Method for purifying tacrolimus crude product

Abstract

The invention relates to a purification method of a tacrolimus crude product. The purification method comprises the following steps: obtaining an upper column liquid containing the tacrolimus crude product; an affinity resin chromatographic column is adopted for carrying out first chromatography treatment on the upper column liquid, ascomycin and dihydro tacrolimus are removed, a first chromatography liquid is obtained, the affinity resin chromatographic column contains metal ions, and the metal ions can be coordinated with tacrolimus; and carrying out secondary chromatography treatment on the first chromatography liquid by adopting macroporous adsorption resin, and removing methyl tacrolimus and a tacrolimus 8-site epimer to obtain purified tacrolimus. The purification method of the tacrolimus crude product has high purity and high yield, and is easy for industrial production.

Description

Technical Field

[0001] The invention relates to the field of drug purification, and in particular to a method for purifying a crude tacrolimus product. Background Art

[0002] Tacrolimus (FK506) is a 23-membered macrolide antibiotic with strong immunosuppressive properties. It is a first-line drug for anti-immune rejection after liver and kidney transplantation in clinical practice. Tacrolimus inhibits the expression of cytokines such as interleukin 2 (IL-2) by inhibiting the activity of calcineurin, indirectly achieving the proliferation and activation of T lymphocytes, thereby exerting an immunosuppressive effect. Compared with cyclosporine A, tacrolimus has stronger efficacy, lower dosage, lower incidence of acute rejection and lower side effects. At the same time, tacrolimus can also be used clinically for the treatment of autoimmune diseases such as atopic dermatitis, eczema, systemic lupus erythematosus, psoriasis, psoriasis, ichthyosis, diabetes, Behcet's disease, rheumatoid arthritis and multiple sclerosis, and has broad application prospects.

[0003] Tacrolimus is produced by fermentation of Streptomyces tsukuba, and then extracted, separated, purified and refined. While the strain ferments and produces tacrolimus, it also produces a large number of isomers and structural analogs, such as ascomycin 19-position diastereomer, ascomycin (FK520), demethyl tacrolimus, tacrolimus 8-position diastereomer, dihydrotacrolimus (2H-FK506), etc. The traditional tacrolimus purification process generally has the problem of difficulty in balancing high yield and high purity. For example, some researchers have used three resin columns in series for purification and multiple crystallization to obtain high-purity tacrolimus, but the yield is only 32.6%. Summary of the invention

[0004] Based on this, it is necessary to provide a method for purifying crude tacrolimus with both high purity and high yield.

[0005] A method for purifying a crude tacrolimus product comprises the following steps:

[0006] Obtaining an upper column liquid containing a crude tacrolimus product, wherein the crude tacrolimus product comprises tacrolimus, ascomycin, dihydrotacrolimus, desmethyltacrolimus and tacrolimus 8-position diastereomer;

[0007] The upper column liquid is subjected to a first chromatography treatment using an affinity resin chromatography column to remove ascomycin and dihydrotacrolimus to obtain a first chromatography liquid, wherein the affinity resin chromatography column contains metal ions that can coordinate with tacrolimus;

[0008] The first chromatographic solution is subjected to a second chromatography treatment using a macroporous adsorption resin to remove demethyl tacrolimus and tacrolimus 8-position diastereomer to obtain purified tacrolimus.

[0009] In some embodiments, the filler of the affinity resin chromatography column is selected from Ag + Coordination resin and Cu 2+ Either or both of the coordination resins.

[0010] In some embodiments, the first eluent used in the first chromatography treatment is selected from any one or a mixed solvent of two of methanol, ethanol, ethyl acetate and acetone;

[0011] Optionally, the flow rate of the first eluent is 0.5 BV / h to 20 BV / h.

[0012] In some embodiments, the first eluent is acetone; or,

[0013] The first eluent is a mixed solvent of ethanol and ethyl acetate, in which the volume percentage of ethanol is 5% to 50%; or,

[0014] The first eluent is a mixed solvent of methanol and ethyl acetate, and the volume percentage of methanol in the mixed solvent is 5% to 50%.

[0015] In some embodiments, the macroporous adsorption resin is selected from any one or more of HP20, HZ820, HZ816, AmberliteXAD-1180 and HZ818.

[0016] In some embodiments, the second eluent used in the second chromatography treatment is a mixed solution of a polar organic solvent and water, and in the second eluent, the volume percentages of the polar organic solvent and the water are 20% to 50% and 50% to 80%, respectively;

[0017] Optionally, the polar organic solvent is selected from any one or more of methanol, ethanol and acetone;

[0018] Optionally, during elution with the second eluent, a gradient elution method is adopted;

[0019] Optionally, the flow rate of the second eluent is 0.5 BV / h to 2 BV / h.

[0020] In some of the embodiments, after the step of performing a second chromatography treatment on the first chromatographic liquid using a macroporous adsorption resin, the method further includes: distilling the second chromatographic liquid obtained after removing demethyl tacrolimus and tacrolimus 8-position diastereomer, removing the polar organic solvent in the second chromatographic liquid, allowing tacrolimus to crystallize in water, and then performing solid-liquid separation and drying.

[0021] In some embodiments, during the first chromatography treatment, the chromatographic solution containing ascomycin in the solute at a mass percentage of less than 1% is collected in sections; and / or,

[0022] During the second chromatography treatment, the chromatographic fluid containing tacrolimus greater than 98.5% by mass in the solute is collected in sections.

[0023] In some embodiments, in the upper column liquid, the concentration of tacrolimus is 50 mg / mL to 300 mg / mL; and / or,

[0024] In the upper column liquid, the solvent is selected from any one or more of methanol, ethanol, acetone and ethyl acetate.

[0025] In some embodiments, before the step of performing a second chromatography treatment on the first chromatographic solution using a macroporous adsorption resin, the first chromatographic solution is concentrated so that the concentration of tacrolimus in the concentrated solution is 50 mg / mL to 300 mg / mL.

[0026] The purification method of the crude tacrolimus product first uses an affinity resin chromatography column for the first chromatographic separation, and utilizes the coordination effect between the metal ions in the affinity resin and the C36 double bond on the macrolide structure of the tacrolimus molecule to specifically adsorb tacrolimus, which can achieve rapid separation of ascomycin and dihydrotacrolimus impurities. The first chromatographic solution is then subjected to macroporous adsorption resin chromatography, which can efficiently remove other impurities such as demethyl tacrolimus and tacrolimus 8-position diastereomers. At the same time, the macroporous adsorption resin chromatography can also remove a small amount of metal ions that fall off in the first step of affinity resin chromatography, avoid the generation of heavy metals or new impurities, and obtain purified tacrolimus. The above purification method improves the separation efficiency, and experiments have shown that the purity of tacrolimus purified by the above method can reach 99.6%, and the contents of ascomycin and dihydrotacrolimus are both less than 0.1%, which meets the USP (United States Pharmacopeia) standard, and the total yield of tacrolimus is greater than 70%, which can take into account high purity and high yield, and is suitable for industrial production.

[0027] In addition, compared with the traditional purification method of connecting multiple macroporous adsorption resin columns in series, the above method shortens the column purification cycle. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1is a process flow chart of the purification method of crude tacrolimus in some embodiments;

[0029] Figure 2 is a liquid chromatogram of crude tacrolimus in Example 1;

[0030] Figure 3 is the liquid chromatogram of the first chromatographic liquid in Example 1;

[0031] Figure 4 is a liquid chromatogram of the purified tacrolimus crystal product in Example 1;

[0032] Figure 5 is a liquid chromatogram of the purified tacrolimus crystal product in Example 2;

[0033] Figure 6 This is the liquid chromatogram of the purified tacrolimus crystal product in Comparative Example 1. DETAILED DESCRIPTION

[0034] In order to facilitate the understanding of the present invention, the present invention will be described more comprehensively below in conjunction with the specific embodiments. Preferred embodiments of the present invention are provided in the specific embodiments. However, the present invention can be implemented in many different forms and is not limited to the embodiments described herein. On the contrary, the purpose of providing these embodiments is to make the understanding of the disclosure of the present invention more thorough and comprehensive.

[0035] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art of the present invention. The terms used herein in the specification of the present invention are only for the purpose of describing specific embodiments and are not intended to limit the present invention. The term "and / or" used herein includes any and all combinations of one or more related listed items.

[0036] Unless otherwise specified or there is a contradiction, the terms or phrases used in this invention have the following meanings:

[0037] In the present invention, "first" and "second" are used only for descriptive purposes and cannot be understood as indicating or implying relative importance or implicitly indicating the number of the indicated technical features. Therefore, the features defined as "first" and "second" may explicitly or implicitly include at least one of the features.

[0038] In the description of the present invention, “plurality” means at least two, for example, two, three, etc., unless otherwise clearly and specifically defined.

[0039] In the present invention, "one or several" refers to any one, any two or any two or more of the listed items. Among them, "several" refers to any two or any two or more.

[0040] In the present invention, the percentage concentration involved, unless otherwise specified, refers to the final concentration. The final concentration refers to the proportion of the added component in the system after the addition of the component.

[0041] The words "preferably", "more preferably", etc. in the present invention refer to embodiments of the present invention that may provide certain beneficial effects in certain circumstances. However, other embodiments may also be preferred under the same circumstances or other circumstances. In addition, the description of one or more preferred embodiments does not imply that other embodiments are not applicable, nor is it intended to exclude other embodiments from the scope of the present invention.

[0042] When a numerical range is disclosed in the present invention, the above range is considered to be continuous and includes the minimum and maximum values ​​of the range, as well as every value between such minimum and maximum values. Further, when a range refers to an integer, every integer between the minimum and maximum values ​​of the range is included. In addition, when multiple ranges are provided to describe features or characteristics, the ranges can be merged. In other words, unless otherwise indicated, all ranges disclosed in the present invention should be understood to include any and all subranges included therein.

[0043] In the present invention, the technical features described in an open manner include closed technical solutions composed of the listed features, and also include open technical solutions containing the listed features.

[0044] The terms "including" and "having" and any variations thereof in the embodiments of the present invention are intended to cover non-exclusive inclusions. For example, a process, method, system, product, or device that includes a series of steps or units is not limited to the listed steps or units, but may optionally include steps or units that are not listed, or may optionally include other steps or components that are inherent to these processes, methods, products, or devices.

[0045] Reference to "embodiments" in the present invention means that a particular feature, structure, or characteristic described in conjunction with the embodiments may be included in at least one embodiment of the present application. The appearance of the phrase in various places in the specification does not necessarily refer to the same embodiment, nor is it an independent or alternative embodiment that is mutually exclusive with other embodiments. It is explicitly and implicitly understood by those skilled in the art that the embodiments described in the present invention may be combined with other embodiments.

[0046] In industry, crude tacrolimus is purified by resin or acidic silica gel to separate impurities, but it is difficult to achieve high purity, high yield and short cycle. For example, some researchers used three resin columns in series for purification and multiple crystallization to obtain high-purity tacrolimus, but the yield was only 32.6%. Other researchers used silver nitrate as the mobile phase to separate ascomycin and dihydrotacrolimus by column chromatography, which improved the purification efficiency, but the product was susceptible to Ag. + pollution problem, and the use of Ag + Modified silica gel or resin column chromatography separation also has the problem of silver loading being loose and easily leaking out, making it difficult to achieve industrial application. In addition, the inventors found that this process has limited separation effect on other impurities, such as demethyl tacrolimus, tacrolimus 8-position diastereomer and tacrolimus, which have closer molecular structures and almost identical groups on the main chain and side chain of the structure, and cannot be removed by this process, making it difficult to meet the USP standard.

[0047] Based on this, the present invention provides a method for purifying crude tacrolimus, please refer to Figure 1 , including the following steps:

[0048] Step S110: obtaining a column liquid containing crude tacrolimus.

[0049] The crude tacrolimus product comprises tacrolimus, ascomycin, dihydrotacrolimus, demethyltacrolimus and tacrolimus 8-dimer. Specifically, the crude tacrolimus product is obtained by fermentation and extraction, and the details can be referred to patent CN108929335A.

[0050] In some embodiments, the concentration of tacrolimus in the upper column liquid is 50 mg / mL to 300 mg / mL. For example, the concentration of tacrolimus in the upper column liquid can be, but is not limited to, 50 mg / mL, 80 mg / mL, 100 mg / mL, 120 mg / mL, 150 mg / mL, 180 mg / mL, 200 mg / mL, 220 mg / mL, 250 mg / mL, 280 mg / mL, 300 mg / mL, or a range consisting of any two of these values. It is understood that the concentration of tacrolimus in the upper column liquid here refers to the concentration of pure tacrolimus without other impurities, which is generally obtained according to liquid chromatography.

[0051] In some embodiments, in the upper column liquid, the solvent is selected from any one or more of methanol, ethanol, acetone and ethyl acetate. Preferably, in the upper column liquid, the solvent includes acetone.

[0052] Specifically, the crude tacrolimus product is dissolved in a solvent to obtain a column liquid containing the crude tacrolimus product.

[0053] Step S120: using an affinity resin chromatography column to perform a first chromatography treatment on the upper column liquid to remove ascomycin and dihydrotacrolimus to obtain a first chromatography liquid. The affinity resin chromatography column contains metal ions that can coordinate with tacrolimus.

[0054] In some embodiments, the filler of the affinity resin chromatography column is selected from Ag + Coordination resin and Cu 2+ Preferably, the filler of the affinity resin chromatography column is Ag. + Coordination resin. Ag + The coordination resin can be purchased from Shanghai Huazhen Technology Co., Ltd.

[0055] Compared with tacrolimus, ascomycin and dihydrotacrolimus molecules have fewer double bonds at C36 on their macrolide structures, so Ag can be used to + , Cu 2+ The coordination effect of transition metal ions and the C36 double bond specifically adsorbs tacrolimus, thereby achieving the separation of ascomycin and dihydrotacrolimus impurities.

[0056] In some embodiments, the first eluent used in the first chromatography treatment is selected from any one or a mixed solvent of two of methanol, ethanol, ethyl acetate and acetone. In some embodiments, the first eluent is a single solvent acetone. In other embodiments, the first eluent is a mixed solvent of ethanol and ethyl acetate, and the volume percentage of ethanol in the mixed solvent is 5% to 50%. In some other embodiments, the first eluent is a mixed solvent of methanol and ethyl acetate, and the volume percentage of methanol in the mixed solvent is 5% to 50%.

[0057] Preferably, the first eluent is acetone. Compared with a double solvent, the use of a single solvent as the eluent of the affinity chromatography resin eliminates the steps of preparing the eluent and adjusting the ratio. The solvent can be used directly after recovery without further preparation, avoiding the cumbersome operation of determining the solvent ratio such as gas phase and liquid phase detection. The concentrated liquid is directly used as the column liquid of the next resin column, without the need for solvent transfer, which simplifies the operation, significantly improves the standardization of process operations, and reduces costs.

[0058] In some embodiments, the flow rate of the first eluent is 0.5 BV / h to 20 BV / h. Wherein, BV / h represents the spatial flow rate, that is, the average amount of liquid flowing through a unit volume of resin per unit time (h) in the column. For example, the flow rate of the first eluent can be, but is not limited to, 0.5 BV / h, 1 BV / h, 2 BV / h, 4 BV / h, 6 BV / h, 8 BV / h, 10 BV / h, 12 BV / h, 14 BV / h, 16 BV / h, 18 BV / h, 20 BV / h or a range consisting of any two of these values.

[0059] In some embodiments, during the first chromatography treatment, the chromatographic solution in which the mass percentage of ascomycin in the solute is less than 1% is collected in segments. Specifically, the eluted effluent is collected in segments, and the eluted effluent in which the mass percentage of ascomycin in the solute is less than 1% is combined to obtain the first chromatographic solution. By controlling the mass percentage of ascomycin in the collected eluted effluent, the purity of tacrolimus in the first chromatographic solution is further improved.

[0060] Step S130: performing a second chromatography treatment on the first chromatographic solution using a macroporous adsorption resin to remove demethyl tacrolimus and tacrolimus 8-position diastereomer to obtain purified tacrolimus.

[0061] Since the molecular structures of demethyl tacrolimus, tacrolimus 8-position diastereomer and tacrolimus are closer, and the groups on the main chain and side chain of the structure are almost the same, it is difficult to remove them by affinity resin column chromatography, and the proportion of impurities in the finished product does not meet the USP standard, and the affinity resin column chromatography method also has the problem of heavy metal pollution caused by the detachment of metal ions. The inventor conducted research on this problem and found that after the affinity resin chromatography step, macroporous resin adsorption can efficiently remove impurities such as demethyl tacrolimus and tacrolimus 8-position diastereomer, and at the same time remove a small amount of metal ions that fall off in the first step of column chromatography, avoiding the generation of heavy metals or new impurities.

[0062] In some embodiments, the macroporous adsorption resin is selected from one of HP20, HZ820, HZ816, Amberlite XAD-1180 or HZ818.

[0063] In some embodiments, the second eluent used in the second chromatography treatment is a mixed solution of a polar organic solvent and water. In the second eluent, the volume percentages of the polar organic solvent and water are 20% to 50% and 50% to 80%, respectively.

[0064] In some embodiments, the polar organic solvent is selected from any one or more of methanol, ethanol and acetone.

[0065] In some embodiments, a gradient elution method is used. In the step of gradient elution, the volume percentage of the polar organic solvent in the second eluent increases from small to large.

[0066] In some embodiments, the flow rate of the second eluent is 0.5 BV / h to 2 BV / h. For example, the flow rate of the second eluent may be, but is not limited to, 0.5 BV / h, 0.6 BV / h, 0.8 BV / h, 1 BV / h, 1.2 BV / h, 1.4 BV / h, 1.5 BV / h, 1.6 BV / h, 1.8 BV / h, 2 BV / h, or a range consisting of any two of these values.

[0067] In some embodiments, during the second chromatography treatment, the chromatographic solution containing more than 98.5% tacrolimus by mass in the solute is collected in stages. Specifically, the eluted effluent is collected in stages, and the eluted effluent containing more than 98.5% tacrolimus by mass in the solute is combined to obtain the second chromatographic solution.

[0068] In some embodiments, before the step of performing a second chromatography treatment on the first chromatographic solution using a macroporous adsorption resin, the first chromatographic solution is further concentrated so that the concentration of tacrolimus in the concentrated solution is 50 mg / mL to 300 mg / mL. For example, in the concentrated solution, the concentration of tacrolimus can be, but is not limited to, 50 mg / mL, 80 mg / mL, 100 mg / mL, 120 mg / mL, 150 mg / mL, 180 mg / mL, 200 mg / mL, 220 mg / mL, 250 mg / mL, 280 mg / mL, 300 mg / mL or a range consisting of any two of these values. It is understood that the concentration of tacrolimus in the concentrated solution here refers to the concentration of pure tacrolimus that does not contain other impurities, which is generally obtained based on liquid chromatography.

[0069] In some of the embodiments, during the concentration process, normal pressure concentration or reduced pressure concentration is adopted.

[0070] In some embodiments, the method further comprises: distilling the second chromatographic solution obtained after removing demethyl tacrolimus and tacrolimus 8-position diastereomer, removing the polar organic solvent in the second chromatographic solution, allowing tacrolimus to crystallize in water, and then performing solid-liquid separation and drying. Refined tacrolimus can be obtained through the above steps.

[0071] The above-mentioned method for purifying crude tacrolimus has at least the following advantages:

[0072] (1) The above-mentioned method for purifying crude tacrolimus uses affinity chromatography resin to quickly separate ascomycin and dihydrotacrolimus, which are difficult to separate by other fillers, and then uses macroporous adsorption resin to quickly separate the remaining impurities, thereby improving the separation efficiency and the purity of the finished product, meeting the USP standard, and shortening the purification cycle. Experiments have shown that the purity of tacrolimus obtained by the above method can reach 99.6%, and the contents of ascomycin and dihydrotacrolimus are both less than 0.1%, exceeding the USP standard. The total yield of the whole process is greater than 70%, which greatly reduces the purification cost of crude tacrolimus.

[0073] (2) In the above-mentioned method for purifying crude tacrolimus, after the affinity resin chromatography removes ascomycin and dihydrotacrolimus, the second macroporous adsorption resin can easily and efficiently separate the remaining impurities, such as demethyl tacrolimus and 8-position diastereomers of tacrolimus, thereby greatly shortening the column purification cycle and improving the yield. At the same time, it can also remove the metal ions that may fall off from the previous affinity resin, avoiding the introduction of heavy metals, and is very suitable for industrial production.

[0074] (3) In the above-mentioned purification method of crude tacrolimus, the first eluent used in affinity resin chromatography can be a single solvent acetone in addition to a mixed solvent. Single solvent elution does not require re-mixing, avoiding tedious operations such as gas phase and liquid phase detection to determine the solvent ratio, and also eliminating the step of transferring the solvent for the second chromatography, which significantly improves the standardization of process operations and greatly reduces labor costs.

[0075] In order to make the purpose and advantages of the present invention clearer, the purification method of crude tacrolimus of the present invention and its effect are further described in detail below in conjunction with specific examples. It should be understood that the specific examples described herein are only used to explain the present invention and shall not be used to limit the present invention. The following examples do not include other components except inevitable impurities unless otherwise specified. The drugs and instruments used in the examples are conventionally selected in the art unless otherwise specified. The experimental methods for which specific conditions are not specified in the examples are implemented according to conventional conditions, such as the conditions described in the literature and books or the methods recommended by the manufacturer.

[0076] The crude tacrolimus used in the following examples and comparative examples was obtained by fermentation and extraction by Fujian Kerui Pharmaceutical Co., Ltd., with specific reference to patent CN108929335A. The organic solvents used, such as methanol, acetone, ethanol, and ethyl acetate, were all commercially available industrial-grade reagents. + The coordination resins and other materials are all produced by Shanghai Huazhen Technology Co., Ltd. XAD-1180 comes from Shanghai Yuanye Biotechnology Co., Ltd.

[0077] The high performance liquid chromatograph used in the following examples and comparative examples is Shimadzu LC2010CHT. In the embodiment, the HPLC detection conditions of tacrolimus crude product and concentrate are as follows: the chromatographic column is Agilent ZORBAX SB-CN column, and the chromatographic column specifications are 5μm, 4.6mm×250mm. The mobile phase volume ratio is a mixed solution of acetonitrile: 0.1% phosphoric acid aqueous solution = 40:60, the detection wavelength is 210nm, the column temperature is 40°C, and the flow rate is 1.5mL / min. Under the chromatographic detection conditions, RT (retention time) tacrolimus = 21.4min, RT ascomycin = 18.7min, RT demethyl tacrolimus = 19.4min, RT dihydro tacrolimus = 24.9min, and RT tacrolimus 8-position isomer = 26.1min.

[0078] The HPLC detection method used for purified tacrolimus in the following examples and comparative examples is as follows: detection wavelength is 220nm, C18 column (4.6mm×150mm, 3μm), flow rate is 1mL / min, column box temperature is 60°C, and injection volume is 20μL. Solution A is a 6mM phosphoric acid solution; solution B is acetonitrile: tert-butyl methyl ether = 81:19 (volume ratio); solution C is solution A: solution B = 4:1 (volume ratio); solution D is solution A: solution B = 1:4 (volume ratio). The mobile phase is solution C and solution D, and the elution method is shown in Table 1 below. Under the chromatographic detection conditions, RT tacrolimus = 27.6min, RT ascomycin = 24.3min, RT dihydrotacrolimus = 34.9min, 14.7min and 17.3min are isomer 1 and isomer 2, respectively, which are included in the tacrolimus content.

[0079] Table 1 Elution method in HPLC detection of purified tacrolimus

[0080] Time (minutes) Solution C (volume percentage) Solution D (volume percentage) 0 72 28 30 72 28 53 15 85 54 72 28 60 72 28

[0081] Example 1

[0082] This embodiment provides a method for purifying crude tacrolimus, comprising the following steps:

[0083] 120 g of crude tacrolimus was weighed and dissolved in 500 mL of acetone to obtain the first column liquid. The concentration of tacrolimus in the first column liquid was measured by liquid phase detection to be 195.1 mg / mL. 50 g (255 mL) of the column liquid was loaded onto an affinity resin chromatography column. The affinity resin chromatography column was filled with 10 kg of Ag +Coordination resin. The first eluent is pure acetone, and the elution flow rate is 4BV / h. Ascomycin and dihydrotacrolimus will be eluted first. After about 2BV, these two impurities are completely eluted. Then the purification begins, and the pure acetone is purified until the elution of tacrolimus is completed, and the purification ends after about 10BV. The purified liquid is collected to obtain the first chromatographic liquid, which is heated and concentrated to 250mL at normal pressure to obtain a concentrated liquid. The concentration of tacrolimus in the concentrated liquid is measured by liquid chromatography to be 194.3mg / mL, and the yield is 97.6%.

[0084] Take 155mL (30g) of the concentrated solution as the second column liquid and perform the second step of macroporous adsorption resin chromatography. The resin filler is HZ818 resin, the resin loading is 7.8L, and the height-to-diameter ratio is 10:1. An acetone / water mixture with an acetone volume percentage of 42% is used as the mobile phase, and the elution flow rate is 1BV / h. After eluting about 10BV, acetone / water with an acetone volume concentration of 50% is used for purification, and HPLC detection is performed on the liquid under the resin column at regular intervals. When the proportion of a single impurity is higher than 0.3%, the purification is terminated. The eluted effluent with a tacrolimus content of more than 98.5% is collected to obtain a second chromatographic solution. The second chromatographic solution is concentrated under reduced pressure, the acetone is evaporated, and tacrolimus crystals are precipitated in the water layer. After solid-liquid separation, it is dried to obtain 23.5g of tacrolimus crystalline finished product, and the tacrolimus yield is about 76%. According to HPLC test, the purity of tacrolimus exceeds 99.7%, and the contents of ascomycin and dihydrotacrolimus are both less than 0.1%, exceeding the USP standard, and almost all of them are separated cleanly. The total yield of tacrolimus is about 74%.

[0085] Example 2

[0086] This embodiment provides a method for purifying crude tacrolimus, comprising the following steps:

[0087] Weigh 65 g of crude tacrolimus and dissolve it in 250 mL of ethyl ester to obtain the first column liquid. The tacrolimus concentration in the first column liquid is 210.3 mg / mL by liquid phase detection. 240 mL of the first column liquid is measured and added to the affinity chromatography column. The affinity chromatography column is filled with 10 kg of Ag. + Coordination resin. The first eluent is an ethanol / ethyl acetate mixture (volume ratio is ethanol:ethyl acetate=30:70), the elution flow rate is 5BV / h, ascomycin and dihydrotacrolimus will be eluted first, and after about 2.5BV, these two impurities are completely eluted. Then, the purification begins until the elution of tacrolimus is completed, and the purified liquid is collected to obtain the first chromatographic liquid, which is heated and concentrated at normal pressure to obtain a concentrated liquid. The concentration of tacrolimus in the concentrated liquid is measured by liquid chromatography to be 204.5mg / mL, and the yield is 97.2%.

[0088] Take 147mL (30g) of the concentrated solution as the second column liquid and perform the second step of macroporous adsorption resin chromatography. The resin filler is Amberlite XAD@1180 non-ionic porous resin, the resin loading is 10L, and the height-diameter ratio is 13:1. An acetone / water mixture with a volume percentage of 40% acetone is used as the second eluent, and the flow rate of the second eluent is 1BV / h. After eluting about 15BV, an acetone / water mixture with a volume percentage of 50% acetone is used for purification, and the liquid under the resin column is regularly subjected to HPLC detection. When the proportion of a single impurity is higher than 0.3%, the purification is terminated. The eluted effluent with a tacrolimus content of more than 98.5% is collected to obtain a second chromatographic solution. The second chromatographic solution is concentrated under reduced pressure to evaporate the acetone, and tacrolimus crystals are precipitated in the water layer. After solid-liquid separation, it is dried to obtain 22.6g of tacrolimus crystalline finished product, and the tacrolimus yield is about 73%. According to HPLC detection, the purity of tacrolimus can reach 99.6%, and the contents of ascomycin and dihydrotacrolimus are both less than 0.1%, meeting the USP standard. The total yield of tacrolimus is about 73%.

[0089] Comparative Example 1

[0090] Comparative Example 1 provides a method for purifying a crude tacrolimus product, comprising the following steps:

[0091] Weigh an appropriate amount of tacrolimus crude product and dissolve it in acetone to obtain an upper column liquid. Liquid phase detection determines that the tacrolimus concentration in the upper column liquid is 195.1 mg / mL. Measure 15 mL (30 g) of the upper column liquid and directly perform macroporous adsorption resin chromatography. The resin filler is Amberlite XAD-1180 non-ionic porous resin, the resin loading is 10 L, and the height-to-diameter ratio is 13:1. Three columns are connected in series. A tetrahydrofuran / water solution with a tetrahydrofuran volume concentration of 34% is used as the eluent, and the flow rate of the eluent is 1 BV / h. After eluting about 50 BV, the liquid under the resin column is regularly tested by HPLC. When the ascomycin ratio is lower than 0.5%, purification begins, and the eluted effluent with a tacrolimus content of more than 98.5% is collected. When the dihydrotacrolimus ratio is higher than 0.5%, purification ends to obtain a chromatographic liquid. The chromatographic solution is concentrated under reduced pressure, the organic solvent is evaporated, and the tacrolimus in the extraction water layer is dissolved in ethyl acetate. After reduced pressure concentration, an appropriate amount of cyclohexane is added to precipitate crystals. The crystals are collected and vacuum dried to obtain the tacrolimus product. According to liquid phase detection, the purity of tacrolimus can reach 99.5%, the ascomycin content is higher than 0.1%, and the yield is only 27.5%.

[0092] The liquid chromatogram of the crude tacrolimus in Example 1 is as follows: Figure 2 As shown, the liquid chromatogram of the first chromatographic liquid is as follows Figure 3 As shown, the liquid chromatogram of the purified tacrolimus crystals is as shown Figure 4 In Example 2, the liquid chromatogram of the purified tacrolimus crystals is as shown in Figure 5In Comparative Example 1, the liquid chromatogram of the purified tacrolimus crystals is shown in Figure 6 shown.

[0093] By comparing the first chromatographic solution and the finished crystallization liquid chromatography in Example 1, it can be seen that the use of Ag ion coordination resin or modified silica gel alone can only specifically remove impurities such as ascomycin and dihydrotacrolimus, but cannot remove all impurities, and there is about 1% of tacrolimus 8-position diastereomer and other impurities. However, through the combined purification of affinity chromatography resin and non-ionic macroporous adsorption resin, all impurities can be effectively separated to obtain ultra-high purity tacrolimus exceeding the USP standard.

[0094] By comparing Example 2 with Comparative Example 1, it can be seen that the non-ionic macroporous adsorption resin used in the background technology requires multiple resins to be connected in series to effectively separate impurities and obtain tacrolimus that meets the USP standard, but the purity is not as good as that of Example 1 and Example 2 of the present invention. At the same time, the cycle is long, the yield is extremely low, the cost is high, and it is not suitable for industrial production.

[0095] It can be seen from the comparison of the above embodiments and comparative examples that:

[0096] (1) In the above purification method, the first chromatography treatment uses affinity chromatography resin, which can quickly separate ascomycin and dihydrotacrolimus. The second chromatography treatment uses macroporous resin, which can remove the remaining impurities that cannot be separated by the first chromatography, and can also remove the silver ions eluted from the first chromatography column, thereby avoiding heavy metal pollution. At the same time, the production process is simplified and the cost is reduced.

[0097] (2) Pure acetone is used as the eluent for affinity resin chromatography. Compared with dual solvents, the steps of preparing and adjusting the eluent are omitted. The solvent can be directly used after recovery without further preparation. The concentrated solution is directly used as the column liquid for the next macroporous adsorption resin column without the need for solvent transfer.

[0098] (3) Compared with the traditional purification method, such as comparative example 1, the macroporous adsorption resin in the second chromatography treatment only needs one column instead of three columns in series to achieve the same or even higher purity. At the same time, the yield is greater than 70%, which is much higher than the yield of 27.5% of pure macroporous resin purification, and the cycle is shortened.

[0099] The technical features of the above-described embodiments may be arbitrarily combined. To make the description concise, not all possible combinations of the technical features in the above-described embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0100] The above-described embodiments only express several implementation methods of the present invention, which is convenient for understanding the technical solutions of the present invention in detail, but cannot be understood as limiting the scope of protection of the invention patent. It should be pointed out that, for ordinary technicians in this field, several variations and improvements can be made without departing from the concept of the present invention, which all belong to the protection scope of the present invention. It should be understood that the technical solutions obtained by those skilled in the art through logical analysis, reasoning or limited experiments on the basis of the technical solutions provided by the present invention are all within the protection scope of the claims attached to the present invention. Therefore, the protection scope of the patent of the present invention shall be based on the contents of the attached claims, and the description and drawings can be used to interpret the contents of the claims.

Claims

1. A method for purifying crude tacrolimus, characterized in that: The steps include: Obtaining an upper column liquid containing a crude tacrolimus product, wherein the crude tacrolimus product comprises tacrolimus, ascomycin, dihydrotacrolimus, desmethyltacrolimus and tacrolimus 8-position diastereomer; The upper column liquid is subjected to a first chromatography treatment using an affinity resin chromatography column to remove ascomycin and dihydrotacrolimus to obtain a first chromatography liquid, wherein the affinity resin chromatography column contains metal ions that can coordinate with tacrolimus; The first chromatographic solution is subjected to a second chromatography treatment using a macroporous adsorption resin to remove demethyl tacrolimus and tacrolimus 8-position diastereomer to obtain purified tacrolimus.

2. The method for purifying crude tacrolimus according to claim 1, characterized in that: The filler of the affinity resin chromatography column is selected from Ag + Coordination resin and Cu 2+ Either or both of the coordination resins.

3. The method for purifying crude tacrolimus according to claim 1 or 2, characterized in that: The first eluent used in the first chromatography treatment is selected from any one of methanol, ethanol, ethyl acetate and acetone or a mixed solvent of two thereof; Optionally, the flow rate of the first eluent is 0.5 BV / h to 20 BV / h.

4. The method for purifying crude tacrolimus according to claim 3, characterized in that: The first eluent is acetone; or, The first eluent is a mixed solvent of ethanol and ethyl acetate, in which the volume percentage of ethanol is 5% to 50%; or, The first eluent is a mixed solvent of methanol and ethyl acetate, and the volume percentage of methanol in the mixed solvent is 5% to 50%.

5. The method for purifying crude tacrolimus according to claim 1, characterized in that: The macroporous adsorption resin is selected from any one or more of HP20, HZ820, HZ816, Amberlite XAD-1180 and HZ818.

6. The method for purifying crude tacrolimus according to claim 1 or 5, characterized in that: The second eluent used in the second chromatography treatment is a mixed solution of a polar organic solvent and water, wherein the volume percentages of the polar organic solvent and the water in the second eluent are 20% to 50% and 50% to 80% respectively; Optionally, the polar organic solvent is selected from any one or more of methanol, ethanol and acetone; Optionally, during elution with the second eluent, a gradient elution method is adopted; Optionally, the flow rate of the second eluent is 0.5 BV / h to 2 BV / h.

7. The method for purifying crude tacrolimus according to claim 6, characterized in that: After the step of performing a second chromatography treatment on the first chromatographic liquid using a macroporous adsorption resin, the method also includes: distilling the second chromatographic liquid obtained after removing demethyl tacrolimus and tacrolimus 8-position diastereomer, removing the polar organic solvent in the second chromatographic liquid, allowing tacrolimus to crystallize in water, and then separating the solid and liquid and drying.

8. The method for purifying crude tacrolimus according to claim 1, characterized in that: During the first chromatography treatment, the chromatographic solution containing ascomycin in the solute at a mass percentage of less than 1% is collected in stages; and, During the second chromatography treatment, the chromatographic fluid containing tacrolimus greater than 98.5% by mass in the solute is collected in sections.

9. The method for purifying crude tacrolimus according to claim 1, characterized in that: In the upper column liquid, the concentration of tacrolimus is 50 mg / mL to 300 mg / mL; and / or, In the upper column liquid, the solvent is selected from any one or more of methanol, ethanol, acetone and ethyl acetate.

10. The method for purifying crude tacrolimus according to claim 1, characterized in that: Before the step of performing a second chromatography treatment on the first chromatographic solution using a macroporous adsorption resin, the method further includes: concentrating the first chromatographic solution so that the concentration of tacrolimus in the concentrated solution is 50 mg / mL to 300 mg / mL.

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