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

Original text

A potency test should be carried out as provided in Part A, section A.3.3.7, on each final lot, if such a test has not been done on the final bulk.

New text

Original text

Each final lot should be tested for unexpected toxicity (sometimes called 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 well 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.

New text

Original text

The evidence that vaccines shown to protect mice against intracerebral challenge also protected immunized children against whooping cough when such children were exposed to the disease in the home by infection from a sibling was published in the 1950s. This correlation was the basis for the establishment of the current potency test (11). Although the potency test has a long record of use, it has often been criticized, especially on its reproducibility. However, a recent WHO proficiency study involving 13 laboratories in 12 countries confirmed that the intracerebral challenge assay was effective in distinguishing potent and sub-potent batches of vaccine and gave consistent results both between repeat tests and between different laboratories (12) Nevertheless, the mouse protection test is technically demanding and efforts have been made to develop alternative in vitro potency assays, such as serological assays. However, the lack of understanding of the mechanisms of protection in humans afforded by whole-cell pertussis vaccines, in particular, of the value of neutralizing antibodies, the nature of the critical antigens and the role of cell-mediated immunity, it is diffi cult to design an acceptable alternative. The serological approach was extensively discussed at an European Directorate for the Quality of Medicines/European Centre for the Validation of Alternative Methods (EDQM/ECVAM) consultation in 2005 (13) where the issue of the relevance of simple antibody measurements to human clinical protection was considered. It was concluded that such tests cannot yet be considered as validated alternatives to the mouse protection potency test for whole-cell pertussis vaccines. However, correlation between production of agglutinins in mice and protection in children demonstrated as early as the Medical Research Council (MRC) trials in the 1950s should be further explored as a potential alternative or a complementary test to the currently recommended potency test. There was also a strong recommendation from the EDQM /ECVAM consultation to use validated humane end-points in the mouse protection test

New text

Original text

Multiple-dilution in vivo potency testing of combined vaccines requires a considerable number of laboratory animals. A significant reduction in the use of laboratory animals could be achieved through the development and use of simplified in vivo models (e.g. single-dilution models) and particularly through those that would allow for the concurrent serological testing of multiple components (e.g. concurrent testing of purified pertussis antigens and diphtheria and tetanus toxoids) (30–34). A laboratory that intends to introduce an alternative method should perform adequate validation studies to enable comparisons to be made with the multiple-dilution in vivo model (32–34).

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

In general, potency values determined by a test in guinea-pigs, as described in the WHO Recommendations to assure the quality, safety and efficacy of diphtheria vaccines (2), are significantly lower in the absence of a whole-cell pertussis component than the values found in vaccines containing this component.

New text

Original text

Similar to diphtheria toxoid, potency values for tetanus toxoid determined by the tests described in the WHO Recommendations to assure the quality, safety and efficacy of tetanus vaccines (adsorbed) (3) are significantly higher in the presence of a wP component and in the presence of a Hib component produced with a tetanus toxoid carrier than the values found in the absence of such components, particularly when assayed in mice.

New text

Original text

In the presence of aluminium-based adjuvants, the in vitro Chinese hamster ovary (CHO) cell-based assay may not be applicable for testing the formulated product and for some chemically detoxified antigens. In addition, the in vivo test may be sensitive to other components in the formulation rather than to any residual native pertussis toxin (PT) (e.g. aluminium-based adjuvants or IPV). Proper standardization of the in vivo test, and the development and introduction of alternative test methods, are strongly encouraged.

New text

The PTx inactivation process should be controlled and demonstrated to consistently reduce the active PTx to levels which are found to be safe in clinical trials. Inactivation of PTx can be monitored using a CHO cell clustering assay or similar in vitro method. The CHO cell clustering response has been demonstrated to have greater sensitivity and lower variability than the mouse histamine sensitization test (HIST) and this test is no longer recommended. Although a modified CHO cell clustering method can be used to monitor residual PTx activity in the presence of an adjuvant, inactivation of PTx should be controlled and verified prior to adsorption. The PTx 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 PTx should not be necessary once stability of the PTd has been demonstrated. The testing for residual PTx activity or activity from reversion is not necessary for PTd derived from genetic inactivation. The genetic insert should be confirmed to be stable, and the cell line shown to be absent of an active PTx gene.

Original text

Each final lot of the combined vaccine should be tested to assess the identity of each component, and the sterility, pyrogenicity or endotoxin content, adjuvant content, preservative content, the potency of each component and innocuity in accordance with the recommendations for each individual vaccine.

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

Repeating the tests – particularly those such as potency testing, which involve animals – on the reconstituted combined vaccine is not required provided that during development duly validated studies demonstrating the compatibility of the two components following reconstitution have been conducted by the manufacturer, and that due consideration has been given to issues of batch consistency, batch size and the frequency of production.

New text

Repeating the tests – particularly those such as potency testing – on the reconstituted combined vaccine is not required provided that during development duly validated studies demonstrating the compatibility of the two components following reconstitution have been conducted by the manufacturer, and that due consideration has been given to issues of batch consistency, batch size and the frequency of production.

Original text

Specify the number, strain and sex of animals used – this test is not necessary for a product obtained by genetic modification

New text