Time course of SDS-alkaline lysis of recombinant bacterial cells for plasmid release

SDS-alkaline lysis of recombinant Escherichia coli cell suspensions was carried out in a coaxial cylinder rheometer, and the data were used to establish the time course of lysis reaction. The results of the experiments showed that cell lysis reaction time depended on cell strain but was unaffected by plasmid size and plasmid copy number. The high molecular weight globular proteins and chromosomal DNA were denatured, and the resulting changes in rheometric measurements characterised the denaturation time.     

Removal of contaminant nucleic acids by nitrocellulose filtration during pharmaceutical-grade plasmid DNA processing

Pharmaceutical-grade plasmid DNA for use in vaccines and gene therapy requires the development of reproducible and scaleable downstream processes. Shearing of chromosomal DNA at the commencement of the purification results in fragments that are difficult to separate from supercoiled plasmid DNA. Regulatory standards will probably require that the level of chromosomal DNA contamination is kept below 0.01 mg mg(-1) plasmid DNA. This work reports the use of nitrocellulose membranes to decrease chromosomal DNA contamination in plasmid DNA preparations derived from a 450-l bioreactor. Clarified lysates, resuspended PEG precipitates and anion exchange chromatography elutes were filtered through nitrocellulose. In all the cases, chromosomal DNA was selectively retained by the membrane while most supercoiled plasmid DNA was recovered in the filtrate. Contamination levels dropped from over 27% to below 1% as measured by Southern analysis. Under ionic strength conditions equal to or above 1.5 M NaCl, a fraction of the contaminant RNA was also retained by the nitrocellulose membrane.     

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.     

Biophysical characterization of an integrin-targeted lipopolyplex gene delivery vector

Nonviral gene delivery vectors now show good therapeutic potential: however, detailed characterization of the composition and macromolecular organization of such particles remains a challenge. This paper describes experiments to elucidate the structure of a ternary, targeted, lipopolyplex synthetic vector, the LID complex. This consists of a lipid component, Lipofectin (L) (1:1 DOTMA:DOPE), plasmid DNA (D), and a dual-function, cationic peptide component (I) containing DNA condensation and integrin-targeting sequences. Fluorophore-labeled lipid, peptide, and DNA components were used to formulate the vector, and the stoichiometry of the particles was established by fluorescence correlation spectroscopy (FCS). The size of the complex was measured by FCS, and the sizes of LID, L, LD, and ID complexes were measured by dynamic light scattering (DLS). Fluorescence quenching experiments and freeze-fracture electron microscopy were then used to demonstrate the arrangement of the lipid, peptide, and DNA components within the complex. These experiments showed that the cationic portion of the peptide, I, interacts with the plasmid DNA, resulting in a tightly condensed DNA-peptide inner core; this is surrounded by a disordered lipid layer, from which the integrin-targeting sequence of the peptide partially protrudes.     

Targeting lipopolyplexes using bifunctional peptides incorporating hydrophobic spacer amino acids: synthesis, transfection, and biophysical studies

We have developed efficient synthetic routes to two hydrophobic amino acids, suitably protected for solid-phase peptide synthesis, and have successfully synthesized peptides containing these or other hydrophobic amino acids as spacers between a Lys16 moiety and an integrin-targeting motif. These peptides have in turn been used to formulate a range of lipopolyplex vectors with Lipofectin and plasmid DNA. The transfection efficiencies of these vectors and their aggregation behavior in buffers and in serum have been studied. We have shown that vectors containing peptides incorporating long linkers that are entirely hydrophobic are less efficient transfection agents. However, linkers of equivalent length that are in part hydrophobic show improved transfection properties, which is probably due to the improved accessibility of the integrin-binding motif.     

Design,construction, and optimization of a novel,modular, and scalable incubation chamber for continuous viral inactivation

We designed, built or 3D printed, and screened tubular reactors that minimize axial dispersion to serve as incubation chambers for continuous virus inactivation of biological products. Empirical residence time distribution data were used to derive each tubular design's volume equivalent to a theoretical plate (VETP) values at a various process flow rates. One design, the Jig in a Box (JIB), yielded the lowest VETP, indicating optimal radial mixing and minimal axial dispersion. A minimum residence time (MRT) approach was employed, where the MRT is the minimum time the product spends in the tubular reactor. This incubation time is typically 60 minutes in a batch process. We provide recommendations for combinations of flow rates and device dimensions for operation of the JIB connected in series that will meet a 60‐min MRT. The results show that under a wide range of flow rates and corresponding volumes, it takes 75 ± 3 min for 99% of the product to exit the reactor while meeting the 60‐min MRT criterion and fulfilling the constraint of keeping a differential pressure drop under 5 psi. Under these conditions, the VETP increases slightly from 3 to 5 mL though the number of theoretical plates stays constant at about 1326 ± 88. We also demonstrated that the final design volume was only 6% ± 1% larger than the ideal plug flow volume. Using such a device would enable continuous viral inactivation in a truly continuous process or in the effluent of a batch chromatography column. Viral inactivation studies would be required to validate such a design. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:954–965, 2017     

Effect of shear on plasmid DNA in solution

This study was designed to evaluate the effect of shear on the supercoiled circular (SC) form of plasmid DNA. The conditions chosen are representative of those occurring during the processing of plasmid-based genes for gene therapy and DNA vaccination. Controlled shear was generated using a capillary rheometer and a rotating disk shear device. Plasmid DNA was tested in a clarified alkaline lysate solution. This chemical environment is characteristic of the early stages of plasmid purification. Quantitative data is reported on shear degradation of three homologous recombinant plasmids of 13, 20 and 29 kb in size. Shear sensitivity increased dramatically with plasmid molecular weight. Ultrapure plasmid DNA redissolved in 10 mM Tris/HCl, 1 mM EDTA pH 8 (TE buffer) was subjected to shear using the capillary rheometer. The shear sensitivity of the three plasmids was similar to that observed for the same plasmids in the clarified alkaline lysate. Further experiments were carried out using the 20 kb plasmid and the rotating disk shear device. In contrast with the capillary rheometer data, ultrapure DNA redissolved in TE buffer was up to eight times more sensitive to shear compared to plasmid DNA in the clarified alkaline lysate. However, this enhanced sensitivity decreased when the ionic strength of the solution was raised by the addition of NaCl to 150 mM. In addition, shear damage was found to be independent of plasmid DNA concentration in the range from 0.2 7g/ml to 20 7g/ml. The combination of shear and air-liquid interfaces caused extensive degradation of the plasmid DNA. The damage was more evident at low ionic strength and low DNA concentration. These findings show that the tertiary structure of plasmid DNA can be severely affected by shear forces. The extent of damage was found to be critically dependent on plasmid size and the ionic strength of the environment. The interaction of shear with air-liquid interfaces shows the highest potential for damaging SC plasmid DNA during bioprocesses.     

Sex-specific paternal age effects on offspring quality in Drosophila melanogaster

Advanced paternal age has been repeatedly shown to modulate offspring quality via male- and/or female-driven processes, and there are theoretical reasons to expect that some of these effects can be sex-specific. For example, sex allocation theory predicts that, when mated with low-condition males, mothers should invest more in their daughters compared to their sons. This is because male fitness is generally more condition-dependent and more variable than female fitness, which makes it less risky to invest in female offspring. Here, we explore whether paternal age can affect the quality and quantity of offspring in a sex-specific way using Drosophila melanogaster as a model organism. In order to understand the contribution of male-driven processes on paternal age effects, we also measured the seminal vesicle size of young and older males and explored its relationship with reproductive success and offspring quality. Older males had lower competitive reproductive success, as expected, but there was no difference between the offspring sex ratio of young and older males. However, we found that paternal age caused an increase in offspring quality (i.e., offspring weight), and that this increase was more marked in daughters than sons. We discuss different male- and female-driven processes that may explain such sex-specific paternal age effects.     

High pressure disruption of yeast cells: The use of scale down operations for the prediction of protein release and cell debris size distribution

Experiments were carried out aimed at establishing the effects of equipment scale down on the disruption of Baker's yeast cells in high pressure homogenisers. Data are reported on the cell debris particle size distribution (PSD) and on total protein release as a function of the applied pressure for two valve geometries and three scales of operation covering flow rates of 28, 60 and 280 L/h. A comparison of the results from the experiments indicates that over the range of parameters investigated both the total protein release and the cell debris PSDs are independent of valve geometry and flow rate through the homogeniser. These observations are discussed in the light of relevant previous publications. The cell debris PSDs have been simulated by using a recently published model and the total protein release data are described by the well-established Hetherington expression (Hetherington et al., 1971). (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 55: 642-649, 1997.     

The effects of fermentation conditions on yeast cell debris particle size distribution during high pressure homogenisation

Experiments were performed to characterize the particle size distribution of bakers' yeast cells during high pressure homogenisation. Results were obtained for mechanically agitated batch and continuously grown cultures under a range of operating conditions. It was found that the dependency of cell debris size distribution on the number of passes through the homogeniser and the homogeniser pressure was independent of the cell properties and culture conditions, but for a fixed pressure and number of passes the extent of disruption was strongly affected by the operating conditions in the fermenter. The entire cell debris size distributions were successfully simulated using the mean and variance of the distributions and a previously published model equation which related these parameters to the operating pressure and number of passes through the homogeniser.List of Symbols k breakage coefficient in Eq. 1 - d cell diameter - d ₅₀ median diameter of homogenate size distribution - d ₅₀ dimensionless d ₅₀ defined as - D dilution rate - F(d _NP) cumulative undersize distribution (volume basis) - N number of passes - P total pressure - P _threshold threshold pressure - P (P-P _threshold) - w Boltzmann parameter, Eq. 4 - w dimensionless standard deviation defined as Greek Letters exponent in Eq. 1 - exponent in Eq. 1 UCL is the Biotechnology and Biological Sciences Research Council's Interdisciplinary Research Centre for Biochemical Engineering and the Council's support to the participating UCL departments is gratefully acknowledged. The provision of continuous fermentation material from Dr. M. Gregory, Process System Engineering IRC, is gratefully acknowledged.