Universal label-free in-process quantification of influenza virus-like particles
Universal label-free in-process quantification of influenza virus-like particles
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DOI：
10.1002/biot.201700031
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发表时间：
2017-08-01
影响因子：
4.7
通讯作者：
Carrondo, Manuel J. T.
中科院分区：
文献类型：
--
作者：
Carvalho, Sofia B.;Moleirinho, Mafalda G.;Carrondo, Manuel J. T.
病毒样颗粒（VLP）正在成为疫苗，特别是流感大流行的疫苗，增加了对VLP生产生物工艺开发的兴趣。然而，对于复杂的VLP，用于定量的分析工具还不能跟上生物过程的进展。目前，流感的定量依赖于传统方法：血凝试验或单向免疫扩散。这些分析技术耗时、繁琐，并且不支持高效的下游工艺开发和监测。在此，我们报告了一种无标记的工具，该工具使用生物层干涉法（BLI）技术应用于Octet平台上，以在生物过程的所有阶段定量流感病毒VLP。使用与链霉亲和素生物传感器相关的人（2，6-连接的唾液酸）和禽（2，3-连接的唾液酸）生物素化受体来定量几种单价和多价流感病毒VLP中的血凝素含量。所应用的方法能够定量从粗样品到最终生物加工VLP产品的血凝素。BLI技术证实了其作为高通量分析工具的价值，与传统方法相比具有高灵敏度和更高的检测限。这种简单快速的方法可以获得实时结果，这对于下游加工的在线监测、改进工艺开发、控制和优化至关重要。
Virus-like particles (VLPs) are becoming established as vaccines, in particular for influenza pandemics, increasing the interest in the development of VLPs manufacturing bioprocess. However, for complex VLPs, the analytical tools used for quantification are not yet able to keep up with the bioprocess progress. Currently, quantification for Influenza relies on traditional methods: hemagglutination assay or Single Radial Immunodiffusion. These analytical technologies are time-consuming, cumbersome, and not supportive of efficient downstream process development and monitoring. Hereby we report a label-free tool that uses Biolayer interferometry (BLI) technology applied on an Octet platform to quantify Influenza VLPs at all stages of bioprocess. Human (2,6-linked sialic acid) and avian (2,3-linked sialic acid) biotinylated receptors associated with streptavidin biosensors were used, to quantify hemagglutinin content in several mono- and multivalent Influenza VLPs. The applied method was able to quantify hemagglutinin from crude samples up to final bioprocessing VLP product. BLI technology confirmed its value as a high throughput analytical tool with high sensitivity and improved detection limits compared to traditional methods. This simple and fast method allowed for real-time results, which are crucial for in-line monitoring of downstream processing, improving process development, control and optimization.