Description:FORM 2

THE PATENTS ACT, 1970
(39 of 1970)
&
The Patents Rules, 2003

COMPLETE SPECIFICATION
(See section 10 and rule 13)

METHOD FOR PURIFICATION OF LEUPRORELIN OR PHARMACEUTICALLY ACCEPTABLE SALTS THEREOF

PIRAMAL PHARMA LIMITED, a company incorporated under the Companies Act 2013, of Ground floor, Piramal Ananta, Agastya Corporate Park, Kamani Junction, LBS Marg,Kurla West, Mumbai, Maharashtra – 400070, India.

The following specification particularly describes the invention and the manner in which it is to be performed.

FIELD OF THE INVENTION:

This invention relates to a process for purification and isolation of leuprorelin (compound of formula (I)) or pharmaceutically acceptable salt(s) thereof;

Formula (I)
the said process comprising the steps of analytical purity confirmation by SFC, and further purification and isolation of leuprorelin or salt(s) thereof, to provide purity of leuprorelin or salt(s) thereof more than about 98% by preparative Supercritical Fluid Chromatography (SFC).

BACKGROUND OF THE INVENTION:
Leuprorelin belongs to a gonadotrophin-releasing hormone (GnRH) analogue used to treat a wide range of hormone-related disorders including advanced prostatic cancer, endometriosis and precocious puberty.

There are several processes available for purification of leuprorelin or salt(s) thereof either by HPLC or preparative HPLC but still a substantial need exists to develop an efficient and streamlined process or method to provide highly pure leuprorelin or salt(s) thereof. The inventors of the present invention have surprisingly found that the preparative SFC is an extremely useful technique for separation and purification of peptides like leuprorelin or salt(s) thereof.

Chinese patent publication CN101597325A relates to a method of purifying leuprorelin belonging to the HPLC technical field, and especially relates to a method for mass-producing and purifying leuprorelin.

Journal of Chromatography A, volume 1642 (2021), by Gioacchino Luca Losacco et. al. discloses “screening of different column chemistries available for UHPSFC analysis was performed, in combination with additives of either basic or acidic nature. The combination of an acidic additive (13 mM TFA) with a basic stationary phase (Torus DEA and 2-PIC) was found to be the best for a series of six synthetic peptides possessing either acidic, neutral or basic isoelectric points. Secondly, methanesulfonic acid (MSA) was evaluated as a potential replacement for TFA. Due to its stronger acidity, MSA gave better performance than TFA at the same concentration level”. Further it discloses a systematic comparison between UHPSFC and UHPLC for analysis of peptides.

“Chromatography of pharmaceutical peptides contrasting SFC and HPLC” Uppsala Universitet, 2019 by Joakim Bagge, discloses a comparison of a well-established and a novel, "green" and efficient technique to separate peptides of pharmaceutical interest. The results showed that the solvents tert-butanol and methanol performed best between 20-30 and 50 volumetric percent water as additive in SFC and HPLC, respectively. These diluents were then used for the peptides within a set of chosen therapeutic peptides to evaluate the retention and selectivity in HPLC and SFC. The SFC method was able to separate Leuprolide and Triptorelin while HPLC was not. A comparison was also made in between the two stationary phases Kromasil CN and Kromasil XT, where a global selectivity was shown to be higher in case of Kromasil CN.
United State patent publication US20150166602A1 relates to a process for the preparation of leuprolide or pharmaceutical acceptable salt(s) thereof by a combination of solid phase synthesis and post assembly solution phase amidation. The invention also relates to applying a non-protected leuprolide precursor to prepare leuprolide or pharmaceutically acceptable salt(s) thereof.

The inventors of the present invention have surprisingly found that, use of analytical preparative SFC systems have advantages over the conventional chromatography systems, wherein the advantages include less separation time during the process, less solvent consumption and the said process provides high purity of leuprorelin or salt(s) thereof. Further, as compared to the conventional SFC techniques which uses strong acids as mobile phase, the instant invention employs cheaper and mildly acidic mobile phase, which makes the process economical and easy to handle, particularly on commercial scale.

SUMMARY OF THE INVENTION:

The present invention relates to an improved process for purification and isolation of peptides like leuprorelin or pharmaceutically acceptable salt(s) thereof by preparative SFC method, the said process comprises an analytical purity confirmation of crude peptide by SFC method and further, its purification and isolation by preparative SFC, wherein the said process provides more than 98% purity of leuprorelin or pharmaceutically acceptable salt(s) thereof. The present invention has advantages over the conventional chromatography systems, wherein the process provides high purity peptide, requires less separation time, and less solvent consumption. Also, this process is superior to the existing processes of purification either by high-performance liquid chromatography (HPLC) or preparative HPLC.

In one general aspect, the present invention provides a process for purification and isolation of peptides, comprising the steps of;
(i) confirming analytical purity of peptides by SFC,
(ii) purifying crude peptides of step (i) by preparative SFC,
(iii) isolating peptide of step (ii) from pure fraction, wherein the process comprises use of carbon dioxide as supercritical fluid.

In another general aspect, the present invention provides a process for purity confirmation of leuprorelin or pharmaceutically acceptable salt(s) thereof and further includes purification and isolation by analytical SFC method and preparative SFC method, wherein process comprises use of a supercritical fluid as a mobile phase.

In another general aspect, the present invention provides a process for purification and isolation of leuprorelin as compound of formula (I) or salt(s) thereof by preparative supercritical fluid chromatography, the said process comprises carbon dioxide as a supercritical fluid.

In another general aspect, the present invention provides a process for purification and isolation of peptides, wherein the peptide is leuprorelin or salt(s) thereof.

In a further aspect, the present invention provides a process for purification and isolation of peptides, wherein the peptide is leuprorelin or its acetate salt or its trifluoroacetate salt.

In another general aspect, the present invention provides a process for purification and isolation of peptides, comprising the steps of;
(i) confirming analytical purity of peptides by SFC using a supercritical fluid,
(ii) purifying crude peptides of step (i) by preparative SFC using a supercritical fluid,
(iii) isolating peptide of step (ii) from pure fraction,
(iv) distillation of the pure fraction obtained in step (iii),
(v) lyophilisation of the crude obtained in step (iv) and
(vi) storing the crude obtained in the step (v) as pure peptide at a temperature in the range of about -10°C to about -30°C to avoid any decomposition,
wherein the said peptide is leuprorelin or salt(s) thereof.

The present invention provides use of at least one supercritical fluid, wherein the supercritical fluid is carbon dioxide.

The details of one or more embodiments of the present invention are set forth in the description below.

BRIEF DESCRIPTION OF THE DRAWINGS
Fig. 1: Chromatogram of crude leuprorelin or salt(s) thereof for analytical purity confirmation
Fig. 2: Chromatogram of purified and isolated leuprorelin or salt(s) thereof by analytical purity confirmation using analytical SFC

Fig. 3: Chromatogram of crude leuprorelin or salt(s) thereof by preparative SFC

DETAILED DESCRIPTION OF THE INVENTION

The term “about” means within 30%, 25%, 20%, 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, or 0.05% of a given value or range.

The present invention provides a process for purification and isolation of leuprorelin as compound of formula (I),

Formula (I)
or salt(s) thereof.
The present invention also provides a process for the purification and isolation of leuprorelin or salt(s) thereof, wherein the said process comprises analytical purity confirmation by SFC, and the said process further provides purification and isolation of leuprorelin by preparative SFC. Accordingly, the said method provides more than about 98% of purity of leuprorelin or salt(s) thereof.

The synthetic methods used in laboratory employ trifluoroacetic acid during preparation of leuprorelin, which results in preparation of crude leuprolide trifluoroacetate. The crude leuprolide trifluoroacetate requires purification, which is conventionally carried out by reverse phase column chromatography. The process of reverse phase chromatography requires very long elution times, running into several hours using water or mixture of water and organic miscible phase like acetonitrile. The large volumes of water with eluted compound need lyophilisation in large number of batches to afford the pure leuprorelin trifluoroacetate. These problems with the conventional method is overcome using preparative SFC method. The eluting time is reasonably less (about 20 minutes) as compared to conventional reverse phase chromatography. Also the supercritical fluid is carbon dioxide and the compound eluted with mobile phase will have much lower content of water, which is removable by distillation and followed by lyophilisation. The efficiency of SFC method is higher than conventional reverse phase chromatography in terms of percentage purity and recovery rate, hence provides a solution to the problems posed by conventional reverse phase chromatography. The trifluoroacetate salt in a state of high purity can then be exchanged to phosphate salt and further to acetate salt on ion exchange columns; since the acetate salt is the approved commercial product.

In an embodiment, the process for purification and isolation of peptides, comprising the steps of;
(i) confirming analytical purity of crude peptides by SFC,
(ii) purifying crude peptides of step (i) by preparative SFC,
(iii) isolating peptide of step (ii) from pure fraction, wherein the process comprises use of supercritical fluid as a mobile phase.

In another embodiment the present invention provides a process for purification and isolation of leuprorelin as compound of formula (I) or salt(s) thereof by preparative supercritical fluid chromatography, the said process comprises use of carbon dioxide as a supercritical fluid.

In another embodiment, the present invention provides purity of leuprorelin or salt(s) thereof of more than about 98% by preparative supercritical fluid chromatography.

In another embodiment, the present invention provides purity of leuprorelin or salt(s) thereof of about 99.6% by preparative supercritical fluid chromatography, wherein the analytical purity of recovered compound (I) or salt(s) thereof is confirmed by supercritical fluid chromatography as provided in figure 2, wherein the supercritical fluid is carbon dioxide.

In another embodiment, the present invention provides purity of leuprorelin or salt(s) thereof of about 99.6% by preparative SFC, wherein the salts include trifluoroacetate salt of leuprorelin as compound of formula (II),

•

Formula (II)

In another embodiment, the present invention provides a process for purification and isolation of leuprorelin or salt(s) thereof, wherein the salt purified and isolated is trifluoroacetate (TFA) salt, which is optionally converted into acetate salt on ion exchange columns.

In another embodiment, the present invention provides a process for purification and isolation of peptides, comprising the steps of;
(i) confirming analytical purity of peptides by SFC,
(ii) purifying crude peptides of step (i) by preparative SFC,
(iii) isolating peptide of step (ii) from pure fraction,
(iv) distillation of the pure fraction obtained from step (iii),
(v) lyophilisation of the pure fraction obtained from step (iv), and
(vi) storing of the pure peptide obtained from step (v) at a temperature in the range of about -10°C to about -30°C to avoid any decomposition,
wherein the process comprises passing of crude compound (I) at a temperature in the range of about -10°C to about -30°C through a preparative SFC containing one or more mobile phases and at least one stationary phase, wherein the said mobile phase comprises carbon dioxide as a supercritical fluid.

In a further embodiment, the present invention provides a process for purification and isolation of leuprorelin or salt(s) thereof, comprising the steps of;
(i) confirming analytical purity of crude leuprorelin or salt(s) thereof by SFC using carbon dioxide as a supercritical fluid,
(ii) purifying crude leuprorelin or salt(s) thereof of step (i) by preparative SFC using carbon dioxide as a supercritical fluid,
(iii) isolating the pure fraction of leuprorelin or salt(s) thereof obtained from step (ii),
(iv) distillation of the pure fraction obtained from step (iii),
(v) lyophilisation of the pure fraction obtained from step (iv),
(vi) storing of the pure leuprorelin or salt(s) thereof obtained from step (v) at a temperature in the range of about -10°C to about -30°Cto avoid any decomposition, and
(vii) optionally, performing ion exchange chromatography to the pure leuprorelin or salt(s) thereof, obtained in step (vi) to convert to desired acetate salt.

Further, the invention provides process for analytical purity confirmation of crude leuprorelin or salt(s) thereof by SFC, comprising the steps of;
preparing mobile phase for SFC, and
preparing sample in mobile phase B (diluent) for SFC,
wherein the process comprises carbon dioxide as a supercritical fluid, and
confirming analytical purity of crude leuprorelin or salt(s) thereof by SFC.

In another embodiment, the invention provides storing of the pure leuprorelin or salt(s) thereof at a temperature in the range of about -10°C to -30°C, wherein the exemplary temperature is about -20°C.

In another embodiment, the present invention provides use of a stationary phase, wherein the analytical SFC stationary phase is 2-ethylpyridine 100 A? with a column length of 250 mm; and a diameter of 4.6 mm, with particle size of 5 µm.

In another embodiment, the present invention provides use of a stationary phase, wherein the preparative SFC stationary phase is 2-ethylpyridine 100 A? with a column length of 250 mm; and a diameter of 30 mm, with particle size of 5 µm.

Further, the invention provides use of one or more mobile phases, wherein at least one mobile phase comprises carbon dioxide, and the other mobile phase(s) optionally comprise glacial Acetic Acid, dichloromethane (DCM), methanol, water or a combination thereof. The flow rate for the mobile phase is about 3 mL/minute to about 6 mL/minute, preferably about 4 mL/minute.

The present invention provides use of one or more mobile phases, wherein the mobile phase A comprises CO2 and mobile phase B comprises one or more solvents selected from the acetic acid, dichloromethane, Milli-Q water or methanol or a combination thereof.

In further embodiment, the invention provides a process for distillation of the pure leuprorelin or salt(s) thereof, wherein the process comprising distillation of the pure fraction at about 38°C - 44°C till a light yellow liquid with trace amount of fraction of mobile phase B is obtained.

In further embodiment, the invention provides a process for lyophilisation of the pure leuprorelin or salt(s) thereof to obtain white to off-white solid crystals of leuprorelin or salt(s) thereof.

The present invention provides use of SFC Investigator, wherein crude leuprorelin or salt(s) thereof, is run through a SFC investigator, optionally SFC investigator with PDA detector, the said process or method comprises use of mobile phase A and mobile phase B with flow rate of about 4 mL/minute with column oven temperature at about 40°C and run time is about 5 minutes to about 30 minutes, preferably about 17 minutes and wavelength of about 220 nm.

In another embodiment, the present invention provides storage of pure leuprorelin or salt(s) thereof at a temperature of about -20°C to avoid any decomposition.

The present invention is further illustrated by the following example which is provided to be exemplary of the invention and do not limit the scope of the invention. While the present invention has been described in terms of its specific embodiments, certain modifications and equivalents will be apparent to those skilled in the art and are intended to be included within the scope of the present invention.

Example 1: Preparation of Mobile Phases.
The “mobile phase A” comprising CO2 and “mobile phase B” comprising one or more solvents selected from the acetic acid, dichloromethane, Milli-Q water or methanol. The preparation of mobile phase B comprising the steps of;
(i) Mixed half quantity HPLC gradient grade methanol with about 1mL glacial acetic acid; about 20 mL dichloromethane; about 50 mL Milli-Q water and made up to 1000 ml volume with the methanol,
(ii) Sonicated the mobile phase obtained in step (i) for about 2 minutes to about 10 minutes, wherein the process is performed on SFC Investigator with photodiode array (PDA) detector, with flow rate of about 4.0 mL per minute at temperature of about 40°C and run time of about 17 minutes. The sample was prepared with approximately 2000 ppm in the diluent or mobile phase B.

Example 2: Parameters for the SFC method are provided in the table 1.
Table 1 provides the chromatographic parameter for SFC investigator.
Sample Crude leuprorelin or salt(s) thereof
Instrument SFC Investigator with PDA Detector
Mobile Phase (A) Liquid CO2
Mobile Phase (B) 0.1% Glacial Acetic Acid
2% Dichloromethane
5% water
Methanol
Flow Rate 4.0 mL per minute
Column Oven Temperature 40°C
Column Princeton SFC 2-Ethylpyridine 100Å
(Column Length – 250 mm; Diameter – 4.6 mm, and particle size – 5 µm)
Run Time 17 minutes
Diluent Mobile Phase B
Sample Preparation Approximately 2000 ppm in diluent
Wavelength 220 nm
Backpressure 100 Bar
Gradient Time %A %B
0 70 30
12 50 50
17 50 50

The leuprorelin trifluoroacetate crude sample was prepared using 2 mg crude leuprorelin trifluoroacetate in the diluent which provided sample of 2000 ppm which is further sonicated.

The figure 1 provides analytical purity check of crude leuprorelin trifluoroacetate, which provided peak on the chromatogram with single absorbance at 220 nm and retention time of leuprorelin trifluoroacetate is about 6.23 minutes

The figure 2 provides analytical purity confirmation of purified and isolated leuprorelin trifluoroacetate by analytical SFC and provided peak on the chromatogram with single absorbance at 220 nm and retention time of leuprorelin is about 6.36 minutes.

Example 3: Parameters for the preparative SFC method are provided in the table 2.

Table 2 provides the chromatographic parameter for preparative SFC investigator.
Preparative Column Princeton SFC 2-Ethylpyridine 100 A?
(Column Length – 250 mm; Diameter – 30 mm, and particle size – 5 µm) and
(Part no:- 250300-03577)
Sample Crude leuprorelin or salt(s) thereof (sonicated)
Instrument Waters SFC 200 with waters 2489 UV/Visible Detector.
Mobile Phase (A) Liquid CO2
Mobile Phase (B) or Diluent 0.1 mL Glacial Acetic Acid
20 mL Dichloromethane
50 mL water (Mili-Q)
930 mL Methanol
Flow Rate 80g/min (gram/minute)
Isocratic Mode Line A: Line B
% of A Line 58%
% of B Line 42%
Column Oven Temperature 40°C
Run Time 17 minutes
Diluent Mobile Phase B
Sample Preparation Approximately 5000 ppm in diluent
Wavelength 220 nm
Backpressure 100 Bar

The figure 3 provides preparative SFC chromatogram of the crude leuprorelin trifluoroacetate with absorbance at 220 nm and retention time of the leuprorelin trifluoroacetate is about 8.57 minutes, whereas W2489 is the instrument code.

Example 4: Solvent evaporation and product isolation from pure fraction - distillation process and storage.
Post preparative SFC purification, transferred the pure fraction of leuprorelin trifluoroacetate in to 2 L (Liter) round bottom flask (RBF) and distilled the pure fraction via Bauchi Rotavapor at 40-42°C till light yellow liquid with trace amount of fraction of mobile phase–B is observed.
Transferred this light yellow liquid from 2 L round bottom flask to small 100 mL RBF and add 50-60mL water. Lyophilize the sample from 100 mL RBF. After lyophilisation, white to off-white solid of leuprorelin trifluoroacetate was obtained. The pure Leuprorelin trifluoroacetate sample obtained from distillation followed by lyophilisation process is stored at a temperature of about -20°C to avoid any decomposition.

The present invention thus provides SFC purity by area percentage of about 99.68% @ 220 nm, wherein the said method provides an actual recovery of leuprorelin trifluoroacetate of about 99.59 %.
, C , C , Claims:We Claim:

1. A process for purification and isolation of leuprorelin as compound of formula (I)

(I)
or pharmaceutically acceptable salt(s) thereof by preparative supercritical fluid chromatography (SFC), wherein carbon dioxide is used as a supercritical fluid.

2. The process as claimed in claim 1, comprising the steps of;
(i) confirming analytical purity of leuprorelin or salt(s) thereof by supercritical fluid chromatography,
(ii) purifying crude leuprorelin or salt(s) thereof of step (i) by preparative supercritical fluid chromatography,
(iii) isolating the pure fraction of leuprorelin or salt(s) thereof obtained from step (ii),
(iv) distillation of the pure fraction obtained from step (iii),
(v) lyophilisation of the pure fraction obtained from step (iv), and
(vi) storing of the pure leuprorelin or salt(s) thereof obtained from step (v) in at a temperature in the range of about -10°C to – 30°C to avoid any decomposition,
wherein the said process comprises mobile phase A containing carbon dioxide as supercritical fluid and a mobile phase B.

3. The process as claimed in claim 2, wherein the mobile phase B comprises one or more solvents selected from acetic acid, dichloromethane, Milli-Q water or methanol or a combination thereof.

4. The process as claimed in claim 2, wherein the obtained product of pure leuprorelin (I) or salt(s) thereof has a purity of more than about 98% by preparative supercritical fluid chromatography.

5. The process according to claim 2, wherein the obtained product of pure leuprorelin (I) or salt(s) thereof, has a purity of more than about 99.6% by preparative supercritical fluid chromatography.

6. The process according to claim 2, wherein the pharmaceutically acceptable salt of leuprorelin is a trifluoroacetate salt as compound of formula (II),

•

which is optionally converted into acetate salt of leuprorelin.