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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 (2021):
A Generic Procedure for the Isolation of pH- and Magnesium-Responsive Chicken scFvs for Downstream Purification of Human Antibodies. (Publisher's Version)
In: Frontiers in Bioengineering and Biotechnology, 8, Frontiers, e-ISSN 2296-4185,
DOI: 10.26083/tuprints-00019238,
[Article]

Abstract

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 MgCl2 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.

Item Type: Article
Erschienen: 2021
Creators: Hinz, Steffen C. ; Elter, Adrian ; Rammo, Oliver ; Schwämmle, Achim ; Ali, Ataurehman ; Zielonka, Stefan ; Herget, Thomas ; Kolmar, Harald
Origin: Secondary publication via sponsored Golden Open Access
Status: Publisher's Version
Title: A Generic Procedure for the Isolation of pH- and Magnesium-Responsive Chicken scFvs for Downstream Purification of Human Antibodies
Language: English
Abstract:

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 MgCl2 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.

Journal or Publication Title: Frontiers in Bioengineering and Biotechnology
Journal volume: 8
Publisher: Frontiers
Collation: 13 Seiten
Divisions: 07 Department of Chemistry
07 Department of Chemistry > Fachgebiet Biochemie
Date Deposited: 04 Aug 2021 08:01
DOI: 10.26083/tuprints-00019238
Official URL: https://tuprints.ulb.tu-darmstadt.de/19238
URN: urn:nbn:de:tuda-tuprints-192389
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