Biochemical engineering approaches to the challenges of producing pure plasmid DNA

Levy, M. Susana; O’Kennedy, Ronan; Ayazi-Shamlou, Parviz; Dunnill, P.

doi:10.1016/s0167-7799(00)01446-3

Cited by 170 publications

(126 citation statements)

References 49 publications

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“…As reviewed by Lengsfeld and Anchordoquy [2], the most important parameters that influence shear-induced breaks of macro-DNA are strain rate, ionic strength, gas-liquid interface and turbulence, in addition to DNA molecular weight, size, and flexibility. Recently, concerns about DNA degradation have been raised in relation to the mass production of high quality plasmids for use in DNA therapeutic strategies [3,10,11,14]. Various bio-processing steps utilized in mass production such as lysis, solid removal, purification, and finishing would impart physical stresses that can induce undesirable degradation of target plasmids [3].…”

Section: Introductionmentioning

confidence: 99%

“…Recently, concerns about DNA degradation have been raised in relation to the mass production of high quality plasmids for use in DNA therapeutic strategies [3,10,11,14]. Various bio-processing steps utilized in mass production such as lysis, solid removal, purification, and finishing would impart physical stresses that can induce undesirable degradation of target plasmids [3]. Although a substantial amount of knowledge has been accumulated regarding the physical degradation of DNA, it does not seem to be readily applicable to routine laboratory environments.…”

Section: Introductionmentioning

confidence: 99%

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Flow cytometric investigation on degradation of macro-DNA by common laboratory manipulations

Yoo¹,

Lim²,

Yang³

et al. 2011

JBPC

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The degree and characteristics of physical degradation of macro-DNA molecules by common laboratory manipulations are reported. With linearized lambda-phage viral DNA as the model DNA, fragmentation of macro-DNA by various indispensable laboratory manipulations were investigated using a high sensitivity flow cytometric setup. Investigated manipulations included pipetting, vortexing, rocking, freezethawing, ultrasonication and ultrafiltration. "Exhaustive counting" of the intact lambda DNA molecules following such manipulations enabled a quantitative assessment of the resulting fragmentation, which also revealed the type of degradation reflected in the fragmentation patterns. The use of high sensitivity flow cytometry was especially suited to investigate the degradation of dilute DNA solutions that may not be suitable for analysis using traditional methods. Notable findings of this study included: the boarderline-size of DNA chains in terms of susceptibility to shear stresses by such manipulations; discernable instability of nicked DNAs; shattering-fragmentation of DNAs by freezethawing or ultrasonication; effectiveness of some protection media; marked "self-protection effect" of concentrated DNA solutions. These findings support and refine our traditional knowledge on how to maintain the physical integrity of macro-DNA molecules against inevitable laboratory manipulations.

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Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Flow cytometric investigation on degradation of macro-DNA by common laboratory manipulations

Yoo¹,

Lim²,

Yang³

et al. 2011

JBPC

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“…Convective transport through the pores has been hypothesized [12]. Theoretical studies suggest that a certain fraction of the liquid passes the pores at very high velocities, this happens at intraparticle Peclet numbers higher than 50. Irrespective of that phenomenon these materials seem a priori well suited for plasmid separation, since pore diameter exceeds approximately five times the hydrodynamic diameter of an average sized plasmid [13]. Tiainen et al [11] introduced nonporous silica fibres suited for column chromatography and having a surface area of 2.4 m 2 /g.…”

Section: Introductionmentioning

confidence: 99%

Adsorption of plasmid DNA on anion exchange chromatography media

Tarmann

Jungbauer²

2008

J of Separation Science

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Original PaperAdsorption of plasmid DNA on anion exchange chromatography media Anion exchange chromatography (AEC) is a useful and effective tool for DNA purification, but due to average pore sizes between 40 and 100 nm most AEC resins lack truly useful binding capacities for plasmid DNA (pDNA). Equilibrium binding capacities and uptake kinetics of AEC media including conventional media (Source 30 Q, Q Sepharose HP), a polymer grafted medium (Fractogel EMD DEAE (M)), media with large pores (Celbeads DEAE, PL SAX 4000 30 lm) and a monolithic medium (CIM-DEAE) were investigated by batch uptake or shallow bed experiments at two salt concentrations. Theoretical and experimental binding capacities suggest that the shape of the pDNA molecule can be described by a rod with a length to diameter ratio of 20:1 and that the molecule binds in upright position. The arrangement of DNA like a brush at the surface can be considered as entropy driven, kind of selfassembly process which is inherent to highly and uniformly charged DNA molecules. The initial phase of adsorption is very fast and levels off, associated with a change in mass transfer mechanism. Feed concentrations higher than 0.1 mg/mL pDNA pronounce this effect. Monolithic media showed the fastest adsorption rate and highest binding capacity with 13 mg pDNA per mL.

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“…The potential of widespread use of therapeutic genes for treatment of acquired diseases such as cancer, hepatitis and HIV, and the potential for high plasmid doses, means that the need to resolve issues related to the processing of plasmid DNA on a production scale is becoming increasingly important (Levy et al, 2000;Prazeres et al, 1999).…”

Section: Introductionmentioning

confidence: 99%

Impact of engineering flow conditions on plasmid DNA yield and purity in chemical cell lysis operations

Meacle

Lander

Shamlou

et al. 2004

Biotech & Bioengineering

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Chemical lysis of bacterial cells using an alkaline solution containing a detergent may provide an efficient scalable means for selectively removing covalently closed circular plasmid DNA from high-molecular-weight contaminating cellular components including chromosomal DNA. In this article we assess the chemical lysis of E. coli cells by SDS in a NaOH solution and determine the impact of pH environment and shear on the supercoiled plasmid and chromosomal DNA obtained. Experiments using a range of plasmids from 6 kb to 113 kb determined that in an unfavorable alkaline environment, where the NaOH concentration during lysis is greater than 0.15 F 0.03 M (pH 12.9 F 0.2), irreversible denaturation of the supercoiled plasmid DNA occurs. The extent of denaturation is shown to increase with time of exposure and NaOH concentration. Experiments using stirred vessels show that, depending on NaOH concentration, moderate to high mixing rates are necessary to maximize plasmid yield. While NaOH concentration does not significantly affect chromosomal DNA contamination, a high NaOH concentration is necessary to ensure complete conversion of chromosomal DNA to single-stranded form. In a mechanically agitated lysis reactor the correct mixing strategy must balance the need for sufficient mixing to eliminate potential regions of high NaOH concentrations and the need to avoid excessive breakage of the shear sensitive chromosomal DNA. The effect of shear on chromosomal DNA is examined over a wide range of shear rates (10 1 À10 5 s À1) demonstrating that, while increasing shear leads to fragmentation of chromosomal DNA to smaller sizes, it does not lead to significantly increased chromosomal DNA contamination except at very high shear rates (about 10 4 À105 s À1). The consequences of these effects on the choice of lysis reactor and scale-up are discussed.

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Biochemical engineering approaches to the challenges of producing pure plasmid DNA

Cited by 170 publications

References 49 publications

Flow cytometric investigation on degradation of macro-DNA by common laboratory manipulations

Flow cytometric investigation on degradation of macro-DNA by common laboratory manipulations

Adsorption of plasmid DNA on anion exchange chromatography media

Impact of engineering flow conditions on plasmid DNA yield and purity in chemical cell lysis operations

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