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Alternatives to Monoclonal Antibody Production (Proceedings)

Alternatives in Monoclonal Antibody Production

William S. Stokes, DVM
Associate Director
 Animals and Alternatives
 National Institute of Environmental Health Sciences and Director
 Interagency Coordinating Committee on the Validation of Alternative Methods

Validation and Acceptance Considerations for New Research Methods1

The 1993 NIH Revitalization Act, Public Law 103-43, (USC, 1993) directed the NIH to prepare a plan to support research on methods for medical research and experimentation that do not require the use of animals, that reduce the number of animals needed, and that produce less pain and distress in animals. It further stated that NIH should establish the validity and reliability of such methods. The law directed the National Institute of Environmental Health Sciences (NIEHS), which administers the U.S. National Toxicology Program, to establish criteria for the validation and regulatory acceptance of alternative test methods. An ad hoc Interagency Coordinating Committee on the Validation of Alternative Methods (ICCVAM), with representatives from 15 Federal regulatory and research programs and agencies, subsequently developed a report outlining the criteria and processes for the validation and regulatory acceptance of alternative toxicological test methods (NIEHS, 1997).

While these criteria were developed for toxicological testing methods, most of the principles of validation and acceptance also apply to any type of method being used to accomplish a specific objective. In this paper, I will discuss how these test method validation and acceptance criteria might be applied to the evaluation of in vitro techniques for monoclonal antibody production.


When discussing new methods, it is important to understand what is meant by terms such as validation, reliability, and acceptance. Validation has been defined as the process by which the reliability and relevance of a method are established for a specific purpose. Reliability is a measure of the degree to which a method can be performed reproducibly within and among laboratories over time. Relevance is the extent to which a method is useful for a specific purpose. Acceptance is the process whereby a given method is determined to be suitable for a specific purpose.

Criteria for Validationof a New Method

In order to determine the acceptability of a method, the method must have undergone adequate validation to determine the extent to which it is relevant and reliable for a specified purpose. In order to adequately describe the usefulness of a method, the following validation criteria, adopted from the ICCVAM Report (NIEHS, 1997) should generally be addressed. The extent to which each criterion is met will vary with the specific method and its proposed use.

  1. The scientific rationale for the method should be provided, including a clear statement of its proposed use. For instance, is the method being proposed as a method that can generate all types of monoclonal antibodies, or only certain types?
  2. The biological basis of the in vitro method and its relationship to the existing in vivo method should be explained.
  3. A detailed protocol for the method should be available that includes a description of the materials needed, and is sufficiently detailed that results can be satisfactorily replicated in other laboratories.
  4. The limitations of the method should be described. For instance, are there limitations on the type of monoclonal antibodies that can be produced by the method, or limitations related to the use of certain hybridoma cell lines?
  5. The reliability of the method should be described. This will include the extent of variability inherent to the method, and the variability of the method within and among laboratories.
  6. The relevance of the method should have been assessed. That is, performance of the method for the proposed range of applications should be demonstrated. This performance should be compared with the performance of the method for which the new method is being proposed as a substitute or replacement.
  7. The data supporting the validity of a new method should be available for review, including detailed protocols. The data and protocol(s) should be readily available and in the public domain. Methods and results should be published in independent peer-reviewed publications.
  8. The quality of the data supporting the validity of new methods should be described. For instance, there should be an indication of the extent that data was collected in accordance with Good Laboratory Practices (GLPs), and other quality assurance measures related to the materials and method.
  9. The methodology and results should have been subjected to independent scientific peer review. Publication in peer-reviewed scientific journals is one way of achieving a certain level of peer review.

Criteria for Acceptance of a New Method

Methods which have been evaluated in validation studies are not automatically accepted for use. The extent of acceptability of a new method will depend on the outcome of the validation studies undertaken to assess the usefulness of the method. A new method proposed for acceptance should generally be supported by the following criteria, which were adapted from the regulatory acceptance criteria for new test methods (NIEHS, 1997).

  1. The method should have undergone independent scientific peer review by disinterested persons who are experts in the field, knowledgeable in the method, and financially unencumbered with the outcome of the evaluation.
  2. There should be a detailed protocol with standard operating procedures (SOPs), a list of operating characteristics, and criteria for assessing results and performance of the method.
  3. The method should be at least as useful as the method that it is proposed to be used as a substitute or replacement.
  4. There should be adequate data for the range of proposed uses of the method. For instance, for an in vitro method for monoclonal antibody production, data should support the proposed range of applications.
  5. The strengths and limitations of the method should be described.
  6. The method should be robust, i.e., relatively insensitive to minor changes in protocol, and transferable among properly equipped and staffed laboratories.
  7. The method should be time and cost effective, and likely to be used.
  8. For methods that involve the use of animals, there should have been adequate consideration of the refinement, reduction, and replacement of animal use.

Considerations Relevant to In Vitro Methods for Monoclonal Antibody Production

After reviewing the literature and information available on in vitro methods for monoclonal antibody production, I identified eight issues that warrant further attention by participants in this workshop, particularly scientists developing the methods, as well as IACUCs and OPRR. These are as follows:

  1. What is the current validation status of each of the in vitro methods that we have heard about today? Much useful information has been presented today that further supports the usefulness of several methods. However, a comprehensive review of the validation status of each method has not been readily available prior to this meeting. Such information is essential in order for scientists to make an informed decision on the usefulness of these methods for their particular need.
  2. Are there specific applications for which in vitro methods have been shown to be as useful as in vivo methods? Based on information presented at this workshop, the answer is clearly yes. Is there evidence that in vitro methods are useful for all monoclonal antibody production? Again, based on information presented today, it appears that the answer at this time is no, although we have heard estimates from experts that the in vitro methods can be used for 85-90% of such production at this time.
  3. Are validation studies needed to further determine the usefulness of in vitro monoclonal antibody methodologies? The answer here is obviously yes. The extent of such further validation studies should be determined by scientists that are experts in the field.
  4. Are additional research and development efforts needed to improve the in vitro methods? The answer here is obviously yes. Continued development and application of new technologies can be expected to further enhance the efficiency of the in vitro methods discussed here today. The NIH Small Business Innovative Research Grants Program and the Small Business Technology Transfer Grants Program might be appropriate mechanisms by which additional method development and validation could be accomplished.
  5. Are the in vitro methods "reasonably and practicably available&qupt; available, in terms of time, cost, and equipment? Based on information presented today, the answer appears to be yes, provided that all direct and indirect cost factors are considered.
  6. What advice should be presented to scientists and IACUCs at this time? Should the in vitro methods always be attempted first, and mice only approved for use if the in vitro method fails? Or should the in vitro methods only be expected to be used initially for certain applications, and mice used initially if a scientific rationale is provided for why the in vitro method is not likely to be useful? Or, as we have heard from at least one institution today, do the IACUCs simply encourage the use of in vitro methods, but approve in vivo methods if requested by the investigator. Finally, should there be a total ban on the use of mice for monoclonal antibody production? As Dr. Sibal and others have discussed today, this would not be appropriate at this time.
  7. What is the best way to disseminate information on valid new in vitro methods? There is obviously a need to provide scientifically accurate information about the concept, usefulness, availability, cost, and known limitations of in vitro methods to the scientific community.
  8. What is the most efficient and effective way to provide training on valid new in vitro methods? Institutions are required by the PHS Policy on the Humane Care and Use of Laboratory Animals (PHS, 1996) to assure that scientists, animal technicians, and other personnel involved with animal care and use have available to them instruction or training on the concept, availability, and use of research methods that limit the use of animals. Institutions should ensure that the spirit of this requirement is fulfilled with regard to in vitro methods for monoclonal antibody production, as well as other alternative methods that can refine, reduce, and replace the use of animals (Stokes and Jensen, 1995).

In conclusion, the presentations today have demonstrated how new technologies and scientific knowledge can be incorporated into innovation methods to support biomedical research and also reduce the need for animals. Based on the rapid improvement of these in vitro methods over the past several years, it is likely that their efficiency and usefulness will continue to evolve at a rapid pace. While increased efficiency in terms of time and cost will speed acceptance by the scientific community, acceptance will also be facilitated by the timely dissemination of accurate information regarding their validation status and availability.


  • National Institute of Environmental Health Sciences (NIEHS). Validation and regulatory acceptance of toxicological test methods: a report of the ad hoc Interagency Coordinating Committee on the Validation of Alternative Methods. NIH Publication No. 97-3981. NIEHS, Research Triangle Park, NC, USA:, 1997.
  • Public Health Service (PHS). Public Health Service Policy on Humane Care and Use of Laboratory Animals. U.S. Department of Health and Human Services. Washington, D.C., 1996.
  • Stokes, W.S., and Jensen, D.J.: Guidelines for institutional animal care and use committees: consideration of alternatives. Contemporary Topics in Laboratory Animal Science, 34(3):51-60, 1995.
  • United States Code (USC). National Institutes of Health Revitalization Act of 1993. Public Law 103-43. 42 USC. U.S. Government Printing Office, Washington, DC. 1993.

For Additional Information, contact:
William S. Stokes, D.V.M., Diplomate ACLAM
Chair, Interagency Coordinating Committee on the Validation of Alternative Methods (ICCVAM)
Environmental Toxicology Program
National Institute of Environmental Health Sciences
MD EC-17, P.O. 12233
Research Triangle Park, NC 27709
tel. 919-541-7997 or -3138
fax 919-541-0947 or -2242

1Presented at the "Alternatives in Monoclonal Antibody Production: A Workshop of The Johns Hopkins Center for Alternatives to Animal Testing and the Office of Protection from Research Risks, National Institutes of Health". September 24-25, 1997, Baltimore, Maryland.

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