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Stop Animal Exploitation NOW!
S. A. E. N.
"Exposing the truth to wipe out animal experimentation"

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

Introduction - History

In 1975 George Kohler and Cesar Milstein (13) published a short paper describing their new method for producing monoclonal antibodies (MAbs). Their work included the observations that: (1) "the manufacture of predefined specific antibodies by means of permanent tissue culture cell lines is of general interest," (2) "such cells can be grown in vitro in massive cultures," and (3) "such cultures could be valuable for medical and industrial use." These comments were prophetic, since their new alternative technique was subsequently adopted in virtually every field of biomedical research and biotechnology and in many areas of clinical diagnosis and therapy. So important was this approach to antibody production that Kohler and Milstein received the 1984 Nobel Prize for their discovery.

Although the original research was principally an in vitro technique, it was also apparent that monoclonal antibodies could be produced by injecting the hybridoma cells into the abdominal cavities of different species of rodents. This was the initial use of the ascites method. Since these MAbs were easily made in any laboratory and the ascites process, widely viewed as both simple and inexpensive, 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 being questioned in Europe. Milstein noted that "in later years, both on practical and humane grounds, I became concerned with the use of ascitic fluids" (personal communication, 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. It simply was not possible to humanely produce MAbs using animal-based procedures.

In 1989, The Netherlands government introduced a Code of Practice for the Production of Monoclonal Antibodies (1), which provided detailed descriptions of the veterinary problems and pathophysiology associated with the ascites process and placed restrictions on its use. The resulting increased humane awareness among Dutch researchers provided further encouragement for adoption of in vitro approaches to MAb production. In 1995, a symposium held in Bilthoven, The Netherlands, concluded that progress in development of such alternatives (both in efficacy and cost) was sufficient that the use of ascites could no longer be justified. 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.

By 1996 in vitro production of MAbs was the method of choice in Europe for commercial concerns and others needing large quantities and on the increase for the smaller-scale needs of individual researchers. This latter group made up about 60 percent of European MAb users, primarily for basic research and some diagnostic procedures. Their MAb needs were often met using the ascites technique. A similar situation exists in the United States.

Scientist representatives from several member states of the European Union met in October 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 different types of research and commercial needs for MAbs and the available production options, they concluded that "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." (15) The group further called for a Europe-wide prohibition on the routine use of ascites methods of MAb production. In Europe the trend is toward adoption of in vitro alternatives at all stages of the MAb process.

There are three principal steps in production of monoclonal antibodies: 1) immunization, 2) hybridoma formation and 3) MAb production. Each has its own potential for adoption of alternative methods. Although currently done primarily as an in vivo procedure using a few animals, it is possible, especially with human MAbs, to conduct the immunization process entirely in cell cultures. Some technical difficulties remain to be solved before this becomes a routine non-animal-based procedure.

Formation of the hybridoma cells has always been an in vitro technique. However, final production of the monoclonal antibodies involves use of either the ascites or in vitro alternative approaches. The present review briefly focuses on this last step, with an emphasis on the availability of multiple alternative MAb production techniques, suitable for the small-to-medium-scale research and commercial laboratories.

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