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Column-free optical deconvolution of intrinsic fluorescence for a monoclonal antibody and its product-related impurities

Uçan, Deniz; Hales, John E; Aoudjane, Samir; Todd, Nathan; Dalby, Paul A; (2023) Column-free optical deconvolution of intrinsic fluorescence for a monoclonal antibody and its product-related impurities. Journal of Chromatography A , 1711 , Article 464463. 10.1016/j.chroma.2023.464463. Green open access

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Abstract

The quantification of monoclonal antibody (mAb) aggregates and fragments using high pressure liquid chromatography-size exclusion chromatography (HPLC-SEC) typically requires off-line measurements that are time-consuming and therefore not compatible with real-time monitoring. However, it has been crucial to manufacturing and process development, and remains the industrial standard in the assessment of product-related impurities. Here we demonstrate that our previously established intrinsic time-resolved fluorescence (TRF) approach can be used to quantify the bioprocess critical quality attribute (CQA) of antibody product purity at various stages of a typical downstream process, with the potential to be developed for in-line bioprocess monitoring. This was directly benchmarked against industry-standard HPLC-SEC. Strong linear correlations were observed between outputs from TRF spectroscopy and HPLC-SEC, for the monomer and aggregate-fragment content, with R2 coefficients of 0.99 and 0.69, respectively. At total protein concentrations above 1.41 mg/mL, HPLC-SEC UV-Vis chromatograms displayed signs of detector saturation which reduced the accuracy of protein quantification, thus requiring additional sample dilution steps. By contrast, TRF spectroscopy increased in accuracy at these concentrations due to higher signal-to-noise ratios. Our approach opens the potential for reducing the time and labour required for validating aggregate content in mAb bioprocess stages from the several hours required for HPLC-SEC to a few minutes per sample.

Type: Article
Title: Column-free optical deconvolution of intrinsic fluorescence for a monoclonal antibody and its product-related impurities
Open access status: An open access version is available from UCL Discovery
DOI: 10.1016/j.chroma.2023.464463
Publisher version: https://doi.org/10.1016/j.chroma.2023.464463
Language: English
Additional information: © 2023 The Author(s). Published by Elsevier B.V. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).
Keywords: Analytical instrumentation, Process analytical technology, Antibody, Time-resolved intrinsic fluorescence, Biophysics
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/10179728
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