Discussion
The techno-economic process simulation in this work demonstrates the
potential cost-savings for production of a moderate volume drug
substance in a two-stage semicontinuously-operated plant cell suspension
culture. It also illustrates viability of batch-mode operation of plant
cell suspension culture for commercial manufacturing and highlights
significant differences in facility design between these two modes of
operation. This simulation uses recombinant BChE as a model product,
which has long been a challenging and costly molecule to produce but
could represent any complex biologic molecule needed at moderate
production levels (10’s of kg per year).
In this analysis, two-stage semicontinuous operation yields 4% lower
COGS than two-stage batch operation in the CMO scenario, 11% lower in
the new facility scenario, and 9% lower in the new facility scenario
excluding depreciation costs. Based on the product of interest and the
stability of the product in the cell culture environment (e.g.
resistance to protease degradation, pH denaturation), semicontinuous
operation may provide significant benefits over batch operation which
are not captured in this model since the product was assumed to be
cell-associated.
We found that semicontinuous operation may be particularly favorable for
facilities with high upstream costs; the economic benefits of
semicontinuous operation realized in these models are in the 31- 63%
lower upstream operating costs. As compared to two-stage batch
operation, there are 100 fewer executions of the seed train per year.
The higher starting biomass density in the “steady state”
semicontinuous growth phase results in production reactor cycles every 6
days as opposed to every 13 days in batch. However, raw material costs
are 58% higher than in two-stage batch operation. Media requirements
are 97% volumetrically higher in semicontinuous operation wherein a
full 20,000 L of each growth and expression media are consumed for a
return on only 10,000 L of culture harvested in each cycle.
Interestingly, the CIP costs of semicontinuous operation are 70% higher
than the batch case despite 100 fewer executions of production
bioreactor cleaning. This is due to the lower harvest size of
semicontinuous (10,000 L) compared to batch (20,000 L) resulting in
twice as many annual downstream processing batches.
We demonstrate that a simple induction strategy to let the sugar in the
media naturally deplete could provide additional benefits to batch
operation, reducing COGS to within 1% of that of two-stage
semicontinuous operation. However, there is appreciable uncertainty as
to whether the assumptions of comparable growth and expression kinetics
between the medium exchange and simple induction strategies are
appropriate. Simple induction is a promising avenue for research and
development to improve manufacturing of rrBChE, or other recombinant
products under the control of the RAmy3D promoter, particularly in case
gravity sedimentation and medium exchange in large-scale conventional
bioreactors may be difficult to implement. The benefit of simple
induction with the Ramy3D promoter would also be expected to increase
substantially with the cost of culture media.
The semicontinuous process modeled here has some similarities and
differences to the one used by Protalix for production of their product
Elelyso®, an orphan drug used for treatment of
Gaucher’s disease. Elelyso® is produced
intracellularly in carrot root cell culture and uses a semicontinuous
process (Grabowski, Golembo, & Shaaltiel, 2014). Thus, Protalix’s
process provides an additional reference point to justify the
feasibility of the process described in this model. Another major hurdle
overcome by Protalix was initial establishment of the regulatory pathway
for plant-made recombinant human biologics. The mammalian viral
contamination-related shutdown of a competing mammalian cell culture
production facility, along with the competing product’s market
exclusivity at the time, served to accelerate regulatory evaluation of
Protalix’s product and establish a more trusting and favorable view of
plant-made pharmaceuticals (Mor, 2015).
Despite this, a few hurdles remain for mainstream adoption of plant cell
culture technologies. Pharmaceutical manufacturing processes require
stably preserved cell-banking to supply a well-defined starting material
and prevent genetic drift in the culture. Cryopreservation techniques
have been established for plant cell cultures (Kwon, Jeong, Choi, Pak,
& Kim, 2013; Mustafa, de Winter, van Iren, & Verpoorte, 2011), but
there is no protocol that can be universally applied to all species
(Santos, Abranches, Fischer, Sack, & Holland, 2016). There is also an
ongoing literature debate as to the potential immunogenicity of plant
glycan structures. While some studies indicate a potential for an immune
response to plant glycans on human therapeutics (Chung et al., 2008),
several other studies of actual in vivo administration indicate
that this does not occur in practice (Rup et al., 2017; Shaaltiel &
Tekoah, 2016). However, the difficulty in proving that something does
not occur will likely continue to challenge regulatory approval and
mainstream acceptance of this technology.
For BChE specifically, this study provides manufacturing models which
demonstrate a substantial improvement over current production technology
in terms of product safety, reliability, and cost. To date, no form of
BChE has been approved for therapeutic use in humans. Recombinant hBChE
produced in transgenic goats (Protexia®, product by
PharmAthene, now Altimmune) reached Phase I clinical trials
(ClinicalTrials.gov Identifier: NCT00744146), and results indicated that
it was well-tolerated (Jurchison, 2009). However, the project was
discontinued after project funding expired in 2010 and the production
facilities were sold (PharmAthene, 2015). No production cost analysis
was reported. Aside from Protexia®, the most
well-developed technology for BChE production involves purification of
hBChE from human blood plasma. This product, too, has had success in
Phase I clinical trials (ClinicalTrials.gov Identifier: NCT00333528).
Though many technical aspects of pilot scale purification of hBChE have
been documented (Saxena, Tipparaju, Luo, & Doctor, 2010), to our
knowledge, no cost analyses have been publicly reported for this process
either. However, in February of 2012, the Defense Advanced Research
Projects Agency (DARPA 2012) released a call for research proposals
titled “Butyrylcholinesterase Expression in Plants.” In this document,
DARPA cites a BChE dose size of 400 mg and estimates a cost per dose of
hBChE as ~$10,000 (DARPA, 2012), though no references
are given for this value. In addition to the extremely high cost of
plasma-derived hBChE, availability is extremely limited: the entire
theoretically available blood supply in the US could only produce 1 to 2
kg of pure hBChE, or 2,500 to 5,000 doses, per year (Ashani, 2000).
Therefore, cost-effective production of recombinant BChE has been a
long-standing goal. Our models suggest that plant cell suspension
culture manufacturing has the potential to reduce the COGS to less than
3% of the 2012 DARPA manufacturing estimate.
To that end, we have not only studied rrBChE production in rice cell
culture, but have also evaluated production of recombinant BChE using
transient expression in N. benthamiana plants through
agroinfiltration (Alkanaimsh et al., 2016), and published a
techno-economic analysis of this system (Tusé et al., 2014). In this
work, a single dose of recombinant BChE is estimated to cost $234 when
produced in an existing facility and $474 when a new facility is
constructed. Overall, these values are lower than, but comparable to,
our findings for rrBChE production in rice cell. However, the two models
differ in several important ways. Tusé et al. (2014) assume an
expression level of 500 mg BChE/kg FW of plant tissue, which is
significantly higher projection than what is assumed in the rice cell
culture model. The Tusé et al. (2014) model assumes a low downstream
recovery of 20%, which is supported by literature surrounding
purification of BChE from N. benthamiana whole plant systems
(Geyer et al., 2010). Much of the BChE loss occurs in the initial
recovery steps; assumptions regarding the costs and binding capacities
of the chromatography steps are comparable to this model.
While these two plant-based systems appear to give similar product
costs, the choice of expression host depends on other factors, in
addition to cost. Transient expression avoids the long lead times
associated with development of a transgenic line, which can be essential
in rapid response applications. However, transgenic bioreactor-based
systems benefit from increased process controllability, reproducibility,
and compatibility with existing infrastructure and regulatory
guidelines. For BChE and similar targets, a combination of both these
strategies may prove beneficial in meeting global defense needs for both
stockpiling and rapid response situations. For other products, such as
orphan drugs to treat rare disease, cell culture systems may be
preferred for the regulatory process familiarity.