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Steegmüller, T; Kratky, T; Gollwitzer, L; Schwaminger, SP; Berensmeier, S.
Development of a New Affinity Gold Polymer Membrane with Immobilized Protein A.
Membranes (Basel). 2024; 14(2): Doi: 10.3390/membranes14020031 [OPEN ACCESS]
Web of Science PubMed PUBMED Central FullText FullText_MUG

 

Führende Autor*innen der Med Uni Graz
Schwaminger Sebastian
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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.

Find related publications in this database (Keywords)
affinity membrane chromatography
protein A
antibody purification
protein immobilization
gold membrane
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