Biomedical Engineering Reference
In-Depth Information
experience. Production costs vary with degree of desired purity. For high purity material
(95% pure), a cost of about $4 e $8/g can be expected. For higher purity material (99%)
with full quality assurance and control, a cost of about $20 e $30/g is reasonable.
At least two enzymes are being produced commercially from transgenic plants. Large-scale
production of monoclonal antibodies (e.g. 500 kg/year) for topical uses is being considered.
The use of plant cell cultures is also being explored. The primary advantage of such
cultures over transgenic plants is the much higher level of control that can be exercised
over the process. Plant cell cultures, compared to animal cell cultures, grow to very high
cell density, use defined media, and are intrinsically safer.
14.7.8. Comparison of Strategies
The choice of host e vector system is complicated. The characteristics desired in the protein
product and the cost are the critical factors in the choice. The dominant systems for commer-
cial production are E. coli and CHO cell cultures. The process economics of these two systems
for production of tissue plasminogen activator, tPA, has been given by Datar, Cartwright, and
Rosen ( Biotechnology 11:349, 1993). Their analysis was for plants making 11 kg/year of product.
The CHO cell process was assumed to produce 33.5 mg/L of product, while E. coli made
460 mg/L. The CHO cell product was correctly folded, biologically active, and released into
the medium. The E. coli product was primarily in the form of inclusion bodies and thus bio-
logically inactive, misfolded, and insoluble. The process to resolubilize and refold the E. coli
product into active material requires extra steps. The recovery process for the CHO cell mate-
rial requires five steps, while 16 steps are required for the E. coli process. The larger the number
of steps, the greater the possibility of yield loss. Total recovery of 47%with the CHO-produced
material was possible compared to only 2.8% for the E. coli -produced material.
The extra steps in the E. coli process are for cell recovery, cell breakage, recovery of inclu-
sion bodies, resolubilization of inclusion bodies, concentration, sulfination, refolding, and
concentration of the renatured protein. The difficulty of these processes depends on the
nature of the protein; tPA is particularly difficult. With tPA the concentration of tPA had to
be maintained at 2.5 mg/L or less, and refolding is slow, requiring 48 h. A 20% efficiency
for renaturation was achieved. Many proteins can be refolded at higher concentrations (up
to 1 g/L) and much more quickly. For tPA, the result is unacceptably large tanks and very
high chemical usage. In this case, 5 tons of urea and 26 tons of guanidine would be necessary
to produce only 11 kg of active tPA.
For tPA, the required bioreactor volumes were 14,000 L for the CHO process and 17,300 L
for the E. coli process. The capital costs were $11.1 million for the CHO process and $70.9
million for the E. coli process, with 75% of that capital cost being required for the refolding
tanks. Under these conditions, the unit production costs are $10,660/g for the CHO process
versus $22,000/g for the E. coli process. The rate of return on investment (ROI) for the CHO
process was 130% versus only 8% for the E. coli process. However, if the refolding step yield
was 90% instead of 20%, the overall yield would improve to 15.4%, and the unit production
cost would fall to $7530 with an ROI of 85% for the improved E. coli process at production of
11 kg/year. If the E. coli plant remained the original size (17,300-L fermentor) so as to produce
61.3 kg/year, the unit production cost would drop to $4400/g. The cost of tPA from the CHO
process is very sensitive to cost of serum in the medium. If the price of media dropped from
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