Accurate Quantification and Characterization of Adeno-Associated Viral Vectors
Accurate Quantification and Characterization of Adeno-Associated Viral Vectors
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DOI：
10.3389/fmicb.2019.01570
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发表时间：
2019-07-17
影响因子：
5.2
通讯作者：
Ravnikar, Maja
中科院分区：
文献类型：
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作者：
Dobnik, David;Kogovsek, Polona;Ravnikar, Maja
基因治疗病毒载体开发中的主要挑战之一是建立用于其大规模生产的优化工艺。这需要优化上游和下游工艺以及能够对病毒进行逐步分析表征的方法，其结果为产量、纯度和效价的生产迭代优化提供信息。这里最大的问题是过多的病毒载体制剂，其中许多干扰分析技术。我们以腺相关病毒（AAV）为例，并显示了结合使用分子方法和透射电子显微镜（TEM）的病毒载体的表征和定量的好处。分析结果表明，液滴数字PCR（ddPCR）在稳健性和测定方差方面优于定量实时PCR（qPCR），这与部分纯化（过程中）样品尤其相关。此外，我们证明了PCR分析前样品制备的重要性。我们通过TEM分析评价了病毒结构、聚集体和杂质的存在，发现这些影响了通过qPCR和ddPCR观察到的病毒滴度差异，并且可以通过样品制备进行改变。这些结果可作为建立分析方法的指南，这些方法是提供腺相关病毒载体鉴别和纯度测量所需的。
One of the main challenges in the gene therapy viral vector development is to establish an optimized process for its large scale production. This requires optimization for upstream and downstream processes as well as methods that enable the step-by step analytical characterization of the virus, the results of which inform the iterative refinement of production for yield, purity and potency. The biggest problem here is a plethora of viral vector formulations, many of which interfere with analytical techniques. We took adeno-associated virus (AAV) as an example and showed benefits of combined use of molecular methods and transmission electron microscopy (TEM) for viral vectors' characterization and quantification. Results of the analyses showed that droplet digital PCR (ddPCR) performs better than quantitative real-time PCR (qPCR), in terms of robustness and assay variance, and this was especially relevant for partially purified (in-process) samples. Moreover, we demonstrate the importance of sample preparation prior to PCR analysis. We evaluated viral structure, presence of aggregates and impurities with TEM analysis and found that these impacted the differences in viral titers observed by qPCR and ddPCR and could be altered by sample preparation. These results serve as a guide for the establishment of the analytical methods required to provide measures of identity and purity for AAV viral vectors.