In the present work, a novel agarose–nickel bilayer composite (ANBC) adsorbents modified with Cibacron Blue 3GA (CB3) dye-ligand were fabricated via the three-phase emulsion method. The surface morphology of the novel adsorbents was evaluated using an optical microscope, scanning electron microscopy, and atomic force microscopy. Also, the commercially purchased adsorbents were modified with the same ligand and compared to novel composite (ANBC-CB3). Also, Lactoferrin (Lf) nanoparticles synthesized and employed as a protein model. Residence time distribution experiments were performed to consider the effect of particle density and fluid viscosity. The results established that the ANBC-CB3 bilayer composite follows a typical size range of 60–300 μm and a mean diameter of 125 μm with a spherical appearance. In batch adsorption, ANBC-CB3 revealed a higher dynamic binding capacity (DBC) of Lf versus Streamline™-CB3. Furthermore, the experimental data were consistent with the Langmuir isotherm model. Finally, The DBC at 10% breakthrough and the two-fold flow rate was more than 70% for the novel adsorbent. It was proved that ANBC-CB3 as high selective adsorbents for protein nanoparticle purification is suitable for high flow rate operations in an expanded bed column.
