Purification of GLP-1 analogues

Abstract

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

Description

[Technical field]

[0001] Related Applications: This application claims the benefit of priority to our Indian Patent Application No. 201941004693, filed on February 6, 2019, which is incorporated herein by reference.

[0002] The present invention relates to a method for purifying crude GLP-1 analog, Liraglutide, inter alia, represented by Formula-I. Formula-I [ka] [Background technology]

[0003] Background and Prior Art of the Disclosure Liraglutide (VICTOZA®) is a glucagon-like peptide-1 (GLP-1) receptor agonist used as an adjunct to diet and exercise to improve glycemic control in adults with type 2 diabetes.

[0004] Liraglutide is a long-acting analogue of naturally occurring human glucagon-like peptide-1 (GLP-1(7-37)) in which the lysine at position 34 is replaced by arginine and a palmitoyl group is attached to the lysine at position 26 via a glutamoyl spacer. Liraglutide (VICTOZA®), developed by Novo Nordisk, first gained approval in the United States in 2010 as a subcutaneous injection. Liraglutide is highly difficult to purify due to its long peptide chain and high hydrophobicity due to the palmitoyl group. Several attempts at purification of GLP-1 analogues, including liraglutide, have been reported previously.

[0005] The Journal of Medicinal Chemistry 43, 1664-1669, 2000 discloses a purification process of liraglutide by reversed-phase-high performance liquid chromatography (RP HPLC) using a cyanopropyl column (Zorbax 300SB-CN) and a standard acetonitrile / TFA system. The above disclosed method results in a reduced purification yield of 35%. WO2013117135 discloses a purification process of liraglutide by RP HPLC using an isopropyl alcohol / TFA system. The disclosed method involves multiple purification steps involving 3RP HPLC operations, which is a laborious process.

[0006] GLP-1 peptides are produced either synthetically or by recombinant approaches and often have closely related impurities that are difficult to separate on RP-HPLC. These impurities are isomers or deletion / addition based impurities that have similar characteristics to the parent molecule. These closely related impurities pose purification challenges.

[0007] It is well known that the use of RP-HPLC is limited for the separation and identification of complex mixtures having components with large variations in pKa values. Thus, the resolution of precisely eluting impurities has always been a challenge in chromatographic purification. In the resolution of organic ions with traditional HPLC methods, the use of ion-pairing reagents can enhance peak shape and retention time when common remedies such as changing the eluent ratio or altering the stationary phase fail. This technique is often referred to as ion-pairing chromatography (IPC).

[0008] IPC is a type of RP-HPLC in which an ion-pairing reagent is added to the mobile phase to drive the formation of ion pairs with the charged analyte, which makes the reversed-phase column suitable for the separation of ionic molecules. Retention / separation follows a dynamic combination of reversed-phase and ion-pairing-ion exchange mechanisms.

[0009] The ion-pairing reagents comprise long linear alkyl chains (C3 to C16) and ionic groups that can reversibly adsorb onto the alkyl chains (C8 or C18) of the RP phase to form dynamic ion exchangers, where ionic compounds can be separated. There are two main types of ion-pairing reagents: anionic alkylsulfonates for basic compounds and cationic quaternary amines for acidic compounds. The commonly used alkylsulfonates in IPC are 1-hexanesulfonic acid sodium salt, 1-heptasulfonic acid sodium salt, 1-octanesulfonic acid sodium salt, etc. The selectivity of the system strongly depends on the choice and amount of the former ion pair in the mobile phase. Reagents with longer chain lengths are significantly better adsorbed onto the RP phase, which has a positive effect on retention.

[0010] The separation of liraglutide from these closely related impurities was studied on RP-HPLC in the absence and presence of ion pairing agents, i.e., sodium salts of 1-octanesulfonic acid, hexanesulfonic acid. It was observed that the resolution of closely related impurities was more effective in the presence of ion pairing agents, resulting in an overall better purity compared to purification runs in which no ion pairing agents were used.

[0011] The present invention provides a process for the purification of liraglutide from these closely related impurities, which was studied on RP-HPLC in the absence and presence of ion pairing agents, namely sodium salts of propanesulfonic acid, butanesulfonic acid, pentanesulfonic acid, hexanesulfonic acid, heptanesulfonic acid, octanesulfonic acid, nonanesulfonic acid, decanesulfonic acid, undecanesulfonic acid, dodecanesulfonic acid, tridecanesulfonic acid.

[0012] It was observed that the resolution of closely related impurities was more effective in the presence of the ion pairing agent resulting in an overall better purity compared to RPHPLC purification where no ion pairing agent was used. Summary of the Invention

[0013] An aspect of the present application provides a process for the purification of liraglutide. One aspect of the present invention discloses a process for purifying crude liraglutide, the process comprising: a. obtaining a solution of liraglutide by dissolving crude liraglutide in a mixture comprising an aqueous acidic solution and acetonitrile; b. subjecting the solution of crude liraglutide to a first HPLC purification using an aqueous acidic solution and an ion pairing agent as mobile phase A and acetonitrile containing an alcohol as mobile phase B; c. subjecting the liraglutide from the first HPLC purification to a second HPLC purification; and d. Isolating purified liraglutide. Another aspect of the present invention discloses a method for purifying crude liraglutide, wherein the ion pairing agent is selected from salts of alkanesulfonic acids.

[0014] Another aspect of the present invention discloses a method for purifying crude liraglutide, wherein the salt of an alkane sulfonic acid is selected from the group consisting of 1-octanesulfonic acid sodium salt or 1-heptanesulfonic acid sodium salt. Another aspect of the present invention discloses a process for purifying crude liraglutide, wherein the salt of an alkane sulfonic acid is 1-hexanesulfonic acid sodium salt. Another aspect of the present invention discloses a method for purifying crude liraglutide, wherein the aqueous acidic solution is selected of citric acid, acetic acid, trifluoroacetic acid, or formic acid. Another aspect of the present invention discloses a method for purifying crude liraglutide by HPLC using an ion pairing agent. Equipment parameters: HPLC instrument parameters: Column: C8, 150 x 4.6 mm, 2.7 μm Column temperature: 60℃ Detection: UV Wavelength: 215nm

[0015] Advantages of this invention: Crude liraglutide powder (assay 20-25%; purity 30-50%) is subjected to two successive RP-HPLC purification steps, each under different conditions, followed by lyophilization to produce pure liraglutide. The present invention involves the use of a selective ion-pairing agent in one of the RP-HPLC steps to purify crude liraglutide from closely related impurities. The absence of an ion-pairing agent in the process results in no or poor resolution of related impurities, leading to impurities in the final API.

[0016] Comparison of Liraglutide Purity Profiles Without and With Octane-1-Sulfonic Acid as an Ion Pairing Agent: The table below provides a comparison of liraglutide purity profiles with respect to closely related impurities present at RRTs of 0.93, 0.98, and 1.06, without and with the use of octane-1-sulfonic acid as an ion pairing agent.

[0017] [Table 1] [Brief description of the drawings]

[0018] So that the disclosure may be readily understood and substantially reduced to practice, reference will now be made to exemplary embodiments as illustrated in the accompanying drawings, in which: Figure 1 illustrates a block diagram of a microprocessor according to the present disclosure; Figure 2 illustrates a block diagram of a microprocessor according to the present disclosure; [Figure 1] FIG. 1 illustrates the HPLC pattern of crude liraglutide of formula I. [Diagram 2] FIG. 2 illustrates the HPLC pattern of liraglutide of formula I after purification without the use of OSA. [Diagram 3] FIG. 3 illustrates the HPLC pattern of liraglutide of formula I after purification with the use of OSA. [Figure 4] FIG. 4 illustrates the HPLC pattern of liraglutide of formula I after purification with the use of HSA. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0019] Detailed Description of the Invention Aspects of the present invention are further described herein below using specific examples. The examples are provided for a better understanding of certain embodiments of the invention and do not limit their scope in any way. Possible modifications and equivalents that are obvious to a person skilled in the art using the teachings of the present description and general skills in the technical field of the present invention are also intended to form part of this specification and be included within its scope.

[0020] example: Example 1: 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.

[0021] RP-HPLC-1: The crude liraglutide solution was loaded onto 20 ml of a C8-substituted silica column (particle size 10-13 μm) equilibrated with approximately 60 ml of 100 mM citric acid containing 0.05% w / v octane-1-sulfonic acid sodium salt (mobile phase A), 25% acetonitrile:isopropanol (7:3) (mobile phase B) pH 2.0. After loading, the column was washed with 8:2 buffer A:buffer B.

[0022] The product was eluted by applying a gradient up to 60% B. The detection wavelength was kept at 215 nm. The chromatographic temperature was kept at 25° C. Fractions with a purity of >91% were pooled and the average pooled purity in RP-1 was >95% with less than 0.50% closely related impurities. The liraglutide product from RP-HPLC-1 was further used in RP-HPLC-2.

[0023] Example 2: 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.

[0024] RP-HPLC-1: The crude liraglutide solution was loaded onto 20 ml of a C8-substituted silica column (particle size 10-13 μm) equilibrated with approximately 60 ml of 100 mM citric acid containing 0.05% 1-hexanesulfonic acid sodium salt (mobile phase A), 25% acetonitrile:isopropanol (7:3) (mobile phase B) pH 2.0.

[0025] After loading, the column was washed with 8:2 buffer A:buffer B. The product was eluted by applying a gradient up to 60%. The detection wavelength was kept at 215 nm. The chromatographic temperature was kept at 25° C. Fractions were collected and analyzed for purity. Fractions with a purity of >91% were pooled. The purity of the RP-HPLC-1 purified liraglutide HPLC chromatogram is shown in FIG. The HPLC purity of RP-HPLC-1 purified liraglutide was >95% with less than 2.0% of closely related impurities.

[0026] The pH of these pools was adjusted to 7.8 and the organic solvent was removed by distillation at 35° C. Precipitation was performed at pH-4.9 and RP-HPLC-1 purified liraglutide was isolated. The liraglutide product from RP-HPLC-1 was further used in RP-HPLC-2.

[0027] Example 3: 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.

[0028] RP-HPLC-1: The crude liraglutide solution was loaded onto 20 ml of a C8-substituted silica column (particle size 10-13 μm) equilibrated with approximately 60 ml of 100 mM citric acid (mobile phase A), 25% acetonitrile:isopropanol (7:3) (mobile phase B) pH 2.0. After loading, the column was washed with 8:2 buffer A:buffer B. The product was eluted by applying a gradient up to 60% B. The detection wavelength was kept at 215 nm. The chromatographic temperature was kept at 25° C. Fractions were collected and analyzed for purity. Fractions with a purity of >91% were pooled. The HPLC purity of RP-HPLC-1 purified liraglutide was >95% with less than 2.0% of closely related impurities. The purity of the RP-HPLC-1 purified liraglutide HPLC chromatogram is shown in FIG. The liraglutide product from RP-HPLC-1 was further used in RP-HPLC-2.

[0029] Example 4: 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.

[0030] RP-HPLC-1: The crude liraglutide solution was loaded onto 2.4 L of a C8-substituted silica column (particle size 10-13 μm) equilibrated with approximately 7.2 L of 100 mM citric acid containing 0.05% octane-1-sulfonic acid sodium salt (mobile phase A), 25% acetonitrile:isopropanol (7:3) (mobile phase B) pH 2.0. After loading, the column was washed with 8:2 buffer A:buffer B. The product was eluted by applying a gradient up to 60% B. The detection wavelength was kept at 215 nm. The chromatographic temperature was kept at 25° C. Fractions were collected and analyzed for purity. Fractions with a purity of >91% were pooled. The purity of the RP-HPLC-1 purified liraglutide HPLC chromatogram is shown in FIG. The HPLC purity of RP-HPLC-1 purified liraglutide was >94% with closely related impurities less than 0.50%.

[0031] The pH of these pools was adjusted to 7.8 and the organic solvent was removed by distillation at 35° C. Precipitation was performed at pH-4.9 and RP-HPLC-1 purified liraglutide was isolated. The liraglutide product from RP-HPLC-1 was further used in RP-HPLC-2.

[0032] RP-HPLC-2: 3.1 L of RP-HPLC-1 purified liraglutide dissolved in 50 mM disodium hydrogen phosphate containing 25% methanol at 3 mg / ml was loaded onto 2.4 L of a C8-substituted silica column (particle size 10-13 μm) equilibrated with approximately 7.2 L of 50 mM sodium phosphate buffer pH 7.5 containing 5% acetonitrile. The product was eluted by applying a gradient up to 41% B. The detection wavelength was kept at 215 nm.

[0033] The chromatographic temperature was maintained at 25° C. Individual fractions were collected and analyzed for purity. Fractions with purity >98.0% were pooled and distilled at 35° C. to remove the organic solvent. Precipitation was performed at pH 4.9. The purified liraglutide was subjected to lyophilization. The HPLC purity of the lyophilized powder was >99% with no impurities greater than 0.20%.

Claims

1. 1. A process for purifying crude liraglutide, comprising: a. Obtaining a solution of crude liraglutide by dissolving crude liraglutide in a mixture comprising an aqueous acidic solution and acetonitrile; b. subjecting the solution of crude liraglutide to a first HPLC purification using an aqueous acidic solution and an ion pairing agent as mobile phase A and acetonitrile containing an alcohol as mobile phase B; c. subjecting the liraglutide from the first HPLC purification to a second HPLC purification; and d. Isolating the purified liraglutide wherein the ion pairing agent is a salt of an alkane sulfonic acid.

2. 2. The method of claim 1, wherein the salt of an alkanesulfonic acid is selected from the group consisting of 1-octanesulfonic acid sodium salt and 1-heptanesulfonic acid sodium salt.

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

4. 2. The method of claim 1, wherein the aqueous acidic solution is selected from citric acid, acetic acid, trifluoroacetic acid, or formic acid.

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