Comparison of analytical techniques for the quantification of bacterial endotoxins in injectable preparations using the additive color method

Introduction
Injectable preparations are subject to apyrogenicity requirements, particularly with regard to bacterial endotoxins. This testing is primarily performed using the Limulus Amebocyte Lysate (LAL) test, but recombinant methods are also available, such as the recombinant Factor C (rFC) plate assay or the recombinant cascade reagent (rCR) plate (rCRp) and cartridge (rCRc) assays. The objective was to compare these 3 methods, taking into account their analytical and environmental performance, as well as their productivity and practical effectiveness.

Materials and methods
The comparison of methods was performed using the RGB tool developed by Nowak and Kościelniak (1), based on experimental results and literature data, using samples of lipidic and non-lipidic parenteral nutrition, according to 3 criteria: analytical performance (red = R), environmental sustainability (green = G), and productivity (blue = B). The criteria selected for analytical performance were: spike recovery, coefficients of variation, and the difference between measured and expected endotoxin concentrations. For environmental impact: animal mortality, reagent consumption, and energy consumption. For productivity: preparation and analysis time, and the number of false positives and negatives. Plate-based techniques were evaluated under optimal conditions (full plate) and unfavorable conditions (a single sample per plate). Overall performance is determined based on measured brightness (MB) and color.

Results et discussion
All 3 methods met the established analytical criteria. The rCRc was classified as white (=R+V+B) because it exhibited the highest brightness (MB 89%) due to the absence of animal-derived products, good productivity, and an average spike recovery of 109%, despite its lower precision. Due to its inferior analytical performance, the rCRp under optimal conditions is classified as cyan (=V+B) (MB 78%). Next are the rFC under optimal conditions (MB 65%) and the LAL cartridge (MB 61%), classified as magenta (=R+B) due to the use of fetal calf serum (rFC) and horseshoe crab blood (LAL). Under unfavorable conditions, the rCRp (MB 64%) and the rFC (MB 51%) are classified as V and R, respectively, since they meet only one of the 3 criteria. Optimizing plate filling reduces analysis time from 60 to 3 minutes per sample, and cartridge-based techniques reduce preparation time.

Conclusion
This study identifies the rCR technique as the most balanced alternative in terms of the evaluated performance criteria. It also highlights the importance of optimizing plate filling to increase productivity, but above all, the need to eliminate the use of animal-based resources to improve environmental impact.

Reference : (1) Nowak et Kościelniak, Anal. Chem. 2019, 91, 10343−10352

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