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

Original text

For routine testing, the potency of diphtheria vaccine may be determined using guinea-pigs or mice. When potency tests are carried out in mice instead of guineapigs, transferability should be demonstrated for the product being tested.
Titration of immune serum samples may be performed using an in vivo or in vitro TNT – such as a Vero cell assay – or using another in vitro method, such as the enzyme-linked immunosorbent assay (ELISA), subject to validation.

New text

As biological assays (e.g. humoral antibody response in sera from a suitable species) with the titration of Ab by in vitro methods (in vitro TNT, ELISA, MIT depending on the component tested) and/or physicochemical tests have been developed and are considered to be more precise and reproducible than the challenge test, a biological and/or physicochemical assay should be used if approved by the NRA. In some countries the titration of Abs are performed using multiplex immunological methods for combined DTaP vaccines. If several final lots are issued from one final bulk product, the potency assay should be carried out on the final bulk product and omitted on the final lots. After the demonstration of consistency of production by the biological and/or physicochemical assay on an appropriate number of final bulk products, a single dilution assay approved by the NRA should be carried out. Biological and/or physicochemical assays are preferred. However, if an in vivo challenge test for Diphtheria, Pertussis & Tetanus is carried out, it must be scientifically justified and should be approved by the NRA. A single dilution assay may be implemented for Diphtheria, Pertussis & Tetanus components after demonstration of consistency of production on an appropriate number of final bulk products and should be approved by the NRA. In the context of a 3Rs strategy, development of a package of appropriately validated in vitro assays for the characterization of the drug product is strongly encouraged in order to replace animal models. For all components, in vitro antigenicity assays are being developed and may be considered as potency assays once they are appropriately validated.

Original text

A potency test should be carried out on each final lot as described in Part A, section A.3.5.2.6, if such a test has not been performed on the final bulk.

New text

Original text

Each final lot should be tested for innocuity by intraperitoneal injection of 1 human dose (but not more than 1 ml) into each of five mice (weighing 17–22 g) and by intraperitoneal injection of at least 1 SHD (but not more than 1 ml) into each of two guinea-pigs (weighing 250–350 g). The tests should be approved by the NRA. The final product is considered to be innocuous if the animals survive for at least seven days without showing significant signs of toxicity. This test is also referred to as the abnormal toxicity test or the general safety test.
If the NRA approves, the innocuity test on the final lot may be omitted from routine lot release once the consistency of production has been demonstrated.

New text

Original text

The assay to detect diphtheria toxin as part of in-process safety testing can be performed using guinea-pigs or using an in vitro cell culture system. The purpose of the potency test is to demonstrate, using a suitable animal model, the capacity of the product being tested to induce an immune response analogous to that of toxoid shown to be efficacious in humans.

New text

The assay to detect diphtheria toxin as part of in-process safety testing can be performed using a suitable assay in agreement with the NRA. The Vero cell assay is highly sensitive and is considered superior to existing in vivo test methods.

Original text

Studies that support stability of a vaccine for the purpose of licensing have to be performed as real-time studies under the intended storage conditions. Stability-indicating parameters should be carefully selected. They should always include, but should not be limited to, the potency test. Tests should be conducted to determine the loss of potency at appropriate time intervals during storage. Final containers from at least three batches of vaccine derived from different bulks should be tested on the expiry date to demonstrate
stability during storage.

New text

Original text

Each final bulk should be tested for toxicity using the mouse weight gain test. The final bulk is considered satisfactory if the following conditions are met:
(a) at the end of 72 hours the average weight of the group of vaccinated mice is not less than that preceding the injection,
(b) at the end of 7 days the average weight gain per mouse is not less than 60% of that per control mouse, and
(c) no deaths occur when 10 mice are used and no more than one death occurs when 20 mice are used.

New text

Each final bulk should be tested for toxicity using a suitable assay. The assay must be sensitive to all potential toxins that may be present. The presence of toxins should be determined during product development. The mouse weight gain test is considered imprecise and in need of replacement. Manufacturers should develop alternatives or refinement of the mouse weight gain test.

Original text

The endotoxin content of vaccines can be estimated by the limulus amoebocyte lysate assay or the rabbit pyrogen test.

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

A Chinese hamster ovary cell (CHO-cell) assay, based on the clustering of cells after treatment with pertussis toxin is used in some countries to measure pertussis toxin in vaccine. A pertussis toxin standard is included in the assay, and a vaccine reference is used as a positive control. All samples are serially diluted to obtain an endpoint and the concentration of the pertussis toxin in the test sample is calculated in relation to the toxin reference. Tests for histamine sensitizing activity in mice may also be used.

New text

If required by the NRA, a specific toxicity test may be performed using a suitable, validated cell-based assay (e.g. CHO-cell assay). The assay should be performed using diluted vaccine. A histamine sensitizing activity test in mice may only be used where alternatives are not possible.

Original text

The potency of each final bulk (or of each final lot) should be determined by comparison with that of a reference vaccine calibrated against the International Standard for Pertussis Vaccine or an equivalent standard vaccine approved by the national regulatory authority. The assay should be performed by the intracerebral mouse protection test. The assay method and the method of calculating the results should be approved by the national regulatory authority. The potency is estimated in terms of IU in the volume recommended for a single human dose. The vaccine passes the recommendations for potency if the result of a statistically valid test shows that the estimated potency of the vaccine is not less than 4.0 IU in the volume recommended for a single human dose and if the lower fi ducial limit (P = 0.95) of the estimated potency is not less than 2.0 IU. Additional tests may be done, but in this case the results of all valid tests must be combined in the weighted geometric mean estimate and its lower fiducial limit.

New text

Alternatives to the Kendrick test, based on biological assay systems (e.g. humoral antibody response in sera from a suitable species ) have been explored. When shown to be more sensitive and reproducible an alternative assay should be used if 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. After the demonstration of consistency of production by the biological assay/Kendrick test on an appropriate number of final bulk products, a single dilution assay approved by the NRA should be carried out. Biological assays are preferred. However, if the Kendrick test is carried out instead of a serological assay, it must be scientifically justified and should be approved by the NRA. In the context of a 3Rs strategy, development of a package of appropriate in vitro biochemical methods, validated for the characterization of the drug product, are strongly encouraged. If an in vitro assay has been developed, it should be implemented as the potency test if approved by the NRA.

Original text

The identity test may be based on an immunological reaction (for example, agglutination of the organisms) with a specifi c antipertussis serum. Alternatively, vaccines may also be inoculated into animals to show that pertussis-specifi c antibodies (e.g. agglutinins) are present in their serum.

New text

An identification test shall be carried out using a suitable immunochemical method.