A juice extractor can simplify the downstream processing of plant-derived biopharmaceutical proteins compared to blade-based homogenizers
Introduction
The advantages of plant-based expression systems for the manufacture of biopharmaceutical proteins include low costs, ease of process scale-up and product safety [1], [2], [3]. The relevance of product safety became evident when the Israel-based company Protalix Biotherapeutics began producing glucocerebrosidase (for the treatment of Gaucher disease) in carrot cells [4] and outcompeted the previous market leader Genzyme because the latter had to cease their production due to virus contamination [5], [6]. The upstream capacity can be increased easily by providing larger greenhouses or by open field cultivation. In contrast, downstream processing (DSP) is a major cost factor for plant-based production systems [7], [8], mainly reflecting the large number of unit operations required for clarification due to the high particle burden in extracts prepared by blade-based homogenizers, hereafter described as blenders [7], [9], [10]. The scalability of DSP equipment can also be limited by technical and economic constraints, e.g. the size of a blender is restricted by the power and maximum speed of the motor supplied with the device, which depends on the cost/benefit ratio to the manufacturer. These constraints ultimately reduce the economy of scale because scale-up becomes associated with increasing costs. Even before technical constraints apply, numbering-up may become necessary to prevent lag times and delays during processing that result from discontinuous blending steps. To avoid these drawbacks, we tested the ability of a continuously operational juice extractor to extract protein-containing plant sap from transgenic tobacco leaves expressing two model proteins (the monoclonal antibody 2G12 and the fluorescent reporter protein DsRed), thereby integrating extraction and initial solid–liquid separation in a single device. We investigated the compatibility of this method with blanching and flocculation, two techniques that facilitate the purification of recombinant proteins and the clarification of plant extracts, respectively [11], [12]. We also used a previously described cost model to evaluate the impact of juice extraction on production costs compared to the use of a conventional blender [8].
Section snippets
Plant cultivation and protein extraction
Transgenic tobacco plants expressing monoclonal antibody 2G12 and the fluorescent protein DsRed were cultivated in a greenhouse as previously described [10]. Leaves were harvested 50 days after seeding, and the intact leaves were blanched as previously described [11] if this step was included in the design-of-experiments (DoE) setup. Proteins were then released from the tissue using either a blender [10], or a bench-top juice extractor (8006 Nutrition Center Masticating Juicer, Omega,
Juicer extraction increases the protein concentration but reduces the overall yield
Transgenic tobacco leaves expressing 2G12 and DsRed were harvested from greenhouse-grown plants as previously described [10]. The concentrations of TSP, DsRed and 2G12 in the extract were 2.9, 2.4 and 1.4 times higher, respectively, when juice extraction was used instead of blending (Fig. 1a). Furthermore, the extract volume was reduced by 80% when juice extraction was used instead of blending. In contrast, the yield per unit biomass was 40–65% lower for juice extraction (Fig. 1b) and was not
A juicer setup streamlines extraction but reduces protein recovery
The higher protein concentrations in leaf extracts produced by juice extraction are advantageous, particularly for the purification of low-abundance proteins, because initial target enrichment can be as challenging as the final polishing steps [16]. The elevated concentrations reflected the absence of any diluting extraction buffer, which also reduced the time required for buffer preparation and thus labor and consumables costs. Furthermore, the lower extract volume facilitates process-scale
Conclusions
In summary, juice extraction offers an economical alternative to blenders, especially for target proteins with low expression levels, because the costs for DSP consumables and labor can be lowered by integrating the extraction and solid–liquid separation steps and reducing the extract volume that must be processed. Even for products expressed at high levels, juicers offer several advantages over blenders because of their continuous mode of operation and the larger amount of biomass they can
Acknowledgements
The authors acknowledge Dr. Thomas Rademacher for providing the transgenic tobacco seeds and Ibrahim Al Amedi for cultivating the tobacco plants. We wish to thank Dr. Richard M Twyman for editorial assistance. This work was funded in part by the European Research Council Advanced Grant “Future-Pharma”, proposal number 269110, and the Fraunhofer-Zukunftsstiftung (Fraunhofer Future Foundation). The authors have no conflict of interest to declare.
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