Process for the preparation of pegcetacoplan and its purification using chromatography

Abstract

The present invention relates to a process for the preparation of Pegcetacoplan by solid-phase synthesis and its purification using Ion exchange chromatography.

Description

[0001] PROCESS FOR THE PREPARATION OF PEGCETACOPLAN AND ITS PURIFICATION USING CHROMATOGRAPHY

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS

[0003] This application claims the benefit of earlier Indian provisional patent application IN 202441078209 filed on October 15, 2024, and Indian provisional patent application IN 202541001434 filed on January 07, 2025, which are hereby incorporated by reference in its entirety.

[0004] FIELD OF THE INVENTION

[0005] The present invention relates to a process for the preparation of Pegcetacoplan by solid-phase synthesis and its purification using Ion exchange chromatography.

[0006] BACKGROUND OF THE INVENTION

[0007] Pegcetacoplan, a complement inhibitor and is developed by Apellis Pharmaceuticals. Pegcetacoplan is marketed under the brand name EMPAVELI® in the U.S for the treatment of adult patients with paroxysmal nocturnal hemoglobinuria (PNH). Pegcetacoplan is also marketed under the brand name SYFOVRE® in the U.S for the treatment of geographic atrophy (GA) secondary to age-related macular degeneration (AMD).

[0008] Pegcetacoplan is aa symmetrical mmoolleeccuullee comprised of two identical pentadecapeptides covalently bound to the ends of a linear 40-kiloDalton (kDa) PEG molecule. The peptide portions of Pegcetacoplan contain 1-methyl-L-tryptophan (Trp(Me)) in position 4 and amino(ethoxyethoxy)acetic acid (AEEA) in position 14 and is structurally represented by the following chemical Formula I:

[0009] Formula I U.S. Pat. No. 7,888,323, U.S. Pat. No. 7,989,589, and U.S. Pat. No. 9,169,307 disclosed pentadecapeptide fragment of Pegcetacoplan. Further U.S. Pat. No.

[0010] 1,003,5822, U.S. Pat. No. 10,125,171, U.S. Pat. No. 10,875,893, US 11,292,815 disclosed Pegcetacoplan.

[0011] The inventors of the present invention developed a solid phase peptide synthesis of Pegcetacoplan, which minimizing or avoiding the use of highly toxic, pungent and / or volatile reagents.

[0012] SUMMARY OF THE INVENTION

[0013] The present invention relates to a process for the preparation of Pegcetacoplan by solid-phase synthesis.

[0014] The main aspect of the present invention relates to a process for obtaining Pegcetacoplan by means of solid phase synthesis using Rink amide AM resin. The process will involve the coupling of appropriate protected amino acids in a required sequence, cleavage, Intramolecular cyclization and pegylation of peptide followed by purification to get Pegcetacoplan.

[0015] One aspect of the present invention is to provide a solid phase peptide synthesis of Penta decapeptide of Pegcetacoplan comprising the steps of: a) anchoring first amino acid (Fmoc- Lys (Boc)-OH) to a resin, b) capping the unreacted resin in step (a), c) coupling carboxyl terminus of the next amino acid to the amine group in presence of a coupling reagent, d) repeating step (c) by sequential addition of remaining amino acids to form a protected pentadecapeptide with resin, and e) cleaving the resin bound peptide with a cocktail mixture from the resin and deprotecting the protecting groups to isolate the peptide.

[0016] Another aspect of the present invention is to provide synthesis of cyclized peptide of Pegcetacoplan by cyclizing linear pentadecapeptide comprising the steps of: a) dissolving linear pentadecapeptide in a solvent, b) adding cyclizing reagents to the peptide solution of step (a) to obtain cyclic peptide, and c) isolating the cyclic peptide of Pegcetacoplan.

[0017] Another aspect of the present invention is to provide a process for the preparation of Pegcetacoplan comprising the steps of: a) dissolving cyclized pentadecapeptide in a solvent, b) adding pegylation reagent to the step (a), c) adding an inorganic base to step (b), d) isolating the obtained Pegcetacoplan, and e) optionally purifying the obtained Pegcetacoplan

[0018] Another aspect of the present invention is to provide a process for the preparation of Pegcetacoplan comprising the steps of: a) dissolving cyclized pentadecapeptide in a solvent, b) adding an inorganic base to step (a), c) adding pegylation reagent to the step (b), d) isolating the obtained Pegcetacoplan, and e) optionally purifying the obtained Pegcetacoplan.

[0019] Another aspect of the present invention is to provide a solid phase peptide synthesis of Pegcetacoplan comprising the steps of: a) anchoring first amino acid (Fmoc- Lys (Boc)-OH) to a resin, b) capping the unreacted resin in step (a), c) coupling carboxyl terminus of the next amino acid to the amine group in presence of a coupling reagent, d) repeating step (c) by sequential addition of remaining amino acids to form a protected pentadecapeptide with resin, e) cleaving the resin bound peptide with a cocktail mixture from the resin and deprotecting the protecting groups to isolate crude peptide, f) cyclization of obtained peptide in step (e) to form cyclic peptide and is purifying by preparative HPLC, and g) pegylation of the obtained cyclic peptide in step (f) to get Pegcetacoplan. The present invention also relates to a process for the purification of Pegcetacoplan using ion exchange chromatography.

[0020] One of the main aspects of the present invention is to provide a process for the purification of Pegcetacoplan from a mixture containing Pegcetacoplan and impurities using ion exchange chromatography comprising the steps of: a) loading Pegcetacoplan onto ion exchange material, b) eluting Pegcetacoplan mixture using an eluting solution, c) collecting the eluent comprising Pegcetacoplan.

[0021] Another aspect of the present invention is to provide a method for purifying Pegcetacoplan using ion exchange chromatography, wherein the ion exchange chromatography is a cation exchange chromatography.

[0022] Another aspect of the present invention is to provide a method for purifying Pegcetacoplan using ion exchange chromatography, wherein the ion exchange chromatography is an anion exchange chromatography.

[0023] DETAILED DESCRIPTION OF THE INVENTION

[0024] The present invention relates to a process for the preparation of Pegcetacoplan by solid-phase synthesis.

[0025] The present invention also relates to a process for the purification of Pegcetacoplan using ion exchange chromatography.

[0026] Abbreviations:

[0027] AcOH acetic acid t-Bu tert-butyl

[0028] DCC N,N'-dicyclohexyl carbodiimide

[0029] DCM dichloromethane

[0030] DIG N,N'-diisopropylcarbodiimide

[0031] DMF N,N'-Dimethylformamide DIEA Diisopropylethylamine

[0032] AM resin Rink amide AM resin

[0033] Fmoc 9-fluorenylmethoxycarbonyl

[0034] HOBt N -hydroxybenzo triazole

[0035] HBTU O-Benzotriazole-N,N,N',N'-tetramethyl-uronium- hexafluoro-phosphate

[0036] MTBE Methyl tert-butyl ether

[0037] SPPS Solid phase peptide synthesis

[0038] Phe Phenylalanine

[0039] Cys Cystine

[0040] Trp Trp tophan

[0041] Lys Lysine

[0042] Thr Threonine

[0043] Arg Arginine

[0044] His Histidine

[0045] Ala Alanine

[0046] Gly Glycine

[0047] Asp Aspartic acid

[0048] Gin Glutamine

[0049] Vai Valine

[0050] AEEA Aminoethylethanolamine lie Isoleucine

[0051] TFA Trifluoroacetic acid

[0052] TIS Triisopropylsilane

[0053] DODT 3 ,6-dioxa- 1 , 8-octanedithiol

[0054] Definitions

[0055] To facilitate a clearer understanding of the present disclosure, it is essential to define certain terms in advance.

[0056] The term "ion-exchange" aanndd "ion-exchange chromatography" refer to a chromatographic process in which an ionizable solute of interest ( e.g a protein of interest in a mixture) interacts with an oppositely charged ligand linked (e.g., by covalent attachment) to a solid phase ion exchange material under appropriate conditions of pH and conductivity, such that the solute of interest interacts non- specifically with the charged compound more or less than the solute impurities or contaminants in the mixture. The contaminating solutes in the mixture can be washed from a column of the ion exchange material or are bound to or excluded from the resin, faster or slower than the solute of interest. "Ion-exchange chromatography" specifically includes cation exchange (CEX), anion exchange (AFX), and mixed mode chromatography. Ion exchange chromatography is interchangeably referred herein as IEC and IEX.

[0057] A "cation exchange resin" or "cation exchange membrane" refers to a solid phase which is negatively charged, and which has free cations for exchange with cations in an aqueous solution passed over or through the solid phase. Any negatively charged ligand attached to the solid phase suitable to form the cation exchange resin can be used, e.g, a carboxylate, sulfonate and others as described below.

[0058] Commercially available cation exchange resins include, but are not limited to, for example, those having a sulfonate based group (e.g, YMC-Gel BioPro IEX Smartsep S30, MonoS, Minis, Source 15S and 30S, SP SEPHAROSE® Fast Flow, SP

[0059] SEPHAROSE® High Performance, Capto S, Capto SP ImpRes from GE Healthcare, TOYOPEARL® SP-650S and SP-650M from Tosoh, MACRO-PREP® High S from BioRad, Ceramic HyperD S, TRISACRYL® M and LS SP and Spherodex LS SP from Pall Technologies); a sulfoethyl based group (e.g, FRACTOGEL® SE, from EMD, POROS® S-10 and S-20 from Applied Biosystems); a sulphopropyl based group (e.g, TSK Gel SP 5PW and SP-5PW-HR from Tosoh, POROS® HS-20, HS 50, and POROS® XS from Life Technologies); a sulfoisobutyl based group (e.g, FRACTOGEL® EMD SO3 from EMD); a sulfoxyethyl based group (e.g, SE52, SE53 and Express-Ion S from Whatman), a carboxymethyl based group (e.g, CM SEPHAROSE® Fast Flow from GE Healthcare, Hydrocell CM from Biochrom Labs Inc., MACRO-PREP® CM from BioRad, Ceramic HyperD CM, TRISACRYL® M CM, TRISACRYL® LS CM, from Pall Technologies, Matrx CELLUFINE® C500 and C200 from Millipore, CM52, CM32, CM23 and Express-Ion C from Whatman, TOYOPEARL® CM-650S, CM-650M and CM-650C from Tosoh); sulfonic and carboxylic acid based groups (e.g, BAKERBOND® Carboxy-Sulfon from J. T. Baker); a carboxylic acid based group (e.g, WP CBX from J. T Baker, DOWEX®. MAC-3 from Dow Liquid Separations, AMBERLITE® Weak Cation Exchangers, DOWEX® Weak Cation Exchanger, and DIAION® Weak Cation Exchangers from Sigma-Aldrich and FRACTOGEL® EMD COO — from EMD); a sulfonic acid based group (e.g, Hydrocell SP from Biochrom Labs Inc., DOWEX® Fine Mesh Strong Acid Cation Resin from Dow Liquid Separations, UNOsphere S, WP Sulfonic from J. T. Baker, SARTOBIND® S membrane from Sartorius, AMBERLITE® Strong Cation Exchangers, DOWEX® Strong Cation and DIAION® Strong Cation Exchanger from Sigma- Aldrich); or a orthophosphate based group (e.g, Pl 1 fromWhatman).

[0060] Other cation exchange resins include carboxy-methyl-cellulose, BAKERBOND

[0061] ABXTM, Ceramic HyperD Z, Matrex Cellufme C500, Matrex Cellufme C200.

[0062] An "anion exchange resin" or "anion exchange membrane" refers to a solid phase which is positively charged, thus having one or more positively charged ligands attached thereto. Any positively charged ligand attached to the solid phase suitable to form the anionic exchange resin can be used, such as quaternary amino groups.

[0063] Commercially available anion exchange resins include DEAE cellulose, POROS® PI 20, PI 50, HQ 10, HQ 20, HQ 50, D 50 from Applied Biosystems, SARTOBIND® Q from Sartorius, MonoQ, MiniQ, Source 15Q and 30Q, DEAE and ANX SEPHAROSE® Fast Flow, Q SEPHAROSE® High Performance, QAE SEPHADEX® and FAST Q SEPHAROSE® (GE Healthcare), WP PEI, WP DEAM, WP QUAT from J. T. Baker, Hydrocell DEAE and Hydrocell QA from Biochrom Labs Inc., UNOsphere Q, MACRO-PREP®. DEAE and MACRO-PREP® High Q from Biorad, Ceramic HyperD Q, ceramic HyperD DEAE, TRISACRYL® M and LS DEAE, Spherodex LS DEAE, QMA SPHEROSIL® LS, QMA SPHEROSIL®. M and MUSTANG® Q from Pall Technologies, DOWEX® Fine Mesh Strong Base Type I and Type II Anion Resins and DOWEX® MONOSPHER E 77, weak base anion from Dow Liquid Separations, INTERCEPT® Q membrane, Matrex CELLUFINE® A200, A500, Q500, and Q800, from Millipore, FRACTOGEL® EMD TMAE, FRACTOGEL® EMD DEAE and FRACTOGEL® EMD DMAE from EMD,

[0064] AMBERLITE® weak strong anion exchangers type I and II, DOWEX® weak and strong anion exchangers type I and II, DIAION® weak and strong anion exchangers type I and II, DUOLITE® from Sigma- Aldrich, TSK gel Q and DEAE 5PW and 5PW-HR, TOYOPEARL® SuperQ-650S, 650M and 650C, QAE-550C and 650S, DEAE-650M and 650C from Tosoh, QA52, DE23, DE32, DE51, DE52, DE53, Express-Ion D or Express-Ion Q from Whatman, and SARTOBIND® Q (Sartorius Corporation, New York, USA).

[0065] Other anion exchange resins include POROS XQ, SARTOBIND® Q, Q SEPHAROSE™ XL, Q SEPHAROSE™ big beads, DEAE Sephadex A-25, DEAE Sephadex A-50, QAE Sephadex A- 25, QAE Sephadex A-50, Q SEPHAROSE™ high performance, Q SEPHAROSE™ XL, Resource Q, Capto Q, Capto DEAE, Toyopearl GigaCap Q, Fractogel EMD TMAE HiCap, Nuvia Q, or PORGS PL

[0066] The term "buffer" as used herein, refers to a substance which, by its presence in solution, increases the amount of acid or alkali that must be added to cause unit change in pH. A buffered solution resists changes in pH by the action of its acid-base conjugate components. Buffered solutions for use with biological reagents are generally capable of maintaining a constant concentration of hydrogen ions such that the pH of the solution is within a physiological range. Traditional buffer components include, but are not limited to, organic and inorganic salts, acids and bases.

[0067] The term "chromatography" refers to any kind of technique which separates a protein of interest ( e.g a PEGylated protein) from other molecules (e.g, contaminants) present in a mixture. Usually, the protein of interest is separated from other molecules (e.g, contaminants) as a result of differences in rates at which the individual molecules of the mixture migrate through a stationary medium under the influence of a moving phase, or in bind and elute processes.

[0068] The term "chromatography column" or "column" in connection with chromatography as used herein, refers to a container, frequently in the form of a cylinder or a hollow pillar which is filled with the chromatography matrix or resin. The chromatography matrix or resin is the material which provides the physical and / or chemical properties that are employed for purification. One embodiment of the present invention is to provide a solid phase peptide synthesis of pentadecapeptide of Pegcetacoplan comprising the steps of: a) anchoring first amino acid (Fmoc- Lys (Boc)-OH) to a resin, b) capping the unreacted resin in step (a), c) coupling carboxyl terminus of the next amino acid to the amine group in presence of a coupling reagent, d) repeating steps (c) by sequential addition of remaining amino acids to form a protected pentadecapeptide with resin, and e) cleaving the resin with a cocktail mixture from the resin and deprotecting the protecting groups to isolate the peptide.

[0069] According to the present embodiment, the first amino acid Fmoc-Lys (Boc)-OH is anchored to the resin in the presence of coupled reagent.

[0070] Within this context of this embodiment, the resin used is selected from Rink amide

[0071] AM resin, Wang resin and 2-chlorotrityl resin. The resin undergoes swelling in the presence of a solvent selected from dichloromethane, N, N-dimethylformamide, N- methyl-2-pyrrrolidone or mixtures. The coupling agent used for the coupling of this first amino acid is selected from DIC / 6-Cl-HOBt, DIC / HOBt, HBTU / HOBt / DIEA or

[0072] DlC / Oxyma.

[0073] After completion of the reaction, the deprotection of Fmoc is performed by addition of 20% piperidine in N,N-dimethyl formamide. After deprotection, the resin is washed with N,N-dimethyl formamide.

[0074] After anchoring the first amino acid to resin, before proceeding to the next steps, the unreacted linkers on the resin are appropriately protected (capped) in order to avoid the undesired peptide chain formation. The reagent used for the capping such as pyridine and acetic anhydride in MDC.

[0075] Next according to the present embodiment, the sequential addition of remaining amino acids namely Fmoc-AEEA-OH, Fmoc-Thr(tBu)-OH, Fmoc-Cys (Trt)-OH, Fmoc-Arg (Pbf)-OH, Fmoc-His (Trt)-OH, Fmoc-Ala-OH, Fmoc-Gly-OH, Fmoc-Trp (Boc)-OH, Fmoc-Gln (Trt)-OH, Fmoc-Asp(OtBu)-OH, Fmoc-Trp (Me)-OH, Fmoc- Val-OH, Fmoc-Cys (Trt)-OH Fmoc-Ile-OH is performed using a coupling reagent such as DIC and Oxyma Pure to form protected pegcetacoplan peptide with resin.

[0076] The obtained protected peptide resin obtained is as follows.

[0077] Ac-Ile-Cys (Trt)-Val-Trp (Me)-Gln (Trt)-Asp (OtBu)-Trp (Boc)-Gly-Ala-His (Trt)-

[0078] Arg (Pbf)-Cys(Trt)-Thr(tBu)-AEEA-Lys(Boc)-Rinkamide AM Resin.

[0079] The coupling efficiency after each coupling step is monitored during the synthesis by means of a Kaiser test or any other suitable test. If the coupling efficiency is low, the steps of individual coupling are to be repeated prior to the deprotection and coupling with next amino acid sequence, deprotected the amino group is coupled with next N- protected amino acid in a solvent in presence of a coupling reagent.

[0080] The solvent used for the coupling reaction is selected from dichloromethane, tetrahydrofuran, dimethylformamide, N-methylpyrolidone or mixture thereof. The coupling agent used for the coupling of the amino acids is selected from DIC / 6-C1- HOBt, DIC / HOBt, HBTU / HOBt / DIEA or DIC / Oxyma.

[0081] Next according to the present embodiment, after the completion of the reaction, the resin is optionally washed with solvents such as DMF and DCM to remove residual reagents and byproducts. The process is repeated if desired and before proceeding to next step.

[0082] Next according to the present embodiment, the cleavage and deprotection of the peptide is carried out with a cocktail mixture. The cleavage of the peptide from the resin involves treating the protected peptide anchored to the resin with an acid having at least one scavenger. The acid utilized in the cleavage reagent is TEA. The amount of TEA used for the purpose of cleavage of peptide from the resin and deprotection in the cocktail mixture may range from 80-90%. The scavengers used are selected from TIS, TIPS, DODT, Phenol, water or in any combination thereof. The particular Cocktail mixture used for the cleavage of the peptide from resin is TEA, TIS, TIPS, DODT, anisole, phenol and water. The temperature for the cleavage and deprotection is carried out at about 10-30°C, preferably at 25°C. After completion of the cleavage reaction, the cleavage mixture is filtered to remove the spent resin. The crude, peptide is precipitated and washed with methyl tertbutyl ether (MTBE), and then dried under vacuum.

[0083] Another embodiment of the present invention is to provide synthesis of cyclized peptide of Pegcetacoplan by cyclizing linear pentadecapeptide comprising the steps of: a) dissolving linear pentadecapeptide in a solvent b) adding cyclizing reagents to the peptide solution of step (a) to obtain cyclic peptide, and c) isolating the cyclic peptide of Pegectacoplan.

[0084] According to present embodiment, the linear pentadecapeptide is dissolved in a solvent. The solvent useful in this embodiment may be, for example, an alcohol solvent, ether solvent, nitrile solvent, water, or a miscible mixture thereof. Examples of suitable alcohol solvents include methanol, ethanol, propanol, isopropanol, n- butanol, sec-butanol, 2-butanol, t-butanol, 1 -pentanol, 2 -pentanol, 3 -pentanol, 2- methyl-1 -butanol, 2-methyl-l -butanol, 2-2-methyl-2-butanol, 3-methyl-2-butanol, 2,2-dimethyl-l -propanol, 1,1, dimethyl- 1 -propanol. Examples of suitable nitrile solvents include acetonitrile, propionitrile and / or mixtures thereof. Examples of suitable ether solvents include tetrahydrofuran, 1,4-dioxane and mixtures thereof. In some particularly useful embodiment, water is used as the solvent.

[0085] Next according to present embodiment, adding cyclizing reagents to the above linear peptide solution in step (a). In this step, the peptide thiol from step (a) is oxidized to form a disulfide bridge using oxidizing (cyclization) reagents such as such as Iodine in methanol. The disulphide bond also obtained in air oxidation or KaFe / CNje under basic conditions. The peptide formed after oxidation is subjected to passing through HI with Indion 830 OH Resin to quench the excess iodine and filter the spent resin and isolating the filtrate having the cyclized peptide.

[0086] Next according to the present embodiment, the resultant cyclized peptide purification process is carried out on reverse phase preparative HPLC using C18 silica as a stationary phase followed by lyophilization to get pure peptide. Another embodiment of the present invention is to provide a process for the preparation of Pegcetacoplan comprising the steps of: a) dissolving cyclized pentadecapeptide in a solvent, b) adding pegylation reagent to the step (a), c) adding an inorganic base to step (b) to adjust the pH, d) isolating the obtained Pegcetacoplan, and e) optionally purifying the obtained Pegcetacoplan.

[0087] According to the present embodiment, cyclized pentadecapeptide is dissolved in a solvent, Within the context of the present disclosure, the solvent may be, for example, an alcohol solvent, ether solvent, water, or a miscible mixture thereof. Examples of suitable alcohol solvents include methanol, ethanol, propanol, isopropanol, n-butanol, sec -butanol, 2 -butanol, t-butanol, 1 -pentanol, 2 -pentanol, 3-pentanol, 2-methyl-l- butanol, 2-methyl-l -butanol, 2-2-methyl-2-butanol, 3-methyl-2-butanol, 2,2-dimethyl- 1 -propanol, 1,1, dimethyl- 1 -propanol. Examples of suitable ether solvents include tetrahydrofuran, 1,4-dioxane and mixtures thereof. In some particularly useful embodiment, THE and water is used as the solvent.

[0088] Next according to the present embodiment, the said pegylation process involves hydroxy succinimide protected Polyethylene glycol (SC-PEG-SC 40K) is dissolved in THE and water and simultaneously dissolve the peptide in THE and water. Polyethylene glycol solution is slowly added to the peptide.

[0089] Next according to the present embodiment, adding an inorganic base to adjust the pH. Suitable inorganic bases for use in this reaction include, but are not limited to, alkaline metal hydroxides, alkaline metal bicarbonates, alkaline metal carbonates, alkaline alkoxides, and mixtures thereof. Suitable alkaline metal hydroxides for use in this reaction include, but are not limited to, sodium hydroxide, potassium hydroxide, and mixtures thereof. Suitable alkaline metal bicarbonates useful in this reaction include, but are not limited to, sodium bicarbonate, potassium bicarbonate, and mixtures thereof. Suitable alkaline metal carbonates useful in this reaction include, but are not limited to, sodium carbonate, potassium carbonate, cesium carbonate, and mixtures thereof. Suitable alkaline alkoxides useful in this reaction include, but are not limited to, sodium methoxide, sodium ethoxide, potassium methoxide, potassium ethoxide, potassium propoxide, sodium tert-butoxide, potassium tert-butoxide, and mixtures thereof. In some particularly useful embodiment, the use of sodium carbonate as a base was found to be effective.

[0090] In some particularly useful embodiment, polyethylene glycol solution is slowly added to the peptide and adjust the pH to 8.0 with diluted sodium bicarbonate solution and stir the reaction for 2-3 Hrs. The progress of the reaction is monitored by HPLC.

[0091] After completion of the reaction, distill the reaction mass under vacuum to remove THF. Residual volume is diluted with purified water. The pegylated peptide undergoes two sequential purifications using preparative HPLC followed by lyophilization to get Pegcetacoplan. The obtained pegylated peptide mass conformed by MALDI-TOF analysis.

[0092] Another aspect of the present invention is to provide a process for the preparation of Pegcetacoplan comprising the steps of: a) dissolving cyclized pentadecapeptide in a solvent, b) adding an inorganic base to step (a), c) adding pegylation reagent to the step (b), d) isolating the obtained Pegcetacoplan, and e) optionally purifying the obtained Pegcetacoplan.

[0093] According to the present embodiment, cyclized pentadecapeptide is dissolved in a solvent, Within the context of the present disclosure, the solvent may be, for example, an alcohol solvent, ether solvent, nitrile solvent, water, or a miscible mixture thereof.

[0094] Examples of suitable alcohol solvents include methanol, ethanol, propanol, isopropanol, n-butanol, sec-butanol, 2-butanol, t-butanol, 1-pentanol, 2-pentanol, 3- pentanol, 2-methyl-l -butanol, 2-methyl-l -butanol, 2-2-methyl-2-butanol, 3-methyl-2- butanol, 2,2-dimethyl-l -propanol, 1,1, dimethyl- 1 -propanol. Examples of suitable nitrile solvents include acetonitrile, propionitrile and / or mixtures thereof. Examples of suitable ether solvents include tetrahydrofuran, 1,4-dioxane and mixtures thereof. In some particularly useful embodiment, water and acetonitrile is used as the solvent. Next according to the present embodiment, adding an inorganic base to adjust the pH. Suitable inorganic bases for use in this reaction include, but are not limited, to, alkaline metal hydroxides, alkaline metal bicarbonates, alkaline metal carbonates, alkaline alkoxides, and mixtures thereof. Suitable alkaline metal hydroxides for use in this reaction include, but are not limited to, sodium hydroxide, potassium hydroxide, and mixtures thereof. Suitable alkaline metal bicarbonates useful in this reaction include, but are not limited to, sodium bicarbonate, potassium bicarbonate, and mixtures thereof. Suitable alkaline metal carbonates useful in this reaction include, but are not limited to, sodium carbonate, potassium carbonate, cesium carbonate, and mixtures thereof. Suitable alkaline alkoxides useful in this reaction include, but are not limited to, sodium methoxide, sodium ethoxide, potassium methoxide, potassium ethoxide, potassium propoxide, sodium tert-butoxide, potassium tert-butoxide, and mixtures thereof. In some particularly useful embodiment, the use of sodium carbonate as a base was found to be effective.

[0095] In some particularly useful embodiment, polyethylene glycol solution is slowly added to the peptide and adjust the pH to 8.0 with diluted sodium bicarbonate solution and stir the reaction for 2-3 Hrs.

[0096] Next according to the present embodiment, the said pegylation reagent hydroxy succinimide protected Polyethylene glycol (SC-PEG-SC 40K) is either dissolved in acetonitrile and added slowly to the peptide solution or adding pegylation reagent hydroxy succinimide protected Polyethylene glycol (SC-PEG-SC 40K) is slowly added to the peptide solution. The progress of the reaction is monitored by HPLC.

[0097] After completion of the reaction, the reaction mass is quenched with 0.5% formic acid in water (30 volumes) and stirred for 20-30 minutes and distill the reaction mass under vacuum to remove acetonitrile. Residual volume is diluted with purified water. The pegylated peptide undergoes two sequential purifications using preparative HPLC followed by lyophilization to get Pegcetacoplan. The obtained pegylated peptide mass conformed by MALDI-TOF analysis.

[0098] Another embodiment of the present invention is to provide a solid phase peptide synthesis of Pegcetacoplan comprising the steps of: a) anchoring first amino acid (Fmoc- Lys (Boc)-OH) to a resin, b) capping the unreacted resin in step (a), c) coupling carboxyl terminus of the next amino acid to the amine group in presence of a coupling reagent, d) repeating step (c) by sequential addition of remaining amino acids to form a protected pentadecapeptide with resin, e) cleaving the resin bound peptide with a cocktail mixture from the resin and deprotecting the protecting groups to isolate crude peptide, and f) cyclization of obtained peptide in step (e) to form cyclic peptide and is purifying by preparative HPLC, g) pegylation of the obtained cyclic peptide in step (f) to get Pegcetacoplan.

[0099] The schematic description of the process is as shown in scheme 1.

[0100] Scheme-1 Resin

[0101] Fmoc-Lys (Boc)-OH Fmoc-AEEA-OH Fmoc-Thr(tBu)-OH Fmoc-Cys (Trt)-OH Fmoc-Arg (Pbf)-OH Fmoc-His (Trt)-OH Fmoc-Ala-OH Fmoc-Gly-OH Fmoc-Trp (Boc)-OH Fmoc-GIn (Trt)-OH Fmoc-Asp(OtBu)-OH Fmoc-Trp (Me)-OH Fmoc-Val-OH Fmoc-lle-OH Dichloromethane , , Dimethyl formamide

[0102] Acetic anhydride Pyridine

[0103] Ac-lle-Cys (Trt)-Val-Trp(Me)-Gln(Trt)-Asp(OtBu)-Trp(Boc)-Gly-Ala-His(Trt)-Arg(Pbf)-Cys(Trt)-Thr(tBu)-AEEA-Lys(Boc)- Rinkamide AM Resin (stage-1) TEA TIPS DODT Phenol Anisole Water MTBE

[0104] AC-lle-Cys-Val-Trp(Me)-Gln-Asp-Trp-Gly-Ala-His-Arg-Cys-Thr-AEEA-Lys-C0NH2 (stage-2)

[0105] Water

[0106] Methanol

[0107] Iodine

[0108] Indion 830 OH Resine

[0109] AC-lle-Cy Is-Val-Trp(Me)-Gln-Asp-Trp-Gly-Ala-His-Arg-Cys-Thr- IAEEA-Lys-C0NH2 (Crude)

[0110] TEA Ammonium acetate Ammonium bicarbonate Water

[0111] YMC CIS media

[0112] AC-lle-Cy Is-Val-Trp(Me)-Gln-Asp-Trp-Gly-Ala-His-Arg-Cys-Thr- IAEEA-Lys-C0NH2 (Pure;Stage-3)

[0113] l.usO-OC-O-[CH2-CH2-O]899-CH2-CH2-O-Co-Osu

[0114] 2. Sodium bicarbonate

[0115] 3.THF

[0116] 4.water

[0117] Another aspect of present invention relates to a process for the purification of

[0118] Pegcetacoplan using ion exchange chromatography.

[0119] The synthesis of Pegcetacoplan typically yields a complex mixture of byproducts, including hydrolysis products and PEGylation reaction catalysts, necessitating the subsequent separation of these substances to isolate the desired PEGylated product. One embodiment of the present invention is to provide a process for the purification of Pegcetacoplan from a mixture containing Pegcetacoplan and impurities using ion exchange chromatography comprising the steps of: a) loading Pegcetacoplan onto ion exchange material, b) eluting Pegcetacoplan mixture using an eluting solution, and c) collecting the eluent comprising Pegcetacoplan.

[0120] In accordance with the present invention, ion exchange resin is packed in low pressure glass column. The Pegcetacoplan reaction mass is diluted with a solvent and absorbed in low pressure glass column. The absorbed Pegcetacoplan is eluted from the column by applying an eluting solution (i.e eluent buffer) and followed by collecting the eluent comprising Pegcetacoplan.

[0121] Within the context of this embodiment, the ion exchange chromatography may be a cation exchange chromatography. Cation exchange chromatography uses a negatively charged ion exchange resin with an affinity for molecules having net positive surface charges.

[0122] According to the present invention, the ion exchange chromatography comprises a CEX resin include, but are not limited to, for example, those having a sulfonate based group (e.g, YMC-Gel BioPro IEX Smartsep S30, MonoS, Minis, Source 15S and 30S, SP SEPHAROSE® Fast Flow, SP SEPHAROSE® High Performance, Capto S, Capto SP ImpRes from GE Healthcare, TOYOPEARL® SP-650S and SP-650M from Tosoh, MACRO-PREP® High S from BioRad, Ceramic HyperD S, TRISACRYL® M and LS SP and Spherodex LS SP from Pall Technologies); a sulfoethyl based group (e.g, FRACTOGEL® SE, from EMD, POROS® S-10 and S-20 from Applied Biosystems); a sulphopropyl based group (e.g, TSK Gel SP 5PW and SP-5PW-HR from Tosoh, POROS® HS-20, HS 50, and POROS® XS from Life Technologies); a sulfoisobutyl based group (e.g, FRACTOGEL® EMD SO3 from EMD); a sulfoxyethyl based group (e.g, SE52, SE53 and Express-Ion S from Whatman), a carboxymethyl based group (e.g, CM SEPHAROSE® Fast Flow from GE Healthcare, Hydrocell CM from Biochrom Labs Inc., MACRO-PREP® CM from BioRad, Ceramic HyperD CM, TRISACRYL® M CM, TRISACRYL® LS CM, from Pall Technologies, Matrx CELLUFINE® C500 and C200 from Millipore, CM52, CM32, CM23 and Express-Ion C from Whatman, TOYOPEARL® CM-650S, CM-650M and CM-650C from Tosoh); sulfonic and carboxylic acid based groups (e.g, BAKERBOND® Carboxy-Sulfon from J. T. Baker); a carboxylic acid based group (e.g, WP CBX from J. T Baker, DOWEX®. MAC-3 from Dow Liquid Separations, AMBERLITE® Weak Cation Exchangers, DOWEX® Weak Cation Exchanger, and DIAION® Weak Cation Exchangers from Sigma-Aldrich and FRACTOGEL® EMD COO — from EMD); a sulfonic acid based group (e.g, Hydrocell SP from Biochrom Labs Inc., DOWEX® Fine Mesh Strong Acid Cation Resin from Dow Liquid Separations, UNOsphere S, WP Sulfonic from J. T. Baker, SARTOBIND® S membrane from Sartorius, AMBERLITE® Strong Cation Exchangers, DOWEX® Strong Cation and DIAION® Strong Cation Exchanger from Sigma- Aldrich); or a orthophosphate based group (e.g, Pl 1 fromWhatman). Carboxy-methyl-cellulose, BAKERBOND ABXTM, Ceramic HyperD Z, Matrex Cellufme C500, Matrex Cellufme C200 and any combination thereof.

[0123] According to the present embodiment, the ion exchange chromatography may be an anion exchange chromatography. Anion exchange chromatography uses a positively charged ion exchange resin with an affinity for molecules having net negative surface charges.

[0124] According to the present embodiment, the anion exchange resin include, but are not limited to, for example, DEAF cellulose, POROS® PI 20, PI 50, HQ 10, HQ 20, HQ 50, D 50 from Applied Biosystems, SARTOBIND® Q from Sartorius, MonoQ, MiniQ, Source 15Q and 30Q, DEAF and ANX SEPHAROSE® Fast Flow, Q SEPHAROSE® High Performance, QAE SEPHADEX® and FAST Q SEPHAROSE® (GE Healthcare), WP PEI, WP DEAM, WP QUAT from J. T. Baker, Hydrocell DEAF and Hydrocell QA from Biochrom Labs Inc., UNOsphere Q, MACRO-PREP®. DEAF and MACRO-PREP® High Q from Biorad, Ceramic HyperD Q, ceramic HyperD DEAF, TRISACRYL® M and LS DEAF, Spherodex LS DEAF, QMA SPHEROSIL® LS, QMA SPHEROSIL®. M and MUSTANG® Q from Pall Technologies, DOWEX® Fine Mesh Strong Base Type I and Type II Anion Resins and DOWEX® MONOSPHER E 77, weak base anion from Dow Liquid Separations, INTERCEPT® Q membrane, Matrex CELLUFINE® A200, A500, Q500, and Q800, from Millipore, FRACTOGEL® EMD TMAE, FRACTOGEL® EMD DEAE and FRACTOGEL® EMD DMAE from EMD, AMBERLITE® weak strong anion exchangers type I and II, DOWEX® weak and strong anion exchangers type I and II, DIAION® weak and strong anion exchangers type I and II, DUOLITE® from Sigma-Aldrich, TSK gel Q and DEAE 5PW and 5PW-HR, TOYOPEARL® SuperQ-650S, 650M and 650C, QAE-550C and 650S, DEAE-650M and 650C from Tosoh, QA52, DE23, DE32, DE51, DE52, DE53, Express-Ion D or Express-Ion Q from Whatman, and SARTOBIND® Q (Sartorius Corporation, New York, USA), POROS XQ, SARTOBIND® Q, Q SEPHAROSE™ XL, Q SEPHAROSE™ big beads, DEAE Sephadex A-25, DEAE Sephadex A-50, QAE Sephadex A- 25, QAE Sephadex A-50, Q SEPHAROSE™ high performance, Q SEPHAROSE™ XL, Resource Q, Capto Q, Capto DEAE, Toyopearl GigaCap Q, Fractogel EMD TMAE HiCap, Nuvia Q, or PORGS PL and any combination thereof.

[0125] Next according to the present embodiment, the resin used is ion exchange chromatography is selected from cation exchange resins or anion exchange resins or Polymeric Chromatography Media UniPS 10-300.

[0126] Next according to the present invention embodiment, Pegcetacoplan reaction mass may be diluted with a solvent and absorbed in low pressure glass column. Within the context of this embodiment, the used solvent used is selected from group consisting of an alcohol solvent, ether solvent, nitrile solvent, water, or a miscible mixture thereof, wherein alcohol solvents include methanol, ethanol, propanol, isopropanol, n-butanol, sec -butanol, 2 -butanol, t-butanol, 1 -pentanol, 2 -pentanol, 3-pentanol, 2-methyl-l- butanol, 2-methyl-l -butanol, 2-2-methyl-2-butanol, 3-methyl-2-butanol, 2,2-dimethyl- 1 -propanol, 1,1, dimethyl- 1 -propanol, nitrile solvents include acetonitrile, propionitrile and / or mixtures thereof and ether solvents include tetrahydrofuran, 1,4-dioxane and mixtures thereof.

[0127] Next according to the present invention embodiment, following the loading of a Pegcetacoplan solution onto the ion exchange material, the subsequent elution of Pegcetacoplan using an elution buffer.

[0128] The elution buffer is designed to recover or collect the polypeptide bound to the ion exchange material. Generally, the ionic strength, i.e., the conductivity, of the buffer / solution passing through the ion exchange column is increased. This can be accomplished either by an increased buffer salt concentration or by the addition of other salts, so called elution salts, to the buffer solution.

[0129] In the context of this embodiment, the used buffers include, but are not limited to, organic and inorganic salts, acids and bases. The pharmaceutically acceptable buffer substances are used, such as, e.g, formic acid or salts thereof, acetic acid or salts thereof, phosphoric acid or salts thereof, citric acid or salts thereof, morpholine, 2-(N- morpholino) ethanesulfonic acid or salts thereof, histidine or salts thereof, glycine or salts thereof, or tris (hydroxymethyl) aminomethane (TRIS) or salts thereof are used as the buffer substance. Optionally the buffered solution can comprise an additional salt, such as, e.g, sodium chloride, sodium sulphate, potassium chloride, potassium sulfate, sodium citrate, or potassium citrate.

[0130] In some particularly useful embodiment, the elution is carried by using buffer 0.1% formic Acid and 1 M NaCl, wherein in the buffer, 0.1% formic acid is for an absorption and 1 M NaCl is for the elution with a gradient program of absorption buffer is from 100% to 0% and elution buffer is from 0% to 100%. In some another particular useful embodiment, the elution is carried for Polymer Microparticles Purification is 20 mmole Potassium Hexafluorophosphate and 0.1% Perchloric acid and Acetonitrile: THE: Water (75:5:20).

[0131] According to the present embodiment the collected main fractions are pooled together and subjected to remove slats and followed by lyophilization to get pure Pegcetacoplan.

[0132] The present invention describes a novel methodology for the effective separation of impurities, specifically demonstrating that the use of strong cation exchange resin facilitates the successful isolation of pegylated peptides particularly Pegcetacoplan, with empirical data indicating that the polyethylene glycol (PEG) component significantly influences the resin binding affinity in cation exchange chromatography, thereby allowing for the elution of mono-peptide peg impurities at the solvent front while retaining the desired peptide and Pegcetacoplan within the column, thus ensuring comprehensive removal of all impurities generated during the PEGylation process.

[0133] The following example is provided to illustrate the process of the present invention.

[0134] How ever, they are not intended to limit the scope of an invention.

[0135] Examples

[0136] Stage-1: Solid phase synthesis of the protected peptide

[0137] Rink amide AM resin was swelled in Dichloromethane (8.0 v) and washed the resin with DMF 2 x 8.0 v). Fmoc- group was deprotected with 20% piperidine in DMF (2 x 8 vol), followed by DMF Washings (4 x 8 vol). The first amino acid Fmoc- Lys (Boc)-OH (2.0 meq) was coupled to the Resin in presence of DIC (3.0 Moles) and Oxyma Pure (2.0 moles). The unreacted functional sites of the resin were capped using pyridine (0.6 v) and Acetic anhydride (0.6 v ) in MDC (9.0 v). Elongation of peptide was carried out by sequential addition of remaining amino acids as per Pegcetacoplan peptide sequence. The protected peptide resin obtained is as follows. Ac-Ile-Cys (Trt)-Val-Trp (Me)-Gln (Trt)-Asp (OtBu)-Trp (Boc)-Gly-Ala-His (Trt)- Arg (Pbf)-Cys(Trt)-Thr(tBu)-AEEA-Lys(Boc)-Rink amide AM Resin.

[0138] Stage-2: Cleavage of the peptide from the resin, together with the removal of the side chain protecting groups.

[0139] The above resin bound peptide (Stage- 1) was cleaved with cocktail mixture TEA (8.0 v) and scavengers TIPS (0.5 v), DODT (0.5 v), Phenol (0.5 v) and Water (0.5 v). The peptide was detached from the resin with concomitant cleavage of the side chain protecting groups. Upon completion of the cleavage reaction, the cleavage mixture is filtered to remove the spent resin. The crude, peptide is precipitated and washed with methyl tertbutyl ether (MTBE), and then dried under vacuum to a constant weight. The isolated compound is purity is -70% by HPLC.

[0140] Stage-3: Intramolecular Cyclization of peptide and purification by preparative HPLC. 100g Linear peptide was dissolved in water (200 v) and added slowly Iodine (0.8 mole) dissolved in Methanol (2 v), after completion of reaction, Quench the excesses of iodine and HI with Indion 830 OH Resin. Filter the spent resin and discard. Collected Filtrate having the cyclized peptide was purified by reverse phase preparative HPLC using C18 silica as a stationary Phase followed by Lyophilization to get pure peptide. The compound purity is -98% by HPLC.

[0141] Stage-.4: Pegylation of the Peptide using polyethylene glycol (PEG)-N- hydroxysuccinimide (NHS) active esters.

[0142] Example 1:

[0143] Hydroxy succinimide protected Polyethylene glycol (SC-PEG-SC 40K, 1.0 mole) dissolved in THE: Water (1: 1, 50 v). Simultaneously, dissolve the Peptide (2.0 moles) In THE: Water (1: 1, 50 v) and slowly add into Polyethylene glycol solution and adjust the pH to 8.0 with diluted sodium bicarbonate solution and stir the reaction for 2-3 Hrs. Check the progress of the reaction by HPLC (-90%). Distill the reaction mass under vacuum to remove THE. Residual volume is diluted with purified water. The pegylated peptide undergoes two sequential purifications using preparative HPLC followed by lyophilization to get Pegcetacoplan. The pegylated peptide mass conformed by MALDI-TOF analysis and the compound purity is -99% by HPLC.

[0144] Example 2:

[0145] The peptide (2.0 moles) was dissolved in water (30 volumes), followed by the addition of acetonitrile (100 volumes). The mixture was stirred for 2-5 minutes, and the pH was adjusted to 8.0 using diluted sodium bicarbonate solution. Hydroxy succinimide-protected polyethylene glycol (SC-PEG-SC, 40K, 1.0 mole) was then added to the reaction mixture in portions over 10-15 minutes. The reaction was stirred for 2-3 hours, and progress was monitored by HPLC (-80% completion). The reaction mass was quenched with 0.5% formic acid in water (30 volumes) and stirred for 20-30 minutes. Acetonitrile was removed under vacuum distillation, and the residual volume was diluted with purified water. The pegylated peptide was purified in two sequential steps using preparative HPLC, followed by lyophilization to yield Pegcetacoplan. The pegylated peptide mass was confirmed by MALDLTOF analysis, and the final compound showed -99% purity by HPLC.

[0146] Example 3:

[0147] The peptide (2.0 moles) was dissolved in acetonitrile (20 volumes), and the pH was adjusted to 8.0 using DIPEA. Separately, hydroxy succinimide-protected polyethylene glycol (SC-PEG-SC, 40K, 1.0 mole) was dissolved in acetonitrile (30 volumes) and then added to the peptide solution. The reaction mixture was stirred for 2-3 hours, and progress was monitored by HPLC (-70% completion). Upon completion, MTBE (400 volumes) was added to the reaction mixture and stirred for 45-60 minutes. The precipitated solids were filtered and washed twice with MTBE (2 x 50 volumes). The solids were dried under vacuum to obtain crude pegylated peptide. The crude material was purified in two sequential steps using preparative HPLC, followed by lyophilization to yield Pegcetacoplan. The pegylated peptide mass was confirmed by MALDLTOF analysis, and the compound purity was determined to be -95% by HPLC.

[0148] Pegcetacoplan purification by using IEX Chromatography

[0149] Example 1: Pegcetacoplan by IEX Purification

[0150] Materials:

[0151] • Instrument: Knauer Prep HPLC

[0152] • Column: Low Pressure Glass Column, Packed with YMC-Gel BioPro IEX

[0153] Smartsep S30 (Cation exchange Resin)

[0154] • Column Temp: Ambient

[0155] Detector: UV-220 nm

[0156] Flow rate: 10 ml / min.

[0157] Mobile Phase A: 0.1% Formic Acid in MQ Water (Absorption

[0158] Buffer / Charging Buffer)

[0159] • Mobile Phase B: 1 M NaCl in MQ Water (Elution Buffer) Method:

[0160] The hydrophilic polymer beads with a strong cation exchanger (YMC-Gel BioPro IEX Smartsep S30) are packed in pressure glass column. Pegcetacoplan reaction mass is diluted with water and absorbed in low pressure glass column. The absorbed Pegcetacoplan is eluted from the column by applying a gradient of IM NaCl in dilute aqueous formic acid. The main fractions are collected. The collected main fractions are pooled together and subjected for TFF to remove slats and lyophilized.

[0161] (Mobile Phase A: is 0.1% Formic acid in MQ Water, Mobile Phase B: 1 M NaCl in MQ Water, 100% A in 30 minutes, then 0% to 50% B for 60 minutes and then hold until the product is eluted). Purity -99%.

[0162] Gradient Program:

[0163] Time Flow Mobile Phase A% Mobile Phase B% min ml (Absorption

[0164] Buffer / Charging Buffer) (Elution Buffer)

[0165] 0 0 100 0

[0166] 2 10 100 5

[0167] 20 10 100 0

[0168] 30 10 100 0

[0169] 50 10 55 45

[0170] 55 10 50 50

[0171] 60 10 40 60

[0172] 70 10 30 70

[0173] 80 10 20 80

[0174] 85 10 20 80

[0175] 90 10 80 20

[0176] 105 10 98 2 Example 2: Pegce tacoplan Polymer Microparticles Purification

[0177] Materials:

[0178] • Instrument: Knauer Prep HPLC

[0179] • Column: EC 100 mm column dimensions of 100 x 250 mm packed with

[0180] UniPS 10-300 Polymer Microparticles.

[0181] • Column Temp: Ambient

[0182] Detector: UV-220 nm

[0183] Flow rate: 150 ml / min.

[0184] Mobile Phase A:

[0185] • 20 mmole Potassium Hexafluorophosphate LR grade in Purified water / milli Q water and 0.1% Perchloric acid.

[0186] • Mobile Phase B: Acetonitrile :THF: Water (75:5:20)

[0187] • Gradient Program:

[0188] Time Flow Mobile Phase A% Mobile Phase B % min ml

[0189] 0 0 98 2

[0190] 150 150 30 70

[0191] 151 150 80 20

[0192] 160 150 98 2

Claims

We claim:

1. A process for the preparation of pentadecapeptide of Pegcetacoplan comprising the steps of: a. anchoring first amino acid (Fmoc- Lys (Boc)-OH) to a resin, b. capping the unreacted resin in step (a), c. coupling carboxyl terminus of the next amino acid to the amine group in presence of a coupling reagent, d. repeating step (c) by sequential addition of remaining amino acids to form a protected pentadecapeptide with resin, e. cleaving the resin bound peptide with a cocktail mixture from the resin and deprotecting the protecting groups to isolate the peptide.

2. The process according to claim 1, wherein the resin used in step a selected from Rink amide AM resin, Wang resin and 2-chlorotrityl resin.

3. The process according to claim 1, wherein a coupling reagent used in step c is selected from DIC / 6-Cl-HOBt, DIC / HOBt, HBTU / HOBt / DIEA orDIC / Oxyma.

4. The process according to claim 1, wherein coupling reaction is carried out in a solvent, wherein the solvent is selected from dichloromethane, tetrahydrofuran, dimethylformamide, A-methylpyrolidone or mixture thereof.

5. The process according to claim 1, wherein Cocktail mixture is TFA, TIS,TIPS, DODT, anisole, phenol, water and mixture thereof or TFA, thioanisole, water, phenol, ethanedithiol (EDT) and mixture thereof or TFA, thioanisoleand mixture thereof or TFA, thioanisole, water, phenol and mixture thereof orTFA, thioanisole, ethanedithiol, trifluoromethane sulfonic acid (TFMSA) and mixture thereof or TFA, water and mixture thereof.

6. A process for the preparation of cyclized peptide of Pegcetacoplan by cyclizing linear Penta decapeptide comprising the steps of: a. dissolving linear Penta decapeptide in a solvent, b. adding cyclizing reagents to the peptide solution of step (a), and c. isolating the cyclic peptide of Pegectacoplan.

7. The process according to claim 6, wherein solvent in step (a) is selected from alcohol solvent, ether solvent, nitrile solvent, water, or a miscible mixture thereof.

8. The process according to claim 7, the wherein alcohol solvents include methanol, ethanol, propanol, isopropanol, n-butanol, sec -butanol, 2-butanol, t- butanol, 1 -pentanol, 2-pentanol, 3 -pentanol, 2-methyl-l -butanol, 2-methyl-l- butanol, 2-2-methyl-2-butanol, 3-methyl-2-butanol, 2,2-dimethyl-l -propanol,1,1, dimethyl- 1 -propanol, nitrile solvents include acetonitrile, propionitrile and / or mixtures thereof and ether solvents include tetrahydrofuran, 1,4- dioxane and mixtures thereof.

9. The process according to claim 6 wherein cyclization reagent in step (b) isIodine in methanol10. The process according to claim 6, wherein the cyclic peptide in step c is further comprising the purification, using reverse phase preparative HPLC.

11. A process for the preparation of Pegcetacoplan comprising the steps of: a. dissolving cyclized Penta decapeptide in a solvent, b. adding pegylation reagent to the step (a), c. adding an inorganic base to step (b), d. isolating the obtained Pegcetacoplan, and e. purifying the obtained Pegcetacoplan.

12. A process for the preparation of Pegcetacoplan comprising the steps of: a. dissolving cyclized Penta decapeptide in a solvent, b. adding an inorganic base to step (a), c. adding pegylation reagent to the step (b), d. isolating the obtained Pegcetacoplan, and e. purifying the obtained Pegcetacoplan13. The process according to claim 11 and 12, wherein the pegylation of the cyclic peptide in step (c) is carried out using polyethylene glycol (PEG)-N- hydroxysuccinimide (NHS) active ester.

14. The process according to claim 13, the wherein solvent is selected from group consisting of an alcohol solvent, ether solvent, nitrile solvent, water, or a miscible mixture thereof.

15. The process according to claim 14, the wherein alcohol solvents include methanol, ethanol, propanol, isopropanol, n-butanol, sec -butanol, 2-butanol, t- butanol, 1 -pentanol, 2-pentanol, 3 -pentanol, 2-methyl-l -butanol, 2-methyl-l- butanol, 2-2-methyl-2-butanol, 3-methyl-2-butanol, 2,2-dimethyl-l -propanol,1,1, dimethyl- 1 -propanol, nitrile solvents include acetonitrile, propionitrile and / or mixtures thereof and ether solvents include tetrahydrofuran, 1,4- dioxane and mixtures thereof.

16. The process according to claim 11 and 12, wherein the in-organic base is selected from group consisting of alkaline metal hydroxides, alkaline metal bicarbonates, alkaline metal carbonates, alkaline alkoxides, and. mixtures thereof.

17. The process according to claim 16, wherein alkaline metal hydroxides include, but are not limited to, sodium hydroxide, potassium hydroxide, and. mixtures thereof, alkaline metal bicarbonates include, but are not limited to, sodium bicarbonate, potassium bicarbonate, and mixtures thereof, alkaline metal carbonates include, but are not limited to, sodium carbonate, potassium carbonate, cesium carbonate, and mixtures thereof, alkaline alkoxides include, but are not limited to, sodium methoxide, sodium ethoxide, potassium methoxide, potassium ethoxide, potassium propoxide, sodium tert-butoxide, potassium tert-butoxide, and mixtures thereof.

18. The process according to claim 11 and 12, wherein pegylated reagent is either dissolved in a solvent and added slowly to the peptide solution or adding pegylated reagent to the peptide solution.

19. The process according to claim 18, wherein solvent used is selected from group consisting of an alcohol solvent, ether solvent, nitrile solvent, water, or a miscible mixture thereof20. The process according to claim 19, wherein alcohol solvents include methanol, ethanol, propanol, isopropanol, n-butanol, sec -butanol, 2-butanol, t-butanol, 1- pentanol, 2-pentanol, 3 -pentanol, 2-methyl-l -butanol, 2-methyl-l -butanol, 2-2-methyl-2-butanol, 3-methyl-2-butanol, 2, 2-dimethyl- 1 -propanol,1,1, dimethyl- 1 -propanol, nitrile solvents include acetonitrile, propionitrile and / or mixtures thereof and ether solvents include tetrahydrofuran, 1,4- dioxane and mixtures thereof.

21. A solid phase peptide synthesis of Pegcetacoplan, comprising the steps of: a. anchoring first amino acid (Fmoc- Lys (Boc)-OH) to a resin, b. capping the unreacted resin in step (a), c. coupling carboxyl terminus of the next amino acid to the amine group of first amino acid in presence of a coupling reagent, d. repeating step (c) by sequential addition of remaining amino acids to form a pentadecapeptide with resin, e. cleaving the resin bound peptide with a cocktail mixture from the resin and deprotecting the protecting groups to isolate peptide, f. cyclization of obtained peptide in step (e) to form cyclic peptide and is purifying by preparative HPLC, and g. pegylation of the obtained cyclic peptide in step (f) to getPegcetacoplan22. A process for the purification of Pegcetacoplan from a mixture containingPegcetacoplan and impurities using ion exchange chromatography comprising the steps of: a) loading Pegcetacoplan onto ion exchange material, b) eluting Pegcetacoplan mixture using an eluting solution, and c) collecting the eluent comprising Pegcetacoplan.

23. The process according to claim 22, wherein ion exchange chromatography is selected from a cation exchange chromatography or anion exchange chromatography.

24. The process according to claim 22, the wherein resin used is ion exchange chromatography is selected from cation exchange resins or anion exchange resins or Polymeric Chromatography Media UniPS 10-300.

25. The process according to claim 24, the wherein cation exchange resins is selected from group consist of sulfonate based group (e.g, YMC-Gel BioProIEX Smartsep S30, MonoS, Minis, Source 15S and 30S, SP SEPHAROSE®Fast Flow, SP SEPHAROSE® High Performance, Capto S, Capto SP ImpRes from GE Healthcare, TOYOPEARL® SP-650S and SP-650M from Tosoh,MACRO-PREP® High S from BioRad, Ceramic HyperD S, TRISACRYL®M and LS SP and Spherodex LS SP from Pall Technologies); a sulfoethyl based group (e.g, FRACTOGEL® SE, from EMD, POROS® S-10 and S-20 from Applied Biosystems); a sulphopropyl based group (e.g, TSK Gel SP5PW and SP-5PW-HR from Tosoh, POROS® HS-20, HS 50, and POROS®XS from Life Technologies); a sulfoisobutyl based group (e.g,FRACTOGEL® EMD SO3 from EMD); a sulfoxyethyl based group (e.g,SE52, SE53 and Express-Ion S from Whatman), a carboxymethyl based group(e.g, CM SEPHAROSE® Fast Flow from GE Healthcare, Hydrocell CM fromBiochrom Labs Inc., MACRO-PREP® CM from BioRad, Ceramic HyperDCM, TRISACRYL® M CM, TRISACRYL® LS CM, from Pall Technologies,Matrx CELLUFINE® C500 and C200 from Millipore, CM52, CM32, CM23 and Express-Ion C from Whatman, TOYOPEARL® CM-650S, CM-650M and CM-650C from Tosoh); sulfonic and carboxylic acid based groups (e.g,BAKERBOND® Carboxy-Sulfon from J. T. Baker); a carboxylic acid based group (e.g, WP CBX from J. T Baker, DOWEX®. MAC-3 from Dow LiquidSeparations, AMBERLITE® Weak Cation Exchangers, DOWEX® WeakCation Exchanger, and DIAION® Weak Cation Exchangers from Sigma-Aldrich and FRACTOGEL® EMD COO from EMD); a sulfonic acid based group (e.g, Hydrocell SP from Biochrom Labs Inc., DOWEX® Fine MeshStrong Acid Cation Resin from Dow Liquid Separations, UNOsphere S, WPSulfonic from J. T. Baker, SARTOBIND® S membrane from Sartorius,AMBERLITE® Strong Cation Exchangers, DOWEX® Strong Cation andDIAION® Strong Cation Exchanger from Sigma- Aldrich); or a orthophosphate based group (e.g, Pl 1 fromWhatman). Carboxy-methyl- cellulose, BAKERBOND ABXTM, Ceramic HyperD Z, Matrex CellufmeC500, Matrex Cellufme C200 and any combination thereof.

26. The process according to claim 24, the wherein anion exchange resin include, but are not limited to, for example, DEAF cellulose, POROS® PI 20, PI 50,HQ 10, HQ 20, HQ 50, D 50 from Applied Biosystems, SARTOBIND® Qfrom Sartorius, MonoQ, MiniQ, Source 15Q and 30Q, DEAF and ANXSEPHAROSE® Fast Flow, Q SEPHAROSE® High Performance, QAESEPHADEX® and FAST Q SEPHAROSE® (GE Healthcare), WP PEI, WPDEAM, WP QUAT from J. T. Baker, Hydrocell DEAF and Hydrocell QA from Biochrom Labs Inc., UNOsphere Q, MACRO-PREP®. DEAF andMACRO-PREP® High Q from Biorad, Ceramic HyperD Q, ceramic HyperDDEAF, TRISACRYL® M and LS DEAF, Spherodex LS DEAF, QMASPHEROSIL® LS, QMA SPHEROSIL®. M and MUSTANG® Q from PallTechnologies, DOWEX® Fine Mesh Strong Base Type I and Type II AnionResins and DOWEX® MONOSPHER E 77, weak base anion from DowLiquid Separations, INTERCEPT® Q membrane, Matrex CELLUFINE®A200, A500, Q500, and Q800, from Millipore, FRACTOGEL® EMD TMAE,FRACTOGEL® EMD DEAF and FRACTOGEL® EMD DMAE from EMD,AMBERLITE® weak strong anion exchangers type I and II, DOWEX® weak and strong anion exchangers type I and II, DIAION® weak and strong anion exchangers type I and II, DUOLITE® from Sigma-Aldrich, TSK gel Q andDEAF 5PW and 5PW-HR, TOYOPEARL® SuperQ-650S, 650M and 650C,QAE-550C and 650S, DEAE-650M and 650C from Tosoh, QA52, DE23,DE32, DE51, DE52, DE53, Express-Ion D or Express-Ion Q from Whatman, and SARTOBIND® Q (Sartorius Corporation, New York, USA), POROSXQ, SARTOBIND® Q, Q SEPHAROSE™ XL, Q SEPHAROSE™ big beads, DEAF Sephadex A-25, DEAF Sephadex A-50, QAE Sephadex A- 25,QAE Sephadex A-50, Q SEPHAROSE™ high performance, QSEPHAROSE™ XL, Resource Q, Capto Q, Capto DEAF, Toyopearl GigaCapQ, Fractogel EMD TMAE HiCap, Nuvia Q, or PORGS PL and any combination thereof.

27. The process according to claim 22 wherein, the elution in step (b) is carried by using buffer such as 0.1% formic Acid and 1 M NaCl, wherein, 0.1% formic acid buffer is for an absorption and 1 M NaCl buffer is for the elution with a gradient program of absorption buffer is from 100% to 0% and elution buffer is from 0% to 100%.

28. The process according to claim 27 wherein, the elution in step (b) is carried by using buffer, wherein the buffer for polymer microparticles purification resin is 20 mmole Potassium Hexafluorophosphate and 0.1% Perchloric acid andAcetonitrile: THE: Water (75:5:20).

29. The process according to claim 22 wherein, the collecting Pegcetacoplan fractions are lyophilized to produce Pegcetacoplan.

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