Comparison of Limulus Amoebocyte Lysate and Recombinant Factor C Assays for Endotoxin Detection in Four Human Vaccines with Complex Matrices

Endotoxins, heat-stable lipopolysaccharides from Gram-negative bacteria, are potential contaminants that can be introduced during manufacturing of pharmaceutical products, including vaccines. Parental pharmaceutical products undergo endotoxin testing because endotoxins are pyrogenic in humans and can induce severe physiological reactions. Currently, animal-derived Limulus amoebocyte lysate (LAL) assays are widely used. Endotoxins, heat-stable lipopolysaccharides from Gram-negative bacteria, are potential contaminants that can be introduced during manufacturing of pharmaceutical products, including vaccines. Parental pharmaceutical products undergo endotoxin testing because endotoxins are pyrogenic in humans and can induce severe physiological reactions. Currently, animal-derived Limulus amoebocyte lysate (LAL) assays are widely used. Assays using recombinant factor C (rFC), a nonanimal-derived reagent, have been proposed as alternatives. Some components in the matrices of pharmaceutical products can interfere with these assays. We compared two LAL- and two rFC-based assays for endotoxin detection in four complex human vaccine matrices. We showed that the results for the rFC-based assays were at least equivalent to those for the LAL-based assays, although the rFC-based assays were found to be adequate but slightly less suitable for one of the products that contained proteases as the methods used to inactivate the proteases reduced the assay performance. Likewise, LAL was adequate but less suitable for another product that contained glucans. The rFC assays offer a number of benefits, including compliance with the principles of the 3Rs, i.e., replacement, reduction, and refinement of animal testing by safeguarding animal welfare and promoting more ethical and sustainable use of animals for testing. After they are fully validated, as per the compendial requirements, they could be considered as suitable replacement assays for the detection of endotoxin in the manufacturing processes of pharmaceutical products. In summary, we demonstrated that both LAL and rFC assays are adequate for testing and releasing four vaccine products.