CN113912624B - Tacrolimus separation and purification method - Google Patents

Abstract

The invention provides a method for separating and purifying tacrolimus, comprising: 1) filtering the tacrolimus fermentation liquid with a ceramic membrane to obtain a fermented liquid concentrated liquid; 2) adding ethanol to the fermented liquid concentrated liquid to obtain a mixed system, and then add resin to the mixed system for adsorption; 3) use ethanol solution to elute the adsorbed resin to obtain the crude product of tacrolimus chromatography; 4) sequentially process the crude product of tacrolimus chromatography Perform primary crystallization, preparative column separation, and secondary crystallization to obtain pure tacrolimus. The separation and purification method is simple in operation, low in cost, and can obtain tacrolimus with high purity and high yield.

Description

A kind of separation and purification method of tacrolimus

technical field

The invention belongs to the field of biopharmaceuticals and relates to a method for separating and purifying tacrolimus.

Background technique

Tacrolimus, also known as FK506, is a fermentation product isolated from Streptomyces. It is a macrolide antibiotic and a powerful new immunosuppressant, mainly by inhibiting interleukin-2 (IL -2), thereby comprehensively inhibiting T lymphocytes, and its immunosuppressive activity is 10-100 times that of the traditional immune agent cyclosporin A. Although the total synthesis method of tacrolimus has been reported in the literature, the steps are cumbersome, costly, and difficult, and it is difficult to apply it to industrial production.

At present, tacrolimus is mainly obtained by isolation and extraction from fermentation broth, but most of the strains capable of producing tacrolimus will also produce ascomycin and some other macrolide impurities during the fermentation process, which are related to The structure of tacrolimus is very similar, which greatly increases the difficulty of extracting tacrolimus from the fermentation broth. In recent years, there are more and more methods for the research and separation and purification of tacrolimus, mainly focusing on the extraction of fermented mycelia, separation of macroporous resins, silica gel chromatography, high-speed countercurrent chromatography, reversed-phase column chromatography, Silver salt modified silica gel or resin chromatography, normal phase column preparation, etc. However, these methods will undoubtedly use high-cost fillers and complex mixed solvent systems, and some even use acetonitrile, tetrahydrofuran and other toxic solvents , is only suitable for laboratory research and is difficult to apply to industrial production. Therefore, how to separate and purify tacrolimus from the fermentation broth with high purity and high yield simply and at low cost has always been an urgent problem to be solved in this field.

Contents of the invention

The invention provides a method for separating and purifying tacrolimus. The method is simple to operate, can obtain high-purity and high-yield tacrolimus at low cost, and is expected to be used in the industrialized production of tacrolimus.

The invention provides a method for separation and purification of tacrolimus, comprising the following steps:

1) Filtrating the tacrolimus fermentation broth with a ceramic membrane to obtain a fermented broth concentrate;

2) adding ethanol to the fermented broth concentrate to obtain a mixed system, and then adding resin to the mixed system for adsorption;

3) eluting the adsorbed resin with ethanol solution to obtain the crude product of tacrolimus chromatography;

4) The chromatographic crude product of tacrolimus is sequentially subjected to primary crystallization, preparative column separation, and secondary crystallization to obtain pure tacrolimus.

The above separation and purification method, wherein, in step 2), the volume concentration of ethanol in the mixed system is 30-35%.

The separation and purification method as described above, wherein, in step 3), the use of ethanol solution to elute the adsorbed resin includes:

1) packing the adsorbed resin into a column to obtain a first resin column, and sequentially connecting the first resin column with a second resin column and a third resin column;

The second resin column is the same as the resin in the first resin column and is a polar macroporous resin;

The resin in the third resin column is a non-polar macroporous resin;

2) The first ethanol solution is used to elute the adsorbed resin, and after tacrolimus is desorbed from the second resin column, the second ethanol solution is used to elute the third resin column alone to obtain the Chromatographic crude product of tacrolimus;

The volume concentration of the first ethanol solution is smaller than the volume concentration of the second ethanol solution.

The separation and purification method as described above, wherein the polar macroporous resin is selected from at least one of Huazhen HZ806, Huazhen HZ826, and Huazhen HP2MG.

The separation and purification method as described above, wherein the volume concentration of the first ethanol solution is 40-60%; and/or,

The volume concentration of the second ethanol solution is 65-80%.

The separation and purification method as described above, wherein the volume ratio of the resin in the first resin column to the resin in the second resin column and the resin in the third resin column is 1:(2～3) : (5～8).

The separation and purification method as described above, wherein, the non-polar macroporous resin is selected from at least one of Mitsubishi HP20, Huazhen Chromatographic No. 3, Huazhen HZ818, Huazhen HZ816, and Huazhen HZ820.

The separation and purification method as described above, wherein the primary crystallization includes: dissolving the crude tacrolimus chromatography product in toluene at 50°C to a mass concentration of 10-20 mg/L, and then cooling down to room temperature for chromatographic analysis. After 12-15 hours of crystallization, a crude product of primary crystallization was obtained.

The separation and purification method as described above, wherein the preparative column separation includes: dissolving the crude product obtained after the primary crystallization, using polyamide resin as a preparative column filler to perform the preparative column separation, and collecting the tacrolimus fraction ;

The chromatographic conditions for the separation of the preparative column are: wavelength: 214nm; chromatographic column size: 50mm×250mm; mobile phase: butyl acetate or a mixed solvent of toluene and acetone; sample loading: 20g/L.

The separation and purification method as described above, wherein the secondary crystallization comprises: concentrating the tacrolimus fraction to a tacrolimus concentration of 180-220 mg/mL, standing for crystallization at 30°C, and filtering to obtain The pure tacrolimus.

The tacrolimus separation and purification method provided by the present invention can remove most of the pigments and polar impurities in the tacrolimus fermentation broth through ceramic membrane filtration, and then dilute with ethanol, resin adsorption, elution, primary crystallization, Technical means such as preparative column separation and secondary crystallization can further remove pigments and other impurities in the fermentation broth, greatly improving the purity of tacrolimus. The tacrolimus with a purity of more than 99% can be obtained with a relatively high yield by using the method of the invention.

Description of drawings

Figure 1 is a liquid chromatogram of pure tacrolimus in Example 3.

Detailed ways

In order to make the purpose, technical solutions and advantages of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are part of the implementation of the present invention. example, not all examples. Based on the embodiments of the present invention, all other embodiments obtained by persons of ordinary skill in the art without making creative efforts belong to the protection scope of the present invention.

The invention provides a method for separation and purification of tacrolimus, comprising the following steps:

1) Filtrating the tacrolimus fermentation broth with a ceramic membrane to obtain a fermented broth concentrate;

2) adding ethanol to the fermented broth concentrate to obtain a mixed system, and then adding resin to the mixed system for adsorption;

3) eluting the adsorbed resin with ethanol solution to obtain the crude product of tacrolimus chromatography;

4) The chromatographic crude product of tacrolimus is subjected to primary crystallization, preparative column separation, and secondary crystallization to obtain pure tacrolimus.

The tacrolimus fermentation broth used in the present invention is a Streptomyces fermentation broth, and the purity of Tacrolimus in the Streptomyces fermentation broth is usually 55-65%. In addition to pigments and large polar impurities, it also includes ascomycin and Tacrolimus isomers and other impurities that are very similar in structure to tacrolimus greatly increase the difficulty of separation and purification of tacrolimus.

In step 1), the pigment and large polar impurities in the fermentation broth can be separated by using ceramic membrane filtration. After filtering through the ceramic membrane, the concentrated fermented liquid with increased concentration and concentrated volume can be obtained. The yield of tacrolimus in the concentrated fermented liquid obtained after being filtered through the ceramic membrane is almost no loss, and the content of tacrolimus in the concentrated fermented liquid is consistent with that in the fermented liquid.

In step 2), ethanol is added to the concentrated solution of the fermentation broth to obtain a mixed system, and then resin is added to the mixed system for adsorption, so that the system can form a dynamic equilibrium of dissolution-adsorption. Specifically, ethanol can dissolve a certain amount of tacrolimus in the concentrated solution of the fermentation broth. At this time, the tacrolimus dissolved in the ethanol system can be adsorbed by the resin, and the adsorption of the resin can promote the concentration of tacrolimus in the concentrated solution of the fermentation solution. The dissolution of crolimus in ethanol forms a reciprocating cycle, and most of the tacrolimus in the fermented liquid concentrate can be adsorbed by the resin.

When it is detected that the content of tacrolimus in the concentrated solution of the fermentation broth is less than 20 μg/mL, it can be determined that the adsorption is complete. At this time, the adsorbed resin can be filtered out with a sieve, and the pigment and large polar impurities adsorbed on the resin surface can be washed with a small amount of water.

In step 3), ethanol solution is used to elute the adsorbed resin. This elution is a gradient elution process, which can further remove some impurities and pigments. It can be understood that the eluate obtained after elution needs to be concentrated to obtain the crude product of tacrolimus chromatography. After elution, the purity of the obtained crude tacrolimus is 90-94%.

In step 4), the crude product of tacrolimus chromatography is first crystallized once, and the pigment impurities in the crude product can be further removed by the first crystallization, so that the color and purity of tacrolimus can be further improved. After the first crystallization, tacrolimus The content of limus can reach the same chromatographic purity as tacrolimus, but impurities similar in structure to tacrolimus, such as ascomycin isomers and dihydrotacrolimus, cannot be separated. The primary crystallized crude product is separated by a preparative column, and impurities such as dihydrotacrolimus can be separated, and the purity of tacrolimus can reach 96-99%. The crude tacrolimus separated by the preparative column is subjected to secondary crystallization to obtain a pure tacrolimus with a purity >99%.

It should be noted that for the purity of tacrolimus, the Pharmacopoeia standard is to combine tacrolimus, tacrolimus 19-epimer and open-ring tacrolimus, so the And the pure tacrolimus with a purity >99% means that the total content of tacrolimus, tacrolimus 19-epimer and ring-opened tacrolimus is >99%.

The tacrolimus separation and purification method provided by the present invention does not need to use high-cost fillers such as silver salt-modified silica gel and resin, and can be filtered through ceramic membranes, resin adsorption, gradient elution, primary crystallization, preparation column separation, and secondary crystallization. and other steps, the pure tacrolimus can be obtained by separating and obtaining pure tacrolimus with high purity and high yield with simple operation and low cost.

Furthermore, when the pore size of the ceramic membrane is 50-100 μm, it is more conducive to the removal of pigments and large polar impurities.

The inventors have found that when the volume concentration of ethanol in the mixed system is 30-35% in step 2), it is more conducive to the selective adsorption of tacrolimus in the fermentation broth by the resin, and higher purity and yield can be obtained. pure tacrolimus.

In a specific embodiment, in step 3), ethanol solution is used to elute the adsorbed resin, including:

1) packing the adsorbed resin into a column to obtain a first resin column, and connecting the first resin column in series with the second resin column and the third resin column;

Wherein, the resin in the second resin column is the same as the resin in the first resin column and is a polar macroporous resin; the resin in the third resin column is a non-polar macroporous resin;

2) The first ethanol solution is used to elute the adsorbed resin, and after tacrolimus is desorbed from the second resin column, the second ethanol solution is used to elute the third resin column alone to obtain tacrolimus Chromatographic crude;

Wherein, the volume concentration of the first ethanol solution is smaller than the volume concentration of the second ethanol solution.

Tacrolimus is adsorbed in the first resin column, which is the component to be eluted, and the second resin column and the third resin column are used for tacrolimus in the resin for development layer chromatography. Use the first ethanol solution with a smaller volume concentration to elute the adsorbed resin until tacrolimus is desorbed from the second resin column. At this time, most of the weakly polar impurities are retained in the first resin column. Strong impurities and some non-polar impurities. Separate the resin columns connected in series, and use the second ethanol solution with higher volume concentration to elute the third resin column alone, so as to obtain the separated and purified tacrolimus crude product by chromatography.

Further, the polar macroporous resin is selected from at least one of HZ806, Huazhen HZ826, and Huazhen HP2MG. The above-mentioned resins are medium-polar macroporous resins, which are more conducive to the adsorption of tacrolimus in the mixed system.

Further, the volume concentration of the first ethanol solution is 40-60%; and/or, the volume concentration of the second ethanol solution is 65-80%.

Further, when the resin volume ratio in the first resin column, the second resin column, and the third resin column is 1:(2-3):(5-8), better elution effect can be obtained, and the obtained The crude product of tacrolimus chromatography was more pure.

The non-polar macroporous resin used in the third resin column can be selected from non-polar macroporous resins commonly used in this field, including but not limited to Mitsubishi HP20, Huazhen chromatography No. 3, Huazhen HZ818, Huazhen HZ816, Huazhen At least one of the vibration HZ820.

In a specific embodiment, the primary crystallization includes: dissolving the chromatographic crude product of tacrolimus in toluene at 50°C to a mass concentration of 10-20 mg/L, and then cooling down to room temperature for 12-15 hours of crystallization, A crystalline crude product was obtained. Carrying out the primary crystallization under the above conditions can make the purity of the primary crystallization product higher.

In a specific embodiment, the preparative column separation includes: dissolving the product obtained after primary crystallization, using polyamide resin as a preparative column packing for preparative column separation, and collecting tacrolimus fractions.

Among them, the chromatographic conditions for preparative column separation are: wavelength: 214nm; chromatographic column size: 50mm×250mm; mobile phase: butyl acetate or a mixed solvent of toluene and acetone; sample loading: 20g/L.

The inventors found in the research that the preparative column separation under the above conditions can not only obtain a higher purity and yield of tacrolimus, but also have a higher separation efficiency. And after the collected fractions of tacrolimus are tested by liquid phase, it can be detected that the purity of tacrolimus is >96%, and the content of other impurities in the fractions is <0.5% except for tacrolimus ascomycin isomers .

Specifically, when the mobile phase is selected from a mixed solvent of toluene and acetone during column separation, a better separation effect can be obtained when the volume ratio of toluene to acetone is 2:1.

In a specific embodiment, the secondary crystallization includes: concentrating the tacrolimus fraction to a tacrolimus concentration of 180-220 mg/mL, standing for crystallization at 30°C, and obtaining tacrolimus after filtration. Pure products. Under this crystallization condition, pure tacrolimus can be obtained with higher purity and yield.

Hereinafter, the separation and purification method of tacrolimus provided by the present invention will be further introduced in combination with specific examples. In the following examples, unless otherwise specified, all raw materials can be obtained commercially or by conventional methods.

Example 1

The separation and purification method of tacrolimus in this embodiment comprises the following steps:

1) Use a ceramic membrane to filter 30L tacrolimus fermentation broth (the content of tacrolimus in the fermentation broth is 1.054mg/mL, as determined by HPLC, the fermentation broth contains 31.62g of tacrolimus, and the tacrolimus in the fermentation broth The purity is 63.45%), and use 90L of water to wash the filter cake to obtain ceramic membrane dope 10L. Wherein, the pore diameter of the ceramic membrane is 50 nm.

2) Add ethanol to the ceramic membrane dope until the volume concentration of ethanol in the dope is 30%, then add 3 L of Huazhen HZ806 resin, stir for 2 hours and then filter, rinse the filtered resin with a small amount of water, and wash the 3 L of Huazhen Pack the column with Zhen HZ806 resin to get the first resin column, then use 6L Huazhen HZ806 resin to pack the column to get the second resin column, and then use 15L Huazhen HZ818 resin to get the third resin column, and put the three resin columns according to the first resin column- The sequence of the second resin column-the third resin column is connected in series to obtain a series resin column, and the ethanol with a volume concentration of 40% is used as the eluent to elute the series resin column, and the output liquid eluted to the second resin column is detected After less than tacrolimus, the series resin column was separated, and the third resin column was eluted with ethanol with a volume concentration of 75%, and after the discharge liquid of the third resin column could not detect tacrolimus, the Take off is over.

3) After concentrating the eluent, obtain the crude tacrolimus product from chromatography, add toluene at 50°C to dissolve the crude product of tacrolimus, and control the concentration of tacrolimus in the solution to 10-20 mg/mL , then lower the temperature of the solution to 40 °C at a cooling rate of 1 °C/h, and then continue to cool the solution to 10-15 °C at a cooling rate of 3 °C/h, stand for 12 hours to crystallize, and obtain a crude crystallization product after filtration .

4) The primary crystallized crude product was dissolved in butyl acetate, washed with 0.5% NaHCO ₃ , 0.1% oxalic acid, and saturated brine respectively, dried over anhydrous sodium sulfate, and filtered, and the filtrate Concentrate, the concentrate is separated by a preparative column, eluted with butyl acetate as the mobile phase, and tacrolimus fractions are collected, and the yield of tacrolimus after separation by the preparative column is 80.67%;

Among them, the filler of the preparation column is polyamide resin, and the resin particle size is 30 μm, and the separation conditions of the preparation column are:

Wavelength: 220nm; Chromatographic column: 50×250mm; Flow rate: 20mL/min; Sample load: 20g/L.

5) Concentrate the combined liquid until the concentration of tacrolimus is 180-220 mg/mL, let it stand at 30°C for 48 hours to precipitate crystals, filter to obtain the secondary crystallization product, and vacuum-dry the secondary crystallization product at 40°C, 22.1 g of tacrolimus was obtained with a purity of 99.35% and a total yield of 69.89%.

Example 2

The separation and purification method of tacrolimus in this example is basically the same as that in Example 1, except that:

In step 1), the tacrolimus content in the tacrolimus fermentation broth used was 1.07 mg/mL, the fermentation broth contained 32.1 g of tacrolimus, and the tacrolimus purity in the fermentation broth was 64.12%.

In step 2), ethanol with a volume concentration of 50% is used as the eluent to elute the series resin column.

Finally, 22.1 g of tacrolimus was obtained with a purity of 99.09% and a total yield of 68.56%.

Example 3

The separation and purification method of tacrolimus in this example is basically the same as that in Example 1, except that:

In step 1), the content of tacrolimus in the tacrolimus fermentation broth used was 1.01 mg/mL, the fermentation broth contained 30.3 g of tacrolimus, and the purity of tacrolimus in the fermentation broth was 63.77%.

In step 2), ethanol with a volume concentration of 45% is used as the eluent to elute the series resin column.

Finally, 20.75 g of tacrolimus was obtained with a purity of 99.59% and a total yield of 68.48%.

In this example, after obtaining the crude product of tacrolimus chromatography, the resin in the first resin column was regenerated with absolute ethanol, the regenerated solution appeared red, and the regenerated resin returned to white.

Fig. 1 is the pure liquid chromatogram of tacrolimus of embodiment 3, after processing the spectrogram of Fig. 1, relevant information in Table 1 can be obtained:

Table 1

Example 4

The separation and purification method of tacrolimus in this example is basically the same as that in Example 1, except that:

In step 1), the content of tacrolimus in the tacrolimus fermentation broth used was 1.14 mg/mL, the fermentation broth contained 34.2 g of tacrolimus, and the purity of tacrolimus in the fermentation broth was 63.97%.

In step 2), the macroporous resin in the third resin column is selected from Mitsubishi HP20 resin.

Finally, 23.94 g of tacrolimus was obtained with a purity of 99.45% and a total yield of 70.0%.

Example 5

The separation and purification method of tacrolimus in this example is basically the same as that in Example 4, except that:

In step 1), the tacrolimus content in the tacrolimus fermentation broth used was 1.113 mg/mL, the fermentation broth contained 33.39 g of tacrolimus, and the tacrolimus purity in the fermentation broth was 64.58%.

In step 2), the macroporous resin in the first resin column and the second resin column is selected from Huazhen HP2MG resin, and ethanol with a volume concentration of 55% is used as the eluent to elute the series resin column.

Finally, 21.32 g of tacrolimus was obtained with a purity of 99.10% and a total yield of 63.85%.

Example 6

The separation and purification method of tacrolimus in this example is basically the same as that in Example 3, except that:

In step 1), the content of tacrolimus in the tacrolimus fermentation broth used was 1.215 mg/mL, the fermentation broth contained 36.45 g of tacrolimus, and the purity of tacrolimus in the fermentation broth was 64.78%.

In step 4), a mixed solvent of toluene and acetone with a volume ratio of 2:1 is used as the mobile phase for elution.

Finally, 22.57 g of tacrolimus was obtained with a purity of 99.24% and a total yield of 61.92%.

Examples 7-10

The separation and purification methods of tacrolimus in Examples 7 to 10 are basically the same as in Example 3, except that the sample loading of the preparation column in Example 7 is 10 g/L, and the sample loading of the preparation column in Example 8 is 15g/L, the sample loading capacity of the preparation column of Example 9 is 20g/L, and the sample loading capacity of the preparation column of Example 10 is 25g/L.

Wherein, the purity and yield of the tacrolimus products obtained by separation and purification in Examples 7-10 are shown in Table 2.

Table 2

Sample loading g/L purity% Yield% Example 7 10 99.88 69.11 Example 8 15 99.78 68.87 Example 9 20 99.70 68.05 Example 10 25 99.20 66.89

It can be seen from Table 2 that when the sample loading gradually increased, the purity and yield of tacrolimus also decreased, and when the sample loading was 25g/L, the yield of tacrolimus decreased significantly. When the sample load is 20g/L, it can not only ensure high separation efficiency of the preparative column, but also obtain high purity and yield of tacrolimus after separation.

Example 12

The separation and purification method of tacrolimus in this example is basically the same as in Example 3, except that in step 2), non-polar Mitsubishi HP20 resin is added for adsorption, and the resin in the second resin column is also selected From non-polar Mitsubishi HP20 resin.

The tacrolimus obtained through separation and purification in this example has a purity of 99.10% and a total separation yield of 62.30%.

Example 13

The separation and purification method of tacrolimus in this example is basically the same as that in Example 3, except that in step 3), butyl acetate is used to dissolve tacrolimus and perform primary crystallization.

The tacrolimus obtained through separation and purification in this example has a purity of 99.31%, and a total separation yield of 58.42%.

Compared with Example 3 in this example, the purity of the separated and purified tacrolimus is basically the same, but due to the better solubility of butyl acetate, the total yield of tacrolimus is significantly lower than that of Example 3.

Example 14

The separation and purification method of tacrolimus in this example is basically the same as that in Example 3, except that in step 4), the mixed solvent of ethyl acetate and n-hexane is used as the mobile phase preparation column for separation in the preparative column separation, wherein The volume ratio of ethyl acetate to n-hexane is 55:45.

The purity of the tacrolimus obtained through separation and purification in this example was 99.38%, and the total yield of separation was 55.44%.

Compared with Example 3 in this example, the purity of the isolated and purified tacrolimus is roughly the same, the total yield of separation is significantly lower than that of Example 3, and the recycling cost of the mixed solvent is higher than that of acetic acid in Example 3 Butyl ester mobile phase.

Example 15

The separation and purification method of tacrolimus in this example is basically the same as that in Example 3, except that in step 2), ethanol is added to the fermented broth concentrate to the volume of ethanol in the mixed system of the fermented broth concentrate and ethanol The concentration is 20%.

The purity of the tacrolimus obtained through separation and purification in this example was 99.47%, and the total yield of separation was 59.07%.

Example 16

The separation and purification method of tacrolimus in this example is basically the same as that in Example 3, except that in step 2), ethanol is added to the fermented broth concentrate to the volume of ethanol in the mixed system of the fermented broth concentrate and ethanol The concentration is 50%.

The purity of the tacrolimus obtained through separation and purification in this embodiment was 99.01%, and the total separation yield was 67.99%.

Comparative example 1

The separation and purification method of tacrolimus in this comparative example is basically the same as in Example 3, the difference is that in step 1), filter paper is used to filter the tacrolimus fermentation broth, and the filter residue obtained after filtration is extracted with ethanol to extract The volume concentration of ethanol in the liquid is 30%, and then resin is added to the extract for adsorption.

After the chromatographic crude product of tacrolimus was obtained, the resin in the first resin column was regenerated with absolute ethanol. Wherein, the regenerated solution is black, and the regenerated resin is light red. Continue to use sodium hydroxide solution for treatment, and the color of the resin returns to white.

The purity of the tacrolimus obtained by separation and purification in this comparative example was 99.13%, and the total separation yield was 66.03%.

Through the comparison of Example 3 and Comparative Example 1, it can be known that the filter residue after filtering and washing with ceramic membranes removed most of the pigments and impurities, reduced the probability of resin adsorption of impurities, and the color of the resin desorption solution became significantly lighter. The efficiency and purity are also significantly higher than those obtained by filter paper filtration.

However, the filter paper filtration method cannot remove most of the pigments in the fermentation broth during filtration, resulting in a significantly lower yield and purity of tacrolimus, and the resin cannot be restored to white after regeneration with absolute ethanol, and hydrogen oxidation is required. The sodium solution continues to be regenerated, resulting in increased separation costs.

Comparative example 2

The separation and purification method of tacrolimus in this comparative example is basically the same as that in Example 3, the difference is that in step 2) in this comparative example, ethanol is not added to dilute the tacrolimus fermented liquid concentrate, and the resin is directly used for adsorption.

The purity of the tacrolimus obtained through separation and purification in this comparative example was 99.15%, and the total separation yield was 43.21%.

Through the comparison of Example 3 and Comparative Example 2, it can be seen that if ethanol is not added to the dope before resin adsorption, it is difficult for the resin to adsorb tacrolimus in the dope, and the purity and yield of the finally separated tacrolimus are all low. Low.

Comparative example 5

The separation and purification method of tacrolimus in this comparative example is basically the same as that in Example 3, except that in step 2), ethanol is directly added to the concentrated solution of the fermentation broth until the volume concentration of ethanol is 30%, stirred for 2 hours, filtered, and the filtrate is directly passed through The second and third resin columns were subjected to expansion chromatography.

The purity of the tacrolimus obtained by separation and purification in this comparative example was 98.14%, and the total separation yield was 57.21%.

The above embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those of ordinary skill in the art should understand that: it can still be applied to the foregoing embodiments The technical solutions described in the examples are modified, or some or all of the technical features are equivalently replaced; and these modifications or replacements do not make the essence of the corresponding technical solutions depart from the scope of the technical solutions of the embodiments of the present invention.

Claims (9)

1. A method for separating and purifying tacrolimus, comprising the following steps: 1) Filtrating the tacrolimus fermentation broth with a ceramic membrane to obtain a fermented broth concentrate; 2) adding ethanol to the fermented broth concentrate to obtain a mixed system, and then adding resin to the mixed system for adsorption; 3) eluting the adsorbed resin with ethanol solution to obtain the crude product of tacrolimus chromatography; 4) performing primary crystallization on the chromatographic crude product of tacrolimus in sequence, separation on a preparative column, and secondary crystallization to obtain pure tacrolimus; The primary crystallization includes: dissolving the chromatographic crude product of tacrolimus in toluene at 50°C to a mass concentration of 10-20 mg/L, and then cooling down to room temperature for crystallization for 12-15 hours to obtain the crude product of primary crystallization; The preparative column separation includes: dissolving the crude product obtained after the primary crystallization, using polyamide resin as a preparative column filler to perform the preparative column separation, and collecting tacrolimus fractions; The chromatographic conditions for the preparation column separation are: wavelength: 214nm; chromatographic column size: 50mm×250mm; mobile phase: butyl acetate or a mixed solvent of toluene and acetone, sample loading: 20g/L; The secondary crystallization includes: concentrating the tacrolimus fraction to a tacrolimus concentration of 180-220 mg/mL, standing for crystallization at 30° C., and obtaining the pure tacrolimus after filtration. 2. The separation and purification method according to claim 1, characterized in that, in step 2), the volume concentration of ethanol in the mixed system is 30-35%. 3. The separation and purification method according to claim 1 or 2, characterized in that, in step 3), the use of ethanol solution to elute the adsorbed resin comprises: 1) packing the adsorbed resin into a column to obtain a first resin column, and sequentially connecting the first resin column with a second resin column and a third resin column; The second resin column is the same as the resin in the first resin column and is a polar macroporous resin; The resin in the third resin column is a non-polar macroporous resin; 2) The first ethanol solution is used to elute the adsorbed resin, and after tacrolimus is desorbed from the second resin column, the second ethanol solution is used to elute the third resin column alone to obtain the Chromatographic crude product of tacrolimus; The volume concentration of the first ethanol solution is smaller than the volume concentration of the second ethanol solution. 4. The separation and purification method according to claim 3, wherein the polar macroporous resin is selected from at least one of Huazhen HZ806, Huazhen HZ826, and Huazhen HP2MG. 5. The separation and purification method according to claim 3, characterized in that, the volume concentration of the first ethanol solution is 40-60%; and/or, The volume concentration of the second ethanol solution is 65-80%. 6. The separation and purification method according to claim 4, characterized in that, the volume concentration of the first ethanol solution is 40-60%; and/or, The volume concentration of the second ethanol solution is 65-80%. 7. separation and purification method according to claim 3, is characterized in that, the volume ratio of the resin in the described first resin column and the resin in the described second resin column and the resin in the described 3rd resin column is 1:(2～3):(5～8). 8. The separation and purification method according to any one of claims 4-6, characterized in that, the resin in the first resin column and the resin in the second resin column and the resin in the third resin column The volume ratio of the resin is 1:(2-3):(5-8). 9. separation and purification method according to claim 3, is characterized in that, described nonpolar macroporous resin is selected from at least one of Mitsubishi HP20, No. 3 Huazhen chromatography, Huazhen HZ818, Huazhen HZ816, Huazhen HZ820 kind.

Images