Development of a New Affinity Gold Polymer Membrane with Immobilized Protein A
New and highly selective stationary phases for affinity membrane chromatography have the potential to significantly enhance the efficiency and specificity of therapeutic protein purification by reduced mass transfer limitations. This work developed and compared different immobilization strategies fo...
| Main Authors: | , , , , |
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| Format: | Article |
| Language: | English |
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MDPI AG
2024-01-01
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| Series: | Membranes |
| Subjects: | |
| Online Access: | https://www.mdpi.com/2077-0375/14/2/31 |
| _version_ | 1797297533795434496 |
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| author | Tobias Steegmüller Tim Kratky Lena Gollwitzer Sebastian Patrick Schwaminger Sonja Berensmeier |
| author_facet | Tobias Steegmüller Tim Kratky Lena Gollwitzer Sebastian Patrick Schwaminger Sonja Berensmeier |
| author_sort | Tobias Steegmüller |
| collection | DOAJ |
| description | New and highly selective stationary phases for affinity membrane chromatography have the potential to significantly enhance the efficiency and specificity of therapeutic protein purification by reduced mass transfer limitations. This work developed and compared different immobilization strategies for recombinant Protein A ligands to a gold-sputtered polymer membrane for antibody separation in terms of functionalization and immobilization success, protein load, and stability. Successful, functionalization was validated via X-ray photoelectron spectroscopy (XPS). Here, a recombinant Protein A ligand was coupled by N-hydroxysuccinimide (NHS)/N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide (EDC) chemistry to carboxy-functionalized, gold-sputtered membranes. We achieved a binding capacity of up to 104 ± 17 mg of the protein ligand per gram of the gold-sputtered membrane. The developed membranes were able to successfully capture and release the monoclonal antibody (mAb) Trastuzumab, as well as antibodies from fresh frozen human blood plasma in both static and dynamic setups. Therefore, they demonstrated successful functionalization and immobilization strategies. The antibody load was tested using bicinchoninic acid (BCA), ultraviolet-visible spectroscopy (UV-vis) measurements, and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The outcome is a fully functional affinity membrane that can be implemented in a variety of different antibody purification processes, eliminating the need for creating individualized strategies for modifying the surface to suit different substrates or conditions. |
| first_indexed | 2024-03-07T22:21:38Z |
| format | Article |
| id | doaj.art-4810f5bcf964437e96e879b9af2a1660 |
| institution | Directory Open Access Journal |
| issn | 2077-0375 |
| language | English |
| last_indexed | 2024-03-07T22:21:38Z |
| publishDate | 2024-01-01 |
| publisher | MDPI AG |
| record_format | Article |
| series | Membranes |
| spelling | doaj.art-4810f5bcf964437e96e879b9af2a16602024-02-23T15:26:53ZengMDPI AGMembranes2077-03752024-01-011423110.3390/membranes14020031Development of a New Affinity Gold Polymer Membrane with Immobilized Protein ATobias Steegmüller0Tim Kratky1Lena Gollwitzer2Sebastian Patrick Schwaminger3Sonja Berensmeier4Chair of Bioseparation Engineering, TUM School of Engineering and Design, Technical University of Munich, Boltzmannstraße 15, 85748 Garching, GermanyAssociate Professorship Physical Chemistry with Focus on Catalysis, TUM School of Natural Sciences, Technical University of Munich, Lichtenbergstraße 4, 85748 Garching, GermanyChair of Bioseparation Engineering, TUM School of Engineering and Design, Technical University of Munich, Boltzmannstraße 15, 85748 Garching, GermanyChair of Bioseparation Engineering, TUM School of Engineering and Design, Technical University of Munich, Boltzmannstraße 15, 85748 Garching, GermanyChair of Bioseparation Engineering, TUM School of Engineering and Design, Technical University of Munich, Boltzmannstraße 15, 85748 Garching, GermanyNew and highly selective stationary phases for affinity membrane chromatography have the potential to significantly enhance the efficiency and specificity of therapeutic protein purification by reduced mass transfer limitations. This work developed and compared different immobilization strategies for recombinant Protein A ligands to a gold-sputtered polymer membrane for antibody separation in terms of functionalization and immobilization success, protein load, and stability. Successful, functionalization was validated via X-ray photoelectron spectroscopy (XPS). Here, a recombinant Protein A ligand was coupled by N-hydroxysuccinimide (NHS)/N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide (EDC) chemistry to carboxy-functionalized, gold-sputtered membranes. We achieved a binding capacity of up to 104 ± 17 mg of the protein ligand per gram of the gold-sputtered membrane. The developed membranes were able to successfully capture and release the monoclonal antibody (mAb) Trastuzumab, as well as antibodies from fresh frozen human blood plasma in both static and dynamic setups. Therefore, they demonstrated successful functionalization and immobilization strategies. The antibody load was tested using bicinchoninic acid (BCA), ultraviolet-visible spectroscopy (UV-vis) measurements, and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The outcome is a fully functional affinity membrane that can be implemented in a variety of different antibody purification processes, eliminating the need for creating individualized strategies for modifying the surface to suit different substrates or conditions.https://www.mdpi.com/2077-0375/14/2/31affinity membrane chromatographyprotein Aantibody purificationprotein immobilizationgold membrane |
| spellingShingle | Tobias Steegmüller Tim Kratky Lena Gollwitzer Sebastian Patrick Schwaminger Sonja Berensmeier Development of a New Affinity Gold Polymer Membrane with Immobilized Protein A Membranes affinity membrane chromatography protein A antibody purification protein immobilization gold membrane |
| title | Development of a New Affinity Gold Polymer Membrane with Immobilized Protein A |
| title_full | Development of a New Affinity Gold Polymer Membrane with Immobilized Protein A |
| title_fullStr | Development of a New Affinity Gold Polymer Membrane with Immobilized Protein A |
| title_full_unstemmed | Development of a New Affinity Gold Polymer Membrane with Immobilized Protein A |
| title_short | Development of a New Affinity Gold Polymer Membrane with Immobilized Protein A |
| title_sort | development of a new affinity gold polymer membrane with immobilized protein a |
| topic | affinity membrane chromatography protein A antibody purification protein immobilization gold membrane |
| url | https://www.mdpi.com/2077-0375/14/2/31 |
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