Insulin amyloid morphology is encoded in H-bonds and electrostatics interactions ruling protein phase separation.

Ion-protein interactions regulate biological processes and are the basis of key strategies of modulating protein phase diagrams and stability in drug development. Here, we report the mechanisms by which H-bonds and electrostatic interactions in ion-protein systems determine phase separation and amyloid formation. Using microscopy, small-angle X-ray scattering, circular dichroism and atomistic molecular dynamics (MD) simulations, we found that anions specifically interacting with insulin induced phase separation by neutralising the protein charge and forming H-bond bridges between insulin molecules.

Ligand binding to proteins - When flawed fluorescence quenching methodology and interpretation become the new norm.

Intrinsic protein fluorescence quenching measurements have become a widespread methodology to determine ligand-binding properties of in particular serum albumin. Particularly common is the use of double log equations to extract parameters like binding constant and stoichiometry and/or number of binding sites. In this article we discuss that the methodology has several significant and often unrecognized pitfalls, and the double log equations are improperly derived for their purported use.

Leucine as a Moisture-Protective Excipient in Spray-Dried Protein/Trehalose Formulation.

The incorporation of leucine (Leu), a hydrophobic amino acid, into pharmaceutically relevant particles via spray-drying can improve the physicochemical and particulate properties, stability, and ultimately bioavailability of the final product. More specifically, Leu has been proposed to form a shell on the surface of spray-dried (SD) particles. The aim of this study was to explore the potential of Leu in the SD protein/trehalose (Tre) formulation to control the water uptake and moisture-induced recrystallization of amorphous Tre, using lysozyme (LZM) as a model protein.

Amino acids as stabilizers for lysozyme during the spray-drying process and storage.

Amino acids (AAs) have been used as excipients in protein formulations both in solid and liquid state products due to their stabilizing effect. However, the mechanisms by which they can stabilize a protein have not been fully elucidated yet. The purpose of this study was to investigate the effect of AAs with distinct physicochemical properties on the stability of a model protein (lysozyme, LZM) during the spray-drying process and subsequent storage.