title: Two-dimensional difference fluorescence gel electrophoresis to verify the scale-up of a non-affinity-based downstream process for isolation of a therapeutic recombinant antibody.
abstract: For therapeutic antibody production Protein A chromatography is often replaced by non-affinity-based purification sequences, which are considered as more economical. 2-D DIGE was applied for evaluation of scale-up of non-affinity based process of a humanized monoclonal antibody, anti-Rh(D) IgG(1), in comparison with other conventional analytical methods, like SDS-PAGE, Western blot, or SEC. Due to a high sensitivity of this technique (125 pg protein/spot) and high dynamic range of five orders of magnitude, low molecular weight impurities were detected in purified samples. Cation exchange chromatography was efficient capture step for IgG(1) purification in laboratory and pilot scale. The differences between samples after first purification step in laboratory and pilot scale were compensated with second purification step where almost the same protein pattern was observed. 2-D DIGE is a helpful tool for monitoring of purification effects and for scale-up verification of downstream processes.
authors.affiliation: Department of Biotechnology, University of Natural Resources and Applied Life Sciences Vienna, Vienna, Austria.
authors.forename: Julita K
authors.initials: JK
authors.lastname: Grzeskowiak
authors.forename: Anne
authors.initials: A
authors.lastname: Tscheliessnig
authors.forename: Ming Wei
authors.initials: MW
authors.lastname: Wu
authors.forename: Poh Choo
authors.initials: PC
authors.lastname: Toh
authors.forename: Janet
authors.initials: J
authors.lastname: Chusainow
authors.forename: Yih Yean
authors.initials: YY
authors.lastname: Lee
authors.forename: Niki
authors.initials: N
authors.lastname: Wong
authors.forename: Alois
authors.initials: A
authors.lastname: Jungbauer
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology / Q000302:isolation & purification*; D015153:Blotting, Western / Q000379:methods; D016466:CHO Cells; D002850:Chromatography, Gel / Q000379:methods; D002852:Chromatography, Ion Exchange; D006224:Cricetinae; D003412:Cricetulus; D015180:Electrophoresis, Gel, Two-Dimensional / Q000379:methods*; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods; D005453:Fluorescence; D005456:Fluorescent Dyes; D006801:Humans; D007074:Immunoglobulin G / Q000276:immunology / Q000302:isolation & purification; D010865:Pilot Projects; D011994:Recombinant Proteins / Q000276:immunology / Q000302:isolation & purification*; D015203:Reproducibility of Results; D012204:Rh-Hr Blood-Group System / Q000276:immunology; D013050:Spectrometry, Fluorescence
chemical_list: D000911:Antibodies, Monoclonal; D005456:Fluorescent Dyes; D007074:Immunoglobulin G; D011994:Recombinant Proteins; D012204:Rh-Hr Blood-Group System; C026410:Rho(D) antigen