Immobilizing calcium-dependent affinity ligand onto iron oxide nanoparticles for mild magnetic mAb separation

•Calcium-dependent magnetic mAb separation aims to enhance downstream processing.•Affinity ligand ZCa-cys was immobilized onto three iron oxide nanoparticle types.•Physical immobilization enabled higher but less oriented loadings than covalent.•High mAb binding capacities were reached (up to 196 mg g-1).•High mAb recoveries were achieved at pH 5.5 (up to 88 %). Current downstream processing of monoclonal antibodies (mAbs) is limited in throughput and requires harsh pH conditions for mAb elution from Protein A affinity ligands. The use of an engineered calcium-dependent ligand (ZCa) in magnetic separation applications promises improvements due to mild elution conditions, fast processability, and process integration prospects. In this work, we synthesized and evaluated three magnetic nanoparticle types immobilized with the cysteine-tagged ligand ZCa-cys. Ligand homodimers were physically immobilized onto bare iron oxide nanoparticles (MNP) and MNP coated with tetraethyl orthosilicate (MNP@TEOS). In contrast, ZCa-cys was covalently and more site-directedly immobilized onto MNP coated with (3-glycidyloxypropyl)trimethoxysilane (MNP@GPTMS) via a preferential cysteine-mediated epoxy ring opening reaction. Both coated MNP showed suitable characteristics, with MNP@TEOS@ZCa-cys demonstrating larger immunoglobulin G (IgG) capacity (196 mg g−1) and the GPTMS-coated particles showing faster magnetic attraction and higher IgG recovery (88 %). The particles pave the way for the development of calcium-dependent magnetic separation processes. [Display omitted]