Identification and characterization of oxidation and deamidation sites in monoclonal rat/mouse hybrid antibodies

Oxidation of methionine residues and deamidation of asparagine residues are the major causes of chemical degradation of biological pharmaceuticals. The mechanism of these non-enzymatic chemical reactions has been studied in great detail. However, the identification and quantification of oxidation and deamidation sites in a given protein still remains a challenge. In this study, we identified and characterized several oxidation and deamidation sites in a rat/mouse hybrid antibody. We evaluated the effects of the sample preparation on oxidation and deamidation levels and optimized the peptide mapping method to minimize oxidation and deamidation artifacts. Out of a total number of 18 methionine residues, we identified six methionine residues most susceptible to oxidation. We determined the oxidation rate of the six methionine residues using 0.05% H(2)O(2) at different temperatures. Methionine residue 256 of the mouse heavy chain showed the fastest rate of oxidation under those conditions with a half life of approximately 200 min at 4 degrees C and 27 min at 37 degrees C. We identified five asparagine residues prone to deamidation under accelerated conditions of pH 8.6 at 37 degrees C. Kinetic characterization of the deamidation sites showed that asparagine residue 218 of the rat heavy chain exhibited the fastest rate of deamidation with a half live of 1.5 days at pH 8.6 and 37 degrees C. Analysis of antibody isoforms using free flow electrophoresis showed that deamidation is the major cause of the charged variants of this rat/mouse hybrid antibody.