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 consi st of three steps: capture, purification, and polishing. Special empha sis 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 stren gth. CM-HyperD, a cation-exchanger composed of an inorganic macroporou s support filled with a viscoelastic gel with a high charge density wa s used. Capture of monoclonal antibodies from clarified hybridoma cell culture grown in media supplemented with fetal calf serum was investi gated. Screening of different pH conditions and buffers for the load s tep showed that monoclonal antibodies were efficiently bound by CM-Hyp erD at pH 4.0 and 5.0 at an ionic strength equivalent to culture super natant. Combination of negative purification with Q-Sepharose FF and c apturing with CM-HyperD gave sufficient yield and resolution. Implemen tation of wash steps with higher conductivity did not improve the puri ty, but decreased the yield. Interestingly, high flow rates improved t he purity. When antibodies were captured from serumfree culture supern atant the antibody could be eluted in a single peak with substantial r eduction of contaminants. Capturing of antibodies by ion-exchange sorb ents from culture supernatant is possible despite the high salt conten t. (C) 1998 John Wiley & Sons, Inc.