Monash University

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Novel small molecule ligand affinity chromatography systems: high-throughput screening and scale-up

posted on 2017-02-06, 03:15 authored by Petzold, Martin
The industrial purification of recombinant proteins for therapeutic use is a highly complex and expensive multi-stage procedure that requires critical evaluation and adjustment for every single product. In addition to other adsorptive techniques, affinity chromatography methods with a defined target molecule specificity are commonly used, e.g. for the separation of monoclonal antibodies by exploiting their biological recognition by immobilised Protein A. In contrast to these protein-based adsorbents, immobilised low molecular weight affinity ligands offer a number of advantages including low cost, chemical and physical stability, broad specificity, low risk of contamination and high reusability. The focus of this project has been on the characterisation and development of two distinct purification strategies: (i) Immobilised metal ion affinity chromatography (IMAC) of peptide-tagged proteins using a new cassette system, and (ii) mixed-mode affinity separation of monoclonal antibodies with a novel family of small-molecule N-heteroaromatic ligands. With the help of a robotic liquid handling platform, a variety of high-throughput screening experiments were conducted to analyse the protein binding behaviour of the two systems and determine potential candidates for large-scale application. In addition to miniaturised batch adsorption methods using 96-well multititer plates, a recently developed automated approach to miniaturised parallel pressure-driven frontal chromatography was employed. These studies were followed by conventional column chromatography experiments including a scale-up of the bed volume. For both affinity systems, promising ligand candidates and viable process conditions could be identified. The new small molecule ligand families meet industrial requirements regarding safety and robustness and can be used as generic platform technologies to streamline complicated purification processes. The cost as well as the environmental impact of biopharmaceutical production could be reduced by using these cheaper materials which can be synthesised under mild conditions.


Campus location


Principal supervisor

Milton Thomas William Hearn

Additional supervisor 1

Reinhard Boysen

Year of Award


Department, School or Centre


Additional Institution or Organisation

Centre for Green Chemistry


Doctor of Philosophy

Degree Type



Faculty of Science