1. Introduction
Plant-based biopharmaceuticals are an alternative for producing human recombinant proteins. Plants provide several advantages over other expression systems, including the ability to grow in simple, low-cost, and chemically defined medium, efficient protein folding and post-translational modification, and the lack of susceptibility to human pathogens (Huang and McDonald, 2009; Santos et al., 2016). Additionally, plant cell suspension cultures grown in a controlled and sterile bioreactor containing animal-free chemically defined medium in an aseptic environment are compatible with regulatory guidelines such as current good manufacturing practices (cGMP). Taliglucerase alfa (TGA) from transgenic carrot cell suspension cultures grown in disposable bioreactors by Protalix Biotherapeutics (Carmiel, Israel) is the first plant-made recombinant therapeutic protein for human use approved by the FDA in 2012; it is used for enzyme replacement therapy of type 1 Gaucher disease (Fox, 2012; Tekoah et al., 2015). The market price of TGA is 25% lower than its primary competitor, CHO cell-made imiglucerase (Cerezyme®) from Genzyme (Fox, 2012) perhaps indicating the cost-effectiveness of plant cell cultures. Importantly, there is no definitive proof of adverse effects from plant-specific glycans in patients treated with TGA (Shaaltiel and Tekoah, 2016), and the immunogenicity risk associated with anti-plant glycan antibodies in human trials was not apparent (Rup et al., 2017).
Human butyrylcholinesterase (hBChE) circulating in blood plasma is a tetrameric hydrolase enzyme with four ~85 kDa identical monomers containing 9-N -glycosylation sites each (Lockridge, 2015). Human BChE can be used for therapeutic and prophylactic treatment against the exposure to organophosphate (OP) nerve agents and pesticides (Lockridge, 2015). However, the use of purified hBChE from blood plasma for clinical purposes is limited due to its high cost, low yield, and limited availability (Alkanaimsh et al., 2019; Corbin et al., 2016). Among several systems aiming to produce recombinant BChE (rBChE) as a replacement for hBChE, metabolically-regulated transgenic rice cell culture, where the rice α-amylase 3D (RAmy3D) is up-regulated under sugar starvation (Corbin et al., 2016; Huang et al., 1993; Huang et al., 2001; Terashima et al., 1999; Trexler et al., 2002; Trexler et al., 2005) leading to the production of rrBChE, is a promising cost-effective platform most likely due to its low-cost, chemically defined medium. A recent study by our group using technoeconomic analysis showed that the cost of production and purification of rice-made rBChE (rrBChE) was dramatically reduced from the estimated $20,000 for a 400 mg dose from blood plasma to less than $300/dose of pure rrBChE using semicontinuous transgenic rice cell suspensions (Corbin et al., 2020). Here, we demonstrate the use of a 40-L conventional stainless-steel stirred bioreactor (STB) for scaling-up the production of rrBChE in transgenic rice cell suspension cultures under single-stage operations (rice cells naturally deplete sugar in the culture medium without medium exchange) in an uncontrolled dissolved oxygen (DO) condition at a constant aeration rate (Macharoen et al., 2020).
Wild-type plant cell suspension cultures in pilot-scale and industrial scale STBs for the production of plant metabolites have been widely reported (Katō et al., 1972; Noguchi et al., 1977; Schiel and Berlin, 1987; Ulbrich et al., 1985), including Taxus chinensis suspended cells grown in a 75,000-L STB at Phyton Biotech®(Ahrensburg, Germany) for the production of paclitaxel, a potent cancer treatment chemical (Imseng et al., 2014). However, the studies of pilot-scale and commercial scale bioreactors for recombinant therapeutic protein productions from transgenic plant cell cultures are scarce. So far, there is only one report using 20-L and 600-L STBs for the scale-up of recombinant green fluorescence protein-hydrophobin fusion (GFP-HFBI) production in transgenic tobacco BY-2 suspension cell cultures (Reuter et al., 2014). In this study, for the first time, we demonstrate the robustness of metabolically regulated transgenic rice cells that can be maintained over long time periods, 80 days of cultivation with four cycles of cyclical semicontinuous operation in a conventional 40-L pilot-scale STB to produce rrBChE. We also show comparable batch culture results in the 40-L pilot-scale bioreactor compared to the 5-L lab-scale bioreactors (Macharoen et al., 2020) using kLa as a scale-up parameter.