Probing effects of pressure release on virus capture during virus filtration using confocal microscopy
Probing effects of pressure release on virus capture during virus filtration using confocal microscopy
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DOI：
10.1002/bit.25614
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发表时间：
2015-10-01
影响因子：
3.8
通讯作者：
Zydney, Andrew L.
中科院分区：
文献类型：
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作者：
Dishari, Shudipto K.;Venkiteshwaran, Adith;Zydney, Andrew L.
在生物治疗药物的生产中，病毒过滤被用来确保药物安全。最近的几项研究表明，由于过程中断，例如瞬时压力释放，病毒滞留显著减少。在这项工作中，发展了一种新的双标记荧光技术，利用共聚焦显微镜探测病毒滤膜中的病毒捕获。实验使用Ultipor((R))DV20、ViResolve((R))Pro和ViResolve((R))NFP膜，以噬菌体Phi X174为模型病毒。用两批荧光标记的噬菌体挑战过滤器：一批用红色染料(Cy5)标记，另一批用绿色染料(SYBR Gold)标记，以显示压力释放前后捕获的噬菌体。在共聚焦图像中看到的捕获图案是底层膜形态和孔结构的强烈函数。DV20和ViResolve((R))NFP显示先前捕获的噬菌体进一步迁移到过滤器中，这与观察到的压力释放后病毒滞留的损失一致。相比之下，捕获的病毒在ViResolve((R))Pro膜中没有迁移，而且这些过滤器也是唯一在压力释放后显示稳定病毒滞留的过滤器。使用双标记荧光技术的病毒捕获的直接可视化提供了对控制不同孔结构的病毒过滤器的保留特性的因素的独特见解。生物技术。比昂斯。2015；112：2115-2122。(C)2015年威利期刊公司。
Virus filtration is used to ensure drug safety in the production of biotherapeutics. Several recent studies have shown a dramatic decrease in virus retention as a result of a process disruption, e.g., a transient pressure release. In this work, a novel two-label fluorescence technique was developed to probe virus capture within virus filtration membranes using confocal microscopy. Experiments were performed with Ultipor((R)) DV20, Viresolve((R)) Pro, and Viresolve((R)) NFP membranes using bacteriophage phi x174 as a model virus. The filters were challenged with two batches of fluorescently labeled phage: one labeled with red dye (Cy5) and one with green dye (SYBR Gold) to visualize captured phage from before and after the pressure release. The capture patterns seen in the confocal images were a strong function of the underlying membrane morphology and pore structure. The DV20 and Viresolve((R)) NFP showed migration of previously captured phage further into the filter, consistent with the observed loss of virus retention after the pressure release. In contrast, there was no migration of captured virus in the Viresolve((R)) Pro membranes, and these filters were also the only ones to show stable virus retention after a pressure release. The direct visualization of virus capture using the two-label fluorescence technique provides unique insights into the factors controlling the retention characteristics of virus filters with different pore structure. Biotechnol. Bioeng. 2015;112: 2115-2122. (c) 2015 Wiley Periodicals, Inc.