Effects of Annealing Lyophilized and Spray-Lyophilized Formulations of Recombinant Human Interferon-g SERENA D. WEBB, 1 JEFFREY L. CLELAND, 2 JOHN F. CARPENTER, 3 THEODORE W. RANDOLPH 1 1 Department of Chemical Engineering, University of Colorado, Department of Engineering, Center for Pharmaceutical Biotechnology, Engineering Center, Room ECCH 111, Boulder, Colorado 80309-0424 2 Genentech, Inc., South San Francisco, California 94080 3 Department of Pharmaceutical Sciences, School of Pharmacy, University of Colorado Health Sciences Center, Denver, Colorado 80262 Received 29 April 2002; revised 13 September 2002; accepted 22 October 2002 ABSTRACT: The purpose of this study was to examine the effects of adsorption of recombinant human interferon-g (rhIFN-g) on ice surfaces and subsequent drying during processing by spray-lyophilization and lyophilization. Ice/liquid interfacial areas were manipulated by the freezing method as well as by the addition of an annealing step during lyophilization; that is, rhIFN-g adsorption was modified by the addition of nonionic surfactants. rhIFN-g was lyophilized or spray-lyophilized at a concentration of 1 mg/mL in 5% sucrose, 5% hydroxyethyl starch (HES)  0.03% polysorbate 20 in 140 mM KCl, and 10 mM potassium phosphate, pH 7.5. After the samples were frozen, half were annealed on the lyophilizer shelf. Recovery of soluble protein was measured at intermediate points during processing. On drying, the secondary structure of rhIFN-g was determined by second-derivative infrared (IR) spectroscopy, specific surface areas (SSAs) were measur- ed, scanning electron micrographs (SEM) were taken, and dissolution times were re- corded. Adsorption of rhIFN-g to ice/liquid interfaces alone was not responsible for aggregation. Rather, drying was necessary to cause aggregation in lyophilized sucrose formulations. Addition of an annealing step to the lyophilization cycle resulted in more native-like secondary protein structure in the dried solid, eliminated cracking of the dried cakes, and suppressed both the formation of air/liquid interfaces and rhIFN-g aggregation on reconstitution. ß 2003 Wiley-Liss, Inc. and the American Pharmaceutical Association J Pharm Sci 92:715–729, 2003 Keywords: rhIFN-g; surfactant, Tween 20; polysorbate 20; lyophilization; freeze drying; spray-lyophilization; annealing; ice/liquid interface; air/liquid interface INTRODUCTION A large ice/liquid interfacial area (0.1–2 m 2 /mL) forms during the freezing step of a lyophilization or spray-lyophilization process. This interfacial area has been implicated in aggregation of proteins, 1,2 which may adsorb to the ice/liquid interface and undergo subsequent unfolding and aggregation. In previous studies of interfacial adsorption of protein at the ice/liquid interface, fast or slow cooling protocols have been used to manipulate the size of the interface. 1–5 Chang et al. 2 noted that rapid cooling in liquid nitrogen caused nearly the same increase in turbidity in solutions containing one of a variety of proteins as 11 slow freeze – thaw cycles. This result was explained in terms of the larger ice/liquid interfacial surface area in the rapidly frozen samples. Presumably, however, the cumulative total surface area to which the JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 92, NO. 4, APRIL 2003 715 Correspondence to: Theodore W. Randolph (Telephone: 303- 492-4776; Fax: 303-492-4341; E-mail: Randolph@pressure3.colorado.edu) Journal of Pharmaceutical Sciences, Vol. 92, 715–729 (2003) ß 2003 Wiley-Liss, Inc. and the American Pharmaceutical Association