Functionalization of magnetic nanoparticles with high-binding capacity for affinity separation of therapeutic proteins
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Magnetic nanoparticles with immobilized metal ligands were prepared for the separation of antibody fragments. First, iron oxide nanoparticles were produced in a solvothermal synthesis using triethylene glycol as solvent and iron(III) acetylacetonate as organic precursor. Via functionalization of the particles with priorly reacted 3-glycidoxypropyltrimethoxysilane and N α,N α-bis(carboxymethyl)-l-lysine (NTA), and charging with Ni2+, magnetic affinity adsorbents were obtained. The particles were applied to separate a His-tagged antibody fragment from a heterogeneous protein mixture of a microbial cultivation supernatant. Binding properties and specificity for purification of the target product ABF D1.3 scFv were optimized regarding the GNTA concentration and were found superior as compared to commercially available systems. A molar ratio of 1:2 Fe2O3:GNTA was most beneficial for the specific purification of the antibody fragment.
KeywordsIron oxide nanoparticles Protein purification His-tag Bacillus megaterium Antibody fragment Non-aqueous synthesis
The authors thank Prof. G. Goya and G. Antorrena Pardo, University of Zaragoza, Spain, for the XPS analysis, and D. Menzel, Institute for Condensed Matter Physics, TU Braunschweig, for the Raman analysis. R. Pitschke and Prof. M. Antonietti, Max Planck Institute for Colloids and Interfaces, Potsdam, are gratefully acknowledged for the TEM measurements. We also thank Miriam Steinwand and Prof. Stefan Dübel, Institute of Biochemistry, Biotechnology and Bioinformatics, for provision of the D1.3 scFv antibody standard. This work was kindly supported by the German Research Foundation via the Collaborative Research Center (SFB 578-From Gene to Product) at TU Braunschweig.
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