Abstract

The positively charged amino acids are commonly used excipients in biopharmaceutical formulations for stabilization of therapeutic proteins, yet the mechanisms for their modulation of protein stability are poorly understood. In this study, both lysine and histidine are shown to affect the thermal stability of myoglobin, bovine serum albumin, and lysozyme through a combination of mechanisms governed by their respective functional side chains and glycine, similar to arginine. This study provides evidence that at low concentrations, lysine and histidine interact with proteins by a combination of (1) direct electrostatic interactions with negatively charged side chains, (2) possible binding to high-affinity hydrophobic binding sites, and (3) glycine-mediated weak interactions with peptide backbone and polar side chains. At high concentrations, lysine and histidine act via (4) glycine-mediated competition for water between the unfolding protein and the excipient and (5) sidechain-mediated interaction with apolar regions exposed during unfolding (histidine). Lysine and histidine are useful for biopharmaceutical formulations as they were less destabilizing of the protein structures tested than arginine at concentrations above 100 mM.

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