DESC:FIELD OF INVENTION
The present invention relates to an improved process for the preparation of Liraglutide.

BACKGROUND OF THE INVENTION
Liraglutide, having structure of Formula-I is an acylated human Glucagon-Like Peptide-1 (GLP-1) receptor agonist with 97% amino acid sequence homology to endogenous human GLP-1(7-37). GLP-1(7-37) represents < 20% of total circulating endogenous GLP-1.
H-His-Ala-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys(N-e-(N-a-Palmitoyl-L-?-glutamyl))-Glu-Phe-Ile-Ala-Trp-Leu-Val-Arg-Gly-Arg-Gly-OH
Formula-I
Liraglutide is indicated as an adjunct to diet and exercise to improve glycemic control in adults with type 2 diabetes mellitus.
US 6,268,343 claims Liraglutide and its preparation process in which reverse-phase HPLC is required for the purification of the intermediate GLP-1(7-37)-OH, followed by reaction with Na-alkanoyl-Glu(ONSu)-OtBu in liquid phase.
US 9,260,474 discloses a process of preparation Liraglutide by solid phase synthesis, involving sequential addition of amino acids to the supported resin including Fmoc-Lys (Alloc)-OH is employed for lysine; removal of Alloc protecting group of lysine side chain and coupling with Palmitoyl-Glu-OtBu; followed by de-protection and cleavage of resin to obtain crude Liraglutide and further purification to get pure Liraglutide.
CN 103864918B discloses fragment based solid phase synthesis of Liraglutide, involving sequential addition of 1-10 amino acids to the supported resin and coupling with Palmitoyl-Glu-OtB; followed by coupling of 11-19 amino acid and 20-31 amino acid sequences, de-protection and cleavage of resin to obtain crude Liraglutide and further purification to get pure Liraglutide.
In drug substance synthesis there is always a continuing need for preparation process which can obtain drug substance in high yields, more purity and has low impurities; and which is commercially feasible as well.

SUMMARY OF THE INVENTION
The present invention relates to an improved process of preparation of Liraglutide comprising;
a. coupling of Fmoc-Gly-OH to resin;
b. sequential coupling of amino acids with N-terminal protection and side chain protection based on the sequence of peptide backbone of Liraglutide till Lys20 wherein PG-Lys(Fmoc)-OH is employed for lysine to obtain PG-Lys(Fmoc)-Glu(OtBu)-Phe-Ile-Ala-Trp(Boc)-Leu-Val-Arg(Pbf)-Gly-Arg(Pbf)-Gly-Resin;
c. de-protection of Fmoc from lysine;
d. coupling of Na-Palmitoyl-L-?-glutamyl-OtBu on side chain of Lysine;
e. de-protection of PGfrom lysine;
f. coupling rest of the amino acid to obtain H-His(Trt)-Ala-Glu(Otbu)-Gly-Thr(tBu)-Ser-(tBu)-Asp(OtBu)-Val-Ser(tBu)-Ser(tBu)-Tyr(tBu)-Leu-Glu(OtBu)-Gly-Gln(Trt)-Ala-Ala-Lys(Pal-Glu(Otbu)-Glu(OtBu)-Phe-Ile-Ala-Trp(Boc)- Leu-Val- Arg(Pbf)- Gly- Arg(Pbf)- Gly-CTC-Resin;
g. cleavage of Liraglutide from the solid support and removal of amino acid protecting group using cleavage reagent to obtain crude Liraglutide;
h. Pure Liraglutide is obtained by purification and lyophilisation.

According to preferred embodiment resin is selected from the group of chlorotrityl resin (CTC), Sasrin, Wang Resin, 4-methytrityl chloride, TentaGel S, TentaGel TGA, Rink acid resin, NovaSyn TGT resin,HMPB-AM resin, 4-(2-(amino methyl)-5-methoxy)phenoxy butyric acid anchored to polymeric resin MBHA and 4-(4-(amino methyl)-3-methoxy)phenoxy butyric acid anchored to polymeric resin MBHA.
According to preferred embodiment PG is selected from the group of Alloc, Mtt, Dde and ivDde.
According to preferred embodiment cleavage reagent is TFA: TIPS: Phenol: Water.
According to preferred embodiment step h) further comprises:
i. first purification of the crude Liraglutide by dissolving in ammonium bicarbonate at pH 9-10 followed by RP-HPLC using 0.1% TFA/acetonitrile;
ii. second purification by RP-HPLC using ammonium bicarbonate in water pH:7.5-8.5/acetonitrile;
iii. lyophilisation of purified Liraglutide.
The present invention relates to an improved process of Liraglutide with more purity and high yield.
BRIEF DESCRIPTION OF ABBREVIATIONS:
Dde - 1-(4,4-dimethyl-2,6- dioxacyclohexylidene)ethyl
ivDde-1-(4,4-Dimethyl- 2,6-dioxocyclohex-1-ylidene)-3-mehtylbutyl
HBTU - 0 -Benzotriazole-N,N,N',N'-tetramethyluroniumhexafluorophosphate
Cl-HOBt - 6-chloro-hydroxy-benzotriazole
HOBt -Hydroxy benzotriazole
TBTU- o-(benzotriazol- 1-yl)-N,N,N',N'-tetramethyluronium tetrafluoroborate,
DCC- 1,3-dicyclohexylcarbodiimide
DIC – Diisopropylcarbodiimide
HBTU - o-(benzotriazol-l-yl)-l ,l ,3,3-tetramethyluroniumhexafluorophosphate
BOP-Benzotriazol-l-yl-oxy-tris(dimethylamino)phosphonium hexafluorophosphate
PyBOP - Benzotriazol- l-yloxytri(pyrrolidino)phosphonium hexafluorophosphate
PyBrOP - Bromotri(pyrrolidino)phosphonium hexafluorophosphate
PyClOP - Chlorotri(pyrrolidino)phosphonium hexafluorophosphate (PyClOP),
Oxyma - Ethyl-2-cyano-2-(hydroxyimino)acetate (Oxyma Pure),
COMU - 1-Cyano-2-ethoxy-2-oxoethylidenaminooxy)dimethylaminomorholinocarbeniumhexa fluorophosphate
DMF - N,N -dimethylformamide
DCM - Dichloromethane
THF - Tetrahydrofuran
NMP - N -Methyl pyrrolidine
TFA - Trifluoro acetic acid
EDT - Ethanedithiol
TIPS/TIS - Triisopropyl silane
DTT - Diothreitol
DMS - Dimethyl sulfide
DMSO - Dimethyl sulfoxide
MTBE - Methyltert-butylether
MeOH - Methanol
IPA - Isopropyl alcohol
CTC - Chlorotrityl chloride
Fmoc - 9-fluorenylmethoxycarbonyl
Boc- tert-butoxycarbonyl
Cbz – Benzyloxycarbonyl
Alloc- Allyloxycarbonyl
Mtt- 4-methyltrityl
DETAILED DESCRIPTION OF THE INVENTION:
The present invention provides processes for the preparation of Liraglutide of formula (I).
In one embodiment the present invention relates to an improved process of preparation of Liraglutide comprising;
b. coupling of N-terminal protected glycine to resin;
c. sequential coupling of amino acids with N-terminal protection and side chain protection based on the sequence of peptide backbone of Liraglutide till Lys20 wherein PG-Lys(Fmoc)-OH is employed for lysine to obtain PG-Lys(Fmoc)-Glu(OtBu)-Phe-Ile-Ala-Trp(Boc)-Leu-Val-Arg(Pbf)-Gly-Arg(Pbf)-Gly-Resin;
d. de-protection of Fmoc from lysine;
e. coupling of Na-Palmitoyl-L-?-glutamyl-OtBu on side chain of Lysine;
f. de-protection of PG from lysine;
g. coupling rest of the amino acid to obtain protected Liraglutide attached to resin;
h. cleavage of Liraglutide from the solid support and removal of amino acid protecting group to obtain crude Liraglutide;
i. optionally, purifying the resulting Liraglutide.
In step (a), Fmoc-Gly-resin is obtained by coupling N-terminal protected glycine to a resin; by solid-phase synthesis, followed by de-protection of Fmoc to obtain H2N-Gly-resin.
The N-terminal protecting group in present invention is selected from but not limited to a group selected from Fmoc, Boc, Cbz, and the like. Preferably Fmoc protected solid phase peptide synthesis is used.
The solid phase synthesis in present invention is carried out on a resin i.e. insoluble polymer which is acid sensitive. An acid sensitive resin is selected from a group comprising chlorotrityl resin (CTC), Sasrin, Wang Resin, 4-methytrityl chloride, TentaGel S, TentaGel TGA, Rink acid resin, NovaSyn TGT resin,HMPB-AM resin, 4-(2-(amino methyl)-5-methoxy)phenoxy butyric acid anchored to polymeric resin MBHA, 4-(4-(amino methyl)-3-methoxy)phenoxy butyric acid anchored to polymeric resin MBHA and 4-(2-(amino methyl)-3,3-dimethoxy)phenoxy butyric acid anchored to polymeric resin MBHA include, most preferred super acid labile resin is 2-chlorotrityl resins (2-CTC resin).
In one embodiment of the invention PG is selected from the group of Alloc, Mtt, Dde and ivDde. In one embodiment of the invention, in step (b) amino acids with N-terminal Fmoc protection and side chain protection are sequentially coupled followed by Fmoc de-protection till Glu21 based on the sequence of peptide backbone of Liraglutide, wherein Dde-Lys(Fmoc)-OH is employed for lysine to obtain Dde-Lys(Fmoc)-Glu(OtBu)-Phe-Ile-Ala-Trp(Boc)-Leu-Val-Arg(Pbf)-Gly-Arg(Pbf)-Gly-Resin.
The present invention relates to an improved process of preparation of Liraglutide comprising use of Dde-Lys(Fmoc)-OH as one of the starting material or as a building block in the synthesis of Liraglutide.
Dde protected primary amines are stable to 20% piperidine and TFA but are cleaved with 2% hydrazine in DMF. Thus, amino groups protected by these groups can be selectively unmasked on the solid phase without affecting the side-chain protecting groups of other residues, facilitating subsequent site-specific modification. Furthermore, the reaction can be monitored by spectrophotometry since the indazole cleavage product absorbs strongly at 290 nm.
In step (c), Fmoc de-protection from lysine is carried out to obtain Dde-Lys(NH2)-Glu(OtBu)-Phe-Ile-Ala-Trp(Boc)-Leu-Val-Arg(Pbf)-Gly-Arg(Pbf)-Gly-Resin.
In step (d), Pal-Glu(OH)-Otbu is coupled on side chain NH2 of Lysine to obtain Dde-Lys(Pal-Glu-Otbu)-Glu(OtBu)-Phe-Ile-Ala-Trp(Boc)-Leu-Val-Arg(Pbf)-Gly-Arg(Pbf)-Gly-CTC -Resin.
In step (e), de-protection of Dde on lysine from fragment obtained in step (c), using 2% hydrazine hydrate in DMF to obtain NH2-Lys(Pal-Glu-Otbu)-Glu(OtBu)-Phe-Ile-Ala-Trp(Boc)-Leu-Val-Arg(Pbf)-Gly-Arg(Pbf)-Gly- CTC-Resin. Dde deprotection also obtained by use of NH2OH‚ HCl/imidazole in NMP/ CH2Cl2 instead of 2% hydrazine hydrate in DMF.
In step (f), sequentially or in fragment coupling of following amino acids with N-terminal Fmoc protection and side chain protection followed by de-protection of Fmoc to obtain protected Liraglutide.
Fmoc-Ala-OH, Fmoc-Ala-OH, Fmoc-Gln(Trt)-OH, Fmoc-Gly-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Leu-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Val-OH, Fmoc-Asp(OtBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Phe-OH, Fmoc-Thr(tBu)-OH, Fmoc-Gly-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Ala-OH and Boc-His(Trt)-OH.
The coupling reagent used in step (a), (b) (d) and (f) of the above process of sequential coupling of amino acid comprises o-(7-azabenzotriazol-l-yl)-l,l,3,3-tetramethyluronium hexafluorophosphate (HATU), o-(benzotriazol-l-0)-l,l,3,3-tetramethyluronium hexafluoro phosphate (HBTU), o-(benzotriazol-l-yl)-l,l,3,3-tetramethyluronium tetrafluoroborate (TBTU), benzotriazole-l-yl-oxy-tris (dimethyl amino)phosphoniumhexafluorophosphate (BOP), benzo triazole-l-yl-oxy-tris-pyrrolidino phosphonium hexafluorophosphate (PyBOP), N,N-bis-(2-oxo-3-oxazolidinyl)phosphonic dichloride (BOP-C1), bromo-trispyrrolidino-phosphonium hexa fluorophosphate (PyBroP), 1,3-diisopropyl-carbodiimide (DIC), PyClOP, Oxyma pure, COMU, HOBt; preferably HOBt/DIC.
The amount of individual coupling agents used may range from about 1 to about 6 molar equivalents, per molar equivalent of resin with respect to resin loading capacity. Preferably, 3 molar equivalents of individual coupling agents per molar equivalent of the resin with respect to resin loading capacity may be used.
The solvent used in said coupling reaction is selected from DMF, DCM, NMP or DMSO in combination between them; preferably DMF.
The coupling temperature is usually in the range of from 15 to 30°C; preferably at room temperature.
De-protection of Fmoc in step in step (a), (b) (c) and (f) of the above process of the above process may be carried out with 20% piperidine in N-methyl pyrrolidone (NMP), dichloromethane (DCM) or dimethylformamide (DMF); preferably DMF. Both organic apolar aprotic solvents are routinely applied in the art for all steps of solid-phase synthesis.
Further, the following alternative protecting groups or protective groups well known in the art can be used to protect amino acids or their derivatives, which are used in the process of the present invention. Commonly employed carboxy-protection groups for Glu, Asp are e.g. Mpe, O-1-Adamantyl, O-benzyl and even simply alkyl esters may be used, though less commonly used. For sake of ease, typically and preferably tert.butyl groups are used. Tyrosine may be protected by different protection groups, e.g. tert.butyl ether or Z- or more preferably 2-Bromo-Z esters. It is equally possible to use tritylalcohol protection groups such as 2-chloro-trityl or 4-methoxy or 4, 4' methoxy-trityl groups. Preferably, it is a trityl or a tert.butyl protection group. More preferably, it is a tertiary butyl (tBu) protection group, meaning the tyrosyl side chain is modified to tertiary-butyl ether. The tBu group is only efficiently removed under strongly acidic condition. Arginine protection group may be preferably selected from the group consisting of 2,2,4,6,7-pentamethyldihydrobenzofuranyl-5-sulfonyl (Pbf), adamantyloxy-carbonyl and isobornyl-oxy-carbonyl, 2,2,5,7,8-pentamethylenchromanesulfonyl-6-sulfonyl (Pmc), 4-methoxy-2,3,6-trimethylbenzenesulfonyl (Mtr) and its 4-tert.butyl-2,3,5,6-tetramethyl homologue (Tart) or Boc, which are only cleaved under strongly acidic conditions as defined above. More preferably, it is Pbf, Pmc, Mtr, most preferably, it is Pbf; upon global deprotection of side chains under strongly acidic conditions, in usually aqueous medium, bystander-alkylation of deprotected tyrosine is not observed with Pmc,Mtr and esp. Pbf. Pbf's cleavage rate is the highest ever. Ser, Thr typically may be typically and preferably protected by e.g. tert.-butyl or trityl, most preferably tert-butyl. Other modes of protection are equally feasible, e.g. with benzyl, though less preferred since eventually requiring hydrogenolytic removal or extended incubation at strongly acidic incubation, which is both equally undesirable. Similar considerations apply to protection of Lys; typically and preferably, Lys is protected with Boc, Alloc, Mtt, ivDde, TCP. Trp must not necessarily be protected during solid-phase synthesis, though protection with typically Boc is preferred.
After the completion of the reaction, the resin may be optionally washed with solvents such as dichloromethane, dimethylformamide to remove residual reagents and byproducts. The process may be repeated, if desired.
The coupling efficiency after each coupling step may be monitored during synthesis by means of a Kaiser Test (Ninhydrin Test) or any other suitable test (HPLC). The individual coupling steps, if showing unexpectedly low coupling efficiency may also be repeated prior to proceeding for de-protection and coupling with next amino acid of the sequence.
In step (g), cleavage of Liraglutide from the solid support and removal of amino acid protecting group is carried to obtain crude Liraglutide.
Removal of amino acid side chain protection and polymer support of the peptide from the resin involves treating the protected peptide anchored to the resin with an acid and at least one scavenger. The peptide cleavage reagent used in the process of the present invention is a cocktail mixture of acid, scavengers and solvents. The acidic is preferably based on an acidic material such as TFA, and contains scavenger reagents including, but not limited to, EDT, DTT, triisopropylsilane, 2, 2’-(ethylenedioxy)diethane, acetyl cystein, DMS, phenol, cresol and thiocresol or mixture thereof and water; preferably TFA: TIS: phenol: water. The relative ratio of acidic material to scavenger to water may be from about 90% to about 95% acidic material, from about 2.5% to about 5% scavenger, and from about 2.5% to about 5% water by weight. Preferably the ratio of TFA: TIS: phenol: water is 92.5%: 2.5%: 2.5%: 2.5%.
The temperature at which the cleavage and global de-protection may be carried out ranging from about 15° C. to about 40° C. Preferably, at room temperature.
After the completion of the reaction, the reaction mixture may optionally be filtered and washed with acid or an organic solvent. Crude Liraglutide may be isolated by combining the reaction mass with an organic solvent, preferably by combining with an ether solvent. Ether solvents that may be used include but are not limited to diethyl ether, diisopropyl ether, tert-butyl methyl ether, tert-butyl ethyl ether, tert-amyl methyl ether, isopropyl ether and the like or combinations thereof. Preferably, tert-butyl methyl ether is used.
The obtained suspension may be maintained at a temperature of about 0° C. to about 15° C., preferably at a temperature of about 0° C. to about 5° C. to effect the complete precipitation of the product. The obtained precipitate may be separated using conventional techniques known in the art. One skilled in the art may appreciate that there are many ways to separate a solid from the mixture, for example it can be separated by using techniques such as filtration by gravity or by suction, centrifugation, decantation, and the like. The obtained crude product may be optionally washed with an organic solvents preferably ether and subjected to drying.
In step (h), the purification of crude Liraglutide is performed by reverse-phase high performance liquid chromatography followed by Lyophilization.
The Liraglutide is purified by applying reversed phase high performance liquid chromatography (RP-HPLC) using reverse-phase C8 column comprising a first and second chromatography steps with a mixture of an aqueous buffer or aqueous acid with an organic solvent for elution.
Purification of Liraglutide is carried out by successive Reverse Phase HPLC. The RP-HPLC is expediently performed using a commercially available silica gel sorbent as stationary phase. The elution is carried out either by isocratic condition or by gradient mode. Common mobile phases used for elution include, but not limited to, aqueous buffer comprises ammonium acetate buffer or ammonium bicarbonate, water or water containing acid such as acetic acid (0.1% to 5%) or trifluoro acetic acid (TFA), or any miscible combination of water with various organic solvents like THF, acetonitrile and methanol. The purification system preferably employs gradient elution; preferably gradient elution is performed by either increasing or decreasing the amounts of an organic modifier. Suitable organic modifiers include, but are not limited to, acetonitrile, THF, ethanol, methanol, ethanol, n-propanol or iso-propanol. Alternatively the purification also carried out by other known chromatographic purification methods
In other embodiment of the present invention, the first chromatography step is preferably carried out using TFA/ACN (acetonitrile), second chromatography step using ammonium bicarbonate in water /ACN.
In one embodiment the process according to the present invention is shown in scheme-1.
Scheme-I
Alternatively, Liraglutide can be prepared in similar fashion using Fmoc-Lys (ivDDe)-OH with required modification.
The invention is further exemplified by the following non-limiting examples, which are illustrative representing the preferred modes of carrying out the invention. The invention's scope is not limited to these specific embodiments only but should be read in conjunction with what is disclosed anywhere else in the specification together with those information and knowledge which are within the general understanding of the person skilled in the art.
Examples
Stage I: Preparation of H-His(Trt)-Ala-Glu(Otbu)-Gly-Thr(tBu)-Ser-(tBu)-Asp(OtBu)-Val-Ser(tBu)-Ser(tBu)-Tyr(tBu)-Leu-Glu(OtBu)-Gly-Gln(Trt)-Ala-Ala-Lys(Pal-Glu(Otbu)-Glu(OtBu)-Phe-Ile-Ala-Trp(Boc)- Leu-Val- Arg(Pbf)- Gly- Arg(Pbf)- Gly-CTC-Resin
2-CTC resin (25.0 g) with a substitution degree of 0.96mmol/g was added to the solid-phase reaction vesicle. Subsequently, the resin was washed twice with DCM, and swollen in DCM for 30-45 min.
Fmoc-Gly-OH (11.0 g, 1.0 eq) was dissolved in of DCM (200 ml) and activated by adding DIEA (12.8 mL, 3.0 eq) at low temperature.
The obtained activated amino acid solution was added to reaction vesicle containing swollen resin. After 2 h, resin was washed with DCM, blocked for 30 min by methanol and subsequent drying, Fmoc-Gly-CTC resin was obtained, with a detected substitution degree of 0.45-0.56 mmol/g.
Fmoc-Gly-CTC resin was washed with DMF, and left for swelling (in DMF) for 30 min.
Fmoc protection was removed by 20% Piperdine in DMF, and resin was then washed with DMF and DCM and again with DMF. The resin was tested by ninhydrin / bromophenol test, in which the removal of Fmoc was indicated by the appearance of colour of the resin.
Fmoc-Arg(Pbf)-OH (24.3g, 3.0eq), HOBt (5.0g, 3.0eq), DIC (5.8ml, 3.0eq) were dissolved in DMF (200 ml), loaded to the solid-phase reaction column and reacted at room temperature for 2-3 h. The endpoint of the reaction was determined by ninhydrin / bromophenol test, in which colourless and transparent resin indicated a complete reaction; while a colour developed by the resin indicated an incomplete reaction, for which another 1 h reaction was required. Such criteria were applied to the endpoint determination by ninhydrin / bromophenol test herein below.
The above Fmoc de-protection step and corresponding amino acid coupling step were repeated up to position Lys20 using following protected amino acid.
Fmoc-Gly-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Val-OH, Fmoc-Leu-OH, Fmoc-Trp(Boc)-OH, Fmoc-Ala-OH, Fmoc-Ile-OH, Fmoc-Phe-OH, Fmoc-Glu(OtBu)-OH, Dde-Lys(Fmoc)-OH,
The next amino acid Palmitoyl-L-Glu -Otbu was coupled using HOBt (5.0g, 3.0eq), DIC (5.8ml, 3.0eq) in DMF (200 ml). The completion of the coupling was confirmed by a ninhydrin / bromophenol test.
After coupling of Palmitoyl-L-Glu -Otbu, Dde-de-protection step was completed by using 2% hydrazine hydrate in DMF and the resin was then washed with DMF, DCM and again with DMF. The resin was tested by ninhydrin / bromophenol test, in which the removal of Dde was indicated by the appearance of colour of the resin. The above corresponding amino acid coupling step and Fmoc de-protection step were repeated sequentially and based on the sequence of peptide backbone of Liraglutide using following protected amino acids.
Fmoc-Ala-OH,Fmoc-Ala-OH, Fmoc-Gln(Trt)-OH, Fmoc-Gly-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Leu-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Val-OH, Fmoc-Asp(OtBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Phe-OH, Fmoc-Thr(tBu)-OH, Fmoc-Gly-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Ala-OH and Boc-His(Trt)-OH.
Stage II: Cleavage of Liraglutide from Resin Along with Global Deprotection
To protected Liraglutide-CTC resin (67 g), the cleavage reagent TFA: TIPS: Phenol: Water (92.5: 2.5: 2.5: 2.5) (0.6 L) was poured and the reaction was performed at room temperature for 3-4 h. After completion of the reaction, the resin was filtered out and the filtrate was collected. The resin was washed by a small amount of TFA. The obtained filtrates were combined and concentrated. The concentrated mass was precipitated with MTBE (3-4 L). The obtained precipitated product was filtered and was washed MTBE and dried under vacuum to give crude Liraglutide. (Yield: 33.0 g).
Stage III: Purification of Crude Liraglutide Using RP HPLC.
Crude Liraglutide (33.0 gm) was dissolved in 10 mM ammonium bicarbonate (50 mL) followed by addition of liq. NH3 to adjust pH 9-10.
The crude Liraglutide was purified by RP-HPLC system equipped with 80 X 250 mm reverse phase C8 DAC column using routine 0.1% TFA/acetonitrile as mobile phase at detection wavelength of 214 nm. The target fraction was collected to give the purified peptide with purity greater than 95%.
The target fraction was collected and concentrated by rotary evaporation, reloaded the concentration mass in RP-HPLC system equipped with 80 X 250 mm reverse phase C8 DAC column using 10mM ammonium bicarbonate in water pH:7.5-8.5/acetonitrile, the target fraction was collected to give the purified peptide with a purity greater than 99.0%.
The target fraction was collected and concentrated by rotary evaporation.
After lyophilisation, purified Liraglutide was obtained. (HPLC purity of > 99.0%; yield: 4.0gm).

Dated this: 28th day of October 2021 Dr. S. Ganesan
Alembic Pharmaceutical Ltd.
,CLAIMS:1. An improved process of preparation of Liraglutide comprising;
a. coupling of Fmoc-Gly-OH to resin;
b. sequential coupling of amino acids with N-terminal protection and side chain protection based on the sequence of peptide backbone of Liraglutide till Lys20 wherein PG-Lys(Fmoc)-OH is employed for lysine to obtain PG-Lys(Fmoc)-Glu(OtBu)-Phe-Ile-Ala-Trp(Boc)-Leu-Val-Arg(Pbf)-Gly-Arg(Pbf)-Gly-Resin;
c. de-protection of Fmoc from lysine;
d. coupling of Na-Palmitoyl-L-?-glutamyl-OtBu on side chain of Lysine;
e. de-protection of PGfrom lysine;
f. coupling rest of the amino acid to obtain H-His(Trt)-Ala-Glu(Otbu)-Gly-Thr(tBu)-Ser-(tBu)-Asp(OtBu)-Val-Ser(tBu)-Ser(tBu)-Tyr(tBu)-Leu-Glu(OtBu)-Gly-Gln(Trt)-Ala-Ala-Lys(Pal-Glu(Otbu)-Glu(OtBu)-Phe-Ile-Ala-Trp(Boc)- Leu-Val- Arg(Pbf)- Gly- Arg(Pbf)- Gly-CTC-Resin;
g. cleavage of Liraglutide from the solid support and removal of amino acid protecting group using cleavage reagent to obtain crude Liraglutide;
h. Pure Liraglutide is obtained by purification and lyophilisation.

2. An improved process of preparation of Liraglutide according to claim 1, wherein resin is selected from the group of chlorotrityl resin (CTC), Sasrin, Wang Resin, 4-methytrityl chloride, TentaGel S, TentaGel TGA, Rink acid resin, NovaSyn TGT resin,HMPB-AM resin, 4-(2-(amino methyl)-5-methoxy)phenoxy butyric acid anchored to polymeric resin MBHA and 4-(4-(amino methyl)-3-methoxy)phenoxy butyric acid anchored to polymeric resin MBHA.

3. An improved process of preparation of Liraglutide according to claim 1, wherein PG is selected from the group of Alloc, Mtt, Dde and ivDde.

4. An improved process of preparation of Liraglutide according to claim 1, wherein deprotection of Dde involves use of hydrazine hydrate.
5. An improved process of preparation of Liraglutide according to claim 1, wherein cleavage reagent is cocktail mixture of acid, scavengers and solvents.

6. An improved process of preparation of Liraglutide according to claim 6, wherein cocktail mixture is TFA: TIPS: Phenol: Water.

7. An improved process of preparation of Liraglutide according to claim 1, wherein step h) further comprises:
i. first purification of the crude Liraglutide by dissolving in ammonium bicarbonate at pH 9-10 followed by RP-HPLC using 0.1% TFA/acetonitrile;
ii. second purification by RP-HPLC using ammonium bicarbonate in water pH:7.5-8.5/acetonitrile;
iii. lyophilisation of purified Liraglutide.