Turula Vincent E, Kim John, Michon Francis, Pankratz James, Zhang Yuwen, Yoo Chul
BioScience Division, Baxter Healthcare Corp., 12140 Indian Creek Ct., Beltsville, MD 20705, USA.
Anal Biochem. 2004 Apr 15;327(2):261-70. doi: 10.1016/j.ab.2004.02.001.
The development of an analytical procedure for the evaluation of a conjugate vaccine's structural wholeness or integrity is described. The principle component of the vaccine was the N-propionylated group B meningococcal polysaccharide (NPr-GBMP) covalently attached to a carrier protein. The goal of the procedure was to determine whether any whole polysaccharide, oligosaccharide, or monosaccharide, from minute to moderate levels, became detached off the conjugate. Free saccharide was isolated from the formulation, which included an aluminum hydroxide adjuvant for analysis. Due to its linkage, the NPr-GBMP did not release sialic acid efficiently with acid hydrolysis to the extent necessary for accurate quantitation. To accomplish depolymerization, the NPr-GBMP was subjected to methanolysis, 3N hydrochloric acid in methanol for 16h at 80 degrees C. The main product of the methanolysis reaction was a de-N-acylated methyl glycoside of sialic acid. N-acetylneuraminic acid oligomers and colominic acid were used to confirm the methanolysis depolymerization efficiency of the alpha(2 --> 8) saccharides; with the treatment all oligomers produced a common methyl glycoside. For this determination anion exchange chromatography and size exclusion chromatography were both interfaced to an integrated pulsed amperometric detector. Sensitivity and linearity were demonstrated to be sufficient for the application with vaccine dose formulations with low total saccharide concentrations.
本文描述了一种用于评估结合疫苗结构完整性的分析方法的开发。该疫苗的主要成分是共价连接到载体蛋白上的N-丙酰化B群脑膜炎球菌多糖（NPr-GBMP）。该方法的目的是确定是否有任何从微量到中等水平的完整多糖、寡糖或单糖从结合物上脱离。从制剂中分离出游离糖，制剂中包括用于分析的氢氧化铝佐剂。由于其连接方式，NPr-GBMP在酸水解时不能有效地释放出准确定量所需的唾液酸。为了实现解聚，将NPr-GBMP进行甲醇解，即在80℃下用甲醇中的3N盐酸处理16小时。甲醇解反应的主要产物是唾液酸的脱N-酰化甲基糖苷。使用N-乙酰神经氨酸寡聚物和结肠酸来确认α(2→8)糖类的甲醇解解聚效率；经过处理，所有寡聚物都产生了一种共同的甲基糖苷。为了进行这种测定，阴离子交换色谱和尺寸排阻色谱都与集成脉冲安培检测器相连。结果表明，对于总糖浓度较低的疫苗剂量制剂，该方法的灵敏度和线性足以适用。
