Screening of L-histidine based ligands to purify the supercoiled plasmid DNA isoform

摘要

Summary form only given. Cervical cancer and high risk human papillomavirus (HPV) are highly associated and currently, the preventive vaccines only induce the antibody immunity. The urge to attenuate lesions and tumour progressions led to development of DNA vaccines, able to induce both humoural and cellular immune responses. Plasmid DNA (pDNA) is being widely explored as a non-viral vector due to its lack of toxicity, low cost manufacture and high expression levels. To produce an effective pDNA vaccine, a biotechnology platform is required and every single step has to be optimized in order to obtain this molecule with high purity degree, able to efficiently transfect eukaryotic cells with satisfactory expression of the target gene. Amino acid-affinity chromatography has been considered a promissory approach that selectively recognizes the biologically active pDNA topology, the supercoiled (sc) isoform. Thus, the aim of this work was to perform a screening of L-histidine-based amino acids employing the surface plasmon resonance (SPR) technique in order to modify a monolithic support with the suitable ligand for sc pDNA purification. Several experiments were performed with three plasmids of different sizes (6.05, 8.70 and 14 Kbp) and different isoforms (open circular, sc and linear), separately. The SPR results revealed that the overall affinity of plasmids to L-histidine-based ligands was high (KD >10 M) and the highest affinity was found for HPV-16 E6/E7. Therefore, L-histidine and 1-benzyl-L-histidine ligands were immobilized on epoxy monoliths and several chromatographic studies were accomplished with the aforementioned samples. In general, the sc isoform developed strong interactions with the modified supports and the separation of plasmid isoforms was achieved by decreasing the ammonium sulfate concentration. Lower salt content was required in the 1-benzyl-L-histidine monolith because of stronger interactions promoted with pDNA. The separation- of plasmid isoforms remained unchanged by flow rate variations. The breakthrough experiments of L-histidine monolith revealed satisfactory dynamic binding capacity when compared to other matrices [1]. Overall, purification methodologies can benefit from affinity analysis provided by SPR biosensor. The combination of L-histidine and 1-benzyl-L-histine ligands with monolithic supports can be a promising strategy to purify the sc pDNA with the desirable purity degree for pharmaceutical applications, such as DNA vaccines.