Production of mouse monoclonal antibody with galactose-extended sugar chain by suspension cultured tobacco BY2 cells expressing human beta(1,4)-galactosyltransferase.

Previously, we developed a transgenic tobacco BY2 cell line (GT6) in which glycosylation was modified by expressing human beta(1,4)-galactosyltransferase (betaGalT). In this study, we produced a mouse monoclonal antibody in GT6 cells, and determined the sugar chain structures of plant-produced antibodies. Galactose-extended N-linked glycans comprised 16.7%, and high-mannose-type and complex-type glycans comprised 38.5% and 35.0% of the total number of glycans, respectively. N-linked glycans with the plant-specific sugars beta(1,2)-xylose and alpha(1,3)-fucose comprised 9.8%. The introduction of human betaGalT into suspension cultured tobacco cells resulted in the production of recombinant proteins with galactose-extended glycans and decreased contents of beta(1,2)-xylose and alpha(1,3)-fucose.

N-linked glycan structures of a mouse monoclonal antibody produced from tobacco BY2 suspension-cultured cells.

cDNA encoding H- and L-chains from a mouse monoclonal antibody was introduced into tobacco BY2 cells, and the resulting sugar chain structures of plant-produced antibodies were analyzed by a combination of HPLC, exoglucosidase digestion and matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry. The glycan structures determined were Man(5-6)GlcNAc2 (22.3%), GlcNAcMan5GlcNAc2 (3.1%), GlcNAcMan3FucXylGlcNAc2 (24.4%), GlcNAcMan3XylGlcNAc2 (17.8%), Man3FucXylGlcNAc2 (24.3%), and Man3XylGlcNAc2 (8.1%). The major glycan structures of the antibodies produced by transgenic suspension-cultured cells contain typical plant bisecting beta(1,2)-xylose and alpha(1,3)-fucose residues, suggesting the posttranslational modification of a recombinant antibody in the late Golgi apparatus.