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 be established for processing virus-like particles (VLPs) 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.

Fig. 1

Facilities for cGMP production of NVCP VLP vaccine and other plant-made pharmaceuticals. (a) The 3600 ft² BSL-2 greenhouse facility for plant biomass generation and NVCP expression. (b) The Plant Biopharmaceutical Center that houses the Central Bioprocessing Suite, the QA/QC laboratory, and the Process Development laboratory. (c) The floor plan of the Central Bioprocessing Suite. The arrows indicate the unidirectional flow of in-process materials (green), purified final product (red) and people (yellow).

Fig. 2

Biomass generation with Jiffy-7 peat pellets and growth trays under production conditions optimized in this study. Wild-type N. benthamiana plants one (a), three (b), four (c), and five (d) weeks after seed sowing.

Fig. 3

Optimization of biomass generation and NVCP expression. (a) Biomass and NVCP yield under natural and artificial light. N. benthamiana plants were growth either under natural or artificial light for 5 weeks. Leaf biomass (green square) and NVCP expression level (red column) were measured by weighing and ELISA, respectively. (b) Temporal pattern of biomass yield and NVCP expression. Plants were grown under artificial light for 5 weeks, and biomass production (green square) and NVCP accumulation (red column) were examined. For both (a) and (b), mean ± standard deviation (SD) of samples (N>10) from three independent infiltration experiments are presented. (c) and (d) Visualization of GFP expression in Agroinfiltrated leaves. N. benthamiana plants were infiltrated either with GV3101 cultures that carrying the three MagnICON vectors for GFP expression (c) or with infiltration buffer as a negative control (d). Leaves were examined and photographed 7 dpi under UV light. One representative of at least three independent experiments is shown.

Fig. 4

Process flow for the production of NVCP VLP vaccine under cGMP regulations. C of A: certificate of analysis, UF/DF: ultrafiltration and diafiltration.

Fig. 5

Purification of NVCP from N. benthamiana plants. Leaf protein extract was purified and analyzed on a 4-20% SDS-PAGE gel under reducing condition. Lane 1, Insect cell-produced NVCP as a reference standard; Lane 2, Clarified leaf extract from uninfiltrated plants; Lane 3, Clarified leaf extract from NVCP-producing plants; Lane 4, Supernatant of low-pH precipitation; Lane 5, Purified plant-derived NVCP from DEAE anion-exchange chromatography. ◄: RuBisCo large and small subunits; : NVCP.