EP4181946A1

EP4181946A1 - Improved purification process of semaglutide

Info

Publication number: EP4181946A1
Application number: EP21846746.2A
Authority: EP
Inventors: Colin Thomas DEWAR; Peter Mccormack; Philip Gaffney; Karthik Ramaswamy; Ravi Ram Chandrashekar ELATI; Yagna Kiran Kumar Komaravolu; Ramesh BHUPATHIRAJU
Original assignee: Dr Reddys Laboratories Ltd
Current assignee: Dr Reddys Laboratories Ltd
Priority date: 2020-07-18
Filing date: 2021-07-16
Publication date: 2023-05-24
Also published as: WO2022018748A1

Abstract

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.

Description

IMPROVED PURIFICATION PROCESS OF SEMAGLUTIDE FIELD OF THE INVENTION

BACKGROUND OF THE INVENTION

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. (1.34 mg/mL) injection for subcutaneous use is available in:(i) Single -patient-use pen that delivers 0.25 mg or 0.5 mg per injection, & (ii) Single-patient-use pen that delivers 1 mg per injection. Semaglutide, proprietary name RYBELSUS®, developed by Novo Nordisk was first approved by USFDA on 20 Sep 2019 as a GLP-1 receptor agonist for oral use in strength 3 mg, 7 mg and 14 mg.

Semaglutide is chemically known as N- ²⁶-[2-(2-[2-(2-[2-(2-[4-(17-Carboxyhepta- decanoylamino)-4(S)-carboxybutyrylamino]ethoxy)ethoxy]acetylamino)ethoxy]ethoxy)acetyl]

[Aib⁸,Arg³⁴]GLP-l-(7-37)peptide. The molecular formula is C187H291N45O59 and the molecular weight is 4113.58 g/mol. Semaglutide can be represented by the following structural Formula I:

H

Formula I US8129343B2 reported solid phase peptide synthesis of Semaglutide. Other patents/applications such as US8637647, US9732137, W02013098191A1, WO2016046753A1, WO2018032843A1, WO2017114191A1, WO2019170895A1, W02019170918A1,

WO2019120639A1 and W02020074583A2 reported the preparation of semaglutide via fermentation, solid phase peptide synthesis or fragmentation approaches.

Purification of glucagon-like peptides is 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.

Even though, the above mentioned prior art discloses diverse processes for purification of semaglutide, there is still a need for improved methods enabling the industrial production of highly pure semaglutide. Surprisingly, a simple method for the preparation of highly pure semaglutide has been found. It has been found that the purification process of the present application is advantageous in terms of affording peptide drug substance which is compatible for formulation such as having physical stability at large scale specifically during holding or in-use period. Other than purification of peptides, physical stability of therapeutic peptides plays an important role as they are required to have a shelf life of several years in order to be suitable for common use.

All references cited herein are incorporated by reference in their entireties for all purposes.

SUMMARY OF THE INVENTION

In the first embodiment, 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 peptide fractions obtained in step b) to a third 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; d) isolating, the pure semaglutide from the pooled desired peptide fractions obtained in step c).

In one aspect of the first embodiments, the pure isolated semaglutide comprising phosphate content in the range between about 5 to about 15% w/w.

In another aspect of first embodiment, 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.

In another aspect of first embodiment, basic phosphate buffer can be selected from sodium dihydrogen phosphate, disodium hydrogen phosphate or sodium phosphate.

In second embodiment, the present invention is related to the pure isolated semaglutide comprising phosphate content in the range between about 5 to about 15% w/w.

In third embodiment, 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.

In fourth embodiment, 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.

In fifth embodiment, 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.

DETAILED DESCRIPTION

The step (a) of the first embodiment, 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 step (a) of the first embodiment, 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. In an embodiment, 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 step (b) of the first embodiment, the mobile phase B is organic solvent which can be selected from acetonitrile, C1-C4 alcohols, or suitable mixtures thereof.

In an aspect of the first embodiment, 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 step (c) of the first embodiment, 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 step (c) of the first embodiment, the mobile phase B is organic solvent which can be selected from acetonitrile, Cl- C4 alcohols or suitable mixture thereof.

The 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.

In one aspect of the first embodiment, pure semaglutide has purity greater than 99.0%.

In an aspect of the first embodiment, 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. In an aspect the crude semaglutide was obtiained by Solid Phase Peptide Synthesis (SPPS) as reported in US8129343B2, CN104356224A with or without microwave technology. Alternatively, it can be obtained by combination of recombinant and chemical steps as reported in W02009083549A1. The crude liquid sample of semaglutide obtained by above method can be isolated as dried peptide by well-known methods.

In an aspect, 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. In an aspect the crude sample of semaglutide in buffer solution can be subjected to purification without drying and isolation as solid. In an aspect, 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:

(i) dissolving the crude dried semaglutide with a purity of around 60% - 70% in suitable buffer;

(ii) heating the solution as obtained in step (i) to remove unwanted adduct;

(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:

(i) diluting a crude liquid sample in the suitable solvent;

(ii) obtaining the composition comprising semaglutide and one or more impurities

The suitable solvent can be selected but not limited from a group consisting of from acetonitrile, C1-C4 alcohols, water or suitable mixtures thereof.

In an aspect of the first embodiment, 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. These 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.

In fourth embodiment, 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.

According to the present invention, 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. Surprisingly, present inventor found that the stable semaglutidehas improved physical stability at large scale specifically during holding or in-use period. Also, the stable semaglutide has improved solubility of about 5.5 mg/ml in water. Importantly, improved physical stability and solubility of stable semaglutide makes it more compatible for making injectable product.

Definitions

The following definitions can be used in connection with the words or phrases used in the present application unless the context indicates otherwise. Amino acids may be referred to herein by either their commonly known three letter symbols or by the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Likewise nucleotides may be referred to by their commonly accepted single-letter codes.

The term “Aib” as used herein refers to a-aminoisobutyric acid.

The term “TFA” as used herein refers to trifluoroacetic acid.

The term “H3PO4” as used herein refers to phosphoric acid.

In the context of the present invention, the term "purification" is used to designate a process by which a composition comprising semaglutide and one or more impurities is purified.

In the context of the present invention, semaglutide purity is indicated herein as "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. In other words, 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. This measure is common practice in the field, and the skilled person will routinely devise a product specific RP-HPLC protocol and perform the quantification according to the established guidelines set out in the United States Pharmacopeia. The suitability of the RP-HPLC protocol for the detection of peptidic contaminations is routinely assessed by determining the peak purity by LCMS. Under the assumption that, due to their similar structure, all peptidic components have the same absorption, the RP-HPLC purity can be used as a proxy for purity expressed as mass percentage [% (w/w)]. The skilled person is well aware of how to prepare samples for chromatographic purification.

In the context of the present application, the expression "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. For example, 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. Preferred types of 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.

In the context of the present application, the expression "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. Likewise, the terms "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.Examples of stationary phases which can be used in present invention are Daisogel™ C18 ODS, Daiso ODS-Bio, Daiso-ODS-A-HG C18, Daisogel™ C8-Bio, YMC ODS-A, YMC Triart C8-L, Luna C8, Luna Cl 8, Kromasil™ Cl 8, and Kromasil™ 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.In a preferred embodiment of the invention, 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.

Although the exemplified procedures herein illustrate the practice of the present invention in some of its embodiments, the procedures should not be construed as limiting the scope of the invention. Modifications from consideration of the specification and examples within the ambit of current scientific knowledge will be apparent to one skilled in the art.

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. The 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.

Example 2: Purification of semaglutide

The liquid crude semaglutide was diluted with water and loaded onto the column. a) First Purification (Basic):

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:

Mobile Phase A: 4m M 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)

Mobile Phase B: 100% Acetonitrile

The desired fractions having purity >90% were pooled and diluted with water and were subjected to second purification. b) Second Purification (Acidic):

The pure fractions obtained from first purification step were loaded onto reverse phase media C18 bonded silica (10 micron) column after equilibration with 90:10, 25mM ammonium formate 0.2% TFA buffer of pH 2.8 (pH adjusted using liquid ammonia) : acetonitrile. The separation was performed with gradient elution with following mobile phases: Mobile Phase A: 25mM Ammonium Formate 0.2%TFA, pH 2.8 (pH adjusted by liquid ammonia)

Mobile Phase B: 100% Acetonitrile

The desired fractions having purity >97.5% were pooled and diluted with water and were subjected to third purification. c) Third Purification (Basic):

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:

Mobile Phase A: 4m M Disodium Hydrogen Phosphate, pH 8 (pH adjusted by H3PO4)

Mobile Phase B: 100% Acetonitrile

The desired fractions having purity > 98.0% of semaglutide was pooled together and subjected to desalting and desolvatization through rotary evaporation & the concentrated solution further lyophilized to afford pure semaglutide having purity of > 98.0% (Phosphate content 5.7%w/w & Sodium content: 4.2% w/w).

Example 3: Purification of semaglutide a) Preparation of the composition comprising semaglutide and one or more impurities from dried peptide:

The crude semaglutide of around 60%purity was dissolved in 200 mM ammonium bicarbonate buffer to give a solution with a concentration of 25mg/ml crude peptide. The resulting solution was heated at 60°C for 1 hour, cooled and diluted with an 80:20 mixture of water: acetonitrile to give a solution of 12.5mg/ml peptide in 90:10 100m ammonium bicarbonate: acetonitrile. b) Preparation of the composition comprising semaglutide and one or more impurities from liquid crude semaglutide sample:

The liquid crude semaglutide was loaded directly into the HPLC pump and then onto the column at a 90:10 mix with acetonitrile. The concentration of the feed is typically ~ 2mg/mL semaglutide peptide. c) First Purification (Acidic): The reverse phase media C18 bonded silica (10 micron) media was equilibrated with 90:10 35mM ammonium formate 0.2% TFA buffer of pH 2.9 (pH adjusted using TFA) : acetonitrile. Further, above prepared solution was loaded onto the column. After loading, the column was washed with 90:10 35mM ammonium formate 0.2% TFA buffer of pH 2.9 (pH adjusted using TFA) : acetonitrile. The separation was performed with gradient elution with following mobile phases:

Mobile Phase A: 35mM Ammonium Formate 0.2%TFA, pH 2.9 (pH adjusted by TFA)

Mobile Phase B: 100% Acetonitrile

The desired fractions having purity >90% were pooled and diluted with 2M ammonium bicarbonate solution to give pure fractions of semaglutide having pH 8. d) Second Purification (Basic):

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:

Mobile Phase A: 4m M Disodium Hydrogen Phosphate, pH 8 (pH adjusted by H3PO4)

Mobile Phase B: 100% Acetonitrile

The desired fractions having purity > 99.0% of semaglutide was pooled together and subjected to desolvatization through rotary evaporation & the concentrated solution further lyophilized to afford pure semaglutide having purity of 99% (Phosphate content 5.7%w/w & Sodium content: 4.2% w/w).

Claims

Claim 1 : A process for purification of Semaglutide on reverse phase high performance liquid chromatography (RP-HPLC) comprising: a) optionally, a first purification step characterized in that chromatography purification is performed using an mobile phase comprising aqueous basic buffer at a pH between about 7.5-8.5 and organic solvents; b) a second purification step characterized in that chromatography purification is performed using a mobile phase comprising aqueous acid buffer and optionally inorganic salts at a pH <3.5 and organic solvents; and c) a third purification step characterized in that chromatography purification is performed using an mobile phase comprising aqueous basic buffer at a pH between about 7.5-8.5 and organic solvents.

Claim 2: A process for purification of Semaglutide on reverse phase high performance liquid chromatography (RP-HPLC) comprising: a) optionally, a first purification step characterized in that chromatography purification is performed using an mobile phase comprising a aqueous phosphate buffer at a pH between about 7.5-8.5 and organic solvents; b) a second purification step characterized in that chromatography purification is performed using an mobile phase comprising aqueous acid buffer and optionally inorganic salts at a pH <3.5 and organic solvents; and c) a third purification step characterized in that chromatography purification is performed using an mobile phase comprising a aqueous phosphate buffer at a pH between about 7.5-8.5 and organic solvents.

Claim 3: The process according to claims 1 or 2, wherein the aqueous acid buffer is selected from orthophosphoric acid, TFA and inorganic salt is selected from sodium chloride, sodium sulphate, ammonium chloride, ammonium acetate, ammonium sulphate, ammonium formate, potassium chloride, potassium sulphate.

Claim 4: The process according to claims 1 or 2, wherein the aqueous acid buffer optionally further comprises organic acid selected from the group citric acid, formic acid, trifluoroacetic acid, acetic acid.

Claim 5: The process according to claim 1, wherein the aqueous basic buffer is selected 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.

Claim 6: The process according to claim 2, wherein the aqueous phosphate buffer is selected from sodium dihydrogen phosphate, disodium hydrogen phosphate, sodium phosphate, potassium dihydrogen phosphate, dipotassium hydrogen phosphate, potassium phosphate, ammonium phosphate or a combination thereof.

Claim 7 : The process of claims 1 or 2, where the stationary phase in steps a) and c) are selected from Cl 8, C8 and in step b) are selected from C4, Cl 8 and C8.

Claim 8: The process of claim 6 wherein the stationary phase in steps a) and c) are C18 and in step b) is C4.

Claim 9: A process for purification of Semaglutide on reverse phase high performance liquid chromatography (RP-HPLC) comprising purifications with a mixture of aqueous buffer and an organic solvent for elution, characterized in that at least one chromatography purification is performed using an aqueous mobile phase comprising acidic buffer, optionally in combination with inorganic salts at a pH <3.5 and elution with an organic solvent.

Claim 10: The process according to Claim 8, stationary phase is run with basic buffer before elution with acid buffer.

Claim 11 : The process according to claims 1 and 2, wherein the purified fractions comprising Semaglutide after elution has a pH of 6.5-8.0.

Claim 12: The process according to claim 6, further comprising isolation of GLP-1 analogue or its derivatives by processes selected from lyophilization, pi precipitation, by addition of suitable anti-solvent or combinations thereof.

Claim 13: A method for increasing the shelf-life of Semaglutide, the method comprising treating Semaglutide with 1-6 mM aqueous basic phosphate buffer at pH 7.0-8.5.

Claim 14: Semaglutide comprising anion between 2.5-9.0% w/w and cation between 1.5- 5.0% w/w relative to the total weight of dried material.

Claim 15: A gelation/fibrillation/aggregation resistant solution, comprising Semaglutide having 2.5-9.0% w/w of phosphate and 1.5-5.0% of sodium, relative to the total weight of dried material.

Claim 16: Semaglutide according to claim 14, wherein anion is phosphate & cation is sodium. Claim 17: Isolated Semaglutide comprising about 2.5-9.0% w/w of phosphate and 1.5-5.0% w/w of sodium relative to the total weight of dried material which before lyophilization in water has a pH between 6.5-8.0.

Claim 18: A composition of Semaglutide comprising D-enantiomer content of amino acids- Aspartic acid, Phenylalanine, Glutamic acid, Tyrosine or 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.

Claim 19: A pharmaceutical composition comprising Semaglutide prepared according to claims 1 or 2, and pharmaceutically acceptable excipients. Claim 20: A pharmaceutical composition prepared by combining isolated Semaglutide comprising about 2.5-9.0% w/w of phosphate and 1.5-5.0% w/w of sodium relative to the total weight of dried material with a pharmaceutically acceptable carrier.

Claim 21: Semaglutide manufactured by a process comprising the steps of a) purifying Semaglutide using the method according to claims 1 or 2 and b) isolating Semaglutide. Claim 22: A pharmaceutical composition prepared by a process comprising the steps of a) purifying Semaglutide using the method according to claims 1 or 2, b) drying said purified Semaglutide, and c) admixing said dried Semaglutide with a pharmaceutically acceptable excipient.

Claim 23: A pharmaceutical composition comprising Semaglutide according to claim 17 and pharmaceutically acceptable excipients.