Developments in biological standardization最新文献

We have found that our MDCK-derived cell line (BV-5F1) is non-tumorigenic in tests conducted in accordance with FDA guidelines, and thus may be suitable for producing live, attenuated or inactivated vaccine. The cell line has been extensively tested for the presence of contaminating microorganisms. No infectious agents of viral or other microbial origin were present. Using the BV-5F1 cell line, we have now designed a process for the large-scale production of influenza virus for the manufacture of a vaccine. The production system involves expansion of cells anchored on a microcarrier using stirred fermenters, followed by virus infection. Viral particles are purified in a way similar to the licensed egg-derived vaccine Fluviral SF and mainly involves ultracentrifugation, ultrafiltration and formaldehyde inactivation. The final product is a split inactivated vaccine. A randomized, double-blind clinical study was made in healthy adults using the new split influenza vaccine derived from viruses grown in cell culture (bivalent formulation). The results of this Phase I study have demonstrated that the split influenza vaccine derived from cell culture is highly immunogenic and safe in adults.

Safety tests in animals are an important part of the licensing procedures for human and veterinary medicines. Pharmacopoeial and other legal regulations require immunobiologicals to be approved for safety on a batch-to-batch basis. A large number of animals is needed to perform these general and specific safety tests. The search for alternatives according to the famous 3R concept proposed by Russel & Burch [1] is also relevant to safety tests. This session of the conference will highlight recent progress in the application of this concept. However, before considering potential alternatives to a test, it is advisable to re-evaluate the necessity of the safety test in question. Many animal-based safety test procedures were introduced because the production of biologicals was difficult and methods to control this production were limited. Nowadays much progress has been achieved due to a better knowledge of the prophylaxis of infectious diseases and the standardisation of methods of production and control. Therefore, some animal tests may no longer be necessary. A good example of this is the deletion of the abnormal toxicity test from the European Pharmacopoeia. Efforts to limit the use of animals in the quality control of immunobiologicals should therefore include a reassessment of the value of the safety test. Consequently, reassessment should be the first R used to evaluate whether an animal test can be removed. The 3R concept of Russel & Burch [1] to replace, reduce and refine animal tests has to be considered if that does not prove to be possible.

The United States Code of Federal Regulations requires that all influenza virus vaccines produced for use in the United States adhere to specific regulatory standards including the demonstration of safety and efficacy. For vaccines produced in cell lines, rigorous characterization for manufacturing is particularly important. Influenza vaccines produced by the passage of viruses in mammalian cell lines will require careful evaluation to ensure the removal or inactivation of potential adventitious agents.

Currently the only accepted method for the detection of botulinum neurotoxin in contaminated samples is the mouse bio-assay. Although highly sensitive this test has a number of drawbacks: it is expensive to perform, lacks specificity and involves the use of animals. With increasing resistance to such animal tests there is a need to replace the bio-assay with a reliable in vitro test. Over the past six years it has been demonstrated that all the botulinum neurotoxins act intracellularly as highly specific zinc endoproteases, cleaving proteins involved in the control of secretion of neurotransmitters. In the work described, this enzymatic activity has been utilised in assay formats for the detection in foods of neurotoxin from the serotypes involved in food-borne outbreaks in man. These assays have been shown to have a greater sensitivity, speed and specificity than the mouse bio-assay. It is envisaged that such assays will prove realistic alternatives to animal based tests.

Alternative tests have a role in vaccine testing, especially to confirm production consistency. Given the characteristics of these alternative tests and of the products for which they may be used, there are several factors which will influence their use. These include a good understanding of the test and the product to be tested, strong national regulatory infrastructure, a laboratory run in accordance with the principles of laboratory quality systems, and the ability to validate the alternative method. This means that national regulatory authorities will need strong expertise in epidemiology and quality assurance to complement laboratory experience.

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.