Cobalt(III)-induced hexamerization of PEGylated insulin

Insulin conjugates in which the B1Phe residue has been chemically modified often exhibit a reduced tendency to associate into hexamers due to weakened interactions between subunits. The purpose of this study was to prepare a hexamer formulation for such insulin conjugates by using Co(III) as a coordinating metal ion. PEGylated insulin in which monomethoxypoly(ethylene glycol) (mPEG, M_r 5000 or 20,000) had been site-specifically attached to B1Phe was chosen as a model conjugate. Hexamerization of mPEG-insulin upon H₂O₂-mediated oxidation of Co(II) was kinetically and quantitatively analysed by visible spectrometry and size-exclusion HPLC. Co(III) mPEG-insulin hexamers thus obtained were extremely stable, existing mostly as a hexameric form even at nanomolar concentrations. A remarkable increase in hydrodynamic volumes was observed for Co(III) mPEG(20k)-insulin hexamers (1600 kDa), as well as Co(III) mPEG(5k)-insulin hexamers (300 kDa). Our results demonstrate the potential benefits of Co(III) hexamer formulation for weakly associating insulin conjugates in the treatment of diabetes.