Recombinant humanized monoclonal antibody HER2, rhuMAb HER2, in liquid formulations undergoes oxidation when exposed to intense light and elevated temperatures (30 & 40 degrees C). Met-255 in the heavy chain of the Fc region of the antibody is the primary site of oxidation. Met-431 of the Fc fragment can also be oxidized under extreme conditions. The amount of oxidation was determined by cleaving the Fab and Fc fragments by papain digestion, and the oxidized Fc fragment was detected by hydrophobic interaction chromatography. Oxidation of rhuMAb HER2 was also formulation dependent. The presence of NaCl in the rhuMAb HER2 formulation 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 oxidation of rhuMAb HER2 occurred by the formation of free radicals, and light-induced oxidation of rhuMAb HER2 occurred via single oxygen pathway. Antioxidants, such as methionine, sodium thiosulfate, catalase, or platinum, prevented Met oxidation in rhuMAb HER2, presumably as free radicals or oxygen scavengers. The minimum effective levels (molar ratios of protein to antioxidant) required to inhibit temperature-induced oxidation were 1:5 and 1:25 for methionine and thiosulfate, respectively. A thiosulfate adduct of rhuMAb HER2 was observed by cation-exchange chromatography. These studies demonstrate that stoichiometric amounts of methionine and thiosulfate are sufficient to eliminate temperature-induced oxidation of rhuMAb HER2 caused by free radicals that were generated by the presence of metal ion and peroxide impurities in the formulation.

Protein aggregation is a complex phenomenon that can occur in vitro and in vivo, usually resulting in the loss of the protein's biological activity. While many aggregation studies focus on a mechanism due to a specific stress, this study focuses on the general nature of aggregation. Recombinant human interferon-gamma (rhIFN-gamma) provides an ideal model for studying protein aggregation, as it has a tendency to aggregate under mild denaturing stresses (low denaturant concentration, temperature below the Tm, and below pH 5). All of the aggregates induced by these stresses have a similar structure (high in intermolecular beta-sheet content and a large loss of alpha-helix) as determined by infrared and circular dichroism spectroscopy. Thermally induced and denaturant-induced aggregation processes follow first-order kinetics under the conditions of this study. Spectroscopic and kinetic data suggest that rhIFN-gamma aggregates through an intermediate form possessing a large amount of residual secondary structure. In contrast to the aggregates formed under denaturing stresses, the salted-out protein has a remarkably nativelike secondary structure.

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