Abstract
Pyrogen content is a key quality feature that must be checked in all injectable products, including vaccines. Four tests are currently available in the European Pharmacopoeia to monitor pyrogen/endotoxin presence: the rabbit pyrogen test (RPT), the bacterial endotoxin test, the recombinant factor C test, and the monocyte activation test (MAT). Here, we explored the possibility to replace the RPT with the MAT in the quality control of a vaccine against tick-borne encephalitis virus (TBEV). The testing was carried out using cryopreserved peripheral blood mononuclear cells as cell source. IL-6 release was selected as readout for the detection of both endotoxin and non-endotoxin contaminants. MAT applicability for pyrogen testing of the TBEV vaccine was assessed through preparatory tests and resulted in the establishment of a very sensitive assay (limit of detection (LOD) = 0.04 EU/mL; sensitivity = 0.1 EU/mL). Both quantitative Method A and semiquantitative Method B were used for data analysis. Our studies revealed that for a vaccine without intrinsic pyrogenicity, such as that against TBEV, sensitivity (the lowest endotoxin value of the standard curve) should be used instead of LOD to define a stable maximum valid dilution of the product. In conclusion, we describe the challenges of MAT implementation for anti-TBEV vaccine following the current Ph. Eur. chapter 2.6.30 and propose a re-evaluation of the validity criteria of Methods A and B in order to set a semi-quantitative or limit test suitable for those products for which a reference lot comparison analysis is not applicable or favorable.
Highlights
Tick-borne encephalitis (TBE) is an illness caused by tick-borne encephalitis virus (TBEV) infection, whose clinical manifestations range from febrile illness to highly aggressive downstream neurological symptoms (Ruzek et al, 2019)
3.1 Comparison of TNF-α, interleukin 6 (IL-6) and IL-1β as monocyte activation test (MAT) read-outs for the detection of both endotoxin and non-endotoxin contaminants To define which cytokine is the most appropriate read-out in our setting, a simultaneous cytokine measurement was performed on peripheral blood mononuclear cells (PBMC) supernatants stimulated with both endotoxin and non-endotoxin stimuli
PBMC were treated with RSE, Fig. 1: Pro-inflammatory cytokine release in PBMC cultures stimulated with endotoxin and non-endotoxin contaminants Peripheral blood mononuclear cells (PBMC) were stimulated with RSE (A) (0.2 and 0.4 EU/mL), R848 (B) (0.15 and 0.3 mg/mL), FSL-1 (C) (0.02 and 0.04 ng/mL) or left untreated, and TNF-α, IL-6 and IL-1β production was assessed in culture supernatants by cytometric bead assay
Summary
Tick-borne encephalitis (TBE) is an illness caused by tick-borne encephalitis virus (TBEV) infection, whose clinical manifestations range from febrile illness to highly aggressive downstream neurological symptoms (Ruzek et al, 2019). Two European vaccines have been produced: FSME-IMMUN (Pfizer, USA), prepared from the Neudoerfl strain of the European subtype (Barrett et al, 2003), and Encepur (GSK), based on the Karlsruhe (K23) strain (Harabacz et al, 1992; Girgsdies and Rosenkranz, 1996). These vaccines have been used for more than 30 years and are highly effective in preventing TBE (Barrett et al, 2003)
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