Immobilization of nonviral vectors during the freezing step of lyophilization

The development of nonviral vectors as commercial therapeutics will require formulations that are sufficiently stable to allow shipping and storage for prolonged periods. Given the well-known instability of these systems as aqueous suspensions, it would be desirable to develop lyophilized formulations that are resistant to shipping stress and can be stored for extended periods at ambient temperatures. Previous studies have shown that aggregation and structural changes resulting in reduced transfection rates can occur during the freezing step of lyophilization. While it has been clearly demonstrated that freezing-induced damage is promoted by vector crowding that results from the reduced volume of unfrozen solution, the precise mechanism of damage has yet to be fully elucidated, i.e., damage may occur due to ice formation and/or during incubation in the frozen state. In this study, we investigate the time- and temperature-dependence of damage during freezing and demonstrate that aggregation can occur while frozen vector suspensions are incubated at a constant temperature. Aggregation is not seen during incubation at temperatures below T-g', and can also be avoided above the glass transition temperature under some conditions. Our data are consistent with a model describing the mobility of vectors in the unfrozen sucrose solution being sufficiently restricted such that inter-particle interactions are prevented in the frozen state. Furthermore, the protection achieved during freezing at temperatures above T-g' is applicable to a complete lyophilization cycle (i.e., freezing and drying), and provides stabilization at higher primary drying temperatures. (C) 2004 Wiley-Liss, Inc. and the American Pharmacists Association.