Chromatography process for purification of insulin analogues

A two step process for purification of non-glycosylated insulin analogue(s) from a complex mixture comprising both non-glycosylated, and glycosylated insulin analogue(s), wherein the said purification process comprises the steps:

• a1) performing RP-HPLC with the said complex mixture in the presence of an ion pairing agent in combination with acetonitrile as an organic modifier at acidic pH ranging from about 3.5 to about 3.9 to yield an a1 first mixture comprising partially purified non-glycosylated insulin analogue;
• and
• b1) performing RP-HPLC on the a1 first mixture in the presence of an ion pairing agent that is different from step a1 in combination with acetonitrile as the organic modifier at alkaline pH ranging from about 7.3 to about 8.7 to obtain at least 99.95% purified non-glycosylated insulin analogue, wherein the ion pairing agent is selected from the group consisting of octane sulphonic acid, sodium perchlorate and tetrabutyl ammonium bisulphate (TBAB);
• or;
• a2) performing RP-HPLC on said complex mixture in the presence of an ion pairing agent in combination with acetonitrile as-an organic modifier at alkaline pH ranging from about 7.3 to about 8.7 to yield an a2 first mixture comprising partially purified non-glycosylated insulin analogue;
• and
• b2) performing RP-HPLC with the a2 first mixture in the presence of an ion pairing agent that is different from step a2 in combination with acetonitrile as the organic modifier at acidic pH ranging from about 3.5 to about 3.9 to obtain at least 99.95% purified non-glycosylated insulin analogue, wherein the ion pairing agent is selected from the group consisting of octane sulphonic acid, sodium perchlorate and tetrabutyl ammonium bisulphate (TBAB).