(228df) On the Improvement of Immobilized Metal Affinity Chromatography (IMAC): Preparation, Characterization and Validation of a Superporous Agarose Monolith

E.M. Martín del Valle*, M. Elviro, , M.A. Galán

*Corresponding author: E. Martín del Valle. Department of Chemical Engineering, University of Salamanca P/Los Caidos, 37008 Salamanca, Spain.

Mail: emvalle@usal.es Phone: +34 923 294479

Purification of biomolecules is a key step in the biotechnology industry as it often represents the major manufacturing costs. Affinity chromatography is a high performance purification step with high selective separation as it uses a reversible binding between biomolecules wanted to purify and a specific ligand joined to a chromatographic support.

Immobilized metal ion affinity chromatography (IMAC) is a type of affinity chromatography based in the affinity of some side chain of amino acids for metal ions bound to a metal-chelating agent immobilized on a chromatographic matrix. Since the interaction between the immobilized metal ions and the side chain of amino acids has a reversible character, it can be utilized for adsorption and then be recovered using nondenaturing conditions.

In the last years, new kinds of chromatographic supports have emerged to improve the chromatographic properties and industrial scale-up of the known matrix, as higher mass transfer, and specially a lower pressure drop.

Monolithic superporous agarose beds were prepared by an emulsion method prior to use. The agarose matrix is formed by a homogeneous distribution of macropores. This bed is first used as support in catalase and BSA adsorption and purification by IMAC chromatography using Cu²⁺ as metallic ion. The monolithic matrix was used in order to achieve high superficial area and low pressure drop.

Furthermore, catalase and BSA adsorption equilibrium and elution were studied. The obtained results were compared in order to determine the influence of the adsorbed protein in adsorption and elution processes. The matrix was reused fourteen times and it can be concluded there is not any lose of yield, and any regeneration of Cu²⁺ was required.

In summary, a monolithic superporose agarose have been developed and used in catalase and BSA purification by IMAC. This support had high superficial area available for protein adsorption, it had improved the amount of protein adsorbed, it had decreased the needed time for adsorption and elution and it had been reused fourteen times without any losing of yield neither Cu²⁺ regeneration. Furthermore, influence of the kind of target compound was evaluated in order to understand better the chromatographic process inside the monolith, necessary to scale up.