Purpose: The objective of this study was to elucidate the different mechanisms of action of different excipients on the oxidation of Met1, Met122, Met127, and Met138 in granulocyte colony-stimulating factor (G-CSF) by using hydrogen peroxide as the oxidant.
Methods: The oxidation of Met1, Met127, and Met138 was quantified by peptide mapping analysis. The oxidation of Met122 has biphasic oxidation kinetics with a faster second phase. Therefore, the oxidation of Met122 was quantified by two different methods: peptide mapping analysis for the first phase of oxidation and direct reverse-phase HPLC for the second phase of oxidation.
Results: The current work reveals that the preferential excluding excipients sorbitol, sucrose, and trehalose, in the concentration range 0-30% (w/v), and the preferential binding excipients urea and guanidine hydrochloride, in the concentration range 0-0.8 M, do not affect the oxidation of methionine residues in G-CSF at pH 4.5. The chelating agents citrate and EDTA have different effects on the rates of oxidation of methionine residues in G-CSF. At low concentrations, citrate decreases the rates, while at high concentrations, citrate increases the rates. EDTA decreases the rates of oxidation of methionine residues in G-CSF, such that its effect becomes more and more as its concentration is increased from 0 to 200 mM. The efficacy of EDTA on the rates of oxidation of the four methionine residues in G-CSF follows the order Met122 > Met127 > Met138 > Met1.
Conclusions: Our results indicate that EDTA can protect the methionine residues in G-CSF against oxidation induced by hydrogen peroxide. The more exposed the methionine residue is, the more difficult it is to be protected by EDTA. The mechanism may be due to the specific ion binding of EDTA to proteins.