Xavier Lemercinier

Full 1H NMR assignment and detailed O-acetylation patterns of capsular polysaccharides from Neisseria meningitidis used in vaccine production

We report essentially complete 1H NMR assignments for the capsular polysaccharides from Neisseria meningitidis serotypes A, C, W-135, and Y. These polysaccharides are components of current polysaccharide vaccines against meningococcal infection and of the polysaccharide-protein conjugate vaccines under development. From these NMR data the pattern of O-acetylation was determined. O-Acetylation of the W-135 polysaccharide is reported for the first time. We also show that, for the Types C and W-135 polysaccharides a migration of O-acetyl groups occurs during storage in solution, and demonstrate that high field 1H NMR represents a simple and sensitive method to define the O-acetylation pattern of individual batches of these polysaccharides.

Use and validation of NMR assays for the identity and O-acetyl content of capsular polysaccharides from Neisseria meningitidis used in vaccine manufacture

We describe a validated NMR (nuclear magnetic resonance) spectroscopic assay for the identity of the capsular polysaccharides (CPSs) from Neisseria meningitidis Groups A, C, W135 and Y used in vaccine manufacture, and to determine the proportion of residues carrying an O-acetyl substituent. Proof of structural identity and quantitation of the O-acetyl content are key control parameters for these vaccines. The meningococcal CPSs have variable levels of O-acetylation, present at multiple sites in the repeat unit, leading to complex NMR spectra. Base-catalysed de-O-acetylation of the Groups A, C, W135 and Y CPSs yields simplified and reproducible spectra suitable for comparison with reference data. The degree of O-acetylation of the original CPS can be determined by integration of the acetate anion resonance and a suitable resonance from the saccharide moiety. The assay was validated using 46 independent samples from five manufacturers, and is shown to be robust and reproducible.

Structural analysis of the Lactobacillus rhamnosus strain KL37C exopolysaccharide.

The exopolysaccharide from the lactic acid bacterium Lactobacillus rhamnosus strain KL37C isolated from human intestinal flora was prepared by sonication of bacterial cell mass suspended in water followed by centrifugation and cold ethanol precipitation of the supernatant. The polysaccharide material was purified by gel permeation chromatography on an TSK HW-50 column and characterised using chemical and enzymatic methods. On the basis of sugar and methylation analysis and 1H, 13C, 1D and 2D NMR spectroscopy the exopolysaccharide was shown to be composed of the following pentasaccharide repeating unit:-->3)-alpha-D-Glcp-(1-->2)-beta-D-Galf-(1-->6)-alpha-D-Galp-(1-->6)-alpha-D-Glcp-(1-->3)-beta-D-Galf-(1-->

Physico-chemical and immunological examination of the thermal stability of tetanus toxoid conjugate vaccines

The thermal stability of meningococcal C (MenC)- and Haemophilus influenzae b (Hib)-tetanus toxoid (TT) conjugate vaccines was investigated using spectroscopic and chromatographic techniques and immunogenicity assays in animal models. In this stability study, both the bulk concentrate and final fills were incubated at -20, 4, 23, 37 or 55 degrees C for 5 weeks or subjected to cycles of freeze-thawing. The structural stability, hydrodynamic size and molecular integrity of the treated vaccines were monitored by circular dichroism (CD), fluorescence and nuclear magnetic resonance (NMR) spectroscopic techniques, size exclusion chromatography (FPLC-SEC), and high performance anion exchange chromatography coupled with pulsed amperometric detection (HPAEC-PAD). Only storage at 55 degrees C for 5 weeks caused some slight unfolding and modification in the tertiary structure of the carrier protein in the MenC-TT conjugate. Substantial loss of saccharide content from the MenC conjugates was observed at 37 and 55 degrees C. Unexpectedly, the experimental immunogenicity of MenC-TT vaccine adsorbed to Alhydrogel was significantly reduced only by repeated freeze-thawing, but not significantly decreased by thermal denaturation. Neither the molecular integrity nor the immunogenicity of the lyophilised Hib-TT vaccines was significantly affected by freeze-thawing or by storage at high temperature. In conclusion, the MenC- and Hib-TT conjugate vaccines were relatively stable when stored at higher temperatures, though when MenC-TT vaccine was adsorbed to Alhydrogel, it was more vulnerable to repeated freeze-thawing. When compared with CRM(197) conjugate vaccines studied previously using similar techniques, the tetanus toxoid conjugates were found to have higher relative thermal stability in that they retained immunogenicity following storage at elevated temperatures.

Full assignment of the proton and carbon NMR spectra and revised structure for the capsular polysaccharide from Streptococcus pneumoniae Type 17F

Full proton, 13C and 31P NMR assignments for the capsular polysaccharide from Streptococcus pneumoniae Type 17F are reported, and a revised structure differing in the anomeric configuration of the sidechain beta-Galp residue proposed. This polysaccharide is a component of the current 23-valent polysaccharide vaccine. The implications of this revised structure for published work are discussed.

Solution stability studies of the subunit components of meningococcal C oligosaccharide–CRM197 conjugate vaccines

Spectroscopic methods were used to detect modifications in the structures of CRM197, the mutant diphtheria toxin, and meningococcal C capsular oligosaccharide following their conjugation and incubation at various temperatures. Meningococcal C oligosaccharide–CRM197 conjugate vaccines obtained from two different manufacturers were incubated at −20, 4, 23, 37 or 55 °C for 5 weeks or subjected to ten cycles of freeze–thawing. The CRM197 carrier protein and the saccharide components of the treated vaccines were monitored by CD and NMR spectroscopic techniques. CD data indicated incubation temperature‐dependent conformational changes in the carrier protein from vaccine A. Modifications appeared in both secondary and tertiary structures of the conjugated CRM197 when incubated at 23 °C or above. This was characteristic of the ‘open’ conformation previously observed for this protein component. The NMR spectra also indicated modification of the structure of the conjugated CRM197 component of vaccine A when incubated at 23 °C or above, but failed to show any modification in the conjugated oligosaccharide. On the other hand, the structure of the oligosaccharide chains in vaccine B appeared to be degraded following incubation at 55 °C, even though the thermal effect on the conjugated CRM197 was less apparent. Repeated freeze–thawing did not affect the CD or NMR spectra. In conclusion, the two meningococcal C oligosaccharide–CRM197 conjugate vaccines were stable when stored at their recommended temperatures, but were differently affected by elevated temperatures. The conjugates differ in their conjugation chemistry, attachment positions, oligosaccharide chain length and loading, as well as recommended pH and storage buffer, and their different stability properties can probably be attributed to a combination of these factors.

Direct preparation of cyclodextrin monophosphates

Aqueous solutions of cyclodextrins and inorganic metaphosphate at pH 4, upon drying and subsequent warming, produce mixtures of isomeric monophosphate esters which are amenable to separation by anion-exchange chromatography. The products are characterised by enzymatic, mass, and NMR spectroscopic analysis. The methodology provides a route to these derivatives by a single reaction.