Bioprocess Engineering
products entered clinical trials. Current
frontiers include stem cell research for
tissue, organ, and bone replacements; in-
tegrated fermentation and downstream
of protein from transgenic plants and
Another important precursor of present-
day bioprocessing is the fractionation of
human plasma proteins for the production
of its components: mainly, serum albu-
min, immunoglobulins, Fbrinogen, and
various clotting factors. Use of puriFed
proteins is a far more efFcient utilization
of human plasma – an expensive national
resource – than total plasma transfusion.
The industry originated during World War
II and is based on fractional precipitation
of plasma component proteins by the addi-
tion of ethanol under controlled conditions
of temperature, pH, and ionic strength.
The ethanol process developed by Cohn
and his numerous associates is used world-
wide with remarkably few changes since
its introduction.
Modern puriFcation of therapeutic pro-
teins that are produced through recom-
binant DNA technology is not based
on ethanol precipitation; rather, it relies
mainly on chromatography. Nevertheless,
protein puriFcation beneFted enormously
from the older plasma fractionation in-
dustry, which developed such essential
ancillary equipment and processes as cen-
trifuges, Flters, freeze-dryers, sterile oper-
ation, pyrogen testing, and quality control.
Plasma fractionation still handles much
larger volumes of proteins and at a lower
cost than any other form of therapeutic
protein processing.
The use of radioisotopes and electron
microscopes, also around World War II,
contributed signiFcantly to advances in
bioprocessing. The use of these techniques
to track isotopes in the body and to
visualize cell components when combined
analytical tools of today including nuclear
magnetic resonance machines, gas and
liquid chromatographs, ultrasound, laser
and magnetic imaging techniques, and X-
ray diffraction tools. These advances in
instrumentation allowed for a switch from
‘‘trial-and-error’’ medicine and products to
physiology-based biotechnology.
The current bioprocessing industry re-
lies on several complementary technolo-
gies. The backbone of the industry is
the use of recombinant proteins, obtained
by inducing a host organism to express
foreign proteins. To this end, a gene reg-
ulating the production of this protein is
inserted into a plasmid, an autonomous,
self-replicating piece of DNA that is in-
serted into a living cell. The host organism
can be a bacterium, yeast, plant, mam-
malian, or insect cell, as well as an intact
plant or higher animal. The Frst recom-
binant plasmid was patented by Cohen
and Boyer in 1973. The plasmid is con-
structed to cause a cell to manufacture a
large quantity of foreign or product pro-
tein (up to 50 g of protein per liter of cell
medium). The excess protein is, in many
cases, expressed in a denatured form as
a densely aggregated particle. This step
facilitates the puriFcation of the protein,
but it then requires solubilization and re-
folding in the biologically active form – an
often daunting problem. Obtaining the ac-
tive protein usually dictates the choice of
plasmid and host cell.
The formulation of the original gene (i.e.
the sequence of the nucleotides forming
the DNA) is greatly facilitated by the
polymerase chain reaction (PCR) method,
Frst developed in the mid-1980s by the
Cetus Corporation and commercialized
by Perkin-Elmer. PCR is an enzymatic
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