Comparative Study of the N-Glycans of Human Monoclonal Immunoglobulins M Produced by Hybridoma and Parental Cells

Abstract Cell–cell hybridization is one method of establishing cell lines capable of producing an abundance of antibodies. In order to clearly characterize antibodies produced by hybridomas, the influence of cell–cell hybridization on the glycosylation of produced antibodies should be studied. In this report, we describe structural changes of the N -glycans in immunoglobulin M (IgM) produced by a hybridoma cell line termed 3-4, which was established through hybridization of an IgM-producing Epstein–Barr virus transformed human B-cell line termed No. 12, and a human myeloma cell line termed P109. We analyzed the structures of sugar chains on the constant region of the μ-chain of IgMs produced by parental No. 12 cells and hybridoma 3-4 cells. In both parental cells and hybridoma cells, the predominant structures at Asn171, Asn332, and N395 were fully galactosylated biantennary complex types, with or without core fucose and/or bisecting GlcNAc. However, the amount of bisecting GlcNAc was markedly decreased in the hybridoma cells. Therefore, the activity of UDP- N -acetylglucosamine:β- d -mannoside β-1,4- N -acetylglucosaminyltransferase (GnT-III) responsible for the formation of bisecting GlcNAc was measured in parental cells and hybridoma cells. No. 12 cells showed some GnT-III activity, whereas P109 cells showed no such activity. The corresponding level of activity observed in hybridoma 3-4 cells was much lower than that in No. 12 cells. The above results demonstrated a reduction in the intracellular activity of GnT-III in the hybridoma cells, which was largely due to the influence of P109 cells. Moreover, the sugar chain structures of IgMs produced by the cells reflected the level of GnT-III activity.