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

Abstract

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.

Keywords:

• bioanalyzer
• GCSF
• hydrogen peroxide
• NFS-60 cell proliferation assay
• oxidation

Acknowledgments

The authors thank Dr. Kamal Sharma, managing director, Lupin Limited, for his constant support and encouragement. We are also thankful to the Indian Institute of Science, Bangalore, India, for helping us with the CD spectra data.

Notes

Notes. NFS-60 cells were exposed for 48 h to rhGCSF treated with increasing concentrations of H₂O₂. Degree of cell proliferation was assessed using MTS, and data were expressed considering OD₄₉₀ of untreated (0% H₂O₂) rhGCSF as 100%.

Notes. NFS-60 cells were exposed for 48 h to rhGCSF treated with H₂O₂ in the presence or absence of DTT. Degree of cell proliferation was assessed using MTS, and data were expressed considering OD₄₉₀ of untreated (0%) rhGCSF as 100%.