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

Original text

Each virus master and working seed lot should be tested for neurotropism, viscerotropism and immunogenicity in nonhuman primates, as described in Appendix 2.

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 genetic stability 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 genetic stability 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 genetic stability of the strain (evaluated through the sequence at different stages 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 required, 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

Each final lot should be tested for the absence of abnormal toxicity in mice and guinea-pigs, using a general safety (innocuity) test approved by the NRA, and should pass the test. This test may be omitted for routine lot release once consistency of production has been established to the satisfaction of the NRA.

New text

Original text

The vaccine in the final container should be tested for endotoxin with a Limulus amoebocyte lysate test. The endotoxin content should be consistent with levels found to be acceptable in vaccine lots used in clinical trials and approved by the NRA.

New text

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

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

New text

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

The virus working seed should have a well-defined relationship to the virus master seed with respect to passage level and method of preparation, such that the virus working seed retains all of the in vitro and in vivo phenotypes and the genetic character of the virus master seed. Nonclinical and clinical data are needed to support this relationship.
Full in vitro and in vivo testing for detecting adventitious agents should be conducted on either master or working seed lots.

New text

The virus working seed should have a well-defined relationship to the virus master seed with respect to passage level and method of preparation, such that the virus working seed retains all of the phenotypes and the genetic character of the virus master seed. Nonclinical and clinical data are needed to support this relationship. Appropriate testing for detecting adventitious agents should be conducted on either master or working seed lots.

Original text

Each seed should be characterized by full-length nucleotide sequence determination and by other relevant laboratory and animal tests, which will provide information on the consistency of each virus seed.

New text

Each seed should be characterized by full-length nucleotide sequence determination and by other relevant laboratory tests, which will provide information on the consistency of each virus seed.

Original text

Seed lots should be shown to be free from mycobacteria by a method approved by the NRA. Nucleic acid amplification techniques may be used as an alternative to the microbiological culture method for mycobacteria and/or to the in vivo guinea-pig test for the detection of mycobacteria after suitable validation and agreement from the NRA (33).

New text

A test for the absence of virulent mycobacteria should be performed. Where available and approprately validated, an in vitro test should be used (for example a validated nucleic acid amplification test or culture method). If in vitro assays are not available or appropriate, a suitable compendial in vivo test may be used.

Original text

Monkey and human cell cultures inoculated with the virus antibody mixture should be observed microscopically for cytopathic changes. For virus grown in monkey or human cells, the neutralized virus is tested on a separate culture of these cells. If other cell systems are used, cells of that species, but from a separate batch, are also inoculated. At the end of the observation period, the cells should be tested for haemadsorbing viruses. Each virus master or working seed lot should also be tested in animals that include guinea-pigs, adult mice, and suckling mice.

New text

Monkey and human cell cultures inoculated with the virus antibody mixture should be observed microscopically for cytopathic changes. For virus grown in monkey or human cells, the neutralized virus is tested on a separate culture of these cells. If other cell systems are used, cells of that species, but from a separate batch, are also inoculated. At the end of the observation period, the cells should be tested for haemadsorbing viruses and other adventitious agents using a suitable method approved by the NRA

Original text

Tests in nonhuman primates:all vaccine candidates should be evaluated, at least once during nonclinical development, for neurovirulence in nonhuman primates, as detailed in Part B (nonclinical evaluation)…..the master seed should be tested in
nonhuman primates. If these tests were not performed at the master seed level, they should be performed at working seed level.....Recent data suggest that certain small animal models for neurovirulence may serve as a surrogatefor nonhuman primates, at least where viruses expressing yellow fever strain 17D genes are concerned (40).

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

Test for neurovirulence: to provide assurance that a candidate vaccine virus is not unexpectedly neurovirulent, each vaccine strain of each serotype, or a tetravalent formulation if agreed by the NRA, should be tested for neurovirulence in monkeys by inoculation of Macaca mulatta (rhesus), Macaca fascicularis (cynomolgus) or other susceptible species of monkey, in the course of preclinical evaluation.

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.