Comparison of mono Q, superose-6, and ABx fast protein liquid chromatograhy for the purification of IgM Monoclonal antibodies

Abstract

Nine immunoglobulin M (IgM) monoclonal antibodies (MAbs) produced in ascites fluids or in cell culture supernatants, have been purified on a fast protein liquid chromatography (FPLC) system using anion-exchange, size-exclusion, or mixed-mode chromatography matrices. The use of a mixed-mode ABx column provided an IgM that had a purity of greater than 99% after a single purification step. Anion-exchange chromatography using a Mono Q column, provided a partial purification fo the IgM which could subsequently be purified to a product of ca. 90% purity (determined from sodium dodecyl sulfate polyacrylamide gel electrophoresis) by size-exclusion chromatography on a Superose-6 column. Alternatively, the ascites containing the IgM was ammonium sulfate precipitated and chromatographed on the Superose-6 column under normal-as well as high-ionic strength conditions, which also yielded a product of ca. 90% purity. The purification of IgM from concentrated cell culture supernatants was evaluated using the Superose-6 or the ABx column. IgM purified from this source was greater than 99% pure when chromatographed on the mixed-mode column and ca. 60% pure on the size-exclusion column. MAbs from each of the procedures retained their immunoreactivity, as shown by indirect immunofluorescence staining of fixed cell preparations. A comparison of these methods revealed that mixed mode chromatography was simple, efficient, and yielded a product of high purity. The optimization of these methods facilitates the large-scale purification of mouse IgM MAbs and provides practical procedures for generating IgMs for use as diagnostic and therapeutic reagents.