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

Area of Science:

• Biotechnology
• Chromatography
• Materials Science

Background:

• Immobilized metal affinity chromatography (IMAC) media are essential for recombinant protein purification.
• Optimizing IMAC media enhances purification efficiency.

Purpose of the Study:

• To design and optimize NTA-Ni2+ agarose-based microspheres (ABM) for efficient protein purification.
• To establish optimal conditions for ABM preparation using a Box-Behnken design.

Main Methods:

• Agarose-based microspheres prepared using a three-factorial Box-Behnken design.
• One-step crosslinking of epichlorohydrin (ECH)-nitrilotriacetic acid (NTA) for ligand coupling.
• Characterization using SEM, AFM, DSC, FTIR, and SDS-PAGE.

Main Results:

• Optimal conditions determined: 8.12 wt% emulsifier PP, 1624.46 rpm stirring speed, 53.86 °C oil-phase temperature (span value Y=0.50684).
• Structurally stable NTA-Ni2+ ABM with uniform cross-linking network confirmed by SEM, AFM, DSC, and FTIR.
• High binding capacity (15.2 ± 0.8 mg/mL) for His-tagged proteins and efficient purification demonstrated by SDS-PAGE.

Conclusions:

• The study provides a rational design and practical solution for IMAC medium development.
• Optimized NTA-Ni2+ ABM offers enhanced performance for recombinant protein purification.