Figure 3.
Generation of a CPSXiv-based glycoconjugate. (a) To ensure matching of the oligosaccharide fraction generated from CPSXiv and CPSXn, 1H NMR spectra were recorded and showed perfect correspondence. (b) Schematic representation of CPSX hydrolysis and coupling of the resulting oligosaccharides to the protein carrier CRM197. The reducing end sugars are marked in red. (c) The efficacy of oligosaccharide coupling to CRM197 was controlled by HPLC size exclusion chromatography with detection of the conjugates via absorption at 214 nm. Similar to the benchmark (CPSXn-CRM197), CPSXiv-CRM197 elutes in two major peaks. In line with the lower avDP of CPSXiv oligosaccharides (avDP10, Pool 2), the second peak showed a mild shift to lower molecular masses. Importantly, no free CRM197 was detected. CPSX, NmX capsule polysaccharide; CPSXiv, in vitro produced CPSX; CPSXn, CPSX obtained from natural source; HPLC, high-performance liquid chromatography; p.p.m., parts per million.