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Abstract: 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 300μg/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. [Copyright &y& Elsevier]

Abstract: 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; 2mm×2mm×2mm; pore diameter 30–50μm) in a 2.5-l stirred-tank bioreactor. The immobilized cells were grown to a density of over 107 cells/cm3-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 serum-supplemented 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. [Copyright &y& Elsevier]

Abstract: We describe the secretory production of a mouse anti-bovine ribonuclease A single-chain variable fragment (scFv) fused with the Fc region of human IgG1 in stably transformed lepidopteran insect cells. Use of the insect-derived Bip and melittin signal peptides resulted in higher yields of the secreted scFv–Fc fusion protein than use of the baculovirus gp64 signal peptide. After cotransfection with an expression vector that contains the Bip signal sequence upstream of the DNA encoding the scFv–Fc and a selection vector that carries a neomycin resistance gene, Trichoplusia ni BTI-TN-5B1-4 (High Five) cells were cultured in the presence of G418. Colonies of resistant cells were obtained around 2 weeks after adding G418, and clonal cells were screened by enzyme-linked immunosorbent assay (ELISA) of the culture supernatant. The yield of the scFv–Fc protein secreted from the most productive clone was around 60mg/l in a shake-flask culture. To improve the productivity, we investigated the effect of medium supplements of sodium butyrate (NaBu), dimethyl sulfoxide (DMSO), and sericin. Supplementing culture medium with sericin increased the scFv–Fc protein yield to 82mg/l, but productivity was not increased by either NaBu or DMSO. These results indicate that the stably transformed insect cells may allow for the efficient production of scFv–Fc and other Fc fusion proteins. [Copyright &y& Elsevier]

• Influenza A virus-like particles (VLPs) were produced using recombinant insect cells. • VLPs were produced using insect cells as host cells without using a baculovirus. • A secretory form of VLPs consists of hemagglutinin and matrix protein 1. • The VLP productivity is comparable to that of the baculovirus–insect cell system. Virus-like particles (VLPs) are hollow nanoparticles composed of recombinant viral surface proteins without a virus genome. In the present study, we investigated the production of influenza VLPs using recombinant insect cells. DNA fragments encoding influenza A virus hemagglutinin (HA) and matrix protein 1 (M1) were cloned with the Drosophila BiP signal sequence in plasmid vectors containing a blasticidin and a neomycin resistance gene, respectively. After Trichoplusia ni BTI-TN-5B1-4 (High Five) cells were co-transfected with a pair of constructed plasmid vectors, stably transformed cells were established via incubation with blasticidin and G418. Western blot analyses showed that recombinant High Five cells secreted HA and M1 proteins into the culture supernatant. Immunoprecipitation of the culture supernatant with an anti-HA antibody and transmission electron microscopy suggested that secreted HA and M1 proteins were in a particulate structure with a morphology similar to that of an influenza virus. Hemagglutination assay indicated that expressed HA molecules retained hemagglutination activity. In a shake-flask culture, recombinant cells achieved a high HA yield (≈ 10 μg/ml) comparable to the yields obtained using the baculovirus–insect cell system. Recombinant insect cells may serve as excellent platforms for the efficient production of influenza VLPs for use as safe and effective vaccines and diagnostic antigens. [ABSTRACT FROM AUTHOR]