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.