KR20080024286A - A method for purification of tacrolimus with high yield and high purity - Google Patents

Abstract

A method for purification of tacrolimus as an immune-suppressant is provided to improve economical efficiency, purity by removing impurities and color ingredients, simplicity and yield of purification without side effects by using no organic solvent which is harmful to human, so that the tacrolimus is mass produced. A method for purification of tacrolimus comprises the steps of: adsorbing the filtered solution of cultured medium of a tacrolimus-producing microorganism selected from Streptomyces hygroscopicus DKT523(KCTC 10964BP), Streptomyces tsukubaensis No. 9993(USP 5,624,842) and Streptomyces sp. MA6858 (USP 5,116,756) to porous adsorption resin and eluting it to obtain the crude purified solution of tacrolimus; treating the crude purified solution with active carbon to obtain the filtrate; adsorbing the filtrate to porous adsorption resin and reverse phase resin and eluting it; and crystallizing and recrystallizing the eluate to obtain the tacrolimus powder. Further, the filtered solution of cultured medium of a tacrolimus-producing microorganism is produced by mixing microorganism cultured medium and methanol at 1:1, extracting the microorganism cultured medium and filtering it through diatomaceous earth.

Description

A method for purification of tacrolimus with high yield and high purity}

1 is a diagram showing the HPLC of tacrolimus purified by final recrystallization according to the present invention.

The present invention relates to a method for purifying tacrolimus, which can be used as an antibacterial agent and an immunosuppressant, in high yield and high purity.

Tacrolimus has a molecular formula of C ₄₄ H ₆₉ NO ₁₂ .H ₂ O, a molecular weight of 822, and is called "1-benzyl-4-((5,6-dimethoxy-1-indanon)-" called FK-506 or Fujimycin. 2-yl) methylpiperidine hydrogen chloride ”is a macrolide compound having 23 rings. Tacrolimus has an immunosuppressant action that suppresses the immune response and is used for organ transplantation, prevention of neonatal hemolytic disease, and treatment of autoimmune disorder. Tacrolimus is a drug that has recently been in the spotlight because it has fewer side effects after renal toxicity, hypertension, and diabetic transplantation. Tacrolimus is a drug developed by Fujisawa, Co., Ltd., Japan, and was first isolated from fermentation broth of Streptomyces tsukubaensis , isolated from Tsukuba, northern Japan. It was subsequently sold by Fujisawa Healthcare in 1994 under the trade name Prograf as oral and injectables. Inc.). First reported in 1987 ( The Journal of Antibiotics , 16, No. 9, pp. 1249-1255, 1987), and since then, various methods for separation and purification have been proposed in several papers and patents. In general, fermentation products using microbial culture are present in the culture medium together with the culture medium components and various metabolites. Therefore, several steps of separation and purification are required to obtain tacrolimus in high purity. At this time, the development of efficient (high purity, high yield, low cost, ease of operation, etc.) refining process is a key factor for mass production.

Specifically, WO 00/71546 discloses a lactone-containing high molecular compound having alkenyl groups as a side chain using a strong cationic ion exchange resin containing sulfonic acid groups pretreated with silver ions, having a common basic chemical structure. Diaion R RCP160M, Capcell Pak SCX series resin column in which a mixture of high molecular weight compounds such as tacrolimus and ascomycin is pretreated with silver ions such as silver nitrate by separating lactone-containing compounds having alkyl groups from the lactone-containing high molecular compounds A method of recovering tacrolimus by filling and eluting a strong base resin column containing a benzene sulfonic acid group such as is described. The purification method described above is impossible to apply to mass production due to the increase of manufacturing cost by using Diaion R RCP160M and Capcell Pak SCX, which are very expensive resins.

US patent application USP 6,576,135; The purification method known from WO 01/18007 discloses a method for separating a lactone-containing polymer compound, which is adsorbed onto a nonionic adsorption resin to separate tacrolimus-like flexible material, and contains a silver ion. And a method of recovering tacrolimus by adsorbing and eluting with activated alumina to separate a single compound from a lactone-containing polymer compound. The above refining method is difficult to apply to mass production by using a very expensive resin due to the increase in manufacturing cost, and also contains a large amount of silver nitrate in the eluting solvent, which is an inevitable additional process for its removal.

WO 05/54253 discloses a method for purifying macrolide antibiotics, which transfers tacrolimus to an organic solvent as a solvent capable of layer separation, and is treated with ammonia gas to remove impurities from the phase or reverse phase chromatography. A method of recovering tacrolimus by repeatedly carrying out a silica gel column chromatography method pretreated with and silver ions is described. However, there are problems in that the process is not environmentally friendly by the use of organic solvents, and low yields due to the reverse phase resin and the silica gel resin.

Korean Patent Registration 0485877 describes a strain patent and a simple purification method of tacrolimus as an example. As shown in the examples, the present patent describes a process for refining by simply solvent extraction and repetition of silica gel, but it is not suitable for high purity tacrolimus production because it does not completely remove the flexible material and colored impurities.

The tacrolimus purification method described in the above-mentioned patent does not provide a high purity purified product free of impurities and colored components, an organic solvent which is undesirable for the safety of the human body during the process, and a limitation in obtaining a high purity purified product. It has problems such as having it, having a somewhat complicated process, having a low purification yield, and having a high manufacturing cost.

Accordingly, the present inventors have felt the necessity of developing a high yield and high purity purification method of tacrolimus, and through the continuous research and development confirmed a purification method that is more economical and efficient than the existing tacrolimus purification method and can mass-produce tacrolimus in high purity and high yield. The present invention was completed.

An object of the present invention is to provide a method for purifying tacrolimus in high yield and high purity.

In accordance with the above object, the present invention provides a method for purifying tacrolimus in high yield and high purity.

Specifically, the present invention comprises the first step of adsorbing and eluting a filtrate of a fermentation culture of tacrolimus producing microorganisms to a porous adsorptive resin to obtain a tacrolimus crude liquid; Treating the crude liquid with activated carbon and obtaining a filtrate; A third step of adsorbing and eluting the filtrate of the second step into the porous adsorptive resin and the reverse phase resin or by adsorbing and eluting the acidic and basic adsorbent pretreated with silver ions; It provides a purification method of obtaining a high yield and high purity tacrolimus through the fourth step of the purification step of determining and recrystallizing the eluate to obtain tacrolimus powder.

The filtrate of the fermentation culture of tacrolimus producing microorganism in the first step may be obtained by mixing and extracting methanol in a volume of 1: 1 to the microbial fermentation culture and filtered with diatomaceous earth.

Activated carbon in the second step is added 1 to 10 times, preferably 1 to 5 times, more preferably 2 to 4 times the tacrolimus weight, 0.1 to 10 to 50 ℃, preferably 15 to 30 ℃ temperature It is characterized in that the stirring for 5 hours, preferably 0.5 to 3 hours, and after stirring, the filter paper is filtered 1 to 10 times, preferably 1 to 3 times.

In addition, the activated carbon in the second step is preferably AQUA NUCHAR, NUCHAR SA, NUCHAR SA-20, NUCHAR SA-30, NORIT A SUPRA EUR, NORIT B SUPRA EUR, NORIT CN 1, DARCO G 60, DARCO KB-B , NORIT ESUPRA USA, NORIT SX 1, NORIT SX 1G, NORIT SX 2, NORIT SX ULTRA, CAL 12X40 and GW 12X40.

In the third step, the porous adsorptive resin has a low particle size with a small particle size range, preferably a porous neutral adsorptive resin having a particle size range of 75 to 150 μm, more preferably AMBERITE XAD 7, AMBERITE XAD 16, AMBERITE XAD 1600T At least one porous adsorbent resin selected from DIAION HP20, DIAION HP2MG, DIAION HP20SS, SEPABEADS SP825, SEPABEADS SP850 and SEPABEAD SP700, most preferably DIAION HP20 SS and SEPHABEADS, which are polymers of polystyrene and divinyl benzene It is characterized by using SP20 SS resin.

In the third step, the reverse phase resin is characterized by having a particle size of 5 ~ 170㎛, preferably 15 ~ 150㎛ reverse phase resin, more preferably DIASOGEL 3001A, LiChrospher RP or Vydac 238TP RP.

In the third step, the acidic and basic adsorbent resins are silica gel having a particle size density range of 0.030 μm to 0.073 μm, preferably 0.040 μm to 0.063 μm, and an particle size density range of 35 to 160 μm, preferably 45 to 150 μm. It is characterized in that the alumina (activated alumina), aluminum oxide (aluminum oxide) or alumina (alumina).

In the third step, the elution is adsorbed on the porous adsorptive resin, washed with purified water and eluted using a water miscible ketone having 3 to 6 carbon atoms of 20 to 80% (v / v) of pH 6.0 to pH 8.0. It is done.

In the fourth step, the crystallization and recrystallization are performed to mix the nonpolar alkane solvent and the alkyl acetate at a ratio of 1 to 200% (v / v), preferably 1 to 100% (v / v) to increase the purity of tacrolimus. And then dissolve 30 to 450 mg / ml, preferably 50 to 400 mg / ml of tacrolimus and stir at 70 to 350 rpm, preferably 100 to 300 rpm at a temperature of 1 to 70 ° C., preferably 4 to 50 ° C. After primary crystallization for 7 to 53 hours, preferably 10 to 48 hours, and then mixing the nonpolar alkane solvent and alkyl acetate in the same ratio as in the primary crystals, and the temperature of 0 to 70 ℃, preferably 2 to 40 ℃ In, characterized in that the secondary crystals are carried out for 1 to 30 hours, preferably 2 to 24 hours while stirring at 70 to 450 rpm, preferably 100 to 400 rpm.

The tacrolimus producing microorganism is characterized by being a strain of Streptomyces hygroscopic picus DKT523 (S. hygroscopicus DKT523, KCTC 10964BP), Streptomyces Tsukubaensis No.9993 (USP 5,624,842) and the genus Streptomyces genus MA6858 (USP 5,116,756). do.

When explaining the present invention more specifically, the preferred culturing method of tacrolimus producing strain for purifying tacrolimus with high purity and high yield of the present invention is as follows, but is not limited thereto.

The fermentation medium of the present invention is preferably carbon fiber, cottonseed foil as nitrogen source, fermentation conditions are 25 ~ 35 ℃, aeration amount 1.0 ~ 2.0vvvm, agitation rate is 100 ~ 400rpm gradually depending on the state of the strain sequentially Is preferably increased. After the fermentation process, the pH of the fermentation broth is adjusted to a neutral pH of pH 6.0 to pH 8.0 using caustic soda and hydrochloric acid.

In addition, "porous adsorptive resin" as defined in the present invention means a synthetic adsorbent made of a high molecular polymer without an ion exchange group, and in detail, a polymer of a styrene and divinylbenzene having a pore radius of 50 to 300 microns and a large network structure. Crosslinked aromatic or aliphatic polymer and methacrylic adsorption resin.

Purification method according to the present invention overcomes the difficulties of mass production due to the economic burden, low purity, complex process and low purification yield of the tacrolimus purification method generally performed, and can be widely used for antibacterial and immunosuppressant, etc. It has the advantage of being able to purify the economic burden, high purity, high yield and simple process.

For reference, the purification method of the present invention is a part and all of the process is described in International Patent Application Publication No. WO 00/71546, US Patent Application USP 6,576,135; It is not the same as or similar to the process claimed in WO 01/18007, WO 05/54253, and Korean Patent Registration 0485877, and the patent rights of the existing patents are equally or equally. You can confirm that it does not infringe.

Hereinafter, the present invention will be described in detail by the following Examples and Experimental Examples.

However, the following Examples and Experimental Examples are only illustrative of the present invention, and the content of the present invention is not limited by the following Experimental Examples.

Example  One. Tacrolimus Adjustment  Produce

1-1. Tacrolimus Adjustment Production Example (One)

120 liters of Streptomyces hygroscopicus DKT523 (S. hygroscopicus DKT523, KCTC 10964BP) culture solution was added 120 liters of methanol and filtered through a drum filter to recover 168 L of the filtrate. The concentration of tacrolimus was 152 mg / ml, the total amount of tacrolimus was 25.5 g, and the pH of the filtrate was 7.6. The filtrate was adsorbed at a flow rate of 3 BV (Bed Volume) / hr in a column (5 × 30 cm) packed with 20 ml (3.06 g of tacrolimus) of the filtrate and filled with 300 ml of AMBERITE XAD 7 resin, a porous adsorptive resin. 5BV (1.5 L) at 4BV / hr flow rate with 40% acetone. 4BV (1.2 L) was eluted at 4BV / hr flow rate with 75% acetone. HPLC analysis of the eluate gave 2.76 g of tacrolimus with a purity of 70.3% and a recovery of 90.2%.

1-2. Tacrolimus Adjustment Production Example (2)

The filtrate was adsorbed at a flow rate of 3 BV / hr in a column (5 × 30 cm) filled with 300 ml of SEPABEADS SP850 resin, which is 20 L (tacrolimus 3.06 g) of the filtrate of Example 1-1. After washing 5BV (1.5 L) at 4BV / hr flow rate with 40% acetone, 4BV (1.2L) was eluted at 4BV / hr flow rate with 75% acetone. HPLC analysis of the eluate gave 2.75 g of tacrolimus with a purity of 76.3% and a recovery of 90.00%.

1-3. Tacrolimus Adjustment Production Example (3)

20 l (3.06 g of tacrolimus) in the filtrate of Example 1-1 was adsorbed on a column (5 × 30 cm) filled with 300 ml of DIAION HP20 resin, a porous adsorptive resin, at a flow rate of 3 BV / hr, and 50% After washing 5BV (1.5L) at 4BV / hr flow rate with acetone, 4BV (1.2L) was eluted at 4BV / hr flow rate with 75% acetone. HPLC analysis of the eluate yielded 2.90 g of tacrolimus with a purity of 70.0% and a recovery of 95.00%.

Example  2. Activated Carbon Treatment

Before treatment of activated carbon, the adjustment liquid and activated carbon treatment liquid were dispensed into 96 well plates, and absorbance was measured at a wavelength of 405 nm with THERMOmax microphone plate reader (Molcular Devices Co.). It was confirmed that the eluate was obtained. 8.28 g of activated carbon (NORIT A SUPRA EUR) was added to 1200 ml of the eluent (tacrolimus 2.76 g) passed through the porous adsorptive resin of Example 1-1 and stirred at 200 rpm for 1 hour while maintaining the temperature at 25 to 30 ° C. It was. After passing through the KS filter to remove activated carbon, 2.48 g of tacrolimus with a purity of 88.6% were obtained.

Example  3. Tacrolimus  purchase

3-1. Tacrolimus Example (One)

30 l (4.59 g of tacrolimus) of the culture filtrate obtained in Example 1-1 was adsorbed and eluted in a porous adsorptive resin, and treated with the same amount of activated carbon NORIT A SUPRA EUR as compared to tacrolimus in the same manner as in Example 2. A column filled with 300 ml of DIAION HP20 SS resin, a product of Mitsubishi Chemical Co., a porous adsorptive resin having a density particle in the range of 75 to 150 µm, was obtained. 30 cm), the filtrate was adsorbed at 2 BV (Bed Volume) / hr flow rate, eluted 5 BV (Bed Volume) at 2BV (Bed Volume) / hr flow rate with 55% acetone, and then purified liquid containing tacrolimus The other aliquot was diluted to a concentration of 50% acetone and the same operation was repeated three more times. Tacrolimus obtained by the method of the porous adsorption column had a recovery amount of 2.56 g and a purity of 90.1%. The recovered solution was concentrated under reduced pressure, transferred to ethyl acetate, and concentrated again. The chromaticity of the concentrated tacrolimus modifier solution was dissolved in an acetone solution at a concentration of 200 mg / ml, and the absorbance at 405 nm was measured to confirm that the absorbance was reduced by 15 times. The concentrated recovery solution was flowed 10 ml / min on an MPLC column (2 × 30 cm) filled with DIASOGEL 3001A (Daiso Co.) with reversed phase resin (with C _{1 to} C ₁₈ non-polar side chains as silica substrate). The mixture was adsorbed with, eluted with 60% acetonitrile, concentrated under reduced pressure at 40 ° C, transferred to ethyl acetate, and then concentrated under reduced pressure. Concentrated purity yielded 2.89 g of 93.30% tacrolimus.

3-2. Tacrolimus Example (2)

30 l (4.59 g of tacrolimus) of the culture filtrate obtained in Example 1-1 was adsorbed and eluted in a porous adsorptive resin, and treated with the same amount of activated carbon NORIT A SUPRA EUR as compared to tacrolimus in the same manner as in Example 2. After the activated carbon was removed, the filtrate was poured into a column (5 × 30 cm) filled with 300 ml of activated alumina (Wakko Co.) pretreated with silver ions having a particle size density of 45 to 150 μm. Volume) / hr, and 10BV (Bed Volume) was eluted using 50% (v / v) mixed solvent of hexane and acetone. The eluted eluate was concentrated under reduced pressure at 40 ° C. to obtain 3.68 g of tacrolimus with a purity of 93.50%.

3-3. Tacrolimus Example (3)

30 l (4.59 g of tacrolimus) of the culture filtrate obtained in Example 1-1 was adsorbed and eluted on a porous adsorption resin (DIAION HP20), and treated with the same amount of activated carbon (NORIT A SUPRA EUR) compared to tacrolimus, through a KS filter. Activated carbon was removed. The filtrate was adsorbed at a flow rate of 1.5 BV (Bed Volume) / hr on a column (5 × 30 cm) filled with 300 ml of silica gel (Merck Co.) pretreated with silver ions having a particle size density of 0.040 μm to 0.063 μm, 10BV (Bed Volume) was eluted with 25% (v / v) mixed solvent of hexane and ethyl acetate. The eluted eluate was concentrated under reduced pressure at 40 ° C to obtain 3.07 g of tacrolimus with a purity of 95.50%.

3-4. Tacrolimus Example (4)

30 l (4.59 g of tacrolimus) of the culture filtrate obtained in Example 1-1 was adsorbed and eluted on a porous adsorption resin (DIAION HP20), and treated with the same amount of activated carbon (NORIT A SUPRA EUR) compared to tacrolimus, through a KS filter. Activated carbon was removed. Column filled with 300 ml of porous adsorption resin (DIAION HP20 SS resin) manufactured by Mitsubishi Chemical Co., a polymer of polystyrene and divinyl benzene having a particle size density range of 75 to 150 μm The filtrate was adsorbed at (5 × 30 cm) at a flow rate of 1.5 BV (Bed Volume) / hr, and a mixed solvent 10 BV (Bed Volume) containing 0.3 M silver nitrate was eluted in 50% acetone. The eluted eluate was concentrated under reduced pressure at 40 ° C. to obtain 2.98 g of tacrolimus with a purity of 95.50%.

The yields and purity of Examples 3-1 to 3-4 are shown in comparison to Table 1 below.

Suzy Recovery water Example 3-1 DIAION HP20 SS, RP 62.96% 93.30% Example 3-2 Alumina 80.17% 93.50% Example 3-3 Silica gel 66.80% 95.00% Example 3-4 DIAION HP20SS 64.90% 95.50%

Example  4. Tacrolimus  Decision and recrystallization

4-1. According to the solvent composition ratio Tacrolimus Example

The tacrolimus obtained in Example 3-1 was adjusted to 200 mg / ml with ethyl acetate, and then the mixed solvent having a ratio of ethyl acetate to nucleic acid of 10, 5, 2, 1.4, and 1% (v / v). The mixture was dissolved in and dissolved for 5 minutes at 50 ° C, and then crystallized at 15 ° C for 24 hours. The solvent concentration results are shown in Table 2 below.

Condition Recovery water 10% (V / V) Acetate Acetate / Hexane 63.1% 89.34% 5% (V / V) Acetate Acetate / Hexane 64.3% 90.20% 2% (V / V) Acetate Acetate / Hexane 65.30% 95.23% 1% (V / V) Acetate Acetate / Hexane 63.90% 91.20%

4-2. Molten Tacrolimus  Concentration Example

The tacrolimus obtained in Example 3-1 was adjusted to 200 mg / ml, 150 mg / ml, 100 mg / ml with ethyl acetate, and the ratio of ethyl acetate to the nucleic acid was 2% (v / v). Each was mixed with a mixed solvent, dissolved at 50 ° C. for 5 minutes, and crystals were formed at 15 ° C. for 24 hours. Concentration results are shown in comparison with Table 3 below.

Condition Recovery water 200mg / ml Acetate Acetate / Hexane 2% (V / V) 64.2% 93.65% 150mg / ml Acetate Acetate / Hexane 2% (V / V) 65.33% 95.67% 100mg / ml Acetate Acetate / Hexane 2% (V / V) 65.45% 95.84% 50 mg / ml Acetate Acetate / Hexane 2% (V / V) 62.95% 95.21%

4-3. According to the crystallization temperature Tacrolimus Example

The tacrolimus obtained in Example 3-1 was adjusted to 150 mg / ml with ethyl acetate, and the mixture was mixed with nucleic acid in a mixed solvent having a ratio of ethyl acetate of 2% (v / v) for 5 minutes at 50 ° C. After dissolution, crystals were formed at 4, 10, 15 and 20 ° C. for 10 hours. Experimental results for each crystal temperature are shown in Table 4 below.

Condition Recovery water 4 ℃ Acetate Acetate / Hexane 2% (V / V) 99.03% 93.02% 10 ℃ Acetate Acetate / Hexane 2% (V / V) 92.30% 95.64% 15 ℃ Acetate Acetate / Hexane 2% (V / V) 70.12% 95.66% 20 ℃ Acetate Acetate / Hexane 2% (V / V) 63.42% 95.78%

4-3. Tacrolimus  Decision and recrystallization Example

2.75 g of tacrolimus obtained in Example 3-1 was dissolved in a mixed solvent of ethyl acetate and hexane ratio of 2% (v / v), dissolved at 50 ° C for 5 minutes, and then crystallized at 15 ° C for 24 hours. Was formed. The precipitated crystals were filtered through Whatman filter paper No. 4, and then dried in a vacuum dryer at 40 ° C. for 4 hours to obtain 2.47 g of tacrolimus having a purity of 98.2%. The obtained tacrolimus was again crystallized at 4 ° C. for 4 hours by the recrystallization method described above. The precipitated crystals were filtered through Whatman filter paper No. 4 and vacuum dried for 24 hours in a 40 ° C. vacuum dryer to obtain 2.22 g of tacrolimus with a purity of 99.0% with a final yield of 48.3%, and HPLC analysis in FIG. 1. The results are shown.

The method for purifying the tacrolimus of the present invention overcomes the difficulties of mass production due to the economic burden, low purity, complex process and low purification yield of the tacrolimus purification method which is generally performed, and can be widely used for antibacterial and immunosuppressive agents. Present tacrolimus can be purified with reduced economic burden, high purity, high yield and simple process.

Claims (14)

A first step of adsorbing and eluting a filtrate of a fermentation culture of tacrolimus producing microorganisms to a porous adsorptive resin to obtain a tacrolimus crude liquid; Treating the crude liquid with activated carbon and obtaining a filtrate; A third step of adsorbing and eluting the filtrate of the second step into the porous adsorptive resin and the reverse phase resin; A method for purifying tacrolimus comprising a series of continuous processes comprising a fourth step of determining and recrystallizing the eluate to obtain tacrolimus powder. A first step of adsorbing and eluting a filtrate of a fermentation culture of tacrolimus producing microorganisms to a porous adsorptive resin to obtain a tacrolimus crude liquid; Treating the crude liquid with activated carbon and obtaining a filtrate; Adsorbing and eluting the acidic and basic adsorbents pretreated with silver ions; A method for purifying tacrolimus comprising a series of continuous processes comprising a fourth step of determining and recrystallizing the eluate to obtain tacrolimus powder. According to claim 1 or 2, wherein the tacrolimus producing microorganisms are Streptomyces hygroscopicus DKT523 (S. hygroscopicus DKT523, KCTC 10964BP), Streptomyces Tsukubaensis No.9993 (USP 5,624,842) and Streptomyces genus Purification method characterized in that the MA6858 (USP 5,116,756) strain. According to claim 1 or 2, wherein the filtrate of the fermentation culture of tacrolimus producing microorganism of the first step is obtained by mixing and extracting 1: 1 volume of methanol in the microbial fermentation culture and filtered with diatomaceous earth Purification method to do. The method according to claim 1 or 2, wherein the activated carbon of the second step is added 1 to 5 times the tacrolimus weight and stirred at 15 to 30 ° C for 0.5 to 3 hours. The purifying method according to claim 1 or 2, wherein the removal after the activated carbon treatment in the second step is performed by filtration with filter paper one to three times. According to claim 1 or 2, wherein the activated carbon of the second stage is AQUA NUCHAR, NUCHAR SA, NUCHAR SA-20, NUCHAR SA-30, NORIT A SUPRA EUR, NORIT B SUPRA EUR, NORIT CN 1, DARCO G 60 At least one activated carbon selected from DARCO KB-B, NORIT ESUPRA USA, NORIT SX 1, NORIT SX 1G, NORIT SX 2, NORIT SX ULTRA, CAL 12X40 and GW 12X40. The method of claim 1, wherein the porous adsorptive resin of the third step is a porous neutral adsorptive resin having a particle size density range of 75 to 150 μm. 9. The method of claim 8, wherein the porous neutral adsorptive resin is selected from AMBERITE XAD 7, AMBERITE XAD 16, AMBERITE XAD 1600T, DIAION HP20, DIAION HP2MG, DIAION HP20SS, SEPABEADS SP825, SEPABEADS SP850 and SEPABEAD SP700. The refining method according to claim 1 or 2, wherein the reverse phase resin of the third step is a reverse phase resin having a particle size of 15 to 150 µm. The method of claim 10, wherein the reverse constant resin is DIASOGEL 3001A, LiChrospher RP, or Vydac 238TP RP. According to claim 1 or 2, wherein the acidic and basic adsorption resin of the third step, silica gel having a particle size density range of 0.040㎛ ~ 0.063㎛, activated alumina (45 ~ 150㎛ particle size range), Purification method characterized in that the aluminum oxide (aluminum oxide) or alumina (alumina). According to claim 1 or 2, wherein the elution of the third step is adsorbed on the porous adsorptive resin, washed with purified water and 20 to 80% (v / v) of 3 to 6 carbon atoms of pH 6.0 to pH 8.0 Purification method using a water-miscible ketone eluting. The method of claim 1 or 2, wherein the crystallization and recrystallization of the fourth step is 50 to 400 mg / ㎖ tacrolimus after mixing the non-polar alkane solvent and alkyl acetate at a ratio of 1 to 100% (v / v) After dissolving, primary crystallization for 10 to 48 hours with stirring at 100 to 300rpm at a temperature of 4 to 50 ℃, mixed with a non-polar alkane solvent and alkyl acetate in the same ratio as in the primary crystals and at a temperature of 2 to 40 Purification method characterized in that the secondary crystals are carried out for 2 to 24 hours while stirring at 100 to 400rpm.

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