Efficient purification and desalting of nucleic acids are essential unit operations in downstream processing (DSP). Conventional methods offer good solutions for either desalting (e.g., tangential flow filtration, dialysis and size exclusion) or purification (e.g., chromatography and precipitation), but integrating both into a single step on preparative scale has remained a big challenge. Here, we present a novel chromatographic method using a multimodal anion-exchanging (AEX) Convective Interaction Media (CIM) Swiper column that enables binding of nucleic acids at slightly acidic pH and elution at neutral pH with low salt concentration. Swiper desalting capability was first demonstrated for purified plasmid DNA (pDNA) samples from two standard pDNA DSP elution buffers: Tris-EDTA (TE), 0.9 M NaCl, pH 7.2 (AEX) and TE, 0.9 M ammonium sulphate (AS) and pH 7.2 (hydrophobic interaction chromatography). A scalable desalting process of enriched and partially purified pDNA samples was developed; with desalting capacity of >1 mg pDNA per mL of column and pDNA recovery of >90%, achieved with a simple TE, pH 7.2 elution buffer. Building from here, a single-step DSP combining purification and desalting of pDNA directly from clarified bacterial lysate was evaluated. Dynamic binding capacity (DBC) for pDNA from lysate using 4 mL CIM Swiper column was 1.0 mg/mL with the elution recovery of 90%. RNA, proteins and endotoxins in the pDNA elution fractions were below or close to the FDA-specified guidelines for pDNA purity, whereas host cell DNA and open circular (OC) pDNA were not efficiently depleted. Although the demonstrated process does not reach the specified product quality parameters, we pave the way to simplifying pDNA DSP flow by demonstrating an integration of two previously separate process steps at preparative scale, reducing footprint and costs.
Miklavčič et al. (2026) studied this question.