Binding and elution strategy for improved performance of arginine affinity chromatography in supercoiled plasmid DNA purification

New interesting strategies for plasmid DNA (pDNA) purification were designed, exploiting affinity interactions between amino acids and nucleic acids. The potential application of arginine-based chromatography to purify pDNA has been recently described in our work; however, to achieve higher efficiency and selectivity in arginine affinity chromatography, it is essential to characterize the behaviour of binding/elution of supercoiled (sc) isoforms. In this study, two different strategies based on increased sodium chloride (225-250 mm) or arginine (20-70 mm) stepwise gradients are described to purify sc isoforms. Thus, it was proved that well-defined binding/elution conditions are crucial to enhance the purification performance, resulting in an improvement of the final plasmids yields and transfection efficiency, as this could represent a significant impact on therapeutic applications of the purified sc isoform.     

Application of monoliths for plasmid DNA purification development and transfer to production

The demand of high-purity plasmid DNA (pDNA) for gene-therapy and genetic vaccination is still increasing. For the large scale production of pharmaceutical grade plasmids generic and economic purification processes are needed. Most of the current processes for pDNA production use at least one chromatography step, which always constitutes as the key-step in the purification sequence. Monolithic chromatographic supports are an alternative to conventional supports due to their excellent mass transfer properties and their high binding capacity for pDNA. Anion-exchange chromatography is the most popular chromatography method for plasmid separation, since polynucleotides are negatively charged independent of the buffer conditions. For the implementation of a monolith-based anion exchange step into a pDNA purification process detailed screening experiments were performed. These studies included supports, ligand-types and ligand-densities and optimization of resolution and productivity. For this purpose model plasmids with a size of 4.3 and 6.9 kilo base pairs (kbp) were used. It could be shown, that up-scaling to the production scale using 800 ml CIM Convective Interaction Media radial flow monoliths is possible under low pressure conditions. CIM DEAE was successfully implemented as intermediate step of the cGMP pDNA manufacturing process. Starting from 2001 fermentation aliquots pilot scale purification runs were performed in order to prove scale-up and to predict further up-scaling to 8 1 tube monolithic columns. The analytical results obtained from these runs confirmed suitability for pharmaceutical applications.     

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

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.     

Supercoiled plasmid DNA purification by integrating membrane technology with a monolithic chromatography

The present study describes the integration of membrane technology with monolithic chromatography to obtain plasmid DNA with high quality. Isolation and clarification of plasmid DNA lysate were first conducted by a microfiltration step, by using a hydrophilic nylon microfiltration membrane, avoiding the need of centrifugation. For the total elimination of the remaining impurities, a suitable purification step is required. Monolithic stationary phases have been successfully applied as an alternative to conventional supports. Thus, the sample recovered from the membrane process was applied into a nongrafted CarbonylDiImidazole disk. Throughout the global procedure, a reduced level of impurities such as proteins and RNA was obtained, and no genomic DNA was detectable in the plasmid DNA sample. The chromatographic process demonstrated an efficient performance on supercoiled plasmid DNA purity and recovery (100 and 84.44%, respectively). Thereby, combining the membrane technology to eliminate some impurities from lysate sample with an efficient chromatographic strategy to purify the supercoiled plasmid DNA arises as a powerful approach for industrial‐scale systems aiming at plasmid DNA purification.     

Supercoiled plasmid quality assessment by analytical arginine-affinity chromatography

Supercoiled plasmids are an important component of gene-based delivery vehicles, applied in new therapeutic strategies such as gene therapy or DNA vaccination. However, aiming at the general distribution of plasmid DNA (pDNA) therapeutics requires a procedure to easily and efficiently assess the purity and recovery yield of the supercoiled (sc) plasmid isoform. Based on affinity interactions between amino acids and nucleic acids, an arginine affinity methodology with UV detection was established to quantify and to control the quality of sc plasmid biopharmaceuticals. The fact that this new technique allows to distinguish between plasmid isoforms represents an advantage, since it allows the selective quantification of the biologically active pDNA topology, and a more accurate analysis of the quality of the isolated plasmid. The analytical experiments were performed in 12 min and the method was found to be accurate, precise, reproducible and linear for a sc plasmid concentration range between 2 and 150 μg/mL. In comparison with other established methods used in the quantification of native pDNA (oc+sc), the main advance introduced by this new method is the possibility to quantify the sc plasmid in a sample containing other plasmid topologies, ensuring the purity of plasmid products to be therapeutically applied.     

Dynamic binding capacity of plasmid DNA in histidine-agarose chromatography

The use of histidine-agarose chromatography in the purification of supercoiled (sc) plasmid DNA (pDNA) from Escherichia coli lysates has been reported recently. In the current work we describe a set of breakthrough experiments which were designed to study the effect of parameters such as flow-rate, temperature, concentration and conformation on the dynamic binding capacity of pDNA to the histidine support. One of the most striking results shows that the dynamic binding capacity for sc pDNA decreases linearly from 250.8 to 192.0 microg sc pDNA/mL when the temperature is varied from 5 to 24 degrees C. This behaviour was attributed to temperature-induced, pre-denaturation conformational changes which promote the removal of negative superhelical turns in sc pDNA molecules and decrease the interaction of DNA bases with the histidine ligands. The capacity for sc pDNA was highly improved when using feeds with higher pDNA concentrations, a phenomenon which was attributed to the fact that pDNA molecules in more concentrated solutions are significantly compressed. A maximum capacity of 530.0 microg pDNA/mL gel was obtained when using a 125 microg/mL pDNA feed at 1 mL/min and 5 degrees C, a figure which is comparable to the plasmid capacity values published for other chromatographic supports. Finally, a more than 2-fold increase in capacity was obtained when changing from open circular to sc pDNA solutions. Overall, the results obtained provide valuable information for the future development and implementation of histidine chromatography in the process scale purification of pDNA.     

Monolith-based immobilized metal affinity chromatography increases production efficiency for plasmid DNA purification

Immobilized metal affinity monolith column as a new class of chromatographic support is shown to be superior to conventional particle-based column as plasmid DNA (pDNA) purification platform. By harnessing the affinity of endotoxin to copper ions in the solution, a majority of endotoxin (90%) was removed from the alkaline cell lysate using CuCl(2)-induced precipitation. RNA and remaining endotoxin were subsequently removed to below detection limit with minimal loss of pDNA using either monolith or particle-based column. Monolith column has the additional advantage of feed concentration and flowrate-independent dynamic binding capacity for RNA molecules, enabling purification process to be conducted at high feed RNA concentration and flowrate. The use of monolith column gives three fold increased productivity of pDNA as compared to particle-based column, providing a more rapid and economical platform for pDNA purification.     

Characterisation of grafted weak anion-exchange methacrylate monoliths

A weak ion-exchange grafted methacrylate monolith was prepared by grafting a methacrylate monolith with glycidyl methacrylate and subsequently modifying the epoxy groups with diethylamine. The thickness of the grafted layer was determined by measuring permeability and found to be approximately 90nm. The effects of different buffer solutions on the pressure drop were examined and indicated the influence of pH on the permeability of the grafted monolith. Protein separation and binding capacity (BC) were found to be flow-unaffected up to a linear velocity of 280cm/h. A comparison of the BC for the non-grafted and grafted monolith was performed using beta-lactoglobulin, bovine serum albumin (BSA), thyroglobulin, and plasmid DNA (pDNA). It was found that the grafted monolith exhibited 2- to 3.5-fold higher capacities (as compared to non-grafted monoliths) in all cases reaching values of 105, 80, 71, and 17mg/ml, respectively. It was determined that the maximum pDNA capacity was reached using 0.1M NaCl in the loading buffer. Recovery was comparable and no degradation of the supercoiled pDNA form was detected. Protein z-factors were equal for the non-grafted and grafted monolith indicating that the same number of binding sites are available although elution from the grafted monolith occurred at higher ionic strengths. The grafted monolith exhibited lower efficiency than the non-grafted ones. However, the baseline separation of pDNA from RNA and other impurities was achieved from a real sample.     

Effect of chromatographic conditions and plasmid DNA size on the dynamic binding capacity of a monolithic support

DNA therapies are becoming recognized alternatives for the treatment and prevention of severe pathologies. Although most current trials have used plasmids <10 kbp, in the future larger plasmids would be required. The purpose of this work was to study the chromatographic behavior of nongrafted carbonyldiimidazole monolithic disks using plasmids with different sizes under hydrophobic conditions. Thereunto, the purification of several plasmids was performed. Higher size plasmids needed lower ammonium sulfate concentration, due to the greater number of interactions between the plasmids and monolith. The dynamic binding capacity experiments for the different plasmids revealed a lower capacity for bigger plasmids. It was also verified that the increase of salt concentration from 2.5 to 3 M of ammonium sulfate increased the capacity. At the highest salt concentration, a slight improvement in the capacity using lower flow rate was observed, possibly due to compaction of plasmid molecules and its better organization on the monolith channels. Finally, a low pH also had a positive effect on the capacity. So, this monolithic support proved to be appropriate to purify the supercoiled isoform of different plasmids with different sizes, providing a valuable instrument as a purification technique.     

Pressurized liquid extraction with in-cell clean-up followed by gas chromatography-tandem mass spectrometry for the selective determination of parabens and triclosan in indoor dust

This work studied the possibility of using polyethyleimine (PEI) as an affinity ligand for the purification of plasmid DNA (pDNA) from alkaline lysates using aqueous two-phase systems (ATPSs). The goal was to find conditions under which this cationic polymer could steer the partition of pDNA to the phase where less impurities accumulate. In poly(ethylene glycol) (PEG)/ammonium sulphate systems, neither free nor PEGylated PEI (pPEI) were able to change the partition of pDNA. This is probably due to the high salt concentration present in these systems that impair the interaction between pDNA and PEI. In PEG 3350/dextran 110 systems, the desired effect could be observed but 0.2-0.5M ammonium sulphate had to be added to prevent the co-partition of RNA to the same phase. These results were used to develop a methodology to obtain polyplexes from alkaline lysates in a two-step ATPSs extraction process. In the first step, a PEG 600/ammonium sulphate system is used to remove most impurities to the top phase. The pDNA-containing bottom phase is then isolated and contacted with a second PEG 3350/dextran 110 system supplemented with a small amount of pPEI (0.2%). Plasmid yield was 100% and the final preparation had no RNA and only small amounts of contaminant protein. Additionally, pDNA was obtained in the form of 53nm-sized polyplexes which are likely to suit specific gene delivery applications.     

Hydrophobic interaction membrane chromatography for plasmid DNA purification: Design and optimization

Chromatography is one of the key operations in the downstream processing of plasmid DNA (pDNA). However, the increased demand for highly purified pDNA experienced in recent years has made clear the need for alternative processes capable of retaining the advantages of conventional chromatography, such as selectivity, while providing increased throughput at a lower cost. The work presented in this article outlines the development and optimization of an alternative hydrophobic interaction membrane chromatography process for the purification of pDNA. The studies included the modification of functionalized membrane supports with a linear alkyl chain ligand and the testing of chromatographic performance of these membranes. Three modification procedures were tested and the membranes were screened for their capacity and selectivity. The modified membranes could separate the model plasmid pVAX1‐LacZ (6050 bp) from impurities in clarified Escherichia coli cell lysates (specifically RNA), with good resolution. Subsequent optimization of elution profiles with the best‐performing modified membrane, resulted in a high purification factor of 4.7, competitive with its bead process counterpart, and a plasmid yield of 73%.     

Chromatography of plasmid DNA

Liquid chromatography plays a central role in process-scale manufacturing of therapeutic plasmid DNA (pDNA) for gene therapy and DNA vaccination. Apart from its use as a preparative purification step, it is also very useful as an analytical tool to monitor and control pDNA quality during processing and in final formulations. This paper gives an overview of the use of pDNA chromatography. The specificity of pDNA purification and the consequent limitations to the performance of chromatography are described. Strategies currently used to overcome those limitations, as well as other possible solutions are presented. Applications of the different types of chromatography to the purification of therapeutic pDNA are reviewed, and the main advantages and disadvantages behind each technique highlighted.     

Current trends in separation of plasmid DNA vaccines: A review

Plasmid DNA (pDNA)-based vaccines offer more rapid avenues for development and production if compared to those of conventional virus-based vaccines. They do not rely on time- or labour-intensive cell culture processes and allow greater flexibility in shipping and storage. Stimulating antibodies and cell-mediated components of the immune system are considered as some of the major advantages associated with the use of pDNA vaccines. This review summarizes the current trends in the purification of pDNA vaccines for practical and analytical applications. Special attention is paid to chromatographic techniques aimed at reducing the steps of final purification, post primary isolation and intermediate recovery, in order to reduce the number of steps necessary to reach a purified end product from the crude plasmid.     

Why is less cationic lipid required to prepare lipoplexes from plasmid DNA than linear DNA in gene therapy?

The most important objective of the present study was to explain why cationic lipid (CL)-mediated delivery of plasmid DNA (pDNA) is better than that of linear DNA in gene therapy, a question that, until now, has remained unanswered. Herein for the first time we experimentally show that for different types of CLs, pDNA, in contrast to linear DNA, is compacted with a large amount of its counterions, yielding a lower effective negative charge. This feature has been confirmed through a number of physicochemical and biochemical investigations. This is significant for both in vitro and in vivo transfection studies. For an effective DNA transfection, the lower the amount of the CL, the lower is the cytotoxicity. The study also points out that it is absolutely necessary to consider both effective charge ratios between CL and pDNA and effective pDNA charges, which can be determined from physicochemical experiments.     

Immobilisation of a repressor protein for binding of plasmid DNA

The use of plasmid DNA in gene therapy and genetic vaccination has increased the need for scalable and sustainable production processes. One key challenge for bioprocess engineering is the separation of plasmid DNA from structurally related impurities. Affinity purification procedures allow a highly selective capturing of the target molecule. In this paper, we present the isolation of a his-tagged lac repressor, its non-covalent immobilisation to different matrices and binding of DNA, thus enabling us to screen for combinations of ligands and stationary phases by using a building block principle.     

Tuning the Topological Landscape of DNA–Cyclodextrin Nanocomplexes by Molecular Design

Original molecular vectors that ensure broad flexibility to tune the shape and surface properties of plasmid DNA (pDNA) condensates are reported herein. The prototypic design involves a cyclodextrin (CD) platform bearing a polycationic cluster at the primary face and a doubly linked aromatic module bridging two consecutive monosaccharide units at the secondary face that behaves as a topology-encoding element. Subtle differences at the molecular level then translate into disparate morphologies at the nanoscale, including rods, worms, toroids, globules, ellipsoids, and spheroids. In vitro evaluation of the transfection capabilities revealed marked selectivity differences as a function of nanocomplex morphology. Remarkably high transfection efficiencies were associated with ellipsoidal or spherical shapes with a lamellar internal arrangement of pDNA chains and CD bilayers. Computational studies support that the stability of such supramolecular edifices is directly related to the tendency of the molecular vector to form noncovalent dimers upon DNA templating. Because the stability of the dimers depends on the protonation state of the polycationic clusters, the coaggregates display pH responsiveness, which facilitates endosomal escape and timely DNA release, a key step in successful transfection. The results provide a versatile strategy for the construction of fully synthetic and perfectly monodisperse nonviral gene delivery systems uniquely suited for optimization schemes.     

Large-volume methacrylate monolith for plasmid purification. Process engineering approach to synthesis and application

The extent of exothermicity associated with the construction of large-volume methacrylate monolithic columns has somewhat obstructed the realisation of large-scale rapid biomolecule purification especially for plasmid-based products which have proven to herald future trends in biotechnology. A novel synthesis technique via a heat expulsion mechanism was employed to prepare a 40 mL methacrylate monolith with a homogeneous radial pore structure along its thickness. Radial temperature gradient was recorded to be only 1.8 degrees C. Maximum radial temperature recorded at the centre of the monolith was 62.3 degrees C, which was only 2.3 degrees C higher than the actual polymerisation temperature. Pore characterisation of the monolithic polymer showed unimodal pore size distributions at different radial positions with an identical modal pore size of 400 nm. Chromatographic characterisation of the polymer after functionalisation with amino groups displayed a persistent dynamic binding capacity of 15.5 mg of plasmid DNA/mL. The maximum pressure drop recorded was only 0.12 MPa at a flow rate of 10 mL/min. The polymer demonstrated rapid separation ability by fractionating Escherichia coli DH5alpha-pUC19 clarified lysate in only 3 min after loading. The plasmid sample collected after the fast purification process was tested to be a homogeneous supercoiled plasmid with DNA electrophoresis and restriction analysis.     

质粒DNA在灌注色谱基质上的吸附行为

Anion exchange chromatography is the most popular chromatographic method for plasmid separation.POROS R1 50 is a perfusio n chro mato graphic suppo rt w hich is a reversed phase matrix and is an alternative to co nventio nal o nes due to its mass transfer pro perties.The adso rptio n and elutio n o f the pIDKE2 plasmid o nto reversed phase POROS R1 50 w as studied.Langmuir iso therm mo del w as adjusted in o rder to get the max imum adso rptio n capacity and the disso ciatio n co nstant fo r POROS R1 50-plasmid DNA(pDNA) system.Breakthro ugh curves w ere o btained fo r vo lumetric flo w s betw een 0.69-3.33 mL/min,given dynamic capacity up to 2.3 times higher than tho se repo rted fo r io nic ex change matrix used during the purificatio n pro cess o f plasmids w ith similar size to that o f pIDKE2.The efficiency w as less than 45% fo r the flo w co nditio ns and initial co ncentratio n studied,w hich means that the suppo rt w ill no t be o perated under saturatio n circumstances.     

Adsorption Strategy of Plasmid DNA Nanoparticulate: Preparative Purification by a Simple Custom Expanded Bed Column

This paper summarizes the critical examination of the hydrodynamic performance of the NBG expanded bed contactor operated with streamline-DEAE adsorbent under various operating conditions for expanded bed adsorption of plasmid DNA nanoparticles from alkaline lysate. The purification process is not RNase-free. In this study, a rapid and efficient scaleable purification protocol obtaining, plasmid DNA nanoparticles (average size of 40 nm) with a high purity level for use as therapeutic agent in customized NBG expanded bed columns was developed. This technique allows efficient levels of binding to the column media and vector purification without centrifugation or filtration steps. Residence time distribution (RTD) studies were exploited to achieve the optimal condition of plasmid DNA nanoparticle (pDNA) recovery upon anion exchange adsorbent in this contactor. In addition, the purification experiments were carried out in the expanded bed columns with settle bed height of 6.0 ± 0.2 cm. NaCl gradient elution enabled the isolation of supercoiled plasmid from low-Mr RNA, cDNA and plasmid variants. Subsequently dynamic binding capacity of the adsorbent was calculated while these values decreased with increase in flow velocity. Moreover, the effect of pH upon the performance of this recovery process and the feedstock volume upon the expanded bed anion exchange purification was investigated. The results demonstrated that separation of low-Mr RNA from plasmid DNA isoforms in the range of pH between 5.5 and 7.5 is achievable in this column. The yield of recovery of pDNA in optimal condition was higher than 88.51% which was a superior result in one-pass frontal chromatography. The generic application of simple customized NBG expanded bed column and its potential for the purification and recovery of plasmid DNA as a nanoparticulate bioproduct is strongly discussed.