Purification of supercoiled pDNA using orthogonal HIC and AEX bind-and-elute steps Purifying plasmids for cell and gene therapy vectors requires chromatography systems where sequence size is not a limitation. This method describes a two-step orthogonal purification process that enables efficient pDNA capture from large volumes of lysate and high resolution of supercoiled plasmid DNA (sc pDNA), with consistent performance for both short and long sequences (for example, 34 kb). The capture step is performed with a hydrophobic interaction chromatography (HIC) adsorbent, pDNAHERO®, followed by polish with Q PuraBead® Edge anion exchange chromatography resin.
Methods
pDNA capture Details of the capture were as follows: HIC capture 1. Alkaline lysis process 2. Calcium chloride precipitation 3. Diafiltration into 50 mM TRIS, 10 mM EDTA, pH 7.5 (TE) 4. Adjust clarified lysate to 2.85 M ammonium sulphate in TE by dilution with 3 M ammonium sulphate in TE 5. Load onto pDNAHERO® Conditions for HIC bind-and-elute Equilibration buffer A1: 3 M ammonium sulphate, 50 mM TRIS, 10 mM EDTA, pH 7.5 Elution buffer B1: 50 mM TRIS, 10 mM EDTA, pH 7.5 CIP A2: RO water A3, 1 M NaOH
Issue date: 1 July 2025
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