Jasulaityte, Greta; (2021) Characterisation of ion exchange chromatography resins for therapeutic protein manufacture. Doctoral thesis (Eng.D), UCL (University College London).

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Abstract

This thesis, completed in collaboration with Purolite Life Sciences, aims to optimise agarose-based ion exchange chromatography resin design to address increased complexity of current feed materials: high titres and associated impurities. Resins with different average particle sizes: 45, 65 and 90 µm, and ligand density: 0.05-0.28 mol/L, were designed and manufactured using a traditional stirred tank and a novel jetting process. Jetting method employing a porous membrane aimed to achieve a narrower particle size distribution than the alternative. Resin designs were evaluated according to the key performance indicators: dynamic binding capacity (DBC), adsorption rate, lifetime, and resolution. DBC was performed at different flow rates with BSA feed, adsorption kinetics were observed with the confocal laser scanning microscope, whilst a fluorescent-dye assay was developed to measure aggregate accumulation in resins. Resolution studies were accomplished with ovalbumin, and monoclonal antibodies, IgG1 and IgG4. Resins of 45 µm size and <0.10 mol/L ligand density, irrespective of resin manufacturing method, achieved the highest DBC at 125 cm/h flow rate, 50% faster protein adsorption rate, increased lifetime, and highest recovery in IgG1 and IgG4 separations. Matrix’s performance was found to increase and then plateau with increasing ligand density. Whilst strong multipoint protein-ligand interactions caused by higher ligand density availability provided an increased retention time and improved ovalbumin isoform separation, steric hindrance effects were also a reason for poor protein recovery and reduced resins’ lifetime. This was of particular importance for the unstable molecule IgG4: ligand density below 0.20 mol/L generated up to 20% higher yield at low buffer pH, whilst additional ligand resulted in peak splitting, oligomerisation and fouling. In contrast, IgG1 being more stable than IgG4 was not affected by these conditions. This work demonstrates that selecting an appropriate combination of particle size and ligand density is crucial for optimal resin performance and throughput, and can be customised to suit unstable biopharmaceuticals’ needs.

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