Critical Analysis of the Commercial Potential of Plants for the Production of Recombinant Proteins

Abstract

Over the last three decades, the expression of recombinant proteins in plants and plant cells has been promoted as an alternative cost-effective production platform. However, the market is still dominated by prokaryotic and mammalian expression systems, the former offering high production capacity at a low cost, and the latter favored for the production of complex biopharmaceutical products. Although plant systems are now gaining widespread acceptance as a platform for the larger-scale production of recombinant proteins, there is still resistance to commercial uptake. This partly reflects the relatively low yields achieved in plants, as well as inconsistent product quality and difficulties with larger-scale downstream processing. Furthermore, there are only a few cases in which plants have demonstrated economic advantages compared to established and approved commercial processes, so industry is reluctant to switch to plant-based production. Nevertheless, some plant-derived proteins for research or cosmetic/pharmaceutical applications have reached the market, showing that plants can excel as a competitive production platform in some niche areas. Here, we discuss the strengths of plant expression systems for specific applications, but mainly address the bottlenecks that must be overcome before plants can compete with conventional systems, enabling the future commercial utilization of plants for the production of valuable proteins.

Keywords: CHO cells; Pseudomonas fluorescens; cell-free biosynthesis; molecular farming; plant-made pharmaceuticals.

Figure 1

Production of a 19-kDa phenylalanine-free protein in P. fluorescens. After extraction and the removal of cell debris, the product was purified from the clarified extract by single-stage immobilized metal-ion affinity chromatography. Due to the high protein concentration, representative samples of the load and elution fraction were analyzed by SDS-PAGE at dilution ratios of 1:80, 1:160, and 1:320. The strong band at ~32 kDa represents the phenylalanine-free protein – the larger size of the protein probably reflects a combination of its high surface charge and generally high stability, which prevents full de-folding during sample preparation. This example shows the high efficiency of the purification step: only traces of other proteins are found in addition to the target protein in the final elution fraction, corresponding to a purity of >95%.

Figure 2

Comparison of different eukaryotic cell-free expression systems with the tobacco BY-2 cell-free lysate (BYL). Cell-free reaction modes (coupled batch or continuous feeding of substrates and removal of inhibitory byproducts by dialysis) are indicated. The data represent the highest protein yields reported for each system including the information which target protein has been used to determine maximum product levels and are sourced from the following references and company information: rabbit reticulocyte lysate (RLL, luciferase) (promega.de), Pichia pastoris yeast extract (YeE, human serum albumin) (Aw and Polizzi, 2018), CHO cell extract (luciferase) (Brodel et al., 2014), Spodoptera frugiperda insect cell extract (ICE, procaspase 3) (promega.de), HeLa cell extract (HCE, model protein not indicated) (thermofisher.com), wheat germ extract (WGE, model protein not indicated) (biotechrabbit.com), Leishmania tarentolae extract (LTE, eGFP) (jenabioscience.com), HCEd (model protein not indicated) (thermofisher.com), CHOd (epidermal growth factor-eYFP fusion), and WGEd (model protein not indicated) (biotechrabbit.com), and BYL (eYFP).

Figure 3

SDS-PAGE analysis of total soluble proteins in N. benthamiana leaf extracts (A), tobacco BY-2 culture medium (B), and CHO culture medium (C) without (left) and with (right) secreted monoclonal antibody (100 μg/ml). The positions of the antibody light chain (LC) and heavy chain (HC) are indicated. M: PageRuler Pre-stained Protein Marker.

Figure 4

Costs for the production and purification of the human M12 antibody produced in transgenic tobacco plants in a contained greenhouse. The duration of the whole process from sowing to analysis of the purified protein was 10 weeks. We processed 200 kg of leaf material to provide 77 g of highly-purified M12 antibody. FLW, fresh leaf weight; DSP, downstream processing.