Purification of Liraglutide

Abstract

The present invention provides a new and improved purification process for liraglutide precursor, which uses selective unit operation steps and a selective pH gradient in reversed-phase high performance liquid chromatography to purify the crude liraglutide precursor from closely related impurities.

Description

[Technical field]

[0001] Related Applications: This application claims priority to our Indian patent application IN 202141036153, filed on August 10, 2021, which is incorporated herein by reference.

[0002] Technical Field The present invention relates to a method for purifying crude GLP-1 analogues, particularly the precursor of liraglutide represented by formula-I. [ka] Formula-I [Background technology]

[0003] Background and Prior Art of the Disclosure Liraglutide (VICTOZA®) is a glucagon-like peptide-1 (GLP-1) receptor agonist indicated as an adjunct to diet and exercise to improve glycemic control in adults with type 2 diabetes. Liraglutide is a long-acting analogue of the 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 the lysine at position 26 via a glutamoyl spacer.

[0004] Liraglutide (VICTOZA®), developed by Novo Nordisk, was first approved in the United States in 2010 as a subcutaneous injection solution. 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 previously reported.

[0005] The Journal of Medicinal Chemistry 43, 1664-1669, 2000 discloses a purification process for liraglutide by reversed-phase high performance liquid chromatography (RP HPLC) using a cyanopropyl column (Zorbax 300SB-CN) and a standard acetonitrile / TFA system. The process as disclosed above results in a reduced purification yield of 35%.

[0006] WO2013117135 discloses a purification process for liraglutide by RP HPLC using an isopropyl alcohol / TFA system. The method as disclosed involves multiple purification steps involving three RP HPLC operations in a laborious process.

[0007] GLP-1 peptides are produced by either synthetic or recombinant approaches and often have closely related impurities that are difficult to separate on RP-HPLC. These impurities are either isomeric impurities or deletion / addition based impurities with similar characteristics as the parent molecule. These closely related impurities pose challenges to purification. It is well known that the use of RP-HPLC is limited in its ability to separate and identify complex mixtures with components that vary widely in pKa value, thus making the resolution of closely eluting impurities a challenging chromatographic purification.

[0008] Also, when the liraglutide precursor is produced by a recombinant approach, purification challenges involve separation of cells and fermentation medium components from the precursor, removal of host cell proteins and host cell DNA and related impurities, hexose unit added impurities and deletion impurities. It has been observed in the present invention that the resolution of the above impurities required a purification process involving various steps to achieve the desired purity. Summary of the Invention

[0009] SUMMARY OF THE PRESENT APPLICATION An aspect of the present application provides a process for the purification of liraglutide precursor. One aspect of the present invention discloses a method for purifying crude recombinant liraglutide precursor, the method comprising: a. subjecting the fermentation broth to microfiltration; b. subjecting the product of step a) to diafiltration; c. solubilizing the product of step b) followed by centrifugation; d. subjecting the product of step c) to a depth filtration step; d. subjecting the product of step c) to a depth filtration step; e. the filtered supernatant from step d) is subjected to a cation exchange chromatography purification step; f. subjecting the product of step e) to reverse phase high pressure liquid chromatography (RP-HPLC); and g. Isolating the purified liraglutide precursor.

[0010] Another aspect of the present invention discloses a method for purifying liraglutide precursor, wherein the microfiltration is carried out at pH 3.0-6.0. Another aspect of the present invention discloses a method for purifying liraglutide precursor, wherein diafiltration is carried out at pH 3.0-6.0.

[0011] Another aspect of the present invention discloses a method for purifying liraglutide precursor, wherein solubilization is performed by addition of urea. Another aspect of the present invention discloses a method for purifying liraglutide precursor, wherein the mobile phase gradient is a buffer in the pH range of 3.0 to 5.0.

[0012] Another aspect of the present invention discloses a method for purifying liraglutide precursor, wherein the buffer is selected from glycine-HCL buffer, citrate buffer, acetate buffer, citrate-phosphate buffer, succinate buffer, maleate buffer.

[0013] Another aspect of the present invention discloses a method for purifying crude recombinant liraglutide precursor, the method comprising: a. subjecting the fermentation broth to microfiltration at pH 3.0-6.0; b. subjecting the product of step a) to diafiltration at pH 3.0-6.0; c. solubilizing the product of step b) using urea followed by centrifugation; d. subjecting the product of step c) to a depth filtration step; e. the filtered supernatant from step d) is subjected to a cation exchange chromatography purification step; f. subjecting the product of step e) to reverse phase high pressure liquid chromatography (RP-HPLC); and g. Isolating the purified liraglutide precursor. Here the mobile phase gradient is a buffer in the pH range of 3.0-5.0.

[0014] Advantages of this invention: 1. The liraglutide precursor is expressed extracellularly, but is not accompanied by lysis. 2. The initial volume of the broth is significantly reduced (3.5-4 fold) thanks to the use of microfiltration / diafiltration operations. 3. No solvent is used in the capture chromatography, i.e. cation exchange chromatography step, thus making the process economical. 4. The purified precursor has a purity of >98% and is taken up for acylation. 5. The overall yield of the process up to the precursor purification is significantly high and economically viable, i.e., 45%. The yield of acylated liraglutide purification is also high, >60%, with a purity of >99.5%. Each step of the processes disclosed herein is contemplated both individually and in the context of the multi-step sequence in which it is described. [Brief description of the drawings]

[0015] Brief description of the figure So that the present disclosure may be readily understood and put into practical effect, reference will now be made to exemplary embodiments as described with reference to the accompanying drawings, which together with the detailed description below are incorporated into and form a part of this specification and serve to further explain the embodiments and clarify various principles and advantages in accordance with the present disclosure, where:

[0016] [Figure 1] FIG-1: Illustrates the preparative profile of the cation exchange chromatographic step prepared according to Example 1. [Diagram 2] Figure 2: Illustrates the preparative profile of the RP-HPLC I step, prepared according to Example 2. [Diagram 3] FIG-3: Illustrates SDS-PAGE images giving comparative purity profiles across different unit operations up to the precursor purified according to Example 2.

[0017] [Figure 4] Figure 4: Illustrates the preparative profile of the RP-HPLC II step, prepared according to Example 4. [Diagram 5] Figure 5: Illustrates the preparative profile of the RP-HPLC III step, prepared according to Example 5. [Figure 6] Figure-6: Illustrates the SDS-PAGE image of the final purified bulk material (silver stained) according to Example 6. [Figure 7] Figure 7: Illustrates total ion chromatogram (TIC) overlay profiles of CEX pellets vs. RP pellets according to Example 6. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0018] Detailed Description of the Invention Aspects of the present invention are further described below using specific examples. The examples are provided for a better understanding of certain aspects of the present invention, but are not provided to limit its scope in any way. Possible modifications and equivalents will be obvious to those skilled in the art using the teachings of the present description, and general techniques in the field of the present invention are also intended to form part of this specification and be included within its scope.

[0019] example: Example 1: 530 kg of fermentation broth with a titer of 0.4 g / L was subjected to MF and DF followed by urea solubilization and centrifugation. The HPLC purity of the precursor determined at the end of centrifugation was 8%. It was then loaded onto a pre-equilibrated cation exchange (CEX) column followed by washing and pH-based elution. The pooled purity of the CEX fractions was found to be 70-75%. The pH of the CEX fractions was adjusted from 3.5 to 5.5 to produce a CEX pellet. The detection wavelength was kept at 280 nm. The chromatograph temperature was kept at 25° C. The preparative chromatogram is as shown in FIG. 1.

[0020] Example 2: The CEX pellet obtained from Example 1 was then purified on a RP-HPLC-I using a 2.4 L C8 column. The bound precursor was eluted using a step gradient of the mobile phase (A: acetate buffer; B: ACN). The detection wavelength was kept at 280 nm. The chromatograph temperature was kept at 25° C. The preparative chromatogram is shown in FIG. 2. The fractions with a purity >97% were concentrated under vacuum, followed by isoelectric precipitation. Centrifugation of the suspension produced a precipitate of the purified precursor. The precipitate was washed with water, centrifuged and stored at −20° C., and had an HPLC purity of >98%. The SDS-PAGE image shown in FIG. 3 gives a comparative purity profile across the different unit operations to the purified precursor.

[0021] [Table 1]

[0022] Example 3: 59 g of purified precursor with a purity of 98.3% obtained from Example 2 was subjected to the acylation step. The acylation yield was >70% and the resulting crude liraglutide had an assay of >65% and an HPLC purity of 77%.

[0023] Example 4: The acylated crude was dissolved in equilibration buffer with pH 2.0-4.0 and loaded onto a 2.4 L pre-equilibrated C8 column. The bound product was eluted using a gradient (A: equilibration 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, fractions were pooled, achieving a pool purity of ≥99% with a step yield of 75-80%. The detection wavelength was kept at 215 nm. The chromatograph temperature was kept at 25 °C. The preparative chromatogram is shown in Figure 4.

[0024] Example 5: The RP-HPLC-II elution pool was further purified by reversed-phase high pressure chromatography (RP-HPLC-III). The RP-2 pool was diluted by adjusting the pH to 6.5-8.0 and loaded onto a pre-equilibrated 2.4 L, C8 column. The bound product was eluted using a gradient (A: equilibration buffer; B: ACN) and analyzed for HPLC purity and product content. The detection wavelength was kept at 215 nm. The chromatograph temperature was kept at 25° C. The preparative chromatogram is shown in FIG. 5.

[0025] The pH of the fractions was diluted using citrate buffer and stored at 2-8°C. Finally, the fractions were pooled, achieving a pool purity of ≥99.5% with a step yield of >90%. The pool was centrifuged to separate the pellet, followed by a water wash to isolate the purified pellet. The isolated pellet was lyophilized to produce purified liraglutide. [Table 2]

[0026] Example 6: 12170 kg of fermentation broth with a titer of 0.4 g / L was subjected to microfiltration (MF) and diafiltration (DF) followed by urea solubilization and centrifugation. The HPLC purity of the precursor determined at the end of centrifugation was 8%. It was then loaded onto a pre-equilibrated cation exchange column followed by washing and pH-based elution. The pooled purity of the CEX fractions was found to be 68-73%. Adjusting the pH of the CEX fractions from 3.5 to 5.5 produced a CEX pellet, which had a purity of 78-80%. The detection wavelength was kept at 280 nm. The chromatograph temperature was kept at 25°C. The preparative chromatogram was similar to that shown in Figure 1. The CEX pellet was then purified on a RP-HPLC-I using a 18 L C8 column. The bound precursor was eluted using a step gradient of mobile phase (A: acetate buffer; B: ACN). The detection wavelength was kept at 280 nm. The chromatograph temperature was kept at 25° C. The preparative chromatogram was similar to that shown in FIG. 2. The fractions with purity >93% were concentrated under vacuum followed by isoelectric precipitation. Centrifugation of the suspension produced a precipitate of purified precursor. The precipitate was washed with water, centrifuged and stored at −20° C. and had an HPLC purity of >98%. The CEX pellet and RP pellet were analyzed by high resolution mass spectrometry (HR-MS) to understand the identity of the impurities. The total ion chromatogram (TIC) overlay profile of CEX pellet vs RP pellet is shown in FIG. 7. Based on the HR-MS profile of the CEX pellet, the addition of hexose units (mono-hexose, dihexose, trihexose, tetrahexose) was at 0.90 RRT (12.50 min by TIC), several deletion impurities at 1.03-1.16 RRT (14.2-16 min by TIC), and high molecular weight protein (HMWP) impurities at 1.24-1.39 RRT (17-19.2 min by TIC). Most of these impurities were resolved in the RP-HPLC I step, and only small levels of deletion impurities were present in the RP-HPLC pellet, which was >98% pure.

[0027] Example 7: RP-1 pellet obtained from Example 6 was subjected to acylation step. The acylation yield was 60% and the resulting crude liraglutide had an assay of 70% and HPLC purity of >80%. The acylated crude was then purified on a 18L C8 column. The acylated crude was dissolved in equilibration buffer with pH of 2.0-4.0 and loaded onto a pre-equilibrated C8 column. The coupled product was eluted using a gradient (A: equilibration 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, fractions were pooled, achieving a pool purity of ≥99% with a step yield of 80-85%. The detection wavelength was kept at 215 nm. The chromatograph temperature was kept at 25°C. The preparative chromatogram was similar to that shown in Figure 4.

[0028] Example 8: The elution pool from Example 7 was diluted by adjusting the pH to 6.5-8.0 and loaded onto a pre-equilibrated 18 L, C8 column. The bound product was eluted using a gradient (A: equilibration buffer; B: ACN) and analyzed for HPLC purity and product content. The detection wavelength was kept at 215 nm. The chromatograph temperature was kept at 25° C. The preparative chromatogram was similar to that shown in FIG. 5. The pH of the fractions was diluted using citrate buffer and stored at 2-8° C. The fractions were finally pooled, achieving a pool purity of ≧99.5% with a step yield of 93%. The pool was centrifuged to separate the pellet, followed by a water wash to isolate the purified pellet. The isolated pellet was lyophilized to produce purified liraglutide.

Claims

1. 1. A method for purifying a crude recombinant liraglutide precursor, comprising: a. subjecting the fermentation broth to microfiltration; b. subjecting the product of step a) to diafiltration; c. solubilizing the product of step b) followed by centrifugation; d. subjecting the product of step c) to a depth filtration step; e. the filtered supernatant from step d) is subjected to a cation exchange chromatography purification step; f. subjecting the product of step e) to reverse phase high pressure liquid chromatography (RP-HPLC); and g. Isolating the purified liraglutide precursor The method comprising:

2. 2. The method of claim 1, wherein the microfiltration is carried out at a pH of 3.0 to 6.

0.

3. 2. The method of claim 1, wherein the diafiltration is carried out at a pH of 3.0 to 6.

0.

4. The method of claim 1, wherein the solubilization is carried out by the addition of urea.

5. 2. The method of claim 1, wherein the mobile phase gradient is buffered in the pH range of 3.0 to 5.

0.

6. 6. The method of claim 5, wherein the buffer is selected from glycine-HCL buffer, citrate buffer, acetate buffer, citrate-phosphate buffer, succinate buffer, and maleate buffer.

7. 1. A method for purifying a crude recombinant liraglutide precursor, comprising: a. subjecting the fermentation broth to microfiltration at pH 3.0-6.0; b. subjecting the product of step a) to diafiltration at pH 3.0-6.0; c. solubilizing the product of step b) using urea followed by centrifugation; d. subjecting the product of step c) to a depth filtration step; e. the filtered supernatant from step d) is subjected to a cation exchange chromatography purification step; f. subjecting the product of step e) to reverse phase high pressure liquid chromatography (RP-HPLC); and g. Isolating the purified liraglutide precursor wherein the mobile phase gradient is a buffer in the pH range of 3.0 to 5.0.

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