Pentavalent meningococcal conjugate vaccine(PATH)

The pentavalent meningococcal polysaccharide conjugate vaccine, which includes serogroup A, C, Y, W, and X, has recently been prequalified by the WHO and is currently the only vaccine available against serogroup X. The amount of free saccharide in conjugate vaccines is a crucial factor that directly impacts the stability and immunogenicity of the vaccine. Therefore, precise estimation of free saccharide content is particularly important in multivalent conjugate vaccines. This study focuses on the development of conditions for estimating free saccharide in monovalent meningococcal X conjugate bulk. Accuracies were demonstrated at 5 %, 10 %, 25 %, and 40 % of the test specification, with recoveries ranging from 70 to 130 %. Repeatability analysis showed intra-assay variation ranging from 2 to 6 %, while inter-assay variation ranged from 2 to 14 %. Specificity studies indicated that there was no interference from assay components such as sample excipients, DOC, or acids. When these established conditions were applied to the finished product for free saccharide estimation, an interesting observation was noted. It was found that the unconjugated meningococcal X saccharide interacted with other biomolecules present in the vaccine formulation, leading to decreased free saccharide recovery for serogroup X. It is believed that aggregation, possibly due to carrier proteins (TT and CRM) and/or associated polysaccharides, may be responsible for trapping the free saccharide from serogroup X. Efforts were made to control these biomolecular interactions by adjusting solubilities, buffering, and physico-chemical conditions to separate free saccharide. Phosphate buffers, ionic-nonionic detergent solutions, salt buffers, and 32 biomolecule formulations were explored in various combinations before identifying the root cause for the decreased X free saccharide recoveries.

Molecular size distribution (MSD) of polysaccharides serves as a key parameter that directly correlates to the immunogenicity of vaccine. MSD at meningococcal polysaccharide (A, C, Y and W) or conjugate bulk level is well established under detailed pharmacopeial and WHO guidelines. We report here, a newly developed method for determination of molecular size distribution of pentavalent Meningococcal conjugate vaccine comprising of A, C, Y, W and X (MenFive). Although serogroup specific molecular size could not be estimated here; lot to lot consistency monitoring, molecular aggregates distribution in final lot, are key takeaways of this method. Determination of MSD in pentavalent fill finished product was quite challenging. Various columns/detectors combination, buffers, physico-chemical conditions (temperature, 2-8 °C, 25 °C, 40 °C and 60 °C; flow rate, 0.3 mL to 0.8 mL), liquid/lyophilized formulations, were explored. Polymer-based packed columns were explored for estimation for MSD by aqueous size exclusion chromatography, using combinations of- Shodex OHPAK SB 807 HQ, Shodex OHPAK SB 806 HQ, G6000 PWXL, coupled with guard Shodex OHPAK SB-G-6B. MenFive showed heterogenous distribution of molecules ranging from 200 to 19000 kDa, indicating its complex nature. However, 1000-8000 kDa was dominant range, comprising of ≥ 50 % distribution of molecules, in both liquid as well as lyophilized formulations, with average molecular weight around 6000-6500 kDa. The molar mass distribution after slicing would provide an insight to the conformation of molecules through its presentation as HMW, LMW, aggregates and subsequently, the presence of dominant population of molecules of a particular molecular weight and its total contribution in the sample.