Purification of plasmid DNA vectors by aqueous two-phase extraction and hydrophobic interaction chromatography (original) (raw)
Related papers
2009
In this paper, the efficiency of an aqueous two-phase system (ATPS) for purification of nanometer-sized bioparticles, plasmid DNA (pDNA), was studied. Polymer-salt ATPS consisting of polyethylenglycol (PEG)-K 2 HPO 4 was used for the purification of 7 kb and 14 kb plasmid vectors. PEG-300 and PEG-1450 were applied to investigate the influence of different molecular mass of PEG on partitioning behavior of pDNA. The Taguchi design of experiments has been applied in order to optimize the significant system characteristics including PEG/salt ratio, temperature, lysate mass fraction and size of plasmid for pDNA separation by using ATPS. The results indicated that PEG/salt ratio has a considerable contribution on pDNA recovery both in the presence of PEG-300 and PEG-1450. It is also obtained that the size of pDNA in the range of 7 kb to 14 kb is not a significant factor on its partitioning. Furthermore, pDNA is easily partitioned to polymer-rich top phase in PEG300/salt system; and in sal...
Separation and Purification Technology, 2009
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.
Journal of Chromatography A, 2007
This work studied the possibility of using polyethyleimine (PEI) as an affinity ligand for the purification of plasmid DNA (pDNA) from alkaline lysates using aqueous two-phase systems (ATPSs). The goal was to find conditions under which this cationic polymer could steer the partition of pDNA to the phase where less impurities accumulate. In poly(ethylene glycol) (PEG)/ammonium sulphate systems, neither free nor PEGylated PEI (pPEI) were able to change the partition of pDNA. This is probably due to the high salt concentration present in these systems that impair the interaction between pDNA and PEI. In PEG 3350/dextran 110 systems, the desired effect could be observed but 0.2-0.5 M ammonium sulphate had to be added to prevent the co-partition of RNA to the same phase. These results were used to develop a methodology to obtain polyplexes from alkaline lysates in a two-step ATPSs extraction process. In the first step, a PEG 600/ammonium sulphate system is used to remove most impurities to the top phase. The pDNA-containing bottom phase is then isolated and contacted with a second PEG 3350/dextran 110 system supplemented with a small amount of pPEI (0.2%). Plasmid yield was 100% and the final preparation had no RNA and only small amounts of contaminant protein. Additionally, pDNA was obtained in the form of 53 nm-sized polyplexes which are likely to suit specific gene delivery applications.
Journal of Chromatography A, 2013
Purification of high quantities of human grade plasmid DNA is one of the most intensive production steps. Because of that several methods have been proposed, among them also chromatographic purification using methacrylate monoliths. Recently, a process comprising the combination of hydrophobic interaction (HIC) monolith and ion-exchange monolith was developed. In this work both chemistries were tried to be introduced on a single monolith. Methacrylate monoliths bearing octylamine groups, combination of butyl (C4) grafted methacrylate groups and diethylaminoethyl (DEAE) groups as well as grafted chains bearing both C4 and DEAE groups were prepared. All monoliths were investigated for their ionic and protein capacity and compared to conventional epoxy, C4, and DEAE methacrylate monoliths. Octylamine monolith and monolith bearing combination of C4 grafted methacrylate groups and DEAE groups were found to be the most promising candidates and were further tested for plasmid DNA (pDNA) dynamic binding capacity under ion-exchange (IEX) and HIC binding conditions and ability to separate open circular (OC) from supercoiled (SC) pDNA forms and RNA from pDNA. Since monolith bearing combination of grafted C4 methacrylate groups and DEAE groups was superior in all three tested features, exhibiting pDNA dynamic binding capacity of 4.7 mg/ml under IEX conditions and 2.1 mg/ml under HIC conditions, it was used for the development of a single step purification method and tested with pure pDNA as well as with cell lysate. Developed method removed over 99% of RNA, host cell proteins (HCP) and genomic DNA (gDNA) demonstrating capacity to purify around 1.5 mg of pDNA/ml of monolith from cell lysate.
Nucleic Acids: Innovative Methods for Recovery, Clarification and Purification
2014
The importance of nucleic acids in pure form for preparative and analytical perspectives, have increased constantly, demanding the development of new and more efficient methods for their recovery and isolation. This thesis describes a series of different innovative methods for recovery and purification of these biomolecules. In a general overview of a downstream processing, there are several critical steps that may influence the yield and quality of the final product, especially in the lysis, clarification and purification steps. The lysis process is the first critical step, since it can be detrimental to the nucleic acids and it also can release all the content from the cells by its disruption. A new approach was developed in order to minimize this effect and reduce the main host cell contaminants. The electropermeability approach for extraction of small nucleic acids from bacteria avoids complete cell disruption and thereby reduces contaminants for the next stages. The clarification and concentration steps are normally done to remove most of the contaminants from the cell lysis steps. In this thesis an aqueous two-phase system (ATPS) approach based on poly(ethylene glycol)/poly(acrylate)/salt two phase system is described. This method allows the handling of large volumes, which is important in the preparative industrial scale. ATPS resulted in a pure plasmid DNA directly from crude cell lysates. In addition, smaller DNA fragments from Polymerase Chain Reaction (PCR) can be isolated using this method. The regular downstream process is normally finished by chromatographic approaches, which often are the main final purification step. DNA molecules harbour some intrinsic chemical properties that render them suitable for chromatographic separations. These include a negatively charged phosphate backbone as well as a hydrophobic character originating mainly from the major groove of DNA which exposes the base pairs on the surface of the molecule. In addition, single stranded DNA often allows for a free exposure of the hydrophobic aromatic bases. In this thesis, multimodal chromatography (MMC) was evaluated as an alternative tool for complex separations of nucleic acids. MMC embraces more than one kind of interaction between the chromatographic ligand and the target molecules. This resin demonstrated a superiority for DNA isolation, including in the purification of pDNA from crude cell lysate and for DNA fragments from PCR samples.
Scalable recovery of plasmid DNA based on aqueous two-phase separation
Biotechnology and Applied Biochemistry, 2005
Future developments in gene therapy and DNA vaccination depend on cost-effective large-scale production of pharmaceutical-grade pDNA (plasmid DNA). Given the large amount of impurities present in the feedstock, purification processes that have high specificity and capacity at a moderate cost are required. In the present study, we describe a non-chromatographic procedure based on aqueous two-phase extraction allowing a fast and simply scalable capture step. PEG [poly(ethylene glycol)] in combination with potassium citrate or potassium phosphate was tested as phase component for extraction. By increasing either PEG or salt concentration, the partitioning of nucleic acids changed from bottom to top phase. Phase systems with a composition of 15 % PEG 800 and 20 % potassium phosphate at pH 7.0 showed a strong partitioning of pDNA to the bottom phase, linked to a clear decrease in open circular pDNA, while proteins, genomic DNA and RNA remain at the top or at the interphase. A great advantage of the current process is that the complete procedure of lysis, precipitation, clarification and extraction can be performed in a single vessel. The number of denatured and sheared genomic DNAs in a spiking experiment was found to be depleted by more than 99 %.
Isolation of plasmid DNA from cell lysates by aqueous two-phase systems
Biotechnology and Bioengineering, 2002
This work presents a study of the partitioning of a plasmid vector containing the cystic fibrosis gene in polyethylene glycol (PEG)/salt (K2HPO4) aqueous two-phase systems (ATPS). The plasmid was extracted from neutralized alkaline lysates using PEG with molecular weights varying from 200 to 8000. The effects of the lysate mass loaded to the ATPS (20, 40, and 60% w/w) and of the plasmid concentration in the lysate were evaluated. The performance of the process was determined by qualitative and quantitative assays, carefully established to overcome the strong interference of impurities (protein, genomic DNA, RNA), salt, and PEG.Plasmid DNA partitioned to the top phase when PEG molecular weight was lower than 400. The bottom phase was preferred when higher PEG molecular weights were used. Aqueous two-phase systems with PEG 300, 600, and 1000 were chosen for further studies on the basis of plasmid and RNA agarose gel analysis and protein quantitation.The recovery yields were found to be proportional to the plasmid concentration in the lysate. The best yields (>67%) were obtained with PEG 1000. These systems (with 40 and 60% w/w of lysate load) were able to separate the plasmid from proteins and genomic DNA, but copartitioning of RNA with the plasmid was observed. Aqueous two-phase systems with PEG 300 concentrated both plasmid and proteins in the top phase. The best system for plasmid purification used PEG 600 with a 40% (w/w) lysate load. In this system, RNA was found mostly in the interphase, proteins were not detected in the plasmid bottom phase and genomic DNA was reduced 7.5-fold. © 2002 Wiley Periodicals, Inc. Biotechnol Bioeng 78: 376–384, 2002.
DNA extraction methods such as plasmid minipreps, gel, and PCR purifications, are indispensable techniques for genetic manipulations. There are numerous factors that contribute to the efficiency of these processes, which determine the success of complex downstream molecular analytics and diagnostic tests. To study and optimize these factors, we compared our own proprietary buffers to commercially available column-based kits, utilizing their spin columns and protocols. Through systematic substitution of the buffers in the kits with our own proprietary buffers, we selected the highest DNA yielding buffer recipes. Further analysis of the differences between the buffers showed that high concentrations and presence of certain chaotropic agents and cations are necessary for good plasmid miniprep, gel extraction, and PCR purification kits.
Chromatographic purification of plasmid DNA on hydrophobic methacrylate monolithic supports
Separation and Purification Technology, 2015
Due to stringent requirements for the purity and efficacy of the plasmid DNA (pDNA) as a pharmaceutical product, chromatography is often used in the downstream process. Monolithic stationary phases exhibit several advantages over the conventional beaded supports mainly due to the convective flow and higher surface accessibility and are efficient chromatographic supports for separation of large biomolecules such as pDNA. High ligand density butyl-modified (C4 HLD) monolithic support is currently used in a polishing step of a pDNA purification process in CIM™ HiP 2 Plasmid Process Pack and the goal of the present work was a development of a hydrophobic methacrylate monolith with improved resolution for pDNA isoforms separation and removal of host contaminants. After a detailed search for an appropriate ligand, a pyridine-modified monolithic support was chosen and tested under descending ammonium sulfate linear gradient. The purification process was optimized according to the most efficient pDNA isoforms separation and the quantification of the main impurities during the purification steps was performed, as well as the purity and the recovery of eluted supercoiled (sc) pDNA isoform. Usage of pyridine-modified monoliths resulted in a more efficient separation between pDNA isoforms, with a similar dynamic binding capacity and recovery as C4 HLD monoliths (over 3 mg/mL and 90%, respectively). Pyridine and C4 HLD monoliths were equally efficient for the removal of the main process impurities, but pyridine exhibited higher purity in terms of sc pDNA homogeneity (98%) comparing with C4 HLD (95%), showing to be a suitable alternative to C4 HLD for this polishing step.