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

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.

Systematic investigation of the effect of lyophilizate collapse on pharmaceutically relevant proteins I: stability after freeze-drying.

The objective of this work was to investigate the effect of cake collapse during freeze-drying on the stability of protein lyophilizates containing a monoclonal IgG(1)-antibody or a second pharmaceutically relevant protein, referred to as PA01. In addition, L-lactic dehydrogenase was investigated because of its well-documented sensitivity towards freeze-drying stresses. Collapse was induced by two different means. First, by varying the ratio of the crystalline bulking agent mannitol to the amorphous stabilizer sucrose, different extents of collapsed cakes were generated. Second, formulations were freeze-dried using an aggressive collapse-cycle and a conventional freeze-drying protocol and collapsed and noncollapsed cakes of identical formulation were produced. Lyophilizates were analyzed using a comprehensive set of analytical techniques to monitor protein stability in terms of formation of soluble and insoluble aggregates, the biological activity and the conformational stability. The stability of excipients, namely the glass transition temperature, crystallinity, reconstitution behavior, and the residual moisture content was analyzed as well. In addition, the extent of collapse was quantified using the decrease of the specific surface area (SSA). Collapsed cakes had comparable residual moisture levels to noncollapsed lyophilizates. Reconstitution times were not increased. Protein stability was not relevantly different between collapsed and noncollapsed cakes.