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High-Yield Production and Cost-Effective Purification of an Alkali-Resistant Hexameric Protein A Variant for Antibody Affinity Chromatography

2026-06Artigo científico Acesso aberto
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dc.contributor.authorNatal, Carolina Mota
dc.contributor.authorLacombe, Claudia
dc.contributor.authorZollner, Alexander
dc.contributor.authorDomingos, Catarina
dc.contributor.authorMendes, José
dc.contributor.authorNascimento, André
dc.contributor.authorDias, Ana Margarida Gonçalves Carvalho
dc.contributor.authorBarbosa, Arménio Jorge Moura
dc.contributor.authorPeixoto, Cristina
dc.contributor.authorWiltschi, Birgit
dc.contributor.authorJungbauer, Alois
dc.contributor.authorRoque, Ana Cecília Afonso
dc.contributor.institutionDQ - Departamento de Química
dc.contributor.institutionUCIBIO - Applied Molecular Biosciences Unit
dc.contributor.institutionInstituto de Tecnologia Química e Biológica António Xavier (ITQB)
dc.contributor.pblWiley
dc.date.accessioned2026-07-20T13:49:04Z
dc.date.available2026-07-20T13:49:04Z
dc.date.issued2026-06
dc.descriptionPublisher Copyright: © 2026 The Author(s). Biotechnology Journal published by Wiley-VCH GmbH.
dc.description.abstractRecombinant Staphylococcal Protein A is the most widely used affinity ligand for industrial antibody purification, as well as for research and discovery of new antibodies. It has become a platform technology throughout the antibody manufacturing industry for the direct capture of products from a clarified cell culture supernatant. However, protein A adsorbents contribute significantly to manufacturing costs in the early stage of process development. In this work, we aimed to increase the production yield of an engineered protein A ligand comprising six repeats of the alkali-resistant protein A-derived Z domain, with a terminal cysteine residue for adsorbent immobilization (Z6AlkC). We coupled the extracellular production of Z6AlkC via secretion in high-cell-density, carbon-limited bioreactor cultures, with a simple and fast ultradiafiltration step for purification. Our bioprocess achieved 4 g/L of secreted Z6AlkC with an 89% purity, which is an improvement from previous reports that obtained intracellular expression and lower yield. We further purified the Z6AlkC protein by IgG-based affinity chromatography (Z6AlkC-P) and ultradiafiltration (Z6AlkC-F) and used it as an affinity ligand for antibody purification, as a proof of functionality. Both ligands yielded similar purification antibody behavior, showing that high-performance protein A-based ligands can be produced in scalable, high-productivity, and cost-effective methods.en
dc.description.versionpublishersversion
dc.description.versionpublished
dc.format.extent14
dc.format.extent2773757
dc.identifier.doi10.1002/biot.70247
dc.identifier.issn1860-6768
dc.identifier.otherPURE: 166469821
dc.identifier.otherPURE UUID: da1e6bc3-a119-4b98-82c4-882a46729e20
dc.identifier.otherScopus: 105040400885
dc.identifier.otherPubMed: 42205065
dc.identifier.otherWOS: 001776982200001
dc.identifier.otherORCID: /0000-0002-9756-4958/work/221295749
dc.identifier.otherORCID: /0000-0001-7416-7215/work/221295992
dc.identifier.urihttp://hdl.handle.net/10362/204670
dc.identifier.urlhttps://www.scopus.com/pages/publications/105040400885
dc.identifier.urlhttps://www.webofscience.com/wos/woscc/full-record/WOS:001776982200001
dc.language.isoeng
dc.peerreviewedyes
dc.subjectAffinity chromatography
dc.subjectAntibody purification
dc.subjectProtein A
dc.subjectZ-domain
dc.subjectApplied Microbiology and Biotechnology
dc.subjectMolecular Medicine
dc.titleHigh-Yield Production and Cost-Effective Purification of an Alkali-Resistant Hexameric Protein A Variant for Antibody Affinity Chromatographyen
dc.typejournal article
degois.publication.firstPage1
degois.publication.issue6
degois.publication.lastPage14
degois.publication.titleBiotechnology Journal
degois.publication.volume21
dspace.entity.typePublication
rcaap.rightsopenAccess

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