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

Original text

For vaccines produced in embryonated eggs or cell cultures, a sample of at least 5 ml of the monovalent virus pool should be tested for freedom from adventitious agents (as defined in section A.1.4) using cell cultures as described below. After neutralization of the influenza virus by hyperimmune antibody preparation, the monovalent virus pool should be inoculated on cell cultures of human cells, simian cells, chicken cells, or cells of the species used for vaccine production.

New text

Each virus seed lot propagated in avian tissues and each virus harvest propagated in primary avian tissues should be tested for avian viruses if the risk assessment, approved by the NRA, indicates that this test provides a risk mitigation taking into account the overall testing package. Relevant culture methods and/or molecular biology or broad molecular methods approved by the NRA should be part of the overall testing package. Animal testing (including fertilised SPF eggs) may only be used to qualify virus seed lots if the risk assessment indicates that such testing provides a risk mitigation taking into account the overall testing package. Animal testing is not performed on virus harvest for routine batch release.

Original text

A test for endotoxin should be included (e.g. the Limulus amoebocyte lysate
(LAL) test) as a test of safety and manufacturing consistency of the final lot.

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

At the end of the observation period a fraction of control cells comprising not less than 25% of the total should be tested for the presence of haemadsorbing viruses, using guinea-pig red blood cells. 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.

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 sample of at least 10 ml of each single pool produced in primary or serially passaged monkey cell culture shall be tested for the presence of SV40 virus or other adventitious agents. The single pool shall be neutralized by a high-titred antiserum against RVF.
The immunizing antigen used for the preparation of the antiserum should be shown to be free from extraneous agents and grown in cell cultures free from extraneous microbial agents that might elicit antibodies that could inhibit the growth of any adventitious agents present in the RVF virus pool.

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.

Original text

A sample of at least 30 ml of each single pool shall be tested as soon as possible after pooling by inoculation into three healthy rab­bits, each weighing between 1.5 and 2.5 kg; proportionately larger volumes shall be used if more animals are inoculated. The inoculations shall be made at multiple sites, each rabbit being given a total of 1 ml of the single pool by intradermal injection and 9 ml by sub­cutaneous injection. The animals shall be observed for at least three weeks. All rabbits that die after the first 24 h of the test or that show signs of illness shall be examined by autopsy, with removal of the brain and organs for detailed inspection. The single pool passes the test if at least 2 of the rabbits remain healthy and if none of the rabbits shows lesions of any kind at the sites of inoculation or shows evidence of infection with B virus or with any adventitious transmissible agent attributable to the single pool.

New text

Original text

Each final lot shall be tested for abnormal toxicity by appropriate tests involving injection into mice and guinea-pigs. The tests shall be approved by the national control authority.

New text

Original text

Each vaccine shall be tested for immunizing potency by tests ap­proved by the national control authority. Such tests shall include an in vitro assay for antigen content and an in vivo assay for immune response. The potency of the vaccine shall be approved by the national control authority.

New text

Quantitative in vitro assays (e.g. ELISA) have been developed and are considered appropriate for assessing potency during quality control and batch release testing. Therefore, a quantitative in vitro test, approved by the NRA and using appropriately characterized monoclonal antibodies, should be performed using samples representative of each final vaccine lot. If a biological assay using in vitro methods for the Ab titration is carried out instead of an in vitro assay, it must be scientifically justified and should be approved by the NRA. If several final lots are issued from one final bulk product, the serological assay should be carried out on the final bulk product and omitted on the final lots to reduce animal use. After the demonstration of consistency of production by the multidose serological assay on an appropriate number of final bulk products, a single dilution assay approved by the NRA should be carried out.

Original text

At the end of the observation period, at least 25% of the control cells should be tested for the presence of haemadsorbing viruses using guinea-pig red blood cells. If the latter cells have been stored, the duration of storage should not have exceeded 7 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. Some NRAs require, as a test for haemadsorbing viruses, that other types of red blood cells, including cells from humans, monkeys and chickens (or other avian species), are also used instead of guinea-pig cells alone.

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 final bulk should be tested using an in vivo assay for immunogenicity and in vitro antigen content assay approved by the NRA, unless this is to be performed on final product. Product-specific reference preparations may be used in these tests.
Once consistency of production has been established for a suitable number of consecutive final bulks, the in vivo assay may be omitted for the purpose of routine lot release, with the agreement of the NRA

New text

Quantitative in vitro assays (e.g. ELISA) have been developed and are considered appropriate for assessing potency during quality control and batch release testing. Therefore, a quantitative in vitro test, approved by the NRA and using appropriately characterized monoclonal antibodies, should be performed using samples representative of each final vaccine lot. If a biological assay using in vitro methods for the Ab titration is carried out instead of an in vitro assay, it must be scientifically justified and should be approved by the NRA. If several final lots are issued from one final bulk product, the biological assay should be carried out on the final bulk product and omitted on the final lots to reduce animal use. After the demonstration of consistency of production by the biological assay on an appropriate number of final bulk products, a single dilution assay approved by the NRA should be carried out.

Original text

The potency of each final lot should be determined using an in vivo assay and a validated in vitro antigen content assay (see section A.4.6.2 above) if such a test has not been performed on the final bulk. Potency should be calculated using a reference vaccine calibrated against the First WHO International Standard for EV71 inactivated vaccine (see International reference materials above) or other reference preparation.

New text

The potency of each final lot should be determined using a suitable assay (see section A.4.6.2 above) if such a test has not been performed on the final bulk. Potency should be calculated using a reference vaccine calibrated against the First WHO International Standard for EV71 inactivated vaccine (see International reference materials above) or other reference preparation.