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.