Monoclonal antibodies, following the development of this technology in 1975, have become an extraordinarily important resource for medical research, diagnosis, therapy, and basic science. In recognizing the overwhelming importance of this technology to modern medical science, it is also important to recognize that monoclonal antibody production is still largely dependent on the use of experimental animals.

In November 1997, The Office for Protection from Research Risks (OPRR) at the National Institutes of Health, forwarded a letter to all Public Health Service (PHS) awardee institutions and Institutional Animal Care and Use Committees (IACUCs) on avoiding or minimizing discomfort, distress, and pain in the care and use of animals for the production of monoclonal antibodies using mouse ascites method since there is evidence that the ascites method of monoclonal antibody production causes discomfort, distress, or pain. Accordingly, the IACUCs at all Public Health Service awardee institutions must critically evaluate the proposed use of the ascites method. The Committee must determine that a) the proposed use is scientifically justified, b) methods that avoid or minimize discomfort, distress, and pain (including in vitro methods) have been considered, and c) the latter have been found unsuitable. Fulfillment of this three-part IACUC responsibility, with appropriate documentation, is considered central to an institution's compliance with its Animal Welfare Assurance and the PHS Policy .

In keeping with the need to minimize both the use and the discomfort and pain inflicted on experimental animals, the following considerations and recommendations in the application of monoclonal antibody technology are proposed.

Monoclonal antibody technology relies on experimental animals for two basic steps. The first step involves immunizing mice. Although there are alternatives to this step (either by in vitro immunization steps or using recombinant libraries), these alternatives have not proven to provide the same level of efficiency, specificity, or affinity of antibody available from traditional immunization protocols. In addition, with the advent of alternatives to Freund's adjuvant, less than minimal or slight pain or discomfort accompanies this step and therefore there is no compelling reason to consider alternatives at this time. Careful consideration of the total number of required animals should be the investigators main concern in justification of this step .

A second common step that uses animals comes in antibody production. In order to produce large quantities of monoclonal antibody, it has been traditional to grow hybridoma cell lines as an ascites tumor in vivo. In general, this procedure produces antibody titers approximately 1000-10,000-fold higher than those obtained in tissue culture. Since 1-10 ml of ascites fluid containing 1-5 mg/ml specific antibody can be obtained per animal while hybridoma cells in culture produce only 0.5-5 µg/ml, one mouse can produce antibody equivalent to between 1 and 100 liters of tissue culture fluid. The main advantages of ascites are the extremely high yield of antibody and that the method is not excessively labor-intensive. However, its main disadvantage is the potential pain and discomfort caused to animals, due to painful peritonitis, abdominal tension, and infiltratively-growing tumors. It should also be noted that the monoclonal antibody produced by this method is contaminated by endogenous immunoglobulin and has the potential for contamination by viruses or bioreactive cytokines that may interfere with later use.

There are in vitro alternatives to monoclonal antibody production by ascites. These include standard static or agitated suspension cell cultures, membrane-based and matrix-based culture systems, and high cell-density bioreactors. The disadvantages to these systems include their substantially greater effort and higher labor costs, increased costs due to the components of tissue culture media, and the poor growth and/or antibody secretion of some hybridoma lines in vitro. In particular, high cell-density bioreactors are probably beyond the capability of most laboratories due to the high initial and ongoing material and labor costs and the specialized expertise required.

In light of the above considerations, the New York University IACUC requests that investigators consider the following recommendations in designing monoclonal antibody production.

In general, in vitro methods for monoclonal antibody production are considered standard and accepted practice. The use of the ascites method requires rigorous and well-documented justification. Justifications based solely on cost or convenience will not be considered adequate.

In addition, the NYU IACUC strongly urges the Medical Center to establish a monoclonal antibody core facility with high-density cell culture bioreactor capability that would obviate any need for continued use of the ascites method.

• Most applications require only small quantities and low concentrations of antibodies. Examples of common applications requiring only small quantities of antibody include immunoblots, immunoprecipitations, immunocytochemistry, flow cytometry, and small-scale affinity columns. Such applications are easily accommodated by use of unpurified tissue culture supernatants or by monoclonal antibodies purified from tissue culture supernatants. For purposes requiring up to approximately 10-50 mg of antibody, standard tissue culture methods involving growth of up to 50 liters of hybridoma cells should be considered the method of choice. Proposed use of ascites for applications of this kind would require specific justification (e.g., use of a hybridoma with unfavorable growth characteristics in vitro) in addition to the considerations described in 3 (below).
• Small-scale membrane-based culture systems are available that facilitate production of monoclonal antibodies in the range of 10-100 mg per culture in 5-30 days. These systems are relatively inexpensive and do not require specialized facilities or expertise. Investigators with applications that require up to 1 g of monoclonal antibody should consider the use of this in vitro alternative for production. Use of ascites production for such applications would require specific justification demonstrating that special circumstances warrant use of the in vivo method. In addition to the considerations described in 3 (below), justification must demonstrate that the disadvantages of ascites production (including pain and discomfort to the animals and potential in vivo contamination) are outweighed by the specific requirements of the individual project.
• Production of greater than 1 g of monoclonal antibody by in vitro methods is probably outside the current capabilities of most laboratories. For these applications, ascites production is presently the only alternative for in-house production. Approval of such projects will require adequate justification for the required amounts of antibody . In addition, the investigator must demonstrate the required expertise for working with tumor-bearing mice, including an adequate daily monitoring system to insure that animals do not experience unnecessary pain or discomfort. Assurance must be made that no animal will be allowed to develop tumors larger than 20% of host body weight. Ascites fluid must be harvested on a single occasion only, either under terminal anesthesia or post mortem. Animals must be killed without delay if they show more than mild distress, overt tumor deposits or spread, or significant dehydration or cachexia.
• The specific guidelines for consideration by Principal Investigators when developing animal study proposals and for Animal Care and Use Committees when reviewing proposals involving the mouse ascites method are:

  a. The volume of the priming agent should be reduced to as small a volume as necessary to elicit the growth of ascitic tumors and at the same time reduce the potential for distress caused by the irritant properties of the priming agent. Although 0.5 ml Pristane has been standard for adult mice, 0.1-0.2 ml has been found to be as effective for many hybridomas.

  b. The time interval between priming and inoculation of hybridoma cells as well as the number of cells in the inoculum are determined empirically. Inocula range from 105 -107 cells in volumes of 0.1 - 0.5 ml and are usually administered 10 -14 days after priming. Generally, very high concentrations are associated with greater mortality and concentrations < 1 x 105 cells elicit fewer ascitic tumors and these tend to have a smaller volume yield. Cell suspensions should be prepared under sterile conditions in physiological solutions.

  c. Hybridomas should be MAP (mouse antibody production) or PCR tested before introduction into the animal host to prevent potential transmission of infectious agents from contaminated cell lines into facility mouse colonies and possibly to humans handling the animals.

  d. Animals should be monitored at least once daily, seven days a week by personnel familiar with clinical signs associated with ascites production and circulatory shock.

  e. Ascites pressure should be relieved before abdominal distension is great enough to cause discomfort or interfere with normal activity. Manual restraint or anesthesia may be used for tapping. Aseptic technique should be used in withdrawing ascitic fluid. The smallest needle possible that allows for good flow.

In accordance with the Animal Welfare Assurance and the Public Health Service Policy, and to appropriately document that investigators proposing the use of monoclonal antibodies have considered alternatives to minimize discomfort, distress, and pain, in future please refer to these recommendations before submitting protocols to the IACUC. Additionally, subsequent to the November 1997 OPRR Letter, the IACUC expects that investigators will incorporate these recommendations into all new applications submitted for funding.