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Articles and Reports

Alternatives to Animal Research and Testing

Alternatives to Monoclonal Antibody Production (Proceedings)
Alternatives to Ascites Production of Monoclonal Antibodies: The Issues
John McArdle, PhD
Alternatives Research and Development Foundation

In 1975 George Kohler and Cesar Milstein published a paper describing their method for producing monoclonal antibodies (MABs). Their work included the observations that "the manufacture of predefined specific antibodies by means of permanent tissue culture cell lines is of general interest" and "such cells can be grown in vitro in massive cultures."

Although the original research was principally an in vitro technique, monoclonal antibodies can be produced by injecting the hybridoma cells into the abdominal cavities of rodents. This was the initial use of the ascites method. Since MABs were easily made in any laboratory and the ascites process was introduced early, its use rapidly expanded. Unfortunately, the original possibility of MAB production replacing uses of laboratory animals was and often continues to be overlooked or ignored. In the decades that followed the original discovery, tens of millions of animals suffered and died despite the availability of more humane alternatives.

During this same period of time the appropriateness of using the ascites method was increasingly questioned in Europe. Milstein noted that "in later years, both on practical and humane grounds, I became concerned with the use of ascitic fluids," (Pers. com., 1997). As new in vitro alternative techniques were developed and validated, it became more difficult to justify the suffering associated with the use of ascites.

There are three principal stages in the production of monoclonal antibodies: immunization (in vitro or in vivo), hybridoma formation (in vitro), and MAB production (in vitro or in vivo). If done in vivo, the first and last procedures are routinely associated with animal suffering and therefore are prime candidates for replacement alternatives.

Support for the continued use of ascites methods are usually based on claims of: (1) more rapid production and high yields of concentrated MABs; (2) minimal requirements for materials, labor, and technical expertise; (3) most hybridomas will grow in mice; (4) supposedly less expensive; and (5) either a generic resistance to or lack of familiarity with in vitro methods.

With increasing expenses of caring for laboratory animals and decreasing costs for in vitro alternatives, the differential (if it exists) between animal and non-animal-based methods is rapidly disappearing.

The most compelling arguments against the use of ascites are derived from the suffering of the animals. In human clinical experience the growth of abdominal tumors is very painful. Ascites fluid accumulation in human patients causes discomfort and pain, while never allowed to progress to advanced stages routinely seen during ascites production in animals. Such procedures in the United States involve 500,000 to 1 million animals per year.

Animals utilized for ascites frequently exhibit a spectrum of clinical symptoms ranging from anorexia and dehydration to difficulty walking, respiratory distress, circulatory shock and classical peritonitis. These symptoms become increasingly severe the longer the animals are allowed to survive (Jackson et al., 1996; Anon., 1996.).

Pathological changes associated with ascites are known for each step in the process. Use of adjuvants produces mild to severe peritonitis and inflammation. Fluid removal may cause hemorrhage, edema, and death. As expected, growth of the ascitic tumors creates: (1) adhesions throughout the abdomen; (2) lymphatic obstruction; (3) tumors with extensive hemorrhagic and necrotic areas; (4) centro-lobular liver necrosis; and other pathologies. (Anon., 1989; Jackson, et al. 1996).

The significance of the list of abnormalities associated with ascites production is further emphasized by observations that the animals may experience severe pathologic changes in their abdomens and chests, but appear to be clinically normal.

From all of the above, it is apparent that animals used for ascites production of monoclonal antibodies are routinely subjected to chronic pain and distress. Simple common sense dictates that it is not possible to humanely produce MABs using rodent ascites.

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 (1) should have expendable material costs similar to that of a mouse; (2) be a simple technique; (3) not require prior adaptation of the hybridomas or special culture conditions; (4) have significantly higher concentration and quality of MABs than for simple stationery cultures; (5) be a closed, reusable system; (6) be affordable to all laboratories; and (7) produce adequate quantities of MABs in a reasonable period of time (Falkenberg, 1995).

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 (Kamp & de Leeuw, 1996).

In contrast, ascites is subject to criticism on technical criteria, including: (1) ascitic fluids contaminated with rodent plasma proteins, immunoglobulins, infectious agents and bioreactive cytokines; (2) the need for extensive animal facilities, associated support services with individual animals requiring daily monitoring; (3) some hybridomas are difficult to grow in rodents; (4) rodents only produce MABs for a few days; (5) from 60 to 80% of mice may not produce ascites due to premature death, development of solid tumors, or failure to establish hybridoma growth; and (6) individual batches of ascites may vary significantly in quality and quantity.

Because in vivo production of monoclonal antibodies involves the largest number of animals and greatest degree of suffering, it receives the most attention for the development of humane replacement alternatives. There are, however, three additional aspects of the process for which alternatives are appropriate and needed.

In vitro immunization needs further development so it can be applied to the broad range of MAB needs. Due to serious humane concerns related to its production, the use of animal serum represents another candidate for appropriate replacement in all in vitro alternatives. Finally, there is a need to replace the use of animal cells with those derived from humans.

Although ascites production can be refined (limiting the weight gain, number of animals and tappings, requiring anesthetics, changing the types of adjuvants, etc.), there really is no need to do so. Replacement is the preferred and available option.

The issues discussed in this workshop have already been addressed in considerable detail in Europe. In June 1988, the Dutch Society for Laboratory Animal Science, the Animal Experimentation Department of the Veterinary Chief Inspectorate and the Dutch Societies for Biochemistry, Immunology, Microbiology, Pathology and Toxicology held an inquiry, which determined that ascites involved "considerable distress for the animals."

As a result of this meeting, in 1989 The Netherlands government introduced a Code of Practice for the Production of Monoclonal Antibodies (Anon., 1989), which placed reasonable restrictions on the use of ascites. The resulting increased humane awareness among Dutch researchers provided further encouragement for adoption of in vitro approaches to MAB production. In November 1995, a symposium held in Bilthoven, The Netherlands, concluded that progress in the development of such alternatives (both in efficacy and cost) was sufficient that the use of ascites could no longer be justified (Hendriksen et al., 1996). The resulting prohibition of animal-based MAB production caused no serious difficulties within the Dutch biomedical research community. Despite initial academic resistance, bans on ascites in Germany and Switzerland experienced similar results, as did the restrictions placed on ascites use in Sweden and The United Kingdom.

Scientist representatives from several member states of the European Union met in November 1996 at the European Center for the Validation of Alternative Methods to discuss the current status of in vitro and in vivo methods of monoclonal antibody production. After careful consideration of the research and commercial needs for MABs and the available production options, they concluded "for all levels of MAB production, there are one or more in vitro methods which are not only scientifically acceptable, but are also reasonably and practically available; and as a consequence, in vivo production can no longer be justified and should cease." (Marx et al. 1997.) The group further called for a Europe-wide prohibition on the routine use of ascites methods.

In the United States another important issue is the responsibilities of the Institutional Animal Care and Use Committees (IACUCs) in relation to alternatives generally, and rodent ascites specifically. IACUCs have a statutory and moral responsibility to require investigators to search for suitable alternatives to painful procedures and to require their use, if found. Since multiple alternatives to ascites are readily available for all routine MAB production needs, no IACUC should approve an ascites protocol. Investigators should be aware of these alternatives and be required to use them. Failure to do so suggests an incomplete or non-existent search for appropriate alternatives.

It is important to stress that a requirement to use alternatives to ascites has no more to do with experimental design, than does a requirement to use a particular anesthetic or veterinary procedure.

If current trends continue, the use of animals to produce monoclonal antibodies will be replaced with in vitro alternatives.

More than 20 years ago a new, in vitro alternative was developed for the production of monoclonal antibodies. Because of its simplicity and importance, this technique was widely adopted. For similar reasons, a new form of laboratory animal cruelty and suffering was also created -- the use of ascites.

For the last decade researchers in Europe and the United States systematically developed and adopted multiple in vitro replacements for the use of rodent ascites. This process has now progressed to the point that it is possible to prohibit the use of ascites in all but the most unusual and rare circumstances.

The American Anti-Vivisection Society decided it was time for researchers and regulatory bodies in the United States to join their European colleagues in ending such widespread, systematic and unnecessary animal suffering. The Antibodies Without Animals Campaign was initiated to finally implement the humane possibilities of Kohler and Milstein's original work and to initiate a new perspective among U.S. regulatory agencies regarding the promotion and use of alternatives to traditional forms of laboratory animal usage. These petitions are good for the animals. good for promoting alternatives, and good for biomedical sciences. Further, they represent an ideal opportunity for the animal advocacy and biomedical research communities to work together. The case for replacement of ascites is so strong, that this should be a win-win situation for both groups (McArdle, 1997).

Alternatives have been described as the gentle hand of science. The topics of this workshop represent a perfect opportunity to promote that perception.


1. Anon (1989) Code of Practice for the Production of Monoclonal Antibodies. Rijswijk, The Netherlands: Veterinary Health Inspectorate.

2. Hendriksen, C., J. Rozing. M. der Kamp and W. de Leeuw (1996) The production of monoclonal antibodies: Are animals still needed? ATLA. 24. 109-110.

3. Jackson, L.R., L.J. Trudel, J.G. Fox and N.S. Lipman (1996) Clinicopathologic features and production parameters of monoclonal antibody production in murine ascites. (In preparation.)

4. Kamp, M. and W. de Leeuw (1996) Short review of in vitro production methods for monoclonal antibodies. NCA Newsletter. 3. 10-11.

5. Kohler, G. and C. Milstein (1975) Continuous cultures of fused cells secreting antibody of predefined specificity. Nature. 256. 495-497.

6. Marx, U., M.J. Embleton, R. Fisher, F.P. Gruber, U. Hansson, J. Heuer, W.A. de Leeuw, T. Logtenberg, W. Merz, D. Portetelle, J-L Romette and D.W. Straughan (1997) Monoclonal antibody Production; the report and recommendations of ECVAM Workshop 23. ATLA. 25. 121-137.

7. McArdle, J.E. (1997) Alternatives to ascites production of monoclonal antibodies. AWIC Newsletter. In press.

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