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Evaluation of fluorescent dyes to measure protein aggregation within mammalian cell culture supernatants

Oshinbolu, S; Shah, R; Finka, G; Molloy, M; Uden, M; Bracewell, DG; (2018) Evaluation of fluorescent dyes to measure protein aggregation within mammalian cell culture supernatants. Journal of Chemical Technology and Biotechnology , 93 (3) pp. 909-917. 10.1002/jctb.5519. Green open access

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Abstract

BACKGROUND: A current challenge in bioprocessing is the ability to analyse critical quality attributes such as aggregation without prior purification. This study evaluated the use of fluorescent dyes (Bis-ANS, SYPRO Orange, Thioflavin T and ProteoStat) to characterise mAb aggregates in Chinese hamster ovary clarified cultures. RESULTS: The null and mAb culture supernatants showed an increase in fluorescence intensity over the duration of the culture. The null cultures on day 14 saw a rapid increase in fluorescence intensity; day 10 to day 14, Bis-ANS and Thioflavin T had average increases of 21% and 48%, respectively, whereas ProteoStat and SYPRO Orange showed an average increase of 60%. Higher fluorescence intensity on day 14 with the null cultures, also correlated with loss of viability. CONCLUSION: Fluorescent dyes are not a specific indicator of mAb aggregation, but rather an indicator of overall protein aggregation or high molecular weight species. SYPRO Orange was more sensitive at detecting very large molecular weight species and ProteoStat seemed better suited to smaller aggregates. Although the assay cannot be used to measure mAb aggregates in cell culture, it could be used to aid cell line selection in maximising viabilities and minimising the amount of aggregates.

Type: Article
Title: Evaluation of fluorescent dyes to measure protein aggregation within mammalian cell culture supernatants
Open access status: An open access version is available from UCL Discovery
DOI: 10.1002/jctb.5519
Publisher version: http://onlinelibrary.wiley.com/wol1/doi/10.1002/jc...
Language: English
Additional information: Copyright © 2017 The Authors. Journal of Chemical Technology & Biotechnology published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry. This is an open access article under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits use, distribution and reproduction in any medium, provided the original work is properly cited.
Keywords: monoclonal antibodies; CHO cells; aggregation; fluorescence; extrinsic dyes
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/10044048
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