Methods of enhancing the recovery of plasmid genes from neutralised cell lysate

In this study we have investigated the use of flotation and filtration, singly and combined, to enhance the separation of plasmid containing liquors from neutralised lysates with very different levels of solids. Filtration of crude neutralised lysates, containing roughly 100 g l^у solids, through various diatomaceous earth and cellulose precoat materials was invariably accompanied by severe loss of plasmid through adsorption and/or absorption. The use of more refined and inert filter aids did not alleviate these problems. The finest filter aid, Celatom FP-1SL, gave the best compromise of filtrate clarity (solids content of 0.05 g l^у) and plasmid purity (71%) and was selected for further studies involving combined use of flotation and filtration. Removing the vast bulk of solids prior to filtration by flotation of the floc and draining of the plasmid liquor beneath, impacted dramatically on the filtration performance. Though systematic reductions in the solids challenge per unit filter area were accompanied by increased flux, elevated levels of solids extrusion, chromosomal DNA and protein contamination were also observed, and losses of plasmid to filter aids were still high. We have observed that increasing the scale of operation during lysis and neutralisation from 0.3 or 0.6 l to 15 l is accompanied by significant improvements in separation of cell debris solids from the plasmid and increased recoveries of the plasmid containing liquor. At the latter scale, the drained liquor contained ~80% of the plasmid and the solids content was only 0.2 g l^у.     

Integration of bacteria capture via filtration and in situ lysis for recovery of plasmid DNA under industry-compatible conditions

Combining capture and lysis of the bacteria with partial purification of the plasmid DNA is beneficial for the design of efficient plasmid production processes at larger scale. Such an approach is possible when the bacteria are captured by filtration. Taking industrial requirements into account, however, such a capture requires complex filtration mixtures containing retentive additives such as bentonite and polycations. This makes the straightforward transfer of established lysis protocols to in situ lysis difficult. In this contribution, the different steps of such a protocol are designed for complex filter cakes, including fragilization (by lysozyme), lysis (alkaline pH/acidic pH, 70/37 degrees C, urea/NaCl/Triton), and specific elution (pH, NaCl, CaCl2, guanidinium hydrochloride). Results are compared in regard to plasmid quality (topoisomeric form) and quantity (compared to the yield obtained by a commercial miniprep of a small aliquot of the bacteria suspension from the bioreactor). Best results in these terms were obtained by the Triton lysis protocol performed at 37 degrees C (30 min of contact with a lysis buffer composed of 50 mM Tris pH 8, 1% Triton, 1 g/L lysozyme, and 6 M guanidinium hydrochloride) followed by the specific elution of the plasmid DNA in 50 mM Tris buffer pH 8.     

Sterilizing filtration of plasmid DNA: effects of plasmid concentration, molecular weight, and conformation

Plasmid DNA purification development has been driven by the increased need for large quantities of highly purified, sterile plasmid DNA for clinical studies. Detailed characterization and development of the terminal sterile filtration process step is often limited due to time constraints and the scarcity of sufficient quantities of purified plasmid. However, the large size of the plasmid molecule and variations in conformation can lead to significant yield losses if this process step is not optimized. In this work, the gradual pore-plugging model of flow decay was found to be valid for plasmid DNA by using an ultra scaledown apparatus (1-4 cm(2) filter area). Filtration capacity was found to be insensitive to pressure. Multiple filter types were screened and both source and composition of materials were found to affect filter capacity dramatically. The filter capacity for plasmid was improved by increasing plasmid concentrations as well as by modifying buffer conditions to reduce the apparent size of the plasmid. Filtration capacities varied over a greater than 2 log range when plasmids with sizes ranging from 5.5 to 11 kb and supercoiled plasmid content of 55-95% were explored. Larger plasmids and feeds with lower supercoiled contents led to reduced capacities. These results can be used to define conditions for scale-up of plasmid sterile filtration, as evidenced by processing a 30 g lot using a filtration area of 1,000 cm(2), with a 96% yield, based on filtration capacity data from 4 cm(2) test filters.     

A mass balance study to assess the extent of contaminant removal achieved in the operations for the primary recovery of plasmid DNA from Escherichia coli cells

Mass balances were performed on an alkaline lysis operation for the primary recovery of supercoiled plasmid DNA as part of a process for plasmid gene preparation. Escherichia coli DH5alpha/pSVbeta was cultured in defined medium by fed-batch fermentation and harvested at the end of the exponential phase. Alkaline lysis of the recombinant cells was performed at fixed shear rates ranging between 46 and 461 s(-1), with neutralization 100 and 300 s after the initiation of the lysis. Mass balance calculations were used to optimize the operating conditions for carrying out the alkaline lysis operation. The results indicated that a plasmid yield of 75% and purity with respect to total DNA of 60% were achievable during the primary recovery operation. The influences of key contaminants, including the soluble proteins and the suspended solids, as they bear on the subsequent purification operations, were evaluated and discussed.     

Use of macroporous polypropylene filter to allow identification of bacteria by PCR in human fecal samples

The detection of pathogenic bacteria directly in human fecal specimens by PCR, requires removal of PCR-inhibitory substances. To investigate whether five different macroporous filters (polypropylene, nylon, polyester, polyethylene, fluorocarbon) could retain polysaccharides, major PCR inhibitors, an in vitro model and human fecal samples were used. The in vitro model consisted of Xanthum gum solutions (3 mg/ml PBS), a bacterial polysaccharide, to which Helicobacter pylori cells were added. Fecal samples from healthy volunteers were spiked with H. pylori and Mycobacterium paratuberculosis cells. Polysaccharide concentrations were significantly reduced only by the polypropylene but not by the other filters. Accordingly, both Xanthum gum solutions and spiked fecal specimens became PCR positive only after filtration with the polypropylene filter. We conclude that this filter can be used to prepare a bacterial DNA template suitable for PCR analysis from human feces.     

Proposal for a better integration of bacterial lysis into the production of plasmid DNA at large scale

The paper addresses the question of how to achieve bacterial lysis in large-scale plasmid DNA production processes, where conventional alkaline lysis may become awkward to handle. Bacteria were grown in shaker flasks and a bioreactor. Suboptimal growth conditions were found advantageous for stable plasmid production at high copy numbers (up to 25mg/L could be achieved). Cells were harvested by filtration in the presence of a filter aid. A linear relationship between the biomass and the optimal filter aid concentration in terms of back pressure could be established. Bacteria-containing filter cakes were washed with isotonic buffer and lysis was achieved in situ by a two-step protocol calling for fragilisation of the cells followed by heat lysis in a suitable buffer. RNA and other soluble cell components where washed out of the cake during this step, while the plasmid DNA was retained. Afterwards a clear lysate containing relatively pure plasmid DNA could be eluted from the cake mostly as the desired supercoiled topoisomer, while cell debris and genomic DNA were retained. Lysis is, thus, integrated not only with cell capture but also with a significant degree of isolation/purification, as most impurities were considerably reduced during the procedure.     

Recycling of wood chips and wheat dregs for sludge processing

A Buchner filtration study was conducted to investigate the effect on sludge dewatering of adding organic waste solids (wood chips or wheat dregs) to sludge after chemical preconditioning (with ferric chloride or alum). Increasing the dose of wood chips or wheat dregs enhanced sludge filtration performance and increased the energy content of the filter cake, but did not consistently increase the total filtrate removed. The additional filtrate removal was found to balance the inert solids load only when the chemical preconditioner used did not result in sufficient coagulation of the sludge and the skeleton builder dose was low (< or = 90%). Accordingly, various dose ranges of wood chips and wheat dregs are suggested for different sludge management schemes.     

Capture of bacteria from fermentation broth by body feed filtration: a solved problem?

The direct capture of bacteria produced in high cell density fermentation by filtration is not possible once the milliliter-scale has been surpassed. Filtration in the presence of a filter aid (body feed filtration) constitutes a putative and scalable alternative, but only if conditions proposed by industry for large-scale filtration processes, namely, flow rates (for aqueous solutions) in the range of 500-1,500 L/(m(2) x h) and a filter aid concentration of 相似文献   

Choice of Pore Size Can Introduce Artefacts when Filtering Picoeukaryotes for Molecular Biodiversity Studies

Published results of studies based on samples size fractionated by sequential filtration (e.g. 0.2–3 μm) indicate that many ciliate, dinoflagellate and rhizarian phylotypes are found among marine picoeukaryotes. This is somewhat surprising as these protists are typically known as being large organisms (often >10 μm) and no picoplanktonic species have so far been identified. Here, the abundances of ciliate and dinoflagellate phylotypes in published molecular studies of picoeukaryotes are shown to correlate negatively with the pore size chosen for the end filter in the sequential filtrations (i.e. the filter used to collect the microbial biomass). This suggests that extracellular DNA adhering to small particles may be the source of ciliate and dinoflagellate phylotypes in picoplanktonic size fractions. This hypothesis was confirmed using real-time qPCR, which revealed significantly less dinoflagellate 18S rDNA in a 0.8–3-μm size fraction compared to 0.2–3 μm. On average, the abundance of putative extracellular phylotypes decreased by 84–89 % when a 0.8-?μm end filter was used rather than a 0.2-μm end filter. A 0.8-μm filter is, however, not sufficient to retain all picoeukaryotic cells. Thus, selection of filter pore size involves a trade-off between avoiding artefacts generated by extracellular DNA and sampling the entire picoeukaryotic community. In contrast to ciliate and dinoflagellate phylotypes, rhizarian phylotypes in the picoplankton size range do not display a pattern consistent with an extracellular origin. This is likely due to the documented existence of picoplanktonic swarmer cells within this group.     

Characteristics of flux and gel layer on microfilter and non-woven fabric filter surface based on anoxic–aerobic MBRs

Non-woven fabric filter- (NWFF) and microfilter-MBR modules were made using 100?μm polypropylene and 0.25?μm polyethylene materials, respectively. The performances and mechanisms of the two processes were investigated, including additional batch filtration tests to find the function of the dynamic gel layer on the membrane surface. The HRT of both MBRs was 9?h and the operating permeate flux was 13?L/m(2)/h. The two MBRs consisted of an anoxic and aerobic reactor. The NWFF or microfilter (MF) was submerged in each of the aerobic reactors. The two MBRs showed similar performances for the removal of organic matters, suspended solids and nitrogen. Cake formation on the NWFF contributed to major resistance, while the gel layer on the microfilter or internal fouling of the pores played a key role in the fouling of the membrane surface. The amount of soluble extracellular polymer substances (EPS) (13?mg/L) of the attached sludge on the NWFF surface was larger than that (11?mg/L) of that suspended sludge. Consequently, the functional gel layer for the coarse and microfilter is established based on the relationship among the EPS, transmembrane pressure and MLSS.     

Pathogen safety profile of a 10% IgG preparation manufactured using a depth filtration-modified process

Gamunex^®-C is a highly purified liquid 10% IgG preparation manufactured by a process that includes caprylate precipitation and incubation, and chromatography steps. In the original process, caprylate precipitation was followed by cloth filtration to remove impurities. The highly porous cloth filter has since been replaced with a tight depth filter. The impact of this process modification on pathogen reduction and product is presented.Virus and prion reduction was determined under set-point conditions using scaled-down models of the manufacturing process, and at or outside operating limits to determine robustness. Product protein compositions before and after the process modification were compared directly using manufacturing data.Filtration through a tight depth filter substantially increased nonenveloped virus reduction, and virus reduction was maintained even when a compromised depth filter was used. In addition, prion reduction was improved by about three logs. The product IgG content, purity, and IgG subclass distribution remained comparable to the original cloth filtration process.The replacement of cloth filtration with depth filtration increased the pathogen safety margin of the manufacturing process without impacting the product composition.     

Transport Speed of the Mucous Feeding Filter in Clavelina lepadiformis (Aplousobranchiata,Tunicata)

The transport speed of latex spheres trapped in the mucous feeding filter of 30 specimens of Calvelina lepadiformis (Müller 1776) was measured by microscopical examination and a stop watch. The speed across the gill screen increased from 24 μm s^?1 in small animals to 220 μm s^?1 in large ones. The net translocation speed of the food roll behind the dorsal Lamina was generally much slower but revealed a similar trend (7–78 μm s^?1). The increased feeding filter speed in larger animals was parallelled by an increased branchial circumference in such a way that the duration of water filtration through each part of the filter remained almost constant (between 38 and 56 s as means for all groups of animals). The amount of filter secreted by large animals approached 150 cm² h^?1 or 2.5 m² week^?1. Based on available data on water filtration rates it could be calculated that the speed of water through the filter was of the same order of magnitude as the observed translocation speed of the filter.     

Prefiltration enhances performance of sterile filtration for glycoconjugate vaccines

Recent studies have reported very low capacity during sterile filtration of glycoconjugate vaccines due to rapid fouling of the sterile filter. The objective of this study was to explore the potential for significantly increasing the capacity of the sterile filter through the use of an appropriate prefilter. Data were obtained using prefilters with different pore size and chemistry, with the sterile filtration performed at constant filtrate flux using 0.22 μm nominal pore size Durapore® polyvinylidene difluoride membranes. Prefiltration through 5 μm pore size Durapore® or Nylon prefilters nearly eliminated the fouling of the sterile filter, leading to more than a 100-fold reduction in the rate of pressure increase for the sterile filter. This dramatic improvement in sterile filter performance was due to the removal of large components (greater than 1 μm in size) as confirmed by dynamic light scattering. These results demonstrate the potential of using large pore size prefilters to significantly enhance the performance of the sterile filtration process for the production of important glycoconjugate vaccines.     

Isolation of a single-stranded plasmid from Clostridium acetobutylicum NCIB 6444.

The cryptic plasmid pDM6 was isolated from late exponential-phase cells of Clostridium acetobutylicum NCIB 6444 by either alkaline lysis or electroporation. The application of high voltage during electroporation resulted in higher DNA yield than did the alkaline lysis procedure. However, electroporation-induced plasmid release generated high amounts of single-stranded DNA compared with the alkaline lysis procedure, which generated both double-stranded DNA (monomer and dimer forms) and single-stranded DNA.     

Separation and purification of phycocyanin from Spirulina sp. using a membrane process

The highest purity ratio of phycocyanin extract was obtained when fresh biomass was used as raw material. The crude extract was purified by membrane process using microfiltration and ultrafiltration. Membrane of pore sizes 5 μm, at feed flow rate of 150 mL min⁻¹, permeate flux of 58.5 L h⁻¹ m⁻² was selected for coarse filtration and membrane with pore size 0.8/0.2 μm at the flow rate of 100 mL min⁻¹, permeate flux of 336 L h⁻¹ m⁻² was selected for fine filtration, giving phycocyanin recovery of 88.6% and 82.9%, respectively. For ultrafiltration, membrane with MWCO at 50 kDa, 69 kPa and 75 mL min⁻¹ of flow rate with a mean permeate flux 26.8 L h⁻¹ m⁻² and a retention rate of 99% was found to be optimal. Under these filtration conditions, food grade phycocyanin with the purity around 1.0 containing c-phycocyanin as the major component was obtained.     

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

Plasmid DNA for biopharmaceutical applications is produced easily in Escherichia coli bacteria. The cell lysis is the most crucial step for purification of plasmid DNA. In this paper, we describe a continuous cell alkaline lysis, neutralization, and clarification combination process for production of plasmid pUDK-HGF using hollow fiber ultrafiltration column as a lysis chamber and compare the plasmid DNA yield and homogeneity with the T-connector and manual processes, respectively. The results show that the plasmid pUDK-HGF yield of the combination process is 13% higher than manual lysis, twice higher than using T-connector. When the proportion of lysed cells and neutralization solution is 3:1, the plasmid pUDK-HGF yield can improve by 70%. This process could be easily scaled up to meet the industrial scale for cell lysis.     

Mechanisms for tolerance to diatomaceous earth between strains of Tribolium castaneum

Fourteen strains of Tribolium castaneum (Herbst) (Coleoptera: Tenebrionidae) had mortalities ranging from 5 to 100% when exposed to diatomaceous earth at 600 ppm for seven days. The most tolerant strain had a lethal dose for 50% of the population (LD₅₀) of 413 ppm and the most susceptible strain had a LD₅₀ of 238 ppm. Adults of the tolerant strain were lighter (2.0 mg) than the susceptible strain (2.6 mg). Tolerant adults lost water at lower rate (6 g h^–1 than susceptible adults (12 g h^–1), when held in wheat treated with 600 ppm diatomaceous earth for 24 h, than held at 5% r.h. with no food. Tolerant adults that were not exposed to diatomaceous earth lost water at a lower rate (3 g h^–1) than susceptible adults (5 g h^–1). Both strains, exposed and not exposed to diatomaceous earth died when their water content was between 33 and 37% of their total weight. Insects taken directly from the cultures had 52% (tolerant) and 53% (susceptible) of their total weight as water. Tolerant adults moved slower through grain and across filter paper than susceptible adults. Tolerant adults avoided wheat treated with diatomaceous earth at concentrations as low as 75 ppm, whereas the adults from the susceptible strain did not avoid diatomaceous earth, even at 600 ppm. The consequences of a strain tolerant to diatomaceous earth is discussed with respect to the use of diatomaceous earth to control stored-product insect infestations.     

Mineralization of pentachlorophenol in a contaminated soil by Pseudomonas sp UG30 cells encapsulated in κ-carrageenan

A fermentation process in Escherichia coli for production of supercoiled plasmid DNA for use as a DNA vaccine was developed using an automated feed-back control nutrient feeding strategy based on dissolved oxygen (DO) and pH. The process was further automated through a computer-aided data processing system to regulate the cell growth rate by controlling interactively both the nutrient feed rate and agitation speed based on DO. The process increased the total yield of the plasmid DNA by approximately 10-fold as compared to a manual fed-batch culture. The final cell yield from the automated process reached 60 g L⁻¹ of dry cell weight (OD₆₀₀ = 120) within 24 h. A plasmid DNA yield of 100 mg L⁻¹ (1.7 mg g⁻¹ cell weight) was achieved by using an alkaline cell lysis method. Plasmid yield was confirmed using High Performance Liquid Chromatography (HPLC) analysis. Because cells had been grown under carbon-limiting conditions in the automated process, acetic acid production was minimal (below 0.01 g L⁻¹) throughout the fed-batch stage. In contrast, in the manual process, an acid accumulation rate as high as 0.36 g L⁻¹ was observed, presumably due to the high nutrient feed rates used to maintain a maximum growth rate. The manual fed-batch process produced a low cell density averaging 10–12 g L⁻¹ (OD₆₀₀ = 25–30) and plasmid yields of 5–8 mg L⁻¹ (approximately 0.7 mg g⁻¹ cells). The improved plasmid DNA yields in the DO- and pH-based feed-back controlled process were assumed to be a result of a combination of increased cell density, reduced growth rate (μ) from 0.69 h⁻¹ to 0.13 h⁻¹ and the carbon/nitrogen limitation in the fed-batch stage. The DO- and pH-based feed-back control, fed-batch process has proven itself to be advantageous in regulating cell growth rate to achieve both high cell density and plasmid yield without having to use pure oxygen. The process was reproducible in triplicate fermentations at both 7-L and 80-L scales. Received 22 March 1996/ Accepted in revised form 20 September 1996     

Rapid isolation and sequencing of purified plasmid DNA from Bacillus subtilis.

We report two methods for isolation of plasmid DNA from the gram-positive bacterium Bacillus subtilis. The protoplast alkaline lysis procedure was developed for general use, and the protoplast alkaline lysis magic procedure was developed for isolation of DNA for sequencing. Both procedures yielded large amounts of high-quality DNA in less than 1 h, while current protocols require 4 to 7 h to perform and give lower yields and quality. Plasmid DNA was obtained from strains containing either high- or low-copy-number plasmids. In addition, the procedures were easily adapted to yield large amounts of plasmid DNA suitable for sequencing from another gram-positive organism, Staphylococcus aureus. Further, we demonstrated that neither chloramphenicol, used for plasmid selection, nor the mutation recE4 reduced plasmid DNA yield from the strains we examined.     

Dewatering of phosphorus extracted from liquid swine waste

Phosphorus (P) recovery from liquid swine manure is an attractive technology when on-farm application of liquid swine manure is not an option. We developed a technology that enables separation of this P, but its high moisture content makes transportation difficult. In this work, we investigated dewatering procedures to concentrate the P product. Sludge rich in calcium phosphate (> 20% P2O5) was obtained using a field prototype, and it was further dewatered using a combination of polymer treatment and filter bags. Anionic polyacrylamide polymer treatment (> or = 20 mg/L) was effective to flocculate the P-rich sludge, which enhanced filtration and dewatering. Without polymer, filtration was incomplete due to clogging of filters. Non-woven polypropylene and monofilament filter bag fabrics with mesh size < or = 200 microm retained > 99% of suspended solids and total P. Solids content dramatically increased from about 1.5% to > 90%. These dewatered solids can be transported more economically off the farm for use as a valuable fertilizer material.