Stabilization of protein structure in freeze-dried amorphous organic acid buffer salts

The purpose of this study was to elucidate the physical properties and protein-stabilizing effects of some pH-adjusting excipients (carboxylic acids and their sodium salts) in frozen solutions and in freeze-dried solids. Thermal and powder X-ray diffraction (XRD) analysis indicated a high propensity of sodium citrates to form glass-state amorphous solids upon freeze-drying. Some salts (e.g., sodium succinate) crystallized in the single-solute frozen solutions. FT-IR analysis of bovine serum albumin (BSA) and bovine immunoglobulin G (IgG) in the aqueous solutions and the freeze-dried solids showed that some glass-forming salts (e.g., monosodium citrate) protected the secondary structure from lyophilization-induced perturbation. Freeze-drying of BSA at different concentrations indicated retention of the secondary structure at similar monosodium citrate/protein concentration ratios, suggesting stabilization through direct interaction that substitute water molecules inevitable for the conformation integrity. The carboxylic acid salts should provide rigid hydrogen bonds and electrostatic interactions that raise the glass transition temperature of the amorphous solids and stabilize protein structure. The relevance of the structural stabilization to the protein formulation design was discussed.