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

Recombinant 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.

Descrição

Palavras-chave

Affinity chromatography Antibody purification Protein A Z-domain Applied Microbiology and Biotechnology Molecular Medicine

URI

http://hdl.handle.net/10362/204670

DOI

10.1002/biot.70247

Coleções

Home collection (FCT)

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