Separation and Characterization of Deamidated Isoforms in Insulin Analogue and its Underlying Mechanism

Deamidation of proteins has remained an elusive phenomenon largely down to the gap that exists in its understanding. One of the primary reasons, is that the experimentation involved is time consuming. However, it remains one of the most important phenomenon in proteomics both in-vitro as well as in-vivo. Deamidation results in the quick conversion of an asparagine residue to a mixture of isoaspartate and aspartate. In glutamine residues deamidation occurs at a much lower rate. Deamidation of asparagine residues is one of the most common post-translational modifications occurring in therapeutic proteins produced using recombinant DNA technology and is the major cause for degradation of bio- pharmaceuticals. Herein, we present the separation and characterization of deamidated Isoforms in a recombinantly produced Insulin analogue by ion exchange chromatography and enzymatic digestion. The deamidation site was confirmed by using state of the art technique: LC-ESI-MS-MS. Additionally secondary structural differences were observed between the unmodified and deamidated variant of the protein.