title: Stabilization of protein structure in freeze-dried amorphous organic acid buffer salts.
abstract: 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.
authors.affiliation: National Institute of Health Sciences, 1-18-1 Kamiyoga, Setagaya-ku, Tokyo 158-8501, Japan. izutsu@nihs.go.jp
authors.forename: Ken-ichi
authors.initials: K
authors.lastname: Izutsu
authors.forename: Saori
authors.initials: S
authors.lastname: Kadoya
authors.forename: Chikako
authors.initials: C
authors.lastname: Yomota
authors.forename: Toru
authors.initials: T
authors.lastname: Kawanishi
authors.forename: Etsuo
authors.initials: E
authors.lastname: Yonemochi
authors.forename: Katsuhide
authors.initials: K
authors.lastname: Terada
mesh_terms: D000818:Animals; D002021:Buffers; D002264:Carboxylic Acids / Q000737:chemistry* / Q000494:pharmacology; D002417:Cattle; D005612:Freeze Drying; D006860:Hydrogen Bonding; D007074:Immunoglobulin G / Q000737:chemistry*; D033362:Powder Diffraction; D011487:Protein Conformation / Q000187:drug effects; D011489:Protein Denaturation / Q000187:drug effects; D055550:Protein Stability / Q000187:drug effects; D012710:Serum Albumin, Bovine / Q000737:chemistry*; D012996:Solutions; D055672:Static Electricity; D013696:Temperature
chemical_list: D002021:Buffers; D002264:Carboxylic Acids; D007074:Immunoglobulin G; D012996:Solutions; D012710:Serum Albumin, Bovine