Bioprocessing of plant-derived virus-like particles of Norwalk virus capsid protein under current Good Manufacture Practice regulations

Abstract

Despite the success in expressing a variety of subunit vaccine proteins in plants and the recent stride in improving vaccine accumulation levels by transient expression systems, there is still no plant-derived vaccine that has been licensed for human use. The lack of commercial success of plant-made vaccines lies in several technical and regulatory barriers that remain to be overcome. These challenges include the lack of scalable downstream processing procedures, the uncertainty of regulatory compliance of production processes, and the lack of demonstration of plant-derived products that meet the required standards of regulatory agencies in identity, purity, potency and safety. In this study, we addressed these remaining challenges and successfully demonstrate the ability of using plants to produce a pharmaceutical grade Norwalk virus (NV) vaccine under current Good Manufacture Practice (cGMP) guidelines at multiple gram scales. Our results demonstrate that an efficient and scalable extraction and purification scheme can established for processing virus-like particles (VLP) of NV capsid protein (NVCP). We successfully operated the upstream and downstream NVCP production processes under cGMP regulations. Furthermore, plant-derived NVCP VLP demonstrates the identity, purity, potency and safety that 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 plant-produced vaccines to become a commercial reality.

Agroinfiltration as an Effective and Scalable Strategy of Gene Delivery for Production of Pharmaceutical Proteins

“…Our laboratory and others have extensively employed agroinfiltration, for the transient expression of pharmaceutical proteins in several plant species (Table 1). While agroinfiltration can be performed on most plant materials, we found that the specific condition of plant material is the most likely factor in causing variability of recombinant protein yield between production batches and between laboratories [5]. In fact, the physiological state and the developmental stage of plants significantly influence their competency in producing the target protein through agroinfiltration.…”

“…Finally, this system integrates the posttranslational processing capacity of eukaryotic plant cells, for producing complex proteins. Along with others, our laboratory has expressed numerous proteins from small subunit vaccines to large immune complexes, with this system [5,6,28,29]. The collective results the delivered DNA [3].…”

“…The collective results the delivered DNA [3]. The ability to deliver vectors to a majority of the have demonstrated that this system is robust and drives high levels of target protein accumulation up to 1 mg per gram of leaf fresh weight (LFW), within 7 to 10 days after agroinfiltration [5,6,24,28,29]. This system retains the speed and expression amplification of viral vectors, while gaining the flexibility of nuclear gene expression.…”

“…Plants offer several advantages over current bioreactor-based platforms, for protein production. For example, plant-made biologics cost significantly less than current cell culture-based systems because mammalian cell cultures require considerable startup investment and expensive growth media [5]. Plants also outpace the scalability of other expression systems, as recombinant protein-expressing biomass can be produced on an agricultural scale, without the need to build duplicated bioreactors and associated facilities [2].…”

Expression, purification, and characterization of theNecator americanusaspartic protease-1 (Na-APR-1 (M74)) antigen, a component of the bivalent human hookworm vaccine

“…Facilities which utilize controlled plant growth and infiltration processes for heterologous protein expression in whole plants that conform to current Good Manufacturing Practice (cGMP) guidelines have been described. 32,33 Although our system is using a non-transgenic plant which does not require the permits and strict containment plans required for transgenic plants, 31 the system has yet to be tested at commercial manufacturing scale. With plant expression technology, there are additional hurdles which must be overcome including issues related to safety of potential contaminating proteins, cultivation, and downstream processing.…”