Novel RP-HPLC based assay for selective and sensitive endotoxin quantification

Bucsella, Blanka (University of Zurich, Department of Chemistry, Zürich, Switzerland ; School of Engineering, HES-SO Valais-Wallis, HEI, HES-SO // University of Applied Sciences Western Switzerland) ; Hoffmann, Anika (School of Engineering, HES-SO Valais-Wallis, HEI, HES-SO // University of Applied Sciences Western Switzerland) ; Zollinger, Mathieu (School of Engineering, HES-SO Valais-Wallis, HEI, HES-SO // University of Applied Sciences Western Switzerland) ; Stephan, Fabio (School of Engineering, HES-SO Valais-Wallis, HEI, HES-SO // University of Applied Sciences Western Switzerland ; Lanza AG, Visp, Switzerland) ; Pattky, Martin (School of Engineering, HES-SO Valais-Wallis, HEI, HES-SO // University of Applied Sciences Western Switzerland ; Lanza AG, Visp, Switzerland) ; Daumke, Ralph (FILTROX AG, St. Gallen, Switzerland) ; Heiligtag, Florian J. (FILTROX AG, St. Gallen, Switzerland) ; Bassas-Galia, Mònica (School of Engineering, HES-SO Valais-Wallis, HEI, HES-SO // University of Applied Sciences Western Switzerland ; Acrostak AG, Winterthur, Switzerland) ; Zinn, Manfred (School of Engineering, HES-SO Valais-Wallis, HEI, HES-SO // University of Applied Sciences Western Switzerland) ; Kalman, Franka (School of Engineering, HES-SO Valais-Wallis, HEI, HES-SO // University of Applied Sciences Western Switzerland)

The paper presents a novel instrumental analytical endotoxin quantification assay. It uses common analytical laboratory equipment (HPLC-FLD) and allows quantifying endotoxins (ETs) in different matrices from about 109 EU / mL down to about 40 EU / mL (RSE based). Test results are obtained in concentration units (e.g. ng ET / mL), which can then be converted to commonly used endotoxin units (EU / mL) in case of known pyrogenic activity. During endotoxin hydrolysis, the endotoxin specific rare sugar acid KDO is obtained quantitatively. After that, KDO is stoichiometrically reacted with DMB, which results in a highly fluorescent derivative. The mixture is separated using RP-HPLC followed by KDO-DMB quantification with a fluorescence detector. Based on the KDO content, the endotoxin content in the sample is calculated. The developed assay is economic and has a small error. Its applicability was demonstrated in applied research. ETs were quantified in purified bacterial biopolymers, which were produced by Gram-negative bacteria. Results were compared to LAL results obtained for the same samples. A high correlation was found between the results of both methods. Further, the new assay was utilized with high success during the development of novel endotoxin specific depth filters, which allow efficient, economic and sustainable ET removal during DSP. Those examples demonstrate that the new assay has the potential to complement the animal-based biological LAL pyrogenic quantification tests, which are accepted today by the major health authorities worldwide for the release of commercial pharmaceutical products.


Keywords:
Article Type:
scientifique
Faculty:
Ingénierie et Architecture
School:
HEI-VS
Institute:
Institut Technologies du vivant
Date:
2020-08
Pagination:
10 p.
Published in:
Analytical Methods
Numeration (vol. no.):
2020, no. 38
DOI:
Appears in Collection:

Note: The file is under embargo until: 2021-08-31


 Record created 2021-04-14, last modified 2021-05-17

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