Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07D—HETEROCYCLIC COMPOUNDS
  • C07D305/00—Heterocyclic compounds containing four-membered rings having one oxygen atom as the only ring hetero atoms
  • C07D305/14—Heterocyclic compounds containing four-membered rings having one oxygen atom as the only ring hetero atoms condensed with carbocyclic rings or ring systems
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P35/00—Antineoplastic agents

Definitions

• the present invention relates to an improved process for the isolation and purification of Paclitaxel from natural sources.
• the process disclosed in this U.S. patent No. 6,452,024 basically comprises the following steps: a) extracting a raw material comprising Paclitaxel from a natural source of taxanes with an organic solvent; b) contacting the raw material with a basic medium or an acidic medium to obtain a biomass by precipitation, isolating and drying said biomass; c) removing resin and natural pigments from the isolated and dried biomass by dissolving the biomass in acetone and then adding to it at least one non-polar solvent such as hexane or heptane, until a Paclitaxel-enriched oily phase is obtained; d) contacting the Paclitaxel-enriched oily phase recovered in the preceding step with an acidic medium when step (b) was carried out with a basic medium, or with a basic medium when step (b) was carried out with an acidic medium, in order to obtain a precipitate by precipitation, isolating the precipitate and drying it; and e
• step (e) preferably comprises: ei) a first chromatographic purification comprising dissolving the precipitate isolated ion step (d) in a volatile solvent, preparing a mixture of the so-obtained solution with silica gel, treating the mixture in a chromatographic column containing silica gel, and recovering Paclitaxel-enriched fractions; ez) a second chromatographic purification comprising by evaporating to dryness the Paclitaxel-enriched fractions recovered in the preceding step until a residue is obtained, and preparing a mixture by solubilizing said residue in a volatile solvent, the mixture being repurified by chromatography under the same conditions as in the preceding sub-step in order to obtain other Paclitaxel-enriched fractions; ⁇ 3> a first crystallization comprising evaporating to dryness the other Paclitaxel-enriched fractions obtained in the preceding sub-step until a residue is obtained, preparing a mixture of this residue in
• the object of the present invention is to provide an improved process which solves most of the problems mentioned hereinabove.
• the object of the invention is to provide an improved process which permits: - to obtain a solution which contains more Paclitaxel and analogs after extraction of the barks, needles and/or branches of Taxus of different species;
• Paclitaxel comprises the following basic steps: a) washing with deionized or pure water, a raw material comprising
• Paclitaxel said raw material coming from said natural source of taxanes, such a washing allowing removal of soluble impurities from said raw material; b) extracting with an organic solvent from said washed raw material, a wet raw material comprising Paclitaxel; c) contacting said wet raw material with a salt to obtain a biomass by precipitation, and isolating and drying said biomass; d) removing resin and natural pigments from the so isolated and dried biomass by dissolving said biomass in acetone or an acetone-hexane mixture and then adding thereto at least one-polar solvent until a Paclitaxel- enriched oil phase is obtained; e) purifying by chromatography at least once the Paclitaxel-enriched oily phase obtained in the preceding step in a volatile solvent to obtain a purified solution and crystallization at least once the purified solution obtained by chromatography.
• the crystallized product that is so obtained is actually a mixture of Paclitaxel crystals, which, after filtration and drying of the crystals
• crystals having a purity higher than 92% ⁇ 98%).
• the crystals having purities less than 99% can be separated, mixed together and subjected to subsequent purification by chromatography in order to get more of the final product having a purity higher than 99%.
• Figures 1 a and 1 b are flow charts in which the basic steps of the process according to the invention and the one disclosed in the closest prior art, namely U.S. patent No. 6,452,024 are compared.
• the improved process according to the invention is intended to be used for the extraction and purification of Paclitaxel from a raw material coming from a natural source of taxanes containing the Paclitaxel to be extracted.
• the natural source of taxanes used as starting material for carrying out the process according to the invention is of Taxus gender. More particularly, it consists of any one of the species of conifers that contain Paclitaxel and its derivatives. Such species of conifer containing Paclitaxel may consist of Taxus brevifolia, Taxus baccata, Taxus canadensis, Taxus wallichiana, Taxus yunnanensis ? Taxus densiforrnis, Taxus hicksii, Taxus wardii, Taxus cuspidata, Taxus capitate or Taxus brown//.
• the process according to the invention has the advantage of being usable with any part of the natural source of taxanes which contains Paclitaxel.
• use is made of barks of the selected conifer(s).
• use can be made of branches and needles of the selected conifers.
• Step 1 - Washing The first step of the improved process according to the invention consists of washing a raw material containing Paclitaxel and its analogs coming from a natural source of taxanes, using a deionized or pure water to do so.
• the raw material which may consist of barks, branches, needles and the like is completely covered with the water with or without stirring at a temperature comprised between 20 °C and 100°C (preferably at a temperature of 20-25 °C) for a period of 2 to 24 hours (preferably 3 hours). After that time, water is drained. Such permits to remove water-soluble impurities from the raw material.
• the second step of the improved process consists of extracting with an organic solvent, a wet raw material containing Paclitaxel and its analogs from the washed raw material obtained in the first step.
• the organic solvent that is used during this extraction step is preferably selected from the group consisting of alcohols, cetones and mixtures thereof.
• preferred solvents reference can be made to methanol, acetone and mixtures of methanol with acetone.
• the alcohol and cetone are preferably present in volume ratio comprised between 9: 1 and 1 :9. More preferably, the volume ratio in the mixture is equal to about 1 : 1 .
• the extract that is so obtained is filtered to remove deposits, and then transferred in a double wall tank in which hot water preferably at a temperature of 65-70 °C, is fed.
• the organic solvent is distilled from this tank.
• the collected solvent amounts to about 70% from the starting volume.
• the remaining solution containing Paclitaxel is then drained into another tank. This remaining solution is actually a non-concentrated extract because of the residual water contained therein.
• the third step of the improved process according to the invention consists of isolating a biomass from the solution obtained in the previous step.
• the non-concentrated extract obtained from the previous step is diluted in methanol and water and then salted-out to obtain precipitation of a biomass.
• Sodium chloride is preferably used as salt for salting-out the extract.
• other salts could be used for the same reaction, like ammonium chloride, ammonium sulfate, sodium or potassium acetate, potassium chloride, sodium or potassium phosphate or sodium or potassium citrate, all of these salts being solubilizable in an aqueous solution.
• Sodium chloride (or any other selected salt), is preferably quickly added to the non-concentrated extract under heavy stirring.
• the requested biomass is formed rapidly by addition of sodium chloride in a concentration comprised between 10 and 200 grams per liter of solution.
• sodium chloride is added at a concentration comprised between 50 and 100, and more preferably between 50 and 75 grams per liter of the non-concentrated extract solution.
• the biomass that is formed and has precipitated is separated from the solution by filtration or by centrifugation.
• the so separated biomass which is wet can be submitted immediately to the next step or dried at ambient air or under vacuum, preferably by ventilation or lyophilization.
• the fourth step of the improved process according to the invention consists of treating the biomass isolated in the preceding step in order to remove resin and natural pigments contained therein.
• this step depends on the kind of biomass obtained in the previous step, namely on whether it is dried or wet.
• the dried biomass is put back in solution by first adding the mixture of acetone and hexane and then adding 1 .5 additional volumes of pure hexane.
• the final ratio of acetone to hexane is 1 volume of acetone for 4 volumes of hexane.
• the pure water is added to the obtained solution to form a Paclitaxel-enriched oily phase.
• Water is preferably added at a ratio of 2 to 10 volumes, and more preferably 5 to 7 volumes, per 100 volumes of added acetone.
• At least one non-polar solvent is added to the obtained solution to form a Paclitaxel-enriched oily phase which is separated from hexane in the same way.
• the non-polar solvent(s) used in this purpose is (are) preferably selected from the group consisting of hydrocarbons miscible with acetone, such as pentane, hexane or heptane.
• hydrocarbons miscible with acetone such as pentane, hexane or heptane.
• pentane hexane
• heptane heptane
• the volume of hexane that is used is usually 3 to 4 times the one of the acetone solution.
• the obtained Paclitaxel-enriched oily phase must be treated to remove residual water before coating it on silica gel and subjecting the so coated silica el to chromatography in order to recover and purify Paclitaxel and its analogs. Removal of water permits to dry more quickly the coated silica gel before loading it onto the chromatography column.
• the residual water in the Paclitaxel-enriched oily phase can be removed by extraction with a water-immiscible solvent.
• the mixture is then decanted and the organic phase is separated from the water.
• the water- immiscible solvent that is used is preferably selected from the group consisting of halogenated hydrocarbons or ether.
• halogenated hydrocarbons or ether As examples of such solvents, reference can be made to chloromethylene, trichloromethane and to diethyl ether.
• the fifth and last step of the process according to the invention consists in chromatographically purifying at least once a solution of the Paclitaxel-enriched oily phase obtained in the preceding step, and crystallizing at least once the purified solution obtained by chromatography.
• the concentrated Paclitaxel-enriched oily phase obtained in the fourth step is submitted to at least one chromatographic purification and to the purified solution obtained by chromatography at least one crystallization.
• the concentrated Paclitaxel-enriched oily phase obtained in the fourth step is submitted to several chromatographic purifications and several crystallizations, the number of chromatographic purifications being preferably equal to two and the number of crystallization being preferably equal to three.
• the Paclitaxel-enriched oily phase obtained in the fourth step of the process is mixed with silica gel and dried under ventilation.
• the silica gel coated with the oily phase is loaded onto a chromatographic column containing the same type of gel.
• the Paclitaxel is purified with an elution mixture comprising from 30 to 40% of acetone and from 60 to 70% of hexane.
• the elution mixture comprises about 35% of acetone and about 65% of hexane.
• the column that is used preferably has a height of 142 cm and inside diameter of 7.6 cm or 1 5.2 cm depending of the amount of Paclitaxel to be purified.
• a Paclitaxel-enriched oily phase containing 4 to 6 g of Paclitaxel is preferably purified in a column having a 7.6 cm diameter.
• An oily phase containing 20 to 24 g of Paclitaxel uses a column having a 1 5.2 cm column diameter.
• the smaller column (7.6 cm diameter) contains 2,2 to 2.3 kg of silica gel while the bigger column (1 5.2 cm diameter) contains 8 to 9 kg.
• the silica gel of the column is washed and balanced with the elution mixture consisting of acetone and hexane.
• Elution of the fraction is carried out with the same solvent mixture preferably at a flow rate of about 100 ml/min in the 7.6 cm diameter column, and a flow rate of 400 ml/min in the 1 5.2 cm diameter column.
• the volume is preferably kept under a pressure varying from 0 to 30 psi.
• the fractions containing Paclitaxel obtained by chromatography in the preceding step are evaporated to dryness and put back in solution in acetone.
• the amount of acetone is adjusted to obtain an absorbency at 228 nm of 1 .0 to 1 .5 O.D. for the maximum of the peak corresponding to Paclitaxel by HPLC analysis.
• the Paclitaxel is crystallized by adding from 3 to 4 volumes of hexane in the acetone solution.
• the fractions containing Paclitaxel obtained by chromatography in the preceding step are reduced by evaporating to 1 /5 of initial volume or until obtention of 1 .0 to 1 .5 O.D. for the maximum of the peak corresponding to Paclitaxel by HPLC analysis.
• the Paclitaxel thus remains in the solvent mixture of acetone-hexane (35-65%). Then, the Paclitaxel is crystallized by adding from 2 to 3 volumes of hexane in the solution.
• the crystals obtained by crystallization in the preceding step are separated by filtration or centrifugation and put back in solution in acetone with a volume of acetone adjusted to obtain an absorbency of the solution varying from 1 .0 to 1 .5 O.D. for the peak corresponding to Paclitaxel by HPLC analysis.
• the Paclitaxel contained in this solution is then recrystallized by adding to the acetone solution from 3 to 4 volumes of hexane per volume of solution.
• the crystals obtained in this step have a Paclitaxel purity comprised between 85 -95% by HPLC analysis.
• the hexane phase is mixed with the fractions obtained from the first chromatography corresponding to the peak which is identified as 9-dihydro 1 3-acetylbaccatin III.
• This component is eluted in several fractions before reaching the Paclitaxel peak.
• the mixture is then evaporated to dryness.
• the so obtained residue is converted into light yellow crystals of 9-dihydro-13-acetylbaccatin III by addition of methanol.
• the crystals are separated by filtration, then put back in solution in acetone and crystallized rapidly by adding 4 volumes of hexane.
• the crystals of 9-dihydro-1 3- acetylbaccatin III obtained in this step by filtration can be recrystallized by the same method as disclosed above and have a purity higher than 98% by HPLC analysis.
• the Paclitaxel crystals obtained in the preceding step are filtered and then put back in solution in acetone.
• the Paclitaxel solution is filtered to remove particles undissolved in the acetone.
• the so obtained solution is then mixed with silica gel and dried under ventilation.
• the silica gel coated with Paclitaxel is loaded onto a chromatographic column containing the same type of gel. Paclitaxel is then repurified for a second time with an organic-solvent based elution mixture.
• the elution mixture comprises from 30 to 40% acetone and from 70 to 60% hexane.
• the crystals obtained in step C are dissolved with acetone, mixed with silica gel and dried.
• the gel impregnated with Paclitaxel is loaded onto a chromatographic column (preferably 142 cm long with a 7.6 cm or 1 5.2 cm inside diameter, depending on the amount of Paclitaxel to be purified).
• the 7.6cm diameter column may contain 2,2 to 2.3 kg of silica gel while the 1 5.2 cm diameter column may contain 8 kg of silica gel.
• the gel in the column is washed and equilibrated with a solvent consisting of acetone and hexane (preferably 35 : 65% per volume).
• Elution of the fractions is carried out with the same mixture of solvents, preferably at a flow rate of about 100 ml/min within the 7.6 cm diameter column or 400 ml/min within the 1 5.2 cm diameter column. Both columns are operated at a pressure varying from 0 to 30 psi.
• the Paclitaxel crystals obtained in the preceding step C are filtered and then put back in solution in methylene chloride.
• Paclitaxel solution is filtered to remove particles undissolved in the chloride methylene.
• the so obtained solution is then mixed with silica gel and dried under ventilation.
• the silica gel coated with Paclitaxel is loaded onto a chromatographic column containing the same type of gel. Paclitaxel is then repurified for the second time with an organic-solvent based elution mixture.
• the elution mixture comprises from 95 to 98% chloromethylene and from 2 to 5% isopropanol. More preferably, the crystals obtained in step C are dissolved with chloromethylene, mixed with silica gel and dried.
• the gel impregnated with Paclitaxel is loaded onto a chromatographic column (preferably 142 cm long with a 7.6 cm or 15.2 cm inside diameter, depending on the amount of Paclitaxel to be purified).
• the 7.6cm diameter column may contain 2.2 to 2.3 kg of silica gel while the 1 5.2 cm diameter column may contain 8 kg of silica gel.
• the gel in the column is washed and equilibrated with a solvent consisting of chloromethylene and isopropanol (preferably 97.5 : 2.5% per volume). Elution of the fractions is carried out with the same mixture of solvents, preferably at a flow rate of about 100 ml/min with the 7.6 cm diameter column or 400 ml/min with the 1 5.2 cm diameter column.
• the enriched fractions containing Paclitaxel recovered by chromatography in step D are combined according to their purity, preferably according to purities of 98 to 99% and 90 to 98%. Then, they are evaporated to dryness and put back in solution in acetone, alcohol (ethanol) ethyl acetate or diethyl ether.
• the volume of added acetone is adjusted to obtain an absorbency varying from 1 .0 to 1 .5 O.D. for the peak corresponding to Paclitaxel according to HPLC analysis.
• the Paclitaxel concentration is much more important, in general 5 times more than concentrated than Paclitaxel in acetone solution.
• the Paclitaxel is then recrystallized for a third time, preferably as follows:
• addition of hexane in the Paclitaxel solution can be carried out at ambient temperature. This will slow down the formation of crystals but crystallization will be completed overnight at 2-4°C or at ambient temperature.
• the crystals are filtered and dried under vacuum, in order to obtain a fine, detached powder.
• the crystals are then dissolved in alcohol (methanol or ethanol) or in acetone and then put back in suspension in water and lyophilized during 66 to 72 hours at a temperature of about -60°C.
• the obtained fractions of taxanes can then be analyzed by HPLC chromatography (Waters system) using an Autosampler (Waters 717 plus), a Photodiode Array Detector (Waters 996), a Multisolvent Delivery System (Waters 600 E ) and a C18 Nova-Pak column, 60 A, 4 ⁇ m (3.9 x 1 50 mm).
• Analysis of the fractions can be carried out by injecting a volume of 5 ⁇ l.
• the column is eluted at a flow rate of 1 ml/min using a solvent gradient of acetonitrile-water-methanol (25:50:30 in volume at the beginning and 35:35:30 at the end).
• the peaks of the compounds are detected at 228nm and the time of analysis of a sample is about 36 minutes.
• the retention time for the Paclitaxel peak is about 18.9 ⁇ 0.2 minutes and 6.5 ⁇ 0.2 minutes for the 9-dihydro-13-acetylbaccatin III peak.
• the volatile solvent used in the chromatographic purification step for the solubilization of the residue is selected from the group consisting of cetones, C1 -C3 light alcohols, ethyl acetate, chloromethylene or a mixture of these solvents.
• This mixture of Paclitaxel crystals consists of: 60% of crystals having a purity higher than 99%,
• Figures 1 a and 1 b are flow charts in which the basic steps of process disclosed and claimed in US patent No. 6,452,024 are compared with those of the present invention:
• the raw material containing the Paclitaxel to be extracted which preferably consists of dried needles and twigs, is washed in purified water before extraction by organic solvent such as methanol or acetone.
• organic solvent such as methanol or acetone.
• the water obtained after such a washing contains some hydrosoluble components which are discarded.
• the wet needles and twigs are then "covered” with methanol or acetone, or a mixture of both, and subjected to extraction for a period of, for example, 1 6 hours at 20°C.
• the extract is then passed through a cartridge filter and pumped into a double wall tank where the solvent is distilled by circulation of heated water in the jacket.
• the extract containing some residual water is called "non-concentrated extract”.
• Paclitaxel-enriched oily phase is ready to be purified in a chromatographic column with silica-gel at low pressure.
• the obtained Paclitaxel-enriched oily phase must be submitted to a second precipitation in acidic or basic medium.
• the precipitate is isolated by centrifugation after having been salted-out by addition of sodium chloride.
• the obtained precipitate is dried and then dissolved in acetone. It is only at that time that the acetone solution is ready to be purified in a chromatographic column with silica-gel at low pressure.
• the Paclitaxel-enriched oily phase is mixed with silica gel and dried under ventilation. Then the silica gel covered with the biomass is loaded onto a chromatographic column.
• the Paclitaxel is purified with an elution mixture of acetone and hexane. The fractions containing Paclitaxel obtained by chromatography are grouped together and then crystallized. The crystals obtained by filtration or centrifugation are dissolved in acetone and then recrystallized for a second time. The crystals are isolated by filtration or centrifugation and ready to be repurified by a second and last chromatographic column with silica gel at low pressure.
• Paclitaxel is purified with an elution mixture of acetone and hexane.
• the fractions containing Paclitaxel obtained by chromatography are grouped together and then repurified by a second chromatographic column in the conditions used for the preceding step.
• the fractions containing the Paclitaxel obtained by the second chromatography are grouped together and then crystallized.
• the crystals obtained by filtration or centrifugation are dissolved in acetone and then recrystallized for a second time.
• the crystals are isolated by filtration or centrifugation and ready to be repurified by third and last chromatographic column with silica-gel at low pressure.
• 6,452,024 are grouped together and then crystallized to form a final product.
• a main advantage of the present invention as compared with the one of US patent No. 6,452,024 are that the process according to the invention comprises less preparation steps from the one of starting extraction of the needles to the obtention of the biomass, and less purification steps: two chromatographies in place of three as described in US patent No. 6,452,024.
• the process according to the invention also uses much less solvents and needs much less raw materials for obtaining the same amount of final product. Therefore, the production cost with the improved process according to the present invention is much lower than not only the usual and traditional processes used for Paclitaxel purification but also the one of US patent No. 6,452,024.
• this washing procedure may be repeated once or twice, the wet needles being kept in the tank by the cotton bag.
• the non-concentrated extract was clarified with 10 L of methanol (about 10% of methanol as compared to the volume of non-concentrated extract volume).
• the biomass was isolated by precipitation by adding rapidly sodium chloride under stirring.
• the concentration of sodium chloride was about 50g per liter of the extract solution. A precipitate was formed immediately and the mixture was kept without stirring overnight. The supernatant was then drained and the heavy precipitate was easily recovered without filtration or centrifugation.
• the biomass obtained by adding sodium chloride may be recovered rapidly by filtration or centrifugation at a speed of 4,200 rpm at 20° C during 30 minutes (J6 MC Beckman Centrifugal machine, 4.2 JS rotor). It is understood that a continuous flow centrifugal machine may preferably be used to process large amount of precipitate.
• a supernatant containing 10-deacetylbaccatin III and Baccatin III was extracted by methylene chloride or diethylether.
• the organic phase was concentrated by evaporation and may be treated to extract these analogs of taxane. Removal of resin and pigments
• the obtained precipitate was air dried. Alternatively, it could have been vacuum dried or lyophilized (Freeze dryer - FTS System). The weight of the precipitate was about 1 .3kg-1 .5kg. This precipitate was solubilized in 6L of an acetone-hexane mixture (1 :1 ) and filtered (or alternatively centrifuged at 4,200 rpm) at 0-2° C during 30 minutes, in order to remove the insoluble particles contained therein.
• the acetone-hexane solution was transferred into a beaker and mixed with 1 .5 volumes of pure hexane (9L) by successive .addition of 1 /2 volume of hexane each under agitation for few minutes, the oily phase was formed by addition of between 5% and 20% of pure water, preferably 5% to 10%. The amount of water was calculated according to the volume of the acetone used.
• the mixture was then fed into a separating flask and allowed to partition for about 30 minutes.
• the Paclitaxel-enriched phase was recovered at the bottom of the flask and concentrated by evaporation to 1 /10 of the initial volume of the solution obtained before percolorization. This preparation was ready to be adsorbed on silica gel for a chromatography purification step.
• the so obtained precipitate was dissolved in 3 L of acetone and filtered (or, alternatively centrifuged at 4,200 rpm) at 0-2 °C during 30 minutes, in order to remove the insoluble particles contained therein. Then, the acetone solution was transferred into a beaker and mixed with 4 volumes of hexane (1 2 L) by successive addition of one volume of hexane each under agitation for a few minutes. The oily phase was formed quickly. The mixture was then fed into a separating flask and allowed to partition for about 30 minutes. The Paclitaxel-enriched phase was recovered at the bottom of the flask. This solution contained some residual water and thus was extracted by adding 1 volume of chloromethylene.
• the water was partitioned from this mixture and the lower chloromethylene phase containing the Paclitaxel was concentrated to 1 /5 of the initial volume of the acetone solution obtained before removal of the resin and pigments or evaporated to dryness. In the latter case, the dried residue was dissolved in 0.5 L of acetone.
• the obtained acetone solution containing Paclitaxel and analogs after removal of the resin and pigments in example 1 was mixed with 500 g of silica gel (230-400 mesh).
• the gel impregnated with the extract was air dried under ventilation (or under vacuum).
• the total weight after drying was about 900-950 g and half of this material was loaded onto a column (142 x 7.6 cm inside diameter) containing 2.2 kg of silica gel (230-400 mesh).
• the gel was washed and balanced with a mixture of acetone and hexane (35 : 65%, volume per volume). Elution was carried out with the same solvent, using a Dynama ⁇ ⁇ solvent delivery system.
• the flow rate of the elution was about 100 ml/min under a pressure between 0 - 30 psi.
• the volume of solvent mixture was about 40 L and each fraction was collected in batches of 1 L.
• HPLC analysis indicated that there were 9 to 10 fractions, from the 26 th or 27 th to 35 th or 36 h , which contained about 0.2 mg/ml to 0.7 mg/ml of Paclitaxel and their purity varied from 10 to 58%.
• the fractions that contained more Paclitaxel have higher purity.
• the quantity of Paclitaxel for each fraction was determined from its peak area by comparison with the area of a Paclitaxel standard.
• fractions containing Paclitaxel can be offset with respect to the others from one purification to the other.
• the obtained acetone solution containing Paclitaxel and analogs after removal of resin and pigments in example 1 was mixed with 500 g of silica gel (230-400 mesh).
• the gel impregnated with the extract was air dried under ventilation (or under vacuum).
• the total weight after drying was about 920 g and half of this material was loaded onto a column (142 x 7.6 cm inside diameter) containing 2.2 kg of silica gel (230-400 mesh).
• the gel was washed and balanced with a mixture of acetone and hexane (40 : 60%, volume per volume).
• the elution was carried out with the same solvent, using a Dynamax ® solvent delivery system.
• the flow rate of the elution was about 100 ml/min under a pressure between 0 - 30 psi.
• the volume of solvent mixture was about 30 L and each fraction was collected in batches of 1 L.
• HPLC analysis indicated that there were 7 to 8 fractions, from the 1 6 th or 1 7 th to 23 th or 24 th , which contained about 0.1 mg/ml to 0.7 mg/ml of Paclitaxel and their purity varied from 8 to 45%.
• the fractions that contained more Paclitaxel have higher purity.
• the quantity of Paclitaxel for each fraction was determined from its peak area by comparison with the area of a Paclitaxel standard.
• Example 4 First crystallization After the first chromatography purification in examples 2 and 3, the fractions with a concentration of Paclitaxel higher than 0.3 mg/ml were combined and evaporated to dryness. The residue was dissolved in acetone and the volume of acetone was adjusted to obtain a maximum peak of Paclitaxel in the 1 .0 to 1 .5 O.D range by HPLC. 4 volumes of hexane were then added to the acetone solution to start the crystallization during the following hour. The mixture was kept at 2-8° C or at room temperature overnight to complete the crystallization.
• the obtained crystals were filtered (or centrifuged) and dissolved in acetone.
• the volume of acetone was adjusted to obtain a Paclitaxel peak by HPLC with a maximum O.D. in the range of 1 .0 to 1.5. Hexane was then added at a ratio of 4 volumes by respect to the acetone solution. Crystals were formed in the following hour. The mixture was kept at a temperature comprised between 2-8° C or at ambient temperature overnight to complete the crystallization. The crystals were filtered or centrifuged and dried under air or vacuum. HPLC analysis indicated that the Paclitaxel content was about 85% or higher.
• fractions containing 9-dihydro-13-acetylbaccatin III obtained from the first purification by chromatography in example 2 or fraction 1 3 th to 1 5 th in example 3 were combined together with the hexane/acetone solutions (mother liquors) obtained from the first and second crystallization steps, containing 9-Dihydro-13-acetylbaccatin III and evaporated to dryness.
• the crystals were formed by adding methanol and recuperated by filtration or centrifugation. The obtained crystals were dissolved in acetone and crystallized by 3 volumes of hexane.
• the product was identified as 9-dihydro-13-acetylbaccatin III with a purity > 95%.
• the Paclitaxel crystals obtained after the second crystallization in example 4 were dissolved in 75 to 100 ml of acetone, then filtered to remove insoluble particles and adsorbed on 75 to 100g of silica gel.
• the gel coated with Paclitaxel was air dried under ventilation or under vacuum.
• the dried gel was loaded on top of a column (142 x 7.6 cm inside diameter) containing 2.2 kg of silica gel (230-400 mesh).
• the gel was washed and balanced with a mixture of acetone and hexane (35 : 65 volume per volume).
• the elution was carried out with the same solvent, using a Dynamax 65 solvent delivery system at a flow rate of about 100 ml/min under a pressure between 0 - 30 psi.
• the volume of solvent mixture was about 40 L and fractions were collected in batches of 1 L.
• HPLC analysis indicated that there were 9 to 10 fractions, from the 26 th or 27 th to 34 th or 35 th , which contained about 0.2 mg/ml to 0.6 mg/ml of Paclitaxel and their purity varied from 85% to 99%.
• the fractions containing Paclitaxel having a purity higher than 98% were combined together for a third and last crystallization.
• fractions containing Paclitaxel can be offset with respect to the others from one purification step to the other.
• the Paclitaxel crystals obtained after the second crystallization in the example 4 were dissolved in 75 to 100 ml of methylene chloride, then filtered to remove insoluble particles and then put into contact with 75g to 100g of silica gel.
• the gel coated with Paclitaxel was air dried under ventilation or under vacuum.
• the dried gel was loaded on top of a column (142 x 7.6 cm inside diameter) containing 2.2 kg of silica gel (230-400 mesh). The gel was washed and balanced with a mixture of chloromethylene and isopropanol (97.5 : 2.5 volume per volume).
• the elution was carried out with the same solvent, using a Dynamax ® solvent delivery system at a flow rate of about 100 ml/min under a pressure between 0 - 30 psi.
• the volume of solvent mixture was about 50 L and fractions were collected in batches of 1 L.
• HPLC analysis indicated that there were 20 fractions, from the 28 th to 48 th , which contained about 0.1 mg/ml to 0.3 mg/ml of Paclitaxel and their purity varied from 98 to > 99%.
• the fractions containing Paclitaxel having a purity higher than 98% were combined together for a third and last crystallization.
• the white crystals were recovered by filtration or centrifugation and dried under ventilation or vaccum.
• the purities of the crystals analysed by HPLC were as follows:
• the residues obtained from the fractions containing Paclitaxel after the second chromatographic purification may be dissolved in ethanol, ethyl acetate or diethyl ether.
• the volume of those solvents is 5 times less than acetone used previously, its means the Paclitaxel concentration is 5 times higher (about 10 mg/ml).
• 1 to 2 volumes of hexane was then added to the ether solution; and, alternatively
• the mixture was kept at 2-8° C or at room temperature overnight to complete the crystallization.
• the purities of those crystals were very similar while using acetone for crystallization.
• the crystals which had a purity inferior to 98% were kept and repurified together by chromatography in the same conditions as in the second purification described above. This repurification allows to still obtain around 75% of the total quantity of these crystals with a purity superior to 99%.
• the crystals were dissolved in a minimum volume of ethanol or methanol or acetone and then put back in the vial containing the pure water.
• the volume of solvent was about 10 to 1 5% in relation to the volume of pure water.
• the Paclitaxel was lyophilized during 72 hours and the temperature was ramped from -60°C to + 20°C at 0.02°C per minute and under a pressure at 100 millitorrs.

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• Animal Behavior & Ethology (AREA)
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• Epoxy Compounds (AREA)
• Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
• Nitrogen And Oxygen Or Sulfur-Condensed Heterocyclic Ring Systems (AREA)
• Treatment Of Liquids With Adsorbents In General (AREA)

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• 2003
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• 2004
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