Hinz, Steffen C. ; Elter, Adrian ; Rammo, Oliver ; Schwämmle, Achim ; Ali, Ataurehman ; Zielonka, Stefan ; Herget, Thomas ; Kolmar, Harald (2020)
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, 8
doi: 10.3389/fbioe.2020.00688
Article, Bibliographie
This is the latest version of this item.
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: | 2020 |
| Creators: | Hinz, Steffen C. ; Elter, Adrian ; Rammo, Oliver ; Schwämmle, Achim ; Ali, Ataurehman ; Zielonka, Stefan ; Herget, Thomas ; Kolmar, Harald |
| Type of entry: | Bibliographie |
| Title: | A Generic Procedure for the Isolation of pH- and Magnesium-Responsive Chicken scFvs for Downstream Purification of Human Antibodies |
| Language: | English |
| Date: | 2020 |
| Publisher: | Frontiers |
| Journal or Publication Title: | Frontiers in Bioengineering and Biotechnology |
| Volume of the journal: | 8 |
| Collation: | 13 Seiten |
| DOI: | 10.3389/fbioe.2020.00688 |
| Corresponding Links: | |
| 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. |
| Classification DDC: | 500 Science and mathematics > 540 Chemistry |
| Divisions: | 07 Department of Chemistry 07 Department of Chemistry > Clemens-Schöpf-Institut > Fachgebiet Biochemie 07 Department of Chemistry > Clemens-Schöpf-Institut |
| Date Deposited: | 02 Aug 2024 12:35 |
| Last Modified: | 02 Aug 2024 12:35 |
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| Suche nach Titel in: | TUfind oder in Google |
Available Versions of this Item
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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)
- A Generic Procedure for the Isolation of pH- and Magnesium-Responsive Chicken scFvs for Downstream Purification of Human Antibodies. (deposited 02 Aug 2024 12:35) [Currently Displayed]
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