A monoclonal IgG-1 was produced by culture of a murine hybridoma (3.8.6) by three different methods, namely culture in ascites, in serum-free media and in serum-supplemented media. IgG-1 was purified to homogeneity (as judged by SDS/PAGE under reducing conditions) from each medium by ion-exchange chromatography and h.p.l.c. Protein A chromatography. Oligosaccharides were released from each IgG-1 preparation by hydrazinolysis and radiolabelled by reduction with alkaline sodium borotritide, and 'profile' analysis of the radiolabelled oligosaccharide alditols was performed by a combination of paper electrophoresis and gel-filtration chromatography. This analysis indicated clear and reproducible differences in the glycosylation patterns of the three IgG-1 preparations. Sequential exoglycosidase analysis of individual oligosaccharides derived from each IgG-1 preparation was used to define these differences. Ascites-derived material differed from serum-free-culture-derived material only with respect to the content of sialic acid. IgG-1 derived from culture in serum-containing media had an intermediate sialic acid content and a lower incidence of outer-arm galactosylation than the other two preparations. These differences in glycosylation could not be induced in any IgG-1 preparation by incubating purified IgG-1 with ascites or culture medium. It is concluded that the glycosylation pattern of a secreted monoclonal IgG is dependent on the culture method employed to obtain it.

Methodology for the purification of monoclonal antibodies from large‐scale mammalian cell culture systems has been investigated. The concentration of monoclonal antibodies in conditioned cell culture media was generally found to be in the milligram per liter range, requiring 100–1,000 liters for production of gram quantities. To reduce the volumes, several ultrafiltration systems, including hollow fiber, plate, and frame, and spiral cartridges were investigated and found to be effective for large‐scale work. Once a concentrated product was obtained, several methods including ammonium sulfate precipitation, ion exchange, protein. A agarose, and size exclusion chromatograhy were utilized and compared. The best results were obtained when concentrated conditioned medium was either diluted or diafiltered (to reduce the high ionic strength of conditioned medium) and fractionated by cation exchange chromatography. Variations in isoelectric points for different monoclonal antibodies require that specific pH and ionic strength parameters be determined to optimize binding and elution. Additional purification was achieved by further ion exchange steps or the use of ammonium sulfate precipitation. For particularly difficult purifications, protein A affinity chromatography was used. Once a purity of 90–95% was achieved, size exclusion chromatography was used as a final step to remove aggregates, process chemicals, contaminant proteins, and to exchange the antibody into the formulation buffer.

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