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Plasmid purification by hydrophobic interaction chromatography using sodium citrate in the mobile phase

机译：通过疏水相互作用色谱法在流动相中使用柠檬酸钠纯化质粒

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The most significant impurities in plasmid DNA (pDNA)-containing solutions after intermediate recovery are host RNA and genomic DNA. Hydrophobic interaction chromatography (HIC) with ammonium sulfate-based buffers has been used to explore the higher hydrophobicity of those impurities when compared with pDNA. Although successful at lab scale, the large amounts of ammonium sulfate involved constitute an important limitation in terms of waste treatment/disposal if HIC is to be used at process scales. In this work, different HIC ligands (phenyl, butyl, octyl), salts and salt concentrations were screened with the specific goal of replacing ammonium sulfate with an environmentally friendly salt. The combination of phenyl-Sepharose with sodium citrate at high concentrations (≥1.2 M) not only allowed the separation of pDNA from RNA, gDNA, endotoxins and protein, but also enabled the separation of supercoiled from open circle isoforms. Alternatively, by exploring negative HIC with the phenyl matrix and 1.0 M sodium citrate, it was possible to purify total pDNA from solutions obtained by tangential flow filtration and pre-conditioned with sodium citrate. At the highest loading tested (2 mL feed equivalent to ca. 1 mg pDNA per mL of bed) despite high-level removal of endotoxins (>98.5%) and proteins (>82%) the purity of recovered plasmid DNA determined by HIC-HPLC was just 73% (primarily due to traces of RNA and gDNA). Much higher purities (close to 100%) were achieved when lower loadings were employed. In summary, this study clearly shows that despite lower purification factors achieved with sodium citrate compared to ammonium sulfate-based HIC, replacing the latter salt with sodium citrate is indeed feasible.
机译：中间回收后，含质粒DNA（pDNA）的溶液中最重要的杂质是宿主RNA和基因组DNA。与基于pDNA的疏水相互作用色谱（HIC）和基于硫酸铵的缓冲液已被用于研究那些杂质的更高疏水性。尽管在实验室规模上取得了成功，但如果要在过程规模上使用HIC，则所涉及的大量硫酸铵就废物处理/处置而言是一个重要的限制。在这项工作中，筛选了不同的HIC配体（苯基，丁基，辛基），盐和盐浓度，其特定目标是用环保盐替代硫酸铵。苯基-琼脂糖与高浓度（≥1.2 M）柠檬酸钠的组合不仅可以从RNA，gDNA，内毒素和蛋白质中分离出pDNA，而且还可以从开环同种型中分离出超螺旋。或者，通过用苯基基质和1.0 M柠檬酸钠研究阴性HIC，可以从通过切向流过滤和柠檬酸钠预处理的溶液中纯化总pDNA。尽管高水平去除了内毒素（> 98.5％）和蛋白质（> 82％），但在测试的最高负载下（2 mL进料相当于每mL床约1 mg pDNA），回收的质粒DNA的纯度由HIC- HPLC仅为73％（主要归因于痕量RNA和gDNA）。当采用较低的负载量时，可获得更高的纯度（接近100％）。总而言之，这项研究清楚地表明，尽管与基于硫酸铵的HIC相比，使用柠檬酸钠获得的纯化因子较低，但用柠檬酸钠代替后者的盐的确是可行的。

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