is 6.5, the particle size, potential and pH of AP are 403nm, -46.58mV and 6.50 respectively; when the pH of the buffer is 7, the particle size, potential and pH of AP are 426nm, -48.58mV and 6.65 respectively; when the pH of the buffer is 7.5, the particle size, potential and pH of AP are 417nm, -48.91mV and 6.67 respectively; when the pH of the buffer is 8, the particle size, potential and pH of AP are 409nm, -47.12mV and 6.69 respectively; in the Cit system, citrate and aluminum ions are complexed, the system potential is very stable, and the isoelectric point is <2.
[0087] Furthermore, when the PB buffer system was used, when the pH of the buffer was 6.5, the particle size, potential and pH of AP were 581 nm, -30.2 mV and 6.18, respectively; when the pH of the buffer was 7, the particle size, potential and pH of AP were 489 nm, -34.79 mV and 6.56, respectively; when the pH of the buffer was 7.5, the particle size, potential and pH of AP were 450 nm, -37.40 mV and 6.76, respectively; when the pH of the buffer was 8, the particle size, potential and pH of AP were 450 nm, -39.42 mV and 6.9, respectively; the isoelectric point was 2.26.
[0088] Furthermore, when a Tris buffer system was used and the pH of the buffer was 8, the particle size, potential and pH of AP were 442 nm, -27.37 mV, and 6.42, respectively; the isoelectric point was 5.19.
[0089] The fourth aspect of the present invention provides a vaccine containing the aluminum phosphate adjuvant provided according to the third aspect of the present invention.
[0090] Among them, the vaccine can be an HPV vaccine (such as Watson Bio's domestically produced 2-valent HPV vaccine (containing 450 μg / mL aluminum phosphate)), a 13-valent pneumonia conjugate vaccine (such as Pfizer's 13-valent pneumonia conjugate vaccine (containing 250 μg / mL aluminum phosphate)).
[0091] Among them, the concentration of aluminum phosphate adjuvant in the vaccine is less than 0.85 mg / dose (0.5 mL per dose) in terms of aluminum content. (Internationally, the safety limit standards for aluminum adjuvants are clearly stated in the form of regulations or general rules, all of which are measured in units of aluminum content. The World Health Organization and the European Union require no more than 1.25 mg / dose, and the United States requires no more than 0.85 mg / dose. The aluminum adjuvant of the currently listed vaccines is mostly 0.3-0.5 mg / dose, and the 0.85 mg / dose used in some vaccines is the highest dose of aluminum adjuvant used in listed vaccines.)
[0092] The various reagents, materials, etc. used in the following examples, unless otherwise specified, are all products that can be obtained from commercial channels; the various tests and detection methods used in the following examples, unless otherwise specified, are all conventional tests and detection methods in the art and can be obtained from textbooks, reference books or academic journals.
[0093] In the following preparation examples and embodiments:
[0094] Zeta potential: The pH of AP was adjusted with sodium hydroxide and hydrochloric acid to obtain AP with pH of 3, 4, 5, 6 and 7, and the Zeta potential of AP at different pH was measured by dynamic light scattering. When the Zeta potential was 0, the pH was considered to be the isoelectric point of the protein or the zero charge point of the aluminum adjuvant.
[0095] Particle size: A dynamic light scattering instrument (Zetasizer pro purchased from Malvern Panalytical) was used to detect the aluminum phosphate adjuvant to obtain the particle size of the aluminum phosphate particles in the aluminum phosphate adjuvant.
[0096] pH: A pH meter (purchased from Mettler-Toledo) was used to detect the pH of the buffer system and the aluminum phosphate adjuvant.
[0097] PDI: A dynamic light scattering instrument (Zetasizer pro purchased from Malvern Panalytical) was used to detect the aluminum phosphate adjuvant to obtain the PDI of the aluminum phosphate particles in the aluminum phosphate adjuvant.
[0098] Characterization of protein adsorption rate: The protein adsorption of AP in Cit, PB, Tris, PBS, and H2O systems was determined by the Lorry method.
[0099] Absorbance measurement: Protein solutions of different concentrations were prepared, and the absorbance at 650 nm was measured using an enzyme reader (Varioskan LUX purchased from Thermo Fisher Scientific) to obtain a standard curve.
[0100] Mix the corresponding concentrations of aluminum phosphate and protein solution to obtain aluminum phosphate-protein mixtures with Al:Protein (based on the weight of aluminum element and the weight of protein) of 1:4, 1:2, 1:1, 2:1 and 4:1. After mixing by vortexing, continue to shake for 2 hours using a universal shaker, and then take a certain volume of solution and centrifuge (8000rpm, 10min). Take a certain volume of supernatant and place it in a centrifuge tube. Add an equal volume of color developing solution and vortex mix for 10 minutes. Then add 2 times the volume of Fehling's reagent under light-proof conditions, vortex and place for 30 minutes, and then measure the absorbance at 650nm using an enzyme marker.
[0101] Sedimentation rate determination: Sedimentation experiments were performed by storing different suspensions in glass bottles of the same size and observing the sedimentation of the suspensions over time. During this period, the changes in the suspensions were recorded by a camera. The sedimentation height (H sed ), which is the height of sediment settlement, from which the sediment volume fraction of each sample is derived according to Eq. In the following formula, H represents the total height of the suspension:
[0102]
[0103] Stability test:
[0104] Dynamic light scattering was used to measure the potential and particle size of AP over time to detect the stability of aluminum phosphate adjuvant in buffer systems with different pH values.
[0105] Protein adsorption rate test: The protein adsorption rate of AP was determined by the lorry method. The specific operation is as follows: prepare lysozyme protein solutions of different concentrations (C 初始蛋白 ), the absorbance at 650nm was measured by an enzyme-labeled instrument to obtain a standard curve. The corresponding concentrations of AP and protein solution were mixed to obtain aluminum phosphate-protein mixtures with Al:Protein ratios of 1:4, 1:2, 1:1, 2:1 and 4:1 based on the weight of the aluminum element and the weight of the protein. After mixing by vortexing, continue to shake for 2 hours using a universal shaker, and then take 1mL of the solution and centrifuge (8000r, 10min). Take 0.5mL of the supernatant and place it in a centrifuge tube. Add an equal volume of 0.5mL of the color developing solution and vortex mix for 10 minutes. Then, add 2mL of Fehling's reagent in a dark environment, vortex and place for 30 minutes, and then measure the absorbance at 650nm using an enzyme-labeled instrument. Compare the absorbance values using the above-mentioned standard curve to obtain the protein concentration in the supernatant (i.e., the concentration of unadsorbed protein C 未吸附蛋白 ), and the protein adsorption rate was calculated according to the following calculation method:
[0106]
[0107] Preparation Example
[0108] This preparation example is used to illustrate the preparation of the aluminum phosphate precipitate used in the following examples, and to characterize the initial aluminum phosphate adjuvant prepared in this preparation example.
[0109] In the following examples, the aluminum phosphate precipitates used were prepared by the following method:
[0110] Accurately weigh 0.4481g (1.85mmol) of aluminum chloride hexahydrate and 0.7019g (1.85mmol) of trisodium phosphate dodecahydrate, respectively, and dissolve them in 80mL of deionized water, stirring continuously to dissolve until they are clear and transparent. At 60°C, use a peristaltic pump to control the flow rate to 5mL / min, use a magnetic stirrer to control the stirring speed to 450rpm, and add the aluminum salt solution and phosphate solution to 40mL of deionized water at the same time. After the reaction solution is added, the solution changes from transparent to milky white, and the reaction time is controlled at 30-35min. After the reaction is completed, stop heating and stirring, and cool naturally at room temperature to obtain the initial aluminum phosphate adjuvant. The particle size distribution of the initial aluminum phosphate adjuvant prepared above is characterized by a laser particle size meter. Figure 1 The initial aluminum phosphate adjuvant had a particle size of 4 μm, a pH of 5.6, a potential of -28 mV, and an isoelectric point of 5.
[0111] like Figure 2 As shown, the microscopic morphology of the initial aluminum phosphate adjuvant was characterized by scanning electron microscopy, and it can be seen that many spherical primary nanoparticles agglomerated to form fluffy porous aggregates with an overall irregular shape.
[0112] like Figure 3 As shown, X-ray diffraction analysis shows that the initial aluminum phosphate adjuvant has a large peak when 2θ is about 30°, and the aluminum phosphate has an amorphous structure, which is similar to the peak of commercial AP.
[0113] like Figure 4 As shown, FT-IR analysis showed that the -1 The characteristic bands are shown at 3700-2700cm -1 In the region of 3700-2700cm, due to the combined effect of structural hydroxyl groups and adsorbed water, -1 The strong and broad asymmetric band at 1640 cm was attributed to -OH stretching. -1 The band at 1085 cm is the -OH bending band of adsorbed water. -1 The PO expansion zone is at 640cm. -1 and 540cm -1 There are two low-frequency bands, 640cm -1The band is related to the bending band of the structural -OH, 540 cm -1 The above data indicate that the synthesized adjuvant is a hydroxy amorphous aluminum phosphate.
[0114] The parameters and characteristics of the above initial aluminum phosphate adjuvant are as follows:
[0115] Zeta potential: -28 mV; particle size: 3917 nm; pH: 5.6; PDI: 0.27; sedimentation rate: 40%.
[0116] According to the aforementioned method, the stability of the initial aluminum phosphate adjuvant was tested, and the results are shown in Table 1.
[0117] Table 1 Stability of initial aluminum phosphate adjuvant
[0118]
[0119] According to the aforementioned method, the protein adsorption rate of the initial aluminum phosphate adjuvant was tested, and the results are shown in Table 2.
[0120] Table 2 Protein adsorption rate of initial aluminum phosphate adjuvant
[0121]
[0122] The above initial aluminum phosphate adjuvant can be used as the raw material for preparing the following examples.
[0123] Example 1
[0124] This example is used to illustrate the preparation of aluminum phosphate adjuvant using Cit buffer system as resuspension liquid.
[0125] Take 15 mL of the initial aluminum phosphate adjuvant obtained in the above preparation example, centrifuge at 4500 rpm, discard the supernatant, wash the aluminum phosphate precipitate three times with water, then add the aluminum phosphate precipitate to 15 mL of Cit buffer system of different pH shown in Table 3, and ultrasonically treat for 10 minutes to fully resuspend the aluminum phosphate precipitate in the Cit buffer system, so as to obtain the aluminum phosphate adjuvant with the Cit buffer system as the resuspending liquid.
[0126] According to the aforementioned method, the Zeta potential, particle size, PDI and sedimentation rate of the aluminum phosphate adjuvant in the Cit system at different pH were measured respectively. The results are shown in Table 3.
[0127] Table 3 Parameter characteristics of aluminum phosphate adjuvant in Cit system with different pH
[0128] pH Zeta potential (mV) Particle size (nm) pH(AP) PDI Sedimentation rate 8 -47.12 409 6.69 0.26 0％ 7.5 -48.91 417 6.67 0.28 0％ 7 -48.58 426 6.65 0.27 0％ 6.5 -46.58 403 6.50 0.29 0％ 6 -45.53 405 6.22 0.30 0％
[0129] According to the above method, the stability of aluminum phosphate adjuvant in Cit system with different pH was tested respectively. The results are shown in Table 4.
[0130] Table 4 Stability of aluminum phosphate adjuvant in Cit system with different pH
[0131]
[0132] According to the above method, the protein adsorption rate of aluminum phosphate adjuvant in Cit system with different pH was tested respectively. The results are shown in Table 5.
[0133] Table 5 Protein adsorption rate of aluminum phosphate adjuvant in Cit system with different pH
[0134]
[0135] It can be seen from the data in Tables 3-5 above that by resuspending micron-sized aluminum phosphate in a Cit buffer system with a pH of 6-8, an aluminum phosphate adjuvant with a particle size of 400nm-430nm can be obtained.
[0136] Compared with the initial aluminum phosphate adjuvant: the particle size of AP in the Cit system is nanometers, which is 10 times smaller; the potential changes from -28mV to -48mV, and the absolute potential increases by 20mV; the properties are more stable, mainly reflected in the particle size, Zeta potential, and PDI remain stable within six months, and the 24h sedimentation rate of the particles is close to 0%, indicating that the particles are not easy to agglomerate; the protein adsorption rate is greatly improved, specifically when Al:Protein(mass)=1:1, the adsorption rate is greatly improved.
[0137] Example 2
[0138] This example is used to illustrate the preparation of aluminum phosphate adjuvant using PB buffer system as resuspension liquid.
[0139] Take 15 mL of the initial aluminum phosphate adjuvant obtained in the above preparation example, centrifuge at 4500 rpm, discard the supernatant, wash the aluminum phosphate precipitate three times with water, then add the aluminum phosphate precipitate to 15 mL of the PB buffer system of different pH shown in Table 6, and ultrasonically treat for 10 minutes to fully resuspend the aluminum phosphate precipitate in the PB buffer system, so as to obtain the aluminum phosphate adjuvant with the PB buffer system as the resuspending liquid.
[0140] According to the above method, the Zeta potential, particle size, PDI and sedimentation rate of the aluminum phosphate adjuvant in the PB system with different pH were measured respectively. The results are shown in Table 6.
[0141] Table 6 Parameter characteristics of aluminum phosphate adjuvant in PB system with different pH
[0142] pH(PB) Zeta potential (mV) Particle size (nm) pH(AP) PDI Sedimentation rate 8 -39.42 450 6.9 0.29 3.5％ 7.5 -37.40 450 6.76 0.30 3.5％ 7 -34.79 489 6.56 0.26 4.3％ 6.5 -30.20 581 6.18 0.24 6.2％
[0143] According to the above method, the stability of aluminum phosphate adjuvant in PB systems with different pH values was tested. The results are shown in Table 7.
[0144] Table 7 Stability of aluminum phosphate adjuvant in PB system with different pH
[0145]
[0146] According to the above method, the protein adsorption rate of aluminum phosphate adjuvant in PB systems with different pH was tested respectively. The results are shown in Table 8.
[0147] Table 8 Protein adsorption rate of aluminum phosphate adjuvant in PB system with different pH
[0148]
[0149] It can be seen from the data in Tables 6-8 above that by resuspending micron-sized aluminum phosphate in a PB buffer system with a pH of 6.5-8, an aluminum phosphate adjuvant with a particle size of 445nm-585nm can be obtained.
[0150] Compared with the initial aluminum phosphate adjuvant: the particle size of AP in the PB system is nanometers, which is 9.5 times smaller; the potential changes from -28mV to -35mV, and the absolute potential increases by 7mV; and the absolute potential increases with the increase of buffer pH; the properties are stable, mainly reflected in the particle size, Zeta potential, and PDI remain stable within six months, and the sedimentation rate of the particles is low, indicating that the particles are not easy to agglomerate; the protein adsorption rate is improved, but compared with the Cit system, the AP in the Cit system is more stable and the protein adsorption rate is improved more significantly.
[0151] Example 3
[0152] This example is used to illustrate the preparation of aluminum phosphate adjuvant using Tris buffer system as resuspension solution.
[0153] Take 15 mL of the initial aluminum phosphate adjuvant obtained in the above preparation example, centrifuge at 4500 rpm, discard the supernatant, wash the aluminum phosphate precipitate three times with water, then add the aluminum phosphate precipitate to 15 mL of Tris buffer system with a pH of 8, and ultrasonically treat for 10 minutes to fully resuspend the aluminum phosphate precipitate in the Tris buffer system, so as to obtain the aluminum phosphate adjuvant with the Tris buffer system as the resuspending liquid.
[0154] According to the aforementioned method, the Zeta potential of the aluminum phosphate adjuvant in the Tris buffer system at pH 8 was determined to be -27.37 mV, the particle size was 442 nm, the PDI was 0.29, and the sedimentation rate was 8.0%.
[0155] According to the aforementioned method, the stability of the aluminum phosphate adjuvant in the Tris buffer system with a pH of 8 was tested. The results are shown in Table 9.
[0156] Table 9 Stability of aluminum phosphate adjuvant in Tris buffer system at pH 8
[0157]
[0158] According to the aforementioned method, the protein adsorption rate of the aluminum phosphate adjuvant in the Tris buffer system with a pH of 8 was tested. The results are shown in Table 10.
[0159] Table 10 Protein adsorption rate of aluminum phosphate adjuvant in Tris buffer system with pH 8
[0160]
[0161] It can be seen from the above data that by resuspending micron-sized aluminum phosphate in a Tris buffer system at pH 8, an aluminum phosphate adjuvant with a particle size of 442 nm can be obtained.
[0162] Compared with the initial aluminum phosphate adjuvant: the particle size of AP in the Tris system is nanometers, which is 9.5 times smaller; the potential does not change; the properties are stable, mainly reflected in the particle size, Zeta potential, and PDI remaining stable within six months, and the sedimentation rate of the particles is low, indicating that the particles are not easy to agglomerate; the protein adsorption rate is close to the initial AP level.
[0163] Example 4
[0164] This example is used to illustrate the process stability of the preparation method of the aluminum phosphate adjuvant provided by the present invention.
[0165] Three batches of aluminum phosphate adjuvant (AP), AP-1, AP-2, and AP-3, were prepared repeatedly as follows:
[0166] Accurately weigh 2.24g (9mmol) of aluminum chloride hexahydrate and 3.50g (9mmol) of trisodium phosphate dodecahydrate, dissolve them in 400mL of deionized water, and stir them continuously to dissolve until they are clear and transparent. At 60°C, use a peristaltic pump to control the flow rate to 42mL / min, use a magnetic stirrer to control the stirring speed to 500rpm, and add the aluminum salt solution and phosphate solution to 40mL of deionized water at the same time. After the reaction solution is added, the solution changes from transparent to milky white, and the reaction time is controlled at 30-35min. After the reaction is completed, stop heating and stirring, and cool naturally at room temperature to obtain the initial aluminum phosphate adjuvants AP-1, AP-2, and AP-3.
[0167] Take 30 mL of the initial aluminum phosphate resuspension obtained in the above preparation example, centrifuge at 4500 rpm for 10 min, add them to 30 mL of the buffer systems of different pH values shown in Tables 11-13, and ultrasonically treat for 10 min to allow the aluminum phosphate precipitate to be fully resuspended in the buffer system, thereby obtaining aluminum phosphate adjuvants with different buffer systems as resuspensions.
[0168] According to the above method, the particle size, potential and PDI of AP were measured respectively, and the results are shown in Tables 11-13.
[0169] Table 11 Particle size, potential and PDI of AP-1 and its dispersion in different pH buffer systems
[0170]
[0171] Table 12 Particle size, potential and PDI of AP-2 and its dispersion in different pH buffer systems
[0172]
[0173] Table 13 Particle size, potential and PDI of AP-3 and its dispersion in different pH buffer systems
[0174]
[0175] It can be seen from the data in Tables 11-13 above that the particle size, potential and PDI of AP in the Cit system are close to those in Example 1; the particle size, potential and PDI of AP in the PB system are close to those in Example 2; the particle size, potential and PDI of AP in the Tris system are close to those in Example 3; this shows that the process stability of the method provided by the present invention is very good and can be promoted and used on a large scale.
[0176] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.
Claims
1. A method for adjusting the particle size of an aluminum phosphate adjuvant, characterized in that: The method comprises the operation of resuspending micron-sized aluminum phosphate in any of the following ways: 1) When using Cit buffer system as resuspension solution: Resuspending the micron-sized aluminum phosphate in a Cit buffer system at a pH of 6-8, so that the particle size of the aluminum phosphate in the aluminum phosphate adjuvant is 400nm-430nm; 2) When using PB buffer system as resuspension solution: Resuspending micron-sized aluminum phosphate in a PB buffer system with a pH of 6.5-8, so that the particle size of the aluminum phosphate in the aluminum phosphate adjuvant is 445nm-585nm; 3) When using Tris buffer system as resuspension solution: Resuspending the micron-sized aluminum phosphate in a Tris buffer system at a pH of 7.8-8.2, so that the particle size of the aluminum phosphate in the aluminum phosphate adjuvant is 420nm-460nm; Resuspending micron-sized aluminum phosphate in a Tris buffer system at a pH of 6.0-7.5, so that the particle size of aluminum phosphate in the aluminum phosphate adjuvant is 3000nm-3500nm; 4) When using PBS buffer system as resuspension solution: Resuspending micron-sized aluminum phosphate in a PBS buffer system at a pH of 6-6.5, so that the particle size of aluminum phosphate in the aluminum phosphate adjuvant is 3300nm-4200nm; The micron-sized aluminum phosphate is resuspended in a PBS buffer system with a pH of 6.8-8, so that the particle size of the aluminum phosphate in the aluminum phosphate adjuvant is 2600nm-3300nm.
2. A method for preparing an aluminum phosphate adjuvant, the method comprising resuspending micron-sized aluminum phosphate in any of the following ways: 1) When using Cit buffer system as resuspension solution: Resuspending the micron-sized aluminum phosphate in a Cit buffer system at a pH of 6-8, so that the particle size of the aluminum phosphate in the aluminum phosphate adjuvant is 400nm-430nm; 2) When using PB buffer system as resuspension solution: Resuspending micron-sized aluminum phosphate in a PB buffer system at a pH of 6.5-8, so that the particle size of the aluminum phosphate in the aluminum phosphate adjuvant is 445-585 nm; 3) When using Tris buffer system as resuspension solution: The micron-sized aluminum phosphate is resuspended in a Tris buffer system with a pH of 7.8-8.2, so that the particle size of the aluminum phosphate in the aluminum phosphate adjuvant is 420nm-460nm.
3. The method for preparing the aluminum phosphate adjuvant according to claim 2, characterized in that: The micron-sized aluminum phosphate is prepared by a chemical precipitation method; The chemical precipitation method for preparing micron-sized aluminum phosphate comprises: uniformly mixing an aluminum salt solution and a phosphate solution at a P / Al molar ratio of 1:1 to obtain an initial aluminum phosphate adjuvant, and then performing a precipitation treatment, collecting the precipitate to obtain a micron-sized aluminum phosphate precipitate; Preferably, the chemical precipitation method for preparing micron-sized aluminum phosphate is operated at 60°C; Preferably, the aluminum salt is at least one of aluminum chloride, potassium aluminum sulfate, and aluminum nitrate; Preferably, the phosphate is at least one of trisodium phosphate, disodium hydrogen phosphate, sodium dihydrogen phosphate, tripotassium phosphate, dipotassium hydrogen phosphate, and potassium dihydrogen phosphate.
4. The method for preparing the aluminum phosphate adjuvant according to claim 2 or 3, characterized in that: The resuspension treatment is as follows: the micron-sized aluminum phosphate precipitate is resuspended in the resuspension solution at a ratio of 1:(0.5-5), preferably 1:(1-3), and more preferably 1:1, of the volume of the micron-sized aluminum phosphate before precipitation to the volume of the resuspension solution.
5. The method for preparing the aluminum phosphate adjuvant according to claim 4, characterized in that: The preparation method of the aluminum phosphate adjuvant also includes: washing the micron-sized aluminum phosphate precipitate with water and then resuspending it; Preferably, the water washing is performed 2-3 times.
6. The method for preparing the aluminum phosphate adjuvant according to claim 5, characterized in that: The preparation method of the aluminum phosphate adjuvant also includes: sterilizing the micron-sized aluminum phosphate precipitate and then resuspending it; Preferably, the sterilization treatment is any one of ozone sterilization, radiation sterilization, and high-temperature steam sterilization.
7. The method for preparing the aluminum phosphate adjuvant according to claim 6, characterized in that: The Cit buffer system is a citrate buffer; it is prepared using citric acid, sodium citrate and water, and the pH of the Cit buffer system is adjusted by adjusting the relative amounts of citric acid and sodium citrate; The PB buffer system is a sodium dihydrogen phosphate / disodium hydrogen phosphate buffer; it is prepared using sodium dihydrogen phosphate, disodium hydrogen phosphate and water, and the pH of the PB buffer system is adjusted by adjusting the relative amounts of sodium dihydrogen phosphate and disodium hydrogen phosphate; The Tris buffer system is a Tris-HCl buffer; it is prepared using Tris, HCl and water, and the pH of the Tris buffer system is adjusted by adjusting the relative amounts of Tris and HCl; The PBS buffer system is a phosphate buffered saline solution, which is prepared from sodium dihydrogen phosphate, disodium hydrogen phosphate, sodium chloride and water. The pH of the PBS buffer system is adjusted by adjusting the relative amounts of sodium dihydrogen phosphate and disodium hydrogen phosphate.
8. The aluminum phosphate adjuvant prepared by the preparation method of the aluminum phosphate adjuvant according to any one of claims 2 to 7.
9. The aluminum phosphate adjuvant according to claim 8, characterized in that: When the Cit buffer system was used, when the pH of the buffer was 6, the particle size, potential and pH of AP were 405nm, -45.53mV and 6.22, respectively; when the pH of the buffer was 6.5, the particle size, potential and pH of AP were 403nm, -46.58mV and 6.50, respectively; when the pH of the buffer was 7, the particle size, potential and pH of AP were 426nm, -48.58mV and 6.65, respectively; when the pH of the buffer was 7.5, the particle size, potential and pH of AP were 417nm, -48.91mV and 6.67, respectively; when the pH of the buffer was 8, the particle size, potential and pH of AP were 409nm, -47.12mV and 6.69, respectively; When the PB buffer system was used, when the pH of the buffer was 6.5, the particle size, potential and pH of AP were 581nm, -30.2mV and 6.18, respectively; when the pH of the buffer was 7, the particle size, potential and pH of AP were 489nm, -34.79mV and 6.56, respectively; when the pH of the buffer was 7.5, the particle size, potential and pH of AP were 450nm, -37.40mV and 6.76, respectively; when the pH of the buffer was 8, the particle size, potential and pH of AP were 450nm, -39.42mV and 6.9, respectively; When the Tris buffer system was used and the pH of the buffer was 8, the particle size, potential and pH of AP were 442 nm, -27.37 mV and 6.42, respectively.
10. A vaccine comprising the aluminum phosphate adjuvant according to claim 8 or 9.