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Alternatives to Monoclonal Antibody Production (Proceedings)

The European Perspective

Coenraad Hendriksen,1 Jan Rozing,2 Margot van der Kamp,3 and Wim de Leeuw4
1RIVM, Antonie van Leeuwenhoelaan 9, P.O. Box 1, 3720 BA Bilthoven, The Netherlands; 2TNO, P.O. Box 2215, 2301 CE Leiden, The Netherlands; 3NCA, Yalelaan 17, DE Uithof, 3584 CL Utrecht, The Netherlands; 4VHI, P.O. Box 5406, 2280 HK Rijswijk, The Netherlands

One of the most distressing procedures carried out in laboratory animals is the in vivo production of monoclonal antibodies. This procedure leads to a number of pathophysiological effects, such as the formation of large tumorous masses in the peritoneal cavity within 10-20 days of inoculation, peritonitis, and the production of ascites, a process which ultimately leads to a state of cachexia and dehydration.

A Code of Practice on the production of monoclonal antibodies has existed in The Netherlands since 1989. This code, issued by the Veterinary Health Inspectorate (VHI), limits the maximum number of animals to be used per hybridoma to 10, sets guidelines for ascites collection and urges researchers to justify their protocols.

On 24 November 1995, a symposium was held at the National Institute of Public Health and Environmental Protection (RIVM) in Bilthoven, The Netherlands, with the title, "The Production of Monoclonal Antibodies: Are Animals Still Needed?" This symposium, which was organized by the RIVM Center for Alternatives, the TNO Center for Alternatives, and The Netherlands Center for Alternatives (NCA), had a two-fold goal: to evaluate the implementation of the code; and to find out whether recent innovations in in vitro production technology justify a modification of the code. This symposium attracted about 120 participants from different disciplines, including animal welfare officers, research scientists and immunologists. The main topics and conclusions can be summarized as follows.

The 1989 code has had a tremendous effect on the policy toward monoclonal antibody production in some institutions, in that in vivo production has been completely or almost completely replaced by in vitro techniques. However, in some other institutions, in vivo production is still a common procedure. It was stressed by several speakers that a central facility for in vitro production within an institution increases expertise and thereby facilitates the replacement of animals for production purposes. In this regard, it is interesting to note that the Dutch Ministry for Education, Cultural Affairs, and Science has given financial support for the establishment of such central production facilities in a few universities.

Problems involved in in vitro production were discussed extensively. The selection of a stable sub-clone and optimal culture conditions form a prerequisite to the generation of a cell line with constant production characteristics. Other aspects which might interfere with in vitro culture include contamination of the cell line with mycoplasma, and sheer forces. However, data was presented which showed that, in general, in vitro culture fails only very rarely (in less than 3% of attempts). It was concluded by all the experts at the symposium that, from a technical point of view, all of the in vitro systems currently available are no less capable of monoclonal antibody production than are in vivo production methods.

A cost/benefit analysis of in vivo and in vitro production was presented by one of the speakers (Table 1). In general, in vitro systems for small volume culture, such as roller bottles and hollow fiber systems, are slightly more expensive and laborious than ascites production, although recent experiences with new technical developments, such as the introduction of the miniPERM system, show that costs can be reduced substantially. It was stressed at the meeting that some expertise on in vitro culture is a prerequisite.

In one of the presentations, results from the use of embryonated chicken eggs as a culture system for mouse monoclonal hybridomas were discussed. The best compartment for production was the allantoic cavity, giving a maximum of 30ug of monoclonal antibody per egg after culture for 3-5 days. However, compared to production in the mouse ascites system, that in the embryonated egg was suboptimal and cannot be considered to be a viable alternative at present.

Another approach to the production of monoclonal antibodies, which might lead to the total replacement of laboratory animals for this purpose in the future, is the Phage Display technique. In this in vitro technique, which is based entirely on in vitro technology, recombinant DNA techniques are used to generate large panels of monoclonal antibodies. Selected monoclonals can then be produced in bacterial expression systems.

At the end of the meeting, it was concluded that, although the scale use of animals is relatively small compared to other procedures in biomedical research (in The Netherlands, less than 0.2% of the total), in vivo monoclonal antibody production is a very explicit example of a technique to which Article 7.2 of EU Council Directive 86/609/EEC can be applied. Article 7.2 states that "an experiment shall not be performed if another scientifically satisfactory method of obtaining the results sought, not entailing the use of animal, is reasonably and practically available." Based on the results of the discussion, the VHI, which is the named national authority with regard to the Experiments on Animals Act, now considers in vivo ascites production to be an obsolete technique, and will be taking steps to ban its use. The next step should be a common European policy.

It is recognized that some scientists might (and even do) avoid this ban by buying ascites in countries where no regulations exist. As an example, it was indicated that ascites production laboratories exist in south-east Asia, which produce ascites for less than $8 per ml.

Table 1: Comparison of in vivo and in vitro systems for the production of monoclonal antibodies

Production systemMonoclonal antibody amount/unitProduction time (weeks)Estimated cost/gram ($)Quality
Ascites20-250 mg31000-6000low
Roller bottles< 2 g42000-6000high
Hollow fiber0.5-30 g8-202700-4000high
Fermentor2-100 g2-81000-6000high

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