Purification of O-antigen for polysaccharide vaccine development against Salmonella paratyphi

Enteric fever caused by Salmonella enterica serovar Paratyphi remains a critical public health challenge due to rising incidence and increasing antimicrobial resistance. The development of effective polysaccharide conjugate vaccines targeting Salmonella Paratyphi requires high-purity O-antigen polysaccharide (PS) conjugated to immunogenic carrier proteins. This study optimized a robust and scalable purification-conjugation methodology, specifically tailored for O-antigen from Salmonella Paratyphi. Initial purification utilized ultrafiltration via tangential flow filtration (TFF), followed by sequential cation- and anion-exchange chromatography steps, significantly reducing protein, nucleic acid, and endotoxin contaminants. Optimized conditions included acid precipitation at pH 3.7, dilution prior to hydroxyapatite chromatography (CHT HA Type-I), and the use of a 10 kDa TFF membrane, enhancing antigen recovery and medium molecular weight species retention (>90%). Polysaccharide yield at 80 L scale was 180 mg/L with a recovery efficiency of 38%. Subsequent conjugation of the purified O-antigen with CRM197 was successfully achieved using controlled CDAP-mediated activation and monitored via gel permeation chromatography (GPC), confirming efficient high-molecular-weight conjugate formation. Analytical characterization consistently demonstrated impurity levels well within acceptable vaccine standards (<0.6% protein, <0.2% DNA), and reproducible molecular weight profiles suitable for immunogenic efficacy. Process scalability was validated at laboratory and pilot scales, confirming adaptability for industrial vaccine manufacturing.