Application of plasmid DNA for gene therapy and vaccination has gained substantial interest in the last two decades. Topological homogeneity and impurity content are crucial for therapeutic usage of pDNA. Downstream processing has major influence on achieving regulatory demands in pDNA production and in order to get optimal purity different purification techniques have to be applied. It was demonstrated that methacrylate monoliths can be used for efficient purification process of plasmid DNA. High dynamic binding capacities and high flow rates of methacrylate monolith enable excellent purity and productivity.
Anion-exchange chromatography is fundamental in downstream processing of plasmids both as a process and analytical technique. CIM anion-exchange monolithic columns have already been successfully used for the industrial scale purification of pharmaceutical grade small plasmid DNA .
In this work we report about the use of the newly developed monolithic analytical column intended for plasmid DNA determination in terms of its analytical performance. Higher degree of sensitivity, precision and accuracy is necessary in order to determine the quality of clinical grade DNA intended for therapeutic use. Plasmids purified from Escherichia coli fermentation exist predominantly in the supercoiled form (SC) the other two topoisomers present in the final product are mostly the open circular (OC) and linear forms . Different chromatographic conditions were tested and the separation was optimized in terms of buffer and pH selection as well as in terms of gradient slope and column length. The results were compared to the results obtained with established analytical methods.
Membrane based anion exchangers are being used increasingly for purification of monoclonal antibodies. The transition from particle-based anion exchangers is driven partly by the convenience of membranes and partly by the cost saving associated with their disposability, however the feature that makes them functionally superior is more effective mass transport.
Gene therapy has already shown some great results in treatment and cure of some monogene diseases, such as diabetes. While the use of genetically modified viruses raises safety concerns, synthetic formulations of genes inserted in plasmids are regarded as safer. At present, most clinical trials involve plasmids smaller than 10 kb. However, the concern that regulation of the functioning of the gene is ensured together with the expectation of the progression of gene therapy to multigene disfunctions, like cancer or complex nevrodegenerative disfunctions (Alzheimer disease), will require the production of larger plasmids .
Commercially available CIM® disk monolithic columns are intended for very fast analyzes and laboratory purification. Their shape is a compromise to achieve acceptable resolution and binding capacity what make them suitable for wide range of laboratory applications. Separations of complex protein mixtures can be carried out within just a few seconds because of flow unaffected resolution and, on the other hand, purification can be effectuated with high productivity due to flow-unaffected dynamic binding capacity . However, in many cases in the field of molecular biology, only a limited amount of sample is available. In such a case it is beneficial to work with small columns having high resolution or they can be used as affinity columns or bioreactors saving significant amount of valuable ligand. Having this goal in mind we developed CIM® disks with the volume of 1/10th and 1/100th of original volume. In comparison to conventional CIM® disks, they exhibit higher resolution and lower limit of detection, therefore smaller concentrations of target macromolecules can be detected. The separation ability and the protein capacity were tested on anion and cation exchange 3.4 mL and 34m L mini disk monolithic columns.
Analysis of a large numbers of samples requires chromatographic supports that not only enable fast separation and purification of a target biomolecules from a complex matrix but are also involved in an automation process. The 96 – microtiter plate format enables both. Although they are routinely used for decade's only recently few reports about the microtiter plates bearing monoliths as a separation media, were reported . Because of advantageous properties such as flow unaffected dynamic binding capacity and resolution 96 - microtiter plates with methacrylate based monolith were prepared. Characterisation of such plate demonstrated that uniform flow rate can be achieved through all wells and no leakage is present. Efficient separation of proteins was achieved within minute. Furthermore CLC (Conjoined Liquid Chromatography) concept  originally derived for analytical columns on CIM disk, can easily be extrapolated to microtiter plates. We demonstrated that multidimensional chromatography with 96 – well plate is feasible and can further accelerate screening processes.
The analysis of molecular interactions is a key part of the drug discovery process, and analytical techniques are available for studying in vitro the ligand/target complex since the early stage of the drug development process.
With regard to the assessment of the activity of chemical libraries, the affinity chromatography on HPLC immobilized-enzyme column (or immobilized enzyme reactors, IMER) is one of most promising methodologies for HTS applications.
Human recombinant acetylcholinesterase (hAChE) represents a well-known target for drug-discovery in Alzheimer’s Disease.
Plasmids are excellent genetic vectors and have been widely used in gene manipulation and recombinant DNA technology for a long time. In recent years, plasmids are intensively investigated for gene therapy purposes and genetic vaccination. In this case, plasmid DNA (pDNA) of high purity is required. To follow such demands, several chromatographic steps are commonly needed. In the case of buffer compatibility, columns can be connected in-line to overcome time consuming and yield lowering multiple chromatographic steps. Since each of the unit operations contributes to the dispersion, the resolution is further decreased by each chromatographic step. This drawback might be surmounted by combining several chromatography steps into a single chromatography column. This approach is known as multidimensional or conjoint liquid chromatography (CLC).
Affinity chromatography is a key method for protein purification. Its main advantage is in the high specificity which enables purification of a single protein from complex biological mixtures. For practical use the specific ligand should be immobilised on insoluble matrix. As a matrix, standard chromatographic supports are commonly used. They are normally in form of small (some m in diameter) particles containing pores to provide high specific surface resulting in high binding capacity. The pores are normally closed on one side, thus the liquid inside them is stagnant and the molecules are transported to the active site by diffusion. Since the diffusion coefficients for macromolecules, such as proteins, are very low, diffusion determines the overall process dynamics. As a consequence, separation or purification of the proteins takes normally 0.5 to 1h even on analytical scale.
By using a combination of two CIM® tube monolithic columns, OH and DEAE chemistry, we were able to successfully purify plasmid DNA from bacterial culture without using RNase. Purified plasmid DNA is very pure, since common contaminants, such as proteins, genomic DNA, endotoxins and RNA were under the detection limit. The scale up units produced according to cGMP standard are already used for the purification of plasmid DNA for gene therapy purposes on industrial scale.
Tissue plasminogen activator (t-PA) is serine protease which converts plasminogen into plas-min dissolving the major component of blood clots, fibrin. So, it can be extremely useful in clinical practice to help curing of heart attack victims. The most available way protein producing is genetic engineering where separation and purification of goal protein are one of the important steps in protein producing process.
Recently developed High performance monolithic disk chromatography, HPMDC, seems to be a very attractive way for study quantitative affinity parameters of recombinant proteins with different ligands as well as for protein separations and purifications. High process speed prevents the denatura-tion due to temperature and solvents influence. The better mass transfer mechanism (convection rather than diffusion) allows to consider only the biospecific reaction as time limiting.
It is known that plasminogen, which is the natural substratum for t-PA, can be successfully used as affinity ligand to separate t-PA from cellular media. However, the use of synthetic ligands for affinity chromatography is more preferable due to their higher stability and lower total cost.
Plasmids are episomes that have been recognized in few eukaryotic and most prokaryotic species. Some plasmids are excellent genetic vectors and they have been widely used in gene manipulation and recombinant DNA technology for a long time. In recent years plasmids were intensively used for gene therapy purposes (1). Most often purification starts with the cells harvest followed by alkaline lysis step in which ribonuclease A (RNase) is typically used. After that, plasmid DNA can be precipitated and used directly or can be further purified by different methods (2). Currently, several chromatographic methods, such as ion-exchange, size exclusion, affinity, and hydrophobic chromatography, have been demonstrated in plasmid purification (3). Until now a limited number of small scale purification methods without use of RNase were published. Convective Interaction Media CIM® is a monolithic chromatographic support for which has been shown that is very efficient for the separation of large molecules, such as proteins, DNA and viruses (4).
Traces of DNA in RNA samples represent impurities that could affect results of mRNA quantification and cDNA synthesis. In most cases, the DNA impurities in RNA samples are removed using enzyme deoxyribonuclease (DNase), which specifically breaks down DNA. In order to avoid the addition of DNase into the analyzing sample, the use of immobilized DNase on solid support is recommended. Because of the DNA size, very few supports available on the market enable efficient interaction between immobilized enzyme and DNA.
In recent years a new group of supports named monoliths was introduced. Because of enhanced exchange between mobile and stationary phase separation and bioconversion processes are significantly accelerated. Therefore also the efficiency of DNA removal using immobilised enzyme might be competitive to the degradation with free enzyme.
Plasmids are episomes that have been recognized in few eukaryotic and most prokaryotic species. Some plasmids are excellent genetic vectors and they have been widely used in gene manipulation and recombinant DNA technology for a long time. In recent years plasmids were intensively used for gene therapy purposes (1).Most often purification starts with the cells harvest followed by alkaline lysis step in which ribonucleaseA (RNase) is typically used. After that plasmid DNA can be precipitated and used directly or can be further purified by different methods (2).Currently, several chromatographic methods, such as ion-exchange, size exclusion, affinity, and hydrophobic chromatography, have been demonstrated in plasmid purification (3). Until now a limited number of small scale purification methods without use of RNase were published. Convective Interaction Media CIM®is a monolithic chromatographic support for which has been shown that is very efficient for the separation of large molecules, such as proteins, DNA and viruses (4).
The only four drugs approved for the clinical treatment of Alzheimer’s Disease (tacrine, rivastigmine, donepezil and galantamine) are acetylcholinesterase inhibitors which act by maintaining high levels of acetylcholine at the muscarinic and nicotinic receptors in the central nervous system. Human acetyicholinesterase (HuAChE) represents a widely studied target enzyme and it is still object of research for the development of new drugs as enzyme inhibitors.
In a previous paper il] we reported the immobilisation of AChE on a silica based chromatographic column (50 x 4.6 mm I.D.) The yield of immobilization and the stability of the AChE—IMEN were considered satisfactory, hut some problems arose. The length of the IMER and the large amount of enzyme covalently bound to the chromatographic support resulted in catalysis product long elution times and some inhibitors aspecific matrix absorption with delayed enzyme activity recovery. In order to avoid these complications and considering the high rate of AChE enzymatic reaction, we decided to reduce the dimension of the solid support for immobilization, hence the amount of immobilized enzyme, by selecting a monolithic matrix disk (12 x 3 min I.D.).
CIMa (Convective Interaction Media) monolithic supports (Bia Separations, Ljubljana) represent a novel generation of stationary phases used for liquid chromatography, bioconversions, and solid phase synthesis. As opposed to individual particles packed into chromatographic columns, CIM supports are cast as continuous homogeneous phases and provide high rates of mass transfer at lower back pressure.
In the present work a CIM® disk with immobilised human recombinant acetylcholinesterase (HuAChECIM€ Disk) was developed. The activity of immobilised enzyme, the long term stability and reproducibility were tested. HuAChECIM disk was applied as an immobilised enzyme micro-reactor (micro-IMER) in on-line HPLC system for inhibitory potency determination of known AChE inhibitors.
The progress in gene-therapy and DNA vaccination leads to a growing demand of therapeutic applicable plasmid DNA (pDNA). To guarantee the supply for the clinical trials and finally for the market new pDNA production processes, which meet all regulatory requirements, have to be developed. Conventional small scale techniques can not easily be transferred to the manufacturing scale (technical reasons and safety considerations). We developed a generic large scale process for highly purified plasmids “free” of bacterial contaminants which works without enzymes, detergents (except SDS during the cell lysis) and organic solvents.
Most commonly plasmids are manufactured by fermentation of E. coli. In the cells several isoforms of the plasmid are generated: supercoiled (sc), open circular (oc) and linear as well as dimeric forms. After alkaline lysis plasmids are accompanied in solution by genomic DNA (gDNA), RNA, proteins and other cell compounds . In addition to these impurities, the plasmid isoforms have to be separated efficiently in order to get a final product containing > 95 % of ccc form . Chromatographic resins used in biotechnology are usually designed for the separation of polypeptides, providing only low capacity for polynucleotides (< 1 mg/mL).
In this work we present an optimised purification step for large scale purification of therapeutic applicable pDNA, based on an alternative chromatography resin (CIM Convective Interaction Media®).
We have developed a screening procedure for peptide ligands for affinity chromatography on the same monolithic support. CIM® monolithic columns used conventionally for analytical and preparative separation of proteins and polynucleotides were minimized to fit into 96 well solid phase extraction plates. Peptide synthesis and screening were performed on the same format using a vacuum manifold for liquid throughput.
The development of new chromatographic supports with the aim to improve their chromatographic, hydrodynamic and mechanical properties is continually going on.
CIM Convective Interaction Media® monolithic columns represent a new chapter in every mode of the chromatography. Monolithic columns consist of a single piece of a highly porous polymer with a bimodal pore size distribution, forming flow-through channels . Since all of the mobile phase flows through the pores, molecules to be separated are transported to the active sites by convection . Therefore, the entire analysis can be completed in a very short time.
In this work, the performance of novel semi-preparative CIM® RP-SDVB disk monolithic column for separating proteins and peptides has been investigated. Since the column length in the case of gradient separations commonly used for large molecules, does not play a significant role, CIM® RP-SDVB disk monolithic column are extremely short, typically of only 3 mm. The effect of decreasing column length on the resolution under the conditions of a linear gradient has been presented.
Finally, a 1 minute purification of oligodeoxynucleotide from the synthetic mixture has been performed.
Gene therapy which is becoming more and more important in human health care requires the purification of high molecular mass compounds, so called nanoparticles (e. g. viruses and plasmids). The method of choice to ensure proper purity would be chromatography.
Most of the chromatographic supports available on the market at the moment can not follow the requests for such work due to low binding capacity for large molecules, limitation with regards to the time of the separation process and requests for CIP (cleaning in place) and SIP (sanitation in place).
Monolithic supports represent a new generation of chromatographic supports. In contrast to conventional particle supports, where the void volume between individual porous particles is unavoidable, these supports consist of a single monolith highly interconnected with larger and smaller open flow-through channels. Due to the structure, molecules to be separated are transported to the active sites on the stationary phase by convection, resulting in very short separation times. This is especially true for large molecules.
In this work we will present the use of monolithic supports for the separation of different nanoparticles on analytical and preparative scales. It will be shown that monolithic supports can overcome the limitations of particle-based supports for the analytics and isolation of big molecules and represent a major step towards the safe and efficient purification or production of nanoparticles.