Nitrogen heterocycles: novel tools for protein purification

Thesis Summary: Proteins such as antibodies, non-glycosylated proteins including cytokines as well as glycosylated proteins such as transferrin play an important role in clinical diagnosis and treatment of diseases, which together constitute a multibillion dollar per year bioindustry. Traditionally, purification of these biomolecules can be done by a combination of hydrophobic interaction chromatography (HIC), immobilised metal affinity chromatography (IMAC), ion exchange chromatography, size exclusion chromatography (molecular sieving) and occasionally electrophoretic methods. The work carried out in this thesis was to develop an efficient, cost effective and greener method for protein purification by using adsorbents based on substituted nitrogen heterocycles. Several thioamine pyridine and bipyridine derivatives were synthesised using green chemical methodologies. These compounds contained a primary amino, hydroxyl, carboxyl or thiol group, which is required for attachment of ligand to a solid support, and a sulphur atom for enhanced binding interactions with proteins. Figure 1: General structure of ligands The general structure of the ligands tested is shown in Figure 1. These ligands were immobilised onto a suitable solid support material, such as agarose, using the general procedure for activation and ligand-loading as shown in the Scheme 1. Experimental parameters such as reaction time, solvent and temperature were investigated to optimise the coupling conditions for the ligand with the activated solid support. R = -CH2-CH2-S- R' Scheme 1: General synthetic route for activation of Sepharose with epichlorohydrin and immobilisation of ligands (A) PSEA isomers and PSEA derivatives, (B) PSESH and (C) PSEOH onto the activated Sepharose through a side chain The adsorbents were evaluated for their binding (suitable reactive performance) for different antibody, cytokine and transferrin species by batch adsorption assays using a range of pH, buffer and salt concentration conditions. The optimal protein binding conditions were then used to calculate the thermodynamic interaction parameters such as dissociation constant (KD) and maximal binding capacity (Qmax) of the adsorbents. Purification of proteins such as monoclonal antibodies, transferrin and cytokines using column chromatographic techniques was performed on a laboratory scale and mAb binding was compared with a commercial adsorbent, Protein A. As the new methods for the synthesis of these ligands are relatively simple, greener and inexpensive, the purification method has the potential to be developed to large scale processes suitable for commercial applications in the biotechnology industry.