Effects of solutes on empirical phase diagrams of human fibroblast growth factor 1

Abstract

A variety of solutes are commonly used to increase the stability of protein in therapeutic formulations. An empirical phase diagram approach is used to evaluate the effects of different types of additives on the solution behavior of a protein of pharmaceutical interest, human fibroblast growth factor 1 (FGF-1). A specific stabilizer, heparin, and a nonspecific stabilizer, sucrose, were used in this work. The protein was characterized as a function of pH (3-8) and temperature (10-85 degrees C) using Far-UV circular dichroism (Far-UV CD), intrinsic and extrinsic fluorescence as well as second derivative UV absorption spectroscopy. Empirical phase diagrams were constructed to summarize the biophysical characterization data obtained with FGF-1 alone, in the presence of a threefold weight excess of heparin (3x heparin) or 10% sucrose (w/v). Three phases are observed in the low temperature regions at pH 3, 4, and 5-8. Phase boundaries corresponding to major heat-induced transitions are detected in the physiological temperature range. The highest thermal stabilities are observed near neutral pH (pH 6 and 7). Both heparin and sucrose appear to enhance the thermal stability of FGF-1, although their effects on the phase diagram are quite distinct. The greatest stabilization is observed at pH 8. Only heparin appears to protect FGF-1 from acid-induced unfolding to any extent.