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Solvent Effects on the Solubility and Physical Stability of Human Insulin-Like Growth Factor I

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Abstract

Purpose. The solubility and physical stability of human Insulin-like Growth Factor I (hIGF-I) were studied in aqueous solutions with different excipients.

Methods. The solubility of hIGF-I was determined by UV-absorption and quantification of light blocking particles. The physical stability of hIGF-I was studied with differential scanning calorimetry (DSC) and circular dichroism (CD) spectroscopy.

Results. Human IGF-I precipitated at low temperature in the presence of 140 mM benzyl alcohol and 145 mM sodium chloride. CD data showed that the tertiary structure of hIGF-I during these conditions was perturbed compared to that in 5 mM phosphate buffer. In the presence of benzyl alcohol 290 mM mannitol stabilized hIGF-I. Sodium chloride or mannitol by themselves had no effect on either the solubility or the tertiary structure. Benzyl alcohol was attracted to hIGF-I, whereas sodium chloride was preferentially excluded. The attraction of benzyl alcohol was reinforced by sodium chloride leading to salting-out of hIGF-I. The CD-data indicated interactions of benzyl alcohol with phenylalanine in hIGF-I. Thermal denaturation of hIGF-I occurred in all solutions with sodium chloride, whereas mannitol or benzyl alcohol had no effect on the thermal stability. The thermal stability of hlGF-I was thus decreased in 145 mM sodium chloride although it was excluded from hIGF-I.

Conclusions. The self-association and thermal aggregation of hIGF-I is driven by hydrophobic interactions. Benzyl alcohol is attracted to hIGF-I and induces changes in the tertiary structure causing hydrophobic attraction of the protein at low temperatures.

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Authors and Affiliations

  1. Department of Pharmaceutical Technology, Pharmacia & Upjohn, S-, 112 87, Stockholm, Sweden

    Jonas Fransson & Ebba Florin-Robertsson

  2. Department of Pharmaceutics, Faculty of Pharmacy, Uppsala University, Box 570, S-, 751 23, Uppsala, Sweden

    Jonas Fransson

  3. Department of Structural Chemistry, Pharmacia & Upjohn, S-l, 12 87, Stockholm, Sweden

    Dan Hallén

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Fransson, J., Hallén, D. & Florin-Robertsson, E. Solvent Effects on the Solubility and Physical Stability of Human Insulin-Like Growth Factor I. Pharm Res 14, 606–612 (1997). https://doi.org/10.1023/A:1012101027814

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