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

Original text

Test for neurovirulence in weanling mice:
Both master seed and working seed should be tested for neurovirulence in weanling mice.
Test for reversion in suckling mice:
Both master seed and working seed should be tested for reversion to virulence in suckling mice.
Test for neuroinvasiveness in weanling mice:
Master seed should be tested for neuroinvasiveness in weanling mice.
Test for neurovirulence in monkeys:
New virus master seed lots of SA14-14-2 should be tested for neurovirulence in monkeys. To avoid the unnecessary use of monkeys, virus master seed lots should be prepared in large quantities. A reference preparation should be included in each test.
An alternative test may be used, with the agreement of the NRA, if equal or greater sensitivity has been demonstrated. Testing of the working seed of SA14-14-2 in monkeys is not required.

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 in mice:
Both master seed and working seed should be tested for neurovirulence in mice.
Test in monkeys:
Because JE-CV vaccine is based on YF vaccine, both master seed and working seed should be tested in monkeys by following the WHO Recommendations for YF vaccine (17).

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

At the end of the observation period, 25% of the control cells should be tested for the presence of haemadsorbing viruses using guinea-pig red blood cells.
In some countries, the NRA requires that other types of red cells, including cells from humans (blood group O), monkeys and chickens (or other avian species) be used in addition to guinea-pig cells.

New text

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

NAT-based assays may be used as an alternative to microbiological methods for culturing mycobacteria or to the in vivo guinea-pig test for detection if they have been validated and the NRA agrees (8).

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

Each final bulk should be tested for neurovirulence in mice. This test should be validated if it has not been tested previously with final bulk.
The test may be performed on the final product or the bulk material, whichever is acceptable to the NRA, and as described in sections A.3.2.5.6.1 and A.3.2.5.6.2.
The test for neuroinvasiveness is not required for single harvests, the pooled harvest or the final product.

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

Each final lot should be tested for unexpected toxicity (i.e. abnormal toxicity) using a general safety test approved by the NRA. This test may be omitted for routine lot release once the consistency of production has been established to the satisfaction of the NRA and when
good manufacturing practices are in place. Each lot, if tested, should pass a general safety test.

New text

Original text

The pyrogen content of the purified polysaccharide should be determined and shown to be within acceptable limits agreed by the NRA. A recognized pyrogenicity test can be performed in rabbits; alternatively, the Limulus amoebocyte lysate test can be performed.

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

To ensure an acceptable level of endotoxin in the final product, the endotoxin content of the monovalent bulk may be determined and shown to be within acceptable limits agreed 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

The bulk conjugate should be tested for the absence of specific toxicity of the carrier protein where appropriate (e.g. when tetanus or diphtheria toxoids have been used). Absence of specific toxicity of the carrier protein may also be assessed through validation of the production process.

New text

The carrier protein should be tested for the absence of specific toxicity of the carrier protein where appropriate (e.g. when tetanus or diphtheria toxoids have been used). Absence of specific toxicity of the carrier protein may also be assessed through validation of the production process. Where available and appropriately validated and with approval from the NRA, an in vitro test should be used. If in vitro assays are scientifically justified as unavailable and inappropriate, a suitable in vivo test may be used.

Original text

The bulk conjugate should be tested for the absence of specific toxicity of the carrier protein where appropriate (e.g. when tetanus or diphtheria toxoids have been used). Absence of specific toxicity of the carrier protein may also be assessed through validation of the production process.

New text

The carrier protein should be tested for the absence of specific toxicity of the carrier protein where appropriate (e.g. when tetanus or diphtheria toxoids have been used). Absence of specific toxicity of the carrier protein may also be assessed through validation of the production process. Where available and appropriately validated and with approval from the NRA, an in vitro test should be used. If in vitro assays are scientifically justified as unavailable and inappropriate, a suitable in vivo test may be used.