Rapid, large-scale production of novel protein therapeutics would be invaluable in emergency situations, such as an infectious disease outbreak or bioterrorist attack. We are investigating transient recombinant protein production in Nicotiana benthamiana plant cell suspension cultures as a platform for rapid response applications. Our system uses genetically engineered Agrobacterium tumefaciens to transfer DNA encoding a protein of interest to plant cells in suspension culture. Plants are a promising alternative expression system because they can produce complex proteins, similar to those produced in mammalian cells, while also enhancing product safety and efficacy in certain cases. Additionally, this system could reduce the initial development time for a novel therapeutic protein manufacturing process because the Agrobacterium constructs could be produced and screened faster than a transgenic plant or mammalian cell line. We have produced a recombinant anthrax toxin receptor-Fc fusion protein by co-culturing recombinant Agrobacterium and N. benthamiana cells in suspension culture. The maximum expression level observed was 0.7 mg per kg of plant cell fresh weight at day 9 of co-culture, which corresponded to 0.19% of total soluble protein extracted from biomass. Expression levels in this system need to be increased for this process to be a viable production platform. Strategies to increase gene transfer efficiency will be tested, such as reducing media volume, increasing Agrobacterium concentration, and increasing cell density to increase physical contact between the Agrobacterium and plant cells. Expression levels could also be impacted by plant cell defense responses induced by Agrobacterium. By day 9 of co-culture, intracellular total protein levels are, on average, reduced to 40% of control plant cell intracellular total protein levels, suggesting effects of Agrobacterium infection on plant cell health. An increase in reactive oxygen species levels, measured using 2′,7′-dichlorofluorescin diacetate, was also observed after addition of Agrobacterium to plant cell cultures. Plant immune responses such as reactive oxygen species production could impact recombinant protein production by affecting plant cell and/or Agrobacterium viability. The impact of these plant cell defense responses on recombinant protein production will be assessed and process conditions will be optimized to minimize these effects.

Enhancing Agrobacterium mediated transient recombinant protein production in Nicotiana benthamiana plant cell suspension culture

253rd National Meeting (2017)

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