Meningococcal meningitis is a bacterial form of meningitis caused by the gram-negative bacteria N. meningitidis. Meningitis is a serious disease, causing inflammation of the tissues surrounding the brain and the spinal cord which leads to long-term consequences and death if remain untreated (Stephens et al., 2007; Rouphael and Stephens, 2012). The disease primarily affects infants and teenagers and has a notable mortality rate among them (Rouphael and Stephens, 2012; Wang et al., 2019). The capsular polysaccharides (Ps) of the bacteria provide the basis for classifying its serogroups. Out of the 12 serogroups that have been identified so far, six (A, B, C, W, X, and Y) are mostly responsible for human disease (Meningitis (who.int), 2025; Pizza et al., 2020). Serogroup-specific vaccinations are given as preventive measures and also as part of immunization programs in several countries for adults and children (Parikh et al., 2020; Tapia et al., 2021). The World Health Organization (WHO) also recommends these vaccines as part of their immunization schedule for infants in the Sub-Saharan regions also known as the “meningitidis belt” of Africa (WHO, 2015; WHO, 2017; Agnememel et al., 2016). Thus, vaccination has been recommended as the best preventive measure to curb meningococcal infection (https://www.who.int/initiatives/defeating-meningitis-by-2030, n.d.; Borrow et al., 2017). Meningococcal polysaccharide (MnPs) vaccines derived from combinations of serogroups A, C, W, and Y are available commercially in monovalent, bivalent, and tetravalent forms (Vogel and Claus, 2011; Dretler et al., 2018). Conjugate polysaccharides vaccines (conjugation of meningococcal polysaccharides to a carrier protein) are preferred over the polysaccharide vaccine as they produce T cell dependent immune response which gives long lasting immunogenicity. Many of the available vaccine against Meningitis are unable to provide protection against serogroup X (https://www.who.int/initiatives/defeating-meningitis-by-2030, n.d.; Dretler et al., 2018). Vaccine against Serogroup B is challenging to develop due to its backbone of polysaccharides; which has strong resemblance to human sialic acids (Dretler et al., 2018).

Despite the advancement in the vaccine development against meningitis, the recurring epidemics of serogroups W, X, and C remain a worry in the Sub-Saharan regions. There have been recent reports of serogroup X outbreaks in Burkina Faso, Togo, Ghana, Kenya, Niger, and Uganda, which necessitates the development of vaccine against serogroup X (Gagneux et al., 2002; Nicolas et al., 2007; Mutonga et al., 2009; Chilukuri et al., 2014). Inclusion of serogroup X in vaccines against epidemic meningitis is highly encouraged by WHO, which has increased demand for the development of an effective conjugate vaccine with Men X serogroup (https://www.who.int/initiatives/defeating-meningitis-by-2030, n.d.; Micoli et al., 2013).

Conjugate vaccines contain carrier protein and thus have less antigenic content (i.e., capsular polysaccharide) compared to polysaccharide vaccines (Joshi et al., 2009). The accurate and precise qualitative and quantitative estimation of such low polysaccharide (the antigen content) in, conjugated vaccine is an arduous task for the vaccine manufacturing units. To verify that every batch is monospecific, the primary test on purified polysaccharides is the serological identification of MnPs (Vipond et al., 2017; Requirements for meningococcal polysaccharide vaccine (Requirements for Biological Substances No. 23), 1981). Also, the same manufacturing facility can be used to produce polysaccharides of different serogroups (Feavers, 2001; Guidelines for good clinical practices (GCP) for trials on pharmaceutical products, 1995), which makes it mandatory as per WHO guidelines to determine that the purified monovalent serogroup specific polysaccharides do not show the presence of any heterologous polysaccharides at >1 % level of dry weight of homologous polysaccharide (Requirements for meningococcal polysaccharide vaccine (Requirements for Biological Substances No. 23), 1981; Feavers, 2001; Guidelines for good clinical practices (GCP) for trials on pharmaceutical products, 1995). It is therefore obligatory to test the presence of heterologous polysaccharides as contaminants in serogroup specific purified polysaccharides.

The purified polysaccharides of N. meningitidis serogroups are identified, and characterized using chemical as well as immunological methods. The chemical methods mainly include estimation of total phosphorous and sialic acid content of the meningococcal polysaccharides, capillary zone electrophoresis (CZE), and Anion Exchange Chromatography with Pulsed Amperometric Detector (HPAEC-PAD) (Svennerholm, 1957; Cook et al., 2013; Gudlavalleti et al., 2014; Lamb et al., 2005). HPAEC-PAD is a precise and sensitive technique for capsular polysaccharide component determination where the method requires breakdown of polysaccharides to monosaccharides which are then separated, and quantified using a multistage linear gradient eluent profile in one column (Gudlavalleti et al., 2014). The immunological and molecular methods employed to characterize the capsular polysaccharides are, Enzyme Linked Immunosorbent Assay (ELISA) (Trad et al., 2011; Inzana and Champion, 2007), nephelometry, radioimmunoassay (RIA), rocket line immunoelectrophoresis (R-LIE), Polymerase Chain Reaction (PCR), Nuclear Magnetic Resonance (NMR), Near Infra-Red (NIR) (Lee, 1983; de Marie et al., 1984; Fabijanić et al., 2019; Danielsson and Olcén, 1979; Yu et al., 2008; Abeygunawardana et al., 2000). ELISA works on the specific antigen-antibody reaction, which is then measured calorimetrically. Rate nephelometry works on large aggregation (nephelos formation) of antigen and antibody, then scattering or reflecting of light towards the detector cell (Lee, 1983). Radio-immunoassay works on a competitive displacement reaction between radioactively labeled antigens and unlabeled samples for specific antibody sites (de Marie et al., 1984; Beuvery et al., 1984). NIR spectroscopy in combination with either soft independent modelling of class analogy (SIMCA) or partial least squares discriminant analysis relies on NIR spectral profiles of polysaccharide samples (Fabijanić et al., 2019). PCR and R-LIE, on the other hand identify the serogroups by the method of serotyping (Danielsson and Olcén, 1979; Yu et al., 2008). Since the majority of immunological techniques rely on a single antigen-antibody interaction, each of the aforementioned approaches has its own sets of limitations. Several studies support the multiplex immunoassays, which confer advantages over monoplex assays with increased accuracy, sensitivity, and repeatability ().

Bead Based Competitive Inhibition Assay (BBCIA) is a Luminex xMAP- based technology that allows simultaneous multiplexing of test samples against multiple antigens. It is a multiplex flow cytometric method that uses different color-coded beads. Each bead set is internally dyed with different ratios of two spectrally distinct fluorophores which are then coupled with a unique antigen, or polysaccharide. This differently color-coded luminex microspheres beads coupled with unique antigens allows the multiplexing of the assay. The fluorescent dye conjugated to detection antibodies generates fluorescence (Median Fluorescence Intensity i.e. MFI) when microspheres coincide with red and green lasers (Van Gageldonk et al., 2008; Rajam et al., 2018; MagPlex® Microspheres, 2025; AMG™ Universal Coupling Kit (AI-VMPAKMP-01.00), 2025; Caboré et al., 2016; Leyva et al., 2008).

In this work, BBCIA was used to identify and quantify the novel pentavalent meningococcal ACWYX conjugate vaccine. BBCIA was used to identify all five different polysaccharides (A, C, W, Y, and X) of N. meningitidis at different stages of vaccine manufacturing, i.e. purified polysaccharides, conjugated bulk, and final vaccine. BBCIA was used to quantify and detect the presence of any heterologous polysaccharides at a level of <1 % of dry weight of homologous polysaccharide (Requirements for meningococcal polysaccharide vaccine (Requirements for Biological Substances No. 23), 1981; Feavers, 2001; Guidelines for good clinical practices (GCP) for trials on pharmaceutical products, 1995; Svennerholm, 1957) which establish the serological specificity and identity of meningococcal polysaccharides. In this work, various validation parameters were applied, in accordance with ICH guidelines to assess the specificity, sensitivity and accuracy of the BBCIA (Requirements for meningococcal polysaccharide vaccine (Requirements for Biological Substances No. 23), 1981; Leyva et al., 2008; Labre et al., 2018). The method, supported by the solid matrix, helped with simultaneous measurement of multiple analytes from a single reaction, thus reducing time, cost, and sample volume. The method developed in this study, offers a vital and necessary tool that makes it possible to quickly identify polysaccharide serogroups, and quantify heterologous MnPs impurities during the production of pentavalent A, C, W, Y, and X meningococcal polysaccharide conjugate vaccines.