Ectoine and hydroxyectoine inhibit thermal-induced aggregation and increase thermostability of recombinant human interferon Alfa2b

This study is to investigate whether ectoines (ectoine and hydroxyectoine) can reduce aggregation of rhIFN alpha 2b in aqueous solutions on thermal stress. The effect of thermal stress condition on the stabilitywas therefore investigated using size exclusion-high performance liquid chromatography (SE-HPLC), different spectroscopic measurements, dynamic light scattering (DLS), electrophoresis, and differential scanning calorimetry (DSC). All experiments were performed in a sodium phosphate buffer system (100 mM, pH 7). The protein samples (100 mu g/ml) were incubated at 50 degrees C for 14 days in the absence or presence (1, 10, 20, and 100mM) of ectoines. In summary, thermal-induced aggregation was reduced in the presence of ectoines, regardless of the ectoines concentration in different periods of incubation time by analyzing with SE-HPLC and turbidity measurement. The inhibitory effect of ectoines on the aggregation was shown by other techniques used. The optimal ectoines concentration was 10 mM for aggregation reduction, so samples containing of 10 mMof ectoines were selected for further evaluation. Secondary structural and conformational stability increased in presence of ectoines as measured by far-UV circular dichroism and fluorescence spectroscopy, respectively. DSC showed slight increase in Tm of interferon in the presence of ectoines. Hydroxyectoine had superior protein-stabilizing properties than ectoine. In conclusion, this study demonstrates that ectoine and hydroxyectoine are highly effective excipients which can significantly reduce the thermal-induced aggregation of rhIFN alpha 2b at low concentration. (C) 2016 Elsevier B. V. All rights reserved.