Production of Biopharmaceuticals in Nicotiana benthamiana-Axillary Stem Growth as a Key Determinant of Total Protein Yield.

Data are scarce about the influence of basic cultural conditions on growth patterns and overall performance of plants used as heterologous production hosts for protein pharmaceuticals. Higher plants are complex organisms with young, mature, and senescing organs that show distinct metabolic backgrounds and differ in their ability to sustain foreign protein expression and accumulation. Here, we used the transient protein expression host Nicotiana benthamiana as a model to map the accumulation profile of influenza virus hemagglutinin H1, a clinically promising vaccine antigen, at the whole plant scale. Greenhouse-grown plants submitted to different light regimes, submitted to apical bud pruning, or treated with the axillary growth-promoting cytokinin 6-benzylaminopurine were vacuum-infiltrated with agrobacteria harboring a DNA sequence for H1 and allowed to express the viral antigen for 7 days in growth chamber under similar environmental conditions. Our data highlight the importance of young leaves on H1 yield per plant, unlike older leaves which account for a significant part of the plant biomass but contribute little to total antigen titer. Our data also highlight the key contribution of axillary stem leaves, which contribute more than 50% of total yield under certain conditions despite representing only one-third of the total biomass. These findings underline the relevance of both considering main stem leaves and axillary stem leaves while modeling heterologous protein production in N. benthamiana. They also demonstrate the potential of exogenously applied growth-promoting hormones to modulate host plant architecture for improvement of protein yields.

Transient expression of antibodies in plants using syringe agroinfiltration.

The improvements in agroinfiltration methods for plant-based transient expression now allow the production of significant amounts of recombinant proteins in a matter of days. While vacuum-based agroinfiltration has been brought to large scale to meet the cost, speed and surge capacity requirements for vaccine and therapeutic production, the more accessible and affordable syringe agroinfiltration procedure still represents a fast and high-yielding approach to recombinant protein production at lab scale. The procedure exemplified here has proven its reproducibility and high-yield capacity for the production of proteins with varying levels of complexity, including monoclonal antibodies.