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Optical Deconvolution of Multiple Co-eluting Proteins in Chromatography

Ucan, Deniz; (2023) Optical Deconvolution of Multiple Co-eluting Proteins in Chromatography. Doctoral thesis (Eng.D), UCL (University College London).

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The in-line time-resolved fluorescence spectroscopy-based approach explored in this thesis for protein quantification in monoclonal antibody biomanufacture eliminates the need for a size exclusion chromatography (SEC) column and the application of formulation constraints to downstream chromatography feeds unlike other analytical techniques. This reduces the time, labour and associated costs for critical quality attribute (CQA) validation and offers superior analytical flexibility in the quantifiable protein species when compared to the current industrial standards of high-pressure liquid chromatography – size exclusion chromatography (HPLC-SEC). Linear correlations of 0.999 and 0.69 have been respectively achieved for monomer and aggregate-fragment quantity analysis between TRF spectroscopy and HPLC-SEC for concentrations below 2.8 mg/mL. The proof of concept for in-line host cell protein (HCP) quantification using TRF spectroscopy demonstrates intrinsic fluorescence decay intensity contributions that are reflective of monoclonal antibody (mAb) and the overall HCP content of separate and co-eluting species, as well as the capability for implementation between multiple bioprocess stages. This project has demonstrated that TRF spectroscopy can resolve independent species-quantity describing decay associated chromatogram (DAC) signal intensities for mAb and HCP-rich Protein A flow-through formulations, when run on their own and mixed together. The lower limit of measurement for HCP in a formulation with mAb was found to be equivalent to the HCP content in industrial Protein A flow-through (1E5-1E6 ng/mL). TRF spectroscopy was found to offer a far greater dynamic range, with measurable total protein concentrations over 10-fold of the concentrations that could be tested on industry-standard HPLC-SEC without UV absorbance peak truncation. Quantitation measurements could be performed on samples with total protein concentrations over 10000 times above those that were required to fall within the standard range for enzyme-linked immunosorbent assays (ELISAs). Thus setting the precedent for future strides in evaluating this technology in an industrial setting and expanding testing to include more protein products.

Type: Thesis (Doctoral)
Qualification: Eng.D
Title: Optical Deconvolution of Multiple Co-eluting Proteins in Chromatography
Language: English
Additional information: Copyright © The Author 2023. Original content in this thesis is licensed under the terms of the Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International (CC BY-NC-ND 4.0) Licence (https://creativecommons.org/licenses/by-nc-nd/4.0/). Any third-party copyright material present remains the property of its respective owner(s) and is licensed under its existing terms. Access may initially be restricted at the author’s request.
UCL classification: UCL
UCL > Provost and Vice Provost Offices > UCL BEAMS
UCL > Provost and Vice Provost Offices > UCL BEAMS > Faculty of Engineering Science
UCL > Provost and Vice Provost Offices > UCL BEAMS > Faculty of Engineering Science > Dept of Biochemical Engineering
URI: https://discovery.ucl.ac.uk/id/eprint/10183254
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