Production of pharmaceutical‐grade recombinant aprotinin and a monoclonal antibody product using plant‐based transient expression systems

Abstract

SummaryPlants have been proposed as an attractive alternative for pharmaceutical protein production to current mammalian or microbial cell-based systems. Eukaryotic protein processing coupled with reduced production costs and low risk for mammalian pathogen contamination and other impurities have led many to predict that agricultural systems may offer the next wave for pharmaceutical product production. However, for this to become a reality, the quality of products produced at a relevant scale must equal or exceed the predetermined release criteria of identity, purity, potency and safety as required by pharmaceutical regulatory agencies. In this article, the ability of transient plant virus expression systems to produce a wide range of products at high purity and activity is reviewed. The production of different recombinant proteins is described along with comparisons with established standards, including high purity, specific activity and promising preclinical outcomes. Adaptation of transient plant virus systems to large-scale manufacturing formats required development of virus particle and Agrobacterium inoculation methods. One transient plant system case study illustrates the properties of greenhouse and field-produced recombinant aprotinin compared with an US Food and Drug Administration-approved pharmaceutical product and found them to be highly comparable in all properties evaluated. A second transient plant system case study demonstrates a fully functional monoclonal antibody conforming to release specifications. In conclusion, the production capacity of large quantities of recombinant protein offered by transient plant expression systems, coupled with robust downstream purification approaches, offers a promising solution to recombinant protein production that compares favourably to cell-based systems in scale, cost and quality.

Transient Virus Expression Systems for Recombinant Protein Expression in Dicot- and Monocotyledonous Plants

“…Several products have been purified using differential separation and standard chromatographic separations and tested in various model systems of human disease. TMV-based vectors, especially the well-developed GENEWARE ® system, have been used to express a large range of recombinant proteins under research and Current Good Manufacturing Practice (cGMP) compliance [14]. One class of products has been successfully tested in human clinical trials, as described below.…”

“…Each vector replicates independently and expresses mAb chains in the same cells that self-assemble functional mAbs at yields up to 1 g/kg fresh weight [33,54,55]. These vectors have been used for efficient large scale production of multi-gram batches of mAbs under Current Good Manufacturing Practices that have been tested in several challenge model systems, including non-human primates [14,74].…”

“…Several reviews have provided surveys of these products [12,14]. In general, expression results were obtained in Nicotiana hosts and proteins were extracted from leaf tissues using total homogenization and clarification methods or leaf infiltration and isolation of interstitial fluids [46,47].…”

“…The handling of plant biomass and its initial extraction requires unique biomanufacturing solutions [14]. Virus vector expression systems offer significant advantages at considerably reduced costs to current cell-based manufacturing systems, such as employed by Protalix and Dow AgroSciences, while avoiding concerns associated with stable plant transformation [29,30].…”

“…The rapid replication cycle of the virus systems provided amplification of messenger RNA and the resulting proteins providing for a "burst" of recombinant expression that can provide impressive yields (reviewed in [12][13][14][15]). While these early vectors were useful in plant cell systems to produce recombinant protein products with potential market value [16], these early systems could not support large scale manufacturing nor did they exploit the advantages of agriculture to provide cost-effective products.…”