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.
In: Frontiers in Bioengineering and Biotechnology, 2020, 8
doi: 10.26083/tuprints-00019238
Artikel, Zweitveröffentlichung, Verlagsversion
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Kurzbeschreibung (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.
| Typ des Eintrags: | Artikel |
|---|---|
| Erschienen: | 2021 |
| Autor(en): | Hinz, Steffen C. ; Elter, Adrian ; Rammo, Oliver ; Schwämmle, Achim ; Ali, Ataurehman ; Zielonka, Stefan ; Herget, Thomas ; Kolmar, Harald |
| Art des Eintrags: | Zweitveröffentlichung |
| Titel: | A Generic Procedure for the Isolation of pH- and Magnesium-Responsive Chicken scFvs for Downstream Purification of Human Antibodies |
| Sprache: | Englisch |
| Publikationsjahr: | 2021 |
| Publikationsdatum der Erstveröffentlichung: | 2020 |
| Verlag: | Frontiers |
| Titel der Zeitschrift, Zeitung oder Schriftenreihe: | Frontiers in Bioengineering and Biotechnology |
| Jahrgang/Volume einer Zeitschrift: | 8 |
| Kollation: | 13 Seiten |
| DOI: | 10.26083/tuprints-00019238 |
| URL / URN: | https://tuprints.ulb.tu-darmstadt.de/19238 |
| Zugehörige Links: | |
| Herkunft: | Zweitveröffentlichung aus gefördertem Golden Open Access |
| Kurzbeschreibung (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. |
| Status: | Verlagsversion |
| URN: | urn:nbn:de:tuda-tuprints-192389 |
| Sachgruppe der Dewey Dezimalklassifikatin (DDC): | 500 Naturwissenschaften und Mathematik > 540 Chemie |
| Fachbereich(e)/-gebiet(e): | 07 Fachbereich Chemie 07 Fachbereich Chemie > Clemens-Schöpf-Institut > Fachgebiet Biochemie 07 Fachbereich Chemie > Clemens-Schöpf-Institut |
| Hinterlegungsdatum: | 04 Aug 2021 08:01 |
| Letzte Änderung: | 02 Aug 2024 12:35 |
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| Suche nach Titel in: | TUfind oder in Google |
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- A Generic Procedure for the Isolation of pH- and Magnesium-Responsive Chicken scFvs for Downstream Purification of Human Antibodies. (deposited 04 Aug 2021 08:01) [Gegenwärtig angezeigt]
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