Sandro D'ascenzi

Size fractionation of bacterial capsular polysaccharides for their use in conjugate vaccines.

We have developed a chromatographic method suitable for the fractionation of polysaccharides having a negatively charged group. The method permits the removal of all those polysaccharide fragments having a short sequence and which are likely unsuitable for conjugate vaccine construction. The selected polysaccharide fragments can be used to produce glycoconjugate vaccines containing a restricted saccharide polydispersion. We have applied this chromatographic method to three different antigens, Haemophilus influenzae type b and Neisseria meningitidis group A and group C polysaccharides. The method is easily adapted for manufacturing purposes.

Development of a new method for the quantitative analysis of the extracellular polysaccharide of Neisseria meningitidis serogroup A by use of high-performance anion-exchange chromatography with pulsed-amperometric detection.

A new method for the quantitative determination of Neisseria meningitidis group A (MenA) capsular polysaccharide (CPS) has been developed. The method is based on trifluoracetic acid (TFA) hydrolysis of the CPS (2 M at 80 degrees C for 3 h), followed by chromatographic separation and quantification of the liberated mannosamine-6-phosphate from the area of the peak obtained using an IonPac AS11 column coupled to the sensitive pulsed amperometric detector ED40. The highly selective nature of this method circumvents the interference problems associated with the classical method based on a colorimetric assay for phosphorus. Provided that suitable hydrolysis conditions can be found, this chromatographic approach might be applicable to the quantification of other bacterial antigens containing phosphorylated sugars such as meningococcal groups H, L, X and Z, and pneumococcal serotypes 6, 10A and 19.

Isolation of oligosaccharides from a partial-acid hydrolysate of pneumococcal type 3 polysaccharide for use in conjugate vaccines

A series of well-defined oligosaccharide fragments of the capsular polysaccharide of Streptococcus pneumoniae type 3 has been generated. Partial-acid hydrolysis of the capsular polysaccharide, followed by fractionation of the oligosaccharide mixture by Sepharose Q ion-exchange chromatography yielded fragments containing one to seven [-->3)-beta-D-GlcpA-(1-->4)-beta-D-Glcp-(1-->] repeating units. The isolated fragments were analysed for purity by high-pH anion-exchange chromatography with pulsed amperometric detection (HPAEC-PAD) using an IonPac AS11 column, and their structures were verified by 1H NMR spectroscopy and nano-electrospray mass spectrometry. The oligosaccharides can be used to produce neoglycoprotein vaccines with a defined carbohydrate part.