Different culture methods lead to differences in glycosylation of a murine IgG monoclonal antibody.
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.
Patel
T P
TP
Oxford GlycoSystem Ltd., Abingdon, Oxen, U.K.
Parekh
R B
RB
Moellering
B J
BJ
Prior
C P
CP
D000818:Animals; D000911:Antibodies, Monoclonal / Q000032:analysis / Q000378:metabolism*; D001202:Ascitic Fluid; D001769:Blood; D002240:Carbohydrate Sequence; D002478:Cells, Cultured; D002850:Chromatography, Gel; D003470:Culture Media; D004591:Electrophoresis, Polyacrylamide Gel; D006026:Glycoside Hydrolases / Q000378:metabolism; D006031:Glycosylation; D006825:Hybridomas / Q000276:immunology; D007074:Immunoglobulin G / Q000032:analysis / Q000378:metabolism*; D051379:Mice; D008969:Molecular Sequence Data; D019158:N-Acetylneuraminic Acid; D009844:Oligosaccharides / Q000032:analysis / Q000737:chemistry / Q000378:metabolism*; D012794:Sialic Acids / Q000378:metabolism
D000911:Antibodies, Monoclonal; D003470:Culture Media; D007074:Immunoglobulin G; D009844:Oligosaccharides; D012794:Sialic Acids; D006026:Glycoside Hydrolases; D019158:N-Acetylneuraminic Acid