Optimizing Storage Stability of Lyophilized Recombinant Human Interleukin-11 with Disaccharide/Hydroxyethyl Starch Mixtures WILLIAM GARZON-RODRIGUEZ, 1 REBECCA L. KOVAL, 2 SUCHART CHONGPRASERT, 1 SAMPATHKUMAR KRISHNAN, 1 THEODORE W. RANDOLPH, 3 NICHOLAS W. WARNE, 2 JOHN F. CARPENTER 1 1 Center for Pharmaceutical Biotechnology, School of Pharmacy, University of Colorado Health Sciences Center, Denver, Colorado 80262 2 Wyeth BioPharma, Drug Product Development Group, Andover, Massachusetts 01810 3 Department of Chemical Engineering, University of Colorado, Boulder, Colorado 80302 Received 31 July 2003; revised 8 September 2003; accepted 8 September 2003 ABSTRACT: Optimal storage stability of a protein in a dry formulation depends on the storage temperature relative to the glass transition temperature (T g ) of the dried formulation and the structure of the dried protein. We tested the hypothesis that optimizing both protein structure and T g —by freeze-drying recombinant human inter- leukin-11 (rhIL-11) with mixtures of disaccharides and hydroxyethyl starch (HES)— would result in increased storage stability compared with the protein lyophilized with either disaccharide or hydroxyethyl starch alone. The secondary structure of the protein in the dried solid was analyzed immediately after lyophilization and after storage at elevated temperatures by infrared spectroscopy. After rehydration, aggregation was monitored by size exclusion chromatography. Oxidation levels and cleavage products were quantified by reversed-phase chromatography. For the formulation with HES alone, which has a relatively high T g , storage stability of rhIL-11 was poor, because HES failed to inhibit lyophilization-induced unfolding. The sugar formulations inhibited unfolding, and had intermediate T g values and storage stabilities. Addition of hydro- xyethyl starch to sucrose or trehalose increased T g without affecting the capacity of the sugar to inhibit protein unfolding during lyophilization. Optimal storage stability of lyophilized rhIL-11 was achieved by using a mixture of disaccharide and polymeric carbohydrates. ß 2004 Wiley-Liss, Inc. and the American Pharmacists Association J Pharm Sci 93:684–696, 2004 Keywords: interleukin-11; proteins; stability; oxidation; protein aggregation; glass transition; infrared spectroscopy INTRODUCTION Freeze-drying is often the method of choice for production of stable formulations of therapeutic proteins because water participates in and/ or mediates physical and chemical degradation of proteins. 1 A rational choice of stabilizing excipients is needed to protect the protein from denaturation during freezing and dehydration, as well as to provide a glassy matrix that is required for long-term storage stability in the dried solid. 2,3 Disaccharides, such as sucrose and trehalose, frequently have been used as effective stabilizers for proteins during freezing, drying, and subse- quent storage in the dried solid. 4–9 These sugars thermodynamically protect proteins during freez- ing by increasing the free energy of unfolding. 10 684 JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 93, NO. 3, MARCH 2004 Correspondence to: John F. Carpenter (Telephone: 303-315- 6074; Fax: 303-315-6281; E-mail: John.Carpenter@UCHSC.edu) Journal of Pharmaceutical Sciences, Vol. 93, 684–696 (2004) ß 2004 Wiley-Liss, Inc. and the American Pharmacists Association