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

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 +/- 0.03 M (pH 12.9 +/- 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)-10(5) s(-1)) 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)-10(5) s(-1)). The consequences of these effects on the choice of lysis reactor and scale-up are discussed.

Formation of LID vector complexes in water alters physicochemical properties and enhances pulmonary gene expression in vivo.

There is currently an urgent need to develop efficient gene-delivery systems for the lung that are free of inflammatory effects. The LID vector is a synthetic gene delivery system, comprised of lipofectin (L), an integrin-targeting peptide (I) and DNA (D) that has previously been shown to have high transfection efficiency in the lung. We have assessed the effect of alternative methods of complex preparation on structural features of the complex, levels and duration of reporter gene expression and the host response to the LID vector. We have demonstrated that making the complex in water affects the structure of the LID complexes making them smaller and more stable with a more cationic surface charge than complexes prepared in phosphate-buffered saline (PBS). When the LID vector was constituted in water and instilled intratracheally into the lungs of mice there was a 10-fold increase in luciferase activity compared with preparation in PBS. Furthermore, luciferase activity was still evident 1 week following vector instillation. This enhancement may be because of altered complex structure, although effects of the hypotonic vector solution on the lung cannot be excluded. The inflammatory effects of instilling the LID vector in water were minimal, even after three administrations of the LID vector, with only mild alterations in cytokine and broncho-alveolar lavage fluid (BALF) cell profiles. These results demonstrate that the LID vector can generate high, and prolonged, levels of gene expression in the lung from small quantities of DNA and that careful attention to synthetic polyplex structure may be important to optimize efficiency of gene expression in vivo.

Ultra scaledown to predict filtering centrifugation of secreted antibody fragments from fungal broth.

Extracellularly expressed anti-hen egg lysozyme single-chain antibody fragments (scFv) produced by Aspergillus awamori were recovered using filtering centrifugation. Two filtering centrifuges with 0.5- and 30-L capacities were used to represent laboratory- and pilot-scale equipment, respectively. Critical regime analysis using the computational fluid dynamics (CFD) technique provided information about the local energy dissipation rates in both units. Experimental data indicated loss of scFv activity for energy dissipation rates above about 2.0 x 10(4) W kg(-1). This loss of activity increased in the presence of gas-liquid interfaces during filtering centrifugation. An ultra scaledown filtering centrifuge with a maximum working volume of 35 mL was designed to mimic the operating conditions identified by the critical regime analysis for the laboratory- and pilot-plant-scale units. The recovered scFv activity levels and the separation performance of the three units were comparable when operated at equal maximum energy dissipation rates.

Scaleable downstream recovery of nematodes used as biopesticides.

This study assesses the suitability of sieving as a scaleable technique for the separation of adult nematodes from infective juveniles, the latter is an effective bioinsecticide whereas the former is waste material resulting from the fermentation process. Batch and semibatch experiments using conventional flow-assisted wet sieving and a novel cross-flow sieving technique were used to study the separation of juveniles from adult nematodes. The experiments were carried out using small-scale devices and the data were analyzed in terms of the screen effectiveness factor. The results were used to identify the sieve size and operating conditions for optimum juvenile recovery. It was found that, for a given species of nematode, optimum recovery was achieved when sieving was carried out in the cross-flow mode, the maximum recovery being a function of the size of the screen. Industrial-scale self-cleaning equipment capable of large-scale continuous screening was used to confirm the capacity of the small-scale operation for scale-up. Experimental results with this unit showed that in continuous operation sieving time is an additional parameter that influences separation performance.

Biochemical engineering approaches to the challenges of producing pure plasmid DNA.

Plasmid-based genes offer promise for a new generation of vaccines and for gene therapy, but the size and character of plasmids pose new challenges to biochemical engineers. By acknowledging these and using bioprocess-design information based on fundamental studies of the system's properties, it will be possible to create efficient and consistent processes for these materials. This review addresses the purity required, the key issue of the sensitivity of the chromosomal DNA contaminant and larger plasmids to hydrodynamic forces, and the impact of this and other characteristics of plasmids on the recovery and purification of DNA for pharmaceutical purposes.