Chemistry: natural resins or derivatives; peptides or proteins; – Peptides of 3 to 100 amino acid residues – Separation or purification
Reexamination Certificate
1999-03-26
2001-07-10
Low, Christopher S. F. (Department: 1653)
Chemistry: natural resins or derivatives; peptides or proteins;
Peptides of 3 to 100 amino acid residues
Separation or purification
C530S345000, C530S412000, C530S416000, C530S417000
Reexamination Certificate
active
06258933
ABSTRACT:
CROSS-REFERENCE TO RELATED APPLICATION
This application claims priority from German Application No. 19813849.0, filed on Mar. 27, 1998, the complete disclosure of which is incorporated herein by reference.
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates to a one-stage process for the resalting and purification of oligopeptides.
2. Background Information
Oligopeptides frequently display biological activity and are therefore used as therapeutic agents. LHRH agonists and antagonists may be mentioned by way of example, which are used, inter alia, to treat certain types of cancer.
The oligopeptides to be purified may be prepared according to processes known in the prior art. Suitable processes include, among others, the Merrifield peptide synthesis on solid support materials or the conventional synthesis in solution. Both in the Merrifield solid phase synthesis and in synthesis in solution it is essential to provide certain regions in the molecule with protective groups that are split off at the end of the preparation. In the solid phase synthesis it is moreover necessary to remove the oligopeptide from the solid support. For further details of the synthesis of peptides reference may be made to the relevant literature (Houben-Weyl, Methoden der organischen Chemie, Vol. 15/1 and 15/2; M. Bodanszky, Principles of Peptide Synthesis, Springer Verlag 1984).
If the peptide to be prepared is a pharmaceutical, then it is often desirable for the oligopeptide to be present in the form of its acetate salt in order not to have to give the patient any foreign or other potentially harmful substances in conjunction with the administration of the drug.
It is often the case, however, that the oligopeptide, owing to circumstances connected with the synthesis, does not necessarily exist in the form of the acetate salt, either because acids other than acetic acid have to be used for the final cleavage of the protective groups, or because the free form of the peptide cannot be prepared or can be prepared only with difficulty and it is not possible to perform a simple conversion to the acetate by means of acetic acid. In order to cleave the protective groups or to cleave the peptide from the resin required for the synthesis, recourse generally has to be made to relatively strong acids such as trifluoroacetic acid, hydrochloric acid or hydrobromic acid. For further details of these cleavage processes reference may be made once again to the standard textbooks (Houben-Weyl, Methoden der organischen Chemie, Band 15/1 and 15/2; M. Bodanszky, Principles of Peptide Synthesis, Springer Verlag 1984).
In order to prepare the required acetate of the relevant oligopeptide for use in animals or humans, one is required in the aforementioned cases to resalt the oligopeptide.
The oligopeptide to be tested as active substance, or that is already available commercially as a therapeutic agent, must satisfy particular requirements as regards its purity. Because of the lack of a suitable conventional purification method, the product mixture formed in the synthesis is generally purified by means of chromatography, in particular high pressure liquid chromatography. For this purpose the oligopeptide must be taken up in a solvent, preferably in the solvent mixture of the mobile solvent chosen as eluent, before it is applied to the column.
For oligopeptides several processes have previously been described in the literature that relate to their resalting and purification. According to Gabriel (Int. J. Peptide Protein Res. 1987, 30, 40-43) the oligopeptide GRF (1-44) —NH
2
can be converted from its trifluoroacetate into the acetate by means of high pressure liquid chromatography using pyridine-containing and acetic acid-containing solvents. With regard to the pyridine residues that inevitably remain in the oligopeptide after such a procedure, there is concern, of course, about the toxicological properties of this substance. Also, a purification process that involves relatively large amounts of dangerous pyridine is undesirable from industrial safety aspects.
Hoeger et al. (Biochromatography 1987, 2, 134-142) have attempted to resalt and purify GnRH peptides while avoiding the use of a pyridine-containing solvent system. Starting from the fluoride salts, a two-stage reversed phase gradient chromatography was carried out in triethylammonium phosphate (TEAP) and trifluoroacetate (TFA) buffers with acetonitrile as modifier. Lyophilisation of the purified peptide fractions was followed by conversion to the acetate salts via anion exchange chromatography with dilute acetic acid or reversed phase chromatography in an ammonium acetate/acetonitrile gradient.
EP 0145258 describes, inter alia, the purification of HF salts of nonapeptides and decapeptides of the group of LHRH agonists. Here, too, the resalting takes place separately from the purification step, first via anion exchange chromatography followed by final purification on an octadecyl-silanised silicic gel phase by means of an eluent consisting of ammonium acetate and acetonitrile under high pressure conditions.
SUMMARY OF THE INVENTION
The object of the present invention is to provide a further process for the resalting and purification of oligopeptides that combines these two operational steps in one step and avoids the use of pyridine.
This and other objects, which are not identified more precisely but which however are obvious from the prior art to the person skilled in the art, are the subject of the characterising part of claim 1. Prefered modifications of the process according to the invention are the subject of the subclaims dependent on claim 1.
By purifying the oligopeptide, in the form of its hydrochloride salt, that is to be resalted and purified, via liquid chromatography by means of an acetate-containing solvent, practically chloride-free purified oligopeptide acetates are obtained in an extremely simple but nevertheless advantageous manner. It is thus possible with the process according to the invention to combine the effective purification and resalting of the oligopeptides in question, which hitherto could only be achieved in two stages or by using toxicologically harmful pyridine, in one single operational step without the addition of pyridine. The product fractions obtained by the present process are advantageously combined and dried by lyophilisation. The acetate is obtained in a yield of ca. 85% from the chloride. The pure oligopeptide acetate that is thus obtained in a percentage of up to 99.5% can after appropriate formulation be used as an active agent for medical treatment and therapy.
For the present process the oligopeptides must be used in the form of their chlorides. The simplest way to achieve this is to use hydrochloric acid for the protective group cleavage. On the other hand, the peptide hydrolysis and other secondary reactions occurring at side-chain groups as a result of the acid strength of the cleavage agent constitute undesirable competition reactions. For these and other reasons (for example the solubility of the peptide in TFA) weaker anhydrous acids such as trifluoroacetic acid or anhydrous strong acid mixtures such as HBr/acetic acid are often used for such protective group cleavages.
It has now completely surprisingly but nevertheless advantageously been found that protected oligopeptides can also be de-protected with concentrated aqueous hydrochloric acid and that the salts of the oligopeptides formed therefrom contain a significantly lower proportion of by-products compared to the more conventional cleavage with the less strong, anhydrous trifluoroacetic acid, possibly mixed with organic solvents, or with the HBr/acetic acid system. This was neither obvious nor forseeable.
The aforedescribed cleavage of the protective groups from the oligopeptide is preferably carried out in a temperature range from −25° C. to 30° C., more preferably from −10° to 10° C., and even more preferably from 0° to 5° C.
The chloride salt of the oligopeptide may be used in the form of its concentrated hydrochloric
Drauz Karlheinz
Gunther Kurt
Kunz Franz-Rudolf
Muller Thomas
Degussa-Huls Aktiengesellschaft
Low Christopher S. F.
Pillsbury Madison & Sutro LLP
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