Screening of l‐histidine‐based ligands to modify monolithic supports and selectively purify the supercoiled plasmid DNA isoform

Abstract

The growing demand of pharmaceutical-grade plasmid DNA (pDNA) suitable for biotherapeutic applications fostered the development of new purification strategies. The surface plasmon resonance technique was employed for a fast binding screening of l-histidine and its derivatives, 1-benzyl-L-histidine and 1-methyl-L-histidine, as potential ligands for the biorecognition of three plasmids with different sizes (6.05, 8.70, and 14 kbp). The binding analysis was performed with different isoforms of each plasmid (supercoiled, open circular, and linear) separately. The results revealed that the overall affinity of plasmids to l-histidine and its derivatives was high (KD > 10(-8) M), and the highest affinity was found for human papillomavirus 16 E6/E7 (K(D) = 1.1 × 10(-10) M and KD = 3.34 × 10(-10) M for open circular and linear plasmid isoforms, respectively). L-Histidine and 1-benzyl-L-histidine were immobilized on monolithic matrices. Chromatographic studies of L-histidine and 1-benzyl-L-histidine monoliths were also performed with the aforementioned samples. In general, the supercoiled isoform had strong interactions with both supports. The separation of plasmid isoforms was achieved by decreasing the ammonium sulfate concentration in the eluent, in both supports, but a lower salt concentration was required in the 1-benzyl-L-histidine monolith because of stronger interactions promoted with pDNA. The efficiency of plasmid isoforms separation remained unchanged with flow rate variations. The binding capacity for pDNA achieved with the l-histidine monolith was 29-fold higher than that obtained with conventional L-histidine agarose. Overall, the combination of either L-histidine or its derivatives with monolithic supports can be a promising strategy to purify the supercoiled isoform from different plasmids with suitable purity degree for pharmaceutical applications.

Chromatographic HPV‐16 E6/E7 plasmid vaccine purification employing L‐histidine and 1‐benzyl‐L‐histidine affinity ligands

“…Due to ability of L‐histidine and 1‐benzyl‐L‐histidine‐modified monoliths to recognize and separate pDNA isoforms from a pre‐purified sample , a thorough study of RNA removal from pDNA in a lysate sample was conducted, bringing new insights about the interactions promoted between histidine‐based ligands and nucleic acids. Initially, the elution strategies previously implemented for L‐histidine‐modified and 1‐benzyl‐L‐histidine‐modified monoliths were tested with the lysate sample and repeated at least three times by using stepwise gradients based on decreasing (NH 4 ) 2 SO 4 concentration from 2.94 to 0 M and from 2 to 0.8 and to 0 M of (NH 4 ) 2 SO 4 in 10 mM Tris‐HCl‐EDTA, pH 8.0, respectively. However, the co‐elution of sc pDNA and RNA was observed under these experimental conditions (Fig.…”

“…Decarboxylated histidine (histamine ligand) has been successfully applied for pDNA isoforms separation using ion‐exchanging mechanism (ascending NaCl gradient) as well as HIC mechanism (descending ammonium sulfate gradient), proving its multimodality ). Moreover, a screening of the binding affinity of histidine‐based ligands (L‐histidine, 1‐benzyl‐L‐histidine and 1‐methyl‐L‐histidine) was recently performed by surface plasmon resonance biosensor for the biorecognition of three plasmids with different sizes, being achieved high affinity ( K D > 10 −8 M) of all ligands, especially with supercoiled pDNA isoform . The 1 H NMR spectra suggested that the imidazole ring of L‐histidine‐based ligands has an important role in interactions established with pDNA.…”

“…The 1 H NMR spectra suggested that the imidazole ring of L‐histidine‐based ligands has an important role in interactions established with pDNA. Following these results, L‐histidine and 1‐benzyl‐L‐histidine‐modified CIM® monoliths were used to separate pDNA isoforms (sc and open circular (oc)) from a pre‐purified sample with decreasing stepwise gradients of ammonium sulfate which mainly promoted hydrophobic interactions . Recently, a principle of sample displacement chromatography was proven to be an alternative approach for large‐scale pDNA purification, achieving higher productivities compared to classical bind‐elute chromatography .…”

HPV‐16 targeted DNA vaccine expression: The role of purification

“…In the past few years, our research group has been focused on the production and purification of the recombinant pDNA vaccine encoding E6 and E7 HPV16 oncoproteins . Thereby, the main purpose of this work was to conduct a differential in vitro comparison study to assess the expression of E6 and E7 proteins, resorting to the transfection of mammalian cells with pDNA samples obtained by different methods.…”

“…In the past few years, our research group has been focused on the production and purification of the recombinant pDNA vaccine encoding E6 and E7 HPV16 oncoproteins. [8][9][10] Thereby, the main purpose of this work was to conduct a differential in vitro comparison study to assess the expression of E6 and E7 proteins, resorting to the transfection of mammalian cells with pDNA samples obtained by different methods. For this, it was our interest to compare a pDNA with 100% sc isoform homogeneity obtained with arginine monolith with a native pDNA sample, containing oc and sc isoform, retrieved by a commercial anion-exchange purification kit.…”