Note: Descriptions are shown in the official language in which they were submitted.
CA 02536000 2009-11-25
PROCESS FOR THE EXTRACTION OF PACLITAXEL AND
9-DIHYDRO-13-ACETYLBACCATIN III FROM TAXUS
BACKGROUND OF THE INVENTION
FIELD OF THE INVENTION
[0001] This invention relates to the isolation and purification of taxane
derivatives from a naturally occurring Taxus species.
DESCRIPTION OF THE PRIOR ART
[0002] TaxolTM was first isolated in 1971 from the western yew, Taxus
brevifolia by Wani, et al. TaxolTM is a member of the taxane family of
diterpenes.
Taxanes are diterpene compounds which find utility in the pharmaceutical
field. Taxanes
may be found in plant materials, and having been isolated therefrom.
Paclitaxel is a well
known chemotherapeutic drug for treatment of various metastatic cancers. It
has been
approved for the treatment of ovarian and breast cancers.
[0003] TaxolTM and various taxane derivatives are highly cytotoxic and possess
strong in vivo activities in a number of leukemic and tumor systems. TaxolTM
is
considered to be an exceptionally promising cancer chemotherapeutic agent.
[0004] . The only currently available source for taxolTM is several species of
very
slow growing yew (genus Taxus, family Taxaceae). Paclitaxel is a natural
product,
primarily extracted from the bark of the Pacific yew tree, Taxus brevifolia,
and is also
found in T baccata, T walichiana, T. yunnanensis and T. canadensis. The
isolation
procedures currently practised are very difficult and low-yielding. The
extraction of
taxolTM from trees of the Taxus genus using liquid methanol has been reported.
However,
taxanes are generally present in plant materials in relatively small amounts
so that, in the
case of taxolTM, for example, large numbers of the slow-growing yew trees
forming a
source for the compound may be destroyed. Further, large amounts of organic
solvents
may be employed in a conventional liquid extraction, which may be time
consuming as
well.
[0005] The concentration of paclitaxel in various raw materials is typically
low,
for example, on the order of between about 0.0004 and about 0.01% (w/w) in the
bark of
Pacific yew. Such low concentration renders the extraction and purification of
the
compound to pharmaceutical grade from raw materials very challenging, and
heretofore
impractical on a commercial scale. Various normal phase chromatography
techniques
have been developed to purify paclitaxel from a crude extract of raw material.
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CA 02536000 2006-02-10
[0006] The success of low pressure chromatography greatly depends on the
nature
of the column. Various problems are associated with the use of silica gel and
alumina
trioxide, all of which are classical supports of the stationary phase in
partition system.
They form a stable stationary phase with most solvent systems, but is a strong
absorbent
and may participate in the separation process to the extent that
chromatographic behaviour
and recovery of samples are affected.
[0007] Chromatography methods have been developed to detect and isolate
paclitaxel from various Taxus species on analytical and preparative basis.
These isolation
processes are mainly conducted on a small laboratory scale and suffer from low
selectivity,
recovery and high production cost, thereby presenting a serious and
unfulfilled need for an
economically practicable method for separating the valuable anti-tumor
compound
paclitaxel from its close analog cephalomanine as well as other closely
related taxanes.
[0008] The use of cultivable and renewable plant parts, e.g., the leaves
(needles)
and twigs of Taxus species should be the most practical and attractive way of
increasing
the supply of paclitaxel. The needles of several Taxus species, including
Taxes canadensis,
have been investigated and found to contain paclitaxel in amounts comparable
to the bark
of Taxus brevifblia.
[0009] Taxes canadensis is an evergreen shrub found principally in Eastern
Canada and Northeastern United States. This species is unique in its taxane
content. The
needles contain a major taxane, 9-dihydro-13-acetylbaccatin III (9-DRAB III,
4) along
with paclitaxel (0.009-0.05%), 10-deacetylbaccatin III (10-DAB III, 6),
baccatin III, (5),
cephalomannine, (3), and other minor taxanes. The concentration of 9-DRAB III
in the
needles is reportedly seven to ten times the concentration of paclitaxel. It
appears that 9-
DHAB III may be become an important precursor to a new class of semi-synthetic
chemotherapeutic agents with increased water solubility.
[00010] Prior art methods disclose the use of various types of chromatographic
techniques to separate paclitaxel and related taxanes, including normal phase
and reverse
phase chromatography on a silica gel or bonded silica gel column. The prior
art methods
end up at low yields, high production costs or involved multiple steps which
were difficult
to scale up to large industrial scale production.
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[000111 The production of taxol from ornamental yew needles, barks and roots
at
present is not economical due to an extremely high percentage of unwanted
impurities
carried forward in the extract (about 40 to 50% by weight of the dried plant
material)
during the extraction. This unusually high percentage of impurities in the
solvent extract at
the needles of ornamental yew makes it very expensive and uneconomical to
purify taxol
and taxanes from this source in addition to the high cost in drying the
needles.
[00012] The current procedures are lengthy, costly, or are practically limited
to
analytical scale. Since paclitaxel occurs in low levels in needles and the
needles contains
large amounts of waxes, the isolation and purification of paclitaxel from
needles to a
clinically acceptable purify pose additional challenges. A daunting task is
the separation of
paclitaxel from its closely related analogue cephalomannine which occurs in
the needles
and bark. The two analogues have been separated by selective chemical
transformation of
cephalomannine in a mixture containing both cephalomannine and paclitaxel.
Disadvantages associated with these procedures include additional cost from
the use of
expensive, sometimes toxic, reagents, additional chromatography required to
separate the
transformed cephalomannine from paclitaxel, the destruction of cephalomannine
and
sometimes paclitaxel is during the process, and additional chemical
transformations which
are necessary for recovery of cephalomannine.
[00013] The most relevant prior art is believed to be U. S. Patent No.
5,969,165,
issued October 19, 1999, to Liu, which provided a high yield and high purity
method for
obtaining taxane analogues from a source containing taxanes. The method
employed a
polymeric resin absorbent for separating the analogues under low pressure
without the use
of complex and extensive separation/purification steps currently provided in
the art. That
method for isolating and purifying taxane analogues from a source containing
taxanes
included the first step of extracting a source of taxanes in an organic
extractant, e.g.,
dichioromethane. The second step involved contacting an absorbent medium with
the
extractant and loading the medium in a column. The column contained an
absorbent agent,
e.g., aluminium oxide. The next step involved eluting, with an organic solvent
mixture,
e.g., a mixture of hexane and acetone, at a pressure of between about 10 and
20 psi to
generate fractions containing taxane compounds. The next step involved
crystallizing the
fractions to provide a solid taxane compound and a mother liquor;
concentrating said
mother liquor. The next step involved eluting, with a polar solvent mixture
and mother
liquor through a polymeric resin to provide at least a second taxane compound.
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[00014] Thus, the art has continued to search for ever more efficient and
environmentally safe methods for obtaining taxanes which minimize the use of
plant
materials and organic solvents.
[00015] The number of publications and patents describing the isolation and
purification of paclitaxel and taxanes from Taxus specie is increasing, but
the procedures
currently known for isolation paclitaxel are very complex and difficult with a
low yield. A
list of typical such patents, below, describe various isolation technology,
from normal
phase chromatography to reverse phase chromatography. The yields of paclitaxel
from
various species of Taxus genus range from 0.005-0.017%. A list of some of
those patents
follows:
U.S. Patent No. 5,019,504 issued 1991, to Christen;
U.S. Patent No. 5,380,916, issued January 10, 1995, to Rao;
U.S. Patent No. 5,380,916, issued January 10, 1998 to Rao;
U S. Patent No 5,407,674, issued April 18, 1995, to Gabetta, et al.;.
U.S. Patent No. 5,620,875, issued April 15, 1997, to Hoffman et al.;
U.S. Patent No. 5,670,673, issued September 23, 1997, to Rao;
U.S. Patent No. 6,503,396, issued January 7, 2003, to Kim, et al.;
[00016] Canadian Patent No. 2,126,698, issued November 8, 1993, in the name of
Nair;
Canadian Patent No. 2,157,905, issued March 18, 1994, in the name of Durand
et al; and
[00017] Canadian Patent 2,213,952, issued June 15, 1999, in the name of G.
Caron.
[00018] The invention in its general form will first be described, and then
its
implementation in terms of specific embodiments will be detailed with
reference to the
drawings following hereafter. These embodiments are intended to demonstrate
the
principle of the invention, and the manner of its implementation. the
invention in its
broadest sense and more specific forms will then be further described, and
defined, in each
of the individual claims which conclude this Specification.
SUMMARY OF THE INVENTION
[00019] It would be advantageous to provide a more efficient and
environmentally safe
method for obtaining taxanes which minimize the use of plant materials and
organic
solvents.
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[00020] The present invention achieves such desired result and provides for
the
isolation of commercially important natural products from readily available
biomass; the
provision of an efficient method for obtaining taxanes from mixtures with
other
compounds or materials; the provision of an isolation procedure for paclitaxel
and other
taxanes from bark, needles, or cell culture of Taxus species which is
amendable to
industrial scale production; the isolation and separation of taxol and other
taxanes from
plant materials, preferably fresh material from ornamental yew, in high
yields; the
provision of a method which significantly reduces the cost of production of
the taxane
derivatives; and the provision of a method which provides commercial
quantities of the
above-referenced natural products from readily-available, renewable sources.
STATEMENT OF INVENTION
[000211 In a first aspect, the present invention broadly teaches an
improvement in a
method for isolating and purifying taxane analogues from a source containing
the taxane
analogues including the step of passing a solution containing the taxane
derivatives
through a chromatographic column. The improvement comprises compressing the
packing
media in the column substantially to avoid voids therein which provides an
organic
solution containing impure taxane derivatives. The chromatographic column is
maintained
under a high pressure of, e.g., about 250 to 400 psi or even as high as about
5000 psi.
[00022] In a second aspect, the present invention provides a method which
includes
the preliminary step of passing an organic solution containing impure taxanes
through a
chromatographic column in which the packing media in the column has been
compressed
substantially to avoid voids therein, and is maintained at a high pressure of,
e.g,., about
300 to 400 psi, or even as high as 1000 psi.
[00023] In a third aspect, the present invention provides a method which
includes
passing a syrup of extracted paclitaxel in an organic solvent through a
chromatographic
column in which a packing medium has been compressed substantially to avoid
voids
therein and is maintained at a pressure of up to about 1000 psi, e.g., about
30-50 psi.
In a fourth aspect, the present invention provides a method which includes
extracting a
biomass of Taxus with an organic solvent and partitioning the solution between
hexane
and water, then re-partitioning the solution between dichloromethane or
chloroform and
water, and recovering a dichloromethane or chloroform solution of impure
paclitaxel.
CA 02536000 2006-02-10
OTHER FEATURES OF THE INVENTION
[00024] One feature of the first aspect of the present invention includes
carrying out
the additional step of passing the organic solution containing the impure
taxane
derivatives through a chromatographic column in which a packing media in said
column
has been compressed to obviate voids in said column and is then maintained at
a pressure
of up to about 400 psi.;
[00025] Another feature of the first aspect of the present invention includes
carrying
out the additional steps of passing that organic solution containing extracted
said impure
taxane derivatives through a chromatographic column in which a packing media
in said
column has been compressed to obviate voids in said column and is then
maintained at a
pressure of up to about 400 psi, thereby to provide an extracted paclitaxel in
an organic
solvent; and passing a syrup of said extracted paclitaxel in an organic
solvent through a
chromatographic column in which a packing medium is compressed to obviate
voids in
said column, and is maintained at a pressure of up to about 50 psi.
[00026] Another feature of the first aspect of the present invention includes
carrying
out the preliminary steps of extracting a biomass of Tax us with an organic
solvent;
partitioning said solution between hexane and water; then repartitioning the
solution
between dichloromethane or chloroform and water; and finally recovering a
dichloromethane or chloroform solution of crude paclitaxel.
[00027] Another feature of the first aspect of the present invention includes
the
selection of the organic solution as methanol, ethanol, a mixture of methanol
and ethanol,
a mixture of dichloromethane and methanol or a mixture of dichloromethane and
ethanol.
[00028] Another feature of the first aspect of the present invention includes
the
selection of the packing media as diphenyl bonded silica gel or C-18 bonded
silica gel.
[00029] Another feature of the first aspect of the present invention includes
the
elution of the column with a gradient solution of acetone/water,
methanol/water or
acetonitrile/water.
[00030] Another feature of the first aspect of the present invention includes
the
selection of the flow rate to be about 350 ml/minute, and the selection of the
pressure to be
maintained at about 600 to 800 psi.
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[000311 Another feature of the first aspect of the present invention includes
the step
of crystallizing and recovering substantially-pure paclitaxel.
[00032] A feature of the second aspect of the present invention includes the
selection of the organic solution as acetone/water, ethanol/water or
isopropanol/water;
[00033] Another feature of the second aspect of the present invention includes
the
selection of the packing medium in the column as a polystyrene-DVB resin, or a
polymethacrylate resin or a polyaromatic resin.
[00034] Another feature of the second aspect of the present invention includes
the
elution of the column with gradient solvent of acetone/water, methanol/water
or
acetonitrile/water, e.g., at a flow rate of about 1 L/minute.
[00035] Another feature of the second aspect of the present invention includes
the
step of crystallizing impure paclitaxel from the eluate of the column.
[00036] A feature of the third aspect of the present invention includes the
selection
of the packing medium as polystyrene-DVB resin, or a polymethacrylate resin or
a
polyaromatic resin.
[00037] Another feature of the third aspect of the present invention includes
the
selection of the elution of the column is with step gradients of
acetone/water,
methanol/water or acetonitrile/water.
[00038] Another feature of the third aspect of the present invention includes
the
selection of the flow rate of eluent through the column as about 350 ml/min
and at a
pressure of about 600 to 800 psi, or even as high as 1000 psi.
[00039] Another feature of the third aspect of the present invention includes
the
additional step of crystallizing 9-dihydro-1,3-acetylbaccatin HI from a
portion of the eluent
from the column.
[00040] A feature of the fourth aspect of the present invention includes the
selection
of the solution of taxane analogues as a solution of crude crystalline
paclitaxel in ethanol.
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[00041] In summary and in more general teens, this invention teaches a
purification
technology to extract an organic solution of paclitaxel by using a "Load and
Lock Axial
Compression Column" for industrial scale preparative high performance liquid
chromatography.
[00042] The successful use of a medium and large diameter column in
preparative
high performance liquid chromatography requires appropriate hardware. The
"Load and
Lock Axial Compression Column" is one in which a piston is used to pack and
unpack the
chromatography bed and to maintain bed compression during use. This
effectively
substantially prevents the formation of voids in the bed. The column can be
packed with
any packing material, including small particle size (about 10 m) media, and
very high
plate numbers are generated. The bed length can be adjusted by controlling the
amount of
packing material used to prepare the column.
[00043] Through the use of such "Load and Lock Axial Compression Column" the
present invention provides a commercially-economical procedure for the
extraction of
paclitaxel from Taxus sp. The procedure involves a sequence of procedural
steps. The last
essential step may be carried out on an impure paclitaxel solution which may
or may not
have been prepared by the third step. Similarly, the third step may be carried
out on an
impure paclitaxel solution which may or may not have been prepared by the
second step.
Additionally, the second step may be carried out on an impure paclitaxel
solution which
may or may not have been prepared by the first step.
[00044] In a preferred embodiment, the first step involves the extraction from
the
plant material, Taxus sp, preferably Taxus canadensis, (Canadian Yew). Any
part of the
plant containing one or more taxanes may be employed, e.g., the bark, roots,
leaves or
needles, branches, twigs, seeds or whole seedlings. Preferably clipping of
needles and
twigs, should be dried and ground. Grinding of the plant material may be
achieved by
conventional means, e.g., through use of a chipper and/or a grinding mill. The
taxanes can
be extracted from the whole plant or from separated parts, e.g., steps, roots,
leaves
(needles), seeds, or mixtures thereof. The material to be extracted can be
either fresh or
dried. Preferably, the needles are used.
[00045] The plant material is preferably first dried and ground to a suitable
particle
size usually ranging from about 0.001 to about 10 mm3. The plant material is
extracted
using an organic solvent, e.g., methanol, ethanol, a mixture of methanol and
ethanol, a
mixture of dichloromethane and methanol, or a mixture of dichloromethane and
ethanol.
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The filtrate from the extractant is mixed with water and is concentrated in
order to reduce
the volume of the liquid. The concentrate is partitioned between hexane and
water in order
to affect the step of defatting. The aqueous liquor is repartitioned between
an organic
solvent, e.g., dichloromethane, chloroform or ethyl acetate and water, and is
concentrated
to a thick syrup.
[00046] In a preferred embodiment, the second step involves passing the thick
syrup
through a column. The preferred solution for this ion exchange is the thick
syrup prepared
in the first step, which is diluted with an organic solvent, e.g., acetone,
ethanol or
isopropanol. However, any suitable organic solution containing extracted
paclitaxel may
be the starting material in this second step.
[00047] The syrup in, e.g., dichloromethane/acetone/water, is separated into
several
fractions by pouring through a medium pressure column containing a polymeric
absorbent
resin. The polymeric absorbent resin may be, e.g., a polystyrene DVB resin, a
polymethacrylate resin or any other suitable polyaromatic resin. The most
common type of
resin used in polymeric absorbent resin are polystyrene polymers with
crosslinking
divinylbenzenes. The polymeric absorbent resin should have a suitable particle
size, which
is thus suitable for the column size and the designated working pressure and
flow rate. One
non-limiting example is a particle size of about 5 to 100 mesh.
[00048] The eluent solution is passed through the column with step gradients
of a
suitable water-miscible solvent, e.g., acetone/water, methanol/water,
ethanol/water or
acetonitrile/water. The flow rate is variable and can be changed according to
the various
operating conditions. One non-limiting example is a flow rate of about 2L/min.
[00049] The fractions containing up to about 45% acetone/water are allowed to
form crystals of 9-dihydro-1,3-acetylbaccatin III.
[00050] The fractions containing more than about 45% acetone/water are
concentrated to remove most of the acetone. They are then extracted with a
suitable
organic solvent, e.g., ethyl acetate, CH3CL or CH2Cl2 and is concentrated to
provide a
crude solid residue of paclitaxel.
(000511 The third step involves the purification of crude solid paclitaxel.
Preferably,
this crude solid paclitaxel is the product of the second step. However, crude
solid
paclitaxel however produced may be the starting material for this third step.
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[00052] The crude solid paclitaxel is dissolved in a suitable water-miscible
solvent,
e.g., acetone, in order to affect the step of defatting, and the water-
miscible organic
solvent/water solution, e.g., an acetone/water solution thereof, is eluted by
way of axis
compression column chromatography. This system is a high performance liquid
chromatography system, with means to provide and maintain a high pressure,
e.g., about
250 to 400 psi or even as high as about 5000 psi.
[00053] This column containing a suitable ion exchange resin as described
above is
loaded with the paclitaxel solution and is eluted with gradients of, e.g.,
acetone/water, at
the above high pressure. The fractions containing paclitaxel in such
acetone/water were
allowed to crystallize, and dried to provide semi-pure paclitaxel.
[00054] The final step involves the purification of the semi-pure paclitaxel.
Preferably, the semi-pure paclitaxel is the product of the third step.
However, semi-pure
paclitaxel, however produced, may be the starting material for the final step.
[00055] The column used in the final step is the same column as used in the
third
step. The ion exchange resin, or packing medium in the column may be diphenyl
bonded
silica gel or C-18 bonded silica gel or any other reverse phase packing
material.
[00056] The semi-pure paclitaxel is dissolved in a suitable water-miscible
solvent,
e.g., acetonitrile, ethanol or methanol. This solution is loaded into the
packed column and
is eluted with gradient solvents of the water-miscible organic solvent/water
solution, e.g.,
acetonitrile/water, at a suitable rate, e.g., about 350 ml/min and a suitable
pressure
maintained at, e.g., about 600 to 800 psi or even as high as about 1000 psi.
[00057] The fraction containing pure paclitaxel was allowed to crystallize,
and the
crystals were then recrystallized from acetone/hexane. A pure white powder
paclitaxel is
provided.
[00058] The foregoing summarizes the principal features of the invention and
some
of its optional aspects. The invention may be further understood by the
description of the
preferred embodiments which now follow.
EXAMPLES OF THE CARRYING OUT OF THE INVENTION
Example 1:
CA 02536000 2006-02-10
Extraction of Biomass:
[00059] The plant material (Canadian yew, Taxus canadensis) was harvested in
the
Canadian provinces of P.E.I. and New Brunswick in the fall 2002, and contained
about
0.032% of paclitaxel. The fresh clippings of the Canadian yew were dried in a
drying kiln
at about 60 to 70 C before grinding.
[00060] 600 kilograms of the ground needles and twigs of biomass was placed
into
a 3000 extractor which equipped with a reflecting condenser. 2400 L of
methanol were
then added. The biomass was refluxed with methanol for about 3 hours, the
solvent was
filtered, and the first filtrate was collected. 2000 L of methanol was then
added to the
biomass reside after filtration, was reflexed for about 2 hours, and was then
filtered. The
second filtrate was collected and the biomass was discharged. The first and
second filtrates
were combined and about 10% water was added. The aqueous solution was
concentrated
in an evaporator under vacuum to about 15% of its original volume.
[000611 The concentrate was twice partitioned between hexane and water to
defat
the concentrate. The aqueous layer was collected and was twice re-partitioned
between
dichloromethane (or chloroform) and water. The organic layer was collected and
concentrated in an evaporator under vacuum to a thick syrup. The hexane layer
was
recovered, and the water layer was discharged.
[00062] Approximately 45 kilograms of syrup was obtained from 600 kilograms of
biomass. The dichloromethane syrup contained approximately 1 to 1.3% of
paclitaxel.
Example 2:
Primary Purification of Paclitaxel:
[00063] 23 Kilograms of the dichloromethane syrup from Example 1 were diluted
with 10 L of acetone in a container equipped with a mechanical stirrer, with
warming if
necessary. The stirring was adjusted to 1.5 rotations per second and 3 L of
deioned water
was added gradually over a period of 10 minutes. The aqueous solution of the
dichloromethane syrup was separated into several fractions by a stainless
steel, industrial
scale, medium pressure column which was equipped with a medium pressure
metering
diaphragm pump.
1l
CA 02536000 2006-02-10
[00064] A slurry of 120 kilograms of polystyrene-DVB resin (Rohm-Hass XAD-
1600), with a particle size of about 50 to 100 mesh, in methanol, was poured
into the
column which was 300 mm diam. and 3000 mm long. After the column had settled
while
the solvent was pumped at about 30 to 50 psi, the methanol was replaced with
about 30%
acetone in water until the column was equilibrated.
[00065] The diluted syrup sample was transferred to the top of the column. The
container was rinsed with a few L of 30% acetone in water and the slurry was
transferred
to the column. The column was then sealed and started to flow as the elution
solvent was
pumped into the column. The column was then eluted with step gradients of 35,
45, 50, 60,
65, 70 and 80% acetone/water. The flow speed was controlled at about 2 L per
minute.
Fractions of approx. 20 liter each were collected with the pressure maintained
between
about 30 to 50 psi.
[00066] After first washing with 80% acetone/water, the column was washed with
pure acetone, followed by washing with a mixture of acetone/ethyl acetate
(1:1) until the
effluent, which was initially very dark, become colorless.
After washing, the column was equilibrated again with 30% acetone in water, at
which
point it was regenerated and was ready for next run.
[00067] The fractions which were collected were monitored by UV absorbance at
227/nm with analytical HPLC, and with TLC monitored by spraying, 10% H2O in
ethanol.
The fractions (most in 45% acetone/water) containing 9-dihydro- I 3-
acetylbaccatin III
were combined and concentrated to remove most of acetone and left in a hood
overnight
until crystallization completed. The crystals were filtered out and re-
crystallized from
methanol to yield 240 grams of pure 9-dihydro-13-acetylbaccatin III as white,
needle like
crystals. Yield: 0.08%.
[00068] The fractions (most in 65% acetone/water) containing paclitaxel were
combined and concentrated to remove most of acetone. The aqueous solution was
extracted with ethyl acetate. The organic phase was collected and concentrated
to dryness.
The residue was analyzed by analytical HPLC. The residue (crude paclitaxel
material)
contained about 10 to 15% paclitaxel. The recovery of paclitaxel was between
about 90 to
100% from the dichloromethane syrup.
12
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Example 3:
Secondary Purification of Paclitaxel:
[00069] The secondary purification used a preparative high performance liquid
chromatography system, which is called "axis compression column
chromatography". The
column used was a Varian Load and Lock axis compression column (250 mm diam. X
1000 mm long). The chromatographic system was a Varian ST-4000 system, which
was
equipped with two high pressure diaphragm pumps, a sample inlet port, a UV
detector, and
a Varian control software system. The column was equipped with a hydraulic
piston for
packing and unpacking the column and lock adaptor. After full packing the
column with
50 liter of polystyrene-DVB resin (Rohm-Hass CG-161 in, particle size about 75
m) in
ethanol, the hydraulic piston was turned on. The piston slowly moves out to
compress the
column packing media at a pressure of about 3000 psi. While the column was
compressed,
the lock was switched to its locking position to lock the column. Thus, the
column can
generate a very high plate number. After the column had settled while the
solvent was
pumped at about 250 to 300 psi, the ethanol was replaced with about 30%
acetone in water
until the column was equilibrated.
[00070] The residue from Example 2, approx. 1.5 kilograms, was dissolved in 4
L
of acetone until all solid was dissolved. 2 L of water was gradually added
with stirring, and
was filtered if necessary. The aqueous solution was then pumped into the
column though
the sample inlet port.
[00071] The column was then eluted with gradient solvents of acetone/water
(starting from 50% acetone in water, ending at 100% acetone). The flow rate
was
controlled at 1 L per minute. Fractions of approx. 5 L each were collected
with the
pressure maintained at between about 300 to 400 psi.
[00072] The fractions collected were monitored by U.V. absorbance at 227/mm
with
analytical HPCL and with TLC monitored by spraying 10% H2O in ethanol. The
fractions
(most in 70% acetone/water) containing paclitaxel were combined and
concentrated to
remove most of acetone and were left in a hood overnight until crystallization
completed.
The crystals were filtered out and re-crystalized from 70% methanol in water
and dried in
a vacuum oven to yield 235 grams of slightly white, needle-like crystals. This
semi-pure
paclitaxel had a purity of about 70 to 80%. Yield: about 90 to 95% from the
crude
paclitaxel material.
13
CA 02536000 2006-02-10
Example 4:
Final Purification of Paclitaxel:
[00073] The final purification used a preparative high performance liquid
chromatography system, and an axis compression chromatographic column. The
column
used was a Varian Load and Lock axis compression high pressure column (100 mm
diam.
X 1000 mm long). The chromatographic system was a Varian SD-2 preparative HPLC
system, which was equipped with a sample inlet port, a UV detector, and a
Varian control
software. The column was equipped with a hydraulic piston for packing and
unpacking the
column and with a lock adaptor. The packing media used was diphenyl bonded
silica gel
or C-18 bonded silica gel.
[00074] After full packing the column with 6 liter of packing media in
ethanol, the
hydraulic piston was turned on. The piston slowly moved out to compress the
column
packing media at a pressure of about 3000 psi. While the column was
compressed, the
Lock was switched to its locking position to lock the column. Before using the
column for
purification, the ethanol was replaced with about 30% acetonitrile in water
until the
column was equilibrated.
[00075] The crude paclitaxel from Example 3, approx. 50 grams, was dissolved
in
200 ml of acetonitrile until all solid was dissolved. 100 ml of water was
gradually added
with stirring and was filtered if necessary. The aqueous solution was then
pumped into the
column through the sample inlet port.
[00076] The column was then eluted with gradient solvents of
acetonitrile/water
(starting from 30% acetonitrile in water, ending at 100% acetonitrile). The
flow rate was
controlled at about 350 ml per minute. Fractions of approx. 1 L each were
collected with
the pressure maintained between about 600 to 800 psi.
[00077] The fractions collected were monitored by U.V. detector with an
absorbance at 227/nm, and was monitored with TLC by spraying about 10% H2O in
ethanol. The fractions containing pure paclitaxel were combined and
concentrated to
remove most of acetonitrile and left in a hood overnight until crystallization
was
completed. The crystals were filtered out and re-crystallized from acetone:
hexane (1:1)
and dried in a vacuum oven to yield 36.5 grams of white powder. This pure
paclitaxel has
14
CA 02536000 2011-05-04
a purity of greater than about 99%. Yield: about 85 to 90% from the about 70
to 80% pure
crude paclitaxel.
[00078] The total overall yield is greater than about 73%.
[00079] As described above, the method of aspects of the present invention is
simple. It provides paclitaxel and other taxanes in high yield and purity on
industrial scale.
The method of aspects of the present invention therefore, provides cost-
effective processes
for mass production of paclitaxel and other related taxanes from a vegetal
source or tissue
culture, particularly from T. canadensis.
[00080] The method of aspects of the present invention provides mass
production of
paclitaxel and related taxanes from plants of the genus Tax us (Taxaceae).
[000811 The isolation and purification method of aspects of the present
invention
permits a highly efficient recovery of taxane derivatives in pure form from a
naturally-
occurring Taxus species. The improvement with this invention over the prior
art is
reflected in the high overall recovery yield of the taxanes as well as in the
purity of the
taxanes isolated.
CONCLUSION
[00082] The foregoing has constituted a description of specific embodiments
showing how the invention may be applied and put into use, these embodiments
are only
exemplary. The invention in its broadest, and more specific aspects is further
described
and defined in the claims which follow.
