Rapid purification and monitoring of immunoglobulin M from ascites by perfusion ion-exchange chromatography.
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.
McCarthy
E
E
PerSeptive Biosystems, Inc., Framingham, MA 01701, USA.
Vella
G
G
Mhatre
R
R
Lim
Y P
YP
D000818:Animals; D000837:Anion Exchange Resins; D000911:Antibodies, Monoclonal / Q000276:immunology; D001201:Ascites / Q000276:immunology*; D002411:Cation Exchange Resins; D002852:Chromatography, Ion Exchange / Q000379:methods*; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay; D005260:Female; D019084:Fluorescent Antibody Technique, Indirect; D006825:Hybridomas / Q000166:cytology / Q000276:immunology; D007075:Immunoglobulin M / Q000302:isolation & purification*; D051379:Mice; D008807:Mice, Inbred BALB C; D009862:Online Systems; D016622:Silver Staining; D012967:Sodium Dodecyl Sulfate / Q000737:chemistry; D013997:Time Factors
D000837:Anion Exchange Resins; D000911:Antibodies, Monoclonal; D002411:Cation Exchange Resins; D007075:Immunoglobulin M; D012967:Sodium Dodecyl Sulfate