A 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:
a) performing RP-HPLC with the said complex mixture in the presence of an ion pairing agent in combination with an organic modifier such as acetonitrile, in acidic pH to yield a first mixture comprising partially purified non-glycosylated insulin analogue; and
b) performing RP-HPLC on the first mixture in the presence of an ion pairing agent in combination with an organic modifier such as acetonitrile, in alkaline pH to obtain at least 99.95% purified non-glycosylated insulin analogue.
or;
a) performing RP-HPLC on the first mixture in the presence of an ion pairing agent in combination with an organic modifier such as acetonitrile in alkaline pH to yield a first mixture comprising partially purified non-glycosylated insulin analogue; and
b) performing RP-HPLC with the said first mixture in the presence of an ion pairing agent in combination with an organic modifier such as acetonitrile, in acidic pH to obtain at least 99.95% purified non-glycosylated insulin analogue.
The process as claimed in claim 1, wherein the insulin analogue is selected from the group consisting of insulin Glargine, and insulin Lispro.
The process as claimed in claim 1, wherein the first mixture comprising partially purified non-glycosylated insulin analogue obtained by low pH-based RP-HPLC comprises the following steps:
a) packing RP-HPLC column with silica-based resin, preferably C8;
b) loading of column at a capacity of 4-12g/L;
c) washing of column with an ion pairing agent, preferably Octane Sulphonic Acid (OSA), in combination with an organic modifier, preferably acetonitrile, at

acidic pH ranging from 3.5 to 3.9; and
d) performing a linear gradient of 24% to 31% for eluting the first mixture of partially purified non-glycosylated insulin analogue.
4. The process as claimed in claim 1, wherein the at least 99.95% purified non-
glycosylated insulin analogue obtained by high pH-based RP-HPLC following the
low pH based RP-HPLC step comprises the following steps:
a) packing RP-HPLC column with silica-based resin, preferably C8;
b) loading of column by first mixture at a capacity of 4-12g/L;
c) washing of column with an ion pairing agent in combination with acetonitrile at alkaline pH ranging from 8.3 to 8.7; and
d) performing a linear gradient of 25% to 29% for eluting at least 99.95% purified non-glycosylated insulin analogue.
5. The process as claimed in claim 1, wherein the first mixture comprising
partially purified non-glycosylated insulin analogue is obtained by high pH-based
RP-HPLC comprising the following steps:
a) packing RP-HPLC column with silica-based resin, preferably C8;
b) loading of column by first mixture at a capacity of 4-12g/L;
c) washing of column with an ion pairing agent in combination with acetonitrile at alkaline pH ranging from 7.3 to 8.7; and
d) performing a linear gradient of 25% to 29% for eluting the first mixture of partially purified non-glycosylated insulin analogue.
6. The process as claimed in claim 1, wherein the at least 99.95% purified
insulin analogue is obtained by low pH-based RP-HPLC following the high pH
based RP-HPLC step comprises the following steps:
a) packing RP-HPLC column with silica-based resin, preferably C8;
b) loading of column at a capacity of 4-12g/L;
c) washing of column with an ion pairing agent, preferably Octane Sulphonic Acid (OSA), in combination with organic modifier, preferably acetonitrile, at

acidic pH ranging from 3.5 to 3.9; and
d) performing a linear gradient of 24% to 31% for eluting at least 99.95% purified non-glycosylated insulin analogue.
7. The process as claimed in claim 1, wherein the ion pairing agent is selected from the group consisting of sodium salt of Octane Sulphonic Acid (OSA), sodium perchlorate, and Tetra butyl ammonium bisulphate (TBAB).
8. The process as claimed in claim 4 and 5, wherein said ion pairing agent is sodium perchlorate when insulin analogue is insulin Glargine, and wherein said ion pairing agent is TBAB when insulin analogue is insulin Lispro.

9. The process as claimed in claiml, wherein insulin analog is insulin Glargine comprising less than 0.05% glycosylated variants of insulin Glargine.
10. The process as claimed in claim 1, wherein insulin analog is insulin Lispro comprising less than 0.05% glycosylated variants of insulin Lispro.
11. Non-glycosylated insulin Glargine at least 99.95% free of glycosylated variants of insulin Glargine.
12. Non-glycosylated insulin Lispro at least 99.95% free of glycosylated variants
of insulin Lispro.