EP4181946A1 - Procédé amélioré de purification de sémaglutide - Google Patents

Procédé amélioré de purification de sémaglutide

Info

Publication number
EP4181946A1
EP4181946A1 EP21846746.2A EP21846746A EP4181946A1 EP 4181946 A1 EP4181946 A1 EP 4181946A1 EP 21846746 A EP21846746 A EP 21846746A EP 4181946 A1 EP4181946 A1 EP 4181946A1
Authority
EP
European Patent Office
Prior art keywords
semaglutide
phosphate
sodium
purification
buffer
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
EP21846746.2A
Other languages
German (de)
English (en)
Inventor
Colin Thomas DEWAR
Peter Mccormack
Philip Gaffney
Karthik Ramaswamy
Ravi Ram Chandrashekar ELATI
Yagna Kiran Kumar Komaravolu
Ramesh BHUPATHIRAJU
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Dr Reddys Laboratories Ltd
Original Assignee
Dr Reddys Laboratories Ltd
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Dr Reddys Laboratories Ltd filed Critical Dr Reddys Laboratories Ltd
Publication of EP4181946A1 publication Critical patent/EP4181946A1/fr
Pending legal-status Critical Current

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Classifications

    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the present application relates to improved and effective purification processes for semaglutide.
  • the present invention is also related to stable semaglutide.
  • the present invention is also related to stable semaglutide comprising phosphate content in the range between 5% to 15% w/w and sodium content in the range between about 4% to about 10% w/w.
  • Semaglutide is a GLP-1 analogue with 94% sequence homology to human GLP-1.
  • GLP-1 is a physiological hormone that has multiple actions on glucose, mediated by the GLP-1 receptors.
  • Semaglutide proprietary name OZEMPIC ® , developed by Novo Nordisk and first approved by USFDA on 05 December 2017 as a GLP-1 receptor agonist and indicated as an adjunct to diet and exercise to improve glycemic control in adults with type 2 diabetes mellitus. OZEMPIC ® , 2 mg/1.5 ml.
  • Semaglutide is chemically known as N- 26 -[2-(2-[2-(2-[2-(2-[2-(2-[4-(17-Carboxyhepta- decanoylamino)-4(S)-carboxybutyrylamino]ethoxy)ethoxy]acetylamino)ethoxy]ethoxy)acetyl]
  • Semaglutide can be represented by the following structural Formula I:
  • WO2019120639A1 and W02020074583A2 reported the preparation of semaglutide via fermentation, solid phase peptide synthesis or fragmentation approaches.
  • glucagon-like peptides are particularly demanding due to their propensity to aggregate. It is known that glucagon and glucagon-like peptides tend to aggregate at acidic pH (e.g. European J. Biochem. 11 (1969) 37-42).
  • the present invention provides methods for the production and purification of semaglutide.
  • Literature reported various purification methods like cation and anion-exchange purification process reported in US6451987B1, US6444788B1, ion-exchange chromatography in W02005019261A1, combination of ion-exchange and RP-HPLC by employing Tris as a buffering agent or an additive and organic modifiers in loading solution in US8710181, counter-current purification system in US9441028, RP-HPLC under involving pH adjustment in a step-wise manner in US9422330, using metal ions in US9447163, simulated chromatographic separations using mathematical model in US9766217.
  • the present invention is related to a process for purification of semaglutide from a composition comprising semaglutide and one or more impurities, comprising the steps of: a) subjecting a composition comprising semaglutide and one or more impurities to first reversed phase HPLC purification, wherein a hydrocarbon bonded silica is used as a stationary phase, using mobile phase A, comprising aqueous basic phosphate buffer at a pH between about 7.5 to 8.5, and mobile phase B comprising organic solvent, and then eluting the desired peptide fractions; b) subjecting the pooled desired peptide fractions obtained in step a) to a second reversed phase HPLC purification, wherein a hydrocarbon bonded silica is used as a stationary phase, using mobile phase A, comprising acidic purification at a pH between about 2.5 to 3.5, and mobile phase B comprising organic solvent, and then eluting the desired peptide fractions; c) subjecting the pooled desired bonded silica
  • the pure isolated semaglutide comprising phosphate content in the range between about 5 to about 15% w/w.
  • the pure isolated semaglutide comprising phosphate content in the range between about 5 to about 15% w/w and sodium content in the range between about 4 to about 10% w/w.
  • basic phosphate buffer can be selected from sodium dihydrogen phosphate, disodium hydrogen phosphate or sodium phosphate.
  • the present invention is related to the pure isolated semaglutide comprising phosphate content in the range between about 5 to about 15% w/w.
  • the present invention is related to the pure isolated semaglutide comprising phosphate content in the range between about 5 to about 15% w/w and sodium content in the range between about 4 to about 10% w/w.
  • the present invention is related to stable semaglutide comprising phosphate content in the range between 5 to 15% w/w and sodium content in the range between about 4 to about 10% w/w.
  • the present invention is related to the pure stable semaglutide comprising D-enantiomer content of amino acids-Aspartic acid, Phenylalanine, Glutamic acid, Tyrosine, Histidine in the range between less than about 0.05% to less than about 0.30% w/w, more preferably less than about 0.05% to less than about 0.20% w/w.
  • the present invention is related to a process for purification of semaglutide from a composition comprising semaglutide and one or more impurities, comprising the steps of: a) subjecting a composition comprising semaglutide and one or more impurities to first reversed phase HPLC purification, wherein a hydrocarbon bonded silica is used as a stationary phase, using mobile phase A, comprising aqueous basic phosphate buffer at a pH between about 7.5 to 8.5, and mobile phase B comprising organic solvent, and then eluting the desired peptide fractions; b) subjecting the pooled desired peptide fractions obtained in step a) to a second reversed phase HPLC purification, wherein a hydrocarbon bonded silica is used as a stationary phase, using mobile phase A, comprising acidic purification at a pH between about 2.5 to 3.5, and mobile phase B comprising organic solvent, and then eluting the desired peptide fractions; c) subjecting the pooled desired bonded silica
  • the mobile phase A comprising aqueous basic phosphate buffer at a pH between about 7.5 to 8.5.
  • the suitable basic buffer can be selected but not limited from a group consisting of sodium dihydrogen phosphate, disodium hydrogen phosphate or sodium phosphate.
  • the mobile phase B is organic solvent which can be selected from acetonitrile, C1-C4 alcohols or suitable mixture thereof.
  • the step (b) is performed at acidic pH between about 2.5 to about 3.5.
  • the acidic purification is performed in the presence of acidic buffer and suitable ion pairing agent.
  • the suitable acidic buffer can be selected from ammonium formate and suitable ion pairing agent can be selected from TFA.
  • the mobile phase B is organic solvent which can be selected from acetonitrile, C1-C4 alcohols, or suitable mixtures thereof.
  • the pH of pooled desired peptide fractions as obtained in step (b) is adjusted to 8.0 ⁇ 0.5 by using suitable buffer.
  • the suitable buffer can be selected but not limited from a group consisting offrom sodium dihydrogen phosphate, disodium hydrogen phosphate, sodium phosphate, potassium dihydrogen phosphate, ammonium bicarbonate or a combination thereof.
  • the mobile phase A comprising aqueous basic phosphate buffer at a pH between about 7.5 to 8.5.
  • the suitable basic buffer can be selected but not limited from a group consisting offrom sodium dihydrogen phosphate, disodium hydrogen phosphate or sodium phosphate.
  • the mobile phase B is organic solvent which can be selected from acetonitrile, Cl- C4 alcohols or suitable mixture thereof.
  • step (d) of the first embodiment involves isolation of pure semaglutide from the desired pooled fractions as obtained in step (c).
  • the suitable isolation process for pure semaglutide can be selected from lyophilization or spray drying.
  • pure semaglutide has purity greater than 99.0%.
  • a crude liquid sample of semaglutide may be obtained from Solid Phase Peptide Synthesis (SPPS) or Liquid Phase Peptide Synthesis (LPPS) or a combination thereof.
  • SPPS Solid Phase Peptide Synthesis
  • LPPS Liquid Phase Peptide Synthesis
  • the crude semaglutide was obtiained by Solid Phase Peptide Synthesis (SPPS) as reported in US8129343B2, CN104356224A with or without microwave technology.
  • SPPS Solid Phase Peptide Synthesis
  • the crude liquid sample of semaglutide obtained by above method can be isolated as dried peptide by well-known methods.
  • a crude liquid sample of semaglutide is obtained by cleavage and deprotection of resin bound protected semglutide using DODT, TIPS, anisole in TFA and water.
  • the crude sample of semaglutide thus obtained maybe extracted with organic solvent(s) such as Methyl tert-butyl ether (MTBE) and the like.
  • organic solvent(s) such as Methyl tert-butyl ether (MTBE) and the like.
  • the crude sample of semaglutide in buffer solution can be subjected to purification without drying and isolation as solid.
  • the present invention relates to storage stable solution of semaglutide in buffer solution.
  • the step (b) of the first embodiment involves purification of semaglutide from a composition comprising semaglutide and one or more impurities, wherein a composition comprising semaglutide and one or more impurities may be prepared from the crude dried semaglutide or the crude liquid sample of semaglutide.
  • the step (b) of the first embodiment involves preparation of a composition comprising semaglutide and one or more impurities from crude dried semaglutide, comprising the step of:
  • step (ii) heating the solution as obtained in step (i) to remove unwanted adduct
  • step (iii) diluting the solution obtained in step (ii) with suitable solvent to give the composition comprising semaglutide and one or more impurities.
  • the suitable buffer can be selected but not limited from a group consisting of from sodium dihydrogen phosphate, disodium hydrogen phosphate, sodium phosphate, potassium dihydrogen phosphate, dipotassium hydrogen phosphate, potassium phosphate, ammonium phosphate, ammonium carbonate, ammonium chloride, ammonium bicarbonate, ammonium sulphate, ammonium hydroxide, sodium acetate, sodium carbonate, sodium chloride, sodium bicarbonate, sodium phosphate and sodium sulphate, potassium carbonate, potassium acetate, or a combination thereof.
  • the suitable solvent can be selected but not limited from a group consisting of from acetonitrile or C1-C4 alcohols, water or suitable mixtures thereof.
  • the step (b) of the first embodiment involves preparation of a composition comprising semaglutide and one or more impurities from the crude liquid sample of semaglutide, comprising the step of:
  • the suitable solvent can be selected but not limited from a group consisting of from acetonitrile, C1-C4 alcohols, water or suitable mixtures thereof.
  • one or more impurities present in the composition comprising semaglutide are unwanted components which may formed during the synthesis of semaglutide.
  • Particularly preferred types of impurities which formed during synthesis of semaglutide may exemplarily be selected from the group consisting of amino acids, peptides and derivatives thereof.
  • impurities may also result from: premature chain termination during peptide synthesis, omission or unintended addition of at leastone amino acid during peptide synthesis, incomplete removal of protecting groups, side reactions occurring during amino acid coupling or Fmoc deprotection steps, inter or intramolecular condensation reactions, side reactions during peptide cleavage from a solid support, racemization, any other type of isomer formation, deamidation, (partial)hydrolysis, and aggregate formation.
  • the pure isolated semaglutide comprising phosphate content in the range between about 5 to about 15% w/w.
  • the pure isolated semaglutide comprising phosphate content in the range between about 5 to about 15% w/w and sodium content in the range between about 4 to about 10% w/w.
  • the present invention is related to the pure isolated semaglutide comprising phosphate content in the range between about 5 to about 15% w/w.
  • the present invention is related to the pure isolated semaglutide comprising phosphate content in the range between about 5 to about 15% w/w and sodium content in the range between about 4 to about 10% w/w.
  • the present invention is related to the pure stable semaglutide comprising phosphate content in the range between about 5 to about 15% w/w and sodium content in the range between about 4 to about 10% w/w.
  • the present invention is related to the pure stable semaglutide comprising D-enantiomer content of amino acids-Aspartic acid, Phenylalanine, Glutamic acid, Tyrosine, Histidine in the range between less than about 0.05% to less than about 0.30% w/w, more preferably less than about 0.05% to less than about 0.20% w/w.
  • the pure isolated semaglutide is stable semaglutide which comprises phosphate content in the range between about 5 to about 15% w/w and sodium content in the range between about 4 to about 10% w/w.
  • the stable semaglutide has improved physical stability at large scale specifically during holding or in-use period.
  • the stable semaglutide has improved solubility of about 5.5 mg/ml in water.
  • improved physical stability and solubility of stable semaglutide makes it more compatible for making injectable product.
  • Nib refers to a-aminoisobutyric acid.
  • TFA trifluoroacetic acid
  • H3PO4 refers to phosphoric acid.
  • purification is used to designate a process by which a composition comprising semaglutide and one or more impurities is purified.
  • HPLC purity i.e.as relative peak area observed in analytical reversed phase high performance liquid chromatography (RP-HPLC) with UV detection at a wavelength between 205 and 230 nm, i.e. at the absorption maximum of the peptide bond.
  • RP-HPLC reversed phase high performance liquid chromatography
  • the value is determined as % area of a given peak area divided by the sum of the areas of all observed peaks in a chromatogram obtained by analytical RP-HPLC with UV detection at a wavelength between 205 and 230 nm.
  • hydrocarbon bonded silica refers to stationary chromatographic phases made from porous silica particles or silica gels having chemically bonded hydrocarbon moieties at their surface. It is understood that the type of chemical bond as well as the chemical nature of the bonded hydrocarbon moieties may vary.
  • a stationary phase for use with the present application may be made from porous silica particles having chemically bonded hydrocarbon moieties of 4 to 18, preferably 8 to 18, carbon atoms. Such hydrocarbon moieties are preferably linear alkyl chains.
  • hydrocarbon bonded silica have hydrocarbon moieties with four (C4), six (C6), eight (C8), ten (CIO), twelve (C12), fourteen (C14), sixteen (C16), or eighteen (C18) carbon atoms.
  • Particularly preferred types of hydrocarbon bonded silica have unbranched alkyl chains of four (C4), eight (C8), twelve (C12) or eighteen (Cl 8) carbon atoms, i.e. butyl, octyl, dodecyl, or octadecyl moieties.
  • C8 bonded silica, in particular n-octyl bonded silica, and/or Cl 8 bonded silica, in particular n-octadecyl bonded silica are even more preferred stationary phases for use in steps a) and b) of a method according to the present invention.
  • the stationary phase used in steps a) and b) may be the same or different in each of the steps.
  • Preferably the stationary phase is the same.
  • C8 bonded silica is used to designate stationary chromatographic phases made from porous silica particles or silica gels having at their surface chemically bonded C8 hydrocarbon moieties, preferably linear octyl, i.e. n-octyl, moieties.
  • Cl 8 bonded silica or “ODS” are used herein interchangeably to refer to stationary chromatographic phases made from porous silica particles or silica gels having at their surface chemically bonded C18 hydrocarbon moieties, preferably linear octadecyl, i.e.
  • n-octadecyl, moieties A wide range of hydrocarbon bonded silica materials is commercially available.
  • stationary phases which can be used in present invention are DaisogelTM C18 ODS, Daiso ODS-Bio, Daiso-ODS-A-HG C18, DaisogelTM C8-Bio, YMC ODS-A, YMC Triart C8-L, Luna C8, Luna Cl 8, KromasilTM Cl 8, and KromasilTM C8 produced by Daiso, YMC, Phenomenex, and AkzoNobel, respectively.
  • the silica particles may be of 2 to 200 micrometer, preferably 2.5 to 20micrometer, preferably 5- 15 micrometer, and most preferably 10 micrometer, indiameter and may have a pore size of 50 to 1000 A, preferably of 80 to 400 A, preferably of 100 to 300 A, most preferably of (about) lOOA.
  • all or parts of the chromatographic purification are carried out at a temperature selected from the range of 10-30 °C, preferably 15-25°C.
  • Example 1 Cleavage and extraction of crude semaglutide Resin bound protected semglutide (6 g) was charged into reactor containing DODT (2.52 mL), TIPS (0.36 mL), anisole (2.52 mL) in TFA (42 mL) and water (0.84 mL). The reaction mass was stirred for 5-20 minutes at 0-10 °C. The reaction mass temperature was raised to 10-20 °C and the reaction mass was maintained for 4-6 hours. The reaction mass was cooled to 0-10°C and water (120 mL) was charged into reaction mass. Ammonia solution (25% v/v, 54 mL) was charged and the reaction mass was quenched at 0-40°C.
  • reaction mass was cooled to 25-35 °C.
  • MTBE 60 x 3 mL was charged into the reaction mass and the reaction mass was stirred for 5-20 minutes at 25-35°C.
  • the reaction mass was filtered and the filtrate was heated with stirring to 35-45°C for 1-2 hour.
  • the layers were separated.
  • Water 120 mL was charged into the aqueous layer and the reaction mass was cooled to 25-35°C.
  • the pH of reaction mass was adjusted to pH 8.0 using 10% TFA solution (10% v/v, 4.5 mL) at 25-35°C to obtain crude semaglutide which was subjected to purification.
  • the reverse phase media C18 bonded silica (10 micron) media was equilibrated with 90:10, 4m M disodium hydrogen phosphate pH 8.25 : acetonitrile.
  • the feed containing crude semaglutide diluted with water is loaded onto the column.
  • the crude peptide was then eluted from the column using a gradient elution, with the following mobile phases:
  • Disodium Hydrogen Phosphate prepared by adding Disodium hydrogen phosphate, trifluoro acetic acid and sodium chloride into purified water), pH 8.25 (pH adjusted by liquid ammonia)
  • the pure fractions obtained from second purification step were loaded onto the reverse phase Cl 8 (10 micron) column after equilibration with 90:10 4m M disodium hydrogen phosphate pH 8: acetonitrile.
  • the feed is loaded onto the column and eluted from the column using a gradient elution, with the following mobile phases:
  • Example 3 Purification of semaglutide a) Preparation of the composition comprising semaglutide and one or more impurities from dried peptide:
  • the pure fractions obtained from first purification step were loaded onto the reverse phase C18 (10 micron) column after equilibration with 90:10 4mM disodium hydrogen phosphate pH 8: acetonitrile.
  • the feed is loaded onto the column and washed with 90:10 4m M disodium hydrogen phosphate pH 8: acetonitrile for 30 min.
  • the peptide was then eluted from the column using a gradient elution, with the following mobile phases:

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  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Biophysics (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Zoology (AREA)
  • Biochemistry (AREA)
  • Toxicology (AREA)
  • General Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Medicinal Chemistry (AREA)
  • Molecular Biology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Endocrinology (AREA)
  • Peptides Or Proteins (AREA)

Abstract

La présente invention concerne des procédés améliorés et efficaces de purification de sémaglutide. La présente invention concerne également un sémaglutide stable. La présente invention concerne en outre un sémaglutide stable comprenant une teneur en phosphate dans la plage de 5 % à 15 % p/p et une teneur en sodium dans la plage d'environ 4 % à environ 10 % p/p.
EP21846746.2A 2020-07-18 2021-07-16 Procédé amélioré de purification de sémaglutide Pending EP4181946A1 (fr)

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
IN202041030673 2020-07-18
IN202141010313 2021-03-11
PCT/IN2021/050691 WO2022018748A1 (fr) 2020-07-18 2021-07-16 Procédé amélioré de purification de sémaglutide

Publications (1)

Publication Number Publication Date
EP4181946A1 true EP4181946A1 (fr) 2023-05-24

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EP21846746.2A Pending EP4181946A1 (fr) 2020-07-18 2021-07-16 Procédé amélioré de purification de sémaglutide

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EP (1) EP4181946A1 (fr)
WO (1) WO2022018748A1 (fr)

Family Cites Families (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP3000482B1 (fr) * 2009-12-16 2021-04-21 Novo Nordisk A/S Derives glp-1 double-acylates
CN109456402A (zh) * 2018-12-31 2019-03-12 江苏诺泰澳赛诺生物制药股份有限公司 一种索玛鲁肽的合成方法

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