Detection of flow-induced perturbations of antibody structure using mass spectrometry

Monoclonal antibody biopharmaceuticals have emerged over the last few decades as a powerful class of therapies, with exquisite specificity and safety. However, these protein-based therapies are prone to environmental stresses which can trigger unfolding and aggregation, causing major roadblocks to their manufacture at many stages. Hydrodynamic flow, which describes the flow of fluid, can cause stress to proteinaceous molecules in solution, as they encounter forces which apply stress (including shear and extensional stress) to the proteins as they move with velocity in a particular environment. Hydrodynamic flow-induced can cause aggregation of proteins, and there is an urgent need to characterise the mechanisms involved in unfolding and aggregation to inform the manufacture of therapies more resistant to these issues in the future. This thesis presents a set of three monoclonal antibodies (mAbs) with similar sequences but strikingly different physiochemical properties. Previously, an anti-nerve growth factor mAb, MEDI-1912 (WFL herein), was developed with picomolar affinity for its target but exhibited poor biophysical properties (including low solubility, a long retention time in a high performance size exclusion chromatography (HP-SEC) column, and self-association to form higher order species analysed by analytical ultracentrifugation (AUC)). The region of this mAb responsible for the poor properties was identified, leading to the development of a triple mutant, STT, with improved properties. Aggregation-prone WFL and its less-aggregation-prone counterpart STT were studied, alongside an additional variant 114 identified through a directed evolution screen of WFL. 114 scored more highly than WFL and STT in an assay previously used to rank aggregation-prone species, indicating its reduced aggregation potential. A combination of chromatographic, spectroscopic, and mass spectrometric techniques were employed, with the intention to contribute to an understanding of what may cause the difference in properties of these three therapeutically-relevant highly homologous mAbs. Even through 114’s retention of aggregation hotspot residues W, F and L, its 4 additional mutations rescue the beneficial physiochemical properties seen for STT. The susceptibility of these mAbs to flow-induced aggregation were ranked, and formed the basis of the method built upon in the second and third results chapters. To build a method capable of fingerprinting the stages of unfolding with a covalent labelling approach, various digestion methods and liquid chromatography (LC) methods were compared, iteratively building a robust liquid chromatography-tandem mass spectrometry (LC-MS/MS) methodology for use in the final chapter. Tandem mass spectrometry refers to the use of the mass spectrometer for fragmentation of introduced peptides into individual amino acids, allowing for the sequencing of the introduced sample and detection of any modification to each individual amino acid by piecing together the resulting spectra. Thus, the fast photochemical oxidation of proteins (FPOP) procedure for conformation-sensitive labelling was applied to STT and successfully combined with the optimised LC-MS/MS protocol, for the generation of modified peptides which could be compared with un-modified peptides. Finally, the FPOP-LC-MS/MS analysis of the three mAbs in parallel revealed strikingly different oxidation patterns between the complementarity determining regions (CDRs). The peptide-level analysis of the oxidation patterns of these mAbs revealed that regions in the heavy chain complementarity determining regions (CDRs) showed the most variation in labelling pattern between the three mAbs. Additionally, the mAbs were also subjected to hydrodynamic flow, and this ‘stressed’ sample was compared to the native sample using the optimized FPOP-LC-MS/MS protocol. WFL demonstrated the most protection from oxidation in CDRs, whereas 114 showed slight increases in oxidation for most peptides, with STT remaining the most constantly labelled before and after flow stress. These experiments demonstrate the applicability of using peptide-level FPOP analysis to begin to unpick the mechanisms of flow-induced unfolding. Overall, the data presented here provides a springboard for future exploration of the flow-FPOP-LC-MS/MS methodology. Understanding the flow-induced structural perturbations of proteins will ultimately aid the design of more unfolding-resistant variants, and contribute to the economic production of current and next generation high-value biopharmaceuticals.