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 o f methionine in human Insulin-like Growth Factor I (hIGF-I)(I) in aque ous solution and in the solid state by the aid of quantification of ox ygen. Methods. The oxidized form of hIGF-I was characterized by trypti c peptide analysis, RP-HPLC and FAB-MS and quantified by RP-HPLC. The oxygen content was quantified polarographically by a Clark-type electr ode. 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) mo nth(-1) and had an activation energy of 95 (+/- 4) kJ/mole. Light expo sure, storage temperature and oxygen content were found to have a cons iderable impact on the oxidation rates. No significant difference in r eaction rates was found for the oxidation of NGF-I in aqueous solution or in the solid state. A method for decreasing the oxygen content in aqueous solution without purging is described. Conclusions. Polarograp hic 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.