A number of physicochemical techniques have been utilized over the last decade to characterize the state of insulin aggregation, namely; cryo-electron microscopy, nano-spray ESI MS, small angle X-ray scattering, and NMR.

Various attempts were undertaken to find conditions for the monomeric structure in solution. The water/acetic acid ( 80/20 vol %), trifluoroethanol and water/actonitrile ( 65/35 vol %) were found appropriate media to observe good quality 1D-NMR spectra suitable for undertaking the structural studies. However, none of these solvents was characterized in detail with respect to aggregation state and existing equilibria. The solution structure of native human insulin was so far only solved in the water/acetic acid ( 80/20 vol %).

The PFGSE (Pulsed Field Gradient Spin Echo) technique was used to monitor the aggregation in solution by means of establishing the diffusion coefficient, D_i, for each species in solution.

The water/actonitrile mixture, as a solvent, was found to simultaneously satisfy requirements of high resolution insulin spectrum and monomeric conditions over the wide concentration range of insulin. These topics wil be presented in a lecture.

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