A kind of high-purity tacrolimus and preparation method thereof

Abstract

The present invention relates to the field of medical technology, and specifically discloses a high-purity tacrolimus and a preparation method thereof. The present invention soaks the mycelium obtained by separating the tacrolimus fermentation broth in a short-chain alcohol solvent and a salt solution, preliminarily removes the pigment and other protein impurities contained in the mycelium, and then uses a nanofiltration concentrator with a cut-off molecular weight of 200Da-300Da to concentrate and remove ions and small molecular impurities, and then adds sodium dodecylbenzene sulfonate to the concentrated solution, changes the distribution ratio of tacrolimus and impurities in the mother liquor and crystals, increases the solubility of impurities in the mother liquor, makes tacrolimus fully crystallize and precipitate, further removes secondary metabolites generated in the fermentation process, improves the quality of the target product, and finally purifies and separates through a dynamic axial compression column, fully removes ascomycin and dihydrotacrolimus and other analog impurities, greatly improves the purity of tacrolimus, and finally obtains high-purity tacrolimus with a content of more than 99.98%.

Description

Technical Field

[0001] The present invention relates to the technical field of biomedicine, and in particular to high-purity tacrolimus and a preparation method thereof. Background Art

[0002] Tacrolimus is a macrolide immunosuppressant widely used in anti-rejection treatment after solid organ transplantation such as liver, pancreas, kidney, heart and lung. Tacrolimus was first approved for marketing in Japan on June 7, 1993 under the trade name Prograf, and subsequently launched in dozens of countries including the United Kingdom, the United States, and Germany. The developed dosage forms include capsules, injections and ointments.

[0003] Modern industry mainly uses microbial fermentation of Streptomyces tsukubaensis and Streptomyces sp. to extract and purify the fermentation broth to obtain tacrolimus. Due to the complex structure of tacrolimus, the fermentation broth also contains tacrolimus analogs, such as ascomycin and dihydrotacrolimus. Therefore, it is difficult to purify tacrolimus with high purity by conventional purification technology.

[0004] At present, the preparation method of high-purity tacrolimus includes: after the fermentation broth is purified by XAD1180 resin for multiple times, the high-purity part of tacrolimus is collected, extracted with ethyl acetate, concentrated, ethyl acetate, cyclohexane and water are added for crystallization, and the crystallization is repeated 2-3 times to obtain a tacrolimus product with a purity of 99.84%, and the yield is only 32.6%. The method has complicated steps, and needs to be purified and recrystallized by resin for multiple times, and the product yield is very low, only about 32.6%. There are also literature reports that the tacrolimus fermentation broth is extracted with dichloromethane, and the extract is concentrated and then crystallized with ethyl acetate. The disadvantage of this method is that the extractant dichloromethane is not only highly toxic, but also has a low boiling point and a low recovery rate of the extractant, resulting in high production costs. Therefore, developing a preparation method of tacrolimus that is simple to operate and has high yield and purity is of great significance for expanding the further development and application of tacrolimus in the field of medicine. Summary of the invention

[0005] In view of the problems of complicated operation and high cost in the existing method for preparing tacrolimus, as well as low yield and purity of the prepared tacrolimus, the present invention provides a high-purity tacrolimus and a preparation method thereof, which mainly removes impurities and pigments in tacrolimus in sequence by filtering and soaking the tacrolimus fermentation broth, concentrating by nanofiltration, crystallizing, and separating by high-pressure chromatography-liquid chromatography, thereby effectively improving the purity and yield of tacrolimus.

[0006] In order to solve the above technical problems, the technical solution provided by the present invention is:

[0007] A method for preparing high-purity tacrolimus comprises the following steps:

[0008] Step a, adding a short-chain alcohol solvent to soak the mycelium obtained by separating the tacrolimus fermentation liquid to obtain an alcohol soaking liquid; adding an inorganic salt solution to the alcohol soaking liquid, continuing to soak, separating the solid and the liquid, and obtaining a salt soaking liquid;

[0009] Step b, concentrating the salt soaking solution using a nanofiltration concentrator with a cut-off molecular weight of 200Da-300Da to obtain a nanofiltrate;

[0010] Step c, adding sodium dodecylbenzene sulfonate to the nanofiltrate, mixing evenly, adding purified water, crystallizing, solid-liquid separation, washing, and drying to obtain tacrolimus crude powder;

[0011] Step d, dissolving the tacrolimus coarse powder with a polar solvent, adding purified water, mixing evenly, adding to a dynamic axial compression column, eluting with a mixed solution of a polar solvent and purified water as a chromatography liquid, collecting the target component, and obtaining a layer washing liquid; wherein the filler of the dynamic axial compression column is UniSil10-120C8 UItra Plus, and the polar solvent is at least one of ethanol, methanol or acetonitrile;

[0012] Step e, concentrating the chromatographic liquid to a non-polar solvent to obtain a concentrated liquid; adding an acetate solvent to the concentrated liquid for extraction, concentrating the extract to dryness, adding an ethanol solution to dissolve, crystallize, separate the solid and the liquid, wash, and dry to obtain a tacrolimus product.

[0013] Compared with the prior art, the preparation method of high-purity tacrolimus provided by the present invention comprises the following steps: soaking mycelium obtained by separating tacrolimus fermentation broth in a short-chain alcohol solvent and an inorganic salt solution to preliminarily remove pigments and other protein impurities contained in the mycelium, and then concentrating and removing ions and small molecular impurities using a nanofiltration concentrator with a cut-off molecular weight of 200Da-300Da, adding sodium dodecylbenzene sulfonate to the concentrated solution, changing the distribution ratio of tacrolimus and impurities in the mother liquor and crystals, increasing the solubility of impurities in the mother liquor, allowing tacrolimus to fully crystallize and precipitate, further removing secondary metabolites generated in the fermentation process, and improving the quality of the target product, and finally performing purification and separation using a dynamic axial compression column filled with UniSil 10-120C8 UItra Plus to fully remove ascomycin and dihydrotacrolimus and other analog impurities, thereby greatly improving the purity of tacrolimus, and finally obtaining a high-purity tacrolimus product with a content of more than 99.98%.

[0014] The present invention can separate and remove pigments, ions, proteins and similar impurities in tacrolimus fermentation broth only through simple processes such as soaking, nanofiltration, crystallization and high-pressure chromatography, and has high purification efficiency, which is conducive to achieving the purpose of rapid and large-scale production of refined tacrolimus products. The product purity can reach more than 99.98%, which improves the safety of clinical drug use, and does not need to be purified multiple times by macroporous adsorption resin, which effectively simplifies the preparation process of tacrolimus, reduces production costs, and has extremely high promotion and application value.

[0015] Preferably, in step a, the short-chain alcohol solvent is at least one of methanol, ethanol or isopropanol.

[0016] Preferably, in step a, the inorganic salt solution is at least one of an aluminum sulfate solution, a ferrous sulfate solution or a zinc sulfate solution with a mass concentration of 20%-25%.

[0017] Preferably, in step a, the content of the short-chain alcohol solvent in the alcohol soaking solution is 70%-75%.

[0018] Preferably, in step a, the amount of the inorganic salt solution added is 4%-6% of the volume of the alcohol soaking solution.

[0019] Preferably, in step a, the soaking time is 2h-3h.

[0020] The preferred short-chain alcohol solvent, inorganic salt and added amount can fully remove the pigment and other protein impurities contained in the mycelium.

[0021] Preferably, in step b, the concentration is to a potency of 100,000 μg / mL-150,000 μg / mL.

[0022] The preferred concentrated solution concentration is beneficial to improving the purity and yield of tacrolimus, and is also beneficial to reducing the amount of purified water added during subsequent crystallization.

[0023] Preferably, in step c, the mass volume ratio of the sodium dodecylbenzene sulfonate to the nanofiltrate is 0.02-0.05:1, wherein the mass unit is g and the volume unit is mL.

[0024] Preferably, in step c, the volume ratio of the purified water to the nanofiltrate is 0.75-1.0:1.

[0025] Preferably, in step c, the purified water is added slowly, and the adding time is controlled to be 1h-2h.

[0026] Preferably, in step c, the crystallization temperature is 15°C-35°C, and the crystallization time is 12h-24h.

[0027] The preferred addition amounts of sodium dodecylbenzene sulfonate and purified water and the crystallization conditions can fully crystallize tacrolimus and reduce the content of impurities in the crystals, thereby improving the separation effect of tacrolimus and impurities, and further facilitating the improvement of the purification effect and efficiency of subsequent high pressure chromatography.

[0028] Furthermore, in step c, the filter cake is washed with 45wt%-50wt% ethanol solution.

[0029] Preferably, in step d, the loading amount of the tacrolimus crude powder is 0.8%-1.0% of the column volume, wherein the unit of the loading amount is g and the unit of the column volume is mL.

[0030] Preferably, in step d, the volume-to-mass ratio of the polar solvent to the tacrolimus crude powder is 8-12:1, wherein the volume unit is mL and the mass unit is g.

[0031] Preferably, in step d, the amount of purified water added is 20%-25% of the volume of the dissolved solution.

[0032] Preferably, in step d, the content of the polar solvent in the chromatography fluid is 55wt%-65wt%.

[0033] Preferably, in step d, the chromatography flow rate is 5 BV / h-6 BV / h.

[0034] Preferably, in step d, the diameter of the dynamic axial compression column is 80 mm, the particle size of the filler is 10 μm, and the pore size of the filler is The height of the packing is 250 mm.

[0035] The preferred high pressure chromatography conditions are conducive to effectively separating tacrolimus from impurities, especially ascomycin and dihydrotacrolimus and other analogs, achieving better separation effects in a shorter time and improving separation efficiency.

[0036] It should be noted that in step d, the filling steps of the dynamic axial compression column are: 900g of chromatographic filler is homogenized with 2.5L of isopropanol to obtain a homogenate, the homogenate is added to the dynamic axial compression column, the isopropanol is discharged and the chromatographic filler is compacted to complete the filling of the dynamic axial compression column, and then the dynamic axial compression column is balanced with the chromatography fluid.

[0037] It should be noted that in step d, the chromatography process is monitored by high performance liquid chromatography, and the chromatographic solution containing ascomycin <0.1% and dihydrotacrolimus <0.1% is collected.

[0038] Preferably, in step e, the acetate solvent is one or both of ethyl acetate or butyl acetate.

[0039] Preferably, in step e, the volume ratio of the acetate solvent to the concentrated solution is 0.8-1.0:1.

[0040] Preferably, in step e, the concentration of the ethanol solution is 45wt%-50wt%, and the potency of tacrolimus in the system after dissolution is 15μg / mL-300,000μg / mL.

[0041] Preferably, in step e, the crystallization temperature is 2°C-8°C, and the crystallization time is 4h-6h.

[0042] Exemplarily, in step e, the filter cake is washed with 45 wt % to 50 wt % ethanol solution.

[0043] Optimal extraction solvent and crystallization conditions can further improve the purity of tacrolimus.

[0044] It should be noted that, in step e, an alcohol meter is used to detect whether the concentrated solution contains a polar solvent.

[0045] The present invention also provides high-purity tacrolimus, which is prepared by any of the above-mentioned methods for preparing high-purity tacrolimus.

[0046] The present invention effectively improves the purity of tacrolimus through a simple process, so that the purity of tacrolimus reaches more than 99.98%, thereby improving the safety of clinical application of tacrolimus, being suitable for industrial-scale production, and having very important significance for expanding the development and application of tacrolimus in the medical field, and having broad market prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0047] Figure 1 is the preparative chromatogram of step d of Example 1;

[0048] Figure 2 This is a chromatogram of the tacrolimus product prepared in step e of Example 1;

[0049] Figure 3 is the preparative chromatogram of step d of Example 2;

[0050] Figure 4 This is a chromatogram of the tacrolimus product prepared in step e of Example 2;

[0051] Figure 5 is the preparative chromatogram of step d of Example 3;

[0052] Figure 6 This is the chromatogram of the tacrolimus product prepared in step e of Example 3. DETAILED DESCRIPTION

[0053] In order to make the purpose, technical solution and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with the embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0054] In order to better illustrate the present invention, further examples are given below.

[0055] The preparation method of the dynamic axial compression column used in the following examples is as follows: 900 g of UniSil10-120C8 UItra Plus silica gel filler with a particle size of 10 μm is homogenized with 2.5 L of isopropanol to obtain a homogenate, the homogenate is added to a dynamic axial compression column (height 650 mm, diameter 80 mm), the isopropanol is discharged and the chromatographic filler is compacted to a filling height of 250 mm, the dynamic axial compression column is filled, and then the dynamic axial compression column is balanced with a chromatographic fluid.

[0056] Example 1

[0057] This embodiment provides a method for preparing high-purity tacrolimus, comprising the following steps:

[0058] Step a, taking 50L of tacrolimus fermentation broth with a fermentation unit of 1024 μg / mL, vacuum filtering the tacrolimus fermentation broth, retaining mycelium, adding the mycelium to a soaking tank, adding 40L of anhydrous ethanol to the soaking tank, detecting the ethanol content in the soaking liquid with an alcohol meter to 72%, soaking at room temperature for 2 hours, then adding 2L of aluminum sulfate solution (450g of aluminum sulfate dissolved in 2L of deionized water) to the fermentation tank, soaking at room temperature for 2 hours, filtering with a plate and frame filter, and retaining 40L of the soaking liquid;

[0059] Step b, adding the soaking liquid to a D2540 nanofiltration concentrator for pre-concentration, and then transferring to a D1812 nanofiltration concentrator for concentration to a potency of 138450 μg / mL, with a concentrated liquid volume of 300 mL;

[0060] Step c, add 6g of sodium dodecylbenzene sulfonate to the concentrated solution, stir, slowly add 225mL of purified water, the addition time is controlled to be 1h, crystallize at room temperature for 14h after the addition of water, filter, rinse the filter cake with 50% ethanol solution, and dry in a vacuum drying oven at 40°C to obtain 41.95g of crude powder;

[0061] Step d, 10 g of each batch of crude powder was taken, dissolved in 100 mL of anhydrous ethanol, and then 25 mL of purified water was added, and then loaded into a 1.25 L dynamic axial compression column, and chromatographed with 60% ethanol aqueous solution at a chromatography flow rate of 5 VB / h, and the peak platform part of the chromatographic solution was collected, and all the chromatographic solutions were combined to a volume of 8200 mL with a titer of 4095 μg / mL;

[0062] Step e, concentrating the chromatographic liquid until there is no ethanol, adding 700 mL of ethyl acetate for extraction, concentrating the extract to dryness, adding 172 mL of 50% ethanol aqueous solution to dissolve while hot, cooling to 6° C., crystallizing for 5 hours, filtering with suction, rinsing the filter cake with 50% ethanol solution, and drying in a vacuum drying oven at 40° C. to obtain 23.15 g of tacrolimus product with HPLC purity of 99.99%, 0.01% of dihydrotacrolimus, and a total yield of 45.12%.

[0063] The preparation chromatogram of step d of this example is as follows Figure 1 As shown, the chromatogram of the tacrolimus product prepared in step e is as shown Figure 2 shown.

[0064] Example 2

[0065] This embodiment provides a method for preparing high-purity tacrolimus, comprising the following steps:

[0066] Step a, taking 50L of tacrolimus fermentation broth with a fermentation unit of 1117 μg / mL, vacuum filtering the tacrolimus fermentation broth, retaining mycelium, adding the mycelium to a soaking tank, adding 45L of anhydrous methanol to the soaking tank, using an alcohol meter to detect that the methanol content in the soaking liquid is 75%, soaking at room temperature for 2 hours, then adding 2L of yellow blood salt solution (475g of aluminum sulfate dissolved in 2L of deionized water) to the fermentation tank, soaking at room temperature for 2 hours, filtering with a plate and frame filter, and retaining 45L of the soaking liquid;

[0067] Step b, adding the soaking liquid to a D2540 nanofiltration concentrator for pre-concentration, and then transferring to a D1812 nanofiltration concentrator to concentrate to a potency of 148450 μg / mL, and the volume of the concentrate is 300 mL;

[0068] Step c, adding 15g of sodium dodecylbenzene sulfonate to the concentrated solution, stirring, slowly adding 300mL of purified water, the adding time is controlled to be 2h, crystallizing at room temperature for 16h after the addition of water, filtering, eluting the filter cake with 50% ethanol solution, and drying in a vacuum drying oven at 40°C to obtain 40.95g of coarse powder;

[0069] Step d, 10 g of each batch of crude powder was taken, dissolved in 100 mL of anhydrous methanol, and then 25 mL of purified water was added, and then loaded into a 1.25 L dynamic axial compression column, chromatographed with 60% methanol aqueous solution, the chromatography flow rate was 6VB / h, the peak platform part of the chromatographic solution was collected, and all the chromatographic solutions were combined to a volume of 9000 mL, with a titer of 3892 μg / mL;

[0070] Step e, concentrating the chromatographic liquid until there is no methanol, adding 700 mL of ethyl acetate for extraction, concentrating the extract to dryness, adding 175 mL of 50% ethanol aqueous solution to dissolve while hot, cooling to 8° C., crystallizing for 6 hours, filtering with suction, eluting the filter cake with 50% ethanol solution, and drying in a vacuum drying oven at 40° C. to obtain 25.18 g of tacrolimus product with HPLC purity of 99.98%, 0.02% ascomycin, and a total yield of 45.01%.

[0071] The preparation chromatogram of step d of this example is as follows Figure 3 As shown, the chromatogram of the tacrolimus product prepared in step e is as shown Figure 4 shown.

[0072] Example 3

[0073] This embodiment provides a method for preparing high-purity tacrolimus, comprising the following steps:

[0074] Step a, taking 50L of tacrolimus fermentation broth with a fermentation unit of 1098 μg / mL, vacuum filtering the tacrolimus fermentation broth, retaining mycelium, adding the mycelium to a soaking tank, adding 50L of isopropanol to the soaking tank, detecting the isopropanol content in the soaking liquid with an alcohol meter to be 71%, soaking at room temperature for 2h, then adding 2L of zinc sulfate solution (500g of zinc sulfate dissolved in 2L of deionized water) to the fermentation tank, soaking at room temperature for 2h, filtering with a plate and frame filter, and retaining 50L of the soaking liquid;

[0075] Step b, adding the soaking liquid to a D2540 nanofiltration concentrator for pre-concentration, and then transferring to a D1812 nanofiltration concentrator to concentrate to a potency of 145450 μg / mL, and the volume of the concentrate is 300 mL;

[0076] Step c, adding 8 g of sodium dodecylbenzene sulfonate to the concentrated solution, stirring, slowly adding 275 mL of purified water, the adding time is controlled to be 1.5 h, after the water addition is completed, crystallizing at room temperature for 12 h, filtering, eluting the filter cake with 50% ethanol solution, and drying in a vacuum drying oven at 40° C. to obtain 40.05 g of crude powder;

[0077] Step d, 10 g of each batch of crude powder was taken, dissolved in 100 mL of acetonitrile, and then 25 mL of purified water was added, and then loaded into a 1.25 L dynamic axial compression column, and chromatographed with 55% acetonitrile aqueous solution at a chromatography flow rate of 5VB / h, and the peak platform part of the chromatographic solution was collected, and all the chromatographic solutions were combined to a volume of 7900 mL with a titer of 4495 μg / mL;

[0078] Step e, concentrating the chromatographic liquid until there is no acetonitrile, adding 700 mL of ethyl acetate for extraction, concentrating the extract to dryness, adding 168 mL of 50% ethanol aqueous solution to dissolve while hot, cooling to 6° C., crystallizing for 5 hours, filtering with suction, rinsing the filter cake with 50% ethanol solution, and drying in a vacuum drying oven at 40° C. to obtain 25.08 g of tacrolimus product with HPLC purity of 99.98%, 0.02% of dihydrotacrolimus, and a total yield of 45.68%.

[0079] The preparation chromatogram of step d of this example is as follows Figure 5 As shown, the chromatogram of the tacrolimus product prepared in step e is as shown Figure 6 shown.

[0080] Comparative Example 1

[0081] This comparative example provides a method for preparing high-purity tacrolimus, which has the same steps as those in Example 1, except that the filler of the dynamic axial compression column is different. The specific steps are as follows:

[0082] Step a, taking 50L of tacrolimus fermentation broth with a fermentation unit of 1024 μg / mL, vacuum filtering the tacrolimus fermentation broth, retaining mycelium, adding the mycelium to a soaking tank, adding 40L of anhydrous ethanol to the soaking tank, detecting the ethanol content in the soaking liquid with an alcohol meter to 72%, soaking at room temperature for 2 hours, then adding 2L of aluminum sulfate solution (450g of aluminum sulfate dissolved in 2L of deionized water) to the fermentation tank, soaking at room temperature for 2 hours, filtering with a plate and frame filter, and retaining 40L of the soaking liquid;

[0083] Step b, adding the soaking liquid to a D2540 nanofiltration concentrator for pre-concentration, and then transferring to a D1812 nanofiltration concentrator to concentrate to a potency of 141450 μg / mL, and the volume of the concentrate is 300 mL;

[0084] Step c, add 6g of sodium dodecylbenzene sulfonate to the concentrated solution, stir, slowly add 225mL of purified water, the addition time is controlled to be 1h, crystallize at room temperature for 14h after the addition of water, filter, rinse the filter cake with 50% ethanol solution, and dry in a vacuum drying oven at 40°C to obtain 40.95g of crude powder;

[0085] Step d: Take 10 g of each batch of crude powder, dissolve it in 100 mL of anhydrous ethanol, add 25 mL of purified water, and then load it onto a 1.25 L dynamic axial compression column (COSMOSIL C18 10 μm, ), chromatographed with 60% ethanol aqueous solution at a flow rate of 5VB / h, collecting the peak platform portion of the chromatographic solution, combining all chromatographic solutions, the volume was 8500mL, and the titer was 3075μg / mL;

[0086] Step e, concentrating the chromatographic liquid until there is no ethanol, adding 700 mL of ethyl acetate for extraction, concentrating the extract to dryness, adding 170 mL of 50% ethanol aqueous solution to dissolve while hot, cooling to 6° C., crystallizing for 5 hours, filtering with suction, rinsing the filter cake with 50% ethanol solution, and drying in a vacuum drying oven at 40° C. to obtain 20.45 g of tacrolimus product with HPLC purity of 99.25%, 0.32% of dihydrotacrolimus, 0.43% of ascomycin, and a total yield of 39.94%.

[0087] The preparation method of the dynamic axial compression column in step d is as follows: 900 g of COSMOSIL C18 silica gel filler with a particle size of 10 μm is homogenized with 2.5 L of isopropanol to obtain a homogenate, the homogenate is added to a dynamic axial compression column (height 650 mm, diameter 80 mm), the isopropanol is discharged and the chromatographic filler is compacted, the filling height of the filler is 250 mm, the filling of the dynamic axial compression column is completed, and then the dynamic axial compression column is balanced with a chromatographic fluid.

[0088] Comparative Example 2

[0089] This comparative example provides a method for preparing high-purity tacrolimus, which has the same steps as those in Example 3, except that sodium dodecylbenzene sulfonate is not added during crystallization in step c. The specific steps are as follows:

[0090] Step a, taking 50L of tacrolimus fermentation broth with a fermentation unit of 1098 μg / mL, vacuum filtering the tacrolimus fermentation broth, retaining mycelium, adding the mycelium to a soaking tank, adding 50L of isopropanol to the soaking tank, detecting the isopropanol content in the soaking liquid with an alcohol meter to be 71%, soaking at room temperature for 2h, then adding 2L of zinc sulfate solution (500g of zinc sulfate dissolved in 2L of deionized water) to the fermentation tank, soaking at room temperature for 2h, filtering with a plate and frame filter, and retaining 50L of the soaking liquid;

[0091] Step b, adding the soaking liquid to a D2540 nanofiltration concentrator for pre-concentration, and then transferring to a D1812 nanofiltration concentrator to concentrate to a potency of 135450 μg / mL, and the volume of the concentrate is 300 mL;

[0092] Step c, slowly adding 275 mL of purified water to the concentrated solution, the adding time is controlled to be 1.5 h, after the water addition is completed, crystallize at room temperature for 12 h, filter, rinse the filter cake with 50% ethanol solution, and dry in a vacuum drying oven at 40° C. to obtain 30.05 g of crude powder;

[0093] Step d, 10 g of each batch of crude powder was taken, dissolved in 100 mL of acetonitrile, and then 25 mL of purified water was added, and then loaded into a 1.25 L dynamic axial compression column that was the same as Example 3, and chromatographed with 55% acetonitrile aqueous solution at a chromatography flow rate of 5VB / h, and the peak platform portion of the chromatographic solution was collected, and all chromatographic solutions were combined to a volume of 5900 mL with a titer of 4095 μg / mL;

[0094] Step e, concentrating the chromatographic liquid until there is no acetonitrile, adding 700 mL of ethyl acetate for extraction, concentrating the extract to dryness, adding 170 mL of 50% ethanol aqueous solution to dissolve while hot, cooling to 6° C., crystallizing for 5 hours, filtering with suction, rinsing the filter cake with 50% ethanol solution, and drying in a vacuum drying oven at 40° C. to obtain 14.08 g of tacrolimus product with HPLC purity of 99.59%, 0.15% of dihydrotacrolimus, 0.23% of ascomycin, 0.03% of other impurities, and a total yield of 25.65%.

[0095] Comparative Example 3

[0096] This comparative example provides a method for preparing high-purity tacrolimus, and the specific steps are as follows:

[0097] Step a, taking 50L of tacrolimus fermentation broth with a fermentation unit of 1024 μg / mL, vacuum filtering the tacrolimus fermentation broth, retaining mycelium, adding the mycelium to a soaking tank, adding 40L of anhydrous ethanol to the soaking tank, detecting the ethanol content of the soaking liquid with an alcohol meter to 72%, soaking at room temperature for 2 hours, then adding 2L of aluminum sulfate solution (450g of aluminum sulfate dissolved in 2L of deionized water) to the fermentation tank, soaking at room temperature for 2 hours, filtering with a plate and frame filter, and retaining the soaking liquid;

[0098] Step b, adding the above soaking liquid into a concentrator for vacuum concentration, concentrating to an ethanol content of 1%, and the volume of the concentrated liquid is 7L; adding 7L of ethyl acetate to the concentrated liquid, stirring for 1.5h to extract, the volume of the extract is 5.5L, the potency is 8820μg / mL, the extract is vacuum concentrated to a paste with a concentrator, adding 250mL of anhydrous ethanol to dissolve, the potency is 132450μg / mL, the volume is 300mL

[0099] Step c, add 6g of sodium dodecylbenzene sulfonate to the concentrated solution, stir, slowly add 225mL of purified water, the addition time is controlled to be 1h, crystallize at room temperature for 14h after the addition of water, filter, rinse the filter cake with 50% ethanol solution, and dry in a vacuum drying oven at 40°C to obtain 31.24g of crude powder;

[0100] Step d, 10 g of each batch of crude powder was taken, dissolved in 100 mL of anhydrous ethanol, and then 25 mL of purified water was added, and then loaded into a 1.25 L dynamic axial compression column that was the same as in Example 1, and chromatographed with a 60% ethanol aqueous solution at a chromatography flow rate of 5 VB / h, and the chromatographic liquid of the peak platform was collected, and all the chromatographic liquids were combined to a volume of 6200 mL with a titer of 4395 μg / mL;

[0101] Step e, concentrating the chromatographic liquid until there is no ethanol, adding 700 mL of ethyl acetate for extraction, concentrating the extract to dryness, adding 166 mL of 50% ethanol aqueous solution to dissolve while hot, cooling to 6° C., crystallizing for 5 hours, filtering with suction, rinsing the filter cake with 50% ethanol solution, and drying in a vacuum drying oven at 40° C. to obtain 14.25 g of tacrolimus product with HPLC purity of 99.56%, 0.13% of dihydrotacrolimus, 0.31% of ascomycin, and a total yield of 27.83%.

[0102] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modification, equivalent substitution or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for preparing high-purity tacrolimus, characterized in that: The steps include: Step a, adding a short-chain alcohol solvent to soak the mycelium obtained by separating the tacrolimus fermentation liquid to obtain an alcohol soaking liquid; adding an inorganic salt solution to the alcohol soaking liquid, continuing to soak, separating the solid and the liquid, and obtaining a salt soaking liquid; Step b, concentrating the salt soaking solution using a nanofiltration concentrator with a cut-off molecular weight of 200Da-300Da to obtain a nanofiltrate; Step c, adding sodium dodecylbenzene sulfonate to the nanofiltrate, mixing evenly, adding purified water, crystallizing, solid-liquid separation, washing, and drying to obtain tacrolimus crude powder; Step d, dissolving the tacrolimus crude powder with a polar solvent, adding purified water, mixing evenly, adding to a dynamic axial compression column, eluting with a mixed solution of a polar solvent and purified water as a chromatography liquid, collecting the target component, and obtaining a layer washing liquid; wherein the filler of the dynamic axial compression column is UniSilC8 UItra Plus, and the polar solvent is at least one of ethanol, methanol or acetonitrile; Step e, concentrating the chromatographic solution to a non-polar solvent to obtain a concentrated solution; adding an acetate solvent to the concentrated solution for extraction, concentrating the extract to dryness, adding an ethanol solution to dissolve, cooling and crystallizing, solid-liquid separation, washing, and drying to obtain a tacrolimus product; In step a, the short-chain alcohol solvent is at least one of methanol, ethanol or isopropanol; in step e, the acetate solvent is one or both of ethyl acetate or butyl acetate.

2. The method for preparing high-purity tacrolimus according to claim 1, characterized in that: In step a, the inorganic salt solution is at least one of an aluminum sulfate solution or a zinc sulfate solution with a mass concentration of 20%-25%.

3. The method for preparing high-purity tacrolimus according to claim 2, characterized in that: In step a, the content of the short-chain alcohol solvent in the alcohol soaking solution is 70%-75%; and / or In step a, the amount of the inorganic salt solution added is 4%-6% of the volume of the alcohol soaking solution; and / or In step a, the soaking time is 2h-3h.

4. The method for preparing high-purity tacrolimus according to claim 1, characterized in that: In step b, the titer is concentrated to 100,000 μg / mL-150,000 μg / mL.

5. The method for preparing high-purity tacrolimus according to claim 1, characterized in that: In step c, the mass volume ratio of the sodium dodecylbenzene sulfonate to the nanofiltrate is 0.02-0.05:1, wherein the mass unit is g and the volume unit is mL; and / or In step c, the volume ratio of the purified water to the nanofiltrate is 0.75-1.0:

1.

6. The method for preparing high-purity tacrolimus according to claim 1 or 5, characterized in that: In step c, the purified water is added slowly, and the adding time is controlled to be 1h-2h; and / or In step c, the crystallization temperature is 15° C.-35° C., and the crystallization time is 12 h-24 h.

7. The method for preparing high-purity tacrolimus according to claim 1, characterized in that: In step d, the loading amount of the tacrolimus crude powder is 0.8%-1.0% of the column volume, wherein the unit of the loading amount is g and the unit of the column volume is mL; and / or In step d, the volume-to-mass ratio of the polar solvent to the tacrolimus crude powder is 8-12:1, wherein the volume unit is mL and the mass unit is g; and / or In step d, the amount of purified water added is 20%-25% of the volume of the dissolved solution.

8. The method for preparing high-purity tacrolimus according to claim 1 or 7, characterized in that: In step d, the content of the polar solvent in the chromatographic solution is 55wt%-65wt%; and / or In step d, the chromatography flow rate is 5BV / h-6BV / h; In step d, the diameter of the dynamic axial compression column is 80 mm, the particle size of the filler is 10 μm, and the pore size of the filler is The height of the packing is 250 mm.

9. The method for preparing high-purity tacrolimus according to claim 1, characterized in that: In step e, the volume ratio of the acetate solvent to the concentrated solution is 0.8-1.0:1; and / or In step e, the concentration of the ethanol solution is 45wt%-50wt%, and the potency of tacrolimus in the system after dissolution is 15μg / mL-300,000μg / mL; and / or In step e, the temperature of the cooling crystallization is 2°C-8°C, and the crystallization time is 4h-6h.

Images