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...
| Main Authors: | , , , , , , , |
|---|---|
| Format: | Article |
| Language: | English |
| Published: |
Frontiers Media S.A.
2020-06-01
|
| Series: | Frontiers in Bioengineering and Biotechnology |
| Subjects: | |
| Online Access: | https://www.frontiersin.org/article/10.3389/fbioe.2020.00688/full |
| _version_ | 1828444345576980480 |
|---|---|
| author | Steffen C. Hinz Steffen C. Hinz Adrian Elter Adrian Elter Oliver Rammo Achim Schwämmle Ataurehman Ali Stefan Zielonka Thomas Herget Harald Kolmar Harald Kolmar |
| author_facet | Steffen C. Hinz Steffen C. Hinz Adrian Elter Adrian Elter Oliver Rammo Achim Schwämmle Ataurehman Ali Stefan Zielonka Thomas Herget Harald Kolmar Harald Kolmar |
| author_sort | Steffen C. Hinz |
| collection | DOAJ |
| 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 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. |
| first_indexed | 2024-12-10T21:42:14Z |
| format | Article |
| id | doaj.art-d9f77b27f59a460fa48f4fde4750675b |
| institution | Directory Open Access Journal |
| issn | 2296-4185 |
| language | English |
| last_indexed | 2024-12-10T21:42:14Z |
| publishDate | 2020-06-01 |
| publisher | Frontiers Media S.A. |
| record_format | Article |
| series | Frontiers in Bioengineering and Biotechnology |
| spelling | doaj.art-d9f77b27f59a460fa48f4fde4750675b2022-12-22T01:32:28ZengFrontiers Media S.A.Frontiers in Bioengineering and Biotechnology2296-41852020-06-01810.3389/fbioe.2020.00688545334A Generic Procedure for the Isolation of pH- and Magnesium-Responsive Chicken scFvs for Downstream Purification of Human AntibodiesSteffen C. Hinz0Steffen C. Hinz1Adrian Elter2Adrian Elter3Oliver Rammo4Achim Schwämmle5Ataurehman Ali6Stefan Zielonka7Thomas Herget8Harald Kolmar9Harald Kolmar10Institute for Organic Chemistry and Biochemistry, Technische Universität Darmstadt, Darmstadt, GermanyMerck Lab @ Technische Universität Darmstadt, Darmstadt, GermanyInstitute for Organic Chemistry and Biochemistry, Technische Universität Darmstadt, Darmstadt, GermanyMerck Lab @ Technische Universität Darmstadt, Darmstadt, GermanyLife Science Division, Merck KGaA, Darmstadt, GermanyLife Science Division, Merck KGaA, Darmstadt, GermanyInstitute for Organic Chemistry and Biochemistry, Technische Universität Darmstadt, Darmstadt, GermanyProtein Engineering and Antibody Technologies, Merck KGaA, Darmstadt, GermanyStrategy und Transformation, Merck KGaA, Darmstadt, GermanyInstitute for Organic Chemistry and Biochemistry, Technische Universität Darmstadt, Darmstadt, GermanyMerck Lab @ Technische Universität Darmstadt, Darmstadt, GermanyAffinity 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.https://www.frontiersin.org/article/10.3389/fbioe.2020.00688/fullaffinity chromatographyyeast displayprotein purificationimmune librarychicken antibodysingle chain fragment variable |
| spellingShingle | Steffen C. Hinz Steffen C. Hinz Adrian Elter Adrian Elter Oliver Rammo Achim Schwämmle Ataurehman Ali Stefan Zielonka Thomas Herget Harald Kolmar Harald Kolmar A Generic Procedure for the Isolation of pH- and Magnesium-Responsive Chicken scFvs for Downstream Purification of Human Antibodies Frontiers in Bioengineering and Biotechnology affinity chromatography yeast display protein purification immune library chicken antibody single chain fragment variable |
| 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 | affinity chromatography yeast display protein purification immune library chicken antibody single chain fragment variable |
| url | https://www.frontiersin.org/article/10.3389/fbioe.2020.00688/full |
| work_keys_str_mv | AT steffenchinz agenericprocedurefortheisolationofphandmagnesiumresponsivechickenscfvsfordownstreampurificationofhumanantibodies AT steffenchinz agenericprocedurefortheisolationofphandmagnesiumresponsivechickenscfvsfordownstreampurificationofhumanantibodies AT adrianelter agenericprocedurefortheisolationofphandmagnesiumresponsivechickenscfvsfordownstreampurificationofhumanantibodies AT adrianelter agenericprocedurefortheisolationofphandmagnesiumresponsivechickenscfvsfordownstreampurificationofhumanantibodies AT oliverrammo agenericprocedurefortheisolationofphandmagnesiumresponsivechickenscfvsfordownstreampurificationofhumanantibodies AT achimschwammle agenericprocedurefortheisolationofphandmagnesiumresponsivechickenscfvsfordownstreampurificationofhumanantibodies AT ataurehmanali agenericprocedurefortheisolationofphandmagnesiumresponsivechickenscfvsfordownstreampurificationofhumanantibodies AT stefanzielonka agenericprocedurefortheisolationofphandmagnesiumresponsivechickenscfvsfordownstreampurificationofhumanantibodies AT thomasherget agenericprocedurefortheisolationofphandmagnesiumresponsivechickenscfvsfordownstreampurificationofhumanantibodies AT haraldkolmar agenericprocedurefortheisolationofphandmagnesiumresponsivechickenscfvsfordownstreampurificationofhumanantibodies AT haraldkolmar agenericprocedurefortheisolationofphandmagnesiumresponsivechickenscfvsfordownstreampurificationofhumanantibodies AT steffenchinz genericprocedurefortheisolationofphandmagnesiumresponsivechickenscfvsfordownstreampurificationofhumanantibodies AT steffenchinz genericprocedurefortheisolationofphandmagnesiumresponsivechickenscfvsfordownstreampurificationofhumanantibodies AT adrianelter genericprocedurefortheisolationofphandmagnesiumresponsivechickenscfvsfordownstreampurificationofhumanantibodies AT adrianelter genericprocedurefortheisolationofphandmagnesiumresponsivechickenscfvsfordownstreampurificationofhumanantibodies AT oliverrammo genericprocedurefortheisolationofphandmagnesiumresponsivechickenscfvsfordownstreampurificationofhumanantibodies AT achimschwammle genericprocedurefortheisolationofphandmagnesiumresponsivechickenscfvsfordownstreampurificationofhumanantibodies AT ataurehmanali genericprocedurefortheisolationofphandmagnesiumresponsivechickenscfvsfordownstreampurificationofhumanantibodies AT stefanzielonka genericprocedurefortheisolationofphandmagnesiumresponsivechickenscfvsfordownstreampurificationofhumanantibodies AT thomasherget genericprocedurefortheisolationofphandmagnesiumresponsivechickenscfvsfordownstreampurificationofhumanantibodies AT haraldkolmar genericprocedurefortheisolationofphandmagnesiumresponsivechickenscfvsfordownstreampurificationofhumanantibodies AT haraldkolmar genericprocedurefortheisolationofphandmagnesiumresponsivechickenscfvsfordownstreampurificationofhumanantibodies |