Development and characterization of an efficient, organic solvent-free capsular polysaccharide purification process...

Streptococcus pneumoniae serotype 22F is a significant concern in causing pneumococcal disease, and it is associated with increased rates of invasive pneumococcal disease (IPD) like septicaemia and meningitis. Capsular polysaccharide (CPS) of S. pneumoniae is the main causative agent for virulence, hence it is considered for vaccine development. The development of effective vaccine requires a high-purity CPS. Current purification methods utilize organic solvents, which raises the vaccine efficacy and safety concerns. In current study, we have established a novel, multi-step purification process for serotype 22F CPS that is entirely free of organic solvents. It integrates the treatment of fermentation harvest culture to deoxycholate (DOC), nuclease digestion, pH precipitation, and hydrophobic-interaction chromatography (HIC) using Octyl Sepharose to effectively remove major impurities viz. nucleic acids, proteins, and cell wall common polysaccharides (C-Ps). This optimized process yielded high capsular polysaccharide recovery of approximately 65 % with molecular weight 840 kDa. The final CPS structural identity was confirmed by ¹H NMR and it meets stringent purity criteria as defined by WHO, with protein levels at 0.4 %, nucleic acids below 0.1 %, and cell wall polysaccharide (C-Ps) at 2.29 mol%. This solvent-free method provides a safe and scalable platform for the industrial production of pneumococcal polysaccharide antigens.