Research conducted by this laboratory focuses on the investigation of modified, new or alternative procedures for determining the safety and potency of a number of biological products. Information obtained from new methods is compared to the current and historical data provided from existing methods to establish equivalency. Suitable standards and reference preparations must be developed and prepared if studies reveal that current materials are not appropriate. Particular emphasis is placed on alternative procedures that reduce the number of animals required for product testing and provide comparable results in less time than existing methods. In Vitro Potency Assay for Diphtheria Antibody Response. Production of a specific quantity of protective antibodies capable of neutralizing active toxin following immunization with vaccines containing a Diphtheria component is required for release of the vaccine lot. Currently, a second animal test is employed to evaluate the efficacy of the animal response to the test vaccine. This research has focused on replacing this second animal test with an in-vitro cell test. Results of the six laboratory collaborative study were presented in December, 1997 at the FDA Science Forum. These data were also reviewed in several international meetings, and plans to develop and implement a collaborative study continue to be discussed. Discussions about modifications of the STS proposed method are continuing. These discussions involve the amount of toxin used in the in-vitro assay, and the use of a hyper-immune horse serum vs. sera from a single immunization of guinea pigs. Development of an In Vitro Potency Assay for Tetanus Antibody Response. This project relates to the investigation of an in vitro ELISA potency assay to replace the guinea pig death test portion of the official release test for the tetanus component in vaccines. Reports of an ELISA method in the literature, and the use of this method in the laboratory are dependent on a source of purified Tetanus Toxin. Preliminary data and proposals for an international collaborative study were discussed at a meeting in June 2000. Problems related to the quantitiation of a response and the correlation between animal and laboratory tests continue to be discussed. Investigation of the Microstar Rapid Sterility Test Method. For biological products claimed to be sterile, testing requires a 14 day incubation period, with observations based on visual examination of product-inoculated media, to conclude with reasonable confidence that the product is free from extraneous bacterial or fungal contamination. A group of newly emerging "rapid" methods suggests that such bacterial contamination can be detected in less than 14 days by other methods. The Microstar system, made by Millipore Corporation, utilizing bioluminescence to detect viable contaminating organisms, was selected for evaluation based on the laboratory's current use of existing Millipore Steritest equipment and supplies. Prior incubation in USP and CFR specified sterility test media appears to enhance detection of multiple types of organisms; using the MicroStar filters, individual CFU's can be transferred and cultured for subsequent identification using selective media or enzyme based analysis. The MicroStar system has shown the capacity for early detection of small quantities of environmental contaminants in conventional, adsorbed and cell based products. This year, efforts to detect bacterial contaminants in whole blood products resulted in the development of a lysing procedure to negate background activity of blood cells. Work continues in this area. Comparison of LAL Test with the Rabbit Pyrogen Test. Determination of pyrogen or endotoxin content may be measured by the Rabbit Pyrogen test as described in 21 CFR 610.13 or, if validated as an equivalent method, use of the Limulus Amebocyte Lysate (LAL) as described in the USP 26 Bacterial Endotoxins Test (BET). Allowable endotoxin content for a number biological products can be found in the "FDA Guideline On Validation Of The Limulus Amebocyte Lysate Test As An Endproduct Test For Human and Animal Parental Drugs, Biological Products, And Medical Devices"; alternatively, product or manufacturer specific limits are established in conjunction with licensing. Chromogenic, Turbidimetric and gel clot methods for the in-vitro BET test are commercially available; the gel clot method is considered definitive in the absence of enhancement or inhibition. In general, advantages of BET methods over the Rabbit test include requiring less sample and the ability to perform repeat or confirmatory tests quickly. This year, several manufacturers of blood products have provided data to support conversion from Rabbit testing to BET. Studies continue to expand BET testing to additional biological products. This project incorporates FY2002 projects 1Z01BR003003-04, 1Z01BR003004-15, 1Z01BR003005-12, and 1Z01BR003006-03.