Analysis of the precipitation and aggregation of engineered proteins.
Doctoral thesis, UCL (University College London).
The study of aggregation in proteins in this work demonstrates the need to understand its mechanism and moderate it as it is a bottleneck in bioprocessing and formulation where it reduces product yield. It explores the analytical techniques used to detect and analyze aggregation, puts forward thermodynamic parameters that can help predict precipitation in small scale bioprocess and determines aggregation prone regions and regions that may increase stability via site-directed mutagenesis. Thermodynamic parameters such as delta G and m values for precipitation were determined from global fit data of hen egg white lysozyme and alcohol dehydrogenase. The free energy was used to predict when a protein is likely to precipitate and the correlating m values were used to measure when a protein is likely to precipitate according to the dependence on ammonium sulphate. In addition, the curve fitting would allow the packing and nucleation of precipitated molecules to be determined which can be used to further correlate with aggregation mechanisms. The impact of sodium citrate and polyethylene glycol precipitation on fragment antibody binding was also examined. This identified a complex protein-polymer interaction between polyethylene glycol and fragment antibody binding whereby the interaction blocked sodium citrate fragmentation but promoted fragment antibody binding oligomerisation. The proportion of species of fragment antibody binding was further confirmed with analytical ultracentrifugation and small angle x-ray scattering analysis where results indicated that dimer was mostly present, which may act as the reactive species leading to aggregation. Site-directed mutagenesis of fragment antibody binding was also carried out, where an increase in hydrophobicity correlated with increased aggregation rates. The leucine to lysine mutant differed as this mutation was at the interface of the heavy and light chain, leading to fragmentation. The serine to lysine mutant on the other hand was the most stable, but did begin to aggregate after one month due to peptide hydrolysis and non-specific interactions. Further work with nuclear magnetic resonance indicated the pseudo wild type fragment antibody binding was correctly folded but had some dynamic conformational rearrangement indicative of some unfolding. This work highlights the need to understand the aggregation mechanisms under different conditions in order to moderate it in bioprocess. It also discusses the different models of aggregation where these are applied to the case study and whether aggregation can be engineered out of proteins.
|Title:||Analysis of the precipitation and aggregation of engineered proteins|
|Additional information:||Permission for digitisation not received|
|UCL classification:||UCL > School of BEAMS > Faculty of Engineering Science > Biochemical Engineering|
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