The effects of flow on therapeutic protein aggregation

• Main Author: Willis, Leon Fitzroy
• Other Authors: Brockwell, David J. ; Kapur, Nikil ; Ashcroft, Alison E.
• Published: University of Leeds 2018
• Subjects: 570
• Online Access: https://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.758310

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.