Komla, E., et al. Red blood cells as probes for determining free QS21 in liposomal adjuvant formulations to support product safety and stability. Anal Methods, 2025 Nov 20, 17(45):9162-9170. 

Komla, E., et al. Red blood cells as probes for determining free QS21 in liposomal adjuvant formulations to support product safety and stability. Anal Methods, 2025 Nov 20, 17(45):9162-9170.  PMID: 41196220

• The goal of this study was to develop and validate a straightforward, reliable cell-based assay utilizing human red blood cells to detect and quantify free QS-21 in liposomal vaccine formulations, thereby supporting product safety and stability assessments. QS-21, a potent adjuvant used in various licensed vaccines, exhibits inherent toxicity and hemolytic properties when free. To mitigate these issues, QS-21 is incorporated into lipid-based matrices such as liposomes, which act as QS-21 sinks to suppress its adverse effects. However, ensuring the absence of free QS-21 in these formulations is critical for safety, necessitating robust analytical methods to distinguish between bound and unbound QS-21. This background underscores the importance of developing a sensitive, specific, and accessible assay to monitor free QS-21, ultimately aiming to enhance vaccine safety and quality control during manufacturing, release, and stability studies.
• The developed hemolysis-based assay effectively detected and quantified free QS-21 in liposomal vaccine formulations with good specificity, accuracy, and precision, exhibiting a quadratic relationship between hemolytic activity and free QS-21 concentration (R^2 ranging from 0.91 to 0.99). The method’s sensitivity was in the micromolar range, with a lower limit of quantitation around 25 mg/mL. Validation studies across multiple blood donors showed consistent performance, and the assay successfully monitored free QS-21 levels in multiple batches of the ALFQ vaccine, revealing minimal to no detectable free QS-21. It indicated formulation stability and batch-to-batch consistency. Additionally, the assay could detect QS-21 leakage during long-term storage, providing a useful tool to ensure product safety and stability during manufacturing and storage.
• The assay’s reliance on human blood donors introduces variability due to differences in donor sensitivity, requiring careful donor selection to ensure consistent results. Additionally, the method’s sensitivity has a lower limit of approximately 25 mg/mL, which may not detect very low levels of free QS-21 below this threshold. The quadratic relationship between hemolytic activity and QS-21 concentration, while robust within the tested range, may complicate quantitation at concentrations approaching the assay’s detection limits. Future work could focus on enhancing assay sensitivity to detect lower QS-21 levels, possibly by optimizing RBC concentration or detection methods. Developing standardized donor panels or artificial RBC mimetics might reduce variability and improve consistency. Expanding the assay’s applicability to other liposomal adjuvants or vaccine formulations and validating it across different laboratories would strengthen its utility. Additionally, integrating the assay into a broader stability-indicating testing panel could further support product development and regulatory approval processes.

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