Dorota Stadnik

Structure and pharmaceutical formulation development of a new long-acting recombinant human insulin analog studied by NMR and MS

&NA; A monomer structure of a novel human insulin analog A22S‐B3K‐B31R (SK3R) has been characterized by NMR in water/acetonitrile solution and compared with the structure of human insulin (HIS) established in the same medium. The composition of the oligomer ensemble for neat insulins in water was qualitatively assessed by monitoring, derived from NMR experiment, translational diffusion coefficient Di x 10−10 m2 s−1, whose value is a population averaged of individual coefficients for species in oligomeric ensemble. Nanospray ESI/MS experiment was used to establish the masses of oligomers in pharmaceutical formulation of the SK3R insulin. The pharmacodynamic data were established and compared to insulin glargine characterized by the same profile of action in diabetics. The oligomerization process of insulin during development of pharmaceutical formulation with routinely used excipients has been studied using translation diffusion coefficient Di x 10−10 m2 s−1 established in water solution. These properties were compared with those of human insulin (HIS) which is a standard reference for novel recombinant insulins. Graphical abstract Figure. No caption available. HighlightsNMR structure of novel long acting recombinant humn insulin analog A22S‐B3K‐B31R established in water/acetonitrile solutionDevelopment studies on pharmaceutical formulation are presented and compared to human insulin formulation.Pharmacokinetic data are presented and compared to glargine insulin characterized by the same profile of action.

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

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.