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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