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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.

Gellan gum is a polysaccharide polymer used in the pharmaceutical and food industries due to its gelling ability. In this work, it was intended to innovatively develop a new chromatographic matrix based on this polymer. This new chromatographic approach takes into account properties like the porosity and hydrophilicity of gellan gels as well as its anionic nature in order to study the interactions established with three model proteins. The versatility of interactions of this new matrix was also tested applying hydrophobic interaction chromatography conditions. Binding/elution studies with three model proteins were performed using gellan cross-linked with calcium. These studies established the elution patterns of the studied biomolecules, separating simple mixtures of proteins with different interaction profiles. Finally, it was made experiments with nucleic acids in hydrophobic conditions with the same purposes, proving the versatility of interactions in the gellan matrix. The structure of the formulation applied in the chromatographic steps was evaluated by confocal laser microscopy. This technique showed the microscopic organization of the gels correlating it with its macroscopic porosity and permeability. Due to the necessity of the presence of calcium in the chemical environment of the stationary phase it was studied the stability that 3 different ions (calcium, copper and zinc) conferred to the matrix. Of the three studied ions it was copper who gave the biggest stability to the matrix. These preliminary results show a new potential application of gellan gum as a chromatography matrix, taking advantage of its natural charge without the necessity of functionalizing it with specific ligands.