Plant biomass production
N. benthamiana is a wild Australian relative of cultivated tobacco (i.e., Nicotiana tabacum), but has no agronomic or food use. N. benthamiana plants are grown in controlled and contained conditions. A high-quality, soilless, sterilized root-substrate medium provides
physical support and optimal water retention. Apart from fertilization and substrate moisture, which are adjusted during plant
development, all growth conditions remain unchanged during the production cycle (i.e., 40 days). All components used for biomass
production are released under standard operating procedures. Special care is taken to control the presence of heavy metals
in all liquids and solids that contact the biomass. Fungal, microbial, and algae growth are strictly controlled during all
phases of growth.
Plant development is monitored from seeding to infiltration to ensure proper and reproducible biomass development. Plants
are characterized during their growth and must meet specific criteria to be accepted for inoculum infiltration.
Inoculum infiltration and incubation
The authors' pilot facility in Québec City, Canada, has an inoculum infiltration capacity of 1200 plants per day. Infiltration
is performed in a stainless-steel tank, 15 plants at a time. The vacuum infiltration lasts only 1 min, and the procedure is
computer-controlled to ensure reproducibility. At a plant under construction in North Carolina, the infiltration unit will
accommodate 15,000 plants per day. Transportation of the biomass to and from the infiltration unit will be completely automated.
Following infiltration, plants are again maintained in controlled and contained conditions during the three to six days required
for transient expression to take place.
Harvest, extraction, and conditioning
Plant leaves are collected and mixed with a depolymerization solution. Medicago has developed a proprietary technology that
treats the biomass with cell-wall depolymerization agents. This treatment acts on structural or cross-linking components of
the cell wall, which loosens the rigid extracellular matrix of the leaf cells, and subsequently releases the VLPs into the
solution. The procedure releases more than 90% of intact VLPs with less than ~5% of the host-cell components (i.e., protein
and DNA), and is thus an extremely efficient primary recovery step.
Because VLPs are large particles (130 nm in diameter), they can be concentrated by tangential-flow filtration (TFF). This
concentration step also eliminates most of the remaining host-cell components, including low molecular-weight compounds. Diafiltration
at this stage conditions the VLPs in preparation for chromatography.
Chromatography and formulation
VLPs are purified by ion-exchange chromatography, concentrated, and diafiltered against the formulation buffer through a final
TFF. Chromatography also allows the removal of residual DNA and endotoxins.
CHARACTERISTICS OF PURIFIED INFLUENZA VLPS
The purified influenza VLP vaccines used in clinical trials were released under the criteria used for licensed influenza vaccines
and have met all standards for purity and identity. DNA and endotoxins were always below acceptable limits, and these preparations
were tested for sterility and subjected to General Safety Testing in guinea pigs before being released.
Table I: Characteristics of purified H5 viruslike particles (VLPs).
Mass spectrometry helps to identify trace protein contaminants. Most of the contaminants are naturally associated with plant
plasma membrane and are neither immunogenic nor induce an allergic response in animal and human models. Because this product
arises from a new platform, an extensive complementary physicochemical characterization was performed on purified preparations.
Table I presents the measured attributes of the pandemic H5 VLP vaccine product analyzed on multiple lots. The narrow range
of measurements for mean particle size, HA purity, and lipid–protein ratio demonstrates the high lot-to-lot reproducibility
of the manufacturing process. The Coomasie-stained SDS-PAGE and Western blot analysis of the pandemic H5 VLP vaccine product
in Figure 3 further illustrate the purity level of the product (i.e., 98–99% purity).
Figure 3: Purity and identity of an H5 viruslike-particle (VLP) vaccine: Coomassie-stained SDS-PAGE of (1) molecular-weight
marker and (2) 2.5-μg of influenza H5 VLP vaccine, and Western blot analysis using polyclonal antibodies against influenza
H5N1 virus (strain A/Indonesia/5/05) of (3) 50 ng and (4) 500 ng of the influenza H5 VLP vaccine.