GLP-1 analog purification

Abstract

The present invention provides a method for purifying liraglutide using a selective ion pair reagent in reverse-phase high-performance liquid chromatography to purify raw liraglutide from closely associated impurities.

Description

Technology Field

[0001] 1. Related applications

[0002] This application claims the benefit of priority of Indian patent application IN 201941004693 filed on February 6, 2019, which is incorporated herein by reference.

[0003] 2. Technology Field

[0004] The present invention relates to a method for purifying an unprocessed GLP-1 analog, in particular a liraglutide represented by the chemical formula I.

[0005] Background Technology

[0006] Liraglutide (VICTOZA®) is a glucagon-like peptide-1 (GLP-1) receptor agonist used as an adjunct to diet and exercise to improve blood glucose control in adults with type 2 diabetes.

[0007] Liraglutide is a naturally occurring sustained-action analog of human glucagon-like peptide-1 (GLP-1(7-37)), in which lysine at position 34 is substituted with arginine and a palmitoyl group is attached to lysine at position 26 via a glutamoyl spacer.

[0008] Liraglutide (VICTOZA) developed by Novo Nordisk ® ) received its first approval as a subcutaneous injection in the United States in 2010.

[0009] Liraglutide is very difficult to purify due to its long peptide chain and high hydrophobicity caused by the palmitoyl group.

[0010] Several attempts to purify GLP-1 analogs including liraglutide have been reported in the past.

[0011] Journal of Medicinal Chemistry 43, 1664-1669, 2000 discloses a process for purifying liraglutide using reverse-phase high-performance liquid chromatography (RP HPLC) with a cyanopropyl column (Zorbax 300SB-CN) and a standard acetonitrile / TFA system.

[0012] The method disclosed above results in a 35% reduced purification yield.

[0013] WO2013117135 discloses a purification process of liraglutide by RP HPLC using an isopropyl alcohol / TFA system.

[0014] The method disclosed above includes multiple purification steps, including a 3 RP HPLC operation, which is a laborious process.

[0015] GLP-1 peptides are produced by synthetic or recombinant approaches and often contain closely related impurities that are difficult to separate on RP-HPLC. These impurities are isomers or deletion / addition-based impurities that possess characteristics similar to the parent molecule. These closely related impurities are difficult to purify.

[0016] It is well known that the use of RP-HPLC is limited in the separation and identification of complex mixtures containing components with large variations in pKa values. Therefore, the separation of closely eluted impurities has always been a difficult problem in chromatographic purification. When separating organic ions using conventional HPLC methods, the use of ion pair reagents can improve peak shape and retention time when common improvements, such as modifying the elution rate or changing the stationary phase, have failed. This technique is often referred to as ion pair chromatography (IPC).

[0017] IPC is a type of reverse-phase column RP-HPLC suitable for the separation of ionic molecules, in which an ion pair reagent is added to a mobile phase to promote ion pair formation with a charged analyte. Retention / separation follows a dynamic combination of reverse phase and ion pair-ion exchange mechanisms.

[0018] Ion pair reagents include long linear alkyl chains (C3 to C16) and ionic groups that can reversibly adsorb to alkyl chains (C8 or C18) on RP that can form dynamic ion exchangers when ionic compounds can be separated.

[0019] There are two main types of ion pair reagents: basic compounds, namely anionic alkyl sulfons, and acidic compounds, namely cationic quaternary amines. Alkyl sulfons commonly used in IPC include sodium 1-hexanesulfonate, sodium 1-heptasulfonate, and sodium 1-octanesulfonate. The selectivity of the system depends significantly on the selection and amount of ion pair formants in the mobile phase. Reagents with long chain lengths are better adsorbed onto the RP and have a positive effect on retention.

[0020] The separation of liraglutide from these closely related impurities was studied on RP-HPLC in the absence and presence of ion pair reagents, namely the sodium salt of 1-octanesulfonic acid and hexanesulfonic acid. It was observed that the separation of closely related impurities is more effective in the presence of ion pair reagents and generally yields better purity compared to purification performed without the use of ion pair reagents.

[0021] The present invention provides a method for purifying liraglutide from very closely related impurities studied by RP-HPLC in the absence and presence of ion pair reagents, namely, sodium salts of propanesulfonic acid, butanesulfonic acid, pentanesulfonic acid, hexanesulfonic acid, heptanesulfonic acid, octanesulfonic acid, nonanesulfonic acid, decanesulfonic acid, undecanesulfonic acid, dodecanesulfonic acid, and tridecanesulfonic acid.

[0022] It was observed that the separation of closely related impurities is more effective in the presence of ion pair reagents, which generally show better purity compared to RP HPLC purification without ion pair reagents. means of solving the problem

[0023] An aspect of the present invention is to provide a purification process for liraglutide.

[0024] One aspect of the present invention discloses a method for purifying unprocessed liraglutide comprising the following:

[0025] a. A step of obtaining a liraglutide solution by dissolving raw liraglutide in a mixture containing an aqueous acid solution and acetonitrile;

[0026] b. A step of performing primary HPLC purification of the raw liraglutide solution using an aqueous acid solution and an ion pair reagent as mobile phase A, and acetonitrile containing an alcohol as mobile phase B;

[0027] c. A step of treating the liraglutide from primary HPLC purification to secondary HPLC purification; and

[0028] d. A step of separating the purified liraglutide.

[0029] Another aspect of the present invention discloses a method for purifying raw liraglutide in which the ion pair reagent is selected from salts of alkanes sulfonic acids.

[0030] Another aspect of the present invention discloses a method for purifying raw liraglutide, wherein the salt of the alkane sulfonic acid is selected from the group consisting of 1-octane sulfonate sodium salt or 1-heptane sulfonate sodium salt.

[0031] Another aspect of the present invention discloses a method for purifying unprocessed liraglutide, wherein the salt of the alkane sulfonic acid is sodium 1-hexanesulfonate.

[0032] Another aspect of the present invention discloses a method for purifying raw liraglutide, wherein the aqueous acid solution is selected from citric acid, acetic acid, trifluoroacetic acid, or formic acid.

[0033] Another aspect of the present invention discloses a method for purifying raw liraglutide by HPLC using an ion pair reagent.

[0034] Mechanical parameters:

[0035] HPLC Instrument Parameters: Column: C8, 150 x 4.6 mm, 2.7 μm column

[0036] Temperature: 60°C Detection: UV Wavelength: 215 nm Effects of the invention

[0037] Unprocessed liraglutide powder (assay 20-25%; purity 30-35%) is freeze-dried after undergoing two sequential RP-HPLC purification steps under various conditions to obtain pure liraglutide. The present invention comprises using an optional ion pair reagent in one of the RP-HPLC purification steps to purify the closely related unprocessed liraglutide. The absence of the ion pair reagent in the process results in no or poor decomposition of the related impurities, leading to impurities in the final API.

[0038] Comparison of liraglutide purity profiles with and without octane-1-sulfonic acid as an ion-pair reagent:

[0039] The table below provides a comparison of liraglutide purity profiles with and without octane-1-sulfonic acid in relation to the presence of closely related impurities at RRTs of 0.93, 0.98, and 1.06.

[0040] Brief explanation of the drawing

[0041] To ensure that the present disclosure is easily understood and to achieve practical effects, reference will be made to exemplary embodiments illustrated with reference to the accompanying drawings. Together with the following detailed description, the drawings are incorporated into and form part of this specification and serve to further illustrate embodiments according to the present disclosure and to explain various principles and advantages: Fig. 1: Chemical Formula I This shows the HPLC pattern of unprocessed liraglutide. Fig. 2: After purification without using OSA Chemical Formula I This shows the HPLC pattern of liraglutide. Fig. 3 : After purifying using OSA Chemical Formula I This shows the HPLC pattern of liraglutide. Fig. 4 : After purification using HSA Chemical Formula I This shows the HPLC pattern of liraglutide. Specific details for implementing the invention

[0042] Embodiments of the present invention are further described below using specific examples. The embodiments are provided to aid in understanding specific embodiments of the present invention and are not intended to limit the scope of the invention. Modifications and equivalents that are clearly apparent to those skilled in the art using the teachings of this description and the general art in the field of the invention are also intended to form part of this specification and be included within the scope of the invention.

[0043] Example:

[0044] Example 1:

[0045] 275.5 mg of raw liraglutide obtained from solid-phase synthesis was dissolved in 250 mM citric acid monohydrate containing 10% acetonitrile (v / v), filtered, and purified by 2-step RP-HPLC.

[0046] RP-HPLC-1: The above raw liraglutide solution was loaded onto a 20 ml C8-substituted silica column (particle size 10-13 μm) equilibrated with approximately 60 ml of 100 mM citric acid containing 0.05% w / v sodium octane-1-sulfonate (mobile phase A), 25% acetonitrile:isopropanol (7:3) (mobile phase B) and pH 2.0. After loading, the column was washed with an 8:2 buffer A:buffer B. The product was eluted by a gradient up to 60% B. The detection wavelength was maintained at 215 nm. The chromatographic temperature was maintained at 25°C. Fractions with a purity greater than 91% (>91%) were collected, and the average pool purity in RP-1 was greater than 95% (>95%), with closely associated impurities being less than 0.50%.

[0047] RP-HLC-2 was additionally performed on the liraglutide product of the above RP-HPLC-1.

[0048] Example 2:

[0049] 275.5 mg of raw liraglutide obtained from solid-phase synthesis was dissolved in 250 mM citric acid monohydrate containing 10% acetonitrile (v / v), filtered, and purified by 2-step RP-HPLC.

[0050] RP-HPLC-1:

[0051] The above raw liraglutide solution was loaded onto a 20 ml C8-substituted silica column (particle size 10-13 μm) equilibrated with approximately 60 ml of 100 mM citric acid containing 0.05% 1-hexanesulfonate sodium salt (mobile phase A), 25% acetonitrile:isopropanol (7:3) (mobile phase B) at pH 2.0. After loading, the column was washed with an 8:2 buffer A:buffer B. The product was eluted by applying a gradient up to 60% B. The detection wavelength was maintained at 215 nm. The chromatographic temperature was maintained at 25°C. Fractions were collected and their purity analyzed. Fractions with a purity greater than 91% (>91%) were collected.

[0052] The purity of the liraglutide HPLC chromatogram purified by the above RP-HPLC-1 is Fig. 4 As shown in [it].

[0053] The HPLC purity of liraglutide purified by RP-HPLC-1 was greater than 95% (>95%), and the closely related impurities were less than 2.0%.

[0054] The pH of these pools was adjusted to 7.8 and distilled at 35°C to remove organic solvents.

[0055] RP-HLC-2 was additionally performed on the liraglutide product of the above RP-HPLC-1.

[0056] Example 3:

[0057] 275.5 mg of raw liraglutide obtained from solid-phase synthesis was dissolved in 250 mM citric acid monohydrate containing 10% acetonitrile (v / v), filtered, and purified by 2-step RP-HPLC.

[0058] RP-HPLC-1:

[0059] The above raw liraglutide solution was loaded onto a 20 ml C8-substituted silica column (particle size 10-13 μm) equilibrated to pH 2.0 with approximately 60 ml of 100 mM citric acid (mobile phase A) and 25% acetonitrile:isopropanol (7:3) (mobile phase B). After loading, the column was washed with (8:2) buffer A:buffer B. The product was eluted by a gradient up to 60% B. The detection wavelength was maintained at 215 nm. The chromatographic temperature was maintained at 25°C. Fractions were collected and their purity analyzed. Fractions with a purity greater than 91% (>91%) were collected.

[0060] The HPLC purity of liraglutide purified by RP-HPLC-1 was greater than 95% (>95%), and the closely related impurities were less than 2.0%.

[0061] The purity of the liraglutide HPLC chromatogram purified by the above RP-HPLC-1 is Fig. 2 As shown in [it].

[0062] RP-HLC-2 was additionally performed on the liraglutide product of the above RP-HPLC-1.

[0063] Example 4:

[0064] 32.6 g of raw liraglutide obtained from solid-phase synthesis was dissolved in 250 mM citric acid monohydrate containing 10% acetonitrile (v / v), filtered, and purified by 2-step RP-HPLC.

[0065] RP-HPLC-1:

[0066] The above raw liraglutide solution was loaded onto a 2.4 L C8-substituted silica column (particle size 10–13 μm) equilibrated with approximately 7.2 L of 100 mM citric acid containing 0.05% sodium octane-1-sulfonate (mobile phase A), 25% acetonitrile:isopropanol (7:3) (mobile phase B) at pH 2.0. After loading, the column was washed with an 8:2 buffer A:buffer B. The product was eluted by applying a gradient up to 60% B. The detection wavelength was maintained at 215 nm. The chromatography temperature was maintained at 25°C. Fractions were collected and their purity analyzed. Fractions with a purity greater than 91% (>91%) were collected. The purity of the HPLC chromatogram of liraglutide purified by the above RP-HPLC-1 is Fig. 3 As shown in [it].

[0067] The HPLC purity of liraglutide purified by RP-HPLC-1 was greater than 94% (>95%), and the amount of closely related impurities was less than 0.50%.

[0068] The pH of these pools was adjusted to 7.8 and distilled at 35°C to remove organic solvents. Precipitation was performed at pH 4.9 and purified liraglutide was isolated using RP-HPLC-1.

[0069] RP-HLC-2 was additionally performed on the liraglutide product of the above RP-HPLC-1.

[0070] RP-HPLC-2:

[0071] 3.1 L of RP-HPLC-1 purified liraglutide dissolved in 50 mM disodium hydrogen phosphate containing 3 mg / ml of 25% methanol was loaded onto a 2.4 L C8-substituted silica column (particle size 10–13 μm) equilibrated to pH 7.5 in approximately 7.2 L of 50 mM sodium phosphate buffer containing 5% acetonitrile. The product was eluted by a gradient up to 41% B. The detection wavelength was maintained at 215 nm. The chromatography temperature was maintained at 25°C. Individual fractions were collected and their purity analyzed. Fractions with a purity greater than 98% (>98%) were collected and distilled at 35°C to remove organic solvents. Precipitation occurred at pH 4.9. The purified liraglutide was freeze-dried. The HPLC purity of the freeze-dried powder was greater than 99% (>99%), and there were no impurities exceeding 0.20%.

Claims

Claim 1 A method for purifying raw liraglutide, comprising: a. dissolving raw liraglutide in a mixture containing an aqueous acid solution and acetonitrile to obtain a liraglutide solution; b. purifying the raw liraglutide solution by primary high-pressure liquid chromatography using acetonitrile containing an aqueous acid solution and an ion pair reagent as mobile phase A and an alcohol as mobile phase B; c. purifying the liraglutide obtained by purification by the primary high-pressure liquid chromatography of step b by secondary high-pressure liquid chromatography; and d. separating the purified liraglutide obtained by purification by the secondary high-pressure liquid chromatography of step c; wherein the ion pair reagent is a salt of an alkane sulfonic acid. Claim 2 delete Claim 3 A method for purifying unprocessed liraglutide according to claim 1, wherein the salt of the alkane sulfonic acid is selected from the group consisting of 1-octane sulfonate sodium salt, 1-heptane sulfonate sodium salt, and 1-hexane sulfonate sodium salt. Claim 4 A method for purifying unprocessed liraglutide according to claim 1, wherein the salt of the alkane sulfonic acid is sodium 1-octane sulfonate. Claim 5 A method for purifying raw liraglutide according to claim 1, wherein the aqueous acid solution used in step a) is selected from the group consisting of citric acid, acetic acid, trifluoroacetic acid, and formic acid. Claim 6 delete Claim 7 A method for purifying unprocessed liraglutide, wherein the alcohol in claim 1 is isopropanol.

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