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.
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.
Grzeskowiak
Julita K
JK
Department of Biotechnology, University of Natural Resources and Applied Life Sciences Vienna, Vienna, Austria.
Tscheliessnig
Anne
A
Wu
Ming Wei
MW
Toh
Poh Choo
PC
Chusainow
Janet
J
Lee
Yih Yean
YY
Wong
Niki
N
Jungbauer
Alois
A
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
D000911:Antibodies, Monoclonal; D005456:Fluorescent Dyes; D007074:Immunoglobulin G; D011994:Recombinant Proteins; D012204:Rh-Hr Blood-Group System; C026410:Rho(D) antigen