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

Abstract

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.

A continuous cell alkaline lysis, neutralization, and clarification combination process for production of plasmid pUDK‐HGF

“…In laboratory scale, cell debris, proteins, and chrDNA are coprecipitated with SDS and formed suspended flocculent precipitates by manually gentle mixing. Centrifugation and filtration are most frequently used to separate the precipitate from the plasmid containing solution [8], [13][14][15]. But these methods often result in low yield due to plasmid being trapped within the precipitates.…”

“…To overcome local extreme pH and the shearing force problems, the processes using optimized tanks and stirrers were developed in alkaline-cell lysis [14][15][16], [23]. But these techniques lack reproducibility in industrial-scale production.…”

“…In this lysis process, local extreme pH and shear force decreased the plasmid purity and yield. Local extreme pH (higher than 12.5) will lead to irreversible plasmid denaturation and formation of undesired isoforms [14]. Chromosomal DNA (chrDNA) is extremely sensitive to shear and is easily broken into small DNA fragments (∼10 kb) comparable to the size of the plasmid DNA [6].…”

“…Chemical [13][14][15][16], thermal [17][18][19][20], and mechanical [21] methods can be used; the most common method is alkaline lysis based on the work of Birnboim and Doly [22] using 0.2 N NaOH and 1% sodium dodecyl sulfate (SDS) as lysis reagents. In this lysis process, local extreme pH and shear force decreased the plasmid purity and yield.…”

Rapid, Large-Scale Production of Full-Length, Human-Like Monoclonal Antibodies

“…The broth is concentrated 20 fold from 1000-L to 50-L. Subsequent lysis of E. coli cells are carried out using a modified version of previously reported methods (Ciccolini et al 2002;Meacle et al 2004;Clemson and Kelly 2010). Modifications to this step are especially important due to the potential for pDNA damage from caustic solvents and shear stresses (Lengsfeld and Anchordoquy 2001;Freitas et al 2007).…”