Comparison of mono Q, superose-6, and ABx fast protein liquid chromatography for the purification of IgM monoclonal antibodies.
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 of 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.
Chen
F M
FM
Department of Pathology, School of Medicine, University of Southern California, Los Angeles 90033.
Naeve
G S
GS
Epstein
A L
AL
D000837:Anion Exchange Resins; D000911:Antibodies, Monoclonal / Q000302:isolation & purification*; D002478:Cells, Cultured; D002846:Chromatography, Affinity; D002853:Chromatography, Liquid; D004591:Electrophoresis, Polyacrylamide Gel; D006825:Hybridomas; D007074:Immunoglobulin G / Q000302:isolation & purification*; D007475:Ion Exchange Resins; D012685:Sepharose; D013347:Subcellular Fractions / Q000032:analysis
D000837:Anion Exchange Resins; D000911:Antibodies, Monoclonal; D007074:Immunoglobulin G; D007475:Ion Exchange Resins; C043127:Superose 6b; D012685:Sepharose