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Structural characterisation of diphtheria toxoid

By Bernardus Metz


The primary concern of the vaccine industry is to produce vaccines with consistent high quality. To that end, control tests are performed in all stages of manufacturing from starting materials through final vaccine lots. Many animal-based tests are included to determine the potency and safety of human vaccines. This thesis presents the results of a study to structurally characterise diphtheria toxoid with physicochemical and immunochemical techniques. The modifications that result from the formaldehyde treatment were investigated for a better understanding of the detoxification process. This knowledge may help to develop new in vitro tests, as an alternative for the current in vivo potency test. \ud \ud A general overview is given about the opportunities and problems in the reduction of animal use for human vaccine quality control (chapter 2). Progress in the reduction of animal tests with regard to in vivo potency and safety testing is discussed. Many analytical techniques are described that are suitable to investigate properties of antigens, i.e. the identity, size, secondary and tertiary structure, and purity. \ud Five physicochemical and immunochemical tests has been developed to monitor the quality of diphtheria vaccines and could possibly serve as an alternative to established in vivo tests (chapter 3 and 4). A clear correlation was found between these tests and the standard potency and safety tests. In the study a number of criteria were proposed that diphtheria toxoid has to meet, i.e. an apparent shift of the B-fragment in SDS-PAGE, a reduction of the number of primary amine groups, an increased resistance to denaturation, an increased circular dichroism signal in the near-UV region, and a reduced binding to selected monoclonal antibodies. Before implementation, the selected set of in vitro analyses has to undergo extensive validation studies and regulatory acceptance. \ud \ud One of the most important steps in the production of the diphtheria toxoid vaccine is the detoxification of diphtheria toxin by formaldehyde. The unknown chemical modifications occurring during formaldehyde treatment on reactive amino acid residues were identified by using synthetic model peptides (chapter 5). The study revealed that formaldehyde forms labile methylol adducts and Schiff-bases with N-terminal amino acid residues and the side-chains of arginine, cysteine, histidine, lysine and trypthophan residues. Importantly, formaldehyde treatment causes stable cross-links between primary amino groups and several amino acid residues including arginine, asparagine, glutamine, histidine, tyrosine and tryptophan. The information obtained with these model peptides was utilised to study the complex formaldehyde-protein chemistry in the model component bovine insulin (chapter 6) and in diphtheria toxoid itself (chapter 7). \ud \ud Finally, a study was performed in which a basis is provided for an antigenicity assay as a possible substitute for in vivo immunogenicity measurements (chapter 8). For that purpose, monoclonal antibodies were selected that bind to immunodominant epitopes. Five monoclonal antibodies were selected and recommended for usage in immunochemical assays, which might serve as a possible substitute for the in vivo potency test of diphtheria vaccines

Topics: Farmacie, diphtheria toxoid, diphtheria toxin, structure, characterization, vaccine, quality control, animal test, alternatives, chemical modification, formaldehyde
Publisher: Utrecht University
Year: 2005
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