Manufacturing method of linaclotide

Abstract

The present invention relates to a method for producing linaclotide and its acetate salt, more particularly to a method for obtaining amorphous high-purity linaclotide or its acetate salt, having a chromatographic purity of 99.9% or more and containing IMD-linaclotide and Cys-1-α-ketone-linaclotide impurities of 100 ppm or less, preferably 50 ppm or less, more preferably 40 ppm or less, respectively.Furthermore, the present invention relates to an analytical method for detecting IMD-linaclotide and Cys-1-α-ketone linaclotide even at the 40 ppm level (LOD) and quantifying IMD-linaclotide and Cys-1-α-ketone linaclotide even at the 50 ppm level (LOQ) by ion pair chromatography (IPC).

Description

[Technical field]

[0001] The present invention relates to a method for producing linaclotide and its acetate salt, and more specifically, to a method for obtaining amorphous high-purity linaclotide or its acetate salt having a chromatographic purity of 99.9% or more and containing IMD-linaclotide and Cys-1-α-ketone-linaclotide impurities of 100 ppm or less, preferably 50 ppm or less, and more preferably 40 ppm or less, respectively.

[0002] Furthermore, the present invention relates to an analytical method by ion pair chromatography (IPC) that can detect IMD-linaclotide and Cys-1-α-ketone-linaclotide even at a level of 40 ppm (LOD) and can quantify IMD-linaclotide and Cys-1-α-ketone-linaclotide even at a level of 50 ppm (LOQ). [Background technology]

[0003] Linaclotide acetate is the active pharmaceutical ingredient marketed as Linzess® in the US market and Constella® in the EU market in 72, 145 and 290 mcg capsule dosage forms.

[0004] The active pharmaceutical ingredient is a complex Cys-rich cyclic peptide, represented by formula (I), consisting of 14 amino acids and containing three site-specific disulfide bonds between Cys[1-6], [2-10], and [5-13].

[0005] [ka]

[0006] This drug belongs to the agonists of guanylate cyclase and acts locally on the guanylate cyclase subtype C receptor present on the inner surface of the intestinal epithelium. Activation of guanylate cyclase by linaclotide increases cGMP levels, which in turn activates the secretion of chloride and bicarbonate into the intestinal lumen, increasing intestinal fluid secretion and promoting bowel movements.

[0007] The structure of linaclotide was first reported in US Patent No. 7,304,036, which generally states that production of the peptide can be achieved by fermentation of appropriately modified bacterial vectors or by solid-phase peptide synthesis (SPPS), but does not provide a complete description of the chemical procedures. Meanwhile, the first detailed synthesis published for linaclotide is found in Peptide Science 96 (1), 69-80 (2010), which reports an SPPS manufacturing process and proposes an oxidative method for disulfide bridge formation.

[0008] The chemical preparation procedure of linaclotide is described, for example, in WO 2014 / 188011 (Lonza), in which a linear peptide backbone is prepared on the resin by SPPS using a one-by-one aminoacid assembly strategy, followed by cleavage of the linear peptide from the resin and simultaneous deprotection; three disulfide bridges are formed by a random strategy by air oxidation in dimethylsulfoxide, and then crude linaclotide is purified by reversed-phase chromatography and finally isolated by lyophilization from 50% aqueous tert-butanol with an undisclosed purity.

[0009] WO 2015 / 022575 (Auro Peptides) also describes a chemical synthesis procedure in which two fragments are prepared and coupled in a SPPS stepwise strategy to obtain a linear peptide backbone on the resin, followed by cleavage from the resin and simultaneous deprotection, cyclization in a random strategy using air and an oxidizing agent (e.g. hydrogen peroxide) to form three disulfide bridges, and finally purification and lyophilization to obtain linaclotide with an HPLC purity of up to 98.9%.

[0010] Furthermore, WO 2016 / 038497 (Auro Peptides) reports a method for preparing linaclotide, which applies SPPS with a sequential amino acid coupling strategy to generate a linear peptide backbone on the resin, followed by release from the resin and deprotection (simultaneous or sequential), cyclization with a random strategy using air and an oxidizing agent (e.g. hydrogen peroxide), and finally purification by reversed phase chromatography and lyophilization to obtain a final product with 98.9% HPLC purity or over 99% general-purpose HPLC purity. In this method, the details of impurities in the product are not reported.

[0011] WO 2017 / 101810 (Hybio Pharmaceuticals) describes the regioselective synthesis of linaclotide. The synthesis consists of the preparative preparation of a linear peptide backbone on the resin by SPPS via sequential amino acid linkage, the oxidative formation of the first disulfide bridge [1-6] on the peptide while still attached to the resin, the oxidative formation of the second disulfide bridge [2-10] in solution, and finally the oxidative formation of the third disulfide bridge [5-13] after deprotection of the two methylated cysteines. The crude linaclotide is purified by reversed-phase chromatography and lyophilized. Although the synthesis is described as proceeding regioselectively, the oxidative nature of the reaction used to form the necessary disulfide bonds does not allow the avoidance of the formation of peptide dimer and multimer impurities. Furthermore, in addition to the use of industrially less suitable solvents such as diethyl ether, the HPLC purity of the final linaclotide is described as 99.5%, but the content of impurities, e.g. IMD-linaclotide and Cys-1-α-ketone-linaclotide, is not mentioned. However, further purification of the intermediate bis-disulfide peptide by reversed-phase chromatography is described.

[0012] Also, WO 2017 / 134687 (Cipla) describes a method for the preparation of linaclotide, which involves the initial preparation of a linear peptide backbone on a resin by SPPS via sequential amino acid coupling, followed by simultaneous cleavage from the resin and removal of the S-phenylacetamidomethyl protecting group, and finally oxidation of the product in aqueous solvent in a random strategy, purification by reversed-phase chromatography, and lyophilization to obtain a final product with >99% general purpose HPLC purity, free of dimeric and multimeric impurities.

[0013] Similarly, WO 2019 / 113872 (Shenzhen) also details a process for the preparation of linaclotide, via the preparation of a linear peptide backbone on a resin by SPPS or a stepwise strategy via sequential amino acid coupling, which is cyclized by oxidation with N-arogenyl succinimide without cleavage from the resin; the crude linaclotide on the resin is then cleaved, purified by reversed-phase chromatography, and lyophilized. Summary of the Invention [Problem to be solved by the invention]

[0014] With reference to the methods for preparing linaclotide used in the art, the inventors have found that these methods do not provide amorphous linaclotide having a high purity of at least 99.9% and containing less than 100 ppm, preferably less than 50 ppm, and more preferably less than 40 ppm of IMD-linaclotide and Cys-1-α-ketone-linaclotide impurities, respectively. [Means for solving the problem]

[0015] The present inventors have found a simplified, industrially applicable and robust method for the preparation of linaclotide and its acetate salts, which are prepared entirely by liquid phase peptide synthesis (LPPS) without intermediate purification. More specifically, this method can produce amorphous linaclotide or its acetate salts, characterized by a chromatographic purity of 99.9% or more and a content of impurities, particularly IMD-linaclotide and Cys-1-α-ketone-linaclotide, of 100 ppm or less, preferably 50 ppm or less (LOQ of ion pair analysis), more preferably 40 ppm or less (LOD of ion pair analysis), respectively.

[0016] In a first aspect, the present invention provides a method for producing a pharmaceutical composition comprising the following general steps: 1. Initial steps in the construction of linear protected peptides; 2. Deprotection step; 3. Producing crude linaclotide via disulfide bridge formation; 4. Optionally, a purification step; and 5. Optionally, isolating the amorphous high purity linaclotide; The present invention relates to a liquid phase process for preparing linaclotide or an acetate salt thereof, comprising:

[0017] The present method offers significant improvements over the prior art, the advantages of which are summarized below: 1. Cyclically repeating an identical set of standardized steps to obtain a protected linear peptide; 2. Protected linear peptides are prepared throughout by liquid phase peptide synthesis (LPPS); 3. No chromatographic purification of intermediates is performed in the chemical synthesis of linear protected peptide backbones and linear advanced intermediates; 4. The formation of disulfide bridges is carried out in a non-oxidative manner, with a molar cyclization yield of about 70%; 5. The optional purification is preferably carried out by a combination of two techniques consisting of preparative reversed-phase high-speed sample displacement (RP-HPSD) and preparative reversed-phase high-performance liquid chromatography (RP-HPLC), with an overall yield of chromatographic purification of about 55%; 6. The optional final step of isolation of high purity linaclotide is carried out by freeze-drying a t-BuOH-water solution of linaclotide or a water suspension of linaclotide; and 7. The final high purity linaclotide is an amorphous lyophilized powder.

[0018] According to a second aspect, the present invention provides a chromatographic purity of 99.9% or more, which is free of the impurities listed below: - IMD-linaclotide (Impurity 1); - Cys-1-α-ketone (impurity 2) and the amount of each of these is less than 100 ppm, preferably less than 50 ppm, and more preferably less than 40 ppm.

[0019] Preferably, the linaclotide or acetate salt thereof has a multimer content of 0.1% area % or less.

[0020] According to a third aspect, the present invention relates to an ion pair chromatography (IPC) method for analyzing linaclotide or its acetate salt, which is capable of detecting (LOD of 40 ppm) and quantifying (LOQ of 50 ppm) trace amounts of the impurities IMD-linaclotide and Cys-1-α-ketone-linaclotide in amorphous high purity linaclotide. The structures of the impurities IMD-linaclotide and Cys-1-α-ketone-linaclotide are set out below.

[0021] [ka]

[0022] [ka] [Brief description of the drawings]

[0023] [Figure 1] FIG. 1 shows an ion-pair chromatogram (for evaluating chromatographic purity) of amorphous high-purity linaclotide (chromatographic purity: 99.94%) obtained by production option 1. [Diagram 2] FIG. 2 shows an ion-pair chromatogram (for evaluating chromatographic purity) of amorphous high-purity linaclotide (chromatographic purity: 99.91%) obtained by production option 2. [Diagram 3] FIG. 1 shows ion pair chromatograms of IMD-linaclotide and linaclotide spiked with Cys-1-α-ketone-linaclotide impurities (IMD-linaclotide: RRT 0.84-0.85: 1.0% by mass, Cys-1-α-ketone-linaclotide: RRT 0.91-0.92: 1.0% by mass). [Figure 4]Figure 1 shows ion pair chromatograms (for assessment of IMD-linaclotide and Cys-1-α-ketone-linaclotide content) of amorphous high purity linaclotide obtained by manufacturing option 1. IMD-linaclotide: RRT 0.84-0.85: LT 40 ppm (LT LOD). Cys-1-α-ketone-linaclotide: RRT 0.91-0.92: LT 40 ppm (LT LOD). [Diagram 5] Figure 1 shows ion pair chromatograms (for assessment of IMD-linaclotide and Cys-1-α-ketone-linaclotide content) of amorphous high purity linaclotide obtained by manufacturing option 2. IMD-linaclotide: RRT 0.84-0.85: LT 40 ppm (LT LOD). Cys-1-α-ketone-linaclotide: RRT 0.91-0.92: LT 40 ppm (LT LOD). [Figure 6] FIG. 1 shows the GPC chromatogram (for evaluating the multimer content) of amorphous high-purity linaclotide obtained by manufacturing option 1. Multimer (sum of peaks): RRT≦0.81-0.82: 0.07%. [Figure 7] FIG. 1 shows the GPC chromatogram (for evaluating the multimer content) of amorphous high-purity linaclotide obtained by manufacturing option 2. Multimer (sum of peaks): RRT≦0.81-0.82: 0.06%. [Figure 8] FIG. 1 shows the powder X-ray diffraction (PXRD) pattern of amorphous high-purity linaclotide obtained by manufacturing option 1. [Figure 9] FIG. 1 shows the differential scanning calorimetry (DSC) curve of amorphous high-purity linaclotide obtained by manufacturing option 1. Water-solvent loss occurs in the range of 30-100° C., and no endothermic / exothermic phenomena associated with the crystalline form are observed up to 250° C. [Figure 10] FIG. 1 shows the powder X-ray diffraction (PXRD) pattern of amorphous high-purity linaclotide obtained by manufacturing option 2. [Figure 11] Figure 11: Differential scanning calorimetry (DSC) curve of amorphous high-purity linaclotide obtained by manufacturing option 2. No endothermic / exothermic events associated with the crystalline form are observed up to 250°C. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0024] (abbreviation) ACN: Acetonitrile AcOH: Acetic acid DCHA : Dicyclohexylamine DCM: dichloromethane DEA: Diethylamine DIPE: Diisopropyl ether DMF: N,N-dimethylformamide DSC: Differential scanning calorimetry EDC·HCl : N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride Fmoc: Fluorenylmethoxycarbonyl GPC: Gel Permeation Chromatography IPA: Isopropanol iPrOAc : Isopropyl acetate IPC: Ion pair chromatography LOD: Limit of detection LOQ: Limit of quantification LPPS: Liquid Phase Peptide Synthesis MTBE: Methyl tert-butyl ether NMM: N-methylmorpholine NMP: N-methylpyrrolidone OAll: Allyl ester OtBu: tert-butyl ester Oxyma: Ethyl cyano(hydroxyimino)acetate Oxyma-B: 5-(hydroxyimino)-1,3-dimethylpyrimidine-2,4,6(1H,3H,5H)-trione Pd / C: Palladium on carbon Pd(PPh3)4: Tetrakis(triphenylphosphine)-palladium(0) PhSiH3: Phenylsilane PSD: particle size distribution PyOxim: [Ethyl cyano(hydroxyimino)acetato-O 2 ]Tri-1-pyrrolidinylphosphonium hexafluorophosphate RP-HPLC: Reversed-phase high-performance liquid chromatography RP-HPSD: Reversed-phase rapid sample displacement tBu: tert-butyl tBuOH: tert-butanol TFA: Trifluoroacetic acid TIS: Triisopropylsilane TOTU: O-(ethoxycarbonyl)cyanomethyleneamino-N,N,N',N'-tetramethyluronium tetrafluoroborate Trt: Trityl, triphenylmethyl PXRD: Powder X-ray diffraction [Ψ(Dmp,H)pro] : Dimethoxyphenyl-pseudoprolines

[0025] (definition) Unless otherwise defined, all terms, expressions and other scientific terms used herein are intended to have the meaning commonly understood by those skilled in the art to which this disclosure pertains.In some cases, for the purpose of clarity and / or quick reference, terms with commonly understood meanings are defined herein.Therefore, the inclusion of such definitions in this specification should not be interpreted as representing substantial differences from what is commonly understood in the art.

[0026] As used herein, the terms "approximately" and "about" refer to the range of experimental error that occurs in measurements.

[0027] As used herein, "high purity" means having a chromatographic purity of 99.9% or more, and the contents of IMD-linaclotide impurities and Cys-1-α-ketone-linaclotide impurities are each 100 ppm or less, preferably 50 ppm or less, and more preferably 40 ppm or less.

[0028] As used herein, the term "HPLC purity" or "chromatographic purity" refers to the area under the chromatogram curve.

[0029] Purity (%) in this specification is based on chromatographic purity (chromatographic area %).

[0030] The terms "comprising," "having," "including," and "containing" are to be construed as open-ended terms (i.e., meaning "including, but not limited to"). These terms are deemed to provide support for terms such as "consist essentially of," "consisting essentially of," "consist of," or "consisting of."

[0031] The terms "consist essentially of" and "consisting essentially of" are to be construed as semi-close-end terms and mean free of other components (which may include optional excipients) that materially affect the basic and novel characteristics of the invention.

[0032] The terms "consist of" and "consisting of" are to be construed as close-ended terms.

[0033] (Detailed Description) The object of the present invention is a liquid phase process for the preparation of linaclotide, preferably in the form of its acetate salt, comprising the following steps: a1) coupling of dipeptide fragment B[7-8] of formula (II) Fmoc-Asn(Trt)-Pro-OH with hexapeptide fragment C[9-14] of formula (III) Fmoc-Ala-Cys[Ψ(Dmp,H)pro]-Thr(tBu)-Gly-Cys[Ψ(Dmp,H)pro]-Tyr(tBu)-OtBu to obtain peptide [7-14]; a2) coupling peptide [7-14] with an amino acid derivative at position 6 having Fmoc-Cys(Trt)-OH of formula (IV) to obtain peptide [6-14]; a3) coupling peptide [6-14] with an amino acid derivative at position 5 having formula (V) Fmoc-Cys(SO3Na)-ONa to obtain peptide [5-14]; a4) coupling of peptide [5-14] with tetrapeptide fragment A[1-4] of formula (VI) Fmoc-Cys(SO3Na)-Cys(SO3Na)-Glu(tBu)-Tyr(tBu)-OH to obtain linear protected peptide of formula (VII) Fmoc-Cys(SO3Na)-Cys(SO3Na)-Glu(tBu)-Tyr(tBu)-Cys(SO3Na)-Cys(Trt)-Asn(Trt)Pro-Ala-Cys[Ψ(Dmp.H)pro]-Thr(tBu)-Gly-Cys[Ψ(Dmp,H)pro]-Tyr(tBu)-OtBu; b) deprotecting compound (VII) by reaction with a secondary base and trifluoroacetic acid to obtain an intermediate of formula (VIII) H-Cys(SO3H)-Cys(SO3H)-Glu-Tyr-Cys(SO3H)-Cys-Asn-Pro-Ala-Cys-Thr-Gly-Cys-Tyr-OH TFA salt; c) carrying out non-oxidative cyclization of intermediate (VIII) to obtain crude linaclotide; d) optionally purifying the crude linaclotide to obtain purified linaclotide; e) optionally, salifying the purified linaclotide with acetic acid to form the corresponding acetate salt; Includes.

[0034] In a preferred embodiment of the process of the invention, the secondary base in step b) is a secondary amine, preferably DEA, piperidine, piperazine or morpholine, and the trifluoroacetic acid is an at least 80% by volume aqueous solution.

[0035] In a preferred embodiment of the process of the invention, step c) is carried out by intramolecular nucleophilic substitution.

[0036] Preferably, step c) is carried out in a hydroalcoholic buffer of pH 7-8.

[0037] In a further preferred embodiment, in step c), crude linaclotide is obtained as an aqueous solution.

[0038] In a preferred embodiment of the method of the invention, the coupling of formula (II) with formula (III) and / or the coupling of peptide [7-14] with formula (IV) and / or the coupling between peptide [6-14] with formula (V) and / or the coupling between peptide [5-14] with formula (VI) is carried out in the presence of ethyl cyano(hydroxyimino)acetate, also called Oxyma, or a derivative thereof such as PyOxim or TOTU, or a 1,3-dimethylbarbituric acid derivative such as Oxyma-B.

[0039] In a further preferred embodiment of the method of the present invention, the coupling of formula (II) with formula (III) and / or the coupling of peptide [7-14] with formula (IV) and / or the coupling of peptide [6-14] with formula (V) and / or the coupling of peptide [5-14] with formula (VI) is carried out in an organic polar solvent, preferably in an organic polar solvent selected from DMF, NMP, ACN.

[0040] In a further preferred embodiment of the method of the present invention, the coupling of formula (II) with formula (III) and / or the coupling of peptide [7-14] with formula (IV) and / or the coupling of peptide [6-14] with formula (V) and / or the coupling of peptide [5-14] with formula (VI) is carried out at a temperature range of -10°C to 35°C, preferably 0°C to 25°C.

[0041] In a preferred embodiment, the purification step d) is carried out by reversed-phase high-speed sample displacement (RP-HPSD), reversed-phase high-performance liquid chromatography (RP-HPLC) or a combination thereof.

[0042] In a preferred embodiment of purification step d), the elution phase consists of an aqueous solution, a polar organic solvent or a mixture thereof, preferably the polar organic solvent is selected from trifluoroacetic acid, acetic acid, acetonitrile and a mixture thereof. Optionally, a buffer solution, more preferably a phosphate buffer solution, is added to the elution phase.

[0043] In a preferred embodiment, the purified linaclotide is in a solution, preferably said solution comprises water, acetic acid and acetonitrile.

[0044] Preferably, the method of the invention makes it possible to obtain amorphous linaclotide having a chromatographic purity of greater than or equal to 99.9%.

[0045] The method of the invention may further comprise an isolation step f) by lyophilization starting from a hydroalcoholic solution or an aqueous suspension.

[0046] Preferably, the hydroalcoholic solution contains tert-butanol, water, acetic acid or a mixture thereof.

[0047] Preferably, the aqueous suspension is obtained by evaporation of a purified linaclotide solution, more preferably, the aqueous suspension contains acetic acid.

[0048] A further object of the present invention is to provide a method for producing a chromatographic purity of 99.9% or more and a purity of 100% or less of the following impurities: - IMD-linaclotide (Impurity 1); - Cys-1-α-ketone (impurity 2). and m is less than 100 ppm, preferably less than 50 ppm (LOQ), and more preferably less than 40 ppm (LOD).

[0049] Preferably, the amorphous highly pure linaclotide or acetate salt thereof has a multimer content of 0.1% area % or less.

[0050] A further object of the present invention is a method for detecting (LOD of 40 ppm) and quantifying (LOD of 50 ppm) the impurity content of IMD-linaclotide and Cys-1-α-ketone in the product, amorphous high-purity linaclotide or its acetate salt. The method comprises eluting the product through an ion pair chromatography (IPC) column having a silica stationary phase containing alkyl chains and an elution phase consisting of an aqueous solution, a polar organic solvent, or a mixture thereof. Optionally, a buffer, preferably a phosphate buffer, is added to the elution phase.

[0051] In a preferred embodiment of the process according to the invention, said alkyl chain is of the octadecyl type (C18), octyl type (C8) or butyl type (C4), preferably C18.

[0052] Preferably, the polar organic solvent is selected from tetrahydrofuran, dioxane, dichloromethane, methanol, ethanol, n-propanol, isopropanol, butanol, pentane, hexane, toluene, trifluoroacetic acid, acetonitrile, or mixtures thereof; more preferably, selected from trifluoroacetic acid, acetonitrile, or mixtures thereof.

[0053] In a further preferred embodiment, the process of the invention is carried out under the following operating conditions: Stationary phase: Support silica particles containing C18 alkyl chains; Eluent A: Phosphate buffer pH 6.2 Eluent B: ACN with the following gradient elution: %B: 7-7(2 minutes);7-15(19 minutes);15-60(25 minutes) is applied

[0054] The elution phase of the method according to the invention comprises an ion pairing reagent, preferably a heptanesulfonated salt.

[0055] [1. Chemical synthesis of crude linaclotide] In the process of preparation that is the object of the present invention, crude linaclotide is the first key intermediate and is synthesized by a liquid phase peptide synthesis (LPPS) procedure, which comprises: 1. A first step of preparing a linear protected peptide; 2. A second step of deprotection to give the chain-like advanced intermediate; 3. Cyclization with the formation of disulfide bridges leads to the final step of obtaining crude linaclotide. and according to Scheme 1 reported below.

[0056] [ka]

[0057] One of the key points in the synthesis of complex peptides with multiple disulfide bridges such as linaclotide is to make the cyclization step proceed in an appropriate manner and set conditions to obtain the correct regioisomers according to the desired disulfide configuration, avoiding the formation of misfolded impurities and multimers.

[0058] To achieve this goal, a nonoxidative cyclization method was developed in which an intramolecular nucleophilic substitution is carried out in hydroalcoholic buffer at pH 7–8, involving three nucleophilic sites (thiol groups of free Cys) and three leaving groups (sulfonated groups on the remaining Cys).

[0059] A series of preliminary trials was carried out with the aim of selecting the optimal positions of the three sulfonated Cys groups along the backbone: all possible combinations of linear advanced peptides were chemically prepared and subjected to cyclization.

[0060] The best results in chromatographic purity and analysis of the desired linaclotide final product were obtained by testing sulfonation groups placed on Cys positions 1, 2 and 5. However, other combinations gave the worst results, even giving complex chromatographic patterns of positional isomers with no major peak in some cases.

[0061] Thus, surprisingly, intramolecular nucleophilic substitution is not only a favorable synthetic method for the formation of one disulfide bridge, but is particularly efficient for the formation of two or more disulfide bridges (e.g., for the production of complex multi-disulfide bridged peptides such as linaclotide).

[0062] Another important point in the synthesis of complex multi-disulfide bridged peptides such as linaclotide is the selection of protecting groups on the above sulfonated Cys (positions 6, 10, and 13 in the specific invention) and the remaining Cys that need to be cyclized.

[0063] A series of preliminary tests was performed and the best results in terms of final chromatographic purity, orthogonality, side reactions and ease of handling were obtained when Cys at position 6 was functionalized with Trt and Cys at positions 10 and 13 with Cys-pseudoproline.

[0064] Cys-pseudoproline represents a masked form of cysteine ​​with a five-membered thiochetalic hindered ring that is less prone to racemization upon coupling activation. The introduction of this moiety along the backbone has the advantage that it not only ensures higher solubility for the growing peptide, but also reduces aggregation effects, leading to improved work-up.

[0065] Protection of the thiol group of Cys as pseudoproline is a recent discovery. Currently, the only literature available is on SPPS applications, and its relative application in peptide synthesis is still in the early stages.

[0066] For this reason, and taking into consideration that the target of synthesis is a complex Cys-rich peptide with multiple disulfide bridges such as linaclotide, the use of Cys-pseudoproline in the LPPS production method described here represents an innovative application.

[0067] After defining the positions of the sulfonation groups along the linear protected peptide, as well as the type and position of the protecting groups on the remaining Cys, which need to be deprotected before the cyclization step, an overall solution phase peptide synthesis (LPPS) was designed.

[0068] As a pre-synthetic step for preparing the linear protected peptide, the following three fragments must be synthesized: Fragment A(VI): a tetrapeptide containing amino acids 1→4, in which Cys at positions 1 and 2 are derivatized as sulfonated sodium salts. Fmoc-Cys(SO3Na)-Cys(SO3Na)-Glu(tBu)-Tyr(tBu)-OH (VI) Fragment B(II): a dipeptide containing the amino acid at positions 7→8. Fmoc-Asn(Trt)-Pro-OH (II) Fragment C(III): a hexapeptide containing amino acids in positions 9→14, with Cys in positions 10 and 13 protected as Cys-pseudoproline (symbol Cys[Ψ(Dmp,H)pro]). Fmoc-Ala-Cys[Ψ(Dmp,H)pro]-Thr(tBu)-Gly-Cys[Ψ(Dmp,H)pro]-Tyr(tBu)-OtBu (III) Each of the three fragments is characterized by an HPLC purity of ≦90%.

[0069] For the subsequent step of preparation of linear protected peptide, a convergent ligation of fragment A (VI), fragment B (II), fragment C (III) and two amino acid derivatives at position 6, Fmoc-Cys(Trt)-OH (IV) and two amino acid derivatives at position 5, Fmoc-Cys(SO3Na)-ONa (V), is carried out to obtain the following 14 amino acid-containing (14-mer) linear protected peptide (VII). Fmoc-Cys(SO3Na)-Cys(SO3Na)-Glu(tBu)-Tyr(tBu)-Cys(SO3Na)-Cys(Trt)-Asn(Trt)-Pro-Ala-Cys[Ψ(Dmp,H)pro]-Thr(tBu)-Gly-Cys[Ψ(Dmp,H)pro]-Tyr(tBu)-OtBu (VII)

[0070] To obtain a linear protected peptide by linking fragment A (VI), fragment B (II), fragment C (III) and two amino acid derivatives, Fmoc-Cys(Trt)-OH (IV) at position 6 and Fmoc-Cys(SONa)-ONa (V) at position 5, we repeat the simple, industrially applicable, and robust synthetic protocol reported in Scheme 2 below.

[0071] [ka]

[0072] The linear protected peptide is characterized by an HPLC purity of 65% or greater.

[0073] In the subsequent steps of the preparation of the chain-advanced intermediate, the removal of the protecting groups is carried out in two separate steps. Secondary base-mediated Fmoc removal, and Removal of acid-labile protecting groups (e.g., tBu ester, Trt, pseudoproline) mediated by at least 80% by volume of aqueous TFA As a result, the chain-like advanced intermediate (VIII): TFA salt of H-Cys(SO3H)-Cys(SO3H)-Glu-Tyr-Cys(SO3H)-Cys-Asn-Pro-Ala-Cys-Thr-Gly-Cys-Tyr-OH (VIII) The HPLC purity of the chain advanced intermediate is at least 55%.

[0074] In the final step of producing crude linaclotide in solution, cyclization of the chain advanced intermediate is carried out by a process having the following characteristics: a. A non-oxidative method in which folding is carried out by a method based on intramolecular nucleophilic substitution, where the free thiol groups of Cys at positions 6, 10 and 13 act as nucleophilic sites and the sulfonated groups of Cys at positions 1, 2 and 5 act as leaving groups. b. A semi-regioselective process in which the number of resulting regioisomers is smaller than the statistically possible combinations, the goal of which is achieved by a unique step reaction that is different and more advantageous than the sequential cyclization reactions described in existing semi-regioselective processes, making it possible to obtain a solution of crude linaclotide.

[0075] [ka]

[0076] Crude linaclotide in solution is typically obtained with an HPLC purity of 45-55%, which is particularly surprising and advantageous because this purity is reached without intermediate purification, allowing for time savings, cost reductions, and productivity improvements compared to existing methods.

[0077] [2. Purification and isolation of high-purity linaclotide] The crude linaclotide is further processed by combining two purification techniques: reversed-phase high-speed sample displacement (RP-HPSD) and reversed-phase high-performance liquid chromatography (RP-HPLC). Finally, it is isolated by applying one of two distinct preparation options starting from a purified linaclotide solution (ACN-water solution containing AcOH), according to the flow chart reported in Scheme 3: (Manufacturing Option 1) The solvent is replaced from ACN to t-BuOH, and freeze-drying is performed from a t-BuOH-water (containing AcOH) solution. (Manufacturing Option 2) Concentration and lyophilization are performed from an aqueous (AcOH-containing) suspension.

[0078] [ka]

[0079] In detail, the chromatographic purification step is carried out by applying the combination of two separate techniques (RP-HPSD+RP-HPLC) to the crude linaclotide solution.

[0080] The RP-HPSD method is based on the concept that sample molecules bind to the column and separate from each other depending on their affinity with the stationary phase. The technique foresees sample charge overloading on the column and ensures the separation of linaclotide and its impurities by a gentle gradient with a low percentage of organic solvent.

[0081] The overall chromatographic purification process can be depicted in Scheme 4 reported below.

[0082] [ka]

[0083] After the purification process is completed, a purified linaclotide solution with a minimum 99.9% chromatographic purity is obtained from the crude linaclotide solution (approximately 45-55% HPLC purity). The remaining amounts of IMD-linaclotide and Cys-1-α-ketone-linaclotide impurities do not exceed 100 ppm, with each value typically being less than 50 ppm (LOQ) and more typically less than 40 ppm (LOD).

[0084] Once a purified linaclotide solution is obtained, one of the following alternative manufacturing options can be applied to isolate the final amorphous high purity linaclotide.

[0085] (Manufacturing Option 1) The purified linaclotide solution is loaded onto a RP column and eluted from the column with a 1:1 mixture of phase 1 (water-AcOH 100 mM) and phase 2 (t-BuOH) to exchange ACN for t-BuOH. The t-BuOH-water (containing AcOH) solution is then lyophilized to obtain amorphous high-purity linaclotide powder.

[0086] (Manufacturing Option 2) The purified linaclotide solution is concentrated under vacuum to remove ACN. The resulting aqueous (AcOH) suspension is then transferred to trays and lyophilized to obtain amorphous high-purity linaclotide powder.

[0087] [3. API Characterization] Amorphous high-purity linaclotide is characterized as follows:

[0088] A. Purity, Analysis, and Impurity Assessment of IMD-Linaclotide and Cys-1-α-Ketone-Linaclotide by Ion Pair Chromatography (IPC) This analytical method is based on the principle of ion-pair chromatography (IPC). This technique allows the separation of ionic analytes using a mobile phase containing specific modifiers consisting of lipophilic ions with an opposite charge to the analytes. The lipophilic ions of the mobile phase interact with the analytes and balance the ionic charge.

[0089] Optimization of the analytical variables also allows for the separation of complex sample mixtures containing both ionic / ionizable species and neutral analytes.

[0090] (Explanation of analytical method) Column: Gemini-NX, 5 μm, 4.6 × 250 mm Eluent A: 20mM (NH4)3PO4 buffer (pH6.2)+ 5mM Sodium 1-heptanesulfonate monohydrate (ion pair reagent) Eluent B: ACN Gradient %B: 7-7 (2 min); 7-15 (19 min); 15-60 (25 min) Flow rate: 1.0 ml / min, T: 30℃, UV: 220nm IMD-linaclotide: RRT 0.84-0.85 Cys-1-α-ketone-linaclotide RRT 0.91-0.92 Limit of detection (LOD): 40 ppm Limit of quantification (LOQ): 50 ppm

[0091] (B. Multimer Content) By gel permeation chromatography (GPC).

[0092] (Explanation of analytical method) Column: TSK Gel G2000SWXL, 5 μm, 7.8 × 300 mm Eluent: 70% ACN + 0.02% TFA Gradient: Isocratic (40 min) Flow rate: 0.6ml / min, T: 30℃, UV: 215nm Multimer (sum of peaks) RRT ≦0.81-0.82

[0093] (C. Physical form) The physical form of the amorphous high purity linaclotide was analyzed by the following: PXRD (Powder X-ray Diffraction) for evaluation of crystal structure, DSC (differential scanning calorimetry) to evaluate endothermic / exothermic events associated with the presence of possible crystalline contaminants; Evaluate by.

[0094] The following examples are intended to further illustrate the present invention but are not intended to limit it. EXAMPLES

[0095] Example 1 Synthesis of Fragment A [1-5] Fmoc-Cys(SO3Na)-Cys(SO3Na)-Glu(tBu)-Tyr(tBu)-OH [Molecular weight 1055.14, disodium salt].

[0096] (Step I) Synthesis of TFA salt of H-Glu(tBu)-Tyr(tBu)-OAll 1240 grams of DCHA salt of Trt-Glu(tBu)-OH was dissolved in 8.7 L of iPrOAc, the organic solution was washed with an aqueous solution of KHSO4, then concentrated, and the residue was dissolved in NMP (5.6 L). H-Tyr(tBu)-OAll hydrochloride (590 grams) was added, and the coupling reaction was carried out in a PyOxim / NMM system.

[0097] The reaction mixture was diluted with iPrOAc, and the peptide-rich organic solution was washed with aqueous NaHCO3 and aqueous NaCl, then concentrated and the residue was dissolved in DCM (6.1 L). Acidic deprotection was carried out using a TFA-TIS mixture. The reaction was concentrated and precipitated with a mixture of n-heptane-DIPE, filtered, washed and dried.

[0098] The TFA salt of H-Glu(tBu)-Tyr(tBu)-OAll was obtained with 99.8% HPLC purity and 89% yield.

[0099] (Step II) Synthesis of Fmoc-Cys(SO3Na)-Cys(SO3Na)-Glu(tBu)-Tyr(tBu)-OH 952 grams of the TFA salt of H-Glu(tBu)-Tyr(tBu)-OAll and 810 grams of Fmoc-Cys(SO3Na)-ONa were dissolved in 4.8 L of DMF, and the coupling reaction was carried out using the PyOxim / 2,4,6-collidine system.

[0100] The reaction mixture was diluted with iPrOAc, and the peptide-rich organic solution was washed with aqueous KHSO4, NaHCO3, and NaCl, then concentrated, and the residue was dissolved in iPrOAc. Basic deprotection was performed with DEA.

[0101] The reaction mixture was diluted with iPrOAc and the peptide-rich organic solution was washed with aqueous NaHCO3, KHSO4, and NaCl, then concentrated and the residue was dissolved in DMF (7.9 L).

[0102] Fmoc-Cys(SO3Na)-ONa (695 grams) was added and the coupling reaction was carried out with the PyOxim / 2,4,6-collidine system. The reaction mixture was diluted with DCM and the peptide-rich organic solution was washed with aqueous KHSO4, NaHCO3, and NaCl, then concentrated and the residue was dissolved in DCM (19.7 L).

[0103] Allyl ester removal was performed using PhSiH3 and Pd(PPh3)4. The reaction mixture was quenched by the addition of aqueous NaCl, the two phases were separated and aqueous NaHCO3 was added to the organic phase. The peptide was extracted from the aqueous phase by further addition of DCM. The organic phase was concentrated and precipitated with MTBE, filtered, washed and dried.

[0104] Fmoc-Cys(SO3Na)-Cys(SO3Na)-Glu(tBu)-Tyr(tBu)-OH was obtained with an HPLC purity of 94.3% and a yield of 65%.

[0105] Example 2 Synthesis of Fragment B [7-8] Fmoc-Asn(Trt)-Pro-OH [Molecular weight 693.80].

[0106] 177 grams of H-Pro-OBzl hydrochloride was suspended in 1.8 L of DMF. Fmoc-Asn(Trt)-OH (350 grams) was added and the coupling reaction was carried out using the PyOxim / 2,4,6-collidine system. The reaction mixture was precipitated with aqueous NaHCO3, filtered, washed and dried.

[0107] The dried product was hydrogenated to remove the benzyl ester protection using IPA (17.0 L) and water (1.0 L) as the reaction solvent and 5% Pd / C (50% wet) (120 g) as catalyst. The reaction was maintained under H2 for not less than 3 hours and 50 minutes. The suspension was filtered, the catalyst cake was washed with a mixture of IPA-water, and the collected solution was precipitated into aqueous KHSO4. The suspension was filtered, washed, and dried.

[0108] Fmoc-Asn(Trt)-OH was obtained with an HPLC purity of 97.0% and a yield of 79%.

[0109] Example 3 Synthesis of Fragment C [9-14] Fmoc-Ala-Cys[Ψ(Dmp,H)pro]-Thr(tBu)- Gly-Cys[Ψ(Dmp,H)pro]-Tyr(tBu)-OtBu [Molecular weight 1303.60].

[0110] (Step I) Synthesis of Fmoc-Gly-Cys[Ψ(Dmp,H)pro]-Tyr(tBu)-OtBu 950 g of Fmoc-Gly-Cys[Ψ(Dmp,H)pro]-OH and 582 g of H-Tyr(tBu)-OtBu hydrochloride were dissolved in 4.8 L of NMP and the coupling reaction was carried out in a TOTU / NMM system. The reaction mixture was precipitated with an aqueous solution of KHSO4, filtered, washed and dried.

[0111] Fmoc-Gly-Cys[Ψ(Dmp,H)pro]-Tyr(tBu)-OtBu was obtained with a purity of 98.8% by HPLC and a step yield of 90%.

[0112] (Step II) Synthesis of H-Gly-Cys[Ψ(Dmp,H)pro]-Tyr(tBu)-OtBu 1277 grams of Fmoc-Gly-Cys[Ψ(Dmp,H)pro]-Tyr(tBu)-OtBu was dissolved in 5.7 L of ACN. Basic deprotection was performed using DEA.

[0113] The reaction mixture was diluted with iPrOAc, and the peptide-rich organic solution was washed with aqueous KHSO4 and NaCl, then concentrated and the residue was dissolved in iPrOAc. The solution was precipitated by the addition of n-heptane, filtered, washed, and dried.

[0114] H-Gly-Cys[Ψ(Dmp,H)pro]-Tyr(tBu)-OtBu was obtained with an HPLC purity of 95.1% and a yield of 93%.

[0115] (Step III) Synthesis of Fmoc-Thr(tBu)-Gly-Cys[Ψ(Dmp,H)pro]-Tyr(tBu)-OtBu 865g of H-Gly-Cys[Ψ(Dmp,H)pro]-OH and 572g of Fmoc-Thr(tBu)-OH were dissolved in 4.3L of NMP and the coupling reaction was carried out using a PyOxim / NMM system. The reaction mixture was diluted with IPA, and precipitated by adding an aqueous solution of NaHCO3, filtered, washed, and dried.

[0116] Fmoc-Thr(tBu)-Gly-Cys[Ψ(Dmp,H)pro]-Tyr(tBu)-OtBu was obtained with an HPLC purity of 93.9% and a yield of 87%.

[0117] (Step IV) Synthesis of Fmoc-Ala-Cys[Ψ(Dmp,H)pro]-Thr(tBu)-Gly-Cys[Ψ(Dmp,H)pro]-Tyr(tBu)-OtBu 1224 grams of Fmoc-Thr(tBu)-Gly-Cys[Ψ(Dmp,H)pro]-Tyr(tBu)-OtBu was dissolved in 6.1 L of iPrOAc. Basic deprotection was performed using DEA.

[0118] The reaction mixture was diluted with iPrOAc and the peptide-rich organic solution was washed with aqueous KHSO4 and aqueous NaCl, then concentrated and the residue was dissolved in NMP (6.1 L).

[0119] Fmoc-Ala-Cys[Ψ(Dmp,H)pro]-OH (688 grams) was added and the coupling reaction was carried out by TOTU / NMM system. The reaction mixture was diluted with IPA, precipitated with aqueous NaHCO3, filtered and washed. The product was then dried.

[0120] Fmoc-Ala-Cys[Ψ(Dmp,H)pro]-Thr(tBu)-Gly-Cys[Ψ(Dmp,H)pro]-Tyr(tBu)-OtBu was obtained with a purity of 91.9% by HPLC and a yield of 93%.

[0121] Example 4 Synthesis of linear protected linaclotide Fmoc-Cys(SO3Na)-Cys(SO3Na)-Glu(tBu)-Tyr(tBu)-Cys(SO3Na)-Cys(Trt)-Asn(Trt)-Pro-Ala-Cys[Ψ(Dmp,H)pro]-Thr(tBu)-Gly-Cys[Ψ(Dmp,H)pro]-Tyr(tBu)-OtBu [Molecular weight 3122.68].

[0122] (Step I) Synthesis of H-Ala-Cys[Ψ(Dmp,H)pro]-Thr(tBu)-Gly-Cys[Ψ(Dmp,H)pro]-Tyr(tBu)-OtBu 1476 g of fragment C was dissolved in 6.6 L of ACN. Basic deprotection was performed using DEA. The reaction mixture was diluted with iPrOAc, and the peptide-rich organic solution was washed with aqueous KHSO4 and NaCl, then concentrated and the residue was dissolved in iPrOAc. The solution was precipitated by adding DIPE, filtered, washed and dried.

[0123] H-Ala-Cys[Ψ(Dmp,H)pro]-Thr(tBu)-Gly-Cys[Ψ(Dmp,H)pro]-Tyr(tBu)-OtBu was obtained with an HPLC purity of 90.3% and a yield of 91%.

[0124] (Step II) Synthesis of Fmoc-Asn(Trt)-Pro-Ala-Cys[Ψ(Dmp,H)pro]-Thr(tBu)-Gly-Cys[Ψ(Dmp,H)pro]-Tyr(tBu)-OtBu 1087 g of H-Ala-Cys[Ψ(Dmp,H)pro]-Thr(tBu)-Gly-Cys[Ψ(Dmp,H)pro]-Tyr(tBu)-OtBu and 698 g of fragment B were dissolved in 5.4 L of ACN and the coupling reaction was carried out in an Oxyma-EDC·HCl / NMM system. The reaction mixture was precipitated with aqueous NaHCO3, filtered, washed and dried.

[0125] Fmoc-Asn(Trt)-Pro-Ala-Cys[Ψ(Dmp,H)pro]-Thr(tBu)-Gly-Cys[Ψ(Dmp,H)pro]-Tyr(tBu)-OtBu was obtained with an HPLC purity of 87.3% and a yield of 98%.

[0126] (Step III) Synthesis of H-Asn(Trt)-Pro-Ala-Cys[Ψ(Dmp,H)pro]-Thr(tBu)-Gly-Cys[Ψ(Dmp,H)pro]-Tyr(tBu)-OtBu 1727 grams of Fmoc-Asn(Trt)-Pro-Ala-Cys[Ψ(Dmp,H)pro]-Thr(tBu)-Gly-Cys[Ψ(Dmp,H)pro]-Tyr(tBu)-OtBu was dissolved in 8.6 L of iPrOAc. Basic deprotection was performed using DEA.

[0127] The reaction mixture was diluted with iPrOAc, and the peptide-rich organic solution was washed with aqueous KHSO4 and NaCl, then concentrated and the residue was dissolved in iPrOAc. The solution was precipitated with DIPE, filtered and washed, and the product was then dried.

[0128] H-Asn(Trt)-Pro-Ala-Cys[Ψ(Dmp,H)pro]-Thr(tBu)-Gly-Cys[Ψ(Dmp,H)pro]-Tyr(tBu)-OtBu was obtained with an HPLC purity of 89.0% and a yield of 89%.

[0129] (Step IV) Synthesis of Fmoc-Cys(Trt)-Asn(Trt)-Pro-Ala-Cys[Ψ(Dmp,H)pro]-Thr(tBu)-Gly-Cys[Ψ(Dmp,H)pro]-Tyr(tBu)-OtBu 1339g of H-Asn(Trt)-Pro-Ala-Cys[Ψ(Dmp,H)pro]-Thr(tBu)-Gly-Cys[Ψ(Dmp,H)pro]-Tyr(tBu)-OtBu and 512g of Fmoc-Cys(Trt)-OH were dissolved in 6.7L of DMF and the coupling reaction was carried out using a TOTU / NMM system. The reaction mixture was precipitated with aqueous NaHCO3, filtered, washed and dried.

[0130] Fmoc-Cys(Trt)-Asn(Trt)-Pro-Ala-Cys[Ψ(Dmp,H)pro]-Thr(tBu)-Gly-Cys[Ψ(Dmp,H)pro]-Tyr(tBu)-OtBu was obtained with an HPLC purity of 84.3% and a yield of 95%.

[0131] (Step V) Synthesis of H-Cys(SO3Na)-Cys(Trt)-Asn(Trt)-Pro-Ala-Cys[Ψ(Dmp,H)pro]-Thr(tBu)-Gly-Cys[Ψ(Dmp,H)pro]-Tyr(tBu)-OtBu 1733 grams of Fmoc-Cys(Trt)-Asn(Trt)-Pro-Ala-Cys[Ψ(Dmp,H)pro]-Thr(tBu)-Gly-Cys[Ψ(Dmp,H)pro]-Tyr(tBu)-OtBu was dissolved in 8.7 L of iPrOAc. Basic deprotection was carried out using DEA. The reaction mixture was diluted with iPrOAc and the peptide-rich organic solution was first washed with aqueous KHSO4, NaHCO3 and NaCl, then concentrated and the resulting residue was dissolved in DMF (8.7 L).

[0132] Fmoc-Cys(SO3Na)-ONa (404 grams) was added and the coupling reaction was carried out using the PyOxim / 2,4,6-collidine system. The reaction mixture was precipitated with aqueous NaHCO3, filtered, washed, and dried. The dried product was dissolved in 8.7 L of iPrOAc. Basic deprotection was carried out using DEA.

[0133] The reaction mixture was diluted with iPrOAc, the peptide-rich organic solution was washed with aqueous KHSO4 and aqueous NaCl, then concentrated, and the resulting residue was dissolved in iPrOAc, precipitated with DIPE, filtered, and washed, and the product was then dried.

[0134] H-Cys(SONa)-Cys(Trt)-Asn(Trt)-Pro-Ala-Cys[Ψ(Dmp,H)pro]-Thr(tBu)-Gly-Cys[Ψ(Dmp,H)pro]-Tyr(tBu)-OtBu was obtained with an HPLC purity of 82.9% and a yield of 83%.

[0135] (Process VI) Chain protected peptide Fmoc-Cys(SO3Na)-Cys(SO3Na)-Glu(tBu)-Tyr(tBu)-Cys(SO3Na)-Cys(Trt)-Asn Synthesis of (Trt)-Pro-Ala-Cys[Ψ(Dmp,H)pro]-Thr(tBu)-Gly-Cys[Ψ(Dmp,H)pro]-Tyr(tBu)-OtBu

[0136] 1400 g of H-Cys(SO3Na)-Cys(Trt)-Asn(Trt)-Pro-Ala-Cys[Ψ(Dmp,H)pro]-Thr(tBu)-Gly-Cys[Ψ(Dmp,H)pro]-Tyr(tBu)-OtBu and 850 g of fragment A were dissolved in 7.0 L of ACN and the coupling reaction was carried out using Oxyma / B-EDC·HCl / 2,4,6-collidine system. The reaction mixture was precipitated with aqueous NaHCO3, filtered, washed and dried.

[0137] The linear protected peptide was obtained with 66.7% HPLC purity and quantitative yield.

[0138] Example 5 Synthesis of advanced linear intermediates TFA salt of H-Cys(SO3H)-Cys(SO3H)-Glu-Tyr-Cys(SO3H)-Cys-Asn-Pro-Ala-Cys-Thr-Gly-Cys-Tyr-OH [Molecular weight 1772.98, free base].

[0139] (Process I) Synthesis of H-Cys(SO3Na)-Cys(SO3Na)-Glu(tBu)-Tyr(tBu)-Cys(SO3Na)-Cys(Trt)-Asn(Trt)-Pro-Ala-Cys[Ψ(Dmp,H)pro]-Thr(tBu)-Gly-Cys[Ψ(Dmp,H)pro]-Tyr(tBu)-OtBu 2126 grams of the linear protected peptide was dissolved in 10.6 L of ACN. Basic deprotection was performed using DEA. The reaction mixture was diluted with iPrOAc, and the peptide-rich organic solution was washed with aqueous KHSO4 and NaCl, then concentrated, and the resulting residue was dissolved in iPrOAc. The solution was precipitated by the addition of n-heptane, filtered, washed, and dried.

[0140] H-Cys(SO3Na)-Cys(SO3Na)-Glu(tBu)-Tyr(tBu)-Cys(SO3Na)-Cys(Trt)-Asn(Trt)-Pro-Ala-Cys[Ψ(Dmp,H)pro]-Thr(tBu)-Gly-Cys[Ψ(Dmp,H)pro]-Tyr(tBu)-OtBu was obtained with an HPLC purity of 61.7% and a yield of 89%.

[0141] (Step II) Synthesis of the TFA salt of the chain-type advanced intermediate H-Cys(SO3H)-Cys(SO3H)-Glu-Tyr-Cys(SO3H)-Cys-Asn-Pro-Ala-Cys-Thr-Gly-Cys-Tyr-OH 450 grams of H-Cys(SO3Na)-Cys(SO3Na)-Glu(tBu)-Tyr(tBu)-Cys(SO3Na)-Cys(Trt)-Asn(Trt)-Pro-Ala-Cys[Ψ(Dmp,H)pro]-Thr(tBu)-Gly-Cys[Ψ(Dmp,H)pro]-Tyr(tBu)-OtBu was treated with TFA-water-TIS solution (20.3 L-2.3 L-0.5 L). The reaction mixture was precipitated by the addition of MTBE, filtered, washed, and dried.

[0142] The chain advanced intermediate was obtained with 57.9% HPLC purity and 92% yield.

[0143] Example 6 Synthesis of crude linaclotide solution

[0144] [ka]

[0145] 90 grams of the chain-advanced intermediate (estimated 49 grams of 100% peptide) was added to a mixture of phosphate buffer (pH=7.4) and IPA, and the reaction mixture was stirred for 16-32 hours. The mixture was concentrated to remove the IPA and filtered.

[0146] A crude linaclotide solution (peptide content 30 grams) was obtained with an HPLC purity of 53.8% and a cyclization yield of 72%.

[0147] Example 7 Purification of crude linaclotide solution

[0148] The crude linaclotide solution (linaclotide content 75.4 g) was loaded onto a C18 column (diameter 100 mm×maximum height 350 mm) and purified applying three separate and consecutive chromatographic steps. - 1st process Gradient: 0% to 100% of eluent B in 180 min. Eluent A: 0.15% TFA Eluent B: Eluent A-ACN 1-1 - 2nd process Gradient: 0% to 100% of eluent B in 180 min. Eluent A: 20mM NH4H2PO4 buffer (pH 6) Eluent B: Eluent A-ACN 1-1 - 3rd process Gradient: 0% to 100% of eluent B in 180 min. Eluent A: 20~100mM AcOH Eluent B: Eluent A-ACN 1-1

[0149] Finally, 41.7 grams of purified linaclotide solution was obtained from 75.4 grams with 99.97% chromatographic purity and 55% yield (3 steps).

[0150] Example 8 Isolation of high-purity linaclotide from tBuOH-water solution (Option 1) Purified linaclotide solution (41.7 grams, linaclotide concentration 4.2 g / L) was treated as follows: · ACN to tBuOH exchange was performed by the following method: the above solution was loaded onto the C18 column of the previous example and eluted with AcOH (100 mM)-tBuOH 1-1 solution, finally obtaining a solution of linaclotide in water (AcOH) and tBuOH; Cycle the above solution as follows: Freezing of the matrix at -50°C under normal pressure; A primary freeze-drying step at −20° C. under vacuum; and A secondary freeze-drying step in which the freeze-dried product is dried under vacuum at +20°C. The mixture was freeze-dried according to the procedure described above.

[0151] 46.4 grams of amorphous high purity linaclotide powder was obtained (assay 88.1%), equivalent to 40.9 grams of 100% linaclotide.

[0152] The resulting product has the following analytical attributes: Chromatographic purity (IPC) 99.94% (Figure 1); IMD-linaclotide LT 40ppm (LT LOD) (Figure 4), Cys-1-α-ketone-linaclotide LT 40 ppm (LT LOD) (Figure 4), - Polymer content 0.07% (Figure 6), -Isolated yield 98%.

[0153] PXRD and DSC analysis of the resulting product confirmed that the powder was completely amorphous (Figures 8 and 9).

[0154] Example 9 Isolation of high-purity linaclotide from aqueous suspension (Option 2) The purified linaclotide solution (linaclotide content 42.9 g, concentration 4.3 g / L) was treated as follows: Concentration under vacuum was performed to remove ACN to obtain an aqueous suspension; Cycle the above solution as follows: Freezing of the matrix at -50°C under normal pressure; A primary freeze-drying step at −20° C. under vacuum; and A secondary freeze-drying step in which the freeze-dried product is dried under vacuum at +20°C. The mixture was freeze-dried according to the procedure described above.

[0155] 40.9 g of amorphous high purity linaclotide powder was obtained (assay 95.7%), equivalent to 39.1 g of 100% linaclotide.

[0156] The resulting product has the following analytical attributes: Chromatographic purity (IPC) 99.91% (Figure 2), IMD-linaclotide LT 40 ppm (LT LOD) (Figure 5), Cys-1-α-ketone-linaclotide LT 40 ppm (LT LOD) (Figure 5), - Polymer content 0.06% (Figure 7), Isolated yield 91%.

[0157] PXRD and DSC analysis of the resulting product confirmed that the powder was a completely amorphous solid (Figures 10 and 11).

Claims

1. 1. A liquid phase process for producing linaclotide, preferably in the form of an acetate salt, said process comprising: a1) coupling dipeptide fragment B[7-8] of formula (II), Fmoc-Asn(Trt)-Pro-OH, with hexapeptide fragment C[9-14] of formula (III), Fmoc-Ala-Cys[Ψ(Dmp,H)pro]-Thr(tBu)-Gly-Cys[Ψ(Dmp,H)pro]-Tyr(tBu)-OtBu, to obtain peptide [7-14]; a2) coupling peptide [7-14] with an amino acid derivative at position 6 having Fmoc-Cys(Trt)-OH of formula (IV) to obtain peptide [6-14]; a3) Peptide [6-14] is reacted with Fmoc-Cys(SO 3 coupling with an amino acid derivative at position 5 having Na)-ONa to obtain peptide [5-14]; a4) Peptide [5-14] is converted to Fmoc-Cys(SO 3 Na)-Cys(SO 3 Na)-Glu(tBu)-Tyr(tBu)-OH to form the tetrapeptide fragment A[1-4] of formula (VII), Fmoc-Cys(SO 3 Na)-Cys(SO 3 Na)-Glu(tBu)-Tyr(tBu)-Cys(SO 3 obtaining a linear protected peptide of Na)-Cys(Trt)-Asn(Trt)-Pro-Ala-Cys[Ψ(Dmp,H)pro]-Thr(tBu)-Gly-Cys[Ψ(Dmp,H)pro]-Tyr(tBu)-OtBu; b) Deprotecting compound (VII) by reaction with a secondary base and trifluoroacetic acid to give H-Cys(SO) of formula (VIII) 3 H)-Cys(SO 3 H)-Glu-Tyr-Cys(SO 3 H) obtaining an intermediate of -Cys-Asn-Pro-Ala-Cys-Thr-Gly-Cys-Tyr-OH TFA salt; c) carrying out non-oxidative cyclization of intermediate (VIII) to obtain crude linaclotide; d) optionally purifying the crude linaclotide to obtain purified linaclotide; e) optionally salting out the purified linaclotide with acetic acid to form the corresponding acetate salt; A method comprising:

2. 2. The process according to claim 1, wherein the secondary base in step b) is a secondary amine, preferably DEA, piperidine, piperazine or morpholine, and the trifluoroacetic acid is an at least 80% by volume aqueous solution.

3. 2. The method of claim 1, wherein step c) is carried out by intramolecular nucleophilic substitution.

4. 2. The method of claim 1, wherein step c) is carried out in a hydroalcoholic buffer solution with a pH of 7-8.

5. The method according to claim 1, characterized in that in step c), crude linaclotide is obtained as an aqueous solution.

6. 2. The process according to claim 1, wherein one or more of steps a1) to a4) are carried out in the presence of ethyl cyano(hydroxyimino)acetate or a 1,3-dimethylbarbituric acid derivative.

7. 2. The process according to claim 1, characterized in that one or more of steps a1) to a4) are carried out in an organic polar solvent, preferably a solvent selected from DMF, NMP, ACN.

8. 2. The process according to claim 1, characterized in that one or more of steps a1) to a4) are carried out at a temperature ranging from -10°C to 35°C, preferably from 0°C to 25°C.

9. 2. The method of claim 1, wherein the purification step d) is performed by reversed-phase high performance sample displacement (RP-HPSD), reversed-phase high performance liquid chromatography (RP-HPLC) or a combination thereof.

10. 10. The method according to claim 9, wherein the elution phase consists of an aqueous solution, a polar organic solvent or a mixture thereof, preferably the polar organic solvent is selected from trifluoroacetic acid, acetic acid, acetonitrile and a mixture thereof, optionally with the addition of a buffer solution, more preferably a phosphate buffer solution.

11. 10. The method of claim 9, wherein the purified linaclotide is in a solution, preferably the solution comprises water, acetic acid and acetonitrile.

12. 10. The method of claim 9, wherein the linaclotide has an HPLC purity of 99.9% or more, and the contents of IMD-linaclotide and Cys-1-α-ketone-linaclotide impurities are each less than 100 ppm, preferably less than 50 ppm, more preferably less than 40 ppm.

13. 2. The method according to claim 1, further comprising an isolation step f) by lyophilization starting from a hydroalcoholic solution or aqueous suspension.

14. 14. The method of claim 13, wherein the hydroalcoholic solution contains tert-butanol, water, acetic acid, or a mixture thereof.

15. 14. The method of claim 13, wherein the aqueous suspension is obtained by evaporation of a purified linaclotide solution, more preferably the aqueous suspension contains acetic acid.

16. Amorphous linaclotide or an acetate salt thereof, characterized in that it has an HPLC purity of 99.9% or more, and the contents of IMD-linaclotide (impurity 1) and Cys-1-α-ketone-linaclotide (impurity 2) are each less than 100 ppm, preferably less than 50 ppm, and more preferably less than 40 ppm.

17. Amorphous linaclotide or its acetate salt according to claim 16, characterized in that it has a polymer content of 0.1% area % or less.

18. A method for detecting and quantifying the impurity contents of IMD-linaclotide and Cys-1-α-ketone-linaclotide in amorphous high-purity linaclotide or its acetate salt, the method comprising the step of eluting the product through an ion pair chromatography (IPC) column having a silica stationary phase containing alkyl chains and an elution phase consisting of an aqueous solution, a polar organic solvent, or a mixture thereof, and optionally adding a buffer, preferably a phosphate buffer, to the elution phase.

19. 19. The method of claim 18, wherein the alkyl chain is of the octadecyl type (C18), octyl type (C8) or butyl type (C4), preferably C18.

20. 19. The method of claim 18, wherein the polar organic solvent is selected from tetrahydrofuran, dioxane, dichloromethane, methanol, ethanol, n-propanol, isopropanol, butanol, pentane, hexane, toluene, trifluoroacetic acid, acetonitrile, or a mixture thereof; more preferably, selected from trifluoroacetic acid, acetonitrile, or a mixture thereof.

21. The following operating conditions: Stationary phase: carrier silica particles containing C18 alkyl chains; Eluent A: Phosphate buffer pH 6.2 Eluent B: ACN with the following gradient elution: %B: 7-7 (2 minutes); 7-15 (19 minutes); 15-60 (25 minutes) 20. The method of claim 18, wherein:

22. 19. The method of claim 18, wherein the elution phase comprises an ion pairing reagent, preferably a heptane sulfonate salt.

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