HK1087109B - Process for isolation and purification of paclitaxel from natural sources - Google Patents

Description

Method for separating and purifying paclitaxel from natural raw material

Technical Field

The present invention relates to an improved process for the isolation and purification of paclitaxel from natural sources.

Background

The process of the previous type disclosed and claimed by the applicant's Chaichem PHARMACEUTICAL INTERNATIONAL US patent No.6,452,024 and its corresponding foreign patent, is of particular interest compared to all previous processes disclosed as prior art in the preamble of this US patent. More precisely, the method disclosed in this us patent is of interest because it makes it possible to:

biomass (bioglass) is more easily obtained after extraction of bark, needles and/or branches of taxus species of different species;

-increasing the amount of biomass thus obtained and which has to be purified by chromatography;

-reducing the purification steps;

-increasing the amount of paclitaxel obtained; and

reduction of production costs to a more economical level.

The method disclosed in this U.S. patent No.6,452,024 basically comprises the steps of:

a) extracting a paclitaxel-containing material from a natural source of taxanes with an organic solvent;

b) contacting the raw material with an alkaline or acidic medium, precipitating to obtain biomass, separating and drying the biomass;

c) resins and natural pigments are removed from the separated and dried biomass by dissolving the biomass in acetone, to which at least one non-polar solvent (such as hexane or heptane) is then added until a paclitaxel-rich oil phase is obtained.

d) Contacting the oily phase enriched with paclitaxel collected in the preceding step with an acidic medium (when step (b) is carried out with a basic medium) or with a basic medium (when step (b) is carried out with an acidic medium) in order to obtain a precipitate by precipitation, separating the precipitate and drying it; and

e) the solution of the isolated precipitate in the volatile solvent is purified at least once by chromatography, and the purified solution obtained by chromatography is then crystallized at least once.

In the above-mentioned U.S. patent, it is also disclosed that step (e) preferably includes:

e1) a first chromatographic purification comprising dissolving the precipitate separated in step (d) in a volatile solvent to prepare a mixture of the solution thus obtained and silica gel, treating the mixture in a chromatographic column containing silica gel and recovering a fraction enriched in paclitaxel;

e2) a second chromatographic purification consisting in evaporating the fraction enriched in paclitaxel recovered from the previous step to dryness until a residue is obtained, then in dissolving said residue in a volatile solvent to prepare a mixture which is re-purified by chromatography under the same conditions as the previous substep so as to obtain another fraction enriched in paclitaxel;

e3) a first crystallization comprising evaporating the other fraction enriched in paclitaxel obtained in the preceding substep to dryness until a residue is obtained, dissolving said residue in acetone to prepare a mixture, and crystallizing the paclitaxel contained in said mixture with a non-polar solvent;

e4) a second crystallization comprising dissolving the paclitaxel crystals obtained in the preceding substep in acetone and then recrystallizing the paclitaxel crystals under the same conditions as in the preceding substep;

e5) a third chromatographic purification comprising dissolving the crystals obtained in the preceding substep of recrystallization in a volatile solvent to obtain a solution, preparing a mixture of said solution and silica gel, treating said mixture in a column containing silica gel so as to obtain a further fraction enriched in paclitaxel with an eluting solvent; and

e6) a third crystallization comprising evaporating to dryness the further fraction enriched in paclitaxel obtained in the preceding substep until a residue is obtained, dissolving said residue in an alcohol, ketone, or alcohol-ketone mixture to obtain a further mixture, and crystallizing the paclitaxel contained in said further mixture with water.

As previously mentioned, the process disclosed in the above-mentioned U.S. Pat. No.6,452,024 is of great interest because it is simpler, more efficient and more economical than existing processes. However, this method still requires many separation steps for purification by chromatography and crystallization, and when extraction is performed with needles and twigs of natural sources of taxanes, a large amount of solvent-soluble impurities needs to be removed. This still results in high production costs, especially because of the low content of paclitaxel in the different taxanes. In addition, the amount of biomass that can be purified is very limited due to the small size of the column and the low yield of paclitaxel obtained after purification.

Disclosure of Invention

The object of the present invention is to provide an improved method which solves most of the above mentioned problems.

More specifically, it is an object of the present invention to provide an improved method, which is capable of:

-obtaining a solution containing more paclitaxel and analogues thereof after extraction of the bark, needles and/or branches of different taxus species;

-reducing impurities and reducing the volume of the extract of paclitaxel and analogues thereof obtained from the biomass by precipitation;

-further increase of the amount of biomass that has to be purified by chromatography;

-further reduction of purification steps;

-further increasing the amount of paclitaxel obtained; and is

Further reducing the production costs to a more economical level.

The improved process for extracting and purifying paclitaxel from natural sources of taxanes containing paclitaxel according to the present invention comprises the following basic steps:

a) washing a starting material comprising paclitaxel with deionized or purified water, said starting material being derived from a natural source of said taxane, said washing being capable of removing soluble impurities from said starting material;

b) extracting a wet material containing paclitaxel from the washed material with an organic solvent;

c) contacting the wet feedstock with salt to obtain a biomass by precipitation, and then separating and drying the biomass;

d) removing pitch and natural pigments from the thus separated and dried biomass by dissolving the biomass in acetone or an acetone-hexane mixture, and then adding at least one polar solvent thereto until a paclitaxel-rich oil phase is obtained;

e) the paclitaxel-enriched oil phase obtained in the previous step is chromatographically purified at least once in a volatile solvent to obtain a purified solution, and then the purified solution obtained by chromatography is crystallized at least once.

The crystalline product thus obtained is in fact a mixture of paclitaxel crystals, which, after filtration and drying, essentially consist of:

-crystals of about 60% purity higher than 99%;

-about 30% of crystals with a purity higher than 98% (< 99%); and

-about 10% of crystals with a purity higher than 92% (< 98%).

Crystals with a purity of less than 99% can be separated, mixed together and then purified by chromatography to obtain more final product with a purity of more than 99%.

The fundamental differences of the invention with respect to the closest prior art, its advantages and the way it can be simplified to implement, can be better understood by reading the following non-limiting description with reference to the attached drawings and the attached examples.

Drawings

FIGS. 1a and 1b are comparisons of the basic steps of the method according to the invention and the flow chart of the method disclosed in the closest prior art (i.e., U.S. Pat. No.6,452,024).

Detailed Description

As previously mentioned, the improved process according to the present invention is used to extract and purify paclitaxel from starting materials derived from natural sources of the taxane containing the paclitaxel to be extracted.

The natural source of taxane used as starting material for carrying out the process according to the invention is of the genus taxus. More specifically, it includes any conifer species containing paclitaxel and its derivatives. Such paclitaxel-containing conifer species may include Taxus brevifolia, Taxus baccata, Taxus canadensis, Taxus himalaica, Taxus yunnanensis, Taxus densiformis, Taxus hicksii, Taxus wardii, Taxus cuspidata, Taxus capita, or Taxus brownii.

An advantage of the method according to the invention is that any fraction of the natural source of paclitaxel-containing taxanes may be used. Preferably, the bark of one or more selected conifers is used. Alternatively, shoots and needles of selected conifers are used.

Detailed description of the individual steps of the method according to the invention

Step 1-washing

The first step of the improved process according to the present invention comprises washing the starting material containing paclitaxel and its analogs from a natural source of taxanes with deionized or purified water. The raw material may comprise bark, twigs, needles, etc., which are completely immersed in water, with or without stirring (preferably at 20-25 ℃) at a temperature between 20-100 ℃ for 2 to 24 hours (preferably 3 hours). The water was then drained. This enables water-soluble impurities to be removed from the feedstock.

Step 2-extraction

The second step of the improved process involves extracting the wet material containing paclitaxel and its analogs from the washed material obtained in the first step with an organic solvent.

The organic solvent used in the extraction step is preferably selected from the group consisting of alcohols, ketones, and mixtures thereof. As such preferred solvents, mention may be made of methanol, acetone and mixtures of methanol and acetone.

In the case of mixtures of alcohols and ketones, the alcohols and ketones are preferably present in a volume ratio of between 9: 1 and 1: 9. More preferably, the volume ratio of the mixture is equal to about 1: 1.

The extract thus obtained is preferably also filtered to remove precipitates and then transferred into a double-walled tank, between which hot water, preferably at 65-70 ℃ is passed. The organic solvent is distilled from the tank. Typically, the amount of solvent collected is about 70% of the starting volume. The remaining paclitaxel-containing solution is then drained into another tank. The raffinate is effectively a non-concentrated extract because it contains residual water.

Step 3-separation of Biomass

The third step of the improved process according to the invention comprises the separation of the biomass from the solution obtained in the preceding step.

For this purpose, the non-concentrated extract from the previous step is diluted with methanol and water and then salted out to obtain a biomass precipitate. Sodium chloride is preferred as the salt used in the salting-out extract. However, other salts may be used in the same reaction, such as ammonium chloride, ammonium sulfate, sodium or potassium acetate, potassium chloride, sodium or potassium phosphate or citrate, all of which are soluble in aqueous solution.

Preferably, sodium chloride (or any other selected salt) is added to the non-concentrated extract rapidly with vigorous stirring. By adding sodium chloride in a concentration of between 10 and 200 grams per liter of solution, the desired biomass is formed rapidly. The concentration of sodium chloride added is preferably 50-100 g, preferably 50-75 g per liter of non-concentrated extraction solution.

The biomass that forms and has precipitated out is separated from the solution by filtration or centrifugation. The biomass thus separated is wet and can be sent immediately to the next step or dried under air or vacuum, preferably by aeration or freeze-drying.

Step 4-removal of resin and Natural pigment

The fourth step of the improved process according to the invention comprises treating the biomass separated in the preceding step in order to remove the resins and natural pigments contained therein.

This step can be carried out according to the type of biomass obtained in the preceding step, i.e. depending on whether it is dry or wet.

A-when the biomass obtained in the preceding precipitation step is dry, it is returned to solution by adding thereto a mixture of acetone and hexane (preferably 1/1 by volume), preferably equal to about 1/25 of the volume of the non-concentrated extract obtained in step 2 before precipitation.

More preferably, the dried biomass is returned to solution by first adding a mixture of acetone and hexane and then adding 1.5 volumes of additional pure hexane. The final ratio of acetone to hexane was 1 volume of acetone to 4 volumes of hexane. After such dissolution, pure water is added to the resulting solution to form a paclitaxel-rich oil phase. Water is preferably added in an amount of 2 to 10 volumes, more preferably 5 to 7 volumes, per 100 volumes of acetone added.

The mixture thus obtained is then fed to a decanter. Recovering the oil phase containing paclitaxel and other taxanes deposited at the bottom of the bottle. The oil phase is then evaporated and is ready for purification by chromatography on silica gel.

B-when the biomass obtained after centrifugation in the third step is wet (i.e.it is not dried), this wet biomass is returned to the solution formed by the mixture of acetone and hexane (preferably A in a volume ratio of 1: 1) without any addition of water. The volume of acetone is preferably equal to about 1/20 of the volume of the non-concentrated extract obtained in the second step before precipitation.

After such dissolution, at least one non-polar solvent is added to the resulting solution to form a paclitaxel-rich oil phase, which is separated from hexane in the same manner.

The non-polar solvent or solvents used for this purpose are preferably selected from hydrocarbons miscible with acetone, such as pentane, hexane or heptane. When hexane is used, the volume of hexane used is usually 3 to 4 times the volume of the acetone solution.

In the case where the wet biomass contains too much water, the resulting paclitaxel-enriched oil phase must be treated to remove residual water before it is applied to silica gel, which is then subjected to chromatography to recover and purify paclitaxel and the like. Removal of water allows the coated silica gel to be dried more quickly before loading onto the chromatography column.

The residual water in the paclitaxel-rich oil phase can be removed by extraction with a water-immiscible solvent. The mixture was then decanted and the organic phase was separated from the water. The water-insoluble solvent used is preferably selected from halogenated hydrocarbons or ethers. As examples of such solvents may be mentioned dichloromethane, trichloromethane and diethyl ether.

The resulting extract is recovered, concentrated under vacuum and as such is ready for purification by chromatography on silica gel.

Step 5-purification by chromatography

The fifth and final step of the process according to the invention consists in chromatographically purifying at least once the oily phase enriched in paclitaxel obtained in the preceding step, and then crystallizing at least once the purified solution obtained by chromatography.

For this purpose, the concentrated paclitaxel-enriched oily phase obtained in the fourth step is subjected to at least one chromatographic purification and the purified solution obtained by chromatography is then crystallized at least once. Preferably, however, the concentrated paclitaxel-enriched oil phase obtained in the fourth step is subjected to several chromatographic purifications and several crystallizations, preferably two chromatographic purifications and preferably three crystallizations.

These stepwise purifications and crystallizations are now described as substeps A to E.

A-first chromatographic purification

In the first chromatographic purification step, the paclitaxel-enriched oil phase obtained in the fourth step of the process is mixed with silica gel and then dried by aeration. The silica gel coated with the oil phase was loaded onto a column containing the same type of silica gel. In the column, paclitaxel is purified using an elution mixture containing 30-40% acetone and 60-70% hexane. Preferably, the elution mixture contains about 35% acetone and about 65% hexane.

The column used is preferably 142 cm high and 7.6cm or 15.2 cm internal diameter, depending on the amount of paclitaxel to be purified. Preferably, the paclitaxel-enriched oil phase containing 4-6 grams of paclitaxel is purified in a column having a diameter of 7.6 cm. The oil phase containing 20-24 g of paclitaxel was applied to a column with a column diameter of 15.2 cm. The smaller column (7.6 cm diameter) contained 2.2-2.3 kg of silica gel and the larger column (15.2 cm diameter) contained 8-9 kg of silica gel.

The silica gel of the column was washed and equilibrated with an elution mixture consisting of acetone and hexane. Elution of the fractions is carried out with the same solvent mixture, preferably at a flow rate of about 100 ml/min in a 7.6cm diameter column and 400 ml/min in a 15.2 cm diameter column. In both cases, the volume is preferably controlled at a pressure of 0-30 psi.

B-first crystallization

B.1-in this step, the fractions containing paclitaxel obtained by chromatography in the preceding step are evaporated to dryness and then returned to acetone to form a solution. The amount of acetone is adjusted so that the absorbance of the solution at 228nm for the maximum corresponding to the peak of paclitaxel HPLC analysis is 1.0-1.5o.d. Paclitaxel is then crystallized by adding 3-4 volumes of hexane to the acetone solution.

B.2-alternatively, the fraction containing paclitaxel obtained by chromatography in the previous step is reduced by evaporation to 1/5 of the initial volume or until the maximum value of the peak for HPLC analysis corresponding to paclitaxel reaches 1.0-1.5o.d. Thus, paclitaxel remained in the acetone-hexane (35-65%) solvent mixture. Then, paclitaxel is crystallized by adding 2-3 volumes of hexane to the solution.

Crystals form rapidly. The mixture was left overnight at room temperature or at a temperature of 2-8 ℃ to complete the crystallization.

C-second crystallization

In this step, the crystals obtained by crystallization in the previous step are separated by filtration or centrifugation and then returned to acetone to form a solution, and the volume of acetone is adjusted so that the absorbance of the solution is 1.0 to 1.5O.D. for the peak corresponding to HPLC analysis of paclitaxel.

The paclitaxel contained in the solution is then recrystallized by adding 3-4 volumes of hexane per volume of acetone solution to the acetone solution.

The crystals obtained at this step were analyzed by HPLC and the paclitaxel purity was between 85-95%.

After separating the crystals from the above two crystallization steps by filtration, the hexane phase was mixed with the fraction obtained from the first chromatography, and the corresponding peak of the fraction was identified as 9-dihydro-13-acetylbaccatin (acetylbaccatin) III. This component is eluted in several fractions before reaching the paclitaxel peak. The mixture was then evaporated to dryness. The residue thus obtained was converted into pale yellow crystals of 9-dihydro-13-acetylbaccatin III by adding methanol. The crystals were isolated by filtration and then returned to acetone to form a solution, which was rapidly crystallized by the addition of 4 volumes of hexane. The crystals of 9-dihydro-13-acetylbaccatin III obtained by filtration at this stage can be recrystallized in the same manner as disclosed above, and the purity is higher than 98% by HPLC analysis.

D-second chromatographic purification

D.1 in this step, the paclitaxel crystals obtained in the previous step are filtered and then returned to the acetone to form a solution. The paclitaxel solution was filtered to remove acetone insoluble particles. The solution thus obtained is then mixed with silica gel and dried by venting.

The silica gel coated with paclitaxel was loaded onto a column containing the same type of silica gel. Paclitaxel is then re-purified a second time with an organic solvent based on the elution mixture. Preferably, the elution mixture comprises 30-40% acetone and 70-60% hexane.

More preferably, the crystals obtained in step C are dissolved in acetone, mixed with silica gel and then dried. The silica gel impregnated with paclitaxel is loaded onto a chromatography column (preferably 142 cm long, 7.6cm or 15.2 cm inner diameter, depending on the amount of paclitaxel that is purified). A7.6 cm diameter column may contain 2.2 to 2.3 kg of silica gel and a 15.2 cm diameter column may contain 8 kg of silica gel. The silica gel in the column is washed and equilibrated with a solution consisting of acetone and hexane (preferably 35: 65% per volume). Elution of the fractions is carried out with the same solvent mixture, preferably at a flow rate of about 100 ml/min in a 7.6cm diameter column or 400 ml/min in a 15.2 cm diameter column. Both columns were operated at pressures of 0-30 psi.

D.2 alternatively, filtering the paclitaxel crystals obtained in the previous step C and then returning the paclitaxel crystals to the dichloromethane to form a solution. The paclitaxel solution was filtered to remove methylene chloride insoluble particles.

The solution thus obtained is then mixed with silica gel and dried by venting. The silica gel coated with paclitaxel was loaded onto a column containing the same type of silica gel. Paclitaxel is then re-purified a second time with an organic solvent based on the elution mixture. Preferably the elution mixture comprises 95-98% dichloromethane and 2-5% isopropanol. More preferably, the crystals obtained in step C are dissolved in dichloromethane, mixed with silica gel and then dried. The silica gel impregnated with paclitaxel is loaded onto a chromatography column (preferably 142 cm long, 7.6cm or 15.2 cm inner diameter, depending on the amount of paclitaxel that is purified). A7.6 cm diameter column may contain 2.2 to 2.3 kg of silica gel and a 15.2 cm diameter column may contain 8 kg of silica gel. The silica gel in the column is washed and equilibrated with a solvent consisting of dichloromethane and isopropanol (preferably 97.5: 2.5% per volume). Elution of the fractions is carried out with the same solvent mixture, preferably at a flow rate of about 100 ml/min in a 7.6cm diameter column and 400 ml/min in a 15.2 cm diameter column.

E-third crystallization

In this step, the fractions enriched in paclitaxel recovered by chromatography in step D are combined according to their purity, preferably according to 98-99% and 90-98% purity. It is then evaporated to dryness and then dissolved in acetone, alcohol (ethanol), ethyl acetate or diethyl ether to form a solution.

The volume of acetone added was adjusted so that the absorbance was 1.0-1.5o.d. relative to the corresponding peak solution of paclitaxel according to HPLC analysis. When the other solvents mentioned above are used, i.e. alcohol (ethanol), ethyl acetate or diethyl ether, the paclitaxel concentration is more important, usually more than 5 times the concentration of the paclitaxel in acetone solution.

Paclitaxel is then subjected to a third recrystallization, preferably as follows:

1. by adding 3-4 volumes of hexane per volume of acetone to the acetone solution.

2. By adding at least 3 volumes of hexane per volume of alcohol or ethyl acetate to the alcohol solution or ethyl acetate solution.

3. By adding at least 2 volumes of hexane per volume of ether to the ether solution.

In the final purification and recrystallization steps disclosed above, the addition of hexane to the paclitaxel solution may be performed at room temperature. This slows down the formation of crystals, but will crystallize completely after 2-4 ℃ or overnight at room temperature. The crystals were filtered and dried under vacuum to give a fine isolated powder. The crystals are then dissolved in an alcohol (methanol or ethanol) or acetone, and then added to water to form a suspension, which is freeze-dried at a temperature of about-60 ℃ over a period of 66-72 hours.

The resulting taxane fractions were then analyzed by HPLC chromatography (Waters system) using an autosampler (Waters 717 plus), photodiode matrix detector (Waters 996), multi-solvent delivery system (Waters 600)^E) And C18 Nova-Pak column, 604 μm (3.9X 150 mm).

The analysis of the fractions can be carried out by injecting 5 ul. The column was eluted with a solvent gradient of acetonitrile-water-methanol (initially 25: 50: 30 by volume and finally 35: 30 by volume) at a flow rate of 1 ml/min.

The peak of the compound was determined at 228nm and the analysis time of the sample was about 36 minutes. The retention time of the paclitaxel peak was about 18.9 + -0.2 minutes, while the 9-dihydro-13-acetylbaccatin III peak was 6.5 + -0.2 minutes.

Preferably, the volatile solvent used to dissolve the residue in the chromatographic purification step is selected from acetone, light alcohols from C1 to C3, ethyl acetate, dichloromethane or mixtures of these solvents.

At the end of this step E, after crystallization of paclitaxel in hexane and after filtration and drying, a mixture of paclitaxel crystals is obtained. The composition of the paclitaxel crystal mixture is as follows:

-60% of crystals with a purity higher than 99%,

-30% of crystals with a purity higher than 98% and lower than 99%, and

-10% crystals with a purity higher than 92% and lower than 98%.

The fundamental difference between the present invention and the method disclosed and claimed in applicant's U.S. patent No.6,452,024

As previously mentioned, FIGS. 1a and 1b are flow charts comparing the basic steps of the method disclosed and claimed in U.S. Pat. No.6,452,024 with the method of the present invention:

1. as shown in these schemes, in the method according to the present invention, the starting material containing paclitaxel to be extracted is preferably composed of dried needles and twigs, which are washed with pure water before extraction with an organic solvent such as methanol or acetone. The water obtained after this washing contains some water-soluble discarded components. The wet needles and shoots are then "mulched" with methanol or acetone or a mixture of the two, followed by extraction at 20 ℃ for a period of time, for example 16 hours. The extract was then passed through a cartridge filter and fed into a double-walled tank where the solvent was distilled through heated water in a circulating jacket. As previously mentioned, the extract containing some residual water is referred to as the "non-concentrated extract".

In contrast, in U.S. patent No.6,452,024, dried needles and shoots are directly extracted with a solvent mixture of methanol and methylene chloride. The concentrated extract obtained after distillation is very viscous (visquous) and contains a large amount of resins and natural pigments. This extract may be referred to as "concentrated extract".

2. In the process according to the invention, sodium chloride is added to the non-concentrated extract and the biomass formed precipitates out immediately. The biomass was separated by centrifugation.

In U.S. Pat. No.6,452,024, a concentrated extract previously diluted in methanol is precipitated in an alkaline or acidic medium to give biomass. The precipitate formed is very fine and light and is salted out by adding sodium chloride before separation.

3. Then, in the process according to the invention and disclosed in U.S. Pat. No.6,452,024, the biomass is dissolved in acetone and a given amount (preferably 4 volumes) of a non-polar solvent (such as hexane) is added to the acetone solution to form a paclitaxel-rich oil phase.

However, in the process according to the invention, the paclitaxel-enriched oil phase thus obtained is easily purified in a chromatographic column with silica gel at low pressure. In contrast, in the process disclosed in U.S. Pat. No.6,452,024, the resulting paclitaxel-enriched oil phase must be subjected to a second precipitation in an acidic or basic medium. After salting out by addition of sodium chloride, the precipitate was separated by centrifugation. The resulting precipitate was dried and then dissolved in acetone. Only then was the acetone solution easily purified with silica gel at low pressure in the column.

4. In the first chromatographic purification according to the process of the invention, the oil phase enriched in paclitaxel is mixed with silica gel and then dried by aeration. The silica gel covered with biomass was then loaded on a chromatography column. Paclitaxel was purified using an elution mixture of acetone and hexane. Fractions containing paclitaxel obtained by chromatography were pooled together and then crystallized. The crystals obtained by filtration or centrifugation are dissolved in acetone and then recrystallized a second time. The crystals are separated by filtration or centrifugation, and are easily repurified a second time and finally purified on a low pressure chromatographic column using silica gel.

In the first chromatographic purification step of the process disclosed in U.S. Pat. No.6,452,024, the paclitaxel-rich oil phase is mixed with silica gel and dried by aeration. The silica gel covered with biomass was then loaded on a chromatography column. Paclitaxel was purified using an elution mixture of acetone and hexane. The fractions containing paclitaxel obtained by chromatography are pooled together and then re-purified by a second chromatography using the conditions used in the previous step. The fractions containing paclitaxel obtained by the second chromatography were pooled together and then crystallized. The crystals obtained by filtration or centrifugation were dissolved in acetone and then recrystallized for a second time. The crystals are separated by filtration or centrifugation, and are easily repurified for the third time and finally purified by chromatography on silica gel at low pressure.

Thus, in the method disclosed in U.S. Pat. No.6,452,024, the number of chromatographies to be performed is greater (3 times rather than 2 times).

5. Of course, in both cases, the paclitaxel containing fractions from the second and last columns in the present invention or those from the third and last column in U.S. patent No.6,452,024 are pooled and then crystallized to form the final product.

Thus, it can now be better understood that the main advantage of the present invention compared to the process of us patent No.6,452,024 is that the process according to the invention comprises fewer preparation steps, and fewer purification steps from the initial needle leaf extraction step to the obtaining of biomass: the three chromatographies of U.S. Pat. No.6,452,024 were replaced by two chromatographies.

The process according to the invention also uses less solvent and requires less starting material to obtain the same amount of final product. Thus, the improved process according to the present invention is not only much less expensive to produce than the conventional and traditional processes for purifying paclitaxel but also much less expensive than U.S. Pat. No.6,452,024.

Detailed Description

The following examples are given for the purpose of illustration only and should not be construed as limiting the scope of the invention.

Example 1

Washing machine

100 kg of dried and ground needles and branches of Taxus canadensis were packed into a cotton bag. The bag was placed in a stainless steel jar to which 400 liters of distilled water was added. The cotton cloth bag was completely immersed in water at room temperature for 3 hours, and the solution containing water-soluble impurities was drained.

In practice, the washing process may be repeated once or twice, with the wet needles retained in the tank by a cotton cloth bag.

Extraction of

250 liters of methanol was added to the pot containing the wet needles and shoots. Extraction was carried out at room temperature for 16 hours. The extraction fluid is pumped through a filter into a second double-walled tank. Methanol was distilled with hot water at 80 ℃ circulating in a double-walled tank. The recovered methanol was transferred to a tank containing needles for rinsing, and the volume of pure methanol reached 400 liters. The rinsed methanol was then pumped and distilled until about 75% of the methanol volume was recovered. About 100-120 liters of residual solution, hereinafter referred to as "non-concentrated extract," is introduced into another tank and held until the extract temperature is reduced to room temperature.

Precipitation of biomass by salting out

The non-concentrated extract was clarified with 10 liters of methanol (about 10% methanol compared to the volume of the non-concentrated extract). Sodium chloride was added rapidly with stirring and the biomass was separated by precipitation. The concentration of sodium chloride is about 50 grams per liter of extract solution. A precipitate formed immediately and the mixture was left without stirring overnight. The supernatant was then drained and the heavy precipitate was easily recovered without filtration or centrifugation.

If necessary, the biomass obtained by addition of sodium chloride can be recovered rapidly by filtration or centrifugation at 4200rpm for 30 minutes at 20 ℃ (J6 MC Beckman centrifuge, 4.2 JProtor). It should be understood that a continuously operating centrifuge is preferably used to process large volumes of sediment.

The supernatant containing 10-deacetylbaccatin III and baccatin III was extracted with dichloromethane or diethyl ether. These taxane analogs can be processed by extraction by concentrating the organic phase by evaporation.

Removing resin and pigment

1. The resulting precipitate was air dried. Alternatively, it may be vacuum dried or freeze dried (freeze dryer-FTS System). The precipitate weighed about 1.3 kg to 1.5 kg. The precipitate was dissolved in 6 liters of acetone-hexane mixture (1: 1) and then filtered (or centrifuged at 4200 rpm) at 0-2 ℃ during 30 minutes to remove insoluble particles contained therein. The acetone-hexane solution was then transferred to a beaker and mixed with 1.5 volumes of pure hexane (9 liters) by stepwise addition of 1/2 volumes of hexane each with stirring for several minutes, adding 5% -20% pure water, preferably 5% to 10%, to form an oil phase. The amount of water was calculated from the volume of acetone used. The mixture was then transferred to a liquid separation bottle and dispensed for about 30 minutes. The paclitaxel-enriched phase was recovered at the bottom of the vial and the resulting solution was concentrated by evaporation to its starting volume of 1/10 prior to decolorization (decolorization). The product is readily adsorbed on silica gel for the chromatographic purification step.

2. After centrifugation without drying, the precipitate thus obtained was dissolved in 3 liters of acetone and then filtered (or centrifuged at 4200 rpm) at 0-2 ℃ during 30 minutes to remove insoluble particles contained therein. The acetone solution was then transferred to a beaker and mixed with 4 volumes of hexane (12 liters) by stirring for several minutes by stepwise addition of 1 volume of hexane, respectively. The oil phase formed rapidly. The mixture was then transferred to a liquid separation bottle and dispensed for about 30 minutes. The paclitaxel-enriched phase was recovered at the bottom of the vial. This solution contained some residual water and was therefore extracted by addition of 1 volume of dichloromethane.

The water was separated from the mixture and the lower dichloromethane phase containing paclitaxel was concentrated to 1/5 of the starting volume of the resulting acetone solution before removing the resin and pigments or evaporating to dryness. In the latter case, the dry residue was dissolved in 0.5 l of acetone.

Example 2

First chromatographic purification on silica gel under reduced pressure

The acetone solution containing paclitaxel and its analogs obtained after removal of the resin and pigments in example 1 was mixed with 500g of silica gel (230-400 mesh). The silica gel impregnated with the extract is dried by aeration (or under vacuum). The total weight after drying was about 900-) On a column (142 x 7.6cm inner diameter). The silica gel was washed and equilibrated with a mixture of acetone and hexane (35: 65%, v/v). With the same solvent, Dynamax was usedThe solvent delivery system performs the elution. The flow rate for elution at a pressure of 0-30psi is approximately 100 ml/min. The volume of the solvent mixture was about 40 liters and fractions were collected in portions of 1 liter each. HPLC analysis indicated that 9 to 10 fractions (from 26 or 27 to 35 or 36) contained about 0.2 mg/ml to 0.7 mg/ml paclitaxel with a purity change of 10% -58%. The fractions containing more paclitaxel had higher purity. The amount of paclitaxel in each fraction was determined by comparing the area with that of a standard sample of paclitaxel.

Notably, from one purification to another, the paclitaxel-containing fraction may deviate from the other fractions.

The fraction with the highest amount of impurities is discarded. Typically, the first fraction containing less than 0.3 mg/ml paclitaxel is discarded.

Example 3

First chromatographic purification on silica gel under reduced pressure

The acetone solution containing paclitaxel and its analogues obtained after removal of the resin and pigments in example 1 was mixed with 500g of silica gel (230-400 mesh). The silica gel impregnated with the extract is dried by aeration (or under vacuum). The total weight after drying was about 920 g, and half of the material was loaded onto a column (142X 7.6cm ID) containing 2.2 kg of silica gel (230 and 400 mesh). The silica gel was washed and equilibrated with a mixture of acetone and hexane (40: 60%, v/v). With the same solvent, Dynamax was usedThe solvent delivery system performs the elution. The flow rate for elution at a pressure of 0-30psi is approximately 100 ml/min. The volume of the solvent mixture was about 30 liters and each stage was collected in portionsIn batches, 1 liter. HPLC analysis indicated that there were 7 to 8 fractions (from 16 th or 17 th to 23 th or 24 th fraction) containing about 0.1 mg/ml to 0.7 mg/ml of paclitaxel with a purity change of 8-45%. The fractions containing more paclitaxel had higher purity. The amount of paclitaxel in each fraction was determined by comparing the area with that of a standard sample of paclitaxel.

Again, it is worth noting that from one purification to another, the fraction containing paclitaxel may be biased relative to the other fractions.

The fraction with the highest amount of impurities is discarded. Typically, the first fraction containing less than 0.3 mg/ml paclitaxel is discarded.

Example 4

First crystallization

After the first chromatographic purification in examples 2 and 3, the fractions with a paclitaxel concentration higher than 0.3 mg/ml are combined and then evaporated to dryness. The residue was dissolved in acetone, and the volume of acetone was adjusted so that the peak of paclitaxel by HPLC was 1.0 to 1.5o.d. Then 4 volumes of hexane were added to the acetone solution and crystallization started during the next 1 hour. The mixture was kept at 2-8 ℃ or at room temperature overnight until complete crystallization.

Second crystallization

Then, the resulting crystals were filtered (or centrifuged), and then dissolved in acetone. The volume of acetone was adjusted so that the maximum o.d. of the paclitaxel peak by HPLC was in the range of 1.0 to 1.5. Hexane was then added in a ratio to 4 volumes of the acetone solution. Crystals formed in the next 1 hour. The mixture is kept at 2-8 ℃ or overnight at room temperature until complete crystallization. The crystals are filtered or centrifuged and dried under air or vacuum. HPLC analysis indicated a paclitaxel content of about 85% or greater. Direct isolation and first crystallization of 9-dihydro-13-acetylbaccatin III

The 9-dihydro-13-acetylbaccatin III-containing fraction obtained in example 2 by the first chromatographic purification (fraction No. 20 to 25) or the fraction No. 13 to 15 in example 3 was combined with the hexane/acetone solution (mother liquor) containing 9-dihydro-13-acetylbaccatin III obtained from the first and second crystallization steps and then evaporated to dryness. Crystals were formed by adding methanol and then recovered by filtration or centrifugation. The resulting crystals were dissolved in acetone and crystallized from 3 volumes of hexane. The product was identified as 9-dihydro-13-acetylbaccatin III with a purity of > 95%.

Second crystallization of 9-dihydro-13-acetylbaccatin III

The thus obtained crystals of 9-dihydro-13-acetylbaccatin III were dissolved in acetone, and 1 volume of hexane was then added to the acetone solution. The mixture was stirred slowly and 2 volumes of hexane were added. The solution was slowly crystallized. The white crystals are recovered by filtration or centrifugation, dried by aeration or under vacuum. HPLC analysis of the crystals showed a 9-dihydro-13-acetylbaccatin III content equal to or greater than 98%.

Example 5

Second chromatographic purification on silica gel at low pressure

The paclitaxel crystals obtained after the second crystallization in example 4 were dissolved in 75 to 100 ml of acetone, and then filtered to remove insoluble particles, followed by adsorption on 75 to 100 g of silica gel. The paclitaxel-coated silica gel is either air dried or dried under vacuum. The dried silica gel was loaded on top of a column (142X 7.6cm inner diameter) containing 2.2 kg of silica gel (230-. The silica gel was washed and equilibrated with a mixture of acetone and hexane (35: 65 v/v). Dynamax was used with the same solvent at a pressure of 0-30psi and a flow rate of about 100 ml/minThe solvent delivery system performs the elution. The volume of the solvent mixture was about 40 liters and fractions were collected in portions of 1 liter each. HPLC analysis showed that 9 to 10 fractions (from 26 or 27 to 34 or 35) contained aboutPaclitaxel at 0.2 mg/ml to 0.6 mg/ml, and their purity varies from 85% to 99%. Fractions containing paclitaxel with a purity higher than 98% were pooled together for the third and last crystallization.

Again, the paclitaxel-containing fraction may be biased relative to the other fractions from one purification step to another.

Example 6

Second chromatographic purification on silica gel at low pressure

The paclitaxel crystals obtained after the second crystallization in example 4 were dissolved in 75 to 100 ml of dichloromethane, and then filtered to remove insoluble particles, followed by contacting with 75 to 100 g of silica gel. The paclitaxel-coated silica gel is either air dried or dried under vacuum. The dried silica gel was loaded on top of a column (142X 7.6cm inner diameter) containing 2.2 kg of silica gel (230-. The silica gel was washed and equilibrated with a mixture of dichloromethane and isopropanol (97.5: 2.5 v/v). Dynamax was used with the same solvent at a pressure of 0-30psi and a flow rate of about 100 ml/minThe solvent delivery system performs the elution. The volume of the solvent mixture was approximately 50 liters and fractions were collected in portions of 1 liter each. HPLC analysis indicated that 20 fractions (from fraction 28 to fraction 48) contained about 0.1 mg/ml to 0.3 mg/ml of paclitaxel with a purity change of 98% to > 99%. Fractions containing paclitaxel with a purity higher than 98% were pooled together for the third and last crystallization.

Third crystallization

After the second chromatographic purification in example 5 or example 6, the fractions with a purity of paclitaxel higher than 98% are combined and then evaporated to dryness. The residue was dissolved in acetone, and the volume of acetone was adjusted so that the peak of paclitaxel was 1.0 to 1.5o.d. as analyzed by HPLC. Then 4 volumes of hexane were added to the acetone solution and crystallization started during the next 1 hour. The mixture was kept at 2-8 ℃ or at room temperature overnight until complete crystallization. The white crystals are recovered by filtration or centrifugation, dried by aeration or under vacuum. The purity of the crystals analyzed by HPLC was as follows:

99.20 to 99.50% for example 5; and

from 99.50 to 99.90% in example 6.

As disclosed above, the residue obtained from the paclitaxel-containing fraction after the second chromatographic purification may be dissolved in ethanol, ethyl acetate or diethyl ether. The volume of those solvents was 5 times smaller than the acetone used previously, which means that the paclitaxel concentration was 5 times higher (10 mg/ml).

In practice:

-1 to 2 volumes of hexane are subsequently added to the ether solution; or

3 to 4 volumes of hexane are subsequently added to the ethanol or ethyl acetate solution, and crystallization begins during the next 1 hour.

The mixture was kept at 2-8 ℃ or at room temperature overnight until complete crystallization. The purity of those crystals was very similar to that when crystallized from acetone.

The crystals with a purity of less than 98% were kept and purified again together by chromatography using the same conditions as described for the second purification. This re-purification again yielded about 75% of the total amount of these crystals, with a purity greater than 99%.

The crystals were dissolved in a minimum volume of ethanol or methanol or acetone and then returned to a vial containing pure water. The volume of solvent is about 10-15% of the volume of pure water. Paclitaxel was lyophilized over 72 hours at a temperature varying from-60 ℃ to +20 ℃ at 0.02 ℃ per minute and a pressure of 100 millitorr.

Claims (21)

1. A process for extracting and purifying paclitaxel from a natural source of taxanes containing the paclitaxel to be extracted, said process comprising the steps of:

a) washing a starting material containing paclitaxel with deionized or purified water, said starting material being derived from a natural source of said taxane, said washing removing soluble impurities from said starting material;

b) extracting a wet paclitaxel-containing material from said washed material with an organic solvent;

c) contacting the wet feedstock with salt to obtain a biomass by precipitation, and then separating and drying the biomass;

d) removing pitch and natural pigments from the thus separated and dried biomass by dissolving the biomass in acetone or an acetone-hexane mixture, and then adding at least one polar solvent thereto until a paclitaxel-rich oil phase is obtained;

e) the paclitaxel-enriched oil phase obtained in the previous step is chromatographically purified at least once in a volatile solvent to obtain a purified solution, and then the purified solution obtained by chromatography is crystallized at least once.

2. The method according to claim 1, wherein the natural source of paclitaxel-containing taxanes consists of conifers selected from the group consisting of: taxus brevifolia, Taxus baccata, Taxus canadensis, Taxus himalaica, Taxus yunnanensis, Taxus densiformis, Taxus hicksii, Taxus wardii, Taxus cuspidata, Taxus capita, or Taxus brownii.

3. A process according to claim 1 or 2, wherein in step (a) the starting material is selected from the bark, branches and needles of the coniferous tree or mixtures thereof.

4. The process according to claim 1 or 2, wherein in step (b) the organic solvent is selected from alcohols or ketones and mixtures of alcohols and ketones.

5. The process according to claim 4, wherein in step (b), the organic solvent is methanol, acetone or a mixture of methanol and acetone present in a volume ratio of 9: 1 to 1: 9.

6. The process according to claim 1 or 2, wherein in step (c) the salt used to obtain the biomass by precipitation is selected from the group consisting of sodium chloride, ammonium sulfate, sodium acetate, potassium acetate and mixtures thereof.

7. The process according to claim 6, wherein in step (c), the salt is sodium chloride; is used in a concentration of 10 to 100 g per liter of wet raw material obtained by the extraction of step (b).

8. The method according to claim 6, wherein in step (c), the precipitated biomass is separated by filtration or centrifugation, and then directly dried with air or freeze-dried.

9. The process according to claim 1 or 2, wherein in step (d) the dried biomass is returned to solution by adding a mixture of acetone and hexane first and then adding 1.5 volumes of additional pure hexane; the final ratio of acetone to hexane was 1 volume of acetone to 4 volumes of hexane; after such dissolution, pure water is added to the resulting solution to form a paclitaxel-rich oil phase; water is added in an amount of 2 to 10 volumes per 100 volumes of acetone added.

10. A method according to claim 1 or 2, wherein step e) comprises the following sub-steps:

e.1) preparing the mixture of the oil phase enriched in paclitaxel and silica gel obtained in step (d), treating said mixture in a silica gel-containing chromatography column with an eluting solvent to obtain a first paclitaxel-enriched fraction;

e.2) collecting the enriched fractions containing paclitaxel obtained by chromatography in step (e.1) together, evaporating to dryness and then dissolving in acetone to form a solution, the amount of acetone being adjusted so that the absorbance of the solution for the peak corresponding to HPLC analysis is 1.0 to 1.5 optical density; then crystallizing paclitaxel by adding 3-4 volumes of hexane to the solution;

e.3) dissolving the paclitaxel crystals obtained in sub-step (e.2) in acetone and then recrystallizing the paclitaxel crystals with a non-polar solvent;

e.4) dissolving the crystals obtained in substep (e.3) in a volatile solvent to obtain a solution, preparing a mixture of said solution and silica gel, and then treating said mixture with an eluting solvent in a silica gel-containing chromatographic column to obtain a second paclitaxel-enriched fraction with eluting solvent; and

e.5) evaporating the second paclitaxel-enriched fraction obtained in substep (e.4) to dryness until a residue is obtained, dissolving the residue in acetone, alcohol, ether, ethyl acetate or a mixture thereof, and then crystallizing paclitaxel with a non-polar solvent.

11. The method of claim 10, wherein:

in step (e.1), the paclitaxel-enriched oil phase obtained in step (d) is mixed with silica gel and then dried in air; recovering the silica gel and loading in a chromatographic column also containing silica gel; then purifying paclitaxel with an elution mixture comprising 30% -40% acetone and 60% -70% hexane; and

in the step (e.3), the crystals obtained in the step (e.2) are separated by filtration or centrifugation, dissolved in acetone to form a solution, the volume of acetone is adjusted so that the absorbance of the solution for the peak corresponding to HPLC analysis is 1.0 to 1.5 optical density, and then paclitaxel is recrystallized by adding 3 to 4 volumes of hexane to the solution.

12. A method according to claim 10 or 11, wherein in step (e.2): collecting the enriched fractions containing paclitaxel obtained by chromatography in step (e.1) together and evaporating until the absorbance of said enriched fractions reaches an optical density of 1.0 to 1.5 for the peak corresponding to HPLC analysis; paclitaxel is then crystallized by adding 1.5-2 volumes of hexane to the enriched solution.

13. The process according to claim 10 or 11, wherein in step (e.4), the crystals obtained in step (e.3) are filtered or centrifuged and then dissolved in acetone to form a solution; the solution was then mixed with silica gel and dried under ventilation; the silica gel covered with paclitaxel was loaded into a column also containing silica gel; paclitaxel is then re-purified using an organic solvent-based elution mixture comprising 30% to 40% acetone and 60% to 70% hexane.

14. The process according to claim 10 or 11, wherein in step (e.4), the crystals obtained in step (e.3) are filtered or centrifuged and then dissolved in acetone to form a solution; the solution was then mixed with silica gel and dried under ventilation; the silica gel covered with paclitaxel was loaded into a column also containing silica gel; paclitaxel is then re-purified using an organic solvent-based elution mixture comprising 95% to 98% methylene chloride and 2% to 5% isopropanol.

15. The method according to claim 10 or 11, wherein in step (e.5), the enriched fractions containing paclitaxel obtained by chromatography in step (e.4) are combined according to their purity, evaporated to dryness and then dissolved in acetone to form a solution, the amount of acetone being adjusted so that the absorbance of the solution for the peak corresponding to HPLC analysis is 1.0 to 1.5 optical density; paclitaxel is then crystallized by adding 3-4 volumes of hexane to the solution.

16. The method according to claim 10 or 11, wherein in step (e.5), the enriched fraction containing paclitaxel obtained by chromatography in step (e.4) is collected together and evaporated until the absorbance of the enriched fraction reaches an optical density of 1.0 to 1.5 for the peak corresponding to HPLC analysis; paclitaxel is then crystallized by adding 1.5-2 volumes of hexane to the enriched solution.

17. The method according to claim 10 or 11, wherein in step (e.5), the enriched fractions containing paclitaxel obtained by chromatography in step (e.4) are combined according to their purity, evaporated to dryness and then dissolved in ethanol to form a solution, the amount of ethanol being adjusted so that said solution reaches a paclitaxel concentration of 5-10mg/ml according to HPLC analysis; paclitaxel is then crystallized by adding 3-4 volumes of hexane to the solution.

18. The method according to claim 10 or 11, wherein in step (e.5), the enriched fractions containing paclitaxel obtained by chromatography in step (e.4) are combined according to their purity, evaporated to dryness and then dissolved in ethyl acetate to form a solution, the amount of ethyl acetate being adjusted so as to bring the solution to a paclitaxel concentration of 5-10mg/ml according to HPLC analysis; paclitaxel is then crystallized by adding 3-4 volumes of hexane to the solution.

19. The method according to claim 10 or 11, wherein in step (e.5), the enriched fractions containing paclitaxel obtained by chromatography in step (e.4) are combined according to their purity, evaporated to dryness and then dissolved in diethyl ether to form a solution, the amount of diethyl ether being adjusted so that the solution reaches a paclitaxel concentration of 5-10mg/ml according to HPLC analysis; paclitaxel is then crystallized by adding 1-2 volumes of hexane to the solution.

20. The method according to claim 10 or 11, wherein in step (e.4), the volatile solvent is selected from acetone, C₁-C₃Light alcohol, ethyl acetate, diethyl ether, dichloromethane, and mixtures thereof.

21. A method according to claim 10 or 11, comprising the additional step of: extracting crystals of 9-dihydro-13-acetylbaccatin III with methanol from the elution fraction before paclitaxel isolation in step (e.1) and from the mixture obtained after crystallization in steps (e.2) and (e.3); and the thus extracted crystals were recrystallized from a mixture of acetone-hexane.