PURIFICATION OF GLP-1 ANALOGUES

Abstract

The present invention provides for purification of liraglutide using selective ion-pairing agents in the reversed phase-high performance liquid Chromatography, for purifying crude liraglutide from closely related impurities.

Claims

1. A method for purifying crude liraglutide, the method comprising: a. obtaining a solution of liraglutide by dissolving crude liragluide in a mixture comprising an aqueous acid solution and acetonitrile; b. subjecting the solution of liraglutide to a first HPLC purification using an aqueous acid solution and an ion pairing agent as mobile phase A and acetonitrile containing an alcohol as mobile phase B; c. subjecting the liraglutide obtained from first HPLC purification in step b. to a second HPLC purification; and d. isolating the purified liraglutide obtained from the second HPLC purification in step c.

2. The method of claim 1, wherein the ion-pairing agent is a salt of an alkane sulfonic acid.

3. The method of claim 2, wherein the salt of an alkane sulfonic acid is selected from the group consisting of 1-octane sulfonic acid sodium salt, 1-hepta sulfonic acid sodium salt and 1-hexane sulfonic acid sodium salt.

4. The method of claim 2, wherein the salt of an alkane sulfonic acid is 1-octane sulfonic acid sodium salt.

5. The method of claim 1, wherein the aqueous acid solution is selected from citric acid, acetic acid, trifluoroacetic acid and formic acid.

6. A method for purifying liraglutide comprising HPLC using an ion pairing agent.

Description

BRIEF DESCRIPTION OF THE FIGURES

[0041] In order that the disclosure may be readily understood and put into practical effect, reference will now be made to exemplary embodiments as illustrated with reference to the accompanying figures. The figures together with a detailed description below, are incorporated in and form part of the specification, and serve to further illustrate the embodiments and explain various principles and advantages, in accordance with the present disclosure wherein:

[0042] FIG. 1: Illustrates the HPLC pattern of crude Liraglutide of Formula I.

[0043] FIG. 2: Illustrates the HPLC pattern of Liraglutide of Formula I after purification without usage of OSA.

[0044] FIG. 3: Illustrates the HPLC pattern of Liraglutide of Formula I after purification with usage of OSA.

[0045] FIG. 4: Illustrates the HPLC pattern of Liraglutide of Formula I after purification with usage of HSA.

DETAILED DESCRIPTION OF THE INVENTION

[0046] The embodiments of the present invention are further described using specific examples herein after. The examples are provided for better understanding of certain embodiments of the invention and not, in any manner, to limit the scope thereof. Possible modifications and equivalents apparent to those skilled in the art using the teachings of the present description and the general art in the field of the invention shall also form the part of this specification and are intended to be included within the scope of it.

EXAMPLES

Example 1

[0047] 275.5 mg of crude Liraglutide, obtained from solid-phase synthesis was dissolved in 250 mM Citric acid monohydrate containing 10% Acetonitrile (v/v) filtered and subjected to a two-step RP-HPLC purification.

[0048] RP-HPLC-1:

[0049] The crude liraglutide solution was loaded onto a 20 ml of C8-substituted silica column (particle size 10-13 μm) equilibrated with about 60 ml of 100 mM citric acid containing 0.05% w/v octane-1-sulphonic acid sodium salt (Mobile Phase A), 25% Acetonitrile:Isopropanol (7:3) (Mobile Phase B) pH 2.0. Post loading the column was washed with 8:2 Buffer A:Buffer B. Product was eluted by applying a gradient upto 60% B. Detection wavelength was kept at 215 nm. The chromatographic temperature was kept at 25° C. Fractions with purity of >91% were pooled and the average pool purity at RP-1 was >95% with closely related impurities less than 0.50%.

[0050] The liraglutide product from RP-HPLC-1 was taken further for RP-HLC-2.

Example 2

[0051] 275.5 mg of crude Liraglutide, obtained from solid-phase synthesis was dissolved in 250 mM Citric acid monohydrate containing 10% Acetonitrile (v/v) filtered and subjected to a two-step RP-HPLC purification.

[0052] RP-HPLC-1:

[0053] The crude liraglutide solution was loaded onto a 20 ml of C8-substituted silica column (particle size 10-13 μm) equilibrated with about 60 ml of 100 mM citric acid containing 0.05% 1-hexane sulphonic acid sodium salt (Mobile Phase A), 25% Acetonitrile:Isopropanol (7:3) (Mobile Phase B) pH 2.0. Post loading the column was washed with 8:2 Buffer A:Buffer B. Product was eluted by applying a gradient upto 60%. Detection wavelength was kept at 215 nm. The chromatographic temperature was kept at 25° C. Fractions were collected and analyzed for purity. Fractions with purity of >91% were pooled.

[0054] The purity of the RP-HPLC-1 purified liraglutide HPLC chromatogram is as shown in FIG. 4.

[0055] HPLC purity of the RP-HPLC-1 purified liraglutide was >95% with closely related impurities less than 2.0%

[0056] pH of these pool was adjusted to 7.8 and distilled at 35° C. to remove organic solvent. Precipitation was done at pH-4.9 and RP-HPLC-1 purified liraglutide was isolated.

[0057] The liraglutide product from RP-HPLC-1 was taken further for RP-HLC-2.

Example 3

[0058] 275.5 mg of crude Liraglutide, obtained from solid-phase synthesis was dissolved in 250 mM Citric acid monohydrate containing 10% Acetonitrile (v/v) filtered and subjected to a two-step RP-HPLC purification.

[0059] RP-HPLC-1:

[0060] The crude liraglutide solution was loaded onto a 20 ml of C8-substituted silica column (particle size 10-13 μm) equilibrated with about 60 ml of 100 mM citric acid (Mobile Phase A), 25% Acetonitrile:Isopropanol (7:3) (Mobile Phase B) pH 2.0. Post loading the column was washed with (8:2) Buffer A:Buffer B. Product was eluted by applying a gradient upto 60% B. Detection wavelength was kept at 215 nm. The chromatographic temperature was kept at 25° C. Fractions were collected and analyzed for purity. Fractions with purity of >91% were pooled.

[0061] HPLC purity of the RP-HPLC-1 purified liraglutide was >95% with closely related impurities less than 2.0%.

[0062] The purity of the RP-HPLC-1 purified liraglutide HPLC chromatogram is as shown in FIG. 2.

[0063] The liraglutide product from RP-HPLC-1 was taken further for RP-HLC-2.

Example 4

[0064] 32.6 g of crude Liraglutide, obtained from solid-phase synthesis was dissolved in 250 mM Citric acid monohydrate containing 10% Acetonitrile (v/v) filtered and subjected to a two-step RP-HPLC purification.

[0065] RP-HPLC-1:

[0066] The crude liraglutide solution was loaded onto a 2.4 L of C8-substituted silica column (particle size 10-13 μm) equilibrated with about 7.2 L of 100 mM citric acid containing 0.05% octane-1-sulphonic acid sodium salt (Mobile Phase A), 25% Acetonitrile:Isopropanol (7:3) (Mobile Phase B) pH 2.0. Post loading the column was washed with 8:2 Buffer A:Buffer B. Product was eluted by applying a gradient upto 60% B. Detection wavelength was kept at 215 nm. The chromatographic temperature was kept at 25° C. Fractions were collected and analyzed for purity. Fractions with purity of >91% were pooled. The purity of the RP-HPLC-1 purified liraglutide HPLC chromatogram is as shown in FIG. 3.

[0067] HPLC purity of the RP-HPLC-1 purified liraglutide was >94% with closely related impurities less than 0.50%.

[0068] pH of these pool was adjusted to 7.8 and distilled at 35° C. to remove organic solvent. Precipitation was done at pH-4.9 and RP-HPLC-1 purified liraglutide was isolated.

[0069] The liraglutide product from RP-HPLC-1 was taken further for RP-HLC-2.

[0070] RP-HPLC-2:

[0071] 3.1 L of the RP-HPLC-1 purified liraglutide dissolved in 50 mM Di-Sodium hydrogen phosphate containing 25% Methanol at 3 mg/ml was loaded onto a 2.4 L of C8-substituted silica column (particle size 10-13 μm) equilibrated with about 7.2 L of 50 mM Sodium phosphate buffer pH 7.5 containing 5% Acetonitrile. Product was eluted by applying a gradient upto 41% B. Detection wavelength was kept at 215 nm. The chromatographic temperature was kept at 25° C. Individual fractions were collected and analyzed for purity. Fractions with purity of >98.0% were pooled and distilled at 35° C. to remove organic solvent. Precipitation was done at pH 4.9. The purified Liraglutide was subjected to lyophilization. HPLC purity of the lyophilized powder was >99% with no impurity more than 0.20%.