Peroxide formation in polysorbate 80 and protein stability.

Nonionic surfactants are widely used in the development of protein pharmaceuticals. However, the low level of residual peroxides in surfactants can potentially affect the stability of oxidation-sensitive proteins. In this report, we examined the peroxide formation in polysorbate 80 under a variety of storage conditions and tested the potential of peroxides in polysorbate 80 to oxidize a model protein, IL-2 mutein. For the first time, we demonstrated that peroxides can be easily generated in neat polysorbate 80 in the presence of air during incubation at elevated temperatures. Polysorbate 80 in aqueous solution exhibited a faster rate of peroxide formation and a greater amount of peroxides during incubation, which is further promoted/catalyzed by light. Peroxide formation can be greatly inhibited by preventing any contact with air/oxygen during storage. IL-2 mutein can be easily oxidized both in liquid and solid states. A lower level of peroxides in polysorbate 80 did not change the rate of IL-2 mutein oxidation in liquid state but significantly accelerated its oxidation in solid state under air. A higher level of peroxides in polysorbate 80 caused a significant increase in IL-2 mutein oxidation both in liquid and solid states, and glutathione can significantly inhibit the peroxide-induced oxidation of IL-2 mutein in a lyophilized formulation. In addition, a higher level of peroxides in polysorbate 80 caused immediate IL-2 mutein oxidation during annealing in lyophilization, suggesting that implementation of an annealing step needs to be carefully evaluated in the development of a lyophilization process for oxidation-sensitive proteins in the presence of polysorbate. Copyright 2002 Wiley-Liss Inc. and the American Pharmaceutical Association