Despite the tremendous progress in the research and development of plant-made vaccines, currently there is still no licensed plant-derived vaccine for human use. The history and current status of plant-made NVCP as a vaccine candidate clearly illustrates the progress and remaining challenges in the PMP field in general. The first attempt of expressing NVCP in plants was performed with stable transgenic plants (Zhang, et al. 2006
). As for other early PMPs expressed in transgenic plants, it took several months to a year to generate and select NVCP-expressing plant lines and the yield of NVCP was very low (~10-20μg/g tissue fresh weight). We have successfully resolved these issues associated with stable transgenic plants by using transient expression systems based on plant virus replicons. For example, our previous reports indicate that these transient expression systems allow us to achieve high-level NVCP accumulation (~400 μg/g LFW) within 1-2 weeks of vector infiltration. However, like for other PMPs, there are remaining technical and regulatory hurdles that must be overcome before plant-derived NVCP can be accepted as a licensed vaccine product. These challenges include the development of scalable downstream processing procedures to recover NVCP VLP plant leaves, the compliance of FDA cGMP regulation or the operation, and ultimately the demonstration of the identity, purity, potency and safety of plant-made NVCP VLP vaccine that meet the required standards of regulatory agencies. In this study, we tackled these remaining challenges and successfully demonstrated the ability of using plants to produce pharmaceutical grade vaccines under cGMP guidelines at multiple gram scales. Our results specifically show that (1) an efficient, scalable purification process was established for NVCP VLP to address the scalability issue (2) we successfully operated the upstream and downstream NVCP production process under cGMP guidelines, and (3) The identity, purity, potency and safety of plant-derived NVCP VLP produced at scale meet the preset release specifications. This material is being tested in a Phase I human clinical trial. This research provides the first report of producing a plant-derived vaccine at scale under cGMP regulations in an academic setting and an important step for PMPs to become a commercial reality.
Downstream processing is an important component of pharmaceutical protein production. As expression NVCP levels increase, purification costs become an increasingly significant proportion (>80%) of the total cost of NVCP and other vaccine production (Chen 2011
; Chen 2011
; Faye and Gomord 2010
; Rybicki 2010
). Thus, the development of methods that can efficiently purify NVCP from plant tissue at large-scale is essential for plant expression system to be considered a viable production platform candidate and for the full realization of economic effectiveness of PMP technology.
Traditionally, NVLP and other VLP have been recovered based only on a few variations of centrifugation and precipitation methods (Jiang, et al. 1992
; Prasad, et al. 2000
; Santi, et al. 2008
). VLP are routinely purified on the basis of their size and density using gradient ultracentrifugation techniques with sucrose and cesium chloride (CsCl) (Estes 2004
). Ultracentrifugation and density gradient methods are effective isolation tools for isolating bench-scale quantities of NVLP for research. However, they are not practical for large-scale commercial vaccine manufacture because they are difficult to scale up, time-consuming, and produce poor yields (Rolland, et al. 2001
). The unique properties of plant tissues also require specific considerations for purification method development. For example, plants produce more solid debris, therefore, direct loading of plant extracts onto chromatographic columns often causes resin fouling and poor binding of NVCP to the resin (Chen 2008
). The carbon assimilation enzyme RuBisCo is the major contaminating protein in plant leaves, therefore, should be removed from the extract prior to the chromatographic steps.
This research has allowed us to successfully develop an extraction and purification process that effectively circumvents the scale limitations of traditional VLP purification methods and allow efficiently purification of NVCP from N. benthamiana
leaves. Our results show that the low-pH precipitation step effectively eliminates plant solid debris and RuBisCo from the downstream processing feed stream, therefore, effectively resolves the specifically challenges presented by the plant tissue. We also explored column chromatography for its ability to achieve high purity and recovery rates, and its facile adaptability for scaling up and cGMP manufacturing (Chen 2011
). Our results showed that DEAE anion-exchange chromatography is an optimal method for the scalable purification of NVCP VLP. Collectively, we demonstrated that our downstream process that bases on low pH precipitation and a DEAE chromatographic step is highly scalable, and can produce fully assembled VLP with high product purity (>95%), yet eliminates the laborious and time-consuming steps of sucrose and CsCl gradients. This newly-developed downstream process will increase manufacturing productivity, reduce cost of operations, enhance scalability for processing large volumes of NVLP preparations with high yield, and preserve the stability of the VLP. Since precipitation and DEAE chromatography have been successfully employed for cGMP production of licensed pharmaceutical products, this ensures the compliance of our manufacturing procedures with the FDA's cGMP regulations. SOPs based on this processing scheme have been established and used in cGMP production of NVLP for a Phase I human clinical trial.
The demonstration of regulatory compliance of the production process and product quality is another remaining challenge presented to the PMP field (Chen 2011
; Faye and Gomord 2010
). This critical issue has been difficult to resolve due to the lack of interest from pharmaceutical industry for “unproven” technology and the unusual high financial demand for academia. As the first step, we have successfully established cGMP-compliant greenhouses, a central bioprocessing suite, and a QA/QC facility. The design and construction of these facilities have been certified in full compliance of cGMP regulations for pharmaceutical products of both US and EU markets. The establishment of these dedicated facilities laid the foundation for producing NVCP VLP vaccine that can be use for human clinical trials and its future commercial application. We next established three A. tumefaciens
master stock cell banks, and an N benthamiana
seed bank and their corresponding working banks for providing cGMP upstream materials. These banks have been extensively characterized for their identity and stability, and they have been shown to meet the required specifications. We also established the optimal conditions for large-scale biomass generation and infiltration with A. tumefaciens
strains that are carrying the NVCP expression vectors. As we discussed earlier, the downstream processing procedure developed from this study is not only effective and scalable, but also uses materials and techniques that are routinely used for the production of licensed pharmaceutical products, therefore, is fully in compliance with cGMP regulations. Our study also demonstrated the low-cost nature of our plant-based production system. For example, the material cost for generating the plant biomass for one gram of NVCP is approximately $15 at our production scale. In contrast, the material cost (protein-free culture media and a disposable bioreactor) would be at least $390 if the biomass for the same amount of NVCP were generated in a mammalian cell culture. Furthermore, the capital investment required for building our greenhouses is significant less than that of an equivalent mammalian cell culture fermentation facility (Elbehri 2005
In addition to show the cGMP-compliance of the production process, we also aim to demonstrate that NVCP VLP produced by this process exhibits qualities that meet the requirements of regulatory agencies. First, we identified and established analytical assays for in-process samples and the final product. Results indicate that the identity and structure of plant-derived NVCP VLP is indistinguishable from the insect-cell derived NVCP VLP standard. The purity of the final purified NVCP VLP is >95% and was shown to effectively induce potent NV specific immune responses in mice (Velasquez, et al. 2011
). Therefore, the purity, potency and other characteristics also meet the preset release specifications for Phase I human clinical trials. As part of the QMS, we have established a documentation system that documents all aspects of NVCP production process. The dossier includes SOPs for facility validation, bacterial and seed bank characterizations, operations for upstream and downstream processing, and QA/QC analytical procedures.
In summary, we demonstrate that a vaccine candidate based on NVCP VLP can be successfully produced in plants at scale and in compliance of FDA's cGMP regulations. The plant-derived NVCP VLP demonstrated the quality that meets the preset release specifications and is being tested in a Phase I human clinical trial. Such demonstration has brought us one step closer to overcome the remaining challenges of the PMP field, and illustrated the feasibility of using plants as a cost-effective and scalable production platform for commercial vaccines. The establishment of cGMP-compliant PMP production facilities and a documentation dossier in an academic setting will facilitate translational research for other PMP products.