Defining the mechanism behind the self-association of therapeutic monoclonal antibodies using mass spectrometric techniques

Protein aggregation is responsible for a vast array of life-threatening protein based diseases as well as being an economic hurdle in biopharmaceutical development and manufacturing. Monoclonal antibodies represent the fastest growing class of biotherapeutics, with 53 antibodies in late phase clinical trials as of late 2015. Antibodies serve as ideal therapeutics due to their exquisite specificity and favourable safety profile. However, further therapeutic antibody development is hamstringed by uncontrolled self-association and aggregation which can occur at all stages of biotherapeutic development. Therefore, there is an urgent need for methods to dissect the mechanisms that drive uncontrolled self-association and protein aggregation. This thesis presents techniques which were applied to address the identification of aggregated material of a therapeutically relevant monoclonal antibody, and to characterise the mechanism responsible for driving oligomerisation. A combination of mass spectrometric techniques were employed to visualise the oligomeric species. Ion mobility spectrometry coupled to nanoelectrospray ionisation mass spectrometry was utilised to identify the oligomeric species formed under native conditions and to define the oligomers in terms of their mass and collision cross-sectional area. To characterise the regions responsible for driving oligomer formation, chemical cross-linking was employed to capture the oligomeric species in solution which were then analysed using tandem mass spectrometry. The initial dimer interaction was modelled using distance restraints obtained from the chemical cross-linking results and a model proposed that explains the oligomerisation events, and how runaway polymerisation can occur at higher concentrations. Finally, a powerful in vivo assay in the E. coli periplasm was developed to differentiate between aggregation and non-aggregation-prone sequences using single chain variable fragments (scFv) of the antibodies studied. The results presented demonstrate the applicability of the assay to molecules relevant to the biopharmaceutical sector; as an upstream platform for the identification of aggregation-prone sequences, prior to antibody production and development. Overall, the work presented within this thesis describes techniques that can be successfully applied to define the mechanism that underpins the self-association of a therapeutically-relevant monoclonal antibody. Furthermore, the study presents a novel in vivo assay that can be used to identify aggregation-prone sequences, and to develop them further by mutagenesis, which could be useful in protein development in the biopharmaceutical sector.