~ 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
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 ~
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 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 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.