Plasmid Recovery Research Articles

Efficient Construction of a Heteroduplex Plasmid Containing a DNA Damage Lesion for In Vivo Mismatch Repair Activity Assay

Background. Current models have proposed that part of the DNA damage response (DDR) is dependent on the DNA mismatch repair (MMR) system, but there are no good cellular models that demonstrate that MMR proteins recognize DNA damage to subsequently signal cell-cycle checkpoints or apoptosis machinery. For measurement of DNA MMR activity in a living cell, un-nicked circular heteroduplex plasmid DNA is reported to be useful as a substrate. However, multiple factors significantly affect the yield of heteroduplex plasmid DNA, which voids the ability to demonstrate MMR action for DDR. Aims: To establish a method for efficient preparation of a heteroduplex plasmid containing a DNA damage lesion. Methods. We used 5fluorodeoxyuracil (5FdU) as a DNA damage lesion, which models 5-fluorouracil incorporated into DNA as we previously demonstrated for In Vitro MMR binding assay. We first compared the efficacy between two methods of 5FdU-containing heteroduplex plasmid (5FdU plasmid) construction; one was constructed by a ligation reaction between linearized double-strand DNA (dsDNA) plasmid and annealed synthetic oligonucleotide containing 5FdU (ligation method), and the other was by primer extension/ ligation reaction by using both T4 DNA polymerase and T4 DNA ligase at one time after annealing a 5FdU-containing phosphorylated oligomer with a pGEM7Zf(+) single-strand DNA (ssDNA) plasmid (primer extension method). Based on the results, we further analyzed determinant factors of each process to purify high quality 5FdU plasmid. Results. With the ligation method, we yielded less than 1.0 μg from 10 μg of parental dsDNA plasmid, whereas with the primer extension method, 7.4-12 μg could be purified from the 10 μg ssDNA plasmid. We found it easier to detect the heteroduplex site by direct sequencing with the primer extension method compared to the ligation method. To improve quality and quantity of 5FdU plasmid preparation, we realized the following determinant factors: 1) It is easier and more efficient to isolate ssDNA plasmid using M13K07 helper phage through generating individual transformed colonies of JM109/template dsDNA plasmid/M13K07 than conventional M13K07 infection; 2) Phosphorylation of 5FdU-containing oligomer improved the closed 5FU plasmid recovery rate; 3) A shorter time for primer extension/ligation reaction had higher yield due to T4 polymerase having delayed 3'-5' exonuclease activity. The purified 5FdU plasmids could be transfected, and enabled us to observe cellular cytotoxicity when compared to positive and the negative control plasmids, which suggests usefulness for further analysis of DDR response.Conclusion. We established efficient production of a heteroduplex plasmid containing DNA damage lesion, which will be useful to analyze a pure DDR effect in live cells.

Anion exchange chromatography of 4.2 kbp plasmid based vaccine (pcDNA3F) from alkaline lysed E. coli lysate using amino functionalised polymethacrylate conical monolith

New strategies to economically manufacture large quantities of vaccines in less time are greatly needed to cater for the increasing global human population. This study aimed at developing a strategy to efficiently manufacture a highly purified plasmid vaccine via anion exchange monolithic chromatography. Diethylamine and triethylamine were used to provide the functional groups for polymethacrylate monoliths and used in anion exchange chromatography of plasmid pcDNA3F. Five chromatographic settings as described in the first table of this article were studied and it was concluded that the triethylamine functionalised conical monolith combined with optimum buffers’ and pH conditions produced the highest quality of pcDNA3F. Plasmid yield (3003.55 mg/L), plasmid recovery (90.25%), protein (0.01 mg/L), LPS (0.12 EU/mg) with no detectable gDNA and RNA were obtained at a low NaCl concentration of 0.25 M. Apparently, this technology will have a great impact on the overall plasmid vaccine production and particularly on the development of axial flow monolithic plasmid vaccine purification.

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.

Analysis of Selective Metal-Salt-Induced Endotoxin Precipitation in Plasmid DNA Purification Using Improved Limulus Amoebocyte Lysate Assay and Central Composite Design

Recent advancements in plasmid DNA (pDNA) production involve the development of innovative and cost-effective methods as well as reduced number of unit operations. This study investigates the feasibilities of using a metal salt to selectively remove endotoxins from clarified cell lysates containing plasmid DNA. Screening of endotoxin precipitation in various metal salt solutions and optimization of process conditions (pH, ion concentration, temperature, and incubation time) using central composite design experiments have been carried out successfully. Results show that selective endotoxin precipitation (<0.05 EU/μg) can economically be carried out during the alkaline cell lysis procedure (neutralization step) at a pH condition similar to that of alkaline-lysed cell lysate, a low ZnSO(4) concentration (0.5 M), a minimum incubation time (30 min), and a temperature of 15 °C. In summary, this method provides ease of subsequent plasmid DNA purification due to reduced bulk impurities and cost-effective and most importantly high endotoxin removal (>80%) and plasmid recovery (>90%).

Antibiotic-free selection in E. coli: new considerations for optimal design and improved production

BackgroundThe increasing regulatory requirements to which biological agents are subjected will have a great impact in the field of industrial protein expression and production. There is an expectation that in a near future, there may be "zero tolerance" towards antibiotic-based selection and production systems. Besides the antibiotic itself, the antibiotic resistance gene is an important consideration. The complete absence of antibiotic-resistance gene being the only way to ensure that there is no propagation in the environment or transfer of resistance to pathogenic strains.ResultsIn a first step, we have designed a series of vectors, containing a stabilization element allowing a complete elimination of antibiotics during fermentation. Vectors were further improved in order to include alternative selection means such as the well known poison/antidote stabilization system. Eventually we propose an elegant positive pressure of selection ensuring the elimination of the antibiotic-resistance gene through homologous recombination. In addition, we have shown that the presence of an antibiotic resistance gene can indirectly reduce the amount of expressed protein, since even in absence of selection pressure the gene would be transcribed and account for an additional stress for the host during the fermentation process.ConclusionsWe propose a general strategy combining plasmid stabilization and antibiotic-free selection. The proposed host/vector system, completely devoid of antibiotic resistance gene at the end of construction, has the additional advantage of improving recombinant protein expression and/or plasmid recovery.

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.

Droplet enhanced microfluidic-based DNA purification from bacterial lysates via phenol extraction

Microfluidic platforms have been developed to demonstrate DNA purification via liquid extraction techniques at the microscale using an aqueous phase containing either protein, DNA, or a complex cell lysate and an immiscible receiving organic (phenol) phase. Initially, a serpentine device was used to investigate protein partitioning between the aqueous and organic phase, and DNA purification when both protein and DNA were mixed in the aqueous phase and infused conjunctly with the phenol phase. This two-phase system was studied using both stratified and droplet-based flow conditions. The droplet-based flow resulted in a significant improvement of protein partitioning from the aqueous phase into the organic phase due to the convective flow recirculation inside each droplet improving material transport to the organic–aqueous interface. A second device was designed and fabricated to specifically extract plasmid DNA from bacterial lysates using only droplet-based flows. The plasmid recovery using the microdevice was high (>92%) and comparable to the recovery achieved using commercial DNA purification kits and standard macroscale phenol extraction. This study presents the initial steps toward the miniaturization of an efficient on-chip DNA sample preparation using phenol extraction which could be integrated with post-extraction DNA manipulations for integrated genomic analysis modules.

Generation of chromosomal DNA during alkaline lysis and removal by reverse micellar extraction

The separation of structurally related impurities from pharmaceutical plasmid DNA by highly scalable purification techniques is a challenge for biochemical engineering. Next to RNA, proteins, and lipopolysaccharides, the chromosomal DNA of the plasmid replicating host has to be removed. Here, we describe the application of reverse micellar extraction for the separation of chromosomal from plasmid DNA. By applying different procedures for alkaline lysis, bacterial lysates with different amounts of chromosomal DNA were generated. A reverse micellar extraction step enabled us to deplete the concentration of this impurity below the required level of 50 mg g(-1) of plasmid DNA with almost complete plasmid recovery.

Improved electroporation-mediated non-integrative transformation of Thermomyces lanuginosus

The development of an efficient fungal expression system for recombinant proteins requires an improved transformation system for the host organism. We report a facile, efficient and highly reproducible electroporation-mediated transformation system for Thermomyces lanuginosus with a transformation efficiency of 1.27 × 10 3 transformants/µg DNA. Conidia of T. lanuginosus were stably transformed to hygromycin B resistance using the pBC-hygro plasmid construct. Optimal electroporation conditions for maximum transformation of 10 8 conidia ml − 1 in 1.2 M sorbitol buffer (15 mM DTT, 5% DMSO) were a field strength of 5.5 kV/cm for 10 ms and a DNA concentration of 0.5 µg µl − 1 . Transformants were recovered in prewarmed potato dextrose broth supplemented with 1.2 M sorbitol for 1–2 h at 50 °C. The presence of the hygromycin B phosphotransferase ( hph) gene and non-integrative transformation was confirmed by PCR, Southern hybridization analysis and plasmid recovery. Transformants exhibited altered phenotype with reduced pigmentation and transformants were found to be mitotically stable after 15 sequential transfers on nonselective media without selective pressure.

Alternatives for the intermediate recovery of plasmid DNA: Performance, economic viability and environmental impact

Robust cGMP manufacturing is required to produce high-quality plasmid DNA (pDNA). Three established techniques, isopropanol and ammonium sulfate (AS) precipitation (PP), tangential flow filtration (TFF) and aqueous two-phase systems (ATPS) with PEG600/AS, were tested as alternatives to recover pDNA from alkaline lysates. Yield and purity data were used to evaluate the economic and environmental impact of each option. Although pDNA yields > or = 90% were always obtained, ATPS delivered the highest HPLC purity (59%), followed by PP (48%) and TFF (18%). However, the ability of ATPS to concentrate pDNA was very poor when compared with PP or TFF. Processes were also implemented by coupling TFF with ATPS or AS-PP. Process simulations indicate that all options require large amounts of water (100-200 tons/kg pDNA) and that the ATPS process uses large amounts of mass separating agents (65 tons/kg pDNA). Estimates indicate that operating costs of the ATPS process are 2.5-fold larger when compared with the PP and TFF processes. The most significant contributions to the costs in the PP, TFF and ATPS processes came from operators (59%), consumables (75%) and raw materials (84%), respectively. The ATPS process presented the highest environmental impact, whereas the impact of the TFF process was negligible.

Stable expression of a GFP-BSD fusion protein in Babesia bovis merozoites

Transfection has been a valuable technique for elucidating gene function in many pathogens. While transient transfection of Babesia spp. has been reported previously, stable integration of exogenous genes in Babesia has proven difficult. In this study, a plasmid was designed to target integration of a gfp-bsd gene into the Babesia bovis ef-1α locus. Babesia bovis-infected erythrocytes of the biologically cloned Mo7 strain were transfected by electroporation with either circular or linear plasmids and selected in cultures with varying amounts of blasticidin 24 h after electroporation. Several blasticidin-resistant B. bovis transfected cell lines emerged at different rates, ranging from 5 to 26 days after the start of selection. One transfected parasite line (1-2-124) was selected for further analysis based on a rapid growth rate and bright GFP fluorescence in the presence of a lethal concentration of blasticidin. Continued expression of the gfp-bsd fusion gene was confirmed by reverse transcriptase-PCR, Western blot analysis and fluorescence microscopy for longer than 9 months after electroporation. No plasmid or episomal DNA could be detected in this line, and plasmid recovery in Escherichia coli was unsuccessful. Southern blot results and sequencing of PCR amplicons flanking the putative insertion site are consistent with integration of at least one gfp-bsd cassette into the targeted ef-1α locus in the transfected parasite line. Overall the results demonstrate, we believe for the first time, chromosomal integration and stable expression of a foreign gene in B. bovis. With the availability of the B. bovis genome, targeted stable transfection will provide a means to determine the role of specific genes in the biology, clinical disease and immunity of B. bovis, one of the three major tick-borne parasites that limit global livestock production.

Metabolic Engineering of ω3-Very Long Chain Polyunsaturated Fatty Acid Production by an Exclusively Acyl-CoA-dependent Pathway

omega3-Very long chain polyunsaturated fatty acids (VLCPUFA) are essential for human development and brain function and, thus, are indispensable components of the human diet. The current main source of VLCPUFAs is represented by ocean fish stocks, which are in severe decline, and the development of alternative, sustainable sources of VLCPUFAs is urgently required. Our research aims at exploiting the powerful infrastructure available for the large scale culture of oilseed crops, such as rapeseed, to produce VLCPUFAs such as eicosapentaenoic acid in transgenic plants. VLCPUFA biosynthesis requires repeated desaturation and repeated elongation of long chain fatty acid substrates. In previous experiments the production of eicosapentaenoic acid in transgenic plants was found to be limited by an unexpected bottleneck represented by the acyl exchange between the site of desaturation, endoplasmic reticulum-associated phospholipids, and the site of elongation, the cytosolic acyl-CoA pool. Here we report on the establishment of a coordinated, exclusively acyl-CoA-dependent pathway, which avoids the rate-limiting transesterification steps between the acyl lipids and the acyl-CoA pool during VLCPUFA biosynthesis. The pathway is defined by previously uncharacterized enzymes, encoded by cDNAs isolated from the microalga Mantoniella squamata. The conceptual enzymatic pathway was established and characterized first in yeast to provide proof-of-concept data for its feasibility and subsequently in seeds of Arabidopsis thaliana. The comparison of the acyl-CoA-dependent pathway with the known lipid-linked pathway for VLCPUFA biosynthesis showed that the acyl-CoA-dependent pathway circumvents the bottleneck of switching the Delta6-desaturated fatty acids between lipids and acyl-CoA in Arabidopsis seeds.

Purification of plasmid DNA with aqueous two phase systems of PEG 600 and sodium citrate/ammonium sulfate

Polymer/salt aqueous two phase systems (ATPS) based on polyethylene glycol (PEG) 600, sodium citrate and ammonium sulfate were used to partially purify plasmid DNA (pDNA) from Escherichia coli alkaline lysates. The effect of pH and lysate load on the binodal curve was analyzed and tie-lines were determined in order to establish the optimal conditions for ATPS formation. A series of extraction experiments were performed at pH 6.9 using a 20% (w/w) lysate load and systems with 16.5% (w/w) salt, 19.0% (w/w) PEG 600, and a tie-line length of 37.1% (w/w). Under these conditions, plasmid DNA was recovered in the salt-rich bottom phase. However, whereas with sodium citrate-based systems recovery yields closer to 100% were obtained, the use of ammonium sulfate enabled higher purification although with lower recoveries. Thus, the performance of ATPS prepared by combining sodium citrate and ammonium sulfate was also evaluated. A mixture of 25% (w/w) ammonium sulfate and 75% (w/w) sodium citrate offered a good compromise between plasmid recovery (91.1%) and purity (17.2%). Multi-step extraction procedures were evaluated in order to improve the process performance. Although the majority of the impurities were removed in the first step, incremental increases in the purity were obtained with the inclusion of extra steps. The top to bottom phase volume ratio was increased in order to increase plasmid concentration in the bottom phase. Although this was achieved using a phase ratio of 4, it was not possible to concentrate plasmid relatively to the starting lysate. Overall, the results show that ammonium sulfate, a salt which has a high environmental impact, can be partially replaced in ATPS by sodium citrate, without significant decrease in the performance of plasmid purification.

Preparation of inorganic–organic anion-exchange membranes and their application in plasmid DNA and RNA separation

Inorganic–organic anion-exchange membranes were prepared in this study by coating a precursor, the product of N-[3-(trimethoxysilyl)propyl] ethylene diamine reacted with 3-(triethoxysilyl)propyl isocyanate, on macroporous glass fiber and alumina membranes, followed by bromoethane treatment. The FTIR results demonstrated the successful membrane modification, and the resulted anion-exchange capacities were 6.2 and 1.5 μequiv./cm 2, respectively, for modified glass fiber membrane and modified alumina membrane. In batch adsorption process, the corresponding adsorption capacity for plasmid DNA could be sorted by: commercial polymeric SB6407 > modified glass fiber > modified alumina membrane; while for RNA adsorption, the order became: modified glass fiber ≈ SB6407 > modified alumina membrane. The optimal elution condition found from batch desorption performance was: 2 M NaCl in 50 mM Tris–HCl, pH 8 for RNA elution, followed by 1 M NaCl and 20% ethanol in 50 mM Tris–HCl, pH 8 for plasmid DNA elution. In membrane chromatography process, plasmid DNA and RNA could be clearly separated from the feed of 10 μg plasmid DNA + 10 μg RNA mixture or cell lysate by one piece of 47 mm modified glass fiber or SB6407 membrane, but not by the stacked 13 mm modified alumina membranes. The overall plasmid DNA recovery for the modified glass fiber membrane was 98–106%, higher than that of SB6407 membrane (91–96%).

On the application of ‘seeding’ techniques in the primary separation of plasmid DNA from neutralised E. coli lysates

AbstractBACKGROUND: Initial extraction of plasmid DNA from Escherichia coli and its separation from host‐derived contaminants is a difficult task to perform. Here, we examine the application of particle ‘seeding’ solid–liquid separation methods for primary recovery of plasmid DNA from neutralised alkaline cell lysates.RESULTS: Planting magnetic particle ‘seeds’ during cell lysis resulted in enhanced phase separation, facile magnetic separation of the floc, slight improvements in plasmid purity, but diminished plasmid recoveries. When CaCO3‐coated low‐density microspheres were seeded into flocs, phase separation was impaired, shear‐induced floc damage and contamination of the plasmid liquor with genomic DNA and cell debris occurred, but plasmid DNA recovery was improved. Introduction of hydrophobic low‐density microspheres into the floc dramatically improved floc stiffness, phase separation and flotation efficiency, and reduced the solids content in the plasmid liquor 10‐fold. However, strong reinforcement of the cell debris lattice by these microspheres hindered plasmid release into the liquor beneath.CONCLUSION: By incorporating magnetic or buoyant seeds during cell lysis we have identified new routes for separation of shear‐sensitive cell debris solids from crude plasmid‐containing liquors. Effective use of seeding approaches for difficult solid–liquid separation tasks will require evaluation of a wide range of seeds of varying architecture, size, shape, density and chemistry. Copyright © 2007 Society of Chemical Industry

Integration of Bacteria Capture via Filtration and in Situ Lysis for Recovery of Plasmid DNA under Industry-Compatible Conditions

Combining capture and lysis of the bacteria with partial purification of the plasmid DNA is beneficial for the design of efficient plasmid production processes at larger scale. Such an approach is possible when the bacteria are captured by filtration. Taking industrial requirements into account, however, such a capture requires complex filtration mixtures containing retentive additives such as bentonite and polycations. This makes the straightforward transfer of established lysis protocols to in situ lysis difficult. In this contribution, the different steps of such a protocol are designed for complex filter cakes, including fragilization (by lysozyme), lysis (alkaline pH/acidic pH, 70/37 degrees C, urea/NaCl/Triton), and specific elution (pH, NaCl, CaCl2, guanidinium hydrochloride). Results are compared in regard to plasmid quality (topoisomeric form) and quantity (compared to the yield obtained by a commercial miniprep of a small aliquot of the bacteria suspension from the bioreactor). Best results in these terms were obtained by the Triton lysis protocol performed at 37 degrees C (30 min of contact with a lysis buffer composed of 50 mM Tris pH 8, 1% Triton, 1 g/L lysozyme, and 6 M guanidinium hydrochloride) followed by the specific elution of the plasmid DNA in 50 mM Tris buffer pH 8.

A new series of yeast shuttle vectors for the recovery and identification of multiple plasmids from Saccharomyces cerevisiae

The availability of Saccharomyces cerevisiae yeast strains with multiple auxotrophic markers allows the stable introduction and selection of more than one yeast shuttle vector containing marker genes that complement the auxotrophic markers. In certain experimental situations there is a need to recover more than one shuttle vector from yeast. To facilitate the recovery and identification of multiple plasmids from S. cerevisiae, we have constructed a series of plasmids based on the pRS series of yeast shuttle vectors. Bacterial antibiotic resistance genes to chloramphenicol, kanamycin and zeocin have been combined with the yeast centromere sequence (CEN6), the autonomously replicating sequence (ARSH4) and one of the four yeast selectable marker genes (HIS3, TRP1, LEU2 or URA3) from the pRS series of vectors. The 12 plasmids produced differ in antibiotic resistance and yeast marker gene within the backbone of the multipurpose plasmid pBluescript II. The newly constructed vectors show similar mitotic stability to the original pRS vectors. In combination with the ampicillin-resistant pRS series of yeast shuttle vectors, these plasmids now allow the recovery and identification in bacteria of up to four different vectors from S. cerevisiae.

Plasmid adsorption to anion‐exchange matrices: Comments on plasmid recovery

A number of anion-exchange adsorbents were constructed, employing nonporous silica fibers, and examined with the aim of describing factors that influence desorption and recovery of plasmid DNA (pDNA). The fibers were provided with ligands via adsorption of the polymeric amines poly(ethyleneimine) or chitosan, or via graft-polymerization of primary, tertiary, or quaternary amine monomers to vinyl-silanized fibers. Several adsorbents showed an almost irreversible plasmid binding. It was suggested that important factors affecting the DNA releasing ability are (i) type of amine ligand used (primary amines bind plasmids the strongest), (ii) the structure of the nucleic acid (supercoiled pDNA may bind stronger than linear genomic DNA), (iii) shift of ligand pK(a) (due to the proximity of highly charged pDNA), and (iv) the solid support itself (steric factors may lead to kinetically stable complexes). The last factor was derived from several comparisons between support-bound ligand and free soluble ligand. It was thus observed that polyelectrolyte complexes associated with a surface were much more difficult to dissociate than the equivalent soluble complexes.

Plasmid purification using non-porous anion-exchange silica fibres

A new type of fibre-based anion-exchange material for plasmid purification was developed. The basic material consisted of non-porous silica fibres with a mean diameter of 1.5 μm and a surface area of 2.4 m 2 g −1. The fibre surface was provided with several types of ligands, either by adsorption of polymers (chitosan or poly(ethyleneimine)) or by polymerization of amine-containing acrylic monomers onto a propyl methacrylate-silanized surface. The resulting polymer layers contained primary, tertiary or quaternary amines as ion-exchange groups. The packing density could be varied considerably, 9–34% (v/v). The loose packing structure provided excellent flow properties suitable for high-speed operations. The best overall performance was shown by silica fibres provided with tertiary amine polymers, having a plasmid-binding capacity of 0.9 mg ml −1 (pre-purified plasmid) and a plasmid recovery of 62% (performance data remained stable though several adsorption cycles). The high flow rates possible with the fibre material made it especially useful when large volumes of cleared lysate were processed. The columns could be operated with retention of their adsorption properties at speeds of up to 1800 cm h −1, equivalent to 0.5 column volumes per minute. The binding capacity was found to be lower than anticipated from the design of the fibres. Fluorescence imaging showing individual plasmid molecules indicated the fibre population to be heterogeneous with respect to plasmid adsorption, some fibres displaying poor binding properties. Possible reasons for this heterogeneity are discussed.

The suitability of DEAE-Cl active groups on customized poly(GMA-co-EDMA) continuous stationary phase for fast enzyme-free isolation of plasmid DNA

The creation of a commercially viable and a large-scale purification process for plasmid DNA (pDNA) production requires a whole-systems continuous or semi-continuous purification strategy employing optimised stationary adsorption phase(s) without the use of expensive and toxic chemicals, avian/bovine-derived enzymes and several built-in unit processes, thus affecting overall plasmid recovery, processing time and economics. Continuous stationary phases are known to offer fast separation due to their large pore diameter making large molecule pDNA easily accessible with limited mass transfer resistance even at high flow rates. A monolithic stationary sorbent was synthesised via free radical liquid porogenic polymerisation of ethylene glycol dimethacrylate (EDMA) and glycidyl methacrylate (GMA) with surface and pore characteristics tailored specifically for plasmid binding, retention and elution. The polymer was functionalised with an amine active group for anion-exchange purification of pDNA from cleared lysate obtained from E. coli DH5α-pUC19 pellets in RNase/protease-free process. Characterization of the resin showed a unique porous material with 70% of the pores sizes above 300nm. The final product isolated from anion-exchange purification in only 5min was pure and homogenous supercoiled pDNA with no gDNA, RNA and protein contamination as confirmed with DNA electrophoresis, restriction analysis and SDS page. The resin showed a maximum binding capacity of 15.2mg/mL and this capacity persisted after several applications of the resin. This technique is cGMP compatible and commercially viable for rapid isolation of pDNA.