A method for purifying ascomycin and its application

Abstract

This invention proposes a purification method for ascomycin and its application. The method includes acidifying and filtering the ascomycin fermentation broth to obtain a bacterial residue; adding organic solvent I to the bacterial residue for extraction to obtain an extract filtrate; sequentially adding a salt solution, an alkaline solution, and an acid solution to the extract filtrate for treatment, and decolorizing the product to obtain a decolorized organic phase; evaporating the decolorized organic phase under reduced pressure, and sequentially treating it with ethyl acetate and n-hexane, followed by cooling and filtration to obtain wet crystals 1, which are then washed with n-hexane to obtain wet crystals 2; drying the wet crystals 2 sequentially, dissolving them in organic solvent II to obtain a pre-crystallization liquid, crystallizing by low-temperature stirring, filtering, and then vacuum drying the solid phase. This method can obtain ascomycin with high purity (not less than 99.2%), a yield higher than 61%, and is simple to operate, easy to scale up for industrial production, and has low production costs, making it highly valuable for application.

Description

Technical Field

[0001] This invention relates to the field of fermentation broth purification technology, specifically to a method for purifying ascomycin and its application. Background Technology

[0002] Ascomycin (FK520) is a macrolide antibiotic produced by the fermentation of *Streptomyces hygroscopicus*. It possesses various biological activities, including strong immunosuppressive activity, and is an ethyl analogue of the immunosuppressant FK-506 (tacrolimus), making it of significant pharmaceutical value. Due to its 23-membered macrolide structure and multiple chiral centers, chemical synthesis of FK520 is difficult and costly; therefore, microbial fermentation is the primary method for its production.

[0003] Most current research on ascomycin focuses on its fermentation, development of derivatives, antibacterial activity, and clinical applications. The main problems in the current isolation and extraction of ascomycin are low yield and high separation costs. Furthermore, due to the isomerism of ascomycin in aqueous phases, current extraction and separation methods often involve an aqueous phase, resulting in the presence of ascomycin structural analogs in the product, affecting purity and morphology. Currently, there are no reports of industrial-scale production of ascomycin in my country, and the market is largely dominated by foreign companies from Japan and the United States. Therefore, there is an urgent need in this field to provide a method for purifying ascomycin that is low-cost, high-yield, simple to operate, and suitable for industrial production. Summary of the Invention

[0004] The present invention aims to solve at least one of the technical problems existing in the prior art to a certain extent.

[0005] Therefore, in a first aspect, the present invention provides a method for purifying ascomycin. According to an embodiment of the present invention, the method includes the following steps:

[0006] (1) Acidify and filter the ascomycin fermentation broth to obtain bacterial residue;

[0007] (2) Add organic solvent I to the bacterial residue for extraction to obtain an extract filtrate;

[0008] (3) Add salt solution to the extraction filtrate for salt washing to obtain a salt-washed organic phase;

[0009] (4) Add an alkaline solution to the salt-washed organic phase for alkaline washing to obtain an alkaline-washed organic phase;

[0010] (5) Add an acid solution to the alkaline-washed organic phase for acid washing to obtain an acid-washed organic phase;

[0011] (6) The acid-washed organic phase is decolorized with activated carbon to obtain the decolorized organic phase;

[0012] (7) The decolorized organic phase is evaporated under reduced pressure and treated with ethyl acetate and n-hexane in sequence, cooled and filtered to obtain wet crystal 1, which is then rinsed with n-hexane to obtain wet crystal 2.

[0013] (8) The wet crystals 2 are dried and dissolved in organic solvent II in sequence to obtain a pre-crystallization liquid. The liquid is then stirred and crystallized at low temperature, filtered, and then vacuum dried in solid phase.

[0014] Step (8) is repeated three times to obtain purified ascomycin.

[0015] The method according to the embodiments of the present invention can obtain ascomycin with high purity (not less than 99.2%), with a yield of more than 61%, and is simple to operate, easy to scale up for industrial production, and has low production cost, thus having high application value.

[0016] According to embodiments of the present invention, the above method may further include at least one of the following additional technical features:

[0017] According to an embodiment of the present invention, the acidification is carried out using an acidification liquid phase, wherein the acidification liquid phase comprises at least one of formic acid, phosphoric acid, and acetic acid.

[0018] According to an embodiment of the present invention, the acidification causes the pH of the ascomycin fermentation broth to be 3.0 to 3.5.

[0019] According to an embodiment of the present invention, a filter aid is used for filtration in step (1) of the filtration process.

[0020] According to an embodiment of the present invention, the filter aid comprises at least one selected from neutral alumina, cellulose, and diatomaceous earth.

[0021] According to some preferred embodiments of the present invention, the filter aid is diatomaceous earth.

[0022] According to an embodiment of the present invention, the organic solvent I comprises at least one selected from methanol and ethyl acetate.

[0023] According to some preferred embodiments of the present invention, the organic solvent I is ethyl acetate.

[0024] According to an embodiment of the present invention, the salt solution comprises an aqueous solution of sodium chloride, sodium sulfate, or potassium chloride.

[0025] According to some preferred embodiments of the present invention, the salt solution is an aqueous solution of sodium chloride.

[0026] According to an embodiment of the present invention, the concentration of the salt solution is 10% (w / v).

[0027] According to an embodiment of the present invention, the volume ratio of the salt solution to the organic solvent I is 1:1.

[0028] According to an embodiment of the present invention, the alkaline solution comprises an aqueous solution of sodium hydroxide, sodium bicarbonate, or ammonia.

[0029] According to an embodiment of the present invention, the concentration of the alkaline solution is 1 mol / L.

[0030] According to an embodiment of the present invention, the volume ratio of the alkaline solution to the organic solvent I is 1:1.

[0031] According to an embodiment of the present invention, the acid solution comprises an aqueous solution of formic acid, phosphoric acid, or acetic acid.

[0032] According to an embodiment of the present invention, the pH of the acid solution is 3.0 to 3.5.

[0033] According to an embodiment of the present invention, the volume ratio of the acid solution to organic solvent I is 1:1.

[0034] According to an embodiment of the present invention, the decolorization is carried out using a decolorizing agent, which includes at least one of pharmaceutical activated carbon, 7440-44-0 activated carbon, and HC-303 activated carbon.

[0035] According to an embodiment of the present invention, the ratio of the decolorizing agent to the total amount is 1:1.

[0036] According to an embodiment of the present invention, the volume ratio of ethyl acetate to n-hexane in step (7) is 1:40 to 1:50.

[0037] According to an embodiment of the present invention, the cooling gradient is as follows: stirring and holding at 30°C for 1 hour, cooling down to 15°C to 20°C, stirring and holding for 2 hours.

[0038] According to an embodiment of the present invention, the organic solvent II includes at least one selected from acetone and diethyl ether.

[0039] According to an embodiment of the present invention, the organic solvent II is acetone.

[0040] According to an embodiment of the present invention, the cooling gradient is: 20℃ for 2 hours of holding and stirring, 10℃ for 2 hours of holding and stirring, 5℃ for 1 hour of holding and stirring, and -5℃ for 2 hours of holding and stirring.

[0041] According to an embodiment of the present invention, after dissolution by organic solvent II, the potency of ascomycin in the pre-crystallization solution is not less than 200,000 μg / ml.

[0042] In a second aspect, the present invention provides a purified ascomycin. According to an embodiment of the present invention, the ascomycin is obtained by the method described in the first aspect. The purified ascomycin according to the present invention has a high purity, reaching over 99.2%, reducing safety risks during subsequent use.

[0043] Additional aspects and advantages of the invention will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by practice of the invention. Attached Figure Description

[0044] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings used in the embodiments will be briefly introduced below. It should be understood that the following drawings only show some embodiments of the present invention and should not be regarded as a limitation on the scope. For those skilled in the art, other related drawings can be obtained based on these drawings without creative effort.

[0045] Figure 1 This is a chromatogram of the fermentation broth according to Example 1 of the present invention;

[0046] Figure 2 The image shows the finished product of 1-asomycin according to an embodiment of the present invention. Detailed Implementation

[0047] Embodiments of the present invention are described in detail below, examples of which are illustrated in the accompanying drawings. The embodiments described below with reference to the accompanying drawings are exemplary and intended to explain the present invention, and should not be construed as limiting the present invention.

[0048] Furthermore, the terms "first" and "second" are used for descriptive purposes only and should not be construed as indicating or implying relative importance or implicitly specifying the number of indicated technical features. Thus, a feature defined as "first" or "second" may explicitly or implicitly include at least one of that feature. In the description of this invention, "multiple" or "a plurality of" means at least two, two types, such as two, two, three, three, etc., unless otherwise explicitly specified.

[0049] In this document, the terms “comprising,” “having,” or “including” are open-ended expressions, meaning they include the contents specified in this invention but do not exclude other aspects.

[0050] In this document, the term “optionally” generally means that an event or condition described below may, but may not, occur, and the description includes both cases in which the event or condition occurs and cases in which the event or condition does not occur.

[0051] In this article, "total billion" is an industry term for material quality, and its unit can be mg, g, or kg.

[0052] The endpoints and any values ​​of the ranges disclosed herein are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of the various ranges, the endpoint values ​​of the various ranges and individual point values, and individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed herein.

[0053] To facilitate understanding of this invention, certain technical and scientific terms are specifically defined herein. Unless otherwise expressly defined elsewhere in this document, all other technical and scientific terms used herein have the meanings commonly understood by one of ordinary skill in the art to which this invention pertains.

[0054] The method for purifying ascomycin provided by this invention includes the following steps:

[0055] (1) Separate the solid and liquid components of the ascomycin fermentation broth, acidify it, add a filter aid, filter and collect the bacterial residue;

[0056] (2) The filter residue was extracted with organic solvent I, and the filtrate was collected by filtration.

[0057] (3) Wash the filtrate obtained in step (2) with brine and collect the organic phase after brine washing;

[0058] (4) Wash the organic phase obtained from the brine in step (3) with alkali and collect the organic phase after alkali washing;

[0059] (5) The organic phase obtained after alkali washing in step (4) is acid washed and the acid-washed organic phase is collected.

[0060] (6) Decolorize the acid-washed organic phase obtained in step (5) with activated carbon, and filter and collect the decolorized organic phase;

[0061] (7) The decolorized organic phase obtained in step (6) is evaporated to dryness under reduced pressure, dissolved in ethyl acetate to obtain the pre-crystallization liquid, n-hexane is slowly added dropwise, the mixture is cooled and filtered, and the wet crystals are washed with n-hexane to obtain wet crystals.

[0062] (8) The wet crystals obtained in step (7) are dried under vacuum, dissolved in organic solvent II to obtain a liquid before crystallization, stirred at low temperature to crystallize, filtered and then dried under vacuum in solid phase. This crystallization step is repeated three times to obtain the finished product of ascomycin.

[0063] In step (1), formic acid is used to acidify the liquid phase and adjust the pH to 3.0-3.5;

[0064] The filter aid mentioned in step (1) is selected from neutral alumina, cellulose and diatomaceous earth, with diatomaceous earth being preferred;

[0065] The organic solvent I mentioned in step (2) is selected from ethyl acetate, methanol, and preferably ethyl acetate.

[0066] The brine mentioned in step (3) is an aqueous solution of sodium chloride with a concentration of 10% (w / v) and a volume ratio of 1:1 with organic solvent I.

[0067] The alkaline solution mentioned in step (4) is an aqueous solution of sodium hydroxide with a concentration of 1 mol / L and a volume ratio of 1:1 with organic solvent I.

[0068] The acid water mentioned in step (5) is an aqueous solution of formic acid with a pH of 3.0 to 3.5 and a volume ratio of 1:1 with organic solvent I.

[0069] The decolorizing agent mentioned in step (6) is pharmaceutical activated carbon, with a ratio of 1:1 to the total amount, and a 0.22μm organic membrane is used for filtration;

[0070] In step (7), the volume ratio of ethyl acetate to n-hexane is 1:40-50, the temperature gradient is 30℃, and the mixture is stirred and kept warm for 1 hour, then cooled to 15-20℃ and stirred and kept warm for 2 hours.

[0071] The organic solvent II mentioned in step (8) is selected from acetone, diethyl ether, preferably acetone. The cooling gradient is 20℃ for 2 hours of stirring, 10℃ for 2 hours of stirring, 5℃ for 1 hour of stirring, and -5℃ for 2 hours of stirring.

[0072] The advantages of the purification method of ascomycin provided by the present invention are mainly reflected in: (1) less organic solvent is used in the purification process of ascomycin, and the safety and environmental protection of the production process are greatly improved; (2) the purification process is simple and controllable, with low energy consumption and short cycle, which is conducive to industrial expansion of production; (3) the purified ascomycin has a purity of over 99%, a content of over 98%, and a yield of over 60%.

[0073] The ascomycin fermentation broth in this embodiment of the invention is the fermentation broth of *Streptomyces hygroscopicus*, which was deposited at the China General Microbiological Culture Collection Center (CGMCC) on July 4, 2024, with accession number CGMCC NO. 31169. The high-yielding strain (CGMCC NO. 31169) was inoculated onto test tube slant agar and cultured at 28°C and 40% relative humidity for 9 days. Mature spores were collected into 20 ml of sterile water, thoroughly dispersed, and then inoculated at a ratio of 2% into liquid seed bottles. These bottles were then cultured at 28°C and 250 rpm on a shaker for 40 hours. After the seed culture matured, it was inoculated at a ratio of 10% into three 2.8L three-baffle liquid fermentation bottles, all of which were cultured at 28°C and 250 rpm on a shaker for 7 days. The specific composition of the liquid seed culture medium was: starch 1%, glucose 4%, yeast extract 4%, ammonium sulfate 0.3%, magnesium sulfate 0.3%, and calcium carbonate 0.1%. The specific composition of the liquid fermentation medium is as follows: starch 15%, glucose 5%, glycerol 3%, yeast extract 4%, yeast peptone 2%, ammonium sulfate 0.3%, magnesium sulfate 0.3%, zinc sulfate 0.05%, sodium chloride 0.2%, calcium carbonate 0.3%, and defoamer 0.12%.

[0074] The present invention will be explained below with reference to embodiments. Those skilled in the art will understand that the following embodiments are for illustrative purposes only and should not be considered as limiting the scope of the invention. Where specific techniques or conditions are not specified in the embodiments, they are performed according to the techniques or conditions described in the literature in the field or according to the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be obtained commercially. Example 1

[0075] Step (1): Add 5% (w / v) diatomaceous earth filter aid to 35L of ascomycin fermentation broth, stir for 1-2 hours, adjust the pH to 3-3.5 with formic acid, filter through a plate and frame filter to obtain the bacterial residue, and dry the bacterial residue at 50℃-60℃.

[0076] Step (2): Cool the bacterial residue to room temperature, add 3 times the volume of ethyl ester to the dry filter residue at room temperature and extract twice. Stir for 2-4 hours during the extraction process, filter, and extract twice to obtain the first and second extraction filtrates.

[0077] Step (3): Combine the primary and secondary extraction filtrates, prepare a 10% (w / v) sodium chloride aqueous solution of equal volume to the combined filtrates, stir thoroughly to dissolve, add to the extraction filtrate, stir for 1-2 hours, discard the aqueous phase after separation, and obtain the upper ethyl ester phase.

[0078] Step (4): Prepare a 0.1M sodium hydroxide solution of the same volume as the ethyl ester phase obtained in step (3). Add 10% (w / v) sodium chloride to the aqueous phase, stir to dissolve, and then pour into the ethyl ester phase. Stir for 30 min and let stand to separate the layers. After separation, discard the aqueous phase to obtain the upper ethyl ester phase.

[0079] Step (5): Prepare a formic acid solution with a pH of 3 to 3.5 of the same volume as the ethyl ester phase obtained in step (4), pour it into the ethyl ester phase, stir for about 30 minutes and let it stand to separate into layers, test the aqueous phase and the ethyl ester phase, discard the aqueous phase after separation, and obtain the ethyl ester phase.

[0080] Step (6): Add 2 times the amount of pharmaceutical-grade activated carbon to the ethyl ester phase obtained in step (5), stir for 30 minutes, add 5% (w / v) anhydrous magnesium sulfate, filter, and then rinse with 2 times the amount of ethyl acetate to obtain the filtrate.

[0081] Step (7): Concentrate the filtrate at 45°C, add 5-7 times the total amount of ethyl acetate to the filtrate, and then slowly add 40-50 times the amount of n-hexane. Stir and keep warm at 30°C for 1 hour, cool down to 15°C-20°C, stir and keep warm for 2 hours, filter, and wash the obtained wet crystals with n-hexane to obtain wet crystals.

[0082] Step (8): Dry the wet crystals obtained in step (7) to constant weight at 45℃ and -0.08MPa, and dissolve them in acetone until the potency is not less than 200,000 μg / ml. Set the cooling crystallization program: keep stirring at 20℃ for 2h, keep stirring at 10℃ for 2h, keep stirring at 5℃ for 1h, keep stirring at -5℃ for 2h, filter while cold, and wash with cold n-hexane to obtain wet crystals.

[0083] Step (9): Dry the wet crystals obtained in step (8) to constant weight at 45℃ and -0.08MPa, and dissolve them in acetone until the potency is not less than 200,000 μg / ml. Set the cooling crystallization program: keep stirring at 20℃ for 2 hours, keep stirring at 10℃ for 2 hours, keep stirring at 5℃ for 1 hour, keep stirring at -5℃ for 2 hours, filter while cold, and wash with cold n-hexane to obtain wet crystals.

[0084] Step (10): Dry the wet crystals obtained in step (9) to constant weight at 45℃ and -0.08MPa, and dissolve them in acetone until the potency is not less than 200,000 μg / ml. Set the cooling crystallization program: keep stirring at 20℃ for 2h, keep stirring at 10℃ for 2h, keep stirring at 5℃ for 1h, keep stirring at -5℃ for 2h, filter while cold, and wash with cold n-hexane to obtain wet crystals.

[0085] Step (11): HPLC was used for detection. The wet crystals obtained in step (10) were dried to constant weight at 45℃ and -0.08MPa to obtain 14.63g of finished product with a purity of 99.5%, a content of 98.7%, and less than 0.5% of impurity FK-523 (demethylascomycin). The yield was 66.35%.

[0086] Example 2

[0087] Step (1): Add 5% (w / v) diatomaceous earth filter aid to 34L of fermentation broth, stir for 1-2 hours, adjust the pH to 3-3.5 with formic acid, filter through a plate and frame filter to obtain the bacterial residue, and dry the bacterial residue at 50℃-60℃.

[0088] Step (2): Add 3 times the volume of methanol to the dry filter residue at room temperature and extract twice. Stir for 2-4 hours during the extraction process, filter, and obtain the first and second extraction filtrates.

[0089] Step (3): Combine the primary and secondary extraction filtrates, concentrate the solvent at 45℃ and -0.08MPa, and drip the concentrated liquid dropwise. Add an equal volume of ethyl acetate to the concentrated product for extraction, stir for 1-2 hours, discard the aqueous phase after separation, and obtain the upper ethyl acetate phase.

[0090] Step (4): Prepare a 0.1M sodium hydroxide solution of the same volume as the ethyl ester phase obtained in step (3). Add 10% (w / v) sodium chloride to the aqueous phase, stir to dissolve, and then pour into the ethyl ester phase. Stir for 30 min and let stand to separate the layers. After separation, discard the aqueous phase to obtain the upper ethyl ester phase.

[0091] Step (5): Prepare a formic acid solution with a pH of 3-3.5 of the same volume as the ethyl ester phase obtained in step (4), pour it into the ethyl ester phase, stir for about 30 minutes and let it stand to separate into layers, detect the aqueous phase and the ethyl ester phase, discard the aqueous phase after separation, and obtain the ethyl ester phase.

[0092] Step (6): Add twice the amount of pharmaceutical-grade activated carbon to the ethyl ester phase obtained in step (5). After stirring for 30 minutes, add approximately 5% (w / v) of anhydrous magnesium sulfate, filter, and then rinse with twice the amount of ethyl acetate to obtain the filtrate.

[0093] Step (7): Concentrate the filtrate at 45°C, add 5-7 times the total amount of ethyl acetate, then slowly add 40-50 times the amount of n-hexane, stir and keep warm at 30°C for 1 hour, cool down to 15-20°C, stir and keep warm for 2 hours, filter, and wash the wet crystals with n-hexane to obtain wet crystals.

[0094] Step (8): Dry the wet crystals obtained in step (7) to constant weight at 45℃ and -0.08MPa, and dissolve them in acetone until the potency is not less than 200,000 μg / ml. Set the cooling crystallization program: keep stirring at 20℃ for 2h, keep stirring at 10℃ for 2h, keep stirring at 5℃ for 1h, keep stirring at -5℃ for 2h, filter while cold, and wash with cold n-hexane to obtain wet crystals.

[0095] Step (9): Dry the wet crystals obtained in step (8) to constant weight at 45℃ and -0.08MPa, and dissolve them in acetone until the potency is not less than 200,000 μg / ml. Set the cooling crystallization program: keep stirring at 20℃ for 2 hours, keep stirring at 10℃ for 2 hours, keep stirring at 5℃ for 1 hour, keep stirring at -5℃ for 2 hours, filter while cold, and wash with cold n-hexane to obtain wet crystals.

[0096] Step (10): Dry the wet crystals obtained in step (9) to constant weight at 45℃ and -0.08MPa, and dissolve them in acetone until the potency is not less than 200,000 μg / ml. Set the cooling crystallization program: keep at 20℃ and stir for 2h, keep at 10℃ and stir for 2h, keep at 5℃ and stir for 1h, keep at -5℃ and stir for 2h, filter while cold, and wash with cold n-hexane to obtain wet crystals.

[0097] Step (11): Dry the wet crystals obtained in step (10) at 45℃ and -0.08MPa to constant weight to obtain the finished product. The finished product is 13.85g, with a purity of 99.3%, a content of 99.2%, and an impurity of less than 0.5% FK-523 (demethylascomycin). The yield is 63.16%.

[0098] Example 3:

[0099] Step (1): Add 5% (w / v) diatomaceous earth filter aid to 32L of fermentation broth, stir for 1-2 hours, adjust the pH to 3-3.5 with formic acid, filter through a plate and frame filter to obtain the bacterial residue, and dry the bacterial residue at 50℃-60℃.

[0100] Step (2): Add 3 times the volume of ethyl ester to the dry filter residue at room temperature and extract twice. Stir for 2-4 hours during the extraction process, filter, and obtain the first and second extraction filtrates.

[0101] Step (3): Combine the primary and secondary extraction filtrates, concentrate the solvent at 45℃ and -0.08MPa, and drip the concentrated liquid droplet; add an equal volume of ethyl acetate to the concentrated product for extraction, stir for 1-2 hours, discard the aqueous phase after separation, and obtain the upper ethyl ester phase.

[0102] Step (4): Prepare a 0.1M sodium hydroxide solution of the same volume as the ethyl ester phase obtained in step (3). Add 10% (w / v) sodium chloride to the aqueous phase, stir thoroughly to dissolve, and then pour it into the ethyl ester phase. Stir for 30 minutes and let it stand to separate into layers. After separation, discard the aqueous phase to obtain the upper ethyl ester phase.

[0103] Step (5): Prepare an equal volume of formic acid solution with pH 3 to 3.5, pour it into the ethyl ester phase, stir for about 30 minutes and let it stand to separate into layers. Detect the aqueous phase and the ethyl ester phase. After separation, discard the aqueous phase to obtain the ethyl ester phase.

[0104] Step (6): Add twice the amount of pharmaceutical-grade activated carbon to the ethyl ester phase obtained in step (5). After stirring for 30 minutes, add approximately 5% (w / v) of anhydrous magnesium sulfate, filter, and then rinse with twice the amount of ethyl acetate to obtain the filtrate.

[0105] Step (7): Concentrate the filtrate at 45°C, add 5-7 times the total amount of ethyl acetate of the concentrated product, and then slowly add 40-50 times the amount of n-hexane. Stir and keep warm at 30°C for 1 hour, cool down to 15°C-20°C, stir and keep warm for 2 hours, filter, and wash the wet crystals with n-hexane to obtain wet crystals.

[0106] Step (8): Dry the wet crystals obtained in step (7) to constant weight at 45℃ and -0.08MPa, and dissolve them in diethyl ether until the potency is not less than 200,000 μg / ml. Set the cooling crystallization program: keep at 20℃ and stir for 2h, keep at 10℃ and stir for 2h, keep at 5℃ and stir for 1h, keep at -5℃ and stir for 2h, filter while cold, and wash with cold n-hexane to obtain wet crystals.

[0107] Step (9): Dry the wet crystals obtained in step (8) to constant weight at 45℃ and -0.08MPa, and dissolve them in diethyl ether until the potency is not less than 200,000 μg / ml. Set the cooling crystallization program: keep stirring at 20℃ for 2h, keep stirring at 10℃ for 2h, keep stirring at 5℃ for 1h, keep stirring at -5℃ for 2h, filter while cold, and wash with cold n-hexane to obtain wet crystals.

[0108] Step (10): Dry the wet crystals obtained in step (9) to constant weight at 45℃ and -0.08MPa, and dissolve them in diethyl ether until the potency is not less than 200,000 μg / ml. Set the cooling crystallization program: keep stirring at 20℃ for 2h, keep stirring at 10℃ for 2h, keep stirring at 5℃ for 1h, keep stirring at -5℃ for 2h, filter while cold, and wash with cold n-hexane to obtain wet crystals.

[0109] Step (11): Dry the wet crystals obtained in step (10) at 45℃ and -0.08MPa to constant weight to obtain 10.83g of finished product with a purity of 98.3%, a content of 99.2%, and less than 0.5% of impurity FK-523 (demethylascomycin), with a yield of 61.3%.

[0110] Example 4:

[0111] Step (1): Add 5% (w / v) diatomaceous earth filter aid to 36L of fermentation broth, stir for 1-2 hours, adjust the pH to 3-3.5 with formic acid, filter through a plate and frame filter to obtain the bacterial residue, and dry the bacterial residue at 50℃-60℃.

[0112] Step (2): Add 3 times the volume of ethyl ester to the dry filter residue at room temperature and extract twice, stirring for 2-4 hours, then filter to obtain the first and second extraction filtrates.

[0113] Step (3): Combine the primary and secondary extraction filtrates, concentrate the solvent at 45℃ and -0.08MPa, and drip the concentrated liquid droplet; add an equal volume of ethyl acetate to the concentrated product for extraction, stir for 1-2 hours, discard the aqueous phase after separation, and obtain the upper ethyl ester phase.

[0114] Step (4): Prepare a 0.1M sodium hydroxide solution of the same volume as the ethyl ester phase obtained in step (3). Add 10% (w / v) sodium chloride to the aqueous phase, stir to dissolve, and then pour into the ethyl ester phase. Stir for 30 min and let stand to separate the layers. After separation, discard the aqueous phase to obtain the upper ethyl ester phase.

[0115] Step (5): Prepare a formic acid solution with a pH of 3 to 3.5 of the same volume as the ethyl ester phase obtained in step (4), pour it into the ethyl ester phase, stir for about 30 minutes and let it stand to separate into layers, detect the aqueous phase and the ethyl ester phase, discard the aqueous phase after separation, and obtain the ethyl ester phase.

[0116] Step (6): Add twice the amount of pharmaceutical-grade activated carbon to the ethyl ester phase obtained in step (5). After stirring for 30 minutes, add approximately 5% (w / v) of anhydrous magnesium sulfate, filter, and then rinse with twice the amount of ethyl acetate to obtain the filtrate.

[0117] Step (7): Concentrate the filtrate at 45°C, add 5-7 times the total amount of ethyl acetate, then slowly add 40-50 times the amount of n-hexane, stir and keep warm at 30°C for 1 hour, cool down to 15-20°C, stir and keep warm for 2 hours, filter, and wash the wet crystals with n-hexane to obtain wet crystals.

[0118] Step (8): Dry the wet crystals obtained in step (7) to constant weight at 45℃ and -0.08MPa, and dissolve them in diethyl ether until the potency is not less than 300,000 μg / ml. Set the cooling crystallization program: keep at 20℃ and stir for 2h, keep at 10℃ and stir for 2h, keep at 5℃ and stir for 1h, keep at -5℃ and stir for 2h, filter while cold, and wash with cold n-hexane to obtain wet crystals.

[0119] Step (9): Dry the wet crystals obtained in step (8) to constant weight at 45℃ and -0.08MPa, and dissolve them in diethyl ether until the potency is not less than 300,000 μg / ml. Set the cooling crystallization program: keep at 20℃ and stir for 2h, keep at 10℃ and stir for 2h, keep at 5℃ and stir for 1h, keep at -5℃ and stir for 2h, filter while cold, and wash with cold n-hexane to obtain wet crystals.

[0120] Step (10): Dry the wet crystals obtained in step (9) to constant weight at 45℃ and -0.08MPa, and dissolve them in diethyl ether until the potency is not less than 300,000 μg / ml. Set the cooling crystallization program: keep stirring at 20℃ for 2h, keep stirring at 10℃ for 2h, keep stirring at 5℃ for 1h, keep stirring at -5℃ for 2h, filter while cold, and wash with cold n-hexane to obtain wet crystals.

[0121] Step (11): Dry the wet crystals obtained in step (10) at 45℃ and -0.08MPa to constant weight to obtain 11.17g of finished product with a purity of 99.8%, a content of 99.4%, impurity FK-523 (demethylascomycin) less than 0.5%, and a yield of 64.97%.

[0122] Comparative Example 1:

[0123] Step (1): Add 5% (w / v) diatomaceous earth filter aid to 33L of fermentation broth, stir for 1-2 hours, adjust the pH to 3-3.5 with formic acid, filter through plate and frame filter to obtain bacterial residue, and dry the bacterial residue at 50℃-60℃.

[0124] Step (2): Add 3 times the volume of ethyl ester to the dry filter residue at room temperature and extract twice, stirring for 2-4 hours, then filter to obtain the first and second extraction filtrates.

[0125] Step (3): Combine the primary and secondary extraction filtrates, concentrate the solvent at 45℃ and -0.08MPa, and drip the concentrated liquid droplet; add an equal volume of ethyl acetate to the concentrated product for extraction, stirring for 1-2 hours during the extraction process, discard the aqueous phase after separation, and obtain the upper ethyl acetate phase.

[0126] Step (4): Prepare a 0.1M sodium hydroxide solution of the same volume as the ethyl ester phase obtained in step (3). Add 10% (w / v) sodium chloride to the aqueous phase, stir to dissolve, and then pour into the ethyl ester phase. Stir for 30 min and let stand to separate the layers. After separation, discard the aqueous phase to obtain the upper ethyl ester phase.

[0127] Step (5): Prepare a formic acid solution with a pH of 3 to 3.5 of the same volume as the ethyl ester phase obtained in step (4), pour it into the ethyl ester phase, stir for about 30 minutes and let it stand to separate into layers, detect the aqueous phase and the ethyl ester phase, discard the aqueous phase after separation, and obtain the ethyl ester phase.

[0128] Step (6): Add 2 times the amount of pharmaceutical-grade activated carbon to the ethyl ester phase obtained in step (5); stir for 30 minutes, add about 5% (w / v) of anhydrous magnesium sulfate, filter, and then rinse with 2 times the amount of ethyl acetate to obtain the filtrate.

[0129] Step (7): Concentrate the filtrate at 45°C, add 5-7 times the total amount of ethyl acetate, then slowly add 40-50 times the amount of n-hexane, stir and keep warm at 30°C for 1 hour, cool down to 15°C~20°C, stir and keep warm for 2 hours, filter, and wash the wet crystals with n-hexane to obtain wet crystals.

[0130] Step (8): Dry the wet crystals obtained in step (7) to constant weight at 45℃ and -0.08MPa, and dissolve them in acetone until the potency is not less than 200,000 μg / ml. Set the cooling crystallization program: keep stirring at 20℃ for 2h, keep stirring at 10℃ for 2h, keep stirring at 5℃ for 1h, keep stirring at -5℃ for 2h, filter while cold, and wash with cold n-hexane to obtain wet crystals.

[0131] Step (9): Dry the wet crystals obtained in step (8) to constant weight at 45℃ and -0.08MPa, and dissolve them in acetone until the potency is not less than 200,000 μg / ml. Set the cooling crystallization program: keep stirring at 20℃ for 2 hours, keep stirring at 10℃ for 2 hours, keep stirring at 5℃ for 1 hour, keep stirring at -5℃ for 2 hours, filter while cold, and wash with cold n-hexane to obtain wet crystals.

[0132] Step (10): Dry the wet crystals obtained in step (9) at 45℃ and -0.08MPa to constant weight to obtain 15.4g of finished product with a purity of 93.6%, a content of 92.7%, and an impurity of more than 1% FK-523 (demethylascomycin) and a yield of 67.9%.

[0133] Comparative Example 1 illustrates that after the wet crystals were dried to constant weight, the crystallization was carried out less than three times with acetone, resulting in impurity FK-523 (demethylascomycin) exceeding 1%.

[0134] Comparative Example 2:

[0135] Step (1): Add 5% (w / v) diatomaceous earth filter aid to 35L of fermentation broth, stir for 1-2 hours, adjust the pH to 3-3.5 with formic acid, filter through a plate and frame filter to obtain the bacterial residue, and dry the bacterial residue at 50-60℃.

[0136] Step (2): Add 3 times the volume of ethyl ester to the dry filter residue at room temperature and extract twice, stirring for 2-4 hours, then filter to obtain the first and second extraction filtrates.

[0137] Step (3): Combine the primary and secondary extraction filtrates, concentrate the solvent at 45℃ and -0.08MPa, and drip the concentrated liquid droplets. Add an equal volume of ethyl acetate for extraction, stir for 1-2 hours, discard the aqueous phase after separation, and obtain the upper ethyl ester phase.

[0138] Step (4): Prepare an equal volume of 0.1M sodium hydroxide solution, add 10% (w / v) sodium chloride to the aqueous phase, stir thoroughly to dissolve, pour into the ethyl ester phase, stir for 30 min and let stand to separate the layers, discard the aqueous phase after separation, and obtain the upper ethyl ester phase.

[0139] Step (5): Prepare an equal volume of formic acid solution with pH 3 to 3.5, pour it into the ethyl ester phase, stir for about 30 minutes and let it stand to separate into layers. Detect the aqueous phase and the ethyl ester phase. After separation, discard the aqueous phase to obtain the ethyl ester phase.

[0140] Step (6): Add twice the amount of pharmaceutical-grade activated carbon to the ethyl ester phase. After stirring for 30 minutes, add approximately 5% (w / v) of anhydrous magnesium sulfate, filter, and then rinse with twice the amount of ethyl acetate to obtain the filtrate.

[0141] Step (7): Concentrate the filtrate at 45°C, add 5-7 times the total amount of ethyl acetate, then slowly add 40-50 times the amount of n-hexane, stir and keep warm at 30°C for 1 hour, cool down to 15-20°C, stir and keep warm for 2 hours, filter, and wash the wet crystals with n-hexane to obtain wet crystals.

[0142] Step (8): Dry the wet crystals obtained in step (7) to constant weight at 45℃ and -0.08MPa, and dissolve them in acetone until the potency is not less than 200,000 μg / ml. Set the cooling crystallization program: keep at -10℃ and stir for more than 6 hours, filter while cold, and wash with cold n-hexane to obtain wet crystals.

[0143] Step (9): Dry the wet crystals obtained in step (8) to constant weight at 45℃ and -0.08MPa, and dissolve them in acetone until the potency is not less than 200,000 μg / ml. Set the cooling crystallization program: keep at -10℃ and stir for more than 6 hours, filter while cold, and wash with cold n-hexane to obtain wet crystals.

[0144] Step (10): Dry the wet crystals obtained in step (9) to constant weight at 45℃ and -0.08MPa, and dissolve them in acetone until the potency is not less than 200,000 μg / ml. Set the cooling crystallization program: keep at -10℃ and stir for more than 6 hours, filter while cold, and wash with cold n-hexane to obtain wet crystals.

[0145] Step (11): Dry the wet crystals obtained in step (10) at 45℃ and -0.08MPa to constant weight to obtain 11.64g of finished product with a purity of 92.5%, a content of 93.7%, an impurity of more than 5% FK-523 (demethylascomycin), and a yield of 67.9%.

[0146] Comparative Example 2 illustrates that the cooling rate was set too fast during the acetone crystallization process, and the impurity FK-523 (demethylascomycin) was not effectively removed.

[0147] Comparative Example 3:

[0148] Step (1): Add 5% (w / v) diatomaceous earth filter aid to 37L of fermentation broth, stir for 1-2 hours, adjust the pH to 3-3.5 with formic acid, filter through plate and frame filter to obtain bacterial residue, and dry the bacterial residue at 50℃-60℃.

[0149] Step (2): Add 3 times the volume of methanol to the dry filter residue at room temperature and extract twice, stirring for 2-4 hours, then filter to obtain the first and second extraction filtrates.

[0150] Step (3): Combine the primary and secondary extraction filtrates, concentrate the solvent at 45℃ and -0.08MPa, and drip the concentrated liquid droplets. Add an equal volume of ethyl acetate for extraction, stir for 1-2 hours, discard the aqueous phase after separation, and obtain the upper ethyl ester phase.

[0151] Step (4): Prepare a 0.1M sodium hydroxide solution of the same volume as the ethyl ester phase obtained in step (3). Add 10% (w / v) sodium chloride to the aqueous phase, stir to dissolve, and then pour into the ethyl ester phase. Stir for 30 min and let stand to separate the layers. After separation, discard the aqueous phase to obtain the upper ethyl ester phase.

[0152] Step (5): Prepare a formic acid solution with a pH of 3 to 3.5 of the same volume as the ethyl ester phase obtained in step (4), pour it into the ethyl ester phase, stir for about 30 minutes and let it stand to separate into layers, detect the aqueous phase and the ethyl ester phase, discard the aqueous phase after separation, and obtain the ethyl ester phase.

[0153] Step (6): Add 2 times the amount of pharmaceutical-grade activated carbon to the ethyl acetate phase. After stirring for 30 minutes, add 5% (w / v) anhydrous magnesium sulfate, filter, and then rinse with 2 times the amount of ethyl acetate to obtain the filtrate.

[0154] Step (7): Concentrate the filtrate at 45°C, add 5-7 times the total amount of ethyl acetate, then slowly add 40-50 times the amount of n-hexane, stir and keep warm at 30°C for 1 hour, cool down to 15°C-20°C, stir and keep warm for 2 hours, filter, and wash the wet crystals with n-hexane to obtain wet crystals.

[0155] Step (8): Dry the wet crystals obtained in step (7) to constant weight at 45℃ and -0.08MPa, and dissolve them in acetone until the potency is not higher than 100,000 μg / ml. Set the cooling crystallization program: keep stirring at 20℃ for 2h, keep stirring at 10℃ for 2h, keep stirring at 5℃ for 1h, keep stirring at -5℃ for 2h, filter while cold, and wash with cold n-hexane to obtain wet crystals.

[0156] Step (9): Dry the wet crystals obtained in step (8) to constant weight at 45℃ and -0.08MPa, and dissolve them in acetone until the potency is not higher than 100,000 μg / ml. Set the cooling crystallization program: keep stirring at 20℃ for 2h, keep stirring at 10℃ for 2h, keep stirring at 5℃ for 1h, keep stirring at -5℃ for 2h, filter while cold, and wash with cold n-hexane to obtain wet crystals.

[0157] Step (10): Dry the wet crystals obtained in step (9) to constant weight at 45℃ and -0.08MPa, and dissolve them in acetone until the potency is not higher than 100,000 μg / ml. Set the cooling crystallization program: keep stirring at 20℃ for 2h, keep stirring at 10℃ for 2h, keep stirring at 5℃ for 1h, keep stirring at -5℃ for 2h, filter while cold, and wash with cold n-hexane to obtain wet crystals.

[0158] Step (11): Dry the wet crystals obtained in step (10) at 45℃ and -0.08MPa to constant weight to obtain the finished product. The finished product weighs 5.9g, with a purity of 92.3% and a content of 89.2%. The impurity FK-523 (demethylascomycin) is greater than 5%, and the yield is 29.26%.

[0159] Comparative Example 3 illustrates that the acetone crystallization potency was too low, the impurity FK-523 (demethylascomycin) was not effectively removed, and the yield was also low.

[0160] Comparative Example 4:

[0161] Step (1): Add 5% (w / v) diatomaceous earth filter aid to 33L of fermentation broth, stir for 1-2 hours, adjust the pH to 3-3.5 with formic acid, filter through plate and frame filter to obtain bacterial residue, and dry the bacterial residue at 50℃-60℃.

[0162] Step (2): Add 3 times the volume of ethyl ester to the dry filter residue at room temperature and extract twice, stirring for 2-4 hours, then filter to obtain the first and second extraction filtrates.

[0163] Step (3): Combine the primary and secondary extraction filtrates, concentrate the solvent at 45℃ and -0.08MPa, and drip the concentrated liquid droplets. Add an equal volume of ethyl acetate for extraction, stir for 1-2 hours, discard the aqueous phase after separation, and obtain the upper ethyl ester phase.

[0164] Step (4): Prepare an equal volume of 0.1M sodium hydroxide solution, add 10% (w / v) sodium chloride to the aqueous phase, stir thoroughly to dissolve, pour into the ethyl ester phase, stir for 30 min and let stand to separate the layers, discard the aqueous phase after separation, and obtain the upper ethyl ester phase.

[0165] Step (5): Prepare an equal volume of formic acid solution with pH 3 to 3.5, pour it into the ethyl ester phase, stir for about 30 minutes and let it stand to separate into layers. Detect the aqueous phase and the ethyl ester phase. After separation, discard the aqueous phase to obtain the ethyl ester phase.

[0166] Step (6): Add 5% (w / v) anhydrous magnesium sulfate to the ethyl ester phase, filter, and then rinse with 2 times the total amount of ethyl acetate to obtain the filtrate.

[0167] Step (7): Concentrate the filtrate at 45°C, add 5-7 times the total amount of ethyl acetate, then slowly add 40-50 times the amount of n-hexane, stir and keep warm at 30°C for 1 hour, cool down to 15°C-20°C, stir and keep warm for 2 hours, filter, and wash the wet crystals with n-hexane to obtain wet crystals.

[0168] Step (8): Dry the wet crystals obtained in step (7) to constant weight at 45℃ and -0.08MPa, and dissolve them in acetone until the potency is not less than 200,000 μg / ml. Set the cooling crystallization program: keep stirring at 20℃ for 2h, keep stirring at 10℃ for 2h, keep stirring at 5℃ for 1h, keep stirring at -5℃ for 2h, filter while cold, and wash with cold n-hexane to obtain wet crystals.

[0169] Step (9): Dry the wet crystals obtained in step (8) to constant weight at 45℃ and -0.08MPa, and dissolve them in acetone until the potency is not less than 200,000 μg / ml. Set the cooling crystallization program: keep stirring at 20℃ for 2 hours, keep stirring at 10℃ for 2 hours, keep stirring at 5℃ for 1 hour, keep stirring at -5℃ for 2 hours, filter while cold, and wash with cold n-hexane to obtain wet crystals.

[0170] Step (10): Dry the wet crystals obtained in step (9) to constant weight at 45℃ and -0.08MPa, and dissolve them in acetone until the potency is not less than 200,000 μg / ml. Set the cooling crystallization program: keep stirring at 20℃ for 2h, keep stirring at 10℃ for 2h, keep stirring at 5℃ for 1h, keep stirring at -5℃ for 2h, filter while cold, and wash with cold n-hexane to obtain wet crystals.

[0171] Step (11): Dry the wet crystals obtained in step (10) at 45℃ and -0.08MPa to constant weight to obtain the finished product. The finished product is 10.61g, with a purity of 97.3%, a content of 93.2%, and an impurity of more than 1% FK-523 (demethylascomycin). The yield is 66.02%.

[0172] Comparative Example 4 illustrates that if the ethyl ester phase is further purified without decolorization after acid extraction, the final product purity does not meet the requirements.

[0173] In the description of this specification, references to terms such as "one embodiment," "some embodiments," "embodiment," or "specific embodiment," etc., indicate that a specific feature, structure, material, or characteristic described in connection with that embodiment is included in at least one embodiment of the invention. In this specification, the illustrative expressions of the above terms do not necessarily refer to the same embodiment. Furthermore, the specific features, structures, materials, or characteristics described may be combined in a suitable manner in any one or more embodiments. Moreover, without contradiction, those skilled in the art can combine and integrate the different embodiments and features described in this specification.

[0174] Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention. Those skilled in the art can make changes, modifications, substitutions and variations to the above embodiments within the scope of the present invention.

Claims

1. A method for purifying ascomycin, comprising the following steps: (1) Acidify and filter the ascomycin fermentation broth to obtain bacterial residue; (2) Add organic solvent I to the bacterial residue for extraction to obtain an extract filtrate; (3) Add salt solution to the extraction filtrate for salt washing to obtain a salt-washed organic phase; (4) Add an alkaline solution to the salt-washed organic phase for alkaline washing to obtain an alkaline-washed organic phase; (5) Add an acid solution to the alkaline-washed organic phase for acid washing to obtain an acid-washed organic phase; (6) The acid-washed organic phase is decolorized with activated carbon to obtain the decolorized organic phase; (7) The decolorized organic phase is evaporated under reduced pressure and treated with ethyl acetate and n-hexane in sequence, cooled and filtered to obtain wet crystal 1, which is then rinsed with n-hexane to obtain wet crystal 2. (8) The wet crystals 2 are dried and dissolved in organic solvent II in sequence to obtain a pre-crystallization liquid. The liquid is then stirred and crystallized at low temperature, filtered, and then vacuum dried in solid phase. Step (8) is repeated three times to obtain purified ascomycin.

2. The method according to claim 1, characterized in that, The acidification is carried out using an acidification liquid phase, wherein the acidification liquid phase includes at least one of formic acid, phosphoric acid, and acetic acid; Optionally, the acidification causes the pH of the ascomycin fermentation broth to be 3.0 to 3.

5.

3. The method according to claim 1, characterized in that, In step (1), a filter aid is used to assist filtration during the filtration process. Optionally, the filter aid comprises at least one selected from neutral alumina, cellulose, and diatomaceous earth; Preferably, the filter aid is diatomaceous earth.

4. The method according to claim 1, characterized in that, The organic solvent I includes at least one selected from methanol and ethyl acetate; Preferably, the organic solvent I is ethyl acetate.

5. The method according to claim 1, characterized in that, The salt solution includes an aqueous solution of sodium chloride, sodium sulfate, or potassium chloride; Optionally, the salt solution is an aqueous solution of sodium chloride; Optionally, the concentration of the salt solution is 10%; Optionally, the volume ratio of the salt solution to the organic solvent I is 1:

1.

6. The method according to claim 1, characterized in that, The alkaline solution includes an aqueous solution of sodium hydroxide, sodium bicarbonate, or ammonia. Optionally, the concentration of the alkaline solution is 1 mol / L; Optionally, the volume ratio of the alkaline solution to the organic solvent I is 1:

1.

7. The method according to claim 1, characterized in that, The acid solution includes an aqueous solution of formic acid, phosphoric acid, or acetic acid; Optionally, the pH of the acid solution is 3.0 to 3.5; Optionally, the volume ratio of the acid solution to organic solvent I is 1:

1.

8. The method according to claim 1, characterized in that, The decolorization is carried out using a decolorizing agent, which includes at least one of pharmaceutical activated carbon, 7440-44-0 activated carbon, and HC-303 activated carbon. Optionally, the ratio of the decolorizing agent to the total amount is 1:

1.

9. The method according to claim 1, characterized in that, The volume ratio of ethyl acetate to n-hexane in step (7) is 1:40 to 1:50; Optionally, the cooling gradient is as follows: stir and keep warm at 30°C for 1 hour, cool down to 15°C to 20°C, stir and keep warm for 2 hours. Optionally, the organic solvent II includes at least one selected from acetone and diethyl ether; Preferably, the organic solvent II is acetone; Optionally, the cooling gradient is: 20℃ for stirring for 2 hours, 10℃ for stirring for 2 hours, 5℃ for stirring for 1 hour, and -5℃ for stirring for 2 hours. Optionally, after dissolution in organic solvent II, the potency of ascomycin in the pre-crystallization solution is not less than 200,000 μg / ml.

10. A purified ascomycin, characterized in that, The ascosomycin is obtained by the method described in any one of claims 1 to 9.

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