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Neutron scattering to characterise protein interactions with solid-liquid interfaces in bioprocessing

Papachristodoulou, Maria; (2020) Neutron scattering to characterise protein interactions with solid-liquid interfaces in bioprocessing. Doctoral thesis (Ph.D), UCL (University College London). Green open access

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Abstract

A deeper understanding of the nanoscale and mesoscale structure of chromatographic adsorbents and the distribution of proteins within the media, is critical to a mechanistic understanding of separation processes using these materials. Characterisation of the media’s architecture at this scale and protein adsorption within, is challenging using conventional techniques. In this study, a novel resin characterisation technique that enables in-situ measurement of the structure of the adsorbed protein layer within the resin, under typical chromatographic conditions was examined. A quartz flow-through cell was designed and fabricated for use with Small-Angle Neutron Scattering, in order to measure the structures of a silica based protein A chromatography resin during the monoclonal antibody sorption process. We were able to examine the pore-to-pore (˜133nm) and pore-size (˜63 nm) correlations of the resin and the in-plane adsorbed antibody molecules (˜4.2nm) correlation at different protein loadings and washing buffers, in real time using a contrast matching approach. The effect of different washing buffers was also investigated. When 0.03M sodium phosphate with 1M urea and 10% isopropanol buffer, pH8, was introduced, it disrupted the system’s order by causing partial unfolding of the adsorbed antibody, as evidenced by a loss of the in-plane protein correlation. Neutron Reflectivity experiments were undertaken on a planar surface to enhance the understanding of how adsorption processes might impact the stability of the antibody molecules. A 56Å antibody size and 13.7% increase in layer thickness when urea was introduced was observed. The effect of temperature and antibody incubation times on the chromatography system were also explored. The methodology developed here offers new ways to investigate the nanoscale structure and ligand immobilisation within chromatography resins. Which allows for a deeper understanding of the in-situ behaviour of adsorbed proteins within the media under different mobile phase conditions within a sample environment replicating that of a chromatography column.

Type: Thesis (Doctoral)
Qualification: Ph.D
Title: Neutron scattering to characterise protein interactions with solid-liquid interfaces in bioprocessing
Event: UCL
Open access status: An open access version is available from UCL Discovery
Language: English
Additional information: Copyright © The Author 2020. Original content in this thesis is licensed under the terms of the Creative Commons Attribution 4.0 International (CC BY 4.0) Licence (https://creativecommons.org/licenses/by/4.0/). Any third-party copyright material present remains the property of its respective owner(s) and is licensed under its existing terms. Access may initially be restricted at the author’s request.
Keywords: antibodies, chromatography, neutron scattering, protein A
UCL classification: UCL
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: https://discovery.ucl.ac.uk/id/eprint/10100939
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