Relating particle formation to salt- and pH-dependent phase separation of non-native aggregates of alpha-chymotrypsinogen A.

Visible and subvisible particle formation during the storage of protein solutions is of increasing concern for pharmaceutical products. Previous work (Li Y, Ogunnaike BA, Roberts CJ. 2010. J Pharm Sci 99:645-662) showed that the model protein, alpha-chymotrypsinogen A (aCgn), forms non-native aggregates under accelerated (heated) conditions, but the size and morphology of the resulting aggregates depended sensitively on pH and NaCl. Here, it is shown that aggregates created as high-molecular-weight soluble aggregates undergo a pH- and salt-dependent reversible phase transition to a condensed or insoluble phase of suspended microparticles, whereas monomers remain completely soluble in the same regime. The location of the phase boundary is quantitatively consistent with the different regimes of kinetic behavior observed previously for aCgn. This suggests that the while kinetics is important for controlling the rates of monomer loss during non-native aggregation, it may be possible to tune solution thermodynamics and phase behavior to suppress otherwise soluble aggregates from propagating to form visible or large subvisible particles. Interestingly, the aggregate phase boundary is sensitive to the identity of salt anions in solution, highlighting the importance of electrostatics and preferential salt interactions in mediating aggregate condensation and particle formation.

L-Arginine reduces thioflavin T fluorescence but not fibrillation of bovine serum albumin.

This work examines the effects of L-arginine (L-Arg) on the aggregation and amyloid fibrillation of bovine serum albumin (BSA). We demonstrate that L-Arg dose-dependently reduces thioflavin T (ThT) fluorescence of BSA within the L-Arg concentration range used (0-1.4 M). However, as revealed by electron microscopy, size exclusion chromatography, and dynamic light scattering results, L-Arg does not prevent amyloid-like fibril formation by BSA. We conclude that L-Arg competes against ThT for binding sites on BSA amyloid-like fibrils, leading to biased results in ThT fluorescence measurements. Moreover, the use of ThT fluorescence assay to screen for potential inhibitors against amyloid fibrillation can give misleading results.