Optimization of Lyophilization Conditions for Recombinant Human Interleukin-2 by Dried-State Conformational Analysis Using Fourier-Transform Infrared Spectroscopy

Purpose. Examination of the dried-state conformation of interleukin-2 (IL-2) was used to determine the pH conditions and stabilizers that provide optimal storage stability for the lyophilized product. Methods. Fourier-transform infrared spectroscopy and accelerated stability studies which examined solubility, aggregate formation, and covalent cross-linking were used. Results. Varying the pH in the absence of excipients resulted in dramatic differences in the dried state conformation of IL-2. At pH 7, IL-2 unfolds extensively upon lyophilization while at pH below 5 it remains essentially native. Additional unfolding was observed upon incubation at elevated temperatures. A strong direct correlation between the retention of the native (aqueous) structure during freeze-drying and enhanced stability is demonstrated. IL-2 prepared at pH 5 is approximately an order of magnitude more stable than at pH 7 with regard to formation of soluble and insoluble aggregates. A similar pH profile was observed in the presence of excipients, although the excipients alter the overall stability profile. Additional accelerated stability studies examined the stabilizers necessary for optimal stability. Conclusions. Excipients with the capacity to substitute for water upon dehydration better preserve the native structure resulting in enhanced stability. Those that have high glass transition temperatures provide the highest level of stability during storage, although they do not prevent dehydration induced unfolding.