Ho M M, Lemercinier X, Bolgiano B, Crane D, Corbel M J
Division of Bacteriology, National Institute for Biological Standards and Control, Blanche Lane, South Mimms, Potters Bar EN6 3QG, U.K.
Biotechnol Appl Biochem. 2001 Apr;33(2):91-8. doi: 10.1042/ba20000078.
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 degrees 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 CRM(197) when incubated at 23 degrees 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 degrees 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 degrees 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.
采用光谱法检测CRM197、突变型白喉毒素和脑膜炎球菌C荚膜寡糖在不同温度下共轭及孵育后结构的变化。将来自两家不同制造商的脑膜炎球菌C寡糖-CRM197共轭疫苗在-20、4、23、37或55摄氏度下孵育5周，或进行十次冻融循环。通过圆二色光谱（CD）和核磁共振光谱（NMR）技术监测处理后疫苗的CRM197载体蛋白和糖类成分。CD数据表明疫苗A的载体蛋白构象变化与孵育温度有关。当在23摄氏度或更高温度下孵育时，共轭CRM(197)的二级和三级结构均出现修饰。这是该蛋白质成分先前观察到的“开放”构象的特征。NMR光谱也表明，疫苗A的共轭CRM197成分在23摄氏度或更高温度下孵育时结构发生修饰，但未显示共轭寡糖有任何修饰。另一方面，疫苗B中的寡糖链在55摄氏度下孵育后结构似乎发生降解，尽管对共轭CRM197的热效应不太明显。反复冻融不影响CD或NMR光谱。总之，两种脑膜炎球菌C寡糖-CRM197共轭疫苗在推荐温度下储存时是稳定的，但在温度升高时受到的影响不同。共轭物在共轭化学、连接位置、寡糖链长度和负载量以及推荐的pH值和储存缓冲液方面存在差异，其不同的稳定性可能归因于这些因素的综合作用。
