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A Generic Procedure for the Isolation of pH- and Magnesium-Responsive Chicken scFvs for Downstream Purification of Human Antibodies

Affinity chromatography provides an excellent platform for protein purification, which is a key step in the large scale downstream processing of therapeutic monoclonal antibodies (Mabs). Protein A chromatography constitutes the gold standard for Mab purification. However, the required acidic conditi...

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Autores principales: Hinz, Steffen C., Elter, Adrian, Rammo, Oliver, Schwämmle, Achim, Ali, Ataurehman, Zielonka, Stefan, Herget, Thomas, Kolmar, Harald
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Frontiers Media S.A. 2020
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7324474/
https://www.ncbi.nlm.nih.gov/pubmed/32656201
http://dx.doi.org/10.3389/fbioe.2020.00688
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author Hinz, Steffen C.
Elter, Adrian
Rammo, Oliver
Schwämmle, Achim
Ali, Ataurehman
Zielonka, Stefan
Herget, Thomas
Kolmar, Harald
author_facet Hinz, Steffen C.
Elter, Adrian
Rammo, Oliver
Schwämmle, Achim
Ali, Ataurehman
Zielonka, Stefan
Herget, Thomas
Kolmar, Harald
author_sort Hinz, Steffen C.
collection PubMed
description Affinity chromatography provides an excellent platform for protein purification, which is a key step in the large scale downstream processing of therapeutic monoclonal antibodies (Mabs). Protein A chromatography constitutes the gold standard for Mab purification. However, the required acidic conditions (2.8–3.5) for elution from the affinity matrix limit their applicability, particularly for next generation antibodies and antibody fusion proteins, since denaturation and irreversible aggregation can occur due to the acidic buffer conditions. Here we describe a generic procedure for the generation of antigen-specific chromatography ligands with tailor-made elution conditions. To this end, we generated a scFv-library based on mRNA from a chicken immunized with human Fc. The antibody repertoire was displayed on yeast Saccharomyces cerevisiae screened via FACS toward pH- and magnesium-responsive scFvs which specifically recognize human IgG antibodies. Isolated scFvs were reformatted, produced in Escherichia coli and immobilized on NHS-agarose columns. Several scFvs were identified that mediated antibody binding at neutral pH and antibody recovery at pH values of 4.5 and higher or even at neutral pH upon MgCl(2) exposure. The iterative screening methodology established here is generally amenable to the straightforward isolation of stimulus-responsive antibodies that may become valuable tools for a variety of applications.
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spelling pubmed-73244742020-07-10 A Generic Procedure for the Isolation of pH- and Magnesium-Responsive Chicken scFvs for Downstream Purification of Human Antibodies Hinz, Steffen C. Elter, Adrian Rammo, Oliver Schwämmle, Achim Ali, Ataurehman Zielonka, Stefan Herget, Thomas Kolmar, Harald Front Bioeng Biotechnol Bioengineering and Biotechnology Affinity chromatography provides an excellent platform for protein purification, which is a key step in the large scale downstream processing of therapeutic monoclonal antibodies (Mabs). Protein A chromatography constitutes the gold standard for Mab purification. However, the required acidic conditions (2.8–3.5) for elution from the affinity matrix limit their applicability, particularly for next generation antibodies and antibody fusion proteins, since denaturation and irreversible aggregation can occur due to the acidic buffer conditions. Here we describe a generic procedure for the generation of antigen-specific chromatography ligands with tailor-made elution conditions. To this end, we generated a scFv-library based on mRNA from a chicken immunized with human Fc. The antibody repertoire was displayed on yeast Saccharomyces cerevisiae screened via FACS toward pH- and magnesium-responsive scFvs which specifically recognize human IgG antibodies. Isolated scFvs were reformatted, produced in Escherichia coli and immobilized on NHS-agarose columns. Several scFvs were identified that mediated antibody binding at neutral pH and antibody recovery at pH values of 4.5 and higher or even at neutral pH upon MgCl(2) exposure. The iterative screening methodology established here is generally amenable to the straightforward isolation of stimulus-responsive antibodies that may become valuable tools for a variety of applications. Frontiers Media S.A. 2020-06-23 /pmc/articles/PMC7324474/ /pubmed/32656201 http://dx.doi.org/10.3389/fbioe.2020.00688 Text en Copyright © 2020 Hinz, Elter, Rammo, Schwämmle, Ali, Zielonka, Herget and Kolmar. http://creativecommons.org/licenses/by/4.0/ This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
spellingShingle Bioengineering and Biotechnology
Hinz, Steffen C.
Elter, Adrian
Rammo, Oliver
Schwämmle, Achim
Ali, Ataurehman
Zielonka, Stefan
Herget, Thomas
Kolmar, Harald
A Generic Procedure for the Isolation of pH- and Magnesium-Responsive Chicken scFvs for Downstream Purification of Human Antibodies
title A Generic Procedure for the Isolation of pH- and Magnesium-Responsive Chicken scFvs for Downstream Purification of Human Antibodies
title_full A Generic Procedure for the Isolation of pH- and Magnesium-Responsive Chicken scFvs for Downstream Purification of Human Antibodies
title_fullStr A Generic Procedure for the Isolation of pH- and Magnesium-Responsive Chicken scFvs for Downstream Purification of Human Antibodies
title_full_unstemmed A Generic Procedure for the Isolation of pH- and Magnesium-Responsive Chicken scFvs for Downstream Purification of Human Antibodies
title_short A Generic Procedure for the Isolation of pH- and Magnesium-Responsive Chicken scFvs for Downstream Purification of Human Antibodies
title_sort generic procedure for the isolation of ph- and magnesium-responsive chicken scfvs for downstream purification of human antibodies
topic Bioengineering and Biotechnology
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7324474/
https://www.ncbi.nlm.nih.gov/pubmed/32656201
http://dx.doi.org/10.3389/fbioe.2020.00688
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