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Yields and product comparison between Escherichia coli BL21 and W3110 in industrially relevant conditions: anti-c-Met scFv as a case study

INTRODUCTION: In the biopharmaceutical industry, Escherichia coli is one of the preferred expression hosts for large-scale production of therapeutic proteins. Although increasing the product yield is important, product quality is a major factor in this industry because greatest productivity does not...

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Autores principales: Arauzo-Aguilera, Klaudia, Buscajoni, Luisa, Koch, Karin, Thompson, Gary, Robinson, Colin, Berkemeyer, Matthias
Formato: Online Artículo Texto
Lenguaje:English
Publicado: BioMed Central 2023
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10197847/
https://www.ncbi.nlm.nih.gov/pubmed/37208750
http://dx.doi.org/10.1186/s12934-023-02111-4
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author Arauzo-Aguilera, Klaudia
Buscajoni, Luisa
Koch, Karin
Thompson, Gary
Robinson, Colin
Berkemeyer, Matthias
author_facet Arauzo-Aguilera, Klaudia
Buscajoni, Luisa
Koch, Karin
Thompson, Gary
Robinson, Colin
Berkemeyer, Matthias
author_sort Arauzo-Aguilera, Klaudia
collection PubMed
description INTRODUCTION: In the biopharmaceutical industry, Escherichia coli is one of the preferred expression hosts for large-scale production of therapeutic proteins. Although increasing the product yield is important, product quality is a major factor in this industry because greatest productivity does not always correspond with the highest quality of the produced protein. While some post-translational modifications, such as disulphide bonds, are required to achieve the biologically active conformation, others may have a negative impact on the product’s activity, effectiveness, and/or safety. Therefore, they are classified as product associated impurities, and they represent a crucial quality parameter for regulatory authorities. RESULTS: In this study, fermentation conditions of two widely employed industrial E. coli strains, BL21 and W3110 are compared for recombinant protein production of a single-chain variable fragment (scFv) in an industrial setting. We found that the BL21 strain produces more soluble scFv than the W3110 strain, even though W3110 produces more recombinant protein in total. A quality assessment on the scFv recovered from the supernatant was then performed. Unexpectedly, even when our scFv is correctly disulphide bonded and cleaved from its signal peptide in both strains, the protein shows charge heterogeneity with up to seven distinguishable variants on cation exchange chromatography. Biophysical characterization confirmed the presence of altered conformations of the two main charged variants. CONCLUSIONS: The findings indicated that BL21 is more productive for this specific scFv than W3110. When assessing product quality, a distinctive profile of the protein was found which was independent of the E. coli strain. This suggests that alterations are present in the recovered product although the exact nature of them could not be determined. This similarity between the two strains’ generated products also serves as a sign of their interchangeability. This study encourages the development of innovative, fast, and inexpensive techniques for the detection of heterogeneity while also provoking a debate about whether intact mass spectrometry-based analysis of the protein of interest is sufficient to detect heterogeneity in a product. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1186/s12934-023-02111-4.
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spelling pubmed-101978472023-05-20 Yields and product comparison between Escherichia coli BL21 and W3110 in industrially relevant conditions: anti-c-Met scFv as a case study Arauzo-Aguilera, Klaudia Buscajoni, Luisa Koch, Karin Thompson, Gary Robinson, Colin Berkemeyer, Matthias Microb Cell Fact Research INTRODUCTION: In the biopharmaceutical industry, Escherichia coli is one of the preferred expression hosts for large-scale production of therapeutic proteins. Although increasing the product yield is important, product quality is a major factor in this industry because greatest productivity does not always correspond with the highest quality of the produced protein. While some post-translational modifications, such as disulphide bonds, are required to achieve the biologically active conformation, others may have a negative impact on the product’s activity, effectiveness, and/or safety. Therefore, they are classified as product associated impurities, and they represent a crucial quality parameter for regulatory authorities. RESULTS: In this study, fermentation conditions of two widely employed industrial E. coli strains, BL21 and W3110 are compared for recombinant protein production of a single-chain variable fragment (scFv) in an industrial setting. We found that the BL21 strain produces more soluble scFv than the W3110 strain, even though W3110 produces more recombinant protein in total. A quality assessment on the scFv recovered from the supernatant was then performed. Unexpectedly, even when our scFv is correctly disulphide bonded and cleaved from its signal peptide in both strains, the protein shows charge heterogeneity with up to seven distinguishable variants on cation exchange chromatography. Biophysical characterization confirmed the presence of altered conformations of the two main charged variants. CONCLUSIONS: The findings indicated that BL21 is more productive for this specific scFv than W3110. When assessing product quality, a distinctive profile of the protein was found which was independent of the E. coli strain. This suggests that alterations are present in the recovered product although the exact nature of them could not be determined. This similarity between the two strains’ generated products also serves as a sign of their interchangeability. This study encourages the development of innovative, fast, and inexpensive techniques for the detection of heterogeneity while also provoking a debate about whether intact mass spectrometry-based analysis of the protein of interest is sufficient to detect heterogeneity in a product. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1186/s12934-023-02111-4. BioMed Central 2023-05-19 /pmc/articles/PMC10197847/ /pubmed/37208750 http://dx.doi.org/10.1186/s12934-023-02111-4 Text en © The Author(s) 2023 https://creativecommons.org/licenses/by/4.0/Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/ (https://creativecommons.org/licenses/by/4.0/) . The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/ (https://creativecommons.org/publicdomain/zero/1.0/) ) applies to the data made available in this article, unless otherwise stated in a credit line to the data.
spellingShingle Research
Arauzo-Aguilera, Klaudia
Buscajoni, Luisa
Koch, Karin
Thompson, Gary
Robinson, Colin
Berkemeyer, Matthias
Yields and product comparison between Escherichia coli BL21 and W3110 in industrially relevant conditions: anti-c-Met scFv as a case study
title Yields and product comparison between Escherichia coli BL21 and W3110 in industrially relevant conditions: anti-c-Met scFv as a case study
title_full Yields and product comparison between Escherichia coli BL21 and W3110 in industrially relevant conditions: anti-c-Met scFv as a case study
title_fullStr Yields and product comparison between Escherichia coli BL21 and W3110 in industrially relevant conditions: anti-c-Met scFv as a case study
title_full_unstemmed Yields and product comparison between Escherichia coli BL21 and W3110 in industrially relevant conditions: anti-c-Met scFv as a case study
title_short Yields and product comparison between Escherichia coli BL21 and W3110 in industrially relevant conditions: anti-c-Met scFv as a case study
title_sort yields and product comparison between escherichia coli bl21 and w3110 in industrially relevant conditions: anti-c-met scfv as a case study
topic Research
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10197847/
https://www.ncbi.nlm.nih.gov/pubmed/37208750
http://dx.doi.org/10.1186/s12934-023-02111-4
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