Oxidation of human insulin-like growth factor I in formulation studies. 3. Factorial experiments of the effects of ferric ions, EDTA, and visible light on methionine oxidation and covalent aggregation in aqueous solution.

The influence of ferric ions, EDTA, and visible light on the oxidation of methionine and the covalent reducible and nonreducible dimerization in human Insulin-like Growth Factor I (hIGF-I) in aqueous (1 mM) phosphate buffer solution were studied. A reduced factorial experiment with two levels of each factor was used. Regression models for the three responses were constructed with partial least square (PLS) analysis. The hIGF-I variants were quantified by reversed-phase high-performance liquid chromatography (RP-HPLC), gel filtration, and reduced sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The formation of the hIGF-I variants in aqueous solution at pH 6.1 exhibited different dependencies on the variables studied. The oxidation of methionine was affected mainly by visible light and the combination of 10 ppm ferric ions and 20 ppm EDTA, whereas ferric ions alone had no significant effect. The covalent dimerization of hIGF-I was correlated to visible light and ferric ions. The interaction effects of ferric ions with either visible light or EDTA were also significant on the dimerization rates. Both reducible and nonreducible soluble covalent dimers were formed, with the reducible dimer being the most prominent. The oxidation of methionine 59 in hIGF-I is catalyzed by light and by ferric ions in combination with EDTA. The covalent dimerization of hIGF-I is mainly affected by light and by ferric ions. Both reducible and nonreducible dimerization increased by oxidative conditions. Human IGF-I appears to dimerize covalently by both disulfide scrambling and by a radical-promoted nondisulfide pathway. EDTA is necessary for ferric ions to be active in the oxidation of methionine in hIGF-I but not for the covalent dimerization.