title: Ectoine and hydroxyectoine inhibit thermal-induced aggregation and increase thermostability of recombinant human interferon Alfa2b.
abstract: This study is to investigate whether ectoines (ectoine and hydroxyectoine) can reduce aggregation of rhIFNα2b in aqueous solutions on thermal stress. The effect of thermal stress condition on the stability was 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 (100mM, pH7). The protein samples (100μg/ml) were incubated at 50°C for 14days 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 10mM for aggregation reduction, so samples containing of 10mM of 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 Tmof 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 rhIFNα2b at low concentration.
authors.affiliation: Department of Pharmaceutics, School of Pharmacy, Alborz University of Medical Sciences, Karaj, Iran; Department of Pharmaceutics, School of Pharmacy, Shahid Beheshti University of Medical Sciences, Tehran, Iran. Electronic address: Salmannejad.f@gmail.com.
authors.forename: Faranak
authors.initials: F
authors.lastname: Salmannejad
authors.affiliation: Department of Pharmaceutical Biotechnology, School of Pharmacy, Shahid Beheshti University of Medical Sciences, Tehran, Iran.
authors.forename: Nastaran
authors.initials: N
authors.lastname: Nafissi-Varcheh
mesh_terms: D000599:Amino Acids, Diamino / Q000737:chemistry / Q000494:pharmacology*; D002460:Cell Line; D004355:Drug Stability; D006358:Hot Temperature / Q000009:adverse effects*; D006801:Humans; D000077190:Interferon alpha-2; D016898:Interferon-alpha / Q000502:physiology; D066329:Protein Aggregates / Q000187:drug effects* / Q000502:physiology; D055550:Protein Stability / Q000187:drug effects; D011994:Recombinant Proteins
chemical_list: D000599:Amino Acids, Diamino; D000077190:Interferon alpha-2; D016898:Interferon-alpha; D066329:Protein Aggregates; D011994:Recombinant Proteins; C121557:hydroxyectoine; C045628:ectoine
keywords: Aggregation; Ectoine; Hydroxyectoine; Thermal stress; rhIFNα2b