Capture of human monoclonal antibodies from cell culture supernatant by ion exchange media exhibiting high charge density.
A shortcut purification sequence for therapeutic proteins should consist of three steps: capture, purification, and polishing. Special emphasis has been put on direct capture of human monoclonal antibodies from culture supernatants with ion-exchangers avoiding pretreatment steps such as desalting, dilution, and other means to reduce the ionic strength. CM-HyperD, a cation-exchanger composed of an inorganic macroporous support filled with a viscoelastic gel with a high charge density was used. Capture of monoclonal antibodies from clarified hybridoma cell culture grown in media supplemented with fetal calf serum was investigated. Screening of different pH conditions and buffers for the load step showed that monoclonal antibodies were efficiently bound by CM-HyperD at pH 4.0 and 5.0 at an ionic strength equivalent to culture supernatant. Combination of negative purification with Q-Sepharose FF and capturing with CM-HyperD gave sufficient yield and resolution. Implementation of wash steps with higher conductivity did not improve the purity, but decreased the yield. Interestingly, high flow rates improved the purity. When antibodies were captured from serumfree culture supernatant the antibody could be eluted in a single peak with substantial reduction of contaminants. Capturing of antibodies by ion-exchange sorbents from culture supernatant is possible despite the high salt content.
Necina
R
R
Institute of Applied Microbiology, University of Agriculture, Forestry and Biotechnology, Nussdorferlände 11, A-1190 Vienna, Austria.
Amatschek
K
K
Jungbauer
A
A
D000911:Antibodies, Monoclonal / Q000302:isolation & purification*; D002850:Chromatography, Gel; D003470:Culture Media; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay; D006801:Humans; D006825:Hybridomas / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D007475:Ion Exchange Resins
D000911:Antibodies, Monoclonal; D003470:Culture Media; D007475:Ion Exchange Resins