A non-protectant added antifreeze aluminum adjuvant formulation and its use in vaccine storage

Abstract

This invention discloses a cryoprotectant-free antifreeze aluminum adjuvant formulation and its application in vaccines, belonging to the field of cryogenic technology. By mediating the interfacial behavior of aluminum nanoparticles during freeze-thaw cycles through solute components, the degree of damage is altered. The freeze-thaw stability of aluminum adjuvant vaccines is significantly improved simply by optimizing the buffer system (without adding any cryoprotectant). In addition to mitigating freeze damage, this engineering strategy can also be effectively extended to lyophilization platforms. These findings establish solute composition as a new approach to improving the colloidal stability and functional properties of aluminum adjuvants, and have broad application potential in various pharmaceutical formulations and preservation processes. This provides a promising solution to the long-standing problem of ice crystal damage to solid particle components in natural and industrial systems.

Description

Technical Field

[0001] This invention belongs to the field of cryogenic technology, specifically relating to reducing the freezing and freeze-drying damage of aluminum adjuvants by regulating the buffer solute composition. Background Technology

[0002] During freezing, ice crystals interact with surrounding particles, ions, and molecules, reshaping the interfacial microstructure during phase transitions, thereby altering the structure, distribution, and function of these components. Therefore, precise control of freeze-thaw processes is crucial across various fields, including climate science, aerospace, food science, and biomedicine. In biomedical applications involving cryogenic phase transitions, vaccines formulated with aluminum adjuvants are essential but also fragile, particularly sensitive to temperature fluctuations. Accidental freezing exposure can impair vaccine efficacy and stability by causing adjuvant dehydration, loss of surface hydroxyl groups, and promoting particle aggregation, potentially leading to vaccine inactivation and significant waste. Therefore, mitigating freezing-induced damage to aluminum adjuvants remains a pressing and unresolved need. In practical applications, the complexity of formulation components hinders the translation of basic cryoprotection strategies into adjuvant-compatible vaccine systems. Solutes, as essential and ubiquitous components in vaccine formulations, provide an attractive and operational model system for systematically studying interfacial regulatory mechanisms. Therefore, this invention provides a method that eliminates the need for additional cryoprotectants, thus avoiding increased formulation complexity and the introduction of additional regulatory and biosafety considerations. Summary of the Invention

[0003] The purpose of this invention is to provide an antifreeze aluminum adjuvant without added protectant and its application in vaccine storage. By elucidating the interfacial behavior of ice growth during freeze-thaw cycles, the solute composition is optimized to mitigate freeze-thaw-induced damage to the aluminum adjuvant. Guided by solute-mediated solid-liquid interface regulation, the solute alters the morphology of the ice-water interface, inhibiting ice recrystallization through the Gibson-Thomson effect and reducing direct interactions between Alhydrogel particles and ice crystals, thereby reducing particle aggregation and structural damage caused by ice encapsulation.

[0004] An antifreeze aluminum adjuvant formulation without added preservatives, wherein the antifreeze aluminum adjuvant formulation contains a solute, and the freezing phase change process is mediated by the composition regulation of the solute, thereby maintaining the surface structure, dispersion stability and adjuvant function of the aluminum adjuvant particles.

[0005] Preferably, the antifreeze aluminum adjuvant formulation design includes: a single solute component dispersion containing aluminum adjuvant, a multi-solute component composite dispersion containing aluminum adjuvant, a single solute component dispersion containing an antigen-aluminum adjuvant complex, and a multi-solute component composite dispersion containing an antigen-aluminum adjuvant complex.

[0006] Preferably, the aluminum adjuvant comprises aluminum hydroxyaluminate (e.g., Alhydrogel), aluminum phosphate, etc., and the concentration range of the aluminum adjuvant is 0.1-4.8 mg Al / mL, preferably 1.7 mg Al / mL.

[0007] Preferably, the solute comprises one or more of sodium chloride (NaCl), disodium hydrogen phosphate (Na₂HPO₄), sodium dihydrogen phosphate (Na₂HPO₄), succinic acid (SA), 4-hydroxyethylpiperazine ethanesulfonic acid (HEPES), tris(hydroxymethyl)aminomethane hydrochloride (Tris-HCl), and 4-hydroxyethylpiperazine ethanesulfonic acid (HEPES), with 4-hydroxyethylpiperazine ethanesulfonic acid being the most preferred. The concentration range of the solute is 5-200 mM, preferably 50 mM.

[0008] Preferably, the antifreeze aluminum adjuvant formulation may contain an antigen, including one or two of hepatitis B antigen (HBsAg) and diphtheria toxoid, wherein the concentration range of the antigen is 0-0.08 µg / µg A1, preferably 0.068 mg / mL.

[0009] Preferably, the antifreeze aluminum adjuvant allows freezing (-80 °C to -196 °C) or freeze-drying (-80 °C) without compromising the stability of the aluminum adjuvant.

[0010] Application of the above-mentioned antifreeze aluminum adjuvant formulation without added protectant in vaccine storage.

[0011] This invention modifies the interfacial behavior of aluminum nanoparticles by mediating the solute component during freeze-thaw cycles, thereby altering the degree of damage. By simply optimizing the solute system (without adding any cryoprotectants), the freeze-thaw stability of aluminum-adjuvanted vaccines is significantly improved. In addition to mitigating freeze damage, this engineering strategy can also be effectively extended to lyophilization platforms. These findings establish solute composition as a new approach to improving the colloidal stability and functional properties of aluminum adjuvants, and have broad application potential in various pharmaceutical formulations and preservation processes. This provides a promising solution to the long-standing problem of ice crystal damage to solid particle components in natural and industrial systems.

[0012] Beneficial effects:

[0013] This invention effectively prevents surface damage and aggregation of aluminum nanoparticles during freezing by regulating solute, while maintaining colloidal stability and antigen adsorption capacity, thus maintaining a strong antigen-specific humoral immune response.

[0014] This invention reveals the key mechanism of freeze-thaw-induced damage and demonstrates that the freeze tolerance of vaccines can be significantly improved by strategically optimizing the solute without the use of cryoprotectants, providing a feasible strategy for enhancing the stability of particulate-based drugs. Attached Figure Description

[0015] Figure 1 For the assessment of freeze-thaw damage of Alhydrogel (before freeze-thaw (RT) and after freeze-thaw (FT)); (A) Schematic diagram of the freeze-thaw process, including ice crystal nucleation, growth, recrystallization and melting stages; (B) Radio electron microscopy (TEM) images of Alhydrogel before and after freeze-thaw treatment in pure water, scale bar at 500 nm; (C) Radio electron microscopy (TEM) images of Alhydrogel before and after freeze-thaw treatment in 200 mM HEPES, scale bar at 500 nm; (D) Hydrodynamic diameter of Alhydrogel measured in different solute environments at concentration gradients of 200 mM, 50 mM and 5 mM; (E) Suspension stability of Alhydrogel dispersions in different solute systems (200 mM, 50 mM) after standing for 1 h before and after freeze-thaw; (F) Zeta potential measurement results of Alhydrogel before and after freeze-thaw in different buffer systems (200 mM, 50 mM, 5 mM).

[0016] Figure 2 (A) Changes in hydrodynamic diameter of Alhydrogel before and after freeze-drying, measured in pure water or 50 mM HEPES (comparison of before freeze-drying (RT) and after freeze-drying (FD)); (B) Changes in Zeta potential of Alhydrogel before and after freeze-drying, measured in pure water or 50 mM HEPES (comparison of before freeze-drying (RT) and after freeze-drying (FD)).

[0017] Figure 3 This study investigates the adsorption behavior of Alhydrogel before and after freeze-thaw cycles. (A) Schematic diagram of antigen adsorption on Alhydrogel before and after freeze-thaw cycles under different environmental conditions; (B) Adsorption curves of bovine serum albumin (BSA) on Alhydrogel in pure water before freeze-thaw (RT) and after freeze-thaw (FT); (C) Adsorption curves of BSA on Alhydrogel before freeze-thaw (RT) and after freeze-thaw (FT) in different solute systems of 200 mM and 50 mM.

[0018] Figure 4 For the immunogenicity study of Alhydrogel before and after freeze-thaw, (A) is a schematic diagram of the vaccination process. Six-week-old female C57BL / 6 mice (n=6) were injected intramuscularly with HBsAg (2μg) or HBsAg / Alhydrogel (2μg / 50μg Al) on day 0, and a booster immunization was given on day 21; (B) and (C) show the HBsAg-specific serum total IgG (B) and IgG1 (C) measured by ELISA. # This indicates no significant difference. p <0.05, p <0.01, p <0.001, p <0.0001, one-way ANOVA combined with Tukey post-hoc test was used for statistical analysis. Detailed Implementation

[0019] The following non-limiting embodiments are intended to enable those skilled in the art to more fully understand the present invention, but do not limit the invention in any way.

[0020] In the examples below, Alhydrogel is a commercially available rod-shaped aluminum adjuvant widely used in various marketed vaccine formulations and was purchased from InvivoGen in the United States.

[0021] Example 1 In this embodiment, Alhydrogel was selected as a representative model for developing antifreeze formulations. To systematically evaluate the influence of solutes on the mechanism of freezing damage, six commonly used solute components were selected—including sodium chloride (NaCl), disodium hydrogen phosphate (Na2HPO4), sodium dihydrogen phosphate (NaH2PO4), succinic acid (SA), tris(hydroxymethyl)aminomethane hydrochloride (Tris-HCl), and 4-(2-hydroxyethyl)-1-piperazine ethanesulfonic acid (HEPES)—and tested at three concentration gradients (5 mM, 50 mM, and 200 mM).

[0022] Preparation of Alhydrogel adjuvant: First, dissolve the six commonly used solutes separately in pure water to prepare 400 mM solutions, which are the solute-containing solutions. Then, take 0.34 mL of Alhydrogel stock solution (10 mg Al / mL, solvent: pure water) and add 0.025, 0.25, and 1 mL of the 400 mM solute-containing solutions, respectively. Finally, add pure water to prepare 2 mL of dispersion (containing 1.7 mg Al / mL Alhydrogel, and 5 mM, 50 mM, or 200 mM solutes), and mix thoroughly. Set up a control group (excluding the six commonly used solutes, denoted as the pure water control group) by mixing 0.34 mL of Alhydrogel stock solution (10 mg Al / mL, solvent: pure water) with 1.66 mL of pure water.

[0023] Freeze-thaw: Place 2 mL of the dispersion or control group 5 cm above the surface of liquid nitrogen to rapidly freeze it, and then... Immerse in liquid nitrogen at 196°C for two hours, then thaw at room temperature (1-2 hours). The dispersion before freeze-thaw treatment is denoted as [RT]; the dispersion after freeze-thaw treatment is denoted as [FT].

[0024] The freeze-thaw process involves multiple phase transition stages. In the initial stage, water molecules aggregate to form ice nuclei in the liquid state; under sustained low temperatures, these ice nuclei continue to grow; during warming, ice crystals recrystallize through Ostwald ripening, forming larger crystals; finally, with external heating input, they return to the liquid phase. Figure 1 A). Transmission electron microscopy (TEM) characterization results showed that the Alhydrogel[RT] in the control group before freeze-thaw treatment in pure water exhibited a well-dispersed rod-like morphology, while after freeze-thaw treatment, severe aggregation of Alhydrogel[FT] was observed. Figure 1 B). However, in the HEPES environment, Alhydrogel did not show significant aggregation after freeze-thaw cycles (B). Figure 1 C). Dynamic light scattering (DLS) was further used to quantitatively analyze the degree of aggregation. The results showed that the hydrodynamic size of Alhydrogel particles in the control group in pure water significantly increased after one freeze-thaw treatment, from 216 nm to 2778 nm. However, in the 200 mM SA and 50 mM HEPES groups, the change in hydrodynamic size before and after freeze-thaw was minimal. Figure 1 D). According to the Delgado-Vive-Overbeck (DLVO) theory, colloidal stability depends on the interaction potential energy between particles; only by overcoming a certain energy barrier can aggregation occur. During freezing, the pressure generated by ice crystal formation is sufficient to push Alhydrogel over this barrier, thus inducing particle aggregation. This aggregation directly affects the suspension stability after freeze-thaw treatment. Specifically, Alhydrogel in the control group in pure water significantly precipitated after freeze-thaw treatment, while the solute groups all showed some improvement, especially in the formulations of 200 mM SA, 200 mM, and 50 mM HEPES, where Alhydrogel maintained excellent colloidal stability. This is highly consistent with the particle behavior revealed by the aforementioned hydrodynamic size analysis. Figure 1 E). The zeta potential of alhydrogel is significantly affected by the solute environment, showing obvious differences among different solutes (E). Figure 1F). Freeze-thaw treatment disrupts the stability of Alhydrogel particles in pure water (control group), manifested as a decrease in Zeta potential of approximately 9 mV. This decrease implies a weakening of interparticle repulsion, partially explaining why aggregation is more likely to occur after freeze-thaw. More importantly, this electrostatic stability impairment effect is effectively mitigated in HEPES systems with different molar intensities. These findings strongly support the superior protective effect of HEPES as a high-quality solute against structural damage caused by freeze-thaw stress, highlighting its crucial role in maintaining particle integrity.

[0025] Example 2 The stability of the antifreeze aluminum adjuvant formulation after lyophilization was evaluated. Preparation of the Alhydrogel adjuvant: First, HEPES solute was dissolved in pure water to prepare a 400 mM solution, which is the HEPES-containing solution. Then, 0.34 mL of Alhydrogel stock solution (10 mg Al / mL, solvent: pure water) was added to 0.25 mL of the 400 mM HEPES-containing solution. Finally, pure water was added to prepare a 2 mL dispersion (containing 1.7 mg Al / mL Alhydrogel and 50 mM solute), and the mixture was thoroughly mixed. A control group (containing no solute, designated as the pure water control group) was set up by uniformly mixing 0.34 mL of Alhydrogel stock solution (10 mg Al / mL, solvent: pure water) with 1.66 mL of pure water. The 2 mL dispersion or control group was placed 5 cm above the surface of liquid nitrogen to rapidly freeze, and then... The sample was immersed in liquid nitrogen at 196°C for two hours, then transferred to a vacuum dryer (Labconco, USA, parameters set to -80°C, 0 mBar) for 24 hours to freeze-dry and obtain a solid powder. Finally, 2 mL of pure water was added to redisperse the powder. The dispersion before freeze-drying is denoted as [RT]; the dispersion after freeze-drying is denoted as [FD].

[0026] Dynamic light scattering measurements showed that after lyophilization in pure water, the hydrodynamic size of Alhydrogel increased to 133.53 times its original size, while the Zeta potential decreased by 10 ± 1 mV, indicating severe particle aggregation and changes in surface charge. In contrast, lyophilization in 50 mM HEPES buffer only increased the size by 1.28 times, and no significant change in Zeta potential was observed. Figure 2 (A and 2B).

[0027] Example 3 The adsorption capacity of Alhydrogel for antigens after freeze-thaw was studied. Figure 3A). In aluminum-adjuvanted vaccines, antigen adsorption onto the adjuvant is crucial for evoking a potent immune response. The adsorption kinetics between Alhydrogel and this model antigen were characterized using bovine serum albumin (BSA).

[0028] Preparation method of aluminum adjuvant-containing vaccine: Take 29.4 μL of the dispersion before and after freeze-thaw in Example 1, and then add different volumes of BSA solution (the final BSA concentration is set to 0, 0.05, 0.1, 0.15, 0.2, 0.3, 0.4, 0.6, and 0.8 mg / mL, respectively) and incubate for 30 min.

[0029] The resulting dispersion was centrifuged at 8000 rpm for 10 min, and the supernatant was used to determine the concentration of BSA. The results showed that the Alhydrogel, after freezing in water, completely lost its ability to adsorb BSA. Figure 3 B). Different solute compositions affect the adsorption capacity of Alhydrogel. When the frozen adjuvant was exposed to a solute environment containing 200 mM NaCl, its BSA adsorption decreased slightly. In solute systems of 200 mM NaH2PO4, Tris-HCl, or HEPES, freeze-thaw treatment did not change the adsorption of the antigen. Figure 3 C). At a concentration of 50 mM, HEPES maintained the adsorption coefficient and capacity of Alhydrogel ( Figure 3 C).

[0030] Example 4 Based on the regulatory effect of solute composition on the ice-interface behavior of Alhydrogel, the immunogenicity of Alhydrogel adjuvant after freeze-thaw was evaluated, and hepatitis B surface antigen (HBsAg) was selected as the model antigen.

[0031] Vaccine preparation method: The HEPES group consisted of physiological saline containing 50 mM HEPES; the HBsAg group consisted of physiological saline containing 0.04 μg / μL HBsAg antigen; the Alhydrogel group consisted of the pure water control group (without freeze-thaw treatment) from Example 1, with the addition of HBsAg antigen and NaCl, and diluted with pure water to finally prepare a dispersion containing 0.1 mg Al / mL Alhydrogel, 0.04 μg / μL HBsAg antigen, and 0.9% NaCl; Alhydrogel （FT） The control group (treated with freeze-thaw) in Example 1 was supplemented with HBsAg antigen and NaCl, and diluted with pure water to prepare a dispersion containing 0.1 mg Al / mL Alhydrogel, 0.04 μg / μL HBsAg antigen, and 0.9% NaCl; Alhydrogel （HEPES-FT）The group consisted of the 50 mM HEPES dispersion (after freeze-thaw treatment) from Example 1, with the addition of HBsAg antigen, NaCl, and HEPES, and diluted with pure water to prepare a dispersion containing 0.1 mg Al / mL Alhydrogel, 0.04 μg / μL HBsAg antigen, 0.9% NaCl, and 50 mM HEPES; Alhydrogel （FD） The control group (lyophilized) in Example 2 was supplemented with HBsAg antigen and NaCl, and diluted with pure water to prepare a dispersion containing 0.1 mg Al / mL Alhydrogel, 0.04 μg / μL HBsAg antigen, and 0.9% NaCl; Alhydrogel （HEPES-FD） The 50 mM HEPES dispersion (lyophilized) from Example 2 was mixed with HBsAg antigen, NaCl, and HEPES, and diluted with pure water to prepare a dispersion containing 0.1 mg Al / mL Alhydrogel, 0.04 μg / μL HBsAg antigen, 0.9% NaCl, and 50 mM HEPES.

[0032] Mice were injected intramuscularly on days 0 and 21, with each injection consisting of 50 μL. Blood was collected from the eyeballs on day 42. Figure 4 A), after placing the sample in an ice box for 1 hour, centrifuge at 3500 rpm for 30 minutes and collect the supernatant serum to determine the titer of hepatitis B-specific antibodies in the serum. If a formulation containing HEPES is used to stabilize Alhydrogel (Alhydrogel (HEPES-FT)) during freezing, the total IgG antibody titer produced by the HBsAg vaccine containing this adjuvant is 2.6 × 10⁵, and the IgG1 antibody titer is 2.8 × 10⁵. Figure 4 B and 4C). In contrast, the humoral immune response was significantly weakened in the Alhydrogel (FT) group, with the total IgG antibody titer decreasing to 0.22 × 10⁵ and the IgG1 antibody titer also decreasing to 0.32 × 10⁵. Figure 4 B and 4C). Statistical validation confirmed that solute-driven adjuvant freeze-thaw interface behavior regulation can maintain humoral immune responses. Furthermore, the Alhydrogel (HEPES-FD) group effectively preserved humoral immune responses, inducing a total IgG antibody titer as high as 3.8 × 10⁵ and an IgG1 titer of 3.3 × 10⁵. Conversely, the vaccine formulated with freeze-dried Alhydrogel without HEPES solute exhibited significantly lower antibody titers, with total IgG levels dropping to 0.56 × 10⁵ and IgG1 titers also decreasing to 0.63 × 10⁵. Figure 4(B and 4C). These results indicate that solute composition plays a crucial role in stabilizing the entire system, meaning that ice-interface regulation mechanisms that effectively prevent nanoparticle damage can be successfully applied to the development of new freeze-drying platforms.

[0033] For anyone skilled in the art, many possible variations and modifications can be made to the technical solutions of this invention based on the disclosed technical content, or equivalent embodiments can be modified accordingly, without departing from the scope of the technical solutions of this invention. Therefore, any simple modifications, equivalent changes, and modifications made to the above embodiments based on the technical essence of this invention without departing from the content of the technical solutions of this invention should still fall within the protection scope of the technical solutions of this invention.

Claims

1. An anti-freezing aluminium adjuvant formulation without the addition of a protective agent, characterized in that, The antifreeze aluminum adjuvant formulation contains a solute.

2. The antifreeze aluminum adjuvant formulation according to claim 1, characterized in that, The formulation of the antifreeze aluminum adjuvant is a single solute component dispersion containing aluminum adjuvant, a multi-solute component composite dispersion containing aluminum adjuvant, a single solute component dispersion containing an antigen-aluminum adjuvant complex, or a multi-solute component composite dispersion containing an antigen-aluminum adjuvant complex.

3. The antifreeze aluminum adjuvant formulation according to claim 1, characterized in that, The aluminum adjuvant comprises one or both of aluminum hydroxyaluminate and aluminum phosphate.

4. The non-stroage additive based anti-freezing aluminium adjuvant formulation as claimed in claim 1, wherein, The concentration of the aluminum adjuvant is 0.1-4.8 mg Al / mL, and the concentration of the solute is 5-200 mM.

5. The non-stroage additive based anti-freezing aluminium adjuvant formulation as claimed in claim 1, wherein, The antifreeze aluminum adjuvant formulation may contain an antigen.

6. The non-stroage additive antifreeze aluminum adjuvant formulation according to claim 5, characterized in that, The antigen contains one or both of hepatitis B antigen and diphtheria toxoid; the concentration of the antigen is 0-0.08 µg / µg A1.

7. The non-stroage additive based anti-freezing aluminium adjuvant formulation as claimed in claim 1, wherein, The solute comprises one or more of sodium chloride, disodium hydrogen phosphate, sodium dihydrogen phosphate, succinic acid, 4-hydroxyethylpiperazine ethanesulfonic acid, tris(hydroxymethyl)aminomethane hydrochloride, and 4-hydroxyethylpiperazine ethanesulfonic acid; the concentration of the solute is 5-200 mM.

8. The non-stroage additive based anti-freezing aluminium adjuvant formulation as claimed in claim 1, wherein, The antifreeze aluminum adjuvant is frozen or freeze-dried.

9. The application of the antifreeze aluminum adjuvant formulation without protective agent addition as described in any one of claims 1-8 in vaccine storage.

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