Determination of tryptophan oxidation of monoclonal antibody by reversed phase high performance liquid chromatography

A pre-peak was separated from the main peak of a monoclonal antibody by size exclusion HPLC (SE-HPLC) analysis using two TSK-Gel G3000SWXL columns connected in series. When compared with molecular weight markers, the product peak elutes as a molecule with apparent molecular weight of 44 kDa, indicating that separation is not strictly on the basis of size exclusion and that longer retention of the antibody by this column probably results from interaction between the stationary phase and the protein molecules during separation. The pre-peak has been characterized as due to the oxidation of one of tryptophan residues on the heavy chains of this monoclonal antibody. Because the SE-HPLC method using tandem TSK-Gel G3000SWXL columns showed unsatisfactory reproducibility amongst column lots, a more reliable and accurate reversed phase HPLC (RP-HPLC) method was developed to detect the tryptophan oxidation on the heavy chains of the monoclonal antibody. The level of tryptophan oxidation determined by the reversed phase method was correlated with the amount of tryptophan oxidation observed as a pre-peak by SE-HPLC. The RP-HPLC method was qualified for precision, accuracy, linearity of the main peak and the minor peak (oxidized isoforms of the heavy chain), detection limit and quantitation limit.