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Development of a New Affinity Gold Polymer Membrane with Immobilized Protein A.
Steegmüller, Tobias; Kratky, Tim; Gollwitzer, Lena; Schwaminger, Sebastian Patrick; Berensmeier, Sonja.
Affiliation
  • Steegmüller T; Chair of Bioseparation Engineering, TUM School of Engineering and Design, Technical University of Munich, Boltzmannstraße 15, 85748 Garching, Germany.
  • Kratky T; Associate Professorship Physical Chemistry with Focus on Catalysis, TUM School of Natural Sciences, Technical University of Munich, Lichtenbergstraße 4, 85748 Garching, Germany.
  • Gollwitzer L; Chair of Bioseparation Engineering, TUM School of Engineering and Design, Technical University of Munich, Boltzmannstraße 15, 85748 Garching, Germany.
  • Schwaminger SP; Chair of Bioseparation Engineering, TUM School of Engineering and Design, Technical University of Munich, Boltzmannstraße 15, 85748 Garching, Germany.
  • Berensmeier S; Division of Medicinal Chemistry, Otto-Loewi Research Center, Medical University of Graz, Neue Stiftingtalstraße 6, 8010 Graz, Austria.
Membranes (Basel) ; 14(2)2024 Jan 24.
Article in En | MEDLINE | ID: mdl-38392658
ABSTRACT
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.
Key words

Full text: 1 Collection: 01-internacional Database: MEDLINE Language: En Journal: Membranes (Basel) Year: 2024 Document type: Article Affiliation country: Germany Country of publication: Switzerland

Full text: 1 Collection: 01-internacional Database: MEDLINE Language: En Journal: Membranes (Basel) Year: 2024 Document type: Article Affiliation country: Germany Country of publication: Switzerland