Monoclonal Antibody Production (ECVAM Reports)

The Report and Recommendations of ECVAM Workshop 231,2

Reprinted with permission from ECVAM and ATLA

Appendix 1

Proposed European Guideline on Monoclonal Antibody Production

Directive 86/609/EEC and Convention ETS 123

The purpose of this guideline is to advise Member States on the application to monoclonal antibody (MAB) production of the Three Rs principles enshrined in Article 7 of Directive 86/609/EEC (1) and Articles 6, 7, and 8 of the European Convention for the Protection of Vertebrate Animals Used for Experimental and Other Scientific Purposes, ETS 123 (2), whilst having regard to the right of the Member States to apply stricter measures (Article 24 of the directive and Article 4 of the convention). In particular, this guideline aims to provide specific advice to scientists and project reviewers on what is currently regarded as best practice by experts in the field.

Article 7.2 of the directive requires that "an experiment shall not be performed if another scientifically satisfactory method of obtaining the result sought, not entailing the use of the animal, is reasonably and practically available." Also, Article 7.3 of the directive states that "in a choice between experiments, those which use the minimum number of animals. . . cause the least pain, suffering, distress, and lasting harm and which are most likely to provide satisfactory results shall be selected," while "as a general principle," Article 7.4 of the directive requires that "all experiments shall be designed to avoid distress and unnecessary pain and suffering to experimental animals." These requirements are also documented in Articles 6.1, 7, and 8a, respectively, of the convention (2).

Article 2 of the directive and Article 1 of the convention cover any use of an animal for experimental or other scientific purposes which may cause it pain, etc., while Article 3 of the directive and Article 2 of the convention apply to the use of experimental animals for purposes including the manufacture of drugs, and other substances or products. Thus, Directive 86/609/EEC and Convention ETS 123 apply unequivocally to all use of live animals in the production of MABs, whether the antibodies are intended for use research tools, for assays, or for therapeutic or diagnostic purposes.

Monoclonal Antibody Production

After an initial immunization in vivo, immunocompetent cells are fused with myeloma cells in vitro to produce single hybridoma cells secreting the specific antibody. Consequently, all existing hybridoma cell lines are initially grown up in a static in vitro culture.

In light of the present knowledge, it can be concluded that, for all levels of MAB production, one or more in vitro methods are scientifically acceptable, reasonably and practically available. Such in vitro methods have the additional advantage of producing antibodies with very high immunoreactivities. A previous objection to the in vitro methodology was that significant practical effort was needed to concentrate spent culture fluid and produce useful amounts of MABs. However, modern technology provides a variety of economically acceptable in vitro systems which enable the generation of both high concentrations and/or high yields of MABs. Thus, most production facilities and up-to-date research institutes are now producing all of their MABs in vitro.

The use of the traditional method, which cases a considerable amount of pain and distress to the animals involved (3), is a matter of great concern. In this method, selected antibody-producing hybridoma cells are injected into the peritoneal cavity of compatible laboratory animals under aseptic conditions to produce rapidly progressive local tumors secreting MABs in high titre in the ascitic fluid. Substantial pain and discomfort result from the following: a) the initial priming with the irritant pristane; b) the subsequent rapidly growing tumor (which may disseminate); c) the rate and volume of ascites production; and d) the procedures for, and frequency of, harvesting. Clearly, the use of this method in the majority of circumstances where it is not necessary and cannot be justified breaches the provisions of Directive 86/609/EEC and the European Convention and, as a consequence, such in vivo production should cease.

Where there is an exceptional need for an emergency therapeutic application, the in vivo production of MABs should be allowed. In those cases where there is an existing regulatory approval for a therapeutic or diagnostic use, the ascites method can only be accepted until the end of the approval period. In addition, the ascites method should be allowed in other very exceptional circumstances, where verifiable efforts have failed to produce the MAB in vitro. In this situation, each animal experiment should be scientifically justified, and limited in terms of time and the number of animals to be used. Continuing efforts to produce the MAB in vitro would be expected. In themselves, convenience, "custom and practice," lack of equipment, and/or lack of familiarity with cell culture methods are not justifications for new or continued use of the ascites method.

Pristane continues to be used to encourage consistent ascitic, rather than solid, tumors. In such cases, it is usually satisfactory to give a single priming injection of 0.2ml pristane intraperitoneally 7-10 days before injecting 106-107 hybridoma cells. However, before resorting to the use of pristane, it must be born in mind that this causes painful peritonitis (3-5) and other malignant effects (6, 7). The Dutch Code of Practice suggests that pristane should not be used (4), and Freund's complete and incompatible adjuvants have been selected as possible alternatives (8, 9).

Animals should be inspected frequently by suitably trained personnel so that their clinical conditions can be assessed. Initially, the animals should be handled and inspected by such personnel twice a day and, if necessary, more frequently later on. Animals must be killed without delay when they show more than mild distress, overt tumor deposits or spread, or significant dehydration or cachexia.

The volume of ascites should not normally exceed 20% of the host body weight in mice and rats, in the absence of overt cachexia. A 20% increase in body weight in indicative of a very small, almost imperceptible, swelling of the abdomen. Ascites fluid must be harvested once only, either under terminal anesthesia or post-mortem.

References:

  1. Anon. (1986). Council Directive 86/609/EEC of 24 November 1986 on the approximation of laws, regulations, and administrative provisions of the Member States regarding the protection of animals used for experimental and other scientific purposes. Official Journal of the European Communities L358, 1-29.
  2. Anon. (1986). European Convention for the Protection of Vertebrate Animals Used for Experimental and other Scientific Purposes, 51 pp. Strasbourg: Council of Europe.
  3. Kuhlmann, I., Kurth, W. & Ruhdel, I. (1989). Monoclonal antibodies: in vivo and in vitro production on a laboratory scale, with consideration of legal aspects of animal protection. ATLA 17, 73-82.
  4. Anon. (1989). Code of Practice for the Production of Monoclonal Antibodies, 6 pp. Rijswijk, The Netherlands: Veterinary Public Health Inspectorats, Department of Animal Experimentation.
  5. Porter, W.P., Quander, R.V. & Rener, J.C. (1990). Laboured breathing in ascites-producing mice. Lab Animal 18, 21-22.
  6. Hendriksen, C., Rozing, J., van der kamp, M. & de Leeuw, W. (1996). The production of monoclonal antibodies: are animals still needed? ATLA 24, 109-110.
  7. McGuill, M.W. & Rowan, A.N. (1989).Refinement of monoclonal antibody production and animal well-being. ILAR News 31, 7-11.
  8. Jackson, L.R. & Fox, J.G. (1995). Institutional policies and guidelines on adjuvants and antibody production. ILAR News 37, 141-152.
  9. Rowan, A.N. (1995). The third R: refinement. ATLA 23, 332-346.

Return to Main Article