Conformational Analysis of Monoclonal Antibodies in Lyophilized and Spray Dried Solids
Monoclonal antibodies (mAbs) constitute a large class of therapeutic proteins that have diverse clinical uses. Like most proteins, mAbs are vulnerable to chemical and/or physical degradation, which can lead to significant losses during manufacturing and storage. To slow down such reactions, mAbs are oftentimes formulated in solid rather than liquid dosage forms. Additionally, antibody-drug conjugates, an emerging therapeutic modality that involves mAbs conjugated to cytotoxic small molecule drugs, are formulated as lyophilized solids. As such, the importance of understanding and predicting stability of mAbs in the solid-state cannot be overstated. To this end, various analytical methods are typically used to probe the structure and dynamics of proteins in the solid-state as metrics to predict stability. Recently, hydrogen-deuterium exchange with mass spectrometric analysis (HDX-MS) emerged as a useful tool for the analysis of the higher order structure and the development of stable liquid formulations of therapeutic proteins. In the solid-state (ss), HDX-MS provides a sensitive probe of the conformation of proteins as well as the extent of the interactions thereof with the components of the formulation matrix at both the intact and peptide levels, a property that cannot be measured by other analytical methods. In this vein, the overarching aim of this dissertation is to evaluate the utility of ssHDX-MS to (i) predict physical stability of mAbs in the solid-state, (ii) to probe local conformational changes of mAbs in different solid matrices, and (iii) to study the effect of different manufacturing processes on the conformation of mAbs in the solid-state. The results of these investigations demonstrate a correlation between several ssHDX parameters and the aggregation of a model mAb in lyophilized formulations, and show that local conformational changes and/or site specific interactions with excipients are evident in a histidine matrix. Following from this result, adding histidine to a lyophilized sucrose matrix is shown to decrease the aggregation of the mAb at different pH conditions. Finally, ssHDX-MS analysis indicates the presence of conformational and/or spatial heterogeneity in the spray dried formulations of the mAb with sucrose or trehalose compared to the lyophilized counterparts. Taken together, these findings demonstrate the sensitivity of ssHDX-MS analysis to conformational changes weakly indicated by or invisible to spectroscopic methods, and support the broader use of ssHDX-MS to probe formulation and process effects on protein structure, and to predict physical stability in solid formulations.
Topp, Purdue University.
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