Related Experiment Videos
Rapid purification and monitoring of immunoglobulin M from ascites by perfusion ion-exchange chromatography
E McCarthy1, G Vella, R Mhatre
1PerSeptive Biosystems, Inc., Framingham, MA 01701, USA.
A purification and on-line monitoring procedure for IgM was developed. Perfusion ion-exchange chromatography was used for rapid purification of IgM from ascites fluid and hybridoma supernatant. Crude ascites was directly loaded onto an ion exchanger. Due to the complexity of IgM, a two-step ion-exchange procedure had to be developed. This procedure involved a rapid cation-exchange chromatography capture step followed by further purification using anion-exchange chromatography. High linear velocities, in excess of 3500 cm/h, enabled separations to be performed under 5 min. Purity of the final product by SDS-PAGE was shown to be greater than 95%. Furthermore, the antibodies retained biological activity as measured by indirect immunofluorescence (IIF) and ELISA. The IgM peak was also monitored on-line using a novel peak tracking approach. This involved placing an antibody column (specific to the IgM) prior to the ion-exchange column and operating the ion-exchange column with and without the antibody column in-line. The missing peak that is identified by comparing the two chromatograms indicates where the IgM elutes.
A purification and on-line monitoring procedure for IgM was developed. Perfusion ion-exchange chromatography was used for rapid purification of IgM from ascites fluid and hybridoma supernatant. Crude ascites was directly loaded onto an ion exchanger. Due to the complexity of IgM, a two-step ion-exchange procedure had to be developed. This procedure involved a rapid cation-exchange chromatography capture step followed by further purification using anion-exchange chromatography. High linear velocities, in excess of 3500 cm/h, enabled separations to be performed under 5 min. Purity of the final product by SDS-PAGE was shown to be greater than 95%. Furthermore, the antibodies retained biological activity as measured by indirect immunofluorescence (IIF) and ELISA. The IgM peak was also monitored on-line using a novel peak tracking approach. This involved placing an antibody column (specific to the IgM) prior to the ion-exchange column and operating the ion-exchange column with and without the antibody column in-line. The missing peak that is identified by comparing the two chromatograms indicates where the IgM elutes.