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Elucidating the aggregation mechanisms of antibody fragments through biophysical analysis

Hilton, DW; (2016) Elucidating the aggregation mechanisms of antibody fragments through biophysical analysis. Doctoral thesis , UCL (University College London).

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Abstract

The spontaneous formation of aggregates presents an obstacle in the developability, manufacturability and long term storage of numerous therapeutic proteins. These aggregates are a manifestation of the drug molecule's physical instability, potentially leading to reduced biological activity, differences in solution properties or increased immunogenic potential. Currently our understanding of the mechanisms behind aggregation and the role of a protein’s extrinsic environment upon the molecule's aggregation propensity remains limited. In this study we investigated the aggregation behavior of a previously unstudied commercial biopharmaceutical, an α-TNF Fab' antibody fragment. Initially we mapped the differences in aggregation kinetics and product morphologies accompanying changes to the protein's aqueous environment, finding that the aggregation pathway adopted by the molecule depended upon the pH of the system. We then employed a combination of intrinsic and colloidal stability measurements to probe the causes of the behavioral differences seen. Together, these biophysical properties quantitatively captured the relative differences in aggregation rates observed. Furthermore, we were able to infer the existence of various partially folded intermediates whose populations govern the types and rates of aggregates formed. Finally, we sought to identify whether observable changes in the native state conformation precluded aggregate formation through low resolution structural analysis using small angle X-ray scattering. We showed that a drop in pH generated a conformationally expanded state which appeared to coincide with the titration of a key histidine residue within the protein’s structure. We predict that an equivalent mechanism will lead to aggregation at low pH of other human derived antigen binding fragments.

Type: Thesis (Doctoral)
Title: Elucidating the aggregation mechanisms of antibody fragments through biophysical analysis
Event: UCL (University College London)
Language: English
UCL classification: UCL > Provost and Vice Provost Offices
UCL > Provost and Vice Provost Offices > UCL BEAMS
UCL > Provost and Vice Provost Offices > UCL BEAMS > Faculty of Engineering Science
UCL > Provost and Vice Provost Offices > UCL BEAMS > Faculty of Engineering Science > Dept of Biochemical Engineering
URI: http://discovery.ucl.ac.uk/id/eprint/1492898
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