Purification of plasmid DNA by tangential flow filtration

A simple, scalable method for purification of plasmid DNA is described. The method includes modification of the classical alkaline-lysis-based plasmid extraction method by extending the solubilization step from less than 30 min to 24 h. The extraction is followed by the novel use of tangential flow filtration (TFF) for purification of the remaining contaminants. The method does not include the use of any organic solvents, RNase, high-speed centrifugation, or column chromatography steps. The method typically yields 15 to 20 mg of plasmid DNA per liter of bacterial culture and results in removal of >99% of RNA and >95% of the protein that remains after the modified alkaline lysis procedure. The procedure has been demonstrated to be effective in the isolation of seven different plasmids. Plasmids isolated using this method had comparable transfection capability relative to plasmid isolated using a classical, cesium chloride gradient-based method.     

Purification of hemoglobin by tangential flow filtration with diafiltration

A recent study by Palmer, Sun, and Harris (Biotechnol. Prog., 25:189–199, 2009) demonstrated that tangential flow filtration (TFF) can be used to produce HPLC‐grade bovine and human hemoglobin (Hb). In this current study, we assessed the quality of bovine Hb (bHb) purified by introducing a 10 L batch‐mode diafiltration step to the previously mentioned TFF Hb purification process. The bHb was purified from bovine red blood cells (RBCs) by filtering clarified RBC lysate through 50 nm (stage I) and 500 kDa (stage II) hollow fiber (HF) membranes. The filtrate was then passed through a 100 kDa (stage III) HF membrane with or without an additional 10 L diafiltration step to potentially remove additional small molecular weight impurities. Protein assays, SDS‐PAGE, and LC‐MS of the purified bHb (stage III retentate) reveal that addition of a diafiltration step has no effect on bHb purity or yield; however, it does increase the methemoglobin level and oxygen affinity of purified bHb. Therefore, we conclude that no additional benefit is gained from diafiltration at stage III and a three stage TFF process is sufficient to produce HPLC‐grade bHb. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009     

Upscaling of lentiviral vector production by tangential flow filtration

BACKGROUND: HIV-1-derived vectors are promising tools for gene transfer into the brain. Application of these vectors for gene therapy or for the creation of animal models for neurodegenerative diseases requires standardization and upscaling of lentiviral vector production methods. METHODS: In this study, serum-free HIV-1 vector production was efficiently upscaled by use of cell factories and the introduction of tangential flow filtration (TFF) prior to centrifugation. RESULTS: Vector titers (TU/ml) and p24 values (pg p24/ml) for a serum-free HIV-1 vector produced in cell factories and using TFF prior to centrifugation were comparable to those of small-scale productions. TFF allowed a 66-fold concentration of the vectors with complete vector recovery. Further concentration of the vector (30-fold) was achieved either by low-speed centrifugation or by ultracentrifugation. Combination of TFF and ultracentrifugation resulted in a vector recovery of 90-100% and titers that increased 1800-fold and 900-fold for transducing units and p24 concentration, respectively. CONCLUSIONS: With this new standardized method for lentiviral vector production and concentration, 1 ml of concentrated vector is routinely produced with titers of 10(9)-10(10) TU/ml starting from 2 l of cell-culture medium. Moreover, stereotactic injection of this vector in mouse striatum resulted in a large transduced brain volume in the absence of any immune response.     

Industrial scale harvest of proteins from mammalian cell culture by tangential flow filtration

An industrial-scale methods for harvest of biologically active proteins form mammalian cell culture has been developed using tangential flow filtration. A robust and economical process capable of processing approximately 5000 L conditioned media/h with protein yields in excess of 99% has been achieved. A completely contained system has been designed in which total cell number and viability are maintained throughout the process. The process has successfully been implemented at 1.25 x 10(4) L scale for the recovery of kilogram quantities of pharmaceutical proteins such as recombinant tissue type plasminogen activator (rt-PA).     

Concentration of viruses and dissolved DNA from aquatic environments by vortex flow filtration.

Vortex flow filtration (VFF) was used to concentrate viruses and dissolved DNA from freshwater and seawater samples taken in Florida, the Gulf of Mexico, and the Bahamas Bank. Recoveries of T2 phage and calf thymus DNA added to artificial seawater and concentrated by VFF were 72.8 and 80%, respectively. Virus concentrations determined by transmission electron microscopy of VFF-concentrated samples ranged from 3.4 x 10(7)/ml for a eutrophic Tampa Bay sample to 2.4 x 10(5) for an oligotrophic oceanic surface sample from the southeastern Gulf of Mexico. Viruslike particles were also observed in a sample taken from a depth of 1,500 m in the subtropical North Atlantic Ocean. Filtration of samples through Nuclepore or Durapore filters (pore size, 0.2 micron) prior to VFF reduced phage counts by an average of two-thirds. Measurement of dissolved-DNA content by Hoechst 33258 fluorescence in environmental samples concentrated by VFF yielded values only ca. 35% of those obtained for samples concentrated by ethanol precipitation (the standard dissolved-DNA method). However, ethanol precipitation of VFF-concentrated extracts resulted in an increase in measurable DNA, reaching 80% of the value obtained by the standard method. These results indicate that a portion of the naturally occurring dissolved DNA is in a form inaccessible to nucleases and Hoechst stain, perhaps bound to protein or other polymeric material, and is released upon ethanol precipitation. Viral DNA contents estimated from viral counts averaged only 3.7% (range, 0.9 to 12.3%) of the total dissolved DNA for samples from freshwater, estuarine, and offshore oligotrophic environments.(ABSTRACT TRUNCATED AT 250 WORDS)     

Concentration of viruses and dissolved DNA from aquatic environments by vortex flow filtration.

Vortex flow filtration (VFF) was used to concentrate viruses and dissolved DNA from freshwater and seawater samples taken in Florida, the Gulf of Mexico, and the Bahamas Bank. Recoveries of T2 phage and calf thymus DNA added to artificial seawater and concentrated by VFF were 72.8 and 80%, respectively. Virus concentrations determined by transmission electron microscopy of VFF-concentrated samples ranged from 3.4 x 10(7)/ml for a eutrophic Tampa Bay sample to 2.4 x 10(5) for an oligotrophic oceanic surface sample from the southeastern Gulf of Mexico. Viruslike particles were also observed in a sample taken from a depth of 1,500 m in the subtropical North Atlantic Ocean. Filtration of samples through Nuclepore or Durapore filters (pore size, 0.2 micron) prior to VFF reduced phage counts by an average of two-thirds. Measurement of dissolved-DNA content by Hoechst 33258 fluorescence in environmental samples concentrated by VFF yielded values only ca. 35% of those obtained for samples concentrated by ethanol precipitation (the standard dissolved-DNA method). However, ethanol precipitation of VFF-concentrated extracts resulted in an increase in measurable DNA, reaching 80% of the value obtained by the standard method. These results indicate that a portion of the naturally occurring dissolved DNA is in a form inaccessible to nucleases and Hoechst stain, perhaps bound to protein or other polymeric material, and is released upon ethanol precipitation. Viral DNA contents estimated from viral counts averaged only 3.7% (range, 0.9 to 12.3%) of the total dissolved DNA for samples from freshwater, estuarine, and offshore oligotrophic environments.(ABSTRACT TRUNCATED AT 250 WORDS)     

Large-scale preparation of bacterial cell membranes by tangential flow filtration

The preparation of cell membranes by ultracentrifugation of bacterial cell lysates, a pre-requisite for the purification of over-expressed membrane proteins, is both time-consuming and difficult to perform on a large scale. To overcome this bottleneck in the structural investigation of such proteins in the UK Membrane Protein Structure Initiative, we have investigated the alternative use of tangential flow filtration for preparation of membranes from Escherichia coli. This method proved to be superior to the conventional use of ultracentrifuges both in speed and in yield of membrane protein. Moreover, it could more readily be scaled up to process larger quantities of bacterial cells. Comparison of the purity and monodispersity of an over-expressed membrane protein purified from conventionally-prepared membranes and from membranes prepared by filtration revealed no substantial differences. The approach described should therefore be of general use for membrane protein preparation for a wide range of applications, including both structural and functional studies.     

Recycle use of immobilized glucoamylase by tangential flow filtration unit

Various properties of glucoamylase immobilized onto corn stover supporting material and separation of immobilized enzyme by tangential flow filtration unit were studied. Optimum pH and temperature of immobilized enzyme were 3.5 and 60 degrees C, respectively. Enzyme stability was studied in a packed-bed column. The starch conversion rate was attained at 81% for 15 days; after that, the hydrolysis rate gradually decreased. Size of supporting material proved to be an important factor, with higher activity and good loading yield resulting from smaller supporting material. Glucoamylase immobilized onto supporting material less than 44 mum was used for hydrolysis of 10% soluble starch at pH 3.5 and 40 degrees C for 3 h. Then immobilized glucoamylase was separated from the product by means of a tangential flow filtration unit using a 0.2-mum pore size Nylon 66 membrane filter. This operation was continued until 180 ml filtrate was obtained from a 260-mL starting volume. Then, the next batch was started by adding 180 mL starch substrate into the reactor. The batchwise experiments were repeated 20 times. The average filtration rate of each batch was determined and found to sharply decline during the first four batches. Thereafter, it gradually decreased from batch to batch. The cause of decreasing filtration rate appeared to be due to retrogradation of starch. The percentage of starch hydrolysis within 20 batches was in the range 89-96%. The filtration rate becomes higher if the hydrolyzation time is extended to 14 h. Resistance to filtration was also investigated. Almost all of the total resistance is related to insoluble materials, with the significant part of this from the resistance due to insoluble materials deposited on a surface of membrane and boundary layer resistance. Using a microscopic method, no microorganisms were found in the filtrate.     

Concentration of live pico- and nanoplankton by means of tangential flow filtration

A tangential flow filtration system for concentrating live pico-and nanoplankton was tested on natural seawater (<20 µm)from Kiel Bight. Twenty- to thirty-fold concentration of samplesranging from 79 to 152 dm³ was easily achieved, the time necessarydepending on sample volume, pumping pressure and filtrate/retentateratio. Losses of different kinds of cells in relation to theconcentration factor were quantified. Recovery rates were negativelycorrelated with the frequency of sample recirculation throughthe filtration unit.     

Loss of pico- and nanoplankton cells during concentration by tangential flow filtration

The efficiency of a tangential flow filtration device to concentratepico- and nanoplankton particles in sea water uniformly overa range of concentrations was evaluated. To calculate the expenmentalrecovery rates for each concentration factor, we measured theincrease in chlorophyll concentration, number of total particles(Coulter counter) and number of autotrophic cells (flow cytometry).The experimental recovery rates for each stage of volume reductionin the concentration process were expressed as percentages ofthe expected theoretical values. Significant losses of cellularmaterial (low recovery rates) occurred between the retentateseawater tank and the seawater filtrate tank. Similar lossesoccurred in repetitive closed-circuit recirculation experimentswhen the circulating volume of sea water was kept constant.The cause of the apparent losses of material appeared to bemechanical fragmentation of organisms during the pumping andfiltering stages; however, experiments with latex beads showedthat most of the missing beads were retained within the filtrationelements. The low efficiencies and extremely varied recoveryrates of tangential flow filtrations measured under the experimentalconditions and for the different types of particles used inthis work preclude its use for quantitative concentrations ofnatural assemblages of particulate material in sea water.     

Purification of infectious adenovirus in two hours by ultracentrifugation and tangential flow filtration

Adenoviruses are excellent vectors for gene transfer and are used extensively for high-level expression of the products of transgenes in living cells. The development of simple and rapid methods for the purification of stable infectious recombinant adenoviruses (rAds) remains a challenge. We report here a method for the purification of infectious adenovirus type 5 (Ad5) that involves ultracentrifugation on a cesium chloride gradient at 604,000g for 15 min at 4 degrees C and tangential flow filtration. The entire procedure requires less than two hours and infectious Ad5 can be recovered at levels higher than 64% of the number of plaque-forming units (pfu) in the initial crude preparation of viruses. We have obtained titers of infectious purified Ad5 of 1.35x10(10) pfu/ml and a ratio of particle titer to infectious titer of seven. The method described here allows the rapid purification of rAds for studies of gene function in vivo and in vitro, as well as the rapid purification of Ad5.     

Intramodule pressure profiles and protein accumulation during tangential flow filtration

Tangential flow filtration (TFF) through a 30 kDa nominal molecular weight cut-off (MWCO) ultrafiltration membrane is widely employed to concentrate purified monoclonal antibodies (mAbs) to levels required for their formulation into injectable biologics. While TFF has been used to remove casein from milk for cheese production for over 35 years, and in pharmaceutical manufacture of biotherapeutic proteins for 20 years, the rapid decline in filtration rate (i.e., flux) at high protein concentrations is a limitation that still needs to be addressed. This is particularly important for mAbs, many of which are 140–160 kDa immunoglobulin G (IgG) type proteins recovered at concentrations of 200 mg/mL or higher. This work reports the direct measurement of local transmembrane pressure drops and off-line confocal imaging of protein accumulation in stagnant regions on the surface of a 30 kDa regenerated cellulose membrane in a flat-sheet configuration widely used in manufacture of biotherapeutic proteins. These first-of-a-kind measurements using 150 kDa bovine IgG show that while axial pressure decreases by 58 psi across a process membrane cassette, the decrease in transmembrane pressure drop is constant at about 1.2 psi/cm along the 20.7 cm length of the membrane. Confocal laser scanning microscopy of the membrane surface at the completion of runs where retentate protein concentration exceeds 200 mg/mL, shows a 50 μm thick protein layer is uniformly deposited. The localized measurements made possible by the modified membrane system confirm the role of protein deposition on limiting ultrafiltration rate and indicate possible targets for improving membrane performance.     

Cyanobacterial assimilatory nitrate reductase gene diversity in coastal and oligotrophic marine environments

Cyanobacteria are important primary producers in many marine ecosystems and their abundances and growth rates depend on their ability to assimilate various nitrogen sources. To examine the diversity of nitrate-utilizing marine cyanobacteria, we developed PCR primers specific for cyanobacterial assimilatory nitrate reductase (narB) genes. We obtained amplification products from diverse strains of cultivated cyanobacteria and from several marine environments. Phylogenetic trees constructed with the narB gene are congruent with those based on ribosomal RNA genes and RNA polymerase genes. Analysis of sequence library data from coastal and oligotrophic marine environments shows distinct groups of Synechococcus sp. in each environment; some of which are represented by sequences from cultivated organisms and others that are unrelated to known sequences and likely represent novel phylogenetic groups. We observed spatial differences in the distribution of sequences between two sites in Monterey Bay and differences in the vertical distribution of sequence types at the Hawai'i Ocean Time-series Station ALOHA, suggesting that nitrogen assimilation in Synechococcus living in different ecological niches can be followed with the nitrate reductase gene.     

Release of periplasmic enzymes fromEscherichia coli using tangential flow filtration

Summary Escherichia coli cells producing the periplasmic enzymes nuclease, -lactamase and alkaline phosphatase were exposed to osmotic shock treatment in a closed system consisting of a tank, a pump and a filtration unit. The enzymes were released by circulating the cell suspensions in the filtration system and separated from cells and spheroplasts by filtration. This novel releasing method was shown to be equally effective as osmotic shock treatments performed by agitation and centrifugation. Since the extraction is performed in a closed system, aerosol formation is avoided. In addition the method may easily be scaled up.     

Investigation of the parameters affecting the separation of bacterial enzymes from cell debris by tangential flow filtration

The effect of organism, enzyme, method of cell breakage and membrane characteristics on the separation of bacterial enzymes from cell debris by tangential flow filtration has been studied. The effectiveness of separation was assessed by process time, enzyme yield and specific activity, and turbidity of the filtrate. For a particular organism and enzyme, method of cell breakage and membrane characteristics significantly influenced separation performance, though results indicate that it is not yet possible to optimize all aspects of performance simultaneously.     

Studies on the separation of intracellular soluble enzymes from bacterial cell debris by tangential flow membrane filtration

Summary The recoveries of genetically engineered human growth hormone (hGH), carboxypeptidase and -galactosidase, fromE. coli broths have been studied, using the process of microfiltration. The suspension superficial velocity was found to significantly influence the yields obtained. However, the molecular weight of the product could be an important factor in microfiltration. Simple washing procedures seem to be adequate for effective rejuvenation of the membrane.     

Understanding and modeling alternating tangential flow filtration for perfusion cell culture

Alternating tangential flow (ATF) filtration has been used with success in the Biopharmaceutical industry as a lower shear technology for cell retention with perfusion cultures. The ATF system is different than tangential flow filtration; however, in that reverse flow is used once per cycle as a means to minimize fouling. Few studies have been reported in the literature that evaluates ATF and how key system variables affect the rate at which ATF filters foul. In this study, an experimental setup was devised that allowed for determination of the time it took for fouling to occur for given mammalian (PER.C6) cell culture cell densities and viabilities as permeate flow rate and antifoam concentration was varied. The experimental results indicate, in accordance with D'Arcy's law, that the average resistance to permeate flow (across a cycle of operation) increases as biological material deposits on the membrane. Scanning electron microscope images of the post‐run filtration surface indicated that both cells and antifoam micelles deposit on the membrane. A unique mathematical model, based on the assumption that fouling was due to pore blockage from the cells and micelles in combination, was devised that allowed for estimation of sticking factors for the cells and the micelles on the membrane. This model was then used to accurately predict the increase in transmembane pressure during constant flux operation for an ATF cartridge used for perfusion cell culture. © 2014 American Institute of Chemical Engineers Biotechnol. Prog., 30:1291–1300, 2014     

Purification of plasmid DNA by an integrated operation comprising tangential flow filtration and nitrocellulose adsorption

There is an increasing interest in the development of scaleable and reproducible plasmid DNA purification protocols for vaccine and gene therapy. The use of an integrated unit operation, comprising tangential flow microfiltration coupled with the adsorption of contaminants onto nitrocellulose membranes as a single processing step was examined in this work. Experiments were performed using a custom-built tangential flow microfiltration rig (membrane area=12.5 cm(2)). Tangential flow filtration-adsorption of E. coli lysates containing a plasmid product removed most solids (>75%) and decreased chromosomal DNA contamination by 75% w/w. Total plasmid DNA concentration and supercoiled content of the permeate were virtually identical to those of the feed, indicating a recovery yield of 100% (transmission equal to 1). Results were similar for E. coli lysates containing either a 6.9 kb or a 20 kb plasmid. Significant reductions in RNA, endotoxin, and protein levels were also observed. The reproducibility and potential for scale up of this integrated filtration-adsorption operation makes it at attractive option for intermediate- to large-scale pharmaceutical-grade plasmid processing.     

Tangential flow filtration and preliminary phylogenetic analysis of marine picoplankton.

A procedure was developed for harvesting gram quantities of microbial biomass from oligotrophic waters, when mixed populations are present in low abundance. Picoplankton from Atlantic Ocean (Hydrostation S, Sargasso Sea) and Pacific Ocean (Aloha Station) sites were collected in a three-stage process: (i) collection of seawater through an intake covered with 10-microns-pore Nytex; (ii) concentration by a tangential flow filtration device equipped with 10 ft2 (0.929 m2) of 0.1-micron-pore fluorocarbon membrane; (iii) collection of cells from concentrate by centrifugation. The overall efficiency of picoplankton recovery was at least 37%. The cellular morphotypes recovered matched those of the original population. DNA was prepared from frozen cell pellets by enzymatic digestion, solvent extraction, and isopycnic centrifugation. As indicated by the binding of kingdom-specific hybridization probes to the purified DNA, the Sargasso Sea picoplankton in this collection were largely eubacteria.