Rapid and Sensitive Method to Detect Oxidized Forms of rhGCSF Using Agilent 2100 Bioanalyzer

Oxidation of methionine of human granulocyte colony stimulating factor (GCSF) results in loss of biological activity. In this study, we report the use of an Agilent 2100 bioanalyzer to detect oxidized forms of rhGCSF after exposure to hydrogen peroxide (H2O2). Our data show that the bioanalyzer is capable of detecting minor changes in rhGCSF after oxidation with 0.01% (w/v) H2O2, which results in nearly 50% loss in biological activity as observed by cell (NFS-60) proliferation assay. Dithiothreitol could largely protect such a H2O2-mediated oxidation, and thus, we conclude that the major modification of GCSF upon methionine oxidation is the conversion of methionines to its sulfoxide.