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Structural modification of proteins to improve their spray drying and solid-state stability
Date
2025
Abstract
The increasing pressure on the biopharmaceutical industry to produce stable and affordable medicines has prompted decades of research into stabilisation of proteins in the solid-state. Spray drying is a well-established, continuous technique for the production of dry formulations of food enzymes and biotherapeutics, yet it inflicts harsh stresses that tend to denature protein products. Excipients, although successful in ameliorating degradation, can pose barriers in spray drying of proteins, due to product interaction, solution viscosity, and lung-related toxicity, when intended for inhalable formulations. The work presented in this thesis aims to conduct comparative studies of structurally modified proteins as an additional, or substitute, route of product stabilisation for processing of large biomolecules by spray drying. A dual approach is taken to explore this research question by examining both primary structure modifications (i.e., amino acid extensions) and polymer conjugation as stabilisation strategies.
Chapter 1 provides an overview of the key concepts of this research area, such as protein structure and mechanisms of denaturation and aggregation, spray drying and its effect on protein stability, as well as current research in using structural modifications to stabilise proteins, particularly proteins used in the biopharmaceutical sector. It also outlines the aimsand research questions of this thesis, with a special focus on filling the gap in the literature concerning the stability of structurally modified proteins at solid state.
In Chapter 2, a unique thermostability tag, or ‘molecular lock’, was identified in an aldehyde dehydrogenase protein originating from the ancient bacterial strain Thermus thermophilus HB27 (ALDHTt). The tag consisted of a 22-amino acid C-terminal extension, corresponding to similar structural features conserved across the Thermus genus, however not found in mesophilic aldehyde dehydrogenases due to genetic drift. By recombinant production and characterization of the ALDHTt protein and a genetically engineered variant, ALDHTt-508, the unique structural and functional role of the C-terminal extension could be discerned. Removal of the C-terminal extension had a positive effect on oxidoreductase activity in solution and allowed for the oxidation of larger, ortho-substituted aldehydes. The C-terminusextension, however, was found to stabilise the oligomeric state of the ALDH homotetramer during downstream purification and during aggregation kinetic studies.
In Chapter 3, a comparative study was performed on the ALDHTt native enzyme and its mutant (ALDHTt-508) to discern the application of the C-terminal extension towards solid-state stabilisation. Removal of the extension promoted the presence of insoluble and soluble aggregation after the spray drying process by destabilising the oligomeric structure and allowing for intermolecular interaction of exposed residues. ALDHTt with the C-terminal protected oligomer retained, on average, 24% more activity than exposed oligomers after spray drying and retained up to 16% more activity during solid state storage. The research conducted in this chapter proposes a mechanism for the protection of oligomeric proteins by the C-terminal extension under the shear, interfacial and thermal stresses involved in spray drying.
Chapter 4 delves into the effect of polymer conjugation on biomolecule solid-state stability by using the model protein lysozyme. Four PEG-protein conjugates of differing molecular weight and chain nature (linear and branched) were successfully produced, purified and experimentally screened for their ability to stabilise lysozyme. The structure and conformation of each conjugate was studied in depth, using CD (Circular Dichroism), Dynamic Light Scattering (DLS) and Differential Scanning Fluorimetry (DSF) in order to form a basis for the mechanisms of protein stabilisation by PEGylation in the solid-state. PEGylation of lysozyme is proven to stabilise against aggregation pathways by provision of steric hinderance and, in some cases, promoting protein refolding. Additionally, the effect of PEGylation extent on Critical Quality Attributes such as moisture content and particle size was explored.
Chapter 5 includes a summary and concluding remarks on the findings of this thesis, with future work and outlook discussed in detail in chapter 6.
Supervisor
Padrela, Luis
Soulimane, Tewfik
Soulimane, Tewfik
Description
Publisher
University of Limerick
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Funding Information
Sustainable Development Goals
External Link
Type
Thesis
Rights
http://creativecommons.org/licenses/by-nc-sa/4.0/