Automation of N-glycan purification using amine-functionalized NiFe₂O₄ magnetic nanoparticles

Magnetic nanoparticles (MNPs) are increasingly utilized in biomedical applications, including nucleic acid isolation, protein purification and glycan enrichment, owing to their high surface-to-volume ratio, tuneable surface chemistry and compatibility with automation. Here, we report the development, systematic optimization and automated implementation of an amine-functionalized NiFe₂O₄-based MNP protocol for N-glycan purification. Critical workflow steps, including particle drying and dispersion, binding efficiency, and elution buffer selection, were comprehensively evaluated. The optimized protocol, employing 0.5 mg/mL NiFe₂O₄-NH₂ particles and 150 mM ammonium-formate (pH 4.4) as elution buffer enabled the reproducible identification of the major N-glycan peaks from human serum and immunoglobulin G (IgG). Importantly, the method was successfully translated to a Hamilton Microlab Prep robotic liquid handling platform in a 96-well format, ensuring scalability and reduced manual variability. Overall, this study advances a previously established MNP-based glycan purification approach into a robust, scalable, and automation-compatible workflow through synthesis redesign, systematic optimization, and successful robotic implementation.