title: Laboratory Scale Production and Purification of a Therapeutic Antibody.
abstract: Ensuring the successful production of a therapeutic antibody begins early on in the development process. The first stage is vector expression of the antibody genes followed by stable transfection into a suitable cell line. The stable clones are subjected to screening in order to select those clones with desired production and growth characteristics. This is a critical albeit time-consuming step in the process. This protocol considers vector selection and sourcing of antibody sequences for the expression of a therapeutic antibody. The methods describe preparation of vector DNA for stable transfection of a suspension variant of human embryonic kidney 293 (HEK-293) cell line, using polyethylenimine (PEI). The cells are transfected as adherent cells in serum-containing media to maximize transfection efficiency, and afterwards adapted to serum-free conditions. Large scale production, setup as batch overgrow cultures is used to yield antibody protein that is purified by affinity chromatography using an automated fast protein liquid chromatography (FPLC) instrument. The antibody yields produced by this method can provide sufficient protein to begin initial characterization of the antibody. This may include in vitro assay development or physicochemical characterization to aid in the time-consuming task of clonal screening for lead candidates. This method can be transferable to the development of an expression system for the production of biosimilar antibodies.
authors.affiliation: Faculty of Pharmacy, University of Sydney.
authors.forename: Zehra
authors.initials: Z
authors.lastname: Elgundi
authors.affiliation: Faculty of Pharmacy, University of Sydney.
authors.forename: Vicki
authors.initials: V
authors.lastname: Sifniotis
authors.affiliation: Faculty of Pharmacy, University of Sydney.
authors.forename: Mouhamad
authors.initials: M
authors.lastname: Reslan
authors.affiliation: Faculty of Pharmacy, University of Sydney.
authors.forename: Esteban
authors.initials: E
authors.lastname: Cruz
authors.affiliation: Faculty of Pharmacy, University of Sydney; veysel.kayser@sydney.edu.au.
authors.forename: Veysel
authors.initials: V
authors.lastname: Kayser
mesh_terms: D000906:Antibodies / Q000235:genetics / Q000302:isolation & purification*; D061249:Batch Cell Culture Techniques / Q000379:methods; D002452:Cell Count; D002846:Chromatography, Affinity / Q000295:instrumentation / Q000379:methods*; D005822:Genetic Vectors; D057809:HEK293 Cells; D006801:Humans; D007074:Immunoglobulin G / Q000235:genetics / Q000302:isolation & purification; D011094:Polyethyleneimine; D014162:Transfection / Q000379:methods*; D000068878:Trastuzumab / Q000302:isolation & purification
chemical_list: D000906:Antibodies; D007074:Immunoglobulin G; D011094:Polyethyleneimine; D000068878:Trastuzumab