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

Original text

Each final lot should be tested for pyrogenic substances. Where appropriate, tests for endotoxin (for example, the limulus amebocyte lysate (LAL) test) should be performed. However, where there is interference in the test – for example, because of the addition of an immunostimulant such as MPL – a test for pyrogens in rabbits should be performed. A suitably validated monocyte-activation test may also be considered as an alternative to the rabbit pyrogen test. The test is conducted until consistency of production is demonstrated, subject to the agreement of 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

Preparation of strain-specific antiserum reagents: the virus is digested with bromelain to remove haemagglutinin from virus particles. The haemagglutinin missing the transmembrane portion is purified and sheep are immunized multiple times to generate hyperimmune antiserum which is tested for suitability in SRID assays in terms of potency and the quality of the SRID zones produced.

New text

Original text

It is obviously important that the strains of virus used to prepare live measles vaccine should show no tendency to produce neurological complications of the type encountered in some cases of natural measles. Present experience indicates that the live vaccines so far used are, indeed, safe in this respect. In the absence of a more satisfactory test, the intracerebral inoculation of monkeys has been used as a laboratory test to evaluate this property; the development of more reliable methods is desirable. Post-measles encephalopathy may be the result of an immunopathogenic reaction, but the underlying mechanism is not known.

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

Tests for neurovirulence: Each master seed or working seed lot shall be shown to be free from neurovirulence by tests in measles-susceptible monkeys of a species approved by the national control authority. To avoid the unnecessary use of monkeys, virus seed lots should be prepared in large quantities.

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

Tests for neurovirulence: Each master seed or working seed lot shall be shown to be free from neurovirulence by tests in rubella-susceptible monkeys of a species approved by the national control authority. To avoid the unnecessary use of monkeys, virus seed lots should be prepared in large quantities.

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

Tests for neurovirulence: Each master seed or working seed lot shall be shown to be free from neurovirulence by tests in mumps-susceptible monkeys of a species approved by the national control authority. To avoid the unnecessary use of monkeys, virus seed lots should be prepared in large quantities.

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 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 cell cultures shall be tested for the presence of haemadsorbing viruses, using guinea-pig red cells. In some countries; the national control authority fequires that tests for haemadsorbing virsses should also be made on control cultares 3-5 days and 12 days after inoculation of the production cultures and that other types of red 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. In all tests readings should be taken after incubation for 30 minutes at 0-4OC, and again after a further incubation for 30 minutes at 20-25°C. For the test with monkey red cells readings should also be taken after a final incubation for 30 minutes at 34-37 C

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

At the end of the observation period, 25% of the control cell cultures shall be tested for the presence of haemadsorbing viruses, using guinea-pig red cells. In some countries; the national control authority fequires that tests for haemadsorbing virsses should also be made on control cultares 3-5 days and 12 days after inoculation of the production cultures and that other types of red 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. In all tests readings should be taken after incubation for 30 minutes at 0-4OC, and again after a further incubation for 30 minutes at 20-25°C. For the test with monkey red cells readings should also be taken after a final incubation for 30 minutes at 34-37 C

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

At the end of the observation period, 25% of the control cell cultures shall be tested for the presence of haemadsorbing viruses, using guinea-pig red cells. In some countries; the national control authority fequires that tests for haemadsorbing virsses should also be made on control cultares 3-5 days and 12 days after inoculation of the production cultures and that other types of red 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. In all tests readings should be taken after incubation for 30 minutes at 0-4OC, and again after a further incubation for 30 minutes at 20-25°C. For the test with monkey red cells readings should also be taken after a final incubation for 30 minutes at 34-37 C

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

Additional 10ml samples of each pool shall be tested in both human and simian cells.

New text

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.