Two-dimensional fluorescence difference gel electrophoresis for comparison of affinity and non-affinity based downstream processing of recombinant monoclonal antibody.
Although Staphylococcus Protein A (SpA) affinity chromatography is the state of the art capture step for antibody purification, non-affinity methods are more economical. We used two-dimensional fluorescence difference gel electrophoresis (2-D DIGE) to evaluate the purification of a recombinant IgG(1) antibody from cultured cells, with two different processes: (1) SpA capture followed by cation-exchange chromatography (CEX); and (2) CEX capture, followed by anion exchanger, then hydrophobic interaction chromatography. Efficiencies were similar in sodium dodecylsulphate polyacrylamide gel electrophoresis and size-exclusion chromatography; however, 2-D DIGE revealed higher efficiency with SpA than with CEX capture. Thus, 2-D DIGE is a valuable tool for downstream process development.
Department of Biotechnology, University of Natural Resources and Applied Life Sciences, Vienna, Muthgasse 18, A-1190 Vienna, Austria.
Julita K
JK
Grzeskowiak
Anne
A
Tscheliessnig
Poh Choo
PC
Toh
Janet
J
Chusainow
Yih Yean
YY
Lee
Niki
N
Wong
Alois
A
Jungbauer
D000818:Animals; D000911:Antibodies, Monoclonal / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D016466:CHO Cells; D002845:Chromatography / Q000379:methods*; D002846:Chromatography, Affinity; D006224:Cricetinae; D003412:Cricetulus; D015180:Electrophoresis, Gel, Two-Dimensional / Q000379:methods*; D005453:Fluorescence; D007074:Immunoglobulin G / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D013205:Staphylococcal Protein A / Q000737:chemistry
D000911:Antibodies, Monoclonal; D007074:Immunoglobulin G; D011994:Recombinant Proteins; D013205:Staphylococcal Protein A