Metal-induced oxidation of polysorbate 80 in the presence of hydrogen peroxide: mechanistic studies.

Polysorbate 80 (PS80) is a commonly used surfactant in protein formulations and is highly susceptible to oxidative degradation. Here, we present mechanistic studies on metal-induced PS80 oxidation in the presence of hydrogen peroxide in acetate buffer. A total of 12 pharmaceutically relevant metal ions were tested, including Mn(II), Cu(II), Cu(I), Mg(II), Zn(II), Ca(II), Al(III), Pb(II), Sn(II), Co(II), Fe(III), Ni(II) and W(IV). The overall PS80 degradation was monitored by a fluorescence micelle assay (FMA) and the oxidation products were characterized by mass spectrometry (MS). Three metal ions, Cu(II), Co(II), and Fe(III), induced significant PS80 degradation and solutions containing these ions were subjected to further mechanistic studies. The extent of oxidation was dependent on both metal and peroxide concentrations. PS80 degradation catalyzed by Cu(II) and Co(II) was completely prevented by 250 µM and 500 µM ethylenediaminetetraacetic acid (EDTA), but only partially inhibited when catalyzed by Fe(III). The role for superoxide radical anion in the initiation of PS80 oxidation was examined by addition of Cu,Zn superoxide dismutase (SOD). The potential of the metal ions to generate free radicals was monitored with the spin trap 5,5-dimethyl-1-pyrroline N-oxide (DMPO), followed by LC-MS analysis. Degradation mechanisms, particularly the initiation of the oxidation chain reactions, are discussed for each metal.