Anthrax is one of the biological weapons most likely to be used in terrorism or warfare. Anthrax spore laced letters caused five deaths in the fall of 2001 in America. Before a new vaccine for anthrax is developed and approved, the most important task is to assure that the current vaccine---Anthrax Vaccine Adsorbed (AVA) is safe and effective. The Laboratory of Analytical Chemistry of OVRR/CBER/FDA is currently involved in efforts to verify that this product meets the specifications. However, some of the currently used procedures for the vaccine analyses have become obsolescent due to the fact that they are based on relatively non-specific and time-consuming colorimetric techniques. In order to monitor the chemical component contents of the vaccine more accurately and precisely, new methods need to be developed with the help of modern instrumentation such as HPLC/MS. The goal of this proposal is to develop a new HPLC method for the determination of formaldehyde and to explore the possibility of detection and determination of the protective antigen in AVA by HPLC/MS. The results of this research are expected to provide better analytical methods for both the quality control by the manufacturer and regulatory monitoring by the FDA. A. Determination of formaldehyde The currently used method for formaldehyde measurement in the vaccine is a tedious colorimetric method.
The aim of HPLC method development for formaldehyde determination is to find a faster, more specific method for AVA that may also be applicable to other vaccines containing formaldehyde. Though there are several existing HPLC methods for the determination of formaldehyde content in air or water, the determination of formaldehyde in vaccines may present difficulties due to the complexity of the biological sample and the existence of different forms of formaldehyde in vaccines. Different forms of formaldehyde may include formaldehyde free in solution and formaldehyde in the form of methylol functions bound to protein or other vaccine components. In this project, efforts will be made to develop a new method using direct injection after derivatization since many of the existing HPLC methods use an extraction procedure after derivatization, which may present problems in analyte recovery as well as operational complexity. An experimental protocol will be optimized for the analysis of low sample volumes. Research on formaldehyde determination will include two main stages.(1) Developing HPLC method with formaldehyde standard. (2)Modifying HPLC method for the formaldehyde in Anthrax vaccine. B. Analysis of Protective Antigen (PA) for anthrax vaccine PA (83 kDa) is the protective component of AVA, which stimulates a protective immune response against infection with B. anthracis. PA structure was determined in 1997. It is a long and flat molecule of dimensions 100x50x30 ?. PA content in the vaccine varies from lot to lot due to the nature of the fermentation procedure. It is believed that impurities in the vaccine are responsible for a high incidence of local reactions. It would be highly desirable to quantitatively measure the PA content and related impurities in the vaccine. PA is still indirectly measured through antibody titers in animals. This method is obviously undesirable for quality-control purposes. With the help of new mass spectrometry (MS) techniques, coupled with HPLC, we may have the opportunity to develop a method that can detect or even quantitatively determine the PA content rapidly. In the beginning of this project, the vaccine sample will be sent out for examination by MALDI-MS in order to obtain a mass spectrum of the PA molecule because the MALDI technique is able to detect proteins in complicated matrices. There are two main challenges in the analysis of PA by HPLC/MS. One challenge is the development of an easy and reliable desorption procedure for the separation of protein components in AVA from its adjuvant---Alhydrogel. Attempts by Bioport investigators to desorb protein from the formulated vaccine have not yet been successful. The difficulty is caused not only by the complicated interactions between protein and adjuvant surfaces but also by the ability of the protein to undergo structural rearrangements over the time of storage. I will attempt to design a suitable desorption procedure. Protein characterization techniques such as UV/Vis spectrophotometric analysis, SDS-PAGE, ELISA, Western Blot and HPLC/MS will be used to analyze the desorbed proteins and monitor the efficiency of different desorption procedures. Another challenge is to ensure that the desorbed sample is compatible with the detector of the mass spectrometer because the designed desorption procedure may use chemical reagents that suppress sample ionization during MS detection.
