[Official testing of immunological veterinary medicinal products]. / Unabhängige Prüfung von immunologischen Tierarzneimitteln.

The testing of immunological veterinary medicinal products (IVMPs) by official medicines control laboratories (OMCLs) is an important contribution to the control of quality, safety and efficacy of these products. Based on the legislation of the European Union (EU) and with the support of the European Directorate for the Quality of Medicines & HealthCare (EDQM) a network of OMCLs of the EU member states and Switzerland has been built. This network has established its own rules allowing the mutual recognition of test results and rapid communication of details regarding batch release or rejection. Annual reports, OMCL meetings and collaborative studies help to foster confidence between the OMCLs. The procedure for official testing is described and an overview of deficits found at testing is presented in the paper. The testing of selected batches of centrally authorized products is also performed by OMCLs and is briefly described. Communication both among OMCLs and with pharmaceutical industry is an important part of the OMCLs' work to compare test results and to optimize existing or develop new test methods. Several OMCLs are also pursuing the development of new test methods, primarily for the reduction, refinement and replacement of animal experiments in routine testing.

The quantitative ELISA for inactivated Newcastle antigen: experience report from an OMCL.

The relative haemagglutinin-neuraminidase (HN) antigen content of inactivated Newcastle disease virus (NDV) vaccines from different manufacturers was determined by means of an Enzyme-Linked Immunosorbent Assay (ELISA) according to Monograph 870 of the European Pharmacopoeia (Ph. Eur.). Wide ranges of reactivity of the different products were observed. When comparing the antibody responses from chickens vaccinated with vaccines showing either high or low reactivity in the antigen ELISA it was found that approximately the same titres of antibodies were induced in the chickens. One hypothesis is that the inactivation procedures used to inactivate the Newcastle disease antigen may alter the antigenic determinant recognised by the monoclonal antibody used. An alteration of the antigen would influence the binding by the monoclonal antibodies used as catching and detection antibodies in the ELISA which may result in a lower ELISA reactivity. It was also found that HN antigen of two inactivated Paramyxovirus 1 (PMV-1) vaccines for pigeons could not be measured in the ELISA. For these vaccines the antigen-ELISA based on monoclonal antibody IDNDV134.1 cannot be used. Our experience shows that a thorough knowledge of the products tested with the ELISA and their influence on the test method is essential to avoid misinterpretations of the test results. The level of ELISA reactivity should not be used for the comparison of vaccines. Furthermore, prediction of the ability of an unknown vaccine to induce antibodies based on the level of ELISA reactivity is not possible. The results (level of reactivity) of the antigen ELISA for the in vitro potency testing of inactivated Newcastle disease vaccines should therefore be carefully interpreted. However, by knowing the performance characteristics of the NDV antigen ELISA and the characteristics of the vaccines to be tested it becomes a valuable tool for the control of inactivated Newcastle disease vaccines in our laboratory. The implementation of this ELISA method for the batch release testing markedly reduces the number of chickens and the time required for batch release testing.

Evaluation of the sensitivity of PCR methods for the detection of extraneous agents and comparison with in vivo testing.

In this study the sensitivity of polymerase chain reaction (PCR) methods for the detection of Newcastle disease virus (NDV), avian reovirus (ARV), avian influenza virus (AIV) and avian infectious bronchitis virus (IBV) was compared to the sensitivity of the corresponding serological tests described in the European Pharmacopoeia (Ph. Eur.). For this purpose, serial 10-fold dilutions of the respective inactivated vaccines were prepared and groups of SPF chickens were vaccinated with a double dose of the vaccine dilutions. After a period of 21 days, the animals were revaccinated with a single dose. Two weeks later, serum samples from each animal were tested for antibodies using an Idexx enzyme linked immunosorbent assay (ELISA). In parallel, samples of the diluted vaccines were tested by PCR. It was found that the sensitivity of the four PCR tests is comparable to or even slightly better than that of the corresponding serological tests. Thus these PCR tests fulfil the sensitivity requirements of the Ph. Eur. and could be used as alternative tests for the detection of extraneous agents in final batches of inactivated vaccines.

Poultry vaccines: an analysis of the animal trials required for vaccine testing and the way to reduce or refine these tests.

The testing of vaccines for use in chickens requires a large number of animal trials. Especially for poultry vaccines, quality testing of each batch consists of testing for extraneous agents in chickens for all products and potency tests for inactivated products. For the licensing of a vaccine a number of safety and efficacy tests is necessary. The safety testing covers dose and overdose studies, and the influence on reproductive performances and immunological functions. For live vaccines some additional trials concerning spread of vaccine strains, dissemination in the vaccinated animals and reversion to virulence are required. Some possibilities for combining several tests are presented. The purpose is to reduce the number of animals needed in these trials. The efficacy testing mostly requires challenge tests to define onset, level and duration of immunity. Serological test systems to replace the challenges are rarely implemented. The example of efficacy testing of infectious bursal disease vaccines demonstrates the possible replacement of a challenge by a serological test system. Parameters are morbidity, mortality, histological findings, bursa/body-ratios, and humoral antibodies detected by serum neutralization and ELISA.

Replacement of challenge procedures in the evaluation of poultry vaccines.

Vaccination of poultry flocks, especially parent flocks, is often performed with the intention of protecting the progeny via maternal antibodies during the first weeks of life. The efficacy of this vaccination schedule, more precisely described as induction of indirect protection, is normally proven by challenging the chickens. To avoid the challenge, some trials were performed, with the intention of establishing a correlation between antibody titres of vaccinated hens, embryonated eggs, and hatched chickens. The parent flocks were divided into several groups which were vaccinated either with Infectious Bursitis (IBD) vaccine or with Newcastle Disease (ND) vaccine. An acceptable correlation could be established. A well described standard or reference serum and a standardized and reproducible test procedures could be used in different laboratories. This approach allows serological test results to be compared. The possible parameters for the reference sera, including antibody titre, protein content and additional parameters, are discussed.

Control of product batches (before and after registration). The German approach.

At present, batch control is performed after a product is licensed. The range of batch control tests and the detailed description of these tests are fixed for every product during the respective authorization procedure. In an increasing number of cases it also seems necessary to test the prelicensing batches, which are used in the clinical trials for efficacy and safety. The batch control performed in the Paul-Ehrlich-Institute consists of the control of the batch protocols and the practical control of product samples. The practical testing includes purity, efficacy and safety of the batch. Considerable quality differences occur from one batch to another, even when the batch-to-batch-consistency is documented during the authorization procedure. Moreover, without previous indication to the state authority, the authorized control tests are frequently modified by the producer or are not performed for each batch. Some examples of these occurrences are presented. In addition it is necessary to bear in mind that the production of vaccines and sera needs biological seed materials, the quality of which can never be so uniform in the way chemical substances normally are. Since most of the incidents which occur when using vaccines and sera are batch-related and not product-related, the continuous batch control by state authorities is considered to be necessary.

[The problems of the estimation of bacterial contamination in viral drinking water vaccines for poultry]. / Untersuchungen zur Problematik der Beurteilung von bakteriologischen Verunreinigungen in virologischen Trinkwasserimpfstoffen für Geflügel.

The definition of saprophytic and non pathogenic germs according to the DAB 9 (German Pharmacopeia) will be explained as follows. Germs should be seen as saprophytic and non pathogenic when they are either classified as a pathogen by biochemical methods or when the safety test in chickens shows no adverse local or clinical reactions. The reason for the different test results in testing the species and the amount of contaminating germs at the laboratories of vaccine producers and state laboratories are explained by the unequal dissemination of germs in each batch and by the discontinuity in cooling the vaccine samples.