The stability of insulin in crystalline and amorphous solids: observation of greater stability for the amorphous form.
OBJECTIVE
Generalizations based upon behavior of small molecules have established that a crystalline solid is generally much more stable toward chemical degradation than is the amorphous solid. This study examines the validity of this generalization for proteins using biosynthetic human insulin as the model protein.


METHODS
Amorphous insulin was prepared by freeze drying the supernate from a suspension of zinc insulin crystals adjusted to pH 7.1. Storage stability at 25 degrees C and 40 degrees C were compared for the freeze dried material, the dried suspended crystals, and the starting batch of crystals. Samples were equilibrated at selected relative humidities between zero and 75% to obtain samples at various water contents. Assays for dimer formation were performed 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. Differential 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 AsnA21 site and co-valent dimer formation, presumably involving the A-21 site. Contrary to expectations, amorphous insulin is far more stable than crystalline insulin under all conditions investigated. While increasing water content increases the rate of degradation of crystalline insulin, rate constants for degradation in the amorphous solid are essentially independent of water content up to the maximum water content studied (approximately 15%).


CONCLUSIONS
Based upon the FTIR and DSC data, both crystalline and amorphous insulin retain some higher order structure when dried, but the secondary structure is significantly perturbed from 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 obscure.
Pikal
M J
MJ
Lilly Research Laboratories, Eli Lilly & Co., Indianapolis, Indiana 46285, USA. pikal@uconnvm.uconn.edu
Rigsbee
D R
DR
D002152:Calorimetry, Differential Scanning; D003460:Crystallization; D004355:Drug Stability; D005612:Freeze Drying; D006801:Humans; D049528:Insulin, Long-Acting / Q000737:chemistry*; D007700:Kinetics; D017433:Protein Structure, Secondary; D017550:Spectroscopy, Fourier Transform Infrared; D013696:Temperature; D014867:Water
D049528:Insulin, Long-Acting; D014867:Water