Optimization of LAL Assay in Plate Reader and Methods to Reduce Interference in Chitosan for Endotoxin Detection

Endotoxin or Lipopolysaccharide (LPS) is a component of gram-negative bacteria cell wall, which causes hazardous symptoms in humans such as septic shock and death after administration. Endotoxin contamination in clinically administered materials/ nanoparticles is prevalent, typically introduced during production and processing steps, which renders the active pharmaceutical Ingredient (API) unsuitable for use. Before releasing the end product in the market, the companies perform endotoxin detection by FDA approved LAL (Limulus Amoebocyte Lysate) tests. Evaluation of endotoxin contamination in chitosan raw materials used for the production of injectable Chitosan-siRNA nanoparticles and Chitosan/Hyaluronic Acid scaffolds in vivo is complicated due to its positive interaction with endotoxin. A false negative result by the LAL test may lead to approval, but may cause the release of life-threatening cytokines such as interferons when injected in human body. We first determined the amount of endotoxin in the chitosan raw material(s) by gel-clot LAL assay, a semi-quantitative assay, which provided an estimated value of endotoxin contamination. We found values <0.3 EU/mg for chitosan with 92% DDA at high and low (10kDa) molar mass, which is under the acceptable limit. If the gel-clot derived endotoxin value are considered as absolute by USP, then this limit amount represents significant risk going into the clinical phase with the higher administration doses. This leads to the use of more sensitive LAL assays for the accurate and absolute quantification of endotoxin, such as kinetic turbidimetric and kinetic chromogenic LAL assays. In the second part, we developed the kinetics assays, where standards are used to establish a standard curve. A linear regression equation of a Log conc. vs. log onset time standard curve is then determined, the onset time being obtained when the measured absorbance at 405nm of each calibrator reaches a threshold O.D., typically 0.1 in turbidimetric and 0.2 for chromogenic LAL assay. Because of the biological origin of LAL enzyme, the inherent variability in the assay is set as + 2-fold defined by the USP, i.e. a quantified value may have a variation of 50-200%. Because of the use of a plate reader to perform the assay, different technical parameters, such as pathlength correction, identification of wells with high evaporation in 96 well plates, pre-treatment of samples and calibration range were then optimized in order to obtained repeatable standard curves equation. In the third part of the kinetic assay, the endotoxin titration in chitosan with 92% DDA at high and low molecular weight by kinetic turbidimetric LAL assay were developed. Because of interaction between chitosan and endotoxin, inhibition of detection of endotoxin called as inhibition interference was observed. In order to reduce this interaction, various pre-treatments were tested, such as NaOH treatment, chitosanase enzyme digestion as well as chemical depolymerisation of chitosan by nitrous acid and chemical treatment by concentrated hydrochloric acid as recommended by USP. We found that all these treatments were deleterious for the free endotoxin and lead to reduced detection, but a protective effect of chitosan on endotoxin was discovered which allowed good but not total recovery of spiked endotoxin. Based on this, the use of a neutral reagent is recommended for dissolving chitosan that does not disintegrate free endotoxin and also releases the bound endotoxin. The use of dispersing agents, detergents and cations to weaken the bonds between chitosan and endotoxin are also recommended for future use.