Oxidation of human insulin-like growth factor I in formulation studies: kinetics of methionine oxidation in aqueous solution and in solid state.

PURPOSE

The aim of this work was to study the kinetics of oxidation of methionine in human Insulin-like Growth Factor I (hIGF-I)1 in aqueous solution and in the solid state by the aid of quantification of oxygen.

METHODS

The oxidized from of hIGF-I was characterized by tryptic peptide analysis, RP-HPLC and FAB-MS and quantified by RP-HPLC. The oxygen content was quantified polarographically by a Clark-type electrode.

RESULTS

Second-order kinetics with respect to amount of protein and dissolved oxygen was found to be appropriate for the oxidation of methionine in hIGF-I. The rate constants ranged from 1 to 280 M-1 month-1 and had an activation energy of 95 (+/-4) kl/mole. Light exposure, storage temperature and oxygen content were found to have a considerable impact on the oxidation rates. No significant difference in reaction rates was found for the oxidation of hIGF-I in aqueous solution or in the solid state. A method for decreasing the oxygen content in aqueous solution without purging is described.

CONCLUSIONS

Polarographic quantification of dissolved oxygen makes it possible to establish the kinetics for oxidation of proteins. The oxidation of methionine in hIGF-I appears to follow second-order kinetics.