Quantitative Analysis of Plasmid Forms by Agarose and Capillary Gel Electrophoresis

Schmidt, Torsten; Friehs, Karl; Schleef, Martin; Voss, Carsten; Flaschel, Erwin

doi:10.1006/abio.1999.4291

Cited by 67 publications

(37 citation statements)

References 14 publications

(14 reference statements)

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“…This is the most powerful technique to separate the different DNA isoforms, and it is described in detail by M. Schleef in this issue, and in [55].…”

Section: Capillary Gel Electrophoresis (Cge)mentioning

confidence: 98%

Plasmid DNA purification

Stadler¹,

Lemmens²,

Nyhammar³

2004

J. Gene Med.

172

113

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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.

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“…This is the most powerful technique to separate the different DNA isoforms, and it is described in detail by M. Schleef in this issue, and in [55].…”

Section: Capillary Gel Electrophoresis (Cge)mentioning

confidence: 98%

Plasmid DNA purification

Stadler¹,

Lemmens²,

Nyhammar³

2004

J. Gene Med.

172

113

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“…The plasmid pUK21 (PlasmidFactory) was used as a reference standard. CGE analyses were performed using a Beckman P/ACE MDQ instrument equipped with an laser induced fluorescence (LIF) detector (488/520 nm) as described (Schmidt, 1999;Walther, 2003). Briefly, coated capillaries (DB-17; J&W Scientific) with an effective length of 30 cm to the detector window, an inner diameter of 100 lm, and a coating thickness of 0.1 lm were used.…”

Section: Cge Analysis Of Plasmid Topologymentioning

confidence: 99%

“…We compared the topology of this 7-year-old plasmid DNA with that of newly produced DNA batches of the same plasmid. Since DNA topology is easily and reliably detectable by CGE, this method was used for the quantification of the eventual topological variations (ccc vs. non-ccc) of the plasmid DNAs and the assessment of DNA quality (Schmidt et al, 1999). CGE analysis defines plasmid isoforms by respective peak migration, and area as area/migration time ratio.…”

Section: Analyses Of Plasmid Dna Topologies By Cge and Agementioning

confidence: 99%

A Seven-Year Storage Report of Good Manufacturing Practice–Grade Naked Plasmid DNA: Stability, Topology, andIn Vitro/In VivoFunctional Analysis

Walther

Schmeer²,

Kobelt³

et al. 2013

Human Gene Therapy Clinical Development

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The great interest for naked plasmid DNA in gene therapy studies is reflected by the fact that it is currently used in 18% of all gene therapy trials. Therefore, validation of topology and functionality of DNA resulting from its long-term stability is an essential requirement for safe and effective gene transfer. To this aim, we analyzed the stability of good manufacturing practice-grade pCMVb reporter plasmid DNA by capillary gel electrophoresis, agarose gel electrophoresis, and atomic force microscopy. The plasmid DNA was produced for a clinical gene transfer study started in 2005 and was stored for meanwhile 7 years under continuously monitored conditions at -20°C. The stability of plasmid DNA was monitored by LacZ transgene expression functional assays performed in vitro and in vivo on the 7-year-old plasmid DNA samples compared with plasmid batches newly produced in similar experimental conditions and quality standards. The analyses revealed that during the overall storage time and conditions, the proportion of open circular and supercoiled or covalently closed circular forms is conserved without linearization or degradation of the plasmid. The in vitro transfection and the in vivo jetinjection of DNA showed unaltered functionality of the long-stored plasmid. In summary, the 7-year-old and the newly produced plasmid samples showed similar topology and expression performance. Therefore, our stable storage conditions are effective to preserve the integrity of the DNA to be used in clinical studies. This is an important prerequisite for the long-term performance of gene transfer materials used in trials of long duration as well as of the reference material used in standardization procedures and assays.

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“…Analytical techniques based on capillary gel electrophoresis (CGE) have been developed to analyze segregative and structural stability of plasmid DNA during cultivation [69,70]. The determination and quantification of plasmid forms by CGE can be achieved with high accuracy and reproducibility which can not be accomplished by conventional techniques like agarose gel electrophoresis.…”

Section: Stability Of Plasmid Dnamentioning

confidence: 99%

Downstream Processing of Plasmid DNA for Gene Therapy and Genetic Vaccination

Voss

2008

Chem Eng & Technol

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Plasmid DNA is used as a cloning vector to deliver recombinant genetic information into microorganisms. Since the 1990s, this principle has also been applied for the delivery of therapeutic genes in gene therapy and genetic vaccination. This non-viral gene delivery is afflicted with fewer safety concerns in comparison to viral systems. Processes for the production of high-quality plasmid DNA at multiand kilogram scale are necessary to meet the needs of clinical trials as well as future therapeutics. Cell disruption, the separation of structurally-related impurities and analytical techniques for process and quality control are the main challenges for bioengineering. This review summarizes the development in these fields over the past recent years.

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Quantitative Analysis of Plasmid Forms by Agarose and Capillary Gel Electrophoresis

Cited by 67 publications

References 14 publications

Plasmid DNA purification

Plasmid DNA purification

A Seven-Year Storage Report of Good Manufacturing Practice–Grade Naked Plasmid DNA: Stability, Topology, andIn Vitro/In VivoFunctional Analysis

Downstream Processing of Plasmid DNA for Gene Therapy and Genetic Vaccination

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