title: Optimized extraction of a single-chain variable fragment of antibody by using aqueous micellar two-phase systems.
abstract: In this work, the purification of a single-chain variable fragment (scFv) of an antibody by using liquid-liquid extraction in aqueous micellar two-phase systems was optimized by means of central composite design. Protein partitioning assays were performed by using the selected system composition in previous works: Triton X-114 at 4% wt/wt, yeast fermentation supernatant at 60% wt/wt, McIlvaine buffer pH 7.00. The other system component concentrations, Cibacron Blue F3GA (CB), Fabsorbent™ F1P HF (HF) and NaCl, were selected as independent variables. ScFv recovery percentage (%R) and purification factor (PF) were selected as the responses. According to the optimization process both, scFv recovery percentage and purification factor were favored with the addition of HF and NaCl in a range of concentrations around the central point of the second central composite design (HF 0.0120% w/w, CB 0.0200% w/w, NaCl 0.200% w/w). These experimental conditions allowed the concentration and pre-purification of scFv in the micelle-rich bottom phase of the systems with a recovery percentage superior to 88% and a purification factor of approximately 3.5. These results improved the previously presented works and demonstrated the convenience of using aqueous micellar two-phase systems as a first step in the purification of scFv molecules.
authors.affiliation: Departamento de Química-física, Instituto de Procesos Biotecnológicos y Químicos (IPROBYQ), Universidad Nacional de Rosario, CP 2000 Rosario, Argentina. Electronic address: lucianapelleg@yahoo.com.ar.
authors.forename: Luciana P
authors.initials: LP
authors.lastname: Malpiedi
authors.affiliation: Departamento de Química-física, Instituto de Procesos Biotecnológicos y Químicos (IPROBYQ), Universidad Nacional de Rosario, CP 2000 Rosario, Argentina. Electronic address: bnerli@fbioyf.unr.edu.ar.
authors.forename: Bibiana B
authors.initials: BB
authors.lastname: Nerli
authors.affiliation: Chemical Engineering Department, Polytechnic School, University of São Paulo, São Paulo, Brazil. Electronic address: mtaqueda@usp.br.
authors.forename: Maria E S
authors.initials: ME
authors.lastname: Taqueda
authors.affiliation: Department of Clinical Analysis and Toxicology, University of São Paulo, São Paulo, Brazil. Electronic address: dspabdalla@gmail.com.
authors.forename: Dulcineia S P
authors.initials: DS
authors.lastname: Abdalla
authors.affiliation: Biochemical and Pharmaceutical Technology Department/FCF, University of São Paulo, Av. Prof. Lineu Prestes, 580 - Bloco 16, CEP 05508-000 São Paulo, Brazil. Electronic address: pessoajr@usp.br.
authors.forename: Adalberto
authors.initials: A
authors.lastname: Pessoa
mesh_terms: D006801:Humans; D006863:Hydrogen-Ion Concentration; D008823:Micelles; D057127:Single-Chain Antibodies / Q000737:chemistry* / Q000302:isolation & purification*
chemical_list: D008823:Micelles; D057127:Single-Chain Antibodies
keywords: Antibody fragments; Aqueous micellar two-phase systems; Central composite design; Surface response