Journal of Microbial & Biochemical Technology

Endotoxin masking: A kinetically controlled reaction mechanism

3^rd World Congress and Expo on Applied Microbiology

November 07-09, 2016 Dubai, UAE

Johannes Reich

Universitat Regensburg, Germany
Hyglos GmbH, Germany

Posters & Accepted Abstracts: J Microb Biochem Technol

Abstract:

Lipopolysaccharides are the major components of the outer membrane of Gram-negative bacteria. Due to their toxic effects after administration into the bloodstream, lipopolysaccharides are also called endotoxins. They are ubiquitously found, often in low concentration, in nearly all natural products, including pharmaceutical products. Thus, it is mandatory to control parenteral drug products in order to avoid endotoxin contaminations. In quality control of biopharmaceutical products, �??Low Endotoxin Recovery�?� (LER) has been observed while using common detection methods, like the Limulus Amebocyte Lysate (LAL) assay. This is of particular importance when surfactants and other ingredients such as salt, urea and other organic substances are present, all molecules representing parameters strongly influencing the critical micelle concentration (CMC). Micelles and other aggregates are assumed to represent the major physicochemical mechanism on the LER effect by masking the toxin, thus precluding correct determination in bacterial endotoxin testing. As a consequence, once the LER effect appears underestimation of potential endotoxin contaminations and therefore, false negative results in the Limulus-based test methods may appear. In pharmaceutical quality control units, such false negative results have to be strictly avoided. Our work was guided by the assumption, that the root cause of LER effect is the interplay of endotoxin with complex forming agents and surfactants, resulting in diverse aggregates of highly complex molecular structures. However, the physicochemical mechanism and principles of LER are still a matter of debate. Due to the time dependency of the LER phenomenon, time dependent reactions of endotoxin recovery have been analyzed in more detail. The results presented here, demonstrate that dilution of a sample delays or avoids the identification of LER but also indicate predominant effects of complex forming agents. Within the used model system, a minimum concentration of citrate to serve as complex forming buffer system could be determined. Furthermore, we could demonstrate that the presence of surfactants is a strong prerequisite but does not determine kinetics of the LER effect. Interestingly, the endotoxin concentration itself had no significant impact on masking kinetics. Based on our results we propose a new theoretical model for simulating masking kinetics, leading to a better understanding of endotoxin masking in aqueous solutions.

Biography :

Email: Johannes.Reich@hyglos.de