Characterisation of metal-chelate methacrylate monoliths

Area of Science:

• Biochemistry
• Chromatography
• Bioseparation

Background:

• Monolithic stationary phases offer flow-unaffected properties, advantageous for large biomolecule purification.
• Metal-chelate chromatography is effective for isolating histidine-tagged proteins in a single step.

Purpose of the Study:

• To investigate the properties of commercial metal-chelate methacrylate monoliths (Convective Interaction Media - CIM).
• To evaluate the purification efficiency and binding capacity of CIM columns for specific histidine-tagged proteins.

Main Methods:

• Utilized analytical CIM disk and 8 ml monolithic columns for protein purification.
• Purified tumor necrosis factor-alpha (TNF-alpha) analog LK-801 and green fluorescence protein with a 6 histidine tag (GFP-6His).
• Assessed purity, dynamic binding capacity, and adsorption isotherms.

Main Results:

• Achieved over 90% purity for both LK-801 and GFP-6His.
• Reported dynamic binding capacities of approximately 17-18 mg/ml for LK-801 and 30 mg/ml for GFP-6His at 10% breakthrough.
• Demonstrated that dynamic binding capacity and resolution are unaffected by flow rate.

Conclusions:

• Commercial CIM metal-chelate monoliths are effective for high-purity isolation of histidine-tagged proteins.
• The purification performance, including capacity and resolution, remains consistent across varying flow rates.
• CIM monoliths provide a robust and efficient solution for bioseparation of histidine-containing proteins.