Base Case Processing
The upstream processing model is shown in Figure 1. It contains five seed train stages prior to the full-scale production bioreactor. Each seed train step represents a ten-fold increase in working volume over the previous step and is operated in batch mode. The culture is inoculated at 10 g fresh weight (FW) per liter (L), then allowed a 10 day growth phase to reach 100 g FW/L. The entire culture is then transferred to the next stage of the seed train to inoculate at 10 g FW/L. At the 20,000 L production bioreactor stage, the culture begins operating semicontinuously in alternating phases of growth and rrBChE expression as shown in Figure 2 for the two-stage semicontinuous operation. The transgenic rice cell culture controls rrBChE expression with an inducible promoter (rice alpha amylase 3D or RAmy3D promoter) that is triggered by sugar starvation. A more in-depth explanation of the two-stage semicontinuous operation is included in the Supplementary Information.
The downstream process flowsheet is shown in Figure 3. The harvested material from the upstream process is composed of mixed rice cell biomass containing 200 mg rrBChE/kg FW and spent expression medium. The medium is separated from the biomass using a decanter, where 95% of the spent medium is removed. The biomass is then mixed in a 1:3 (w/v) ratio with extraction buffer and homogenized in a bead mill. After extraction, the resulting supernatant is clarified using a disk-stack centrifuge followed by two dead-end filtration steps (0.45 µm then 0.2 µm pore size). The clarified extract is concentrated 10-fold before diafiltration with 4 equivalent volumes of buffer in a tangential flow filtration operation and is then passed through a 0.2 µm dead-end filter before the first of two chromatography steps.
An anion exchange resin is first used as a capture chromatography step before being polished using an affinity resin developed specifically for BChE (Hupresin, CHEMFORASE, Rouen, France). The linear flow rate for all chromatography steps for both resin types is 300 cm/hr and they are operated in bind-and-elute mode with the same buffer compositions (20 mM sodium phosphate buffer at pH 7.4) as previously described (Corbin et al., 2018). Each chromatography operation is paired with a holding tank for pooling of elution fractions, which are passed through a 0.2 µm filter before the following unit operation. The pooled, eluted fractions from Hupresin are sent to a final ultrafiltration/diafiltration (UF/DF) operation, where they are concentrated 20-fold, diafiltered into phosphate buffer, and aliquoted in 1 L single-use bioprocess bags stored in totes. The overall rrBChE downstream process recovery, from homogenization through storage, is 57%.
This process was modeled as a single recipe, which involves one seed train, 24 harvest cycles from the production bioreactor, and 24 downstream process cycles. The production bioreactor produces 0.2 kg rrBChE per harvest cycle and 4.8kg rrBChE per entire recipe. After downstream processing, 0.1 kg rrBChE per harvest and 2.7 kg rrBChE per entire recipe are recovered at >95% purity. To reach the target production of 25 kg pure rrBChE per year, 9 recipes are executed to completion.