Use this URL to cite or link to this record in EThOS: https://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.798535
Title: Analysis of critical quality attributes in monoclonal antibodies for upstream process development
Author: Oshinbolu, Sheun
ISNI:       0000 0004 8507 6820
Awarding Body: UCL (University College London)
Current Institution: University College London (University of London)
Date of Award: 2019
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Abstract:
In process development, there is an increasing demand to screen and select cell lines based on a more detailed understanding of the manufacturability and product quality of the candidate molecules. There is also an increasing need to have this detailed understanding available earlier in the process, in order to make better informed decisions as early as possible. One product quality attribute of interest in industry has been the aggregation of monoclonal antibodies (mAb). Thus, this thesis describes the development of an analytical assay to measure monoclonal antibody aggregates using three approaches. The first approach utilised two types of fluorescent dyes: hydrophobic dyes (Bis-ANS and SYPRO Orange) and molecular rotors (Thioflavin T and ProteoStat). The fluorescent dyes measured aggregates on purified mAb down to 5% mAb aggregates, but they were found not to be specific to mAb aggregates in cell culture medium. A second approach used an affinity peptide (shown to be specific to mAb aggregates) conjugated to molecular rotors (Thioflavin T and CCVJ), bright dyes (Tide Fluor 2 and Fluorescein) and biotin for detection. However, the conjugates did not provide enhanced specificity towards mAb aggregates compared to the dyes on their own. Hence, further investigation would be needed to understand the binding site and mechanism of the affinity peptide. In the third approach, a fluorescence resonance energy transfer (FRET) assay was designed. Experimental evidence showed weak energy transfer was due to aggregated mAbs placing the donor and acceptor outside the distance required to achieve FRET. Energy transfer improved when distances were reduced by using smaller proteins (lectin (38 kDa) and dAb (25 kDa)). The FRET assay was able to quantify 5-30% lectin aggregates (R2 > 0.9) in both purified and CHO media/host cell protein background. Overall, this thesis showed the strengths and weaknesses of using fluorescent dyes in different formats to measure protein aggregates. Although further work is needed for both the FRET and affinity peptide assay, the thesis has explored some of the challenges of using FRET with proteins, donor and acceptor free in solution, which could be applied in designing and understanding other FRET assays. As well as highlighting the next steps required to fully develop and understand the labelled-affinity peptide assay system.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID: uk.bl.ethos.798535  DOI: Not available
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