title: Phenylboronic acid as a multi-modal ligand for the capture of monoclonal antibodies: development and optimization of a washing step.
abstract: In this work, phenylboronic acid (PBA) was thoroughly investigated as a synthetic ligand for the purification of an immunoglobulin G (IgG) from a clarified cell supernatant from Chinese Hamster Ovary (CHO) cell cultures. In particular, the study was focused on the development of a washing step and in the optimization of the elution step using a serum containing supernatant. From the different conditions tested, best recoveries - 99% - and purifications - protein purity of 81% and a purification factor of 16 out of a maximum of 20 - were achieved using 100mM d-sorbitol in 10mM Tris-HCl as washing buffer and 0.5M d-sorbitol with 150mM NaCl in 10mM Tris-HCl as elution buffer. The purification outcome was also compared with protein A chromatography that revealed a recovery of 99%, 87% protein purity and 29 out of a maximum of 33 purification factor. Following the main purification, purified IgG was characterized in terms of isoelectric point, size and activity. In the end, a proof of concept was performed using two different mAbs from serum-free CHO cell cultures.
authors.affiliation: Institute for Biotechnology and Bioengineering, Centre for Biological and Chemical Engineering, Department of Bioengineering, Instituto Superior Técnico, Universidade de Lisboa, Av. Rovisco Pais, 1049-001 Lisbon, Portugal.
authors.forename: Raquel
authors.initials: R
authors.lastname: dos Santos
authors.affiliation: Institute for Biotechnology and Bioengineering, Centre for Biological and Chemical Engineering, Department of Bioengineering, Instituto Superior Técnico, Universidade de Lisboa, Av. Rovisco Pais, 1049-001 Lisbon, Portugal.
authors.forename: Sara A S L
authors.initials: SA
authors.lastname: Rosa
authors.affiliation: Institute for Biotechnology and Bioengineering, Centre for Biological and Chemical Engineering, Department of Bioengineering, Instituto Superior Técnico, Universidade de Lisboa, Av. Rovisco Pais, 1049-001 Lisbon, Portugal.
authors.forename: M Raquel
authors.initials: MR
authors.lastname: Aires-Barros
authors.affiliation: Icosagen Cell Factory, Eerika tee 1, Ülenurme vald, 61713 Tartumaa, Estonia.
authors.forename: Andres
authors.initials: A
authors.lastname: Tover
authors.affiliation: Institute for Biotechnology and Bioengineering, Centre for Biological and Chemical Engineering, Department of Bioengineering, Instituto Superior Técnico, Universidade de Lisboa, Av. Rovisco Pais, 1049-001 Lisbon, Portugal. Electronic address: a.azevedo@tecnico.ulisboa.pt.
authors.forename: Ana M
authors.initials: AM
authors.lastname: Azevedo
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology / Q000302:isolation & purification*; D001897:Boronic Acids / Q000737:chemistry*; D016466:CHO Cells; D002846:Chromatography, Affinity / Q000295:instrumentation / Q000379:methods*; D006224:Cricetinae; D003412:Cricetulus; D006801:Humans; D007074:Immunoglobulin G / Q000276:immunology / Q000302:isolation & purification*; D008024:Ligands; D013205:Staphylococcal Protein A / Q000378:metabolism
chemical_list: D000911:Antibodies, Monoclonal; D001897:Boronic Acids; D007074:Immunoglobulin G; D008024:Ligands; D013205:Staphylococcal Protein A; C010686:benzeneboronic acid
keywords: Capture step; MAbs purification; Multimodal chromatography; Phenylboronate ligands; Wash and elution studies