Purification process for cyclosporin

Details Purification process for cyclosporin Purification process for cyclosporin

2001-10-29

2003-09-16

Shah, Mukund J. (Department: 1624)

Liquid purification or separation

Processes

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

Reexamination Certificate

active

06620325

ABSTRACT:

The present invention relates to a purification process, and in particular to a process for purifying a product from impurities having relatively close distribution coefficients one with another.
Extraction processes are well-known for separating hydrocarbons in the petrochemical industry. It is known that distribution coefficients of the hydrocarbons between given phase systems in such applications differ substantially one from another.
Reviews of extraction methods are published, for example, in Kirk-Othmer Encyclopedia of Chemical Technology, 4th edition, Vol. 10, p. 125-180, pub. John Wiley & Sons 1993, and in The Handbook of Solvent Extraction, ed. Lo, Baird & Hanson, pub. Krieger 1991. The contents of these publications are incorporated herein by reference.
Presently large-scale purification of products of biotechnological processes is generally carried out by extraction or adsorption chromatography. Such products include peptides, macrolides and proteins which are generally produced as mixtures of products having closely-related structural and/or physical properties. Fractional extraction processes known in the petrochemical industry have hitherto not been applied in the biotechnology industry on a large scale for purification of active agents, e.g. peptides, having closely-related physical properties one with another. The present applicants consider a major problem to be that the respective distribution coefficients of biotechnological products and impurities between given phases are close one with another.
Accordingly, in one aspect, the present invention provides a process for purifying a product from a feedstock containing one or more impurities having closely-related physical properties to the product, which process comprises
feeding the feedstock into an extraction column under conditions adapted for separating more- or less-polar impurities from the feedstock, wherein a lighter phase flows counter to a heavier phase, thereby forming an output in one phase containing the product containing less more- or less-polar impurities so that the output contains the product in a substantially purified form.
The feedstock for the above process may be provided by output from a chromatographic purification or other pre-purification step, e.g. decantation. This process may be conducted such that the output serves as input for a subsequent chromatographic purification, e.g. in a series arrangement.
In another aspect, this invention provides a process for purifying a product from a feedstock containing one or more impurities having closely-related physical properties to the product, which process comprises
a) feeding the feedstock into a first extraction column under conditions adapted for separating more- or less-polar impurities from the feedstock, wherein a lighter phase flows counter to a heavier phase, thereby forming a first output in one phase containing the product containing less more- or less-polar impurities, and
b) feeding the first output into a second extraction column under conditions adapted for separating less- or more-polar impurities respectively from the first output, wherein the lighter phase flows counter to the heavier phase, thereby forming in one phase a second output, so that the second output contains the product in a substantially purified form.
The feedstock may be prepared by known methods, for example by fermentation. When the product is produced in a fermentation broth, the broth may be filtered and mixed with a solvent from which the product may be precipitated in an impure state, e.g. containing about 15% to about 30% by weight impurities. Typically the fermentation broth may undergo several cleaning and work-up steps prior to use as feedstock in the process of this invention. Initial filtration(s) and precipitation(s) serve to remove, for example, natural dyes and easily separable impurities from the product, and may serve to enhance phase separation in the extraction steps.
As used herein, the term “phase” is understood to mean a system having at least one component. The phase may comprise a single solvent or a mixture of, for example 2, 3 or more solvents.
As used herein, the term “closely-related physical properties” is understood to mean that the product is difficult to separate from the one or more impurities. The closely-related physical properties may include, for example, the respective distribution coefficients of the product and one or more impurities between two phases. For a cyclopeptide, e.g. a cyclosporin, and in particular cyclosporin A, ratios of distribution coefficients, i.e. distribution coefficient of cyclosporin A/distribution coefficient of impurity, may be between about 3 and about 0.4, e.g. between about 1.5 and about 0.8. These ratios are known as selectivities.
As used herein, the term “large scale” as applied to purification plant, is understood to mean a plant having an output of about one or more tonnes of purified product per annum, e.g. 10 tonnes per annum, or more e.g. around 20 to around 40 tonnes.
As used herein, the term “impurity” is understood to mean an undesirable component.
The process of this invention may be applied to a wide variety of products, e.g. peptides, such as cyclosporins, for example Cyclosporin A and derivatives thereof, Cyclosporin D and derivatives thereof, or Cyclosporin G and derivatives thereof. An example of a Cyclosporin D derivative is, for example ([3′-desoxy-3′-oxo-MeBmt]
1
-[Val]
2
-Ciclosporin) as disclosed in EP 296122; or macrolides, for example rapamycins and derivatives thereof, and ascomycins and derivatives thereof, produced for example by fermentation. Examples of macrolides which may be purified using the process of this invention include rapamycin; 40-O-(2-hydroxy)ethyl rapamycin as described in PCT/EP93/02604; ascomycin; 33-epi-chloro-33-desoxyascomycin as described in EP 427680 in Example 66a; ascomycin derivatives disclosed e.g. in EP 569337 and in EP 626385, for example 5,6-dehydro-ascomycin as disclosed in EP 626385; or an ascomycin derivative known as FK506.
A method for producing Cyclosporin A is disclosed, for example, in Example 1 of British patent specification 1,491,509. Methods of preparing FK506 are described in EP 184162.
A variety of columns are known and available commercially. In one embodiment of this invention, a single column is used which is a countercurrent column adapted for mechanical agitation and/or stirring of the feedstock/phase system. In another embodiment of this invention, two or more columns are used, at least one of which is a countercurrent column adapted for mechanical agitation and/or stirring of the feedstock/phase system.
Preferably the column(s) includes mechanical agitation, e.g. rotary agitation or reciprocating plate. An extraction column adapted for mechanical agitation is available commercially from the Kuehni company, Switzerland.
In another aspect, this invention provides a countercurrent liquid-liquid extraction column adapted for rotary agitation having a sufficient number of trays or compartments to effect, in use, separation of a pharmaceutical from impurities.
In a further aspect, this invention provides the use of a countercurrent liquid-liquid extraction column adapted for rotary agitation for separating a pharmaceutical from impurities.
The column(s) may be adapted for temperature adjustment of the phases. A jacket may be provided, for example, to maintain a desired temperature within the column. When purifying, for example a cyclosporin, temperatures may be maintained within each column at between about 0 and about 100° C., preferably between about 20 and about 80° C., and more preferably between about 30 and about 60° C.
It may be advantageous to conduct the process using a first column in series arrangement with a second and a third column, wherein the second column is in parallel arrangement with the third column. The Applicants contemplate such a configuration when, for example, the plant is to be situated in a building having a covering of limited height.
The phases chosen for th

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