Systematic investigation of the effect of lyophilizate collapse on pharmaceutically relevant proteins III: collapse during storage at elevated temperatures

Abstract

This study investigates the effect of lyophilizate collapse on the stability of pharmaceutical proteins. Recently, it was shown that collapse during freeze-drying has no major negative impact on protein stability during storage at elevated temperatures when compared to non-collapsed cakes [1,2]. In this part of the study, lyophilizates that collapsed during the freeze-drying process were compared to cakes that were initially non-collapsed but collapsed during subsequent storage under accelerated stress conditions. Collapsed and non-collapsed lyophilizates of identical formulation and comparable residual moisture levels, containing a monoclonal IgG antibody, were stored at 40 °C and 50 °C for up to 3 months. Protein stability was monitored using a comprehensive set of analytical techniques assessing the formation of soluble and insoluble aggregates as well as protein conformation. The properties of the freeze-dried cake, namely the glass transition temperature, excipient crystallinity, sucrose degradation, reconstitution behavior, and the residual moisture content, were analyzed as well. The incorporated protein was significantly better stabilized in cakes that collapsed during the freeze-drying process when compared to lyophilizates that collapsed during subsequent storage. This effect can be related to the onset of crystallization and hydrolysis of the stabilizer and non-enzymatic browning.

Keywords: Collapse; Crystallization; Freeze-drying; Monoclonal antibody; Non-enzymatic browning; Protein aggregation; Protein conformation; Solid-state stability; Sucrose.