Combined Presence of Ferrous Ions and Hydrogen Peroxide in Normal Saline and In Vitro Models Induces Enhanced Aggregation of Therapeutic IgG due to Hydroxyl Radicals

Therapeutic monoclonal antibodies (mAb) are known to form aggregates and fragments upon exposure to hydrogen peroxide (H₂O₂) and ferrous ions (Fe²⁺). H₂O₂ and Fe²⁺ react to form hydroxyl radicals that are detrimental to protein structures. In this study, aggregation of mAb in the combined presence of Fe²⁺ and H₂O₂ was investigated in saline and physiologically relevant in vitro models. In the first case study, forced degradation of mAb in saline (a fluid used for administration of mAb) was carried out at 55 °C in the combined presence of 0.2 mM Fe²⁺ and 0.1% H₂O₂. The control and stressed samples were analyzed using an array of techniques including visual observation, size-exclusion chromatography (SEC), dynamic light scattering (DLS), microscopy, UV-vis, fluorescence, Fourier transform infrared spectroscopy, and cell-based toxicity assays. At the end of 1 h, samples having the combined presence of both Fe²⁺ and H₂O₂ exhibited more than 20% HMW (high molecular weight species), whereas samples having only Fe²⁺, H₂O₂, or neither resulted in less than 3% HMW. Aggregate-rich samples also exhibited altered protein structures and hydrophobicity. Aggregation increased upon increasing the time, temperature, and concentration of Fe²⁺ and H₂O₂. Samples having both Fe²⁺ and H₂O₂ also showed higher cytotoxicity in red blood cells. Samples of mAb with chlorides of copper and cobalt with H₂O₂ also resulted in multifold degradation. The first case study showed enhanced aggregation of mAb in the combined presence of Fe²⁺ and H₂O₂ in saline. In the second case study, aggregation of mAb was investigated in artificially prepared extracellular saline and in vitro models such as macromolecule free fraction of serum and serum. In the presence of both Fe²⁺ and H₂O₂, %HMW was higher in extracellular saline compared to macromolecule free fraction of serum. Further, in vitro models having the combined presence of Fe²⁺ and H₂O₂ resulted in enhanced aggregation of mAb compared to models that had neither.