FORM2
THE PATENTS ACT, 1970
(39 of 1970)
&
THE PATENTS RULES, 2003
COMPLETE SPECIFICATION
[See section 10 and rule 13]

1. TITLE OF THE INVENTION
"An Improved Process for Fmoc Synthesis of Semaglutide"
2. APPLICANT
a) NAME: USV Private Limited
b) NATIONALITY: Indian Company incorporated under
the Companies Act 1956
c) ADDRESS: Arvind Vithal Gandhi Chowk,
B.S.D. Marg, Govandi,
Mumbai 400 088, Maharashtra, India.
3. PREAMBLE TO THE DESCRIPTION
The following specification particularly describes the invention and the
manner in which it is to be performed.

Field of the invention:
The present invention relates to an improved process for the synthesis of glucagon-like-peptide-1 (GLP-1) and analogues thereof. In particular, the present invention relates to a process for synthesis of Semaglutide using combination of solid and solution phase synthesis.
Background of the invention:
Diabetes was previously considered to only affect more affluent communities and societies. Infectious diseases were more prevalent in developing countries. However, in recent years, chronic noncommunicable diseases (NCDs) such as diabetes, hypertension and cardiovascular disorders have become the most serious health concerns in both developed and developing countries. IDF Diabetes Atlas, 2019 shows there are around 463 million adults currently living with Diabetes. Prolonged uncontrolled Diabetes Mellitus (DM) often leads to severe health complications such as Dibeteic Retinopathy, Diabetic Nephropathy & Peripheral Neuropathy etc.
Various drugs are available in the market to control blood glucose. Earlier, insulin and its various analogs .were used, which gradually shifted to different classes of anti-diabetic drugs. Anti-diabetic drugs mainly belongs to classes like insulin sensitizers (Biguanides, thiazolidinediones), seceretagogues (sulfonylureas or meglitinide derivatives), a-glucosidase inhibitors, bile acid sequestrants, dipeptidyl peptidase inhibitors, amylinomimetics, selective sodium-glucose co-transporter-2 (SGLT-2) inhibitors, glucagon-like peptide-1 agonists (GLP-1) etc. In spite of availability of multiple anti-diabetic drug options, maintaining blood glucose levels, and preventing complications arising due to improper control of blood glucose remains challenging.
GLP-1 or Glucagon like peptide-1 receptor agonists is a one such class of compounds, which stimulates insulin release and decreases the level of the anti-insulin hormone glucagon in response to increases in blood sugar levels. One of

their main advantages over other classes of anti-diabetic molecules is that they have lower risk of causing hypoglycemia. Better control over blood sugar not only prevents complications arising out of it such as dibeteic retinopathy, diabetic nephropathy, peripheral neuropathy but also prevent cardiovascular complications such as systemic atherosclerosis, cerebrovascular diseases and peripheral artery diseases.
Naturally occurring hormone GLP-1 have short duration of action due to it's N-terminal degradation by the dipeptylpeptidase IV enzyme. In order to overcome this limitation, several modifications are being developed in the native sequence of GLP-1. This has lead to prolonging the action of GLP-1 analogues gradually. Early GLP-1 analogues like Exenatide had dosing frequency of twice daily which has reduced to once daily for Liraglutide. It is reduced further for Semaglutide which has to be administered once-weekly.
In order to achieve prolonged duration of action, certain modifications in the native GLP-1 sequence were made to produce Semaglutide. The main protraction mechanism of semaglutide is albumin binding, facilitated by modification of position 26 lysine with a hydrophilic spacer and a C18 fatty di-acid. Furthermore, semaglutide is modified in position 8 to provide stabilization against degradation by the enzyme dipeptidyl-peptidase 4 (DPP-4). A minor modification was made in position 34 to ensure the attachment of only one fatty di-acid. It has the molecular weight of 4113.58 g/mol.
Semaglutide peptide has the following sequence:
H-His-Aib-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-
Ala-Lys(PEG-PEG-y-Glu-Octadeca-nedioicAcid)-Glu-Phe-Ile-Ala-Trp-Leu-Val-
Arg-Gly-Arg-Gly-OH

The structure of the Semaglutide is mentioned by diagrammatic representation as below.

GLP-1 is typically produced by yeast through recombinant gene technology. However, recent attempts have been made to use synthetic chemistry for producing GLP-1 compounds. To the main GLP-1 peptide, side chain often termed as linker is attached. Attachment of side-chain results in bulky peptide which often tends to aggregate. Property to form aggregates further pose challenge in the purification of final compound. GLP-1 analogues are used for multiple indications and market demand for these drugs is quite high. Thus the speed for large scale of production of these compounds is highly crucial. For large scale production of these compounds various approaches are used. Solid-phase peptide synthesis known to have shorter synthesis time compared to recombinant processes. Thus the robust, highly efficient & high productivity process which is equally cost-effective is the need of the hour.
Various approaches are used to produce Semaglutide using synthetic processes. Patent documents such as CN104356224A, CN109456401A, CN109456402A, CN108059666 B uses fragment based approach of GLP-1 peptide. Few other patent documents such as CN109180801A, CN108676087A use continuous synthesis of GLP-1 peptide with side-chain attachment on resin. However the processes disclosed in the prior art results in low yield and very high impurity profile. Further the tendency to aggregate further increases end quality of Semaglutide. Side-chain attachment affects the cost-effectiveness of the process. Incorporation of unnatural

amino acids such as aminoisobutyric acid and racemization prone amino acids such as Histidine further impair yield and final purity of the product.
The invention described in the present application solves the above-mentioned problems with an improved process of the synthesis of GLP-1 peptides and their analogues in general, Semaglutide in particular.
Object of the invention
An object of the present invention is to provide a simple, cost-effective, reproducible, commercially viable and industrially feasible process for preparation of GLP-1 peptide analogues, Semaglutide in particular. Another object of the present invention is to provide substantially pure Semaglutide.
Summary of the invention
According to one aspect of the present invention, there is provided an improved process for the synthesis of Semaglutide by using combination of solid and solution phase synthesis comprising the steps of
a) synthesizing GLP-l(7-37) Arg 34 Aib 8 peptide by using solid phase synthesis of Fmoc protected amino acids in a sequential manner with the application of temperature gradient during coupling,
b) optionally incorporating Boc protected dipeptide for unnatural amino acid in the Sequence of GLP-1 (7-37) Arg 34 Aib 8 peptide with the application of temperature gradient,
c) cleaving GLP-1 (7-37) Arg 34 Aib 8 peptide sequence from the solid resin followed by purification,
d) attaching side-chain to the desired amino acid in the solution phase to synthesize Semaglutide, further desalting, purifying and lyophilizing it to Semaglutide with a purity of at least 99.5% and single largest impurity not more than 0.20% and process yield of 15-20%.
Preferably, the coupling of amino acids is carried out by increasing temperature

gradient from room temperature to 40-85°C, more preferably 40-55°C. Preferably, the coupling of N terminal last 2 amino acids is carried out by increasing temperature gradient from room temperature to 60-85°C, more preferably 75-80°C. Preferably, the purification of GLP-l(7-37) Arg 34 Aib 8 peptide is carried out by RP-HPLC using gradient mode comprising orthophosphoric acid buffer system with pH in the range of 6-8, preferably 6.5 as an aqueous phase and acetonitrile as an organic phase with isolated yield of atleast 95% and purity of atleast 97%. Preferably, the purification of Semaglutide is carried out by RP-HPLC using gradient mode comprising 0.15% trifluoro acetic acid as an aqueous phase and an organic phase comprising of mixture of solvents selected from methanol, ethanol or acetonitrile preferably acetonitrile and methanol in the ratio of 1:1 to purity of ≥ 98%.
Preferably, the further purification of Semaglutide is carried out by RP-HPLC using gradient mode comprising 0.15% trifluoro acetic acid as an aqueous phase and an organic phase comprising of Sodium bicarbonate buffer of pH 8.3 as an aqueous phase and an organic phase comprising of mixture of solvents selected from methanol, ethanol or acetonitrile preferably acetonitrile to purity of > 99.5%.
Brief discription of the accompanying drawings:
Figure 1 shows analytical RP-HPLC profile on 4.6mm X 250 mm C18, 2.6μ column of chemically synthesized crude GLP-1 (7-3 7) Arg 34 Aib 8 peptide after purification by the process of EXAMPLE 4 of the present invention.
Figure 2 shows analytical RP-HPLC profile on 4.6mm X 250 mm C1 8, 2.6μ column of chemically synthesized crude Semaglutide after the process of EXAMPLE 7 of the present invention
Figure 3 shows analytical RP-HPLC profile on 4.6mm X 250 mm C18, 2.6μ column of chemically synthesized purified Semaglutide obtained after the process of EXAMPLE 8 of the present invention

Figure 4 shows analytical RP-HPLC profile on 4.6mm X 250 mm C18, 2.6μ column of chemically synthesized purified Semaglutide obtained after purification by the process of EXAMPLE 9 of the present invention
Detailed description of the invention:
The present invention relates to an improved process for the synthesis of glucagon-like-peptide-1 (GLP-1) and analogues thereof. In particular, the present invention relates to a process for synthesis of Semaglutide using combination of solid and solution phase synthesis of Fmoc protected amino acids in a sequential manner, optionally incorporating Boc protected dipeptide for unnatural amino acid in the sequence of GLP-1, cleaving GLP-1 sequence from the resin followed by purification; attaching side-chain to the desired amino acid in the solution phase to synthesize desired GLP-1 analog, purifying it to Semaglutide having an amino acid sequence as set forth in Formula I.
In a preferred embodiment, the present invention provides an improved process for synthesis of Semaglutide or salt or precursor thereof as set forth in Formula I by an orthogonal Fmoc strategy comprising of:
H-His-Aib-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-
Ala-Lys(PEG-PEG-y-Glu-Octadeca-nedioicAcid)-Glu-Phe-Ile-Ala-Trp-Leu-Val-
Arg-Gly-Arg-Gly-OH
Formula I i. covalently linking a Fmoc-Gly-OH to polystyrene based solid resin support, ii. removing the N-a-NH2 protecting group from Fmoc-Gly-solid resin support
to obtain a free N-a-NH2 group, iii. coupling the second Fmoc-Arg(Pbf)-OH to the Fmoc-Gly-solid resin support, by activating the amino acid by DIC/Oxymapure in the presence of organic solvent DMF or NMP or a combination of both, preferably NMP,

wherein the coupling is carried out by increasing temperature gradient from
room temperature to 40-85°C, more preferably 40-55°C, iv. deprotecting the Fmoc group by dcprotcctant 20% pipcridinc with additive
1% Formic acid or 0.5 M HOBT or 0.5M Oxymapure in DMF or NMP or a
combination of DMF and NMP, v. repeating steps ii), iii), iv) for assembling the GLP-1 sequence of Formula II
attached to solid resin support,
Boc-His(Boc)-Aib-Glu(Otbu)-Gly-Thr(tbu)-Phe-Thr(tbu)-Ser(tbu)-Asp(Otbu)-Val-Ser(tbu)-Ser(tbu)-Tyr(tbu)-Leu-Glu(Otbu)-Gly-Gln(trt)-Ala-Ala-Lys(Boc)-Glu(Otbu)-Phe-Ile-Ala-Trp(Boc)-Leu-Val-Arg(pbf)-Gly-Arg(pbf)-Gly-OH-Wang Resin
Formula II vi. optionally attaching last two amino acids in the sequence of GLP-1 as dipeptide Boc-His(trt)-Aib-OH, wherein the coupling is carried out by increasing temperature gradient from room temperature to 60-85°C preferably 75-80°C to generate GLP-1 sequence of Formula III attached to solid resin support.
Boc-His(trt)-Aib-Glu(Otbu)-Gly-Thr(tbu)-Phe-Thr(tbu)-Ser(tbu)-Asp(Otbu)-Val-Ser(tbu)-Ser(tbu)-Tyr(tbu)-Leu-Glu(Otbu)-Gly-Gln(trt)-Ala-Ala-
Lys(Boc)-Glu(Otbu)-Phe-Ile-Ala-Trp(Boc)-Leu-Val-Arg(pbf)-Gly-Arg(pbf)-Gly-OH- Wang Resin.
Formula III • vii. cleaving the GLP-1 sequence of Formula II or Formula III from solid resin support using cleavage cocktail consisting of TFA in the range of 80 to 95 % V/V, TIS in the range of 2.5 to 10 % V/V, Phenol in the range of 10 to 20 % V/V, preferably TFA: TIS: Phenol in the ratio of 70: 10: 20 (%V/V) at a concentration of 10-30ml/g of peptidyl resin followed by filtration and precipitation to obtain crude GLP-1 of Formula IV of not less than 70%

peptide purity,
H-His-Aib-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys-Glu-Phe-Ile-Ala-Trp-Leu-Val-Arg-Gly-Arg-Gly-OH
Formula IV
viii.purifying sequence of Formula IV of step vii) by reverse phase HPLC to a purity of at least 95%
ix. activating the side chain tBuO-Ste-Glu(AEEA-AEEA-OH)-OtBu by HOSu to generate N-hydroxysuccinimidyl tBuO-Ste-Glu(AEEA-AEEA-OH)-OtBu
x. conjugating activated side-chain of step (ix) at Lysine epsilon amino group in the synthesized GLP-1 sequence of Formula IV in a solvent mixture comprising of acetonitrile and water at GLP-1 concentration of 1-10 mg/ml and a reaction pH of 9-12.5, preferably 11.5, to generate Semaglutide sequence of Formula V
H-HIis-Aib-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-
Gln-Ala-Ala-Lys(tBuO-Ste-Glu(AEEA-AEEA-OH)-OtBu)-Glu-Phe-Ile-
Ala-Trp-Leu-Val-Arg-Gly-Arg-Gly-OH
Formula V xi. cleaving the protected Semaglutide peptide to generate crude Semaglutide
sequence of Formula I, xii. purifying Semaglutide sequence of Formula I obtained from step xi) by
reverse phase HPLC to a purity of at least 98% xiii. further purifying Semaglutide sequence of Formula I obtained from step xii)
by reverse phase HPLC to purity of at least 99.5% and single largest
impurity not more than 0.20% xiv. desalting Semaglutide sequence of Formula I obtained from step xiii) to a
sodium salt by HPLC or nanofiltration preferably by HPLC to a purity of at

least 99.5% and single largest impurity not more than 0.20% with yield of 15-20% followed by lyophilization.
Another embodiment of the. present invention provides a process wherein the activation is carried out in the coupling using DIC/Oxymapure.
Another embodiment of the present invention provides a process wherein the coupling is carried out by increasing temperature gradient from room temperature to 40-85°C, more preferably to 40-55°C.
Another embodiment of the present invention provides a process wherein the N terminal last 2 amino acids coupling is carried out by increasing temperature gradient from room temperature to 60-85°C, more preferably to 75-80°C.
Yet another embodiment of the present invention provides a process wherein cleavage of GLP-1 of sequence of formula II or "III, from the solid resin support is done wherein concentration of peptidyl resin is 10-30 ml/gram.
In yet another embodiment of the present invention provides a process wherein the purification of crude GLP-1 of sequence of formula II, to a purity of > 97% carried by chromatography is by RP-HPLC by isocratic and/or gradient mode.
In another embodiment of the present invention provides the process wherein the eluent for RP-HPLC purification of crude GLP-1 of sequence of formula II, by gradient mode comprises of orthophosphoric acid buffer system with pH in the range of 6-8, preferably 6.5 as an aqueous phase and acetonitrile as an organic phase with isolated yield of atleast 95% and purity of atleast 97%.
In still another embodiment of the present invention provides the process wherein the purification of semaglutide sequence of Formula I to purity of ≥ 98% by

chromatography is by RP-HPLC by isocratic and/or gradient mode.
In yet another embodiment of the present invention provides the process wherein the eluent for RP-HPLC purification of Semaglutide sequence of Formula I by gradient mode comprises of 0.15% trifluoro acetic acid as an aqueous phase and an organic phase comprising of mixture of solvents selected from methanol, ethanol or acetonitrile preferably acetonitrile and methanol in the ratio of 1:1.
In still another embodiment of the present invention provides the process wherein the further purification of semaglutide sequence of Formula I to purity of ≥ 99.5% with single largest impurity <0.20% by chromatography is by RP-HPLC by isocratic and/or gradient mode.
In still another embodiment of the present invention provides the process wherein the eluent for RP-HPLC purification of semaglutide sequence of Formula I by gradient mode comprises of Sodium bicarbonate buffer of pH 8.3 as an aqueous phase and an organic phase comprising of mixture of solvents selected from methanol, ethanol or acetonitrile preferably acetonitrile.
In still another embodiment of the present invention is an improved process for preparation of GLP-1 analogue Semaglutide as continuous straight chain using combination of solid-solution phase approaches of peptide synthesis. Application of temperature gradient during initial coupling of amino acid allows activation of amino acid which facilitate difficult coupling reactions. Moreover heating allows coupling of sterically hindered amino acid like aminoisobutyric acid which is known to be very difficult to couple using commercially available reagents.
In still another embodiment of the present invention is use of dipeptide Boc-His(trt)-Aib-OH for attaching last two amino acid as aminoisobutyric acid is difficult to couple due to it's sterically hindered nature.

In yet another embodiment of the present invention is use of combination of solid-solution phase approaches of peptide synthesis. While the peptide sequence of Semaglutide is synthesized using solid phase peptide synthesis, attachment of side-chain to the peptide at desired amino acid is done using solution phase synthesis. The choice of solution phase synthesis provides low cost alternative to the solid phase attachment as the excess amount of side-chain consumed in the reaction is 6-7 times lower than the amount of side-chain consumed via the traditional solid phase synthesis approach. Said side-chain attachment is carried out using mixture of water and organic solvents such as acetonitrile, ethanol, isopropyl alcohol more preferably
acetonitrile. Conversion efficiencies obtained with the solvent system are greater
.
than 90%. The reaction is favourable at Semaglutide peptide concentration as high
as 10 mg/ml and at pH ranging from 9 to 12.5 which allows for a low volume high
yield reactions output.
In yet another embodiment of the present invention provides the process of purification of Semaglutide with the overall yield of 15-20%. Drawings accompanying the specification gives the analytical purity obtained post purification by RP-HPLC at intermittent steps. Semaglutide obtained by the process of the present invention has a purity of at least 99.5% and single largest impurity not more than 0.20%.
The foregoing descriptions are merely preferred embodiments of the present invention and are not intended to limit the present invention. For those skilled in the art, the present invention may have various changes and modifications. Any modification, equivalent replacement, and improvement made within the spirit and principle of the present invention shall fall within the protection scope of the present invention.

EXAMPLES
The present invention is described in more detail below with reference to some illustrative examples. It should be understood, however, that these examples serve only to facilitate understanding of the invention but not to restrict the scope of the invention. Unless otherwise specified, the reagents and instruments used in the examples are common commercially available products.
Glossary of terms used in the specification:
AA: Amino acid
Aib: 2-aminoisobutyric acid
ACN: Acetonitrile
BOC: tert-Butyloxycarbonyl
CTC: Chloro trityl resin
DBU: l,8-Diazabicycloundec-7-ene
DIC: N, N Di-isopropylcarbodiimide
DIPE: Diisopropyl ether
DIPEA: N,N Di-sisopropyl ethylamine
DM: Diabetes Mellitus
DMAP: 4-Dimethylaminopyridine
DMF: Dimethylformamide
DMS: Dimethyl sulfide
Eq: Equivalent
FMOC: Fluorenylmethyloxycarbonyl
GLP-1: Glucagon like peptide-1
IDF: International Diabetes Federation
IR: Infrared
HBTU: Hydroxybenzotriazole Uronium Salt
HD: Hemodialysis
HoBT: Hydroxybenzotriazole monohydrate
HPLC: High performance liquid chromatography

L: Litre
MBHA: Methylbenzyhydrylamine
MDC: Methylene Dichloride
MTBE: Methyl tertiary butyl ether
NCD: Non communicable disease
NHS: N-hydroxy succinimide
NMM: N-methyl morpholine
NMP: N-Methyl pyrrolidone
pbf: 2,2,4,6,7-Pentamethyldihydrobenzofuran-5-sulfonyl
PEG: Poly ethylene glycol
PTH: Parathyroid hormone
RP-HPLC: Reverse phase high performance liquid chromatography
RT: Room temperature
SGLT-2: Sodium glucose co-transporter-2
SLI: Single largest impurity
tbu: tertiary butyl
tbu-Ste-Glu(AEEA-AEEA-OH)Otbu: (Carboxymethoxy)
ethoxy]ethylamino]-2-oxoethoxy]ethoxy]ethylamino]-1 -[(2-
methylpropan-2-yl)oxy]-1, -5-dioxopentan-2-yl]amino]-18-
oxooctadecanoic acid
trt:: trityl
TCEP: Tris(2-carboxyethyl)phosphine hydrochloride
TIS: Tri isopropyl silane
TFA: Trifluoroacetic acid
As used herein the term "analogue" refers to a polypeptide or protein means a modified polypeptide or protein wherein one or more amino acid residues of the polypeptide or protein have been substituted by other amino acid residues and/or wherein one or more amino acid residues have been deleted from the polypeptide or protein and/or wherein one or more amino acid residues have been deleted from the

polypeptide or protein and or wherein one or more amino acid residues have been added to the polypeptide or protein.
As used herein the term "deprotectant" refers to any reagent used for removing the N-α-amino protecting group in the present invention reference is hereby made to Fmoc.
As used herein the term "glucagon-like peptide" refers to the glucagon family of polypeptides, exendins and analogues thereof.
As used herein the term "GLP-1 is (7-37)Arg 34 Aib 8 peptide" refers to GLP-1 (7-37) analogue wherein the naturally occurring alanine at position 8 is substituted with α-aminoisobutyric acid and lysine at position 34 has been substituted with arginine.
As used herein the term "linker" refcrs to side-chain sequence of tBuO-Ste-Glu(AEEA-AEEA-OH)-OtBu containing fatty diacid.
As used herein the term "orthogonal Fmoc strategy" refers to an approach which uses the base-labile N-Fmoc group for protection of the a-amino function, and acid labile side chain protecting groups.
As used herein the term "polypeptide or protein" refers to a compound composed of at least two constituent amino acids connected by polypeptide bonds.
As used herein the term "protecting group" is to be understood as a protecting group known to the person skilled in the art of peptide chemistry which is introduced into the peptide by chemical modification of an amine group in order to prevent reaction on the very same amine during chemical reaction.

EXAMPLE 1: Attachment of Fmoc-GIy-OH to Wang Resin
120 g (120 mmol) of Wang resin was swollen in 960 ml of DMF for 45 min, drained and 42.81 g(144 mmol) of Fmoc Gly-OH and 22.03 g (144 mmol) of HOBT monohydrate which was dissolved in 960 ml of DMF was added to the resin. 22.61 ml (144 mmol) of DIG was added to the reaction. 2.93 gm (0.2 Eq) of DMAP dissolved in minimum volume of DMF was added to the reaction. The reaction was allowed to continue under stirring for 90 min and drained. The resin was washed with DMF and the unreacted sites on the resin were blocked using Acetic anhydride and NMM (3 Eq). The resin was finally washed with DMF followed with MDC and dried under vacuum. The Fmoc-Gly wang resin obtained was analyzed for the substitution value.
Result: Substitution: 0.24 mmole / gm
Weight of Fmoc Gly Wang resin: 136.0 g
EXAMPLE 2: Synthesis of GLP-1 (7-37)Arg 34 Aib 8 peptide
Fmoc Gly Wang resin 120 g (30 mmol) was swollen-in DMF (8 ml/gm) and the Fmoc group was unblocked using 20% piperidine with 0.5M HOBT. The deblocking cycle was carried out twice for 5 minutes respectively. The resin was washed with DMF for 6 cycles.
Fmoc Arg(pbf)-OH 58.39 g (3 Eq) was weighed along with 12.78 g (3 Eq) of Oxyma Pure and dissolved in 960 ml of DMF. The solution was added to the deblocked resin and maintained under stirring. 13.7 ml (3 Eq) of DIC was added slowly and the reaction was continued under stirring for 10-15 minutes after which the reaction was gradiently heated to obtain a temperature of 50°C within 20-40 minutes. The reaction was carried out for in polar organic solvents like DMF, DMF: MDC, NMP or combination of stated solvents to improve coupling. After attaining the temperature the reaction was continued for 10 minutes at 50°C and reaction completion was checked by Kaiser test. After completion of the coupling reaction the solution was drained and resin was washed with DMF for 6 cycles.
In similar manner the remaining amino acids were coupled sequentially.

After debocking of the Fmoc Group of Glutamic acid at position 3, weighed 52.44 g (3 Eq) of Boc- His (trt) -Aib-OH and 12.78 g (3 Eq) of Oxyma Pure and dissolved in 960 ml of DMF. The dissolved amino acid was added to the solution of the resin and maintained under stirring. 13.7 ml (3 Eq) of DIC was added slowly to the reaction. After the completion of 20 minutes, 1.5 ml of DIPEA was added to the reaction and initiated heating of the reaction to obtain a reaction mass temperature of 80°C. The reaction was maintained at 80°C for upto 2 hours. Completion of coupling was confirmed by Kaiser test.
The resin was washed with 1000 ml of MDC for 6 cycles and dried under vacuum. Weight of peptidyl resin: 280.0 g

Instrument Automated peptide synthesizer
Solid Support Wang Resin
Excess Ratio of Reagents Amino Acid (3.0 Eq) DIC (3.0 Eq) Oxyma Pure
(3.0 Eq)
Coupling solvent DMF
Coupling steps Coupling for 60 mins, 6 washes with DMF for 2 min each
Deprotection 20% piperidinein DMF with 0.5M HOBT
Deprotection step Deprotection for 7, 15 min and 15 min, 6 washes with DMF for 2 min each
EXAMPLE 3: Cleavage of GLP-1 (7-37)Arg 34 Aib 8 peptide
3.87 L of the cleavage cocktail was prepared by mixing 2.70 L of TFA, 387 ml of TIS and 774 ml of Phenol and stored at 2-8°C for 3-4 hours. 258 gram of peptidyl resin was added to the cocktail. Reaction was stirred for 3 hours and filtered. The filtrate was concentrated and precipitated in 10 volumes of cold DIPE/MTBE. The precipitate obtained was filtered and washed with DIPE/MTBE and dried under vacuum to obtain the crude peptide. Crude Purity: 75 % Yield: 110.5 grams
EXAMPLE 4: Purification of crude GLP-1 (7-37)Arg 34 Aib 8 by HPLC
The crude peptide generated in Example 3 was purified using a reverse phase HPLC

column consisting of C4, 10 micron 100 A silica.
The mobile phase consisted of mixture of Mobile phase A which consisted of a
buffer of 0.05- 045% v/v Orthophosphorir, acid, pH 6.5-7.5 with Sodium hydroxide
in water and Mobile phase B which consisted of Acetonitrile.
13.0 gm of crude GLP-1 (7-37)Arg 34 Aib 8 peptide was dissolved in buffer and the
pH was adjusted to 10 until complete dissolution was achieved. The pH was
gradually decreased to 7.5 using Ortho Phosphoric acid. The solution was filtered
and loaded on the RPHPLC column.
The peptide was purified using a gradient composition of Acetonitrile with the
mobile phase A.
The collected fraction were analyzed by an Analytical HPLC.
Purity of GLP-1 Fraction: 91 %
Yield: 3.9 grams
% Yield with respect to the GLP-1 peptide in crude sample: 95%

Instrument Preparative HPLC
Mobile Phase 0.08% Phosphoric acid pH 6.4 with NaOH
HPLC media Reverse phase C4, 10 Micron 100A
Column Dimension 76.5 mm x 250 mm
Flow rate 140ml/min
Detector wavelength 210nm
EXAMPLE 5: Linker Activation to generate NHS-Linker
14.0 g of Semaglutide linker tBuO-Ste-Glu(AEEA-AEEA-OH)-OtBu and 2.1 g of N-Hydroxysuccinimide (NHS) was dissolved in 200 ml of dried ethyl acetate. 3.75 g of DCC was dissolved in 30 ml of dried ethyl acetate and added to the above linker solution. The reaction mixture was maintained under stirring for 12 hours and analyzed for the conversion of linker to NHS linker by HPLC. The reaction mixture was filtered to separate the insoluble urea and the filtrate was concentrated under reduced pressure to obtain the NHS activated semaglutide linker as an oily mass. Conversion efficiency: 98%

EXAMPLE 6: Linker Attachment to GLP-1 (7-37)Arg 34 Aib 8 peptide
The purified GLP-1 peptide pH was adjusted to 9 using NaOH and was concentrated
on a rotary evaporator to obtain a concentration of 20 mg/ml. Acetonitrile was added
to the GLP-1 solution in a 1:1 ratio. Final concentration of GLP-1 peptide in 50%
ACN was 10 mg/ ml.
Alternatively the GLP-1 purified fractions were diluted and loaded on the RPHPLC
column and eluted using a buffer consisting of 0.1% OPA pH 8.0 with NaOH and
ACN elution was done at 50% Acetonitrile concentration.
1.3 L of GLP-1 at 10 mg/ml containing 50 % ACN was maintained under stirring at
a temperature of 10-15°C. The pH of the solution was adjusted to 12.0 using Sodium
hydroxide.
6.38 g of NHS activated Semaglutide linker was diluted 5 times with Acetonitrile
and added drop wise to the GLP-1 solution under stirring. The pH of the reaction
was always maintained above 11.0 and below 12.5. Completion of the linker
attachment reaction was estimated by HPLC analysis for the formation of
Semaglutide protected peptide.
After completion of the reaction the Solution was concentrated upto a final
concentration of 50 mg/ ml.
% Conversion of GLP-1 to Semaglutide protected peptide: 90%
EXAMPLE 7: Cleavage of Semaglutide protected peptide to generate Semaglutide crude
0.26 L of Semaglutide protected peptide (50 mg/ml) was added to a mixture of 2.2 of TFA and 0.117 L of TIS. The reaction was maintained under stirring for 15 minutes and the Solution was concentrated on a Rotary evaporator. To the oily solution, 3 L of 10% Ammonia in cold water was added. The fumes generated were evacuated and the solution was allowed to stir for 10 minutes. The pH of the solution was adjusted to 8.0 using dilute Orthophosphoric acid or ammonia. The Sample was filtered and used for purification of Semaglutide. Yield: 11.23 g

Purity of Semaglutide: 92% %Yield: 86.3%
EXAMPLE 8: Purification of Semaglutide
The semaglutide crude peptide generated in Example 7 was loaded on IIPLC
column. The mobile phase consisted of a buffer of 0.15% Trifluoro Acetic Acid and
ACN: Methanol (1:1). The peptide was purified using a gradient composition of
Acetonitrile: Methanol with the mobile phase A.
The collected fraction were analyzed by an Analytical HPLC.
Purity of Semaglutide Fraction: 98 %
EXAMPLE 9: Salt exchange of Semaglutide
Purified fraction of Semaglutide generated in example 8 was diluted with water and 5 ml of OP A was added to the solution. The pH of the solution was adjusted to 8.0 using Sodium hydroxide. 50 mM Sodium bicarbonate was added to the solution and the sample was purified by RPHPLC. The mobile phase consisted of a buffer of 30 -mM Sodium Bicarbonate pH 8.3 and Acetonitrile. The peptide was purified using a gradient composition of Acetonitrile with the mobile phase A. The collected fraction were analyzed by an Analytical HPLC. Purity of Semaglutide Fraction: 99.8 % SLI < 0.20% Yield: 2.0 grams
EXAMPLE 10: Isolation and Lyophilization of Semaglutide
Purified Semaglutide was diluted with water and loaded onto the HPLC column. The column was washed with 0.5 mM ammonium Bicarbonate. The peptide was eluted from the column using 50% ACN and Buffer A (0.5 mM ammonium Bicarbonate). The collected fraction were concentrated on a rotary evaporator and the sample was lyophilised to obtain Semaglutide. Purity: 99.8% SLI < 0.20% Yield: 2.0 grams

We Claim
1. An improved process for synthesis of Semaglutide or salt or precursor
thereof as set forth in Formula I by an orthogonal Fmoc strategy comprising
of:
H-His-Aib-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-
Gln-Ala-Ala-Lys(PEG-PEG-Y-Glu-Octadeca-nedioicAcid)-Glu-Phe-Ile-Ala-- Trp-Leu-Val-Arg-Gly-Arg-Gly-OH.
Formula I
i. covalently linking a Fmoc-Gly-OH to polystyrene based solid resin support,
ii. removing the N-a-NH2 protecting group from Fmoc-Gly-solid resin support to obtain a free N-a-NH2 group,
iii. coupling the second Fmoc-Arg(Pbf)-OH to the Fmoc-Gly-solid resin support, by activating the amino acid by DIC/Oxymapure in the presence of organic solvent DMF or NMP or a combination of both, preferably NMP, wherein the coupling is carried out by increasing temperature gradient from room temperature to 40-85°C, more preferably 40-55°C,
iv. deprotecting the Fmoc group by deprotectant 20% piperidine with additive 1% Formic acid or 0.5 M HOBT or 0.5M Oxymapure in DMF or NMP or a combination of DMF and NMP,
v. repeating steps ii), iii), iv) for assembling the GLP-1 sequence of Formula II attached to solid resin support,
Boc-His(Boc)-Aib-Glu(Otbu)-Gly-Thr(tbu)-Phe-Thr(tbu)-Ser(tbu)-Asp(Otbu)-Val-Ser(tbu)-Ser(tbu)-Tyr(tbu)-Leu-Glu(Otbu)-Gly-Gln(trt)-Ala-Ala-Lys(Boc)-Glu(Otbu)-Phe-Ile-Ala-Trp(Boc)-Leu-Val-Arg(pbf)-Gly-Arg(pbf)-Gly-OH-Wang Resin
Formula II vi. optionally attaching last two amino acids in the sequence of GLP-1 as dipeptide Boc-His(trt)-Aib-OH, wherein the coupling is carried out by

increasing temperature gradient from room temperature to 60-85°C preferably 75-80°C to generate GLP-1 sequence of Formula III attached to solid resin support,
Boc-His(trt)-Aib-Glu(Otbu)-Gly-Thr(tbu)-Phe-Thr(tbu)-Ser(tbu)-
Asp(Otbu)-Val-Ser(tbu)-Ser(tbu)-Tyr(tbu)-Leu-Glu(Otbu)-Gly-Gln(trt)-Ala-Ala-Lys(Boc)-Glu(Otbu)-Phe-Ile-Ala-Trp(Boc)-Leu-Val-Arg(pbf)-Gly-Arg(pbf)-Gly-OH- Wang Resin
Formula III vii. cleaving the GLP-1 sequence of Formula II or Formula III from solid resin support using cleavage cocktail consisting of TFA in the range of 80 to 95 % V/V, TIS in the range of 2.5 to 10 % V/V, Phenol in the range of 10 to 20 % V/V, preferably TFA: TIS: Phenol in the ratio of 70: 10: 20 (%V/V) at a concentration of 10-30ml/g of peptidyl resin followed by filtration and precipitation to obtain crude GLP-1 of Formula IV with not less than 70% peptide purity,
H-His-Aib-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-
Gly-Gln-Ala-Ala-Lys-Glu-Phe-Ile-Ala-Trp-Leu-Val-Arg-Gly-Arg-Gly-
OH
Formula IV
viii. purifying sequence of Formula IV of step vii) by reverse phase HPLC to a purity of at least 95%,
ix. activating the side chain tBuO-Ste-Glu(AEEA-AEEA-OH)-OtBu by HOSu to generate N-hydroxysuccinimidyl tBuO-Ste-Glu(AEEA-AEEA-OH)-OtBu,
x. conjugating activated side-chain of step (ix) at Lysine epsilon amino group in the synthesized GLP-1 sequence of Formula IV in a solvent mixture comprising of acetonitrile and water at GLP-1 concentration of 1-10 mg/ml and a reaction pH between the range of 9-12.5, preferably

11.5, to generate semaglutide sequence of Formula V
H-His-Aib-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-
Gly-Gln-Ala-Ala-Lys(tBuO-Ste-Glu(AEEA-AEEA-OH)-OtBu)-Glu-
Phe-Ilc-Ala-Trp-Leu-Val-Arg-Gly-Arg-Gly-OH.
Formula V xi. cleaving the protected semaglutide peptide to generate crude Semaglutide
sequence of Formula I, xii. purifying Semaglutide sequence of Formula I obtained from step xi) by
reverse phase HPLC to a purity of at least 98% xiii. further purifying Semaglutide sequence of Formula I obtained from step
xii) by reverse phase HPLC to purity of at least 99.5% and single largest
impurity not more than 0.20% xiv. desalting Semaglutide sequence of Formula I obtained from step xiii) to a
sodium salt by HPLC or nanofiltration preferably by HPLC to a purity of
at least 99.5% and single largest impurity not more than 0.20% with yield
of 15-20% followed by lyophilization.
2. An improved process for synthesis of Semaglutide or salt or precursor thereof as claimed in claim 1, wherein the activation of amino acid is carried out in the coupling using DIC/Oxymapure.
3. An improved process for synthesis of Semaglutide or salt or precursor thereof as claimed in claim 1, wherein the coupling is carried out by increasing temperature gradient from room temperature to 40-85°C, more preferably to 40-55°C.
4. An improved process for synthesis of Semaglutide or salt or precursor thereof as claimed in claim 1, wherein the N terminal last 2 amino acids coupling is carried out by increasing temperature gradient from room temperature to 60-85°C, more preferably to 75-80°C.
5. An improved process for synthesis of Semaglutide or salt or precursor

thereof as claimed in claim 1, wherein cleavage of GLP-1 of sequence of formula II or III, from the solid resin support is done wherein concentration of peptidyl resin is 10-30 ml/gram.
6. An improved process for synthesis of Semaglutide or salt or precursor thereof as claimed in claim 1, wherein the purification of crude GLP-1 of sequence of formula II, to a purity of ≥ 97% carried by chromatography is by RP-HPLC by isocratic and/or gradient mode.
7. An improved process for synthesis of Semaglutide or salt or precursor thereof as claimed in claim 1, wherein the eluent for RP-HPLC purification of crude GLP-1 of sequence of formula II, by gradient mode comprises of orthophosphoric acid buffer system with pH in the range of 6-8, preferably 6.5 as an aqueous phase and acetonitrile as an organic phase with isolated yield of atleast 95% and purity of atleast 97%.
8. An improved process for synthesis of Semaglutide or salt or precursor thereof as claimed in claim 1, wherein the purification of semaglutide sequence of Formula I to purity of ≥ 98% by chromatography is by RP-HPLC by isocratic and/or gradient mode.
9. An improved process for synthesis of Semaglutide or salt or precursor thereof as claimed in claim 1, wherein the eluent for RP-HPLC purification of Semaglutide sequence of Formula I by gradient mode comprises of 0.15% trifluoro acetic acid as an aqueous phase and an organic phase comprising of mixture of solvents selected from methanol, ethanol or acetonitrile preferably acetonitrile and methanol in the ratio of 1:1.
10. An improved process for synthesis of Semaglutide or salt or precursor thereof as claimed in claim 1, wherein the further purification of semaglutide sequence of Formula I to purity of ≥ 99.5% with single largest impurity <0.20% by chromatography is by RP-HPLC by isocratic and/or gradient mode.
11. An improved process for synthesis of Semaglutide or salt or precursor thereof as claimed in claim 1, wherein the eluent for RP-HPLC purification

of Semaglutide sequence of Formula I by gradient mode comprises of Sodium bicarbonate buffer of pH 8.3 as an aqueous phase and an organic phase comprising of mixture of solvents selected from methanol, ethanol or acetonitrile preferably acetonitrile.