Green chemical solutions for the separation of recombinant proteins, including biopharmaceuticals

The purification of recombinant proteins is a rapidly expanding industry which currently relies on multistep purification and multiple techniques to purify a small amount of protein. One of the techniques to purify these recombinant proteins on a laboratory scale is Immobilised Metal Affinity Chromatography (IMAC). However, the technique is not commonly used on an industrial scale. The advantages of IMAC are its low cost, relatively high protein loading, simplicity of regeneration of the matrix and that it only requires mild elution conditions. To be used on an industrial scale the system is required to have limited or no metal leakage, be highly specific to the protein of interest and be able to be reused multiple times; in essence lowering cost. The focus of this thesis is on the synthesis of existing and novel bis(tacn) ligands and sustainable methods for producing these ligands based on the 12 principles of Green Chemistry. It was found that the existing methods could be made approximately four times more efficient by moving away from the traditional route for the synthesis of tacn. These ligands were characterised by many methods. The metal complexes of the ligands were characterised by X-ray crystallography which gave insight into how the metal ions may bind to the tagged protein. The ligands were immobilised onto Sepharose FF and bound to copper(II), nickel(II) and zinc(II). These adsorbents were then tested using high throughput screening, on a Tecan Evo 200 robotic platform, to test for binding using green fluorescent protein (GFP) as the model protein. The ability to test immobilised ligands using the robot allowed for high accuracy of results and minimal waste, along with being able to screen a library of ligands with different metals. The results obtained show an increase in capacity against commercially available ligands and stronger binding of the tagged protein. The adsorbents showed no metal leakage once bound, and could be reused multiple times (> 10) without the need to reload the metal and minimal loss of efficiency. The immobilised ligand metal complexes also showed a high selectivity for the tagged protein. The results shown in this thesis describe more sustainable methods for both synthesising the ligands and purifying proteins, with the purification process being easily scalable