Liquid purification or separation – Processes – Chromatography
Reexamination Certificate
2002-05-15
2004-03-16
Therkorn, Ernest Q. (Department: 1723)
Liquid purification or separation
Processes
Chromatography
C210S198200, C530S317000, C530S321000, C530S413000, C530S417000
Reexamination Certificate
active
06706192
ABSTRACT:
The object of the invention is a process for the chromatographic purification of cyclosporin A from a crude product containing cyclosporin complex by using a normal phase—column filled with silica gel or an equivalent matrix and by the application of multistep chromatography with a column filled with normal phase silica gel or an equivalent matrix and by a solvent or solvent mixture containing toluene as the major component.
Cyclosporins are cyclic undecapeptides being N-methylated at several places and most of them having approved pharmacological effects. Twenty five members of this group of compounds have been known so far which are designated by letters A to Z. At first cyclosporin A was separated among them, which is a natural material and was isolated from the culture broth of Tolypocladium inflatum Gams strain (Helv. Chim. Acta 59 1075(1976)). This compound first became known as a slight antifungal antibiotic and later attracted the attention as an immunosuppressive. (J. F. Borel et al: Immunology 32 1017(1977)). Cyclosporin A is mainly used in organ transplantation from other persons (lung, heart, kidney, bone marrow, skin). Pharmacological examinations proved that it inhibits both the humoral and cellular immune responses by hindering the proliferation of T-cells and interrupting the synthesis of interleukin-2. Cyclosporin A also has been used for treating autoimmune and inflammatory diseases, such as in autoimmune hematological diseases, ulcerous colitis, Graves-illness, multiple sclerosis, psoriasis, rheumatic arthritis as well. Further experiments were done to cure infections caused by protozoa and tumors. The importance of cyclosporins is indicated by the fact, that number of synthetic related compounds call be prepared by building in different amino acids and substitutes. (e.g. EP 56782, CH 630062 and EP 29122 patents)
Cyclosporins are prepared by fermentation. Cylindrocarpon Lucidum Booth (patent specification No. CH 589,716); Trichoderma polysporum Rifai (patent specification No. CH 603,790); Tolypocladium varium (patent specification No. HU 201,577) are used to prepare cyclosporins. At the end of the fermentation, a cyclosporin complex is formed which may contain other impurities too (ingredients of culture media, anti-foaming agent, metabolites, etc.) depending on the character of the process.
Generally the product is isolated from the broth by extraction processes. This can be done by separating the mycelium from the broth by centrifugation or filtration, then dissolving the active ingredient from the mycelium by methanol or acetone and extracting the filtrate by water-immiscible solvents. An other known execution method is a process without filtration, using whole broth extraction with water-immiscible organic solvent. Solvent content of the organic phase is evaporated by vacuum distillation. However during the organic solvent extraction the compounds having lipid characters are also transferred into the organic phase, which cause difficulties in the further purification. In order to separate these compounds there are known processes (e.g. Swiss patent specification No. 589,716 or published German patent application No. 2455859) where after removing the extracting solvent the residue is dissolved in a mixture of methanol-water and then is extracted several times by the same volume of petroleum ether. Petroleum ether portions are combined and cyclosporins are recovered from them by a mixture of methanol-water. The active substance is transferred by multi-extraction from the combined methanol-water phase into ethylene chloride, which is then washed with water and evaporated to dryness. The crude product prepared by the above method and containing cyclosporins can be purified more efficiently by one of the chromatographic methods.
According to a method described in U.S. Pat. No. 4,117,118 the cyclosporin mixture is transferred first to a Sephadex LH-20 column and eluted with methanol, then it is eluted successively in an alumina column with a mixture of toluene and ethyl acetate (15%), and in a silica gel column with a mixture of chloroform and methanol (2%). Despite of the repeated chromatography the resulting product is not pure, but it is a mixture of cyclosporin A and B.
A similar chromatographic process is disclosed among others in the U.S. Pat. No. 4,215,199, where a rough-cleaning is with a 98:2% v/v mixture of chloroform and methanol on a silica gel column. The eluate is then evaporated to dryness. The residue is dissolved in methanol and is subjected to chromatography in a Sephadex LH-20 column using methanol as eluent. The eluate fractions are evaporated to dryness, then the residue is dissolved in 98:2% v/v mixture of chloroform and methanol. It is subjected again to a silica gel chromatography. Cyclosporin A appears first in the eluate. This and the consecutive fractions are separated, and the pure components are obtained by evaporating the elutes.
According to the German Pat. No. DD 298,276 the oily crude product is dissolved in small quantity of chloroform, then subjected to chromatography in an alumina column with chloroform. The fractions containing cyclosporin A are evaporated in vacuum, dissolved in chloroform, subjected to a similar column and then eluted by chloroform. Fractions containing the active substance are evaporated in vacuum again. Hexane is added to the residue and the cyclosporin A is crystallized. Product is washed by hexane then dried and finally recrystallized from a mixture of ether and hexane or acetone.
According to the Hungarian Pat. No. 201,577 the crude product obtained after evaporation can be cleaned on a silica gel column by elution with a mixture of chloroform and methanol gradually increasing concentration of methanol. The process is started by pure chloroform and continued by increasing in 0.5 vol % steps the methanol in the eluate. Cyclosporin A is eluted by 2 vol %, cyclosporin B by 2.5 vol %, cyclosporin C by 3 vol % methanol containing chloroform from the column. Components are obtained by evaporating the fractions.
Processes mentioned above describe mainly cyclosporin fermentation procedures, where the first aim of the purification steps is to identify the product obtained. Thus the product is isolated only in a small quantity and only the physical and chemical characteristics are given without publishing data relating to the purity of the product and the quantities of the impurities.
Rüieger and Co. /Helv.Chim. Acta 59(4), p. 1075-92 (1976) isolated small quantities of pure cyclosporin A and C by repeated chromatographies and by other purification steps for identification and structure analysis. According to this article the crude product received from the fermentation of Trichoderma polysporum Rifai containing mainly cyclosporins A and C is defatted with methanol and petroleum ether. After evaporation the residue is dissolved in chloroform and is subjected to chromatography by gradient elution with 98.5:1.5 v/v mixture of chloroform and methanol as eluent. Pure crystalline cyclosporin A is obtained by further chromatography. The fraction containing cyclosporin A is dissolved in methanol and is subjected to chromatography in a Sephadex LH-20 column by using methanol as eluent. Peak fractions are evaporated, dissolved in toluene and subjected to chromatography in a column packed with aluminum oxide, using toluene as eluent, in the presence of an increasing, concentration of acetic acid. The crystalline product is obtained after the evaporation of fractions and a treatment with activated carbon in all alcoholic solution.
A purification process feasible in industrial scale is described in the U.S. Pat. No. 5,382,655. According to the process the crude product containing different cyclosporin components is subjected to heat treatment prior to chromatography on silica gel column by the mixtures of chloroform-dichloromethane-ethanol or chloroform-ethyl acetate-ethanol. The product obtained is subjected to further chromatography and recrystallization, which resulted in a pure quality of product good for injection p
Deák Lajos
Keri Vilmos
Makó Györgyné
Miskolczy Istvánné
Nagyné Ärvai
Biogal Gyogyszergyar Rt.
Kenyon & Kenyon
Therkorn Ernest Q.
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