Purification of Plasmid DNA by Multimodal Chromatography.

Multimodal (MM) chromatography can be described as a chromatographic method that uses more than one mode of interaction between the target molecule and the ligand to achieve a particular separation. Owing to its advantages over traditional chromatography, such as higher selectivity and capacity, its application for the purification of biomolecules with therapeutic interest has been widely studied. The potential of MM chromatography for the purification of plasmid DNA has been demonstrated. In this chapter, a downstream process for the purification of supercoiled plasmid DNA using MM chromatography with two different ligands-Capto™ adhere and PPA HyperCell™-is described. In both the cases, the purification process yields a high purity and highly homogeneous sc plasmid product.

Purification of supercoiled p53-encoding plasmid using an arginine-modified macroporous support.

p53 is a tumour suppressor gene that has been explored for cancer gene therapy as a possible alternative to the common treatments. The use of plasmid DNA (pDNA) to carry the therapeutic gene has been considered, but it is requisite to preserve its supercoiled (sc) structure, for eliciting a more effective gene expression and therapeutic action. The purification of the sc pDNA using amino acids-based affinity chromatography has been successfully applied, exploring different amino acids and supports. From these studies, it stood out the selectivity of arginine for the recognition of sc pDNA. However, some limitation on the binding capacity was found in the arginine-agarose support, and in the case of monoliths, some fouling and clogging can limit sequential runs. By using macroporous support modified with arginine it was expected to take advantage of the selectivity of the ligand combined with the flow properties and binding capacity offered by the support. The arginine-modified macroporous support was characterized by SEM, EDX and FTIR also to verify the correct immobilization of arginine, and then used for pDNA purification. The support showed to be effective on the sc p53-pDNA isolation, and the robustness was also achieved by accomplishing the purification of plasmids with different sizes, only by slightly adjusting the experimental conditions. Regarding the dynamic binding capacity of the arginine-modified macroporous support, it was achieved an improvement of more than 50% in the pDNA binding capacity when compared with their homologous arginine-agarose commercial matrix, suggesting potential economic feasibility in case of scale-up.

The use of size-exclusion chromatography in the isolation of supercoiled minicircle DNA from Escherichia coli lysate.

Minicircle DNA (mcDNA) is the new cutting-edge technology which researchers have been exploring for gene therapy and DNA vaccination. Although it presents enormous advantages in comparison to conventional plasmid DNA regarding bioactivity and safety, its challenging isolation from parental plasmid and miniplasmid has been setting back its launching in biomedical sciences. In this work, it is demonstrated the use of a simple size exclusion chromatographic method for the isolation of supercoiled mcDNA. Sephacryl S-1000 SF matrix was explored under different conditions (flow, peak fractionation volume and sample loading) to achieve the best performance and retrieve a mcDNA sample devoid of other bacterial contaminants or plasmid species resultant from the recombination process. This isolation methodology resulted in 66.7% of mcDNA recovery with 98.1% of purity. In addition, to show the robustness of the method, the potential of using this matrix for the isolation of a larger mcDNA was also evaluated. Upon adjusting the flow or the column volume, the larger mcDNA molecule was also successfully isolated. Overall, a simple and effective strategy has been established for the isolation of supercoiled mcDNA, underlining the potential of size exclusion chromatography in mcDNA separation.

Quality assessment of supercoiled minicircle DNA by cadaverine-modified analytical chromatographic monolith.

Minicircle DNA (mcDNA) is the ultimate non-viral DNA vector, presenting higher biosafety and therapeutic effect than conventional plasmid DNA (pDNA). However, given the similarity between mcDNA and its precursor, the parental plasmid (PP), analytical methodologies established for pDNA are unable to distinguish mcDNA from PP. Thus, a new need emerged for the implementation of suitable, rapid and non-expensive analytical methodologies for the characterization of mcDNA samples. Recently, our research group was able to develop a purification strategy for the isolation of supercoiled (sc) mcDNA resorting to cadaverine-modified monolith. Considering the promising results obtained with this strategy, a cadaverine-modified analytical monolith was prepared and explored for mcDNA quantification. Thus, a strategy of three-step increasing NaCl gradient was considered to first elute RNA/protein content, then isolate sc mcDNA and finally eliminate PP and other impurities still bounded to the matrix. A calibration curve was constructed with different sc mcDNA standards within a range of 1-25 µg/mL. Linearity, accuracy, precision and selectivity of this method were validated according to the international guidelines and the limit of detection and the lower limit of quantification were determined as 1 µg/mL. For the first time, to the best of our knowledge, an analytical method for mcDNA quantification is described. Besides ensuring the safety of mcDNA application by assessing the product purity, such methodology can be used in the future to control industrial mcDNA production and purification, perhaps aiding in the establishment of optimized and less expensive biotechnological operations.

Minicircle DNA purification: Performance of chromatographic monoliths bearing lysine and cadaverine ligands.

Minicircle DNA (mcDNA) technology is in the vanguard of vectors designed for gene therapy, since the absence of prokaryotic sequences confers to mcDNA higher biosafety in comparison to other DNA vectors. However, the presence of other isoforms and non-recombined parental molecules hampers the isolation of supercoiled (sc) mcDNA with the chromatographic methods already established for plasmid purification. In this work, two monolithic supports were modified with lysine and its decarboxylated derivative, cadaverine, to explore their performance in the sc mcDNA purification. Increasing NaCl gradients and different pH values (from 6 to 9) were tested in both modified monoliths. In general, cadaverine modified support established stronger interactions with mcDNA than lysine modified monolith, at acidic pH. For instance, at pH 6.0 the retention time for RNA and DNA molecules in lysine modified monolith was 11.58 and 14.59, respectively, while for cadaverine modified monolith was 20.32 and 27.12, respectively. The lysine modified monolith was able to successfully isolate sc mcDNA from the lysate sample. However, recovery yield was significantly sacrificed to guarantee high purity levels of sc mcDNA. The cadaverine modified monolith showed better selectivity than the previous monolith, achieving the successful sc mcDNA isolation from the lysate sample. The final sc mcDNA sample, obtained by the column that showed the best performance, was characterized by real-time PCR, presenting 98.4% purity and 78.6% recovery yield. The impurities content, namely genomic DNA, proteins and endotoxins, was found within the criteria established by regulatory agencies. Overall, a simple and practical chromatographic strategy to purify sc mcDNA was for the first time implemented by exploring a modified monolithic column, with no significant reduction on the purity and recovery and without resorting to backbone modification or specific enzymatic digestion. Such features will surely be crucial in the industrial scale-up of this chromatographic strategy since it will not be associated with significant cost-increase.

DoE to improve supercoiled p53-pDNA purification by O-phospho-l-tyrosine chromatography.

P53 is implicated in various cellular functions and several studies have shown that transfection of cancer cells with wild-type p53-expressing plasmids could directly drive cells into growth arrest and/or apoptosis. In the present work, the 6.07 kbp pcDNA3-FLAG-p53 plasmid, which encodes the p53 tumor suppressor, was produced and recovered from a recombinant cell culture of Escherichia coli DH5α. Following plasmid biosynthesis, the O-phospho-l-tyrosine chromatographic matrix was explored to purify the supercoiled p53-encoding plasmid. In order to quickly determine the optimal chromatographic performance and to obtain the required purity degree, maximizing the recovery yield of the supercoiled plasmid DNA, the Composite Central Face design was applied. The model revealed to be statistically significant (p-value < 0.05), with coefficient of determination of 0.9434 for the recovery yield and 0.9581 for purity and the central point was successfully validated. After the chromatographic process optimization by using the design of experiments tool, 49.7% of the supercoiled p53-encoding plasmid was recovered with 98.2% of purity, when a decreasing ammonium sulphate gradient was applied. The dynamic binding capacity of the O-phospho-l-tyrosine agarose column was 0.35 ±â€¯0.02 mg pDNA/mL matrix at 50% of the breakthrough. Finally, the purified sample was analysed to assess the content of endotoxins, proteins and genomic DNA, showing that all these impurity levels were below the recommendations of the regulatory agencies.

Single-molecule imaging of DNA gyrase activity in living Escherichia coli.

Bacterial DNA gyrase introduces negative supercoils into chromosomal DNA and relaxes positive supercoils introduced by replication and transiently by transcription. Removal of these positive supercoils is essential for replication fork progression and for the overall unlinking of the two duplex DNA strands, as well as for ongoing transcription. To address how gyrase copes with these topological challenges, we used high-speed single-molecule fluorescence imaging in live Escherichia coli cells. We demonstrate that at least 300 gyrase molecules are stably bound to the chromosome at any time, with ∼12 enzymes enriched near each replication fork. Trapping of reaction intermediates with ciprofloxacin revealed complexes undergoing catalysis. Dwell times of ∼2 s were observed for the dispersed gyrase molecules, which we propose maintain steady-state levels of negative supercoiling of the chromosome. In contrast, the dwell time of replisome-proximal molecules was ∼8 s, consistent with these catalyzing processive positive supercoil relaxation in front of the progressing replisome.

Recent advances in chromatographic purification of plasmid DNA for gene therapy and DNA vaccines: A review.

The wide spread of infectious diseases have provoked the scientists to develop new types of vaccines. Among the different types of vaccines, the recently discovered plasmid DNA vaccines, have gained tremendous attentions in the last few decades as a modern approach of vaccination. The scientific interest in plasmid DNA vaccines is attributed to their prominent efficacy as they trigger not only the cellular immune response but also the humoral immune responses. Moreover, pDNA vaccines are easily to be stored, shipped and produced. However, the purification of the pDNA vaccines is a crucial step in their production and administration, which is usually conducted by different chromatographic techniques. This review summarizes the most recent chromatographic purification methods provided in the literature during the last five years following our last review in 2013, including affinity chromatography, hydrophobic interaction chromatography, ion exchange chromatography, multimodal chromatography, sample displacement chromatography and miscellaneous chromatographic methods.

Arginine homopeptides for plasmid DNA purification using monolithic supports.

Purification of plasmid DNA targeting therapeutic applications still presents many challenges, namely on supports and specific ligand development. Monolithic supports have emerged as interesting approaches for purifying pDNA due to its excellent mass transfer properties and higher binding capacity values. Moreover, arginine ligands were already described to establish specific and preferential interactions with pDNA. Additionally, some studies revealed the ability of arginine based cationic peptides to condense plasmid DNA, which increased lengthening can result in strongest interactions with higher binding capacities for chromatographic purposes of large molecules such as pDNA. In this work, arginine homopeptides were immobilized in monolithic supports and their performance was evaluated and compared with a single arginine monolithic column regarding supercoiled (sc) plasmid DNA purification. Specific interactions of arginine based peptides with several nucleic acids present in a clarified Escherichia coli lysate sample showed potential for the sc pDNA purification. Effectively, the immobilization of the arginine homopeptides became more functional compared with the single arginine amino acid, showing higher binding capacities, which was also reflected in the intensity of the interactions. The combination of structural versatilities of monoliths with the specificity of arginine peptides raised as a promising strategy for sc pDNA purification.

Minicircle DNA purification using a CIM® DEAE-1 monolithic support.

Minicircle DNA is a new biotechnological product with beneficial therapeutic perspectives for gene therapy because it is constituted only by the eukaryotic transcription unit. These features improve minicircle DNA safety and increase its therapeutic effect. However, being a recently developed product, there is a need to establish efficient purification methodologies, enabling the recovery of the supercoiled minicircle DNA isoform. Thus, this work describes the minicircle DNA purification using an anion exchange monolithic support. The results show that with this column it is possible to achieve a good selectivity, which allows the isolation of the supercoiled minicircle DNA isoform from impurities. Overall, this study shows a promising approach to obtain the minicircle DNA sample with adequate quality for future therapeutic applications.

Size-based separation of supercoiled plasmid DNA using ultrafiltration.

HYPOTHESIS: Most previous studies of membrane-based separations have shown no effect of DNA size on plasmid transmission through small pore size ultrafiltration membranes, consistent with the predicted behavior for flexible polymer chains. However, supercoiled plasmids are known to have a highly "branched" structure with the number of branches dependent on the DNA length. This difference in branching could lead to a significant dependence of the transmission on the plasmid size, providing opportunities for size-based separations using ultrafiltration. EXPERIMENTS: Data were obtained with 3.0, 9.8, and 16.8 kbp plasmids using both cellulosic and polyethersulfone ultrafiltration membranes with different nominal molecular weight cutoffs. Initial experiments were performed with purified samples of the supercoiled and linear isoforms, with the results used to identify appropriate conditions for plasmid separation. FINDINGS: Plasmid transmission increased with increasing filtrate flux due to elongation of the plasmids in the converging flow field. However, the flux dependence was different for each plasmid due to differences in the extent of branching of the twisted supercoiled DNA. This behavior provided a significant selectivity that could be used to separate the 3.0 and 16.8 kbp supercoiled plasmids using small pore size ultrafiltration membranes.

L-tryptophan and dipeptide derivatives for supercoiled plasmid DNA purification.

The present study focus on the preparation of chromatography supports for affinity-based chromatography of supercoiled plasmid purification. Three l-tryptophan based supports are prepared through immobilization on epoxy-activated Sepharose and characterized by HR-MAS NMR. The SPR is employed for a fast screening of l-tryptophan derivatives, as potential ligands for the biorecognition of supercoiled isoform, as well as, to establish the suitable experimental conditions for the chromatography. The results reveal that the overall affinity is high (KD=10(-9) and 10(-8)M) and the conditions tested show that the use of HEPES 100mM enables the separation and purification of supercoiled at T=10°C. The STD-NMR is performed to accomplish the epitope mapping of the 5'-mononucleotides bound to l-tryptophan derivatives supports. The data shows that the interactions between the three supports and the 5'-mononucleotides are mainly hydrophobic and π-π stacking. The chromatography experiments are performed with l-tryptophan support and plasmids pVAX-LacZ and pPH600. The supercoiled isoform separation is achieved at T=10°C by decreasing the concentration of (NH4)2SO4 from 2.7 to 0M in HEPES for pVAX-LacZ and 2.65M to 0M in HEPES for pPH600. Overall, l-tryptophan derivatives can be a promising strategy to purify supercoiled for pharmaceutical applications.

A cell engineering strategy to enhance supercoiled plasmid DNA production for gene therapy.

With the recent revival of the promise of plasmid DNA vectors in gene therapy, a novel synthetic biology approach was used to enhance the quantity, (yield), and quality of the plasmid DNA. Quality was measured by percentage supercoiling and supercoiling density, as well as improving segregational stability in fermentation. We examined the hypothesis that adding a Strong Gyrase binding Site (SGS) would increase DNA gyrase-mediated plasmid supercoiling. SGS from three different replicons, (the Mu bacteriophage and two plasmids, pSC101 and pBR322) were inserted into the plasmid, pUC57. Different sizes of these variants were transformed into E. coli DH5α, and their supercoiling properties and segregational stability measured. A 36% increase in supercoiling density was found in pUC57-SGS, but only when SGS was derived from the Mu phage and was the larger sized version of this fragment. These results were also confirmed at fermentation scale. Total percentage supercoiled monomer was maintained to 85-90%. A twofold increase in plasmid yield was also observed for pUC57-SGS in comparison to pUC57. pUC57-SGS displayed greater segregational stability than pUC57-cer and pUC57, demonstrating a further potential advantage of the SGS site. These findings should augment the potential of plasmid DNA vectors in plasmid DNA manufacture. Biotechnol. Bioeng. 2016;113: 2064-2071. © 2016 The Authors. Biotechnology and Bioengineering Published by Wiley Periodicals, Inc.

Sample displacement chromatography of plasmid DNA isoforms.

Sample displacement chromatography (SDC) is a chromatographic technique that utilises different relative binding affinities of components in a sample mixture and has been widely studied in the context of peptide and protein purification. Here, we report a use of SDC to separate plasmid DNA (pDNA) isoforms under overloading conditions, where supercoiled (sc) isoform acts as a displacer of open circular (oc) or linear isoform. Since displacement is more efficient when mass transfer between stationary and mobile chromatographic phases is not limited by diffusion, we investigated convective interaction media (CIM) monoliths as stationary phases for pDNA isoform separation. CIM monoliths with different hydrophobicities and thus different binding affinities for pDNA (CIM C4 HLD, CIM-histamine and CIM-pyridine) were tested under hydrophobic interaction chromatography (HIC) conditions. SD efficiency for pDNA isoform separation was shown to be dependent on column selectivity for individual isoform, column efficiency and on ammonium sulfate (AS) concentration in loading buffer (binding strength). SD and negative mode elution often operate in parallel, therefore negative mode elution additionally influences the efficiency of the overall purification process. Optimisation of chromatographic conditions achieved 98% sc pDNA homogeneity and a dynamic binding capacity of over 1mg/mL at a relatively low concentration of AS. SDC was successfully implemented for the enrichment of sc pDNA for plasmid vectors of different sizes, and for separation of linear and and sc isoforms, independently of oc:sc isoform ratio, and flow-rate used. This study therefore identifies SDC as a promising new approach to large-scale pDNA purification, which is compatible with continuous, multicolumn chromatography systems, and could therefore be used to increase productivity of pDNA production in the future.

Screening of L-histidine-based ligands to modify monolithic supports and selectively purify the supercoiled plasmid DNA isoform.

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.

Specific recognition of supercoiled plasmid DNA by affinity chromatography using a synthetic aromatic ligand.

Liquid chromatography is the method of choice for the purification of plasmid DNA (pDNA), since it is simple, robust, versatile, and highly reproducible. The most important features of a chromatographic procedure are the use of suitable stationary phases and ligands. As conventional purification protocols are being replaced by more sophisticated and selective procedures, the focus changes toward designing and selecting ligands of high affinity and specificity. In fact, the chemical composition of the chromatographic supports determines the interactions established with the target molecules, allowing their preferential retention over the undesirable ones. Here it is described the selective recognition and purification of supercoiled pDNA by affinity chromatography, using an intercalative molecule (3,8-diamino-6-phenylphenanthridine) as ligand.

3,3'-Diamino-N-methyldipropylamine as a versatile affinity ligand.

Currently, in biomedicine and biotechnology fields, there is a growing need to develop and produce biomolecules with a high degree of purity. To accomplish this goal, new purification methods are being developed looking for higher performance, efficiency, selectivity, and cost-effectiveness. Affinity chromatography is considered one of the most highly selective methods for biomolecules purification. The purpose of this work is to explore a new type of a structurally simple ligand immobilized onto an agarose matrix to be used in affinity chromatography. The ligand in this study, 3,3'-diamino-N-methyldipropylamine has shown low toxicity and low cost of preparation. Moreover, the ability of the ligand to be used in affinity chromatography to purify proteins and nucleic acids was verified. An increasing sodium chloride gradient, using salt concentrations up to 500 mM, was suitable to accomplish the purification of these biomolecules, meaning that the new support allows the recovery of target biomolecules under mild conditions. Thus, the 3,3'-diamino-N-methyldipropylamine ligand is shown to be a useful and versatile tool in chromatographic experiments, with very good results either for proteins or supercoiled plasmid isoform purification.

Electrophoretic mobility of supercoiled, catenated and knotted DNA molecules.

We systematically varied conditions of two-dimensional (2D) agarose gel electrophoresis to optimize separation of DNA topoisomers that differ either by the extent of knotting, the extent of catenation or the extent of supercoiling. To this aim we compared electrophoretic behavior of three different families of DNA topoisomers: (i) supercoiled DNA molecules, where supercoiling covered the range extending from covalently closed relaxed up to naturally supercoiled DNA molecules; (ii) postreplicative catenanes with catenation number increasing from 1 to ∼15, where both catenated rings were nicked; (iii) knotted but nicked DNA molecules with a naturally arising spectrum of knots. For better comparison, we studied topoisomer families where each member had the same total molecular mass. For knotted and supercoiled molecules, we analyzed dimeric plasmids whereas catenanes were composed of monomeric forms of the same plasmid. We observed that catenated, knotted and supercoiled families of topoisomers showed different reactions to changes of agarose concentration and voltage during electrophoresis. These differences permitted us to optimize conditions for their separation and shed light on physical characteristics of these different types of DNA topoisomers during electrophoresis.

Optimization of supercoiled HPV-16 E6/E7 plasmid DNA purification with arginine monolith using design of experiments.

The progress of DNA vaccines is dependent on the development of suitable chromatographic procedures to successfully purify genetic vectors, such as plasmid DNA. Human Papillomavirus is associated with the development of tumours due to the oncogenic power of E6 and E7 proteins, produced by this virus. The supercoiled HPV-16 E6/E7 plasmid-based vaccine was recently purified with the arginine monolith, with 100% of purity, but only 39% of recovery was achieved. Therefore, the present study describes the application of experimental design tools, a newly explored methodology in preparative chromatography, in order to improve the supercoiled plasmid DNA recovery with the arginine monolith, maintaining the high purity degree. In addition, the importance and influence of pH in the pDNA retention to the arginine ligand was also demonstrated. The Composite Central Face design was validated and the recovery of the target molecule was successfully improved from 39% to 83.5%, with an outstanding increase of more than double, while maintaining 100% of purity.

Histamine monolith versatility to purify supercoiled plasmid deoxyribonucleic acid from Escherichia coli lysate.

Preparation of high quantities of supercoiled plasmid DNA of pharmaceutical grade purity is a research area where intensive investigation is being performed. From this standpoint, several downstream methods have been proposed, among them the monolithic chromatographic strategies owing to excellent mass transfer properties of monolithic supports and their high binding capacity for large biomolecules. The present study explores the physicochemical properties of histamine ligand in a supercoiled plasmid DNA purification process from an Escherichia coli clarified lysate, where the emphasis is given to the elution strategy that allows higher selectivity and efficient removal of other impurities besides the open circular isoform. The combination of high NaCl concentration and acidic pH allowed the elimination of 89% of RNA during the preparative loading of the lysate sample. The results of the purification strategy with ascending sodium chloride gradient revealed that 97% of supercoiled plasmid DNA was recovered with a purity degree of 99%. In addition, using a combined purification strategy with ascending sodium chloride (capture step) and then descending ammonium sulfate (polishing step) gradient, it was achieved a lower supercoiled plasmid DNA recovery yield of 79% with a purity degree of 92%, although the dynamic binding capacity under these conditions was higher than in the previous strategy. A significant reduction of host contents, such as proteins, RNA and genomic DNA, was obtained in both purification strategies. Accordingly, histamine is a useful and versatile ligand that allows the desirable supercoiled plasmid purification with high yield and purity level.