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.     

Successful application of monolithic innovative technology using a carbonyldiimidazole disk to purify supercoiled plasmid DNA suitable for pharmaceutical applications

The growing demand on plasmid DNA (pDNA) manufacture for therapeutic applications requires a final product with higher quality and quantity, spending the least time. Most of the current processes for pDNA production use at least one chromatographic step, which often constitutes a key-step in the purification sequence. Monolithic stationary phases are new alternatives to the conventional matrices, which offer fast separation of pDNA due to their excellent mass transfer properties and their high binding capacity for large molecules, as pDNA. However, the efficient recovery of pure pDNA focuses on a suitable balance of the feedstock, adsorbent and mobile phase properties. To satisfy the increasing demand for pharmaceutical grade plasmids, we developed a novel downstream process which overcomes the bottlenecks of common lab-scale techniques while complying with all regulatory requirements. This work reports an integrative approach using the carbonyldiimidazole monolith to efficiently purify the supercoiled (sc) pDNA active conformation from other plasmid topologies and Escherichia coli impurities present in a clarified lysate. The monolith specificity and selectivity was also assessed by performing experiments with plasmids of several sizes of 2.7, 6.05 and 7.4 kilo base pairs (kbp), verifying the applicability to purify different plasmids. Hence, the process yield of the pDNA purification step using the CDI monolith was 89%, with an extremely reduced level of impurities (endotoxins and gDNA), which was reflected in good transfection experiments of the sc plasmid DNA sample. Overall, the analytical results and transfection studies performed with the pDNA sample purified with this monolithic enabling technology, confirmed the suitability of this pDNA to be used in pharmaceutical applications.     

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.     

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.     

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.     

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.     

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.     

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.     

Comparison of CIM~ C4-HLD monolithic column with Sartobind phenyl membrane column for pIDKE2 purification

The main component of the Center for Genetic Engineering and Biotechnology(CIGB)candidate vaccine against Hepatitis C virus(HCV)is the pIDKE2 plasmid.The current designed downstream process for the production of pIDKE2 fulfils all regulatory requirements and renders the required quantities of pharmaceuticalgrade plasmid DNA(pDNA)with 95%purity.The advantages of this procedure include high plasmid purity and the elimination of undesirable additives,such as toxic organic extractants and animal-derived enzymes.However,yields and consequently the productivity of the process are low.Previous work demonstrated that the most critical step of the process is the reverse phase chromatography,where conventional porous particle resins are used.Therefore,to increase the process productivity,alternative technologies such as membranes and chromatographic monoliths were tested as alternative options for this critical step.Here,a comparison between the behaviors of CIM~ C4-HLD and Sartobind phenyl matrices was performed.To obtain higher productivities and purities,the dynamic binding capacities and selectivities were evaluated.The results showed that both matrices had a similar capacity for pIDKE2 plasmid,but the separation of pDNA isoforms using CIM~ technology was much better than that with Sartobind.Additionally,the optimal conditions for loading plasmid DNA on a CIMC4-HLD 800-mL monolithic column in a real production process were determined.These optimizations will allow production levels to satisfy the high plasmid consumption demanded by clinical trials.     

Characterization of methacrylate monoliths for purification of DNA molecules

The suitability of methacrylate based anion exchange monolithic supports for the separation and purification of plasmid and genomic DNA has been explored. The effect of the size of the channels, ionic strength of the solution, and ligand density on the dynamic binding capacity has been investigated. The dynamic binding capacity was found to be flow independent, at least up to a linear velocity of 700 cm h(-1), and exceeded 9 mg mL(-1) for all types of DNA. The recovery depends on the pH value of the mobile phase and its ionic strength as well as on the density of the active groups. Under optimal conditions recoveries exceeding 80% were obtained even for genomic DNA. Finally, the suitability of this approach is demonstrated by purification of a real-life sample.     

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.     

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.     

Fast and efficient separation of immunoglobulin M from immunoglobulin G using short monolithic columns

Certain diagnostic, analytical and preparative applications require the separation of immunoglobulin G (IgG) from immunoglobulin M (IgM). In the present work, different ion-exchange methacrylate monoliths were tested for the separation of IgG and IgM. The strong anion-exchange column had the highest dynamic binding capacity reaching more than 20mg of IgM/ml of support. Additionally, separation of IgM from human serum albumin, a common contaminant in immunoglobulin purification, was achieved on the weak ethylenediamino anion-exchange column, which set the basis for the IgM purification method developed on convective interaction media (CIM) supports. Experiments also confirmed flow independent characteristics of the short monolithic columns.     

Purification of supercoiled plasmid DNA from clarified bacterial lysate by arginine‐affinity chromatography: Effects of spacer arms and ligand density

Efficient loading on a chromatographic column is the dilemma of the process development faced by engineers in plasmid DNA purification. In this research, novel arginine‐affinity chromatographic beads were prepared to investigate the effect of spacer arm and ligand density to their chromatographic performance for the purification of plasmid. The result indicated that dynamic binding capacity for plasmid increased with an increasing ligand density and carbon number of spacer arm, and the highest binding capacity for plasmid of 6.32 mg/mL bead was observed in the column of arginine bead with a ligand density of 47 mmol/L and 10‐atom carbon spacer. Furthermore, this arginine bead exhibited better selectivity to supercoiled (sc) plasmid. The evidence of a linear gradient elution suggested further that the binding of plasmid on arginine beads was driven by electrostatic interaction and hydrogen bonding. Hence, sc plasmid could successfully be purified from clarified lysate by two‐stepwise elution of salt concentration. By the refinement of the elution scheme and loading volume of clarified lysate, the column of arginine bead with a ligand density of 47 mmol/L exhibited the highest recovery yield and a much higher productivity among arginine‐affinity columns. Therefore, reshaped arginine beads provided more feasible and practical application in the preparation of sc plasmid from clarified lysate.     

3种阴离子交换色谱固定相捕获细胞培养上清液中红细胞生成素的效果比较（英文）

Affinity and ion exchange conventional chromatography have been used to capture erythropoietin(EPO)from mammalian cell culture supernatant.Currently,chromatographic adsorbent perfusion is available,however a limited number of applications have been found in the literature.In this work,three anion exchange chromatographic supports(gel,membrane and monolithic)were evaluated in the capture step of the recombinant erythropoietin purification process.The influences of load and flow rate on each support performance were analyzed.Also the purity of the EPO molecules was determined.A productivity analysis,as a decision tool for larger scale implementation,was done.As a conclusion,the evaluated supports are technically suitable to capture EPO with adequate recovery and good purity.However,the monolithic column admits high operating velocity,showing the highest adsorption capacity and productivity.     

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%.     

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.     

Comparison among three anion exchange chromatographic supports to capture erythropoietin from cell culture supernatant Lourdes

Affinity and ion exchange conventional chromatography have been used to capture erythropoietin (EPO) from mammalian cell culture supernatant. Currently, chromatographic adsorbent perfusion is available, however a limited number of applications have been found in the literature. In this work, three anion exchange chromatographic supports (gel, membrane and monolithic) were evaluated in the capture step of the recombinant erythropoietin purification process. The influences of load and flow rate on each support performance were analyzed. Also the purity of the EPO molecules was determined. A productivity analysis, as a decision tool for larger scale implementation, was done. As a conclusion, the evaluated supports are technically suitable to capture EPO with adequate recovery and good purity. However, the monolithic column admits high operating velocity, showing the highest adsorption capacity and productivity.     

Preparation of a monolithic column for weak cation exchange chromatography and its application in the separation of biopolymers

A procedure for the preparation of a monolithic column for weak cation exchange chromatography was presented. The structure of the monolithic column was evaluated by mercury intrusion. The hydrodynamic and chromatographic properties of the monolithic column--such as back pressures at different flow rates, effects of pH on protein retention, dynamic loading capacity, recovery, and stability--were determined under conditions typical for ion-exchange chromatography. The prepared monolithic column might be used in a relatively broad pH range from 4.0 to 12.0 and exhibited an excellent separation to five proteins at the flow rates of both 1.0 and 8.0 mL/min, respectively. In addition, the prepared column was first used in the purification and simultaneous renaturation of recombinant human interferon gamma (rhIFN-gamma) in the extract solution with 7.0 mol/L guanidine hydrochloride. The purity and specific bioactivity of the purified rhIFN-gamma in only one chromatographic step were obtained to be 93% and 7.8 x 10(7) IU/mg, respectively.     

Adsorption behavior of large plasmids on the anion-exchange methacrylate monolithic columns

The objective of this study was to investigate the behavior of large plasmids on the monolithic columns under binding and nonbinding conditions. The pressure drop measurements under nonbinding conditions demonstrated that the flow velocities under which plasmid passing monolith became hindered by the monolithic pore structure depended on the plasmid size as well as on the average monolith pore size; however, they were all very high exceeding the values encountered when applying CIM monolithic columns at their maximal flow rate. The impact of the ligand density and the salt concentration in loading buffer on binding capacity of the monolith for different sized plasmids was examined. For all plasmids the increase of dynamic binding capacity with the increase of salt concentration in the loading solution was observed reaching maximum of 7.1 mg/mL at 0.4M NaCl for 21 kbp, 12.0 mg/mL at 0.4 M NaCl for 39.4 kbp and 8.4 mg/mL at 0.5M NaCl for 62.1 kbp. Analysis of the pressure drop data measured on the monolithic column during plasmid loading revealed different patterns of plasmid binding to the surface, showing "car-parking problem" phenomena under certain conditions. In addition, layer thickness of adsorbed plasmid was estimated and at maximal dynamic binding capacity it matched calculated plasmid radius of gyration. Finally, it was found that the adsorbed plasmid layer acts similarly as the grafted layer responding to changes in solution's ionic strength as well as mobile phase flow rate and that the density of plasmid layer depends on the plasmid size and also loading conditions.