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Oxidation of human insulin-like growth factor I in formulation studies: kinetics of methionine oxidation in aqueous
J Fransson1, E Florin-Robertsson, K Axelsson
1Department of Pharmaceutical Technology, Pharmacia, Stockholm, Sweden. jonas.fransson@sto.pharmacia.se
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.