The number of tests required to control for complete inactivation in inactivated vaccines

The revision of Annex I of Directive 2001/82/EC by the amending Directive 2009/9/EC has been welcomed by regulators and industry. However, some confusion has occurred about the number of controls of inactivation to be done (one or two).

By a letter dated 28th May 2009, IFAH-Europe requested clarification on this point at IWP/CVMP level.

The requirements for tests to confirm complete inactivation of microorganisms in inactivated veterinary vaccines are described in Annex I to Directive 2001/82/EC as amended by Directive 2009/9/EC as well as in the European Pharmacopoeia, which also has legal status by reference in Directive 2001/82/EC as amended.

According to Annex I the following tests are required to control for complete inactivation:

D. CONTROL TESTS DURING THE MANUFACTURING PROCESS

2. For inactivated or detoxified vaccines, inactivation or detoxification shall be tested during each production run as soon as possible after the end of the inactivation or detoxification process and after neutralisation if this occurs, but before the next step of production.

E. CONTROL TESTS ON THE FINISHED PRODUCT

9. Inactivation
For inactivated vaccines, a test to verify inactivation shall be carried out on the product in the final container unless it has been conducted at a late stage in-process.

Monograph 0062 of the European Pharmacopoeia also states the following requirements:

• A test for complete inactivation and/or detoxification is performed immediately after the inactivation and/or detoxification procedure and, if applicable, the neutralisation or removal of the inactivating or detoxifying agent.
• For inactivated vaccines, where the auxiliary substances would interfere with a test for inactivation and/or detoxification, a test for inactivation or detoxification is carried out during preparation of the final bulk, after the different batches of antigen have been combined but before addition of auxiliary substances; the test for inactivation or detoxification may then be omitted on the final bulk and the batch.
  
  Where there is a risk of reversion to toxicity, the test for detoxification performed at the latest stage of the production process at which the sensitivity of the test is not compromised (e.g. after the different batches of antigen have been combined but before the addition of auxiliary substances) is important to demonstrate a lack of reversion to toxicity.

Thus, although the wording of these two texts are different the effect is the same to specify:

a) A test immediately after end of the inactivation or detoxification process, or as soon after the end as possible. Annex I states that this test should be carried out before the next step of production.

b) A test on the finished product, or if this is not possible because of substances that might interfere with such a test, at the latest stage during production that such a test can be carried out.

The number of tests that need to be carried out depends therefore on whether a single test can satisfy both requirements.

In considering the number of inactivation tests required to assure the safety of the finished product it is relevant that inactivated vaccines are manufactured in accordance with GMP using validated inactivation processes that include at least 33% safety margins in excess of the time required for complete inactivation of the antigens. Furthermore, specific examples of vaccines where a second inactivation test on the finished product is considered essential to ensure safety have not been identified.

It should also be noted that, as indicated in the 'Batch Tests' section of the Technical Guide for the Elaboration and Use of Monographs for Immunological Veterinary Medicinal Products, although the batch must comply with the listed requirements 'For the purpose of batch release by the manufacturer, the tests described do not need to be carried out on each batch where in-process or other final product tests give an equal or better guarantee that the batch would comply or where alternative tests validated with respect to the Pharmacopoeia method have been carried out'. All of the tests specified in the Ph. Eur. therefore do not need to be carried out if compliance with the pharmacopoeial standard can be assured in other ways.

From a scientific perspective, therefore, the IWP/CVMP considers that a single test to confirm complete inactivation carried out at the stage after inactivation when detection of any residual live antigen is most likely should give sufficient assurance of complete inactivation and compliance with the pharmacopoeial standard in most cases. This would generally be expected to be soon after the end of the inactivation process and can be considered to be at a sufficiently late stage in the production process to comply with both requirements of Annex I.

Notwithstanding the above, manufacturers should take care to ensure that their quality control procedures fully comply with all of the relevant legal requirements.