Title of Invention

PEPTIDE PURIFICATION

Abstract A process of purification of an otherwise pure nona- or decapeptide from residual organic solvent, comprising the following steps: - dissolving the nona- or decapeptide in a dissolution solvent mixture comprising water and at least one alcohol selected from methanol, ethanol, propanol, isopropanol; - adding the solution of the nona- or decapeptide in said solvent mixture to a vigorously stirred precipitation solvent mixture essentially consisting of one or several polar compounds selected from methyl acetate, ethyl acetate, methyl propionate, ethyl propionate, butyl acetate, isobutyl acetate, t-butyl acetate, ethyl formate, propyl formate, isopropyl formate, and one or several non-polar compounds selected from hexane, heptane, octane, cyclohexane, methylcyclohexane, and, optionally, of up to 5% of acetic or propionic acid; - isolating the precipitated nona- or decapeptide; - washing the isolated nona- or decapeptide with one or a mixture of said polar compounds or a solvent or solvent mixture of similar polarity, - drying the washed nona- or decapeptide, with the proviso that the water content of said solvent mixture comprising water and at least one alcohol is below 8% (v/v), and that the volume ratio of the dissolution solvent mixture and the precipitation solvent mixture is 1:10 or more.
Full Text FORM - 2 THE PATENTS ACT, 1970
(39 of 1970)
COMPLETE
Specification
(Section 10; rule 13)
PEPTIDE PURIFICATION

POLYPEPTIDE LABORATOIES A/S
of Herredsvejen 2, DK-3400 HiUerod, Denmark, a Danish Company

361/MUMNP/2004
28.06.2004

THE FOLLOWING SPECIFICATION PARTICULARLY DESCRIBES THE NATURE OF THIS INVENTION AND THE MANNER IN WHICH IT IS TO BE PERFORMED:-


ORIGINAL
361/MUMNP/2004

GRANTED
24-8-2004

PEPTIDE PURIFICATION
FIELD OF THE INVENTION
The present invention relates to the purification of peptides, in particular intermediate-size peptides, more particularly nona- and decapeptides, such as LHRH-antagonists.
BACKGROUND OF THE INVENTION
Most intermediate size natural and synthetic peptides are amorphous substances. Many of them have pharmacologically interesting properties, such as many nona- and decapeptides which are LHRH (luteinizing hormone-releasing hormone) antagonists. One particular substance of this kind known only in amorphous form is the synthetic decapeptide of the formula (I)
Ac-D-2Nal-D-4ClPhe-D-3Pal-Ser-MeTyr-D-Asn-Leu-Lys(iPr)-Pro-D-Ala-NH2 (I)
which, being a potent LHRH antagonist, has desirable pharmacological properties.
For use in pharmaceutical preparations it is necessary for the LHRH antagonist (I) and nona- and decapeptides of similar structure to be essentially pure. The raw product obtained in the last step of a multiple-step synthesis is purified by chromatographic and other methods. To eliminate residual solvent from the chromatography a thus purified product usually has to be dissolved in an aqueous medium and freeze-dried. This is a costly process producing a voluminous product which is not easy to handle.

A process of purification of an otherwise pure peptide from residual organic solvent by other means than freeze-drying thus is desirable.
OBJECTS OF THE INVENTION
It is an object of the present invention to provide a process of purification of an otherwise pure peptide of the aforementioned kind from residual organic solvent which avoids freeze-drying.
It is another object of the present invention to provide such an otherwise pure peptide which is essentially free from residual organic solvent and.is not in the form of a cryoprecipitate.
Further objects of the invention will become obvious from the following summary of the invention, the description of a preferred embodiment thereof, and the appended claims.
SUMMARY OF THE INVENTION
According to the present invention is provided a process of purification of an otherwise pure peptide, in particular a nona- or decapeptide, most particularly a nona- or decapeptide which is an LHRH antagonist, from residual organic solvent, comprising the following steps:
- dissolving said otherwise pure peptide in a dissolution solvent mixture comprising water and at least one alcohol selected from methanol, ethanol, propanol, isopropanol;
- adding the solution of the otherwise pure peptide in said solvent mixture to a vigorously stirred precipitation solvent mixture essentially consisting of one or several polar compounds selected from methyl acetate, ethyl acetate, methyl

propionate, ethyl propionate, ethyl isopropionate, butyl acetate, isobutyl acetate, t-butyl acetate, ethyl formate, propyl formate, isopropyl formate, and one or several non-polar compounds selected from hexane, heptane, octane, cyclohexane, methylcyclohexane, and, optionally, of up to 5% of acetic or propionic acid;
- isolating the precipitated peptide;
- washing the isolated peptide with one or a mixture of said polar compounds or a solvent or solvent mixture of similar polarity,
- drying the washed peptide,
with the provisio that the water content of said solvent mixture comprising water and at least one alcohol is below 8% (v/v), and that the volume ratio of the dissolution solvent mixture and the precipitation solvent mixture is 1:10 or more.
"An otherwise pure peptide" is a peptide which is sufficiently
pure for use in a medicine except for volatile impurities
which need to be removed or the content of which needs to be
substantially reduced. The otherwise pure peptide will
normally be a substance having undergone purification by
chromatography. Preferably the otherwise pure peptide is
Ac-D-2Nal-D-4ClPhe-D-3Pal-Ser-MeTyr-D-Asn-Leu-Lys(iPr)-Pro-
D-Ala-NH2 (I).
According to a first preferred aspect of the invention the water content of the dissolution solvent mixture is below 5% (v/v) .
According to a second preferred aspect of the invention the volume ratio of the dissolution solvent mixture and the

precipitation solvent mixture is at least 15, in particular at least 20.
According to a third preferred aspect of the invention the alcohol of the dissolution solvent mixture is ethanol.
According to a fourth preferred aspect of the invention the polar component of the precipitation solvent mixture is ethyl acetate.
According to a fifth preferred aspect of the invention the non-polar component of the precipitation solvent mixture is heptane.
In the following the invention will be described in greater detail by reference to a preferred embodiment thereof which should not be understood to limit the invention.
DESCRIPTION OF A PREFERRED EMBODIMENT
Example 1. The fractions containing pure compound (1) (100 g in total) in ethanol-water-acetic acid 40:59:1 (v/v/v) obtained from preparative HPLC by which the synthetically obtained product had been purified were pooled and concentrated in vacuo to an oil which was co-evaporated twice from ethanol. The resulting solid was dissolved in 440 ml of ethanol and the resulting clear solution added over a period of 2 0 min to 8.8 L of ethyl acetate/heptane 1:1 (v/v). Stirring was continued for 1 hour and filtered. The amorphous product was washed with 3 L of ethyl acetate; it was found that this removed nearly all heptane. The washed product was dried in a vacuum oven at 4 0oC at 0.3 bar for 4 8 hrs. Elemental analysis of the dried product indicated that the monoacetate of (I) had be obtained. Cryoprecipitation, in

contrast, produces the corresponding diacetate. In the following Table analytical parameters of the monoacetate of (I) produced according to the invention are compared with those of a corresponding lyophilized product (diacetate).
Table. All percentages are by weight
lyophilized product precipitated product

Example 2. Variation of the composition of the precipitation solvent or solvent mixture; otherwise, procedure as in Example 1. Precipitation in pure ethyl acetate results in from about 3% to about 5% by weight loss of peptide. Precipitation in pure heptane results in a sticky product which is difficult to filter. A 1:1 (v/v) mixture of ethyl-acetate hexane gives a product which is easy to filter and dry; in repeated experiments the loss of peptide was always less than 0.5% by weight.
i
i
Example 3.Variation of water content of the solution of oily product in the dissolution solvent (absolute ethanol); otherwise, procedure as in Example 1. A water content of 10% (v/v) results in a sticky product on precipitation which is difficult to filter. A water content of 15% (v/v) results in an oily product on precipitation. To obtain satisfactory

results the water content must not exceed 8% (v/v) but should preferably be kept below 5% (v/v) . A water content..below 5% is accomplished by co-evaporating the oily product from the chromatography at least twice with ethanol.
Example 4. Variation in precipitation temperature; otherwise, procedure as in Example 1. The precipitation temperature proved to be not critical. It could be varied from 0°C to 20°C without noticeable differences in product yield and morphology.
Example 5. Variation of concentration of (I) in the dissolution solvent; otherwise, procedure as in Example 1. It was found that the concentration of the oily product from the chromatography which had been co-evaporated with ethanol in the dissolution solvent should be as high as possible. Even a concentration of 33 0 g by weight could be used.
Example 6. Variation of ratio between dissolution solvent and precipitation solvent.volumes and other variations; otherwise, procedure as in Example 1. The optimum ratio was found to be about 1:20. It could be shown that ratios from 1:15 to 1:30 gave satisfactory results. A ratio of 1:10 resulted in a sticky product. Precipitation is very fast. The suspension can be filtered 30 min after the last addition of dissolved substance. Washing with ethyl acetate did not result in loss of product but efficiently removed heptane.

We Claim:
1. A process of purification of an otherwise pure nona- or decapeptide from residual
organic solvent, comprising the following steps:
- dissolving the nona- or decapeptide in a dissolution solvent mixture comprising water and at least one alcohol selected from methanol, ethanol, propanol, isopropanol;
- adding the solution of the nona- or decapeptide in said solvent mixture to a vigorously stirred precipitation solvent mixture essentially consisting of one or several polar compounds selected from methyl acetate, ethyl acetate, methyl propionate, ethyl propionate, butyl acetate, isobutyl acetate, t-butyl acetate, ethyl formate, propyl formate, isopropyl formate, and one or several non-polar compounds selected from hexane, heptane, octane, cyclohexane, methylcyclohexane, and, optionally, of up to 5% of acetic or propionic acid;
- isolating the precipitated nona- or decapeptide;
- washing the isolated nona- or decapeptide with one or a mixture of said polar compounds or a solvent or solvent mixture of similar polarity,
- drying the washed nona- or decapeptide,
with the proviso that the water content of said solvent mixture comprising water and at least one alcohol is below 8% (v/v), and that the volume ratio of the dissolution solvent mixture and the precipitation solvent mixture is 1:10 or more.
2. The process as claimed in claim 1, wherein the water content of said solvent mixture comprising water and at least one alcohol is below 5% (v/v).
3. The process as claimed in claim 1, wherein said nona- or decapeptide is an LHRH antagonist.
4. The process as claimed in claim 3, wherein said nona- or decapeptide is Ac-D-2Nal-D-4ClPhe-D-3Pal-Ser-MeTyr-D-Asn-Leu-Lys(iPr)-Pro-D-Ala-NH2(I).
5. The process as claimed in claim 4, wherein Ac-D-2Nal-D-4ClPhe-D-3PaI-Ser-
MeTyr-D-Asn-Leu-Lys(iPr)-Pro-D-Ala-NH2 (I) is obtained in form of the
monoacetate.

6. The process as claimed in claim 1, wherein the volume ratio of the dissolution solvent mixture and the precipitation solvent mixture is at least 15.
7. The process as claimed in claim 1, wherein the alcohol of the dissolution solvent mixture is ethanol.
8. The process as claimed in claim 1, wherein the polar component of the precipitation solvent mixture is ethyl acetate.
9. The process as claimed in claim 1, wherein the non-polar component of the
precipitation solvent mixture is heptane.

Dated this 28th day of Jun
MOHAN DEWAN
OF R.K.DEWAN & COMPANY
APPLICANTS' PATENT ATTORNEY

Documents:

361-mumnp-2004-cancelled page(24-8-2004).pdf

361-mumnp-2004-claim(granted)-(24-8-2004).pdf

361-mumnp-2004-claims(granted)-(24-8-2004).doc

361-mumnp-2004-correspondence(24-8-2006).pdf

361-mumnp-2004-correspondence(ipo)-(21-9-2006).pdf

361-mumnp-2004-form 18(4-5-2005).pdf

361-mumnp-2004-form 1a-(28-6-2004).pdf

361-mumnp-2004-form 2(granted)-(24-8-2004).doc

361-mumnp-2004-form 2(granted)-(24-8-2004).pdf

361-mumnp-2004-form 26(28-6-2004).pdf

361-mumnp-2004-form 3(28-6-2004).pdf

361-mumnp-2004-form 4(29-3-2006).pdf

361-mumnp-2004-form 5(28-6-2004).pdf

361-mumnp-2004-form-pct-ipea-409(28-6-2004).pdf

361-mumnp-2004-form-pct-isa-210(28-6-2004).pdf

361-mumnp-2004-petition under rule 138(11-2-2005).pdf

361-mumnp-2004-petition under rule 138(24-12-2004).pdf


Patent Number 202753
Indian Patent Application Number 361/MUMNP/2004
PG Journal Number 42/2008
Publication Date 17-Oct-2008
Grant Date 19-Sep-2006
Date of Filing 28-Jun-2004
Name of Patentee POLYPEPTIDE LABORATORIES A/S
Applicant Address HERREDSVEJEN 2, DK-3400 HILLEROD, DENMARK,
Inventors:
# Inventor's Name Inventor's Address
1 RASMUSSEN, JON, H. BUDDINGEVEJ 69, DK-2800 LYNGBY,
2 RAMSMUSSEN, PALLE, H. ALDERSHVILEVEJ 121 B, 1TH, DK-2880 BAGSVAERD,
PCT International Classification Number N/A
PCT International Application Number PCT/IBO2/05581
PCT International Filing date 2002-12-23
PCT Conventions:
# PCT Application Number Date of Convention Priority Country
1 0104462-7 2001-12-29 Sweden