Manufacture of glucagon peptides

A method for the preparation of purified glucagon peptide, comprising:

• a) providing a liquid composition C comprising a chemically synthesized glucagon peptide and at least one peptidic impurity selected from a glucagon derivative, a glucagon truncation variant, a derivative of a glucagon truncation variant, a glucagon deletion variant, a derivative of a glucagon deletion variant, and a combination thereof;
• b) subjecting the composition C to a first reversed phase high performance liquid chromatography (RP-HPLC) purification, wherein a hydrocarbon bonded silica is used as a stationary phase, an aqueous mobile phase of pH 6.5 to 7.5 comprising triethylammonium phosphate and acetonitrile is used, and elution is effected by gradually increasing the acetonitrile concentration within the mobile phase while collecting fractions containing the glucagon peptide;
• and
• c) subjecting the pooled glucagon peptide-containing fractions obtained in step b) to a second reversed phase HPLC purification, wherein a hydrocarbon bonded silica is used as a stationary phase, a mobile phase comprising acetic acid and acetonitrile is used, wherein the concentration of acetic acid in the mobile phase of step c) is between 0.5 and 5% v/v, and elution is effected by gradually increasing the acetonitrile concentration within the mobile phase while collecting and pooling fractions containing the purified glucagon peptide,
• wherein the same stationary phase is used in steps b) and c).