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Biotechnology and Applied Biochemistry (2001) 33, (91–98) (Printed in Great Britain)

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

Mei M. Ho*, Xavier Lemercinier†, Barbara Bolgiano*¹, Dennis Crane*† and Michael J. Corbel*

*Division of Bacteriology, National Institute for Biological Standards and Control, Blanche Lane, South Mimms, Potters Bar EN6 3QG, U.K., and †Laboratory for Molecular Structure, National Institute for Biological Standards and Control, Blanche Lane, South Mimms, Potters Bar EN6 3QG, U.K.

Abbreviations used: Hib, Haemophilus influenzae type b.

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.

Received 22 May 2000/11 September 2000; accepted 18 September 2000

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