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

Original text

Additionally, for Vero cells, the MCB or WCB cells should be propagated to or beyond the maximum production level and be examined for the presence of retroviruses and tumorigenicity in an animal test system.

New text

Additionally, for Vero cells, the MCB or WCB cells should be propagated to or beyond the maximum production level and be examined for the presence of retroviruses and tumorigenicity in a suitable test system.

Original text

Each master or working seed lot should also be tested in animals that may include guinea-pigs, mice and embryonated chicken eggs, as appropriate.

New text

A strategy for testing adventitious viruses in vaccines must be developed based on a risk assessment. Relevant culture methods and/or specific molecular biology or broad molecular methods should be part of the overall testing package with the agreement of the NRA. In vivo tests may only be used if the risk assessment indicates that this test provides an additional risk mitigation taking into account the overall testing package.

Original text

At the end of the observation period, cells comprising no less than 25% of the control cells should be tested for the presence of haemadsorbing viruses, using guinea-pig red blood cells…...In some countries, the national regulatory authority requires that additional tests for haemadsorbing viruses be performed using other species of red blood cells including those from humans (blood group O), monkeys and chickens (or other avian species).....A further reading for the test with monkey red blood cells should be taken after an additional incubation for 30 minutes at 34–37 ºC.

New text

At the end of the observation period a fraction of culture comprising not less than 25% of the total should be tested for the presence of haemadsorbing viruses, using red blood cells from guinea-pig or other suitable red blood cells. It is not necessary to use red blood cells from multiple species. If the red blood cells have been stored prior to use in the haemadsorption assay, the duration of storage should not have exceeded 7 days and the temperature of storage should have been in the range of 2–8 °C.

Original text

Other aspects of process consistency may also be monitored and validated, such as process impurities and residuals as residual host cell protein, residual cellular DNA, endotoxin, bovine serum, trypsin and antibiotics.

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 virus concentration in each of at least three final containers of the rotavirus vaccine final lot should be assayed individually for infectivity in a sensitive assay system in which interference or potentiation between the serotypes present in the vaccine does not occur. An immunofocus or plaque assay may be used in MA-104, Vero or other sensitive cells to determine virus concentration. The assay is based on the visualization of infected areas of a cell monolayer directly or by probing with rotavirus serotype-specifi c monoclonal antibodies. Results should be recorded as FFU/ml or PFU/ml.
If immunological based assays are used, the specifi city and lack of cross-reactivity of the antiserum must be verifi ed. Alternatively, quantitative PCR detection of virus replication in a cell culture system may be used to provide an appropriate measure of infectivity. Results should be recorded as units of infectivity (UI/ml).

New text

Original text

Each recombinant baculovirus seed lot should be tested in cell cultures for adventitious viruses appropriate to the origin and the passage history of the seed baculovirus. For tests on recombinant baculovirus-permissive indicator cells, the neutralization of baculovirus is necessary. Antisera used for this purpose should be free from antibodies that may neutralize adventitious viruses, and should preferably be generated by the immunization of specific-pathogen-free animals with an antigen made from a source (other than the production cell line) which has itself been tested for freedom from adventitious agents. The inoculated indicator cells should be examined microscopically for cytopathic changes. At the end of the examination period, the cells should also be tested for haemadsorbing viruses (see section A.4.2.1.1 below).
Each virus master or working seed lot should also be tested in animals such as guineapigs
and/or mice.

New text

A strategy for testing adventitious viruses in vaccines must be developed based on a risk assessment. Relevant culture methods and/or specific molecular biology or broad molecular methods should be part of the overall testing package with the agreement of the NRA. In vivo tests may only be used if the risk assessment indicates that this test provides an additional risk mitigation taking into account the overall testing package.

Original text

At the end of the observation period at least 25% of the control cells should be tested for the presence of haemadsorbing viruses using guinea-pig red blood cells. If the red blood cells have been stored the duration of storage should not have exceeded 7 days, and the temperature of storage should have been in the range of 2–8 °C. In some countries, the NRA requires that additional tests for haemadsorbing viruses are to be performed using red blood cells of other species, including from humans (blood group O), monkeys and/or chickens (or other avian species).

New text

At the end of the observation period a fraction of culture comprising not less than 25% of the total should be tested for the presence of haemadsorbing viruses, using red blood cells from guinea-pig or other suitable red blood cells. It is not necessary to use red blood cells from multiple species. If the red blood cells have been stored prior to use in the haemadsorption assay, the duration of storage should not have exceeded 7 days and the temperature of storage should have been in the range of 2–8 °C.

Original text

The potency of each final bulk should be assessed with an appropriate in vivo or in vitro method. If an in vivo potency test is used to test final lots, this test may be omitted on the final bulk. The methods for detection of antibodies to HPV VLPs and the analysis of data should be approved by the NRA. The vaccine potency should be compared with that of a reference reparation; the NRA should determine the limits of potency and approve the reference preparation used. For ethical reasons, it is desirable to apply the 3R principles (Replacement, Reduction, Refinement) to the use of animals, where scientifically appropriate (44).

New text

In vitro assays for antigen detection (e.g. ELISA) have been developed and are considered to be appropriate for the potency assay. A quantitative in vitro test, approved by the NRA and using appropriately characterized antibodies, should be performed using samples representative of each final vaccine bulk or final lot. If an in vivo assay is carried out instead of an in vitro assay, it must be scientifically justified and should be approved by the NRA.

Original text

An appropriate quantitative test for potency by an in vivo or in vitro method should be performed on samples that are representative of each final vaccine lot. The method and the analysis of data from potency tests should be approved by the NRA. The vaccine potency should be compared with that of a reference preparation, and the limits of potency should be agreed with the NRA. The NRA should approve the reference preparation used. If an in vivo potency test is used, this test may be omitted on the final bulk. The method of testing for antigen potency in an in vitro test could be quantitative with respect to the antigen content or relative to a reference preparation.

New text

In vitro assays for antigen detection (e.g. ELISA) have been developed and are considered to be appropriate for the potency assay. A quantitative in vitro test, approved by the NRA and using appropriately characterized antibodies, should be performed using samples representative of each final vaccine bulk or final lot. If an in vivo assay is carried out instead of an in vitro assay, it must be scientifically justified and should be approved by the NRA.

Original text

The need to test the final lots of the HPV vaccine for unexpected toxicity (also known as abnormal toxicity) should be agreed with the NRA. Some countries no longer require this test (47).

New text