The production of an Fab fragment of the catalytic antibody 6D9 in stably transformed lepidopteran insect cells was investigated. On the basis of an expression vector that utilizes the Bombyx mori cytoplasmic actin promoter, from which foreign gene expression is stimulated with the B. mori nucleopolyhedrovirus (BmNPV) IE-1 transactivator and the BmNPV HR3 enhancer, two plasmid vectors were constructed which contain either a neomycin or a blasticidin resistance gene for use as a selectable marker. The genes encoding the heavy chain (Hc; Fd fragment) and light chain (Lc) of the 6D9 Fab fragment were inserted separately into the expression vectors. After cotransfection with the resulting plasmids to introduce the Hc and Lc genes and the two different antibiotic resistance genes, Trichoplusia ni BTI-TN-5B1-4 (High Five) cells were cultured in the presence of G418 and blasticidin. Colonies of cells resistant to the antibiotics were obtained around 2 weeks after cotransfection. Western blotting and enzyme-linked immunosorbent assay (ELISA) of the cell culture supernatant suggested that the resistant cells stably secrete an Fab fragment which retains an antigen-binding activity. High yields of over 300 μg/ml of Fab fragment were achieved in simple batch shake-flask culture of transfected insect cells. These results indicate that recombinant insect cells may offer a novel approach for efficient production of antibody molecules.
To develop an efficient biological production process using the baculovirus-insect cell system, recombinant protein production was investigated in an immobilized cell culture with medium replacement. Sf9 insect cells were naturally entrapped within reticulated polyvinyl formal resin biomass support particles (BSPs; 2 x 2 x 2 mm cubes; pore diameter 30-50 μm) in a 2.5-l stirred-tank bioreactor. The immobilized cells were grown to a density of over 10 7 cells/cm 3-BSP, with the medium of 10% fetal bovine serum (FBS)-supplemented TNM-FH replaced at appropriate intervals, before infection with a recombinant baculovirus carrying the β-galactosidase gene. When serum-free TNM-FH was used instead of 10% FBS-supplemented TNM-FH for medium replenishment on and after post-infection day 1, the immobilized cells showed a high β-galactosidase yield comparable to that obtained in a culture using the serumsupplemented medium throughout. This finding indicates that immobilized insect cell culture allows not only intensification of cell culture but also recombinant protein production in protein-free basal media.
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