Purification of liraglutide

Abstract

The present invention provides a novel and improved purification process for a liraglutide precursor using a selective unit operation step and a selective pH gradient in reversed phase-high performance liquid chromatography to purify an unprocessed liraglutide precursor from closely associated impurities.

Description

Technology Field

[0001] 1. Related applications

[0002] This application claims the benefit of priority of the Indian patent application (202141036153) filed on August 10, 2021, 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 sustained-release analog of naturally occurring human glucagon-like peptide-1 (GLP-1(7-37)) in which lysine at position 34 is replaced by arginine and a palmitoyl group is attached to lysine at position 26 via a glutamoyl spacer.

[0008] Liraglutide (VICTOZA®), developed by Novo Nordisk, was first approved 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 palmitoyl groups.

[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 purification process of liraglutide by reverse-phase high-performance liquid chromatography (RP HPLC) using 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 disclosed method involves several purification steps, including three RP HPLC operations, which is an arduous process.

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

[0016] It is well known that the use of RP-HPLC is limited for the separation and identification of complex mixtures containing components with large variations in pKa values. Therefore, it is not always easy to separate impurities that elute closely in chromatographic purification.

[0017] In addition, for liraglutide precursors produced by a recombinant method, the task of purification involves separating cell and fermentation medium components from the precursor, removing host cell proteins and host cell DNA and related impurities, and removing impurities with additional hexose units and deletion impurities.

[0018] It has been observed that the solution to the aforementioned impurities in the present invention requires a purification process consisting of various steps to achieve the desired purity. means of solving the problem

[0019] One aspect of the present invention provides a method for purifying a liraglutide precursor.

[0020] One aspect of the present invention discloses a method for purifying an unprocessed recombinant liraglutide precursor:

[0021] a. A step of microfiltration of the fermentation culture medium;

[0022] b. A step of diafiltration of the product of step a);

[0023] c. A step of centrifuging the product of step b) after solubilizing it;

[0024] d. A step of applying the product of step c) to a deep filtration step;

[0025] e. A step of purifying the filtered supernatant from step d) by cation exchange chromatography;

[0026] f. A step of applying the product of step e) to reverse-phase high-pressure liquid-phase chromatography (RP-HPLC); and

[0027] g. Step of isolating the purified liraglutide precursor.

[0028] Another aspect of the present invention discloses a method for purifying a liraglutide precursor, wherein microfiltration is performed at a pH of 3.0 to 6.0.

[0029] Another aspect of the present invention discloses a method for purifying a liraglutide precursor, wherein the volume filtration is performed at a pH of 3.0 to 6.0.

[0030] Another aspect of the present invention discloses a method for purifying a liraglutide precursor, wherein solubilization is performed by the addition of urea.

[0031] Another aspect of the present invention discloses a method for purifying a liraglutide precursor, wherein the mobile phase gradient is a buffer with a pH range of 3.0 to 5.0.

[0032] Another aspect of the present invention discloses a method for purifying a liraglutide precursor, wherein the buffer is selected from glycine-hydrochloride (Glycine-HCl) buffer, citrate buffer, acetate buffer, citrate-phosphate buffer, succinate buffer, and maleate buffer.

[0033] Another aspect of the present invention discloses a method for purifying an unprocessed recombinant liraglutide precursor comprising the following:

[0034] a. A step of microfiltration of the fermentation culture solution at a pH of 3.0 to 6.0;

[0035] b. A step of filtration of the product of step a) at a pH of 3.0 to 6.0;

[0036] c. A step of solubilizing the product of step b) using an urea and then centrifuging;

[0037] d. A step of applying the product of step c) to a deep filtration step;

[0038] e. A step of purifying the supernatant filtered in step d) by cation exchange chromatography;

[0039] f. A step of applying the product of step e) to reverse-phase high-pressure liquid-phase chromatography (RP-HPLC); and

[0040] g. Step of isolating the purified liraglutide precursor.

[0041] Here, the mobile phase gradient is a buffer solution with a pH range of 3.0 to 5.0. Effects of the invention

[0042] 1. Liraglutide precursors are expressed extracellularly and are not accompanied by lysis.

[0043] 2. The initial volume of the culture medium is significantly (3.5-4 times) reduced due to the use of microfiltration / rectification filtration.

[0044] 3. The process is economical because no solvent is used in the capture chromatography, or cation exchange chromatography, step.

[0045] 4. The purity of the purified precursor used for acylation is 98% or higher.

[0046] 5. The overall process yield up to precursor purification is 45%, which is considerably high and economically feasible. The purification yield of acylated liraglutide is also high, exceeding 60% with a purity of over 99.5%.

[0047] Each step of the process disclosed in this specification is considered individually in the context of the described multi-step sequence. Brief explanation of the drawing

[0048] To ensure that the present disclosure is easily understood and put into practical use, reference will be made to exemplary embodiments with reference to the accompanying drawings. Together with the detailed description below, the drawings are incorporated into this specification and constitute part of the specification, further describing embodiments according to the present disclosure and explaining various principles and advantages. Fig. 1: This shows the manufacturing profile of the cation exchange chromatography step prepared according to Example 1. Figure 2: This shows the manufacturing profile of the RP-HPLC I step prepared according to Example 2. Fig. 3: Shows SDS-PAGE images providing comparative purity profiles across various unit operations up to the precursor purified according to Example 2. Fig. 4: This shows the manufacturing profile of the RP-HPLC II step prepared according to Example 4. Fig. 5: This shows the manufacturing profile of the RP-HPLC III step prepared according to Example 5. Fig. 6: SDS-PAGE image of the final purified drug substance according to Example 6 (silver staining). Fig. 7: Shows the Total Ion Chromatogram (TIC) overlay profile of the CEX pellet versus the RP pellet according to Example 6. Specific details for implementing the invention

[0049] Embodiments of the present invention are further described below using specific examples. Embodiments are provided to better understand specific embodiments of the invention and are not intended to limit the scope of the invention. Modifications and equivalents that are obvious to those skilled in the art using the teachings of this description and the general art in the field of the invention also constitute part of this specification and are intended to be included within the scope of the invention.

[0050] Example:

[0051] Example 1: 530 kg of fermentation culture medium with a titer of 0.4 g / L was permeated through MF and DF, followed by urea solubilization and centrifugation. The HPLC purity of the precursor determined at the end of centrifugation was 8%. Subsequently, the sample was loaded onto a pre-equilibrated cation exchange (CEX) column, followed by washing and pH-based elution. The pool purity of the CEX fraction was found to be 70–75%. To obtain the CEX pellet, the pH of the CEX fraction was adjusted to 3.5–5.5. The detection wavelength was maintained at 280 nm. The chromatography temperature was maintained at 25°C. The preparative chromatogram is shown in Figure 1.

[0052] Example 2:The CEX pellet obtained in Example 1 was purified in RP-HPLC-I using a 2.4 L C8 column. The combined precursor was eluted using a mobile phase step gradient (A: acetate buffer, B: ACN). The detection wavelength was maintained at 280 nm. The chromatography temperature was maintained at 25°C. The chromatogram for preparation is shown in Fig. 2. The fraction with a purity greater than 97% (>97%) was concentrated under vacuum, followed by isoelectric point precipitation. The suspension was centrifuged to obtain the precipitate of the purified precursor. The precipitate was washed with water, centrifuged, and stored at -20°C, with an HPLC purity greater than 98% (>98%). The SDS-PAGE images shown in Fig. 3 provide comparative purity profiles across various unit operations up to the purified precursor.

[0053]

[0054] Example 3: 59 g of purified precursor with a purity of 98.3% obtained in Example 2 above was applied to the acylation step. The acylation yield was greater than 70% (>70%), the analysis result of the obtained raw liraglutide was greater than 65% (>65%), and the HPLC purity was 77%.

[0055] Example 4: The acylated raw material was dissolved in equilibrium buffer at pH 2.0–4.0 and loaded onto a pre-equilibrated 2.4 L C8 column. The combined product was eluted using a gradient (A: equilibrium buffer; B: ACN: IPA) and analyzed for HPLC purity and product content. The pH of the fractions was diluted using phosphate buffer and stored at 2–8°C. Finally, the fractions were collected to achieve a pool purity of over 99% (≥99%) with a step yield of 75–80%. The detection wavelength was maintained at 215 nm. The chromatography temperature was maintained at 25°C. The chromatogram for preparation is shown in Figure 4.

[0056] Example 5: The RP-HPLC-II elution pool was further purified by reverse-phase high-pressure chromatography (RP-HPLC-III). The pH of the RP-2 pool was adjusted to 6.5–8.0 and, after dilution, loaded onto a pre-equilibrated 2.4 L C8 column. The combined product was eluted using a gradient (A: equilibration buffer, B: ACN) and analyzed for HPLC purity and product content. The detection wavelength was maintained at 215 nm. The chromatography temperature was maintained at 25°C. The chromatogram for preparation is shown in Figure 5.

[0057] The pH of the fractions was diluted using citric acid buffer and stored at 2–8°C. Finally, the fractions were collected to achieve a pool purity of 99.5% or higher (≥99.5%) with a step yield of over 90% (>90%). The pool was centrifuged to separate the pellets, which were then washed with water, and the purified pellets were separated. The separated pellets were freeze-dried to obtain liraglutide.

[0058]

[0059] Example 6:12,170 kg of fermentation culture medium with a titer of 0.4 g / L was subjected to microfiltration (MF) and direct filtration (DF), followed by urea solubilization and centrifugation. The HPLC purity of the precursor determined at the end of centrifugation was 8%. Subsequently, it was loaded onto a pre-equilibrated cation exchange column, washed, and pH-based elution was performed. The pool purity of the CEX fraction was found to be 68–73%. To obtain the CEX pellet, the pH of the CEX fraction was adjusted to 3.5–5.5, and the purity of the pellet was 78–80%. The detection wavelength was maintained at 280 nm. The chromatography temperature was maintained at 25°C. The chromatogram for preparation is similar to that shown in Figure 1. Next, the CEX pellet was purified in RP-HPLC-I using an 18 L C8 column. The combined precursor was eluted using a mobile phase gradient (A: acetate buffer, B: ACN). The detection wavelength was maintained at 280 nm. The chromatography temperature was maintained at 25°C. The chromatogram for preparation is similar to that shown in Fig. 2. Fractions with a purity greater than 93% (>93%) were concentrated under vacuum, followed by isoelectric point precipitation. The suspension was centrifuged to obtain a precipitate of the purified precursor. The precipitate was washed with water, centrifuged, and stored at -20°C, with an HPLC purity greater than 98% (>98%). The CEX pellet and RP pellet were analyzed using a high-resolution mass spectrometer (HR-MS) to identify the impurities. The total ion chromatogram (TIC) overlay profiles of the CEX pellet versus the RP pellet are shown in Fig. 7. Based on the HR-MS profile of the CEX pellet, the addition of hexose units (monohexose, dihexose, trihexose, tetrahexose) at 0.90 RRT (12.50 min at TIC), and 1.03-1.16 RRT (14.50 min at TIC).It was confirmed that some deletion impurities were present at 2–16 min, and high molecular weight protein (HMWP) impurities were present at 1.24–1.39 RRT (17–19.2 min in TIC). Most of these impurities were degraded in the RP-HPLC I step, and only a small amount of deletion impurities exists in RP-HPLC pellets with a purity greater than 98% (>98%).

[0060] Example 7: The RP-1 pellet obtained in Example 6 was applied to the acylation step. The acylation yield was greater than 60% (60%), the obtained raw liraglutide had an analytical result of 70%, and the HPLC purity was greater than 80% (>80%). This acylated raw material was purified in an 18L C8 column. The acylated raw material was dissolved in equilibrium buffer at pH 2.0–4.0 and loaded onto a pre-equilibrated C8 column. The combined product was eluted using a gradient (A: equilibrium buffer; B: ACN: IPA) and analyzed for HPLC purity and product content. The pH of the fraction was diluted using phosphate buffer and stored at 2–8°C. Finally, the fraction was collected to achieve a pool purity of greater than 99% (≥99%) with a step yield of 80–85%. The detection wavelength was maintained at 215 nm. The chromatography temperature was maintained at 25°C. The chromatogram for preparation is similar to that shown in Figure 4.

[0061] Example 8:The pH of the elution pool from Example 7 was adjusted to 6.5–8.0 and, after dilution, loaded onto a pre-equilibrated 18L C8 column. The combined product was eluted using a gradient (A: equilibration buffer, B: ACN) and analyzed for HPLC purity and product content. The detection wavelength was maintained at 215 nm. The chromatography temperature was maintained at 25°C. The chromatogram for preparation is similar to that shown in Figure 5. The pH of the fractions was diluted using citric acid buffer and stored at 2–8°C. Finally, the fractions were collected to achieve a pool purity of ≥99.5% with a step yield of >93%. The pool was centrifuged to separate the pellet, which was then washed with water, and the purified pellet was separated. The separated pellet was freeze-dried to obtain liraglutide.

Claims

Claim 1 A method for purifying an unprocessed recombinant liraglutide precursor comprising the following steps: a. microfiltration of a fermentation culture medium; b. diafiltration of the product of step a) to reduce the initial volume; c. centrifugation of the product of step b) after solubilization; d. application of the product of step c) to a deep filtration step; e. purification of the supernatant filtered from step d) by cation exchange chromatography; f. purification of the product of step e) by reverse phase high-pressure liquid-phase chromatography (RP-HPLC); and g. separation of the purified liraglutide precursor; wherein the RP-HPLC purification of step f) comprises elution using a mobile phase gradient. Claim 2 A method according to claim 1, wherein the microfiltration is performed at a pH of 3.0 to 6.

0. Claim 3 A method according to claim 1, wherein the volume filtration is performed at a pH of 3.0 to 6.

0. Claim 4 A method according to claim 1, wherein the solubilization is performed by the addition of urea. Claim 5 A method according to claim 1, wherein the mobile phase gradient is a buffer having a pH range of 3.0 to 5.

0. Claim 6 A method according to claim 5, wherein the buffer is selected from glycine-hydrochloride (Glycine-HCl) buffer, citrate buffer, acetate buffer, citrate-phosphate buffer, succinate buffer, and maleate buffer. Claim 7 A method for purifying an unprocessed recombinant liraglutide precursor comprising the following steps: a. microfiltration of a fermentation culture medium at a pH of 3.0 to 6.0; b. reduction of the initial volume of the product of step a) by volume filtration at a pH of 3.0 to 6.0; c. solubilizing the product of step b) using urea and then centrifuging; d. applying the product of step c) to a deep filtration step; e. purifying the supernatant filtered in step d) by cation exchange chromatography; f. purifying the product of step e) by reverse phase high-pressure liquid-phase chromatography (RP-HPLC); and g. separating the purified liraglutide precursor; wherein step f) comprises eluting using a mobile phase gradient of a buffer solution with a pH range of 3.0 to 5.0.

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