AFM-Based Single-Molecule Force Spectroscopy of PEG-Anti-PEG Antibody Interactions.

Poly(ethylene glycol) (PEG) is widely used as a stealth polymer to enhance drug stability and circulation by reducing immune recognition. However, anti-PEG antibodies are increasingly reported in humans, leading to accelerated drug clearance and adverse immune responses. While ensemble assays have clarified the scheme of PEG-antibody binding, they lack the resolution to probe molecular-scale mechanics. Here, we used atomic force microscopy-based single-molecule force spectroscopy (AFM-SMFS) to examine how PEG terminal chemistry and antibody maturation modulate these interactions. Methoxy- (m-PEG) and hydroxy-terminated PEG (HO-PEG) were tested against Fv-clasps from two anti-PEG IgMs: the naïve IgM M9 and the affinity-matured IgM M11. M11 bound PEG more strongly and at shorter rupture distances than M9, with 2D force-distance maps revealing the most intense signatures for M11 and m-PEG pair. Complementary quartz crystal microbalance with dissipation (QCM-D) and Fourier-transform infrared (FTIR) spectroscopy confirmed higher binding by M11 and a terminal preference of M9 for m-PEG. In addition to antibody maturation, we report that the hydrated structure of PEG plays a significant role in PEG-antibody binding. HO-PEG forms extended, hydrated layers, whereas m-PEG adopts compact, collapsed conformations, shaping antibody accessibility and binding mechanics. These results provide molecular-level insight into how antibody structure and PEG hydration state dictate binding, offering design principles for PEGylated therapeutics with reduced immunogenicity and improved performance.