# US 11,993,631 B2 · Process for manufacturing peptide > Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt - Number: US 11,993,631 B2 - Document ID: US11993631B2 - Status: Granted - Kind: B2 - Application number: 18/270,509 - Filed: 2021-12-29 - Published: 2024-03-21 - Granted: 2024-05-28 - Earliest claimed: 2020-12-30 - Assignees: BioLineRx Ltd. - Applicants: BioLineRx Ltd. - Inventors: Efrat Halbfinger - Web page: https://patentgrep.com/patent/US11993631B2 ## CPC - C07K 1/20: Partition-, reverse-phase or hydrophobic interaction chromatography - C07K 7/08: having 12 to 20 amino acids (gastrins C07K14/595; somatostatins C07K14/655; melanotropins C07K14/68) - C07K 1/042: {characterised by the nature of the carrier} - C07K 1/10: using coupling agents {(C07K1/006 takes precedence)} - C07K: PEPTIDES (peptides in foodstuffs A23; obtaining protein compositions for foodstuffs, working-up proteins for foodstuffs A23J; preparations for medicinal purposes A61K; peptides containing beta-lactam rings C07D; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, C07D; ergot alkaloids of the cyclic peptide type C07D519/02; macromolecular compounds having statistically distributed amino acid units in their molecules, i.e. when the preparation does not provide for a specific; but for a random sequence of the amino acid units, homopolyamides and block copolyamides derived from amino acids C08G69/00; macromolecular products derived from proteins C08H1/00; preparation of glue or gelatine C09H; single cell proteins, enzymes C12N; genetic engineering processes for obtaining peptides C12N15/00; compositions for measuring or testing processes involving enzymes C12Q; investigation or analysis of biological material G01N33/00) ## Abstract A large-scale process is described herein for preparing a cyclic peptide as described, comprising solid phase peptide synthesis of a linear peptide and cleaving it from the resin; oxidizing cysteine residues to form an intramolecular disulfide bond; and isolating the cyclic peptide, wherein: (i) coupling uses diisopropylcarbodiimide and ethyl cyanohydroxyiminoacetate and/or N-hydroxybenzotriazole; (ii) cleaving comprises contacting the peptide with a solution comprising TFA and dithioerythritol and/or dithiothreitol; (iii) the peptide is precipitated after cleaving without prior concentration of the peptide by evaporation; (iv) oxidizing comprises contacting an aqueous solution comprising at least 5 mg/mL peptide with hydrogen peroxide; (v) isolating comprises loading the peptide on a reverse phase chromatography at up to 40 grams/kg column, and elution from the column; (vi) isolating comprises lyophilization, followed by grinding the peptide; and/or (vii) substitution of the resin is at least 0.3 milliequivalents/gram, and/or the resin is a Rink aminomethylstyrene resin. ## Claims ### Claim 1 A large-scale process of preparing a cyclic peptide having SEQ ID NO: 1, or a pharmaceutically acceptable salt thereof, the process comprising: (a) sequentially coupling amino acids and 4-fluorobenzoic acid to a resin by solid phase peptide synthesis, thereby obtaining a linear peptide coupled to said resin; (b) cleaving said linear peptide from said resin, thereby obtaining a free linear peptide; (c) oxidizing cysteine residues of said linear peptide to form an intramolecular disulfide bond, thereby obtaining the cyclic peptide having SEQ ID NO: 1 in solution; and (d) isolating the cyclic peptide having SEQ ID NO: 1, or a pharmaceutically acceptable salt thereof, wherein: (i) said coupling is effected using diisopropylcarbodiimide (DIC) in combination with ethyl cyanohydroxyiminoacetate and/or N-hydroxybenzotriazole; (ii) said cleaving is effected by contacting said linear peptide coupled to said resin with a solution comprising trifluoroacetic acid (TFA) and a scavenger selected from the group consisting of dithioerythritol (DTE) and dithiothreitol (DTT); (iii) the process further comprises precipitating said free linear peptide after said cleaving without concentrating said free linear peptide by evaporation prior to said precipitating; (iv) said oxidizing is effected by contacting an aqueous solution comprising said linear peptide at a concentration of at least 5 mg/mL with hydrogen peroxide; (v) said isolating comprises loading said cyclic peptide on a reverse phase chromatography column at a concentration of no more than 40 grams cyclic peptide per kg of said column, and eluting said cyclic peptide from said column; (vi) said isolating the cyclic peptide having SEQ ID NO: 1 comprises lyophilization, and the process further comprises grinding said cyclic peptide following said lyophilization; and/or (vii) a degree of substitution of said resin is at least 0.3 milliequivalents per gram and/or said resin is a Rink aminomethylstyrene resin. ### Claim 2 (depends on 1) The process of claim 1, wherein said coupling is effected using diisopropylcarbodiimide (DIC) in combination with ethyl cyanohydroxyiminoacetate and/or N-hydroxybenzotriazole. ### Claim 3 (depends on 1) The process of claim 1, wherein said DIC and said ethyl cyanohydroxyiminoacetate and/or N-hydroxybenzotriazole are used in a molar excess of about two-fold. ### Claim 4 (depends on 1) The process of claim 1, wherein a concentration of said scavenger in said solution comprising said scavenger is in a range of from 10 mg/mL to 500 mg/mL. ### Claim 5 (depends on 1) The process of claim 1, wherein said cleaving is effected by contacting said linear peptide coupled to said resin with a solution comprising trifluoroacetic acid (TFA) and a scavenger selected from the group consisting of dithioerythritol (DTE) and dithiothreitol (DTT). ### Claim 6 (depends on 5) The process of claim 5, wherein a concentration of dithiothreitol in said solution is about 50 mg/mL. ### Claim 7 (depends on 1) The process of claim 1, further comprising precipitating said free linear peptide after said cleaving without concentrating said free linear peptide by evaporation prior to said precipitating. ### Claim 8 (depends on 1) The process of claim 1, wherein said precipitating is effected by addition of a mixture of tert-butyl methyl ether (MTBE) and hexane at a volume of about 45 mL of said mixture per gram of said resin. ### Claim 9 (depends on 1) The process of claim 1, wherein said oxidizing is effected by contacting said linear peptide with hydrogen peroxide. ### Claim 10 (depends on 9) The process of claim 9, wherein said contacting is effected by contacting an aqueous solution comprising said linear peptide at a concentration of at least 5 mg/mL with hydrogen peroxide. ### Claim 11 (depends on 1) The process of claim 1, wherein said isolating comprises loading said cyclic peptide on a reverse phase chromatography column at a concentration of no more than 40 grams cyclic peptide per kg of said column, and eluting said cyclic peptide from said column. ### Claim 12 (depends on 1) The process of claim 1, wherein said column is a C18 column. ### Claim 13 (depends on 1) The process of claim 1, wherein said eluting is effected with triethylammonium phosphate solution. ### Claim 14 (depends on 1) The process of claim 1, wherein said isolating the cyclic peptide having SEQ ID NO: 1 comprises lyophilization, and the process further comprises grinding said cyclic peptide following said lyophilization. ### Claim 15 (depends on 1) The process of claim 1, wherein a degree of substitution of said resin is at least 0.3 milliequivalents per gram and/or said resin is a Rink aminomethylstyrene resin.