Comprehensive evaluation of etanercept stability in various concentrations with biophysical assessment

The effect of protein concentration on biophysical stability of etanercept was investigated to monitor its effect on protein formulation development. The conformational and accelerated storage stability of etanercept (marketed as Enbrel®) was examined by biophysical analyses including CD, FTIR, DSC, and DLS together with size-exclusion chromatography (SEC). As concentration of etanercept decreased, conformational stability (Tm) decreased with increasing hydrodynamic size and zeta potential. Decreasing secondary structural stability was also observed for relative helix and β-sheet contents. Further investigation examined the accelerated storage stability at different incubation temperatures. Low protein concentration (0.25 and 0.5 mg/mL) at 4 °C and 30 °C exhibited fast monomer loss compared to high concentration (25 and 50 mg/mL). The lowest etanercept concentration of 0.25 mg/mL displayed the fastest monomer loss and increased fragments since it had lowest Tm values. However, at 50 °C, a marked increase in aggregation was observed at high concentrations, as well as accelerated monomer loss into multimers and insoluble aggregates. Induced insoluble aggregation of etanercept was dependent on its concentration and no significant aggregation issues were found at low concentrations such as 0.25 and 0.5 mg/mL. The results indicated that the conformational stability of protein solution involved steric repulsion of neighboring protein molecules. Electrostatic circumstances and structural interactions resulted in low stability at low concentrations of etanercept under heat stress. Therefore, it might be recommended to be less diluted during protein formulation development, even in the earlier stages of investigation, to avoid undesirable results.

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