Fractionation of protein, RNA, and plasmid DNA in centrifugal precipitation chromatography using cationic surfactant CTAB containing inorganic salts NaCl and NH4Cl (original) (raw)
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Biotechnology Progress, 2006
Fractionation of clarified E. coli lysate components in bench-scale and preparative-scale centrifugal precipitation chromatography (CPC), using a solution of cationic surfactant cetyltrimethylammonium bromide (CTAB) containing 0.5 M NaCl as precipitant, are compared here. Step gradient of CTAB from 0.50% to 0.16% (w/v) gave a successful fractionation in bench-scale CPC; however, a linear gradient of lower CTAB concentration, 0.20–0% (w/v), was used in the preparative scale and resulted in similar fractionation. The preparative-scale CPC has a superior sample loading capacity by the use of tubular dialysis membrane inside convoluted tubing as the separation channel. In this study, the quantity of the sample loaded into the preparative CPC was about 15 times more than that in the bench scale, and in a single run the preparative CPC could prepare approximately 3 mg of plasmid DNA with about 96% of RNA removed. The higher surface area per length of the separation channel in the preparative CPC was believed to benefit mass transfer of CTAB across the membrane, leading to less CTAB being required in the process.
Selective precipitation of plasmid with a water-soluble cationic surfactant
Polymers for Advanced Technologies, 2009
The aim of the present study was the selective precipitation of plasmid directly from alkaline lysate produced from bacterial culture with the cationic detergent, N, N' , N'-polyethylene (10)-N tallow-1, 3-di amine propane. Polyethylene groups of peg-10 tallow were functionalized with tertiary amine groups and long alkyl chain. The functional groups of cationic detergent were determined by means of 1 H nuclear magnetic resonance (NMR) spectroscopy. A simple and efficient process for plasmid purification by cationic detergent was found to be dependent on acid concentration. The structure of peg-10 tallow cationic detergent after adding acid solution was characterized using Fourier transform infrared (FT-IR) spectroscopy. Inorganic acid such as HCl was introduced into the solution of peg-10 tallow. Controlling pH of precipitation system by the addition of different concentrations of HCl acid solutions on peg-10 tallow improved the partial separation of RNA from plasmid DNA (pDNA). However, different lysate masses were applied to observe the effect of 7.5% acid solution on the separation of RNA from pDNA with a peg-10 tallow cationic surfactant. The results were realized by 1% agarose gel analysis.
Separation and Purification Technology, 2009
The most significant impurities in plasmid DNA (pDNA)-containing solutions after intermediate recovery are host RNA and genomic DNA. Hydrophobic interaction chromatography (HIC) with ammonium sulfatebased buffers has been used to explore the higher hydrophobicity of those impurities when compared with pDNA. Although successful at lab scale, the large amounts of ammonium sulfate involved constitute an important limitation in terms of waste treatment/disposal if HIC is to be used at process scales. In this work, different HIC ligands (phenyl, butyl, octyl), salts and salt concentrations were screened with the specific goal of replacing ammonium sulfate with an environmentally friendly salt. The combination of phenyl-Sepharose with sodium citrate at high concentrations (≥1.2 M) not only allowed the separation of pDNA from RNA, gDNA, endotoxins and protein, but also enabled the separation of supercoiled from open circle isoforms. Alternatively, by exploring negative HIC with the phenyl matrix and 1.0 M sodium citrate, it was possible to purify total pDNA from solutions obtained by tangential flow filtration and pre-conditioned with sodium citrate. At the highest loading tested (2 mL feed equivalent to ca. 1 mg pDNA per mL of bed) despite high-level removal of endotoxins (>98.5%) and proteins (>82%) the purity of recovered plasmid DNA determined by HIC-HPLC was just 73% (primarily due to traces of RNA and gDNA). Much higher purities (close to 100%) were achieved when lower loadings were employed. In summary, this study clearly shows that despite lower purification factors achieved with sodium citrate compared to ammonium sulfate-based HIC, replacing the latter salt with sodium citrate is indeed feasible.
Journal of Chromatography A, 2005
The current study explores the possibility of using a polyethyleneglycol(PEG)/ammonium sulphate aqueous two-phase system (ATPS) as an early step in a process for the purification of a model 6.1 kbp plasmid DNA (pDNA) vector. Neutralised alkaline lysates were fed directly to ATPS. Conditions were selected to direct pDNA towards the salt-rich bottom phase, so that this stream could be subsequently processed by hydrophobic interaction chromatography (HIC). Screening of the best conditions for ATPS extraction was performed using three PEG molecular weights (300, 400, 600) and varying the tie-line length, phase volume ratio and lysate load. For a 20 % (w/w) lysate load, the best results were obtained with PEG 600 using the shortest tie-line (38.16 % w/w). By further manipulating the system composition along this tie-line in order to obtain a top/bottom phase volume ratio of 9.3 (35 % w/w PEG 600, 6% w/w NH 4 ) 2 SO 4 ), it was possible to recover 100 % of pDNA in the bottom phase with a 3fold increase in concentration. Further increase in the lysate load up to 40 %(w/w) with this system resulted in a 8-fold increase in pDNA concentration, but with a yield loss of 15 %. The ATPS extraction was integrated with HIC and the overall process compared with a previously defined process that uses sequential precipitations with isopropanol and ammonium sulphate prior to HIC. Although the final yield is lower in the ATPS-based process the purity grade of the final pDNA product is higher. This shows that it is possible to substitute the time-consuming two-step precipitation procedure by a simple ATPS extraction.
Precipitation by polycation as capture step in purification of plasmid DNA from a clarified lysate
Biotechnology and Bioengineering, 2004
The demand for highly purified plasmids in gene therapy and plasmid-based vaccines requires large-scale production of pharmaceutical-grade plasmid. Large-scale purification of plasmid DNA from bacterial cell culture normally includes one or several chromatographic steps. Prechromatographic steps include precipitation with solvents, salts, and polymers combined with enzymatic degradation of nucleic acids. No method alone has so far been able to selectively capture plasmid DNA directly from a clarified alkaline lysate. We present a method for selective precipitation of plasmid DNA from a clarified alkaline lysate using polycation poly(N, N V-dimethyldiallylammonium) chloride (PDMDAAC). The specific interaction between the polycation and the plasmid DNA resulted in the formation of a stoichiometric insoluble complex. Efficient removal of contaminants such as RNA, by far the major contaminant in a clarified lysate, and proteins as well as 20-fold plasmid concentration has been obtained with about 80% recovery. The method utilizes a inexpensive, commercially available polymer and thus provides a capture step suitable for large-scale production. B 2004 Wiley Periodicals, Inc.
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.
Journal of Chromatography B, 2012
Several small molecules, like some therapeutic agents, are able to bind DNA with high specificity. These may represent a relevant alternative as ligands in affinity and pseudoaffinity chromatographic processes for plasmid DNA (pDNA) purification. In the present study, berenil (a DNA intercalator used an anti-trypanosomal agent in veterinary applications) was tested as a ligand to specifically purify plasmids with different sizes, pVAX1-LacZ (6.05 Kbp) and pCAMBIA-1303 (12.361 Kbp) from the impurities present in Escherichia coli alkaline lysates. For this purpose, chromatographic experiments were set using Sepharose derivatized with berenil. The results showed that both pDNA molecules are completely purified using smaller amounts of salt in the eluent than those reported before for other pseudo-affinity and hydrophobic chromatography based processes. Total retention of all lysate components was achieved with 1.3 M ammonium sulphate in the eluent buffer and pDNA elution was obtained by decreasing the salt concentration to 0.55 M. All impurities were eluted after decreasing the concentration to 0 M. The recovery yield for the larger pDNA molecule pCAMBIA-1303 (45%) is lower than that obtained for pVAX1-LacZ (85%), presenting however a higher final purity. Furthermore, pVAX1-LacZ purification studies were also performed using non-clarified E. coli process streams, replacing the clarification step with a second chromatographic run on the berenil-Sepharose support. Using the same binding and elution conditions as before, a pure plasmid sample was obtained with a 33% yield. The pDNA fractions were analysed for E. coli host impurities, and all levels were in accordance to the requirements established by the regulatory agencies (FDA). These results suggest that this chromatographic support is a promising alternative to purify pDNA for therapeutic use.
Bioprocess and Biosystems Engineering, 2009
A new bioprocess using mainly membrane operations to obtain purified plasmid DNA from Escherechia coli ferments was developed. The intermediate recovery and purification of the plasmid DNA in cell lysate was conducted using hollow-fiber tangential filtration and tandem anion-exchange membrane chromatography. The purity of the solutions of plasmid DNA obtained during each process stage was investigated. The results show that more than 97% of RNA in the lysate was removed during the process operations and that the plasmid DNA solution purity increased 28-fold. One of the main characteristics of the developed process is to avoid the use of large quantities of precipitating agents such as salts or alcohols. A better understanding of membrane-based technology for the purification of plasmid DNA from clarified E. coli lysate was developed in this research. The convenience of anionexchange membranes, configured in ready-to-use devices can further simplify large-scale plasmid purification strategies.
Journal of Chromatography A, 2011
Plasmid DNA (pDNA) is purified directly from alkaline lysis-derived Escherichia coli (E. coli) lysates by phenyl boronate (PB) chromatography. The method explores the ability of PB ligands to bind covalently, but reversibly, to cis-diol-containing impurities like RNA and lipopolysaccharides (LPS), leaving pDNA in solution. In spite of this specificity, cis-diol free species like proteins and genomic DNA (gDNA) are also removed. This is a major advantage since the process is designed to keep the target pDNA from binding. The focus of this paper is on the study of the secondary interactions between the impurities (RNA, gDNA, proteins, LPS) in a pDNA-containing lysate and 3-amino PB controlled pore glass (CPG) matrices. Runs were designed to evaluate the role of adsorption buffer composition, feed type (pH, salt content), CPG matrix and sample pretreatment (RNase A, isopropanol precipitation). Water was chosen as the adsorption buffer over MgCl 2 solutions since it maximised pDNA yield (96.2 ± 4.9%) and protein removal (61.3 ± 3.0%), while providing for a substantial removal of RNA (65.5 ± 3.5%) and gDNA (44.7 ± 14.1%). Although the use of pH 3.5 maximised removal of impurities (∼75%), the best compromise between plasmid yield (∼96%) and RNA clearance (∼60-70%) was obtained for a pH of 5.2. Plasmid yield was maximal (>96%) when the concentration of acetate and potassium ions in the incoming lysate feed were 1.7 M and 1.0 M, respectively. The pre-treatment of lysates with RNase A deteriorated the performance since the resulting oligoribonucleotides lack the cis-diol group at their 3 termini. Overall, the results support the idea that charge transfer interactions between the boron atom at acidic pH and electron donor groups in the aromatic bases of nucleic acids and side residues of proteins are responsible for the non-specific removal of gDNA, RNA and proteins.