(127a) Transient Recombinant Protein Production in Glycoengineered Plant Cell Suspension Cultures for Rapid Response Applications | AIChE

(127a) Transient Recombinant Protein Production in Glycoengineered Plant Cell Suspension Cultures for Rapid Response Applications


Sukenik, S. C. - Presenter, University of California, Davis
Karuppanan, K., University of California, Davis
Li, Q., University of California, Davis
Lebrilla, C. B., University of California, Davis
Nandi, S., University of California, Davis
McDonald, K. A., University of California, Davis
In a bioterrorist attack or emerging infectious disease outbreak, rapid production of novel protein-based drugs or vaccines would be invaluable. We are developing a transient plant cell culture platform designed for rapid recombinant protein production in emergency situations. Our system uses genetically engineered Agrobacterium tumefaciens to deliver the DNA encoding a protein of interest to Nicotiana benthamiana plant cells in suspension culture. Different proteins can be produced on this platform simply by co-culturing with a different recombinant Agrobacterium. Compared to stable transgenic mammalian cell line development, our process would reduce the initial development time before large-scale protein production can begin since the required Agrobacterium constructs can be produced in as little as two weeks. Additionally, because agrobacteria can self-replicate to high densities and transfer the DNA construct directly to plant cells, there are also advantages compared with transient production in mammalian cell culture which requires expensive reagents, purified plasmid DNA, or use of mammalian viral vectors.

As a model protein, we have successfully produced a recombinant anthrax toxin receptor-Fc fusion protein (a fusion between the extracellular domain of the human capillary morphogenesis receptor protein that binds anthrax protective antigen and the Fc domain of human IgG, referred to as CMG2-Fc) by adding Agrobacterium to plant cells in suspension culture. Initial process development studies will be discussed, which resulted in expression levels up to 10 μg CMG2-Fc per g plant cell fresh weight after 7 days of co-culture. Although plants are capable of a wide variety of post-translational modifications, there are slight differences between plant and mammalian glycosylation. To reduce plant-specific glycosylation patterns, β(1,2)-xylosyltransferase and α(1,3)-fucosyltransferase knockdown glycoengineered host N. benthamiana cell suspension cultures were generated. CMG2-Fc produced by co-culturing Agrobacterium with these glycoengineered N. benthamiana cell suspension cultures resulted in a dramatic reduction in plant-specific N-glycans compared to CMG2-Fc produced in wild type N. benthamiana plants. In addition to mitigating the risk of an immune response to plant-specific glycans, this proof of concept data demonstrates a method that could be further modified to enhance a product’s efficacy and stability by tuning its N-glycan distribution. Finally, data from techno-economic analysis of both upstream and downstream unit operations performed using SuperPro Designer software will be presented. These simulation results will guide process development work by identifying yield and recovery levels required, as well as key cost drivers, for cost-effective, large-scale production using this novel platform.