Method for separating and purifying ascomycin

Abstract

The invention provides a method for separating and purifying ascomycin. The method for separating and purifying ascomycin comprises the following steps: 1) adding inorganic salt into fermentation liquor containing ascomycin, adjusting the pH value of a system to be 1-4, and carrying out first ceramic membrane filtration to obtain first ceramic membrane concentrated liquor; adding alkaline buffer salt into the first ceramic membrane concentrated solution, adjusting the pH value of the system to 8-11, and carrying out second ceramic membrane filtration to obtain a second ceramic membrane concentrated solution; (2) carrying out filter pressing and water washing on the second ceramic membrane concentrated solution to obtain a filter cake, and extracting the filter cake by using an organic solvent to obtain an extracting solution; (3) cooling, crystallizing, filtering and drying the extract liquor to obtain first purified crystallis.By means of the method, cross-linked impurities such as amino acid, protein and pigment in the fermentation liquor can be effectively removed, and the effect of efficiently separating and purifying high-purity ascomycin is achieved.

Description

Technical Field

[0001] This application relates to the field of organic chemistry, and in particular to a method for isolating and purifying ascomycin. Background Technology

[0002] Ascomycin is an important macrolide compound widely used in the pharmaceutical field, especially as an ethyl analogue of the immunosuppressant FK-506 (tacrolimus) and a key intermediate in semi-synthetic drugs (such as pimecrolimus). Its purity directly affects the efficacy and safety of the final drug. Therefore, designing an efficient method for the isolation and purification of ascomycin is a crucial core step in the entire production process.

[0003] Existing technologies primarily involve extracting ascomycin from fermentation broth or bacterial residue, followed by purification using macroporous resin or silica gel, and finally separation and purification of ascomycin using solvent crystallization. However, this method faces significant challenges in practical applications. The complex composition of the fermentation broth results in low efficiency and dark-colored extracts from traditional extraction processes, and the low solvent solubility during crystallization leads to low purity of ascomycin.

[0004] Therefore, developing an efficient method for isolating and purifying ascomycin is of great significance. Summary of the Invention

[0005] This application provides a method for separating and purifying ascomycin, achieving the technical effect of high-efficiency separation and purification of high-purity ascomycin.

[0006] This application provides a method for isolating and purifying ascocin, comprising the following steps:

[0007] 1) Add inorganic salts to the fermentation broth containing ascomycin, adjust the pH of the system to 1-4, perform the first ceramic membrane filtration to obtain the first ceramic membrane concentrate; add alkaline buffer salts to the first ceramic membrane concentrate, adjust the pH of the system to 8-11, perform the second ceramic membrane filtration to obtain the second ceramic membrane concentrate.

[0008] 2) The concentrated liquid from the second ceramic membrane is subjected to pressure filtration and water washing to obtain a filter cake. The filter cake is then extracted with an organic solvent to obtain an extract.

[0009] 3) Cool the extract to crystallize and filter to dry, to obtain the first purified crystal.

[0010] The method described above further includes, before step 3), passing the extract through a polyamide resin column, collecting the column pass solution, concentrating the column pass solution and adding activated carbon for decolorization, filtering to obtain a decolorized solution, cooling and crystallizing the decolorized solution and filtering and drying, and collecting the first purified crystal.

[0011] The method described above further includes recrystallizing the first purified crystal and filtering and drying it after step 3) to obtain a second purified crystal.

[0012] The method described above further includes, after obtaining the second purified crystal, adding a preparative mobile phase to the second purified crystal for liquid chromatography separation, collecting the eluent, concentrating the eluent, precipitating the precipitate, filtering and drying it to obtain purified ascomycin.

[0013] In the method described above, the inorganic salt in step 1) includes at least one of sodium salt and ammonium salt, and the weight-to-volume ratio of the inorganic salt to the fermentation broth is 0.3-3%; and / or,

[0014] The alkaline buffer salt mentioned in step 1) includes at least one of ammonium bicarbonate, sodium bicarbonate, and sodium carbonate, and the weight-to-volume ratio of the alkaline buffer salt to the first ceramic membrane concentrate is 0.3-3%; and / or,

[0015] The organic solvent mentioned in step 2) includes at least one of methanol, ethanol, acetone, ethyl acetate, and butyl acetate.

[0016] In the method described above, the particle size of the polyamide resin is 30-60 mesh, the concentration is alkali pressure concentration, and the concentration is carried out until the ascomycin content is 250-300 mg / mL. The activated carbon decolorization temperature is 60-65℃ and the decolorization time is 30-60 min.

[0017] The method described above, wherein the cooling crystallization in step 3) includes sequentially performing a first-stage cooling, a second-stage cooling, and a third-stage cooling, wherein the first-stage cooling is performed at a rate of 4-6°C / h to 42-45°C, the second-stage cooling is performed at a rate of 1-2°C / h to 30-32°C, and the third-stage cooling is performed at a rate of 3-5°C / h to 3-5°C; and / or,

[0018] In step 3), the water content of the first purified crystal is <2%.

[0019] The method described above includes, after the first stage of cooling, a heat preservation period of 42-45°C for 3-5 hours.

[0020] The method described above, wherein the recrystallization comprises: adding a water-soluble solvent to the first purified crystal to obtain a solution, then adding the solution dropwise to 1.5 to 2 times its volume of water at a rate of 80 to 90 mL / h at 30 to 35°C; pausing the addition when reflective crystals appear in the system, maintaining the temperature and stirring for 1 to 2 hours, then continuing the addition until completion, and then maintaining the temperature and stirring for another 8 to 12 hours; and / or,

[0021] The water content of the second purified crystal is <5%.

[0022] In the method described above, the water-soluble solvent includes at least one of methanol, ethanol, isopropanol, and acetone, and the concentration of the solution is 80-100 mg / mL.

[0023] The method for separating and purifying ascomycin in this application involves adding inorganic salts to a fermentation broth containing ascomycin to adjust the pH of the system to 1-4, performing a first ceramic membrane filtration to obtain a first ceramic membrane concentrate; then adding an alkaline buffer salt to the first ceramic membrane concentrate to adjust the pH of the system to 8-11, performing a second ceramic membrane filtration to obtain a second ceramic membrane concentrate; subsequently, the second ceramic membrane concentrate is pressure filtered and washed with water to obtain a filter cake, which is then extracted with an organic solvent to obtain an extract; finally, the extract is cooled, crystallized, filtered, and dried to obtain a first purified crystal, achieving the technical effect of highly efficient separation and purification of high-purity ascomycin. Attached Figure Description

[0024] Figure 1 The image shows the color of the extract from Example 1.

[0025] Figure 2 This is a color chart of the first purified crystal in Example 1;

[0026] Figure 3 This is a color chart of the second purified crystal in Example 1;

[0027] Figure 4 Color diagram of purified ascomycin in Example 1;

[0028] Figure 5 This is the HPLC chromatogram of the second purified crystal in Example 1;

[0029] Figure 6 The HPLC chromatogram of the eluent from Example 1 is shown below.

[0030] Figure 7 The HPLC chromatogram of purified ascomycin in Example 1;

[0031] Figure 8 The color chart of the second purified crystal in Example 4A;

[0032] Figure 9 This is the color chart of the second purified crystal in Example 4B. Detailed Implementation

[0033] To make the objectives, technical solutions, and advantages of this application clearer, the technical solutions in the embodiments of this application will be clearly and completely described below in conjunction with the embodiments of this application. Obviously, the described embodiments are only some embodiments of this application, not all embodiments. Based on the embodiments of this application, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this application.

[0034] Ascomycin is a macrocyclic lactone compound derived from fungi, renowned for its potent immunosuppressive properties. It specifically inhibits T-lymphocyte activation, and its core applications lie in derivatives such as tacrolimus, which are widely used for organ transplant rejection and the treatment of autoimmune diseases such as atopic dermatitis. Due to the narrow therapeutic window of this class of drugs, their purity is crucial. High purity ensures precise dosage and avoids unknown toxicities caused by impurities, thus being a primary prerequisite for guaranteeing clinical efficacy and patient safety.

[0035] Currently, the industry commonly uses fermentation broth or bacterial residue to extract ascomycin, followed by purification with macroporous resin or silica gel, and finally crystallization with a mixed solvent to separate and purify the ascomycin. However, this method suffers from low extraction efficiency, dark-colored extract, and low ascomycin purity in practical applications. The inventors have investigated this issue and believe that the low extraction efficiency and dark-colored extract are mainly due to the presence of cross-linked impurities such as amino acids, proteins, and pigments in the fermentation broth. Furthermore, the inventors believe that the low solvent solubility and rapid cooling process in traditional crystallization processes easily lead to uneven crystal particles, resulting in low purity of the final ascomycin product.

[0036] Based on this, this application provides a method for isolating and purifying ascosine, comprising the following steps:

[0037] 1) Add inorganic salts to the fermentation broth containing ascomycin, adjust the pH of the system to 1-4, perform the first ceramic membrane filtration to obtain the first ceramic membrane concentrate; add alkaline buffer salts to the first ceramic membrane concentrate, adjust the pH of the system to 8-11, perform the second ceramic membrane filtration to obtain the second ceramic membrane concentrate.

[0038] 2) The concentrate from the second ceramic membrane is filtered and washed with water to obtain a filter cake. The filter cake is then extracted with an organic solvent to obtain an extract.

[0039] 3) Cool the extract to crystallize and filter to dry, to obtain the first purified crystal.

[0040] This application involves adding inorganic salts to a fermentation broth containing ascomycin, adjusting the pH of the system to 1-4 with oxalic acid, and then performing a first ceramic membrane filtration to obtain a first ceramic membrane concentrate. Then, an alkaline buffer salt is added to the first ceramic membrane concentrate to adjust the pH to 8-11, followed by a second ceramic membrane filtration to obtain a second ceramic membrane concentrate. This alternating acid-base ceramic membrane filtration technology utilizes the solubility differences of amino acids and proteins at different pH levels to effectively remove cross-linked impurities and pigments, significantly improving the purity and color stability of the extract. The second ceramic membrane concentrate is then pressure-filtered and washed with water to obtain a filter cake. This filter cake is then extracted with an organic solvent to obtain an extract. The solubility parameters of the organic solvent match the molecular structure of ascomycin, enabling efficient extraction of the target product while avoiding co-extraction of polar impurities. The extract is then cooled, crystallized, filtered, and dried to obtain a first purified crystal. The cooling crystallization process uses gradient cooling to control the crystal growth rate, ensuring uniform crystal morphology and reducing impurity embedding rates. After the above steps, the purity of ascomycin can be effectively improved and the impurity content can be significantly reduced, which can, to a certain extent, ensure clinical efficacy and patient safety.

[0041] In one specific embodiment, before step 3), the extract is passed through a polyamide resin column, the column chromatography solution is collected, the column chromatography solution is concentrated, activated carbon is added for decolorization, and the solution is filtered to obtain a decolorized solution. The decolorized solution is then cooled, crystallized, filtered, and dried to collect the first purified crystals. Polyamide resin does not adsorb the product but adsorbs highly polar impurities. Combining polyamide resin normal-phase chromatography with activated carbon decolorization removes polar impurities and macromolecular pigments in stages, forming a "preliminary decolorization-deep decolorization" dual-layer purification system, which can significantly reduce the impurity load in subsequent crystallization and preparative chromatography.

[0042] Furthermore, step 3) includes recrystallizing the first purified crystal, filtering and drying it to obtain the second purified crystal. Recrystallization, by dissolving and crystallizing the crystal again in pure water, effectively removes residual trace organic impurities, inorganic salts, and colored impurities, and washes away organic solvents trapped during cooling crystallization. Simultaneously, this process optimizes the physical properties of the product, resulting in crystals with more stable crystal forms and more uniform particle sizes, thereby improving product quality to a higher level in terms of chemical purity, solvent residue, and physical properties.

[0043] Further research by the inventors revealed that after obtaining the second purified crystal, the process includes: adding a preparative mobile phase to the second purified crystal for liquid chromatography separation, collecting the eluent, concentrating the eluent, precipitating the precipitate, filtering and drying it to obtain purified ascomycin. Liquid chromatography separation enables precise molecular-level separation based on the slight differences in partition coefficients between ascomycin and other impurities, thus efficiently removing trace impurities with similar structures and properties that are difficult to remove in the previous crystallization step. Subsequent concentration, crystallization, and drying not only effectively enrich the target product but also further purify it, ultimately obtaining high-purity, stable purified ascomycin, significantly improving the purity and quality of the product.

[0044] This application does not limit the inorganic salt. In one specific embodiment, the inorganic salt in step 1) includes at least one of sodium salt and ammonium salt, and the weight-volume ratio of inorganic salt to fermentation broth is 0.3-3%. The addition of sodium salt (such as sodium chloride) or ammonium salt (such as ammonium bicarbonate) directly affects the precipitation efficiency of amino acids and proteins. A concentration range of 0.3-3% ensures sufficient ionic strength to disrupt intermolecular forces while avoiding excessive salting out that could lead to loss of the target product. For example, when the weight-volume ratio of inorganic salt to fermentation broth is 0.3-1%, a good balance is achieved between impurity removal rate and product recovery rate.

[0045] Further, in step 1), the alkaline buffer salt includes at least one of ammonium bicarbonate, sodium bicarbonate, and sodium carbonate, and the weight-to-volume ratio of the alkaline buffer salt to the first ceramic membrane concentrate is 0.3-3%. The selection of the alkaline buffer salt needs to consider both pH adjustment capability and buffering capacity. Sodium bicarbonate can effectively maintain system stability at pH 9.5, avoiding degradation of the target product due to drastic pH fluctuations. For example, after treatment with 0.5% NaHCO3, the retention rate of ascomycin in the ceramic membrane concentrate is high, while the removal rate of alkaline amino acids can be effectively improved.

[0046] Specifically, in step 2), the organic solvent includes at least one of methanol, ethanol, acetone, ethyl acetate, and butyl acetate. Among them, ethyl acetate (polarity parameter 4.4) or butyl acetate (polarity parameter 3.8) can selectively extract the target product while excluding polar impurities (such as pigments) due to their suitable solubility parameters (matching the molecular polarity of ascomycin).

[0047] In one specific embodiment, the polyamide resin has a particle size of 30-60 mesh, and is concentrated by alkaline pressure concentration until the ascomycin content is 250-300 mg / mL. The activated carbon decolorization temperature is 60-65°C, and the decolorization time is 30-60 min. The pore size distribution of the polyamide resin (30-60 mesh) can effectively adsorb polar impurities (such as RRT0.62 impurities) while avoiding non-specific adsorption of the target product. Column chromatography can effectively improve the purity of the crude product. For example, decolorizing with activated carbon at 65°C for 30 min can change the decolorized solution from orange to yellow, significantly improving the pigment removal rate.

[0048] It should be noted that step 3) of cooling crystallization includes a first stage of cooling, a second stage of cooling, and a third stage of cooling performed sequentially. The first stage of cooling is performed at a rate of 4~6℃ / h to 42~45℃, the second stage of cooling is performed at a rate of 1~2℃ / h to 30~32℃, and the third stage of cooling is performed at a rate of 3~5℃ / h to 3~5℃. This gradient cooling strategy controls supersaturation to allow the crystal to form a regular crystal structure under low-speed growth conditions, reducing impurity entrapment.

[0049] Furthermore, in step 3), the water content of the first purified crystal is <2%. By precisely controlling the water content of the first purified crystal within the suitable range of <2%, this step can effectively elute and remove most of the water-soluble inorganic salts, small molecule polar impurities, and residual solvents while ensuring the stability of the crystal morphology and preventing excessive adhesion or clumping. This not only significantly improves the purity of the first purified crystal, laying a good material foundation for subsequent liquid chromatography purification, but also optimizes the crystal flowability, facilitating subsequent filtration, transfer, and other process operations, thus achieving a balance between purification efficiency and process controllability.

[0050] It should be added that the first stage of cooling is followed by a holding period of 42-45℃ for 3-5 hours. During this isothermal stage, solute molecules in the solution system can fully and orderly arrange themselves onto the crystal nuclei, promoting slow crystal growth and perfect maturation. This not only effectively reduces lattice defects or internal stress caused by rapid cooling, resulting in crystals with more uniform particle size and more regular crystal form, but also allows impurities in the mechanically encapsulated mother liquor sufficient time to precipitate and return to the solution, thereby significantly reducing impurity entrapment and improving the purity of the first purified crystal and the quality of the product.

[0051] In one specific embodiment, recrystallization includes: adding a water-soluble solvent to the first purified crystal to obtain a solution, then adding the solution dropwise to 1.5 to 2 times its volume of water at a rate of 80 to 90 mL / h at 30 to 35°C. When reflective crystals appear in the system, the dropwise addition is paused, and the mixture is kept at this temperature and stirred for 1 to 2 hours. Then, the dropwise addition is continued until the solution is completely removed, followed by another 8 to 12 hours of stirring at this temperature. Aqueous phase recrystallization, by changing the solvent polarity, can further remove residual pigments and weakly polar impurities. Exemplarily, recrystallization effectively improves the purity of the finished product and significantly increases the yield.

[0052] Furthermore, the water content of the second purified crystal is <5%. By precisely controlling the water content of the second purified crystal within this specific range of <5%, an appropriate water content ensures that the crystal can be effectively and uniformly dissolved in the prepared mobile phase during subsequent liquid chromatography separation, avoiding excessively high local concentrations or incomplete dissolution, thereby guaranteeing the efficiency and reproducibility of chromatographic separation. Simultaneously, this control also prevents crystal structure shrinkage that may result from excessively low water content or agglomeration and degradation that may occur from excessively high water content, providing a crucial intermediate guarantee for ultimately obtaining high-purity purified ascomycin.

[0053] Specifically, the water-soluble solvent includes at least one of methanol, ethanol, isopropanol, and acetone, with a solution concentration of 80-100 mg / mL. The selection of the water-soluble solvent is based on its good solubility for ascomycin, its ability to be miscible with water in any proportion, thereby rapidly and uniformly changing the solution polarity during antisolvent crystallization to induce crystallization. Simultaneously, its moderate boiling point and volatility facilitate effective removal in subsequent filtration and drying steps. Furthermore, these solvents generally possess the characteristics of low cost, relatively low toxicity, and mature and reliable industrial applications, ensuring efficient purification and obtaining well-defined crystals while also considering the safety, economy, and operability of the process.

[0054] The present application will be further described below through specific embodiments.

[0055] Example 1

[0056] This embodiment provides a method for isolating and purifying ascocin, including the following steps:

[0057] 1) Add sodium chloride (29.7L of fermentation broth, sodium chloride to fermentation broth weight-volume ratio of 0.3%) to the fermentation broth containing 31.9g of ascomycin. Add oxalic acid to adjust the pH of the system to 2. Perform the first ceramic membrane filtration, top with 60L of water, and collect 10L of the first ceramic membrane concentrate. Then add NaHCO3 (NaHCO3 to the first ceramic membrane concentrate weight-volume ratio of 0.5%) to the first ceramic membrane concentrate, adjust the pH of the system to 9.5, perform the second ceramic membrane filtration, top with 60L of water, and collect 10L of the second ceramic membrane concentrate.

[0058] 2) The concentrate from the second ceramic membrane was filtered through a plate and frame filter to obtain 3.5 kg of bacterial residue, which was then dried. 15 L of ethyl acetate was added for extraction, and the extract was yellow-red (see...). Figure 1 It contains 30.3g of ascomycin;

[0059] 3) Pass the extract through a 60-mesh polyamide resin column, wash with ethyl acetate and collect the column buffer. The column buffer is bright yellow and contains 28.7g of ascomycin. Concentrate the column buffer to 100mL (ascomycin content 300mg / mL), add activated carbon for decolorization to obtain a decolorized solution. The decolorization temperature is 65℃ and the decolorization time is 60min.

[0060] 4) The decolorizing solution was subjected to cooling crystallization. The method was as follows: the temperature was lowered to 42℃ at a gradient of 6℃ / h, and then maintained at 42℃ for 3-5 hours with stirring. At this time, fine reflective crystals were present in the solution. The temperature was then further lowered to 30℃ at a gradient of 2℃ / h, and then further lowered to 5℃ at a gradient of 5℃ / h. After filtration, the crystals were collected and dried to obtain the first purified crystal with a water content of 1.82% and containing 23.9g of ascomycin (see...). Figure 2 ).

[0061] 5) Dissolve the first purified crystal in acetone to a final volume of 200 mL. Add the solution dropwise to 400 mL of water at a rate of 80 mL / h at 30°C. Stop adding the solution when reflective crystals appear in the system. Maintain the temperature and stir for 2 hours, then continue adding until the solution is complete. Continue stirring and maintaining the temperature for another 10 hours. Filter and dry to obtain the second purified crystal with a water content of 4.71 g and containing 21.5 g of ascomycin (see...). Figure 3 Its purity is 97.006%, content is 102.71%, RRT is 0.74, and impurities are 2.18% (HPLC chromatogram is shown). Figure 4 );

[0062] 6) Take 14g of the second purified crystals and perform liquid chromatography separation. Using a 0.5% sample loading rate, inject 40ml into the preparative column each time, injecting once every 40 minutes. Collect the eluent after the peak height of 800mV each time. Mix the eluents and determine the purity to be 98.678%, containing 12.887g of ascomycin (HPLC chromatogram shown). Figure 5 The eluent was concentrated, the precipitate was precipitated, filtered and dried to obtain purified ascomycin (see [link to extract]). Figure 6 The purity was 99.942%, the content was 109.1%, and the single impurity (RRT 0.62) was 0.058% (HPLC chromatogram shown). Figure 7 ).

[0063] Example 2

[0064] 1) Add sodium chloride (30.3L of fermentation broth, sodium chloride to fermentation broth weight-volume ratio of 0.3%) to the fermentation broth containing 30.8g of ascomycin. Add oxalic acid to adjust the pH of the system to 2. Perform the first ceramic membrane filtration, top with 60L of water, and collect 10L of the first ceramic membrane concentrate. Then add NaHCO3 (NaHCO3 to the first ceramic membrane concentrate weight-volume ratio of 0.5%) to the first ceramic membrane concentrate, adjust the pH of the system to 9.5, perform the second ceramic membrane filtration, top with 60L of water, and collect 10L of the second ceramic membrane concentrate.

[0065] 2) The concentrate from the second ceramic membrane was filtered through a plate and frame filter to obtain 3.51 kg of bacterial residue, which was then dried. 15 L of ethyl acetate was added for extraction. The extract was yellow-red and contained 28.8 g of ascomycin.

[0066] 3) Pass the extract through a 60-mesh polyamide resin column, wash with ethyl acetate and collect the column buffer. The column buffer is bright yellow and contains 26.7g of ascomycin. Concentrate the column buffer to 90mL (ascomycin content 300mg / mL), add activated carbon for decolorization to obtain a decolorized solution. The decolorization temperature is 65℃ and the decolorization time is 60min.

[0067] 4) The decolorizing solution was subjected to cooling crystallization. The method was as follows: the temperature was lowered to 42℃ at a gradient of 6℃ / h, and then kept at 42℃ for 3-5 hours. At this time, there were fine reflective crystals in the solution. The temperature was then lowered to 30℃ at a gradient of 2℃ / h, and then to 5℃ at a gradient of 5℃ / h. After filtration, the crystals were collected and dried to obtain the first purified crystal with a water content of 1.66 and a content of 24.1g of ascomycin.

[0068] 5) Add acetone to the first purified crystal to dissolve it to 200 mL to obtain a solution. Add the solution dropwise to 400 mL of water at a rate of 80 mL / h at 30 °C. Stop the dropwise addition when reflective crystals appear in the system, keep warm and stir for 2 h, then continue the dropwise addition until the solution is complete. Keep warm and stir for another 10 h, filter and dry to obtain a second purified crystal with a water content of 4.25 g and a content of 22.3 g of ascomycin. Its purity is 96.835%, content is 100.7%, RRT is 0.74 and impurities are 2.69%.

[0069] 6) Take 14g of the second purified crystals for liquid chromatography separation. Using a 0.5% sample loading rate, inject 40ml into the prepared column each time, injecting once every 40 minutes. Collect the eluent after the peak height of 800mV each time. Mix the eluents and determine the purity to be 98.337%, containing 12.3g of ascomycin. Concentrate the eluent, precipitate the precipitate, filter and dry to obtain purified ascomycin, with a purity of 99.933%, content of 105.7%, and single impurity (RRT 0.62) of 0.067%.

[0070] Example 3

[0071] 1) Add sodium chloride (29.9L of fermentation broth, sodium chloride to fermentation broth weight-volume ratio of 0.3%) to the fermentation broth containing 30.7g of ascomycin. Add oxalic acid to adjust the pH of the system to 2. Perform the first ceramic membrane filtration, top with 60L of water, and collect 10L of the first ceramic membrane concentrate. Then add NaHCO3 (NaHCO3 to the first ceramic membrane concentrate weight-volume ratio of 0.5%) to the first ceramic membrane concentrate, adjust the pH of the system to 9.5, perform the second ceramic membrane filtration, top with 60L of water, and collect 10L of the second ceramic membrane concentrate.

[0072] 2) The concentrate from the second ceramic membrane was filtered through a plate and frame filter to obtain 3.42 kg of bacterial residue, which was then dried. 15 L of ethyl acetate was added for extraction. The extract was yellow-red and contained 28.3 g of ascomycin.

[0073] 3) Pass the extract through a 60-mesh polyamide resin column, wash with ethyl acetate and collect the column buffer. The column buffer is bright yellow and contains 26.8g of ascomycin. Concentrate the column buffer to 90mL (ascomycin content 300mg / mL), add activated carbon for decolorization to obtain a decolorized solution. The decolorization temperature is 65℃ and the decolorization time is 60min.

[0074] 4) The decolorizing solution was subjected to cooling crystallization. The method was as follows: the temperature was lowered to 42℃ at a gradient of 6℃ / h, and then kept at 42℃ for 3-5 hours. At this time, there were fine reflective crystals in the solution. The temperature was then lowered to 30℃ at a gradient of 2℃ / h, and then to 5℃ at a gradient of 5℃ / h. After filtration, the crystals were collected and dried to obtain the first purified crystal with a water content of 1.77 and a content of 24.2g of ascomycin.

[0075] 5) Add acetone to the first purified crystal to dissolve it to 200 mL to obtain a solution. Add the solution dropwise to 400 mL of water at a rate of 80 mL / h at 30 °C. Stop the dropwise addition when reflective crystals appear in the system, keep warm and stir for 2 h, then continue the dropwise addition until the solution is complete. Keep warm and stir for another 10 h, filter and dry to obtain a second purified crystal with a water content of 4.56 g and a content of 22.8 g of ascomycin. Its purity is 97.101%, content is 101.2%, RRT is 0.74 and impurities are 2.35%.

[0076] 6) Take 14g of the second purified crystals and perform liquid chromatography separation. Using a 0.5% sample loading rate, inject 40ml into the prepared column each time, injecting once every 40 minutes. Collect the eluent after the peak height of 800mV each time. Mix the eluents and determine the purity to be 98.421%, containing 11.9g of ascomycin. Concentrate the eluent, precipitate the precipitate, filter and dry to obtain purified ascomycin, with a purity of 99.923%, content of 105.3%, and single impurity (RRT 0.62) of 0.077%.

[0077] Example 4A

[0078] Take 5 liters of the extract (processed in the same way as in step 2 of Example 1), containing 9.4 g of ascomycin. Pass the extract through a 60-mesh polyamide resin column, collect the column chromatography solution, concentrate it, and decolorize it with activated carbon. The decolorized solution is yellow. Crystallize it using the same method as in Example 1. After a single cooling crystallization, the first purified crystal is obtained. The HPLC purity is 94.779%, the content is 96.33%, and it contains 8.2 g of ascomycin. Recrystallize it in aqueous phase to obtain the second purified crystal (see...). Figure 8 It contains 7.5g of ascomycin, with an HPLC purity of 97.152% and a content of 99.94%.

[0079] Example 4B

[0080] Take 5 liters of the extract (processed in the same way as in Example 1, step 2), containing 9.4 g of ascomycin. Concentrate the extract directly according to the method in Example 1, add activated carbon for decolorization, the decolorized solution is orange, crystallize in the same way as in Example 1, and obtain the first purified crystals which are light yellow after one cooling crystallization. HPLC purity: 93.985%, content: 94.78%, containing 7.7 g of ascomycin. Recrystallize in aqueous phase to obtain the second purified crystals (see...). Figure 9 It contains 6.8g of ascomycin, with an HPLC purity of 96.113% and a content of 95.92%.

[0081] Example 5A

[0082] Take 16.0 g of the first purified crystal (step 4) of the same treatment as in Example 1, containing 15.35 g of ascomycin, recrystallize according to Example 1, and separate by preparative column liquid chromatography to obtain 11.1 g of purified ascomycin with a purity of 99.931% and a content of 108.2%, which is pure white.

[0083] Example 5B

[0084] Take 16.0g of the first purified crystal (step 4) of the same treatment as in Example 1, containing 15.35g of ascomycin. Without recrystallization, directly perform column liquid chromatography separation as in Example 1 to obtain 10.2g of purified ascomycin with a purity of 99.810% and a content of 107.3%, which is pure white.

[0085] Example 6A

[0086] Take 16.0g of the first purified crystal (step 4) of the same treatment as in Example 1, containing 15.15g of ascomycin, recrystallize according to Example 1, and separate by preparative column liquid chromatography to obtain 11.4g of purified ascomycin, yield: 75.2%, purity: 99.978%, content: 107.9%, pure white.

[0087] Example 6B

[0088] Take 16.0 g of the first purified crystal (in step 4) of the same treatment as in Example 1, containing 15.15 g of ascomycin, recrystallize at 15°C (the crystal particles are finer, making filtration relatively difficult), and separate by preparative column liquid chromatography to obtain 10.9 g of refined ascomycin, yield: 71.9%, purity: 99.921%, content: 107.1, pure white.

[0089] Example 7A

[0090] Take 75 ml of the decolorizing solution from step 3) of the same treatment as in Example 1, containing 17.6 g of ascomycin. Crystallize by cooling at the same temperature gradient as in Example 1. Collect the first purified crystal and recrystallize. The second purified crystal has a purity of 97.215%, a content of 99.934%, and a critical impurity of 2.324%. Pass it through a preparative column and collect it according to the process to obtain 11.8 g of purified ascomycin with a purity of 99.932% and a content of 108.85%, which is pure white.

[0091] Example 7B

[0092] Take 75 ml of the decolorizing solution from step 3) of the same treatment as in Example 1, containing 17.6 g of ascomycin. Heat the decolorizing solution to 65°C, then allow it to cool naturally to 42°C. Keep it warm and stir until crystals appear, then keep it warm for another 2 hours. Allow it to cool naturally to room temperature (approximately 28°C), then continue cooling with cooling water at a gradient of 5°C / h to 5°C. Filter and collect the first purified crystals, then recrystallize them. The second purified crystals have a purity of 95.587%, a content of 98.77%, and a critical impurity of 4.038%. Pass the crystals through a preparative column, collect them according to the process, and proceed with the operation to obtain 10.5 g of purified ascomycin with a purity of 99.887% and a content of 108.05%, which is pure white.

[0093] Comparative Example 1A

[0094] Take 10 liters of fermentation broth containing 10.15 g of ascomycin, filter it through a ceramic membrane as described in Example 1, and press filter to obtain 0.85 kg of bacterial residue. After drying, extract with 4 L of ethyl acetate. The extract contains 9.1 g of ascomycin and is yellow-red in color. After rotary drying, weigh 15.3 g (in clumps, with a small amount of adhesion). The loss on drying is 10.3%, and the ascomycin content is calculated to be 66.3%.

[0095] Comparative Example 1B

[0096] Take 10 liters of fermentation broth containing 10.15 g of ascomycin, filter directly to obtain 1.03 kg of bacterial residue, dry it, and extract with 4 L of ethyl acetate. The extract contains 7.1 g of ascomycin and is red in color. Add 3 L of ethyl acetate for a second extraction. The second extract contains 1.5 g of ascomycin. Mix the two extracts, evaporate to dryness, and weigh 18.8 g (gelled and adhered). The loss on drying is 12.7%. Calculate the ascomycin content: 52.4%.

[0097] Experimental Example 1

[0098] The purified ascomycin from Examples 1, 2, and 3 were tested, and the results are shown in Table 1:

[0099] 1) Purity: Chromatograms were obtained using high performance liquid chromatography (HPLC), and purity was calculated using the area normalization method. Purity % = (Main peak area / Sum of all peak areas) × 100%;

[0100] 2) Content: The chromatogram was obtained by high performance liquid chromatography, and the content was calculated by external standard method. Content % = (concentration of test sample × dilution factor × volume / sample weight) × 100%.

[0101] Table 1:

[0102]

[0103] Experimental Example 2

[0104] The first and second purified crystals of Examples 4A and 4B were tested, and the results are shown in Table 2:

[0105] 1) Yield: Yield % = (Weight of dry product after crystallization / Weight of material fed into crystallization) × 100%;

[0106] 2) Purity: Chromatograms were obtained using high-performance liquid chromatography (HPLC), and purity was calculated using the area normalization method. Purity % = (Main peak area / Sum of all peak areas) × 100%;

[0107] 3) Content: The chromatogram was obtained by high performance liquid chromatography, and the content was calculated by external standard method. Content % = (concentration of test sample × dilution factor × volume / sample weight) × 100%.

[0108] Table 2

[0109]

[0110] As can be seen from the table above, the color, purity, content, and yield of the first and second purified crystals obtained after crystallization by polyamide column treatment are significantly higher than those obtained without column treatment, which greatly benefits subsequent purification operations.

[0111] Experimental Example 3

[0112] The results of tests on the refined ascosomnol of Examples 5A and 5B are shown in Table 3:

[0113] Table 3

[0114]

[0115] As can be seen from the table above, recrystallization can remove pigments and more impurities, resulting in better preparation. The yield, purity, and content of refined ascomycin are significantly higher.

[0116] Test Example 4

[0117] The results of tests on the refined ascosomnol of Examples 6A and 6B are shown in Table 4:

[0118] Table 4

[0119]

[0120] As can be seen from the table above, recrystallization at 30℃, followed by separation using a preparative column, resulted in slightly better purity and content of refined ascomycin compared to low-temperature crystallization, with a slightly higher yield.

[0121] Experimental Example 5

[0122] The second purified crystals and purified ascomycin from Examples 7A and 7B were tested, and the results are shown in Table 5:

[0123] Table 5

[0124]

[0125] As can be seen from the table above, the cooling gradient during primary crystallization is very important and has a significant impact on the quality and yield of the final product.

[0126] Experimental Example 6

[0127] The extracts of Comparative Example 1A and Comparative Example 1B were tested, and the results are shown in Table 6:

[0128] Extraction yield % = (Asomycin content after extraction / Asomycin content before extraction) × 100%.

[0129] Table 6

[0130]

[0131] As can be seen from the table above, this application uses two different pH ceramic membrane filtration methods to remove impurities, which can remove a large amount of alkaline amino acids, acidic amino acids, water-soluble pigments and other cross-linked impurities. The filter residue is then extracted with solvent, and the resulting extract is lighter in color and has a higher content than the traditional bacterial residue extract. The extraction uses less solvent and has a higher yield.

[0132] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of this application, and are not intended to limit them. Although this application has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features therein. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of this application.

Claims

1. A method for isolating and purifying ascomycin, characterized in that, Includes the following steps: 1) Add inorganic salts to the fermentation broth containing ascomycin, adjust the pH of the system to 1-4, and perform the first ceramic membrane filtration to obtain the first ceramic membrane concentrate; Add alkaline buffer salt to the first ceramic membrane concentrate, adjust the pH of the system to 8-11, and perform a second ceramic membrane filtration to obtain the second ceramic membrane concentrate. 2) The concentrated liquid from the second ceramic membrane is subjected to pressure filtration and water washing to obtain a filter cake. The filter cake is then extracted with an organic solvent to obtain an extract. 3) Cool the extract to crystallize and filter to dry, to obtain the first purified crystal.

2. The method according to claim 1, characterized in that, Before step 3), the extract is passed through a polyamide resin column, the column pass solution is collected, the column pass solution is concentrated and then activated carbon is added for decolorization, the decolorized solution is filtered to obtain a decolorized solution, the decolorized solution is cooled and crystallized and then filtered and dried, and the first purified crystal is collected.

3. The method according to claim 1 or 2, characterized in that, Step 3) is followed by recrystallization of the first purified crystal, followed by filtration and drying to obtain a second purified crystal.

4. The method according to claim 3, characterized in that, After obtaining the second purified crystal, the process further includes: adding a preparative mobile phase to the second purified crystal for liquid chromatography separation, collecting the eluent, concentrating the eluent, precipitating the precipitate, filtering and drying it to obtain purified ascomycin.

5. The method according to any one of claims 1-4, characterized in that, The inorganic salt mentioned in step 1) includes at least one of sodium salt and ammonium salt, and the weight-to-volume ratio of the inorganic salt to the fermentation broth is 0.3-3%; and / or, The alkaline buffer salt mentioned in step 1) includes at least one of ammonium bicarbonate, sodium bicarbonate, and sodium carbonate, and the weight-to-volume ratio of the alkaline buffer salt to the first ceramic membrane concentrate is 0.3-3%; and / or, The organic solvent mentioned in step 2) includes at least one of methanol, ethanol, acetone, ethyl acetate, and butyl acetate.

6. The method according to claim 2, characterized in that, The polyamide resin has a particle size of 30-60 mesh, the concentration is alkali pressure concentration, and the concentration is carried out until the ascomycin content is 250-300 mg / mL. The activated carbon decolorization temperature is 60-65℃ and the decolorization time is 30-60 min.

7. The method according to any one of claims 1-6, characterized in that, Step 3) describes cooling crystallization, which includes sequentially performing a first-stage cooling, a second-stage cooling, and a third-stage cooling. The first-stage cooling is performed at a rate of 4-6°C / h to 42-45°C; the second-stage cooling is performed at a rate of 1-2°C / h to 30-32°C; and the third-stage cooling is performed at a rate of 3-5°C / h to 3-5°C; and / or, In step 3), the water content of the first purified crystal is <2%.

8. The method according to claim 7, characterized in that, The first stage of cooling is followed by heat preservation at 42-45℃ for 3-5 hours.

9. The method according to claim 3, characterized in that, The recrystallization includes: adding a water-soluble solvent to the first purified crystal to obtain a solution, then adding the solution dropwise to 1.5 to 2 times its volume of water at a rate of 80 to 90 mL / h at 30 to 35°C; stopping the dropwise addition when reflective crystals appear in the system, maintaining the temperature and stirring for 1 to 2 hours, then continuing the dropwise addition until completion, and then maintaining the temperature and stirring for another 8 to 12 hours; and / or, The water content of the second purified crystal is <5%.

10. The method according to claim 9, characterized in that, The water-soluble solvent includes at least one of methanol, ethanol, isopropanol, and acetone, and the concentration of the solution is 80-100 mg / mL.

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