Simplified process for preparing native and depolymerized capsular polysaccharides of Streptococcus pneumoniae.

Abstract

Streptococcus pneumoniae is a major pathogen of bacterial pneumonia, meningitis, sepsis, and otitis media. The pathogenicity of this bacterium is largely attributed to its polysaccharide capsule, a protective layer around bacterial cell that enables bacteria to resist against host defense. Capsular polysaccharides (CPSs) of S. pneumoniae have been used as antigens to develop a variety of pneumococcal vaccines against invasive pneumococcal disease (IPD). These vaccines have been proven to be effective in reducing the incidence of IPD cases that are caused by vaccine-covered serotypes at the global scale. A crucial step in the manufacture of pneumococcal polysaccharide and conjugate vaccines is to purify native and depolymerized CPSs to meet strict quality standards in purity and structural integrity. The major impurities comprise proteins, nucleic acids and cell wall polysaccharides (CWPS). Traditionally, the removal of impurities to obtain purified native CPSs involves a complex process of purification, after which purified CPSs need to be further size-reduced to obtain depolymerized CPSs by multi-step approaches. In this study, we streamlined the process of CPS purification, which involves firstly ultrafiltration, followed by one-step acid precipitation, and finally diafiltration to obtain pure native CPSs. Furthermore, hydrolysis using trifluoroacetic acid (TFA) was integrated into the process to obtain purified depolymerized CPSs. The native and depolymerized CPSs produced by this optimized process were comparable to the materials obtained by the traditional approaches in purity and structural integrity, which would meet the quality standards of CPSs for vaccine production in the current edition of the European Pharmacopeia.