SOLVENT EFFECTS ON THE SOLUBILITY AND PHYSICAL STABILITY OF HUMAN INSULIN-LIKE-GROWTH-FACTOR-I

Purpose. The solubility and physical stability of human Insulin-like G rowth Factor I (hIGF-I) were studied in aqueous solutions with differe nt excipients. Methods. The solubility of hIGF-I was determined by UV- absorption and quantification of light blocking particles. The physica l stability of hIGF-I was studied with differential scanning calorimet ry (DSC) and circular dichroism (CD) spectroscopy. Results. Human IGF- I precipitated at low temperature in the presence of 140 mM benzyl alc ohol and 145 mM sodium chloride. CD data showed that the tertiary stru cture of hIGF-I during these conditions was perturbed compared to that in 5 mM phosphate buffer. In the presence of benzyl alcohol 290 mM ma nnitol stabilized hIGF-I. Sodium chloride or mannitol by themselves ha d no effect on either the solubility or the tertiary structure. Benzyl alcohol was attracted to hIGF-I, whereas sodium chloride was preferen tially excluded. The attraction of benzyl alcohol was reinforced by so dium chloride leading to salting-out of hIGF-I. The CD-data indicated interactions of benzyl alcohol with phenylalanine in hIGF-I. Thermal d enaturation of hIGF-I occurred in all solutions with sodium chloride, whereas mannitol or benzyl alcohol had no effect on the thermal stabil ity. The thermal stability of hIGF-I was thus decreased in 145 mM sodi um chloride although it was excluded from hIGF-I. Conclusions. The sel f-association and thermal aggregation of hIGF-I is driven by hydrophob ic interactions. Benzyl alcohol is attracted to hIGF-I and induces cha nges in the tertiary structure causing hydrophobic attraction of the p rotein at low temperatures.