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. 2023 Jun 30;13(1):10646.
doi: 10.1038/s41598-023-37832-4.

A universal UHPLC-CAD platform for the quantification of polysaccharide antigens

A Corrado  1 , M De Martino  1 , V Bordoni  1 , S Giannini  1 , F Rech  1 , S Cianetti  1 , F Berti  1 , C Magagnoli  1 , R De Ricco  2
Affiliations

A universal UHPLC-CAD platform for the quantification of polysaccharide antigens

A Corrado et al. Sci Rep. .

Abstract

Several glycoconjugate-based vaccines against bacterial infections have been developed and licensed for human use. Polysaccharide (PS) analysis and characterization is therefore critical to profile the composition of polysaccharide-based vaccines. For PS content quantification, the majority of Ultra High Performance Liquid Chromatography (UHPLC) methods rely on the detection of selective monosaccharides constituting the PS repeating unit, therefore requiring chemical cleavage and tailored development: only a few methods directly quantify the intact PS. The introduction of charged aerosol detector (CAD) technology has improved the response of polysaccharide analytes, offering greater sensitivity than other detector sources (e.g., ELSD). Herein, we report the development of a universal UHPLC-CAD method (UniQS) for the quantification and quality evaluation of polysaccharide antigens (e.g., Streptococcus Pneumoniae, Neisseria meningitidis and Staphylococcus aureus). This work laid the foundation for a universal UHPLC-CAD format that could play an important role in future vaccine research and development helping to reduce time, efforts, and costs.

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Conflict of interest statement

This work was sponsored by GlaxoSmithKline Biologicals SA. All the authors are employees of the GSK group of companies. F.B. is owner of patents on related topics.

Figures

Figure 1
Figure 1
Serotypes cluster subdivision examples: carboxylic acid highlighted in red (left), phosphodiesters highlighted in blue (right), and neutral (middle).
Figure 2
Figure 2
(A) Serotype 22F RP-CAD calibration curve linearity. The baseline rise observed between 22 and 25 min is also present in blank injections, and it is related to the change of mobile phase composition during the gradient elution. (B) Zoomed view (6 to 18 min) and calibration plot.
Figure 3
Figure 3
Comparison of Spn serotype 22F fermentation broth (step 1) with (C) and without events (B), acquired both with PDA (A) and CAD detectors (B and C). For details on instrumental flow state (to waste/to detector) setting, please refer to Materials and Method’s paragraph and Supplementary Fig. 4 for valves configuration).
Figure 4
Figure 4
RP-CAD UniQS-1 profiles of Spn serotype 2, 12F, 8, 9N, 33F, 22F and 3 PSRP-CAD.
Figure 5
Figure 5
(A) RP-CAD UniQS-1 profiles of Spn Serotypes 11A (Black) and 19A (Brown) from phosphodiester cluster; 33F (Blue) from neutral cluster. (B) Zoomed view (6.0 to 14 min).
Figure 6
Figure 6
(A) HILC-CAD UNiQS-2 of Spn Serotypes. (B) Zoomed view (4 to 10.50 min) and calibration plot.
Figure 7
Figure 7
Comparison of serotype 11A fermentation broth (step 1) with (C) and without events (B), acquired both with PDA (A) and CAD detectors (B and C). * = PS related impurity, not present in purified PS (e.g. Step 4) as reported in Fig. 6.
Figure 8
Figure 8
UniQS-2 profiles of Spn Serotypes, 19A, 20B, 17F, 15B, 11A, and 6A.
See this image and copyright information in PMC

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