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Session A4: Vaccines and Biologicals: Application of the Three R'sChairs: Coenraad Hendriksen (The Netherlands) and Klaus Cussler (Germany) A4: ECVAM's Contributions to the Implementation of the Three R's in the Quality Control of Biologicals Biologicals can be defined as products derived from a living organism, including vaccines, immunosera, hormones, polyclonal and monoclonal antibodies, recombinant DNA proteins, cytokines, and blood products. They are important therapeutic and preventive agents in human and animal health care. Due to their origin and the production techniques used, involving the attenuation, inactivation, or detoxification of the virulent microorganism or toxin in the case of vaccines, these products undergo extensive quality control during production and, in particular, each batch of the finished product is tested for purity, safety, and potency, which often involves the use of animals. Due to the large number of animals used, the fact that the animal tests used are often distressful, and the availability of various alternatives, the implementation of the Three R's principles in the production and quality control of biologicals is one of ECVAM's priorities. Recent developments and achievements in Europe will be discussed with a focus on the direct involvement of ECVAM, for example, via workshops, financial contributions to conferences, production of manuals and reports, training, and financial support of and participation in pre-validation, validation, and feasibility studies. A4: Biological Standardization: A Coordinated European Program for the Reduction of Animal Testing A4: Possibilities and Problems of Implementing Three R's Alternatives in Developing Countries Animals have been widely used in the development, production, and quality control of vaccines. The availability of newer vaccines, consisting of well-defined, purified antigens, has facilitated the use of in vitro techniques for establishing vaccine potency. At the same time, increased awareness and social concern has led to attempts in reducing the use of animals, refining animal-based methodologies to decrease distress, and/or replacing animal tests for alternatives. Substitute procedures for "potency testing" of diphtheria and tetanus toxoid-containing vaccines have been developed that require fewer animals and preclude the use of lethal challenge. The Pan American Health Organization has been assisting countries in the Region of the Americas to implement these procedures in their regulatory quality control activities. There have been important advances in this program. Although difficulties may arise in the implementation process, due to the idea that alternatives may be costlier, analysis of the additional cost of adopting "Good Practices for the Use of Animals" may show that the perception that animal testing is cheaper is incorrect. The final decision for implementation will be taken based on common sense and application of the best science available. A4: Development of Alternative Lot Release Tests for Vaccine Products The office of vaccines research and review (OVRR) within FDA's center for biologics evaluation and research is responsible for the licensing and oversight of vaccines for the United States. Part of that oversight involves product testing for lot release. A number of these release tests--primarily those for establishing vaccine potency--are animal-based. These potency tests often require the use of a large number of animals, are time and labor intensive, and have large associated test variances. Several laboratories within OVRR have previously developed alternatives to animal-based tests; OVRR laboratories are continuing this effort. An update of OVRR's present efforts will be highlighted in the current presentation. Areas that will be discussed include alternative tests for: anthrax vaccine, rabies vaccine, diphtheria and tetanus toxoids, and mumps neurovirulence. The alternative to the mumps neurovirulence test utilizes mice instead of monkeys. A4: A New Approach in the Quality Control Testing of Inactivated Bacterial Vaccines In quality control of inactivated vaccines, immunogenicity testing is essential. Current regulatory requirements for immunogenicity testing focus on the uniqueness of each individual vaccine batch, and emphasis is given to the final product. Frequently, large numbers of animals are required to estimate the vaccine potency in International Units (IU) per ml. A new approach in immunogenicity testing of inactivated bacterial vaccines is under study. In this approach, quality control is seen as an instrument to monitor batch-to-batch comparability at the critical steps in the production process and testing of vaccines rather than estimating the potency of the final product in IU/ml. This consistency concept is already in use for well-defined biologicals, such as hormones and pneumonococcal polysaccharide vaccines. Good manufacturing practice resulting in robust production processes and improved characterization methods has brought the consistency concept within reach of immunnogencity testing of inactivated bacterial vaccines. In literature several immunochemical, bio-and physicochemical tests and in vitro functional assays that can be used to assess comparability of vaccine batches are described. The relevance and reliability of the different in vitro tests have to be studied. Finally, it should be possible to control immunogenicity of vaccines for batch release purposes by using a set of in vitro tests, monitoring batch-to-batch consistency in production. A4: Results and Recommendations of an ECVAM Workshop on Alternatives in Rabies Vaccine Quality Control An ECVAM/AGAATI workshop was held earlier this year to enlighten Three R's approaches in the quality control of inactivated rabies vaccines. Several options for the refinement of the current mouse potency test were identified; single-dilution test, use of control animals (virus titration, reference preparation) for several vaccines under test, use of anesthesia for infection of the mice, use of humane instead of lethal endpoints as indicator. Possibilities for the replacement of the mouse potency test with an in vitro test were presented. None of the in vitro tests (antibody induction test, antibody binding test, single radial immunodiffusion, ELISA) is universally applicable, but these tests are suitable for in process testing. It was concluded that the safety tests in animals, which are required by several authorities, could be either discontinued (batch safety test) or replaced by in vitro tests (residual live virus test in tissue cultures, pyrogenicity tests by cytokine determination) after appropriate validation. A4: Serological Methods for Potency Testing of Tetanus Toxoid Vaccines for Human Use A collaborative study has been performed to validate two in vitro serological assays, ELISA and ToBI test, as alternatives to the direct challenge procedure for potency testing of tetanus toxoid vaccines for human use (Ph.Eur. monograph Tetanus vaccine (adsorbed) (0452)). The study was commissioned by the European Directorate for the Quality of Medicines (EDQM) and the European Centre for the Validation of Alternative Methods (ECVAM) as part of the strategy to replace the multi-dose challenge test in mice or guinea pigs by a single-dose serological test in guinea pigs. In six laboratories, guinea pigs were immunized with tetanus toxoid vaccines from different manufacturers and representing various types of combined products, including one product of borderline quality. Blood samples were taken 2-4 days before challenge. Parameters that were analyzed included: correlation of vaccine potencies obtained by direct challenge test and by serological assays, prediction of survival based on antibody concentrations, and correlation of antibody concentrations in ELISA, ToBI test, and in vivo Toxin Neutralization test in mice. In addition, ELISA and ToBI test repeatability and reproducibility were further studied by titration of a total of 28 serum samples in 23 laboratories. This presentation will provide information on the outcome of the collaborative study. From the results of the study, it is concluded that in vitro serological potency tests and the challenge test are similar with regard to relevance and reliability for the vaccines included in the study. The conditions for the implementation of single-dose serological models for routine batch release of tetanus toxoid vaccines for human use will be outlined. It is envisaged that implementation of the serological procedures will result in a marked refinement of the potency test, as well as a substantial reduction of numbers of laboratory animals being used. A4: In Vitro Assays for Biologicals Containing Leptospira interrogans serovars canicola, grippotyphosa, icterohaemorrhagiae, and pomona Current United States Standard Requirements for veterinary biologicals (Title 9, Code of Federal Regulations, Part 113) mandate that the potency of each serial (batch or lot) of certain Leptospira Bacterins be demonstrated by a hamster vaccination-challenge assay. These tests are expensive, and concerns for animal welfare and biosafety make replacement of these in vivo assays with in vitro assays highly desirable. The Center for Veterinary Biologics (USDA-APHIS) has developed antigen-capture enzyme-linked immunosorbent assays (Elisa's) for testing the potency of veterinary biologicals containing Leptospira canicola, grippotyphosa, icterohaemorrhagiae, and pomona. The detection monoclonal antibody in each sandwich ELISA reacts with a serovar-specific lipooligosaccharide moiety (as demonstrated by Western blot). Each detection monoclonal antibody has been shown to provide passive protection against virulent challenge. Serial dilutions of reference and test bacterins are tested in a parallel line assay to quantify the antigen in each preparation. A test bacterin is satisfactory if it has a relative potency of 1.0 or greater when compared to its reference. Veterinary Services Memorandum No. 800.102 has recently been issued, providing biologics firms with guidance for obtaining exemptions to Standard Requirements for hamster potency assays for leptospiral bacterins. Revised Standard Requirements for all four leptospiral serovars have been drafted for incorporation into the U.S. Code of Federal Regulations.
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