title: Evaluating the effects of buffer conditions and extremolytes on thermostability of granulocyte colony-stimulating factor using high-throughput screening combined with design of experiments.
abstract: In this study, we combined a high-throughput screening method, differential scanning fluorimetry (DSF), with design of experiments (DoE) methodology to evaluate the effects of several formulation components on the thermostability of granulocyte colony stimulating factor (G-CSF). First we performed a primary buffer screening where we tested thermal stability of G-CSF in different buffers, pH values and buffer concentrations. The significance of each factor and the two-way interactions between them were studied by multivariable regression analysis. pH was identified as most critical factor regarding thermal stability. The most stabilizing buffer, sodium glutamate, and sodium acetate were determined for further investigations. Second we tested the effect of 6 naturally occurring extremolytes (trehalose, sucrose, ectoine, hydroxyectoine, sorbitol, mannitol) on the thermal stability of G-CSF, using a central composite circumscribed design. At low pH (3.8) and low buffer concentration (5 mM) all extremolytes led to a significant increase in thermal stability except the addition of ectoine which resulted in a strong destabilization of G-CSF. Increasing pH and buffer concentration led to an increase in thermal stability with all investigated extremolytes. The described systematic approach allowed to create a ranking of stabilizing extremolytes at different buffer conditions.
authors: Research Center Pharmaceutical Engineering GmbH, Graz, Austria.
authors: Elisabeth
authors: E
authors: Ablinger
authors: Monika
authors: M
authors: Hellweger
authors: Stefan
authors: S
authors: Leitgeb
authors: Andreas
authors: A
authors: Zimmer
mesh_terms: D000599:Amino Acids, Diamino / Q000737:chemistry; D002021:Buffers; D002241:Carbohydrates / Q000737:chemistry; D002626:Chemistry, Pharmaceutical / Q000379:methods*; D004355:Drug Stability; D016179:Granulocyte Colony-Stimulating Factor / Q000737:chemistry*; D057166:High-Throughput Screening Assays; D006358:Hot Temperature; D006863:Hydrogen-Ion Concentration
chemical_list: D000599:Amino Acids, Diamino; D002021:Buffers; D002241:Carbohydrates; C121557:hydroxyectoine; D016179:Granulocyte Colony-Stimulating Factor; C045628:ectoine