Soluble insulin analogs combining rapid- and long-acting hypoglycemic properties – From an efficient E. coli expression system to a pharmaceutical formulation

Abstract

The discovery of insulin led to a revolution in diabetes management. Since then, many improvements have been introduced to insulin preparations. The availability of molecular genetic techniques has enabled the creation of insulin analogs by changing the structure of the native protein in order to improve the therapeutic properties. A new expression vector pIBAINS for production of four recombinant human insulin (INS) analogs (GKR, GEKR, AKR, SR) was constructed and overexpressed in the new E. coli 20 strain as a fusion protein with modified human superoxide dismutase (SOD). The SOD gene was used as a signal peptide to enhance the expression of insulin. SOD::INS was manufactured in the form of insoluble inclusion bodies. After cleavage of the fusion protein with trypsin, the released insulin analogs were refolded and purified by reverse-phase high performance liquid chromatography (RP-HPLC). Elongation of chain A, described here for the first time, considerably improved the stability of the selected analogs. Their identity was confirmed with mass spectrometric techniques. The biological activity of the insulin derivatives was tested on rats with experimental diabetes. The obtained results proved that the new analogs described in this paper have the potential to generate prolonged hypoglycemic activity and may allow for even less frequent subcutaneous administration than once-a-day. When applied, all the analogs demonstrate a rapid onset of action. Such a combination renders the proposed biosynthetic insulin unique among already known related formulations.

Intein-mediated recombinant expression of monomeric B22Asp desB30 insulin

“…7), indicating no evidence of any misformed disulfides. This result also agrees with previous studies [32,33] where similar recombinantly expressed proinsulin all folded correctly.…”

Identification of Lysine Misincorporation at Asparagine Position in Recombinant Insulin Analogs Produced in E. coli

“…Recombinant human insulin and its analogs are produced at IBA by the precursor method using E. coli expression system by the similar procedure, as described in a literature ( 22 ). One- or two-step enzymatic digestion is applied depending on the type of targeted protein (Fig.…”

“…Lys B31 Arg B32 human insulin, insulin lispro precursor, Ala A22 Lys B31 Arg B32 human insulin, Gly A22 Lys B31 Arg B32 human insulin were from the Institute of Biotechnology and Antibiotics (Warsaw, Poland). Insulin and analogs were produced by recombinant DNA technology in E. coli (K12) essentially as described previously ( 22 ).…”

A non-inferiority study to compare daily fast-acting insulin versus twice a week slow-acting insulin–moderate diabetes mode

“…Fast-acting insulin has an onset of action of 30 minutes and a peak of action between 2 and 4 hours and activity of up to 8 hours. Slow-acting insulin has an onset of 1-2 hours and an action of up to 14h 8,17 .…”