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

Original text

The manufacturer shall show to the satisfaction of the national control authority that the MWCB satisfies the requirements outlined in this section for freedom from bacteria, fungi and adventitious viral agents.

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

These shall be carried out by methods approved by the NCA

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

Aprprove the test used for freedom from abnormal toxicity in the final product

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Original text

Although the use of primary cells may provide a more physiologically relevant model, the criteria of low assay variability and robustness may not be satisfied. Continuously growing cell lines may overcome these limitations in some cases provided they are more sensitive and more capable of detecting minor differences between the RBP and the SBP.

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Original text

WHO recommends that national reference venom collections be established and that these cover each medically important snake species used in antivenom production. Such reference venoms should be prepared as described elsewhere in these Guidelines (see sections 9 and 21), and must be tested for potency at least annually to ensure they comply with the original specifications.

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Original text

This information may include analysis of:
Enzymatic and toxicological activities of the venoms; –– for example, LD50 and, depending on the particular venom, in vitro procoagulant activity, proteinase activity or phospholipase A2 activity;

New text

This information may include analysis of: Enzymatic and toxicological activities of the venoms; –– for example, potency and, depending on the particular venom, in vitro procoagulant activity, proteinase activity or phospholipase A2 activity;

Original text

Antivenoms should comply with the rabbit pyrogen test where required by the local regulations. This test is based on intravenous injection of antivenoms in the ear vein of rabbits. The dose of antivenom must be calculated by dividing the threshold pyrogenic dose in rabbits by the endotoxin although other doses might be used depending on the pharmacopoeia), followed by the measurement of rectal temperature at various time intervals after injection. The detailed procedures are described in various pharmacopoeias. Bacterial lipopolysaccharides can also be detected by the Limulus amoebocyte lysate (LAL) test. The test should be validated for each type of antivenom, since there have been reports of falsepositive and false-negative reactions when testing antivenoms and other plasmaderived products. The sensitivity of this LAL test should be correlated with the rabbit pyrogen test, and the endotoxin limits established (135). When regulation allows, a validated LAL test is used in place of the rabbit pyrogen test.

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 abnormal toxicity test (7 day observation of the effects of intraperitoneal injection of 0.2 mL and 0.5 mL antivenom into mice and guinea-pigs, respectively), is still required by some pharmacopoeias and is performed at the stage of product development.
However, because of the very limited quality control value of this assay, it is increasingly being abandoned by most regulatory authorities. Correct implementation of GMP should provide evidence that the product would comply with the test for abnormal toxicity.

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Original text

These tests determine the capability of an antivenom to neutralize the lethal effect of the snake venom(s) against which the antivenom is designed. It is first necessary to determine the lethal potency of the venom, using the LD50 assay. The exact volume of antivenom required to neutralize venom lethality can then be determined using the antivenom effective dose (ED50) assay.
Consistent use of outbred strains of mice, of a defined weight range (for example, 18–20 g) that receive a defined challenge dose, is recommended for all the assays. Some producers use other test animals, such as guinea-pigs.

New text

These tests determine the capability of an antivenom to neutralize the lethal effect of the snake venom(s) against which the antivenom is designed. It is first necessary to determine the lethal potency of the venom, using an appropriate assay, approved by the NRA. The use of animals for potency testing of antivenoms raises important ethical considerations and it is essential that 3Rs principles are applied, including use of appropriate analgesia, anaesthesia, humane endpoints, high standards of animal housing, husbandry and care and optimization of experimental design to use the minimum number of experimental animals to measure the potency of an antivenom. In this context, developments of appropriate in vitro immunochemical methods validated for replacing animal experiments are strongly encouraged. If an in vitro assay has been developed, it should be implemented as the potency test if approved by the NRA.

Original text

Results from the following quality control tests need to be provided by the manufacturer as part of the batch release documentation:
(a) venom-neutralizing efficacy test against the most relevant venoms to be neutralized;
(b) identity test;
(c) protein concentration;
(d) purity of the active substance;
(e) content of protein aggregates and non-IgG contaminants;
(f ) pyrogen test;
(g) sterility test;
(h) concentration of excipients;
( i ) osmolality;
( j ) pH;
(k) concentration of preservatives;
( l ) determination of traces of agents used in plasma fractionation;
(m) appearance, and, for freeze-dried preparations, residual moisture and solubility; and
(n) labelling validation and confirmation.
Antivenom reference preparations reflecting specific characteristics of antivenoms produced should be prepared by each manufacturer to be used as standards in their laboratory settings, in particular to measure neutralization capacity of their specific antivenom
products against targeted venoms. Relevant standards are also used to establish conformity of purity and integrity. When possible, a national reference antivenom should be established.
- It is the ethical responsibility of the manufacturer to use only the minimum number of experimental animals to measure the efficacy of an antivenom.
-The development of in vitro methods validated for replacing
animal experiments is strongly encouraged.

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