Expression of Recombinant Antibodies

Frenzel, André; Hust, Michael; Schirrmann, Thomas

Insect cells represent a very versatile eukaryotic expression system. They can be efficiently transfected with insect-specific viruses from the family of Baculoviridae, particularly the Autographa californica nuclear polyhedrosis virus (AcNPV). Baculoviruses are highly species-specific and are considered as safe for humans, mammalians and plants. Infection of human hepatocytes and mammalian cell lines including stable transduction has been demonstrated in cell culture without evidence of viral replication or gene expression under the control of baculoviral promoters (129, 130). Non-essential baculovirus genes involved in the viral life cycle, like Polyhedrin, P10, or Basic can be replaced by heterologous genes. The flexible viral envelop allows packaging of large heterologous gene sequences of more than 20 kb. Heterologous genes under the control of the strong polyhedron promoter are expressed at levels ranging from 0.1 to 50% of the total insect cell protein. Baculoviral protein expression is normally performed in insect cell lines like Sf-9 and Sf-21 of Spodoptera frugiperda, DS2 cells of Drosophila melanogaster, or High Five cells (BTI-TN-5B1-4) of Trichopulsia ni. High Five cells have certain advantages over Sf-9 cells for recombinant protein expression because they secrete up to 25-fold higher protein levels (131), have a more rapid doubling time, allow quick adaptation to serum-free medium and grow in suspension culture. In contrast, Sf-9 and Sf-21 cells are recommended for producing high-titer viral stocks due to higher transfection efficiency. Recombinant protein production can be performed in small-scale using plates or shake flasks as well as in large scale using Spinner flasks or bioreactors. Important parameters for optimizing baculoviral protein production are multiplicity of infection (m.o.i.), production length (usually up to 96 h), addition of protease inhibitors due to the release of viral proteases, temperature (usually 25-30°C), and media pH (pH 6.0-6.4).