Red blood cells as probes for determining free QS21 in liposomal adjuvant formulations to support product safety and stability

Analytical method development is critical to establishing and supporting drug product quality. QS21 is a potent adjuvant, which is currently used in several clinical vaccine trials, and is utilized as an adjuvant in licensed vaccines against malaria, shingles and respiratory syncytial virus (RSV). Due to its toxic and hemolytic properties as a stand-alone or free adjuvant, QS21 has been incorporated into heterogeneous matrices containing cholesterol that serve as QS21 sinks to suppress its toxic activity. Hence, to address the safety attribute of the QS21-based adjuvant, it is imperative to ensure the absence of free QS21 in the drug formulation. To date, quantitative analytical methods that definitively discriminate free from bound QS21 in liposomal adjuvant formulations are not available in the literature. In view of this, we have developed and qualified a cell-based assay using human red blood cells (RBCs) as probes for detecting and estimating free QS21 in liposomal formulations. This method exhibits a medium throughput, and does not require any expensive and special instrumentation system. Qualification results showed that the method presents good specificity for free QS21 detection, accuracy and precision, with quantitation sensitivity in the micromolar range (lower limit of ∼25 μg mL^-1), suitable for our intended purpose. The described method was successfully used as part of the regulatory specification tests for the product release and stability studies of Army Liposome Formulation containing QS21 (ALFQ).