Performance of a non-grafted monolithic support for purification of supercoiled plasmid DNA

Abstract

The use of therapeutics based on plasmid DNA (pDNA) relies on procedures that efficiently produce and purify the supercoiled (sc) plasmid isoform. Several chromatographic methods have been applied for the sc plasmid purification, but with most of them it is not possible to obtain the required purity degree and the majority of the supports used present low capacity to bind the plasmid molecules. However, the chromatographic monolithic supports are an interesting alternative to conventional supports due to their excellent mass transfer properties and their high binding capacity for pDNA. The separation of pDNA isoforms, using short non-grafted monolithic column with CarbonylDiImidazole (CDI) functional groups, is described in the current work. The effect of different flow rates on plasmid isoforms separation was also verified. Several breakthrough experiments were designed to study the effect of different parameters such as pDNA topology and concentration as well as flow rate on the monolithic support binding capacity. One of the most striking results is related to the specific recognition of the sc isoform by this CDI monolith, without flow rate dependence. Additionally, the binding capacity has been found to be significantly higher for sc plasmid, probably because of its compact structure, being also improved when using feedstock with increased plasmid concentrations and decreased linear velocity. In fact, this new monolithic support arises as a powerful instrument on the sc pDNA purification for further clinical applications.

Introduction

The requirements in medicine and molecular biology science, demand large quantities of highly pure and homogenous plasmid DNA (pDNA) for different applications such as cloning, large scale protein production, DNA vaccination or gene therapy [1]. The impact of plasmid structure and conformation on the transfection process efficacy has received some attention in the past. Thus, the supercoiled (sc) pDNA isoform is the desired topological form since it induces the most efficient access to the nucleus of the cell and consequently improves gene expression in eukaryotic cells [2]. According to international regulations, the product-quality is defined as a percentage of the sc isoform compared to the total pDNA, being that the purity of the sc pDNA must be 100% with the homogeneity degree higher than 97% of sc isoform from Escherichia coli (E. coli) host [3]. This foreseeable necessity led to amplified efforts within both research and industry to improve the effectiveness of sc pDNA production and purification methodologies [4].

For the purification of pDNA several chromatographic methods based on particulate supports have been reported. Besides conventional techniques such as anion exchange, hydrophobic interaction and size exclusion chromatography [5], other methods were tested with more or less success. The affinity concept has gained impact with the development of new supports combining different and more specific interactions with pDNA. Based on the natural occurrence of protein–DNA complexes in biological systems, and the atomic studies demonstrating the existence of favored interactions between particular amino acids and nucleic acid bases [6], [7], it was recently developed by our research group a new affinity chromatographic methodology using some amino acids immobilized on agarose matrix for the isolation and purification of several nucleic acids [8]. Particularly, histidine [9], arginine [4] and lysine [10] were already used as ligands to efficiently purify sc pDNA, and the recognition of this isoform proved the presence of specific interactions between pDNA molecule and the amino acid-based matrices studied. Despite these results obtained with the affinity matrices, some problems remained to be solved, such as the low capacity of available supports for pDNA and the low diffusivity of pDNA samples due to their high molecular weight. Considering these facts, it is necessary to study and develop more suitable supports to overcome these problems.

Monolithic sorbents represent one of the newest developments of chromatographic stationary phases for biomolecules separation and purification. A typical monolith is a continuous bed constituted by a single piece of highly porous solid material, whose pore size depends on the polymerization process [11], [12]. The ligands immobilized on these chromatographic supports can be several biological agents as antibodies, enzymes, lectins or amino acids [13] and peptides.

The chromatographic support named as Convective Interaction Media (CIM) was developed with the aim of obtaining a short chromatographic layer, ensuring well-defined, narrow pore-size distributions, excellent separation power and exceptional chemical stability [11]. These innovative CIM disks present numerous advantages comparing to conventional supports applied on sc pDNA purification, among which it is important to refer: high binding capacity due to excellent mass transfer properties and a huge quantity of accessible binding sites for large biomolecules as pDNA [14]; capacity to achieve very fast separation and purification with high reproducibility both at small and large scale [15]; simple handling, flow independent resolution [16]; the target molecule can be eluted in a concentrated form with a reduced biomolecule degradation due to short contact times with the chromatographic matrix [17].

This novel chromatographic material has been largely employed to separate immunoglobulins [18], oligonucleotides [19], proteins [19], [20] and recently to purify pDNA [14], [21], [22], [23]. Knowing that an agarose based matrix with immobilized histidine amino acid allows an efficient separation of pDNA isoforms [9], it becomes interesting to study a new non-grafted glycidyl methacrylate monolith (BIA Separations, Ljubljana, Slovenia) since this support is constituted by CarbonylDiImidazole (CDI) chemical groups. Pointing to the possibility of specific recognition mechanisms between imidazole ring and pDNA molecules, the applicability of CDI monolithic support to purify sc isoform seems to present great potential and is exploited in the present work. Additional chromatographic characterization based on breakthrough experiments was also designed to study the effect of parameters such as, pDNA topology and concentration, as well as the different flow-rate on the monolith dynamic binding capacity.