Evaluation of limulus amebocyte lysate and recombinant endotoxin alternative assays for an assessment of endotoxin...

The United States Pharmacopeia (USP), European Pharmacopeia (EP), and Parenteral Drug Association (PDA) provide guidance on the validation of alternative microbiological methods (U.S. Pharmacopeia National Formulary 2019, Parenteral Drug Association Technical Report No. 33 2013, European Pharmacopoeia (Ph. Eur.) 2017). They define "specificity" as the ability to detect a range of microorganisms. In the context of alternative methods to the compendial Bacterial Endotoxin Test (BET) a range of endotoxins must be considered. This range should represent environmental endotoxins that present risks to pharmaceutical manufacturing processes, final products, and to the most important stakeholder: the patient. This study examines several alternative methods for the bacterial endotoxin detection test. It compares the official and harmonized BET test from two Limulus Amebocyte Lysate (LAL) suppliers to three commercially available recombinant Factor C (rFC) reagents that contain only one of the three enzymes in the horseshoe crab clotting cascade. The study also includes a recombinant reagent that has been developed to include all three of the enzymes involved in the LAL coagulation cascade, occurring in the presence of endotoxins. Pharmaceutically relevant water samples from various points in pharmaceutical water purification processes were used as a source of natural environmental endotoxins. While these water samples are not routinely tested for bacterial endotoxins, they do exist within manufacturing facilities and thus present risks to manufacturing operations (Sandle, 2019). A statistical analysis of 128 samples containing environmental endotoxin has shown that at the 5% level of significance, non-inferiority between the two compendial LAL methods was achieved. However, the non-inferiority claim could not be made with any of the recombinant reagents. The link between the BET and recombinant alternatives remains unresolved and, therefore, requires caution, continued development, and testing.