Studies with an immobilized metal affinity chromatography cassette system involving binuclear triazacyclononane-derived ligands: Automation of batch adsorption measurements with tagged recombinant proteins

• Efficient methods are described for selection of new IMAC-tagged protein systems.
• Adsorption isotherms and binding kinetics of new IMAC resins determined.
• Performance attributes of a new type of tag have been compared to the [His]6 tag.
• Impact of ligand structure on binding constants and resin capacities documented.
• Application uses of different metal-charged binuclear tacn ligands are described.

This study describes the determination of the adsorption isotherms and binding kinetics of tagged recombinant proteins using a recently developed IMAC cassette system and employing automated robotic liquid handling procedures for IMAC resin screening. These results confirm that these new IMAC resins, generated from a variety of different metal-charged binuclear 1,4,7-triaza-cyclononane (tacn) ligands, interact with recombinant proteins containing a novel N-terminal metal binding tag, NT1A, with static binding capacities similar to those obtained with conventional hexa-His tagged proteins, but with significantly increased association constants. In addition, higher kinetic binding rates were observed with these new IMAC systems, an attribute that can be positively exploited to increase process productivity. The results from this investigation demonstrate that enhancements in binding capacities and affinities were achieved with these new IMAC resins and chosen NT1A tagged protein. Further, differences in the binding performances of the bis(tacn) xylenyl-bridged ligands were consistent with the distance between the metal binding centres of the two tacn moieties, the flexibility of the ligand and the potential contribution from the aromatic ring of the xylenyl group to undergo π/π stacking interactions with the tagged proteins.