Structural Studies of an Insulin Analog Cocrystal Form by Atomic Force Microscopy. Michael R. DeFelippis¹, Christopher M. Yip¹, Michael D. Ward², Mark L. Brader¹, ¹Lilly Research Laboratories, Eli Lilly and Company, Indianapolis, IN 46285, ²Department of Chemical Engineering and Materials Science and the Center for Interfacial Engineering, University of Minnesota, Minneapolis, MN 55455

Intermediate-acting insulin therapies for the treatment of diabetes rely on subcutaneous injections of microcrystalline insulin forms typically cocrystallized with protamine. Despite the long history of these formulations, numerous questions regarding the structure and molecular packing motifs remain as high resolution diffraction studies have been hampered by the small crystal dimensions. The high resolution in-situ capabilities of scanning probe microscopy have resulted in new insights into the structure of interfaces. Recent studies of protein crystallization by atomic force microscopy have resulted in direct in-situ confirmation of lattice periodicities, space group symmetries, and visualization of growth processes, including the role of aggregate addition in void formation.

Cocrystallization of the insulin analog Lys^B28Pro^B29 with protamine yielded well-defined bipyramidally tipped tetragonal crystals. The ready dissolution of these crystals at slightly elevated temperatures (25 - 30 [ring]C), as are present in the AFM fluid cell during normal imaging, precluded imaging under ambient conditions. However, in situ tapping mode AFM imaging performed in solution at 6[ring]C using a novel refrigeration scheme enabled direct real-space identification of molecular scale surface periodicities ascribed to individual rows of insulin hexamers. Surface aggregates, tentatively identified as protamine, were also observed. The observed packing motifs were in good agreement with previously reported diffraction data on porcine insulin-clupeine cocrystals. Growth of the Lys^B28Pro^B29-protamine cocrystals occurred through expansion of single molecular layer terraces, in direct contrast to the screw dislocation mechanism observed in earlier AFM studies of Lys^B28Pro^B29 crystallization. These studies illustrate the ready application of atomic force microscopy to the high-resolution in situ characterization of protein crystal systems and presage its application to issues regarding crystal dissolution and interfacial structure, which have therapeutic relevance for crystalline pharmaceuticals.