Chromatographic process for obtaining highly purified...

Details Chromatographic process for obtaining highly purified... Chromatographic process for obtaining highly purified...

1997-03-21

2001-10-23

Therkorn, Ernest G. (Department: 1723)

Liquid purification or separation

Processes

Liquid/liquid solvent or colloidal extraction or diffusing...

C210S659000, C530S317000, C530S321000, C530S413000, C530S417000

Reexamination Certificate

active

06306306

ABSTRACT:

BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates to a novel process for the chromatographic purification of cyclosporin A (Cy A) and related cyclosporins, which is suitable for use in the pharmaceutical industry.
In this process, the active compounds are obtained in a pharmaceutically acceptable purity under economically favourable conditions, i.e. in the case of cyclosporin A, for example, the purity requirements of PHARMEUROPA (PHARMEUROPA Vol. 4, No. 4, page 270, December 1992) are fulfilled.
2. Background Information
With the successful isolation of cyclosporin A from Trichoderma polysporum (LINK ex PERS.) Rifai by A. Rüegger and co-workers [Helv. Chim. Acta 59, 112, (1976)], for the first time a novel, strongly immunosuppressant class of substance was available. In the meantime, as a result of intensive investigations more than 25 related cyclosporins having immunosuppressant and antifungal activity became known [R. Traber et al. Helv. Chim. Acta 70, 13 (1987)]. Today, the outstanding importance of cyclosporin A as the agent of choice in the suppression of the immune defence after organ transplantations is undisputed. As a result of this, in the past there has been no lack of efforts to fulfil the continuously increasing requirements with respect to amount and quality for this lifesaving pharmaceutical by improvement of the preparation process.
The technical solutions known until now for the purification of cyclosporin A-containing crude extracts mostly comprise a plurality of chromatographic stages using organic solvents as eluents.
Thus in the abovementioned study of A. Rüegger, chromatography was first carried out on silica gel 60 Merck (0.063-0.2 mm) with chloroform in increasing amounts of methanol. The product obtained was subsequently subjected to gel chromatography on Sephadex LH 20 in methanol and then finally chromatographed on alumina (Brockmann, Act. I) in toluene with increasing amounts of ethyl acetate.
Later studies also make use of a similar procedure (Table 1). The absorbents Sephadex LH 20, silica gel 60 Merck (0.063-0.2 mm) and neutral alumina are frequently used.
The eluents employed are mostly mixtures of organic solvents.
On account of their high toxicity, chloroform and methylene chloride are unsuitable here, both with respect to solvent residues remaining in the active compound and also because of the safety problems resulting therefrom in the processing of relatively large amounts of these substances (distillation, disposal).
Furthermore, when using gradients or complicated, for example ternary, isocratic mixtures a new adjustment of the eluents after distillation of the eluates is troublesome and expensive.
A method described by BIOGAL in Canadian Patent CA 2 096 892, in which the crude extract is subjected to temperature treatment before chromatography, is also to be assessed similarly.
The crude product is heated at about 110° C. here for about 1 hour and subsequently cooled to room temperature over the course of 5 hours under defined conditions. Using a high proportion of chlorinated hydrocarbons, about 15% of the applied amount of cyclosporin A is isolated in a purity of about 97.6% in single-stage chromatography on silica gel 60 in two eluent systems employed in succession. These results, however, may not be satisfactory with respect to industrial use, as regards yield and purity of the active compound obtained. Additionally, experience shows that these complicated natural substances should not be exposed to any temperature stress because of their thermal instability and possible isomerization.
According to the present state of knowledge, only the applications of FUJISAWA (WO 9 213094) and BIOGAL (CA 2 096 892) manage with single-stage chromatographic purification. To do this, however, a complicated eluent combination or a gradient is necessary, which makes eluent regeneration more difficult.
For more accurate assessment of this method, however, details concerning yields and product purities achieved are mostly lacking.
Examples of some presently known chromatographic purification methods for cyclosporin A
Patent
Company
Purification steps
US 4 117 118
SANDOZ
1.
Sephadex LH 20, methanol
2.
Neutral alumina, toluene/
ethyl acetate, gradient
3.
Silica gel 60, chloroform/
methanol 98:2
US 4 215 199
SANDOZ
1.
Silica gel 60, chloroform/
methanol 98:2
2.
Sephadex LH 20, methanol
3.
Silica gel 60, chloroform/
methanol 98:2
BE 879 402
SANDOZ
1.
Sephadex LH 20, methanol
2.
Silica gel 60,
hexane/acetone 66:33
3.
Crystallisation, acetone, −15° C.
WO 9 213 094
FUJISAWA
1.
Silica gel, hexane
Hexane/ethyl acetate, gradient
Acetone
GB 2 227 489
BIOGAL
1.
Silica gel 60, chloroform/
methanol/acetone 92:4:4
2.
Silica gel 60, hexane/acetone,
gradient
CA 2 096 892
BIOGAL
1.
Silica gel 60, chloroform/
dichloromethane/ethanol 48:50:2
Chloroform/ethyl acetate/
ethanol 48:50:2
A brief description of the SMB technique per se is found, for example, in R. M. Nicoud, LC-GC INTL Vol. 5, No. 5, 43-47 and K. K. Unger (Ed.), Handbuch der HPLC [HPLC Handbook], Part 2, GIT Verlag, Darmstadt, 1994. (see
FIG. 1
)
SUMMARY OF THE INVENTION
Starting from the prior art outlined here, the following objectives result for an inventive solution to the chromatographic purification of cyclosporin A with high requirements for product purity and yield:
The new procedure should yield more than 70% of the amount of cyclosporin A employed with a quality corresponding to PHARMEUROPA (based on the chromatographic purification steps).
The procedure should meet the highest requirements with respect to annual throughput and at the same time drastically reduce the need for solvents and materials for the stationary phases.
The technical solution should be simple, rapid and robust, i.e. solvents and adsorbents must be reusable over a period which is as great as possible. Thus the use of solvent mixtures and gradients, used isocratically, which are difficult to adjust or regenerate is also inapplicable.
Chlorinated hydrocarbons should not be used.
The process should offer possibilities for automation, i.e. for continuous operation, and at the same time meet the requirements of production complying with GMP.
The starting material used for the chromatographic purification is, for example, a crude extract obtained according to known methods (e.g. DD 295 872 A5) from cyclosporin A-containing dried mycelium by extraction (ethyl acetate) and defatting (petroleum spirit/methanol/water), which besides a number of mostly unknown yellow- and red-coloured substances and oily products, for example, has the following composition of cyclosporins:
Unstandardized
Cyclosporins
relation %
1)
C
14.9
B
13.7
L
0.2
A
65.1
G
1.2
D
1.2
Other
3.7
1)
HPLC analytical determination according to PHARMEUROPA Vol. 4, No. 4, 270 ff.
In a first chromatography step, the polar cyclosporins (C, B, L, U) are separated from the non-polar cyclosporins (G, D) such that two useful fractions result, which besides cyclosporin A either contain only polar or only non-polar impurities. By this means, in the second chromatographic stage cyclosporin A can advantageously be purified from the respective impurities.
According to the invention, the ultrapurification of cyclosporin A is carried out by means of chromatographic purification by use of conventional HPLC in combination with the simulated moving bed technique (SMB) as follows:
1st chromatography HPLC or SMB technique
2nd chromatography SMB technique
See Schemes 1-4.
Put more precisely, the invention relates to a process for the purification of cyclosporin A and related cyclosporins from a cyclosporin-containing crude extract using chromatographic processes with silica gel as adsorbent, which is distinguished in that
a) in a first chromatographic stage, the crude extract is separated by fraction cuts in the separated concentration profile into a useful fraction 1 containing the non-polar concomitants and into a useful fraction 2 containing the more polar concomitants by means of preparative HPLC or SMB technique and
b) t

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