Use this URL to cite or link to this record in EThOS: https://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.820084
Title: Biotherapeutic structural investigations of formulation parameters using vibrational spectroscopy
Author: Mcavan, Bethan
ISNI:       0000 0004 9354 1286
Awarding Body: University of Manchester
Current Institution: University of Manchester
Date of Award: 2019
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Abstract:
Monoclonal antibodies (mAbs) are now the most common protein therapeutics on the market. The therapeutic properties of mAbs originate from their inherent ability to specifically bind to a chosen target and the subsequent stimulation of effector functions as result of mAb glycosylation. Therefore mAbs are able to act as drug delivery systems, inhibitors or activators. Protein therapeutic characterisation possesses inherent analytical difficulties as mAbs are complex structures made up of multiple domains. The higher order structure of mAbs is essential for their specificity for targeted treatments. During the production of mAbs, the bioformulation process can induce structural perturbations that we separate into two main categories: Post-translational modifications (PTMs) and degradation. The most common PTMs are oxidation, deamidation and glycation. Degradation of mAbs covers general loss of structure through aggregation and fragmentation. Due to the wide variation and number of possible modifications to mAbs, monitoring, identification and quantification is difficult. Current analytical methods are dependent on sampling, are often destructive, and require time and expertise. Vibrational spectroscopic methods have been highlighted as possible analytical methods to overcome previous shortcomings. In particular, Raman spectroscopy has the potential to be developed into an in-line analytical tool due to being minimally invasive, non-destructive, information rich and rapid. Raman and FTIR are already utilised for in situ monitoring of pharmaceutical manufacturing, such as small molecules, but have not yet been extended into the quality control of mAbs. In this thesis we use a range of conditions to force the degradation and increase the PTM levels of three of the most common types of mAbs: IgG1, Fab and IgG4. We use circular dichroism (CD), peptide mapping, and size exclusion chromatography (SEC) to quantify and study the structural implications of the PTMs and degradation, and compare these to the information gained from Raman spectroscopy. Bioformulation parameters probed include: agitation, pH, temperature, sugar excipients, chemical stressors and UV light. Primarily, we explore the sensitivity of Raman spectroscopy to detect, quantify and provide a structural insight into the modifications. Additionally, we study the aggregation of mAbs to investigate the structural consequences on the protein, as well as identify spectral markers that could be used to monitor aggregation.
Supervisor: Doig, Andrew ; Warwicker, James Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID: uk.bl.ethos.820084  DOI: Not available
Keywords: Protein Characterisation ; Monitoring ; Stability ; FTIR ; Protein Structure ; PAT ; Therapeutic antibodies ; Raman Spectroscopy ; Vibrational Spectroscopy
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