JP2515446B2 - Method for fermentative production and isolation of cyclosporin A and novel cyclosporine producers - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method for producing cyclosporin A, which is a cyclic oligopeptide. In particular, the present invention relates to a microbial method using a new production variety and separation and purification of active ingredients.

Cyclosporin A belongs to the group of cyclic undecapeptides with anti-inflammatory, immunosuppressive, antifungal and antiparasitic properties. Of particular interest to human medicine is the finding of profound immunomodulatory properties for transplant surgery and treatment of autoimmune diseases.

In the patent specifications CH-PS 589716 and CH-PS 603790, this new class of substances was first described as cyclosporine, and in Borel's article (Cyclosporine, Advances in Allergy 38, Kargar Press, 1986): Its chemical structure and biological-pharmacological effects have been comprehensively reported.

It is known that the method for producing cyclosporin A utilizes the ability of microorganisms to naturally synthesize cyclosporine, in which a specific cultivar of microorganisms is used as a production strain in a fermentation method to produce nutrients containing complex culture media. Culturing is carried out in a solution while maintaining usual culture conditions, and the active ingredient is separated and purified by a known method.

Extensive screening studies have discovered a large number of fungi capable of producing cyclosporine. Dry-hus (Sydowia, Bd. 39, 1986, 22
-36) is a fungal genus Cylindrocarpo.
n) and Fusarium, but only the ability to form cyclosporine on the liquid side, while the mycelial fungal varieties Tolypocladium geodes, Trichoderma viride, Neocosmospora vaccinfecta, Isaria ferrina, and Vaticillium. Cyclosporine formation has been reported in submerged cultures for cultivars, Acremonium cultivars, and Beauveria nivea (originally reported as Trypocladium inflatum).
The highest productivity of the active ingredient is the fungal variety Bueberia
Achieved by Nivea. Therefore, this type has become particularly important in the mass production of cyclosporin A. Traber et al. (J. Antibiotics, 4th
2, 1989, 591-597), a high-performance mutant of Beauveria nivea (in the article, Tolypocladium inflatum NRRL8044).
Described as a mutant) produces about 500 mg of cyclosporin A per liter. In a chemically defined synthetic medium containing sucrose as the main component and malic acid as a carbon source and ammonium ion as a nitrogen source, Kobel and Traver (Europ. J. Appl.
Microbiol. Biotechnol. Volume 14, 1982, 237-
240) for 14 culture days, 101 per liter.
A yield of mg cyclosporin A has been achieved. Supplementing this producing strain with a carefully selected amino acid at a concentration of 8 g / l improves the yield, but is not an economically productive solution for technically relevant processes.

[0005] Similar drawbacks have been reported by Agatos et al. (Ann.N.
Y. Acad. Sci. 506, 1987, 657-6
62) also showed fermentation, and after 16 days of culture in semi-synthetic culture medium, a yield of 530 mg cyclosporin A per liter was reported, where it is too expensive as a carbon source and technical method. It uses maltose which is considered to be.

The above-mentioned space-time-yield for one technical production method is too inadequate in the sense of economical utilization of the varieties of production, and, in addition, the various cyclosporins which inevitably lead to the loss of the active ingredient. , Especially adversely affecting the isolation and purification of cyclosporin A. above,
The method for separating cyclosporin A from culture dispersions of various bacterial species takes into account in all cases the expected vulnerabilities to peptide bonds within the cyclosporin A molecule, avoiding high temperatures and avoiding culture dispersions. Starting from liquid-liquid extraction or extraction of separated moist mycelium [US-PS 4117118, US-PS 42151.
99, DE-PS 2455859, BE-PS866
810, V.I. Walburg et al., Helv. Chim. Acta 55 (1
976) 1075]. Most of the extraction residue is 3
The material, which has been partitioned between 0% methanol and petroleum ether, and thus previously defatted, is chromatographed repeatedly on silica gel, aluminum oxide and / or Sephadex LH20, partly 1000 times the culture medium. Use a large amount of adsorbent up to (DE
-PS 2455859). The disadvantages of the known techniques are obvious from this high purification cost and the fact that the harvested broth must be treated immediately during the treatment of the wet mycelium. Furthermore, a disadvantage of the presently known methods is the use of large amounts of solvents, which are particularly incompatible with the environment.

The object of the present invention is to expand the number of highly productive cyclosporin-producing bacteria available and use these novel microorganisms to produce cyclosporin A in a synthetic medium using a complex nitrogen medium. Is to provide an economically efficient microbiological method that is achieved without.
Furthermore, the object of the present invention is technically advantageous, compatible with the environment, efficient and high yield of cyclosporin A.
To provide a method for separating and purifying the same. that time,
The amount of auxiliaries used should be kept as low as possible and limited to substances that are compatible with the environment.

In the present invention, a novel and previously unknown microorganism was isolated from the environment of the natural subtropical climatic zone and purely cultivated. In addition to accumulating, these substances and in particular cyclosporin A, after proper varieties adjustment, as a result of mutagenesis and selection treatment, under immersion culture conditions, within the framework of the multi-step fermentation process, were totally unexpected. Produce at a high rate. This result is particularly surprising since hitherto it has been considered that chemically defined fully synthetic media are not suitable for the active ingredient synthesis of production media at such high rates.

The original microorganism is Sierra del Rosario
(Sierra del Rosario) (Cuba) was isolated from a soil sample and phylogenetically isolated from Cesxyliopsis Rosariensis G. Arnold (Sesquicilliopsis rosariensis G.
ARNOLD) is a representative of an incomplete mycelial fungus classified into a new genus and variety.

The microorganism grows on various culture media containing normal fungal nutrients. Malt-a natural day on agar-
By nurturing at night rhythm, weak within 10 days,
Light flaky colonies about 8 cm in size with white-air hyphae extending to the colony edges are produced. Ring formation
Only a few are observed outside the colony. A weak odor like soup seasoning is confirmed. This aerial mycelium consists of branched, branched hyphae that appear white overall. Rising or vertical conidial carriers are generally strong, more or less circulate or irregularly branched, with many colorless, conical, fine infarcts at the ends of the branches. Conidia are formed at the tips of infarcts one after another, elongated, unicellular, transparent, smooth and thin-walled with small heads. No Yuko is observed. All genera with infarcted conidia cells (eg Fusarien forming microconidia, Gliocladium (Gli)
ocladium), Virticillium (Vertic)
illium), Sesquilic
ium), Symposiophora (Sympodioph)
ora)) refers to Sesxyliopsis Rosalieensis G. Arnold differs depending on the above-mentioned peculiarities, especially the type and manner of arrangement of conidia gene cells.
In the following, the fungal species Cessxyliopsis rosaliensis G. The culture and morphological properties of Arnold are described together. 1. Malt extract-agar medium (1) Teleomorph phase: not observed. (2) Morphological properties of anamorph phase Colony surface: See the above description Colony back surface: Yellow to yellowish gray (3) Temperature range: 5 to 28 ° C Optimum temperature: 24 ° C (4) pH range: 2. 0-8.0 Optimum pH: 5.5 (5) Other properties: Cyclosporine formation was not detected. 2. Sabouraud-Agar (1) Teleomorph phase: Not observed. (2) Morphological properties of anamorph phase Colony surface: Clearly raised colonies and thick flocculent aerial mycelia were observed in the group. Very few spores were noted. Rear side of colony: yellow to orange (3) Temperature range: 5 to 28 ° C Optimal temperature: 24 ° C (4) pH range: 2.0 to 8.0 Optimum pH: 5.5 (5) Other characteristics: Clear Intense cyclosporine formation was detected.

This novel microorganism used for the production of cyclosporin in this method is IMET according to the Budapest Treaty.
It was deposited at the depository, Federal Republic of Germany, O-6900 Jena, Voitenberg Rd. 11 with the number IMET 43888, and likewise the high-productivity variety F605 was deposited with the deposit number IME.
Deposited at T43887.

The present invention thus naturally comprises all other variants cultivated by mutation and / or selection of wild varieties or the abovementioned mutants.

Tolypocladium inflatum (Tolypo
The cladium inflatum variant Wb6-5 could be isolated by selection of high performance monospores on a chemically defined nutrient medium, especially by the presence of calcium and zinc ions in the culture medium. What is particularly surprising is that the isolation of this variant has also been described in the literature, "Cyclosporine formation is an amount that allows economical production only in the presence of complex nutrients such as peptone or certain amino acids. It is based on the view that "it will be done in." Variety Tolypocladium inflatum selected variety Wb6-5 has been deposited with the IMET depository in Jena under the Budapest Treaty with registration number IMET43899. In the method according to the present invention, the selected variety Wb6-5 of Tolypocladium infratum or the variant variety F605 of Cesxyliopsis Rosaliensis is
Incubate in a medium containing ammonium salts as the sole nitrogen source, optionally with the addition of citrate, and in the presence of selected divalent cations. In addition, during fermentation of the individual varieties, red-brown or black-brown-purple colored liquid (E on the mineral salt-glucose-agar with ammonium salt) was used.
Starting from a (mers) culture medium has proved to be advantageous as it ensures a stable production of cyclosporin A.
Furthermore, for the variant Wb6-5, it is particularly advantageous to use ammonium sulphate and / or diammonium hydrogen sulphate as inorganic nitrogen compounds in the production medium.
The zinc ion concentration is 0.5 mg / l ~ in this type of culture.
The effective range is 5.0 mg / l. Sorted product W
It is noticeable that the productivity of b6-5 can, surprisingly, be adversely affected by the presence of potassium salts or ions. This is all the more surprising as all culture media published for this variety contain potassium salts. Such dependence is described in Sesxyliopsis Rosalieensis G. et al. It has not been confirmed in the Arnold variety F605. The presence of certain divalent metal ions in the fermentation medium is much more important for this variety, especially magnesium, iron,
The presence of zinc and calcium ions, to varying degrees, has a direct effect on synthetic activity. Therefore, the fermentation of variety F605 preferably has a magnesium concentration of 250 mg / l, an iron concentration of 20 mg / l and a zinc concentration of 0.
Should be done at 7 mg / l. In particular, in the absence of calcium, even if all the remaining conditions are maintained, the productivity of variety F605 is not effective, so it is important to pay attention to the appropriate amount of calcium ions, and at that time, the ion concentration It should preferably be between 10 mg / l and 200 mg / l. At the same time, production cultures of these heteromorphic varieties were 5 g / l to 30
It is clearly advantageous to work in the presence of citrate ions in the g / l range.

While it has been found that there are differences in the requirements of individual cyclosporin A producing varieties with respect to the amount of specific divalent cations in the production medium, all varieties of the process are expensive. Eliminating intricate nitrogen sources, whose composition is heterogeneous, is a very important advance in process engineering. Agatos et al. (J. Ind. Mikrobiol.
Vol. 1, 1986, 39-48) found ammonium and nitrate to be inadequate for cyclosporine production, which led to these results when these researchers adopted a complex nitrogen source as the only N-containing medium. Was unexpected. Results from inorganic N-sources published later (Margaritis and Chahal, Biotechn
ol. Lett. Vol. 11, 1989, 765-768) also proves the above report.

Fermentation is carried out in a submerged main culture medium under aerobic and aseptic conditions after one or two-step preculture, in which case
The various production varieties are cultivated in liquid culture medium at a temperature of 20 to 30 ° C. and an acidity of 3.0 to 7.5 for 5 to 11 days.

All steps of the method are carried out as follows.

A mycelium dense portion obtained from a liquid (Emers) culture medium of variety Wb6-5 and its spores are dispersed in a 0.9% NaCl solution, and a nutrient containing mono- or disaccharide as a carbon source and ammonium sulfate as a nitrogen source. It is spread on agar and cultured at 24-28 ° C, preferably 24 ° C for 14-21 days. The breeding of the cultivar F605 is carried out in the same manner, but the nutrient agar does not contain ammonium sulfate. From the resulting single colony, a red-brown to black-brown sample with less air mycelia is selected, transferred onto mineral salt-carbohydrate-agar, and then used for preparation of a mycelium dense area of 2 to 4 cm. Transfer this as agar content to the first pre-immersion culture passage, which contains 20% of each volume of culture medium 1
Incubate in an Erlenmeyer flask of 00 ml to 500 ml. This nutrient solution contains glucose, maltose or saccharose as a carbon source, organic or inorganic nitrogen culture medium and mineral salts, and 48 to 96 hours, 24 to 2 hours after inoculation.
Incubate at 8 ° C with shaking on a rotary shaker at 100-240 rpm. After initial growth in the liquid culture medium (corresponding to the first preculture), if necessary,
The second inoculation (corresponding to the second preculture) is performed by inoculating 5-10% of the volume of the nutrient solution of the next culture. This step is carried out in a 500 ml Erlenmeyer flask containing 100 ml of the above medium or in a small fermenter with a net volume of 5 to 25 l of agitated and ventilated, which is the usual technical equipment for temperature control and ventilation. After culturing for 48-96 hours, the main culture medium containing ammonium salt as the sole nitrogen source was inoculated with the pre-culture medium in an amount of 5-10% of its volume,
Incubate for a day with agitation and ventilation, or shaking with rotation. Under this condition, the fermentation mixture is treated with HP in a known manner.
The ability to produce up to 3000 mg of cyclosporin A per liter, measured by LC, is reached. This culture dispersion has a reddish brown to blackish brown color until the time of harvest.

The method according to the invention for separating cyclosporin A from a culture dispersion comprises adding a filter aid to this culture dispersion, followed by filtration and moist biological material (Biomasse).
And the resulting mixture of biological material and filter aid is mixed with a lower carboxylic acid ester or supercritical gas, preferably C
Extraction with O ₂ and delipidation by partitioning the extract between methanol and petroleum ether containing water, the extract thus pre-purified is chromatographed on silica gel and / or aluminum oxide. The advantages of this separation method are, inter alia: -a small particle size but a loss-free separation of biological material, a stable, storable, extractable, dryable substance-a low purification cost and a small amount of purification It can be extracted using ballast material, -is compatible with the characteristics of the producing bacteria used in the subsequent manufacturing process, and-can reduce the usage fee of the extractant.

At that time, the biological material of the producing bacterium to be used is not in the mycelial shape which is usually preferred, but has a particle size of 10 to 30 μm.
It is difficult to form a filter cake which becomes granular and has poor filterability and is difficult to separate. According to the present invention, this difficulty is caused by adding a filter aid having sufficient separation effect (for example, recrystallized gypsum = plecosit, calcitomer) to the culture medium dispersion directly or after previously concentrating by centrifugation. It can be solved by filtering. A volume ratio of filter aid / dry biomaterial (1 to 4): 1 ensures good filterability, fast drying and effective residue separation after extraction.

Obtained in this way, still 35 to 60% by weight
The moist biological material containing the culture filtrate of
Despite the nature of the peptide, convective drying (eg vacuum drying ovens, air circulation ovens, swirl bed or band drying equipment) can be carried out at temperatures up to 80 ° C., preferably 40-60 ° C., with virtually no material loss. It can be converted into a storable dry mycelium lumber, but its moisture: solid quantitative ratio is suitably (0.02-0.15): 1.
Alternatively, freeze-drying can also give a similarly stable, extractable dry product.

For the subsequent two or three extractions, the ethyl ester of acetic acid is preferred, since it requires significantly less ballast material than, for example, ethanol or acetone. This is especially true if the number of extractions is limited to two.

The residue after evaporation is distributed in a manner known per se between methanol or ethanol containing water and petroleum ether to a partial degreasing, which already gives a substance content of 40% by weight. . It may be effective to moisten the hyphae with aqueous ammonia before extraction. When treating mycelium species that are difficult to extract,
Swell at 40% by weight, preferably 25% by weight for 8 to 72 hours and the first extraction is carried out with Ultra-Turrax.

As an alternative to solid-liquid extraction with organic solvents, it is also possible to extract the active ingredient from the dry hyphae with a supercritical gas, preferably CO ₂ . The pressure and temperature depend on the gas. A range of about 100 to 300 bar and 35 to 60 ° C. is suitable for CO ₂ . Tractor,
For example, it is also effective to add 0.5 to 1.5% by weight of ethanol to CO ₂ to assist the extraction. impurities,
In particular, fat can be separated by multistage extraction or fractional distillation.

The first column chromatographic purification is
Depending on the choice, aluminum oxide is neutral (50 to 100 times the amount) or silica gel (30 to 60 times the amount), ethyl acetate of acetic acid is used as the developing phase, and the subsequent purification is always silica gel, preferably 40 times the amount. Of ethyl acetate. Subsequent to the supercritical extraction, an active ingredient selection by HPLC can be performed instead of the atmospheric pressure liquid chromatography. Example 1 The mycelium dense part of the surface culture of the Tolypocladium inflatum Wb6-5 variety was first subjected to glucose (in g / l).
_{20, (NH 4) 2 SO} 4 5, KH 2 PO 4 2, Mg
Transfer to glucose-mineral salt-agar consisting of SO ₄ .7H ₂ O 0.5, CaCO ₃ 3.4, agar 20, pH value 5.3-5.7, sterilized at 120 ° C. for 30 minutes. After culturing in a Petri dish at 24 ° C. for 14 to 21 days, a reddish brown-black brown colored portion is selected from the resulting area. The inoculation of the first pre-immersion culture was carried out using a mycelium dense area of about 1 cm ²
Glucose 50 (in g / l), casein peptone 1
0, KH ₂ PO _4, 1, KCl 2.5, distilled water 1
l, pH value 5.3-5.6, sterilization at 120 ° C for 30 minutes,
Transfer to 100 ml of culture medium consisting of When a 500 ml culture flask is cultivated at 24 to 25 ° C. for 72 hours on a rotary shaker at 180 to 220 rpm, it shows a blackish brown color as a standard for aging, and is used in an amount of 5% as an inoculum for the main culture medium. The main culture medium was filled in a 500 ml Erlenmeyer flask with 100 ml of each, and glucose 80 (in g / l unit), (N
_{H 4) 2 SO 4 5,} (NH 4) 2 HPO 4 2, Mg
_{SO 4 · 7H 2 O 0.5,} ZnSO 4 · 7H 2 O
0.008, CaCO ₃ 5.1, distilled water 1 l, pH
Value 5.3-5.9, sterilized at 120 ° C. for 30 minutes. The culture was carried out at 180 rpm on a rotary shaker for 24 to 2
Perform for 11 days at 5 ° C.

After fermentation yield, methanol is added to the culture liquor in a ratio of 5: 1 (v / v) and the extract is used for evaluation. This evaluation is carried out by means of an HPLC column in a manner known per se. The performance of this type of fermentation mixture is 1100 mg cyclosporin A per liter. Example 2 Malt extract agar (20 g / l of malt extract) on liquid of Sesoxylyopsis Rosaliensis F605 varieties (Eme)
rs) From the culture medium, after culturing at 24 ° C. for at least 14 days, prepare the spore dispersion used for inoculation of the preculture. This pre-culture medium contains maltose or glucose (in g / l) 75, casein peptone 25, KH ₂ PO ₄
1, KCl 2.5, deionized water 1 l, pH value 5.
5. Sterilized at 120 ° C. for 30 minutes. Inoculated with 2.10 ⁷ spores before culture medium 100 ml, in a 500ml Erlenmeyer flask, 1 for 72 hours, rotary shaker on at 24 ° C.
Incubate at 90 rpm. Subsequently, the culture medium was fed with glucose 100 (in g / l), diammonium hydrogen citrate 15, KH ₂ PO _4, 1, MgSO ₄ .7H ₂ O 2.
_{5, FeSO 4 · 7H 2 O} 0.1, ZnSO 4 · 7H
₂ O 0.003, deionized water 1 l, pH value 5.5
5: Sterilize at 110 ℃ for 60 minutes
Transfer at a ratio of 10. All culture conditions are selected according to the preculture. The content of cyclosporin A measured on the 11th day of culture was a maximum of 1150 mg / l per liter of culture dispersion.
Met. The measurement of the active ingredient was carried out as described in Example 1. Example 3 Breeding and culturing of cultivar F605 is carried out in a pre-culture medium as described in Example 2, but unlike Example 2, after pre-culturing the production variety, it already contains the main culture medium described in Example 2. , Stirring at 200-500 min- ¹ for 0.25-1.0 /
Transfer to a 5 l laboratory fermentor ventilated with a volume flow of min / l. Maximum 315 due to fermentation for 14 days
A cyclosporin A yield of 0 mg / l was obtained. Example 4 For cultivar F605, after growing the inoculum as in Example 2, glucose 50 (in g / l), coarsely ground soybean 2.5, (NH ₄ ) ₂ SO ₄ 4.1, KH ₂ PO ₄
2.10 ⁸ spores per liter of medium are inoculated into a stirred fermenter containing a nutrient solution consisting of 5.0, pH 5.5, sterilized at 110 ° C. for 60 minutes. Temperature, stirring and ventilation are in accordance with the conditions described for the main culture medium in Example 3. 450, made from alloy steel, thus cultivated for 4 days, containing the main culture nutrient solution described in Example 2 and maintaining the conditions described above for the preculture.
l Used as a 10% inoculum for the production process in fermenters. In the fermentation carried out as described above, after an average of 14 culture days, 2 g of cyclosporin A were formed per liter of culture dispersion.

Example 5 Sesxylyopsis Rosalieensis G. A 6 l culture dispersion of Arnold (IMET 43887) genus F605 is aspirated through a precocito-coated hand filter plate and the resulting moist material is dried at 50 ° C. for 6 hours in a circulating air drying oven.

246 g of a dry mycelium-precocit mixture containing about 50% by weight of precosite are obtained.

The mixture is swollen with 60 ml of 10% aqueous ammonia solution for 15 minutes and a total of 4.2 l of ethyl acetate is divided into 3 portions and shaken for 30 minutes each. 21 g after removing the solvent by distillation from the extract filtered from the solid substance
14 g of a residue are obtained, which is taken up in 100 ml of ethanol containing 5% water, shaken 3 times with 300 ml of petroleum ether each time and the methanol is evaporated.
Decreased to.

1000 g aluminum oxide in ethyl acetate, neutral, column chromatography of activity step 1,
After about 500 ml outflow, 1450 ml main fraction was obtained,
The residue is silica gel 60 Merck (0.063-0.2 m
5.2 g by rechromatography in m) with the same solvent and 7 volumes of n-hexane.
Of almost white, HPLC gives a product containing 99% by weight of cyclosporin A. Example 6 A culture dispersion of the mutant described in Example 5 containing 327.5 l quantity of 21.2% bio-drying material, 13.1
0.5 m ² mixed with 2 kg of plecosit
Filter with a pressure filter (centrifugal disc filter, Seitz type) having a filter area of. The surface of the filter is previously covered with 250 g of plecosito-filter aid at a time. Wet filter cake (weight 28.5
(kg) in a spiral layer dryer at a ventilation temperature of 35 to 55 ° C. to give a dry mycelium-precosito mixture of 1%.
5.4 kg are obtained. The dried product obtained in a stirring funnel,
Stir first with 100 l and then with 70 l of ethyl acetate for 30 minutes each. The clarified, prepurified extract is concentrated first in a vacuum circulation evaporator and then in a vacuum rotary evaporator to an oily residue, which is taken up in 8 l of methanol. After adding 0.4 l of water, stir 3 times with 16 l of petroleum ether each. The separated methanol layer is concentrated on a vacuum rotary evaporator to give 600 g of residue.

24 kg silica gel 60 in acetic ester
Initial purification by column chromatography on Merck gives 275 g of 79% by weight of crude cyclosporin. Rechromatography is carried out with 14 kg of silica gel 60 Merck in methylene chloride containing 2.5% by weight of methanol. Yield: 102 g, content (HPLC) 97.5% by weight Cyclosporin A Example 7 A vacuum rotary cell filter with a 0.3 m ² filter area is coated with 10 kg of plecosit as a filter aid. The mutant culture dispersion described in Example 5 is filtered through the filter. This culture dispersion contains
First, 60 g / l of plecosit are added. Water content 55.4
62.5 kg of% wet filter cake are obtained.

50 kg of this moist material are dried in a swirl layer to give 22.5 kg of a dry mycelium-plecosit mixture with an active ingredient content of 6.3 g / kg. Example 8 20 kg of the dried mycelium-precocytosed mixture obtained in Example 7 are moistened with 5 l of methanol and stored in a closed container at room temperature for 2 hours.
Leave for 4 hours.

The material is then taken up in 50 l of ethyl acetate and spun in an Ultra-Turrax for 40 minutes. After filtering off with suction, the filter residue is stirred for a further 10 minutes with 50 l of ethyl acetate and suctioned off. The extracts are combined and the residue is treated as in Example 6 to give 61.5 g of purified cyclosporin A. Example 9 0.52 kg of the dry mycelium-precocytosed mixture (TMP) obtained in Example 7 is treated with 5.2-6.5 kg / h of supercritical C
Add 50-80 ml of ethanol with O ₂ at 300 bar and 40 ° C. and extract over 6 hours. About 60%
The cyclosporin A contained in TMP is extracted. The extract thus obtained is treated and purified in the same manner as in Example 6.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁶ Identification code Internal reference number FI Technical display location C12R 1: 645) C12R 1: 645) (72) Inventor Ernst-Joachim, Bormann Jena, Federal Republic of Germany, Edwin-Morgner-Strasse, 44 (72) Inventor Gunther, Arnold Weimar, Moskauer, Strasse, 34

Claims (10)

(57) [Claims] 1. A method for fermentatively producing and separating cyclosporin A by aerobic fermentation of a fungal microorganism in a culture medium containing a nitrogen substrate, a carbon substrate and a mineral salt, the method comprising:
For fermentation, fungal species Cesxyliopsis rosaliensis
G. Arnold new breed, or mutation and / or
Alternatively, the heteromorphic variety obtained from the selection is used as a production variety and is cultured in a culture medium containing an ammonium salt as a sole nitrogen source, a carbon source and optionally a citrate, and a divalent cation, and a culture dispersion. The filter aid is added to the mixture, followed by filtration, and the thus obtained mixture of moist biological material and filter aid is dried to give a lower carboxylic acid ester or supercritical gas, preferably C
Extraction with O ₂ and partitioning of this extract between methanol and petroleum ether, optionally containing water, the prepurified extract is then purified in a manner known per se on silica gel and / or aluminum oxide or A method characterized by a chromatographic treatment by preparative HPLC. 2. The fungal species Cesxyliopsis Rosaliensis G. The method according to claim 1, characterized in that a new breed of Arnold IMET 43888 is used. 3. The fungal species Cesxyliopsis Rosaliensis G. The method according to claim 1, characterized in that a new variety of Arnold F605, IMET 43887 is used. 4. A fungal cultivar Cessxylyopsis Rosariensis G. Arnold IMET number 43888. 5. A fungal cultivar Cessxyliopsis Rosariensis F605 G. Arnold IMET No. 4388
7. 6. A process according to claim 1, characterized in that it starts from a red-brown to black-brown colored submerged medium which is placed on a mineral salt-glucose-agar containing ammonium salts as the sole nitrogen source. . 7. The method according to claim 1, characterized in that the fermentation is carried out in the presence of zinc ions, iron ions and / or calcium ions. 8. A process according to claim 1, characterized in that recrystallized gypsum, preferably plecosit or calcitomer, is used as filter aid. 9. Process according to claim 1, characterized in that ethyl ester of acetic acid or a supercritical gas, preferably CO _2, is used as extractant for the dried biomaterial-filter aid mixture. 10. The dried biomaterial-filter aid-mixture is pre-swelled with methanol or aqueous ammonia before extraction, according to claims 1-3 and 6-.
9. The method according to any one of item 9.