KR100624104B1 - Method for separation and purification of paclitaxel from paclitaxel-containing materials - Google Patents

Abstract

The present invention relates to a method for purifying paclitaxel from paclitaxel containing material. In particular, the present invention is extracted by adding an organic solvent to the paclitaxel-containing material and concentrating the extract, separating the organic solvent layer by adding an organic solvent that is not mixed with water, and concentrating it under reduced pressure, performing elution by phase chromatography Separating the eluate in acetone or dichloromethane, adding pentane or hexane to form a precipitate or dissolving the eluate in methanol and adding water to form a precipitate and the precipitate by high-performance liquid chromatography. It provides a paclitaxel purification method comprising the step of purifying. The high purity paclitaxel of 99.5% or more can be easily separated from the paclitaxel-containing material by the method of the present invention, and thus it can be usefully used for the production of paclitaxel.

Paclitaxel, chromatography, precipitation

Description

Method for separation and purification of paclitaxel from paclitaxel-containing materials}

1 is an HPLC chart confirming the purity and recovery of paclitaxel for each step of purification of paclitaxel of the present invention.

FIG. 2A is a HPLC analysis chart after methanol / distilled water precipitation in step (d ′), and FIG. 2B is an HPLC chart confirming purity and recovery of paclitaxel after step (e) of FIG. 2A.

The present invention relates to a method for purifying paclitaxel from a paclitaxel-containing substance.

Paclitaxel was searched through a large-scale screening program for anticancer active substances of the US National Cancer Institute in the 1960s, and is known as the most important anticancer substance in ovarian cancer and breast cancer. Paclitaxel is a widely used anticancer drug approved by the FDA in 1992.

Paclitaxel is isolated and purified from the bark of the yew tree, which has a very low paclitaxel content of 0.02%, which requires a high cost for purification and causes problems of resource and ecosystem destruction. As a method for overcoming the above problems, a method for semi-synthesizing paclitaxel from the leaves of yew and a mass production method using the cell culture method of interest have been developed. 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 degraded into 10-diacetelpaclitaxel and bacatin III under acidic or basic conditions, and it is not only difficult to purify paclitaxel in high yield, but also has a large time-consuming problem due to a complicated process. .

U.S. Patent No. 5,900,367 discloses a method for treating tar by removing a tar component by treating an organic solvent extract with a synthetic adsorbent, precipitating the precipitate in hexane, precipitating the precipitate using methanol and purified water, and obtaining paclitaxel using high performance liquid chromatography. Presenting. The above method has the advantage that it can be extracted and purified in high purity with a small amount of solvent using only three kinds of extraction solvents, dichloromethane, methanol and hexane, but to increase the purity and yield of fractional precipitation using methanol and purified water, There is a problem that a reclassified precipitation has to be carried out.

It is an object of the present invention to provide a method for effectively extracting and purifying paclitaxel from paclitaxel containing material.

It is also an object of the present invention to provide a method for simplifying the purification of paclitaxel and easily purifying paclitaxel with high purity.

The present invention relates to a method for purifying paclitaxel from paclitaxel containing material.

'Paclitaxel-containing material' of the present invention refers to a cell culture including a tissue, cells or medium cultured using a Taus genus plant or cells derived from the plant. The Taxus genus plants include Taxus brevifolia , Taxus canadensis , Taxus cuspidata , Taxus baccata and Taxus globosa Taxus globosa , Taxus floridana , Taxus wallichiana , Taxus media , Taxus chinensis , but are not limited thereto.

Paclitaxel purification method of the present invention

(a) adding and extracting an organic solvent to paclitaxel-containing material and concentrating the extract;

(b) adding an organic solvent to the concentrate of (a) to separate the organic solvent layer and concentrating it under reduced pressure;

(c) separating the eluate by performing normal phase chromatography on the vacuum concentrate of (b);

(d) dissolving the eluate of (c) in acetone or dichloromethane and adding pentane or hexane to form a precipitate;

(e) purifying the precipitate of (d) by high performance liquid chromatography.

Another paclitaxel purification method of the present invention

(a) adding and extracting an organic solvent to paclitaxel-containing material and concentrating the extract;

(b) adding an organic solvent to the concentrate of (a) to separate the organic solvent layer and concentrating it under reduced pressure;

(c) separating the eluate by performing normal phase chromatography on the vacuum concentrate of (b);

(d ') dissolving the eluate of (c) in methanol and adding water to form a precipitate;

(e) purifying the precipitate of (d ') by high performance liquid chromatography.

The organic solvent of step (a) may be selected from the group consisting of methanol, ethanol, propanol and dichloromethane, but is not limited thereto. Preferably the organic solvent is methanol. The organic solvent may be added to paclitaxel-containing material at 20 to 200% (v / w), preferably 40 to 140% (v / w), and the process of stirring and filtering at room temperature for 30 minutes is repeated two or more times. Paclitaxel and taxane derivatives are extracted. The extract can be concentrated by a method such as vacuum concentration.

In step (b), the organic solvent is added to 10% to 50% (v / v), preferably 20% to 30% (v / v) of the extract of (a) or a concentrate thereof, followed by distribution. To separate the organic solvent layer. The organic solvent layer separation may be repeated two or more times, and the separated layer is concentrated under reduced pressure and dried. The organic solvent used in step (b) is an organic solvent that is not mixed with water, and may be, for example, a nonpolar organic solvent. Examples of organic solvents include alkyl halides such as dichloromethane, carbon tetrachloride, chloroform, and 1,2-dichloroethane, di-ethyl ether, and methyl. ethers such as -t-butyl ether, methyl-t-butyl ether, di-iso-propyl ether, and C ₄ -C ₆ alcohols such as n-butanol and n-pentanol; Ethyl acetate, methyl ethyl ketone, and the like. Part (b) removes some of the polar material.

Step (c) is a step of suspending the dried product dried in step (b) in an organic solvent and performing normal phase column chromatography. As a filler for the column, conventional silica gels such as silica gel 40 (63-200 μm), silica gel 60 (63-200 μm) or silica gel 60F254 (200-500 μm) may be used. The organic solvent for suspending the dried material may be at least one organic solvent selected from the group consisting of dichloromethane, methanol, benzene, acetone, hexane and ethyl acetate. Normal column chromatography can elute fractions through isocartic elution or step gradient elution. For isocratic elution, a solvent in which methanol and dichloromethane are mixed in a volume ratio of 0.5 to 10: 99.5 to 90.0 may be used as a developing solvent. Step gradient gradient elution may be a dichloromethane or a solvent mixed with dichloromethane and dichloromethane in a volume ratio of 0.1 to 5: 99.9 to 95, as the elution solvent, 0.5 to 100% (v / v) methanol and the remaining amount A solvent containing dichloromethane of can be used.

The 'isocratic method' is a method of eluting using a solvent having the same composition, and the 'gradual concentration gradient method' means a method of eluting while changing the composition (concentration) of the solvent step by step.

Fractions eluted using the normal phase chromatography in step (c) may be further concentrated under reduced pressure and drying. The normal phase chromatographic step is to replace the conventional synthetic adsorbent treatment and the precipitation process by hexane, and significantly improve the paclitaxel yield while simplifying the process compared to the conventional purification process. In addition, the fraction recovered through normal phase chromatography can be easily recovered as a precipitate of high yield through the precipitation step that is a later step.

In step (d), acetone or dichloromethane is added to the dried product of step (c) to dissolve, and pentane or hexane is added to form a precipitate. 5 to 100 mg of dry matter may be dissolved per ml of acetone or dichloromethane, and pentane or hexane may be mixed in a ratio of 1: 1 to 20, preferably 1: 5 to 10, by volume relative to acetone or dichloromethane. can do. Precipitation time and temperature after addition of pentane or hexane is not particularly limited, but the precipitation time may be 1 day to 5 days, preferably 1 day to 2 days, the temperature is 35 ℃ or less, preferably 0 ℃ to 15 ℃ Can be. The precipitate is obtained by filtration in a conventional manner.

Step (d ') is adding methanol to the dry matter of step (c) to dissolve it, and water to form a precipitate. 5 to 100 mg of dry matter may be dissolved per 1 ml of methanol, and water may be mixed in a ratio of 1: 1 to 20, preferably 1: 5 to 10, by volume based on methanol. The precipitation time and temperature after adding water are not particularly limited, but the precipitation time may be 1 day to 5 days, preferably 2 days to 3 days, and the temperature may be 35 ° C. or lower, preferably 0 ° C. to 15 ° C. have. The precipitate is obtained by filtration in a conventional manner.

Step (e) is a step of purifying the precipitate of step (d) or (d ') by high performance liquid chromatography. The column filler may be a hydrophobic resin, such as ODS (Octadecylsilylated, C ₁₈ ), C ₈ or C ₄ , which removes nonpolar impurities, and may be purified by reverse phase high performance liquid chromatography. As the developing solvent, 55 to 75% (v / v) of methanol and the remaining amount of purified water are used. The fraction eluted through reverse phase high performance liquid chromatography is further purified by normal phase high performance liquid chromatography packed with silica to remove polar impurities after concentration under reduced pressure. Secondary chromatography was carried out using a developing solvent of 95 to 99% (v / v) dichloromethane and the balance of methanol mixed solution, and finally 99.5% high purity paclitaxel can be obtained.

With the process according to the invention, paclitaxel with a purity of at least 99.5% can be obtained in a yield of at least 65.4%, preferably in a yield of at least 80.7%, without prolonged precipitation.

Table 1 shows the range of purity and recovery of paclitaxel in the samples that can be obtained step by step using the method of the present invention.

Purity of Sediment (%) % Recovery Starting paclitaxel-containing substance 0.01-0.05 100 (a) step: methanol extraction 0.1-0.5 93-97 Step (b): liquid-liquid extraction 3.0-9.0 94-96 (c) step: normal phase column chromatography 20-35 95-97 (d) step: acetone / pentane precipitation 65-85 92-97 (d ') step: methanol / water precipitation 52-82 89-92 (e) step: reversed phase high performance liquid chromatography 90-98 93-96 (e) step: normal phase high performance liquid chromatography 99.5-99.9 92-96 final 99.5-99.9 65.4-80.7

Purity and recovery of the paclitaxel is quantitatively analyzed by HPLC under the conditions of Table 2 below.

device Hewlett Packard 1090 HPLC column Curosil PFP 4.6-250 Column temperature 35 ℃ Mobile phase Acetonitrile: water (35-65% concentration gradient) Flow rate 1ml / min Injection volume 10 ul Detector UV (227 nm)

1 is an HPLC chart confirming the purity and recovery of paclitaxel in each step. (A) is the result of HPLC analysis after organic solvent extraction in step (b), (B) is the result of HPLC analysis after normal phase chromatography in step (c), and (C) is after acetone / pentane precipitation in step (d) HPLC analysis results, (D) is the HPLC analysis results after the high-performance liquid chromatography of step (e). FIG. 2A is a HPLC analysis result of methanol / distilled water precipitation of step (d '), and FIG. 2B is an HPLC chart confirming purity and recovery of paclitaxel after step (e) of FIG. 2A.

Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are only for illustrating the present invention more specifically, and the scope of the present invention is not limited to these examples.

Example 1: Organic Solvent Extraction and Liquid-liquid Extraction of Plant Cell Culture Solution

110 L of methanol was added to 100 kg of plant cells obtained by culturing plant cells (SYG-1, KCTC-0232BP) derived from Taxus Chinenssis, and the mixture was stirred at room temperature for 30 minutes and then filtered. The extraction process was repeated four times to obtain a methanol extract, and concentrated under reduced pressure to obtain a concentrate of about 20 L. 5400 ml of dichloromethane was added to the concentrate, stirred, partitioned to separate the dichloromethane layer, and then concentrated under reduced pressure and dried to obtain 737 g of a dried product having a purity of 6.9%.

Example 2 Normal Phase Column Chromatography (Isocratic Elution)

15 g of the dried product of Example 1 was dissolved in 200 ml dichloromethane and the lysate was poured into a column made of 19 mm diameter x 900 mm stainless steel filled with silica gel 60N (Timely, Japan). 1%, 1.5% and 2% (v / v) of methanol / dichloromethane were used as the developing solvent, and the elution fraction was recovered. The purity and recovery rate of paclitaxel in the recovered elution fraction are shown in Table 3 below.

Experiment number % (v / v), methanol / dichloromethane water(%) % Recovery A 1.0 / 99.0 32.6 84.0 B 1.5 / 98.5 31.8 90.5 C 2.0 / 98.0 30.7 97.5

Example 3 Normal Phase Column Chromatography (Stepwise Gradient Elution)

15 g of the dried material of Example 1 was dissolved in 200 ml of dichloromethane and injected into a column made of 19 mm x 900 mm diameter stainless steel filled with silica gel 60N (Timely, Japan). As the developing solvent, dichloromethane, methanol / dichloromethane (0.5: 99.5 volume ratio) mixed solvent and methanol / dichloromethane (1: 99 volume ratio) mixed solvent were used, respectively. The solvent contained was used. The elution fractions were recovered and the purity and recovery of paclitaxel in the fractions are shown in Table 4 below.

Experiment number Developing solvent, methanol: dichloromethane% (v / v) water(%) % Recovery D 0: 100 22.7 96.9 E 0.5: 99.5 24.2 96.5 F 1.0: 99.0 25.5 97.3

Example 4 Normal Phase Column Chromatography (Stepwise Gradient Elution)

The step of Example 3 was carried out in a mass process.

510 g of the dried product of Example 1 was dissolved in 5600 ml of dichloromethane and packed with silica gel 60N (Timely, Japan) filled with a developing solvent containing 1% (v / v) methanol and the remaining amount of dichloromethane ( 100 mm in diameter x 900 mm in length, made of stainless steel). Thereafter, a solvent containing 2% (v / v) of methanol and 98% (v / v) of dichloromethane as an eluting solvent was added to obtain an eluted fraction. The fractions were concentrated under reduced pressure to give 99.9 g of dry matter. Purity of the paclitaxel purified in the above process is 32.2%, the yield is 97.0%.

Example 5: Acetone / Pentane Precipitation

After dissolving 2 ml of acetone to 40 mg of the dried product of Example 4, 6, 8, 10, 12, 14, 16 and 18 mL of pentane were added thereto, and the mixture was left at 4 ° C. for 48 hours. Thereafter, the precipitate was recovered by filtration, and the purity and recovery rate of paclitaxel in the precipitate were shown in Table 5 below.

Experiment number Starting material Acetone, ml Pentane, ml precipitate Purity (%)  Amount, mg Purity (%)  Recovery rate (%) G 32.2 40 2 6 85.1 82.2 H 32.2 40 2 8 81.4 83.9 I 32.2 40 2 10 79.8 95.4 J 32.2 40 2 12 77.4 96.5 K 32.2 40 2 14 77.1 96.6 L 32.2 40 2 16 76.5 97.6 M 32.2 40 2 18 70.4 97.6

Example 6 Acetone / Pentane Precipitation

Precipitation was carried out under the conditions of Table 6, and the experimental method was the same as in Example 5. The results are shown in Table 6 below.

Experiment number Starting material Acetone, ml Pentane, ml precipitate Purity (%)  Amount, mg Purity (%)  % Recovery N 32.2 10 2 12 74.7 83.7 O 32.2 20 2 12 79.5 93.4 P 32.2 40 2 12 77.4 96.5 Q 32.2 80 2 12 54.5 98.9

Example 7: Acetone / Pentane Precipitation

With respect to the dried material of the fractions having different purity obtained by normal phase chromatography, the experiment was carried out in the same manner as in Example 5, under the conditions of Table 7. The results are shown in Table 7 below.

Experiment number Starting material Acetone, ml Pentane, ml precipitate Purity (%) Amount, mg water(%) Recovery rate (%) R 10.2 40 2 12 24.4 85.4 S 15.4 40 2 12 50.2 90.3 T 21.7 40 2 12 62.4 96.5 U 30.1 40 2 12 74.2 98.8 V 50.2 40 2 12 73.8 98.5

Example 8 Acetone / hexane Precipitation

With respect to the dried material of the fractions having different purity obtained by normal phase chromatography, the experiment was carried out in the same manner as in Example 5 under the conditions of Table 8, except that hexane was used instead of pentane. The results are shown in Table 8 below.

Experiment number Starting material Acetone, ml Hexane, ml precipitate Purity (%) Volume (mg) Purity (%) Recovery rate (%) W 30.0 20 2 12 72.1 95.4 X 45.8 20 2 12 75.4 96.2

Example 9 Dichloromethane / Hexane Precipitation

Experiments were carried out in the same manner as in Example 5 for the dried products of the fractions having different purity obtained by normal phase chromatography, except dichloromethane instead of acetone and hexane instead of pentane. . The results are shown in Table 9 below.

Experiment number Starting material Dichloromethane, ml Hexane, ml precipitate Purity (%) Volume (mg) Purity (%) Recovery rate (%) Y 30.0 20 2 20 52.7 93.4 Z 48.3 20 2 20 72.4 91.6

Example 10 Precipitation Using Methanol and Purified Water

With respect to the dried material of the fractions having different purity obtained by the normal phase chromatography, the sample was dissolved in methanol under the conditions of the following Table 10, and purified water was added thereto, followed by standing at 4 ° C. for 3 days. The precipitate was obtained by filtration and the content and purity of paclitaxel contained in the precipitate were measured. The results are shown in Table 10 below.

Experiment number Starting material Methanol, ml Purified water, ml precipitate Purity (%)  Amount, mg Purity (%)  % Recovery One 32.2 1.0 100 250 52.2 90.5 2 34.4 0.2 7 4.7 70.7 89.8 3 49.1 0.2 10 6.7 81.1 91.9

Example 11 High Speed Liquid Chromatography

The dried material of the precipitate of Experiment No. J of Example 5 was dissolved in 70% methanol and injected into reversed phase high performance liquid chromatography filled with ODS (C ₁₈ ). As a developing solvent, a solvent containing 65% (v / v) of methanol and 35% (v / v) of purified water was used, and fractions were obtained by isocratic elution. Fractions comprise paclitaxel with purity 95% and yield 95%.

The fractions were injected into silica-filled high-performance liquid chromatography and poured a developing solvent containing 98% (v / v) of dichloromethane and 2% (v / v) of methanol to obtain an eluted fraction. . The fraction comprises 99.5% high purity paclitaxel with a yield of 95%.

In addition, the precipitate of Experiment No. 1 of Example 10 was purified by the same method as described above to obtain a paclitaxel-containing fraction having a purity of 99.5%, the yield was 94%.

As described above, the method for purifying paclitaxel of the present invention simplifies the process steps and at the same time significantly improves the yield by replacing the conventional synthetic adsorbent treatment and precipitation with hexane with normal phase chromatography. The fraction recovered through can be easily recovered as a precipitate of high yield through the subsequent precipitation step. Therefore, the method of the present invention can easily separate the paclitaxel from the paclitaxel-containing material with high purity and significantly improve the recovery rate, and thus can be usefully used for the production of paclitaxel.

Claims (10)

(a) extracting by adding an organic solvent to a Taxus genus plant, a cell thereof or a cell culture thereof, and concentrating the extract; (b) adding an organic solvent which is not mixed with water to the concentrate of (a) to separate the organic solvent layer, and concentrating this under reduced pressure; (c) separating the eluate by performing normal phase chromatography on the vacuum concentrate of (b); (d) dissolving the eluate of (c) in acetone or dichloromethane and adding pentane or hexane to form a precipitate; (e) purifying the precipitate of (d) by high performance liquid chromatography; Purification method of paclitaxel comprising a. (a) extracting by adding an organic solvent to a Taxus genus plant, a cell thereof or a cell culture thereof, and concentrating the extract; (b) adding an organic solvent which is not mixed with water to the concentrate of (a) to separate the organic solvent layer, and concentrating this under reduced pressure; (c) separating the eluate by performing normal phase chromatography on the vacuum concentrate of (b); (d ') dissolving the eluate of (c) in methanol and adding water to form a precipitate; (e) purifying the precipitate of (d ') by high performance liquid chromatography; Purification method of paclitaxel comprising a. The method of claim 1 or 2, wherein the organic solvent of step (a) is at least one selected from the group consisting of methanol, ethanol, propanol and dichloromethane. The organic solvent of claim 1 or 2, wherein the organic solvent of step (b) is selected from the group consisting of alkyl halides, ethers, alcohols having 4 to 6 carbon atoms, ethyl acetate and methyl ethyl ketone. The method chosen. According to claim 4, wherein the organic solvent of step (b) is dichloromethane, carbon tetrachloride (carbon tetrachloride), chloroform (chloroform), 1,2-dichloroethane (1,2-dichloroethane), di-ethyl ether ( di-ethyl ether, methyl-t-butyl ether, di-iso-propyl ether, n-butanol and n-pentanol Way. The process of claim 1 or 2, wherein the vacuum concentrate in step (c) is suspended in at least one organic solvent selected from the group consisting of dichloromethane, methanol, benzene, acetone, hexane and ethyl acetate, To perform the photography. The method of claim 1 or 2, wherein the normal phase chromatography of step (c) is performed with a developing solvent consisting of 0.5 to 10% (v / v) methanol and 99.5 to 90% (v / v) dichloromethane. How to be. The process of claim 1 or 2, wherein the normal phase chromatography of step (c) comprises dichloromethane or 0.1 to 5% (v / v) methanol and 99.9 to 95% (v / v) dichloromethane. A solvent and an eluting solvent consisting of 0.5 to 100% (v / v) methanol and the remaining amount of dichloromethane. The method of claim 1, wherein the eluate in (d) comprises dissolving 5 to 100 mg per 1 ml of acetone or dichloromethane, and adding pentane or hexane in a volume ratio of 1: 1 to 20 with respect to acetone or dichloromethane. How to. The method of claim 2, wherein the eluate in step (d ') comprises dissolving at 5 to 100 mg per ml of methanol and adding water at a volume ratio of 1: 1 to 20 with respect to methanol.

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