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The effects of flow on therapeutic protein aggregation

Willis, Leon Fitzroy (2018) The effects of flow on therapeutic protein aggregation. PhD thesis, University of Leeds.

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Abstract

To date, over 70 monoclonal antibody (mAb) biopharmaceuticals have been approved, allowing effective treatment of serious diseases such as cancer. In addition to improving human health, these powerful medicines are very valuable, generating billions of dollars in sales annually. Like all proteins, environmental changes can cause mAbs to unfold, misfold and aggregate. Aggregation can block the progress of mAbs to market, as aggregates have been linked to adverse effects in patients. The hydrodynamic forces mAbs encounter during their manufacturing process have long been thought to be one of the causes of aggregation. This link remains tenuous, however, partly due to a lack of knowledge surrounding how specific flow fields (e.g. shear and extensional flows) perturb protein structure. To assess the effects of flow on therapeutic protein aggregation, a recently developed, bespoke Extensional Flow Device (EFD) was characterised, which mimics the hydrodynamic forces mAbs encounter at manufacturing scale. In this thesis, the model proteins BSA and three mAbs (WFL, mAb1 and STT) were subjected to the defined fluid fields present in the EFD, with the resulting aggregates characterised using an array of biophysical techniques. The data show that protein aggregation can be induced by extensional flow. The extent of aggregation depends on a protein’s sequence and topology, in addition to the flow conditions and buffer composition. For example, the mAbs WFL and STT show disparate aggregation behaviour following hydrodynamic stress, despite having >99 % sequence identity, with the generic mAb1 somewhere in between the two. Reinforced by data from a screen of 33 clinically relevant mAbs, the data in this thesis support future use of the EFD to: explore flow-induced protein aggregation mechanisms; improve mAb bioprocessing and; screen mAb candidates to select sequences and/or formulations which are resistant to potentially deleterious hydrodynamic forces, facilitating the development of next-generation mAb therapeutics.

Item Type: Thesis (PhD)
Additional Information: My PhD studentship was funded by the EPSRC Centre for Doctoral Training in Innovative Manufacturing in Emergent Macromolecular Therapies. Materials and reagents were supplied from my industrial collaborators, who are gratefully acknowledged throughout the thesis.
Keywords: Bioprocessing, Aggregation, Biotechnology, Biophysics, Monoclonal Antibodies, Shear flow, Extensional flow.
Academic Units: The University of Leeds > Faculty of Biological Sciences (Leeds)
Identification Number/EthosID: uk.bl.ethos.758310
Depositing User: Mr Leon Fitzroy Willis
Date Deposited: 13 Nov 2018 15:55
Last Modified: 18 Feb 2020 12:49
URI: http://etheses.whiterose.ac.uk/id/eprint/21963

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