Process for manufacturing peptide
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.
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.
2. The process of claim 1 , wherein said coupling is effected using diisopropylcarbodiimide (DIC) in combination with ethyl cyanohydroxyiminoacetate and/or N-hydroxybenzotriazole.
3. 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.
4. 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.
5. 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).
6. The process of claim 5 , wherein a concentration of dithiothreitol in said solution is about 50 mg/mL.
7. 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.
8. 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.
9. The process of claim 1 , wherein said oxidizing is effected by contacting said linear peptide with hydrogen peroxide.
10. 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.
11. 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.
12. The process of claim 1 , wherein said column is a C18 column.
13. The process of claim 1 , wherein said eluting is effected with triethylammonium phosphate solution.
14. 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.
15. 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.