Evidence of intra- and extracellular modifications of monoclonal IgG polypeptide chains generating charge heterogeneity.
The heavy and light chains of IgG monoclonal antibodies (mAbs) can be shown to be heterogeneous, with respect to isoelectric points, when analyzed by two-dimensional electrophoresis (2-DE). The molecular basis for this charge heterogeneity has not been clearly defined but it has been suggested that it could be due, in part, to differences in glycosylation. To investigate this possibility we have compared the 2-DE pattern of glycosylated and aglycosylated forms of the mouse IgG1 mAb (1B7-11), produced in vitro in the presence and absence of tunicamycin. Charge heterogeneity was shown not to be a consequence of glycosylation status. Intracellular and secreted IgG mAbs were also analyzed to investigate the time course of change in charge properties of the heavy and light chains. The charge heterogeneity was found to be generated intracellularly, and alterations in charge properties could be induced during incubation under physiological conditions. Semilogarithmic plots of the density of the principal heavy and light chain spots against incubation time showed linear relationships, suggesting that the charge shifts result from a first-order reaction. The semilogarithmic plot for the light chain correlated well with the time after IgG synthesis. These results suggest that the charge heterogeneity of an IgG mAb is due to intra- and extracellular modifications of the polypeptide chains which reflect "aging" of antibody molecules.
Mimura
Y
Y
The First Department of Biochemistry, Yamaguchi University School of Medicine, Ube, Japan. biochem1@po.cc.yamaguchi-u.ac.jp
Nakamura
K
K
Tanaka
T
T
Fujimoto
M
M
D000818:Animals; D000911:Antibodies, Monoclonal / Q000737:chemistry*; D003911:Dextrans / Q000276:immunology; D004563:Electrochemistry; D016650:Fluorescein-5-isothiocyanate; D006031:Glycosylation; D006863:Hydrogen-Ion Concentration; D007141:Immunoglobulin Fc Fragments; D007074:Immunoglobulin G / Q000737:chemistry*; D007143:Immunoglobulin Heavy Chains / Q000032:analysis; D007145:Immunoglobulin kappa-Chains / Q000737:chemistry; D051379:Mice; D009844:Oligosaccharides / Q000032:analysis; D010455:Peptides / Q000502:physiology; D010851:Picrates / Q000276:immunology; D013696:Temperature; D013997:Time Factors; D014407:Tumor Cells, Cultured
D000911:Antibodies, Monoclonal; D003911:Dextrans; D007141:Immunoglobulin Fc Fragments; D007074:Immunoglobulin G; D007143:Immunoglobulin Heavy Chains; D007145:Immunoglobulin kappa-Chains; D009844:Oligosaccharides; D010455:Peptides; D010851:Picrates; C041999:alpha-1,6-dextran; C005858:picric acid; D016650:Fluorescein-5-isothiocyanate