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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
Author(s) -
Grzeskowiak Julita K.,
Tscheliessnig Anne,
Wu Ming Wei,
Toh Poh Choo,
Chusainow Janet,
Lee Yih Yean,
Wong Niki,
Jungbauer Alois
Publication year - 2010
Publication title -
electrophoresis
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.666
H-Index - 158
eISSN - 1522-2683
pISSN - 0173-0835
DOI - 10.1002/elps.200900781
Subject(s) - chromatography , downstream processing , chemistry , recombinant dna , monoclonal antibody , western blot , affinity chromatography , fluorescence , protein purification , electrophoresis , antibody , gel electrophoresis , enzyme , biochemistry , biology , physics , quantum mechanics , immunology , gene
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.