(575f) The Use of Low Melting Point Organic Salts as Excipients to Increase Protein Stability in Solution
AIChE Annual Meeting
Thursday, November 11, 2010 - 10:20am to 10:40am
Despite the promise of protein pharmaceuticals, the inherent instability of proteins in solution results in challenges with drug delivery, dosing, patient compliance, and therapeutic side-effects. In this study, the stability and functionality of a lysozyme liquid protein formulation was determined to evaluate the utility of low melting point organic salts, some of which can be considered ionic liquids (ILs), as stabilizing excipients in pharmaceutical formulations. We have demonstrated that certain model proteins are readily soluble in hydrated choline dihydrogen phosphate (CDHP) solutions, and that activity is retained over extended times in these formulations. Protein stability was investigated in detail using the model protein lysozyme. The change in Enthalpy (ΔH), Entropy (ΔS), and Gibbs Free Energy (ΔG) of protein unfolding of lysozyme formulated with CDHP as an excipient was measured with microcalorimetry. The temperature of unfolding of lysozyme increased systematically with the addition of CDHP, and the magnitude of the stabilization effect was observed to be pH dependent. CDHP offers more protection as the pH increases towards the pI of lysozyme. The thermodynamic changes observed are not unlike those that have been measured for the ubiquitously used molecule trehalose, a common stabilizer for both protein and cell preservation applications. We have also established through Small-angle x-ray scattering (SAXS) measurements that CDHP appears to modulate the interparticle interference effect of lysozyme at concentrations as low as just 5% (w/w) CDHP. SAXS data was used to characterize the overall globular folds and solvation properties of lysozyme in aqueous buffer and buffer supplemented with CDHP. Lysozyme in pH 3.8 buffer normally exhibits interparticle interference in the low Q range (0.02 to 0.05 Å-1). CDHP eliminates the interparticle interference effect without altering particle shape or inducing aggregation.