Use this URL to cite or link to this record in EThOS: https://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.779448
Title: Stability and aggregation-prone conformations of an antibody fragment antigen-binding (Fab)
Author: Codina Castillo, Nuria
ISNI:       0000 0004 7965 1446
Awarding Body: UCL (University College London)
Current Institution: University College London (University of London)
Date of Award: 2019
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Abstract:
Antibody-based products have become the main drug class of approved biopharmaceuticals, with over 60 drugs on the market and many more in clinical development. However, many never reach the market because protein aggregates form during manufacturing and storage, which lower the efficacy of the product and may cause immune responses in patients. To date, very little is known about the structural conformers that initiate aggregation. Stability of the humanized fragment antigen-binding (Fab) A33 was first studied using molecular dynamic (MD) simulations under two stresses, low pH and high temperature. Results revealed different unfolding pathways, with CL domain partially unfolding at low pH, and CL and VH at high temperature. These conformational changes exposed different predicted aggregation-prone regions (APR), to suggest different aggregation mechanisms. Further salt bridge analysis provided insights into the ionizable residues likely to get protonated first. Mutational study with FoldX and Rosetta predicted that the constant domain interface can be stabilized further, backed by packing density calculations. To experimentally characterize the aggregation-prone conformers, solution structures of Fab A33 under different conditions of pH and salt concentration, were solved using small angle X-ray scattering (SAXS). SAXS revealed an expanded conformation at pH 5.5 and below, with an Rg increase of 2.2% to 4.1%, that correlated with accelerated aggregation. Scattering data were fitted using 45,000 structures obtained from the atomistic MD simulations under the same conditions, to locate the conformational change at low pH to the CL domain. The approach was then validated using intra-molecular single-molecule FRET with a dual-labelled Fab as an orthogonal detection method. The conformational changes were found to expose a predicted APR, which forms a mechanistic basis for subsequent aggregation. Overall, these findings provide a means by which aggregation-prone conformers can be determined experimentally, and thus potentially used to guide protein engineering, or ligand binding strategies, with the aim of stabilizing the protein against aggregation.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID: uk.bl.ethos.779448  DOI: Not available
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