Host cell protein impurities and protein-protein interactions in downstream purification of monoclonal antibodies

Levy, Nicholas E.
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University of Delaware
Monoclonal antibodies (mAbs) have become increasingly important as protein therapeutics over the last few decades. MAbs can be engineered to achieve high binding affinity to a wide array of desired biological targets. Despite having different therapeutic targets, mAbs have nearly identical amino acid sequences and biophysical properties. The high degree of similarity among different mAb products has led to the development of platform manufacturing processes in many companies. Downstream purification processes for mAbs are designed based on a platform process that is empirically tuned to optimize impurity removal for each new product. A more fundamental understanding of impurities and the product itself would provide insights into the rational design of efficient downstream processes. The first part of this thesis is focused on host cell protein (HCP) impurities, and the main objectives are to identify and characterize HCP impurities and their behavior in a typical downstream platform purification process. In protein A affinity chromatography - the capture step used in the majority of mAb platform purification processes - HCP impurities were found to associate to mAb products due to strongly attractive interactions. By coupling cross-interaction chromatography to proteomic analysis, specific HCP impurities that associate with different mAbs were identified. A subset of HCPs associates with all or most mAb products. Additionally, a unique population of HCPs associates with each mAb product; minor changes to the primary amino acid sequence were found to have a potentially significant impact on the population of associated HCPs. HCP impurities were also studied in non-affinity platform chromatographic processes. Product-association was found to be an important mechanism through which HCPs can co-purify with mAbs in all chromatographic modes studied. HCP impurities with similar chromatographic behavior to those of mAbs were also identified. This work has identified many difficult-to-remove HCPs and provided mechanistic insight that will aid future downstream process development. Lipoprotein lipase is a specific HCP impurity that was persistent in many different chromatographic purification processes and was studied in greater detail in this thesis. This particular HCP associates with most mAbs with high affinity. Also, it was found that lipoprotein lipase can enzymatically degrade nonionic surfactants that are commonly included in mAb formulations to prevent product aggregation. Enzymatic degradation of nonionic surfactants by lipoprotein lipase in mAb formulations could negatively affect product quality by inducing mAb aggregation or precipitation. The final aspect of this work was to compare mAb self-interaction strengths and instantaneous phase boundaries in various solution conditions and to explore the underlying molecular basis for such interactions. In solutions of sulfate salts most of the mAbs studied had nearly identical self-interaction trends and instantaneous phase boundaries. However, the divergent behavior of two nearly identical mAbs shows the potential impact of minor structural changes on product molecules. Overall, there is a qualitative correlation between self-interactions and phase boundary location, but due to mAb anisotropy, quantitative correlations and predictions of mAb properties are difficult. Measurements of mAb fragment interactions provide further insight into different oligomerization patterns and the origin of attractive self-interactions for various mAbs. Although many mAbs were found to have similar self-interaction properties, the domain-level interactions have greater variability. Fab-Fab, Fc-Fc and 'hinge' region interactions were identified as the source of highly attractive self-interactions for different mAbs. Because mAbs are large, anisotropic molecules that are sensitive to minor structural changes, detailed domain-based analysis provides greater insight into oligomerization mechanisms and potentially allows engineering of more stable mAbs.