Is any measurement method optimal for all aggregate sizes and types?
Is any measurement method optimal for all aggregate sizes and types?
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DOI：
10.1208/aapsj080365
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发表时间：
2006-01-01
期刊：
影响因子：
4.5
通讯作者：
Philo, John S.
中科院分区：
文献类型：
--
作者：
Philo, John S.
基于蛋白质的药物表现出广泛的聚集现象，这使得几乎不可能找到一种在所有情况下都能很好地工作的分析方法。聚集体的大小涵盖了从小低聚物到可见的“雪”和沉淀物的范围，通常只有较小的物种是可逆的。人们不太广泛地认识到聚集体也表现出广泛的寿命谱，并且寿命对检测方法有重要的影响。测量本身可能破坏或产生聚集体这一事实构成了重大的分析挑战，也是方法选择的关键决定因素。给出了一些有趣的聚集现象的例子和我们所使用的分析方法。在一个案例中，SEC在压力样品中看到的“聚集体”实际上是部分变性的单体，使用在线多角度激光散射的尺寸排除层析(SEC-MALLS)和沉积速度都表明。在第二种情况下，冻结/融化应力会产生瞬时的、亚稳定的低聚物，这些低聚物具有极高的粘性，很难通过SEC测量。以沉降速度为“金标准”，发展了一种改进的SEC方法，并用来研究这些低聚物随温度变化的解离过程。对于可见颗粒的问题，动态光散射在我们手中已经有效地检测到大的可见颗粒的前驱物质，并跟踪特定制造步骤的压力来源或损坏。
Protein-based pharmaceuticals exhibit a wide range of aggregation phenomena, making it virtually impossible to find any one analytical method that works well in all cases. Aggregate sizes cover a range from small oligomers to visible "snow" and precipitates, and generally only the smaller species are reversible. It is less widely recognized that aggregates also exhibit a broad spectrum of lifetimes, and the lifetime has important consequences for detection methods. The fact that the measurement itself may destroy or create aggregates poses a major analytical challenge and is a key determinant for method selection. Several examples of some interesting aggregation phenomena and the analytical approaches we have used are presented. In one case, an "aggregate" seen by SEC in stressed samples was shown to actually be a partially denatured monomer using both size-exclusion chromatography with online multiangle laser light scattering (SEC-MALLS) and sedimentation velocity. In a second case, freeze/thaw stress generates transient, metastable oligomers that are extremely sticky and difficult to measure by SEC. By using sedimentation velocity as the "gold standard" a much improved SEC method was developed and used to investigate the temperature-dependent dissociation of these oligomers. For problems with visible particulates, dynamic light scattering has been effective, in our hands, at detecting the precursors to the large, visible particles and tracking the source of stress or damage to particular manufacturing steps.