After two rounds of antibiotic selection, the resulting plant will contain 100% transformed chloroplasts (Figure 2). A working
seed bank for production lines will be generated ten to eleven months post-bombardment and will be ready for field propagation.
Because the chloroplast genome is maternally inherited in higher plants, 100% of the seed and seedlings will carry the transformed
chloroplast genome in each subsequent generation.
More than 30 recombinant proteins have been expressed using CTT (Table 2), including both therapeutic proteins, and as proteins
that improve agronomic traits in a variety of plants.
Figure 1. The Transformation-Expression Cassette for Single (X) or Multiple-Gene (X, Y, Z) Expression in Chloroplast
There are four major factors that impact the selection of a transgenic plant as a production system for biopharmaceuticals:
cost of goods, scale-up capacity, regulatory hurdles, and time to market. The success of plant-made biopharmaceuticals or
industrial protein production in tobacco is particularly sensitive to these four factors and can be evaluated through the
analysis of discrete steps in the manufacturing process. The primary development model for producing human recombinant proteins
in tobacco is based on green tissue (leaf) biomass processing. The major steps in green tissue processing are illustrated
in Figure 3.
Master seed bank production is less intensive with tobacco than with other plants. Systems that rely on sub-cloning (for example,
in potatoes and sugarcane) require time to amplify cloning stocks and are susceptible to the consequences of pest attacks
or disease devastation as cloning stocks are restored. Seed-based systems rely on agronomic seed-banking practices that have
been practiced for thousands of years. These provide a ready source of production plants, rapid scale-up ability, and a stable
recombinant gene platform. However, a plant such as corn, which is open-pollinated, requires significant field acreage to
generate production seed and large storage facilities due to the relatively large size of corn kernels. Tobacco can produce
as many as one million seeds per plant, which allows for master seed bank production in a contained and controlled greenhouse
environment at a very low cost.15
Field production of biomass (the target protein) provides the primary cost advantage over traditional production technologies
such as mammalian cell culture and bacterial fermentation.16
The lack of facilities, bioreactors, and process technicians significantly reduces upstream costs. However, issues with gene
containment — specifically pollen flow to conventional plants — have caused concern about field production of biopharmaceuticals.
Using chloroplast transformation technology eliminates the concern about pollen flow because chloroplast genomes are inherited
maternally and are not found in pollen. Therefore, tobacco allows for outstanding field containment, resulting in lower regulatory-related
costs. The high expression levels observed in chloroplast-transformed tobacco also impacts the overall cost.
Figure 2. Generation of Tobacco Plants with Transformed Chloroplast
Chlorogen's current transgenic production cultivar is adapted to a wide range of soil types and climates. Field tobacco practices
differ from traditional practices due to the extraordinarily high plant densities and growing and harvesting procedures. A
field study investigated staggered biomass yields for seedlings transplanted at weekly intervals. Yields were not significantly
different for the May 15 through July 15 release dates.17 The current production cultivar possesses a rapid growth rate, excellent ratooning (regeneration) efficiency, and low total
alkaloid concentrations. These characteristics provide tremendous flexibility with respect to biomass generation and processing.
Harvesting of tobacco is relatively straightforward and is generally accomplished with the use of sickle-bar or rotating-disk
type harvesters. The cut biomass (approximately 10 inches above ground level) is then typically loaded into transportation
bins via moving-bed conveyers. This equipment is relatively low-tech and is readily available from farm equipment vendors.
Plants are harvested at the growth stage that results in maximum protein yields, generally at the pre-button stage just before
flowering. Although chloroplast genes are inherited maternally and not through pollen, the harvesting of pre-flowering plants
reduces even the smallest potential of cross-pollination with conventional tobacco cultivars.