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2017

Preparative scale chromatographic separation of open-circular (oc) from supercoiled (sc) plasmid DNA (pDNA) isoforms has been already established on CIM® C4 with high ligand density (C4 HLD) monolithic columns with sample loading in 3.0 M ammonium sulphate (AS). The process requires high molarity of AS, increasing the overall cost of the process. Sample displacement chromatography (SDC) can be used as an alternative to decrease the AS concentration required during loading onto hydrophobic chromatographic supports. This study compares three chromatographic monoliths with different hydrophobic ligands on the surface (C4 HLD, pyridine and histamine) for the purification of different pDNA vectors in SD mode.

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2016

Since plasmid DNA (pDNA) as a pharmaceutical product has stringent requirements of purity and efficacy, one or more chromatographic steps are often used in the downstream processing train. High ligand density butyl-modified (C4 HLD) monolithic support is currently used in a polishing step of a pDNA purification process (1) and is mainly focused to supercoiled (sc) pDNA isoform separation from the open circular (oc) and linear pDNA isoform as well as for removal of remaining gDNA and RNA. The goal of the study was to compare the productivities of two variations of the polishing chromatographic process employing monoliths – classical bind-elute (BE) versus recently described (2) sample displacement purification (SDP). Classical purification requires high concentration of ammonium sulphate (AS) during loading step and elution is then achieved by descending AS gradient. SDP utilises different relative binding affinities of components in a sample mixture and separates pDNA isoforms under overloading conditions, where sc pDNA isoform acts as a displacer of oc or linear pDNA isoform.

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2014

One of the major requirements for pharmaceutical-grade pDNA is its high homogeneity, being mostly in supercoiled (sc) isoform. Chromatographic separation of sc pDNA from open coiled (oc) or linear isoform is challenging due to their similar interactions with the chromatographic phases. Promising separation efficiency of pDNA isoforms was proven on recently developed histamine modified monolithic chromatographic column in descending ammonium sulfate gradient. The aim of the study was to further optimise the chromatographic conditions for sample analysis, where all three isoforms would be baseline separated.

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2012

Monolith chromatography media coupled with metal affinity ligands proved superior to the conventional particle-based matrix as a plasmid DNA (pDNA) purification platform. By harnessing the differential affinity of pDNA, RNA. Host cell proteins and endotoxin to copper ions in the solution a majority of endotoxin (90%) was removed from the alkaline cell lysate using CuCl2-induced precipitation. RNA and remaining endotoxin were subsequently processed by copper immobilized metal affinity column employing either monolith or particle-based matrix where both RNA and endotoxin were removed below detection limit with almost complete recovery of pDNA in the monolith was found to have several advantages in terms of handling feedstocks crowded with RNA in a concentration-independent manner and exhibiting flowrate-independent dynamic binding capacity for RNA. This enabled monolith-based process to be conducted at high feed concentration and flow rate. Resulting in pDNA vaccine purification at a high yield and purity and the process conditions investigated, the use of monolith column gave at least three fold higher productivity for recovery of purified pDNA as compared to the particle- based column, demonstrating its potential as a more rapid and economical platform for pDNA vaccine purification.

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The present study describes a new methodology to quantify and monitor the quality of supercoiled (sc) plasmid DHA (pDLIA), using a monolithic column based on anion-exchange chromatography. This analytical method with UV detection allows distinguishing the plasmid isoforms by a NaCl stepwise gradient. The selectivity, Linearity, accuracy, reproducibility and repeatability of the method have been evaluated, and the lower quantification and detection limits were also established. The validation was performed according to the guidelines, being demonstrated that the method is precise and accurate for a sc plasmid concentration up to 200 µg/mL. The main advance achieved by using this monolithic method is the possibility to quantify the sc plasmid in a sample containing other plasmid topologies, in a 4 minutes experiment. This work also intends to evaluate the possibility to assess the sc pDNA present in more complex samples, allowing the control of the samples recovered from different bioprocess steps.

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2010

Application of plasmid DNA for gene therapy and vaccination has gained huge interest in last two decades. Topological homogeneity and impurity content are crucial for therapeutic usage of pDNA. Major influence on achieving regulatory demands in pDNA production has downstream processing and in order to get optimal purity different purification techniques have to be included. 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 enabled excelent purity and productivity.

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2009

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.

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2008

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 [1].

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 [2]. 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.

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2006

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 [1].

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2005

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).

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2004

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.

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The availability of sufficient quantities of quality DNA is always a crucial point in DNA based methods, i.e. for PCR, DNA sequencing, Southern blotting, and microarrays [1]. The same is true for the PCR-based methods for detection of genetically modified food [2]. During the production chain foods passes several physical, biological, and chemical processes, which all negatively influences on the quantity of available DNA. The phenomenon is especially expressive when high temperature treatment is performed at low pH [3]. The existing methods for DNA isolation from food cannot always fulfill the expectations of quantity and quality of isolated DNA. Furthermore they usually include 100 mg of sample and are difficult to scale-up [4]. Four major chromatographic modes are used for the separation of DNA: size-exclusion, anion-exchange, ion-pair reversephased, and slalom chromatography. Of these, anion-exchange chromatography combined with micropellicular packing is described as the most prominent technique so far [1].
Anion-exchange CIM® (Convective Interaction Media) monolithic columns allow fast and flow unaffected separation of several biomolecules, including nucleic acids [5].

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2003

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).

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The availability of sufficient quantities of quality DNA is always a crucial point in DNA based methods, i.e. for PCR, DNA sequencing, Southern blotting, and microarrays [1]. The same is true for the PCR-based methods for detection of genetically modified food [2]. During the production chain foods passes several physical, biological, and chemical processes, which all negatively influences on the quantity of available DNA. The phenomenon is especially expressive when high temperature treatment is performed at low pH [3].

The existing methods for DNA isolation from food cannot always fulfill the expectations of quantity and quality of isolated DNA. Furthermore they usually include 100 mg of sample and are difficult to scale-up [4]. Four major chromatographic modes are used for the separation of DNA: size-exclusion, anion-exchange, ion-pair reversephased, and slalom chromatography. Of these, anion-exchange chromatography combined with micropellicular packing is described as the most prominent technique so far [1].

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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).

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The availability of sufficient quantities of quality DNA is always a crucial point in DNA-based methods, i.e. for PCR, DNA sequencing, Southern blotting, and microarrays [1]. The same is true for the PCR-based methods of GMO detection in food [2]. During the production chain foods passes several physical, biological, and chemical processes, which all negatively influences on the quantity of available DNA. The phenomenon is especially expressive when high temperature treatment is performed at low pH [3].

The existing methods, for DNA isolation from food, cannot always fulfill the expectations of quantity and quality of isolated DNA. Furthermore they usually include 100 mg of sample and are difficult to scale-up [4]. Four major chromatographic modes are used for the separation of DNA: size-exclusion, anionexchange, ion-pair reverse-phased, and slalom chromatography. Of these, anionexchange chromatography combined with micropellicular packing is described as the most prominent technique so far [1].

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2002

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.

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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 [1]. 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 [2]. 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®).

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2000

Production and downstream processing in biotechnology requires fast and accurate control of each step in the process. Liquid chromatography of biopolymers on so-called soft supports is typically slow, often causing significant product degradation. One way of improving these boundary conditions in liquid chromatography is the use of monolithic adsorbents. The basis for fast separations with such media is a reduced mass transfer resistance owing to the fact that pore diffusion is practically non-existent. Chromatography with compact, porous units such as monolithic columns is being used increasingly for analytical and preparative separations of biopolymers with apparent molecular mass ranging from several thousand to up to several million.

This paper describes the use of a CIM® Convective Interaction Media for fast purification of plasmid DNA as well as for the concentration of viruses. Plasmid DNAs are circular duplex DNA molecules that are maintained stable as episomal genetic information within bacteria. They play an important role in gene technology - they are used for applications such as transformation, sequencing, transfection studies, etc. These applications require satisfactory purity of used plasmid DNA. For purification of plasmid DNA from Escherichia coli, monolithic units as anion-exchangers (CIM® DEAE and QA disks) were used. Separation of RNA from DNA as well as concentration of plasmid DNA were performed on the same disks.

All the methods for concentration of viruses, in general, are expensive, time-consuming and they are frequently not very successful. Therefore an attempt to bind viruses on an anion exchanger (CIM® DEAE disk) and elute bound virions in small volume (concentration) was done. As a model virus, measles was chosen. Using CIM® DEAE disk concentration of the measles viruses was successfully performed in less than 10 minutes.

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Production and down-stream processing in biotechnology requires fast and accurate control of each step in the process. Liquid chromatography of biopolymers on so-called soft supports is typically slow, often causing significant product degradation. One way of improving these boundary conditions in liquid chromatography is the use of monolithic adsorbents. The basis for fast separations with such media is a reduced mass transfer resistance owing to the fact that pore diffusion is practically non-existent. Chromatography with compact, porous units such as monolithic columns is being used increasingly for analytical and preparative separations of biopolymers with apparent molecular mass ranging from several thousand to up to several million.

This paper describes the use of a CIM® Convective Interaction Media for fast purification of plasmid DNA as well as for the concentration of viruses.

Plasmid DNAs are circular duplex DNA molecules that are maintained stable as episomal genetic information within bacteria. They play an important role in gene technology - they are used for applications such as transformation, sequencing, transfection studies, etc. These applications require satisfactory purity of used plasmid DNA. For purification of plasmid DNA from Escherichia coli, monolithic units as anion-exchangers (CIM® DEAE and QA disks) were used. Separation of RNA from DNA as well as concentration of plasmid DNA were performed on the same disks.

All the methods for concentration of viruses, in general, are expensive, time-consuming and they are frequently not very successful. Therefore an attempt to bind viruses on an anion exchanger (CIM® DEAE disk) and elute bound virions in small volume (concentration) was done. As a model virus, measles was chosen. Using CIM® DEAE disk concentration of the measles viruses was successfully performed in less than 10 minutes.

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