Development of a Monocyte Activation Test for Evaluating Recombinant Hepatitis B Vaccine: A Novel Approach for Pyrogen Assessment.

Abstract

Background: Injectable products, particularly human vaccines, must be free from fever-inducing agents and thoroughly tested for pyrogens as part of a quality control. Consequently, manufacturing facilities are required to conduct appropriate pyrogen tests per pharmacopeial standards. This study aimed to evaluate the reliability of the MAT in quantifying pyrogenic content in the recombinant hepatitis B vaccine.

Methods: We assessed pyrogen activity in the API, formulated vaccine, and aluminum hydroxide by comparing the LAL, RPT, and MAT, measuring activity in RPU as per the European Pharmacopeia. Monocytes from healthy donors were isolated and identified via flow cytometry to measure the CD14+ marker frequency.

Results: The study found that the pyrogenic concentration of LTA in the MAT was 50,000 ng/mL (5.19 EEU/mL). In contrast, the same concentration in the RPT was deemed non-pyrogenic based on rectal temperature assessments. The MAT showed sensitivity to the API and adjuvant, with a detection limit of 2.5 EU/mL for IL-6, outperforming the RPT, which had a detection limit of 5 EU/mL.

Conclusion: A strong IL-6 response to both LPS and LTA stimulation was observed, indicating that IL-6 could serve as a valuable marker for pyrogen testing. The MAT appears to be an effective alternative to the RPT for assessing pyrogenicity, demonstrating commendable consistency and accuracy across various testing systems allowed by the Ph. Eur. General MAT Chapter, especially given the RPT's limitations in controlling pyrogenicity in injectable products.