A multimodal histamine ligand for chromatographic purification of plasmid DNA

Abstract

To exploit different chromatographic modes for efficient plasmid DNA (pDNA) purification a novel monolithic chromatographic support bearing multimodal histamine (HISA) groups was developed and characterized. Electrostatic charge of HISA groups depends on the pH of the mobile phase, being neutral above pH 7 and becoming positively charged below. As a consequence, HISA groups exhibit predominantly ion-exchange character at low pH values, which decreases with titration of the HISA groups resulting in increased hydrophobicity. This feature enabled separation of supercoiled (sc) pDNA from other plasmid isoforms (and other process related impurities) by adjusting salt or pH gradient. The dynamic binding capacity (DBC) for a 5.1kbp large plasmid at pH 5 was 4.0mg/ml under low salt binding conditions, remaining relatively high (3.0mg/ml) even in the presence of 1.0M NaCl due to the multimodal nature of HISA ligand. Only slightly lower DBC (2.7mg/ml) was determined under preferentially hydrophobic conditions in 3.0M (NH4)2SO4, pH 7.4. Open circular and sc pDNA isoforms were baseline separated in descending (NH4)2SO4 gradient. Furthermore, an efficient plasmid DNA separation was possible both on analytical as well as on preparative scale by applying the descending pH gradient at a constant concentration (above 3.0M) of (NH4)2SO4.