Formulation of proteins in vacuum-dried glasses. II. Process and storage stability in sugar-free amino acid systems

The purpose of this research was to investigate the freeze- and vacuum-drying behavior of L-amino acids of current/potential use as adjuvants for formulating proteins. The analytical methods used were wide-angle x-ray diffraction, differential scanning calorimetry, and scanning electron microscopy. Protein analysis was performed either as an activity assay (lactate dehydrogenase [LDH]) or by size-exclusion chromatography (granulocyte colony-stimulating factor [rhG-CSF]). After samples were freeze-dried, only the four basic amino acids (arginine, lysine, histidine, and citrulline) formed amorphous solids, which, however, were partially crystalline. The remaining amino acids all formed fully crystalline solids. After samples were vacuum-dried, (20 degrees C, 0.1 mbar, 1 ml fill volume in 2-ml vials) fully crystalline solids were formed by all of the amino acids. For arginine, the addition of either HCl, H3PO4, or H2SO4 sufficient to form the respective salt produced amorphous solids after vacuum-drying, but they had high residual water contents and low glass transition temperatures (Tg). Addition of phenylalanine to arginine base inhibited crystallization of the latter at low concentrations during vacuum-drying procedure, leading to formation of a pure rubbery solid. At higher concentrations the phenylalanine crystallized, producing dry products with glass transition temperatures of > 60 degrees C. The process and storage stability of LDH and rhG-CSF in the vacuum-dried phenylalanine/arginine glasses was greatly improved at temperatures up to 40 degrees C compared with the unprotected proteins. Uptake of moisture during storage was, however, a complicating factor, reducing Tg, promoting crystallization, and leading to decreased protein stability. The PO4 salt of arginine produced especially high glass transition temperatures after it was vacuum-dried. These sugar-free amino acid formulations thus are potential stabilizes for proteins.