KR101125538B1 - Method of micelle-fractional precipitation hybrid process for the purification of paclitaxel - Google Patents

Abstract

The present invention relates to a method for purifying paclitaxel by a micelle-sedimentation sedimentation hybrid process for efficiently pretreatment of paclitaxel, an anticancer substance, from plant cell culture, and the surface area per reaction solution volume using ion exchange resin and glass beads during the fractionation and sedimentation process. The present invention relates to a method for purifying paclitaxel by a micelle-sedimentation precipitation hybrid process which drastically shortens the separation time by increasing the surface area per working volume (S / V).

Purification method of paclitaxel according to the present invention can be easily separated in high yield paclitaxel by a micellar process using an adsorbent treatment and a cationic surfactant in a high yield as well as increase the surface area per volume of the reaction solution during the fractionation and precipitation process to improve the high yield and precipitation The time can be shortened and the production cost can be drastically reduced, and it is also useful for the purification process of secondary metabolites in an efficient and economic way.

Paclitaxel purification, adsorbent, micelle, fractional precipitation, ion exchange resin, glass beads

Description

Method for purifying paclitaxel by a micelle-fractionated hybrid process {Method of micelle-fractional precipitation hybrid process for the purification of paclitaxel}

The present invention relates to a method for purifying paclitaxel by a micelle-sedimentation precipitation hybrid process, and can easily separate high-purity paclitaxel in high yield by a micelle process using an adsorbent treatment and a cationic surfactant. The present invention relates to a method for purifying paclitaxel by a micelle-sedimentation precipitation hybrid process that can increase yield and shorten settling time by increasing surface area, and can drastically reduce production cost.

Paclitaxel is the most effective diterpenoid anticancer substance, and the chemical structure of paclitaxel was identified by Wani et al. In 1971 and the anticancer mechanism was identified by Schiff et al. In 1979. Paclitaxel has attracted attention as a unique anticancer mechanism, and it is the most widely used anticancer agent since the 1990s because of its relatively low toxicity and strong anticancer activity by inhibiting cancer cell division of mitosis, which is different from conventional anticancer agents. US Food and Drug Administration (FDA) approved for treatment of refractory ovarian cancer, breast cancer, kaposi's sarcoma, and non-small cell lung cancer (NSCLC) Indications for head & neck tumors, rheumatoid arthritis and Alzheimer's treatment continue to expand. In addition, due to the ongoing clinical trials of various prescription methods, paclitaxel demand will continue to increase.

First, the main production method of paclitaxel is a method of extracting directly from the yew tree, which separates and purifies from the bark of the yew tree, making it difficult to continuously supply raw materials and requiring high cost in the purification process. And cause problems of destruction of resources and ecosystems. Second, there is a semisynthesis method that obtains precursors from the leaves of the yew tree and chemically bonds the side chains. This method is also not suitable for protecting the yew tree, which is an environmental protection water because the precursor must be obtained directly from the yew tree. . Third, there is a method of inducing callus in yew trees and culturing plant cells in a main bioreactor through seed culture. Plant cell culture method has the advantage that can be produced in a large amount of paclitaxel of a certain quality because it can be stably produced in the bioreactor without being influenced by external factors such as climate, environment.

In order to produce paclitaxel from plant cell culture, there are several stages of separation and purification.In general, the biomass recovered from the cell culture solution is first extracted with an organic solvent, and the product is subjected to final purification through a pretreatment process. Production process. In this process, the pretreatment process in particular has a great impact on the final purification cost. Existing studies have used expensive chromatography as a pretreatment process for purification or adopted a method of final purification of crude product extracted without pretreatment by high performance liquid chromatography (HPLC) to use a large amount of organic solvents. There are many problems in terms of economics such as shortening the life of column packing material (resin), reducing throughput, and many difficulties in scale-up.

Known techniques for the purification of paclitaxel are as follows.

International Patent Publication No. 2000/40573 discloses a low purity concentrate obtained from a liquid / liquid extraction process in a paclitaxel-containing material, first purified through a silica column, and then precipitated using an acetone / water solution. The process of obtaining paclitaxel of high purity by repeating is shown. However, in order to obtain high purity paclitaxel, a total of three silica chromatography steps and two crystallization steps must be performed repeatedly. Therefore, not only a lot of solvent and time are required, but also a precipitation method using acetone / water solution has to be preceded. There is a drawback that the recovery rate is significantly reduced to 49 ~ 73%.

International Patent Publication No. 2000/078741 extracts natural raw materials of taxanes with an organic solvent, forms a precipitate with a base or an acid, melts them with acetone, and percolorizes them with nucleic acid to give resins and pigments. Is removed, a precipitate is formed with a base or an acid, and at least one chromatographic run and at least one crystallization with acetone and nucleic acid are disclosed. However, in general, paclitaxel is known to be decomposed into 10-diacetelpaclitaxel and bacatin III under acidic or basic conditions, making it difficult to purify paclitaxel in high yield, and there is a problem that requires a large amount of time due to a complicated process. .

In addition, an efficient pretreatment method was developed in 2000 to obtain high purity (> 50%) paclitaxel by fractional precipitation process, but it takes much time (~ 3 days) for precipitation and is applied to mass production process. There is difficulty.

The present invention is to solve the problems of the prior art as described above, an object of the present invention to provide a method for purifying paclitaxel by the micelle-sedimentation precipitation hybrid process combining a micelle process using a cationic surfactant and a fractional precipitation process using a difference in solubility. There is this. More specifically, the present invention can easily separate high-purity paclitaxel in high yield by a micelle process using an adsorbent treatment and a cationic surfactant, and increase the yield and shorten the settling time by increasing the surface area per volume of the reaction solution during the fractionation and precipitation process. It is an object of the present invention to provide a method for purifying paclitaxel by a micelle-precipitation hybridization process, which can reduce the production cost significantly.

The present invention has been made to achieve the above object, and provides a method for purifying paclitaxel by a micelle-sedimentation precipitation hybrid process.

Hereinafter, the present invention will be described in more detail.

In this case, unless there is another definition in the technical terms and scientific terms used, it has the meaning that is commonly understood by those of ordinary skill in the art, unnecessarily obscure the subject matter of the present invention in the following description Description of known functions and configurations that may be omitted.

The present invention relates to a method for purifying paclitaxel by a micelle-separation precipitation hybrid process,

(a) after extracting by adding a water-soluble organic solvent to the Taxus genus plant, its cells or its cell culture solution, the extract is concentrated under reduced pressure,

(b) adding a non-polar organic solvent to the concentrate, extracting liquid-liquid, concentrating and drying under reduced pressure,

(c) drying the filtrate obtained by treating the first dried product obtained by extracting the liquid-liquid and then concentrating and drying under reduced pressure with an adsorbent and filtering;

(d) The second dried product obtained by drying the filtrate was put in a mixed solution of a non-aqueous organic solvent and a cationic surfactant, stirred, and then rested to recover a cationic surfactant layer in which micelles were formed after phase separation.

(e) further adding a non-aqueous organic solvent to the recovered cationic surfactant layer, stirring and standing to recover the non-aqueous organic solvent layer after phase separation, concentrating and drying it under reduced pressure,

(f) recovering the non-aqueous organic solvent layer, adding the third dried product obtained by concentrating and drying under reduced pressure to a mixed solvent of water-soluble organic solvent and water, adding ion-exchange resin or glass beads, stirring and stabilizing the precipitate. And recovering the precipitate after the filtration.

Taxus genus plant of the present invention, its cell or cell culture medium thereof refers to paclitaxel-containing material, the Taxus genus plant is Taxus brevifolia (Taxus brevifolia), Texas Taxus canadensis, Taxus cuspidata, Taxus baccata, Taxus globosa, Taxus floridana, Texas Wallichiana Taxus wallichiana), Taxus media, or Taxus chinensis, and the like.

The water-soluble organic solvent used in the process (a) is used to extract paclitaxel from the biomass (biomass), specific examples of the water-soluble organic solvent used in the present invention is selected from methanol, ethanol, propanol, methylene chloride 1 Species or mixtures of two or more species may be used, and as an organic solvent capable of recovering paclitaxel as much as possible from biomass, methanol may be preferably used. The biomass recovered from the Taxus genus plant, its cells or its cell culture in the step (a) is 0.1-100% (w / v) in methanol, preferably 1-50% ( After addition to w / v), extraction and filtration by stirring for 30 minutes at room temperature may be repeated two or more times to extract the biomass-derived paclitaxel derivatives.

The non-polar organic solvent used in the step (b) serves to remove the polar impurities contained in the paclitaxel derivative through phase separation with the water-soluble organic solvent. Specific examples of the non-polar organic solvent used in the present invention include methylene chloride, One or two or more mixtures selected from ethyl acetate and ether may be used, and in the present invention, the polar impurities contained in the paclitaxel derivative may be most effectively removed by performing one to three liquid-liquid extractions. When the water-soluble organic solvent used for extracting paclitaxel derivatives in the process (a) is alcohol, methylene chloride is generally used for liquid-liquid extraction. Methylene chloride can preferably be used with 20-30% (v / v) of the alcohol concentrate, and the use of excess methylene chloride will place a heavy burden on subsequent processes.

The adsorbent used in the step (c) is preferably a silica-based adsorbent, and when using a silica-based adsorbent, it is possible to effectively remove impurities such as biomass-derived wax, tar, and paclitaxel. It is effective in increasing the purity and efficiency of the purification process.

In the adsorbent treatment of step (c), 20 to 100% (w / w) of the first dried material obtained by concentration and drying under reduced pressure after the liquid-liquid extraction of the adsorbent, preferably 20 to 60% (w / w) And methylene chloride, one or more mixtures of two or more selected from chloroform were added and stirred at 20 to 60 ° C. for 30 minutes, followed by drying the filtrate obtained by filtration. It is possible to effectively remove biomass-derived impurities. In addition, the organic solvent used in the adsorbent treatment of step (c) should have a strong affinity for paclitaxel and a weak affinity for impurities such as tar and wax components.

The water-insoluble organic solvent used in the above (d) and (e) process is dichloromethane, carbon tetrachloride, chloroform, 1,2-dichloroethane, di-ethyl One or more selected from ethers such as di-ethyl ether, methyl-t-butyl ether, di-iso-propyl ether, hexane and benzene Mixture may be used, and the water-insoluble organic solvent used in the process (d) is aimed at efficient removal of the nonpolar impurities through phase separation with the cationic surfactant layer, and the water-insoluble organic solvent used in the process (e). The purpose is to recover paclitaxel contained in the micelles. Most preferably, a mixture of methyl-t-butyl ether and hexane may be used as the water-insoluble organic solvent of step (d), and most preferably the water-insoluble organic solvent of step (d). Preferably methyl-t-butyl ether can be used.

Step (d) of the present invention is a method of removing impurities by micelle formation and phase separation using a cationic surfactant in a micelle process. The cationic surfactant used for the micelle formation is one or two or more selected from N-cetylpridinium chloride, hexadecyltrimethylammonium chloride, dodecyltrimethylammonium chloride A mixture may be used, and as shown in FIG. 1, the cationic surfactant is composed of hydrophilic (5) and hydrophobic (hydrophobic), and paclitaxel (7) which is insoluble in water inside the micelle (4). ) Is surrounded by a hydrophobic tail group, and externally there is a hydrophilic head group that can be dissolved in an aqueous solution. In the micelle process of step (d), the water-insoluble organic solvent layer 1 is phase-separated from the cationic surfactant layer 3 including paclitaxel (2) to effectively remove impurities from the cationic surfactant layer containing paclitaxel. Can be. In addition, the cationic surfactant used in the micelle process of the present invention is preferably used in an aqueous solution, and the cationic surfactant is used in an amount of 1 to 20% (w / v) and most preferably 5 to 15% (w / v) of the aqueous solution. In the case of using the aqueous solution of the cationic surfactant in the above range, it is effective to easily separate high purity paclitaxel in high yield.

The process (f) is a fractional precipitation process using a solubility difference of paclitaxel in a water-soluble organic solvent. Fractionation sedimentation is a very simple method to efficiently purify the anticancer substance paclitaxel, and is a representative pretreatment process that can obtain high purity paclitaxel in high yield by using a difference in solubility. However, the conventional fractional precipitation process has a very long time (~ 3 days) required for fractional precipitation and has a great difficulty in mass separation and purification. Therefore, in the present invention, the experiment by increasing the surface area in the reactor required for fractional precipitation using ion exchange resin (14) and glass beads (glass bead) during the fractional precipitation process as shown in FIG. Was performed.

The water-soluble organic solvent used in the process (f) may be one or a mixture of two or more selected from methanol, ethanol, propanol and methylene chloride, and the water-soluble organic solvent used in the process (f) may dissolve paclitaxel. It should be effective in paclitaxel precipitation by causing difference in solubility by addition of distilled water.

In addition, in the step (f), the water-soluble organic solvent 13 is preferably added to the water 12 so as to be 50 to 70% (v / v) of the mixed solvent to perform a fractional precipitation process, and paclitaxel in the above range. This crystallization enables the formation of the precipitate 15.

The ion exchange resin used in the process (f) is preferably a cation exchange resin, an anion exchange resin or a mixture thereof, and the cation exchange resin and the anion exchange resin may have a styrene-based basic structure. The amount of ion exchange resin added is most preferably a surface area (S / V) of 0.428 mm ⁻¹ per volume of reaction solution for fractional precipitation.

In addition, the average particle diameter of the ion exchange resin used in the step (f) is preferably 0.3 ~ 1.5 mm, it is possible to obtain a high purity and yield at a given fractionation time in the above range.

In the process (f) of the present invention, glass beads may be used instead of the ion exchange resin to increase the surface area per working volume (S / V), and the average particle diameter of the glass beads may be 0.5 to 0.8 mm. In this range, it is possible to obtain high purity and yield at a given fractionation time.

In addition, the process (f) of the present invention is carried out at a low temperature of 1 ~ 10 ℃, if the fractional precipitation process in the above temperature range can obtain a high paclitaxel purity and yield.

Purification method of paclitaxel by the micelle-sedimentation sedimentation hybrid process according to the present invention is a micelle-sedimentation sedimentation hybrid process combining a micelle process using a cationic surfactant and a fractional precipitation process using a difference in solubility, and more specifically, an adsorbent treatment and The micellar process using cationic surfactants can easily separate high-purity paclitaxel with high yield, and increase the surface area per volume of reaction solution in the fractionation and precipitation process, resulting in high yield improvement and shortening settling time, which dramatically reduces the production cost. There is an effect that can be mass-produced in a cost-effective and efficient and economic way.

Hereinafter, the present invention will be described in more detail with reference to Examples, which are intended only for understanding the constitution and effects of the present invention, and are not intended to limit the scope of the present invention.

Test Example 1 Analysis Method of Paclitaxel

In order to confirm the form and size measurement of paclitaxel precipitate in the fractional precipitation process, an electron microscope SV-35 Video Microscope system (Some Tech., Korea) was used and observed at high magnification (× 100). Observed paclitaxel precipitate was confirmed by moving images in the IT-Plus system (Some Tech., Korea), through which the form and size of paclitaxel precipitate was confirmed.

In addition, the content of paclitaxel was analyzed using HPLC (Waters), and all the assay samples were taken three times to determine the content as an average value after analysis. The column used for analysis was Capcell Pak C18 UG 120 (250 mm × 4.6 mm, Shiseido, Japan), the column temperature was 40 ° C, the mobile phase was acetonitrile / water (65/35 ~ 35/65, v / v gradient), 1.0 mL / min, sample injection amount was 20 μL, and a UV (227 nm) detector was used. Paclitaxel standard was used as Sigma-Aldrich product (purity: 95%).

Preparation Example 1 Plant Cell Culture

Plant cell cultures used in this experiment were cultured using cell lines obtained from the leaves of Taxus chinensis. Decanter centrifuge (Westfalia, CA 150 Clarifying Decanter) was used to recover plant cells from the culture, and high-speed centrifuge (α-Lavel, BTRX 205 GD-35 CDEFP) was used to recover plant cell debris. . In the present invention, the collected plant cells and the cell pieces are referred to as biomass.

Example 1 Purification of Paclitaxel by a Micell-Sedimentation Precipitation Hybrid Process

The biomass recovered from the cell culture was added to methanol at 1% (w / v), extracted and filtered at room temperature for 30 minutes, and the reaction was repeated four times by adding fresh methanol to the biomass. Extraction was carried out. The extract filtrate was collected and concentrated in a concentrator (CCA-1100, EYELA, Japan) (30% of the stock), methylene chloride was added to the concentrate (25% of the concentrate) and liquid-liquid extraction was performed for 30 minutes at room temperature. Repeated three times to remove the polar impurities, concentrated under reduced pressure and dried. 50% (w / w) of sylopute (Fuji Silysia Chemical Ltd., Japan), an adsorbent, and methylene chloride were added to the first dried product (paclitaxel purity: 8%) obtained by concentrating and drying under reduced pressure after the liquid-liquid extraction (concentrate). 25%) and stirred at 40 ° C. for 30 minutes and then filtered. The filtrate was dried at 30 ° C. under reduced pressure and used in a micelle process. After drying the filtrate, the second dried product was dissolved in 25 mL of MtBE (methyl-t-butyl-ether), followed by 12.5 mL of an aqueous 5% (w / v) cationic surfactant, CPC (N-cetylpridinium chloride) and 25 mL of hexane. Was added and stirred at room temperature. After stirring for 30 minutes, the mixture was suspended to recover a cationic surfactant layer in which micelles were formed after phase separation. Four repeated experiments were performed under the same conditions. Paclitaxel was recovered into the MtBE layer by putting MtBE back into the cationic surfactant layer recovered through four replicate experiments to decompose micelles from the surfactant layer, and the four replicate experiments were performed in the same manner. The third dried product obtained by concentrating and drying the recovered MtBE layer under reduced pressure was dissolved in methanol, and distilled water was dropped until methanol became 61.5% (v / v). Amberlite IR-120 (styrene-divinylbenzene (gel), Rohm and Haas, USA) with an average particle diameter of 0.5 mm was added to the cation exchange resin to increase the surface area per reaction liquid volume. Refrigerated for 12 hours, 15 hours to obtain a paclitaxel precipitate. The paclitaxel precipitate was filtered, vacuum dried at 35 ° C. for 24 hours and analyzed by HPLC.

[Example 2]

The same process as in Example 1 was carried out except that Amberlite 200 (styrene-divinylbenzene (copolymer), Rohm and Haas, USA) having an average particle diameter of 0.5 mm was used as the cation exchange resin.

Example 3

The same procedure as in Example 1 was carried out except that Amberlite IRA-400 (styrene-divinylbenzene (gel), Rohm and Haas, USA) having an average particle diameter of 0.5 mm was used as an anion exchange resin instead of the cation exchange resin.

Example 4

It was carried out in the same manner as in Example 1 except that Amberlite IRA-96 (styrene-divinylbenzene (macroreticular), Rohm and Haas, USA) having an average particle diameter of 0.5 mm was used as the anion exchange resin instead of the cation exchange resin.

Example 5

The same procedure as in Example 1 was carried out except that glass beads (Glastechnigue Mfg, Germany) having an average particle diameter of 0.7 mm were used instead of the cation exchange resin.

Comparative Example 1

The same procedure as in Example 1 was conducted except that no cation exchange resin was used.

Comparative Example 2

The same procedure as in Example 1 was performed except that Amberlite XAD-16 (polystyrene (apolar), Rohm and Haas, USA) having an average particle diameter of 0.5 mm was used as a nonion exchange resin instead of the cation exchange resin.

Comparative Example 3

It was carried out in the same manner as in Example 1 except that DIAION HP 20 (styrene-DVB, Mitsubishi, Japan) having an average particle diameter of 0.25 mm was used as the non-ion exchange resin instead of the cation exchange resin.

Table 1 shows the yield and purity of paclitaxel purified after 9 hours of fractional precipitation in Examples 1 to 5 and Comparative Examples 1 to 3.

[Table 1]

In the present invention, the experiment was performed by fixing 0.428 mm ⁻¹ , the surface area per volume (S / V) of the optimum reaction solution selected through the basic experiment. The paclitaxel was purified by fractional precipitation of paclitaxel samples having a purity of 45% after the micelle process. .

The cation exchange resin (Amberlite IR-120, Amberlite 200), the anion exchange resin (Amberlite IRA 400, Amberlite IRA 96) and glass beads compared to Comparative Example 1 (control) did not increase the surface area per volume of the reaction solution through Table 1 By increasing the surface area per volume of the reaction solution (0.428 mm ^-1 ) by the glass bead), it was found that the yield can be significantly improved at a given fractionation time (9 hours). That is, compared to the case of Comparative Example 1 (control) that did not increase the surface area per volume, it was found that there is a large increase in yield in a given fractional precipitation time (9 hours). In particular, the highest yield (~ 85%) and purity (~ 87%) were obtained for Amberlite 200, a cationic crosslinker. On the other hand, in the case of non-ion exchange resin (Amberlite XAD-16, DIAION HP 20), no precipitation was formed, so it was found that there was no effect by increasing the surface area. This phenomenon indicates that the surface of the resin is nonphobic (hydrophobic) in the case of non-ion exchange resin, so that hydrophobic substances including paclitaxel are strongly adsorbed. In addition, as a result of measuring by electron microscopy to confirm the form of paclitaxel precipitates obtained under each fractional precipitation conditions, as shown in FIG. On the other hand, in the case of non-ion exchange resin, it was confirmed that no precipitation was formed.

Figure 4 shows the yield (a) and purity (b) of paclitaxel according to the fractionation settling time of Examples 1 to 5 and Comparative Examples 1 to 3, cation exchange resin (Amberlite 200) and anion exchange resin (Amberlite IRA- 400), this study is to investigate the precipitation effect with time of precipitation in case of increasing surface area by glass bead addition and control case not increasing surface area. As shown in FIG. 4, when the surface area (S / V) per volume of the reaction solution was increased, a high yield was obtained at the same precipitation time compared to the control. In particular, Amberlite 200, a cationic bridging resin, was able to recover most paclitaxel (> 98%) at about 12 hours of precipitation. Also, after 12 hours of precipitation, it was found that there was almost no effect of increasing yield. On the other hand, purity increased gradually with precipitation time. After 9 hours of precipitation, there was almost no change in purity (~ 85%) regardless of the increase in surface area per volume (S / V). In general, the purity is low in the early stage of precipitation, but the purity tends to increase gradually as the precipitation time passes, which is relatively high in the early stage of precipitation, and the precipitation of impurities decreases as the precipitation time elapses. Judging. In the case of cation exchange resin and anion exchange resin, high purity paclitaxel can be obtained at the initial stage of precipitation (6 hours), compared to glass beads or control. It is believed that charge improves the precipitation efficiency (purity improvement) by adsorbing and removing impurities.

1 shows the principle and schematic of a micelle based separation process for paclitaxel pretreatment.

Description of the main parts of the drawing

1: water-insoluble organic solvent layer 5: hydrophilic

2: phase separation 6: hydrophobic

3: cationic surfactant layer 7: paclitaxel

4: micelle

Figure 2 shows a schematic diagram of a fractional precipitation process according to the present invention.

Description of the main parts of the drawing

11: pump 14: ion exchange resin or glass beads

12: water (distilled water) 15: paclitaxel precipitate

13: water-soluble organic solvent 16: reactor

3 is an electron micrograph of the paclitaxel precipitate 9 hours after fractional precipitation according to Examples 1 to 5 and Comparative Examples 1 to 3, (A) is paclitaxel precipitate of Example 1, (B) is paclitaxel precipitate of Example 2 (C) is paclitaxel precipitate of Example 3, (D) is paclitaxel precipitate of Example 4, (E) is paclitaxel precipitate of Example 5, (F) is paclitaxel precipitate of Comparative Example 1, (G) is compared Paclitaxel precipitate of Example 2, (H) shows a paclitaxel precipitate picture of Comparative Example 3.

Figure 4 shows the yield (a) and purity (b) of paclitaxel according to the fractional precipitation time of Examples 1 to 5 and Comparative Examples 1 to 3.

Claims (11)

(a) after extracting by adding a water-soluble organic solvent to the Taxus genus plant, its cells or its cell culture solution, the extract is concentrated under reduced pressure, (b) adding a non-polar organic solvent to the concentrate, extracting liquid-liquid, concentrating and drying under reduced pressure, (c) drying the filtrate obtained by treating the first dried product obtained by extracting the liquid-liquid and then concentrating and drying under reduced pressure with an adsorbent and filtering; (d) The second dried product obtained by drying the filtrate was put in a mixed solution of a non-aqueous organic solvent and a cationic surfactant, stirred, and then rested to recover a cationic surfactant layer in which micelles were formed after phase separation. (e) further adding a non-aqueous organic solvent to the recovered cationic surfactant layer, stirring and standing to recover the non-aqueous organic solvent layer after phase separation, concentrating and drying it under reduced pressure, (f) recovering the non-aqueous organic solvent layer, and concentrating and drying the resulting third dried product to a mixed solvent of a water-soluble organic solvent and water, and then selected from cation exchange resins, anion exchange resins or mixtures thereof. A method for purifying paclitaxel by a micelle-sedimentation precipitation hybrid process comprising adding one ion-exchange resin or glass beads, stirring, and stabilizing to recover the precipitate precipitated separately. The method of claim 1, The water-soluble organic solvent used in the process (a) and (f) is one, or a mixture of two or more selected from methanol, ethanol, propanol, methylene chloride, (b) the nonpolar organic solvent used in the process is methylene chloride, Ethyl acetate, a mixture of one or two or more selected from ethers, and the water-insoluble organic solvent used in the steps (d) and (e) is dichloromethane, carbon tetrachloride, chloroform, 1, 2-dichloroethane, di-ethyl ether, methylt-butyl ether, di-iso-propyl ether Purification method of paclitaxel by the micelle-separation settling hybrid process, characterized in that one or two or more selected from hexane, benzene. 3. The method of claim 2, The water-insoluble organic solvent used in the step (d) is a mixture of methyl-t-butyl ether and hexane, and the water-insoluble organic solvent used in the process (e) is methyl-t- Purification method of paclitaxel by the micelle-separation settling hybrid process characterized in that it is butyl ether (methyl-t-butyl ether). The method of claim 1, The adsorbent used in the step (c) is a silica-based adsorbent, characterized in that the purification method of paclitaxel by the micelle-separation settling hybrid process. The method of claim 4, wherein In the adsorbent treatment of the step (c), the adsorbent is added at 20 to 100% (w / w) of the first dried product obtained by the liquid-liquid extraction, concentration under reduced pressure, and drying, and at least one selected from methylene chloride and chloroform. A method for purifying paclitaxel by a micelle-separation settling hybrid process characterized by further adding and stirring two or more kinds of mixtures. The method of claim 1, The cationic surfactant used in the step (d) is one selected from N-cetylpridinium chloride (N-cetylpridinium chloride), hexadecyltrimethylammonium chloride, dodecyltrimethylammonium chloride or A method for purifying paclitaxel by a micelle-separation settling hybrid process, characterized in that it is a mixture of two or more kinds. delete The method of claim 1, A method for purifying paclitaxel by a micelle-precipitation hybridization process in which the average particle diameter of the ion exchange resin used in the step (f) is 0.3 to 1.5 mm. The method of claim 1, The method for purifying paclitaxel by the micelle-precipitation hybridization hybrid process, wherein the average particle diameter of the glass beads used in the step (f) is 0.5 to 0.8 mm. The method of claim 1, The process (f) is a method for purifying paclitaxel by the micelle-separation settling hybrid process, characterized in that carried out at a low temperature of 1 ~ 10 ℃. The method of claim 1, The process (f) is a method for purifying paclitaxel by the micelle-precipitation hybridization process, characterized in that water is further added so that the water-soluble organic solvent is 50 to 70% (v / v) of the mixed solvent.

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