This master’s thesis explores the physical optimisation of protein purification conditions and related methods for finding optimal conditions to minimize aggregation. The methodological work is based on a combination of experiments and model analysis of the experiments, with the main experimental methods used being the size-exclusion chromatography and intrinsic tryptophan fluorescence. Several formulations were prepared with different monoclonal antibodies at different pH, ionic strengths and protein concentrations. By measuring the intrinsic fluorescence of tryptophan residues, we determined the influence of individual factors on the conformational stability of proteins in formulations. We observed fundamental differences in the behavior of the monoclonal antibodies, which imply different denaturation and aggregation mechanisms. The presence of aggregates in the formulations after exposure to low pH was determined by size-exclusion chromatography. With the combination of measurements from both methods we determined the aggregation mechanisms of the monoclonal antibodies and the role of conformational stability in the initial steps of aggregation. We found that a shift toward higher emission wavelengths upon exposure to low pH indicates irreversible, whereas a shift toward lower emission wavelengths indicates reversible protein aggregation. This work is a contribution towards the improvement of therapeutic protein purification process, which is necessary for a safe use of biopharmaceuticals.
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