Review of animal use requirements in WHO biologics guidelines – database of suggested guideline revisions

Original text

A suitable quantitative potency test in mice has been developed (3, 4). In brief, groups of 10–20 mice, 5 weeks of age, or in the weight range 17–22 g, are immunized intraperitoneally with a series of at least three dilutions of either the reference or the test vaccine, using a suitable diluent. Some manufacturers use a diluent that contains the same concentration of alum as the vaccine.
Terminal bleeds are taken after 28 or 42 days, or when an adequate antibody response has developed. Individual sera are assayed for antibodies to HBsAg.

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Original text

The vaccine potency (in vivo and in vitro) should be assessed against a product specific reference preparation. Points to consider when establishing a product-specific reference preparation include:
■ evaluation with the mouse potency test;
■ evaluation with an in vitro potency test;

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If an in vitro assay is not suitable for a particular combination, an in vivo assay should be used. This should be performed at the level of the final bulk.

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Test procedures involving the inoculation of animals with live poliovirus (such as neurovirulence tests) should be performed within containment laboratories and special care taken in line with GAPIII recommendations.

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Genetic stability can be evaluated by both biological and molecular methods. Biological methods include passage in vitro and in vivo followed by neurovirulence testing in transgenic mice or monkeys.

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The potential neurovirulence of a new vaccine strain should be assessed during preclinical development and a risk analysis carried out based on available scientific data and information. If molecular consistency has been demonstrated during characterisation, then assessment of neurovirulence may be omitted for subsequent viral seed lots and/or routine manufacturing. For existing products where animal neurovirulence testing is currently prescribed, this test can be waived when safety and genetic stability of the product are sufficiently assured. This can be established by historical / (pre-) clinical, and pharmacovigilance data, and by data generated with nucleic acid amplification and sequencing techniques, to support the molecular consistency of the virus and for the establishment of a link between genetic sequences and in vivo phenotypes. For all products, a risk-based approach should be performed taking into consideration the genetic features and molecular consistency of the strain (sequence evaluated at different manufacturing steps, determined with traditional or new sequencing technologies) and the nature of the vaccine (attenuated, chimeric, genetically modified), to assess whether a neurovirulence assay is required and what animal model is most suitable. If an in vivo assay is scientifically justified, it should be established at what level (Master Seed Lot, Working Seed Lot, monovalent bulk) the test should be performed to avoid unnecessary duplication.

Original text

The sample should be tested in primary Cercopithecus sp. kidney cell cultures, or cells that have been demonstrated to be of equal susceptibility to SV40 virus, and in human diploid cells.

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A strategy for testing adventitious viruses in vaccines must be developed based on a risk assessment. Relevant culture methods and/or specific molecular biology or broad molecular methods should be part of the overall testing package with the agreement of the NRA. In vivo tests may only be used if the risk assessment indicates that this test provides an additional risk mitigation taking into account the overall testing package.

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As an additional monitor of quality, sera may be examined for freedom from bacteriophage and endotoxin.

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The need for pyrogenicity testing should be assessed during the manufacturing development process and be re-evaluated following any significant changes in the production process or relevant reported production inconsistencies that may influence pyrogenicity. A risk-based approach should be implemented which is suitable to the manufacturing process and the product depending on the potential presence of endotoxins and non-endotoxin pyrogens. The endotoxin content of the final product should be determined using a suitable in vitro assay, such as the recombinant factor C (rFC) or limulus/tachypleus amoebocyte lysate (LAL/TAL) tests. The rFC method is strongly recommended due to concerns over the impact on the sustainability of limulus stocks. The endotoxin content should be consistent with levels found to be acceptable in final product lots used in clinical trials and within the limits agreed upon with the NRA. A monocyte activation test (MAT) may be used for pyrogen testing after a product-specific validation. The use of the rabbit pyrogen test should be avoided due to its inherent variability, high retesting rates, and interspecies differences in pyrogenic responses as compared to humans.

Original text

At the end of the observation period, at least 25% of the control cells should be tested for the presence of haemadsorbing viruses using guinea-pig red blood cells. Some NRAs require, as an additional test for haemadsorbing viruses, that other types of red blood cells, including cells from humans (blood group IV O), monkeys and chickens (or other avian species), should be used in addition to guinea-pig cells.

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At the end of the observation period a fraction of culture comprising not less than 25% of the total should be tested for the presence of haemadsorbing viruses, using red blood cells from guinea-pig or other suitable red blood cells. It is not necessary to use red blood cells from multiple species. If the red blood cells have been stored prior to use in the haemadsorption assay, the duration of storage should not have exceeded 7 days and the temperature of storage should have been in the range of 2–8 °C.

Original text

Appropriate in vivo tests which may be used to evaluate the phenotype of virus in purified monovalent pools produced from the Sabin vaccine strains are described in section A.4.4.7.2 of the WHO Recommendations to assure the quality, safety and efficacy of poliomyelitis vaccines (oral, live, attenuated).
For other attenuated strains derived by recombinant DNA technology, the need for testing purified monovalent pools by in vivo neurovirulence tests should be considered and should be scientifically justified with the agreement of the NRA.

New text

The potential neurovirulence of a new vaccine strain should be assessed during preclinical development and a risk analysis carried out based on available scientific data and information. If molecular consistency has been demonstrated during characterisation, then assessment of neurovirulence may be omitted for subsequent viral seed lots and/or routine manufacturing. For existing products where animal neurovirulence testing is currently prescribed, this test can be waived when safety and genetic stability of the product are sufficiently assured. This can be established by historical / (pre-) clinical, and pharmacovigilance data, and by data generated with nucleic acid amplification and sequencing techniques, to support the molecular consistency of the virus and for the establishment of a link between genetic sequences and in vivo phenotypes. For all products, a risk-based approach should be performed taking into consideration the genetic features and molecular consistency of the strain (sequence evaluated at different manufacturing steps, determined with traditional or new sequencing technologies) and the nature of the vaccine (attenuated, chimeric, genetically modified), to assess whether a neurovirulence assay is required and what animal model is most suitable. If an in vivo assay is scientifically justified, it should be established at what level (Master Seed Lot, Working Seed Lot, monovalent bulk) the test should be performed to avoid unnecessary duplication.

Original text

After removal or neutralization of the inactivating agent, the samples should be tested for the absence of infective poliovirus by inoculation into tissue cultures. Kidney cells from some monkey species, such as those of the genera Macaca, Cercopithecus and Papio spp. – as well as mouse L20B cells expressing human poliovirus receptor, and HEp-2 (Cincinnati) cells – all appear to possess adequate sensitivity. If other tissue culture systems are used, they should have been shown to possess at least the same sensitivity to poliovirus as those specified above by inoculating with partially formalin-inactivated virus (as opposed to infectious, untreated virus) as formalin treatment changes the biological properties of poliovirus (see below). When primary monkey kidney cells are used for this test, the two samples should be inoculated into culture vessels of tissue cultures derived from different batches of cells.

New text

After removal or neutralization of the inactivating agent, the samples should be tested for the absence of infective poliovirus by inoculation into tissue cultures. Wherever possible, established and validated continuous cell lines should be used in preference over the use of primary cells.