The Streptococcus pneumoniae bacteria has over 100 known serotypes that display a continuous change in prevalence by patients’ age and geographical location and therefore necessitate continued efforts toward development of new vaccines with broader protection. Glycoconjugate vaccines have been instrumental in reducing global morbidity and mortality caused by Streptococcus pneumoniae infections. In these vaccines, the bacterial polysaccharide is conjugated to a carrier protein to enhance immunogenicity. To ensure well defined immunogenicity and stability of conjugated vaccines, reliable quantification of non-conjugated (free) polysaccharide is a critical, albeit challenging step during vaccine clinical dosing, release and stability monitoring. Multivalent preparations of Cross-reactive material 197 (CRM197)- conjugated pneumococcal polysaccharide materials often contain only nanogram levels of each individual free polysaccharide at final container concentrations. We have developed a novel method for the separation of free polysaccharides from conjugated material that requires no sample derivatization, employing instead an approach of quantitative immunoprecipitation of CRM197 with 3 different monoclonal antibodies and magnetic beads. A mix of antibodies against both linear and conformational epitopes enables successful removal of conjugates regardless of the protein folded state. The remaining free polysaccharide is subsequently measured in a serotype-specific ELISA.

肺炎链球菌有 100 多种已知血清型，其患病率随患者年龄和地理位置的变化而不断变化，因此需要继续努力开发具有更广泛保护作用的新疫苗。糖复合疫苗有助于降低肺炎链球菌感染引起的全球发病率和死亡率。在这些疫苗中，细菌多糖与载体蛋白缀合以增强免疫原性。为了确保结合疫苗明确的免疫原性和稳定性，在疫苗临床给药、释放和稳定性监测过程中，非结合（游离）多糖的可靠定量是一个关键但具有挑战性的步骤。交叉反应物质 197 (CRM197)-缀合肺炎球菌多糖材料的多价制剂通常在最终容器浓度下仅含有纳克级的每种游离多糖。我们开发了一种从缀合材料中分离游离多糖的新方法，无需样品衍生化，而是采用 CRM197 与 3 种不同的单克隆抗体和磁珠的定量免疫沉淀方法。无论蛋白质折叠状态如何，针对线性和构象表位的抗体混合物都能够成功去除缀合物。随后在血清型特异性 ELISA 中测量剩余的游离多糖。