Quantitative NMR for the structural analysis of novel bivalent glycoconjugates as vaccine candidates

Novel unimolecular bivalent glycoconjugates were assembled combining several functionalized capsular poly-saccharides of Streptococcus pneumoniae and Neisseria meningitidis to a carrier protein by using an effective strategy based on the Ugi 4-component reaction. The development of multivalent glycoconjugates opens new opportunities in the field of vaccine design, but their high structural complexity involves new analytical chal-lenges. Nuclear Magnetic Resonance has found wide applications in the characterization and impurity profiling of carbohydrate-based vaccines. Eight bivalent conjugates were studied by quantitative NMR analyzing the structural identity, the content of each capsular polysaccharide, the ratios between polysaccharides, the poly-saccharide to protein ratios and undesirable contaminants. The qNMR technique involves experiments with several modified parameters for obtaining spectra with quantifiable signals. In addition, the achieved NMR re-sults were combined with the results of colorimetric assay and Size Exclusion HPLC for assessing the protein content and free protein percentage, respectively. The application of quantitative NMR showed to be efficient to clear up the new structural complexities while allowing the quantitative assessment of the components.

Automated summary

Novel unimolecular bivalent glycoconjugates were developed and analyzed using quantitative NMR to address the structural complexity and composition of the vaccines, highlighting the efficiency of this technique in vaccine design.