Observing Interactions between Lipopolysaccharide Aggregates and a Monoclonal Antibody Using Differential Fluorination and ¹⁹ F NMR

Controlling lipopolysaccharide (LPS) levels during the manufacture of biopharmaceutical formulations is essential to ensure that final drug products are free from endotoxin contamination. Therefore, formulations are routinely and rigorously tested for the presence of LPS. However, it has been reported that certain formulation components, including biopharmaceuticals such as monoclonal antibodies (mAbs), can interfere with LPS detection. To uncover the molecular basis of this masking effect, it is critical to understand whether and how LPS interacts with mAbs. In this study, we present a novel approach that combines ¹⁹F NMR spectroscopy with differential fluorine labeling of both LPS aggregates and mAbs. This method enables independent and simultaneous observation of the behavior of each species in solution, allowing for direct detection of interactions, monitoring of the kinetics of supramolecular rearrangement, and measurement of changes in aggregate size, all from the distinct perspectives of both LPS and mAb. Our findings show that, upon mixing, the diffusion coefficients of LPS and mAb1 gradually decrease and eventually converge, indicating the formation of a larger, joint supramolecular complex. This methodology provides a framework for future investigations into how various formulation components influence LPS-mAb interactions and LPS aggregation state. Such insights will be critical for understanding the molecular origins of the masking effect and for developing strategies to prevent it in endotoxin assays.