Executive Summary

Monoclonal antibodies (mAb) are important reagents used in biomedical research, in diagnosis of diseases, and in treatment of such diseases as infections and cancer. These antibodies are produced by cell lines or clones obtained from animals that have been immunized with the substance that is the subject of study. To produce the desired mAb, the cells must be grown in either of two ways: by injection into the abdominal cavity of a suitably prepared mouse or by tissue culturing cells in plastic flasks. Further processing of the mouse ascitic fluid and of the tissue culture supernatant might be required to obtain mAb with the required purity and concentration. The mouse method is generally familiar, well understood, and widely available in many laboratories; but the mice require careful watching to minimize the pain or distress that some cell lines induce by excessive accumulation of fluid (ascites) in the abdomen or by invasion of the viscera. The tissue-culture method would be widely adopted if it were as familiar and well understood as the mouse method and if it produced the required amount of antibody with every cell line; but culture methods have been expensive and time-consuming and often failed to produce the required amount of antibody without considerable skilled manipulation. However, culture methods are now becoming less expensive, more familiar, and more widely available.

The American Anti-Vivisection Society (AAVS) petitioned the National Institutes of Health (NIH) in early 1997 to prohibit the use of an animal in the production of mAb. NIH responded late in 1997, asserting that continued use of the mouse method for producing mAb was scientifically required. In a second petition, in early 1998, AAVS did not accept the NIH response. NIH asked the National Research Council to form a committee to study this issue. The Commit-



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Executive Summary Monoclonal antibodies (mAb) are important reagents used in biomedical research, in diagnosis of diseases, and in treatment of such diseases as infections and cancer. These antibodies are produced by cell lines or clones obtained from animals that have been immunized with the substance that is the subject of study. To produce the desired mAb, the cells must be grown in either of two ways: by injection into the abdominal cavity of a suitably prepared mouse or by tissue culturing cells in plastic flasks. Further processing of the mouse ascitic fluid and of the tissue culture supernatant might be required to obtain mAb with the required purity and concentration. The mouse method is generally familiar, well understood, and widely available in many laboratories; but the mice require careful watching to minimize the pain or distress that some cell lines induce by excessive accumulation of fluid (ascites) in the abdomen or by invasion of the viscera. The tissue-culture method would be widely adopted if it were as familiar and well understood as the mouse method and if it produced the required amount of antibody with every cell line; but culture methods have been expensive and time-consuming and often failed to produce the required amount of antibody without considerable skilled manipulation. However, culture methods are now becoming less expensive, more familiar, and more widely available. The American Anti-Vivisection Society (AAVS) petitioned the National Institutes of Health (NIH) in early 1997 to prohibit the use of an animal in the production of mAb. NIH responded late in 1997, asserting that continued use of the mouse method for producing mAb was scientifically required. In a second petition, in early 1998, AAVS did not accept the NIH response. NIH asked the National Research Council to form a committee to study this issue. The Commit-

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tee on Methods of Producing Monoclonal Antibodies was composed of 11 experts with extensive experience in biomedical research, laboratory animal medicine, pain research, animal welfare, and patient advocacy. The committee was asked to determine whether there is a scientific necessity for producing mAb by the mouse method and, if so, to recommend ways to minimize any pain or distress that might be associated with the method. The committee was also to determine whether there are regulatory requirements for the mouse method and to summarize the current stage of development of tissue-culture methods. On the basis of relevant literature, material submitted to the committee, the experience of members of the committee, and presentations at a 1-day workshop attended by 14 speakers and 20 additional observers, as well as two separate working committee meetings, the committee came to specific conclusions and made recommendations. We believe that choosing the method of producing monoclonal antibodies should be consistent with other recommendations in the Guide for the Care and Use of Laboratory Animals. One such recommendation pertains to multiple survival surgery; the Guide states (page 12) that this practice “should be discouraged but permitted if scientifically justified by the user and approved by the Institutional Animal Care and Use Committee (IACUC)" [emphasis added]. Similarly, we recommend that mAb production by the mouse ascites method be permitted if scientifically justified and approved by the relevant IACUC. We further believe that tissue-culture methods should be used routinely for mAb production, especially for most large-scale production of mAb. When hybridomas fail to grow or fail to achieve a product consistent with scientific goals, the investigator is obliged to show that a good-faith effort was made to adapt the hybridoma to in vitro growth conditions before using the mouse ascites method. Recommendation 1: There is a need for the scientific community to avoid or minimize pain and suffering by animals. Therefore, over the next several years, as tissue-culture systems are further developed, tissue-culture method for the production of monoclonal antibodies should be adopted as the routine method unless there is a clear reason why they cannot be used or why their use would represent an unreasonable barrier to obtaining the product at a cost consistent with the realities of funding of biomedical research programs in government, academe, and industry. This could be accomplished by establishing tissue-culture production facilities in institutions. There are several reasons why the mouse method of producing mAb cannot be abandoned: some cell lines do not adapt well to tissue-culture conditions; in applications where several different mouse mAb at high concentrations are required for injection into mice, the in vitro method can be inefficient; rat cell lines usually do not efficiently generate mAb in rats and adapt poorly to tissue-culture conditions but do produce mAb in immunocompromised mice; downstream puri-

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fication or concentration from in vitro systems can lead to protein denaturation and decreased antibody activity; tissue-culture methods can yield mAb that do not reflect the normal modification of proteins with sugars, and this abnormality might influence binding capacity and other critical biologic functions of mAb; contamination of valuable cell lines with fungi or bacteria requires prompt passage through a mouse to save the cell line; and inability of some cell lines that do adapt to tissue-culture conditions to maintain adequate production of mAb poses a serious problem. For these reasons, the committee concludes that there is a scientific necessity to permit the continuation of the mouse ascites method of producing mAb. However, note that over time, as in vitro methods improve, the need for the mouse ascites method will decrease. Recommendation 2: The mouse ascites method of producing monoclonal antibodies should not be banned, because there is and will continue to be scientific necessity for this method. There does not appear to be convincing evidence that significant pain or distress is associated with the injection into the mouse of pristane (a chemical that promotes the growth of the tumor cells), but during the accumulation of ascites there is likely to be pain or distress, particularly when some cell lines that are tissue-invasive are used and in situations of significant ascites development. Therefore, after injection of hybridoma cells, mice should be evaluated at least daily, including weekends and holidays, after development of visible ascites and should be tapped before fluid accumulation becomes distressful. A limit should be placed on the number of taps and multiple taps should be allowed only if the animal does not exhibit signs of distress. Recommendation 3: When the mouse ascites method for producing mAb is used, every reasonable effort should be made to minimize pain or distress, including frequent observation, limiting the numbers of taps, and prompt euthanasia if signs of distress appear. Two of 13 mAb approved by the Food and Drug Administration for therapeutic use cannot be produced by in vitro means, or converting to an in vitro system for their production would require (because of federal regulations) proof of bioequivalence, which would be unacceptably expensive. Furthermore, many commercially available mAb are routinely produced by mouse methods, particularly when the amount to be produced is less than 10 g, another situation where it would be prohibitively expensive to convert to tissue-culture conditions. However, with further refinement of technologies, media, and practices, production of mAb in tissue culture for research and therapeutic needs will probably become comparable with the costs of the mouse ascites method and could replace the ascites method.

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Recommendation 4: mAb now being commercially produced by the mouse ascites method should continue to be so produced, but industry should continue to move toward the use of tissue-culture methods. In a few circumstances, the use of the mouse ascites method for the production of mAb might be required. We suggest the following as examples of criteria to be used by an IACUC in establishing guidelines for the production of mAb in mice by the ascites method. 1.   When a supernatant of a dense hybridoma culture grown for 7–10 days (stationary batch method) yields an mAb concentration of less than 5 µg/ml. If hollow-fiber reactors or semipermeable-membrane systems are used, 500 mg/ml and 300 mg/ml, respectively, are considered low mAb concentrations. 2.   When more than 5 mg of mAb produced by each of five or more different hybridoma cell lines is needed simultaneously. It is technically difficult to produce this amount of mAb since it requires more monitoring and processing capability than the average laboratory can achieve. 3.   When analysis of mAb produced in tissue culture reveals that a desired antibody function is diminished or lost. 4.   When a hybridoma cell line grows and is productive only in mice. 5.   When more than 50 mg of functional mAb is needed, and previous poor performance of the cell line indicates that hollow-fiber reactors, small-volume membrane-based fermentors, or other techniques cannot meet this need during optimal growth and production. We emphasize that those criteria are not all-inclusive and that it is the responsibility of the IACUCs to determine whether animal use is required for scientific or regulatory reasons. Criteria have not been developed to define a cell line that is low-producing or when tissue-culture methods are no longer a useful means of producing mAb.