A method for preparing quillaja saponin 21

Abstract

The application discloses a preparation method of quillaja saponin 21 and relates to the technical field of adjuvant preparation. The method comprises the following steps: pretreatment and impurity removal of saponin raw materials, first reverse purification, and second reverse purification, so that the preparation of quillaja saponin 21 can be quickly and efficiently completed. The preparation method of quillaja saponin 21 is simple and easy to operate, has high efficiency, high production capacity, low production cost, high purity of main peak product, and is suitable for industrialized production.

Description

Technical Field

[0001] The present invention relates to the technical field of adjuvant preparation, and in particular to a method for preparing Quillaja saponin 21. Background Art

[0002] Vaccine adjuvants are substances that can nonspecifically enhance the body's specific immune response to antigens. They can induce long-lasting, highly effective specific immune responses, improving protection while reducing the amount of immune substances used and the cost of vaccine production, making them crucial in modern vaccines.

[0003] AS01 is a liposomal adjuvant system developed by GlaxoSmithKline (GSK). It contains two immunoadjuvant components: monophosphoryl lipid A (MPL) and Quillaja saponin QS-21. QS-21, extracted from the bark of the South American Quillaja tree, has been extensively studied and used for its low toxicity and high content. QS-21 is composed of multiple isomers, a mixture of acylated triterpenoid saponins containing eight sugar groups. Its structure consists of a branched trisaccharide attached to the hydroxyl group at position 3 of the parent nucleus salsaponin, a linear tetrasaccharide attached to the carboxylic acid at position 28, and a fatty acid with an α-L-arabinose terminal saccharide linked to the hydroxyl group at position 4 of the fucosyl group via an ester bond. The structural isomers are QS-21api (65% apiose) and QS-21xyl (35% xylose). In aqueous solution, QS-21 undergoes intramolecular transfer of the ester bond to produce positional isomers. In addition to the main isomer QS-21A (fat chain connected to the 4-position of the fucosyl group), a small amount of isomer QS-21B (fat chain connected to the 3-position of the fucosyl group) is also produced.

[0004] QS-21 enhances antigen-mediated immune responses through multiple mechanisms, including interaction with lectins on the surface of APCs, providing T cell co-stimulatory signals, interacting with cholesterol, and inducing the production of inflammatory cytokines and secretion of IL-1β / IL-18. Years of clinical application have demonstrated that QS-21 is a safe and well-tolerated immunomodulator. Currently, several vaccines and innovative drugs based on QS-21 are in the market or clinical trial stages. The bottlenecks to the commercialization of QS-21 lie in the low content, instability, and large batch-to-batch variability of Quillaja bark extract, as well as its hundreds of structural analogs, which complicate its isolation and purification.

[0005] In recent years, patents and literature have disclosed some methods for purifying QS-21. Kensil et al. described a method for purifying QS-21 in their 1991 patent (US5057540). (Quillaja bark extract) was extracted with methanol, and the extract was purified by silica gel and reverse phase C4 resin in turn to purify QS-21. The obtained product showed a single band in thin layer chromatography (TLC), but fast atom bombardment mass spectrometry (FAB-MS) analysis showed that it was still a rather complex mixture, and no yield data was mentioned. Kensil et al. described two example methods for purifying QS-21 in their 2001 and 2003 patents (US6231859; US6524584). The first method: the Quillaja bark extract was first dialyzed and freeze-dried as the starting extract, and then QS-21 was crudely purified by silica gel column, and finally QS-21 was purified in two steps by reverse phase C18 column and C18 column with larger particle size. According to the patent description, the yield of this method is about 2.95%, but it should be noted that after the starting extract is enriched by dialysis, the QS-21 content is higher than that of the starting extract. High, specific data are not reported. The second method: first use polyvinyl pyrrolidone (PVPP) to remove polyphenol impurities from Quil-A, then perform two consecutive resin purification steps on a phenyl column, and finally purify QS-21 on a C8 column, and the yield is not reported. The purity of QS-21 obtained by the two purification methods is ≥98% (HPLC chromatogram not shown). However, FAB-MS shows that in addition to the main QS-21 ion, there are three major impurity ions. The two methods described in this patent require multiple (3 to 4 times) purifications using different types of chromatographic fillers (involving: silica gel, reverse phase C18, larger particle size C18, PVPP, phenyl, C8). The operation is cumbersome, the purification efficiency is low, the production cost is high, and more importantly, the final product contains unknown impurities and cannot be applied to the industrial production of QS-21.

[0006] In its 2019 patent (CN 111670044 A), GSK described a saponin extract ( and A purification method for AS01, a component of the vaccine. First, the soap tree bark extract is adsorbed by PVPP, then purified by polystyrene column, phenyl column, and C8 column, and freeze-dried to obtain the QS-21 saponin extract. The yield of this method is not reported. UPLC-UV / MS shows that the 'QS-21 main peak' (purity ≥93%) in the product is a mixture of components with molecular weights of 1856, 1988 (QS-21) and 2002. The largest impurity (0.25-3%) is the '2018 component'. The patent focuses on defining the acceptable range of the content of this component in the starting extract, which greatly limits the applicability of the method. Because the content of the '2018 component' varies greatly in different batches of extracts and cannot be removed by the patented method, deviations from the specifications often occur during the production process, resulting in unqualified products. Therefore, GSK revised its 2020 patent (CN 114080393A), describing a saponin extract (QS-21 main peak ≥88%) with a higher percentage of '2018 component' (>3% to 10%) showing similar biological activity characteristics. The final product described in the two patents applied for by GSK is not high-purity QS-21, but a mixture composed of molecular weight 1856, 1988 (QS-21) and 2002, 2018 impurities (0.25-10%) and some other saponin components, namely QS-21 saponin extract. The purity of its main peak (1856, 1988 and 2002) is only ≥88%. The entire production process described in the patent involves multiple purification steps (PVPP + 3 column chromatography), multiple filler types (involving: PVPP, polystyrene, phenyl, C8) and freeze-drying steps, so its production cost is relatively high. Because the CN 114080393 A patent relaxes the release standards for the main peak purity and the 2018 impurity ratio in the product, it is speculated that its yield is not high and the production capacity cannot meet the demand.

[0007] In addition, Qi et al. (Qi Y, Fox C BJ CHROMATOGR A. 2020, 1635: 461705) reported a two-step orthogonal chromatography method for preparing high-purity QS-21. The starting extract does not require any pretreatment and is purified by C18 column and HILIC column. The engineering batch verification results show that the batch yield is 339 mg, the yield is ~2.1%, the product purity is 97.2%, and it does not contain the "2018 component" impurity. One of the innovations of this article is that it does not require any pretreatment. However, the starting extract It contains a lot of plant polyphenols, polysaccharides and protein ingredients. These impurities will cause dead adsorption in C18 filler during column purification, resulting in irreversible damage. This article only reports the engineering batch data of one batch of production with only hundreds of milligrams, so the batch-to-batch stability of the process needs to be verified by multiple batches of data. Another innovation is the use of HILIC as the second chromatography filler. Compared with the conventional reversed-phase C18, the HILIC filler has certain limitations in use, and its filler life and reproducibility are significantly lower than C18, and it is expensive. Therefore, the purification method reported in this article is not suitable for the industrial production of QS-21.

[0008] In summary, the preparation method reported in the QS-21 patent has complex process, low QS-21 purification efficiency, and high production cost. In order to meet the demand for high yield, only the purity of QS-21 can be reduced or the saponin extract containing QS-21 can be used. The method disclosed in the literature is not suitable for industrial production. In addition to saponins in the extract, there are other impurities (polysaccharides, proteins, tannins, polyphenols, etc.). If the extract is not pretreated before being directly purified by column, some impurities will be dead adsorbed in the chromatography filler, resulting in reduced purification effect, even the risk of blocking the chromatography column, and unable to guarantee the quality stability of the product.

[0009] Therefore, the present application is proposed. SUMMARY

[0010] The purpose of the present application is to provide a preparation method of quillaja saponin 21 which is simple and easy to operate, high in efficiency and productivity, low in production cost, high in purity of main peak product, and suitable for industrial production, so as to solve the above technical problems.

[0011] The present application is implemented as follows:

[0012] The present application provides a preparation method of quillaja saponin 21, which comprises the following steps:

[0013] S1: removing impurities by pretreating saponin raw material: contacting an alcohol solvent with a volume fraction of 75%-90% with the saponin raw material to remove insoluble substances to obtain a pretreated and impurity-removed substance;

[0014] S2: performing first reverse purification on the pretreated and impurity-removed substance, and the chromatographic medium for the first reverse purification is C18; the elution condition is: isocratic elution with 0.05-0.3% acid regulator and ethanol according to a volume ratio of 50%±5%:50%±5%, and collecting high-purity samples;

[0015] S3: The high-purity sample obtained in S2 is subjected to a second reverse purification, and the chromatographic medium for the second reverse purification is C18; the elution conditions are: isocratic elution with 0.05-0.3% acidic regulator and acetonitrile in a volume ratio of 62%±5%:38%±5%, and the high-purity sample is collected as the second reverse purification product.

[0016] The present invention has the following beneficial effects:

[0017] The preparation method of Quillaja saponin 21 provided by the present invention is simple and easy to operate, has high efficiency, high production capacity, low production cost, high purity of the main peak product, and is suitable for industrial production. Specifically:

[0018] (1) The pretreatment and impurity removal step of the present invention uses a green and environmentally friendly solvent (alcohol / water system) to dissolve the saponin raw material. The operation is simple, no special equipment is required, and the impurity removal effect is obvious. It can significantly extend the service life of the chromatography packing, ensure the consistency of multiple batches of purification, and ensure the yield and quality of the QS-21 product. Pretreatment and impurity removal can avoid the problem of some impurities being deadly adsorbed in the chromatography packing, resulting in reduced purification effect and even clogging the chromatography column, thereby solving the problem of poor quality stability of multiple batches of products.

[0019] (2) The chromatography process provided by the present invention includes two "isocratic elutions", which have the following advantages: (a) it can meet the needs of continuous sample loading and does not require the chromatographic equilibration time of "gradient elution", thereby saving the amount of elution solvent used and the purification time; (b) "isocratic elution" has lower requirements on the precision of the pump in the high-pressure preparation system, avoiding the change in separation degree caused by the different mixing ratios of elution solvents in "gradient elution", thereby affecting the purity and quality of the product; (c) "isocratic elution" is less affected by environmental, instrumental and operational factors, and has good durability and batch consistency.

[0020] (3) The chromatographic filler (or chromatographic medium) used in the first and second reverse purifications is conventional reverse phase C18. Compared to special fillers such as phenyl and C8, the production process of C18 filler is more mature, the quality controllability is higher, the filler cost is low, and the selectivity is large, which ensures the feasibility of process operation from many aspects and can also reduce the production cost of the product. In addition, C18 filler can be reused, has high separation, and requires fewer purification times. Only two purifications are required to obtain a high-purity target product. C18 filler has a high sample load and can meet the requirements of the industrial preparation of Quillaja saponin 21.

[0021] (4) The preparation method provided by the present invention has completed process validation for multiple batches of 30-50 g / batch and can meet the production of millions of doses of vaccine. Based on the QS-21 content in the saponin raw material, the total yield of QS-21 prepared by the preparation method provided by the present invention is ≥30%, the yield before and after the two-step chromatography is >45%, the purity of QS-21 '1988 component' is ≥98.0%, and it does not contain the '2018 component' impurity, which is safer and has more advantages than the GSK patent. The yield and product quality of the preparation process are at the international leading level. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the following briefly introduces the drawings required for use in the embodiments. It should be understood that the following drawings only illustrate certain embodiments of the present invention and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other relevant drawings can be obtained based on these drawings without paying any creative work.

[0023] Figure 1 It is the structural formula of QS-21;

[0024] Figure 2 Elution chromatograms of acidic modifiers TFA and phosphoric acid;

[0025] Figure 3 HPLC chromatograms of saponin before (A) and after (B) pretreatment;

[0026] Figure 4 This is the HPLC chromatogram after one reverse purification;

[0027] Figure 5 This is the HPLC chromatogram after secondary reverse purification;

[0028] Figure 6 This is the high-resolution mass spectrum of purified QS-21 (batch number QS23002);

[0029] Figure 7 This is the first reverse purification elution chromatogram of the phenyl column;

[0030] Figure 8 This is the chromatogram of the second reverse purification elution on a phenyl column. DETAILED DESCRIPTION

[0031] To make the purpose, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention are described clearly and completely below. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer are used. Where the manufacturer of the reagents or instruments is not specified, all are conventional products that can be purchased commercially.

[0032] In the present invention, "QS-21" is synonymous with "Quillaja saponin 21". "Chromatographic medium" is synonymous with "chromatographic column filler".

[0033] The present invention provides a method for preparing Quillaja saponin 21, which comprises the following steps:

[0034] S1: Pre-treating and removing impurities from the saponin raw material: contacting the saponin raw material with an alcohol solvent having a volume fraction of 75%-90% to remove insoluble matter to obtain a pre-treated and impurity-removed material;

[0035] S2: The pretreated and impurity-removed material is subjected to a first reverse purification using C18 as the chromatographic medium; the elution conditions are: isocratic elution with 0.05-0.3% acidic regulator and ethanol at a volume ratio of 50% ± 5%: 50% ± 5%, and a high-purity sample is collected;

[0036] S3: The high-purity sample obtained in S2 is subjected to a second reverse purification, and the chromatographic medium for the second reverse purification is C18; the elution conditions are: isocratic elution with 0.05-0.3% acidic regulator and acetonitrile in a volume ratio of 62%±5%:38%±5%, and the high-purity sample is collected as the second reverse purification product.

[0037] The saponin raw material is derived from the Chilean soap tree, which varies greatly between batches. In addition to the main product QS-21, it also contains a large number of plant polyphenols, polysaccharides, and protein structures. Without pretreatment, direct chromatographic purification will lead to the dead adsorption of impurities, reducing column efficiency and making continuous production difficult. Therefore, a pretreatment process is added. By taking advantage of the different solubility of QS-21 and the main impurities in alcohol / water solvents, the impurity removal and enrichment effect is achieved. Specifically, 75%-90% alcohol solvent is added to the saponin dry powder, causing a large amount of impurities to precipitate. The precipitate is then removed by natural sedimentation, centrifugation, or filtration. This prevents the impurity precipitate from being dead adsorbed on the chromatographic column, clogging the column and reducing column efficiency.

[0038] QS-21 was purified through the first and second reverse purifications. Reverse chromatography employed isocratic elution. (a) This allows for continuous sample loading, eliminating the chromatographic equilibration time required by gradient elution, thus reducing elution solvent usage and purification time. (b) Isocratic elution places lower demands on the pump precision of the high-pressure preparation system, thus avoiding variations in separation caused by varying elution solvent ratios in gradient elution, which can affect product purity and quality. (c) Isocratic elution is less susceptible to environmental, instrumental, and operational factors, resulting in excellent durability and batch-to-batch consistency.

[0039] The chromatographic packing (or chromatographic medium) in the first reverse purification and the second reverse purification is a conventional reversed-phase C18. Compared with special packing such as phenyl and C8, the production process of C18 packing is more mature, the controllability of quality is higher, the cost of packing is low, and the selectivity is large, which can ensure the feasibility of process operation in many aspects, and also can reduce the production cost of product. In addition, the C18 packing can be repeatedly used, has high separation degree, and only two purifications are needed to obtain a high-purity target product. The C18 packing has a high loading capacity, which can meet the preparation of industrialized quillaja saponin 21.

[0040] The QS-21 product obtained by using the technical route of the present application has a purity of '1988 component' ≥98.0%, a yield ≥30%, does not contain '2018 component' impurities, has higher safety, and the product quality is at an international leading level. Preferably, the purity of '1988 component' is ≥99.0%, and the yield is ≥30%.

[0041] In step S1, an alcohol solvent with a volume fraction of 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89% or 90% is contacted with the saponin raw material.

[0042] In step S2: isocratic elution is performed with 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, 0.1%, 0.15%, 0.2%, 0.25% or 0.3% of the acidic regulator and ethanol according to a volume ratio of 50%±5%:50%±5%. For example, isocratic elution is performed with the acidic regulator and ethanol according to a volume ratio of 45%:55%, or 46%:54%, or 47%:53%, or 48%:52%, or 49%:51%, or 50%:50%, or 51%:49%, or 52%:48%, or 53%:47%, or 54%:46% or 55%:45%.

[0043] In step S3: isocratic elution is performed with 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, 0.1%, 0.15%, 0.2%, 0.25% or 0.3% of the acidic regulator and acetonitrile according to a volume ratio of 62%±5%:38%±5%. For example, isocratic elution is performed with the acidic regulator and acetonitrile according to a volume ratio of 62%:38%, or 63%:37%, or 64%:36%, or 65%:35%, or 66%:34%, or 67%:33%, or 57%:43%, or 58%:42%, or 59%:41%, or 60%:40% or 61%:39%.

[0044] In preferred embodiments of the present invention, the alcohol solvent includes, but is not limited to, at least one of a monohydric alcohol, a dihydric alcohol, and a trihydric alcohol. Replacing the solvent with an aqueous solution containing 30% acetonitrile and 5 mM citric acid at pH 5.0 fails to remove impurities from saponin. However, alcohol solvents do have a moderate impurity removal effect.

[0045] In a preferred embodiment of the present invention, the monohydric alcohol includes but is not limited to at least one of ethanol, methanol, propanol and butanol.

[0046] In a preferred embodiment of the present invention, the diol includes but is not limited to at least one of ethylene glycol, propylene glycol and butanediol;

[0047] In a preferred embodiment of the present invention, the triol is glycerol.

[0048] In a preferred embodiment of the present invention, the saponin raw material is dissolved in 75%-80% ethanol by volume, or in 80% ± 5% methanol by volume. After treatment with these solvent ratios, QS-21 recovery rates are >98%, and the solvent exhibits a significant impurity removal effect. In particular, treatment with 80% ethanol or 80% methanol significantly increases impurity removal rates; treatment with 80% ethanol increases impurity removal rates to twice that of treatment with 75% ethanol.

[0049] In a preferred embodiment of the present invention, the alcohol solvent is mixed with the saponin raw material at a volume ratio of (15-30) ml:1 g, such as 15-20 ml:1 g, 18-25 ml:1 g, 20-25 ml:1 g, 22-28 ml:1 g, or 20-30 ml:1 g.

[0050] In a preferred embodiment of the present invention, the alcohol solvent is mixed with the saponin raw material at a volume ratio of 25 ml to 1 g.

[0051] In a preferred embodiment of the present invention, the pre-treated impurity-removed material is obtained by removing insoluble matter by at least one of natural sedimentation, centrifugation, and filtration. Filtration includes but is not limited to removing the precipitate by membrane or filter press equipment.

[0052] In a preferred embodiment of the present invention, after the alcohol solvent is mixed with the saponin raw material, it is allowed to stand at 2-8° C. for more than 10 hours, and then the insoluble matter is removed by centrifugation or filtration.

[0053] In a preferred embodiment of the present invention, the mixture is allowed to stand at 2-8° C. for more than 12 hours after mixing.

[0054] In a preferred embodiment of the application, the chromatographic medium for the first reverse purification and the chromatographic medium for the second reverse purification are SP-100-10-ODS-P. The column efficiency of the SP-100-10-ODS-P is optimized to be no less than 15000 N / m.

[0055] If the yield is to be improved, the collected substandard components (purity: 85% to 98%) can be subjected to the two-step purification procedure again, and the quality standard of the final QS-21 product can be met.

[0056] In a preferred embodiment of the application, the acidic regulator for the first reverse purification is formic acid, acetic acid, trifluoroacetic acid or phosphoric acid.

[0057] In a preferred embodiment of the application, the concentration of the acidic regulator is 0.1 to 0.3%. The concentration of the acidic regulator of 0.1 to 0.3% can adjust the resolution of the purification.

[0058] In a preferred embodiment of the application, in the first reverse purification, the pre-processed and impurity-removed material is diluted with water to an ethanol concentration of 40% ± 5%; the sample is pre-diluted so that the target non-polar substance in the sample can be combined with the stationary phase for a longer time; when ethanol is used for elution in the first reverse purification, the interaction between the target non-polar substance and the stationary phase can be weakened, and the target non-polar substance can be quickly eluted, thereby improving the resolution after chromatography.

[0059] In a preferred embodiment of the application, the flow rate for the first reverse purification is 500 to 600 mL / min.

[0060] In a preferred embodiment of the application, the collection of the high-purity sample in the first reverse purification comprises: combining the samples with a purity of ≥70% and an impurity of ≤28% after the main peak.

[0061] In a preferred embodiment of the application, the acidic regulator for the second reverse purification is formic acid, acetic acid, trifluoroacetic acid or phosphoric acid.

[0062] Preferably, the concentration of the acidic regulator is 0.1 to 0.3%.

[0063] In a preferred embodiment of the application, in the second reverse purification, the pre-processed and impurity-removed material is diluted with water to an acetonitrile concentration of 38% ± 5%; the eluent for the second reverse purification is acetonitrile, and therefore, by pre-diluting the sample with acetonitrile, the target non-polar substance in the sample can be combined with the stationary phase for a longer time, which facilitates the subsequent elution and improves the resolution.

[0064] In a preferred embodiment of the application, the flow rate for the second reverse purification is 900 to 960 mL / min.

[0065] In a preferred embodiment of the present invention, collecting high-purity samples during the second reverse purification step includes combining samples having a purity of ≥98% and impurities ≤1.2% after the main peak. Preferably, collecting high-purity samples during the second reverse purification step includes combining samples having a purity of ≥99% and impurities ≤1.2% after the main peak.

[0066] In a preferred embodiment of the present invention, the preparation method further comprises: step S4: solvent replacement. S4: the second reverse purification product obtained in S2 is loaded onto a reverse phase chromatography column for solvent replacement, and 30% to 95% ethanol is used for isocratic or gradient solvent replacement, and the eluted product is collected as the solvent replacement solution.

[0067] The purpose of solvent exchange is to replace TFA and acetonitrile in the purified sample with ethanol and reduce the sample volume. Therefore, conventional reversed-phase chromatography media (e.g., C4, C8, C18, etc.) can be used. The eluent is 30% to 95% aqueous ethanol, either isocratically or in a gradient elution, ensuring a QS-21 yield of ≥95% in this step.

[0068] In a preferred embodiment of the present invention, during the solvent replacement in step S4, isocratic or gradient solvent replacement is first performed with 30% to 40% ± 5% ethanol, and then isocratic or gradient elution is performed with 80% ± 5% to 95% ethanol; the eluted product is collected as the solvent replacement solution.

[0069] QS-21 is acid-sensitive. Prolonged storage in acidic conditions can lead to degradation, resulting in sugar chain breakage and increased intramolecular ester transfer, resulting in the isomer QS-21B. Furthermore, the sample solution volume after the second purification step is large, making subsequent operations inconvenient. The present invention uses a solvent exchange step to replace TFA and acetonitrile in the sample solution with ethanol, while simultaneously concentrating and enriching QS-21.

[0070] Preferably, isocratic solvent replacement is first performed with 30% to 40% ± 5% ethanol, and then isocratic elution is performed with 80% ± 5% to 95% ethanol; the eluted product is collected as the solvent replacement solution.

[0071] First, use low concentration ethanol for replacement, which can replace the solvent (acetonitrile, trifluoroacetic acid, etc.) on the stationary phase without affecting the binding of the target product to the stationary phase; then use high concentration ethanol for elution. Under high concentration ethanol treatment, the interaction force between the target product and the stationary phase is weakened and it is eluted.

[0072] Gradient elution procedures include but are not limited to:

[0073] 0-25min: 30% ethanol, 25min~25.1min: 30%→80% ethanol, 25.1min~40min: 80% ethanol.

[0074] In a preferred embodiment of the present invention, the chromatographic medium of the reverse phase chromatography column used in the solvent replacement in step S4 is C4, C8, C18 or phenyl.

[0075] In a preferred embodiment of the present invention, the chromatographic medium of the reverse phase chromatography column used in the solvent replacement in step S4 is C18.

[0076] In a preferred embodiment of the present invention, during solvent exchange, the second reverse purification product is diluted with water to an acetonitrile concentration of 30% ± 5% before loading. This initial dilution reduces the acetonitrile concentration in the sample, facilitating subsequent solvent exchange.

[0077] In a preferred embodiment of the present invention, during solvent replacement, the flow rate is set to 900-960 mL / min.

[0078] In a preferred embodiment of the present invention, the preparation method further comprises: step S5 ultrafiltration replacement;

[0079] S5: performing ultrafiltration replacement on the solvent replacement liquid, the ultrafiltration replacement comprising: mixing the solvent replacement liquid with an ultrafiltration replacement liquid, and performing ultrafiltration concentration, wherein the solvent replacement liquid comprises an amino acid solution.

[0080] Adding a certain proportion of ultrafiltration replacement fluid, including an amino acid solution, to the QS-21 stock solution (i.e., the solution after solvent replacement) can effectively prevent QS-21 degradation, increase the stability of the stock solution, extend storage time, and ensure product quality. Ultrafiltration replacement can replace the organic solvent (such as ethanol, acetonitrile, etc.) in the sample solution with the amino acid solution and control the QS-21 concentration within the appropriate range.

[0081] In a preferred embodiment of the present invention, the ultrafiltration replacement fluid is an amino acid solution;

[0082] In a preferred embodiment of the present invention, the ultrafiltration replacement fluid includes but is not limited to at least one of histidine, arginine and lysine;

[0083] In a preferred embodiment of the present invention, the concentration of the ultrafiltration replacement fluid is 1 mM to 25 mM; such as 1 mM, 2 mM, 3 mM, 4 mM, 5 mM, 6 mM, 7 mM, 8 mM, 9 mM, 10 mM, 11 mM, 12 mM, 13 mM, 14 mM, 15 mM, 16 mM, 17 mM, 18 mM, 19 mM, 20 mM, 21 mM, 22 mM, 23 mM, 24 mM or 25 mM.

[0084] In a preferred embodiment of the present invention, the concentration of the ultrafiltration replacement fluid is 5 mM to 25 mM.

[0085] In a preferred embodiment of the present invention, the ultrafiltration replacement fluid is histidine, which is used as a pH buffer.

[0086] Adding a certain proportion (1mM~25mM) of amino acids (such as histidine, arginine, lysine, etc.) to the QS-21 stock solution can effectively prevent its degradation, increase the stability of the stock solution, extend the storage time, and ensure product quality.

[0087] In a preferred embodiment of the present invention, the ultrafiltration replacement comprises: contacting a mixture of a solvent replacement fluid and an ultrafiltration replacement fluid with an ultrafiltration membrane rinsed with the ultrafiltration replacement fluid, ultrafiltration concentration, continuously washing the ultrafiltration membrane with the ultrafiltration replacement fluid, and collecting the ultrafiltration concentrated product and the washing liquid;

[0088] In a preferred embodiment of the present invention, the ultrafiltration membrane is a 1000-3000 Da ultrafiltration membrane. When the ultrafiltration membrane is used for ultrafiltration replacement, the yield of QS-21 in this step can be >90%, the residual ethanol content is <500 ppm, and the residual acetonitrile content is <40 ppm.

[0089] In a preferred embodiment of the present invention, the ultrafiltration and replacement further include aseptic packaging.

[0090] The features and performance of the present invention are further described in detail below with reference to the embodiments.

[0091] Main raw materials: saponin (derived from dried extract of Quilla bark) was purchased from Desert King (name: VET-SAP, CAS number: 8047-15-2);

[0092] According to high performance liquid chromatography testing, the QS-21 content in the raw material is not less than 5%; the moisture content is not higher than 10%, the ash content is not higher than 3%, and the heavy metal content and pesticide residues are in line with the Chinese Pharmacopoeia standards.

[0093] Example 1

[0094] This embodiment provides a method for pretreatment and impurity removal of saponin.

[0095] Saponin contains a large amount of plant polyphenols, polysaccharides and protein components, which form dead adsorption in the filler, so the saponin needs to be pretreated.

[0096] In this example, 75% ethanol was used to dissolve the saponin raw material. Specifically, 200g of saponin and 5L of 75% ethanol were mixed at a ratio of 25:1 (v / w). Dry saponin powder was added to the 75% ethanol and stirred thoroughly to dissolve. After standing overnight at 2-8°C, the insoluble matter was filtered through a 0.22μm pore size filter. The recovery rate and impurity removal efficiency of QS-21 were calculated.

[0097] QS-21 recovery (%): calculated by HPLC external standard method;

[0098] Impurity removal rate (%) = weight of precipitate after dissolution and filtration (g) / weight of saponin (g) × 100%.

[0099] Example 2

[0100] Compared with Example 1, the only difference is that the solvent is different. This example uses 80% ethanol as the solvent.

[0101] Example 3

[0102] Compared with Example 1, the only difference is that the solvent is different. This example uses 90% ethanol as the solvent.

[0103] Example 4

[0104] Compared with Example 1, the only difference is that the solvent is different. This example uses 80% methanol as the solvent.

[0105] Example 5

[0106] This embodiment provides a method for preparing Quillaja saponin 21.

[0107] (1) Mix the raw materials in a ratio of 25:1 (v / w), 1.5 kg of saponin, and 80% ethanol for pretreatment and impurity removal.

[0108] (2) First reverse purification:

[0109] Take the pretreated sample, dilute it with water to 40% ethanol concentration, and follow the steps below;

[0110] Dynamic axial compression column: DAC-150;

[0111] Packing medium: SP-100-10-ODS-P;

[0112] Eluent: Solution A - 0.1% trifluoroacetic acid (TFA); Solution B - ethanol;

[0113] Flow rate: 540 mL / min;

[0114] After the loading is completed, the target peak is eluted isocratically with 50% solution A and 50% solution B, and the samples are collected in sections for purity detection. The samples with purity ≥70% and impurity ≤28% are combined to obtain the sample solution for the first reverse purification.

[0115] (3) Second reverse purification:

[0116] Take the sample from the first reverse purification, dilute it with water to 38% ethanol concentration, and follow the steps below;

[0117] Dynamic axial compression column: DAC-200;

[0118] Packing medium: SP-100-10-ODS-P;

[0119] Eluent: Solution A - 0.1% TFA; Solution B - acetonitrile;

[0120] Flow rate: 960 mL / min;

[0121] After the loading is completed, the target peak is eluted isocratically with 62% solution A and 38% solution B, and the samples are collected in sections for purity detection. The samples with purity ≥98% and impurity ≤1.2% are combined to obtain the sample solution for the second reverse purification.

[0122] (4) Solvent replacement

[0123] Take the sample after the second reverse purification, dilute it with water to 30% acetonitrile concentration, and follow the steps below;

[0124] Dynamic axial compression column: DAC-200;

[0125] Packing medium: SP-100-10-ODS-P;

[0126] Flow rate: 960 mL / min;

[0127] After the loading is completed, 30% ethanol is used for isocratic replacement, and then 80% ethanol is used for isocratic elution. When a chromatographic peak appears, the fractions are immediately collected to the end of the peak to obtain the solvent replacement sample solution.

[0128] (5) Ultrafiltration replacement

[0129] Take the solvent-exchanged sample solution and dilute it with 5 volumes of diluent (10 mM histidine, pH 6.0). Use a 3000Da ultrafiltration membrane with a transmembrane pressure of 0.1-0.8 MPa and rinse the membrane with ultrafiltration replacement solution (5 mM histidine, pH 5.0). Take the diluted solution (~60 L) and concentrate it to 3-5 L by ultrafiltration. Then, perform continuous diafiltration with at least 8 volumes of ultrafiltration replacement solution. After completion, drain and collect the QS-21 ultrafiltration replacement sample, and top-wash the pipes and ultrafiltration membrane pack with 1-10 L of ultrafiltration replacement solution. Collect the washing solution. Combine the QS-21 ultrafiltration replacement sample and washing solution, mix thoroughly, and control the theoretical QS-21 content to 2.8-4.0 mg / mL, the residual ethanol content to <500 ppm, and the residual acetonitrile content to <40 ppm.

[0130] (6) Aseptic packaging: Filter the ultrafiltration sample of saponin after ultrafiltration through a 0.45 μm / 0.2 μm sterilizing filter to obtain 9.67 L of the QS-21 adjuvant solution described in this application (QS-21 content: 3.57 mg / mL, yield: 32%, QS-21 content in saponin: 7.2%), and store at a temperature below -20°C after packaging. The structural formula of QS-21 is shown in Figure 2. Figure 1 shown.

[0131] Example 6

[0132] Compared with Example 5, the only difference is that the chromatographic column used in the solvent replacement in step (4) is C4, and after the sample is loaded, gradient elution is performed.

[0133] 0-25min: 30% ethanol, 25min~25.1min: 30%→80% ethanol, 25.1min~40min: 80% ethanol.

[0134] There is no significant difference between using a C4 column and a C18 column in step (4). In actual production, a C18 column is preferred.

[0135] Example 7

[0136] Compared with Example 5, the only difference is the different eluents in step (2). In this example, the eluents are: Solution A - 0.1% phosphoric acid; Solution B - ethanol.

[0137] The elution chromatogram when the eluent in Example 5 is 0.1% TFA and the eluent in Example B is ethanol is as follows: Figure 2 As shown in (A); according to the eluent of this embodiment, liquid A is 0.1% phosphoric acid; liquid B is ethanol, and the elution chromatogram is as shown in Figure 2 The results show that the acidic regulator is selected from TFA and phosphoric acid, and the separation degree of QS-21 is good.

[0138] Comparative Example 1

[0139] Compared with Example 1, the only difference is that the solvent is different. In this comparative example, an aqueous solution containing 30% acetonitrile, 5 mM citric acid, and pH 5.0 is used as the solvent (patent application number: CN202111627088.6). Saponin dry powder is added at a ratio of 25:1 (v / w) and fully stirred to dissolve. After standing at 2-8°C overnight, the insoluble matter is filtered with a 0.22 μm pore size filter membrane, and the recovery rate and impurity removal efficiency of QS-21 are calculated.

[0140] Experimental Example 1

[0141] The recovery rate and impurity removal rate of QS-21 after treatment of Examples 1-4 and Comparative Example 1 were calculated respectively:

[0142] QS-21 recovery (%): calculated by HPLC external standard method;

[0143] Impurity removal rate (%) = weight of precipitate after dissolution and filtration (g) / weight of saponin (g) × 100%.

[0144] The HPLC detection method is shown in Table 1. The HPLC chromatograms of saponin before (A) and after (B) pretreatment are shown in Table 1. Figure 3 shown.

[0145] Table 1 HPLC detection conditions

[0146]

[0147] Table 2 Statistics of recovery and impurity removal rates of QS-21 in different embodiments and comparative example 1

[0148]

[0149] The results are shown in Table 2. The solvent in Comparative Example 1 had no effect on saponin removal, while different ratios of ethanol / methanol showed some effectiveness. After treatment with both 75% and 80% ethanol, the recovery of QS-21 was >98%, with the 80% ethanol treatment doubling the impurity removal rate.

[0150] Experimental Example 2

[0151] According to the saponin feed amount of 1.5 kg in Experimental Example 5, three batches of experiments were carried out, with batch numbers QS23002, QS23003, and QS23004. The samples of each step were tested by RP-HPLC according to the method of Experimental Example 5 to detect the distribution of the QS-21 main peak and impurity peaks.

[0152] Table 3 Distribution of main peaks and impurity peaks of different batches of QS-21

[0153]

[0154] The results are shown in Table 3. The results show that after pretreatment, the purity of the main peak is 12% to 16%.

[0155] First reverse purification: There are many impurity peaks in the sample, among which the purity of the QS-21 main peak is about 70% to 80%, the impurities before the main peak are about 5%, and the impurities after the main peak are about 20%. Figure 4 This is the HPLC chromatogram of the product after the first reverse purification of batch QS23002.

[0156] The second reverse purification: effectively purifies QS-21, wherein the purity of the QS-21 main peak is greater than 99%, and the purity of QS-21B is 0.2-0.6%. Figure 5 This is the HPLC chromatogram of the product after the second reverse purification of batch QS23002.

[0157] In addition, after solvent replacement, the yield of the three batches in the solvent replacement step was >98%, and the sample purity did not change.

[0158] Experimental Example 3

[0159] QS23002, QS23003, QS23004 three batches of QS-21 products were detected for quality. High resolution mass spectrometry (ESI source, acquisition mode is negative ion full scan, m / z scan range: 500-2500, CDL temperature: 200.0℃, Heat block temperature: 200.0℃, carrier gas flow rate: 1.5L / min) shows that (QS23002 is taken as an example, the results of three batches are consistent) only the component with a molecular weight of 1988 (QS-21) is contained in the main peak, the product does not contain '2018 component' impurities, and has higher safety. Figure 6

[0160] The solvent residue, protein residue, endotoxin content and other items are controlled, and the produced QS-21 adjuvant meets the quality standard requirements.

[0161] Table 4 Quality statistical results of QS-21 products of different batches

[0162]

[0163]

[0164] The present application has completed process verification of 30-50g / batch, multiple batches, which can meet the production of million doses of vaccines. The yield of QS-21 is ≥30%, the yield before and after two-step chromatography is >45%, the purity of QS-21 '1988 component' is ≥99.0%, the ethanol residue is <500ppm, the acetonitrile residue is <40ppm, and there is no '2018 component' impurity, which is safer and has more advantages compared with the GSK patent.

[0165] Comparative Example 2

[0166] Compared with Example 2, the only difference is that the filler is different, and the present comparative example uses a phenyl column as the filler for the first and second reverse purification. The elution chromatograms after the first and second reverse purification are respectively referred to Figure 7 and Figure 8 The results show that when the first and second reverse purification is carried out with a phenyl column as the filler, the resolution is poor, the purification effect is poor, and the purity and yield of the final product are affected.

[0167] ​The foregoing description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Those skilled in the art will readily appreciate that various modifications and variations of the present invention are possible. Any modifications, equivalent substitutions, or improvements made within the spirit and principles of the present invention are intended to be within the scope of protection of the present invention.

Claims

1. A method for preparing Quillaja saponin 21, characterized in that: It includes the following steps: S1: pre-treating and removing impurities from the saponin raw material: contacting the saponin raw material with an ethanol solvent having a volume fraction of 75% or 80% or contacting the saponin raw material with a methanol solvent having a volume fraction of 80% to remove insoluble matter to obtain a pre-treated and impurity-removed material; S2: performing a first reverse purification on the pretreated and impurity-removed material, wherein the chromatographic medium for the first reverse purification is C18; the elution conditions are: isocratic elution with 0.05-0.3% acidic regulator and ethanol in a volume ratio of 50%±5%:50%±5%, and collecting a high-purity sample; S3: performing a second reverse purification on the high-purity sample obtained in S2, wherein the chromatographic medium for the second reverse purification is C18; the elution conditions are: isocratic elution with 0.05-0.3% acidic regulator and acetonitrile in a volume ratio of 62%±5%:38%±5%, and collecting the high-purity sample as the second reverse purification product; S4: The second reverse purification product obtained in S3 is loaded onto a reverse chromatography column for solvent replacement, and 30% to 95% ethanol is used for isocratic or gradient solvent replacement, and the eluted product is collected as the solvent replacement liquid; the chromatographic medium of the reverse chromatography column used in the solvent replacement in step S4 is C18; S5: performing ultrafiltration replacement on the solvent replacement liquid, the ultrafiltration replacement comprising: contacting a liquid obtained by mixing the solvent replacement liquid and the ultrafiltration replacement liquid with an ultrafiltration membrane rinsed with the ultrafiltration replacement liquid, ultrafiltration concentration, continuously washing the ultrafiltration membrane with the ultrafiltration replacement liquid, and collecting the ultrafiltration concentrated product and the washing liquid.

2. The method for preparing Quillaja saponin 21 according to claim 1, wherein The volume of ethanol solvent or methanol solvent is mixed with the saponin raw material in a ratio of (15-30) ml:1g.

3. The preparation method of Quillaja saponin 21 according to claim 2, characterized in that, The volume of ethanol solvent or methanol solvent is mixed with the saponin raw material in a ratio of 25 ml:1 g.

4. The method for preparing Quillaja saponin 21 according to claim 2, wherein The insoluble matter is removed by at least one of natural sedimentation, centrifugation or filtration to obtain the pretreated impurity-removed material.

5. The method for preparing Quillaja saponin 21 according to claim 2, wherein After mixing the ethanol solvent or methanol solvent with the saponin raw material, let it stand at 2-8°C for more than 10 hours, and then remove the insoluble matter.

6. The method for preparing Quillaja saponin 21 according to claim 1, wherein The chromatography medium for the first reverse phase purification and the chromatography medium for the second reverse phase purification were SP-100-10-ODS-P.

7. The method for preparing Quillaja saponin 21 according to claim 6, wherein Repeat steps S2 and S3 once.

8. The method for preparing Quillaja saponin 21 according to claim 6, wherein The acidic regulator used in the first reverse purification is formic acid, acetic acid, trifluoroacetic acid or phosphoric acid.

9. The method for preparing Quillaja saponin 21 according to claim 8, wherein The concentration of the acidic regulator is 0.1-0.3%.

10. The method for preparing Quillaja saponin 21 according to claim 8, characterized in that: During the first reverse purification, the pretreated impurity-removed material is diluted with water to an ethanol concentration of 40%±5%.

11. The method for preparing Quillaja saponin 21 according to claim 10, characterized in that: The flow rate of the first reverse purification was 500-600 mL / min.

12. The method for preparing Quillaja saponin 21 according to claim 10, characterized in that: The collecting of high-purity samples in the first reverse purification comprises: combining samples with a purity of ≥70% and impurities after the main peak of ≤28%.

13. The method for preparing Quillaja saponin 21 according to claim 6, characterized in that: The acidic regulator during the second reverse purification is formic acid, acetic acid, trifluoroacetic acid or phosphoric acid.

14. The method for preparing Quillaja saponin 21 according to claim 13, characterized in that: The concentration of the acidity regulator is 0.1-0.3%; During the second reverse purification, the pre-treated impurity-removed material is diluted with water to an acetonitrile concentration of 38% ± 5%; The flow rate of the second reverse purification is 900-960 mL / min; The collecting of high-purity samples in the second reverse purification comprises: combining samples with a purity of ≥98% and impurities after the main peak of ≤1.2%.

15. The method for preparing Quillaja saponin 21 according to claim 14, characterized in that: The collecting of high-purity samples in the second reverse purification comprises: combining samples with a purity of ≥99% and impurities after the main peak of ≤1.2%.

16. The method for preparing Quillaja saponin 21 according to claim 1, characterized in that: During the solvent replacement in step S4, first perform isocratic or gradient solvent replacement with 30% to 40% ± 5% ethanol, and then perform isocratic or gradient elution with 80% ± 5% to 95% ethanol; the eluted product is collected as the solvent replacement solution.

17. The method for preparing Quillaja saponin 21 according to claim 16, characterized in that: First, perform isocratic solvent replacement with 30%~40%±5% ethanol, and then perform isocratic elution with 80%±5%~95% ethanol; the eluted product is collected as the solvent replacement solution.

18. The method for preparing Quillaja saponin 21 according to claim 16, characterized in that: During the solvent replacement, the second reverse purification product was diluted with water to an acetonitrile concentration of 30% ± 5%, and then loaded; During the solvent replacement, the flow rate was set at 900-960 mL / min.

19. The method for preparing Quillaja saponin 21 according to claim 1, characterized in that: The ultrafiltration replacement fluid is selected from at least one of histidine, arginine and lysine.

20. The method for preparing Quillaja saponin 21 according to claim 19, characterized in that: The concentration of the ultrafiltration replacement fluid is 1 mM to 25 mM.

21. The method for preparing Quillaja saponin 21 according to claim 20, characterized in that: The concentration of the ultrafiltration replacement fluid is 5mM~25mM.

22. The method for preparing Quillaja saponin 21 according to claim 20, characterized in that: The ultrafiltration replacement fluid is histidine.

23. The method for preparing Quillaja saponin 21 according to claim 1, characterized in that: The ultrafiltration membrane is an ultrafiltration membrane of 1000-3000Da.

24. The method for preparing Quillaja saponin 21 according to claim 1, characterized in that: The ultrafiltration and replacement process also includes aseptic packaging.

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