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

Abstract

Therapeutic monoclonal antibodies (mAb) are known to form aggregates and fragments upon exposure to hydrogen peroxide (H 2 O 2 ) and ferrous ions (Fe 2+ ). H 2 O 2 and Fe 2+ react to form hydroxyl radicals that are detrimental to protein structures. In this study, aggregation of mAb in the combined presence of Fe 2+ and H 2 O 2 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 Fe 2+ and 0.1% H 2 O 2 . 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 Fe 2+ and H 2 O 2 exhibited more than 20% HMW (high molecular weight species), whereas samples having only Fe 2+ , H 2 O 2 , 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 Fe 2+ and H 2 O 2 . Samples having both Fe 2+ and H 2 O 2 also showed higher cytotoxicity in red blood cells. Samples of mAb with chlorides of copper and cobalt with H 2 O 2 also resulted in multifold degradation. The first case study showed enhanced aggregation of mAb in the combined presence of Fe 2+ and H 2 O 2 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 Fe 2+ and H 2 O 2 , %HMW was higher in extracellular saline compared to macromolecule free fraction of serum. Further, in vitro models having the combined presence of Fe 2+ and H 2 O 2 resulted in enhanced aggregation of mAb compared to models that had neither.

Purification of As(iii) through oxidation of siderite and As(iii) by dissolved oxygen: behavior and mechanism

“…This indicates that ·OH production depended on Fe( ii ) oxidation, which was consistent with previous research showing that the Fenton-like reaction of Fe( ii ) oxidation caused the occurrence of ·OH. 71 Nevertheless, the concentration of total Fe( iii ) at a DO concentration of 9.0 mg L −1 was identical to that at a DO concentration of 4.6 mg L −1 (Fig. 1), which caused the identical concentrations of ·OH generated in the batches (Fig.…”