Effects of temperature and additives on stability and spectrum of a therapeutic fibroblast growth factor

ABSTRACT

Human fibroblast growth factor 20 [FGF20] is a 16.5 kDa protein containing 154 amino acid residues with reportedly poor thermal stability, and low stability, which are considered to be major factors that can limit its pharmacological applications. Thus, the aim of this study was to enhance the thermal stability and bio activity of a therapeutic FGF20 by addition of sucrose or heparin as additives and also at different temperatures. A variety of biophysical techniques such as far-UV circular dichroism [CD], fluorescence and high resolution derivative UV absorption spectroscopy, were employed to characterize FGF20 and study the effects of heparin and sucrose on its thermal stability and bio activity at pH 7.0. Results of this study suggest that human FGF20 is significantly unstable and induction of heat by increased temperatures results in aggregation and precipitation at pH 7.0. Great changes in the fluorescence intensity and shape were achieved by addition of heparin and sucrose at different temperatures compared to the control. From 10 °C to 60 °C, no significant changes were observed in far-UV CD spectrum compared to the control, but significant changes were observed by adding sucrose when these temperatures are above 45 °C. Upon addition of heparin and sucrose, the mitogenic activity increased significantly at all tested temperatures, and these changes may be related to the roles of heparin and sucrose on the structure and conformation of FGF20. Results of this study suggest that heparin and sucrose as additives seems to benjm sufficient to prevent thermal inactivation of FGF20 and also maintain its conformation stability and bio activity