Development of multiplexed flow based assay for simultaneous identification and estimation of meningococcal capsular polysaccharide serogroups A, C, W, Y and X for conjugate vaccine manufacturing.

The development of a multivalent meningococcal polysaccharide vaccine requires the analysis of active ingredient, i.e., capsular polysaccharides, at various stages of its development. Furthermore, in accordance with WHO guidelines for the release of any batch of purified polysaccharides, it is imperative to determine whether any heterologous polysaccharides, with a threshold of less than 1 % of their dry weight, are present in the sample. We have developed a flow-based multiplexed bead-based competitive inhibition assay (BBCIA), which identifies the meningococcal polysaccharide A, C, Y, W, and X at purified polysaccharides, conjugate bulk, and final vaccines development, and quantified the 1 % heterologous polysaccharides as contaminant in purified polysaccharides. Polysaccharides are coupled to the carboxylated microsphere and incubated with a serogroup-specific antibody-antigen mixture. Unneutralized antibodies bind to respective antigens coupled onto the beads and are expressed in terms of median fluorescence intensity (MFI). The BBCIA identified meningococcal polysaccharides with ≥90 % inhibition during identity, and estimated the heterologous polysaccharides as contaminants with 70-130 % recovery of spiked polysaccharides with a percentage CV < 20 %. Results were validated as per ICH guidelines, such as limit of detection and quantification, linearity, precision, robustness, specificity, and accuracy in accordance to international standards. We are reporting an assay tool that simultaneously identifies and quantifies polysaccharides of the multivalent meningococcal vaccine with the advantages of less time, labour and sample volume in analysis with higher sensitivity and accuracy. With high throughput capabilities and increased sensitivity, BBCIA can be readily adopted to identify and estimate various serogroup-specific antigens for vaccine development.