7
Conclusions and Recommendations

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, followed by 2 days of 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 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 in vitro 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 the animals. Therefore, over the next several years, as in vitro systems are further developed, in vitro methods 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



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7 Conclusions and Recommendations 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, followed by 2 days of 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 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 in vitro 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 the animals. Therefore, over the next several years, as in vitro systems are further developed, in vitro methods 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

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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 in vitro production facilities in institutions. There are several scientifically based reasons why the mouse ascites method for producing mAb should not be abandoned: some hybridoma cell lines do not adapt well to in vitro conditions; when small volumes of mouse mAb at high concentrations are required for injection into mice, the in vitro method often does not yield an acceptable product; rat hybridoma cell lines usually do not efficiently generate ascites in rats and adapt poorly to in vitro conditions but do produce mAb in immunocompromised mice; concentrating mAb from in vitro culture supernatant can lead to protein denaturation and decreased antibody activity; in vitro culture methods can yield mAb that do not reflect normal glycosylation patterns, in contrast with mAb generated by the mouse ascites method, and the lack of natural glycosylation might influence antigen-binding capacity and critical biologic functions; contamination of valuable in vitro clones with fungi or bacteria requires prompt passage through a mouse to save the cell line; and inability of in vitro-adapted cell lines to maintain adequate production of mAb poses a serious problem. 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 is no convincing evidence that significant pain or distress is associated with the priming of a mouse with pristane. During the development of ascites, there is likely to be pain or distress, particularly with some cell lines that are tissue-invasive and in situations of significant ascites fluid accumulation. Therefore, after injection of hybridoma cells, mice should be evaluated at least daily 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. It is incumbent on the IACUC to ensure that those directly responsible for using the animals be well trained and experienced in all phases of the procedure, including observation, handling, injection, and tapping of the animals. 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. It is clear that some mAb used therapeutically cannot be produced by in vitro

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means or that converting to an in vitro system for their production would require proof of bioequivalence, which would be unacceptably expensive. Furthermore, many commercially available mAb are routinely produced by mouse ascites methods, particularly when the amount to be produced is less than 10 g, another situation where it would be prohibitively expensive to convert to in vitro conditions. However, with further refinement of technologies, media, and practices, in vitro production of mAb for research and therapeutic needs will probably become comparable in costs to the mouse ascites method. 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 in vitro methods. In some circumstances, the use of the mouse ascites method for the production of mAb might be required. Examples of criteria to be used by an IACUC in establishing guidelines for the production of mAb in mice by the ascites method are: 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.   Cell lines will not grow and secrete in vitro, or analysis of mAb produced in vitro reveals that a necessary biologic activity is reduced or absent. 4.   More than 50 mg of functional mAb is needed, and previous poor performance of the hybridoma indicates that hollow-fiber reactors, small-volume membrane-based fermentors, other high-density cell systems, or other techniques cannot meet this need during optimal growth and production. 5.   Hybridoma cells producing mAb, contaminated with infectious agents, often must be passed through mice. We emphasize that the listed criteria are not all-inclusive and that it is the responsibility of the IACUC to determine whether animal use is required for scientific or regulatory reasons. Criteria have not been developed to define a low-producing hybridoma cell line or when in vitro methods are no longer a useful means of producing mAb.