Systematic investigation of the effect of lyophilizate collapse on pharmaceutically relevant proteins, part 2: stability during storage at elevated temperatures.

The objective of this work was to investigate the effect of lyophilizate collapse on the stability of freeze-dried protein pharmaceuticals. In the first part of this study, it was shown that collapse has no negative impact either on the properties of the freeze-dried cake or on protein stability [Schersch K, Betz O, Garidel P, Muehlau S, Bassarab S, Winter G. 2010. J Pharm Sci 99(5):2256-2278]. In order to further investigate the effect of collapse, its impact on lyophilizate's long-term stability during storage at various temperatures was evaluated at 2°C-8°C, 25°C, 40°C, and 50°C for up to 6 months. Collapsed and noncollapsed lyophilizates of identical formulation and comparable residual moisture levels containing the following proteins were investigated: (1) a monoclonal immunoglobulin G antibody, (2) tissue-type plasminogen activator, and (3) the sensitive model protein l-lactic dehydrogenase. Protein stability was monitored using a comprehensive set of analytical techniques assessing the formation of soluble and insoluble aggregates, the biological activity, and the protein conformation. The properties of the freeze-dried cake--namely, the glass transition temperature, excipient crystallinity, reconstitution behavior, and the residual moisture content, were analyzed as well. Full protein stability in collapsed cakes was observed, and even enhanced protein stability was detected in collapsed cakes with regard to key stability-indicating parameters.