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

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.