Method for purifying saponin adjuvant

Abstract

The invention belongs to the technical field of vaccine reagent extraction, and particularly relates to a saponin adjuvant purification method which is characterized by sequentially comprising the following steps: crushing bark, adding purified water for soaking, taking out supernate, centrifuging, filtering, carrying out ion exchange chromatography, concentrating and exchanging liquid, roughly purifying, finely purifying and the like to obtain a high-purity QS-21 sample. The purification method provided by the invention is suitable for large-scale production, and uniform and high-purity QS-21 can be obtained.

Description

Technical Field

[0001] The invention belongs to the technical field of vaccine reagent extraction, and in particular relates to a saponin adjuvant purification method. Background Art

[0002] Saponin adjuvant (QS-21) is an active ingredient extracted from the bark of the soap tree (Quillaja Saponaria). It has always been used as a preferred adjuvant for vaccines, and composite adjuvants containing QS-21 are widely used in vaccine development research.

[0003] QS-21 can induce specific antigen-antibody and CD8+ cell immune responses in mice, producing IgG1 and IgG2a antibodies. In contrast, aluminum salt adjuvants mainly stimulate the body to produce IgG1 antibodies. Studies have shown that QS-21 can promote cytotoxic T lymphocytes (CTLs), secrete Th1 cytokines, interleukin 2 and gamma interferon, and IgG2a antibodies against protein antigens. QS-21 adjuvant has significant advantages over other types of adjuvants, including dextran adjuvants, bacterial nucleosides, and bacterial lipopolysaccharide adjuvants, in terms of stimulating antibody responses and T cell responses to target antigens.

[0004] Composite adjuvants containing QS-21, such as AS01, AS-02, etc., have been used in clinical research on human viral vaccines, including influenza vaccine, malaria, hepatitis B, human papilloma, AIDS, tuberculosis, non-small cell lung cancer and melanoma.

[0005] The specification of Chinese invention CN201880077052.5 records a saponin extraction of a crude aqueous extract of Quillaja saponaria Molina containing at least a QS-21 main peak and a 2018 component, wherein the ratio of the 2018 component / QS-21 main peak measured by UV absorbance at 214 nm is ≤0.075, and a method and related aspects of obtaining such an extract; it mentions that a sample of the bark material of the Quillaja saponaria is aqueously extracted at a temperature of about 60-70°C for about 2-5 hours, and the extract is then treated with PVPP, filtered and concentrated, and sodium benzoate is added to adjust the pH to about 3.8-5.2 and sterilized. However, this patent has technical problems such as high temperature and long time in the extraction process, the need to add sodium benzoate preservative, and low purity of the crude extract. Summary of the invention

[0006] In order to solve the above technical problems, the present invention provides a saponin adjuvant purification method, which has simple extraction, low cost, high extraction rate, and high purification rate, and does not require other organic solvents; in the present invention, water extraction can extract 90% or more of QS-21 in the bark, and the yield after 3 steps of purification is 30% or more.

[0007] A method for extracting and purifying a saponin adjuvant of the present invention that solves the above technical problems comprises the following steps: (1) Crush the bark and add purified water to soak the bark powder; (2) Taking out the supernatant and centrifuging it to obtain supernatant I containing QS-21 adjuvant, and then filtering it with a filter to remove insoluble particles to obtain supernatant II; (3) Using Polar MC-CM filler to perform ion exchange chromatography on supernatant II to obtain a purified solution; (4) The purified solution is concentrated and replaced to obtain an ultrafiltrate; the concentration of sodium chloride is reduced during the concentration and replacement to prevent affecting the reverse phase chromatography effect.

[0008] (5) Use Amethyst C18-H reverse phase chromatography medium for reverse phase crude purification, collect the elution peak, and obtain a reverse phase liquid; there are multiple elution peaks in the Amethyst C18-H crude purification, and collect part of the information of one of the elution peaks.

[0009] (6) Use reversed-phase UniSil C8 chromatographic medium for reversed-phase purification and collect the elution peak to obtain a high-purity QS-21 sample. There is one elution peak in UniSil C8 purification, and all elution peak information is collected.

[0010] In the present invention, QS-21 is extracted from the bark, and the purification method of the QS-21 adjuvant is suitable for large-scale production, and can obtain uniform QS-21 with high purity.

[0011] The invention first uses soap tree bark as a raw material, firstly needs to grind the bark into uniform bark fine powder to facilitate extraction, then selects ion exchange filler to capture the sample, removes cellulose, polyphenols, alkaloids, tannins and the like; further selects reverse phase filler to roughly purify and remove saponin analogs such as QS-17 and QS-18; further selects C8 filler for fine purification to obtain high-purity QS-21.

[0012] In the optimized solution, a crusher is used to crush large pieces of bark into powder for later use.

[0013] In the step (1), 80% of the bark powder is crushed to pass through a 40-100 mesh medicinal sieve, and in the optimized solution, it is passed through a 50 mesh medicinal sieve. Different particle sizes have different dissolution rates, and if the particle size is too large, 90% of the powder cannot be dissolved within the specified time.

[0014] In the optimized solution, in step (1), 8-12 times the weight of the bark fine powder is added with purified water and the mixture is soaked for 0.5-1.5 hours.

[0015] In a further optimization scheme, in step (1), 10 times the weight of the bark powder is added with purified water and soaked for 1 hour. If the soaking time is too short, the sample cannot be dissolved, and if the soaking time is too long, it will waste work time. Crushing into smaller particles can reduce work time.

[0016] In step (2), the supernatant is centrifuged at a centrifugal force of ≥5000g, and the centrifuged liquid is filtered through a 0.22um filter to remove insoluble particles, and the supernatant is used for ion exchange. The chromatographic sample needs to be filtered through a 0.22um filter membrane, otherwise it will clog the chromatography system pipeline and damage the filler. In addition, if centrifugation is not performed, the subsequent steps cannot be performed at all; if filtration is not performed after centrifugation, concentration cannot be performed, resulting in pipeline blockage.

[0017] In a further optimized solution, the ion exchange filler selected for ion chromatography in step (3) has a pore size of 30, 60 or 80.

[0018] The ion exchange chromatography specifically comprises the following steps: The ion exchange column was equilibrated with phosphate buffer until the baseline was stable; the supernatant was loaded, and after loading, the column was equilibrated again with phosphate buffer containing 20 mM NaCl until the baseline was stable; then eluted with phosphate buffer containing 400-500 mM NaCl, and the target peak was collected to obtain a purified solution.

[0019] In the optimized solution, in step (4), a 5-30 kDa ultrafiltration membrane package is used to concentrate and replace the purified solution, and the solution is replaced 10 times to obtain an ultrafiltrate.

[0020] The reverse phase chromatography of crude Amethyst C18-H in step (5) specifically includes: The reverse phase chromatography column was equilibrated with a buffer containing 30% acetonitrile + 0.05% TFA until the baseline was stable, and the ultrafiltrate was loaded onto the reverse phase chromatography column. After loading, the column was linearly eluted with a buffer containing 95% acetonitrile + 0.05% TFA, and the target peak was collected to obtain a reverse phase solution; The pH of the buffer in the reverse phase crude purification in step (5) is 5.0-6.0; the crude purification solution A: 30% acetonitrile aqueous solution + 0.05% TFA; solution B: 95% acetonitrile aqueous solution + 0.05% TFA, linear elution is performed, 0-50% B.

[0021] The eluent for the purification in step (6) is: 40% acetonitrile + 0.05% TFA. The pH is 5.0-6.0; the eluent is Amethyst C18-H eluent.

[0022] The purified UniSil C8 reverse phase chromatography in step (6) comprises: The column was equilibrated with a buffer solution containing 30% acetonitrile + 0.05% TFA until the baseline was stable, and the reverse phase solution was loaded onto a C8 reverse phase column. After loading, 40% acetonitrile + 0.05% TFA was used for elution, and the elution peak was collected to obtain a high-purity sample solution containing QS-21.

[0023] The main impurities of fine powder extracted with purified water are cellulose, polyphenols, alkaloids, tannins, etc., which can be separated by different ion exchange binding abilities. The molecular weight of QS-21 is 1990Da, and the molecular weight can reach 60-70kDa after forming micelles in aqueous solution. The 30kDa membrane package is used for ultrafiltration to remove some small molecules (soluble impurities and salts). According to the different hydrophobicity, QS-21 and other saponins are separated by reverse phase chromatography.

[0024] In the present invention, after initial separation by reverse phase chromatography Amethyst C18-H filler, the collected liquid with purity ≥50% is combined and then finely separated by reverse phase chromatography UniSil C8 filler, and the collected liquid with purity ≥95% is combined to obtain a high-purity sample.

[0025] The water extraction in the present invention can extract 90% or more of QS21 in the bark, and the yield after three steps of purification is 30% or more. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 This is a diagram of the detection results after ion exchange in the present invention; Figure 2 This is a diagram showing the detection results after reverse phase crude purification in the present invention;

[0027] Figure 3 This is the test result spectrum of QS-21 after reverse phase purification in the present invention;

[0028] Figure 4 The flowchart is extracted in the present invention. DETAILED DESCRIPTION

[0029] The present invention will be further described below in conjunction with specific embodiments: Embodiment 1:

[0030] The present invention provides a method for extracting QS-21 from bark, the specific steps of which are as follows: (1) Crush the bark until 80% of the bark powder can pass through a 40-mesh medicinal sieve; (2) Add 8 times the weight of the bark powder into the purified water, soak for 0.5 h, and centrifuge the supernatant to obtain supernatant I containing QS-21 adjuvant; the centrifugal force is greater than or equal to 5000 g, and the centrifuge is filtered through a 0.22 μm filter to remove insoluble particles, and the supernatant is used for ion exchange.

[0031] (3) After centrifugation, the supernatant was filtered using a 0.22 μm filter to remove insoluble particles to obtain supernatant II; (4) Using Polar MC-CM filler, the supernatant 2 is subjected to ion exchange chromatography to obtain a purified solution; the filler selected for ion chromatography has a pore size of 30, 60, or 80.

[0032] Ion exchange chromatography specifically includes the following steps: The ion exchange column was balanced with phosphate buffer until the baseline was stable; the supernatant was loaded, and after loading, the column was balanced again with 20mM phosphate buffer until the baseline was stable; then 400-500mM NaCl phosphate buffer was used for elution, and the target peak was collected to obtain a purified solution; the test results after ion exchange were as follows: Figure 1 shown.

[0033] (5) The purified solution 1 was concentrated and replaced to reduce the sodium chloride concentration to prevent affecting the reverse phase chromatography effect, and an ultrafiltrate was obtained; a 5-30 kDa ultrafiltration membrane package was used to concentrate and replace the purified solution, and the solution was replaced 10 times to obtain an ultrafiltrate.

[0034] (6) Performing reverse phase crude purification using Amethyst C18-H reverse phase chromatography medium, collecting the elution peak, and obtaining a reverse phase liquid; the crude purification of Amethyst C18-H reverse phase chromatography specifically includes: The reverse phase column was equilibrated with a buffer solution containing 30% acetonitrile + 0.05% TFA until the baseline was stable, and the ultrafiltrate was loaded onto the reverse phase column. After loading, the column was linearly eluted with a buffer solution containing 95% acetonitrile + 0.05% TFA, and the target peak was collected to obtain a reverse phase solution. The detection results after reverse phase crude purification were as follows: Figure 2 The pH of the reverse phase buffer is 5.0-6.0.

[0035] (7) Perform reverse phase purification using reverse phase UniSil C8 chromatographic medium, collect the elution peak, and obtain a high-purity QS-21 sample; the purification UniSil C8 reverse phase chromatography includes: The column was balanced with a buffer solution containing 30% acetonitrile + 0.05% TFA until the baseline was stable, and the reverse phase solution 1 was loaded onto the C8 reverse phase column. After loading, 40% acetonitrile + 0.05% TFA was used for elution, and the elution peak was collected to obtain a high-purity sample solution containing QS-21. The test result spectrum of QS-21 after reverse phase purification is shown in the figure below. Figure 3 shown. Embodiment 2:

[0036] (1) Centrifugation of bark powder water: The bark is broken into pieces until 80% of the bark powder can pass through a 50-mesh medicinal sieve. Take the bark fine powder, add purified water 10 times its weight, soak for 1 hour, take the upper clear liquid for centrifugation, the centrifugal force is greater than or equal to 5000g, the centrifugation time is 30 minutes, the temperature is 2-8°C, and the centrifuged liquid is filtered with a 0.22um filter to obtain the supernatant for ion exchange.

[0037] (2) Capturing QS-21 from water extract: Chromatographic filler name: Polar MC-CM filler; Column equilibration: equilibrate with 20 mM phosphate buffer (pH 8.0) for 3 to 5 column volumes; Loading: Load the supernatant after centrifugation and filtration directly onto the ion exchange column; After equilibration: After loading, the column was equilibrated again with 20 mM phosphate buffer until the baseline was stable; Elution: Elution was performed with 500 mM NaCl phosphate buffer. The elution peak was collected when UV280 reached 100 mAU and stopped when UV280 dropped to 100 mAU. The water extract was purified by Polar MC-CM chromatography column and QS-21 was collected in binding / elution mode. After ion exchange purification, most of the pigments and a small amount of impurities were removed, and the yield could reach 93.59%.

[0038] (3) Use a 30 kDa ultrafiltration membrane to ultrafilter the purified solution 10 times: Membrane package manufacturer: Hangzhou Kebet 5-30kDa ultrafiltration membrane package; material PES; Membrane cleaning: clean the membrane with injection water and 0.5M NaOH respectively, then circulate with 0.5M NaOH for no less than 30 minutes, and finally clean 10 membrane volumes with injection water; First, add the same volume of water for injection as the eluent, control the pressure at 0.5-1.5 bar, concentrate to the original volume and then add water for injection again. Repeat the ultrafiltration and liquid replacement process 10 times, and test the conductivity (qualified standard ≯500μs / cm), as shown in Table 1 below: Table 1 Conductivity of permeate from 30kD membrane package ultrafiltration concentration at different times: Ultrafiltration times Concentrated liquid conductivity (μs / cm) Ultrafiltration times Concentrated liquid conductivity (μs / cm) 1 21431.7 6 1005.58 2 10998.2 7 744.7 3 5579.3 8 569.1 4 3079.2 9 496.7 5 1724.5 10 343.4 From the above table, it can be seen that the membrane package is used to ultrafilter the ion exchange eluate to reduce the salt concentration of the buffer. The concentration of the buffer salt will affect the adsorption of the stationary phase. Too high or too low buffer salt concentration will affect the separation effect. After the 9th liquid change, it meets the qualified standards. However, in order to ensure the purification effect of the subsequent reverse phase separation, a full 10 liquid changes are performed.

[0039] (4) Rough separation by reverse phase chromatography: Chromatographic filler name: Amethyst C18-H, Solution A: 30% acetonitrile aqueous solution + 0.05% TFA; Solution B: 95% acetonitrile aqueous solution + 0.05% TFA; Column equilibration: Equilibrate the reverse phase chromatography column with 30% acetonitrile + 0.05% TFA buffer until the baseline is stable; Loading: Load the ultrafiltered sample solution; Elution: linear elution from 0%B to 50%B, collecting target peaks in segments, and merging samples with purity greater than 50% after detection; After ultrafiltration of the membrane package, the solution is purified by Amethyst C18-H chromatography column, and the characteristic peaks are collected in segments. The purity after the sample is combined is 80-100%. The Amethyst C18-H medium bonded stationary phase adopts a unique and innovative design, with high-purity silica gel with good mechanical stability as the matrix. It has excellent stability in the pH range of 1.5-10 and has high-pressure resistant rigidity. Amethyst C18-H chromatography filler has a good purification effect on the sample solution and is suitable for the initial purification of QS-21.

[0040] (5) Reverse phase chromatography separation: Chromatographic filler: UniSil C8; Solution C: 40% acetonitrile aqueous solution + 0.05% TFA; Column equilibration: Use 30% acetonitrile aqueous solution + 0.05% TFA to equilibrate the column until the baseline is stable; Sample loading: dilute the Amethyst C18-H eluate with injection water at 1 / 2 of its weight and then load on the sample; Elution: directly use 40% acetonitrile aqueous solution + 0.05% TFA for isocratic elution. Start collecting the elution peak when UV205 reaches 30mAU, and stop collecting the peak when UV205 drops to 200mAU; The Amethyst C18-H eluate was purified by UniSil C8 chromatography column. QS-21 could be successfully bound and eluted. The yield could reach more than 90%, and its purity could reach more than 95%. The purification effect was good and it was more suitable for the fine purification of QS-21. Embodiment 3:

[0041] The other contents are as in Example 2, wherein the bark is crushed until 80% of the bark powder can pass through a 100-mesh medical sieve; then 12 times the weight of the bark fine powder is added with purified water, and after soaking for 1.5 hours, the supernatant is taken and centrifuged to obtain supernatant I containing QS-21 adjuvant.

[0042] Comparative Example 1: The other contents are as in Example 2, wherein the steps of capturing QS-21 from the water extract are as follows: Chromatographic filler name: Nano-UniGel-30CM; Column equilibration: equilibrate with 20 mM phosphate buffer (pH 8.0) for 3 to 5 column volumes; Loading: Load the supernatant after centrifugation and filtration directly onto the ion exchange column; After equilibration: After loading, the column was equilibrated again with 20 mM phosphate buffer until the baseline was stable; Elution: elution was performed with 500 mM NaCl phosphate buffer. The elution peak was collected when UV280 reached 100 mAU and stopped when UV280 dropped to 100 mAU. After the water extract was purified by Nanomicro UniGel-30CM ion exchange chromatography column, QS-21 was collected in bind / elute mode, removing most of the pigments and a small amount of impurities with a yield of 89.34%, which was lower than that of Polar MC-CM filler, so Polar MC-CM filler was selected for capture.

[0043] Comparative Example 2: The other contents are the same as those in Example 2, and the steps of reverse phase chromatography fine separation in the matrix are as follows: Name of chromatography packing material: UniSil C4; the chromatography packing materials are different for the same instrument.

[0044] Column equilibration: Use 30% acetonitrile aqueous solution + 0.05% TFA to equilibrate the column until the baseline is stable; Sample loading: dilute the Amethyst C18-H eluate with water for injection, add 1 / 2 of the eluate weight, and load the sample after dilution; Elution: directly use 40% acetonitrile aqueous solution + 0.05% TFA for isocratic elution. Start collecting the elution peak when UV205 reaches 50mAU, and stop collecting the peak when UV205 drops to 200mAU; The Amethyst C18-H eluate was purified by UniSil C4 chromatography column, and QS-21 could be successfully bound and eluted, with a yield of about 40% and a purity of more than 95%. Compared with C8 in Example 2, although the purity of the eluted sample was also more than 95%, its yield was extremely low and was not suitable for fine purification of QS-21.

[0045] Comparative Example 3: Other contents are as in Example 2. After the purification centrifugal filtration step, there is no centrifugal step or ion exchange chromatography step, and the concentration, liquid replacement, and crude purification are directly performed: Without ion exchange chromatography, the subsequent steps simply cannot be performed.

[0046] Comparative Example 4: The other contents are the same as those in Example 2, except that there is no filtration or ion exchange chromatography in the steps, and the concentration, liquid replacement, and crude purification are directly performed: If there is no filtration after centrifugation, concentration cannot be carried out, causing pipeline blockage.

[0047] Comparative Example 5: The other contents are the same as those in Example 2, except that there is no filtration, ion exchange chromatography and concentration and liquid exchange in the steps, and the crude and refined product is directly purified: It is not possible, the volume is very large, the loading time is long, and it will definitely clog the pipeline.

[0048] Comparative Example 6: Other contents are as in Example 2. When there is no crude purification in the step, the purification is directly carried out: No further processing is possible.

[0049] From the above, it can be seen that the various steps of the method of the present invention are organically combined and synergistic, and have a good effect as a whole, with simple extraction, low cost, high extraction rate, and high purification rate, and no other organic solvents are needed.

[0050] The above implementation / test examples are merely examples for the purpose of clear explanation, and are not intended to limit the implementation methods. For those skilled in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to list all the implementation methods here. The obvious changes or modifications derived therefrom are still within the scope of protection of the invention.

Claims

1. A method for purifying a saponin adjuvant, characterized in that: The following steps are involved: (1) Crush the bark and add purified water to soak the bark powder; (2) Taking out the supernatant and centrifuging it to obtain supernatant I containing QS-21 adjuvant, and then filtering it with a filter to remove insoluble particles to obtain supernatant II; (3) Using Polar MC-CM filler to perform ion exchange chromatography on supernatant II to obtain a purified solution; (4) Concentrating the purified solution and replacing the solution to obtain an ultrafiltrate; (5) Perform reverse phase crude purification using Amethyst C18-H reverse phase chromatography medium, collect the elution peak, and obtain a reverse phase liquid; (6) Use reversed-phase UniSil C8 chromatographic medium for reversed-phase purification and collect the elution peak to obtain a high-purity QS-21 sample.

2. A method for purifying a saponin adjuvant according to claim 1, characterized in that: In the step (1), 80% of the bark powder is crushed to pass through a 40-100 mesh medicinal sieve, and in the optimized solution, it is passed through a 50 mesh medicinal sieve.

3. A method for purifying a saponin adjuvant according to claim 1 or 2, characterized in that: In the step (1), 8-12 times the weight of the bark fine powder is added with purified water and the mixture is soaked for 0.5-1.5 hours.

4. A method for purifying a saponin adjuvant according to claim 3, characterized in that: In the step (1), 10 times the weight of the bark fine powder is added with purified water and the mixture is soaked for 1 hour.

5. A method for purifying a saponin adjuvant according to claim 1, characterized in that: In step (2), the supernatant is centrifuged at a centrifugal force of ≥5000 g, and the centrifuged liquid is filtered through a 0.22 μm filter to remove insoluble particles, thereby obtaining a supernatant for ion exchange.

6. A method for purifying a saponin adjuvant according to claim 1, characterized in that: The ion exchange filler selected for the ion chromatography in step (3) has a pore size of 30, 60 or 80.

7. A method for purifying a saponin adjuvant according to claim 6, characterized in that: The ion exchange chromatography specifically comprises the following steps: The ion exchange column was equilibrated with phosphate buffer until the baseline was stable; the supernatant was loaded, and after loading, the column was equilibrated again with phosphate buffer containing 20 mM NaCl until the baseline was stable; then eluted with phosphate buffer containing 400-500 mM NaCl, and the target peak was collected to obtain a purified solution.

8. A method for purifying a saponin adjuvant according to claim 1, characterized in that: In the step (4), a 5-30 kDa ultrafiltration membrane package is used to concentrate the purified solution and replace the solution 10 times to obtain an ultrafiltrate.

9. A method for purifying a saponin adjuvant according to claim 1, characterized in that: The reverse phase chromatography of crude Amethyst C18-H in step (5) specifically includes: The reverse phase chromatography column was equilibrated with a buffer solution containing 30% acetonitrile + 0.05% TFA until the baseline was stable, and the ultrafiltrate was loaded onto the reverse phase chromatography column. After loading, linear elution was performed with a buffer solution containing 95% acetonitrile + 0.05% TFA, and the target peak was collected to obtain a reverse phase liquid.

10. A method for purifying a saponin adjuvant according to claim 1, characterized in that: The purification of UniSil C8 reverse phase chromatography in step (6) comprises the following steps: The column was equilibrated with a buffer containing 30% acetonitrile + 0.05% TFA until the baseline was stable, and the reverse phase liquid was loaded onto a C8 reverse phase column. After loading, elution was performed with a buffer containing 40% acetonitrile + 0.05% TFA, and the elution peak was collected to obtain a high-purity sample solution containing QS-21.

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