Liquid purification or separation – Processes – Chromatography
Patent
1997-11-12
1999-11-02
Walker, W. L.
Liquid purification or separation
Processes
Chromatography
530317, 435 711, B01D 1508, A61K 3812
Patent
active
059763817
DESCRIPTION:
BRIEF SUMMARY
BACKGROUND AND SUMMARY OF THE INVENTION
The present invention relates to a process for the preparation of pure cyclosporin chromatographically from a mixture of different cyclosporins and other substances prepared by fermentation, using an eluent consisting essentially of high pressure carbon dioxide.
Cyclosporins are neutral, highly lipophilic, cyclic undekapeptides with a variable amino acid composition. At present, 25 different cyclosporin forms (A-Z) are known, from which the A-form has proved to be clinically the most valuable. Cyclosporins are produced by some fungi, e.g. Cylindrocarpon lucidum, Trichoderma polysporum, and various species of the genus Tolypocladium. Cyclosporins have been discovered to have not only of antibiotic activity, but also immunosuppressive properties, and they are currently used in the post-operative treatment of transplantation operations to prevent the rejection of the transplants. Cyclosporins are produced by fermenting fungal strains which produce great amounts of them. When mycclial extracts are obtained they usually contain different cyclosporin forms as a mixture. The desired form of cyclosporin has to be separated from the mixture obtained by fermentation by a purification process.
In the process currently in use, the separation of cyclosporin A is carried out by extracting the mycelial mass obtained by the fermentation first with methanol, whereafter the final separation is carried out in silica columns using different mixtures of organic solvents. The separation method is fairly slow and demands large chromatography columns with respect to the production volumes. Additionally, in a conventional process, great amounts of organic solvents must be handled, the purification and recycling of which is uneconomic and requires large equipment.
It is known that a substance which is in liquid form or in a supercritical state dissolves many compounds substantially better than the same substance in a gaseous state. Supercritical chromatography is indeed used to an ever-increasing amount to extract and separate various substances, especially in analytical applications (White et al., 1988, J. High Resolut. Chrom. & Chrom. Comm. 11:94-98).
In the U.S. Pat. No. 4,478,720, a process for fractionating of mixtures, and especially or purification of hydrocarbon mixtures by elution chromatography is disclosed. In the process, supercritical carbon dioxide without adjuvants is used as the eluent. In the international patent application WO 93/23394 optically pure S-timolol is produced from racemic R,S-timolol. Although the isomeric separation described in said application is in principle of the same kind as the separation disclosed in the present invention, the two processes differ from each other even for their grounds in that in the enantiomer separation of timolol is a question of chiral selectivity between the timolol enantiomers and the chiral column filling, whereas the present invention is based on the different polar interactions between the cyclosporins to be separated and the non-chiral column filling.
We have now found that the desired cyclosporin form can easily be obtained in pure form from a mixture of cyclosporin forms and other substances prepared by fermentation by feeding the mixture into a chromatography column with an eluent consisting of high pressure carbon dioxide and an adjuvant, and recovering from the eluent flow, the fraction which contains the desired form of cyclosporin in adequately pure form.
By the process of this invention any desired form of cyclosporin included in the starting mixture can be prepared in pure form. Preferably cyclosporin A or cyclosporin G is prepared.
The purity of cyclosporin meeting the drug requirements has to be at least 98.5 weight-%. By optimizing the process parameters cyclosporin meeting the drug requirements is directly obtained according to the process of the present invention from a certain part of the eluent flow.
An advantage of the process is in that the separation of the cyclosporin forms can be carried out continuously
REFERENCES:
patent: 4478720 (1984-10-01), Perrut
patent: 4581166 (1986-04-01), Peter et al.
patent: 5256547 (1993-10-01), Rudat et al.
White, C.M., et al., Analysis of Pharmaceuticals and Other Solutes of Biochemical Importance . . . , Journal of High Resolution Chromatography & Chromatography Communications, vol. 11, Jan. 1988, pp. 94-98.
Wong et al. "Supercritical Fluid Chromatography for Therapeutic Drup Monitoring of Immunosuppressants: Selectivity for Cyclosporing A, FK 506 (Tacrolimus), and Rapamycin". Journal of Liquid Chromatography, 17(10), pp. 2093-2109, 1994.
Aaltonen Olli
Alkio Martti
Hase Anneli
Lundell Juhani
Ruohonen Jarkko
Santen Oy
Walker W. L.
Ward Richard W.
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