The most cost-effective extraction method is compression sieving or screw press. Plant material is fed into the screw press
in a continuous manner, and compression under high pressure ruptures the plant cell walls. The compressed material is moved
along a sieve plate, which separates the plant-derived extract from solids. This type of extraction equipment is low-tech
and readily available from equipment vendors. Screw press extraction uses natural plant liquids, eliminating the need to pay
for additional solvents.
Table 3. Selected Component Composition of Tobacco (Green Tissue)
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Clarification
Once the target protein has been extracted from tobacco, residual plant solids, bioburden, oils, and other extraneous matter
must be removed. Typically, the two methods employed are centrifugation and filtration. Centrifugation generally requires
a larger capital investment than filtration and needs to be carefully evaluated during process modeling. Using fine and ultra-fine
membrane filtration, and of course filtration with diatomaceous earth (DE) or polymeric filters can be cost effective with
higher efficiencies and lower capital costs. The target protein can be separated by differential solubility by selectively
tagging the protein, which induces aggregation or binding to specific media.
Purification
Generally, the first step in purification is capture. This is normally accomplished through the use of specific chromatography
resins such as ion exchange or affinity type resins. Resins are used in a variety of configurations such as the standard downflow
system, radial chromatography, or streamline expanded-bed technology from Amersham Biosciences. The choice of capture system
typically depends on the protein of interest and the cost of the resin.
Figure 4. Crude Protein Yield Based on Tobacco Biomass per Acre, and Expressions Levels Measured as % Total Soluble Protein
(TSP)
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Purification may employ several further chromatography steps including ion exchange, affinity, hydrophobic interaction, low-pressure
reverse phase, or size exclusion. Each subsequent chromatography step is targeted for the removal of specific classes of contaminants
such as host proteins, host compounds, host DNA, endotoxin, and environmental contaminants such as residual herbicides, pesticides,
and bacterial and fungal toxins. Attention to interactions between target protein and plant phenolics is especially important
during the purification process. A list of some of the contaminating molecules is shown in Table 3. Water, which is 80 to
90% of the wet weight, is not counted in this analysis of the dry solids.
Process ECONOMICS
The manufacture of tobacco-produced proteins can be cost effective due to the low cost of field production and upstream processing,
relative to the capital burden and operations-intensive processes used in traditional technologies such as mammalian cell
culture. Various companies suggest that by eliminating expensive upstream equipment, the cost of therapeutic proteins made
in transgenic systems may be an order of magnitude lower than the same proteins produced with traditional technologies. Others
have reviewed process models, predicting that there may only be a savings of 10 to 15% over traditional cell culture systems.
This is because upstream costs of traditional technologies are said to contribute less than one-half of the overall production
costs, so an order of magnitude reduction in cost-of-goods using transgenic technology is not reasonable.18 However, the traditional model uses many standard unit operation criteria for both transgenic and cell culture systems that
are not always applicable.
There are many ways to reduce the downstream costs associated with protein manufacturing. Often, when cost allocations are
applied to upstream comparative construction and operations models, support or ancillary cost factors are not subtracted.
Applying modular construction can reduce capital costs. Modular cleanroom components are pre-fabricated and are generally
assembled within a low-cost shell structure. Although the modular components may contribute a higher cost per square foot
to the cleanroom structure, the overall facility cost is generally less due to the simplified construction and readily available
materials.
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