Preparation method of saponin adjuvant

Abstract

The invention discloses a preparation method of a saponin adjuvant, and relates to the technical field of biological medicine, and the preparation method comprises the following steps: dissolving and filtering: dissolving Quil-A freeze-dried powder by using a dissolving solution, and filtering by using a filter to obtain a filtrate; roughly purifying; performing fine purification; performing ultrafiltration replacement, namely adjusting the pH value by using an adjusting solution, and concentrating a purified sample; replacing the liquid by using the replacement liquid through the ultrafiltration membrane bag to obtain an ultrafiltration sample; sterilizing and filtering: filtering the ultrafiltration sample by a filter to obtain a QS-21 stock solution; a dissolving solution is 30% acetonitrile 5mM citric acid, and the pH value is 5.5; the eluent A is a 0.1% TFA solution; the eluent B is a 0.1% TFA acetonitrile solution; the adjusting liquid is 0.5 M Tris; the displacement liquid is 3.2 mM of citric acid, 6.8 mM of sodium citrate and 120 mM of NaCl, and the pH value is 5.5. The method is simple in process operation and low in cost; the prepared QS-21 is strong in immune effect, low in toxicity and good in stability.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technologies, and particularly to a preparation method of a saponin adjuvant. Background Art

[0002] QS-21 is a highly efficient triterpenoid saponin vaccine adjuvant, initially extracted from the bark of Quillaja saponaria Quillaja saponaria Molina. Its unique immunostimulatory properties make it a key component in various vaccine formulations, especially excellent in enhancing humoral and cellular immune responses, and it has been widely used in tumor vaccines and prophylactic vaccines.

[0003] As known from US5057540, the Kensil team used methanol to extract the bark of Quillaja saponaria in early research, and purified it through silica gel and reversed-phase C4 resin. However, FAB-MS analysis showed that the product was still a complex mixture, and the yield data was not disclosed.

[0004] Two improvement schemes are known from US6231859 and US6524584: Dialysis freeze-drying method: Using the extract of Quillaja saponaria as the raw material, after enriching QS-21 through dialysis, it is purified through two steps of silica gel column rough purification and C18 column fine purification, and the claimed yield reaches 2.95%; PVPP co-purification method: Using polyvinylpyrrolidone to remove Quil-A polyphenol impurities, and then purified through two steps of phenyl column and C8 column.

[0005] The above preparation methods are complex in operation, requiring the use of silica gel, reversed-phase C18, PVPP, phenyl, C8, silica gel column, etc., and there are still many impurities in the final extract; in addition, the silica gel column has a short service life and high cost.

[0006] On this basis, Qi Y et al. (Qi Y, Fox C B. J CHROMATOGR A. 2020, 1635:461705) proposed a new preparation method, using Quil-A as the raw material and using C18 column and HILIC column for preparation. Among them, the C18 column is easily damaged by the impurity proteins and carbohydrates in Quil-A, reducing the service life, the amplified production cost is high and the process reproducibility is poor; in addition, the HILIC column requires a long equilibration time, and has strict requirements on the proportion of the aqueous phase and the organic phase in the mobile phase, and has strong use limitations.

[0007] With the development of biomedicine, the demand for QS-21 has gradually increased. Its industrial production mainly relies on the separation and purification from the crude extract Quil-A of Quillaja saponaria bark. However, due to the complex composition of Quil-A and the unstable structure of QS-21, the existing processes still face many challenges.

[0008] Based on this current situation, the present invention is proposed. Summary of the Invention

[0009] To solve the deficiencies of the prior art, the present invention provides a preparation method of a saponin adjuvant, which has simple process operation and low cost; the prepared QS-21 has strong immune effect, low toxicity and good stability.

[0010] To achieve the object of the present invention, the following scheme is proposed: A preparation method of a saponin adjuvant, comprising the following steps: a) Dissolution and filtration: Dissolve Quil-A freeze-dried powder with a dissolution solution, and filter with a filter to obtain a filtrate; b) Coarse purification: Using the filtrate obtained in step a) as a sample, purify according to the following operations: 1) Loading with UniPS 10-300 packing as a chromatography medium; 2) Eluting and separating with eluent A and B, and collecting the samples in segments; 3) Combining the samples; c) Fine purification: Using the coarsely purified sample obtained in step b) as a sample, purify according to the following operations: 1) Loading with UniPS 5-300 packing as a chromatography medium; 2) Eluting and separating with eluent A and B, and collecting the samples in segments; 3) Combining the samples; d) Ultrafiltration and replacement: After adjusting the pH with an adjusting solution, concentrate the finely purified sample; use a replacement solution to perform liquid replacement through an ultrafiltration membrane package to obtain an ultrafiltration sample; e) Sterilization filtration: Filter the ultrafiltration sample with a filter to obtain the QS-21 stock solution; Wherein, The dissolution solution is: 30% acetonitrile 5 mM citric acid pH 5.5; Eluent A is: 0.1% TFA solution; Eluent B is: 0.1% TFA acetonitrile solution; The adjusting solution is: 0.5 M Tris; The replacement solution is: 3.2 mM citric acid 6.8 mM sodium citrate 120 mM NaCl pH 5.5.

[0011] Furthermore, in step a), the QS-21 content of the Quil-A freeze-dried powder is not less than 6%, and the ratio of the Quil-A freeze-dried powder to the dissolution solution is 1:20 (W / V).

[0012] Furthermore, in step a), the pore size of the filter used is 1.0 + 0.45 μm, and the filtration flow rate is 100-300 ml / min.

[0013] Further, in step b), the samples merged in operation 3) are subjected to the purification operation in step b) again to obtain a roughly purified sample, i.e., the sample is passed through the column roughly twice. The rough purification steps are as follows: Test the column efficiency, with the number of theoretical plates per meter ≥ 32000 N / m; Set the system flow rate at 120 - 180 cm / h, the wavelength at 215 nm, the gradient of eluent A at 70% - 58%, and the gradient of eluent B at 30% - 42%; After equilibrating the chromatographic column, load the sample, then equilibrate again, and then start linear elution for 12 column volumes. Collect the eluent in fractions and merge the samples according to the standard to complete the first rough purification. Set the system flow rate at 120 - 180 cm / h, the wavelength at 215 nm, the gradient of eluent A at 70% - 60%, and the gradient of eluent B at 30% - 40%; Adjust the acetonitrile concentration of the merged sample to 30% with water. After equilibrating the chromatographic column, load the sample, then equilibrate again, and then start linear elution for 15 column volumes. Collect the eluent in fractions and merge the samples according to the standard to complete the second rough purification. In the merging of the two samples, the purity of the first merge > 50%, and the purity of the second merge > 70%.

[0014] Further, in step c), the samples merged in operation 3) are subjected to the purification operation in step c) twice again to obtain a highly purified sample, i.e., the sample is passed through the column highly purified three times. The purification parameters for the high - purification step are as follows: Test the column efficiency, with the number of theoretical plates per meter ≥ 35000 N / m; Set the system flow rate at 90 - 150 cm / h, the wavelength at 215 nm, the gradient of eluent A at 70% - 60%, and the gradient of eluent B at 30% - 40%; Adjust the acetonitrile concentration of the merged sample to 30% with water. After equilibrating the chromatographic column, load the sample, then equilibrate again, and then start linear elution for 15 column volumes. Collect the eluent in fractions and merge the samples according to the standard to complete the first high - purification. Repeat this operation to complete the remaining two high - purifications; In the merging of the three samples, the purity of the first merge > 82%, the purity of the second merge > 88%, and the purity of the third merge > 93.5%.

[0015] Further, in step d), the pore size of the ultrafiltration membrane package is 3 KD, and the material is regenerated cellulose.

[0016] Further, in step d), slowly adjust the pH value to 5.5 ± 0.1 with 0.5M Tris. During concentration, control the feed flow rate at 150 - 250 LMH, the ultrafiltration transmembrane pressure at 0.8 - 1.2 bar, and the concentration multiple at 4 - 6 times; during displacement, control the feed flow rate at 150 - 250 LMH, the ultrafiltration transmembrane pressure at 0.8 - 1.2 bar, and continuously displace the sample in a fed-batch manner, with the displacement volume being 9 - 12 times.

[0017] Further, in step e), the pore size of the filter used is 0.45 + 0.2 μm.

[0018] Further, in step e), the QS-21 stock solution is stored below -70°C.

[0019] The QS-21 stock solution obtained by the above preparation method has the following key properties: Purity: the main peak area ≥ 93.0%, the QS-21 component > 98%; Total impurities ≤ 2.0%; Residual solvents: acetonitrile ≤ 500 ppm, trifluoroacetic acid ≤ 10 ppm; The pH value is 5.0 - 6.0.

[0020] The beneficial effects of the present invention are as follows: 1. Simple operation and low difficulty; in the present invention, the eluents used in the purification process are exactly the same and the elution process is basically the same, and it can be completed only by collecting the samples and combining them according to the standard.

[0021] 2. Good process stability; in the present invention, there is no need to pretreat the sample, two kinds of packing materials are used for purification, and the purification parameters are consistent, so the process stability is good.

[0022] 3. Low production cost and easy to scale up; the packing materials used in the present invention are cheap and have a long service life, the whole process is simple, with low difficulty and good stability, and it is very easy to scale up.

[0023] 4. Good impurity removal effect; by detecting the purified intermediate samples, it is determined that the impurity removal effect is good.

[0024] 5. High purity of QS-21; the QS-21 prepared according to the present invention has a main peak purity > 93%, the QS-21 component content ≥ 98%, the total impurities are less than 2%, and the residual organic solvents are less; and it has a strong immune effect, low toxicity and good stability. Description of the Drawings

[0025] Figure 1 Shows the impurity peak position diagram; Figure 2 Shows the first crude purification sample chromatogram; Figure 3 Shows the highly purified sample chromatogram; Figure 4 Shows a comparison chart of the immune stimulation effects of self-made QS-21 and commercially available QS-21; Figure 5 Shows the detection result chart of B2-D42IgG antibody for comparing the immunogenicity of the same type of vaccines; Figure 6 Shows the result chart of B2-D42IgG cellular immune analysis for comparing the immunogenicity of the same type of vaccines. Detailed implementation manners

[0026] The raw materials used in the present invention are Quil-A, which is sourced from the dry extract of Quillaja saponaria bark, with a CAS number of 8047-15-2, and is purchased from Croda in Denmark.

[0027] The quality results of Quil-A show that its QS-21 content is not less than 6%, all meeting the relevant requirements in the fourth part of the Chinese Pharmacopoeia 2020 edition, JY / T 0568-2020, and GB / T 39665-2020.

[0028] The main instruments and equipment involved in the present invention are as shown in Table 1 below: Table 1 Record of main instruments and equipment The reagents and consumables involved in the examples of the present invention are: All the reagents used in the present invention are common reagents. The chromatographic packings UniPS 10-300 and UniPS 5-300 are purchased from Suzhou NanoMicro Technologies Co., Ltd. and have no animal-derived and animal-derived derivative materials.

[0029] The solutions and their preparation methods involved in the examples of the present invention are as shown in Table 2 below: Table 2 Record of solutions and preparation methods The present invention provides a preparation method of a saponin adjuvant, comprising the following steps: a) Dissolution and filtration Accurately weigh 79.98 g of Quil-A sample, add 1600 ml of dissolution solution. The dissolution solution is 30% acetonitrile + 5 mM citric acid with a pH of 5.5. The ratio of Quil-A to the dissolution solution is 1:20 (W / V). Place it on a magnetic stirrer for dissolution, and the stirring time is not less than 30 min; filter with a filter having a pore size of 1.0 + 0.45 μm, and control the filtration flow rate within the range of 100 - 300 ml / min; take the filtered sample to detect the purity and content of QS-21.

[0030] b) Coarse purification First coarse purification: Column packing and column efficiency test: Packed with UniPS 10-300 packing material, 5-10 ml of 10% acetone-acetonitrile solution was loaded, acetonitrile was used as the mobile phase, the wavelength was 254 nm, the system flow rate was 120-180 cm / h, and the column efficiency was tested. The results are shown in Table 3. The qualified standard for the test is: the number of theoretical plates per meter ≥ 32000 N / m.

[0031] Table 3 Column efficiency table of UniPS 10-300 column packing Parameter setting: Set the system flow rate at 120-180 cm / h, the wavelength at 215 nm, the eluent A of pump A is 0.1% TFA solution, the gradient is 70% - 58%, and the eluent B of pump B is 0.1% TFA acetonitrile solution, the gradient is 30% - 42%.

[0032] Equilibration: First, rinse with pump B for 0.5 column volumes, and then rinse with 70% of pump A and 30% of pump B for 1.5 column volumes.

[0033] Sample loading: Take the filtered Quil-A sample and load it using pump C of the high-performance liquid chromatograph.

[0034] Re-equilibration: After sample loading, rinse with 70% of pump A and 30% of pump B for 3 column volumes.

[0035] Elution: Linearly elute from 70% of pump A and 30% of pump B to 58% of pump A and 42% of pump B for 12 column volumes, and collect the eluent in fractions.

[0036] Collection: Detect the purity of QS-21 in the fractionally collected eluent, combine the eluent with a purity greater than 50% to obtain the first crude pure sample.

[0037] Feed amount of the first crude pure: 1590 ml of filtered sample.

[0038] Second crude purification: Parameter setting and solution preparation: Set the system flow rate at 120-180 cm / h, the wavelength at 215 nm, the eluent A of pump A is 0.1% TFA solution, the gradient is 70% - 60%, and the eluent B of pump B is 0.1% TFA acetonitrile solution, the gradient is 30% - 40%.

[0039] Take the first crude pure sample, calculate its theoretical acetonitrile concentration. The calculation method is: Calculate the average acetonitrile gradient of the combined sample from the chromatogram, and subtract 1 column volume forward. The acetonitrile gradient value corresponding to the 1-column volume deduction is the theoretical acetonitrile concentration of the sample. Calculate the theoretical acetonitrile concentration of the sample and dilute it with water until the theoretical concentration of acetonitrile is 30%.

[0040] Equilibration: First, rinse with Pump B for 0.5 column volumes, and then rinse with 70% of Pump A and 30% of Pump B for 1.5 column volumes.

[0041] Sample loading: Take the diluted first crude pure sample and load it using Pump C of the high-pressure liquid chromatograph.

[0042] Re-equilibration: After sample loading, rinse with 70% of Pump A and 30% of Pump B for 1 column volume.

[0043] Elution: Linearly elute from 70% of Pump A and 30% of Pump B to 60% of Pump A and 40% of Pump B for 15 column volumes, and collect the eluate in fractions.

[0044] Collection: Detect the purity of QS-21 in the fractionally collected eluate, combine the eluate with a purity greater than 70% to obtain the crude pure sample.

[0045] Feed amount for the second crude purification: 2700 ml of the first crude pure sample; 545 ml of purified water.

[0046] c) Fine purification First fine purification: Column packing and column efficiency test: Pack using UniPS 5-300 packing, load 5 - 10 ml of 10% acetone - acetonitrile solution, use acetonitrile as the mobile phase, wavelength of 254 nm, system flow rate of 90 - 150 cm / h, test the column efficiency, and the results are shown in Table 4. The qualified standard is: the number of theoretical plates per meter ≥ 35000 N / m.

[0047] Table 4 Column efficiency table for UniPS 5-300 column packing Parameter setting and solution preparation: Set the system flow rate at 90 - 150 cm / h, wavelength of 215 nm, eluent A of Pump A is 0.1% TFA solution, gradient of 70% - 60%, eluent B of Pump B is 0.1% TFA acetonitrile solution, gradient of 30% - 40%.

[0048] Take the crude pure sample, calculate its theoretical acetonitrile concentration. The calculation method is: Calculate the average acetonitrile gradient of the combined sample from the chromatogram, and deduct 1 column volume forward. The acetonitrile gradient value corresponding to the 1-column-volume deduction is the theoretical acetonitrile concentration of the sample. Calculate the theoretical acetonitrile concentration of the sample and dilute it with water until the theoretical concentration of acetonitrile is 30%.

[0049] Equilibration: First, rinse with Pump B for 0.5 column volumes, and then rinse with 70% of Pump A and 30% of Pump B for 1.5 column volumes.

[0050] Sample loading: Take the diluted crude pure sample and load it using Pump C of the high-pressure liquid chromatograph.

[0051] Rebalancing: After sample loading is completed, rinse with 70% of Pump A and 30% of Pump B for 1 column volume.

[0052] Elution: Linearly elute from 70% of Pump A and 30% of Pump B to 60% of Pump A and 40% of Pump B for 15 column volumes, and collect the eluate in fractions.

[0053] Collection: Detect the purity of QS-21 in the fractionally collected eluate, combine the eluate with a purity greater than 82% to obtain the first purified sample.

[0054] Feed amount for the first purification: 2680 ml of crudely purified sample; 500 ml of purified water.

[0055] Second purification: According to the parameters and operations of the first purification, use the first purified sample as the sample and repeat the operation once. Combine the eluate with a purity greater than 88% to obtain the second purified sample.

[0056] Feed amount for the second purification: 2650 ml of the first purified sample; 530 ml of purified water.

[0057] Third purification: According to the parameters and operations of the first purification, use the second purified sample as the sample and repeat the operation once. Combine the eluate with a purity greater than 93.5% to obtain the purified sample.

[0058] Feed amount for the third purification: 3000 ml of the second purified sample; 600 ml of purified water.

[0059] d) Ultrafiltration replacement Cleaning of the ultrafiltration membrane module: Take purified water for injection to clean the ultrafiltration system until the pH of the reflux end and the permeate end is neutral; drain the purified water for injection in the system, and rinse the ultrafiltration system with the ultrafiltration replacement liquid at 2 L / m 2 to rinse the ultrafiltration system, and the volume of the ultrafiltration replacement liquid permeated from the permeate end is not less than 200 ml.

[0060] Take the purified sample and slowly adjust it to pH 5.5 ± 0.1 with 0.5 M Tris.

[0061] Sample concentration: Control the inlet flow rate at 150 - 250 LMH and the ultrafiltration transmembrane pressure at 0.8 - 1.2 bar to concentrate the sample volume to 1 / 4 - 1 / 6 of the original volume.

[0062] Replacement of the buffer solution of the concentrated sample: Control the inlet flow rate at 150 - 250 LMH and the ultrafiltration transmembrane pressure at 0.8 - 1.2 bar, and continuously replace the sample in a fed-batch manner for 12 times the volume of the concentrated sample.

[0063] Sample evacuation: Close the permeate end and evacuate all the samples in the ultrafiltration system; Use 100 - 400 ml of ultrafiltration replacement solution to rinse the residual samples in the ultrafiltration system, circulate for 5 min, and combine the rinse solution with the samples to obtain the QS-21 ultrafiltration sample.

[0064] Dosage for ultrafiltration replacement: 2950 ml of purified sample; 78 ml of adjustment solution.

[0065] e) Sterile filtration Under a C+A clean environment, take the ultrafiltration sample and filter it through a 0.45 + 0.2 μm sterile filter to obtain the QS-21 stock solution, which is stored at -70°C or below.

[0066] Regarding the key process research experiments of the present invention, it is as follows: Experiment 1: Research on impurity removal effect For the intermediate samples of each purification step in three pilot batches, conduct HPLC purity detection. Select the impurity peaks with relatively high contents in the purified samples as characteristic peaks, and select 5 impurity peaks near the main peak of QS-21 as characteristic impurity peaks, and compare and analyze the impurity removal effects of each chromatography step. Since there are too many impurities in the crude purification stage, only the impurity removal research in the purified stage is carried out. The impurity peaks are as Figure 1 shown, and the relevant information of the impurity peaks is shown in Table 5 below: Table 5: Table of relevant information of impurity peaks Calculation formula for impurity peak removal rate: In the formula: S1 is the peak area of the impurity peak in the previous step of operation, mAu.min; V1 is the volume of the collected sample in the previous step of operation, ml; S2 is the peak area of the impurity peak in this step of operation, mAu.min; V2 is the volume of the collected sample in this step of operation, ml.

[0067] Calculation formula for total impurity peak removal rate: In the formula: S3 is the peak area of the impurity peak in the second crude purification sample, mAu.min; V3 is the volume of the second crude purification sample, ml; S4 is the peak area of the impurity peak in the purified sample, mAu.min; V4 is the volume of the purified sample, ml.

[0068] According to the test results, the impurity removal rates of the three pilot batches are shown in Table 6 below, the organic solvent residues of the three pilot batches are shown in Table 7 below, the intermediate purities of the three pilot batches are shown in Table 8 below, and the yields of the three pilot batches are shown in Table 9 below.

[0069] Table 6 Results Table of Impurity Removal Rates for Three Pilot Batches Table 7 Results Table of Residual Organic Solvents for Three Pilot Batches Table 8 Results Table of Intermediate Purities for Three Pilot Batches Table 9 Results Table of Yields for Three Pilot Batches In summary, for three consecutive pilot batches, the test data of each intermediate in the purification of QS-21 were basically consistent, and the process was stable.

[0070] Experiment 2 Quality Research Select a batch of pilot QS-21 for research, as follows: The chromatogram of the first crude pure sample (UV214nm) is as Figure 2 shown, with impurities at retention times of 9.64 min, 10.02 min, and 10.66 min; the chromatogram of the highly purified sample (UV214nm) is as Figure 3 shown, with the retention times of the main peaks of QS-21 being 9.98 min (QS-21-B) and 10.20 min (QS-21), and the impurities at retention times of 9.64 min, 10.02 min, and 10.66 min having been removed. The MS identification results of each chromatographic peak are shown in Table 10 below. Table 10 Results Table of MS Identification of Each Chromatographic Peak Experiment 3 Validation of the Efficacy of QS-21 Compare the QS-21 prepared by the present invention with commercial QS-21, and prepare the vaccine and group according to Table 11 below: Table 11 Record Table 1 of Vaccine Preparation and Grouping MRJ103 adjuvant contains: DOTAP, DOPC, cholesterol, QS-21, CpG 1018; Test procedure: Order C57BL / 6Jx mice, inject 50 μl per mouse (1 / 10 human dose) into the leg muscle, immunize on D0 and D28, collect blood on D21 and D42, and isolate serum to detect IgG antibodies.

[0071] The results are as Figure 4 shown, showing no significant difference between the two, proving that the QS-21 prepared by the present invention has the same immune stimulation effect as commercially available QS-21.

[0072] Experiment 4: Application of QS-21 in Vaccines Table 12 Record Sheet of Vaccine Preparation and Grouping II The MRJ103 adjuvant contains: DOTAP, DOPC, cholesterol, QS-21, CpG 1018; Experimental procedure: Order C57BL / 6J mice, inject 50 μl per mouse (1 / 10 human dose) into the leg muscle, immunize on D0 and D28, collect blood on D21, collect spleen on D42, and isolate serum and spleen cells.

[0073] The detection results of B2-D42 IgG antibodies for comparing the immunogenicity of similar vaccines are as Figure 5 shown, indicating that there is no significant difference in antibodies between the two. The analysis results of B2-D42 IgG cellular immunity (CD4 + T cells) for comparing the immunogenicity of similar vaccines are as Figure 6 shown, and its ability to induce TH1-type cellular immune response is superior to that of the commercial vaccine Xinanlishi.

[0074] Experiment 5: Toxicity Study of QS-21 Experimental method: Dilute the self-made and commercially available QS-21 to final concentrations of 80 μg / ml, 40 μg / ml, 20 μg / ml, 10 μg / ml, and 5 μg / ml respectively. Inject 5 Balb / C mice intraperitoneally at each concentration, observe for 7 days, and record the survival of the mice. The results are shown in Table 13 below.

[0075] Table 13 Record Sheet of Mouse Survival The results show that the toxicity of the self-made QS-21 is lower than that of the commercially available QS-21.

[0076] The above embodiments are only used to illustrate the technical ideas and features of the present invention, and do not represent the only or limit the present invention. Those skilled in the art should understand that without departing from the scope of the present invention, various changes or equivalent replacements made to the present invention all fall within the scope of protection of the present invention.

Claims

1. A method for preparing a saponin adjuvant, characterized in that: The following steps are involved: Dissolution and filtration: Dissolve Quil-A in a dissolving solution and filter to obtain a filtrate; Crude purification: Using the filtrate obtained in step a) as a sample, purify according to the following operation: 1) UniPS 10-300 filler was used as the chromatography medium for loading; 2) Eluents A and B are used for elution separation, and samples are collected in sections; 3) Combine samples; Purification: Using the crude pure sample obtained in step b) as the sample, purify it according to the following operation: 1) UniPS 5-300 filler was used as the chromatography medium for loading; 2) Eluents A and B are used for elution separation, and samples are collected in sections; 3) Combine samples; Ultrafiltration replacement: After adjusting the pH with a regulating solution, the sample is concentrated and purified; Using a replacement fluid to pass through an ultrafiltration membrane bag to replace the fluid, thereby obtaining an ultrafiltration sample; Sterile filtration: Filter the ultrafiltration sample to obtain QS-21 stock solution; in, The dissolving solution was: 30% acetonitrile 5 mM citric acid pH 5.5; Eluent A: 0.1% TFA solution; Eluent B: 0.1% TFA acetonitrile solution; The conditioning solution is: 0.5M Tris; The replacement solution was: 3.2 mM citric acid, 6.8 mM sodium citrate, 120 mM NaCl, pH 5.

5.

2. The preparation method according to claim 1, characterized in that: In step a), the QS-21 content of Quil-A is not less than 6%, and the ratio of Quil-A to the dissolving solution is 1:20 (W / V).

3. The preparation method according to claim 1, characterized in that: In step a), the pore size of the filter used is 1.0+0.45 μm, and the filtration flow rate is 100-300 ml / min.

4. The preparation method according to claim 1, characterized in that: In step b), the sample combined in operation 3) is subjected to the purification operation of step b) again to obtain a crude pure sample; the purity of the first combined sample is >50%, and the purity of the second combined sample is >70%.

5. The preparation method according to claim 1, characterized in that: In step c), the sample combined in operation 3) is subjected to the purification operation of step c) twice to obtain a pure sample, the purity of the first combined sample is >82%, the purity of the second combined sample is >88%, and the purity of the third combined sample is >93.5%.

6. The preparation method according to claim 1, characterized in that: In step d), the pore size of the ultrafiltration membrane package is 3KD and the material is regenerated cellulose.

7. The preparation method according to claim 1, characterized in that: In step d), the pH value is adjusted to 5.5±0.1, the liquid flow rate is controlled to be 150-250LMH, the transmembrane pressure is 0.8-1.2 bar, the concentration multiple is 4-6 times, and the displacement volume is 9-12 times.

8. The preparation method according to claim 1, characterized in that: In step e), the pore size of the filter used is 0.45+0.2 μm.

9. The preparation method according to claim 1, characterized in that: In step e), the QS-21 stock solution is stored below -70°C.

10. The preparation method according to claim 1, characterized in that: QS-21 stock solution has the following key properties: Purity: main peak area ≥93.0%, QS-21 component ≥98%; Total impurities ≤ 2.0%; Residual solvents: acetonitrile ≤410ppm, trifluoroacetic acid ≤10ppm; The pH value is 5.0~6.0.

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