Development of a Specific and Sensitive HPAEC-PAD Method for Quantification of Vi Polysaccharide Applicable to other Polysaccharides Containing Amino Uronic Acids
Development of a Specific and Sensitive HPAEC-PAD Method for Quantification of Vi Polysaccharide Applicable to other Polysaccharides Containing Amino Uronic Acids
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DOI：
10.1021/acs.analchem.9b05107
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发表时间：
2020-05-05
影响因子：
7.4
通讯作者：
Micoli, Francesca
中科院分区：
文献类型：
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作者：
Giannelli, Carlo;Raso, Maria Michelina;Micoli, Francesca
伤寒是发展中国家发病和死亡的主要原因。基于 Vi 荚膜多糖的疫苗已获得许可或正在开发中，用于治疗伤寒。 Vi 含量是疫苗释放的关键质量属性，用于监测疫苗的稳定性并确保适当的免疫反应。 Vi 多糖是 α-1,4-N-乙酰半乳糖氨基糖醛酸的均聚物，在 C-3 位进行 O-乙酰化，可抵抗单体定量前糖链解聚常用的酸水解。我们之前开发了一种基于强碱性水解，然后进行高性能阴离子交换色谱-脉冲电流检测分析的定量方法，但灵敏度较低，用于定量来自多糖解聚的未知产物。在这里，我们描述了一种基于酸水解并同时使用三氟乙酸和盐酸的 Vi 多糖定量方法的开发。使用实验设计方法来确定最佳水解条件。该方法的灵敏度比前一种方法高 100 倍，具体来说，形成了一种已知产物，通过单维和二维核磁共振波谱和质谱证实，该产物是脱 O 和脱 N 乙酰化的 Vi 单体。确定了准确度和精密度，并改进了色谱条件，从而缩短了分析时间。该方法将有助于基于 Vi 的疫苗的表征。此外，类似的方法有可能扩展到含有 2-氨基糖醛酸的其他多糖，正如此处已针对宋内志贺氏菌 O 抗原、肺炎链球菌血清型 12F 以及金黄色葡萄球菌 5 型和 8 型荚膜多糖进行验证的那样。
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 alpha-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.