WO2021129308A1 - Procédé de purification d'un analogue de glp-1 - Google Patents

Procédé de purification d'un analogue de glp-1 Download PDF

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WO2021129308A1
WO2021129308A1 PCT/CN2020/132230 CN2020132230W WO2021129308A1 WO 2021129308 A1 WO2021129308 A1 WO 2021129308A1 CN 2020132230 W CN2020132230 W CN 2020132230W WO 2021129308 A1 WO2021129308 A1 WO 2021129308A1
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glp
purification
phase
purifying
anion exchange
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PCT/CN2020/132230
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Chinese (zh)
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黄嘉成
尹传龙
陶安进
余品香
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翰宇药业（武汉）有限公司
深圳翰宇药业股份有限公司
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Publication of WO2021129308A1 publication Critical patent/WO2021129308A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/575—Hormones
    • C07K14/605—Glucagons
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/14—Extraction; Separation; Purification
    • C07K1/16—Extraction; Separation; Purification by chromatography
    • C07K1/18—Ion-exchange chromatography
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/14—Extraction; Separation; Purification
    • C07K1/16—Extraction; Separation; Purification by chromatography
    • C07K1/20—Partition-, reverse-phase or hydrophobic interaction chromatography

Definitions

  • the invention belongs to the technical field of purification methods, and specifically relates to a purification method of GLP-1 analogs.
  • GLP-1 Glucagon-like peptide-1
  • DPP-IV dipeptidyl peptidase IV
  • the peptide chain of GLP-1 analogues is longer and may be modified by side chains, there will be more impurities in the process of chemical synthesis, such as isomer impurities, amino acid deletion or added impurities, side reaction impurities, etc. .
  • impurities such as isomer impurities, amino acid deletion or added impurities, side reaction impurities, etc.
  • it is mainly purified by two or more steps of reversed-phase chromatography to prepare refined peptides with a purity of ⁇ 99.0% and a single heterogeneity of ⁇ 0.15%.
  • patent CN 106749613A uses three-step reversed-phase purification
  • patent CN 108640985A uses two-step reversed-phase purification to obtain somaglutide peptide
  • patent CN 107903318A and patent CN 109438569A both use two-step reverse-phase purification to obtain liraglutide peptide .
  • reversed-phase chromatography has strong separation capabilities, it is also accompanied by the use of a large amount of organic solvents and the production of organic waste liquids. It does not have an advantage in terms of environmental pollution, waste liquid recovery and treatment, and production costs.
  • anion exchange is used for purification in the first step
  • reverse phase chromatography is used for purification in the second step, so that somaglutide and liraglutide with purity ⁇ 99.0% and single heterogeneity ⁇ 0.15% can be obtained.
  • the anion exchange purification in the first step has a large capacity, and while achieving the enrichment of the target, it is complementary to the reverse phase purification in the second step, and removes the amino acid deletion impurities that are difficult to remove during the reverse phase purification; in addition, the anion exchange purification There is no need to use organic solvents, which reduces the cost of production and waste liquid treatment, and is also more environmentally friendly.
  • high-resolution reversed-phase chromatography was used to purify, and finally somaglutide and liraglutide with purity ⁇ 99.0% and single heterogeneity ⁇ 0.15% were obtained.
  • the purpose of the present invention is to provide a purification method of GLP-1 analogs.
  • anion exchange purification technology that is more environmentally friendly, lower solvent cost, and larger capacity is used, which achieves the enrichment of the target substance and forms a combination with reversed-phase chromatography technology.
  • the technical solution adopted by the present invention is: a purification method of GLP-1 analogs, including the following steps:
  • step 1) it also includes dissolving and filtering the GLP-1 analog.
  • the solvent used for dissolving the GLP-1 analog is one of Tris, dipotassium hydrogen phosphate, disodium hydrogen phosphate, diammonium hydrogen phosphate, ammonium bicarbonate, and ammonium acetate, with a concentration of 10-30 mM, and pH For 7-9.
  • GLP-1 analog is somaglutide or liraglutide.
  • the anion exchange purification adopts a strong anion exchange packing with a particle size of 10-30 ⁇ m and a pore size
  • the buffer salt system is one of Tris, dipotassium hydrogen phosphate, disodium hydrogen phosphate, diammonium hydrogen phosphate, ammonium bicarbonate, and ammonium acetate, with a concentration of 10-30 mM, and a pH of 7-9;
  • the salt used for elution is NaCl, and the elution gradient is 0-1.0M.
  • the acid or base corresponding to the buffer salt system is usually used to adjust the pH.
  • the pH is adjusted by one or two of hydrochloric acid, phosphoric acid, formic acid, acetic acid, potassium hydroxide, sodium hydroxide, and ammonia.
  • the anion exchange purification includes: using a buffer salt system as a balance solution to balance 5 CVs of the strong anion exchange column; after balance, load the sample; after loading the sample, first elute the 5 CVs with the balance solution, and then use the balance
  • the elution gradient of the balance solution containing 1.0M NaCl and the balance solution containing 1.0M NaCl is linear, and the elution gradient of the balance solution containing 1.0M NaCl ranges from 0 to 100%.
  • reversed-phase chromatography uses C8 reversed-phase packing with a particle size of 10 ⁇ m and a pore size
  • the organic phase purified by reverse phase chromatography is acetonitrile, the aqueous phase is 0.05-0.15% (V/V) phosphoric acid, and the elution gradient of the organic phase is 23-51%.
  • ion exchange purification is used, and the capacity is greater than that of traditional reversed-phase purification, which can enrich the target substance.
  • the first step is ion exchange purification, no organic solvents are used, no organic waste liquid is produced, and it is more environmentally friendly; the use of salts for elution is more environmentally friendly and lower in price.
  • Ion-exchange purification and reverse-phase purification are complementary to remove some amino acid deletion impurities that are difficult to remove during reverse-phase purification; after reverse-phase purification, refined peptides with purity ⁇ 99.0% and single impurities ⁇ 0.15% are obtained.
  • Figure 1 is a chromatogram of the refined peptide of Somaglutide in Example 1;
  • Figure 2 is a mass spectrum of the refined peptide of Somaglutide in Example 1;
  • Figure 3 is a chromatogram of the refined peptide of Somaglutide in Example 2.
  • Figure 4 is a mass spectrum of the refined peptide of Somaglutide in Example 2.
  • Figure 5 is a chromatogram of the refined peptide of Somaglutide in Example 3.
  • Figure 6 is a mass spectrum of Somaglutide refined peptide in Example 3.
  • Figure 7 is a chromatogram of liraglutide refined peptide in Example 4.
  • Figure 8 is a mass spectrum of the liraglutide peptide in Example 4.
  • Figure 9 is a chromatogram of liraglutide refined peptide in Example 5.
  • Figure 10 is a mass spectrum of the liraglutide peptide in Example 5.
  • Figure 11 is a chromatogram of liraglutide refined peptide in Example 6.
  • Figure 12 is a mass spectrum of the liraglutide peptide in Example 6.
  • Figure 13 is a chromatogram of somaglutide refined peptide in Comparative Example 1;
  • Figure 14 is a mass spectrum of Somaglutide refined peptide in Comparative Example 1;
  • Figure 15 is a chromatogram of liraglutide refined peptide in Comparative Example 2.
  • Figure 16 is a mass spectrum of liraglutide peptide in Comparative Example 2.
  • Sample treatment Dissolve the crude peptide of Somaglutide with 20mM Tris buffer (pH adjusted by HCl) at a concentration of about 20mg/ml; filter it with a 0.45 ⁇ m microporous membrane before purification.
  • the second step of purification the first step of purification fraction is divided into samples, and octaalkylsilane bonded silica filler is used as the stationary phase (20*250mm, 10 ⁇ m, ), with 0.15% phosphoric acid as the aqueous phase and acetonitrile as the organic phase, gradient linear elution (organic phase 23-43%, elution time 60min), flow rate 20ml/min, detection wavelength 280nm.
  • the target peak was collected, and the purity of somaglutide was ⁇ 99.0%, single impurities ⁇ 0.15%, of which Des-His 1 , Des-Aib 2 and Des-His 1 -Aib 2 impurities were all ⁇ 0.05%, and the purification yield was 55.81% .
  • the chromatogram of the refined peptide of Somaglutide is shown in Figure 1, and the mass spectrum of the refined peptide of Somaglutide is shown in Figure 2.
  • the second step of purification the first step of purification fraction is divided into samples, and the octaalkylsilane-bonded silica gel filler is used as the stationary phase (20*250mm, 10 ⁇ m, ), with 0.10% phosphoric acid as the aqueous phase and acetonitrile as the organic phase, gradient linear elution (organic phase 25-45%, elution time 80min), flow rate 20ml/min, detection wavelength 280nm.
  • the target peak was collected, and the purity of somaglutide was ⁇ 99.0%, single impurities ⁇ 0.15%, of which Des-His 1 , Des-Aib 2 and Des-His 1 -Aib 2 impurities were all ⁇ 0.05%, and the purification yield was 54.74% .
  • the chromatogram of Somaglutide refined peptide is shown in Figure 3, and the mass spectrum of Somaglutide refined peptide is shown in Figure 4.
  • disodium hydrogen phosphate buffer sodium hydroxide and phosphoric acid to adjust the pH
  • the second step of purification the first step of purification fraction is divided into samples, and octaalkylsilane bonded silica filler is used as the stationary phase (20*250mm, 10 ⁇ m, ), with 0.05% phosphoric acid as the aqueous phase and acetonitrile as the organic phase, gradient linear elution (organic phase 26-46%, elution time 100min), flow rate 20ml/min, detection wavelength 280nm.
  • the target peak was collected, and the purity of somaglutide was ⁇ 99.0%, single impurities ⁇ 0.15%, of which Des-His 1 , Des-Aib 2 and Des-His 1 -Aib 2 impurities were all ⁇ 0.05%, and the purification yield was 55.29% .
  • the chromatogram of the refined peptide of Somaglutide is shown in Figure 5, and the mass spectrum of the refined peptide of Somaglutide is shown in Figure 6.
  • the second step of purification the first step of purification fraction is divided into samples, and octaalkylsilane bonded silica filler is used as the stationary phase (20*250mm, 10 ⁇ m, ), with 0.10% phosphoric acid as the aqueous phase and acetonitrile as the organic phase, gradient linear elution (organic phase 29-49%, elution time 80min), flow rate 20ml/min, detection wavelength 280nm.
  • the target peaks were collected, and the purity of liraglutide obtained was ⁇ 99.0%, single impurities ⁇ 0.15%, and Des-His 1 and Des-His 1- Ala 2 impurities were both ⁇ 0.05%, and the purification yield was 53.85%.
  • the chromatogram of liraglutide refined peptide is shown in FIG. 7, and the mass spectrum of liraglutide refined peptide is shown in FIG. 8.
  • the second step of purification the first step of purification fraction is divided into samples, and octaalkylsilane bonded silica filler is used as the stationary phase (20*250mm, 10 ⁇ m, ), with 0.15% phosphoric acid as the aqueous phase and acetonitrile as the organic phase, gradient linear elution (organic phase 28-48%, elution time 90min), flow rate 20ml/min, detection wavelength 280nm.
  • the target peaks were collected, and the purity of liraglutide obtained was ⁇ 99.0%, single impurities ⁇ 0.15%, and the impurities of Des-His 1 and Des-His 1- Ala 2 were both ⁇ 0.05%, and the purification yield was 53.20%.
  • the chromatogram of liraglutide refined peptide is shown in FIG. 9, and the mass spectrum of liraglutide refined peptide is shown in FIG. 10.
  • the second step of purification the first step of purification fraction is divided into samples, and octaalkylsilane bonded silica filler is used as the stationary phase (20*250mm, 10 ⁇ m, ), with 0.05% phosphoric acid as the aqueous phase and acetonitrile as the organic phase, gradient linear elution (organic phase 31-51%, elution time 100min), flow rate 20ml/min, detection wavelength 280nm.
  • the target peaks were collected, and the purity of liraglutide obtained was ⁇ 99.0%, single impurities ⁇ 0.15%, and Des-His 1 and Des-His 1 -Ala 2 impurities were both ⁇ 0.05%, and the purification yield was 52.88%.
  • the chromatogram of liraglutide refined peptide is shown in Figure 11, and the mass spectrum of liraglutide refined peptide is shown in Figure 12.
  • the main difference from Examples 1 to 3 is that the first step uses reversed-phase chromatography for purification.
  • Sample processing dissolve the crude peptide of somaglutide with 0.4% aqueous ammonia solution at a concentration of about 20mg/ml; filter it with a 0.45 ⁇ m microporous membrane before purification.
  • the first step of purification Take the crude peptide solution of Somaglutide as the sample, and use the octaalkylsilane-bonded silica gel filler as the stationary phase (20*250mm, 10 ⁇ m, ), with 0.05% trifluoroacetic acid as the water phase, acetonitrile as the organic phase, the loading amount is 0.3g, and the gradient linear elution (organic phase 31 ⁇ 51%, elution time 50min), flow rate 20ml/min, detection wavelength 280nm; collect the main peak to obtain the first purification fraction of somaglutide.
  • the second step of purification the first step of purification fraction is divided into samples, and octaalkylsilane bonded silica filler is used as the stationary phase (20*250mm, 10 ⁇ m, ), with 0.15% phosphoric acid as the aqueous phase and acetonitrile as the organic phase, gradient linear elution (organic phase 23-43%, elution time 60min), flow rate 20ml/min, detection wavelength 280nm.
  • the difference from Examples 4-6 is that the first step uses reversed-phase chromatography for purification.
  • Sample processing dissolve the crude peptide of liraglutide with 0.5% ammonia solution at a concentration of about 20mg/ml; filter it with a 0.45 ⁇ m microporous membrane before purification.
  • the first step of purification take the liraglutide crude peptide solution as the sample, and use the octaalkylsilane-bonded silica gel filler as the stationary phase (20*250mm, 10 ⁇ m, ), with 0.10% trifluoroacetic acid as the water phase and acetonitrile as the organic phase, the sample volume is 0.3g, and the gradient linear elution (organic phase 37 ⁇ 57%, elution time 60min), flow rate 20ml/min, detection wavelength 280nm; collect the main peak, which is the first purification fraction of liraglutide.
  • the second step of purification the first step of purification fraction is divided into samples, and octaalkylsilane bonded silica filler is used as the stationary phase (20*250mm, 10 ⁇ m, ), with 0.10% phosphoric acid as the aqueous phase and acetonitrile as the organic phase, gradient linear elution (organic phase 29-49%, elution time 80min), flow rate 20ml/min, detection wavelength 280nm.
  • the target peaks were collected, and the purity of liraglutide obtained was ⁇ 99.0%, single impurities ⁇ 0.15%, and the impurities of Des-His 1 and Des-His 1 -Ala 2 were 0.06% and 0.11%, respectively, and the purification yield was 51.34%.
  • the chromatogram of liraglutide refined peptide is shown in FIG. 15, and the mass spectrum of liraglutide refined peptide is shown in FIG. 16.
  • Comparative example 4 Liraglutide purification (CN 109438569A)
  • Comparative Example 4 The two examples in Comparative Example 4 were purified by two-step reverse phase chromatography, and the purity and maximum single impurities of the obtained liraglutide peptide were 99.48%/0.18% and 99.40%/0.25%.

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Abstract

Est divulgué, un procédé de purification d'un analogue de GLP-1, le procédé comprenant les étapes suivantes : 1) la purification d'un analogue de GLP-1 par échange d'anions ; 2) la purification de la substance purifiée obtenue en 1) par chromatographie en phase inverse. Dans la première étape de purification, une technologie de purification par échange d'anions, qui est plus respectueuse de l'environnement, présente un coût de solvant inférieur, et dont la capacité est plus grande est utilisée, laquelle permet l'enrichissement d'une substance cible, et est complémentaire de la technologie de chromatographie en phase inverse, éliminant les impuretés de délétion d'acides aminés qui sont difficiles à éliminer pendant la purification en phase inverse, et réduisant également le coût et la pression pour la protection de l'environnement ; dans la seconde étape de purification, une technologie de purification en phase inverse à haute résolution est utilisée pour éliminer les impuretés d'isomère, les impuretés de réactions secondaires, etc. qui ne peuvent pas être éliminées au moyen d'une purification par échange d'anions, et enfin, un analogue de GLP-1 ayant une pureté ≥ 99,0 % et une impureté unique représentant ≤ 0,15 % est obtenu.
PCT/CN2020/132230 2019-12-27 2020-11-27 Procédé de purification d'un analogue de glp-1 WO2021129308A1 (fr)

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CN107903318A (zh) * 2017-12-29 2018-04-13 江苏诺泰澳赛诺生物制药股份有限公司 一种纯化利拉鲁肽的方法
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WO2005059095A2 (fr) * 2003-12-10 2005-06-30 The General Hospital Corporation Dilatation et differenciation de cellules des ilots pancreatiques
CN105176886A (zh) * 2015-10-21 2015-12-23 湖南师范大学 一株球形赖氨酸芽胞杆菌及其晶体蛋白和活性产物的应用
EP3517543A1 (fr) * 2018-01-30 2019-07-31 Bachem Holding AG Fabrication de peptides de glucagon
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