Unravelling the complexities of protein conformational stability using Raman spectroscopy and two-dimensional correlation analysis

Ettah, Ilokugbe and Ashton, Lorna and Dalby, Paul and Middleton, David (2020) Unravelling the complexities of protein conformational stability using Raman spectroscopy and two-dimensional correlation analysis. PhD thesis, Lancaster University.

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Protein drug development is a risky, lengthy and expensive process. Accordingly, there are significant time and cost implications for stakeholders regardless of the stage at which a prospective candidate is discontinued. Mitigating this risk depends critically on making informed decisions as early as possible during the process. Some of the most indispensable decision-making aids are analytical techniques used in the characterisation of protein stability. Protein stability is a multifaceted and crucial determinant for progressing a candidate through development to commercialisation. The inherent complexity of proteins, the evolving pipeline of engineered protein constructs, as well as limitations of current techniques place a demand for the expansion and development of more analytical probes for protein stability. This thesis focuses on the development of Raman spectroscopy and 2D-correlation analysis (2DCA) for monitoring conformational stability during early protein drug development. Although Raman spectroscopy has been used for many decades to study protein conformational changes in proteins; it is highly underutilized during protein drug development, owing partly to the complexity of Raman protein spectra. 2DCA is a mathematical technique that can significantly improve the visualization of and clarify the interpretation of complicated spectral features arising from an applied perturbation. The specificity of 2DCA in extracting such spectral variations directly linked to a perturbation is apt for protein stability testing, where the identification of a protein’s response to stress is pivotal. In this work, we demonstrate this combined technique as a sensitive means of distinguishing the conformational stability profiles of proteins under an applied perturbation on the basis of post-translation modification, metal-ligand and surface charge mutation. In all cases, we show that the technique is capable of informing the choice of the most stable candidate and or conditions, even where subtle differences exist.

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Thesis (PhD)
?? raman spectroscopyprotein conformationprotein drug developmentprotein stability2d-correlation analysisbiopharmaceuticalsantibody fragmenttransferrinalpha casein ??
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26 Nov 2020 12:45
Last Modified:
22 Dec 2023 00:04