3.1.3 Production, purity, and productivity of rrBChE
With the RAmy3D signal peptide in the gene construct, we previously reported a significant amount rrBChE in both culture medium and rice cell aggregates in the screening process (Corbin et al., 2016) and a 5-L STB using single-stage batch culture and simplified bioreactor processing (Macharoen et al., 2020). Figure 1c shows the accumulation of culture medium and cell-associated rrBChE during the induction phases in cycles 1-4. Total active rrBChE reached its maximum level at 3-day post induction (dpi) in cycle 1 but 4 dpi or later in cycles 2-4. We stopped the induction phases in cycles 2-4 at 4 dpi to be consistent with cycle 1 and to not excessively exhaust rice cell metabolic activity (Figure S1a) since allowing rice cells one day longer in the induction phase could impact the cell growth in the subsequent cycle. The maximum total active rrBChE in cycles 1-4 were comparable at 58.2 ± 13.4, 46.1± 6.7, 49.8 ± 5.5, and 48.2 ± 6.4 µg /g FW, respectively (Table 2), suggesting that the transgenic rice cells are stable and robust over 82 days of cultivation in a cyclical semicontinuous operation. While Gagnon et al. recently reported a constant steady-state volumetric productivity of IgG from CHO-K1 cells in a 100-L continuous stirred-tank bioreactor (CSTR) linked with a perfusion bioreactor operation for 83 days (Gagnon et al., 2019), here, we demonstrate the reproducible production of a plant-made therapeutic recombinant protein in a 40-L STB using simplified-bioreactor processing over 82 days of semicontinuous operation. To best of our knowledge, this is the longest plant cell culture bioreactor run.
The purity of rrBChE is determined by the ratio of the concentration of rrBChE (mg/L) to the total soluble protein (TSP; mg/L) multiplied by 100 to convert to a percent of total protein. The maximum purities of cell-associated rrBChE in cycles 1-4 were 0.91 ± 0.07%, 0.69 ± 0.03%, 0.80 ± 0.06%, and 0.79 ± 0.06%, respectively (Table 2), while our previous study reported 0.27-0.55% g rrBChE/g TSP (Corbin et al., 2016). Improvement in the purity of rrBChE in the crude extract may facilitate the downstream processing. Maximum purities of culture medium rrBChE were also quite high at 0.80 ± 0.06%, 0.72 ± 0.08%, and 0.61 ± 0.01% in cycles 2, 3, and 4, respectively (not available in cycle 1; Table 2). It could be implied that the greater proportion of culture medium rrBChE than cell-associated rrBChE in cycle 2 at 4 dpi (Figure 1c) resulted in higher maximum purity of rrBChE in the culture medium than in the cell extract, and vice versa in cycle 4, whereas the maximum purities of culture medium and cell-associated rrBChE in cycle 3 were comparable due to similar maximum rrBChE levels in both sources.
The volumetric productivities in cycles 1 and 2 using full strength sucrose NB+S were comparable at 76 ± 25 and 92 ± 15 µg L-1day-1, respectively (Table 2). Using half-strength sucrose (NB+0.5xS) in cycles 3 and 4 also gave comparable volumetric productivities at 226 ± 26 and 271 ± 48 µg L-1day-1, respectively (Table 2), which are at least 2.4-fold higher than cycles 1 and 2 and at least 1.2-fold higher than our previous report, 36 ± 8 µg L-1day-1 in cycle 1 and 184 ± 17 µg L-1 day-1 in cycle 2 (Corbin et al., 2016). The specific productivities in cycles 1-4 were 20.5 ± 6.7, 24.0 ± 3.8, 42.4 ± 5.1 and 41.1 ± 7.6 µg g DW-1day-1, respectively, indicating that using NB+0.5xS improved specific productivity at least 1.7-fold. In other words, the use of half-strength sucrose in the cyclical semicontinuous single-stage culture operation with the simplified bioreactor processes improves volumetric productivity and specific productivity primarily due to shortening of growth phase. Altogether, we demonstrate that our transgenic rice cell suspensions cultivated in a pilot-scale STB are robust and stable over long term (82 days) cyclical semicontinuous operation in which cleaning, sterilizing and inoculating are minimized resulting in saving the cost of rrBChE production in which our recent technoeconomic analysis showed that the overall cost of rrBChE production and purification has significantly reduced in semicontinuous operation compared to batch operation (Corbin et al., 2020).