Novel NTA-Ni²⁺ Agarose-Based Microspheres: Structural Features and Chromatographic Capacity

The design and optimization of immobilized metal affinity chromatography (IMAC) media are crucial to enhancing the purification efficiency of recombinant proteins. In this study, the agarose-based microspheres are prepared by using a three-factorial Box-Behnken design followed by NTA-Ni²⁺ agarose-based microspheres (ABM) preparation by the "one-step" crosslinking of epichlorohydrin (ECH)-nitrilotriacetic acid (NTA) to efficiently couple the NTA ligand to the surface of the matrix. After preparation, various sophisticated techniques, including SEM, AFM, DSC, FTIR, and SDS-PAGE, were used to analyze the morphological structure, thermal stability, and chemical composition of NTA-Ni²⁺ ABM. The optimal conditions are identified as an emulsifier PP concentration of 8.12 wt%, a stirring speed of 1624.46 rpm, and an oil-phase temperature of 53.86 °C, giving a span value (Y) of 0.50684. SEM, AFM, DSC, and FTIR results showed that the fabricated NTA-Ni²⁺ ABM were structurally stable and had a uniform cross-linking network for up to 8 h of coupling reaction time. The performance results showed that the beads had a high binding capacity for His-tagged proteins (15.2 ± 0.8 mg/mL), and SDS-PAGE results demonstrated the efficient purification ability for target proteins. These findings provide the theoretical basis and a practical solution for the rational design and application of IMAC medium.