Adsorption Of Plasmid DNA Research Articles

Understanding the adsorption of plasmid DNA and RNA molecules onto arginine-agarose chromatographic resin.

BackgroundThe production of nucleic acids (plasmid DNA or mRNA) in response to the development of new advanced vaccine platforms has greatly increased recently, mostly resulting from the pandemic situation. Due to the intended pharmaceutical use, nucleic acids preparations must fulfill all the required specifications in terms of purity and quality. Chromatography is a standard operation used to isolate these molecules from impurities, playing a central role in the manufacturing processes. However, the mechanism of nucleic acid adsorption in chromatographic resins is poorly understood, often leading to low adsorption capacities and a lack of specificity.Methods and resultsHere we investigated the adsorption of plasmid DNA and RNA molecules onto arginine-agarose, a resin with potential for large-scale application. Equilibrium batch studies were performed through pre-purified samples, using arginine-based ligands by varying the adsorption conditions in the pH value range from 6.0 to 9.0. Langmuir and Freundlich isotherm models were used to describe the adsorption equilibrium. The best fit for both nucleic acids was achieved using the Freundlich model. The correct choice of pH showed critical for controlling the efficacy of arginine-nucleic acid interaction, due to its influence on the nucleic acid structures. This type of analysis is necessary for the improvement of the selectivity and binding capacities of the resins used for plasmid DNA or mRNA purification.ConclusionsThe results presented here indicate that adsorption conditions can be tuned to enhance separation between pDNA and RNA, an important feature in the purification of nucleic acids for vaccine production.

Comparative assessment of poly (D,L-lactide-co-glycolide) nanoparticles modified by either cetyltrimethylammonium bromide or chitosan for plasmid DNA adsorption

Purpose: To evaluate poly (D,L-lactide-co-glycolide) PLGA nanoparticles modified by cetyltrimethyl ammonium bromide (CTAB) or chitosan for plasmid DNA adsorption.Methods: PLGA nanoparticles were prepared by solvent diffusion method and modified by including CTAB in the aqueous (F1) or oil phase (F2), or by including low (F3) or medium (F4) molecular weight chitosan. The nanoparticles were characterised by differential scanning calorimetry (DSC) and Fourier transform infrared spectroscopy (FTIR), as well as for cell toxicity, cell uptake and transfection.Results: CTAB failed to confer positive charge on the nanoparticles. CTAB desorbed easily from F1 surface. This resulted in negative zeta potential, increased cytotoxicity as well as decreased cell uptake and transfection. In F2, CTAB was located mainly in PLGA matrix, resulting in negative charge with decreased cytotoxicity, and increased cell uptake and transfection compared to F1. On the other hand, chitosan-modified nanoparticles (F3 and F4) showed stronger interaction between chitosan and PLGA, leading to positively-charged particles, decreased cytotoxicity, as well as increased cell uptake and transfection. Amongst the four formulations, F4 exhibited the highest transfection.Conclusion: These results should aid in understanding how PLGA nanoparticles are modified by CTAB and chitosan. Modification with chitosan yields PLGA nanoparticles with higher DNA adsorption and transfection with lower cytotoxicity.Keywords: Chitosan, cetyltrimethyl ammonium bromide (CTAB), Nanoparticle, Poly (D,L-lactide-coglycolide) PLGA, Plasmid DNA adsorption, Gene therapy

Detailed adsorption mechanism of plasmid DNA by newly isolated cellulose from waste flower spikes of Thypa latifolia using quantum chemical calculations

Current study was designed to use the newly obtained cellulose from waste flower spikes of Thypa latifolia plant for plasmid DNA adsorption. Cellulose was isolated according to a previously described method including acid and base treatment, and cellulose content was recorded as 17%. T. latifolia cellulose was physicochemically characterized via FT-IR, TGA and SEM techniques. Detailed mechanism of plasmid DNA adsorption by newly isolated cellulose was described using chemical quantum calculations. To check the effect of Cu++ immobilization on the affinity of cellulose for plasmid DNA, copper ions were immobilized onto T. latifolia cellulose. pUC18 plasmid DNA was used for adsorption studies. Membranes prepared with only T. latifolia cellulose and Cu++ immobilized T. latifolia cellulose revealed different adsorption ratios as 43.9 and 86.9% respectively. This newly isolated cellulose from waste flower spikes of T. latifolia can be utilized as a suitable carrier for plasmid DNA.

Single Molecule Catch and Release: Potential-Dependent Plasmid DNA Adsorption along Chemically Graded Electrode Surfaces.

Single molecule detection methods were employed to study the potential dependent adsorption and desorption of dye labeled plasmid DNA along chemical gradients prepared on indium tin oxide (ITO) electrodes. Gradients were formed over silica-base-layer-coated ITO surfaces by exposing them in a directional fashion to aminopropyltrimethoxysilane from the vapor phase. Sessile drop water contact angle measurements, spectroscopic ellipsometry, and X-ray photoelectron spectroscopy were used to verify that a gradient was formed and to characterize its wettability, thickness, and composition as a function of position. The gradient-coated ITO electrode served as both the working electrode and a window into the electrochemical cell used to manipulate DNA adsorption. For single molecule studies, the electrochemical cell was filled with buffer solution containing YOYO-1-labeled plasmid DNA. Fluorescence videos acquired along the gradients depicted clear position-, potential-, and pH-dependent variations in DNA adsorption and desorption. The results demonstrate that DNA adsorption was largely independent of applied potential and irreversible at high amine coverage (i.e., multilayers), under pH ∼ 6 buffer. DNA adsorption became more reversible as the amine coverage decreased and the solution pH increased. Potential dependent control over DNA adsorption and desorption was best achieved at monolayer-to-submonolayer aminosilane coverage under pH ∼ 8 buffer. The knowledge gained in these studies will aid in the development of electrochemical methods for the capture and release of DNA and other polyelectrolytes at electrode surfaces.

Soil properties affecting adsorption of plasmid DNA and its transformation efficiency in Escherichia coli

We examined the soil properties affecting adsorption of plasmid DNA and its transformation efficiency using competent Escherichia coli cells in sterile forested Andosols (n = 2), arable Andosols (n = 3), arable Cambisols (n = 2), and humic acid. Organic matter-removed soil samples were also prepared by subjecting each soil to ignition treatment. The amount of plasmid DNA adsorbed was influenced by individual soil properties rather than soil types and fertilizer management practices. Among the soil properties determined, organic matter content and pH appeared to have the greatest influence on the amount of DNA adsorbed onto soil samples. Adsorption of plasmid DNA greatly repressed the transformation efficiency of E. coli relative to free DNA. Decreased transformation efficiency was observed in samples showing high binding affinity for plasmid DNA, and the affinity appeared to be affected by exchangeable Ca, soil texture, and humic acid.

Adsorption behaviors of DNA by modified magnetic nanoparticles: Effect of spacer and salt

Abstract The isolation of nucleic acid is an essential operation before biochemical and diagnostic processes. Here the adsorption of plasmid DNA and salmon sperm DNA on amine magnetic nanoparticles coated with ligand acridine orange was investigated. Spacers such as 6-bromohexanoic acid, 4-bromobutyric acid, and 2-bromoacetic acid were used to facilitate connection between the magnetic nanoparticles and acridine orange. We carried out adsorption mechanism with a study of adsorption isotherm and adsorption kinetics to find out the strong evidence for the influence of electrostatic and intercalating forces which enhance the DNA separation efficiency. The modified magnetic nanoparticles (MNPs) were characterized using TEM, SQUID, Zeta potential and FTIR. The maximum adsorption capacity of acridine orange modified MNPs using the 6-bromohexanoic acid as the spacer for plasmid DNA was found to be 181.1 μg mg−1. The addition of 0.5 M CaCl2 in the solution could further enhance the adsorption capacity to 335.8 μg mg−1. The adsorption isotherms and kinetics fitted well to the Langmuir model and the pseudo-first-order model. The acridine orange modified MNPs exhibited very short equilibrium time (2 min) for the adsorption of plasmid DNA and salmon sperm DNA. The facile method could facilitate the development of simple and fast separation of nucleic acid.

Adsorption Behaviors of Nucleic Acid by Cationic Magnetic Nanoparticles

Cationic magnetic nanoparticles are prepared by covalently binding spermidine and polyethylenimine onto the surface of nanoparticles via a glutaraldehyde coupling method. Nanoparticles modified by spermidine or polyethylenimine were characterized using Fourier-transformed infrared spectra, transmission electron microscopy and scanning electron microscopy. In this study, cationic magnetic nanoparticles were prepared by covalently conjugating cationic ligands onto the surface of nanoparticles. The plasmid DNA adsorption by the surface-modified magnetic nanoparticles was analyzed by Freundlich, Temkin, and Langmuir models. The maximal adsorption capacities in Langmuir model for polyethylenimine- and spermidine- modified nanoparticles are 341 and 116 μg/mg, respectively. Overall, the results demonstrated that the polyethylenimine-modified magnetic nanoparticles has the potential for purification of plasmid.

Potential application of hydrogel-based strong anion-exchange membrane for plasmid DNA purification

The potential application of a hydrogel-based strong anion-exchange ( Q) membrane to purify plasmid DNAs was evaluated. The maximum binding capacity of plasmid DNA was estimated to be 12.4 mg/ml of membrane volume with a plasmid recovery yield of ∼90%. The effect of the inherent properties of plasmid DNA, membrane adsorbent, and the ionic environment on membrane performance was systematically investigated. Plasmid DNAs with smaller tertiary structure tended to have a better recovery than those with larger tertiary structure. Environmental Scanning Electron Microscopy (ESEM) revealed that the hydrogel structure is more porous on one side of membrane than the other. Membrane pre-treatment significantly improved pore distribution and increased membrane porosity resulting in a better adsorption, recovery, and higher flux. The selection of proper operating pH led to further improvement. The relative contribution of these factors to improve membrane chromatography of plasmid DNAs was analyzed using statistical modeling. It was found that the adsorption of plasmid DNA was mainly affected by the available adsorptive area associated with membrane porosity, whereas the recovery of plasmid DNAs was mainly affected by the environmental pH.

Modelling and simulation of plasmid DNA adsorption on ion‐exchange membrane columns

AbstractA transport model that considers convection, diffusion, and finite kinetic rate on the membrane column as well as the influence of extra column dispersion and lag times, was used in this study to mathematically describe the frontal adsorption behaviour of plasmid DNA in an ion‐exchange membrane chromatography system. The corresponding partial differential equations system was solved using the numerical method of lines (MOL) on a MATLAB platform. Experimental data from literature describing the frontal adsorption of pCI DNA in an ion‐exchange membrane column was used as a model system for validation of MOL solution of the transport model. This approach results in a unique way to predict frontal performance and it is a valuable tool to assess the use of membrane adsorbers in large scale processes for plasmid purification.Un modèle de transport qui considère la convection, la diffusion et la cinétique finie sur la colonne de membrane ainsi que l'influence de la dispersion supplémentaire de colonne et les délais de réponse, a été utilisé dans cette étude pour décrire mathématiquement le comportememt d'adsorption frontale de l'ADN plasmidique dans un système de chromatographie à membrane échangeuse d'ions. Le système correspondant d'équations différentielles partielles a été résolu en utilisant la méthode numérique des lignes sur une plate‐forme MATLAB. Des données expérimentales de la littérature décrivant l'adsorption frontale de l'ADN pCI dans une colonne à membrane échangeuse d'ions ont été utilisées comme système modèle pour la validation d'une solution MOL pour le système de transport. Cette approche aboutit à un moyen unique de prévoir la performance frontale et c'est un outil de valeur pour évaluer l'utilisation des adsorbants à membrane dans des procédés à grande échelle pour la purification des plasmides. © 2010 Canadian Society for Chemical Engineering

Adsorption of plasmid DNA on ceramic hydroxyapatite chromatographic materials

The adsorption of plasmid DNA onto two different types of ceramic hydroxyapatite beads with a particle diameter of 20 μm, namely Ceramic Hydroxyapatite Type II and the Type III, which is not commercially available, were investigated. Type II and the Type III have a pore diameter of 80 and 240 nm, respectively. Equilibrium and dynamic binding capacity for a 4.9 kbp model plasmid on Ceramic Hydroxyapatite Type II and Type III were enhanced by addition of NaCl to the adsorption buffer. This result indicates that the adsorption mechanism cannot be solely explained by electrostatic interaction. The affinities of plasmid DNA for Ceramic Hydroxyapatite Type II (with a K(D) of ≈0.005 mg/mL) and to Hydroxyapatite Type III (with a K(D) of ≈0.045 mg/mL) were not affected by NaCl, whereas the binding capacity was. This observation corroborates the assumption that a change of the shape of the plasmid molecule is affected and could be the reason for increased binding capacity with salt. The maximal binding capacity shows that at least a part of the CHT II bead must be accessible for the plasmid, whereas CHT III can be saturated with the plasmid. In both cases, an extremely hindered transport takes place.

Adsorption of plasmid DNA onto lipid/polymer particle assemblies

In this study, the adsorption of plasmid DNA onto preformed lipid/polymer particle assemblies was investigated. These assemblies, called LipoParticles, with a great potential in the biomedical field, were previously studied and thoroughly characterized. They consist of submicrometer anionic spherical polymer particles composed of either poly(styrene) or poly(lactic acid) which are coated with lipid layers formed by mixtures of zwitterionic 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) and cationic 1,2-dipalmitoyl-3-trimethylammonium-propane (DPTAP) in tunable proportions. The plasmid adsorption onto the LipoParticle surface was clearly established by the inversion of the surface charge of the assemblies, fluorescence microscopy observations, and agarose gel electrophoresis. From a quantitative standpoint, adsorption isotherms were found to be independent on the polymer core nature but rather strongly dependent on the adsorbed lipid shell composition. Furthermore, the data were fitted with the Langmuir model providing interesting information about the plasmid adsorption process such as the affinity constants between species, as well as the saturation levels of adsorbed plasmids onto assemblies. Finally, the compaction state of the adsorbed DNA was examined thanks to displacement experiments of a DNA-intercalated fluorescent dye, and found to be tightly related to the proportion of the cationic lipid in the adsorbed lipid layer. DPTAP-rich lipid formulations indeed displayed an important plasmid compaction that could be linked to a high affinity constant and a high amount adsorbed.

Adsorption of plasmid DNA on anion exchange chromatography media

Anion exchange chromatography (AEC) is a useful and effective tool for DNA purification, but due to average pore sizes between 40 and 100 nm most AEC resins lack truly useful binding capacities for plasmid DNA (pDNA). Equilibrium binding capacities and uptake kinetics of AEC media including conventional media (Source 30 Q, Q Sepharose HP), a polymer grafted medium (Fractogel EMD DEAE (M)), media with large pores (Celbeads DEAE, PL SAX 4000 A 30 microm) and a monolithic medium (CIM-DEAE) were investigated by batch uptake or shallow bed experiments at two salt concentrations. Theoretical and experimental binding capacities suggest that the shape of the pDNA molecule can be described by a rod with a length to diameter ratio of 20:1 and that the molecule binds in upright position. The arrangement of DNA like a brush at the surface can be considered as entropy driven, kind of self-assembly process which is inherent to highly and uniformly charged DNA molecules. The initial phase of adsorption is very fast and levels off, associated with a change in mass transfer mechanism. Feed concentrations higher than 0.1 mg/mL pDNA pronounce this effect. Monolithic media showed the fastest adsorption rate and highest binding capacity with 13 mg pDNA per mL.

Performance of R‐N(R′)‐R′′ functionalised poly(glycidyl methacrylate‐co‐ethylene glycol dimethacrylate) monolithic sorbent for plasmid DNA adsorption

High-throughput plasmid DNA (pDNA) manufacture is obstructed predominantly by the performance of conventional stationary phases. For this reason, the search for new materials for fast chromatographic separation of pDNA is ongoing. A poly(glycidyl methacrylate-co-ethylene glycol dimethacrylate) (GMA-EGDMA) monolithic material was synthesised via a thermal-free radical reaction, functionalised with different amino groups from urea, 2-chloro-N,N-diethylethylamine hydrochloride (DEAE-Cl) and ammonia in order to investigate their plasmid adsorption capacities. Physical characterisation of the monolithic polymer showed a macroporous polymer having a unimodal pore size distribution pivoted at 600 nm. Chromatographic characterisation of the functionalised polymers using pUC19 plasmid isolated from E. coli DH5alpha-pUC19 showed a maximum plasmid adsorption capacity of 18.73 mg pDNA/mL with a dissociation constant (KD) of 0.11 mg/mL for GMA-EGDMA/DEAE-Cl polymer. Studies on ligand leaching and degradation demonstrated the stability of GMA-EGDMA/DEAE-Cl after the functionalised polymers were contacted with 1.0 M NaOH, which is a model reagent for most 'cleaning in place' (CIP) systems. However, it is the economic advantage of an adsorbent material that makes it so attractive for commercial purification purposes. Economic evaluation of the performance of the functionalised polymers on the grounds of polymer cost (PC)/mg pDNA retained endorsed the suitability of GMA-EGDMA/DEAE-Cl polymer.

Breakthrough Performance of Plasmid DNA on Ion-Exchange Membrane Columns

Breakthrough performance of plasmid DNA adsorption on ion-exchange membrane columns was theoretically and experimentally investigated using batch and fixed-bed systems. System dispersion curves showed the absence of flow non-idealities in the experimental arrangement. Breakthrough curves (BTC) were significantly affected by inlet flow rate and solute concentration. In the theoretical analysis, a model was integrated by the serial coupling of the membrane transport model and the system dispersion model. A transport model that considers finite kinetic rate and column dispersed flow was used in the study. A simplex optimization routine, coupled to the solution of the partial differential model equations, was employed to estimate the maximum adsorption capacity constant, the equilibrium desorption constant, and the forward interaction rate constant, which are the parameters of the membrane transport model. The analysis shows that as inlet concentration or flow rate increases, the deviation of the model from the experimental behavior decreases. The BTCs displacement as inlet concentration increases was explained in terms of a greater degree of column saturation reached and more efficient operation accomplished. The degree of column saturation was not influenced by inlet flow rate. It was necessary to consider in the column model the slight variation in the BTC produced by the axial dispersion, in order to accomplish the experimental curve dispersion. Consequently, the design criteria that for Pe > 40 the column axial dispersion can be neglected should be taken with precaution.

Role of Divalent Cations in Plasmid DNA Adsorption to Natural Organic Matter-Coated Silica Surface

The adsorption kinetics of supercoiled and linear plasmid DNA onto a natural organic matter (NOM)-coated silica surface are acquired using a quartz crystal microbalance with dissipation monitoring (QCM-D) in the presence of common divalent electrolytes CaCl2 and MgCl2. The adsorption kinetics of both DNA are noticeably higher in the presence of CaCl2 compared to MgCl2. We hypothesize that specific bridging between the DNA phosphate groups and NOM carboxyl functional groups in the presence of Ca2+ cations may lead to more efficient attachmentthan in the presence of Mg2+ cations, which are only likely to allow for charge neutralization. The influence of background Na+ cations on the adsorption kinetics in the presence of CaCl2 is found to be insignificant, while the presence of Na+ leads to slower attachment kinetics in MgCl2. Rinsing the DNA layer adsorbed in the presence of CaCl2 with a solution of low NaCl concentration followed by deionized water does not result in observable detachment, indicating irreversibility of DNA adsorption. Instead, softening of the DNA layer adsorbed in the presence of CaCl2 with background Na+ occurs with the rinses due to the increase in electrostatic repulsion between the phosphate functional groups along the DNA backbone. In the case of the DNA layer adsorbed in the presence of CaCl2 without background Na+, softening of the layer does not occur with the rinses.

Adsorption of Plasmid DNA to a Natural Organic Matter-Coated Silica Surface: Kinetics, Conformation, and Reversibility

A quartz crystal microbalance with dissipation (QCM-D) has been used to determine the adsorption rate of ampicillin-resistant linear and supercoiled plasmid DNA onto a silica surface coated with natural organic matter (NOM). The structure of the resulting adsorbed DNA layer was determined by analyzing the viscoelastic properties of the adsorbed DNA layers as they formed and were then exposed to solutions of different ionic composition. The QCM-D data were complemented by dynamic light scattering measurements of diffusion coefficients of the DNA molecules as a function of solution ionic composition. The obtained results suggest that electrostatic interactions control the adsorption and structural changes of the adsorbed plasmid DNA on the NOM-coated silica surface. The adsorption of DNA molecules to the NOM layer took place at moderately high monovalent (sodium) electrolyte concentrations. A sharp decrease in solution ionic strength did not result in the release of the adsorbed DNA, indicating that DNA adsorption on the NOM-coated silica surface is irreversible under the studied solution conditions. However, the decrease in electrolyte concentration influenced the structure of the adsorbed layer, causing the adsorbed DNA to adopt a less compact conformation. The linear and supercoiled DNA had similar adsorption rates, but the linear DNA formed a thicker and less compact adsorbed layer than the supercoiled DNA.

Plasmid DNA Adsorption on Silica: Kinetics and Conformational Changes in Monovalent and Divalent Salts

A quartz crystal microbalance with dissipation (QCM-D) is used to determine the adsorption rate of a supercoiled plasmid DNA onto a quartz surface and the structure of the resulting adsorbed DNA layer. To better understand the DNA adsorption mechanisms and the adsorbed layer physicochemical properties, the QCM-D data are complemented by dynamic light scattering measurements of diffusion coefficients of the DNA molecules as a function of solution ionic composition. The data from simultaneous monitoring of variations in frequency and dissipation energy with the QCM-D suggest that the adsorbed DNA layer is more rigid in the presence of divalent (calcium) cations compared to monovalent (sodium) cations. Adsorption rates are significantly higher in the presence of calcium, attaining a transport-limited rate at about 1 mM Ca2+. Results further suggest that in low ionic strength solutions containing 1 mM Ca2+ and in moderately high ionic strength solutions containing 300 mM NaCl, plasmid DNA adsorption to negatively charged mineral surfaces is irreversible.

Preparation and Characterization of Thermosensitive Submicron Particles for Gene Delivery

In this study, a new thermosensitive material was proposed as a carrier for gene delivery. The thermosensitive submicron particles were synthesized by the dispersion copolymerization of N-isopropylacylamide (NIPA) with a relatively new, cationic comonomer, N-3-dimethylaminopropylmethacrylamide (DMAPM) with higher ionization ability with respect to the commonly used cationic comonomers. To achieve particle sizes smaller than 1 microm, suitable for gene delivery, the total monomer concentration in the dispersion copolymerization was kept at a sufficiently low level. The size of poly(NIPA-co-DMAPM) particles was determined as 454 nm, by AFM in dry state. The poly(NIPA-co-DMAPM) particles showed both temperature and pH sensitivity in the aqueous media. The plasmid DNA adsorption onto the thermosensitive cationic particles was investigated at different temperatures and pHs. The adsorbed amount of plasmid DNA onto the particles was significantly increased by the introduction of cationic comonomer. The equilibrium plasmid DNA adsorptions up to 13 mg/g dry particles were achieved at physiological pH. Approximately 36% w/w of adsorbed plasmid could be desorbed from the cationic nanolatex. The results of biocompatibility studies performed with mouse fibroblast cells showed the suitability of thermosensitive cationic particles for intended application.

LacO‐LacI interaction in affinity adsorption of plasmid DNA

Current approaches for purifying plasmids from bacterial production systems exploit the physiochemical properties of nucleic acids in non-specific capture systems. In this study, an affinity system for plasmid DNA (pDNA) purification has been developed utilizing the interaction between the lac operon (lacO) sequence contained in the pDNA and a 64mer synthetic peptide representing the DNA-binding domain of the lac repressor protein, LacI. Two plasmids were evaluated, the native pUC19 and pUC19 with dual lacO3/lacOs operators (pUC19(lacO3/lacOs)), where the lacOs operator is perfectly symmetrical. The DNA-protein affinity interaction was evaluated by surface plasmon resonance using a Biacore system. The affinity capture of DNA in a chromatography system was evaluated using LacI peptide that had been immobilized to Streamline adsorbent. The KD-values for double stranded DNA (dsDNA) fragments containing lacO1 and lacO3 and lacOS and lacO3 were 5.7 +/- 0.3 x 10(-11) M and 4.1 +/- 0.2 x 10(-11) M respectively, which compare favorably with literature reports of 5 x 10(-10)-1 x 10(-9) M for native lacO1 and 1-1.2 x 10(-10) M for lacO1 in a saline buffer. Densitometric analysis of the gel bands from the affinity chromatography run clearly showed a significant preference for capture of the supercoiled fraction from the feed pDNA sample. The results indicate the feasibility of the affinity approach for pDNA capture and purification using native protein-DNA interaction.

Chromatographic Approaches in the Purification of Plasmid DNA for Therapy and Vaccination

AbstractThe need for large scale plasmid DNA purification processes has increased in the last few years due to the rapid evolution of gene therapy and DNA vaccines. Chromatographic purification of plasmid DNA has specific problems which are mostly related to the structural nature of plasmid DNA, the resemblance of plasmid DNA to some impurities, and the lack of capacity and selectivity of the traditional bead adsorbents. In this paper, plasmid DNA purification using chromatographic processes is reviewed. Initially the most relevant structural and chemical properties of supercoiled plasmid DNA and related impurities are described, and the purification objectives are specified. The most significant properties are highlighted and their possible exploitation for plasmid DNA adsorption is described. After a brief description of currently used chromatographic purification strategies, novel approaches centered on the use and development of new stationary phases with higher capacity utilization are described.