Plasmid DNA purification

Abstract

The demand for efficient production methods of plasmid DNA (pDNA) has increased vastly in response to rapid advances in the use of pDNA in gene therapy and in vaccines since the advantageous safety concerns associated with non-viral over viral vectors.A prerequisite for the success of plasmid-based therapies is the development of cost-effective and generic production processes of pDNA. However, to satisfy strict regulatory guidelines, the material must be available as highly purified, homogeneous preparations of supercoiled circular covalently closed (ccc) pDNA. Large-scale production of pDNA for therapeutic use is a relatively new field in bioprocessing. The shift from small-scale plasmid production for cell transfection to large-scale production sets new constraints on the bacterial fermentation, processing of bacterial lysate and final purification and formulation of the plasmid DNA. The choice of bacterial strain used for plasmid cultivation affects the plasmid yield, the proportion of different isoforms and the amount of endotoxins in the starting material. The choice of bacterial strain will be greatly influenced by the production and purification procedures of pDNA. Master and working cell banks need to be characterised and established. Alkaline lysis of the bacteria damages the pDNA, resulting in a reduced recovery of ccc pDNA and an increase in partially denaturated ccc pDNA and open circular (oc) forms. Shear stress in these processes needs to be tightly controlled, and buffer composition and pH need to be optimised. To obtain a homogeneous plasmid DNA preparation, different pDNA purification strategies aim at capturing ccc pDNA and eliminating the oc isoform. A highly purified final product corresponding to the stringent recommendations set forth by health and regulatory authorities can be achieved by (i). different chromatography techniques integrated with ultra/diafiltration to achieve optimal purification results; (ii). the formulation of the final pDNA product, that requires a detailed study of the plasmid structure; and (iii). the development of sensitive analytical methods to detect different impurities (proteins, RNA, chromosomal DNA, and endotoxins). We present here a revue of the whole process to obtain such a plasmid DNA, and report an example of RNAse-free purification of ccc pDNA that could be used for gene therapy.

Good Manufacturing Practices production and analysis of a DNA vaccine against dental caries

“…Size exclusion chromatography (SEC) was done in our study. It is a simple and reproducible method for plasmid purification that separates the plasmid from protein, RNA, host DNA, and endotoxin in a single chromatographic step [33,34] . The commercial matrices Sepharose 6 Fast Flow gel filtration media were developed specifically to meet the highthroughput demands of industrial process separations.…”

Angiogenic and antiangiogenic gene therapy

“…Similarly, the cellular fate of the plasmid is not well understood although convincing data are still being published. [67][68][69] So far, three possible mechanisms of plasmid uptake are under consideration: large membrane disruption, small membrane pores and receptormediated endocytosis. [67][68][69] Generally, gene therapy transfer systems are divided into two groups: viral and nonviral.…”

“…[67][68][69] So far, three possible mechanisms of plasmid uptake are under consideration: large membrane disruption, small membrane pores and receptormediated endocytosis. [67][68][69] Generally, gene therapy transfer systems are divided into two groups: viral and nonviral. The nonviral as well as viral delivery systems have visible obstacles and they are under dynamically enlarged evaluation.…”

“…The naked plasmid DNA effectively transfects the skeletal muscles or heart and successfully expresses angiogenic genes that are due to new vessel formation and the improvement of the clinical state of patients. [67][68][69] It is rather common knowledge that naked DNA is expressed in vivo after simple intramuscular injections. [67][68][69] However, the efficiency of transfection and expression is still low and still limits larger applications of naked DNA strategy in the clinic.…”

“…[67][68][69] It is rather common knowledge that naked DNA is expressed in vivo after simple intramuscular injections. [67][68][69] However, the efficiency of transfection and expression is still low and still limits larger applications of naked DNA strategy in the clinic. As described by numerous studies, electroporation in vivo technique may be considered as a method that results in increased levels of plasmid transfection in vivo.…”