Development and Applications of a Novel Chemical Assay for the Quantification of Bacterial Endotoxins

Diplôme Thèse Development and Applications of a Novel Chemical Assay for the Quantification of Bacterial Endotoxins eng 2023 2023 director Bakker Eric 716236 Département de chimie minérale et analytique (CHIAM) director Kalman Franka author Hoffmann Anika 932378 110 10.13097/archive-ouverte/unige:172351 urn:nbn:ch:unige-1723514 Sc. 5767 <p>Bacterial endotoxin (ET) testing is crucial for the safety of pharmaceutical, biotechnological, and medical products. All official tests currently recognized by health authorities are based on biological immune reactions of either animals, or human cell cultures to ETs or pyrogens in general. Because of their enzymatic nature, these tests struggle with strong effects of the sample matrix that might lead to false results for the ET content of samples that could endanger human health. Therefore, we developed in Chapter 2 a novel chemical reversed-phase liquid chromatography (LC)-based ET quantification assay. It is based on a derivatization reagent that is used for sialic acid determination. The existing method was revised and tested for its applicability to transfer it to ET quantification. The developed assay uses the rare sugar acid 3-deoxy-D-manno-oct-2-ulosonic acid (Kdo) as an ET biomarker. After hydrolysis under mild acidic conditions, Kdo is labelled with 1,2-diamino-4,5-methylenedioxybenzene (DMB), allowing its sensitive fluorescence detection following reversed phase separation of the reaction mixture. It allows for the selective and sensitive detection of ETs. The new Kdo-DMB-LC assay has a large dynamic range of 109 to 40 EU mL-1 and a limit of quantification of 40 EU mL-1 that corresponds to 4 ng ET per mL-1. The maximum relative standard deviation is below 10%. The structural composition and size of ET molecules is very diverse, so are occurring non-stoichiometric modifications of the core and of the Kdo sugar acid. In Chapter 3, the transferability of the S-type ET standards developed Kdo-DMB-LC assay to R-type ETs was demonstrated. S-type ETs have beside Lipid A and the core moiety, long O-antigen chains that are missing in R-type ETs. The resulting differences in physicochemical properties such as aggregation, or solubility in aqueous solution might impact the efficiency of the new assay. Hydrolysis experiments results showed no impact due to the ET size but revealed the high degree of Kdo modifications for R-type ETs. Kdo substitutions with Galactose, L-glycero-D-manno-heptose, and phosphoethanolamine groups were found. A quantitative release of all different Kdo species was observed at 120 min of hydrolysis time for different ET standards. With that the applicability of the assay for different types of ETs is demonstrated. However, the mild acid hydrolysis condition only cleaves distal Kdo and Kdo species. That leaves the Kdo that links Lipid A to the core, bound to the consecutive oligo-/ polysaccharides. It is not retained by the used reverse-phase and therefore lost for quantification. This requires future method development as currently these Kdo-oligo-/polysaccharide fragments are not quantified. An additional HILIC separation or an adjustment of hydrolytic conditions are thought to solve the current shortcoming. Many pharmaceutical products such as antibodies, or proteins are recombinantly produced in Gram- negative bacteria cell cultures. Strains like Escherichia coli and Pseudomonas are popular production hosts also for biotechnological products such as bioplastics e.g., polyhydroxyalkanoates (PHA) or 4 polylactic acid (PLA).</p> Dewey 540 40 Département de chimie minérale, analytique et appliquée 145 false 772 Groupe Bakker da945ce6-3285-4e1d-ac67-23e965384c40 Thesis Restricted PhD_thesis_Hoffmann_Anika_jury.pdf 3119667 application/pdf Private 2024-10-04 af786bbd4571a44188b4cb4d3954f2dfa618ce3d44546bfa08596b27140bfe19 2426d22b-3cdf-418b-b29b-f41995a66c42 Imprimatur Public Hoffmann_Anika_Imprimatur.pdf 205203 application/pdf 9496ef0f318e481b64b9e813a802e52bc2b975b4133851f5267c2446ba42f7ef restrictedAccess Faculté des sciences / Section de chimie et biochimie / Département de chimie minérale, analytique et appliquée

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Development and Applications of a Novel Chemical Assay for the Quantification of Bacterial Endotoxins				2023	304	0

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Hoffmann, Anika