ANTIOXIDANTS FOR PREVENTION OF METHIONINE OXIDATION IN RECOMBINANT MONOCLONAL-ANTIBODY HER2

Recombinant humanized monoclonal antibody HER2, rhuMAb HER2, in liquid formulations undergoes oxidation when exposed to intense light and el evated temperatures (30 & 40 degrees C). Met-255 in the heavy chain of the Fe region of the antibody is the primary site of oxidation. Met-4 31 of the Fe fragment can also be oxidized under extreme conditions. T he amount of oxidation was determined by cleaving the Fab and Fe fragm ents by papain digestion, and the oxidized Fe fragment was detected by hydrophobic interaction chromatography, Oxidation of rhuMAb HER2 was also formulation dependent, The presence of NaCl in the rhuMAb HER2 fo rmulation caused an increase in oxidation at higher temperatures after contact with stainless steel containers or stainless steel components in the filling process. The corrosion of stainless steel by chloride ions at the low pH of the formulation buffer generated iron ions that catalyzed methionine oxidation in rhuMAb HER2, Temperature-induced oxi dation of rhuMAb HER2 occurred by the formation of free radicals, and light-induced oxidation of rhuMAb HER2 occurred via singlet oxygen pat hway, Antioxidants, such as methionine, sodium thiosulfate, catalase, or platinum, prevented Met oxidation in rhuMAb HER2, presumably as fre e radicals or oxygen scavengers. The minimum effective levels (molar r atios of protein to antioxidant) required to inhibit temperature induc ed oxidation were 1:5 and 1:25 for methionine and thiosulfate, respect ively. A thiosulfate adduct of rhuMAb HER2 was observed by cation-exch ange chromatography. These studies demonstrate that stoichiometric amo unts of methionine and thiosulfate are sufficient to eliminate tempera ture-induced oxidation of rhuMAb HER2 caused by free radicals that wer e generated by the presence of metal ions and peroxide impurities in t he formulation.