Comparative study of the N-glycans of human monoclonal immunoglobulins M produced by hybridoma and parental cells

Cell-cell hybridization is one method of establishing cell Lines capable of producing an abundance of antibodies. In order to clearly characterize ant ibodies produced by hybridomas, the influence of cell-cell hybridization on the glycosylation of produced antibodies should be studied. In this report , me describe structural changes of the N-glycans in immunoglobulin M (IgM) produced by a hybridoma cell line termed 3-4, which was established throug h 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 anal yzed the structures of sugar chains on the constant region of the mu-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, A sn332, and N395 were fully galactosylated biantennary complex types, with o r without core fucose and/or bisecting GlcNAc. However, the amount of bisec ting GlcNAc was markedly decreased in the hybridoma cells. Therefore, the a ctivity of UDP-N-acetylglucosamine:beta-D-mannoside beta-1,4-N-acetylglucos aminyltransferase (GnT-III) responsible for the formation of bisecting GlcN Ac was measured in parental cells and hybridoma cells. No. 12 cells showed some GnT-III activity, whereas P109 cells showed no such activity. The corr esponding 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 th e intracellular activity of GnT-III in the hybridoma cells, which was large ly 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. (C) 2000 Academic Press.