Development of a Specific and Sensitive HPAEC-PAD Method for Quantification of Vi Polysaccharide Applicable to other Polysaccharides Containing Amino Uronic Acids.

Carlo Giannelli,Francesca Micoli,Antonia De Felice,Federico Pippi,Maria Michelina Raso,Elena Palmieri

doi:10.1021/acs.analchem.9b05107

Carlo Giannelli, Francesca Micoli + Show 4 more

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https://doi.org/10.1021/acs.analchem.9b05107

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Journal: Analytical Chemistry	Publication Date: Apr 24, 2020
Citations: 17	License type: cc-by

Affiliation: Toscana Life Sciences

Abstract

Typhoid fever is a major cause of morbidity and mortality in developing countries. Vaccines based on the Vi capsular polysaccharide are licensed or in development against typhoid fever. Vi content is a critical quality attribute for vaccines release, to monitor their stability and to ensure appropriate immune response. Vi polysaccharide is a homopolymer of α-1,4-N-acetylgalactosaminouronic acid, O-acetylated at the C-3 position, resistant to the commonly used acid hydrolysis for sugar chain depolymerization before monomer quantification. We previously developed a quantification method based on strong alkaline hydrolysis followed by High Performance Anion Exchange Chromatography-Pulsed Amperometric Detection analysis, but with low sensitivity and use for quantification of an unknown product coming from polysaccharide depolymerization. Here we describe the development of a method for Vi polysaccharide quantification based on acid hydrolysis with concomitant use of trifluoroacetic and hydrochloric acids. A Design of Experiment approach was used for the identification of the optimal hydrolysis conditions. The method is 100-fold more sensitive than the previous one, and specifically, resulting in the formation of a known product, confirmed to be the Vi monomer both de-O- and de-N-acetylated by mono- and bidimensional Nuclear Magnetic Resonance spectroscopy and mass spectrometry. Accuracy and precision were determined, and chromatographic conditions were improved to result in reduced time of analysis. This method will facilitate characterization of Vi-based vaccines. Furthermore, a similar approach has the potential to be extended to other polysaccharides containing 2-amino uronic acids, as already verified here for Shigella sonnei O-antigen, Streptococcus pneumoniae serotype 12F, and Staphylococcus aureus types 5 and 8 capsular polysaccharides.

Highlights

Typhoid fever is a major cause of morbidity and mortality in developing countries
We previously developed a quantification method based on strong alkaline hydrolysis followed by HPAEC-PAD analysis.[22]
HCl concentration, Trifluoroacetic acid (TFA) concentration, time of hydrolysis, and temperature were the factors evaluated in the range 5−8 M, 10−20% v/v, 2−5 h, and 80−110 °C, respectively

Summary

Introduction

Typhoid fever is a major cause of morbidity and mortality in developing countries. Vaccines based on the Vi capsular polysaccharide are licensed or in development against typhoid fever. Acetylated at the C-3 position, resistant to the commonly used acid hydrolysis for sugar chain depolymerization before monomer quantification. We previously developed a quantification method based on strong alkaline hydrolysis followed by High Performance Anion. We describe the development of a method for Vi polysaccharide quantification based on acid hydrolysis with concomitant use of trifluoroacetic and hydrochloric acids. Accuracy and precision were determined, and chromatographic conditions were improved to result in reduced time of analysis. This method will facilitate characterization of Vi-based vaccines. Antibiotic resistance has become a major problem and multidrug-resistant isolates, often associated with the dominant H58 haplotype, are prevalent in parts of Asia and Africa.[4,5]

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