A qs-21 complex adjuvant and a method of preparing the same

Abstract

The application discloses a QS-21 composite adjuvant and a preparation method thereof, and belongs to the technical field of biological medicines. The QS-21 composite adjuvant is prepared by compounding a polyanion material with QS-21 to form nanoparticles and organically combining with a freeze-drying protective agent, has a high retention rate, solves the problems of hydrolysis and oxidation, avoids the demand for a phospholipid carrier, simplifies a vaccine co-preparation process, and retains the immune enhancement activity of QS-21 by adding the freeze-drying protective agent subsequently, reduces the exposure of QS-21 to a degradation environment, has a low hemolysis rate, and thus has significant clinical application value and industrialization potential.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to a QS-21 compound adjuvant and its preparation method. Background Technology

[0002] QS-21 is a natural triterpenoid saponin complex derived from the bark of the soapberry tree (Quillaja saponaria). Due to its significant immunomodulatory properties, it has been widely used in various vaccine adjuvant systems, particularly in prophylactic vaccines, therapeutic tumor vaccines, and recombinant protein vaccines. QS-21 can significantly enhance cellular and humoral immune responses to antigens, exhibiting superior performance in dose reduction, adjuvant synergy, and memory immunity. It is one of the important natural adjuvants that has entered clinical application and has commercialization prospects.

[0003] However, the stability of QS-21 has been a key technical bottleneck in its clinical application and industrialization. Studies have shown that QS-21 is highly susceptible to hydrolysis and oxidative degradation in aqueous solution, and even under refrigeration at 2-8°C, it can only be stably stored for a few months. Once exposed to slightly higher temperatures or air, its activity decreases significantly, leading to a substantial reduction in the formulation's shelf life. This instability not only places higher demands on vaccine storage and transportation but also increases the complexity of formulation development and quality control. Therefore, effectively improving the stability of QS-21 has become one of the core issues in the field of vaccine adjuvant technology.

[0004] Specifically, the instability of QS-21 mainly stems from multiple easily degradable sites in its molecular structure. Firstly, hydrolysis primarily occurs at multiple ester and glycosidic bonds in its structure, particularly the ester bond connecting the fatty acyl group and the fucose residue, which is highly susceptible to breakage under neutral pH conditions. Simultaneously, the glycosidic linkage is also prone to hydrolysis under heating or alkaline conditions, thus disrupting the overall configuration of QS-21. Secondly, oxidation occurs concentrated at the C4 aldehyde group on the triterpene core. This functional group is readily oxidized to a carboxylic acid by atmospheric oxygen, causing QS-21 to transform from its active form to its inactive form. These two degradation pathways often coexist during storage, meaning that even if QS-21 remains transparent and clear in appearance, its actual immunomodulatory activity continuously declines, posing a potential risk to vaccine product stability control. Furthermore, the physicochemical properties of QS-21 also play a crucial role in its degradation process. QS-21 has a flexible macromolecular structure, exhibiting overall amphiphilicity. It combines hydrophilic glycosyl groups with a hydrophobic triterpenoid skeleton, lacking a rigid supporting structure, making it prone to adsorbing moisture and oxygen, thus reducing molecular stability. Furthermore, because QS-21 is a natural extract, even after multi-step purification, residual substances such as Fe may still remain in the final product. 2+ Cu 2+Impurities such as trace metal ions or fatty acids can induce free radical generation and trigger chain oxidation reactions, further accelerating the degradation of aldehyde groups and fatty chains, and exacerbating the risk of adjuvant inactivation. The natural structural characteristics of the QS-21 molecule, its hydrophilic-hydrophobic imbalance, lack of rigid protection, and susceptibility to exogenous oxidation and hydrolysis constitute the systemic problems of its poor stability, easy degradation, and difficulty in preservation. It is urgent to carry out research on stabilization strategies from the perspectives of molecular protection, microenvironment regulation, and structural modification, and to construct innovative delivery carriers that can be prepared on a large scale and adapted to multiple vaccine formulation systems. Summary of the Invention

[0005] The present invention aims to provide a QS-21 composite adjuvant and its preparation method. By constructing a stable nanocomposite structure with QS-21 using a specific polyanionic material, the storage stability and safety of use are effectively improved without affecting its adjuvant function.

[0006] To achieve the above objectives, this invention discloses a method for preparing QS-21 composite adjuvant, as detailed below:

[0007] S1. Weigh 1-10 mg of QS-21 powder and add it to 1 mL of Tris buffer solution with pH 6.0-6.8. Place the solution in an ice bath and shake for 5-20 min.

[0008] S2. Prepare a 5-20 mg / mL polyanionic solution and add it dropwise to the solution prepared in S1 at a rate of 0.1-0.3 mL / min. Stir at 200-500 r / min for 3-10 min.

[0009] S3. After the addition is complete, keep stirring for 20-30 minutes, let stand for 5-10 minutes; adjust the pH and remove impurities using a 0.22μm filter membrane;

[0010] S4. Add freeze-drying protectant and stir at 100-300 r / min for 1-3 min;

[0011] S5. After vacuum freeze-drying, QS-21 composite adjuvant is obtained.

[0012] The polyanion is any one of polyglutamic acid, sodium hyaluronate, alginate oligosaccharide, or polyglutamic acid complex.

[0013] Preferably, the mass ratio of QS-21 to polyanion is 1:1-5.

[0014] The preparation method of the polyglutamic acid complex is as follows: 0.1-1.2 mmol of polyglutamic acid and 0.1-1.8 mmol of the condensate are activated; 0.1-1.2 mmol of L-cysteine ​​hydrochloride is added, and the reaction is carried out in water in the dark; the pH is adjusted and dialysis is performed.

[0015] The condensate is at least one of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, N-hydroxysuccinimide, and 2-carboxylic acid pyridine.

[0016] The aforementioned light-protected reaction in water is carried out under a nitrogen atmosphere for 12-36 hours in the dark.

[0017] The dialysis treatment is as follows: use a dialysis bag with a molecular weight cutoff of 10-15 kDa for dialysis for 48-72 hours, and change the water every 2-4 hours.

[0018] The freeze-drying protectant is selected from any one of trehalose, mannitol, and sucrose; the amount added is 2-5% of the total system mass.

[0019] The vacuum freeze-drying conditions involved in step S5 are a temperature of -60 to -20°C and a drying time of 6 to 20 hours.

[0020] This invention discloses a QS-21 composite adjuvant that can be stored for 7 days at 25-40℃ and 20-30% relative humidity.

[0021] The beneficial effects of this invention are:

[0022] Compared with existing liposome encapsulation methods, the present invention has the following advantages:

[0023] 1. The QS-21 composite adjuvant provided by this invention has better physical stability and can maintain its activity for more than 7 days under non-cold chain conditions at 25-40℃, avoiding the problem of easy oxidation of existing liposomes; it does not use phospholipids, cholesterol and other components, which simplifies the co-preparation process of adjuvant and antigen, and is suitable for a variety of vaccine antigens.

[0024] 2. The thiolized polyanions compounded in this invention are widely available and have mature processing technology, possessing strong potential for industrial scale-up and formulation integration; they can maintain dispersibility and particle size stability after mixing with antigens, and do not affect the immune recognition function of the QS-21 triterpenoid ring structure.

[0025] 3. The present invention prepares QS-21 composite adjuvant and adds a freeze-drying protectant during the freeze-drying process to reduce ice crystal damage during freeze-drying; the synergistic effect of thiolized polyglutamic acid can shield the degradation sites of QS-21, providing key support for industrial freeze-drying process. Detailed Implementation

[0026] A method for preparing QS-21 compound adjuvant is as follows:

[0027] S1. Weigh 1-10 mg of QS-21 powder and add it to 1 mL of Tris buffer solution with pH 6.0-6.8. Place the solution in an ice bath and shake for 5-20 min.

[0028] S2. Prepare a 5-20 mg / mL polyanionic solution and add it dropwise to the QS-21 solution prepared in S1 at a rate of 0.1-0.3 mL / min, while maintaining magnetic stirring at 200-500 r / min for 3-10 min.

[0029] S3. After the addition is complete, continue stirring for 20-30 minutes, let stand for 5-10 minutes; adjust the pH to 6.2-6.5 and remove impurities using a 0.22μm filter membrane;

[0030] S4. Add freeze-drying protectant and stir at 100-300 r / min for 1-3 min;

[0031] S5. After drying at -60 to -20℃ for 6-20 hours, QS-21 composite adjuvant is obtained.

[0032] The parameters and sources of some substances in the examples are as follows:

[0033] QS-21 powder: CAS No.: 141256-04-4; Desert King, USA.

[0034] Sodium hyaluronate: CAS No.: 9067-32-7; molecular weight is 30-200kDa.

[0035] Alginate oligosaccharides: CAS No.: 9005-38-3; molecular weight is 50-100kDa.

[0036] Polyglutamic acid: CAS No.: CAS 25513-46-6; molecular weight is 10-30kDa.

[0037] 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide: CAS No.: 25952-53-8.

[0038] N-Hydroxysuccinimide: CAS No.: 6066-82-6.

[0039] 2-Pyridine carboxylate: CAS No.: 98-98-6.

[0040] L929 mouse fibroblasts: 1×10 6 cells; Kanglang Biotechnology.

[0041] 1% fresh rabbit red blood cells: concentration 1*10 8 Red blood cells / mL; Xinfan Biotechnology.

[0042] Phenol red-free medium: RPMI Medium 1640, Beijing Solarbio.

[0043] Example 1

[0044] A method for preparing QS-21 compound adjuvant is as follows:

[0045] S1. Take 5 mg of QS-21 powder, add it to 1 mL of Tris buffer solution with pH 6.3, place it in an ice bath and shake for 12 min, then set aside.

[0046] S2. Add 5 mg of sodium hyaluronate to 1 mL of water and stir at 300 r / min for 10 min under ice bath conditions; filter sterilize with a 0.22 μm filter membrane and set aside; add 1 mL of the prepared solution dropwise to the QS-21 solution described in step S1 at a rate of 0.1 mL / min and stir at 300 r / min for 5 min.

[0047] S3. After the addition is complete, continue stirring for 30 minutes and let stand for 10 minutes; adjust the pH to 6.4 and remove impurities using a 0.22μm filter membrane;

[0048] S4. The QS-21 composite adjuvant was prepared by vacuum freeze-drying at -55℃ for 12 hours.

[0049] Example 2

[0050] A method for preparing QS-21 compound adjuvant is as follows:

[0051] S1. Weigh 5 mg of QS-21 powder and add it to 1 mL of Tris buffer solution with pH 6.3. Place the solution in an ice bath and shake for 12 min.

[0052] S2. Add 5 mg of alginate oligosaccharide to 1 mL of water and stir at 300 r / min for 10 min under ice bath conditions; filter sterilize with a 0.22 μm filter membrane and set aside; add 1 mL of the prepared solution dropwise to the QS-21 solution described in step S1 at a rate of 0.1 mL / min and stir at 300 r / min for 5 min.

[0053] S3. After the addition is complete, continue stirring for 30 minutes and let stand for 10 minutes; adjust the pH to 6.4 and remove impurities using a 0.22μm filter membrane;

[0054] S4. The QS-21 composite adjuvant was prepared by vacuum freeze-drying at -55℃ for 12 hours.

[0055] Example 3

[0056] A method for preparing QS-21 compound adjuvant is as follows:

[0057] S1. Weigh 5 mg of QS-21 powder and add it to 1 mL of Tris buffer solution with pH 6.3. Place the solution in an ice bath and shake for 12 min.

[0058] S2. Take 5 mg of polyglutamic acid and add it to 1 mL of water. Stir at 300 r / min for 10 min under ice bath conditions. Filter the solution with a 0.22 μm filter membrane for sterilization and set aside. Add 1 mL of the prepared solution dropwise to the QS-21 solution described in step S1 at a rate of 0.1 mL / min and stir at 300 r / min for 5 min.

[0059] S3. After the addition is complete, continue stirring for 30 minutes and let stand for 10 minutes; adjust the pH to 6.4 and remove impurities using a 0.22μm filter membrane;

[0060] S4. The QS-21 composite adjuvant was prepared by vacuum freeze-drying at -55℃ for 12 hours.

[0061] Example 4

[0062] A method for preparing QS-21 compound adjuvant is as follows:

[0063] S1. Weigh 5 mg of QS-21 powder and add it to 1 mL of Tris buffer solution with pH 6.3. Place the solution in an ice bath and shake for 12 min.

[0064] S2. Take 10 mg of polyglutamic acid and add it to 1 mL of water. Stir at 300 r / min for 10 min under ice bath conditions. Filter the solution with a 0.22 μm filter membrane for sterilization and set aside. Add 1 mL of the prepared solution dropwise to the QS-21 solution described in step S1 at a rate of 0.1 mL / min and stir at 300 r / min for 5 min.

[0065] S3. After the addition is complete, continue stirring for 30 minutes and let stand for 10 minutes; adjust the pH to 6.4 and remove impurities using a 0.22μm filter membrane;

[0066] S4. The QS-21 composite adjuvant was prepared by vacuum freeze-drying at -55℃ for 12 hours.

[0067] Example 5

[0068] A method for preparing QS-21 compound adjuvant is as follows:

[0069] S1. Weigh 5 mg of QS-21 powder and add it to 1 mL of Tris buffer solution with pH 6.3. Place the solution in an ice bath and shake for 12 min.

[0070] S2. Add 15 mg of polyglutamic acid to 1 mL of water and stir at 300 r / min for 10 min under ice bath conditions; filter sterilize with a 0.22 μm filter membrane and set aside; add 1 mL of the prepared solution dropwise to the QS-21 solution described in step S1 at a rate of 0.1 mL / min and stir at 300 r / min for 5 min.

[0071] S3. After the addition is complete, continue stirring for 30 minutes and let stand for 10 minutes; adjust the pH to 6.4 and remove impurities using a 0.22μm filter membrane;

[0072] S4. The QS-21 composite adjuvant was prepared by vacuum freeze-drying at -55℃ for 12 hours.

[0073] Example 6

[0074] A method for preparing QS-21 compound adjuvant is as follows:

[0075] S1. Weigh 5 mg of QS-21 powder and add it to 1 mL of Tris buffer solution with pH 6.3. Place the solution in an ice bath and shake for 12 min.

[0076] S2. Take 20 mg of polyglutamic acid and add it to 1 mL of water. Stir at 300 r / min for 10 min under ice bath conditions. Filter the solution through a 0.22 μm filter membrane for sterilization and set aside. Add 1 mL of the prepared solution dropwise to the QS-21 solution described in step S1 at a rate of 0.1 mL / min and stir at 300 r / min for 5 min.

[0077] S3. After the addition is complete, continue stirring for 30 minutes and let stand for 10 minutes; adjust the pH to 6.4 and remove impurities using a 0.22μm filter membrane;

[0078] S4. The QS-21 composite adjuvant was prepared by vacuum freeze-drying at -55℃ for 12 hours.

[0079] Example 7

[0080] A method for preparing QS-21 compound adjuvant is as follows:

[0081] S1. Weigh 5 mg of QS-21 powder and add it to 1 mL of Tris buffer solution with pH 6.3. Place the solution in an ice bath and shake for 12 min.

[0082] S2. Take 15 mg of polyglutamic acid complex and add it to 1 mL of water. Stir at 300 r / min for 10 min under ice bath conditions. Filter the solution with a 0.22 μm filter membrane for sterilization and set aside. Add 1 mL of the prepared solution dropwise to the QS-21 solution described in step S1 at a rate of 0.1 mL / min and stir at 300 r / min for 5 min.

[0083] S3. After the addition is complete, continue stirring for 30 minutes and let stand for 10 minutes; adjust the pH to 6.4 and remove impurities using a 0.22μm filter membrane;

[0084] S4. The QS-21 composite adjuvant was prepared by vacuum freeze-drying at -55℃ for 12 hours.

[0085] The preparation method of the polyglutamic acid complex is as follows:

[0086] Add 0.78 mmol of polyglutamic acid to 8 mL of water and stir at 200 rpm for 8 min. Add 0.93 mmol of N-hydroxysuccinimide and activate for 30 min, then set aside. Dissolve 0.78 mmol of L-cysteine ​​hydrochloride in 2 mL of water and add it dropwise to the activated solution. Adjust the pH to 4.8 and react in the dark under a nitrogen atmosphere for 24 h. Adjust the pH to 5.0 with 0.1 mol / L sodium hydroxide aqueous solution and then dialyze using a 12000 Da dialysis bag for 72 h, changing the water every 3 h.

[0087] Example 8

[0088] A method for preparing QS-21 compound adjuvant is as follows:

[0089] S1. Weigh 5 mg of QS-21 powder and add it to 1 mL of Tris buffer solution with pH 6.3. Place the solution in an ice bath and shake for 12 min.

[0090] S2. Take 15 mg of polyglutamic acid complex and add it to 1 mL of water. Stir at 300 r / min for 10 min under ice bath conditions. Filter with a 0.22 μm filter membrane for sterilization and set aside. Add 1 mL of the prepared solution dropwise to the QS-21 solution described in step S1 at a rate of 0.1 mL / min and stir at 300 r / min for 5 min.

[0091] S3. After the addition is complete, continue stirring for 30 minutes and let stand for 10 minutes; adjust the pH to 6.4 and remove impurities using a 0.22μm filter membrane;

[0092] S4. The QS-21 composite adjuvant was prepared by vacuum freeze-drying at -55℃ for 12 hours.

[0093] The preparation method of the polyglutamic acid complex is as follows:

[0094] Add 0.78 mmol of polyglutamic acid to 8 mL of water and stir at 200 rpm for 8 min. Add 0.93 mmol of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and activate for 30 min. Set aside. Dissolve 0.78 mmol of L-cysteine ​​hydrochloride in 2 mL of water and add it dropwise to the activated solution. Adjust the pH to 4.8 and react in the dark under a nitrogen atmosphere for 24 h. Adjust the pH to 5.0 with 0.1 mol / L sodium hydroxide aqueous solution and dialyze using a 12000 Da dialysis bag for 72 h, changing the water every 3 h.

[0095] Example 9

[0096] A method for preparing QS-21 compound adjuvant is as follows:

[0097] S1. Weigh 5 mg of QS-21 powder and add it to 1 mL of Tris buffer solution with pH 6.3. Place the solution in an ice bath and shake for 12 min.

[0098] S2. Take 15 mg of polyglutamic acid complex and add it to 1 mL of water. Stir at 300 r / min for 10 min under ice bath conditions. Filter sterilely through a 0.22 μm filter membrane and set aside. Add 1 mL of the prepared solution dropwise to the QS-21 solution at a rate of 0.1 mL / min and stir at 300 r / min for 5 min.

[0099] S3. After the addition is complete, continue stirring for 30 minutes and let stand for 10 minutes; adjust the pH to 6.4 and remove impurities using a 0.22μm filter membrane;

[0100] S4. The QS-21 composite adjuvant was prepared by vacuum freeze-drying at -55℃ for 12 hours.

[0101] The preparation method of the polyglutamic acid complex is as follows:

[0102] Add 0.186 mmol of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide to 0.5 mL of anhydrous dimethyl sulfoxide and stir at 100 rpm for 8 min. Separately, dissolve 0.186 mmol of N-hydroxysuccinimide in 0.5 mL of anhydrous dimethyl sulfoxide and add it to the prepared solution at a rate of 0.1 mL / min. Stir at 300 rpm for 15 min under ice bath conditions to obtain the condensate, which is then ready for use.

[0103] Add 0.78 mmol of polyglutamic acid to 8 mL of water and stir at 200 rpm for 8 min. Add the prepared condensate and 0.558 mmol of pyridine 2-carboxylic acid and activate for 30 min. Dissolve 0.78 mmol of L-cysteine ​​hydrochloride in 2 mL of water and slowly add it dropwise to the activated solution. Adjust the pH to 4.8 and react in the dark under a nitrogen atmosphere for 24 h. Adjust the pH to 5.0 with 0.1 mol / L sodium hydroxide and dialyze using a 12000 Da dialysis bag for 72 h, changing the water every 3 h.

[0104] Example 10

[0105] A method for preparing QS-21 compound adjuvant is as follows:

[0106] S1. Weigh 5 mg of QS-21 powder and add it to 1 mL of Tris buffer solution with pH 6.3. Place the solution in an ice bath and shake for 12 min.

[0107] S2. Take 15 mg of polyglutamic acid complex and slowly add it to 1 mL of water. Stir at 300 r / min for 10 min under ice bath conditions. Filter the solution through a 0.22 μm filter membrane for sterilization and set aside. Add 1 mL of the prepared solution dropwise to the QS-21 solution at a rate of 0.1 mL / min and stir at 300 r / min for 5 min.

[0108] S3. After the addition is complete, continue stirring for 30 minutes and let stand for 10 minutes; adjust the pH to 6.4 and remove impurities using a 0.22μm filter membrane;

[0109] S4. Add 100mg trehalose and stir at 300rpm for 1min;

[0110] S5. The QS-21 composite adjuvant was prepared by vacuum freeze-drying at -55℃ for 12 hours.

[0111] The preparation method of the polyglutamic acid complex is as follows:

[0112] Add 0.4185 mmol of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide to 0.5 mL of anhydrous dimethyl sulfoxide and stir at 100 rpm for 8 min. Separately, dissolve 0.4185 mmol of N-hydroxysuccinimide in 0.5 mL of anhydrous dimethyl sulfoxide and add it to the prepared solution at a rate of 0.1 mL / min. Stir at 300 rpm for 15 min under ice bath conditions to obtain the condensate, which is then ready for use.

[0113] Add 0.78 mmol of polyglutamic acid to 8 mL of water and stir at 200 rpm for 8 min. Add the prepared condensate and 0.093 mmol of pyridine 2-carboxylic acid and activate for 30 min. Dissolve 0.78 mmol of L-cysteine ​​hydrochloride in 2 mL of water and slowly add it dropwise to the activated solution. Adjust the pH to 4.8 and react in the dark under a nitrogen atmosphere for 24 h. Adjust the pH to 5.0 with 0.1 mol / L sodium hydroxide and dialyze for 72 h using a 12000 Da dialysis bag, changing the water every 3 h.

[0114] Comparative Example 1

[0115] A method for preparing QS-21 adjuvant is as follows: Take 5 mg of QS-21 powder, add it to 1 mL of Tris buffer solution with pH 6.3, and place it in an ice bath and shake for 12 min; keep stirring at 300 r / min for 5 min, and let it stand for 10 min; adjust the pH to 6.4 and remove impurities using a 0.22 μm filter membrane; freeze dry under vacuum at -55℃ for 12 h to obtain QS-21 composite adjuvant.

[0116] Comparative Example 2

[0117] A method for preparing QS-21 compound adjuvant is as follows:

[0118] S1. Weigh 5 mg of QS-21 powder and add it to 1 mL of Tris buffer solution with pH 6.3. Place the solution in an ice bath and shake for 12 min.

[0119] S2. Take 15 mg of polyglutamic acid complex and add it to 1 mL of water. Stir at 300 r / min for 10 min under ice bath conditions. Filter sterilely through a 0.22 μm filter membrane and set aside. Add 1 mL of the prepared polyglutamic acid solution dropwise to the QS-21 solution at a rate of 0.1 mL / min and keep stirring at 300 r / min for 5 min.

[0120] S3. After the addition is complete, continue stirring for 30 minutes and let stand for 10 minutes; adjust the pH to 6.4 and remove impurities using a 0.22μm filter membrane;

[0121] S4. The QS-21 composite adjuvant was prepared by vacuum freeze-drying at -55℃ for 12 hours.

[0122] The preparation method of the polyglutamic acid complex is as follows:

[0123] Add 0.465 mmol of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide to 0.5 mL of anhydrous dimethyl sulfoxide and stir at 100 rpm for 8 min. Separately, dissolve 0.465 mmol of N-hydroxysuccinimide in 0.5 mL of anhydrous dimethyl sulfoxide and add it to the prepared solution at a rate of 0.1 mL / min. Stir at 300 rpm for 15 min under ice bath conditions to obtain the condensate, which is then ready for use.

[0124] Add 0.78 mmol of polyglutamic acid to 8 mL of water and stir at 200 rpm for 8 min. Add the prepared condensate and activate for 30 min. Dissolve 0.78 mmol of L-cysteine ​​hydrochloride in 2 mL of water and slowly add it dropwise to the activated solution. Adjust the pH to 4.8 and react in the dark under a nitrogen atmosphere for 24 h. Adjust the pH to 5.0 with 0.1 mol / L sodium hydroxide and dialyze for 72 h using a 12000 Da dialysis bag, changing the water every 3 h.

[0125] Comparative Example 3

[0126] A method for preparing QS-21 compound adjuvant is as follows:

[0127] S1. Weigh 5 mg of QS-21 powder and add it to 1 mL of Tris buffer solution with pH 6.3. Place the solution in an ice bath and shake for 12 min.

[0128] S2. Take 15 mg of polyglutamic acid complex and add it to 1 mL of water. Stir at 300 r / min for 10 min under ice bath conditions. Filter sterilely through a 0.22 μm filter membrane and set aside. Add 1 mL of the prepared polyglutamic acid solution dropwise to the QS-21 solution at a rate of 0.1 mL / min and keep stirring at 300 r / min for 5 min.

[0129] S3. After the addition is complete, continue stirring for 30 minutes and let stand for 10 minutes; adjust the pH to 6.4 and remove impurities using a 0.22μm filter membrane;

[0130] S4. The QS-21 composite adjuvant was prepared by vacuum freeze-drying at -55℃ for 12 hours.

[0131] The preparation method of the polyglutamic acid complex is as follows:

[0132] Add 0.465 mmol of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide to 0.5 mL of anhydrous dimethyl sulfoxide and stir at 100 rpm for 8 min. Separately, dissolve 0.465 mmol of N-hydroxysuccinimide in 0.5 mL of anhydrous dimethyl sulfoxide and add it to the prepared solution at a rate of 0.1 mL / min. Stir at 300 rpm for 15 min under ice bath conditions to obtain the condensate, which is then ready for use.

[0133] Add 0.78 mmol of polyglutamic acid to 8 mL of water and stir at 200 rpm for 8 min. Add the prepared condensate and activate for 30 min. Dissolve 0.78 mmol of phenylalanine ethyl ester in 2 mL of water and slowly add it dropwise to the activated solution. Adjust the pH to 4.8 and react in the dark under a nitrogen atmosphere for 24 h. Adjust the pH to 5.0 with 0.1 mol / L sodium hydroxide and dialyze for 72 h using a 12000 Da dialysis bag, changing the water every 3 h.

[0134] Test Example 1

[0135] Particle size distribution detection

[0136] Dynamic light scattering (DLS) was used to detect the adjuvant samples prepared in Examples 1-10 and Comparative Examples 1-3 at 25°C using a Malvern ZetaSizer Nano ZS-90 instrument. Samples were reconstituted with ultrapure water to 1 mg / mL, filtered through a 0.22 μm filter membrane, and measured. The results were repeated three times and the average value was taken. The results are shown in Table 1.

[0137] Table 1. Results of particle size distribution test

[0138]

[0139] Note: PDI is the polydispersity index, and a value < 0.2 ensures the uniformity of particles.

[0140] Compared to Comparative Example 1, the composite adjuvants in Examples 1-6 have certain advantages in terms of particle size distribution and dispersibility; the PDI results of Comparative Example 1 show that QS-21 is prone to aggregation when used alone. Among them, the polyanion added in Examples 1-3 may effectively improve the uniformity of the system.

[0141] In Examples 3-6, as the concentration of the polyglutamic acid solution increased, the hydrogen bonding between the carboxyl groups of polyglutamic acid and the polyhydroxyl groups and C4-aldehyde groups on the QS-21 triterpenoid skeleton and sugar chains strengthened, forming a dynamic equilibrium with the hydrophobic association of the QS-21 triterpenoid skeleton. Considering that Examples 3-4 may have had smaller particle sizes and insufficient encapsulation, resulting in uneven particle dispersion; and that in Example 6, when the polyglutamic acid solution was at a high concentration, excessive molecular chains may have become entangled due to electrostatic repulsion and steric hindrance, causing some QS-21 to be encapsulated in the aggregated polyglutamic acid network, forming localized abnormal particle size regions.

[0142] In Example 5, the polyglutamic acid molecular chain exhibits moderate extension, balancing hydrophobic association and hydrogen bonding with QS-21. This ensures sufficient polyanionic encapsulation to shield degradation sites while avoiding particle aggregation caused by high concentrations. Examples 7-9 utilize a single condensing agent or a complex condensing compound to activate the polyglutamic acid molecular chain. The thiolated polyglutamic acid undergoes a specific thioacetalization reaction with the C4-aldehyde group of QS-21, forming stable covalent bonds. Simultaneously, hydrogen bonds synergistically form between the carboxyl groups of the polyglutamic acid backbone and the polyhydroxyl groups of QS-21, enhancing the stability of the composite structure and reducing particle dissociation and aggregation. Compared to Comparative Examples 2-3, Examples 9-10, using a condensing compound, achieve higher activation efficiency, reduce the production of thiol oxidation side reactions, and result in the lowest PDI and the most uniform particle size distribution in the composite particles. Furthermore, in Example 10, the addition of trehalose resulted in better particle size and dispersibility than in the unprotected Example 9; this may be because during the freeze-drying process, the added trehalose formed hydrogen bonds with the hydroxyl groups of polyglutamic acid, reducing the mechanical damage to the nanostructure caused by ice crystal formation.

[0143] Test Example 2

[0144] Stability test

[0145] Take 10 mg of each lyophilized powder from Examples 1-10 and Comparative Examples 1-3, and dispense them into sealed glass bottles. Place them in a drying oven at 40℃ and relative humidity ≤30% for 7 days to simulate long-term storage degradation. The retention rate of QS-21 is determined by high performance liquid chromatography (HPLC). The mobile phase is acetonitrile-water in a 70:30 ratio, the flow rate is set to 1.0 mL / min, and the detection wavelength is 210 nm. The results are shown in Table 2.

[0146] Table 2 Stability Test Results

[0147]

[0148] Retention rate is one of the indicators for measuring the stability of QS-21. It reflects the ability of QS-21 to maintain its structure and activity during storage and use. The higher the retention rate, the less degradation there is, and the better the stability.

[0149] Compared to Comparative Example 1, Examples 1-7 showed better retention of QS-21 in combination with polyanionic compounds. Example 3 exhibited a better retention rate than Examples 1-2, possibly due to the loose structure of sodium hyaluronate and the reduction of active sites by the terminal groups of alginate oligosaccharides, which failed to effectively shield easily degradable sites such as ester bonds and aldehyde groups. Examples 7-9 and Comparative Examples 2-3 likely involved the activation of polyglutamic acid molecular chains, with intermolecular hydrogen bonds forming between thiol and hydroxyl groups, constructing an outer shell network. QS-21 was then stabilized by hydrophobic association and hydrogen bonds within the shell, further blocking oxygen and moisture, inhibiting oxidation and hydrolysis, and reducing QS-21 exposure to the degradation environment. However, excessive polyglutamic acid molecules entangled due to electrostatic repulsion and steric hindrance, forming irregular aggregates. This resulted in some QS-21 being encapsulated within the aggregate core, unable to fully bind with the protective layer. Furthermore, pH fluctuations accelerated degradation, reducing the specific surface area of ​​the aggregated particles, disrupting the continuity of the protective layer, and decreasing the protection rate.

[0150] Furthermore, Example 10, which added trehalose, showed better retention; the uniform distribution of thiol groups ensured a more balanced interaction with QS-21, effectively blocking external oxygen, moisture, and trace metal ions from corroding, inhibiting hydrolysis and oxidation reactions at the source, reducing structural disintegration during the reconstitution process of freeze-dried products, and providing a reliable solution for the industrial production and subsequent preservation of vaccines.

[0151] Test Example 3

[0152] Cytotoxicity test

[0153] Materials: L929 mouse fibroblasts, tissue-mimicking cells.

[0154] The adjuvant samples from Examples 1-10 and Comparative Examples 1-3 were reconstituted to 100 μL / mL and then reconstituted to 100 μg / mL in DMEM medium containing 10% fetal bovine serum. 100 μL was added to each well of a cell culture plate and incubated at 37°C and 5% CO2 for 24 h. 10 μL of 5 mg / mL MTT solution was added, and the plate was cultured for another 4 h. After discarding the supernatant, 150 μL of DMSO was added, and the plate was shaken for 10 min. The OD value at 570 nm was measured after calibrating the microplate reader. Each sample was tested in triplicate, and the mean value was taken. The calculation formula is: Survival rate = OD 实验组 / OD 对照组 ×100%.

[0155] Hemolysis rate test

[0156] Materials: 1% fresh rabbit red blood cell suspension: Take 800 μL of red blood cell suspension and dilute it to 8 mL with phenol red-free medium. Mix well to prepare the red blood cell stock solution and store at 4℃ for up to 6 hours. Physiological saline is used as a negative control and distilled water is used as a positive control.

[0157] Add 200 μL of the prepared erythrocyte stock solution to the adjuvant sample tubes prepared in Examples 1-10 and Comparative Examples 1-3; transfer to a 6-well plate and incubate at 37°C with 5% CO2 for 4 h, agitating the bottom of the erythrocytes every 15 min during the exposure. After the exposure, transfer each group of samples to a 4 mL centrifuge tube, centrifuge at 2500 rpm, collect the supernatant, and measure the absorbance A value at 541 nm. The exposure conditions for the positive control group were the same as those for the negative control group; after 4 h, centrifuge at 2500 rpm and collect the supernatant; zero the plate with distilled water. The calculation formula is: Hemolysis rate = (A... 实验组 -A 阴性对照 ) / (A 阳性对照 -A 阴性对照 () × 100%. The results are shown in Table 3.

[0158] Table 3 Results of cell viability and hemolysis rate tests

[0159]

[0160] Safety standards for in vivo models: Cell viability safety threshold: ≥70% is considered as not exhibiting significant cytotoxicity; refer to ISO 10993-5 "Biological evaluation of medical devices – Part 5: In vitro cytotoxicity tests". Hemolysis rate: The grading standards are: <2% non-hemolysis, 2–5% slight hemolysis, >5% significant hemolysis; refer to GB / T 16886.4 "Biological evaluation of medical devices – Part 4: Selection of blood interaction tests"; a hemolysis rate exceeding the threshold indicates that the material may damage the red blood cell membrane, posing a risk of hemolysis.

[0161] Comparative Example 1, a single QS-21 system, exhibited low cell viability and high hemolysis rate, while Examples 1-10, with their composite polyanion, showed enhanced safety. QS-21 is an amphiphilic molecule that can insert into the cell membrane via hydrophobic interactions, potentially disrupting cell membrane integrity, damaging lipid structures, causing hemolysis, reducing cell viability, and interfering with cell metabolism.

[0162] In Examples 1-3, the polyanions may form stable composite particles through intermolecular association with multi-point hydrogen bonds via hydrophobic association, reducing potential toxicity. In Examples 3-6, the polyglutamic acid is a linear polypeptide; in Examples 3-4, the polyglutamic acid solution concentration is low, potentially resulting in more free QS-21 and insufficient encapsulation; in Example 6, the high concentration of polyglutamic acid leads to chain entanglement and the formation of large aggregates, thereby increasing contact with the cell membrane. These aggregates easily adsorb onto the cell surface, and large particles may deposit, increasing local stress on the membrane surface, disrupting the extracellular osmotic pressure balance, and thus reducing cell viability; in the optimization of polyglutamic acid concentration, Example 5 shows a moderately stretched polyglutamic acid molecular chain.

[0163] Furthermore, Examples 7-9 and Comparative Examples 2-3 introduced thiol groups into the side chains of polyglutamic acid through a condensation reaction, which weakly bonded to the hydroxyl and aldehyde groups of QS-21, enhancing the interfacial bonding of the polyglutamic acid-QS-21 complex and inhibiting the dissociation of nanoparticles, thereby reducing the hemolysis rate. The trehalose added in Example 10 is a natural sugar, which may reduce residual water migration and minimize structural disintegration caused by drying and subsequent resolution through water substitution and vitrification. In cases where resolution is required, the hydration layer and osmotic pressure buffering effect formed by trehalose help maintain colloidal stability, reduce local concentration peaks, and has good compatibility with cell membranes, resulting in higher cell survival and lower hemolysis rate.

Claims

1. A method for preparing QS-21 composite adjuvant, characterized in that, Includes the following steps: S1. Weigh 1-10 mg of QS-21 powder and add it to 1 mL of Tris buffer solution with pH 6.0-6.

8. Place the solution in an ice bath and shake for 5-20 min. S2. Prepare a 5-20 mg / mL polyanionic solution and add it dropwise to the solution prepared in S1 at a rate of 0.1-0.3 mL / min, while maintaining magnetic stirring for 3-10 min. S3. After the addition is complete, continue stirring for 20-30 minutes, let stand for 5-10 minutes; adjust the pH and remove impurities using a 0.22μm filter membrane; S4. Add freeze-drying protectant and stir at 100-300 r / min for 1-3 min; S5. After vacuum freeze-drying, QS-21 composite adjuvant is obtained; The polyanion is a polyglutamic acid complex; The preparation method of the polyglutamic acid complex is as follows: Add 0.4185 mmol of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide to 0.5 mL of anhydrous dimethyl sulfoxide and stir at 100 rpm for 8 min to obtain a preparative solution. Separately, dissolve 0.4185 mmol of N-hydroxysuccinimide in 0.5 mL of anhydrous dimethyl sulfoxide and add it to the preparative solution at a rate of 0.1 mL / min. Stir at 300 rpm for 15 min under ice bath conditions to obtain the condensate, which is then ready for use. Add 0.78 mmol of polyglutamic acid to 8 mL of water and stir at 200 rpm for 8 min. Add the prepared condensate and 0.093 mmol of pyridine 2-carboxylic acid and activate for 30 min. Dissolve 0.78 mmol of L-cysteine ​​hydrochloride in 2 mL of water and slowly add it dropwise to the activated solution. Adjust the pH to 4.8 and react in the dark under a nitrogen atmosphere for 24 h. Adjust the pH to 5.0 with 0.1 mol / L sodium hydroxide and dialyze using a 12000 Da dialysis bag for 72 h, changing the water every 3 h. The freeze-drying protectant is trehalose.

2. The preparation method of QS-21 composite adjuvant according to claim 1, characterized in that, The mass ratio of QS-21 to polyanion is 1:1-5.

3. The preparation method of QS-21 composite adjuvant according to claim 1, characterized in that, The stirring speed involved in step S2 is 200-500 r / min.

4. The preparation method of QS-21 composite adjuvant according to claim 1, characterized in that, The vacuum freeze-drying conditions described in step S5 are a temperature of -60 to -20°C and a drying time of 6 to 20 hours.

5. A QS-21 compound adjuvant, characterized in that, Prepared by the method described in any one of claims 1-4.

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