Elucidating protein M performance and its binding modes in antibody purification via integrated site-specific immobilization and molecular dynamics

Universal antibody purification remains challenging because conventional ligands (Protein A/G/L) target specific antibody regions, limiting their general application. In contrast, protein M from human mycoplasma exhibits unprecedented broad-spectrum binding to diverse antibody types through interaction with the conserved part of the variable domain of the light chain. Its engineered variant, protein M-8His, introduces pH-responsive histidine residues while maintaining this broad binding capability, but the molecular mechanisms governing its variable performance across different antibodies remain poorly characterized. To investigate this, we developed an antibody purification platform through site-specific immobilization of protein M-8His on Ni 2+ -modified superparamagnetic nanoparticles (NiNPs). NiNPs demonstrated a protein M-8His recoverable binding capacity of 74.41 mg/g. The resulting protein M-8His functionalized NiNPs (MNiNPs) demonstrated broad-spectrum antibody binding, with recoverable binding capacities up to 52.01 mg/g for trastuzumab scFv, while maintaining 76.1 % activity after 15 days of storage. Molecular dynamics simulations suggested two distinct interaction patterns: histidine-dominated interactions driving pH-dependent elution (H337/H338 for trastuzumab scFv) and mixed-interaction binding with reduced histidine contribution (for S2P6 and 76E1 antibodies), explaining the observed performance variations. Energy decomposition analysis showed that trastuzumab scFv (100 %) and 76E1 Fab (85.1 %) predominantly bind via Fv domain while S2P6 Fab mainly binds via constant domain (76.7 %). These findings provide mechanistic insights into protein M-8His interactions and lay a foundation for optimizing protein M-8his for industrial applications.