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

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An immunological activity test (MIT, MICA, or approved alternative) should be carried out as described in section A.3.4.2.7, on each final lot, if such a test has not been conducted on the final bulk.

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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.

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Detailed protocol for MICA assay

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Detailed protocol for HIST assay with temperature change endpoint

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Detailed protocol for HIST assay with lethal endpoint

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Detailed protocol for MIT assay

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To confirm that the vaccine does not revert to toxicity during storage, the specific toxicity test described in Part A, section A.3.5.2.5, should be scheduled up to the expiry date as part of the stability studies. In addition, at the time of the expiry date, the vaccine should meet the requirements for the final product in terms of sterility, potency, adjuvant content, degree of adsorption, preservative content, pH and extractable volume, where applicable (as described in Part A, sections A.5.2, A.5.3 and A.5.5–A.5.9), provided that the vaccine has
been stored at the recommended temperature.

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A suitable in vitro assay (e.g. the BINACLE assay) should be performed on the final purified bulk at the time of the expiry date. If the production method removes the risk of reversion or historical data demonstrate a lack of reversion, the test is not required and could be omitted following discussion and approval from the NRA.

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Harvests should be treated as potentially toxic, and subject to the appropriate safety restrictions until the detoxification has been shown to be complete by performance of a specific toxicity test (as detailed in section A.3.4.4) or any other suitable in vivo method.

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Harvests should be treated as potentially toxic, and subject to the appropriate safety restrictions until the detoxification has been shown to be complete.

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Each bulk purified toxoid, diluted with the same buffer solution as used in the final vaccine, should be tested for the absence of tetanus toxin in guinea-pigs; the guinea-pigs should each weigh 250–350 g and not previously have been used for experimental purposes. At least five guinea-pigs should be injected subcutaneously with 1 ml of a dilution of purified tetanus toxoid containing at least 500 Lf of toxoid; they must be observed daily for signs of tetanic paralysis over a period of 21 days. A suitable method is outlined in the WHO Manual for quality control of diphtheria, tetanus and pertussis vaccines (45).
Animals that die, whatever the cause, will need to be examined by necropsy.

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Each bulk purified toxoid, diluted with the same buffer solution as used in the final vaccine, should be tested for the absence of tetanus toxin. In vitro assays (e.g. BINACLE assay) are being developed and are currently undergoing validation (EDQM BSP 136). Inactivation of tetanus toxin can be monitored using an in vitro assay (once approved by the NRA) or a suitable in vivo assay. The tetanus toxin inactivation process should be demonstrated during process development to produce a stable toxoid which does not undergo reversion during downstream processing steps or under recommended storage conditions. Subsequent testing for reversion of tetanus toxin should not be necessary once stability of the product has been demonstrated with sufficient historical evidence.

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The diluted toxoid sample is incubated at 34–37 °C for a period of six weeks (42 days). At the end of the incubation period, five guinea-pigs are each injected subcutaneously with 5.0 ml (i.e. 10 human doses, using multiple injection sites where necessary) of test sample. The animals are observed for 21 days for signs of ill health. No toxicity should be detected. The bulk purified toxoid passes the test if no guinea-pig shows symptoms of specific paralysis or any other signs of tetanus within 21 days of injection.

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Each final bulk should be tested for specific toxicity in at least five guinea-pigs; each guinea-pig should weigh 250–350 g and not have been used previously for experimental purposes. Each guinea-pig is given a subcutaneous injection of a quantity equivalent to at least 5 SHD, and is observed daily for a period of 21 days. Animals that die from any cause should undergo necropsy and be inspected for symptoms of tetanus paralysis.
Subject to the approval of the NRA, the specific toxicity test used on the final bulk may be omitted from routine lot-release procedures once consistency in production has been established.

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