title: Downstream processing of human antibodies integrating an extraction capture step and cation exchange chromatography.
abstract: In this paper we explore an alternative process for the purification of human antibodies from a Chinese hamster ovary (CHO) cell supernatant comprising a ligand-enhanced extraction capture step and cation exchange chromatography (CEX). The extraction of human antibodies was performed in an aqueous two-phase system (ATPS) composed of dextran and polyethylene glycol (PEG), in which the terminal hydroxyl groups of the PEG molecule were modified with an amino acid mimetic ligand in order to enhance the partition of the antibodies to the PEG-rich phase. This capture step was optimized using a design of experiments and a central composite design allowed the determination of the conditions that favor the partition of the antibodies to the phase containing the PEG diglutaric acid (PEG-GA) polymer, in terms of system composition. Accordingly, higher recovery yields were obtained for higher concentrations of PEG-GA and lower concentrations of dextran. The highest yield experimentally obtained was observed for an ATPS composed of 5.17% (w/w) dextran and 8% (w/w) PEG-GA. Higher purities were however predicted for higher concentrations of both polymers. A compromise between yield and purity was achieved using 5% dextran and 10% PEG-GA, which allowed the recovery of 82% of the antibodies with a protein purity of 96% and a total purity of 63%, determined by size-exclusion chromatography. ATPS top phases were further purified by cation exchange chromatography and it was observed that the most adequate cation exchange ligand was carboxymethyl, as the sulfopropyl ligand induced the formation of multi-aggregates or denatured forms. This column allowed the elution of 89% of the antibodies present in the top phase, with a protein purity of 100% and a total purity of 91%. The overall process containing a ligand-enhanced extraction step and a cation exchange chromatography step had an overall yield of 73%.
authors.affiliation: IBB-Institute for Biotechnology and Bioengineering, Centre for Chemical and Biological Engineering, Instituto Superior Técnico, Lisbon, Portugal.
authors.forename: Ana M
authors.initials: AM
authors.lastname: Azevedo
authors.forename: Paula A J
authors.initials: PA
authors.lastname: Rosa
authors.forename: I Filipa
authors.initials: IF
authors.lastname: Ferreira
authors.forename: J
authors.initials: J
authors.lastname: de Vries
authors.forename: T J
authors.initials: TJ
authors.lastname: Visser
authors.forename: M Raquel
authors.initials: MR
authors.lastname: Aires-Barros
mesh_terms: D000818:Animals; D000906:Antibodies / Q000302:isolation & purification*; D016466:CHO Cells; D002411:Cation Exchange Resins; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D002852:Chromatography, Ion Exchange / Q000379:methods*; D006224:Cricetinae; D003412:Cricetulus; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans
chemical_list: D000906:Antibodies; D002411:Cation Exchange Resins