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

Original text

Each final lot should be tested for the absence of 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 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 general safety.

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Original text

The potency should be determined by titration of the neutralizing antibody produced in immunized mice by the plaque-reduction neutralization test. Five hundred microlitres of each dilution is injected intraperitoneally into at least ten mice, of the same sex, approximately 4 weeks of age and typically having a body weight of 14–18 g, on two occasions 7 days apart. Seven days after the second injection, each animal is bled.

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

An amount of less than 100 International Units of endotoxin per μg of purified polysaccharide can be achieved when measured by the Limulus amoebocyte lysate (LAL) test. Alternatively, a recognized pyrogenicity test can be performed in rabbits (59). Endotoxin content has also been measured by use of a chromatographic technique to determine the profile of LPS-associated lipids (65).

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

The bulk conjugate should be tested for the absence of specific toxicity of the carrier protein where appropriate (when tetanus or diphtheria toxoids have been used). Absence of specific toxicity of the carrier protein may also be assessed through validation of the production process

New text

The carrier protein should be tested for the absence of specific toxicity of the carrier protein where appropriate (e.g. when tetanus or diphtheria toxoids have been used). Absence of specific toxicity of the carrier protein may also be assessed through validation of the production process. Where available and appropriately validated and with approval from the NRA, an in vitro test should be used. If in vitro assays are scientifically justified as unavailable and inappropriate, a suitable in vivo test may be used.

Original text

The requirements to test lots of meningococcal conjugate vaccine for unexpected toxicity (abnormal toxicity) should be agreed with the national regulatory authority.
Such a test may be omitted for routine lot release with the agreement of the national regulatory authority once consistency of production has been well established to the satisfaction of the national regulatory authority and when good manufacturing practices are in place.

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Original text

The vaccine in the final container should be tested for pyrogenic activity by intravenous injection into rabbits or by a Limulus amoebocyte lysate (LAL) test, which should be validated for this purpose. Endotoxin content or pyrogenic activity should be consistent with levels found to be acceptable in vaccine lots used in clinical trials and 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

While immunogenicity testing in animals forms a necessary part of vaccine development, experience gained following the licensure of the Men C conjugate vaccines suggests that a routine animal potency test is not necessary when vaccine consistency has been assured by physicochemical criteria.

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Original text

Each filling lot shall be tested for pyrogenicity by the intravenous injection of rabbits. Three or more healthy rabbits that have not been injected previously shall be used.

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

No fewer than five guinea-pigs (reduced to two in TRS 904 Annex 2) weighing approximately 350g each shall be injected intraperitoneally with 500ug of the pollysaccharide(s). The animals shall be observed for 7 days and the injection shall cause neither significant symptoms nor death during this period.

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Original text

No fewer than five mice weighing approximately 18g each shall be injected intraperitoneally with 100ug of the pollysaccharide(s). The animals shall be observed for 7 days and the injection shall cause neither significant symptoms nor death during this period.

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