Comparison of LAL and rFC Assays-Participation in a Proficiency Test Program between 2014 and 2019

Endotoxin (lipopolysaccharide) testing of drugs is routinely required in pharmaceutical industries. Suitable compendial assays are defined by national pharmacopoeias. At this time, Limulus Amoebocyte Lysate (LAL) assays are the gold standard. LAL is used in vitro for specific detection of endotoxin based on endotoxin-activated Factor C-mediated clotting cascade. However, alternative mediated pathways (e.g., Factor G), impurities, and further factors may influence test results. Some of these influencing factors are eliminated by recombinant Factor C (rFC) test, which represents a promising alternative. rFC not only enables highly specific endotoxin testing, as interfering Horseshoe Crab blood components are eliminated, but also offers ethical and ecological advantages compared to classical LAL assays. However, the question remains whether rFC-based tests are robust test systems, equivalent or superior to LAL and suitable for routine bacterial endotoxin testing. Pharmaceutical test users have validated the test successfully for their specific products, but no long-term studies have been published that combine testing of unknown samples, inter-laboratory, -operator, and -lot changes. Thus, it was of great interest to investigate rFC test performance in a routine setting within a proficiency test program set-up. Over a period of six years comparative endotoxin testing was conducted with one kinetic chromogenic LAL assay and two rFC-based assays. Results of this study demonstrate that both rFC-based assays were comparable to LAL. All results met acceptance criteria defined by compendial bacterial endotoxin testing. RFC-based methods generated results with even better endotoxin recovery rates compared to LAL. Therefore, rFC-based tests were found to represent reliable methods, as equivalent or even superior to LAL assays and suitable for routine bacterial endotoxin testing.