title: Mechanism of protein stabilization by trehalose during freeze-drying analyzed by in situ micro-raman spectroscopy.
abstract: Raman investigations were performed in situ during freeze-drying of two model proteins, lysozyme and chymotrypsinogen. The structures of proteins dissolved in 0-30 wt % solutions of trehalose in D2 O were monitored with the fingerprint (800-1800 cm(-1) ) spectrum, simultaneously with freezing, ice sublimation, and water desorption analyzed in the O-D stretching (2200-2700 cm(-1) ) region. In the absence of trehalose, the main changes were detected at the end of primary drying, and correspond to distortion and disordering of secondary structures. A stabilizing effect of trehalose was evidenced in primary and secondary drying stages. Raman images were calculated after freezing and primary drying, providing the distributions of trehalose, water, and protein which occur during the first two stages of the lyophilization cycle. Raman images show a slight heterogeneity in the degree of protein denaturation at the end of primary drying, in relation with the structure of the frozen product observed during freezing. The ability of trehalose to make the protein more rigid was determined as responsible for the protein stabilization during a lyophilization cycle.
authors.affiliation: Université Lille Nord de France, Lille F-59000, France. alain.hedoux@univ-lille1.fr
authors.forename: Alain
authors.initials: A
authors.lastname: Hedoux
authors.forename: Laurent
authors.initials: L
authors.lastname: Paccou
authors.forename: Samira
authors.initials: S
authors.lastname: Achir
authors.forename: Yannick
authors.initials: Y
authors.lastname: Guinet
mesh_terms: D002919:Chymotrypsinogen / Q000737:chemistry*; D005612:Freeze Drying / Q000379:methods*; D009113:Muramidase / Q000737:chemistry*; D011487:Protein Conformation; D055550:Protein Stability; D013059:Spectrum Analysis, Raman; D014199:Trehalose / Q000737:chemistry*
chemical_list: D002919:Chymotrypsinogen; D014199:Trehalose; D009113:Muramidase
keywords: Raman mapping; Raman spectroscopy; lyophilization; protein structure; proteins; stability; trehalose