Effect of freezing rates and excipients on the infectivity of a live viral vaccine during lyophilization
Effect of freezing rates and excipients on the infectivity of a live viral vaccine during lyophilization
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DOI：
10.1021/bp034362x
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发表时间：
2004-07-01
影响因子：
2.9
通讯作者：
Slater, NKH
中科院分区：
文献类型：
--
作者：
Zhai, SL;Hansen, RK;Slater, NKH
冻干是提高不稳定生物药品稳定性的最常用方法，但在加工过程中，由于在冷冻和干燥阶段发生的压力，产品活性的很大一部分可能会损失。在不同浓度的冷冻保护剂赋形剂存在下，冷冻速率对单纯疱疹病毒2 （HSV-2）传染性恢复的影响被报道在这里。研究的冷冻条件为：货架冷却（223 K），淬火成泥态氮（SN2），再进入熔化的液氮（LN2）冷却的丙烷中。测量了相应的冻结速率，并利用扫描电子显微镜（SEM）测定了样品中形成的冰晶尺寸。病毒活性分析表明，在低（0.25% w/v蔗糖）赋形剂浓度下，氮冷却的病毒滴度回收率最高。在可选的冷却方法中，通过熔融丙烷冷却的样品中病毒滴度的损失始终是最高的。然而，这种损失可以通过在较低温度和较高真空条件下的冻干来最小化。我们认为，这是由于在升温到进行冷冻干燥的温度期间，通过融化丙烷冷却的样品的冰再结晶率较高，因为在升温期间，较小的冰晶容易扩大。在相同的冷冻条件下，含有较高浓度糖赋形剂的制剂可获得较高的病毒滴度回收率。原因被认为是双重的。首先，糖通过氢键与生物分子的极性残基结合来稳定膜和蛋白质，作为水的替代品。其次，浓缩糖溶液降低了病毒膜内水的成核温度，防止病毒和外部介质内形成大冰晶。
Lyophilization is the most popular method for achieving improved stability of labile biopharmaceuticals, but a significant fraction of product activity can be lost during processing due to stresses that occur in both the freezing and the drying stages. The effect of the freezing rate on the recovery of herpes simplex virus 2 (HSV-2) infectivity in the presence of varying concentrations of cryoprotectant excipients is reported here. The freezing conditions investigated were shelf cooling (223 K), quenching into slush nitrogen (SN2) and plunging into melting propane cooled in liquid nitrogen (LN2). The corresponding freezing rates were measured, and the ice crystal sizes formed within the samples were determined using scanning electron microscopy (SEM). The viral activity assay demonstrated the highest viral titer recovery for nitrogen cooling in the presence of low (0.25% w/v sucrose) excipient concentration. The loss of viral titer in the sample cooled by melting propane was consistently the highest among those results from the alternative cooling methods. However, this loss could be minimized by lyophilization at lower temperature and higher vacuum conditions. We suggest that this is due to a higher ratio of ice recrystallization for the sample cooled by melting propane during warming to the temperature at which freeze-drying was carried out, as smaller ice crystals readily enlarge during warming. Under the same freezing condition, a higher viral titer recovery was obtained with a formulation containing a higher concentration of sugar excipients. The reason was thought to be twofold. First, sugars stabilize membranes and proteins by hydrogen bonding to the polar residues of the biomolecules, working as a water substitute. Second, the concentrated sugar solution lowers the nucleation temperature of the water inside the virus membrane and prevents large ice crystal formation within both the virus and the external medium.