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, if appropriate. Tests for bacterial endotoxin (for example, the limulus amoebocyte lysate (LAL) test) should be performed. However, if there is interference in the test – for example, because of the addition of an immunostimulant such as 3-O-desacyl-4ʹ-monophosphoryl lipid A – a test for pyrogens should be performed. The classical rabbit pyrogen test should now be replaced by a validated monocyte-activation test 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

An appropriate in vitro or in vivo quantitative test for potency should be performed using samples representative of each final vaccine lot. In the case of freeze-dried vaccines, potency should be determined after the freeze-dried product has been reconstituted with the approved diluent.

New text

An appropriate quantitative test (in vitro wherever possible) for potency should be performed using samples representative of each final vaccine lot. In the case of freeze-dried vaccines, potency should be determined after the freeze-dried product has been reconstituted with the approved diluent.

Original text

Rabbits, 2–4 weeks old, may be used as the source of kidneys for cell culture.

New text

Original text

If vaccine is prepared in animal skins, animals of a species approved by the national control authority, in good health, and not previously employed for experimental purposes should be used. Manufacturers are encouraged to use animals from closed or intensively monitored colonies where these are available. The animals should be kept in well-constructed and adequately ventilated animal rooms in cages spaced as far apart as possible.

New text

Original text

Suckling 3–5-day-old CD-1 outbred mice are inoculated intracerebrally with 20 ml of the filtered bulk suspension or the comparator vaccine. The target titre of the inoculum is 5.0 log 10 pfu/ml. The titre of virus in the inoculum should be confirmed by titration of the residual inocula, and should be within 0.5 log 10 pfu of the target. The mice are observed for up to 21 days and the mortality ratio and survival times are compared between groups.

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 filling lot should be tested for unexpected toxicity (sometimes called abnormal toxicity) using a general safety (innocuity) test approved by the national regulatory authority. This test may be omitted for routine lot release once consistency of production has been well established to the satisfaction of the national regulatory authority and when good manufacturing practices are in place. Each lot, if tested, should pass a test for abnormal toxicity. However it should be noted that preliminary experiments may be needed to determine the sample volume to use for this product in this test.

New text

Original text

Each filling lot should be tested for endotoxin if this has not been done on the final bulk. The method used and content permitted should be approved by the national regulatory authority.

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

If primary monkey kidney cell cultures were used in the adaption of the virus strain to cell culture, tests for simian viruses should be done.

New text

A strategy for testing adventitious viruses in viral 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 be only used if the risk assessment indicates that this test provides a risk mitigation taking into account the overall testing package.

Original text

At the end of the observation period, 25% of the control cells shall be tested for the presence of haemadsorbing viruses using guinea-pig red cells.
In some countries…other red cells including humans, monkey and chickens shoud 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

A potency test on the final bulk may be performed. The required potency of the vaccine and the assay method shall be approved by the national control authority.

New text

As suitable in vitro assays (e.g. ELISA) have been developed and are considered to be appropriate as a potency assay, a quantitative in vitro test, approved by the NRA or using appropriately characterized monoclonal antibodies or other affinity binders, should be performed on each final vaccine bulk or final lot. In vitro potency assays are preferred. However, an in vivo assay may be used if scientifically justified and approved by the NRA.