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

Original text

Each final bulk should be tested in an in vivo assay for immunogenicity by tests approved by the NRA. An in vivo potency assay in rats has been standardized and shown to be a suitable test for IPV.

New text

In vitro assays for antigen detection (e.g. ELISA) have been developed and are considered to be appropriate for the potency assay. A quantitative in vitro test, approved by the NRA and using appropriately characterized antibodies, should be performed using samples representative of each final vaccine bulk or final lot. If an in vivo assay is carried out instead of an in vitro assay, it must be scientifically justified and should be approved by the NRA.

Original text

If the use of an adjuvant in the final bulk interferes with the assay, a desorption or treatment step may be necessary before performing the D-antigen ELISA. If treatment/desorption is not possible, the interference of the adjuvant should be documented and an in vivo assay should be performed.

New text

Original text

The endotoxin content of each final lot should be determined by a method approved by the NRA. Levels should be consistent with levels found to be acceptable in vaccine lots used in pre-licensure 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

Tests for evaluating the potency of IPVs include an in vivo assay for immune response. A suitable in vivo assay method consists of intramuscular injection into the hind limb(s) of rats of four dilutions of the vaccine to be examined and a reference vaccine, using for each dilution a group of not fewer than 10 rats of a suitable strain and which are free of specific pathogen. The animals are bled after 20–22 days.
Laboratories are encouraged to validate alternative methods for the assay of neutralizing antibody to reduce the use of live polioviruses in laboratories.

New text

Original text

Additional tests may include but are not limited to propagation of the MCB or WCB cells to or beyond the maximum in vitro age for production, and examination for the presence of retroviruses and tumorigenicity in an animal test system.

New text

Additional tests may include but are not limited to propagation of the MCB or WCB cells to or beyond the maximum in vitro age for production, and examination for the presence of retroviruses and tumorigenicity in a suitable assay approved by the NRA

Original text

It is important to show that the cell banks (cell seed, MCB and WCB) are free from adventitious agents relevant to the species used in their derivation. Cell banks should be assessed for the absence of adventitious agents that may have been present during production

New text

Original text

New virus working seeds should be evaluated for neurovirulence. Summaries of the MNVT and TgmNVT, including pass/fail criteria, are given in Appendix 2 along with considerations on the choice of assay. The test should be approved by the NRA for the specific product, and transgenic mice, nonhuman primates, or both, may be used.
If the NRA agrees, then the neurovirulence of the virus working seeds and at least three consecutive monovalent bulks prepared from it should meet the criteria for acceptability given in section A.4.4.7.2 and the appropriate SOP before the working seed can be considered suitable for use in the production of OPV. Historically, four consecutive monovalent bulks prepared from the seed virus have been tested in monkeys to monitor production consistency.

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. If these cells have been stored, the duration of storage should not have exceeded seven days, and the storage temperature should have been in the range of 2–8 °C. In tests for haemadsorbing viruses, calcium and magnesium ions should be absent from the medium.

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

Each single harvest should be identified as the appropriate poliovirus serotype by immunological assay on cell culture using specific antibodies or by a molecular method that has been validated and approved by the NRA. Neutralization tests can distinguish the serotype of polioviruses. Molecular methods, such as sequencing or deep sequencing, can distinguish Sabin virus from wild-type virus. Care should be taken to ensure that the serum samples used are monospecific by titrating them against homotypic and heterotypic viruses of known virus titre. Monoclonal antibodies may be useful in this test.

New text

Original text

Each monovalent bulk should be identified as the appropriate poliovirus serotype by immunological assay on cell culture using specific antibodies, or by a molecular method that has been validated and approved by the NRA. Neutralization tests can distinguish the serotype of polioviruses. Molecular methods, such as sequencing or deep sequencing, can distinguish Sabin virus from wild-type virus. Care should be taken to ensure that the serum samples used are monospecific by titrating them against homotypic and heterotypic viruses of known virus titre. Monoclonal antibodies may be useful in this test.

New text