Volkin D B, Burke C J, Marfia K E, Oswald C B, Wolanski B, Middaugh C R
Department of Vaccine Pharmaceutical Research, Merck Research Laboratories, West Point, PA 19486, USA.
J Pharm Sci. 1997 Jun;86(6):666-73. doi: 10.1021/js960475h.
A variety of biophysical techniques have been employed to examine the size and conformational integrity of highly purified hepatitis A virus (HAV) in solution (purified HAV particles are subsequently formalin-inactivated and adsorbed to aluminum salts for use as the vaccine VAQTA). The size of HAV particles was assessed by a combination of electron microscopy, sedimentation velocity, and dynamic light scattering. The effect of ionic strength and temperature on the overall conformational stability of HAV was determined by a combination of intrinsic HAV protein fluorescence, fluorescent probes of both RNA and protein, and UV-visible spectroscopy. A major structural change in HAV occurs near 60 degrees C with the addition of 0.2 M magnesium chloride enhancing the thermal stability of HAV by approximately 10 degrees C. Salt concentrations above 0.2 M, however, decrease the solubility of HAV. The effect of pH on the physical properties of HAV particles was monitored by dynamic light scattering, analytical size exclusion HPLC, and interaction with fluorescent dyes. HAV particles undergo a substantially reversible association/aggregation at pH values below 6 with the concomitant exposure of previously buried hydrophobic surfaces below pH 4. These results are in good agreement with previous studies of HAV thermal stability under extreme conditions in which the irreversible inactivation of the viral particles was measured primarily by the loss of viral infectivity. The wide variety of biophysical measurements described in this work, however, directly monitor structural changes as they occur, thus providing a molecular basis with which to monitor HAV stability during purification and storage.
已采用多种生物物理技术来检测溶液中高度纯化的甲型肝炎病毒（HAV）的大小和构象完整性（纯化后的HAV颗粒随后用福尔马林灭活并吸附到铝盐上用作疫苗VAQTA）。通过电子显微镜、沉降速度和动态光散射相结合的方法评估HAV颗粒的大小。通过HAV蛋白固有荧光、RNA和蛋白的荧光探针以及紫外可见光谱相结合的方法，确定离子强度和温度对HAV整体构象稳定性的影响。在60摄氏度左右，HAV会发生重大结构变化，添加0.2 M氯化镁可使HAV的热稳定性提高约10摄氏度。然而，盐浓度高于0.2 M会降低HAV的溶解度。通过动态光散射、分析型尺寸排阻高效液相色谱以及与荧光染料的相互作用，监测pH对HAV颗粒物理性质的影响。在pH值低于6时，HAV颗粒会发生基本可逆的缔合/聚集，同时在pH值低于4时，先前埋藏的疏水表面会暴露出来。这些结果与之前在极端条件下对HAV热稳定性的研究结果高度一致，在这些研究中，病毒颗粒的不可逆失活主要通过病毒感染力的丧失来衡量。然而，本研究中描述的多种生物物理测量方法可直接监测结构变化的发生，从而为监测纯化和储存过程中HAV的稳定性提供分子基础。
