Plant cell suspension cultures have several advantages that make them suitable for the production of recombinant proteins. They can be cultivated under aseptic conditions using classical fermentation technology, they are easy to scale-up for manufacturing, and the regulatory requirements are similar to those established for well-characterized production systems based on microbial and mammalian cells. It is therefore no surprise that taliglucerase alfa (Elelyso®)—the first licensed recombinant pharmaceutical protein derived from plants—is produced in plant cell suspension cultures. But despite this breakthrough, plant cells are still largely neglected compared to transgenic plants and the more recent plant-based transient expression systems. Here, we revisit plant cell suspension cultures and highlight recent developments in the field that show how the rise of plant cells parallels that of Chinese hamster ovary cells, currently the most widespread and successful manufacturing platform for biologics. These developments include medium optimization, process engineering, statistical experimental designs, scale-up/scale-down models, and process analytical technologies. Significant yield increases for diverse target proteins will encourage a gold rush to adopt plant cells as a platform technology, and the first indications of this breakthrough are already on the horizon.

Plant cell cultures have been used as expression hosts for recombinant proteins for over two decades. The quality of plant cell culture-produced proteins such as full-size monoclonal antibodies has been shown to be excellent in terms of protein folding and binding activity, but the productivity and yield fell short of what was achieved using mammalian cell culture, in which the key to gram-per-liter expression levels was strain selection and medium/process optimization. We carried out an extensive media analysis and optimization for the production of the full-size human anti-HIV antibody 2G12 in N. tabacum cv. BY-2. Nitrogen source and availability was found to be one key factor for the volumetric productivity of plant cell cultures. Increased amounts of nitrate in the culture medium had a dramatic impact on protein yields, resulting in a 10-20-fold increase in product accumulation through a combination of enhanced secretion and higher stability. The results were scalable from shake flasks to stirred-tank bioreactors, where the maximum yield per cultivation volume was 8 mg L(-1) over 7 days. During the stationary phase, antibody levels were 150-fold higher in nitrogen-enriched medium compared to standard medium. The enhanced medium appeared not to affect antibody quality and activity, as determined by Western blots, surface plasmon resonance binding assays and N-glycan analysis.

Summary Industrial plant biotechnology applications include the production of sustainable fuels, complex metabolites and recombinant proteins, but process development can be impaired by a lack of reliable and scalable screening methods. Here, we describe a rapid and versatile expression system which involves the infusion of Agrobacterium tumefaciens into three‐dimensional, porous plant cell aggregates deprived of cultivation medium, which we have termed plant cell packs ( PCP s). This approach is compatible with different plant species such as Nicotiana tabacum BY 2, Nicotiana benthamiana or Daucus carota and 10‐times more effective than transient expression in liquid plant cell culture. We found that the expression of several proteins was similar in PCP s and intact plants, for example, 47 and 55 mg/kg for antibody 2G12 expressed in BY 2 PCP s and N. tabacum plants respectively. Additionally, the expression of specific enzymes can either increase the content of natural plant metabolites or be used to synthesize novel small molecules in the PCP s. The PCP method is currently scalable from a microtiter plate format suitable for high‐throughput screening to 150‐mL columns suitable for initial product preparation. It therefore combined the speed of transient expression in plants with the throughput of microbial screening systems. Plant cell packs therefore provide a convenient new platform for synthetic biology approaches, metabolic engineering and conventional recombinant protein expression techniques that require the multiplex analysis of several dozen up to hundreds of constructs for efficient product and process development.

Plants provide an advantageous expression platform for biopharmaceutical proteins because of their low pathogen burden and potential for inexpensive, large-scale production. However, the purification of target proteins can be challenging due to issues with extraction, the removal of host cell proteins (HCPs), and low expression levels. The heat treatment of crude extracts can reduce the quantity of HCPs by precipitation thus increasing the purity of the target protein and streamlining downstream purification. In the overall context of downstream process (DSP) development for plant-derived malaria vaccine candidates, we applied a design-of-experiments approach to enhance HCP precipitation from Nicotiana benthamiana extracts generated after transient expression, using temperatures in the 20–80°C range, pH values of 3.0–8.0 and incubation times of 0–60 min. We also investigated the recovery of two protein-based malaria vaccine candidates under these conditions and determined their stability in the heat-treated extract while it was maintained at room temperature for 24 h. The heat precipitation of HCPs was also carried out by blanching intact plants in water or buffer prior to extraction in a blender. Our data show that all the heat precipitation methods reduced the amount of HCP in the crude plant extracts by more than 80%, simplifying the subsequent DSP steps. Furthermore, when the heat treatment was performed at 80°C rather than 65°C, both malaria vaccine candidates were more stable after extraction and the recovery of both proteins increased by more than 30%.