Efficient purification of plasmid DNA for gene transfer using triple-helix affinity chromatography (original) (raw)
Related papers
DNA purification by triple-helix affinity precipitation
Biotechnology and Bioengineering, 2003
Recent advances in DNA-based medicine (gene therapy, genetic vaccination) have intensified the necessity for pharmaceutical-grade plasmid DNA purification at comparatively large scales. In this contribution triplehelix affinity precipitation is introduced for this purpose. A short, single-stranded oligonucleotide sequence (namely (CTT) 7), which is capable of recognizing a complementary sequence in the double-stranded target (plasmid) DNA, is linked to a thermoresponsive Nisopropylacrylamide oligomer to form a so-called affinity macroligand (AML). At 4°C, i.e., below its critical solution temperature, the AML binds specifically to the target molecule in solution; by raising the temperature to 40°C, i.e., beyond the critical solution temperature of the AML, the complex can be precipitated quantitatively. After redissolution of the complex at lower temperature, the target DNA can be released by a pH shift to slightly alkaline conditions (pH 9.0). Yields of highly pure (plasmid) DNA were routinely between 70% and 90%. Non-specific coprecipitation of either the target molecule by the nonactivated AML precursor or of contaminants by the AML were below 7% and presumably due to physical entrapment of these molecules in the wet precipitate. Ligand efficiencies were at least 1 order of magnitude higher than in triple-helix affinity chromatography.
The Journal of Gene …, 2009
Background It is well known that the success of gene transfer to cells and subsequent expression is strictly affected by the vector manufacturing process. Several challenges encountered in the gene therapy field have emphasized the need for the development of novel platforms that allow the recovery of gene vectors and enable efficient transfection of cells. The use of plasmid DNAbased therapeutics relies on procedures that efficiently purify the supercoiled (sc) plasmid isoform. Plasmid DNA (pDNA) purification strategies that use amino acids as immobilized ligands have recently yielded interesting results. Methods The present study describes a strategy that uses argininechromatography to specifically purify sc pDNA from other isoforms and Escherichia coli impurities present in a clarified lysate. Results Control analysis shows that the purity of the sc pDNA is 100% with a homogeneity higher than 97% of sc. Furthermore, no RNA was detectable, the protein content was lower than 10 µg/ml and a 117-fold reduction on genomic DNA contamination and 95% endotoxin removal were accomplished. The chromatographic process demonstrated an impressive performance on sc isoform recovery (79% yield). Furthermore, the sc transfection efficiency of COS-7 cells (62%) was significantly higher compared to the efficiency (25%) achieved with a pDNA control. Conclusions With the simplified sc pDNA purification process, a high yield was achieved, sc pDNA was purified under mild conditions and shown to be extremely efficient with respect to cell transfection. Arginine-chromatography is thus an interesting option for use as a late stage plasmid purification step.
Biomolecular Engineering, 2005
Low cellular uptake and poor nuclear transfer hamper the use of non-viral vectors in gene therapy. Addition of functional entities to plasmids using the Bioplex technology has the potential to improve the efficiency of transfer considerably. We have investigated the possibility of stabilizing sequence-specific binding of peptide nucleic acid (PNA) anchored functional peptides to plasmid DNA by hybridizing PNA and locked nucleic acid (LNA) oligomers as ''openers'' to partially overlapping sites on the opposite DNA strand. The PNA ''opener'' stabilized the binding of ''linear'' PNA anchors to mixed-base supercoiled DNA in saline. For higher stability under physiological conditions, bisPNA anchors were used. To reduce nonspecific interactions when hybridizing highly cationic constructs and to accommodate the need for increased amounts of bisPNA when the molecules are uncharged, or negatively charged, we used both PNA and LNA oligomers as ''openers'' to increase binding kinetics. To our knowledge, this is the first time that LNA has been used together with PNA to facilitate strand invasion. This procedure allows hybridization at reduced PNA-to-plasmid ratios, allowing greater than 80% hybridization even at ratios as low as 2:1. Using significantly lower amounts of PNA-peptides combined with shorter incubation times reduces unspecific binding and facilitates purification. #
Affinity Chromatography Approaches to Overcome the Challenges of Purifying Plasmid DNA
Trends in biotechnology, 2008
The diversity of biomolecules present in plasmid DNA (pDNA)-containing extracts and the structural and chemical similarities between pDNA and impurities are some of the main challenges of improving or establishing novel purification procedures. In view of the unequalled specificity of affinity purification, this technique has recently begun to be applied in downstream processing of plasmids. This paper discusses the progress and importance of affinity chromatography (AC) for the purification of pDNA-based therapeutic products. Several affinity approaches have already been successfully developed for a variety of applications, and we will focus here on highlighting their possible contributions to the pDNA purification challenge. Diverse affinity applications and their advantages and disadvantages are discussed, as well as the most significant results and improvements in the challenging task of purifying plasmids.
Use of locked nucleic acid oligonucleotides to add functionality to plasmid DNA
Nucleic Acids Research, 2003
The available reagents for the attachment of functional moieties to plasmid DNA are limiting. Most reagents bind plasmid DNA in a non-sequence-speci®c manner, with unde®ned stoichiometry, and affect DNA charge and delivery properties or involve chemical modi®cations that abolish gene expression. The design and ability of oligonucleotides (ODNs) containing locked nucleic acids (LNAs) to bind supercoiled, double-stranded plasmid DNA in a sequence-speci®c manner are described for the ®rst time. The main mechanism for LNA ODNs binding plasmid DNA is demonstrated to be by strand displacement. LNA ODNs are more stably bound to plasmid DNA than similar peptide nucleic acid (PNA)`clamps' for procedures such as particlemediated DNA delivery (gene gun). It is shown that LNA ODNs remain associated with plasmid DNA after cationic lipid-mediated transfection into mammalian cells. LNA ODNs can bind to DNA in a sequence-speci®c manner so that binding does not interfere with plasmid conformation or gene expression. Attachment of CpG-based immune adjuvants to plasmid by`hybrid' phosphorothioate±LNA ODNs induces tumour necrosis factor-a production in the macrophage cell line RAW264.7. This observation exempli®es an important new, controllable methodology for adding functionality to plasmids for gene delivery and DNA vaccination.
Preparative purification of supercoiled plasmid DNA using anion-exchange chromatography
Journal of Chromatography A, 1998
Large scale manufacturing of gene vectors such as plasmid DNA is an important issue in gene therapy. Anion-exchange chromatography is fundamental in the downstream processing of plasmids both as a process and analytical technique. This work reports the use of Q-Sepharose columns (1, 10 and 40 ml) for the preparative purification of plasmid pUC18. NaCl gradient elution enabled the isolation of supercoiled plasmid from low-M RNA, cDNA and plasmid variants. A compact r covalently closed, supercoiled form of denatured plasmid carrying large stretches of single-stranded DNA was identified as one of the major contaminants. Anion-exchange HPLC on a Poros QE 20 column was used to quantify plasmid yield. Supercoiled plasmid was recovered in a single fraction with a 6268% yield. Loadings higher than 40 mg / ml gel could be used but at the expense of a loss of resolution between open circular and supercoiled forms. Plasmid quality was evaluated by gel electrophoresis, restriction analysis, transformation experiments and protein assays.
Biotechnology Progress, 2005
In contrast to proteins, many nucleic acids can undergo reversible modification of their conformations, and this flexibility can be used to facilitate purification. Selective renaturation with capture is a novel method of removing contaminating genomic DNA from plasmid samples. Plasmid DNA quickly renatures after thermal denaturation and cooling (or alkaline denaturation followed by neutralization), whereas genomic DNA remains locally denatured after rapid cooling in mismatch-stabilizing high ionic strength buffer. Partially denatured genomic DNA can be selectively bound to a metal chelate affinity adsorbent through exposed purine bases, while double-stranded renatured plasmid DNA is not bound. Using this method we have readily achieved 1,000,000-fold clearance of 71 wt % contaminating E. coli genomic DNA from plasmid samples.
Expanded Bed Chromatography, 1999
We describe a process for the commercial manufacture of therapeutic grade plasmid DNA. The industrially scaleable unit operations employed in this process are: (i) optimized alkaline lysis; (ii) bag filtration; (iii) expanded bed anion exchange chromatography; (iv) ultrafiltration, and (v) size exclusion chromatography. These steps are scaleable alternatives to current approaches to plasmid DNA isolation such as high speed centrifugation for feedstock clarification and solvent precipitation for plasmid concentration, and an efficient alternative to conventional low through-put packed bed chromatography. The process produces plasmid DNA characterized by low level chromosomal DNA, RNA and endotoxin contamination without the use of flammable solvents or toxic reagents and is suitable for therapeutic administration.