(348f) Rapid Bioproduction of Protein Biologics Using Plants in Response to Sars-Cov-2

Problem: As demonstrated by the recent pandemic, the rapid development of scalable protein therapies is critical. At times or places when highly specialized and large-scale equipment is unavailable, e.g. third world countries or space, this need to produce therapeutics and diagnostics is even more imperative. Fortunately, plants are capable of growth in low resource environments for cost-effective and scalable protein production. Plants can be harnessed for biological growth in the same capacity as a bioreactor-based system, however, the current gold standard for recombinant protein production in plants uses gram negative Agrobacterium tumefaciens (A. tumefaciens) and is optimized for expression in Nicotiana benthamiana (N. benthamiana), a relative of tobacco, making biologics produced in this system more difficult to quickly and safely purify for clinical use. In contrast, edible plants that are generally regarded as safe (GRAS) can be used as a host for numerous biologics, like lettuce (Lactuca sativa), which has been extensively studied for space applications and grown on the International Space Station because it can utilize limited in situ resources for growth. Current production systems require multiple days to produce recombinant proteins, thus reducing the ability to keep up with the rapidly changing needs of global health, as well as the amount of therapeutic which can be brought to market quickly. This project focuses on developing bioprocessing technologies for a dynamic plant model using a viral expression system that can be employed to rapidly produce protein biologics and reagents in less than 24 hours under low resource requirements.

Methods: Romaine lettuce (Lactuca sativa var. longifolia) and control N. benthamiana were transfected by particle bombardment; recombinant protein production dynamics were compared to transformation by infiltration of A. tumefaciens. Production of target proteins in leaf tissue extract was identified by visualization on SDS-PAGE and Western blot. The transgene for the protein of interest, the receptor-binding domain protein (RBD) of SARS-CoV-2, was labeled with a His-tag and inserted into a geminiviral vector for rapid production in the plant hosts and used in both particle bombardment and agroinfiltration methods for transient expression.

Results: From the studies, it was identified that bombarded tissue displays variations in protein production depending on age of the plant or growth of the leaf. A. tumefaciens infiltration protein dynamics indicate that the genetic construct can efficiently be expressed in both hosts within 24 hours, with N. benthamiana expressing the protein of interest in as little as 4 hours (Fig. 1).

Implications: While the production of RBD is the focus of this study, the platform is designed to be applicable to produce other protein-based therapeutics and diagnostics within 24 hours. RBD is a valuable initial target as it can be used for screening antibodies and additional therapeutics for diagnostics to combat COVID-19, thus the low-cost and large-scale production of this biologic is imperative. The next step for this project would be to construct a viral vector for systemic infection within the host plants for rapid and enhanced bioproduction of target proteins. This technology has the potential to allow for a mobile therapeutic production system that can be rapidly deployed with limited need for external resources, decreased specialized training for processing, and a large reduction in waste compared to current production platforms.

Figure 1. Time study of agroinfiltrated progression of RBD production in N. benthamiana (A) and romaine (B). Lanes 1: protein standard ladder; 2: 4-hours post infiltration (HPI); 3: 8 HPI; 4: 12 HPI; 5: 16 HPI; 6: 20 HPI; 7: wild type plant crude extract; 8: RBD-His positive control, 30ng. Western blot probed with mouse anti-His antibody at a concentration of [1ng/uL] followed by anti-mouse-HRP at [0.1ng/uL].