Benchmarking polymeric cryogels for immobilized metal affinity chromatography

Abstract

Cryogels are polymers prepared in frozen environment and seen as new separation matrices with their applications in many bioseparation methods. They have significant benefits including supermacroporosity, short diffusion path, low pressure, and low resistance to both adsorption and elution. Macro and connected pores give cryogels a unique spongy structure. Immobilized metal affinity chromatography (IMAC) is a generally employed analytical separation method for the purification of biomolecules. Several transition ions create stable complexes with electron-rich compounds. IMAC sorbent is obtained by complexing first-order transition metal ions over chelating agents. Lysozyme is an enzyme found in various vertebrate cells and secretions. Common applications include its use as a cell disrupting agent, as an anti-bacterial agent, as a food additive, and as a medicine for the treatment of infections and ulcers. In this study, cryogel-based polymeric material prepared by free-radical polymerization method with hydroxyethyl methacrylate/glycidyl methacrylate monomer pair were covalently interacted with iminodiacetic acid metal chelating agent, and regions showing affinity for lysozyme enzyme were formed by binding with Ni(II) ions. The cryogel was first characterized by Fourier transform infrared spectrophotometer, scanning electron microscopy, thermal gravimetric analysis, X-ray photoelectron spectroscopy and swelling test. Then, the effects of pH, lysozyme concentration, temperature, ionic strength and flow rate on lysozyme adsorption capacity were explored and optimum conditions were found.