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Characterisation of the Interaction Between Recombinant Staphylococcal Protein-As and Human Heavy Chain Variable Domains

Shin, Ji Ho; (2022) Characterisation of the Interaction Between Recombinant Staphylococcal Protein-As and Human Heavy Chain Variable Domains. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

The capturing of antibodies and their variant formats by Staphylococcal protein A (SpA; Protein-A) has been reliant on the naturally found strong nanomolar affinity between the fragment crystallisable (Fc) fraction of antibodies from a range of species and SpA. This affinity is core to the foundation of Protein-A affinity chromatography. This single-step purification process can result in over 90% purity of the monoclonal antibody – making it one of the most important process steps in the overall downstream processing of large-scale antibody manufacturing. It is the lack of the Fc portion (being ‘Fc-less’) which often results in certain antibody formats failing to be captured by Protein-A. This thesis presents a structural and mutational variant-level characterisation of the interaction between the fragment antigen-binding (Fab) portion of human antibodies, more specifically their heavy chain variable domains (VHs), and SpA. It is theorised and anticipated that this interaction has the potential to serve as an additional interaction enabling the capture of ‘Fc-less’ antibody therapeutics through Protein-A affinity chromatography. The project first investigated and defined the tolerance of amino acids at the VH-positions at the interaction-site, and then the reason as to why the VHs vary in their affinity towards SpA. It was found that there are non-interface VH-residues and hence positions not facing the interaction-site that affect the VH’s Protein-A binding strength – being the main reason discovered as to why a variation in the interaction is observed. As human VHs and highly homologous camelid VHHs (both consisting of SpA binders) are increasingly used and considered as building blocks for future therapeutics, it is hoped and expected that such findings will help enable the biopharma industry to continue to utilise the highly efficient and well-established Protein-A affinity chromatography to produce safe and cost-effective but novel therapeutic antibodies in various formats for patients in need.

Type: Thesis (Doctoral)
Qualification: Ph.D
Title: Characterisation of the Interaction Between Recombinant Staphylococcal Protein-As and Human Heavy Chain Variable Domains
Language: English
Additional information: Copyright © The Author 2021. Original content in this thesis is licensed under the terms of the Creative Commons Attribution-NonCommercial 4.0 International (CC BY-NC 4.0) Licence (https://creativecommons.org/licenses/by-nc/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.
UCL classification: 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
UCL > Provost and Vice Provost Offices > UCL BEAMS
UCL
URI: https://discovery.ucl.ac.uk/id/eprint/10152084
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