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.