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Diabetes is a chronic metabolic disorder that has affected approximately 463 million populations till date and this number is expected to exponentially rise in the coming decade. Majority of diabetic patients require multiple insulin injections regularly, at the risk of hypoglycemia. As an alternative to conventional insulin, glargine insulin, a long-acting insulin analog that helps in maintaining blood glucose levels, is being preferred. Hence, the present work focuses on glargine insulin production using recombinant E. coliBL-21(DE3) cells through fed-batch fermentation. After fermentation, the cells were lysed through high pressure homogenization to release inclusion bodies containing insulin glargine precursor and the major impurities like Arg (B31)-insulin were reduced by incorporating citraconylation in appropriate concentration. These steps are novel which makes the present study distinct from the other. Furthermore, refolding and enzymatic digestion of insulin glargine precursor was carried out to obtain glargine insulin. Consequently, purification and polishing of glargine insulin were performed by loading onto cation exchange chromatography and reverse-phase high performance liquid chromatography which fetched 98.6% pure product. Through the aforementioned process, from 20 L of culture broth, nearly 0.3 g/L of recombinant glargine insulin with high purity was obtained in two purification steps. Hence, the present study devises an efficient and economical process for large-scale production of glargine insulin.

Insulin is a well-characterized peptide produced by recombinant DNA technology that is widely used in maintaining blood glucose level in the diabetic patients. In this study, we have used vector pET-9a to express the human insulin in E. coliBL-21 DE3. The recombinant E. colicells were cultured by fed-batch fermentation (20.0 L) process which resulted in a dry cell mass of 20 g/L. Pro-Insulin was expressed as inclusion bodies (IBs) and it was 20% of the total protein measured by densitometry. The IBs were then solubilized and refolded to get the native biological structure. The proinsulin form has been converted into active insulin by enzymatic digestion using trypsin and carboxypeptidase-B. The active insulin was purified using ion-exchange and reverse phase HPLC. The final purified insulin has achieved a purity of 98% with Des-Insulin impurity ≤ 0.5%, when analyzed in analytical HPLC as per the United States Pharmacopiea (USP) method. This optimized process of recombinant human insulin may be used as a model for insulin analogues such as glargine, aspart, lispro etc.