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440 Hybridomas, Genetic Engineering of

typical minimum quantity for sale is 200 µg which may
cost around $300. This method of production is conve-
nient because variable quantities of different antibodies
can be produced from batteries of mice.
The production of monoclonal antibodies in vivo, us-
ing mice (or other laboratory animals) has come under
increasing criticism because of the ethical issues posed
by the use of laboratory animals. In Europe, regulatory
approval of this method has been withdrawn except for
cases were alternative methods are shown not to be avail-
able. Mice generating Mabs typically exhibit abdominal
distention, anorexia, anemia, decreased activity, and body
mass, dehydration, difficulty in walking, respiratory dis- FIGURE 15 Stirred-tank bioreactor.
tress, shock, hunched posture, peritonitis, immunosup-
pression, and possibly death. slowly than most bacteria or fungi. They require gentler
Additionally, the ascites method presents problems for culture conditions and control systems that are optimized
product purification. The overall protein content of ascites for lower metabolic rates. Therefore, the design, mode of
fluid is high, posing considerable difficulty in obtaining a operation, and control systems of a stirred tank reactor
pure monoclonal antibody. Furthermore, the ascites fluid used for animal cells are distinctly different from those
contains antibodies secreted by the host mouse and these that would be applicable to bacterial or fungal cells.
are virtually impossible to separate from the monoclonal The stirred tank reactor has been developed commer-
antibody. Thus the final “purified” product has residual cially in large-scale animal cell culture processes up to
activity that may interfere with the application of the mon- a volume of at least 10,000 liters. For laboratory use
oclonal antibody. there are also numerous bench-top stirred tank reactors
(1–5 liters) that are available commercially and that have
been designed specifically for the growth of animal cells in
B. In Vitro Production
suspension. Figure 15 shows a typical design. Bench-top
The basis for commercial production of monoclonal anti- models are generally made of glass with a stainless-steel
bodies from hybridomas is cell culture technology which head-plate, whereas the larger fermenters are made en-
involves the growth of isolated mammalian cells in liq- tirely from stainless steel. The metal head plate of a stirred
uid culture in vitro. Cells are grown in bioreactors, and tank reactor consists of a range of ports and pipes. This
can be produced in high densities if the appropriate phys- allows electrodes to be inserted and tubing to be attached
ical conditions and nutrients are provided. Small vol- for media input or sampling.
ume cultures (<200 ml) are usually set up in T flasks Manufacturers of bench-top models include the follow-
or spinner flasks in temperature-controlled incubators, of- ing: Applikon, New Brunswick, LH Fermentation, Setric
ten without controlling other culture parameters. How- SGI, Braun, Bio-engineering. Each of these companies
ever, in order to obtain high cell densities and maxi- produces uniquely designed and controlled fermenters, all
mize productivity of secreted products at a larger scale of which have been shown to be suitable for animal cell
(>1 liter) other culture parameters are controlled. These culture.
include oxygen supply, temperature, pH, and culture mix-
ing. Three culture system bioreactor designs have been
D. The Airlift Fermenter
used routinely for the production of monoclonal antibod-
ies. These include stirred-tank, air-lift, and hollow fiber This type of fermenter consists of a tall column with an
bioreactors. inner draught tube (Fig. 16). Fluid circulation is provided
by a stream of air which passes through the inside of the
draught tube. This is a simple system without mechanical
C. Stirred-Tank Bioreactor
components and therefore not susceptible to breakdown.
The stirred tank bioreactor is a simple and widely used Bubble or foam damage is minimized by having a long
fermenter design that consists of a cylindrical vessel with column, since it has been shown that maximum cell dam-
a stirrer. The design has been used extensively in all mi- age occurs at the point of bubble bursting at the top of
crobial fermentation and has been the main system used in the liquid column. Airlift fermenters (>1,000 liters) have
yeast fermentation in the brewing industry for centuries. been used routinely for the production of bulk quantities
However, animal cells are more fragile and grow more of monoclonal antibodies from hybridomas.