The current study explores the possibility of using a polyethyleneglycol(PEG)/ammonium sulphate aqueous two-phase system (ATPS) as an early step in a process for the purification of a model 6.1 kbp plasmid DNA (pDNA) vector. Neutralised alkaline lysates were fed directly to ATPS. Conditions were selected to direct pDNA towards the salt-rich bottom phase, so that this stream could be subsequently processed by hydrophobic interaction chromatography (HIC). Screening of the best conditions for ATPS extraction was performed using three PEG molecular weights (300, 400, 600) and varying the tie-line length, phase volume ratio and lysate load. For a 20 % (w/w) lysate load, the best results were obtained with PEG 600 using the shortest tie-line (38.16 % w/w). By further manipulating the system composition along this tie-line in order to obtain a top/bottom phase volume ratio of 9.3 (35 % w/w PEG 600, 6% w/w NH4)2SO4), it was possible to recover 100 % of pDNA in the bottom phase with a 3-fold increase in concentration. Further increase in the lysate load up to 40 %(w/w) with this system resulted in a 8-fold increase in pDNA concentration, but with a yield loss of 15 %. The ATPS extraction was integrated with HIC and the overall process compared with a previously defined process that uses sequential precipitations with isopropanol and ammonium sulphate prior to HIC. Although the final yield is lower in the ATPS-based process the purity grade of the final pDNA product is higher. This shows that it is possible to substitute the time-consuming two-step precipitation procedure by a simple ATPS extraction.
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机译：当前的研究探索了使用聚乙二醇（PEG）/硫酸铵水两相系统（ATPS）作为纯化模型6.1 kbp质粒DNA（pDNA）载体的早期步骤的可能性。将中和的碱性裂解物直接加入ATPS。选择条件将pDNA引向富盐的底部相，以便随后可以通过疏水相互作用色谱（HIC）处理该物流。使用三种分子量（300、400、600）的PEG并改变连接线长度，相体积比和裂解物负载量，筛选出ATPS提取的最佳条件。对于20％（w / w）的裂解物负载量，使用最短连接线（38.16％w / w）的PEG 600可获得最佳结果。通过沿该联系线进一步操纵系统组成以获得9.3的上/下相体积比（35％w / w PEG 600、6％w / w NH4）2SO4，可以回收100％底部相中pDNA的浓度增加了3倍。通过该系统，裂解物负载进一步增加至40％（w / w），导致pDNA浓度增加了8倍，但产量损失了15％。与先前定义的过程相比，ATPS萃取与HIC集成在一起，整个过程与之前定义的过程相比，之前使用HIC之前先后用异丙醇和硫酸铵进行连续沉淀。尽管在基于ATPS的方法中最终收率较低，但最终pDNA产品的纯度等级较高。这表明可以通过简单的ATPS提取代替费时的两步沉淀程序。
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