Manufacture, formulation and dosing of apraglutide

A method of making a sodium salt of apraglutide comprising:

• a) performing solid phase peptide synthesis (SPPS) to synthesize the apraglutide on an Fmoc-Rink-amide-MethylBenzHydril Amine (MBHA)-resin, comprising
• i) preparing an MBHA-resin on which the SPPS will be performed;
• ii) performing an initial Fmoc deprotection reaction followed by a coupling reaction to add a first Fmoc-protected amino acid to the resin, thereby forming a protected peptide on the resin;
• iii) performing an Fmoc deprotection reaction followed by a coupling reaction to append at least one Fmoc-protected amino acid to the protected peptide, comprising:
• (a1) treating the resin with a solution comprising piperidine and oxyma in DMF, wherein the resin is contacted with a first amount of a solution comprising piperidine and oxyma in DMF for 15 minutes followed by contacting the resin with a second amount of a solution comprising piperidine and oxyma in DMF for 30 minutes;
• (b1) washing the resin with DMF;
• (c1) washing the resin with a solution comprising DMF and oxyma;
• (d1) contacting the resin with at least one Fmoc-protected amino acid and a first amount of a solution comprising diisopropylcarbodiimide (DIC) and ethyl cyanohydroxyiminoacetate (oxyma);
• (e1) contacting the resin with a second amount of a solution comprising DIC and oxyma;
• and
• (f1) washing the product formed in step (e2) with DMF;
• iv) repeating step iii until the apraglutide is synthesized on the resin to produce an Fmoc-protected and side-chain protected apraglutide linked to the resin;
• v) performing an Fmoc deprotection reaction to produce a side-chain protected apraglutide linked to the resin;
• and
• vi) drying the side-chain protected apraglutide linked to the resin;
• b) cleaving the synthesized apraglutide off the resin and deprotecting the side chains of the synthesized apraglutide by treating the resin with a solution comprising trifluoroacetic acid (TFA), water, and anisole;
• c) purifying the synthesized apraglutide from step (b) by performing a first preparative reversed-phase high performance liquid chromatography (RP-HPLC) purification using TFA-based mobile phases, thereby producing a solution comprising the apraglutide with a purity of no less than 90% as determined by high-performance liquid chromatography (HPLC);
• d) purifying the product of step (c) by performing a second RP-HPLC purification, using NaHCO3-based mobile phases, thereby producing a solution comprising the apraglutide with a purity of no less than 97% as determined by HPLC;
• and
• e) further purifying the product from step (d) by performing a third RP-HPLC purification using NaOAc-based mobile phases, thereby producing a solution comprising a sodium salt of the apraglutide with a purity of no less than 97% as determined by HPLC.