The protein size, electrical interaction, and conformational stability of etanercept (marketed as Enbrel®) were examined by thermodynamic and light scattering methods with changing pH and buffer concentration. As pH of etanercept increased from pH 6.6 to 8.6, electrical repulsion in the solution increased, inducing a decrease in protein size. However, the size changed less in high buffer concentration and irreversible aggregation issues were not observed; in contrast, aggregates of about 1000 nm were observed in low buffer concentration at the pH range. Three significant unfolding transitions ( T _m) were observed by differential scanning calorimetry (DSC). Unlikely to T _m1, T _m2 and T _m3 were increased as the pH increased. Higher T _m at high buffer concentration was observed, indicating increased conformational stability. The apparent activation energy of unfolding was further investigated since continuous increase of T _m2 and T _m3 was not sufficient to determine optimal conditions. A higher energy barrier was calculated at T _m2 than at T _m3. In addition, the energy barriers were the highest at pH from 7.4 to 7.8 where higher T _m1 was also observed. Therefore, the conformational stability of protein solution significantly changed with pH dependent steric repulsion of neighboring protein molecules. An optimized pH range was obtained that satisfied the stability of all three domains. Electrostatic circumstances and structural interactions resulted in irreversible aggregation at low buffer concentrations and were suppressed by increasing the concentration. Therefore, increased buffer concentration is recommended during protein formulation development, even in the earlier stages of investigation, to avoid protein instability issues.