Developments in biologicals最新文献

Vaccination continues to be the most effective way to control Rift Valley fever (RVF), a zoonotic insect-borne viral disease of livestock. The irregular, cyclical and persistent nature of RVF in its occurrence in enzootic situations suggests that the vaccination strategy to be considered for these regions should be different from what is envisaged for free from risk regions. Currently available RVF vaccines have been extensively used for the control of the disease. However, these vaccines have shortcomings that have encouraged many research groups to develop new vaccine candidates that would address a large number of the current challenges, and be suitable for use both in disease-free regions and in different contingency and emergency preparedness strategies. The characteristics of different RVF vaccines and vaccination strategies are discussed in this report.

Vaccination of domestic animals against rabies creates a critical barrier between wildlife reservoirs and the human population. Ensuring these vaccines are potent and effective is paramount in preventing human exposure to this deadly and costly disease. The National Institutes of Health (NIH) test is, at present, the most widely used and internationally recommended potency assay for batch testing inactivated rabies vaccines. This test has numerous inherent limitations and disadvantages, including a lack of precision. The NIH test requires a large number of animals and involves unrelieved pain and suffering. A relevant in vitro assay should provide a more accurate, reproducible, rapid, safe, and humane rabies vaccine potency test.

The development of an alternative in vitro potency test required experimental studies, which were performed in-house and in collaboration with other laboratories (Official Medicines Control Laboratories, Manufacturers), coordinated by EDQM (European Directorate for the Quality of Medicines & HealthCare). This paper provides background information concerning the development of the quantitative ELISA for inactivated Newcastle disease virus (NDV) antigen, which was added in the European Pharmacopoeia monograph as an in vitro batch potency test.

This paper proposes a program under which the use of animals for requalification of in vitro potency tests could be eliminated. Standard References (USDA/CVB nomenclature) would be developed, characterized, stored and monitored by selected reference laboratories worldwide. These laboratories would employ scientists skilled in protein and glycoprotein chemistry and equipped with state-of-the-art instruments for required analyses. After Standard References are established, the reference laboratories would provide them to the animal health industry as "gold standards". Companies would then establish and validate a correlation between the Standard Reference and the company Master Reference (USDA/CVB nomenclature) using an internal in vitro assay. After this correlation is established, the company could use the Standard References for qualifying, monitoring and requalifying company Master References without the use of animals. Such a program would eliminate the need for animals for requalification of Master References and the need for each company to develop and validate a battery of Master Reference Monitoring assays. It would also provide advantages in terms of reduced costs and reduced time for requalification testing. As such it would provide a strong incentive for companies to develop and use in vitro assays for potency testing.

Testing vaccines involves expensive animal models and extensive in vitro characterization. Techniques such as ELISA and ELISPOT are traditionally used to measure immunogenicity, assess the potency of recombinant vaccines and detect the presence of biological contaminants. However, these time-proven techniques suffer from technical limitations affecting the overall vaccine development process. Limitations include: consumption of large volumes of biological sample (eg. plasma), high variability, and limited dynamic range. Furthermore, ELISA and ELISPOT involve a multitude of blocking and wash steps which limit their automatability. AlphaLISA technology is an exceptionally sensitive non-wash immunoassay platform which alleviates all the aforementioned drawbacks, allowing one to improve biologics development processes. Examples of how AlphaLISA assays can be used to assess the potency of vaccines will be presented.

The Consistency Approach offers the possibility of reducing the number of animals used for a potency test. However, it is critical to assess the effect that such reduction may have on assay performance. Consistency of production, sometimes referred to as consistency of manufacture or manufacturing, is an old concept implicit in regulation, which aims to ensure the uninterrupted release of safe and effective products. Consistency of manufacture can be described in terms of process capability, or the ability of a process to produce output within specification limits. For example, the standard method for potency testing of inactivated rabies vaccines is a multiple-dilution vaccination challenge test in mice that gives a quantitative, although highly variable estimate. On the other hand, a single-dilution test that does not give a quantitative estimate, but rather shows if the vaccine meets the specification has been proposed. This simplified test can lead to a considerable reduction in the number of animals used. However, traditional indices of process capability assume that the output population (potency values) is normally distributed, which clearly is not the case for the simplified approach. Appropriate computation of capability indices for the latter case will require special statistical considerations.

A proposed definition of a stability indicating assay is "a validated quantitative analytical procedure that can detect changes over time in the pertinent properties of the product" (Federal Register/Vol. 75 No. 180/Friday, September 17, 2010/Proposed Rules). In vaccines intended for veterinary usage, the potency assay has traditionally been used as a measure of stability. Some potency assays may be acceptable as stability indicating assays, whereas other potency assay will not meet the criteria for stability indicating assays. For example, an ELISA potency test may or may not detect degradation products depending on the specificity of the antisera. With time, the ELISA may overestimate the antigen as partial degradation occurs or if an aggregated or particulate antigen dissociates. Specific assays parameters and attributes that are required for a potency assay to be indicative of serial or reference stability are discussed.

Ph. Eur. Monograph 0064 "Swine erysipelas vaccine (inactivated)" currently advises mouse serology for batch potency testing. However, technological advances in vaccine production, improved quality control systems and comprehensive post marketing surveillance increasingly promote the acceptance of non-animal approaches for batch release testing. Protein and immune profiles of inactivated swine erysipelas vaccines obtained by SDS-PAGE and Western Blot might offer a convenient global and functional in vitro alternative. Characteristic and consistent protein and immune profiles could be obtained for aluminium-adjuvanted vaccines. Immunoreactivity of polyclonal sera raised in mice differs markedly from reactivity of swine sera.

The biological nature of IVMPs leads to some unavoidable batch to batch variation in production. The potency test is part of the quality control of the finished product intended to confirm consistency of production and that each batch is formulated equivalent to batches that have been demonstrated to be efficacious. Adequate validation of potency tests is essential to ensure that the results of the assays accurately reflect the amount, titre, or potency of the active substance measured and to indicate the limitations on the accuracy of the measurements to be expected from the test used. The CVMP/IWP published their conclusions concerning validation of potency tests in a Reflection Paper in March 2010. The test validation must demonstrate a dose response and the precision of the result should enable reliable detection of a sub-standard batch. However, the inherent variability in experimental animals often leads to unacceptably wide confidence intervals for in vivo tests which limits their ability to detect slight changes of the antigen amount. The development of in vitro methods as alternatives to in vivo potency tests is encouraged.

Despite significant investment and technical efforts, veterinary vaccine manufacturers continue to experience challenges with the transition from historic animal-based potency methods to in vitro potency assays. These challenges have a number of contributing factors, including an inadequate understanding of protective antigens and epitopes, a lack of ruggedness and discriminating capabilities in evolving immunologically-based methods, inconsistencies between methods used for in-process antigen measurement and finished product potency, and a lack of clear methods to characterize the finished formulation (including complex adjuvants). A lack of harmonized guidelines and consistent regulatory expectations further complicates these efforts. There is room for optimism, however. There are numerous examples of successful in vitro potency test implementations. Titrations of modified live viral and bacterial vaccines, immune-based quantitative assays, and the recent application of direct physicochemical methods have allowed the transition from animal testing in many applications globally. Specific challenges for assay development and implementation are discussed in the areas of 1) target antigen selection, 2) complexity of finished product formulation, 3) potency discrimination, and 4) stability-indicating relevance.