title: Storage stability of keratinocyte growth factor-2 in lyophilized formulations: effects of formulation physical properties and protein fraction at the solid-air interface.
abstract: Lyophilized formulations of keratinocyte growth factor-2 (KGF-2) were prepared with a range of disaccharide (sucrose or trehalose) and hydroxyethyl starch (HES) mass ratios. Protein degradation was assessed as a function of time of storage of the dried formulations at 40, 50 and 60°C. Lyophilized and stored samples were rehydrated, and protein degradation was quantified by measuring loss of monomeric protein with size exclusion chromatography and by determining chemical degradation in the soluble fraction with reverse-phase chromatography. The secondary structure of the protein in the lyophilized formulations was studied with infrared spectroscopy. The magnitudes of degradation were compared the key physical properties of the formulations including retention of protein native secondary structure, glass transition temperature (Tg), inverse mean square displacements 〈u(2)〉(-1) for hydrogen atoms (fast β relaxation), and the relaxation time τ(β), which correlates with relaxation due to fast Johari-Goldstein motions in the glass (Xu et al., 2013) [1]. In addition, specific surface areas of the lyophilized formulations were determined by Brunauer-Emmet-Teller analysis of krypton adsorption isotherms and used to estimate the fraction of the KGF-2 molecules residing at the solid-air interface. KGF-2 degradation rates were highest in formulations wherein the protein's structure was most perturbed, and wherein β relaxations were fastest, but the dominant factor governing KGF-2 degradation in freeze-dried formulations was the fraction of the protein found at the glass solid-air interface.
authors.affiliation: Department of Pharmaceutical Sciences, University of Colorado, Denver, CO, USA.
authors.forename: Dilip
authors.initials: D
authors.lastname: Devineni
authors.affiliation: School of Pharmacy, Ludwig-Maximilians-University, Munich, Germany.
authors.forename: Christoph
authors.initials: C
authors.lastname: Gonschorek
authors.affiliation: Polymers Division, National Institute of Standards and Technology, Gaithersburg, MD, USA.
authors.forename: Marcus T
authors.initials: MT
authors.lastname: Cicerone
authors.affiliation: Department of Chemical and Biological Engineering, University of Colorado, Boulder, CO, USA.
authors.forename: Yemin
authors.initials: Y
authors.lastname: Xu
authors.affiliation: Department of Pharmaceutical Sciences, University of Colorado, Denver, CO, USA.
authors.forename: John F
authors.initials: JF
authors.lastname: Carpenter
authors.affiliation: Department of Chemical and Biological Engineering, University of Colorado, Boulder, CO, USA. Electronic address: theodore.randolph@colorado.edu.
authors.forename: Theodore W
authors.initials: TW
authors.lastname: Randolph
mesh_terms: D000388:Air; D002626:Chemistry, Pharmaceutical; D051526:Fibroblast Growth Factor 10 / Q000737:chemistry*; D005612:Freeze Drying; D005898:Glass; D006801:Humans; D010084:Oxidation-Reduction; D017433:Protein Structure, Secondary; D013499:Surface Properties
chemical_list: D051526:Fibroblast Growth Factor 10
keywords: Glass dynamics; Glass transition temperature; Global mobility; KGF-2; Local mobility; Lyophilization; Protein interfacial adsorption; Protein stability; Protein structure; Structural relaxation time