Cell integrity preservation reduces impurity release and enables high-purity capsular polysaccharide production in Streptococcus pneumoniae

Objectives Streptococcus pneumoniae is a major cause of pneumonia, meningitis, sepsis, and otitis media worldwide. Its capsular polysaccharide (CPS) is the principal virulence determinant and a key antigen in pneumococcal vaccines. Large-scale CPS manufacturing requires highly purified products with minimal impurities, particularly cell wall polysaccharides (CWPS). However, conventional sodium deoxycholate (DOC)-induced autolysis disrupts cellular integrity and releases substantial amounts of CWPS and other cellular contaminants, thereby increasing downstream purification complexity. Methods Here, we developed an integrated production strategy combining optimized fermentation with non-lytic inactivation to preserve cellular integrity and suppress impurity release at the source. Electron microscopy was used to evaluate cellular morphology following inactivation. Based on this upstream control, a simplified downstream purification process incorporating ultrafiltration, single-step calcium chloride-assisted acid precipitation, and diafiltration was established for CPS purification. Results The proposed strategy was evaluated across seven pneumococcal serotypes (1, 4, 5, 18C, 19A, 22F, and 33F). Non-lytic inactivation preserved cellular morphology and substantially reduced the release of cell-associated impurities. The resulting CPS preparations showed markedly reduced impurity levels, particularly CWPS, while retaining structural integrity and meeting the relevant specifications of the European Pharmacopoeia (11th Edition). Conclusions These findings demonstrate that controlling impurity generation at the source can substantially reduce the downstream purification burden in pneumococcal CPS manufacturing. The proposed mechanism-driven strategy provides a practical and potentially scalable framework for producing high-purity CPSs and supports the development of increasingly multivalent pneumococcal polysaccharide and conjugate vaccines.