Paper Title
Development of A Monocyte Activation Test for Recombinant Hepatitis B Vaccine To Replace the Rabbit Pyrogentest and LAL Test

Abstract
Pyrogens can compromise the safety of vaccines, making it essential to measure this property during vaccine production. While the Limulus Ambocyte Lysate (LAL) method and Rabbit Pyrogen Test (RPT) are commonly used, they have weaknesses that can be overcome by the Monocyte Activation Test (MAT). The aim of this study is to promote the acceptance and use of the MAT test as a replacement for the RPT and LAL in the industry for controlling the production of intrinsically pyrogenic vaccines. The transition from the RPT to the MAT is in line with the 4Rs principle of replacement, reduction, rehabilitation, and refinement, providing low-cost alternatives for pharmaceutical companies. A specific product was tested using LAL, RPT, and MAT tests against the manufactured HBV. The recombinant hepatitis B surface antigen vaccine was produced at a large-scale fermentation using a C-226 recombinant strain of the methylotrophic yeast Pichiapastoris and has been formulated. Based on the obtained results, a significant difference in IL-6 concentration was observed between samples. The samples containing cell with aluminum hydroxide gel (Cell+Gel), cell with HBV (Cell+HBV), and cell with API (Cell+API) exhibited a statistically significant differences in IL-6 concentration compared to the alone cell cultures with media Dulbecco's Modified Eagle Medium (P<0.0001). However, these differences were within the normal range, and none of the examined samples showed any signs of pyrogenicity based on IL-6 concentration. The MAT test used the IL-6 measurement method, and we found that IL-6 strongly responds to stimulation with LPS and LTA in our study. Based on our findings, the MAT test is suitable for fever vaccine production vaccine production for those vaccines that may cause fever as it does not require the use of animals, is cost-effective, uses fewer samples, and is safe. Therefore, the MAT test can be considered as an alternative method for measuring pyrogens during vaccine production. Keywords - Recombinant Hepatitis B, Pyrogens, Monocyte Activation Test, Rabbit Pyrogen Test