Influenza viruses cause annual epidemics and occasional pandemics and thus represent a significant public health problem together with considerable economic consequences. As current influenza virus vaccines do not provide the best immunological protection, plasmid DNA vaccines have been seen as a potential alternative due to the stimulation of both B- and T-cell responses without the presence of any infectious agent and because they are easily produced, highly stable and safe. From this standpoint, several downstream methods have been proposed to obtain high quantities of pharmaceutical grade supercoiled plasmid DNA. This work describes a rapid process, based on ion exchange chromatography using an ethylenediamine (EDA) monolith for the purification of an experimental DNA influenza vaccine. The purification process allowed a significant reduction of the host contaminants, such as proteins, RNA and genomic DNA and the DNA vaccine recovery with a purity degree of 97.1% and a step yield of 47%. Finally, both transfection efficiency of the influenza vaccine and its cytotoxicity in two types of eukaryotic cell lines were tested in order to compare with the same plasmid DNA purified with a commercial kit. (C) 2015 Elsevier B.V. All rights reserved.