A deep dive into spin-labeled polysorbate's interaction with therapeutic antibody using 2D NMR, EPR and MD...

Polysorbates (PS) are widely used surfactants in biopharmaceutical formulations playing a crucial role in protecting proteins against mechanical stress and interface-induced damage. However, their susceptibility to degradation can compromise their function and lead to particle formation. Recent studies suggest that monoclonal antibodies (mAbs) may mitigate PS degradation catalyzed by histidine chloride buffer, indicating the presence of protein-PS interactions. In this study, we investigated these interactions using NMR, starting with ¹H T₂ CPMG filter experiments and methyl fingerprinting, which failed to detect interactions. To enhance sensitivity, we synthesized spin-labeled PS (SLPS), enabling paramagnetic relaxation enhancement (PRE) NMR experiments, specifically amide fingerprinting, which successfully revealed interactions. Complementary electron paramagnetic resonance (EPR) measurements of SLPS and mAb also detected interactions, but only in the presence of sucrose, underscoring their weak and transient nature. Additionally, molecular dynamics simulations identified potential interaction hotspots on the antibody structure, providing mechanistic insights into these interactions.