Method for preparing liraglutide by using GLP-1 analogue

Abstract

The present invention relates to a method for preparing liraglutide by using a GLP-1 analog. Crude liraglutide is prepared under optimal reaction conditions and with a specific mixing ratio by using a GLP-1 analog, a fatty acid and triethylamine, and is purified through two-step purification, and thus a high-purity liraglutide can be prepared in a high yield.

Description

Method for manufacturing liraglutide using a GLP-1 analogue

[0001] The present invention relates to a method for producing liraglutide using a GLP-1 analogue, and more particularly, to a method for producing liraglutide, comprising the steps of obtaining crude liraglutide using a GLP-1 analogue, a fatty acid, and trimethylamine; and purifying the crude liraglutide by performing a two-step purification process.

[0002] In 1983, Bell et al. discovered glucagon-like peptide-1 (GLP-1) while analyzing the gene sequence of proglucagon (PG). GLP exists in two subtypes: GLP-1 analogs and GLP-2 analogs. These have nearly identical amino acid sequences to glucagon, with approximately 35% homology between the two.

[0003] GLP-1 is a peptide hormone secreted by human enterocytes, resulting from the proteolytic cleavage of proglucagon by L-cells that produce proteases, hence its name glucagon-like peptide-1. Extensive research suggests that exogenous modulation of GLP-1 stimulates its insulin secretory effects. GLP-1 includes GLP-1(1-37), GLP-1(1-36), GLP-1(7-37), a glycine derivative, and GLP-1(7-36)-NH2, as well as other molecular forms. GLP-1 currently exists in humans in two forms: GLP-1(7-36)-NH2, which consists of 30 amino acid residues with an amidated C-terminus, and GLP-1(7-37), which consists of 31 amino acid residues.

[0004] 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. Liraglutide is a long-acting analog 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 lysine at position 26 via a glutamoyl spacer. Liraglutide (VICTOZA) developed by Novo Nordisk ® ) was first approved as a subcutaneous injection in the United States in 2010.

[0005] Liraglutide is very difficult to purify due to its long peptide chain and high hydrophobicity due to the palmitoyl group.

[0006] Several attempts to purify GLP-1 analogues, including liraglutide, have been reported in the past.

[0007] In this regard, Journal of Medicinal Chemistry 43, 1664-1669, 2000 discloses a purification process for liraglutide using reverse phase high performance liquid chromatography (RP HPLC) using a cyanopropyl column (Zorbax 300SB-CN) and a standard acetonitrile / TFA system, but the disclosed method results in a reduced purification yield of 35%.

[0008] In addition, Org. Lett., 2019, 21, 2459-2463 discloses a method for producing crude liraglutide by introducing a similar Wang Linker (4-hydroxymethylphenol) to synthesize a fragment peptide 2, coupling it with a separately synthesized fragment peptide 1, and then removing all protecting groups. However, liraglutide produced by the disclosed method takes a long time to produce, and has a crude purity of less than 20%, so there is a problem of increased production cost.

[0009] In addition, Journal of Peptide Synthesis, 2016, 22, 471-479 discloses a method for manufacturing liraglutide using a fractionated peptide and using Pal-Glu(OSu)-OtBu to introduce a Pal-Glu group to the side chain of lysine. However, the disclosed method has the problem that the purity of crude liraglutide is low (32.9%) and the manufacturing time is long, leading to an increase in unit price.

[0010] In addition, WO2013117135A1 discloses a process for purifying liraglutide by reverse phase high performance liquid chromatography (RP HPLC) using an isopropyl alcohol / TFA system, but the disclosed method includes multiple purification steps including 3 RP HPLC operations, which is a laborious process.

[0011] In addition, WO2018104922A1 discloses a method for producing crude liraglutide using inorganic salts such as MgCl2 and CuCl2 in a solid phase synthesis step. However, the disclosed method has a low yield of crude liraglutide of 6 to 18%, and has problems of increased purification time and unit cost due to multiple purifications using preparative HPLC during the purification process.

[0012] In addition, WO2017162650 discloses another method for preparing liraglutide, which involves precipitation of liraglutide peptide or precursor peptide by mixing it with an anti-solvent comprising diisopropyl ether and acetonitrile, and WO2014199397 discloses a method for obtaining liraglutide by solid phase synthesis using Wang resin, but the methods have limitations such as being highly specific, being complex to be implemented at an industrial level, or having unsatisfactory purity profile and yield of the final product or being commercially viable.

[0013] In this way, conventional methods for manufacturing liraglutide take a long time, and have the problem of low yield due to the long purification process using preparative liquid chromatography (prep-HPLC) to purify crude liraglutide, which increases the price of liraglutide.

[0014] Therefore, although there are many known synthetic methods for preparing liraglutide, there is still a need to explore new synthetic methods that are scalable and economically viable, and in particular, synthetic methods that are suitable for the preparation of liraglutide while overcoming the problems associated with the known techniques by having an improved purity profile and being applicable on an industrial scale.

[0015] Accordingly, the present inventors obtained crude liraglutide using a GLP-1 analogue, fatty acid, and trimethylamine at a specific mixing ratio and optimal reaction conditions, and performed a two-step purification process to purify it, thereby producing highly pure liraglutide in a high yield, and confirmed that the liraglutide produced by the production method of the present invention has a secondary structure equivalent to that of conventional liraglutide, thereby completing the present invention.

[0016] [Prior Art Literature]

[0017] Patent documents

[0018] (Patent Document 0001) PCT Publication No. 2013-117135

[0019] (Patent Document 0002) PCT Publication No. 2018-104922

[0020] (Patent Document 0003) PCT Publication No. 2017-162650

[0021] (Patent Document 0004) PCT Publication No. 2014-199397

[0022] Non-patent literature

[0023] (Non-patent literature 0001) Journal of Medicinal Chemistry 43, 1664-1669, 2000

[0024] (Non-patent literature 0002) Org. Lett., 2019, 21, 2459-2463

[0025] (Non-patent literature 0003) Journal of Peptide Synthesis, 2016, 22, 471-479

[0026] The present invention has been devised to solve the above problems, and provides a method for preparing liraglutide, comprising the steps of obtaining crude liraglutide using a GLP-1 analogue, a fatty acid, and trimethylamine; and purifying the crude liraglutide by performing a two-step purification process.

[0027] In one embodiment of the present invention for achieving such a task, a method for preparing liraglutide is provided, including: 1) preparing a glucagon-like peptide-1 (GLP-1) analogue; 2) reacting a fatty acid with the GLP-1 analogue of step 1); 3) reacting the reaction solution of step 2) by adding triethylamine; 4) acid precipitating the reaction solution of step 3) and recovering the precipitate to obtain crude liraglutide; and 5) purifying the crude liraglutide by performing at least two methods selected from the group consisting of preparative liquid chromatography (Prep-LC), high-performance liquid chromatography (HPLC), column chromatography, and ion exchange chromatography.

[0028] In another embodiment of the present invention, liraglutide manufactured by the above manufacturing method is provided.

[0029] The present invention has the effect of producing high-purity liraglutide in a high yield by obtaining crude liraglutide using a GLP-1 analogue, fatty acid, and trimethylamine at a specific mixing ratio and optimal reaction conditions, and purifying it by performing a two-step purification process.

[0030] Figures 1a to 1e each show the HPLC results measuring the yield of liraglutide according to the reaction ratio (1:0, 1:1, 1:2, 1:3, 1:4) of fatty acid and GLP-1 according to one embodiment (Example 1) of the present invention.

[0031] Figures 2a to 2c each show the HPLC results measuring the yield of liraglutide according to the reaction pH (pH9, pH10, pH11) of fatty acid and GLP-1 according to one embodiment (Example 1) of the present invention.

[0032] Figure 3 shows the results of RP-HPLC purity analysis of unprocessed liraglutide according to one embodiment of the present invention (Example 2-2).

[0033] Figure 4 shows the results of RP-HPLC purity analysis of liraglutide after primary purification according to one embodiment of the present invention (Example 3-1).

[0034] Figure 5 shows the results of RP-HPLC purity analysis of liraglutide after secondary purification according to one embodiment of the present invention (Example 3-2).

[0035] Figure 6 shows the results of secondary structure analysis through circular dichroism (CD) according to one embodiment of the present invention (Experimental Example 1), where Saxenda is a control drug and HKP-01 is a liraglutide sample manufactured by the manufacturing method of the present invention.

[0036] Figure 7 shows the average plasma concentration-time profile of liraglutide after a single intravenous administration of 10 μg / kg of a test substance and a control substance to a male rat according to one embodiment of the present invention (Experimental Example 2).

[0037] Unless otherwise defined, all terms (including technical and scientific terms) used herein may be used in their common sense to those of ordinary skill in the art to which the present invention pertains. Furthermore, terms defined in commonly used dictionaries are not to be interpreted ideally or excessively unless explicitly and specifically defined otherwise.

[0038] As used herein, the singular form may include the plural form unless the context clearly indicates otherwise.

[0039] When a part in this specification is said to “include” a certain component, this does not mean that it excludes other components, but rather that it may include other components, unless otherwise specifically stated.

[0040] In addition, all numbers and expressions indicating the amounts of components, reaction conditions, etc. described in this specification should be understood to be modified by the term “about” in all cases unless otherwise specified.

[0041] Additionally, the experimental procedures specified in this specification are identical to those commonly performed in the art unless specifically described otherwise.

[0042] Hereinafter, the present invention will be described in detail.

[0043] One aspect of the present invention comprises the steps of: 1) preparing a glucagon-like peptide-1 (GLP-1) analogue;

[0044] 2) A step of mixing and reacting fatty acid and the GLP-1 analog of step 1);

[0045] 3) A step of adding triethylamine to the reaction solution of step 2) and causing a reaction;

[0046] 4) A step of acid precipitating the reaction solution of step 3) and recovering the precipitate to obtain crude liraglutide; and

[0047] 5) A method for producing liraglutide is provided, comprising a step of purifying the above-mentioned unprocessed liraglutide by performing at least two methods selected from the group consisting of preparative liquid chromatography (Prep-LC), high-performance liquid chromatography (HPLC), column chromatography, and ion exchange chromatography.

[0048] In one specific example of the present invention, the GLP-1 analogue of step 1) may include an amino acid sequence having 80% or more homology with the amino acid sequence shown in SEQ ID NO: 1 below: HAEGTFTSDV SSYLEGQAAK EFIAWLVRGR G (SEQ ID NO: 1).

[0049] Specifically, it may include an amino acid sequence having a homology of 90% or more, more specifically 95% or more, and particularly specifically 97% or more. As a sequence having such homology, an amino acid sequence that represents a protein that exhibits substantially the same or corresponding efficacy as each of the above proteins is included without limitation. Furthermore, it is self-evident that amino acid sequences having such homology, in which some sequences are deleted, modified, substituted, or added, are also included within the scope of the present invention.

[0050] In one specific example of the present invention, the step of preparing the GLP-1 analogue of step 1) may include a process of purifying the GLP-1 analogue.

[0051] In one specific example of the present invention, in step 2), the fatty acid and the GLP-1 analog may be mixed at a molar ratio of 1:1 to 10, specifically, 1:1 to 9, 1:1 to 8, 1:1 to 7, 1:1 to 6, 1:1 to 5, 1:1 to 4, 1:2 to 4, and more specifically, 1:3, but is not limited thereto.

[0052] In one specific example of the present invention, the reaction of step 2) may be performed at a pH of 8 to 12, specifically at a pH of 8 to 11, at a pH of 9 to 11, more specifically at a pH of 9.1 to 10.9, at a pH of 9.2 to 10.8, at a pH of 9.3 to 10.7, at a pH of 9.4 to 10.6, at a pH of 9.5 to 10.5, at a pH of 9.6 to 10.4, at a pH of 9.7 to 10.3, at a pH of 9.8 to 10.2, at a pH of 9.9 to 10.1, and preferably at a pH of 10, but is not limited thereto.

[0053] In one specific embodiment of the present invention, the reaction of step 2) is carried out at a temperature of 1 to 40°C, specifically 1 to 39°C, 1 to 37°C, 1 to 36°C, 1 to 35°C, 1 to 34°C, 1 to 33°C, 1 to 32°C, 2 to 32°C, 3 to 32°C, 4 to 32°C, 5 to 32°C, 6 to 32°C, 7 to 32°C, 8 to 32°C, 9 to 32°C, 10 to 32°C, 11 to 32°C, 12 to 32°C, 13 to 32°C, 14 to 32°C, 15 to 32°C, 16 to 32°C, 17 to 32°C, 18 to 32°C, 19 to 32°C, 20 to It may be 32°C, 20 to 31°C, 20 to 30°C, 20 to 29°C, 20 to 28°C, 20 to 27°C, 20 to 26°C, 20 to 25°C, 20 to 24°C, 20 to 23°C, 20 to 22°C, 20 to 21°C, preferably room temperature, but is not limited thereto.

[0054] In one specific example of the present invention, the reaction of step 2) may be performed for 10 to 60 minutes, specifically, 10 to 55 minutes, 10 to 50 minutes, 10 to 45 minutes, 10 to 40 minutes, 10 to 35 minutes, 10 to 30 minutes, 11 to 30 minutes, 12 to 30 minutes, 13 to 30 minutes, 14 to 30 minutes, or 15 to 30 minutes, but is not limited thereto.

[0055] In one specific example of the present invention, in step 3), triethylamine may be added at a weight ratio of 1000:1 to 100:1 with respect to the reaction solution, specifically, at a weight ratio of 900:1 to 100:1, 800:1 to 100:1, 700:1 to 100:1, 600:1 to 100:1, 600:1 to 200:1, 600:1 to 300:1, 600:1 to 400:1, and preferably 500:1, but is not limited thereto.

[0056] In one specific example of the present invention, the reaction of step 3) is carried out at a pH of 8 to 12, specifically at a pH of 8 to 11, at a pH of 9 to 11, more specifically at a pH of 9.1 to 11, at a pH of 9.2 to 11, at a pH of 9.3 to 11, at a pH of 9.4 to 11, at a pH of 9.5 to 11, at a pH of 9.6 to 11, at a pH of 9.7 to 11, at a pH of 9.8 to 11, at a pH of 9.9 to 11, at a pH of 10 to 11, at a pH of 10.1 to 11, at a pH of 10.2 to 11, at a pH of 10.3 to 11, at a pH of 10.4 to 11, at a pH of 10.5 to 11, at a pH of 10.6 to 11, at a pH of 10.7 to 11, at a pH of 10.7 to 10.9, preferably at a pH of 10.8. It may be performed, but is not limited to this.

[0057] In one specific embodiment of the present invention, the reaction of step 3) may be carried out at a temperature of 1 to 40°C, specifically, 2 to 39°C, 3 to 38°C, 4 to 37°C, 5 to 36°C, 6 to 35°C, 7 to 34°C, 8 to 33°C, 9 to 32°C, 10 to 31°C, 11 to 30°C, 12 to 29°C, 13 to 28°C, 14 to 27°C, 15 to 26°C, 15 to 25°C, and preferably, room temperature, but is not limited thereto.

[0058] In one specific embodiment of the present invention, the reaction of step 3) may be performed for 30 to 120 minutes, specifically, 30 to 110 minutes, 30 to 100 minutes, 30 to 90 minutes, 30 to 80 minutes, 30 to 70 minutes, 35 to 70 minutes, 40 to 70 minutes, 45 to 70 minutes, 50 to 70 minutes, 55 to 70 minutes, 55 to 65 minutes, 56 to 64 minutes, 57 to 63 minutes, 58 to 62 minutes, 59 to 61 minutes, and preferably 60 minutes, but is not limited thereto.

[0059] In one specific example of the present invention, the acid precipitation of step 4) is carried out at a pH of 2 to 5, specifically, pH 2.1 to 5, pH 2.2 to 5, pH 2.3 to 5, pH 2.4 to 5, pH 2.5 to 5, pH 2.6 to 5, pH 2.7 to 5, pH 2.8 to 5, pH 2.9 to 5, pH 3 to 5, pH 3.1 to 5, pH 3.2 to 5, pH 3.3 to 5, pH 3.4 to 5, pH 3.5 to 5, pH 3.6 to 5, pH 3.7 to 5, pH 3.8 to 5, pH 3.9 to 5, pH 4 to 5, pH 4.1 to 5, pH 4.2 to 5, pH 4.3 to 5, pH 4.4 to 5, pH It may be performed at a temperature of 4.5 to 5, pH 4.6 to 5, pH 4.7 to 5, pH 4.8 to 5, and preferably pH 4.9, but is not limited thereto.

[0060] In one specific example of the present invention, step 4) may additionally include a process of deprotecting the unprocessed liraglutide.

[0061] The above deprotection process includes a) a step of concentrating the recovered precipitate (crude liraglutide); and b) a step of reacting while adjusting the pH and then stopping the reaction.

[0062] Here, in the step a), the raw liraglutide may be concentrated to 2 to 20 mg / ml, specifically, 2 to 19 mg / ml, 2 to 18 mg / ml, 2 to 17 mg / ml, 2 to 16 mg / ml, 2 to 15 mg / ml, 2 to 14 mg / ml, 2 to 13 mg / ml, 2 to 12 mg / ml, 2 to 11 mg / ml, 2 to 10 mg / ml, 3 to 10 mg / ml, 4 to 10 mg / ml, 5 to 10 mg / ml, 6 to 10 mg / ml, 6 to 9 mg / ml, 6 to 8 mg / ml, but is not limited thereto.

[0063] In addition, the pH control of step b) here is pH 8 to 14, specifically, pH 9 to 14, pH 10 to 14, pH 10 to 13, pH 11 to 13, pH 11.1 to 13, pH 11.2 to 13, pH 11.3 to 13, pH 11.4 to 13, pH 11.5 to 13, pH 11.6 to 13, pH 11.7 to 13, pH 11.8 to 13, pH 11.9 to 13, pH 12 to 13, pH 12.1 to 13, pH 12.2 to 13, pH 12.3 to 13, pH 12.4 to 13, pH 12.5 to 13, pH 12.5 to 12.9, pH 12.6 to 12.8, preferably pH 12.7, and reacted for 5 to 30 minutes, 5 to 25 minutes, 5 to 20 minutes, 10 to 20 minutes, 11 to 19 minutes, 12 to 18 minutes, 13 to 17 minutes, 14 to 16 minutes, preferably 15 minutes, and then adjusted to pH 6 to 9, specifically, pH 6.1 to 8.9, pH 6.2 to 8.8, pH 6.3 to 8.7, pH 6.4 to 8.6, pH 6.5 to 8.5, pH 6.6 to 8.4, pH 6.7 to 8.3, pH 6.8 to 8.2, pH 6.9 to 8.1, pH 7 to 8, pH 7.1 to 8, pH 7.2 to 8, pH 7.3 to 8, pH 7.4 to 8, The reaction may be stopped for 1 to 20 minutes, 2 to 19 minutes, 3 to 18 minutes, 4 to 17 minutes, 5 to 16 minutes, 6 to 15 minutes, 7 to 14 minutes, 8 to 13 minutes, 9 to 12 minutes, 9 to 11 minutes, preferably 10 minutes by titrating to pH 7.5 to 8, pH 7.6 to 8, pH 7.6 to 7.9, pH 7.6 to 7.8, preferably pH 7.7, but is not limited thereto.

[0064] In one specific example of the present invention, the raw liraglutide obtained in step 4) may contain liraglutide in an amount of 60% or more, specifically, 70% or more, 71% or more, 72% or more, 73% or more, 74% or more, 75% or more, 76% or more, 77% or more, 78% or more, 79% or more, 80% or more, and more specifically, 80 to 99%, 80 to 98%, 80 to 97%, 80 to 96%, 80 to 95%, 80 to 94%, 80 to 93%, 80 to 92%, 80 to 91%, 80 to 90%, but is not limited thereto.

[0065] In one specific embodiment of the present invention, the purification in step 5) may include a one-step and two-step purification process, and each purification process may be the same or different.

[0066] Here, the first-step purification process may be performed by ion exchange chromatography, and specifically, may be anion exchange chromatography using a salt-tolerant anion-exchange resin, but is not limited thereto.

[0067] The present invention selects anion exchange chromatography using a salt-resistant anion exchange resin as the first-step purification process, thereby shortening the purification time through a simple process and providing advantageous effects in terms of yield and cost compared to conventional purification methods that purify only with HPLC.

[0068] Additionally, the above two-step purification process may be performed using preparative liquid chromatography (Prep-LC), but is not limited thereto.

[0069] In one specific example of the present invention, the liraglutide purified in step 5) may have a purity of 98% or higher.

[0070] In the present invention, the liraglutide manufacturing method can be represented by the following chemical formula (I):

[0071] (Ⅰ).

[0072] In one embodiment of the present invention, liraglutide manufactured by the above manufacturing method is provided.

[0073] Hereinafter, the present invention will be described in more detail with reference to the following examples and experimental examples. However, the following examples and experimental examples are intended only to illustrate the present invention, and the scope of the present invention is not limited to these examples.

[0074] <Example 1> Preparation of glucagon-like peptide-1 (GLP-1) analogue

[0075] A glucagon-like peptide-1 (GLP-1) analogue (GLP-1 analogue having the amino acid sequence of SEQ ID NO: 1) used to manufacture liraglutide of the present invention was produced.

[0076] Specifically, a gene encoding a GLP-1 analogue was cloned into the pACYCDuet-1 (Nvagen) vector to construct a GLP-1 analogue expression vector. Next, the vector was transformed into BL21 (DE3) cells to obtain a cell line containing the GLP-1 analogue expression vector. The obtained production cell line was cultured in a shaking incubator at 37°C in LB liquid medium containing antibiotics. The cells in which expression was induced were pelleted by centrifugation, and the GLP-1 analogue was isolated from the supernatant obtained through cell disruption and centrifugation using affinity and hydrophobic chromatography.

[0077] <Example 2> Optimization of reaction conditions for the production of crude liraglutide.

[0078] 2-1. Optimization of the reaction ratio between fatty acids and GLP-1 analogs

[0079] To obtain high purity liraglutide, the reaction ratio of fatty acids and GLP-1 analogues for the production of crude liraglutide was optimized.

[0080] Specifically, the GLP-1 analogue purified in Example 1 was dissolved in sterilized distilled water to a concentration of 5 mg / ml, and NaOH was added to adjust the pH to 10.0±0.2. Thereafter, the fatty acid and GLP-1 analogue were mixed at different reaction molar ratios (1:0, 1:1, 1:2, 1:3, 1:4) and reacted at room temperature for 20 minutes. Next, triethylamine was added at a weight ratio of 500:1 to the reaction solution. After that, the pH was adjusted to 10.8 using NaOH, and after 1 hour of reaction at room temperature, the pH was adjusted to 4.9±0.1 to perform acid precipitation. The precipitate was then collected using a centrifuge. To deprotect the recovered precipitate (crude liraglutide), the recovered precipitate was concentrated to 7 mg / mL, adjusted to pH 12.7±0.2 with 1 M NaOH, and reacted for 15 minutes. Next, the pH was adjusted to 7.7±0.1 with 10% acetic acid, and the reaction was stopped for 10 minutes. The yield of crude liraglutide was confirmed through RP-HPLC analysis (Figs. 1a to 1e). Table 1 below shows the results.

[0081] Fatty acid:GLP-1 analogue 1:11:21:31:4 Crude liraglutide yield (%) 74828683

[0082] As a result, it was confirmed that the reaction molar ratio of fatty acid and GLP-1 analogue of 1:3, which shows a yield of up to 86%, is the optimal reaction ratio condition.

[0083] 2-2. Optimization of the pH of the reaction between fatty acids and GLP-1 analogs

[0084] To obtain high purity liraglutide, the reaction pH of fatty acids and GLP-1 analogues for the production of crude liraglutide was optimized.

[0085] Specifically, the GLP-1 analogue purified in Example 1 above was dissolved in sterilized distilled water at a concentration of 5 mg / ml, and titrated to pH 9, pH 10, and pH 11 by adding NaOH. Thereafter, the fatty acid and the GLP-1 analogue were mixed at a molar ratio of 1:3, which is the reaction molar ratio of the fatty acid and the GLP-1 analogue optimized in Example 2-1, and then crude liraglutide was prepared in the same manner as described in Example 2-1, and the yield of crude liraglutide was confirmed through RP-HPLC analysis (Figs. 2a to 2c). Table 2 below shows the results.

[0086] pH91011Liraglutide Yield (%)648779

[0087] As a result, it was confirmed that the optimal reaction pH condition was pH 10, which resulted in a yield of up to 87% for the reaction of fatty acids and GLP-1 analogues.

[0088] <Example 3> Preparation of liraglutide

[0089] 3-1. Preparation of crude liraglutide

[0090] After preparing crude liraglutide using the same method as described in Example 2-1 under the reaction molar ratio (1:3) and reaction pH (pH 10) conditions of fatty acid and GLP-1 analogue optimized in Example 2, the yield of crude liraglutide was analyzed through RP-HPLC analysis (Fig. 3).

[0091] As a result, it was confirmed that the yield of raw liraglutide was approximately 90%.

[0092] 3-2. Primary purification: salt-tolerant anion-exchange chromatography

[0093] The crude liraglutide prepared in Example 3-1 was purified and fractionated using a salt-tolerant anion-exchange resin as follows:

[0094]

[0095] After zero equilibration of the column at UV 280 nm using 5 mM ammonium acetate, 60% IPA (washing buffer) equilibration buffer, concentrated crude liraglutide was bound to the column. Substances not bound to the resin were removed by sufficiently flowing with equilibration buffer, and liraglutide was eluted at UV 280 nm using a 0% to 100% concentration gradient of IFA Elution solution. The eluted peak was analyzed by RP-HPLC (Fig. 4).

[0096] As a result, it was confirmed that the purity of liraglutide after the first purification was approximately 93% or higher.

[0097] 3-2. Secondary purification: Prep-LC

[0098] Liraglutide recovered in Example 3-2 was purified by removing impurities through a Prep-LC purification process as follows:

[0099]

[0100] Specifically, after zero equilibration at UV 280 nm, 214 nm on a 10 mM ammonium acetate, 20% EtOH, pH 7.5 column (ZORBAX 300SB-C18, Agilent), the concentrated liraglutide was bound to the column. Impurities were removed using a 0–50% concentration gradient, and liraglutide was eluted through a 50–100% concentration gradient of the eluent. The eluted peak was analyzed by RP-HPLC (Fig. 5).

[0101] As a result, it was confirmed that the purity of liraglutide after secondary purification was approximately 98% or higher.

[0102] <Experimental Example 1> Confirmation of structural equivalence

[0103] In order to confirm the structural equivalence of the purified sample of the present invention and liraglutide, structural analysis was performed using circular dichroism (CD), which is mainly used for studying optical isomers and secondary structures of proteins or nucleic acids.

[0104] Specifically, the secondary structures of liraglutide manufactured through the purification process of Example 3 and the control drug (Saxenda) were compared and analyzed. The equipment used was a JASCO J-1500 model and the analysis was performed under the following analysis conditions.

[0105] <Analysis Conditions>

[0106]

[0107]

[0108] Figure 6 shows the results of secondary structure analysis through circular dichroism, where Saxenda is a control drug and HKP-01 is a liraglutide sample manufactured by the manufacturing method of the present invention.

[0109] As a result of the analysis, as shown in Fig. 6, it was confirmed that the control drug and the test substance had equivalent secondary structures in the overlay CD spectroum.

[0110] <Experimental Example 2> Pharmacokinetic study via intravenous administration using Sprague-Dawley rats

[0111] Using 7-8 week old specific pathogen free (SPF) male rats widely used in various efficacy and toxicity tests, the rats were divided into G1 and G2 groups after a 7-day acclimation period after acquisition and administered a single intravenous dose of 10 μg / kg of the test substance and the control substance. After administration of the two substances to each group, blood samples were collected at 0, 0.25, 0.5, 1, 2, 4, 6, 8, 10, 12, 24, 30, 48, and 72 hours (total 14 points) and stored frozen. The plasma concentration of liraglutide in rat plasma was analyzed for 168 tubes (12 rats x 14 points x 1 tube) of the collected samples, and the mean concentration-time profile was confirmed (Fig. 7). Table 3 below summarizes the pharmacokinetic parameters.

[0112] Test Group C0(ng / mL)AUC last (hr*ng / mL)AUC inf (hr*ng / mL)t 1 / 2 (hr)V ss (mL / kg)CL(mL / hr / kg)G1304.85±37.07898.19±62.37932.38±66.302.637.6±2.810.8±0.8(Reference)(302.98)(896.32)(930.36)(2 .3-2.7)G2337.46±46.171002.88±89.481043.94±90.102.634.7±3.89.6±0.9(Test)(334.78)(999.27)(1040.44)(2.5-2.7)Test / Ref (%)111.49P-value (CI 95%)0.041

[0113] AUC and C0 values ​​are expressed as mean ± standard deviation with geometric mean values ​​in parentheses.

[0114] t 1 / 2 Values ​​are presented as median with range in parentheses.

[0115] Test / Ref(%) values ​​were calculated based on the geometric mean.

[0116] Figure 7 shows the mean plasma concentration-time profile of liraglutide after a single intravenous administration of 10 μg / kg of test and control substances to male rats.

[0117] As a result, the plasma concentration of liraglutide was quantifiable up to 12 hours after administration in all animals, confirming that the bioequivalence between the two substances was equivalent.

[0118] According to the present invention, by obtaining crude liraglutide using a GLP-1 analogue, fatty acid, and trimethylamine at a specific mixing ratio and optimal reaction conditions, and performing a two-step purification process to purify it, high-purity liraglutide can be manufactured in a high yield.

Claims

1. 1) Step of preparing a glucagon-like peptide-1 (GLP-1) analogue; 2) A step of mixing and reacting a fatty acid and the GLP-1 analogue of step 1); 3) A step of adding triethylamine to the reaction solution of step 2) and causing a reaction; 4) a step of precipitating the reaction solution of step 3) and recovering the precipitate to obtain crude liraglutide; and 5) A method for producing liraglutide, comprising a step of purifying the raw liraglutide by performing two or more methods selected from the group consisting of preparative liquid chromatography (Prep-LC), high-performance liquid chromatography (HPLC), column chromatography, and ion exchange chromatography.

2. In paragraph 1, A method for producing liraglutide, wherein the GLP-1 analogue of step 1) comprises an amino acid sequence having at least 80% homology with the amino acid sequence shown in the following sequence number 1: HAEGTFTSDV SSYLEGQAAK EFIAWLVRGR G (SEQ ID NO: 1).

3. In paragraph 1, A method for producing liraglutide, wherein the step of preparing a GLP-1 analogue in step 1) above includes a process for purifying the GLP-1 analogue.

4. In paragraph 1, A method for producing liraglutide, wherein in step 2), fatty acids and GLP-1 analogues are mixed at a molar ratio of 1:1 to 1:

10.

5. In paragraph 4, A method for producing liraglutide, wherein in step 2), fatty acids and GLP-1 analogues are mixed at a molar ratio of 1:1 to 1:

4.

6. In paragraph 1, A method for producing liraglutide, wherein the reaction of step 2) above is performed at pH 8 to 12.

7. In paragraph 1, A method for producing liraglutide, wherein the reaction of step 2) is performed at 1 to 40°C.

8. In paragraph 1, A method for producing liraglutide, wherein the reaction of step 2) above is performed for 10 to 60 minutes.

9. In paragraph 1, A method for producing liraglutide, wherein in the above step 3), triethylamine is added in a weight ratio of 1000:1 to 100:1 with respect to the reaction solution.

10. In paragraph 1, A method for producing liraglutide, wherein the reaction of step 3) above is performed at pH 8 to 12.

11. In paragraph 1, A method for producing liraglutide, wherein the reaction of step 3) above is performed at 1 to 40°C.

12. In paragraph 1, A method for producing liraglutide, wherein the reaction of step 3) above is performed for 30 to 120 minutes.

13. In paragraph 1, A method for producing liraglutide, wherein the acid precipitation of step 4) is performed at a pH of 2 to 5.

14. In paragraph 1, A method for producing liraglutide, wherein the above step 4) further includes a process for deprotecting unprocessed liraglutide.

15. In paragraph 14, the deprotection process a) a step of concentrating the crude liraglutide; and b) A method for producing liraglutide, comprising the step of reacting while controlling pH and then stopping the reaction.

16. In paragraph 15, A method for producing liraglutide, wherein the crude liraglutide is concentrated to 2 to 20 mg / ml in step a).

17. A method for producing liraglutide in claim 15, wherein in step b), the reaction is carried out by titrating to pH 8 to 14 for 5 to 30 minutes, and then titrating to pH 6 to 9 for 1 to 20 minutes.

18. In paragraph 1, A method for manufacturing liraglutide, wherein the purification of step 5) above includes a 1-step and 2-step purification process.

19. In paragraph 18, A method for producing liraglutide, wherein the above step 1 purification process is performed by ion exchange chromatography.

20. In paragraph 19, A method for producing liraglutide, wherein the above ion exchange chromatography is anion exchange chromatography using a salt-tolerant anion-exchange resin.

21. In paragraph 18, A method for producing liraglutide, wherein the above two-step purification process is performed by preparative liquid chromatography (Prep-LC).

22. In paragraph 1, A method for producing liraglutide, wherein the liraglutide purified in the above step 5) has a purity of 98% or higher.

23. In paragraph 1, The above liraglutide is represented by the following chemical formula (Ⅰ), a method for producing liraglutide: (Ⅰ).

24. Liraglutide manufactured by any one of the manufacturing methods of claims 1 to 23.

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