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

Original text

A potency test in mice may be used for this purpose.

New text

Currently, in vitro assays (e.g. ELISA) have been developed or are under development and may be appropriate as a replacement to the in vivo assay for determination of potency. A quantitative in vitro test, approved by the NRA and using well-characterized antibodies, should be performed on each final vaccine lot. In vitro potency assays are preferred, however if an in vivo assay is carried out, it must be scientifically justified and should be approved by the NRA.

Original text

A potency test in mice may be used for this purpose.

New text

Currently, in vitro assays (e.g. ELISA) have been developed or are under development and may be appropriate as a replacement to the in vivo assay for determination of potency. A quantitative in vitro test, approved by the NRA and using well-characterized antibodies, should be performed on each final vaccine lot. In vitro potency assays are preferred, however if an in vivo assay is carried out, it must be scientifically justified and should be approved by the NRA.

Original text

Each final lot shall be tested for abnormal toxicity.

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Original text

Each final lot shall be tested for pyrogenic substances.

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

Relevant safety tests should be described and may include tests for: (a) endotoxins; (b) bacterial and fungal sterility (including demonstration of lack of bactericidal or fungicidal activity of the test article); or (c) bioburden (including quantity, identification and freedom from specified unwanted organisms). Although a test for pyrogenicity may be performed if required by the NRA, animal testing should be avoided whenever alternative satisfactory testing is accepted. For ethical reasons, it is desirable to apply the 3Rs concept of “Replace Reduce Refine” to minimize the use of animals, and consideration should be given to the use of appropriate in vitro alternative methods for safety evaluation. In particular, manufacturers and regulators should take note of the decision of the WHO Expert Committee on Biological Standardization in 2018 to discontinue the inclusion of the general safety (innocuity) test in routine lot release testing requirements for all vaccines in WHO Recommendations, Guidelines and other guidance documents for biological products (82). This test should therefore not be required or requested.

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Original text

Furthermore, a test for endotoxin should be conducted on each lot and appropriate specifications defined. If required by the NRA, a test for pyrogenicity may be performed – however, animal testing should be avoided whenever alternative satisfactory testing is allowed. For ethical reasons, it is desirable to apply the 3Rs concept of “Replace Reduce Refine” to minimize the use of animals and consideration should be given to the use of appropriate in vitro alternative methods for safety evaluation. Pyrogenicity may be determined using the monocyte activation test. The test known as the innocuity, abnormal toxicity or general safety test should not be required or requested.

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Original text

The RTS,S adjuvanted malaria vaccine includes two antigenic determinants, S and CS, for which two quantitative in vivo potency assays are performed for characterization purposes. Both of these assays are immunogenicity assays in mice.
A suitable quantitative potency test in mice is as follows. Groups of 10 mice, between six and eight weeks of age, are immunized two times subcutaneously with a 14-day interval with a series of dilutions of the reference and test vaccine using the vaccine reconstituted with the adjuvant system. The strain of mice used for this test must give a suitable dose-response curve with the reference and test vaccine. Terminal bleeds are taken when an adequate antibody response has developed. Individual sera are assayed for either antibodies to S or antibodies to CS using an enzyme immunoassay.

New text

Original text

An appropriate quantitative test for potency should be performed on samples representative of the final vaccine lot….Methodological considerations regarding in vivo and in vitro potency assays for RTS,S/AS01 are available on the WHO Biologicals web site at: http://who.int/biologicals/vaccines/malaria/en/index.html and will be updated when necessary.

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 (e.g. humoral response in sera from a suitable species) 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 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 AS01 adjuvant system in the final container should be tested for pyrogenic activity by intravenous injection into rabbits. The test for pyrogenic activity may not be required after the consistency of production has been demonstrated to the satisfaction of 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

An identity test should be performed on at least one labelled container from each final lot using methods approved by the NRA. The test used for determining the antigen content will generally be suitable for assessing identity. Alternatively, immunoblots using antigen-specific antibodies could also be used to confirm the molecular identity of the product.

New text