Direct Affinity Ligand Immobilization onto Bare Iron Oxide Nanoparticles Enables Efficient Magnetic Separation of Antibodies

Ines Zimmermann, Yasmin Kaveh-Baghbaderani, Friederike Eilts, Nadja Kohn, Paula Fraga-García, Sonja Berensmeier

Research output: Contribution to journalArticlepeer-review

1 Scopus citations

Abstract

Magnetic separation is a promising alternative to chromatography for enhancing the downstream processing (DSP) of monoclonal antibodies (mAbs). However, there is a lack of efficient magnetic particles for successful application. Aiming to fill this gap, we demonstrate the suitability of bare iron oxide nanoparticles (BION) with physical site-directed immobilization of an engineered Protein A affinity ligand (rSpA) as an innovative magnetic material. The rSpA ligand contains a short peptide tag that enables the direct and stable immobilization onto the uncoated BION surface without commonly required laborious particle activation. The resulting BION@rSpA have beneficial characteristics outperforming conventional Protein A-functionalized magnetic particles: a simple, fast, low-cost synthesis, a particle size in the nanometer range with a large effective specific surface area enabling large immunoglobulin G (IgG) binding capacity, and a high magnetophoretic velocity advantageous for fast processing. We further show rapid interactions of IgG with the easily accessible rSpA ligands. The binding of IgG to BION@rSpA is thereby highly selective and not impeded by impurity molecules in perfusion cell culture supernatant. Regarding the subsequent acidic IgG elution from BION@rSpA@IgG, we observed a hampering pH increase caused by the protonation of large iron oxide surfaces after concentrating the particles in 100 mM sodium acetate buffer. However, the pH can be stabilized by adding 50 mM glycine to the elution buffer, resulting in recoveries above 85% even at high particle concentrations. Our work shows that BION@rSpA enable efficient magnetic mAb separation and could help to overcome emerging bottlenecks in DSP.

Original languageEnglish
Pages (from-to)3942-3952
Number of pages11
JournalACS Applied Bio Materials
Volume7
Issue number6
DOIs
StatePublished - 17 Jun 2024

Keywords

  • downstream processing
  • kinetics
  • magnetic nanoparticles
  • pH buffering of iron oxides
  • protein recovery
  • site-directed Protein A immobilization

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