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Articles and Reports

Alternatives to Animal Research and Testing

Alternatives to Ascites Production of Monoclonal Antibodies
by John McArdle, Ph.D.
Alternatives Research and Development Foundation
Eden Prairie, Minnesota

In Vitro Alternatives

The most appropriate type of in vitro alternative for each research or diagnostic situation depends largely on the quantity and purity of monoclonal antibody needed. There are, however, some general criteria for rating each system. The ideal method (4, 5):

  1. should have expendable material cost similar to that of a mouse.
  2. should be a simple technique, requiring no special expertise beyond that for typical cell culture procedures.
  3. should not require prior adaptation of the hybridomas or special culture conditions.
  4. should have significantly higher concentration and quality of MAbs than for simple stationary cultures.
  5. should be a closed, reusable system; free of contamination.
  6. should be affordable to all laboratories.
  7. should produce adequate quantities of MAbs in a reasonable period of time.

Only a few of the available in vitro methods meet all of these criteria, but there are a wide variety of options available to investigators, ranging from simple, individual cell culture containers to giant, commercial bioreactors.

For individuals needing only small quantities of MAbs, simple, inexpensive stationary cell cultures may be suitable. These involve a variety of flasks and bottles and are simple and easy to use with existing equipment and skills. Batches can be run for days, weeks, or months, depending on the amount of nutrient supplementation and waste removal. These latter processes can be automated. Regardless of the configuration selected, the MAbs must be concentrated after production.

Figure 2. Gas-permeable tissue culture bags are excellent for MAb production.

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 and 2.

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)).

Go on to Table 1. Antibody production in different bioreactor systems
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