Evaluation of etanercept degradation under oxidative stress and potential protective effects of various amino acids

Abstract To evaluate the oxidative stability of proteins, a model protein, etanercept, was exposed to oxidative stress conditions using hydrogen peroxide. Various amino acids were also evaluated on their antioxidant effect. Transition temperature (T_m), secondary structural content, hydrodynamic size, and aggregation and fragmentation of etanercept in solution were assessed using dynamic light scattering (DLS), size exclusion chromatography (SEC), differential scanning calorimetry (DSC), and far-UV circular dichroism (CD). Sample solutions were stored at 4°C, 20°C, and 40°C under oxidative stress. The DLS results exhibited a decrease in the Z-average and intensity peak size of etanercept during the storage, suggesting fragmentation issues rather than aggregation by oxidation. The SEC results exhibited an increase in fragmentation and a decrease in aggregation and monomer content. The monomer content remained higher in histidine than in other amino acids, followed by methionine. There were three T_m of etanercept that were selected as key parameters of conformational stability. Oxidized samples exhibited a significant decrease in T_m values, indicating decreased conformational stability. Methionine exhibited the highest values in T_m1, followed by histidine. The CD spectrum exhibited one unique negative peak of etanercept without amino acids, and changed with oxidation. Only methionine exhibited an enhancement of the stability. All four biophysical analyses results suggest that the histidine and methionine provide a protective effect in the protein solution against oxidative stress. However, histidine was effective as an antioxidant but methionine showed highly enhanced conformational and secondary structural stability.