THE STABILITY OF INSULIN IN CRYSTALLINE AND AMORPHOUS SOLIDS - OBSERVATION OF GREATER STABILITY FOR THE AMORPHOUS FORM

Purpose. Generalizations based upon behavior of small molecules have e stablished that a crystalline solid is generally much more stable towa rd chemical degradation than is the amorphous solid. This study examin es the validity of this generalization for proteins using biosynthetic human insulin as the model protein. Methods. Amorphous insulin was pr epared by freeze drying the supernate from a suspension of zinc insuli n crystals adjusted to pH 7.1. Storage stability at 25 degrees C and 4 0 degrees C were compared for the freeze dried material, the dried sus pended crystals, and the starting batch of crystals. Samples were equi librated at selected relative humidities between zero and 75% to obtai n samples at various water contents. Assays for dimer formation were p erformed by size exclusion HPLC and assays for deamidated product were carried out by reverse phase HPLC. Degradation was found to be linear in square root of time, and the slopes from % degradation vs. square root of time were used to define the rate constants for degradation. D ifferential scanning calorimetry (DSC) and Fourier-transform infrared spectroscopy (FTIR) were used to characterize the state of the protein in the solids. Results. As expected based upon previous results, the primary degradation pathways involve deamidation at the Asn(A21) site and co-valent dimer formation, presumably involving the A-21 site. Con trary to expectations, amorphous insulin is far more stable than cryst alline insulin under all conditions investigated. While increasing wat er content increases the rate of degradation of crystalline insulin, r ate constants for degradation in the amorphous solid are essentially i ndependent of water content up to the maximum water content studied (a pproximate to 15%). Conclusions. Based upon the FTIR and DSC data, bot h crystalline and amorphous insulin retain some higher order structure when dried, but the secondary structure is significantly perturbed fr om that characteristic of the native solution state. However, neither DSC nor FTIR data provide a clear interpretation of the difference in stability between the amorphous and crystalline solids. The mechanism responsible for the superior stability of amorphous insulin remains ob scure.