Use this URL to cite or link to this record in EThOS: https://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.771468
Title: Using chemical denaturants to determine aggregation propensity of biopharmaceuticals
Author: Holloway, Luke
ISNI:       0000 0004 7658 425X
Awarding Body: University of Manchester
Current Institution: University of Manchester
Date of Award: 2019
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Abstract:
The overall aim of this PhD project was to use chemical denaturants such as urea and guanidine hydrochloride (Gdn HCl) to increase the relative population of intermediate and/or unfolded states of proteins as known precursors to protein aggregation. Measurements of protein-protein interactions (PPIs) under denaturing conditions of both the model protein lysozyme and a set of five pharmaceutically relevant monoclonal antibodies (mAbs) were made. Light scattering techniques were employed including static light scattering (SLS) to determine the second osmotic viral coefficient, B22 and dynamic light scattering (DLS) to determine the interaction parameter, kD. SLS and DLS was also used to measure aggregate growth rates under denaturant and/or temperature accelerated conditions and were ratified by measuring monomer loss kinetics using size exclusion chromatography couple with multi-angle laser light scattering (SEC-MALLS). The data generated in the first results chapter highlighted the importance of measuring the refractive index increment of the protein under constant solvent chemical potential, to ensure correct values of B22 and molecular weight at infinite protein dilution are obtained as well as excluding short delay times of an autocorrelation decay in solutions of high co-solvent concentrations to similarly calculate correct values of kD and hydrodynamic radius at infinite protein dilution. The data generated in the second and third results chapter showed that PPIs of intermediate, aggregate prone states could be quantified using SLS at high protein concentrations (20 g/L) and were related to the aggregation propensity of the mAbs under both native and denaturing conditions. Furthermore, addition of 0.5 M arginine HCl under denaturing conditions caused a reduction in aggregation rates and concordantly an increase in repulsive PPIs.
Supervisor: Warwicker, James ; Curtis, Robin Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID: uk.bl.ethos.771468  DOI: Not available
Keywords: refractive index increment ; guanidine HCl ; urea ; denaturants ; mAbs ; monoclonal antibodies ; dynamic light scattering ; protein aggregation ; protein-protein interactions ; static light scattering ; interaction parameter ; second osmotic virial coefficient
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