PREPARATION OF TAXANES

FIELD OF INVENTION

[0001] The present invention relates to a method of isolating a mixture of taxanes from a biomass composition of a species of the genus Taxus or Austrotaxus, or other diterpenoid-containing biomass materials

BACKGROUND OF THE INVENTION

[0002] Taxanes are a group of diterpinoid compounds, which have been demonstrated to be useful in the treatment of cancer and other serious diseases, such as multiple sclerosis and kidney disease. In particular, the taxane compounds paclitaxel, docetaxel, Baccatin III, 10-O-deacetylbaccatin III (10-DAB or DAB), 13-acetyl-9- dihydrobaccatin III (DHB), cephalomannine, and prostratin have been identified as uniquely effective in pharmaceutical applications. Certain taxanes, such as paclitaxel and docetaxel, can be used directly in pharmaceutical applications, without additional chemical modification, while many other taxanes (DAB and DHB, for example) are viewed as precursors for the production of other, more potent taxanes such as paclitaxel and docetaxel. Paclitaxel, whose trade name is Taxol ^® , is the predominant taxane used in cancer treatment. Paclitaxel is a naturally-occurring taxane, and can also be produced from other natural taxane analogs.

[0003] The major sources of the taxanes are the bark, needles and clippings of the yew (hemlock) tree, which belongs to the genus Taxus. Unfortunately, even though taxanes are more concentrated in yew than in other species of trees, the absolute concentrations are very low, for example typically 0.01 % for paclitaxel in the bark and in the range of from 0.003 to 0.015 percent (dry basis) for paclitaxel in the

clippings and needles (Huang et al., J. Nat. Prod., 49:665, 1986.). Furthermore, the yew tree is relatively rare and grows quite slowly, raising valid concerns that reforestation and resource management cannot keep up with the demand. Although synthetic pathways for producing Taxol ^® have been devised, they are extremely complex and generally too costly for commercial production.

[0004] Initially, Taxol ^® (paclitaxel) was derived exclusively from the bark of the yew trees. As demand accelerated, however, it became obvious that the standing resource could not be sustained on a renewable basis, and researchers began to extract paclitaxel and other analogs from cuttings derived from the yew tree, such as the stems and needles. In addition, semi-synthetic processes were also developed, which resulted in the production of paclitaxel and eventually docetaxel from taxane analogs.

[0005] Even with the increased use of non-bark components of the yew tree, and the additional semi-synthetic production of Taxol ^® , the overall recovery and production of paclitaxel and docetaxel remain relatively low, and the sustainability of production remains an issue. Conventional R&D in this area has focused on enhanced taxane production by genetic optimization and by improved recovery, but improvements to date have been relatively modest.

[0006] Current methods of commercial Taxol ^® and taxane production are complex and costly. The unit operations are predominantly physical methods involving: harvesting/collection, grinding, mulching, preliminary solvent extraction and separation to get a crude taxane product. Once the crude taxane product is produced, Taxol ^® is recovered and purified in additional solvent extractions and other refining

steps. In many cases, the other natural taxanes are chemically converted to additional

yields of paclitaxel or to docetaxel.

[0007] Given that the typical concentration of Taxol ^® from the bark of the yew tree is about 0.01% and since Taxol ^® is generally recovered from the bark of the yew tree

with an efficiency of about 70 to 75%, it takes about 14,000 kg of yew bark to yield 1 kg of Taxol. Similarly, about 7,000 and 14,000 kg of yew clippings, such as twigs and needles, yield 1 kg of recoverable Taxol ^® . The yield of Taxol ^® from various species of the yew tree has been reported as being in the range of from 50 mg/kg to 165 mg/kg (i.e., 0.005-0.017%). (see Miller et al., J Org. Chem., 46:1469, 1981; McLaughlin et al., J. Nat. Prod., 44:312, 1981; Kingston et al., J. Nat. Prod., 45:466, 1982, and Senih et al., J. Nat. Prod., 47:131, 1984, U.S. Pat. No. 5,407,674 and U.S.

Pat. No. 5,380,916.)

[0008] Prior art methods for isolating taxanes from yew biomass generally include an initial step of solvent extraction. This solvent extraction step unfortunately removes a large amount of impurities together with the desired taxane compounds. As a result, methods have been developed that involve one or more liquid partitioning steps to enrich the concentrations of taxanes in the extracts, followed by several chromatography steps, (see, for example, Rao WO 92/07842; Nair WO 94/13827; Elsohley et al. WO 92/18492; Carver et al. U.S. Pat. No. 5,281,727, issued Jan. 25, 1994; Pandey and Yankov, U.S. Pat. No. 5,654,448, issued Aug. 5, 1987; Cociancich and Pace, U.S. Pat. No. 5,744,333, issued Apr. 28, 1998; Liu U.S. Pat. No. 5,969,165, issued Oct. 19, 1999; and Snader, "Isolation and Detection," in Taxol-Science and Applications, M. Suffness, ed., CRC Press, Boca Raton, FIa. 1995, pp. 277-286.)

[0009] A drawback of these methods is that they require large amounts of costly and,

sometimes, toxic organic solvents for the extraction and partitioning steps.

[0010] Cass et al. (Cass, B.J., Piskorz, J., Scott, D.S. and Legge, R.L. 2001. Challenges in the isolation of taxanes from Taxus canadensis by pyrolysis. Journal of Analytical and Applied Pyrolysis. 57, 275-285 describe a method of extracting Taxol ^® from the needles or whole clippings of Taxus canadensis by pyrolysis using a

fluidized bed reactor. The amount of Taxol ^® that is recovered according to the disclosed method is about 0.006 % based on the dry weight of the tissue mass subjected to the pyrolysis reaction, or about 20% of the amount that can be recovered using conventional organic solvent protocols. The disclosed pyrolysis reaction produced a number of phenolic compounds that were assumed to interfere in the detection of any other taxanes that may have been produced according to the described method. However, attempts at substantially removing these phenolic impurities from the pyrolysis sample using normal phase or reversed phase column chromatography, liquid-liquid partitioning or size exclusion chromatography, were unsuccessful, and in

fact no additional taxane yields were detected or confirmed to be present.

[0011] U.S. Patent Application Publication No. 2003/0013899, published on January 16, 2003, discloses a method of purifying taxanes extracted from yew biomass using an organic solvent without the need for a liquidrliquid partitioning step. The method involves extracting the biomass with an organic solvent to form an extract containing the taxanes, adsorbing the extract of taxanes onto an adsorption resin and selectively desorbing the taxanes from the resin using an elution solvent to produce a taxane enriched eluate.. Individual taxanes may be separated from the elutate by a further chromatography step. It will be appreciated that large amounts of organic solvent are

required to effect the solvent extraction process. Moreover, there is no disclosure of the application of the disclosed purification method to a taxane-containing sample

produced by pyrolysis of a biomass of the yew tree, which contains pyrolytic lignin

and phenolic impurities.

[0012] There is therefore a need for a new method of isolating taxanes in a purified form from renewable parts of the yew tree in high yield without the requirement of large amounts of toxic and costly organic solvents.

SUMMARY OF THE INVENTION

[0013] The present invention relates to a method of isolating a mixture of taxanes from a bio-oil derived from the destructive distillation of diterpenoid-containing biomass materials, for example, pyrolysis of all or various components of the biomass of a species of the genus Taxus or Austrotaxus . Specifically, the bio-oil is obtained from one or more fractions of the liquid and/or char products produced from pyrolysis of a portion of the biomass of a yew tree.

[0014] A significant increase in the yields of Taxol ^® (paclitaxel) and taxanes are realized using the present invention. For example, paclitaxel yields are increased by a factor of 3 to 5 (i.e., 300 to 500 %), DHB yields are increased by a factor of 2 to 3 (i.e., 200 to 300 %), DAB yields increased by a factor of 8 (i.e., 800%) and overall taxane yields are increased by a factor of about 20 (i.e., 2000%), compared to average taxane yields from yew needles and clippings, as reported in the literature.

[0015] The present invention provides a method of isolating a mixture of taxanes from a bio-oil composition derived from the destructive distillation of biomass of a diterpenoid-containing biomass material, comprising:

mixing a chromatographic resin with the bio-oil composition to form a bio- oil/resin mixture, and

1 T eluting the mixture of taxanes from the bio-oil/resin mixture using an eluting solvent.

[0016] According to the present invention there is also provided a method of isolating a mixture of taxanes from a biomass composition derived from various components of i a species of the genus Taxus or Austrotaxus e.g. a yew tree, or a cultivated species derived from the hybridization of T. buccata and T. cuspitata, or other diterpenoid-rich biomass materials, comprising:

(a) destructive distillation e.g. pyrolysis, of the biomass to form a bio-oil composition containing the mixture of taxanes,

i (b) mixing a chromatographic resin with the bio-oil composition to form a bio- oil/resin mixture, wherein the mixture of taxanes is adsorbed onto the resin, and

(c) eluting the mixture of taxanes from the bio-oil/resin mixture using an eluting solvent.

[0017] The above-defined method may further comprise a step of washing the bio- oil/resin mixture prior to the step of eluting with a washing solvent to elute non-polar impurities, the washing solvent having a lower polarity than the eluting solvent.

[0018] The chromatographic resin used in the above-defined methods can be silica, a polyaromatic resin, a polyacrylate resin, a polymethacrylate resin, a polystyrene resin, a brominated polystyrene resin, alumina, amberlite, cellulose, hydroxypropylated, cross-linked dextran, Dowex ^® 50WX8-200, polyvinylpyrrolidone (PVP), Polyvinylpyrrolidone (PVPP), CELITE ^® , FLORSIL ^® , SEPHADEX ^® LH-20, SEPHADEX ^® LH-60, activated charcoal and a mixture thereof. Another type of chromatographic resin that may be used in the present invention include a hydrophobic-interaction resin, such as non-silica based macroporous hydrophobic

macropolymers, which separate molecules based on hydrophobic-interactions and size exclusion interactions with the resin. Non-limiting examples of such resins include resins comprising polymers and copolymers based on polyaromatic, polyacrylate, polymethacrylate, polystyrene and modified polystyrene, particularly brominated polystyrene.

[0019] Solvents that may be used in the adsorption, washing and elution steps of a method according to the present invention include a mixture of one, or more than one non-polar organic solvent with one, or more than one miscible polar organic solvent. Non-limiting examples of non-polar solvents include, without limitation Cs-C ₈ alkanes and toluene. Examples of polar solvents include Ci-C ₆ alcohols, Ci-C ₄ alkyl ketones, C ₁ -C ₅ alkyl esters of C ₁ -C ₅ carboxylic acids, Ci-C ₄ carboxylic acids, Cj-C ₄ alkyl nitrites, C ₄ -C ₈ alkyl ethers, chloroform, dichloromethane, nitromethane, and mixtures thereof. The specific ratio of polar to non-polar solvents may be determined readily by one of skill in the art depending on the specific resin or combination of resins used, the polarity of the taxanes to be isolated and the source of the biomass, without the necessity of undue experimentation. A non-limiting example of a solvent is from about 5-95% methanol in water, another example is a solvent of about 70% methanol, or about 80% methanol in water.

[0020] Furthermore, the biomass composition used in the above methods may be derived from bark, needles, stems, roots, plant cells in culture, or a mixture thereof. The biomass that is processed to form the bio-oil may be in a freshly harvested state, a dry state, or in a hydrated state. The biomass may also be reduced in size, for example shredded or ground by methods known in the art, prior to being processed into the bio-oil.

[0021] In addition, the taxanes isolated from the bio-oil may comprise one, or more than one taxane selected from the group consisting of: Taxol ^® (paclitaxel),

cephalomannine, baccatin III, 10-deacetyltaxol, 10-deacetylcephalomannine, 10- deacetylbaccatin III (10-DAB or DAB), 13-acetyl-9-dihydrobaccatin III (DHB), 7- xylosyltaxol, 7-xylosylcephalonammine, 7-xylosylbaccatin III, and derivatives and

analogs thereof.

[0022] The method of the present invention is advantageous in that it does not require an initial liquid-phase fractionation procedure, such as, liquid:liquid partitioning or solvent extraction of the bio-oil composition, before one or more taxanes are isolated by chromatographic separation.

[0023] In a particular example of the above-defined method, the bio-oil composition is produced using destructive distillation (eg., pyrolysis) in a standard retort (i.e., a fixed or moving bed either under vacuum or at pressure), a bubbling fluid bed reactor, an upflow reactor, a circulating fluid bed reactor, a transported bed reactor, an ablative reactor, an entrained flow reactor, rotary kiln reactor or a mechanical transport reactor (eg., auger reactor system).

[0024] In the method of the present invention, the pyrolysis is carried out typically in the temperature range of about 300 ⁰ C to about 650 ⁰ C, preferably in the range of 380 ⁰ C to 550 ⁰ C. The residence time of the biomass in the pyrolysis system, from heat-up to recovery (i.e., quench), is in the range of 0.2 to 30 seconds, and preferably in the range of 0.2 to 5 seconds.

[0025] The isolated yield of Taxol ^® obtained by prior art organic solvent extraction techniques as described above from the bark of Taxus is typically in the order of

0.01%, and from clipping and needles, about 0.003 to about 0.015%. However, with

the methods of the present invention, isolated yields of about 0.031 to 0.049 % have been obtained. The isolated yield of DHB from clippings and needles, as obtained by prior art organic solvent extraction techniques, is typically on the order of 0.04%.

However, with the methods of the present invention, isolated DHB yields of between 0.08 and 0.12 % have been obtained. The isolated yield of DAB from clippings and needles, as obtained by prior art organic solvent extraction techniques, is typically on

the order of 0.06 %. However, with the methods of the present invention, isolated DAB yields of between 0.46 and 0.53 % have been obtained. The yield of total taxanes recovered from clippings and needles, as obtained by prior art organic solvent

extraction techniques, is typically on the order of 0.25%. However, with the methods of the present invention, taxane yields of between 5 and 7 % have been measured. Clearly, the taxane yields of the present invention are well in excess of those obtained

using prior art techniques.

[0026] U.S. Patent Application Publication No. 2003/0013899 discloses the extraction of Marsh dried needles obtained from Taxus Canadensis using an organic solvent. The organic solvent extract was adsorbed onto silica resin and eluted with an eluting solvent. In the process according to the present invention, a bio-oil composition, containing pyrolytic lignin and phenolic impurities is formed in a first

step, as a source of taxanes and applied onto a resin for further separation. A bio-oil composition containing pyrolytic lignin and phenolic immpurities is quite different from the disclosed organic solvent extract and as a result, unique analytical problems were encountered. The impurities (phenolics, lignin, etc.) in the bio-oil were found to interfere with the taxanes enriched fraction. The process described in U.S. publication

2003/0013899, developed for organic solvent extracts, proved inadequate for the bio-

oil.

[0027] This summary of the invention does not necessarily describe all features of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

[0028] These and other features of the invention will become more apparent from the following description in which reference is made to the appended drawings wherein:

[0029] FIG. 1 is a schematic illustration of a prior art (see US patent no. 6,844,420) destructive distillation (pyrolysis) system, for example, which is not to be considered limiting in any manner.

[0030] FIG. 2 is a flow chart showing the present invention process. Example 2C: A Taxus feedstock pyrolysed in a thermal processing upflow reactor run at 397°C, and in liquid quench mode, to produce bio-oil samples.

[0031] FIG. 3 shows the thin-layer chromatography (TLC) of the taxane rich fractions from the bio-oil showing that the paclitaxel and other taxanes are produced.

[0032] FIG. 4 shows the HPLC chromatogram of the taxane rich fraction showing that the paclitaxel and other taxanes are produced from the bio-oil.

[0033] FIG. 5 shows the HPLC chromatograms of the taxane fraction from Taxus using the conventional methanolic solvent extraction.

[0034] FIG. 6 shows the HPLC chromatograms of the taxane compounds isolated from the bio-oil.

[0035] FIG. 7 shows the thin-layer chromatography (TLC) (qualitative analysis of Examples IA, IB, 1C, ID, IE, IF, IG and IH) of the taxane rich fractions from the bio-oil showing that the paclitaxel, Baccatin III, 13-Acetyl-9-Dihydro-Baccatin III and other taxanes are produced.

DETAILED DESCRIPTION OF THE INVENTION

[0036] The present invention relates to a method of isolating a mixture of taxanes from a bio-oil derived from the destructive distillation of diterpenoid-containing biomass material, for example, all or various components of the biomass of a species of the genus Taxus or Austrotaxus. The bio-oil maybe obtained from one or more fractions of the liquid and/or char products produced from pyrolysis of a portion of the biomass of a yew tree.

[0037] By "bio-oil", "whole-oil" or "pitch" it is meant the tar, pyroligneous acid- containing mixture or whole liquid fraction obtained following destructive distillation, for example but not limited to, pyrolysis, of wood or other biomass. The bio-oil product is typically obtained from the product vapor that is produced along with char following destructive distillation (e.g. pyrolysis). Upon removal of the char, the product vapor is condensed and collected within one or more recovery units, for example, one or more condensers that may be linked in series. Whole oil, whole bio- oil, or whole pitch refers to the combination of the condensed products obtained from all of the recovery units. However, a fraction of the bio-oil may also be obtained in accordance with the invention as described herein.

[0038] The bio-oil, whole oil or pitch product may be produced by one of several different types of pyrolysis reactions, for example, using:

- a bubbling fluid bed reactor (fluidized bed reactor; see Scott D S, Piskorz J and Radlein D, "Liquid Products from the Continuous Flash Pyrolysis of Biomass", Ind. Eng. Chem. Process Des. Dev. 24, 1985, p 581-588; Scott, D.S.; Piskorz, J.; Majerski, P.; and Radlein, D. [1996] "Fast Pyrolysis of Biomass for Recovery of

Exotic Chemicals," in Developments In Thermochemical Biomass Conversion, Bridgwater AV and Boocock DGB, [Eds.] [Blackie 1997]; Cuevas A, Reinoso C and Scott DS, "Pyrolysis oil production and its perspectives", in Proc. Power production

from biomass II, Espoo, March 1995 [VTT]; Robson A, PyNe newsletter No. 11, pp 1-2, June 2001, ISSN 1470-3521 [Aston University, UK]; and McLellan R, PyNe

Newsletter No. 10, pp 12, December 2000, ISSN 1470-3521 [Aston University,

Birmingham UK], the disclosures of which are incorporated herein by reference);

- a circulating fluid bed, an upflow reactor (thermal pyrolysis) or transported bed reactor (see U.S. Patent No. 5,792,340; International Application No.

PCT/CAOO/00369, published as WO 00/61705; and Boukis I, Gyftopoulou ME, Papamichael I, "Biomass fast pyrolysis in an air-blown circulating fluidized bed

reactor", in Progress in Thermochemical Biomass Conversion, Ed. Bridgwater AV, [Blackwell, Oxford UK, 2001], the disclosures of which are incorporated herein by reference);

- an ablative reactor (see Lede J, Panagopoulos J, Li HZ and Villermaux J, 'Fast Pyrolysis of Wood: direct measurement and study of ablation rate', in Fuel, 1985, vol. 64, pp. 1514-1520; Diebold JP and Scahill J, "Production of Primary Pyrolysis Oils in a Vortex Reactor in Pyrolysis Oils from Biomass", EJ Soltes and TA Milne, eds., ACS, Washington, D.C., 1988, pp 31-40; and Peacocke GVC and Bridgwater AV, "Ablative Plate Pyrolysis of Biomass for Liquids", Biomass and Bionergy, 1995, vol. 7, no. 1-6, pp. 147-154, the disclosures of which are incorporated

herein by reference);

- an entrained flow reactor (see Kovac R.J. and O'Neil DJ, "The Georgia Tech Entrained Flow Pyrolysis Process," Pyrolysis and Gasification, G.L. Ferrero, K. Maniatis, A. Buekens, and A. V. Bridgwater, eds., Elsevier Applied Science 1989, pp. 169-179.; and Maniatis K, Baeyens J, Peeters H and Roggeman G, "The Egemin flash

pyrolysis process: commissioning and results", pp 1257-1264 in Advances in

thermochemical biomass conversion, Ed. AV Bridgwater [Blackie 1993], the disclosures of which are incorporated herein by reference);

- a rotating cone reactor (see Prins W and Wagenaar BM, In Biomass gasification and pyrolysis, Eds. Kaltschmitt MK and Bridgwater AV, pp 316-326 [CPL 1997]; and Wagenaar BM, Venderbosch RH, Carrasco J, Strenziok R, van der Aa BJ, "Rotating cone bio-oil production and applications", in Progress in Thermochemical Biomass Conversion, Ed. Bridgwater AV, [Blackwell, Oxford UK, 2001], the disclosures of which are incorporated herein by reference); or

- a vacuum pyrolysis reactor (see Yang J, Blanchette D, de Caumia B, Roy C "Modelling, scale-up and demonstration of a vacuum pyrolysis reactor", in Progress in Thermochemical Biomass Conversion, Ed. Bridgwater AV, [Blackwell, Oxford UK, 2001], the disclosures of which are incorporated herein by reference)

- a rotary kiln pyrolysis system (see Marty E "Production of Fuels from Waste and Biomass in the EDDIth Thermolysis Process - Recent Industrial Developments", [Institut Francais du Petrole, Rueil-Malmaison (Paris), France, 2002], the disclosures of which are incorporated herein by reference).

- a mechanical transport pyrolysis systems or auger pyrolysis system (see Fransham P "Low cost bio-oil production using a new pyrolysis system and its integration with an efficient rotary system", [Encon Enterprises Inc., Ottawa, Canada, 2004], the disclosures of which are incorporated herein by reference).

- a retort pyrolysis system (see Rocha J D, Coutinho A R, Luengo C A "Biopitch Produced from Eucalyptus Wood ..." [Braz. J. Chem. Eng. VoI 19(2), Sao

Paulo, Apr.-June 2002], and see U.S. Patent No. 4,705,603, the disclosures of which

are incorporated herein by reference).

[0039] By "recovery unit" it is meant a device that collects product vapors produced during pyrolysis. A recovery unit may include, but is not limited to, a condenser, contact or surface condenser that cools and collects a liquid product from the product vapor, or a liquid quench that may also cool and collect a liquid product from the product vapor. A recovery unit may also include demisters, fiber filter beds or other devices used within the art to collect the liquid product from the product vapor. A recovery unit may comprise one or more components, for example, one or more condensers, which are typically linked in series.

[0040] By "selected product fraction", or "fraction of the whole oil" it is meant a fraction of the liquid product that is obtained from a product vapor following removal of char and condensation of the product. For example, which is not to be considered limiting in any manner, the selected product fraction may comprise the liquid product obtained from at least one recovery unit, for example a primary recovery unit, a secondary recovery unit, or a combination thereof. The selected product fraction may be used as the bio-oil from which a mixture of taxanes is isolated according to the present invention.

[0041] The present invention provides a method of isolating a mixture of taxanes from a bio-oil, or a bio-oil fraction, where the bio-oil or bio-oil fraction is derived from pyrolysis of a biomass derived from a species of the genus Taxus or Austrotaxus. The process effectively separates lingocellulose and phenolic contaminating material

from the produced bio-oil to yield an enriched fraction of taxanes, or a substantially

pure mixture of taxanes.

[0042] Destructive distillation, eg. pyrolysis, can be achieved by heat transfer to the feed material, by heating and removal of the product via a vacuum, or by a combination of heat transfer and pyrolysis under vacuum. The bio-oil obtained from pyrolysis of the biomass derived from a yew tree comprises depolymerized lignin, fragmented cellulose- and hemicellulose-derived products, and other reactive components including phenolics, in addition to a mixture of taxane compounds.

[0043] Pyrolysis of wood or other biomass residues results in the production of product vapors and char. After removal of the char components from the product stream, the product vapors are condensed to obtain a whole oil, or bio-oil product from pyrolysis. As mentioned above, a suitable pyrolysis process for preparing such a bio-oil is described in US patent no. 6,844,420 (Freel and Graham); the disclosure of which is incorporated herein by reference, and is diagrammatically presented in Figure 1. Briefly, the system includes a feed system (10), a reactor (20), a particulate inorganic heat carrier reheating system (30), and for the purposes of the invention described herein, at least one recovery unit, which as shown in Figure 1 , and which is not to be considered limiting in any manner, may comprise a primary (40) and a secondary (50) condenser through which the product vapors produced during pyrolysis are cooled and collected for example using a liquid quench (80). The recovery unit may also include a de-mister (60) and a fiber filter bed (70) or other device to collect the liquid product. The bio-oil composition of this invention may be derived from a selected product fraction obtained from at least one recovery unit, for example the primary, or the secondary recovery unit, or a combination thereof, or it may be a

whole oil, obtained from first and second recovery units, including demisters and fiber

filter bed, or a combination thereof. However, it is to be understood that analogous destructive distillation or pyrolysis systems, comprising different number or size of recovery units, or different condensing means may be used for the selective preparation of the oil feedstock for the purpose of the present invention.

[0044] The recovery unit system used within the pyrolysis reactor system, outlined in Figure 1 , which is not to be considered limiting in any manner, may involve the use of direct-liquid contact condensers (80) to cool the pyrolytic oil product. However, it is to be understood that any suitable recovery unit may be used, including surface condensers. In one embodiment, liquid, used within these condensers (80) to cool the pyrolytic product, is obtained from the corresponding cooled primary or secondary condenser product (90; Figure 1). However, as would be evident to one of skill in the art, any other compatible liquid for cooling the product within the primary and secondary recovery units, or a combination thereof, may also be used for this purpose. Furthermore, it is considered within the scope of this invention that other scrubber or cooling means including heat exchangers comprising solid surfaces and the like may also be used for cooling the product vapours.

[0045] The bio-oil composition of the present invention may be produced by a process comprising:

i.) introducing biomass into a pyrolysis reactor, wherein the reactor is run at a temperature of from about 300 ⁰ C to about 65O ⁰ C,

ii.) allowing the biomass feedstock to react in the pyrolysis system with a residence time of less than about 30 seconds, to produce a product stream,

iii.) collecting the taxane-containing products in a liquid and solid product recovery system, and

iv.) collecting the bio-oil or bio-oil fractions from the product stream

[0046] In an example of the above process, the loading ratio is in the range of from about 15:1 to about 100:1 or any subrange thereof; from about 20:1 to about 80:1 or any subrange thereof; from about 20:1 to about 50:1 or any subrange thereof; or from about 20:1 to about 30:1 or any subrange thereof.

[0047] In other examples of the above-defined process, the residence time may vary from about 0.05 to about 30 seconds, from about 0.05 to about 5.0 seconds or any subrange thereof; or from about 0.5 to about 2 seconds or any subrange thereof.

[0048] In further examples, the upflow reactor is run at a temperature in the range of from about 300°C to about 650°C or any subrange thereof, from about 380°C to about 580°C or any subrange thereof, or from about 390°C to 500 ^° C or any subrange thereof.

[0049] In one embodiment of the invention, the heat carrier used within the pyrolysis reactor typically exhibits low catalytic activity and is of a size of about 100 to about

500 μm, or any amount therebetween, for example from about 200 to about 300μm, or

about 250 μm. Such a heat carrier may be an inert particulate solid, such as sand, for

example silica sand. By silica sand it is meant a sand comprising greater than about 80% silica, preferably greater than about 95% silica, and more preferably greater than about 99 % silica. Other components of the silica sand may include, but are not limited to, from about 0.01 % (about 100 ppm) to about 0.04 % (400 ppm) iron oxide, preferably about 0.035% (358 ppm); about 0.00037% (3.78 ppm) potassium oxide; about 0.00688% (68.88 ppm) aluminum oxide; about 0.0027 (27.25) magnesium

oxide; and about 0.0051% (51.14 ppm) calcium oxide. It is to be understood that the above composition is an example of a silica sand that can be used as a heat carrier as described herein, however, variations within the proportions of these ingredients

within other silica sands may exist and still be suitable for use as a heat carrier. Other known inert particulate heat carriers or contact materials, for example kaolin clays, rutile, low surface area alumina, oxides of magnesium aluminum and calcium as described in US 4,818,373 or US 4,243,514, may also be used.

[0050] After the bio-oil is obtained, it is mixed with the chromatographic resin to form a bio-oil/resin mixture. In a particular example, the chromatographic resin, for example but not limited to, polyvinylpolypyrrolidone, is mixed with an amount of bio- oil. If desired, the resulting mixture may be dried to provide a powder. The ratio of the chromatographic resin to the bio-oil may be from about 4:1 to about 1 :2 by weight or any amount therebetween, or from about 2:1 to about 1 :2 by weight or any amount therebetween. For example, the ratio of the chromatographic resin to the bio-oil may be mixed at about 1 : 1 by weight. Optionally the bio-oil may be mixed with the chromatographic resin in the presence of a solvent that induces taxanes in the mixture to adsorb to the chromatographic resin.

[0051 ] The chromatographic resin/bio-oil mixture may be stored until further use. Alternatively, the resin/bio-oil mixture may be placed in Buchner funnel or similar holder, or placed in a column comprising a second portion of chromatographic resin, and washed with one or more than one elution solvent, for example, two, or more than two elution solvents of each progressively increasing or decreasing polarity depending upon the chromatographic resin chosen to elute a mixture of taxanes.

[0052] If the chromatographic resin/bio-oil mixture is applied onto a second portion

of chromatographic resin in a column, then the ratio of column resin to resin/bio-oil

mixture may be from between about 1 :2 and about 10:1 by weight or any amount therebetween, or between about 2:1 and about 10:1, or any amount therebetween. In addition, the resin present in the chromatographic resin/bio-oil mixture may be the

same or different from the second portion of resin present in the column.

[0053] In one example, the resin in the chromatographic resin/bio-oil mixture is

polyvinylpolypyrrolidone, and the mixture is placed in Buchner funnel and sequentially eluted with two, or more solvents of increasing polarity to separate a mixture of one or more taxanes from impurities differing in polarity or hydrophobicity from the taxanes. If the two, or more than two solvents are appropriately chosen it may be possible to separate individual taxanes from the resin, which differ from each other in polarity. For example, a mixture of paclitaxel and cephalomannine may be separated from more polar 10-deacetylated taxane derivatives by the selection of the appropriate elution solvents.

[0054] In a further example, the polyvinylpolypyrrolidone resin/bio-oil mixture is initially washed with a non-polar wash solvent to elute non-polar impurities from the polyvinylpolypyrrolidone resin. The resin/bio-oil mixture may then be eluted with a solvent that is relatively more polar than the wash solvent so that one or more taxanes can be eluted from the resin, while impurities that are relatively more polar than the taxanes remain adsorbed to the polyvinylpolypyrrolidone resin.

[0055] Mixtures of one, or more than one non-polar organic solvent with one, or more than one miscible polar organic solvent may be used for adsorbing, washing and

eluting according to the present invention. Non-limiting examples of non-polar

solvents include, C5-C8 alkanes and toluene. Examples of polar solvents include Cl- C6 alcohols, C1-C4 alkyl ketones, C1-C5 alkyl esters of C1-C5 carboxylic acids, Cl- C4 carboxylic acids, C1-C4 alkyl nitrites, C4-C8 alkyl ethers, chloroform, dichloromethane, nitromethane, and mixtures thereof. The ratio of polar to non-polar solvents may be determined readily by one of skill in the art depending on the resin or combination of resins used, the polarity of the taxanes to be isolated, and the source of the biomass. A non-limiting example of a solvent is from about 5-95% methanol in water, another example is a solvent of about 70% methanol, or about 80% methanol in water.

[0056] Taxanes may also be isolated from the bio-oil by using the non-silica based macroporous hydrophobic macropolymers described above according to the method described in U.S. Patent Application Publication No. 2003/0013899 Al (which is incorporated herein by reference). Essentially such a separation may involve removing the solvent from the eluted fractions obtained by the above-described procedure, reconstituting the samples in a suitable water miscible organic solvent or aqueous mixture thereof, equilibrating the hydrophobic-interaction resin with a loading solvent (wash solvent) comprising a buffered or unbuffered aqueous/organic solvent mixture that induces the adsorption of taxanes to the resin but does not result in any appreciable retention of impurities having a relatively higher hydrophilicity than that of the taxanes, directly loading the reconstituted mixture of taxanes onto the

equilibrated resin or mixing the taxanes with a portion of the equilibrated resin and placing the resulting mixture on a separate portion of equilibrated resin in a column, washing the loaded column with the loading solvent to remove the hydrophilic

impurities, and eluting the column with a buffered or unbuffered aqueous/organic solvent mixture having a relatively higher proportion of organic solvent to water than the wash solvent to cause the desorption of the taxanes.

[0057] In another example, the mixture of taxanes isolated using the silica or non- silica based macroporous hydrophobic macropolymer may be further separated on a second column containing non-silica based macroporous hydrophobic macropolymer, in a manner similar to that described above, to separate and isolate individual taxanes from the mixture of taxanes.

DESCRIPTION OF FEED MATERIAL - CANADA YEW

[0058] Taxus Canadensis Marsh, Canada yew, ground hemlock shrubs were collected in July-September 2004 from Sault Ste. Marie surrounding area, Ontario, Canada (lat. 46.34 N, long. 84.17 W). Pressed voucher specimens are deposited in the Canadian Forest Service-Sault Ste. Marie herbarium as Taxus canadensis Marsh (2004- 4001-10 CFS-SSM # s), Taxaceae - yew family. The fresh T. Canadensis needles and twigs were air dried at room temperature 22-24 ⁰ C. The dried sample was ground to ~0.5 mm particle size in a Thomas- Wiley Laboratory mill, Model 4 (Thomas Scientific, USA).

ANALYTICAL METHODS AND PROCEDURES

Analysis of Taxane Products:

[0059] High performance liquid chromatography was performed using a Waters Delta Prep 4000 Liquid Chromatograph equipped with a computer and Empower software, a Waters ^® 996 autoscan photodiode array spectrophotometric detector; and an analytical

column, for example, a Curosil-PFP Phenomenox (250 x 4.60 mm i.d.). A modified gradient chromatographic technique (Phenomenex) was used at room temperature using an acetonitrile/water solvent system. However, other solvent systems were also used as indicated in the claims. Samples were eluted using an appropriate gradient, for example, a 25/75 to 65/35 gradient of acetonitrile/water over a 40 minute period with a flow rate of 1.0 ml/min. Compounds were detected at a wavelength of 228 nm and resolved peaks were scanned by the photodiode array detector from 200 to 400 nm.

[0060] A dilute solution (10 mg/mL) of extract was filtered through 13 mm GHP 0.45μm Minispike (Waters, EDGE) and lOμL was injected onto an HPLC column with and without spiking with standards. Peaks were identified on the basis of retention times and UV spectra. Peak heights, measured as absorbance at 228 nm, were converted to mg/ml using conversion factors obtained for Taxane standards. Such HPLC analyses were performed in triplicate.

[0061] UV spectra were recorded on a UV- Vis. Beckman DU series 640 spectrophotometer. Taxanes were identified by co-chromatography with authentic samples (ChromaDex, Sanata Ana, CA, USA) using TLC and HPLC.

EXAMPLES

[0062] The present invention will be further illustrated in the following examples.

Example 1 : Production of bio-oil using pyrolysis

[0063] A hemlock feedstock, prepared according to Example 1, was reacted in a pyrolysis reactor essentially as described in US Patent No. 6,844,420 (the disclosure

of which is incorporated herein by reference).. A char product is rapidly separated

from the product vapour/gas stream, and the product vapour rapidly quenched within a primary recovery unit using, a direct liquid contact condenser, or a liquid quench, as described below. The compounds remaining within the product vapour are transferred to a secondary recovery unit linked to the primary recovery unit in series. The product vapour is then quenched within the secondary recovery unit using, a direct liquid contact condenser, or a liquid quench, and the condensed product collected. Any remaining product within the product vapour is collected within the demister and filter bed (see FIG. 1). The primary recovery unit product is collected, as well as the secondary recovery unit product. The yield of liquid product from the recovery unit ranges from about 40 to about 60% (w/w), and is typically about 49% (see FIG. 1).

Example IA:

[0064] A hemlock feedstock (4,715 g) containing 2.52 wt% ash and 8.73 wt% moisture was pyrolysed in an upflow reactor at 490°C using a surface-condenser, to produce a bio-oil sample. The liquid yield on a feed "ash free" basis was 55.49 wt%. Samples of the liquid submitted for further purification and taxane analysis included an aqueous fraction (1024.5g), a tar fraction (440.5g) and pure liquid fraction (17Og) squeezed from a fiberglass filter material in the demister, and submitted for further purification.

Example IB:

[0065] A hemlock feedstock (7,996 g) containing 1.80 wt% ash and 5.20 wt% moisture was pyrolysed in an upflow reactor at 398° C run in surface-condensing

mode, to produce a bio-oil sample. The liquid yield on a feed "ash free" basis was

45.12 wt%, char was 28.54 wt% and the gas was 17.41 wt% (see FIG. 2).

[0066] Samples of the taxane-containing pyrolysis products were taken and submitted for further purification and taxane analysis

Example 1C:

[0067] A hemlock feedstock (5,641 g) containing 1.80 wt% ash and 5.20 wt% moisture was pyrolysed in an upflow reactor run at 397°C, and in liquid quench mode,

to produce bio-oil samples. The quench liquid was an approximate 50/50 mix of the aqueous product from the run of Example IA, and demineralized water. The liquid yield on a feed "ash free" basis was 46.77 wt%, char was 25.87 wt% and the gas was 19.0 wt%. The purpose of this run was to compare product yields obtained using a

liquid quench with that of surface condensing.

[0068] Samples of the taxane-containing pyrolysis products were taken and submitted

for further purification and taxane analysis.

Analyses for Examples IA, IB and 1C (FIG. 2): The analytical procedure that is selected for the identification and quantification of taxanes and individual taxane components is extremely important, since phenolic impurities may cause interference. In this regard, thin-layer chromatography (TLC) of the bio-oil using silica gel and

methylene chloride:MeOH 95:5 as a solvent (see FIG. 3) proved beneficial in obtaining qualitative analysis of the taxanes.

Example 2: Isolation of Taxanes by Gel chromatography

[0069] Isolation Method 1: Bio-oil samples (1 g) obtained as outlined in Examples

IA, IB and 1C were mixed and adsorbed onto 1 g of Polyvinylpolypyrrolidone, PVPP (Sigma Chemical Co., P-6755 [25249-54-1]) and applied to strata-X 33μm Polymeric

Sorbent lg/20ml Giga Tubes (Phenomenex, 8B-S100-JEG). The strata-X was

conditioned with 2OmL methanol and equilibrated with 20 mL D.I. water before loading the above-mentioned sample. Elution was carried out at a slow rate using the following solvent systems:

a) methanol - D.I. water (7:3) [5 x 12mL],

b) methanol : acetonitrile : D.I. water (6:3:1), (6:2:2) or (5:2:3) [5 x 12mL], and

c) methanol : acetonitrile (1 :1) [5 x 12mL].

[0070] The taxanes enriched fractions were analyzed chromatographically by TLC and HPLC (see Table 4 ). TLC was used for qualitative analysis and HPLC was used for quantitative analysis. Solvent system "b" (6:3:1) was effective for the quantitative determination of taxane yields, and the yields reported below were determined using this solvent system.

[0071] The yield of total taxanes and individual taxane components, as produced via pyrolysis and as recovered using Method 1 , are reported in Tables 1 and 2,

respectively. A comparison of total "Method 1" taxane yields with those reported in the literature for conventional solvent recovery processes (e.g. Daoust, G and Sirois, G., 2003, "Canada Yew (Taxus canadensis Marsh.) and Taxanes: A Perfect Species or Filed Production and improvement Through Genetic Selection" Natural Resources Canada, Canadian Forest Service, Sainte-Foy, Quebec; Rao, K. V. "Taxol

and related taxanes. I . Taxanes ofTaxus brevifolia bark", Pharm. Res, 1993 VoI

10(4); April, pp. 521-524; Rao, K. V. et. al., "A new large-scale process for taxol and related taxanes from Taxus brevifolia", Pharm. Res, 1995, VoI 12(7); July, pp. 1003-

1010), is also given in Table 1. A comparison of the "Method 1" yields of individual representative taxanes with the solvent control test yields is also given in Table 2

[0072] From the data in Tables 1 and 2 it is clear that in all cases, the yields of total taxanes and individual representative taxanes are significantly higher using "Method 1 " present invention than yields that occur using conventional solvent extraction methods.

Table 1: "Method 1" Yields of Total Taxanes via Destructive Distillation (Pyrolysis) compared with Conventional Solvent Yields (as reported in the literature*)

*Daoust, G and Sirois, G., 2003, "Canada Yew (Taxus canadensis Marsh.) and Taxanes: A Perfect Species for Filed Production and improvement Through Genetic Selection" Natural Resources Canada, Canadian Forest Service, Sainte-Foy, Quebec

Table 2: "Method 1" Yields of Taxol ^® (paclitaxel) and Other Taxane Compounds via Destructive Distillation (Pyrolysis) compared with Solvent Extraction (Solvent Control*) Yields

*see Example 3

[0073] Isolation Method 2: Bio-oil samples (1 g) obtained as outlined in Examples IA, IB and 1C were mixed and adsorbed onto 1 g of Polyvinylpolypyrrolidone, PVPP (Sigma Chemical Co., P-6755 [25249-54-1]) and applied to strata-X 33μm Polymeric

Sorbent lg/20ml Giga Tubes (Phenomenex, 8B-S 100- JEG). The strata-X was conditioned with 2OmL methanol and equilibrated with 20 mL D.I. water before loading the above-mentioned sample. Elution was carried out at a slow rate using 20 mL of water followed by 20 mL aliquots of increasing concentrations (20, 50, 70, 100%) of methanol. All fractions were analyzed chromatographically with maximum concentration found in the fraction eluting with 50-70% methanol.

[0074] The taxol, cephalomannine, baccatin III, 10-deacetyltaxol, 10- deacetylcephalomannine, 10-deacetylbaccatin III were further purified by column chromatography and crystallization from the taxane-enriched fraction. Taxanes were identified by co-chromatography with authentic samples (ChromaDex, Sanata Ana, CA, USA) using TLC and HPLC (see FIG. 6). Taxane yields are similar to those obtained using Method L.

[0075] Isolation Method 3 (A Comparison of Different Gel Materials): In order to test and rate the efficacy of different gel materials for taxane isolation and recovery, bio-oil samples (1 g) obtained as outlined in Examples IA, IB and 1C were mixed

and adsorbed onto 1 g of the following chromatography gels listed in Table 3, and packed in a Buchner funnel. Elution was carried out at a slow rate using the following

solvent systems: i) CH ₂ Cl ₂ - MeOH - MeCOEt - Me ₂ CO (1 :1 :1 :1), ii) MeOH - MeCOEt - Me ₂ CO - H ₂ O (1 :1 :1 :1) and iii) MeOH - H ₂ O (7:3).

Table 3: Chromatography gel used:

[0076] Yields are determined for each gel.

Example 3: Solvent Extraction of Taxanes (non-pyrolytic Control Test)

[0077] Fresh Taxus canadensis Marsh needles (4417.13 g fr. wt.) were extracted at

room temperature in two steps: first, by steeping for 24 h in 100% MeOH (1 g fr. wt/10 ml solvent), followed by chopping in a commercial Waring blender and decanting the solvent; second, by steeping the chopped residue for an additional 24 h in 60 % aqueous MeOH. The combined methanolic extracts were evaporated under reduced pressure until most or all of the MeOH had been removed. The residue was freeze-dried to obtain 713.88 g of crude extract. Thus from each g fresh weight of needles, 162 mg of T. canadensis crude extract was obtained. Results of this test are presented in Table 2 as "solvent control".

Example 4. Liquid-liquid extraction:

[0078] Prior art includes the initial extraction of taxanes from biomass via a solvent extraction. In the present invention, this initial step is avoided, and the taxane components are effectively recovered, after destructive distillation, using a chromatographic resin adsorption step prior to solvent extraction i.e. by solvent elution from the resin. In this Example, the bio-oil product (produced via pyrolysis) is solvent-extracted directly, without chromatographic resin (gel) addition. The taxanes are extracted directly from bio-oil produced according to Example 1 using the following solvents: acetone, acetonitrile, 1-butanol, 2-butanone, chloroform, dichloromethane, ethyl acetate, ethanol, hexane, methanol, petroleum ether, 2- propanol and water. The crude pyrolysis oil is mixed with methanol, methanol: water (8:2; 7:3; 6:4; 1 :1), acetone, acetone:water (8:2; 7:3; 6:4; 1 :1) and solvent spiked with acid. The data illustrate that taxane recovery is enhanced using a gel (chromatographic resin) addition step.

Table 5 includes the sample information and peak results from Figure 4a.

Table 6 includes the sample information and peak results from Figure 4b.

Table 7 includes the sample information and peak results from Figure 4c.

Table 8 includes the sample information and peak results from Figure 4d.

Table 9 includes the sample information and peak results from Figure 4e.

Table 10 includes the sample information and peak results from Figure 4f.

Table 11 includes the sample information and peak results from Figure 4g.

Table 12 includes the sample information and peak results from Figure 4h.

Table 13 includes the sample information and peak results from Figure 5.

Table 14 includes the sample information and peak results from Figure 6a.

Table 15 includes the sample information and peak results from Figure 6b.

Table 16 includes the sample information and peak results from Figure 6c.

Table 17 includes the sample information and peak results from Figure 6d.

Table 7 (continued)

Peak Results

Analysis for Examples 1A - 1H [Fig 7a to 7d and Table 18]:

[0081] The analytical procedure that is selected for the identification and quantification of taxanes and individual taxane components is extremely important, since phenolic impurities may cause interference. In this regard, thin-layer chromatography (TLC) of the bio-oil using silica gel and methylene chloride:MeOH 95:5 as a solvent proved beneficial in obtaining qualitative analysis of the taxanes.

Isolation Method 5:

[0082] 1O g of samples (original feedstock crude extract or the crude pyrolyzed products, Bio-oil) are adsorbed onto 2 g of Polyvinylpolypyrrolidone , PVPP (Sigma Chemical Co., P-6755 [25249-54-1]) housed in 20 ml SPE tubes (solid phase extraction) (Supelco) packed with 2 g of PVPP. Elution was carried out at a slow rate using Supelco's SPE 24-port Manifold model with suction. The following solvent systems were used:

[0083] hexane, b) methylene chloride : methanol (95:5) and c) methanol.

[0084] The taxanes enriched fractions were analyzed chromatographically by TLC (see FIG 7a to 7d and Table 18). TLC was used for qualitative analysis. Solvent system "b" methylene chloride : methanol (95:5) was best in terms of the quantitative determination of taxanes, and the qualitative analysis reported below were determined using this solvent system fractions.

52

Table 19 includes the process details for Table 18 and Figure 7.

54

Table 20 includes the process conditions for Table 18 and Figure 7.

[0084] All citations are hereby incorporated by reference.

[0085] The present invention has been described with regard to one or more embodiments. However, it will be apparent to persons skilled in the art that a number of variations and modifications can be made without departing from the scope of the invention as defined in the claims.

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