Note: Descriptions are shown in the official language in which they were submitted.
~ 2 1 ~
FIELD OF THE INVENT:CON:
The present invention relates to a process for mycelial extraction of
Cyclosporin using supercritical phase carbon dioxide under reduced lipid
co-e~traction conditions.
E~ACKGROUND OF THE IMVENTION:
Cyclosporin, and in particular cyclosporine (or cyclosporin A) has
been found to have medicinal value, particularly where immune system
intervention is a required element in clinical case management.
Cyclosporin was first identified in compounds produced by Beauv2ria
nivea ATCC 34921 and Cylindrocarpon lucidum. More specifically,
cyclosporins were found to be produced as secondary metabolites of
various fungal strains, such as Cylindrocarpon lucidum Booth; and,
Trichoderma polvsPorum (Link ex Pers.) Rifai - also kno~n as
Tolypocladium inflatum Gams - also known as the above mentioned
Beauvaria nivea strain.
Background on the discovery and early developments relating to
cyclosporins is elaborated in Borel, J., (1983~ Cyclosporin: ~istorical
Perspectives, Tranæplant Proc. 15, Suppl. 1, 2230 - 2241.
rrhe cyclosporins are biologically active monocyclic peptides and
are variously useful as antibiotics and immunosuppressants (particularly
cyclosporin A in human organ transplant applications). Cyclosporin A is
also underyoing tesking in the treatment of juvenile dia~etes and other
auto-immune diseasesO See also Stiller, C., and Keown, P., (1984)
Cyclosporin Therapy_in Perspective, Progress in Transplantation (Morris
and Tilney, Eds.) pp 11 - 45, Churchill Livingston Publishers, London.
CH~ t1
Cyclosporin A is a white
~c amorphous powder (when in
1~ CH~ P~ amorphous -Eorm), composed of
CH2 CH K C ~ CH ~
¦H I 3¦N ~ H3 11 amino acid residues, and
Cl~ N C~C~ C~C~2
3 ~10 ~ 2 ll ~l has a melting point range of
2 ~I q ¦~ ~; 148 to 151 degrees C, an
~3 1 ~ 7 1 6 ~ 5 i ~ I t~ empirical formula of
OC~C~CD~C~;~ ff~C~
~ D I ~H ~H~ l~2 C62H~1N11O12, and an elementary
CH3 CH S CH; 3CH~ Ctt3
IH ,t~ analysis as follows: C
CN3 Cl13 3
61.96%; H 9.24%; N 12.82%;
and, O 15.98%. Cyclosporin A
is characterized in greater
Cyclosporin A Structure
detail in US patent
4,215,199, and in even greater detail still in the following articles:
A. Ruegger, M. Kuhn, H. Lichti, H.R. Loosli, R. Hugenin, C. Quiquerez,
and A. von Wartburg, HELV. CHIM. ACTA., 1976, Vol. 59; and, TJ Petcher,
HP. Weber and A. Ruegger, HELV. CHIM. ACTA., 1976 Vol. 59; and, M.
Dreyfuss, E. Harri, H. Hofmann, H. Kobel, W. Pasche and H. Tscherter,
EUROPEAN J. OF APPL. MICROBIOLOGY, 1976.
Cyclosporirl B has also been extensively characterized. It is also
a white amorphous powder (when in the amorphous form), but has a melting
point range of 127 degrees to 130 degrees C. This polypeptide has an
elementary analysis as follows: C 61.68%; H 9.18%, N 12.97%; and, O
c~
Cl~J ~ CHl C~l,
LIIJ--~H C~l--rl~--C~l
!;--L I I .
~.",_C", I ~C~I~
'1,' I~ - "" " "' " ----C C ll l
CU--CH~--1HL ~ IH ~R C ~
~ 1~ c~ L ~ 'tl~
.,.",...,,..oec
cl~ c~ .~:--cll.
~c~l~cll~--ci~ co--c)~l
Cyclosporin B
16.17%. Its empirical formula is C61H~09N11O12.
In general, cyclosporins are cyclic, eleven-amino-acid, peptides
containing several unique amino acids. They are highly methylated, non-
polar and highly hydrophobic compounds.
Xn addition to being produced by fungi strains, some synthetic
pathways have also been discovered. Only a relatively few such
synthetic schemes for modification of these cyclosporins are known,
however. See for example: Wenger, R., Traber, R.P., Kobel, H., and
Hofmann, H., (1985) Cyclosporin Derivatives and Their Use - French
Patent 561,651. This approach invol~ed in vivo amino acid substitution.
Also see: Wenger, R., (1986) Synthesis of Cyclosporin and Analo~s:
Structural and Conformational Reauirements for Immuno-suppressive
ctivity - Prog. Allergy 38, 46 - 64; Wenger, R., (1983) Synthesis of
Cyclosporin and Analoques: Structure and Activity Relationships of New
Cyclosporin Derivatives - Transplant. Proc. 15, Suppl~1, 2230 - 2241;
and, Wenger, R., (1982~ Chemistrv of Cyclosporin, Cyclosporin A -
2 1 ~
,,~
Proceedings of the International Conference (D. White, Ed.) pp 19 - 34,
Elsevier Biomedical Press, Amsterdam, Netherlands. The approaches set
out in these publications involve the total synthesis of cyclosporin,
starting from tartaric acid.
Notwithstanding the elucidation of these synthetic pathways,
cyclosporins continue to be produced commercially by way of extraction
from the mycelia of one or more of the above mentioned species of fungi.
Current commercial extraction methodology depends on the use of
methanol as a solvent, in a lengthy and typically energy intensive
e~traction process. An archetypical process for the industrial
production of cyclosporine from the fermentation broth entails first
separating the mycelial cake from the culture filtrate. The mycelial
cake is then homogenized, possibly as many as three or more times, in a
9:1 methanol-water mixture. The mixtures are filtered, then vacuum
concentrated to remove the preponderance of the methanol. The residual
water is then extracted with several passes through a 1,2
Dichloroethane, organic solvent evaporation drying step. Only then is
the extractant suspended in methanol and subjected to Sephadex gel
filtration, to separate the cyclosporins (eg A, B, C, D, G) from the
lipid co-extractants. Thereafter, the cyclosporins are separated using
silica gel column chromatography, and eluted with a water saturated
ethyl acetate to elute the cyclosporins in order of their relative
polarity, ~eg D, then G, A, B, and finally, C). The crude, separated
cyclosporins are then purified by crystallization using petroleum ether
for cyclosporin G, and low temperature acetone crystallization for
cyclosporins A,B,C, and D).
Processing costs associated with liquid phase extraction and
subsequent fine purification of Cyclosporin A are the major contributing
factors to the overall final cost and economic limitations on the
current availability of the product.
Liquid phase solvent extraction is only one of a wide variety of
methodologies that are generally known in the extraction technology.
Included among other generally known techniques is supercritical
extraction, which in a sense is really a special case of solvent
extraction in which elevated pressure conditions (ie above the critical
point of the solvent), are employed to augment solvation of the desired
solute.
Supercritical extraction was first reported in the late 1800's, as
applied to dissolving inorganic salts in supercritical phase organic
solvents. Since then it has been applied to petroleum refining and
other organic materials. In the 1970's supercritical solvent technology
became widely accepted as a commercially viable extraction technique for
use in a wide variety of chemical, food and pharmaceutical products.
Supercritical fluid extraction is a technique that exploits the
solvating potential of a fluid under temperature and pressure conditions
above the critical values for that fluid. From the extractive
standpoint, supercritical extraction realizes advantages of both:
a) distillation (in the sense of being applicable to the
separation of components having di:Efering volatilities); and,
b) liquid extraction (in the sense of being applicable to the
2 ~
separation of components either having nominal differences in their
respective volatilities, or which are thermally labile).
Supercritical fluid extraction processes rely on the fact that when
a gas is compressed isothermally to a pressurP that is greater than its
characteristic critical pressure, while being held at a temperature that
is greater than its characteristic critical value, then the fluid in the
resulting state exhibits solvating power above and beyond that otherwise
associated with the solvent.
At least one application of supercritical mycelial extraction has
recently been the subject of a study into a possible alternative to the
conventional liquid phase solvent processes used in typical commercial
cyclosporin production. That study showed that the process was at least
technically feasible. On the other hand, the capital conversion costs
and operating yield inefficiency associated with supercritical
extraction would not be offset by any of the advantages that were
identified over the course of that study.
The study in question was the doctoral thesis of Derk Willem te
Bokkel - entitled SUPERCRITICAL CARBON DIOXIDE EXTRACTION OF
CYCLOSPORINE FROM THE FUNGUS BEAUVARIA NIVEA. In that study a range of
extraction conditions where investigated in the hope of enhancing
extractant yields. In at least one such case, methanol was employed as
a co-extractant in the interests of enhancing the extraction yield. In
model solubility studies using pure cyclosporin A, (see page 97 of the
above identified thesis), the use of methanol as a co-solvent in a
supercritical carbon dioxide solvent mixture resulted in increase
'~ ~ 3 ~
cyclosporin A solubility on the order of twenty ~old, as compared with
pure supercritical carbon dio~ide alone. In studies directed at actual
mycelial extractions, however, te Bokkel noted (see page iii - Abstract,
of the above identified thesis), that the "addition of methanol showed
no effect on the cyclosporine e~traction yields".
The work of te Bokkel also included an examination of the extracted
cyclosporin products. Co-extractant impurities precipitated out with
the extracted cyclosporine as a viscous material, and although amounts
varied depending on the experimental extraction conditions used in each
case, the amounts were nevertheless considerable. A preliminary
qualitative thin layer chromatography test was done on the co-extracted
materials, (see page 126 of the above identified thesis). These TLC
preliminary results suggested the presence of some lipids, which would
have to be separated out from any clinically useful cyclosporine
preparation - a costly and time consuming process ætep that could defeat
the viability o supercritical extraction as an alternative to present
day liquid phase solvent extraction processesO
SUMMARY OF THE INVENTION:
The preæent invention, however, is premised on the realization that
supercritical mycelial extraction processing can yield additional
technical advantages over and above both the current commercial liquid
phase solvent processes, and those associated with the previous
supercritical extraction studies. More particularly, the novel
processing conditions herein described materially reduce the
~"
proportional amount and rate of lipid co-extractants, and thereby reduce
the requirements for downstream purification processing. This opens up
alternative yield/economic management strategies for commercial
producers of cyclosporine.
In accordance with one aspect of the present invention, therefore,
there is provided a process for supercri-tical carbon dioxide extraction
of cyclosporine from mycelia, in which the supercritical extraction
pressure conditions do not fall below about 34 MPa, throughout the
extraction processing. In accordance with this practice, it has been
demonstrated that the lipid co-extraction reported by te Bokkel, can be
substantially avoided, to yield a cyclosporine product that is free from
certain lipid contaminants. This materially influences the economics of
supercritical processing in terms of offering an alternative to
conventional liquid phase solvent extraction in cyclosporine extraction
processes from fungal mycelia.
Pursuant to this aspect of the present invention there is provided
a process for supercritical carbon dioxide extraction of cyclosporine
from fungal xnycelia, wherein supercritical carbon dioxide pressure does
not fall below 34 MPa, throughout cyclosporine extraction from fugal
mycelia. It is particularly advantageous for this process to be carried
out using fungal mycelia which contains about 7 to 10~ moisture.
Processing under supercritical carbon dioxide pressures which are
maintained between 34 to 39 MPa through out the course of the extraction
procedure, is preferred, with a supercritical carbon dioxide density of
0.925 or greater, (preferably the supercritical carbon dioxide density
2 1 ~
,
is in the range of 0.93 to 0.97; and even more preferably in the range
of from 0O935 to 0.965). This generally entails processing temperatures
of 305 degrees K, or greater, (preferably in the range of from 305 to
315 degrees K). In an especially preferred practice under this aspect
of the present invention, the supercritical density is increased from
about o.9 to about 0.96 over the course of said extraction, with the
pressure being increased from about 34.5 up to about 38.5 MPa, and the
supercritical carbon dioxide extraction temperature being increased from
about 305 degrees K up to about 312 degrees K.
In general, the present invention relates to a process wherein
supercritical carbon dioxide extraction of cyclosporine from fungal
mycelia, is carried out at supercritical carbon dioxide pressures at or
above 35 MPa. Here again the mycelia preferably contain about 7-10%
moisture, and the supercritical carbon dioxide pressure is maintained
between 35 to 39 MPa. Supercritical carbon dioxide density is 0.925 or
greater are preferred, (especially in the range of 0.93 to 0.97, and
particularly in the range of from 0.935 to 0.965. To this end,
preferred supercritical carbon dioxide temperatures of 305 degrees K, or
greater are desirable, (and especially in the range of form 305 to 315
degrees K. As in accordance with the particular examples set out
elsewhere herein, a preferred practice of the invention involves the
density being increased from about 0.9 to about 0.96 over the course of
said extraction. Under such a process the pressure is increased from
about 35 up to about 38.5 MPa over the course of the e~traction, while
the temperature is increased from about 305 degrees K up to about 312
5 ~
.
degrees K.
In another respect, the present invention is predicated on the
unexpected finding that the use of ethanol co-solvent in supercritical
carbon dioxide extractions from fun~al mycelia, can au~nent cyclosporine
e~traction yields as compared with supercritical carbon dioxide
extraction alone. This is surprising in light of the findings of te
Bokkel in the case of methanol co-solvent supercritical carbon dio~ide
extractions from fungal mycelia.
In accordance with a particularly preferred aspect of the present
invention, there is provided a process in which processing pressures do
not fall below about 34 MPa, throughout the supercritical carbon dioxide
extraction process, in the presence of ethanol co-solvent, to enhance
cyclosporine extraction yields from mycelia, while concurrently
suppressing the relative proportions of co-extracted lipid materials,
either or both qualitatively (in terms of the kinds of lipids which are
and are not co-extracted), and/or quantitatively (in terms of the
amounts thereof). For example, it was found that while phospholipid co-
extractants are moderately soluble in ethanol, per se, they are much
less soluble when the ethanol is entrained in the supercritical phase
carbon dio~ide. Accordinyly, the combination of the specified
processing pressure-maintenance and the use of the ethanol co-solvent as
set out above cooperate to allow yield enhancing benefits of the ethanol
co-extractant without necessarily undoing all of the lipid-avoidance
advantages of operating under the specified pressure regimen.
Thus, in accordance with this latter aspect of the present
~38~
invention, ihere is provided a process for supercritical carbon dioxide
extraction of cyclosporine from fungal mycelia, wherein an effective
amount of ethanol is included as a cyclosporine extraction yield-
enhancing co-solvent in the supercritical co-solvent fluid. As before,
it is especially desirable that the mycelia contain about 7 to 10~
moisture. Preferably, the ethanol is present in an effective amount of
up to 20, and preferably not more than about 15% on a weight of ethanol
to weight of mycelia basis, (in a particularly preferred practice, the
ethanol is present in an amount of about 10%). In combination with the
previously mentioned processes, it îs preferred that the supercritical
carbon dioxide pressure does not fall below 34 MPa during cyclosporine
extraction from said fungal mycelia, and pressures in the range of 34 to
39 MPa. Generally spea~ing, it is preferred that extraction pressures
of 35 MPa be employed in the present invention, and in particular, that
supercritical carbon dioxide pressure be maintained between 35 to 39
MPa. A supercritical carbon dioxide density of 0.925 or greater is
preferred, and the range o 0.93 to 0.97 is especially so. Even more
particularly, khe supercritical density is desirably in the range of
from 0.935 to 0.965. For that purpose, supercritical carbon dioxide
temperatures of 305 degrees K, or greater, can be employed ( eg.
temperatures in the range of form 305 to 315 degrees K over the range of
pressures mentioned above). As shown in the examples set out below, th0
preferred practice according to the present invention relates to a
process in which the supercr-itical carbon dioxide density is increased
from about 0.9 to about 0.96 over the course of the extraction. This is
2 ~
achieved by increasing the pressure from about 34.5 up to about 38.5
MPa, and collaterally increasing the supercritical carbon dioxide
e~traction temperature from about 305 degrees K up to about 312 degrees
K.
From another perspective, the present invention relates to a
process for supercritical carbon dioxide extraction of cyclosporine from
fungal mycelia, wherein the supercritical carbon dioxide density is
0.925 or greater. The supercritical carbon dioxide density is
preferably in the range of 0.93 to 0.97, and especially in the range of
from 0.935 to 0.965. As above, the mycelia preferably contains about 7
to 10% moisture.
DETAILED DESCRIPTION OF TH~ INVENTION:
INTRODUCTION TO THE DRAWINGS:
Figure 1 is a schematic representation of a supercritical carbon
dioxide, cyclosporine extraction system; and,
Figure 2 is a detailed view of an extractor module employed in the
practice of the present invention as set out in the Examples herein
recited.
A supercritical extraction system in accordance with the practice
o~ the present invention comprises an extraction vessel, a carbon
dioxide compressor, and an oven or the like. The extraction vessel
provides the situs for the solubilization of the solute in the carbon
dioxide, while the compressor provides the necessary gas compression,
5 ~
and the oven provides the required heat.
As illustrated in Figure 1, liquid carbon dioxide was provided in
dip tubbed equipped cylinders 1, containing 22.68 kg of commercial grade
(99.5~ purity), carbon dioxide. The carbon dioxide was chilled in a
shell and tube heat exchanger la, in which the shell side was cooled
using a 50% solution of polyethylene glycol at -15 degrees C. The
carbon dioxide was chilled to a temperature of about -5 to -8 degrees C,
before being passed along at a pressure of about 5.6Mpa (carbon dioxide
density of about 0.174) to a temperature equilibrated Milton Roy
compressor 2. Compressor 2 was employed to compress the carbon dioxide
to a desired pressure above the critical pressure (valve 2a is adjusted
to provide the pressure set point control). Once the balance of the
systems valves are opened, the supercritical carbon dioxide flows
through the system, and ultimately out the atmospheric vent ~.
Referring now in particular to Figure 2, the extractor vessel 4,
was an ~E Autoclave Engineering Model CNLX1606 tubing nipple 4a, and two
Model 6F41686 adaptors 4b, (with a safe pressure handling rating of
about 10,000 p6i ) Extractor 4 was connected to the balance of the
system with Swageloktm Model QF4 quick connects. Extractor 4 is
located within the oven 3, as shown in Figure 1. Oven 4 has a fan (not
shown) which is employed to heat the carbon dioxide prior to its
entering the extractor 4. Various thermocouples are employed to monitor
the temperature of the supercritical carbon dioxide, throughout the
system, and especially as it enters th~ extractor 4, in the
supercritical phase, and as it leaves extractor 4.
J 7,j ~
In practice, the e~tractor is loaded with a predekermined quantity
of mycelium. The mycelium is harvesked from the culture broth, and
dried in an oven until the moisture content is in the range of about 7
to 10% by weight. The system is pressurized, and the pressure adjusted
with the set point valve 2a. Valves downstream of the extractor are
opened and the supercritical solvent flows through the system, over the
mycelium. The carbon dioxide and its entrained solutes are then
depressurized through micrometering or regulating valve 6, and passed
(bubbled) through a collection solvent such as ethanol or methanol, in
a collection tube 7. The carbon dioxide is then vented through vent 8.
Samples may be collected from tube 7, and are analyzed for cyclosporine,
and neutral and polar lipids. Note, that on reaching the endpoint of
any given extraction, the system as a whole is depressurized,, and the
extractor is removed. The system is then flushed with ethanol or other
suitable solvent to collect precipitated solutes on the piping and valve
walls - this can account for up to 30% or more of the total amount of
extracted cyclosporine.
In accordance with one aspect of the present invention, ethanol is
employed as a co-solvent (or entrainer). This can be done in any number
of ways, and was carried out in this case by introducing the ethanol
directly to the moist mycelium or to glass wool or bead packing, in the
extractor vessel, or by injecting the ethanol using a syringe pump.
Example 1
B. nivea was cultured, and the mycelia thereof harvested
from the culture broth. The broth was then oven dried at
14
about llo degrees F. The dried residue was then crushed. A
sample was taken, and extracted with liquid ethanol to
determine its cyclosporine content. The balance of the
mycelial matter, (4.4 grams) was then loaded into the
extractor vessel 4, in a powdered form. Based on the liquid
ethanol extracted sample, the starting material was known to
contain about 7.5 mg of cyclosporine per gram of powdered
mycelium. The extraction was carried out at pressures
beginning at 5000 psi and increasing over the course of
extraction to about 5400 psi. Temperatures began at about
95.6 degrees F, and were increased to about 101.7 degrees F.
After about 775 minutes, 10.31 mg of cyclosporine had be~n
extracted from the mycelium, and after about 800 minutes, that
had risen to about 10.74 mg of cyclosporine. A total of about
800 L of carbon dioxide was used in the extraction. After the
extraction was completed, the system was flushed with ethanol,
and a further 6.66 mg of cyclosporine were recovered, bringing
the total recovery to 17.7 mg of cyclosporine, and a final
extraction yield of 58.0%.
Example 2
~ . nive~ was cultured, and the mycelia thereof harvested
from the culture broth. The broth was then oven dried at
about 110 degrees F. The dried residue was then crushed. A
sample was taken, and extracted with liquid ethanol to
'~ 5 ~3
determine its cyclosporine content. The balance of the
mycelial matter, (4.4 grams) was then mixed with ethanol (10%
by weight of ethanol to weight of mycelium) and then loaded
into the extractor vessel 4. Based on the liquid ethanol
extracted sample, the starting material was known to contain
about 7.5 mg of cyclosporine per gram of powdered mycelium.
The extraction was carried out at pressures beginning at 4950
psi and increasing over the course of extraction to about 5600
psi. Temperatures began at about 89.0 degrees F, and were
increased to about 103.1 degrees F. After about 530 minutes,
17.2 mg of cyclosporine had been extracted from the mycelium,
and after about 550 minutes, that had risen to about 17.5 mg
of cyclosporine. A total of about 584 L of carbon dioxide was
used in the extraction. After the extraction was completed,
the system was flushed with ethanol, and a further 5.3 mg of
cyclosporine were recovered, bringing the total recovery to
22.8 mg of cyclosporine, and a final extraction yield of
76.0%.
Analysis of the extracted cyclosporine from Example 1, showed that
phospholipids where not co-extracted. Analysis of the extracted
cyclosporine from Example 2, showed that phosphatidyl ethanolamine was
extracted (probably due to the increased polarity of the supercritical
phase solvent). Note however, that the use of the ethanol entrainer in
Example 2 resulted in far less carbon dioxide being used during the
2 1 ~ 5
e~traction process, and that the time required to conduct the extraction
was significantly reduced - and most importantly, that the extraction
yield was materially higher than was the case in Example 1. A summary
of some of the results from Examples 1 and 2, and a further trial in
which ethanol was used as an entrainer in an amount of 15% on a weight
of ethanol to weight of mycelium basis, reveals that increasing the
amount of ethanol, results in a collateral increase in the amount of
carbon dioxide that is used in the process, as well as a slight decrease
in the amount of cyclosporine extracted.
Conditions Cyclosporine Time EtOH Volume l
extracted (minutes) (%wt) f CO2 ¦
used
_ _ I
Example 1 58% 800 0 800 l
I
Example 2 76% 550 10 584 l
l _ _ _
Additional 71% 550 15 609
Trial (4950 to
5800 psi and
89.4 to 102.~ .
degrees F _ _
~ _
The extracted cyclosporine from Examples 1 and 2, were analyzed and
. 20 compared relative to cyclosporine extracted using li~uid ethanol at
atmospheric pressure and about 24 degrees C, from the samples mentioned
above. The results of that analysis are presented below in tabular
17
2108~'a
form, where the amounts of extracted compounds from B. nivea mycelia is
shown as mg per gram dry weight of mycelia.
_
Extractant Compound Liquid Example 2 Example 1
EtOH
e~traction
(mg/g Dry
wt.)
Cyclosporine 7.5 5.7 4.35
_
Phospholipids (Polar
Lipids)
1. phosphatidyl choline 15.82 2.69
_ _ _
2. phosphatidyl serine 0 0 0
_
3. phosphatidyl inositol 2.48 0.15 0
. _
4. phosphatidyl 3.66 0.26 0
ethanolamine
_
5. lyso-phosphatidyl 7.93 0 0
choline _
6. lyso-phosphatidyl 8.85 0 0
ethanolamine _ _ _
2~8~i5~
_
Neutral Lipids (non-
polar)
_
7. triglycerides 16.78 O O
8. ergosterol 3.8 2.0 0.66
9. 1,2-diglycerides O O O
. _
10. cholesterol trace O O
amounts
11. free fatty acids O O O
_
The comparison set forth above, reveals some of the advantages of
the present invention, as mentioned earlier herein. The present
invention is, however, amenable to many variations in the hands of
persons skilled in the art, and the scope of the invention is not to be
limited other than in accordance with the claims as set forth herein.
