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

Abstract

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.

The Polysialic Acid-specific O-Acetyltransferase OatC from Neisseria meningitidis Serogroup C Evolved Apart from Other Bacterial Sialate O-Acetyltransferases

“…For NmC-CPS containing only mono-O-acetylated residues, non-enzymatic migration of acetyl groups was observed. In freshly prepared CPS, O-acetylation was found predominantly at O-8, whereas after storage at room temperature, the majority of acetyl groups were shifted to O-7 (17,77,78). Therefore, OatC might be selective for O-8, and acetylation of O-7 might occur exclusively by non-enzymatic migration.…”

Carrier priming with CRM197 or diphtheria toxoid has a different impact on the immunogenicity of the respective glycoconjugates: Biophysical and immunochemical interpretation

“…Far UV spectra of CRM 197 showed intense signals in the region 200-240 nm related to the n → * and → * amide transitions with a shape indicative of a substantial contribution of ␣-helix and ␤-strand forms. The profile of CRM-MenA conjugates in the same region showed a shift of the cross-over toward lower wavelengths, a decreased intensity and a relative increase of the peak at 208 nm compared to that at 220 nm, which may be related to an increase of less folded structures [20]. The deviation from the native CRM 197 profile appears more pronounced for the conjugate with higher glycosylation degree (Fig.…”

“…2a). Conjugation caused a reduction of intensity likely indicating increased mobility of the aromatic side chains compatible with a more open conformation [20,21]. Far UV spectra of CRM 197 showed intense signals in the region 200-240 nm related to the n → * and → * amide transitions with a shape indicative of a substantial contribution of ␣-helix and ␤-strand forms.…”

Bacterial polysaccharides as major surface antigens: interest in O-acetyl substitutions

“…NMR assays are part of the arsenal of analytical methods in use for stability studies. 140 They were validated as potent tools to ensure proper control of the later parameter and respect of the pharmacopeial specifications, which require O-acetylation of more than half CPS repeats 15 in the case of MenC CPS. 115,141 In particular, 1 H NMR spectroscopy, which provides a fingerprint for each novel vaccine lot, is part of the established physicochemical technologies employed by vaccine manufacturers.…”