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首页> 外文期刊>Bioprocess and Biosystems Engineering >Plasmid pVAX1-NH36 purification by membrane and bead perfusion chromatography
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Plasmid pVAX1-NH36 purification by membrane and bead perfusion chromatography

机译：膜和磁珠灌注色谱法纯化质粒pVAX1-NH36

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摘要

The demand for plasmid DNA (pDNA) has increased in response to the rapid advances in vaccines applications to prevent and treat infectious diseases caused by virus, bacteria or parasites, such as Leishmania species. The immunization protocols require large amounts of supercoiled plasmid DNA (sc-pDNA) challenging the development of efficient and profitable processes for capturing and purified pDNA molecules from large volumes of lysates. A typical bioprocess involves four steps: fermentation, primary recovery, intermediate recovery and final purification. Ion-exchange chromatography is one of the key operations in the purification schemes of pDNA owing the chemical structure of these macromolecules. The goal of this research was to compare the performance of the final purification step of pDNA using ion-exchange chromatography on columns packed with Mustang Q membranes or perfusive beads POROS 50 HQ. The experimental results showed that both matrixes could separate the plasmid pVAX1-NH36 (3936 bp) from impurities in clarified Escherichia coli lysates with an adequate resolution. In addition, a 24- and 21-fold global purification factor was obtained. An 88 and 63% plasmid recuperation was achieved with ion-exchange membranes and perfusion beads, respectively. A better understanding of perfusion-based matrices for the purification of pDNA was developed in this research.
机译：随着预防和治疗由病毒，细菌或寄生虫（如利什曼原虫属）引起的传染病的疫苗应用的迅速发展，对质粒DNA（pDNA）的需求已增加。免疫方案需要大量的超螺旋质粒DNA（sc-pDNA），挑战了从大量裂解物中捕获和纯化pDNA分子的有效而有利的过程。典型的生物过程包括四个步骤：发酵，初步回收，中间回收和最终纯化。由于这些大分子的化学结构，离子交换色谱是pDNA纯化方案中的关键操作之一。这项研究的目的是在装有Mustang Q膜或灌注性磁珠POROS 50 HQ的柱子上，使用离子交换色谱法比较pDNA最终纯化步骤的性能。实验结果表明，两种基质都能以足够的分辨率从澄清的大肠杆菌裂解物中的杂质中分离出质粒pVAX1-NH36（3936 bp）。另外，获得了24和21倍的全局纯化因子。用离子交换膜和灌注珠分别实现了88％和63％的质粒回收率。在这项研究中，人们对基于灌注的基质用于pDNA的纯化有了更好的了解。

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