Cargando…

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

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 hamst...

Descripción completa

Detalles Bibliográficos
Autores principales: Oshinbolu, Sheun, Shah, Rachana, Finka, Gary, Molloy, Mike, Uden, Mark, Bracewell, Daniel G
Formato: Online Artículo Texto
Lenguaje:English
Publicado: John Wiley & Sons, Ltd 2018
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5838538/
https://www.ncbi.nlm.nih.gov/pubmed/29540956
http://dx.doi.org/10.1002/jctb.5519
_version_ 1783304280255496192
author Oshinbolu, Sheun
Shah, Rachana
Finka, Gary
Molloy, Mike
Uden, Mark
Bracewell, Daniel G
author_facet Oshinbolu, Sheun
Shah, Rachana
Finka, Gary
Molloy, Mike
Uden, Mark
Bracewell, Daniel G
author_sort Oshinbolu, Sheun
collection PubMed
description 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. © 2017 The Authors. Journal of Chemical Technology & Biotechnology published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.
format Online
Article
Text
id pubmed-5838538
institution National Center for Biotechnology Information
language English
publishDate 2018
publisher John Wiley & Sons, Ltd
record_format MEDLINE/PubMed
spelling pubmed-58385382018-03-12 Evaluation of fluorescent dyes to measure protein aggregation within mammalian cell culture supernatants Oshinbolu, Sheun Shah, Rachana Finka, Gary Molloy, Mike Uden, Mark Bracewell, Daniel G J Chem Technol Biotechnol Research Articles 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. © 2017 The Authors. Journal of Chemical Technology & Biotechnology published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry. John Wiley & Sons, Ltd 2018-01-05 2018-03 /pmc/articles/PMC5838538/ /pubmed/29540956 http://dx.doi.org/10.1002/jctb.5519 Text en © 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 (http://creativecommons.org/licenses/by/4.0/) License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.
spellingShingle Research Articles
Oshinbolu, Sheun
Shah, Rachana
Finka, Gary
Molloy, Mike
Uden, Mark
Bracewell, Daniel G
Evaluation of fluorescent dyes to measure protein aggregation within mammalian cell culture supernatants
title Evaluation of fluorescent dyes to measure protein aggregation within mammalian cell culture supernatants
title_full Evaluation of fluorescent dyes to measure protein aggregation within mammalian cell culture supernatants
title_fullStr Evaluation of fluorescent dyes to measure protein aggregation within mammalian cell culture supernatants
title_full_unstemmed Evaluation of fluorescent dyes to measure protein aggregation within mammalian cell culture supernatants
title_short Evaluation of fluorescent dyes to measure protein aggregation within mammalian cell culture supernatants
title_sort evaluation of fluorescent dyes to measure protein aggregation within mammalian cell culture supernatants
topic Research Articles
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5838538/
https://www.ncbi.nlm.nih.gov/pubmed/29540956
http://dx.doi.org/10.1002/jctb.5519
work_keys_str_mv AT oshinbolusheun evaluationoffluorescentdyestomeasureproteinaggregationwithinmammaliancellculturesupernatants
AT shahrachana evaluationoffluorescentdyestomeasureproteinaggregationwithinmammaliancellculturesupernatants
AT finkagary evaluationoffluorescentdyestomeasureproteinaggregationwithinmammaliancellculturesupernatants
AT molloymike evaluationoffluorescentdyestomeasureproteinaggregationwithinmammaliancellculturesupernatants
AT udenmark evaluationoffluorescentdyestomeasureproteinaggregationwithinmammaliancellculturesupernatants
AT bracewelldanielg evaluationoffluorescentdyestomeasureproteinaggregationwithinmammaliancellculturesupernatants