Modifications of the stationary cell cultures primarily involve
improved nutrient supply and waste removal, continuous MAb harvesting,
and increased hybridoma cell densities. One of the simplest of these is
the use of gas permeable tissue culture bags (fig. 2) which provide a
practical, economical, and flexible system, using readily available
laboratory equipment. In addition to simple convenience, the system is
more efficient than rigid tissue culture flasks on a per cell basis and
is significantly less expensive than rodent ascites on a cost per
milligram basis and total yield . Because the bags are a closed system
with no mechanical parts, they are relatively free of contamination and
require little or no monitoring. MAbs are produced in quantities
sufficient for small, medium, and large-scale needs (17, 23).
Stationary cultures can also be upgraded by a variety of mechanisms
to keep the cells in suspension. These include improvements in stirring
and overflow replacement of media (for example, the cytostat (20)) and
agitation of cell cultures in roller bottles, spinner flasks, and
stirred tanks (2, 14, 19). These alternatives require specific,
relatively inexpensive equipment and are simple, fast, and effective.
Roller bottles produce low to medium monoclonal antibody yields at a
price similar to that of ascites. Comparative studies of this system
that examined 39 different types of hybridomas found that all grew well
and produced MAbs (14). Oscillating bubble systems produce higher yields
of MAbs, with quantities in each tube exceeding that produced by one
ascitic mouse, as well as using equipment that is reusable (19). Due to
shear forces in the culture media, spinner flasks may damage the
hybridoma cells, and thus are a less effective alternative.
To protect fragile hybridoma cells in suspension cultures and some
types of bioreactors, it is possible to encapsulate them in polymers.
This provides high yields of MAbs (comparable to ascites methods), with
ten-fold increases in cell density over simple suspension systems.
Because the cells are isolated from the culture media, it is possible to
establish a long-term, continuous operation (up to 40 days in an
expanded bed bioreactor). Although available for smaller-scale needs,
this technology is primarily used for commercial production of large
quantities of MAbs (21).
Dialysis tubing or chambers are used in several different
configurations to achieve high hybridoma cell densities with MAb
concentrations and purity similar to that found in ascites. The cells
may be placed in dialysis bags or other containers within a nutrient
chamber to produce up to a gram of MAb within a few weeks. This
represents a very inexpensive and simple system which allows several
hybridomas to be grown simultaneously (22).
When dialysis tubing is combined with a simple roller-bottle-like
mechanism, up to four hybridomas may be grown at once at 10 to 30 fold
increase in concentration over stationary cultures. Such compound
devices produce MAbs in quantities and purity often equivalent to that
of ascites (4).
Combination with a tumbling chamber system provides passive gas
exchange with simple, inexpensive, reusable equipment. This approach is
capable of producing up to 300 mg of MAb in 21 days and is "universally
adaptable in any research laboratory" (10).
One of the most promising in vitro methods for producing high yields
of MAbs at a concentration, purity, and cost comparable to mice are the
new modular minifermentors (5). These systems meet all the requirements
for the perfect in vitro alternative. The hybridoma cells are grown in
disposable culture chambers, separated from a reusable media chamber by
a gas-permeable dialysis membrane. From 9 to 159 milligrams of MAb can
be inexpensively produced in 1 to 4 weeks without the use of serum.
Based on current European monoclonal antibody guidelines, these devices
have yields equivalent to that of three ascitic mice.
Airlift (packed-bed) bioreactors provide long-term, serum-free
production of MAbs in gram amounts. They are particularly
cost-effective, since all of the components are reusable (18).
Hollow fiber bioreactors are designed to provide a more
physiologically stable environment for the hybridomas and come in both
small and large-scale units that produce high cell densities and MAb
yields under low or serum-free conditions. Although somewhat more
expensive to initially establish than other types of in vitro
alternatives, their ability to produce concentrated, relatively pure
MAbs at up to half the cost of ascites and in amounts that can be
equivalent to 200 mice, 60 large spinner flasks, or a 60 liter fermentor,
makes them an attractive option for basic biomedical research and
smaller-scale commercial needs. They also require technical expertise to
operate without contamination and equipment failures (7, 8).
As noted by the participants in the 1996 ECVAM meeting, there are a
variety of in vitro approaches to producing MAbs, some of which are
briefly described above. For all levels of consumption, replacements for
the use of ascites methods are available. Some of the principal
characteristics of these alternative options are summarized in tables 1
Table 1. Antibody production in different bioreactor systems (Adapted
from Stoll et al., 1995 (24)).
Table 2. Comparisons of different monoclonal production methods.
(Adapted from Hendriksen et al., 1996 (6); Kamp amd de Leeu 1996 (11)).